EP1326600A2 - Mediateurs de voies de signalisation de hedgehog, compositions et utilisations associees - Google Patents

Mediateurs de voies de signalisation de hedgehog, compositions et utilisations associees

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Publication number
EP1326600A2
EP1326600A2 EP01979788A EP01979788A EP1326600A2 EP 1326600 A2 EP1326600 A2 EP 1326600A2 EP 01979788 A EP01979788 A EP 01979788A EP 01979788 A EP01979788 A EP 01979788A EP 1326600 A2 EP1326600 A2 EP 1326600A2
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EP
European Patent Office
Prior art keywords
formulation
occurrence
independently
hedgehog
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01979788A
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German (de)
English (en)
Inventor
Anthony D. Baxter
Edward A. Boyd
Oivin M. Guicherit
Stephen Price
Lee D. Rubin
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Curis Inc
Original Assignee
Curis Inc
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Priority claimed from PCT/US2001/032054 external-priority patent/WO2002030421A2/fr
Publication of EP1326600A2 publication Critical patent/EP1326600A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil

Definitions

  • 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.
  • 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).
  • inductions usually 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.
  • 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).
  • Hedgehog family of signaling molecules mediate many important short- and long-range patterning processes during invertebrate and vertebrate development.
  • a single hedgehog gene regulates segmental and imaginal disc patterning.
  • a hedgehog gene family is involved in the control of left-right asymmetry, polarity in the CNS, somites and limb, organogenesis, chondrogenesis and spermatogenesis.
  • the first hedgehog gene was identified by a genetic screen in the fruitfly Drosophila melanogaster (N ⁇ sslein-Nolhard, 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.
  • the vertebrate family of hedgehog genes includes at least four members, e.g., paralogs of the single drosophila hedgehog gene.
  • Exemplary hedgehog genes and proteins are described in PCT publications WO 95/18856 and WO 96/17924.
  • Three of these members herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds, and mammals.
  • a fourth member, herein referred to as tiggie-winkle hedgehog (Thh) appears specific to fish.
  • Desert hedgehog (Dhh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (Ihh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Shh, which as described above, is primarily involved in morphogenic and neuroinductive activities.
  • 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.
  • 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.
  • HH has been implicated in short- and long-range patterning processes at various sites during Drosophila development. In the establishment of segment polarity in early embryos, it has short-range effects which appear to be directly mediated, while in the patterning of the imaginal discs, it induces long range effects via the induction of secondary signals.
  • 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) CeU 75:1401-1416 ) , and the shield in the zebrafish (Eldcer et al. (1995) supra; Krauss, S. et ⁇ /.(1993) Cell 75:1431-1444).
  • Shh from the notochord and the floorplate appears to induce ventral cell fates.
  • 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
  • zebrafish Eldcer et al. (1995) supra; Krauss et al.
  • Shh protein induces floorplate and motor neuron development with distinct concentration thresholds, floor plate at high and motor neurons at lower concentrations (Roelink et al. (1995) supra; Marti et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol. 5:651-658).
  • antibody blocking suggests that Shh produced by the notochord is required for notochord-mediated induction of motor neuron fates (Marti et al. (1995) supra).
  • 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.
  • 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).
  • Shh promotes the expression of sclerotome specific markers like Paxl and Twist, at the expense of the dermamyotomal marker Pax3.
  • 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).
  • Shh also induces myotomal gene expression (Hammerschmidt et al. (1996) supra: Johnson, R.L. et al. (1994) Cell 79:1165-1173; M ⁇ nsterberg, 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 (M ⁇ nsterberg et al. (1995) supra).
  • ZPA Zero of polarizing activity
  • Hedgehog proteins and BMPs are likely to have been conserved at many, but probably not all sites of vertebrate Hedgehog expression.
  • Shh has been shown to induce the expression of Bmp4, another vertebrate dpp homologue (Roberts, D.J. et al (1995) Development 121:3163-3174).
  • Bmp4 another vertebrate dpp homologue
  • Shh and Bmp2, 4, or 6 show a striking correlation in their expression in epithelial and mesenchymal cells of the stomach, the urogenital system, the lung, the tooth buds and the hair follicles (Bitgood, M.J. and McMahon, A.P. (1995) Dev. Biol. 172:126-138).
  • 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).
  • 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.
  • Patched was originally identified in Drosophila as a segment polarity gene, one of a group of developmental genes that affect cell differentiation within the individual segments that occur in a homologous series along the anterior-posterior axis of the embryo. See Hooper, J.E. et al. (1989) Cell 59:751; and Nalcano, Y. et al. (1989) Nature 341:508. Patterns of expression of the vertebrate homologue of patched suggest its involvement in the development of neural tube, skeleton, limbs, craniofacial structure, and sldn. Genetic and functional studies demonstrate that patched is part of the hedgehog signaling cascade, an evolutionarily conserved pathway that regulates expression of a number of downstream genes.
  • Patched participates in the constitutive transcriptional repression of the target genes; its effect is opposed by a secreted glycoprotein, encoded by hedgehog, or a vertebrate homologue, which induces transcriptional activation. Genes under control of this pathway include members of the Wnt and TGF-beta families.
  • BCNS basal cell nevus syndrome
  • BCNS also predisposes to multiple tumor types, the most frequent being basal cell carcinomas (BCC) that occur in many locations on the body and appear within the first two decades of life. Most cases of BCC, however, are unrelated to the syndrome and arise sporadically in small numbers on sun-exposed sites of middle-aged or older people of northern European ancestry.
  • BCC basal cell carcinomas
  • the present invention makes available methods and reagents for inhibiting activation of the hedgehog signaling pathway, e.g., to inhibit aberrant growth states resulting from phenotypes such as ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function, comprising contacting the cell with an agent, such as a small molecule, in a sufficient amount to agonize a normal ptc activity, antagonize a normal hedgehog activity, or antagonize smoothened activity, e.g., to reverse or control the aberrant growth state.
  • an agent such as a small molecule
  • Figures 1-31 depict reactions useful for synthesizing compounds according to the present invention.
  • Figure 32a-o illustrates representative compounds according to the present invention.
  • Figure 33 A shows gli-1 mRNA expression in cells treated with vehicle
  • Figure 33B demonstrates that D and jervine inhibited the gli-1 mRNA levels as measured by quantitative real-time PCR.
  • FIG 34A shows that adding Shh protein to cultured sldn explants resulted in ptc activation as indicated by the blue staining of these cultures (X-gal). Histology samples show intensely stained cells with basophilic nuclei and a high nucleus to cytoplasm ratio (H&E [lOx] and H&E [40x]). These structures resemble BCCs in that they are arranged in clusters throughout the dermal layer and are separated by palisades of normal appearing dermal cells. Blue staining indicates that the Patched pathway was active in cells within the BCC-like structures (Eosin+X-gal).
  • Figure 34B illustrates that BCC-like clusters, one of which is indicated by the arrow, in the mouse skin punch expressed keratin- 14 (brown reaction product), a marker of undifferentiated keratinocytes. Undifferentiated basal cells in the epidermis were also keratin- 14-positive. Human BCCs are reported to express keratin- 14.
  • Figure 35A demonstrates that increasing concentrations of D are associated with a dose-dependent decrease in the amount of lacZ reporter enzyme activity. Lower levels of lacZ activity are indicative of decreased Patched pathway activity in the presence of Shh protein.
  • Figure 35B shows staining of D-treated explants and demonstrates that 0.2 ⁇ M D decreased X-gal staining compared with the intense X-gal staining of skin punches treated with Shh protein alone, indicating the downregulation of the expression of t e ptc gene.
  • Figure 35C portrays histology samples of sldn punches treated with D (bottom row), suggesting that treatment inhibited the appearance of Shh-induced BCC-like structures.
  • Figure 36 depicts that skin punches treated for 6 days with exogenous Shh protein alone showed intense X-gal staining compared with those treated with vehicle alone (top row). Skin punches pretreated with D at 10, 20 and 50 ⁇ M for 5 hours before being exposed to exogenous Shh protein demonstrated complete inhibition of Shh protein-induced upregulation of the Patched pathway (bottom row — 3 slides on the right). No inhibition was seen when the skin punches pretreated with vehicle were exposed to exogenous Shh protein, as shown by intense X-gal staining (bottom row on the left).
  • Figure 37B illustrates that after 2 days of exposure to 5 ⁇ M D (right) or vehicle (left), apoptotic nuclei, indicated by the brown color in the slides on the right, appeared within the BCC-like structures.
  • Figure 38A demonstrates that short-term treatment with D reduced the amount of X-gal staining, suggesting a downregulation of pathway activity, compared with vehicle.
  • Figure 38B shows that even at a concentration of 1 ⁇ M, D induced the regression of X-gal-positive BCC-like structures compared with vehicle.
  • Figure 38C portratys that short-term treatment with D completely downregulated gli-1 transcription (left). This effect appeared to be specific to the Patched pathway and was not due simply to general cytotoxicity, as shown by the fairly constant mRNA levels of a housekeeping enzyme, GAPDH (right).
  • Figure 39 A X-gal staining of the treated explants showed that skin punches cultured in the presence of vehicle alone developed intensely stained blue foci indicative of an upregulation of the Patched pathway and BCC structures.
  • Figure 39C In sldn punches from transgenic mice D, at concentrations of 1 and 5 ⁇ M, significantly inhibited the level of gli-1 mRNA compared with skin punches from mice treated with vehicle alone (left). This inhibition did not appear to be caused by non-specific cytotoxicity, as statistical comparison (using ANON A) of the mR ⁇ A levels of the gene that encodes the housekeeping GAPDH enzyme among groups showed no significant difference in general cellular metabolic activity (right).
  • Figure 40A The morphological features characteristic of BCCs, such as islands of undifferentiated basal cells, and in some cases, palisading of peripheral cells and stromal clefting were maintained when cultures were stained with H&E.
  • Figure 40B The GLI-1 gene, a pivotal indicator of Patched signaling, remained active at high levels, as indicated in red.
  • Figure 41 Quantitative in situ hybridization shows that the level of GLI-1 expression is reduced in the D-treated samples as compared to vehicle-treated controls.
  • the present invention relates to the discovery that signal transduction pathways regulated by hedgehog, patched (ptc), gli and/or smoothened can be inhibited, at least in part, by small molecules.
  • the activation of a receptor may be the mechanism by which these agents act.
  • the ability of these agents to inhibit proliferation of patched loss-of-function (ptc lo ) cells may be due to the ability of such molecules to interact with hedgehog, patched, or smoothened, or at least to interfere with the ability of those proteins to activate a hedgehog, ptc, and/or smoothened-mediated signal transduction pathway.
  • these small molecules which intefere with aspects of hedgehog, ptc, or smoothened signal transduction activity will likewise be capable of inhibiting proliferation (or other biological consequences) in normal cells and/or cells having a patched loss-of-function phenotype, a hedgehog gain-of-function phenotype, or a smoothened gain-of- function phenotype.
  • these compounds may be useful for inhibiting hedgehog activity in normal cells, e.g., which do not have a genetic mutation that activates the hedgehog pathway.
  • the subject inhibitors are organic molecules having a molecular weight less than 2500 amu, more preferably less than 1500 amu, and even more preferably less than 750 amu, and are capable of inhibiting at least some of the biological activities of hedgehog proteins, preferably specifically in target cells.
  • the methods of the present invention include the use of small molecules which agonize tc inhibition of hedgehog signalling, such as by inliibitiiig activation of smoothened or downstream components of the signal pathway, in the regulation of repair and/or functional performance of a wide range of cells, tissues and organs, including normal cells, tissues, and organs, as well as those having the phenotype of ptc loss-of-function, hedgehog gain-of-function, or smoothened gain- of-function.
  • the subject method has therapeutic and cosmetic applications ranging from regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, regulation- of skin and hair growth, etc.
  • 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) .
  • the subject method can be to treat epithelial cells having a phenotype of ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function.
  • the subject method can be used in treating or preventing basal cell carcinoma or other hedgehog pathway-related disorders.
  • a subject antagonist may inhibit activation of a hedgehog pathway by binding to smoothened. In certain embodiments, a subject antagonist may inhibit activation of a hedgehog pathway by binding to patched.
  • the subject method can be used as part of a treatment regimen for malignant meduUoblastoma and other primary CNS malignant neuroectodermal tumors.
  • the present invention provides pharmaceutical preparations comprising, as an active ingredient, a hedgehog antagonist, ptc agonist, or smoothened antagonist such as described herein, formulated in an amount sufficient to inhibit, in vivo, proliferation or other biological consequences of ptc loss-of- function, hedgehog gain-of-function, or smoothened gain-of-function.
  • the subject treatments using hedgehog antagonists, patched agonists, or smoothened 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.
  • allelic modification or mutation of a gene refers to such genetic lesions as, for example, deletions, substitution or addition of nucleotides to a gene, as well as gross chromosomal rearrangements of the gene and/or abnormal methylation of the gene.
  • mis-expression of a gene refers to aberrant levels of transcription of the gene relative to those levels in a normal cell under similar conditions, as well as non- wild-type splicing of mRNA transcribed from the gene.
  • Basal cell carcinomas exist in a variety of dinical and histological forms such as nodular-ulcerative, superficial, pigmented, morphealike, fibroepithelioma and provoked syndrome. Basal cell carcinomas are the most common cutaneous neoplasms found in humans. The majority of new cases of nonmelanoma skin cancers fall into this category.
  • “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.
  • 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.
  • carcinomatous epithelial growths are "papillomas”, which refers to benign tumors derived from epithelium and having a papillomavirus as a causative agent; and "epidermoidomas”, which refers to a cerebral or meningeal tumor formed by inclusion of ectodermal elements at the time of closure of the neural groove.
  • papillomas which refers to benign tumors derived from epithelium and having a papillomavirus as a causative agent
  • epidermoidomas which refers to a cerebral or meningeal tumor formed by inclusion of ectodermal elements at the time of closure of the neural groove.
  • 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.
  • Dermatal 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.
  • Dermamal 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.
  • ED 5 ⁇ means the dose of a drug which produces 50% of its maximum response or effect.
  • 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.
  • epidermal refers 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.
  • epithelial tissue includes: olfactory epithelium, which is the pseudostratified epithelium lining the olfactory region of the nasal cavity, and containing the receptors for the sense of smell; glandular epithelium, which refers to epithelium composed of secreting cells; squamous epithelium, which refers to epithelium composed of flattened plate-like cells.
  • 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.
  • epithelialization refers to healing by the growth of epithelial tissue over a denuded surface.
  • epidermis refers to an aggregation of cells associated with the epidermis and specialized to secrete or excrete materials not related to their ordinary metabolic needs.
  • salivaous glands are holocrine glands in the corium that secrete an oily substance and sebum.
  • sebum refers to glands that secrete sweat, situated in the corium or subcutaneous tissue, opening by a duct on the body surface.
  • 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.
  • 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
  • horny layer composed of flattened, cornified non-nucleated cells.
  • the clear layer is usually absent.
  • Excisional wounds include tears, abrasions, cuts, punctures or lacerations in the epithelial layer of the sldn 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.
  • the “growth state” of a cell refers to the rate of proliferation of the cell and/or the state of differentiation of the cell.
  • An “altered growth state” is a growth state characterized by an abnormal rate of proliferation, e.g., a cell exhibiting an increased or decreased rate of proliferation relative to a normal cell.
  • hair refers to a threadlike structure, especially the specialized epidermal structure composed of keratin and developing from a papilla sunk in the corium, produced only by mammals and characteristic of that group of animals. Also, “hair” may refer to the aggregate of such hairs.
  • a “hair follicle” refers to one of the tubular-invaginations of the epidermis enclosing the hairs, and from which the hairs grow.
  • “Hair follicle epithelial cells” refers to epithelial cells which surround the dermal papilla in the hair follicle, e.g., stem cells, outer root sheath cells, matrix cells, and inner root sheath cells. Such cells may be normal non-malignant cells, or transformed/immortalized cells.
  • hedgehog antagonist refers to an agent which potentiates or recapitulates the bioactivity of patched, such as to repress transcription of target genes.
  • Preferred hedgehog antagonists can be used to overcome a ptc loss-of- function and/or a smoothened gain-of-function, the latter also being refered to as smoothened antagonists.
  • the term 'hedgehog antagonist' as used herein refers not only to any agent that may act by directly inhibiting the normal function of the hedgehog protein, but also to any agent that inhibits the hedgehog signalling pathway, and thus recapitulates the function of ptc.
  • hedgehog gain-of-function refers to an aberrant modification or mutation of ay ⁇ tc gene, hedgehog gene, or smoothened gene, or a decrease (or loss) in the level of expression of such a gene, which results in a phenotype which resembles contacting a cell with a hedgehog protein, e.g., aberrant activation of a hedgehog pathway.
  • the gain-of-function may include a loss of the ability of the ptc gene product to regulate the level of expression of Ci genes, e.g., GUI, GH2, and GH3.
  • hedgehog gain-of-function' is also used herein to refer to any similar cellular phenotype (e.g., exhibiting excess proliferation) which occurs due to an alteration anywhere in the hedgehog signal transduction pathway, including, but not limited to, a modification or mutation of hedgehog itself.
  • a tumor cell with an abnormally high proliferation rate due to activation of the hedgehog signalling pathway would have a 'hedgehog gain-of-function' phenotype, even if hedgehog is not mutated in that cell.
  • 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.
  • 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.
  • keratosis refers to proliferative skin disorder characterized by hyperplasia of the horny layer of the epidermis.
  • exemplary ke atotic disorders include keratosis follicularis, keratosis palmaris et plantaris, keratosis pharyngea, keratosis pilaris, and actinic keratosis.
  • LD50 means the dose of a drug which is lethal in 50% of test subjects.
  • the term “nail” refers to the horny cutaneous plate on the dorsal surface of the distal end of a finger or toe.
  • the term “patched loss-of-function” refers to an aberrant modification or mutation of a. ptc gene, or a decreased level of expression of the gene, which results in a phenotype which resembles contacting a cell with a hedgehog protein, e.g., aberrant activation of a hedgehog pathway.
  • the loss-of-function may include a loss of the ability of the ptc gene product to regulate the level of expression of Ci genes, e.g., GUI, GH2 and GU3.
  • 'ptc loss-of-function' is also used herein to refer to any similar cellular phenotype (e.g., exhibiting excess proliferation) which occurs due to an alteration anywhere in the hedgehog signal transduction pathway, including, but not limited to, a modification or mutation of ptc itself.
  • a tumor cell with an abnormally high proliferation rate due to activation of the hedgehog signalling pathway would have a 'ptc loss-of-function' phenotype, even if ptc is not mutated in that cell.
  • a "patient” or “subject” to be treated by the subject method can mean either a human or non-human animal.
  • prodrug is intended to encompass compounds which, under physiological conditions, are converted into the therapeutically active agents of the present invention.
  • a common method for making a prodrug is to include selected moieties which are hydrolyzed under physiological conditions to reveal the desired molecule.
  • the prodrug is converted by an enzymatic activity of the host animal.
  • proliferating and proliferation refer to cells undergoing mitosis.
  • 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.
  • epidermodysplasia is a form of faulty development of the epidermis.
  • epidermolysis which refers to a loosened state of the epidermis with formation of blebs and bullae either spontaneously or at the site of trauma.
  • psoriasis refers to a hyperproliferative skin disorder which alters the sldn's regulatory mechanisms.
  • lesions are formed which involve primary and secondary alterations in epidermal proliferation, inflammatory responses of the sldn, 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 lceratinization, 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.
  • 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.
  • skin may be used, and should be understood to refer generally to attributes of the sldn, as appropriate to the context in which they are used.
  • smoothened gain-of-function refers to an aberrant modification or mutation of a smo gene, or an increased level of expression of the gene, which results in a phenotype which resembles contacting a cell with a hedgehog protein, e.g., aberrant activation of a hedgehog pathway. While not wishing to be bound by any particular theory, it is noted that ptc may not signal directly into the cell, but rather interact with smoothened, another membrane-bound protein located downstream of ptc in hedgehog signaling (Marigo et al, (1996) Nature 384: 1 7- 179).
  • the gene smo is a segment-polarity gene required for the correct patterning of every segment in Drosophila (Alcedo et al., (1996) Cell 86: 221-232). Human homologs of smo have been identified. See, for example, Stone et al. (1996) Nature 384:129-134, and GenBank accession U84401.
  • the smoothened gene encodes an integral membrane protein with characteristics of heterotrimeric G-protein-coupled receptors; i.e., 7-transmembrane regions. This protein shows homology to the Drosophila Frizzled (Fz) protein, a member of the wingless pathway. It was originally thought that smo encodes a receptor of the Hh signal.
  • therapeutic index refers to the therapeutic index of a drug defined as LD 50 /ED 50 .
  • 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.
  • acylamino is art-recognized and refers to a moiety that can be represented by the general formula: 0 N— ⁇ — R '
  • R 9 wherein R 9 is as defined above, and R' ⁇ represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -R8 > where m and Rg are as defined above.
  • 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.
  • 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.
  • alkoxyl or allcoxy 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.
  • 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-allcynyl, -O-(CH2) m -R8, where m and Rg are described above.
  • 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.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer.
  • 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.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
  • a halogen
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • 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, allcylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
  • Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, allcylthios, aminoalkyls, carbonyl- substituted alkyls, -CF3, -CN, and the like.
  • 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.
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-allcynyl, and -S-(CH2) m -R8, wherein m and Rg are defined above.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • 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:
  • R9, RJQ and R'10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2) m -Rg, or R9 and R ⁇ Q taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure;
  • Rg 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.
  • only one of R9 or R10 can be a carbonyl, e.g., R9, R1 Q and the nitrogen together do not form an imide.
  • R9 and RJQ each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2) -R8-
  • 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 R9 and R ⁇ Q s an allcyl group.
  • amino is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
  • R / 10 wherein R9, R1 Q are as defined above.
  • Preferred embodiments of the amide will not include imides which may be unstable.
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaiOmatic group).
  • 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.
  • 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, arallcyl, alkenyl, alkynyl, cycloallcyl, hydroxyl, alkoxyl, amino, nitre, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like.
  • 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 cycloallcyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • Carbocycle refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
  • carbonyl is art-recognized and includes such moieties as can be represented by the general formula:
  • X is a bond or represents an oxygen or a sulfur
  • R ⁇ represents a hydrogen, an alkyl, an alkenyl, -(CH2) m -R8 or a pharmaceutically acceptable salt
  • R'11 represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -R8 > where m and Rg are as defined above.
  • X is an oxygen and or R' ⁇ i is not hydrogen
  • the formula represents an "ester”.
  • X is an oxygen, and R is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R ⁇ is a hydrogen, the formula represents a "carboxylic acid".
  • 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.
  • heterocyclyl or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles.
  • Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiaziiie, furazan, phenoxazine, pyrrolidine,
  • 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, cycloallcyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, Icetone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloallcyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate
  • nitro means -NO2; the term “halogen” designates -
  • a “phosphonamidite” can be represented in the general formula:
  • R9 and R10 are as defined above, Q2 represents O, S or N, and R48 represents a lower alkyl or an aryl, Q2 represents O, S or N.
  • a “phosphoramidite” can be represented in the general formula:
  • R9 and Rio are as defined above, and Q2 represents O, S or N.
  • a "phosphoryl” can in general be represented by the formula:
  • the phosphoryl group of the phosphorylallcyl can be represented by the general formula: wherein Q represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N.
  • Q represents an S
  • the phosphoryl moiety is a "phosphorothioate”.
  • polycyclyl or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloallcynyls, 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, arallcyl, alkenyl, alkynyl, cycloallcyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, Icetone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, arallcyl, alkenyl, alkynyl, cycloallcyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbony
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and lcetals 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 nd ed.; Wiley: New York, 1991).
  • 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-(CH2) m -Rg, m and
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • 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.
  • 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.
  • 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.
  • R41 is as defined above.
  • sulfonate is art-recognized and includes a moiety that can be represented by the general formula: :
  • R41 is an electron pair, hydrogen, alkyl, cycloallcyl, or aryl.
  • sulfoxido or "sulfinyl”, as used herein, refers to a moiety that can be represented by the general formula:
  • R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloallcyl, heterocyclyl, arallcyl, or aryl.
  • Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoallcynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or allcynyls.
  • 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.
  • triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, j ⁇ -toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
  • 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.
  • Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, j 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.
  • 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.
  • a particular enantiomer of a compound of the present invention 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.
  • diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • 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.
  • 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.
  • 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.
  • hydrocarbon is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom.
  • permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted.
  • Rj 5 R2, R3, and R 4 independently for each occurrence, represent H, lower alkyl, -(CH 2 ) n aryl (e.g., substituted or unsubstituted), or -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted);
  • L independently for each occurrence, is absent or represents -(CH2) n -, - alkenyl-, -alkynyl-, -(CH 2 ) n alkenyl-, -(CH 2 ) n alkynyl-, -(CH 2 ) n O(CH 2 ) p -, - (CH 2 )nNR 8 (CH 2 )p-, -(CH 2 )nS(CH 2 )p-, -(CH 2 )nalkenyl(CH 2 ) p -,
  • X and D independently, can be selected from -N(Rg)-, -O-, -S-, -(R 8 )N- N(R 8 )-, -ON(R 8 )-, or a direct bond;
  • Y and Z independently, can be selected from O or S;
  • R 8 independently for each occurrence, represents H, lower alkyl, - (CH 2 ) n aryl (e.g., substituted or unsubstituted), -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or two Rg taken together may form a 4- to 8-membered ring; p represents, independently for each occurrence, an integer from 0 to 10, preferably from 0 to 3; n, individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5; and q and r represent, independently for each occurrence, an integer from 0-2.
  • D does not represent N-lower allcyl. In certain embodiments, D represents an arallcyl- or heteroaralkyl-substituted amine.
  • R ⁇ represents a lower allcyl group, such as a branched allcyl, a cycloalkyl, or a cycloalkylalkyl, for example, cyclopropyl, cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl, cyclobutylmethyl, etc.
  • Y and Z are O.
  • XLR taken together, include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • At least one of R ls R 2 , and R 3 includes an aryl or heteroaryl group. In certain related embodiments, at least two of Ri, R 2 , and R 3 include an aryl or heteroaryl group. In certain embodiments, Rj is lower alkyl. In certain embodiments, L attached to Ri represents O, S, or NR 8 , such as
  • E is NR 8 .
  • E represents an arallcyl- or heteroarallcyl-substituted amine, e.g., including polycyclic R 8 .
  • Rl R , R 3 , R , R 8 , L, X, Y, Z, n, p, q, and r are as defined above;
  • R t represents a lower allcyl group, such as a branched alkyl, a cycloallcyl, or a cycloalkylallcyl, for example, cyclopropyl, cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl, cyclobutylmethyl, etc.
  • the sum of q, r, and s is less than 5, e.g., is 2, 3, or 4.
  • XLR taken together, include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • a cyclic amine such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • L attached to Ri represents O, S, or NR 8 , such as NH.
  • at least one of Ri, R 2 , and R 3 includes an aryl or heteroaryl group.
  • at least two of R ls R 2 , and R 3 include an aryl or heteroaryl group.
  • M is absent.
  • compounds useful in the present invention may be represented by general formula (III):
  • Rl R 2 , R 3 , R 4 , R , L, M, X, Y, Z, n, p, q, and r are as defined above.
  • Y and Z are O.
  • R ⁇ represents a lower alkyl group, preferably a branched alkyl, a cycloalkyl, or a cycloalkylalkyl, for example, cyclopropyl, cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl, cyclobutylmethyl, etc.
  • the sum of q and r is less than 4, e.g., is 2 or 3.
  • XLR_ 4 taken together, include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • At least one of R ls R , and R 3 includes an aryl or heteroaryl group. In certain related embodiments, at least two of Rj, R , and R include an aryl or heteroaryl group. In certain embodiments, Ri is lower allcyl.
  • L attached to R ⁇ represents O, S, or NR 8 , such as NH.
  • M is absent.
  • compounds useful in the present invention may be represented by general formula (IV):
  • R j R 2 , R 3 , R , R ⁇ , L, M, X, n, and p are as defined above.
  • XLR 4 taken together, include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • R ⁇ represents a lower allcyl group, preferably a branched allcyl, a cycloallcyl, or a cycloalkylallcyl, for example, cyclopropyl, cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl, cyclobutylmethyl, etc.
  • At least one of Ri, R , and R 3 includes an aryl or heteroaryl group. In certain related embodiments, at least two of R l3 R 2 , and R 3 include an aryl or heteroaryl group. In certain embodiments, R ⁇ is lower alkyl.
  • L attached to R] represents O, S, or NR 8 , such as NH.
  • M is absent.
  • L represents a direct bond for all occurrences.
  • compounds useful in the present invention may be represented by general formula (V):
  • Y, n, p, q, and r are as defined above;
  • V is absent or represents O, S, or NR 8 ;
  • R 5 represents substituted or unsubstituted allcyl (e.g., branched or unbranched), alkenyl (e.g., branched or unbranched), alkynyl (e.g., branched or unbranched), cycloalkyl, or cycloalkylallcyl;
  • R 6 represents substituted or unsubstituted aryl, aralkyl, heteroaryl, heteroarallcyl, heterocyclyl, heterocyclylalkyl, cycloallcyl, or cycloalkylallcyl, including polycyclic groups; and R 7 represents substituted or unsubstituted aryl, arallcyl, heteroaryl, or heteroarallcyl.
  • Y is O.
  • the sum of q and r is less than 4.
  • NJ N taken together, represent a cyclic diamine, such as a piperazine, etc., which may be substituted or unsubstituted, e.g., with one or more substitutents such as oxo, lower allcyl, lower allcyl ether, etc.
  • NJ 2 or NJR taken together represent a substituted or unsubstituted heterocyclic ring to which the other occurrence of N is attached.
  • one or both occurrences of J are substituted with one or more of lower alkyl, lower allcyl ether, lower allcyl thioether, amido, oxo, etc.
  • a heterocyclic ring which comprises an occurrence of J has from 5 to 8 members.
  • R 5 represents a branched alkyl, cycloallcyl, or cycloalkylallcyl.
  • R includes at least one heterocyclic ring, such as a thiophene, furan, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
  • R represents a phenyl alkyl, such as a benzyl group, optionally substituted with halogen, hydroxyl, lower alkyl, nitro, cyano, lower allcyl ether (e.g., optionally substituted, such as CHF 2 CF O), or lower allcyl thioether (e.g., optionally substituted, such as CF 3 S).
  • a phenyl alkyl such as a benzyl group, optionally substituted with halogen, hydroxyl, lower alkyl, nitro, cyano, lower allcyl ether (e.g., optionally substituted, such as CHF 2 CF O), or lower allcyl thioether (e.g., optionally substituted, such as CF 3 S).
  • R 8 when it occurs in V, represents H or lower allcyl, preferably H.
  • compounds useful in the present invention may be represented by general formula (VI):
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , G, J, V, Y, Z ⁇ n, and p are as defined above.
  • Y is O.
  • NJ 2 N taken together, represent a heterocyclic ring, such as a piperazine, etc., which may be substituted or unsubstituted, e.g., with one or more substitutents such as oxo, lower allcyl, lower allcyl ether, etc.
  • NJ 2 or NJR 9 taken together represent a substituted or unsubstituted heterocyclic ring to which the other occurrence of N is attached.
  • one or both occurrences of J are substituted with one or more of lower alkyl, lower alkyl ether, lower alkyl thioether, amido, oxo, etc.
  • a heterocyclic ring which comprises an occurrence of J has from 5 to 8 members.
  • R 5 represents a branched allcyl, cycloallcyl, or cycloalkylallcyl.
  • R 6 includes at least one heterocyclic ring, such as a thiophene, furan, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
  • R 7 represents a phenyl alkyl, such as a benzyl group, optionally substituted with halogen, hydroxyl, lower allcyl, nitro, cyano, lower allcyl ether (e.g., optionally substituted, such as CHF 2 CF2O), or lower allcyl thioether (e.g., optionally substituted, such as CF 3 S).
  • a phenyl alkyl such as a benzyl group, optionally substituted with halogen, hydroxyl, lower allcyl, nitro, cyano, lower allcyl ether (e.g., optionally substituted, such as CHF 2 CF2O), or lower allcyl thioether (e.g., optionally substituted, such as CF 3 S).
  • R 8 when it occurs in V, represents H or lower alkyl, preferably H.
  • the subject compound is selected from the compounds depicted in Figure 32.
  • the subject antagonists can be chosen on the basis of their selectively for the hedgehog pathway. This selectivity can be for the hedgehog pathway versus other pathways, or for selectivity between particular hedgehog pathways, e.g., ptc- 1 , ptc-2, etc.
  • the subject inhibitors inhibit ptc loss-of- function, hedgehog gain-of-function, or smoothened gain-of-function mediated signal transduction with an ED 5Q of 1 mM or less, more preferably of 1 ⁇ M or less, and even more preferably of 1 nM or less.
  • the subject inhibitors inhibit activity of the hedgehog pathway with a Kj less than 10 nM, preferably less than 1 nM, even more preferably less than 0.1 nM.
  • the small molecule is chosen for use because it is more selective for one patched isoform over the next, e.g., 10-fold, and more preferably at least 100- or even 1000-fold more selective for one patched pathway (ptc- ⁇ ,ptc-2) over another.
  • a compound which is an antagonist of the hedgehog pathway is chosen to selectively antagonize hedgehog activity over protein Idnases other than PKA, such as PKC, e.g., the compound modulates the activity of the hedgehog pathway at least an order of magnitude more strongly than it modulates the activity of another protein kinase, preferably at least two orders of magnitude more strongly, even more preferably at least three orders of magnitude more strongly.
  • a preferred inhibitor of the hedgehog pathway may inhibit hedgehog activity with a Kj at least an order of magnitude lower than its Kj for inhibition of PKC, preferably at least two orders of magnitude lower, even more preferably at least three orders of magnitude lower.
  • the K, for PKA inhibition is less than 10 nM, preferably less than 1 nM, even more preferably less than 0.1 nM.
  • a compound of Formula X may be transformed according to the following scheme:
  • LG represents a leaving group, such as a halogen (e.g., CI, Br, or I) or a sulfonate ester (e.g., tosylate, mesylate, triflate, etc.);
  • a halogen e.g., CI, Br, or I
  • a sulfonate ester e.g., tosylate, mesylate, triflate, etc.
  • A represents an oxygen or sulfur bound to an acid-protecting group or a group having the formula XLR 4 ;
  • B represents a nitrogen-protecting group or a group having the formula MR 3 ;
  • R 3 and R 4 independently for each occurrence, represent H, lower allcyl, -
  • Y can be selected from O and S;
  • X is be selected from -N(R 8 )-, -O-, -S-, or a direct bond;
  • L independently for each occurrence, is absent or represents -(CH 2 )nalkyl-, - alkenyl-, -allcynyl-, -(CH 2 ) n alkenyl-, -(CH 2 ) n alkynyl-, -(CH 2 )nO(CH 2 )p-, - (CH 2 ) n NR 8 (CH 2 ) p -, -(CH 2 ) n S(CH 2 ) p -, -(CH 2 ) n alkenyl(CH 2 ) p -,
  • R 8 independently for each occurrence, represents H, lower allcyl, - (CH 2 ) n aryl (e.g., substituted or unsubstituted), -(CH2) n heteroaryl (e.g., substituted or unsubstituted), or two R 8 taken together may form a 4- to 8-membered ring;
  • step A includes converting the hydroxyl to a leaving group
  • step B includes displacing the leaving group with an azide
  • step C includes reducing the azide to an amine
  • converting the hydroxyl to a leaving group may be performed by reacting the hydroxyl with a sulfonyl halide to generate a sulfonate ester, e.g., using tosyl chloride or tosyl anhydride to generate a tosylate, mesyl chloride or mesyl anhydride to generate a mesylate, or triflyl chloride or triflyl anhydride to generate a triflate, etc.
  • converting the hydroxyl to a leaving group may be performed by reacting the hydroxyl with an halogenating reagent such as a thionyl halide, a phosphorous trihalide, phosphorous pentahalide, phosphorous oxyhalide, etc.
  • an halogenating reagent such as a thionyl halide, a phosphorous trihalide, phosphorous pentahalide, phosphorous oxyhalide, etc.
  • Other techniques for converting a hydroxyl group to a leaving group are well known in the art and may be used in step A.
  • step A further includes displacing a first leaving group with a second leaving group and inverting the stereochemistry of the leaving group-bearing carbon.
  • this mesylate Reaction of this mesylate with a nucleophilic halide reagent, such as Nal, will result in displacement of the mesylate with iodide, generating a compound of Formula XI wherein the leaving group, iodine, and the group bearing Y and A have a trans stereochemical relationship.
  • a nucleophilic halide reagent such as Nal
  • displacing the leaving group with an azide may be performed using an alkali or alkaline earth metal salt of azide anion, such as sodium azide, using a silyl azide reagent, such as trimethylsilyl azide, or using any other azide reagent, e.g., a nucleophilic azide source, as is well known in the art.
  • azide anion such as sodium azide
  • silyl azide reagent such as trimethylsilyl azide
  • any other azide reagent e.g., a nucleophilic azide source, as is well known in the art.
  • reducing the azide to an amine may be performed using a hydride reagent, such as lithium aluminum hydride, lithium triallcylborohydride, etc., using a reducing metal and an acid source, such as zinc metal or samarium diiodide with acetic acid, using catalytic hydrogenation, such as hydrogen and a transition metal catalyst such as platinum or palladium, or by any other suitable means.
  • a hydride reagent such as lithium aluminum hydride, lithium triallcylborohydride, etc.
  • an acid source such as zinc metal or samarium diiodide with acetic acid
  • catalytic hydrogenation such as hydrogen and a transition metal catalyst such as platinum or palladium
  • q+s+r is an integer from 2 to 3.
  • s is 0.
  • q and r each represent 1.
  • A represents an oxygen bound to an acid-protecting group.
  • the acid protecting group may be a substituted or unsubstituted allcyl, alkenyl, allcynyl, aryl, or aralkyl group.
  • examples of such groups include methyl, ethyl, trimethylsilylethyl, methylthiomethyl, allyl, benzyl, p-nitrobenzyl, tetrahydropyranyl (THP), t-butyl, or any other suitable group.
  • THP tetrahydropyranyl
  • A represents an alkylthio group.
  • B represents a nitrogen-protecting group, such as a substituted or unsubstituted acyl, allcyl, alkenyl, allcynyl, aryl, or arallcyl group, or a group which, when taken together with N, forms a carbamate.
  • nitrogen- protecting groups include benzyl, allyl, ⁇ -methoxybenzyl, acetyl, trifluoroacetyl, t- butoxycarbonyl, benzyloxycarbonyl, etc.
  • a wide variety of nitrogen-protecting ' groups are known in the art and may be employed in this method without departing from the scope and spirit of the invention.
  • Y is O.
  • A represents XLR_ 4 , which may, taken together, include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • a cyclic amine such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • R 3 includes an aryl or heteroaryl group.
  • M is absent.
  • the compound of Formula XIII is enriched for the isomer wherein the amine and the substituent including Y and A have a cis relationship, e.g., >75%, >85%, or even >95% of the cis isomer. In other embodiments, the compound of Formula XIII is enriched for the isomer wherein the two substituents have a trans relationship, e.g., >75%, >85%, or even >95% of the trans isomer.
  • such enrichment results from employing an isomerically enriched starting material, e.g., the compound of Formula X is enriched for, >75%, >85%, or even >95% of the cis or trans isomer prior to beginning step A.
  • an isomerically enriched starting material e.g., the compound of Formula X is enriched for, >75%, >85%, or even >95% of the cis or trans isomer prior to beginning step A.
  • a compound of Formula XIV may be transformed according to the following scheme:
  • q and r each represent, independently, an integer in the range of 0 to 2, such that the sum of q+r is an integer in the range of 2-4;
  • LG represents a leaving group, such as a halogen (e.g., CI, Br, or I) or a sulfonate ester (e.g., tosylate, mesylate, triflate, etc.);
  • a halogen e.g., CI, Br, or I
  • a sulfonate ester e.g., tosylate, mesylate, triflate, etc.
  • A represents an oxygen or sulfur bound to an acid-protecting group or a group having the formula NJ 2 N(R 9 ) 2 ;
  • B represents a nitrogen-protecting group or a group having the formula GR 6 ;
  • R 9 independently for each occurrence, is absent or represents H or lower allcyl, or two occurrences of J or one occurrence of J taken together with one occurrence of R , forms a ring of from 5 to 7 members, which ring includes one or both occurrences of N;
  • R 6 represents substituted or unsubstituted aryl, arallcyl, heteroaryl, heteroarallcyl, heterocyclyl, heterocyclylalkyl, cycloalkyl, or cycloalkylallcyl, including polycyclic groups; and
  • Y can be selected from O and S; and wherein step A includes converting the hydroxyl to a leaving group, step B includes displacing the leaving group with an azide, and step C includes reducing the azide to an amine.
  • converting the hydroxyl to a leaving group may be performed by reacting the hydroxyl with a sulfonyl halide to generate a sulfonate ester, e.g., using tosyl chloride or tosyl anhydride to generate a tosylate, mesyl chloride or mesyl anhydride to generate a mesylate, or triflyl chloride or triflyl anhydride to generate a triflate, etc.
  • converting the hydroxyl to a leaving group may be performed by reacting the hydroxyl with an halogenating reagent such as a thionyl halide, a phosphorous trihalide, phosphorous pentahalide, phosphorous oxyhalide, etc.
  • an halogenating reagent such as a thionyl halide, a phosphorous trihalide, phosphorous pentahalide, phosphorous oxyhalide, etc.
  • Other techniques for converting a hydroxyl group to a leaving group are well known in the art and may be used in step A.
  • step A further includes displacing a first leaving group with a second leaving group and inverting the stereochemistry of the leaving group-bearing carbon.
  • Formula XIV has a cis stereochemical relationship with the group bearing Y and A
  • reaction of this compound with mesyl chloride will generate a mesylate in a cis stereochemical relationship with the group bearing Y and A.
  • Reaction of this mesylate with a nucleophilic halide reagent, such as Nal will result in displacement of the mesylate with iodide, generating a compound of Formula XV wherein the leaving group, iodine, and the group bearing Y and A have a trans stereochemical relationship.
  • displacing the leaving group with an azide may be performed using an alkali or alkaline earth metal salt of azide anion, such as sodium azide, using a silyl azide reagent, such as trimethylsilyl azide, or using any other azide reagent, e.g., a nucleophilic azide source, as is well Icnown in the art.
  • azide anion such as sodium azide
  • silyl azide reagent such as trimethylsilyl azide
  • any other azide reagent e.g., a nucleophilic azide source, as is well Icnown in the art.
  • reducing the azide to an amine may be performed using a hydride reagent, such as lithium aluminum hydride, lithium trialkylborohydride, etc., using a reducing metal and an acid source, such as zinc metal or samarium diiodide with acetic acid, using catalytic hydrogenation, such as hydrogen and a transition metal catalyst such as platinum or palladium, or by any other suitable means.
  • a hydride reagent such as lithium aluminum hydride, lithium trialkylborohydride, etc.
  • an acid source such as zinc metal or samarium diiodide with acetic acid
  • catalytic hydrogenation such as hydrogen and a transition metal catalyst such as platinum or palladium
  • q+r is an integer from 2 to 3.
  • q and r each represent 1.
  • A represents an oxygen bound to an acid-protecting group.
  • the acid protecting group may be a substituted or unsubstituted allcyl, alkenyl, alkynyl, aryl, or arallcyl group. Examples of such groups include methyl, ethyl, trimethylsilylethyl, methylthiomethyl, allyl, benzyl, / ⁇ -nitrobenzyl, tetrahydropyranyl (THP), t-butyl, or any other suitable group.
  • a wide variety of acid- protecting groups are Icnown in the art and may be employed in this method without departing from the scope and spirit of the invention.
  • A represents an alkylthio group.
  • B represents a nitrogen-protecting group, such as a substituted or unsubstituted acyl, alkyl, alkenyl, allcynyl, aryl, or aralkyl group, or a group which, when taken together with N, forms a carbamate.
  • nitrogen- protecting groups include benzyl, allyl, /?-methoxybenzyl, acetyl, trifluoroacetyl, t- butoxycarbonyl, benzyloxycarbonyl, etc.
  • a wide variety of nitrogen-protecting groups are Icnown in the art and may be employed in this method without departing from the scope and spirit of the invention.
  • Y is O.
  • B is GR 6 , wherein R 6 includes at least one heterocyclic ring, such as a thiophene, furan, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
  • A represents NJ 2 N, which, taken together, may represent a cyclic diamine, such as a piperazine, etc., which may be substituted or unsubstituted, e.g., with one or more substitutents such as oxo, lower allcyl, lower allcyl ether, etc.
  • NJ 2 or NJR taken together represent a substituted or unsubstituted heterocyclic ring to which the other occurrence of N is attached.
  • one or both occurrences of J are substituted with one or more of lower allcyl, lower allcyl ether, lower allcyl thioether, amido, oxo, etc.
  • a heterocyclic ring which comprises an occurrence of J has from 5 to 8 members.
  • the compound of Formula XVII is enriched for the isomer wherein the amine and the substituent including Y and A have a cis relationship, e.g., >75%, >85%, or even >95% of the cis isomer.
  • the compound of Formula XVII is emiched for the isomer wherein the two substituents have a trans relationship, e.g., >75%, >85%, or even >95% of the trans isomer.
  • such enrichment results from employing an isomerically enriched starting material, e.g., the compound of Formula XIV is emiched for, >75%, >85%, or even >95% of the cis or trans isomer prior to beginning step A.
  • an isomerically enriched starting material e.g., the compound of Formula XIV is emiched for, >75%, >85%, or even >95% of the cis or trans isomer prior to beginning step A.
  • an amine having a structure of Formula XIII or XVII may be further transformed, e.g., by performing additional steps towards generating a compound of at least one of Formulae I- VI.
  • a method according to the present invention might include one or more of the following steps:
  • G coupling to the nitrogen in the ring a group -MR 3 or -GR 6 ; H) removing a protecting group from the nitrogen in the ring; I) removing a protecting group from the group bearing Y; J) placing a nitrogen-protecting group on the exocyclic amine;
  • R 1; R 2 , R 3 , and R4 independently for each occurrence, represent H, lower allcyl, -(CH 2 ) n aryl (e.g., substituted or unsubstituted), or -(CH2) n heteroaryl (e.g., substituted or unsubstituted);
  • Z is O or S
  • V is absent or represents O, S, or NR 8 ;
  • R 5 represents substituted or unsubstituted allcyl (e.g., branched or unbranched), alkenyl (e.g., branched or unbranched), alkynyl (e.g., branched or unbranched), cycloallcyl, or cycloalkylallcyl; and
  • R 7 represents substituted or unsubstituted aryl, arallcyl, heteroaryl, or heteroarallcyl.
  • steps D through K may be performed in any order, depending on the various reactions and protecting groups used, as is well understood in the art.
  • Various protecting groups suitable for use in the present method have been outlined above, and are well Icnown in the art, as are numerous techniques for attaching and removing such protecting groups, and any of these may be employed in the present method without departing from the scope and spirit of the present invention.
  • step D may be performed by reacting the exocyclic amine with an acylating agent, such as an acid halide, an isocyanate, an isothiocyanate, a haloformate, a halothioformate, an anhydride, a dicarbonate, a sulfonyl halide, a sulfinyl halide, a carbamyl chloride, a thiocarbamyl chloride, or an activated acylating moiety prepared in situ.
  • an acylating agent such as an acid halide, an isocyanate, an isothiocyanate, a haloformate, a halothioformate, an anhydride, a dicarbonate, a sulfonyl halide, a sulfinyl halide, a carbamyl chloride, a thiocarbamyl chloride, or an activated acylating moiety prepared in situ
  • An acylating agent may be prepared in situ, for example, by reacting a carboxylic acid with an activating agent, such as a carbodiimide (e.g., diisopropylcarbodiimide, dicyclohexylcarbodiimide, l-(3- dimethylaminopropyl)-3-ethylcarbodiimide, etc.), phosphorous-based reagents (such as BOP-C1, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, or any other reagent that reacts with a carboxylic acid group resulting in a reactive intermediate having an increased susceptibility, relative to the carboxylic acid, towards coupling with an amine.
  • an activating agent such as a carbodiimide (e.g., diisopropylcarbodiimide, dicyclohexylcarbodiimide, l-(3- dimethylaminopropyl)-3-
  • a primary amine or alcohol can be treated with a phosgene equivalent, such as carbonyl diimidazole, phosgene, triphosgene, diphosgene, etc., or a thiophosgene equivalent, such as thiophosgene, thiocarbonyldiimidazole, etc., to generate an acylating agent (e.g., an isocyanate, isothiocyanate, chloroformamide, or chlorothioformamide, for example) capable of reacting with an amine to form a urea or thiourea, without necessitating isolation or purification of the acylating agent.
  • a phosgene equivalent such as carbonyl diimidazole, phosgene, triphosgene, diphosgene, etc.
  • a thiophosgene equivalent such as thiophosgene, thiocarbonyldiimidazole, etc.
  • step G may be performed using reagents and techniques such as those described for step D, above.
  • step G may be performed by reacting the endocyclic amine with an electrophile, such as an allcyl halide or sulfonate, an arallcyl halide or sulfonate, a heteroarallcyl halide or sulfonate, a cycloallcyl halide or sulfonate, a cycloalkylallcyl halide or sulfonate, a heterocyclyl halide or sulfonate, or a heterocyclylalkyl halide or sulfonate.
  • an electrophile such as an allcyl halide or sulfonate, an arallcyl halide or sulfonate, a heteroarallcyl halide or sulfonate, a cycloallcyl halide or
  • step G may be performed by reductive alkylation, e.g., reacting the endocyclic amine with an appropriately substituted aldehyde in the presence of a reducing agent, such as sodium borohydride.
  • step E may be performed using reductive allcylation or by reacting the exocyclic amine with an electrophile, such as a halide or sulfonate.
  • step F may be performed by reacting an ester, thioester, or xanthate with a compound having the formula, for example, of HNJ N(R 9 ) 2 or HX Rj, e.g., in the presence of a Lewis acid, at an elevated temperature, etc.
  • step F may be performed by reacting a carboxylic acid with an activating agent, such as a carbodiimide (e.g., diisopropylcarbodiimide, dicyclohexylcarbodiimide, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide, etc.), a phosphorous-based reagent (such as BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, or any other reagent that reacts with a carboxylic acid group resulting in a reactive intermediate having an increased susceptibility, relative to the carboxylic acid, towards coupling with a nucleophile.
  • an activating agent such as a carbodiimide (e.g., diisopropylcarbodiimide, dicyclohexylcarbodiimide, l-(3-dimethylaminopropyl)-3- e
  • Y and Z are O.
  • R] represents a lower allcyl group, such as a branched allcyl, a cycloallcyl, or a cycloalkylallcyl, for example, cyclopropyl, cyclopropylmethyl, neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl, cyclobutylmethyl, etc.
  • XLR taken together, include a cyclic amine, such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • a cyclic amine such as a piperazine, a morpholine, a piperidine, a pyrrolidine, etc.
  • L attached to Ri represents O, S, or NR 8 , such as NH.
  • at least one of Rj, R 2 , and R 3 includes an aryl or heteroaryl group.
  • at least two of Ri, R 2 , and R 3 include an aryl or heteroaryl group.
  • M is absent.
  • X is not NH.
  • NJ 2 N taken together, represent a cyclic diamine, such as a piperazine, etc., which may be substituted or unsubstituted, e.g., with one or more substitutents such as oxo, lower allcyl, lower allcyl ether, etc.
  • NJ 2 or NJR 9 taken together represent a substituted or unsubstituted heterocyclic ring to which the other occurrence of N is attached.
  • one or both occurrences of J are substituted with one or more of lower alkyl, lower allcyl ether, lower allcyl thioether, amido, oxo, etc.
  • a heterocyclic ring which comprises an occurrence of J has from 5 to 8 members.
  • R 5 represents a branched alkyl, cycloalkyl, or cycloalkylallcyl.
  • R 6 includes at least one heterocyclic ring, such as a thiophene, furan, oxazole, benzodioxane, benzodioxole, pyrrole, indole, etc.
  • R 7 represents a phenyl allcyl, such as a benzyl group, optionally substituted with halogen, hydroxyl, lower allcyl, nitro, cyano, lower allcyl ether (e.g., optionally substituted, such as CHF 2 CF 2 O), or lower alkyl thioether (e.g., optionally substituted, such as CF S).
  • R 8 when it occurs in V, represents H or lower allcyl, preferably H.
  • Another aspect of the present invention relates to a method of modulating a differentiated state, survival, and/or proliferation of a cell having a ptc loss-of- function, hedgehog gain-of-function, or smoothened gain-of-function, by contacting the cells with a hedgehog antagonist according to the subject method and as the circumstances may warrant.
  • a hedgehog antagonist according to the subject method and as the circumstances may warrant.
  • 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.
  • the present method is applicable to cell culture techniques wherein, whether for genetic or biochemical reasons, the cells have a ⁇ tc loss-of- function, hedgehog gain-of-function, or smoothened gain-of-function phenotype.
  • In vitro neuronal culture systems have proved to be fundamental and indispensable tools for the study of neural development, as well as the identification of neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF).
  • NNF nerve growth factor
  • CNTF ciliary trophic factors
  • BDNF brain derived neurotrophic factor
  • One use of the present method may be in cultures of neuronal stem cells, such as in the use of such cultures for the generation of new neurons and glia.
  • 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.
  • the subject method can be used to culture, for example, sensory neurons or, alternatively, motor neurons.
  • Such neuronal cultures can be used as convenient assay systems as well as sources of implantable cells for therapeutic treatments.
  • a method 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.
  • Progenitor cells are thought to be under an inhibitory influence which maintains the progenitors in a suppressed state until their differentiation is required.
  • 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.
  • 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.
  • heterologous is meant a host other than the animal from which the progenitor cells were originally derived.
  • autologous is meant the identical host from which the cells were originally derived.
  • Cells can be obtained from embryonic, post-natal, juvenile or adult neural tissue from any animal.
  • 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.
  • 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.
  • CNS central nervous system
  • PNS peripheral nervous system
  • these areas include regions in the basal ganglia, preferably the sti ⁇ atum 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.
  • regions in the basal ganglia preferably the sti ⁇ atum 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.
  • 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.
  • 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 Icnown procedure, including treatment with enzymes such as trypsin, collagenase and the like, or by using physical methods of dissociation such as with a blunt instrument or by mincing with a scalpel to a allow outgrowth of specific cell types from a tissue.
  • Dissociation of fetal cells can be carried out in tissue culture medium, while a preferable medium for dissociation of juvenile and adult cells is artificial cerebral spinal fluid (aCSF).
  • aCSF artificial cerebral spinal fluid
  • Regular aCSF contains 124 mM NaCl, 5 mM KC1, 1.3 mM MgCl 2 , 2 mM CaCl 2 , 26 mM NaHCO 3 , and 10 mM D-glucose.
  • Ca2 + aCSF contains the same ingredients except for MgCl2 at a concentration of 3.2 mM and CaCl2 at a concentration of 0.1 mM.
  • Dissociated cells can be placed into any Icnown 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.
  • 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.
  • 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 °C-40 °C, more preferably between 32 °C-38 °C, and most preferably between 35 °C-37 °C.
  • 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).
  • 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.
  • 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 °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.
  • cells lift off the floor of the flask and continue to proliferate in suspension forming a hollow sphere of undifferentiated cells.
  • the proliferating clusters are fed every 2-7 days, and more particularly every 2-4 days by gentle centrifugation and resuspension in medium containing growth factor.
  • 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.
  • intracerebral grafting has emerged as an additional approach to central nervous system therapies.
  • 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) JExp 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.
  • 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.
  • Stem cells useful in the present invention are generally Icnown.
  • neural crest cells For example, several neural crest cells have been identified, some of which are multipotent and likely represent uncommitted neural crest cells, and others of which can generate only one type of cell, such as sensory neurons, and likely represent committed progenitor cells.
  • the role of hedgehog antagonists employed in the present method to culture such stem cells can be to regulate differentiation of the uncommitted progenitor, or to regulate further restriction of the developmental fate of a committed progenitor cell towards becoming a terminally differentiated neuronal cell.
  • the present method can be used in vitro to regulate the differentiation of neural crest cells into glial cells, schwann cells, chromaffm 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.
  • 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.
  • a hedgehog antagonist to regulate the growth state of neurons and other neuronal cells in both the central nervous system and the peripheral nervous system.
  • the ability of ptc, hedgehog, and smoothened to regulate neuronal differentiation during development of the nervous system and also presumably in the adult state indicates that, in certain instances, the subject hedgehog antagonists can be expected to facilitate control of adult neurons with regard to maintenance, functional performance, and aging of normal cells; repair and regeneration processes in chemically or mechanically lesioned cells; and treatment of degeneration in certain pathological conditions.
  • 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 method can also be used in generating nerve prostheses for the repair of central and peripheral nerve damage.
  • hedgehog 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. patents 5,092,871 and 4,955,892.
  • the subject method can be used in the treatment of neoplastic or hyperplastic transformations such as may occur in the central nervous system.
  • the hedgehog antagonists can be utilized to cause such transformed cells to become either post-mitotic or apoptotic.
  • the present method may, therefore, be used as part of a treatment for, e.g., malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and ependymomas.
  • the subject method can be used as part of a treatment regimen for malignant meduUoblastoma and other primary CNS malignant neuroectodermal tumors.
  • the subject method is used as part of treatment program for meduUoblastoma.
  • MeduUoblastoma a primary brain tumor, is the most common brain tumor in children.
  • a meduUoblastoma is a primitive neuroectodermal tumor arising in the posterior fossa. They account for approximately 25% of all pediatric brain tumors (Miller). Histologically, they are small round cell tumors commonly arranged in true rosettes, but may display some differentiation to astrocytes, ependymal cells or neurons (Rorlce; Kleihues). PNET's may arise in other areas of the brain including the pineal gland (pineoblastoma) and cerebrum.
  • the subject method is used as part of treatment program for ependymomas.
  • Ependymomas account for approximately 10% of the pediatric brain tumors in children. Grossly, they are tumors that arise from the ependymal lining of the ventricles and microscopically form rosettes, canals, and perivascular rosettes. In the CHOP series of 51 children reported with ependymomas, % were histologically benign. Approximately 2/3 arose from the region of the 4th ventricle. One third presented in the supratentorial region. Age at presentation peaks between birth and 4 years, as demonstrated by SEER data as well as data from CHOP. The median age is about 5 years. Because so many children with this disease are babies, they often require multimodal therapy.
  • compositions comprising hedgehog antagonists can also be utilized for both cell culture and therapeutic methods involving generation and maintenance of non-neuronal tissue.
  • the present invention makes use of the discovery that ptc, hedgehog, and smoothened axe 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.
  • 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.
  • compositions of hedgehog antagonists can be utilized in conjunction with transplantation of such artificial livers, as well as embryonic liver structures, to regulate uptake of intraperitoneal implantation, vascularization, and in vivo differentiation and maintenance of the engrafted liver tissue.
  • the subject method can be employed therapeutically to regulate such organs after physical, chemical or pathological insult.
  • therapeutic compositions comprising hedgehog antagonists can be utilized in liver repair subsequent to a partial hepatectomy.
  • pancreas and small intestine from the embryonic gut depends on intercellular signalling between the endodermal and mesodermal cells of the gut.
  • the differentiation of intestinal mesoderm into smooth muscle has been suggested to depend on signals from adjacent endodermal cells.
  • Sonic hedgehog One candidate mediator of endodermally derived signals in the embryonic hindgut is Sonic hedgehog. See, for example, Apelqvist et al. (1997) Curr Biol 7:801-4.
  • the Shh gene is expressed throughout the embryonic gut endoderm with the exception of the pancreatic bud endoderm, which instead expresses high levels of the homeodomain protein Ipfl/Pdxl (insulin promoter factor 1/pancreatic and duodenal homeobox 1), an essential regulator of early pancreatic development.
  • Ipfl/Pdxl insulin promoter factor 1/pancreatic and duodenal homeobox 1
  • Apelqvist et al., supra have examined whether the differential expression of Shh in the embryonic gut tube controls the differentiation of the surrounding mesoderm into specialised mesoderm derivatives of the small intestine and pancreas. To test this, they used the promoter of the Ipfl Pdxl gene to selectively express Shh in the developing pancreatic epithelium.
  • pancreatic mesoderm developed into smooth muscle and interstitial cells of Cajal, characteristic of the intestine, rather than into pancreatic mesenchyme and spleen.
  • pancreatic explants exposed to Shh underwent a similar program of intestinal differentiation.
  • 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.
  • the subject method could be used as part of a technique to generate and/or maintain such tissue both in vitro and in vivo.
  • modulation of the function of hedgehog can be employed in both cell culture and therapeutic methods involving generation and maintenance ⁇ -cells and possibly also for non-pancreatic tissue, such as in controlling the development and maintenance of tissue from the digestive tract, spleen, lungs, urogenital organs (e.g., bladder), and other organs which derive from the primitive gut.
  • 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.
  • pancreatic cancers are marked by abnormal proliferation of pancreatic cells which can result in alterations of insulin secretory capacity of the pancreas.
  • pancreatic hyperplasias such as pancreatic carcinomas
  • pancreatic carcinomas can result in hypoinsulinemia due to dysfunction of ⁇ -cells or decreased islet cell mass.
  • the subject inhibitors can be used to enhance regeneration of the tissue after anti-tumor therapy.
  • the present invention makes use of the apparent involvement of ptc, hedgehog, and smoothened in regulating the development of pancreatic tissue.
  • the subject method can be employed therapeutically to regulate the pancreas after physical, chemical or pathological insult.
  • the subject method can be applied to to cell culture techniques, and in particular, may be employed to enhance the initial generation of prosthetic pancreatic tissue devices.
  • Manipulation of proliferation and differentiation of pancreatic tissue for example, by altering hedgehog activity, can provide a means for more carefully controlling the characteristics of a cultured tissue.
  • the subject method can be used to augment production of prosthetic devices which require ⁇ -islet cells, such as may be used in the encapsulation devices described in, for example, the Aebischer et al. U.S. Patent No. 4,892,538, the Aebischer et al. U.S. Patent No. 5,106,627, the Lim U.S. Patent No. 4,391,909, and the Sefton U.S. Patent No. 4,353,888.
  • Early progenitor cells to the pancreatic islets are multipotential, and apparently coactivate all the islet-specific genes from the time they first appear. As development proceeds, expression of islet- specific hormones, such as insulin, becomes restricted to the pattern of expression characteristic of mature islet cells.
  • the phenotype of mature islet cells is not stable in culture, as reappearence of embryonal traits in mature ⁇ -cells can be observed.
  • the differentiation path or proliferative index of the cells can be regulated.
  • 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.
  • manipulation of hedgehog function to affect tissue differentiation can be utilized as a means of maintaining graft viability.
  • Bellusci et al. (1997) Development 124:53 report that Sonic hedgehog regulates lung mesenchymal cell proliferation in vivo.
  • the present method can be used to regulate regeneration of lung tissue, e.g., in the treatment of emphysema.
  • Such tumors include, but are by no means limited to, tumors related to Gorlin's syndrome (e.g., basal cell carcinoma, meduUoblastoma, meningioma, etc.), tumors evidenced in pet knock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumors resulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.), tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma, thyroid carcinoma, etc.), Ext-1 -related tumors (e.g., bone cancer, etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.), and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney, bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinal cancer (e.g., stomach, intestin
  • 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.
  • 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.
  • 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.
  • 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 signal transduction, to regulate a cartilage repair response in the comiective tissue by managing the rate of differentiation and/or proliferation of chondrocytes embedded in the tissue.
  • a hedgehog antagonist particularly an antagonist selective for Indian hedgehog signal transduction
  • 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.
  • 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.
  • 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.
  • the subject method can be used to treat a degenerative disorder of a lcnee, 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.
  • 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.
  • the present invention further contemplates the use of the subject method in the field of cartilage transplantation and prosthetic device therapies.
  • 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.
  • meniscal cartilage is used to repair anterior cruciate ligaments, the tissue undergoes a metaplasia to pure fibrous tissue.
  • 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.
  • 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-glycosaminoglycan 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.
  • chondrocytes can be grown in culture on biodegradable, biocompatible highly porous scaffolds formed from polymers such as polyglycolic acid, polylactic acid, agarose gel, or other polymers which degrade over time as function of hydrolysis of the polymer backbone into innocuous monomers.
  • the matrices are designed to allow adequate nutrient and gas exchange to the cells until engraftment occurs.
  • the cells can be cultured in vitro until adequate cell volume and density has developed for the cells to be implanted.
  • One advantage of the matrices is that they can be cast or molded into a desired shape on an individual basis, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices can be used which allow for manipulation at the time of implantation, as in a joint.
  • 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.
  • the implanted device is treated with a hedgehog antagonist in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
  • a hedgehog antagonist in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
  • 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.
  • the subject method is used to enhance attachment of prosthetic devices.
  • 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.
  • 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.
  • 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.
  • preparations comprising hedgehog antagonists can be employed, for example, to control endochondral ossification in the formation of a "model" for ossification.
  • a hedgehog antagonist can be used to regulate spermatogenesis.
  • the hedgehog proteins particularly Dhh, have been shown to be involved in the differentiation and/or proliferation and maintenance of testicular germ cells. Dhh expression is initiated in Sertoli cell precursors shortly after the activation of Sry (testicular determining gene) and persists in the testis into the adult. Males are viable but infertile, owing to a complete absence of mature sperm. Examination of the developing testis in different genetic backgrounds suggests that Dhh regulates both early and late stages of spermatogenesis. Bitgood et al. (1996) Curr Biol 6:298.
  • the hedgehog antagonist can be used as a contraceptive. In similar fashion, hedgehog antagonists of the subject method are potentially useful for modulating normal ovarian function.
  • the subject method also has wide applicability to the treatment or prophylaxis of disorders afflicting epithelial tissue, as well as in cosmetic uses.
  • 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.
  • 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.
  • “promotion of wound healing” can also mean that certain methods of wound healing have improved success rates, (e.g., the take rates of sldn grafts,) when used together with the method of the present invention.
  • 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.
  • 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.
  • lack of treatment results in proliferation of tissue around the periphery of the lesion on an inflammatory basis.
  • the epithelium bordering the ulcer usually demonstrates proliferative activity, resulting in loss of continuity of surface epithelium.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject method provides a means for inhibiting proliferation of the remaining lens epithelial cells.
  • 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.
  • 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.
  • the subject method can also be used in the treatment of corneopathies marked by corneal epithelial cell proliferation, as for example in ocular epithelial disorders such as epithelial downgrowth or squamous cell carcinomas of the ocular surface.
  • 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.
  • Hair “growth” refers to the formation and elongation of the hair fiber by the dividing cells.
  • the common hair cycle is divided into three stages: anagen, catagen and telogen.
  • 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.
  • telogen The resting stage is Icnown 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.
  • 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.
  • the present method can be used in the treatment of trichosis characterized by abnormally rapid or dense growth of hair, e.g. hypertrichosis.
  • 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.
  • 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.
  • hedgehog antagonists can be used for patients undergoing chemo- or radiation- therapies which ordinarily result in hair loss.
  • the subject treatment can protect hair follicle cells from death which might otherwise result from activation of cell death programs.
  • the instant method can also be removed with concommitant relief of the inhibition of follicle cell proliferation.
  • the subject method can also be used in the treatment of foUiculitis, such as foUiculitis decalvans, foUiculitis ulerythematosa reticulata or Iceloid foUiculitis.
  • 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.
  • the subject method can be used to induce differentiation and/or inhibit proliferation of epithelially derived tissue.
  • Such forms of these molecules can provide a basis for differentiation therapy for the treatment of hyperplastic and/or neoplastic conditions involving epithelial tissue.
  • preparations can be used for the treatment of cutaneous diseases in which there is abnormal proliferation or growth of cells of the sldn.
  • 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 epidennis, as for example, caused by psoriasis or atopic dermatosis.
  • psoriasis which is characterized by scaly, red, elevated plaques on the skin
  • the keratinocytes are Icnown to proliferate much more rapidly than normal and to differentiate less completely.
  • the preparations of the present invention are suitable for the treatment of dermatological ailments linlced to keratinization disorders causing abnormal proliferation of skin cells, which disorders may be marked by either inflammatory or non-inflammatory components.
  • a hedgehog antagonist e.g., which promotes quiescence or differentiation
  • a hedgehog antagonist e.g., which promotes quiescence or differentiation
  • 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.
  • keratotic lesions are also candidates for treatment with the subject method.
  • Actinic lceratoses 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.
  • treatment of keratosis can include application, preferably topical, of a hedgehog antagonist composition in amounts sufficient to inhibit hyperproliferation of epidermal/epidermoid cells of the lesion.
  • 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 Staphylo coccus epidermidis bacteria and Pitrosporum ovale, a yeast.
  • Treatment with an antiproliferative hedgehog antagonist, particularly topical preparations, may be useful for preventing the transitional features of the ducts, e.g. hypercornification, which lead to lesion formation.
  • the subject treatment may further include, for example, antibiotics, retinoids and antiandrogens.
  • 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.
  • 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, antipuritics, and antibiotics.
  • the subject method could be used to inhibit angiogenesis.
  • Hedgehog is Icnown to stimulate angiogenesis.
  • Hedgehog protein is also capable of increasing vascularization of the normally avascular mouse cornea.
  • the ptc-1 gene is expressed in normal vascular tissues, including the endothelial cells of the aorta, vascular smooth muscle cells, adventitial fibroblasts of the aorta, the coronary vasculature and cardiomyocytes of the atria and ventricles. These tissues are also sensitive to hedgehog protein. Treatment with exogenous hedgehog causes upregulation of ptc-1
  • hedgehog proteins stimulate proliferation of vascular smooth muscle cells in vivo. Hedgehog proteins also cause fibroblasts to increase expression of angiogenic growth factors such as VEGF, bFGF, Ang-1 and Ang-2. Lastly, hedgehog proteins are known to stimulate recovery from ischemic injury and stimulate formation of collateral vessels.
  • hedgehog antagonists are expected to act as angiogenesis inhibitors, particularly in situations where some level of hedgehog signaling is necessary for angiogenesis.
  • Angiogenesis is fundamental to many disorders. Persistent, unregulated angiogenesis occurs in a range of disease states, tumor metastases and abnormal growths by endothelial cells. The vasculature created as a result of angiogenic processes supports the pathological damage seen in these conditions. The diverse pathological states created due to unregulated angiogenesis have been grouped together as angiogenic dependent or angiogenic associated diseases. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
  • Diseases caused by, supported by or associated with angiogenesis include ocular neovascular disease, age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pteiygium keratitis sicca, Sjogren's, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, poly
  • angiogenesis plays a critical role in cancer.
  • a tumor cannot expand without a blood supply to provide nutrients and remove cellular wastes.
  • Tumors in which angiogenesis is important include solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma, and benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
  • Angiogenic factors have been found associated with several solid tumors. Prevention of angiogenesis could halt the growth of these tumors and the resultant damage to the animal due to the presence of the tumor.
  • Angiogenesis is also associated with blood-born tumors such as leulcemias, any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen. It is believed that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia-like tumors.
  • angiogenesis is important in metastasis. Initially, angiogenesis is important is in the vascularization of the tumor which allows cancerous cells to en,ter the blood stream and to circulate throughout the body. After the tumor cells have left the primary site, and have settled into the secondary, metastasis site, angiogenesis must occur before the new tumor can grow and expand. Therefore, prevention of angiogenesis could lead to the prevention of metastasis of tumors and possibly contain the neoplastic growth at the primary site.
  • Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Prevention of angiogenesis could be used to induce amenorrhea, to block ovulation or to prevent implantation by the blastula. It is anticipated that the invention will be useful for the treatment and/or prevention of respiratory distress syndrome or other disorders resulting from inappropriate lung surface tension. Respiratory distress syndrome results from insufficient surfactant in the alveolae of the lungs. The lungs of vertebrates contain surfactant, a complex mixture of lipids and protein which causes surface tension to rise during lung inflation and decrease during lung deflation.
  • lung deflation surfactant decreases such that there are no surface forces that would otherwise promote alveolar collapse. Aerated alveoli that have not collapsed during expiration permit continuous oxygen and carbon dioxide transport between blood and alveolar gas and require much less force to inflate during the subsequent inspiration.
  • lung surfactant increases surface tension as the alveolar surface area increases. A rising surface tension in expanding alveoli opposes over-inflation in those airspaces and tends to divert inspired air to less well-aerated alveoli, thereby facilitating even lung aeration. Respiratory distress syndrome is particularly prevalent among premature infants. Lung surfactant is normally synthesized at a very low rate until the last six weeks of fetal life.
  • Lung tissue of premature infants shows high activity of the hedgehog signaling pathway. Inhibition of this pathway using hedgehog antagonists increases the formation of lamellar bodies and increases the expression of genes involved in surfactant biosynthesis. Lamellar bodies are subcellular structures associated with surfactant biosynthesis. For these reasons, treatment of premature infants with a hedgehog antagonist should stimulate surfactant biosynthesis and ameliorate RDS. In cases where adult RDS is associated with hedgehog pathway activation, treatment with hedgehog antagonists should also be effective. It is further contemplated that the use of hedgehog antagonists may be specifically targeted to disorders where the affected tissue and/or cells evince high hedgehog pathway activation. Expression of gli genes is activated by the hedgehog signaling pathway, including gli-1, gli-2 and gli-3.
  • gli-1 expression is most consistently correlated with hedgehog signaling activity across a wide range of tissues and disorders, while gli-3 is somewhat less so.
  • the gli genes encode transcription factors that activate expression of many genes needed to elicit the full effects of hedgehog signaling.
  • the Gli-3 transcription factor can also act as a repressor of hedgehog effector genes, and therefore, expression of gli-3 can cause a decreased effect of the hedgehog signaling pathway.
  • Gli-3 acts as a transcriptional activator or repressor depends on post-translational events, and therefore it is expected that methods for detecting the activating form (versus the repressing form) of Gli-3 protein would also be a reliable measure of hedgehog pathway activation
  • gli-2 gene expression is expected to provide a reliable marker for hedgehog pathway activation.
  • the gli-1 gene is strongly expressed in a wide array of cancers, hyperplasias and immature lungs, and serves as a marker for the relative activation of the hedgehog pathway.
  • tissues, such as immature lung, that have high gli gene expression are strongly affected by hedgehog inhibitors. Accordingly, it is contemplated that the detection of gli gene expression may be used as a powerful predictive tool to identify tissues and disorders that will particularly benefit from treatment with a hedgehog antagonist.
  • gli-1 expression levels are detected, either by direct detection of the transcript or by detection of protein levels or activity.
  • Transcripts may be detected using any of a wide range of techniques that depend primarily on hybridization of probes to the gli-1 transcripts or to cDNAs synthesized therefrom.
  • Well known techniques include northern blotting, reverse-transcriptase PCR and microarray analysis of transcript levels.
  • Methods for detecting Gli protein levels include western blotting, immunoprecipitation, two-dimensional polyacrylamide gel electrophoresis (2D SDS-PAGE)(preferably compared against a standard wherein the position of the Gli proteins has been determined), and mass spectroscopy.
  • Mass spectroscopy may be coupled with a series of purification steps to allow high-throughput identification of many different protein levels in a particular sample. Mass spectroscopy and 2D SDS-PAGE can also be used to identify post-transcriptional modifications to proteins including proteolytic events, ubiquitination, phosphorylation, lipid modification etc. Gli activity may also be assessed by analyzing binding to substrate DNA or in vitro transcriptional activation of target promoters. Gel shift assays, DNA footprinting assays and DNA-protein crosslinldng assays are all methods that may be used to assess the presence of a protein capable of binding to Gli binding sites on DNA.
  • gli transcript levels are measured and diseased or disordered tissues showing abnormally high gli levels are treated with a hedgehog antagonist.
  • a hedgehog antagonist Premature lung tissue, lung cancers (e.g., adenocarcinomas, bronchoalveolar adenocarcinomas, small cell carcinomas), breast cancers (e.g., inferior ductal carcinomas, inferior lobular carcinomas, tubular carcinomas), prostate cancers (e.g., adenocarcinomas), and benign prostatic hyperplasias all show strongly elevated gli-1 expression levels in certain cases. Accordingly, gli-1 expression levels are a powerful diagnostic device to determine which of these tissues should be treated with a hedgehog antagonist.
  • cancers of urothelial cells e.g., bladder cancer, other urogenital cancers
  • bladder cancer other urogenital cancers
  • gli-1 levels in certain cases.
  • loss of heterozygosity on chromosome 9q22 is common in bladder cancers.
  • the ptc-1 gene is located at this position and ptc-1 loss of function is probably a partial cause of hyperproliferation, as in many other cancer types. Accordingly, such cancers would also show high gli expression and would be particularly amenable to treatment with a hedgehog antagonist.
  • ptc-1 and ptc-2 are also activated by the hedgehog signaling pathway, but these genes are inferior to the gli genes as markers of hedgehog pathway activation.
  • ptc-1 ox ptc-2 is expressed although the hedgehog pathway is highly active.
  • Indian hedgehog plays an important role and the hedgehog pathway is activated, but only ptc-2 is expressed. Accordingly, these genes are individually unreliable as markers for hedgehog pathway activation, although simultaneous measurement of both genes are contemplated as a useful indicator for tissues to be treated with a hedgehog antagonist.
  • Ailments which may be treated by the subject method are disorders specific to non-humans, such as mange.
  • 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.
  • 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.
  • the subject method is used as part of a treatment ot prophylaxis regimen for treating (or preventing) basal cell carcinoma.
  • the deregulation of the hedgehog signaling pathway may be a general feature of basal cell carcinomas caused by ptc mutations. Consistent overexpression of human ptc mRNA has been described in tumors of familial and sporadic BCCs, determined by in situ hybridization. Mutations that inactivate ptc may be expected to result in overexpression of mutant Ptc, because ptc displays negative autoregulation. Prior research demonstrates that overexpression of hedgehog proteins can also lead to tumorigenesis.
  • That sonic hedgehog (Shh) has a role in tumorigenesis in the mouse has been suggested by research in which transgenic mice overexpressing Shh in the skin developed features of BCNS, including multiple BCC-like epidermal proliferations over the entire skin surface, after only a few days of skin development.
  • a mutation in the Shh human gene from a BCC was also described; it was suggested that Shh or other Hh genes in humans could act as dominant oncogenes in humans.
  • Sporadic ptc mutations have also been observed in BCCs from otherwise normal individuals, some of which are UV-signature mutations.
  • the subject method can also be used to treatment patients with BCNS, e.g., to prevent BCC or other effects of the disease which may be the result of ptc loss-of- function, hedgehog gain-of-function, or smoothened gain-of-function.
  • Basal cell nevus syndrome is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age. BCNS patients are very susceptible to the development of these tumors; in the second decade of life, large numbers appear, mainly on sun-exposed areas of the skin. This disease also causes a number of developmental abnormalities, including rib, head and face alterations, and sometimes polydactyly, syndactyly, and spina bifida.
  • BCNS fibromas of the ovaries and heart
  • cysts of the skin and jaws and in the central nervous system
  • medulloblastomas and meningiomas The subject method can be used to prevent or treat such tumor types in BCNS and non-BCNS patients.
  • Studies of BCNS patients show that they have both genomic and sporadic mutations in the ptc gene, suggesting that these mutations are the ultimate cause of this disease.
  • the present invention provides pharmaceutical preparations comprising hedgehog antagonists.
  • the hedgehog antagonists for use in the subject method may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, controlled release patch, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral and topical administrations are preferred.
  • 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.
  • 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.
  • 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.
  • 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 Icnown to those of skill in the art.
  • 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.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular hedgehog antagonist employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well Icnown in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • 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.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • 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.
  • 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.
  • treatment is intended to encompass also prophylaxis, therapy and cure.
  • 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.
  • the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable and/or sterile carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.
  • Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • compositions 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.
  • the compound included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting.
  • 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.
  • 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.
  • the subject compounds may be simply dissolved or suspended in sterile water.
  • terapéuticaally effective amount means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect by overcoming a ptc loss-of-function, hedgehog gain-of-function, or smoothened gain- of-function in at least a sub-population of cells in an animal and thereby blocking the biological consequences of that pathway in the treated cells, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases "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.
  • pharmaceutically acceptable carrier 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.
  • 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.
  • 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, mamiitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide
  • 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.
  • pharmaceutically acceptable salts 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.
  • sulfate bisulfate
  • phosphate nitrate
  • acetate valerate
  • oleate palmitate
  • stearate laurate
  • benzoate lactate
  • phosphate tosylate
  • citrate maleate
  • fumarate succinate
  • tartrate tartrate
  • napthylate mesylate
  • mesylate glucoheptonate
  • lactobionate lactobionate
  • 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.
  • 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.
  • 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.
  • pharmaceutically acceptable salts 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 tike.
  • 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)
  • wetting agents 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.
  • antioxidants examples 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.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • 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 Icnown in the ait 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.
  • compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • 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.
  • 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.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • 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
  • 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.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention 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.
  • compositions 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.
  • 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.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • 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.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 Icnown in the art to be appropriate.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • aqueous and nonaqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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.
  • 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.
  • 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
  • the absorption of the drug 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.
  • 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.
  • the compounds of the present invention 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.
  • 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.
  • 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.
  • an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed.
  • feed premixes and complete rations can be prepared and administered are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feeds and Feeding” O and B books, Corvallis, Ore., U.S.A., 1977).
  • a subject compound such as compound D or a salt thereof, may be formulated in an aqueous solution, e.g., for topical application.
  • Suitable salts include salts of subject compounds with hydrochloric acid, hydrobromic acid, hydroiodic acid, succinic acid, tartaric acid, lactic acid, methanesulfonic acid, maleic acid, or any other suitable acid, such as one that forms a pharmaceutically acceptable anion in the presence of an amine base.
  • the aqueous solution may contain a pharmaceutically acceptable salt, such as a salt including a cation selected from sodium, potassium, magnesium, and calcium, and an anion selected from acetate, citrate, phosphate, chloride, any other suitable ions, or combinations thereof.
  • a pharmaceutically acceptable salt such as a salt including a cation selected from sodium, potassium, magnesium, and calcium, and an anion selected from acetate, citrate, phosphate, chloride, any other suitable ions, or combinations thereof.
  • the aqueous solution may, additionally or alternatively, include dextrose, lactose, mannitol, or another polyhydroxylated compound, such as a pharmaceutically acceptable carbohydrate, such as a mono- or di-saccharide, or polyol.
  • an aqueous solution may contain solutes to result in an osmolarity between 200 and 400 mOsm, preferably between 250 and 350 Osm, even more preferably between 280 and 300 mOsm, such as 290 mOsm.
  • the pH of the solution will be in the range of 3 to 6, preferably 3.5 to 5, even more preferably between 4 and 4.5.
  • an aqueous solution may comprise up to about 7% of a carbohydrate or polyol such as mannitol, lactose, or dextrose, e.g., up to about 6%, ox about 3% to about 6%, or about 4 to about 5%, up to about 50 mM of a salt selected from sodium acetate and sodium citrate, e.g., up to about 20 mM, or about 2 mM to about 20 mM, or about 5 mM to about 15 mM, and sufficient pharmaceutically acceptable solutes, such as sodium chloride, to result in an osmolarity between about 200 and 400 mOsm, preferably between 250 and 350 Osm, even more preferably between 280 and 300 mOsm.
  • a carbohydrate or polyol such as mannitol, lactose, or dextrose
  • a salt selected from sodium acetate and sodium citrate e.g., up to about 20 mM,
  • the aqueous solution is substantially free of a carbohydrate or polyol, substantially free of sodium acetate and sodium citrate, or both, e.g., may consist essentially of physiological saline and a salt of a subject compound, or is substantially free of salts such as sodium chloride, sodium acetate, and sodium citrate and consists essentially of an aqueous solution of a carbohydrate or polyol and a salt of a subject compound.
  • an aqueous solution of a subject compound such as compound D
  • an aqueous solution may comprise 10 mM sodium acetate in physiological saline at a pH of 4.2.
  • an aqueous solution may comprise 10 mM sodium acetate in a 5% dextrose solution, or simply a 5% dextrose solution.
  • the subject antagonists, 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.
  • the compounds of the present invention 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 antagonist lead compounds, as well as to refine the specificity, toxicity, and/or cytotoxic-ldnetic profile of a lead compound.
  • ptc, hedgehog, or smoothened bioactivity assays such as may be developed using cells with either a ptc loss-of-function, hedgehog gain-of- function, or smoothened gain-of-function, can be used to screen a library of the subject compounds for those having agonist activity toward ptc or antagonist activity towards hedgehog or smoothened.
  • 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.
  • 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.
  • 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 linlced to a polymer bead by a hydrolyzable or photolyzable group, optionally located at one of the positions of the candidate antagonists or a substituent of a synthetic intermediate.
  • the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead.
  • the bead library can then be "plated" with ptc loss-of-function, hedgehog gain-of- function, or smoothened gain-of-function cells for which an hedgehog antagonist is sought.
  • the diversomers can be released from the bead, e.g. by hydrolysis.
  • a series of compounds conforming to the general structures disclosed herein were prepared and tested for biological activity (vide infra).
  • a suitable core structure can be readily prepared from commercially available tr ⁇ r ⁇ -4-hydroxy-L-proline as summarized in the scheme below:
  • the organic layer was washed with 1 M aqueous KHCO 3 (950 mL), 1 M aq. citric acid (2 x 950 mL), and water (750 mL) and dried over MgSO .
  • the solvent was removed in vacuo and the residue crystallized with hexane (1.9 L).
  • the crystalline mesylate was collected on a filter, washed with hexane (2 x 500 mL), and dried at 40 °C in vacuo to give 624 g of the product.
  • a saturated solution of HCl in dioxane (1940 mL) was prepared at 10-16 °C, and a solution of the azide (523 g, 1.94 mmol) in dioxane (480 mL) was added dropwise with stirring and cooling over 30 min. to keep the temperature below 25
  • Subject compounds can be prepared from the above core, or from related compounds or derivatives, using solution-phase or solid-phase techniques, as shown in the schemes below:
  • trans-hydroxy-L-proline compound all isomers of this compound are commercially available, including cis/trans and D/L compounds, providing access to a wide range of diastereomerically pure intermediates and subject compounds.
  • a trans- aminoproline core can be obtained from a tr ⁇ r ⁇ -hydroxyproline starting material by forming an intermediate cz ' -y-bromoproline (by forming, for example, a triflate or mesylate of the hydroxyl and displacing the sulfonate with bromide ion), followed by a second displacement with azide, to provide net retention of the trans stereochemical relationship, as is well Icnown in the art.
  • diastereomeric mixtures may be prepared, as in the above Scheme 3, followed by an optional separation of the isomers.
  • test compounds can also be tested in cell- based assays.
  • cell which have a ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-function phenotype can be contacted with a test agent of interest, with the assay scoring for, e.g., inhibition of proliferation of the cell in the presence of the test agent.
  • 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-xnediated 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.
  • 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 GH3 gene is downregulated in response to hedgehog induction (Marigo et al. (1996) Development 122:1225-1233).
  • 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
  • a transcription based assay which is sensitive to the ability of a specific test compound to modify hedgehog-xnediated signalling pathways. Expression of the reporter gene, thus, provides a valuable screening tool for the development of compounds that act as antagonists of hedgehog.
  • Reporter gene based assays of this invention measure the end stage of the above described cascade of events, e.g., transcriptional modulation.
  • a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on ptc loss-of-function, hedgehog gain-of-function, smoothened gain-of-function, or stimulation by SHH itself.
  • the amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, mRNA expression from the reporter gene may be detected using RNAse protection or RNA- based PCR, or the protein product of the reporter gene may be identified by a characteristic stain or an intrinsic biological activity.
  • the amount of expression from the reporter gene is then compared to the amount of expression in either the same cell in the absence of the test compound or it may be compared with the amount of transcription in a substantially identical cell that lacks the target receptor protein. Any statistically or otherwise significant decrease in the amount of transcription indicates that the test compound has in some manner agonized the normal ptc signal (or antagonized the gain-of-function hedgehog or smoothened signal), e.g., the test compound is a potential hedgehog antagonist.
  • Lithium hydroxide monohydrate (17 mg, 0.405 mmol) was added to a solution of methyl (2S,4i?)-l-(l,3-benzodioxol-5-ylmethyl)-4-[(3,3- dimethylbutanoyl)(3-methoxybenzyl)amino]tetrahydro-lH-2-pyrrolecarboxylate (10) (100 mg, 0.20 mmol) in 66 % v/v methanol in water (1.0 mL). The mixture was stirred overnight at room temperature, then the solvent was removed under reduced pressure and the residue partitioned between dichloromethane (1.0 mL) and water (1.0 ml).
  • the aqueous phase was acidified with 1.0 M aqueous citric acid and the two layers were vigorously stirred for 10 min at room temperature. The layers were separated and the aqueous layer was back-extracted with dichloromethane. The combined dichloromethane extracts were dried (MgSO 4 ) and evaporated under reduced pressure to give the title acid (11) (70 mg, 72%) as an off-white solid: ⁇ H (360 MHz; CDC1 3 ) 1.00 and 1.03 (2xs, 9H, rotamers), 2.17-2.39 (m, 3H), 2.62-2.71 (m, IH), 3.28-3.34 (m.
  • Lithium hydroxide monohydrate (17 mg, 0.405 mmol) was added to a solution of methyl (2S,4S)-l-(l,3-benzodioxol-5-ylmethyl)-4-[(3,3- dimethylbutanoyl)(3-methoxybenzyl)amino]tetrahydro-lH-2-pyrrolecarboxylate (20) (100 mg, 0.20 mmol) in 66 % v/v methanol in water (1.0 mL). The mixture was stirred overnight at room temperature, then the solvent was removed under reduced pressure and the residue partitioned between dichloromethane (1.0 mL) and water (1.0 ml).
  • the aqueous phase was acidified with 1.0 M aqueous citric acid solution and the two layers were vigorously stirred for 10 min at room temperature. The layers were separated and the aqueous layer was back-extracted with dichloromethane. The combined dichloromethane extracts were dried (MgSO 4 ) and evaporated under reduced pressure. The residue was purified by flash column cliromatography on silica gel using dichloromethane/ethyl acetate (1:1, v/v) and then dichloromethane/methanol (9:1, v/v) as eluents to give the title acid (21) (88 mg, 91%>) as an off-white solid:
  • Variations of the protecting group scheme can increase the efficiency and speed with which compounds of the subject invention may be prepared.
  • the schemes below which can be readily executed by one of skill in the art based on the disclosure above together with Icnown methods in the art, provide rapid, efficient routes to compounds which may inhibit hedgehog activity.
  • the particular moieties, groups, and reactions e.g., electrophilic or reductive alkylation of the amine
  • Method 2 dichloromethane, hexane, N,N-dimethylformamide, dichloromethane, hexane, dichloromethane and hexane;
  • Method 3 water, N,N-dimethylformamide, water, 1.0 M aqueous sodium hydroxide solution, water, N,N-dimethylformamide, water, 1.0 M aqueous sodium hydroxide solution, water, N,N-dimethylformamide, dichloromethane, methanol, dichloromethane and methanol
  • Method 4 N,N-dimethylformamide, dichloromethane, N,N- dimethylformamide, dichloromethane, methanol, dichloromethane , methanol (2x) and ether (2x).
  • Step A The preparation of (Nitrophen-4 '-yloxycarboxy)benz-4- yloxymethyl polystyrene-(Wang PNP carbonate polystyrene)
  • Step C Building Block loading onto Wang Diamine:
  • Step D Reductive Animation at C-4
  • Step E Reductive Alkylation or Capping
  • Resin analogues were suspended in a 20% v/v solution of piperidine in N,N- dimethylformamide and gently agitated for 30 min at room temperature. The resin suspension was subsequently filtered and washed with N,N-dimethylformamide. This treatment of the resin with piperidine in N,N-dimethylformamide was repeated once more to ensure complete N-Fmoc-deprotection. After standing for 30 min, the resin was filtered, washed using the sequence listed in method 4 and dried under vacuum at 40 °C.
  • Step G Reductive Alkylation or Capping atNl
  • the appropriate resin ( ⁇ 60mg per well in a 2 ml filter block) was swollen in a 50% v/v mixture of anhydrous tetrahydrofuran and chloroform (1 mL), then gently agitated and treated with the appropriate acid chloride (5 equivalents) and N,N- diisopropylethylamine ( ⁇ 150 ⁇ l, 10 equivalents). After gentle agitation of the filter blocks overnight at room temperature, the resins were filtered and washed using the sequence listed in method 5.
  • Step H Cleavage of Final Product from Wang resin using TFA:
  • Compounds to be tested are dissolved in DMSO to a concentration of 10 mM, and stored at -20 °C.
  • an octylated (lipid-modified) form of the N-terminal fragment of the Sonic Hedgehog protein (OCT-SHH) is used. This N-temiinal SHH fragment is produced bacterially.
  • Compounds may be tested in the "Gli-Luc" assay below, using the cell line 10T(sl2), wherein the cells contain a Hedgehog-responsive reporter construct utilizing Luciferase as the reporter gene. In this way, Hedgehog pathway signaling activity can be measured via the Gli-Luc response.
  • 10tl/2(sl2) cells are plated in a 96-well micro-titer plate (MTP) at 20,000 cells/well in full medium [DMEM with 10%) FBS]. Then plates are placed in the incubator for incubation overnight (O/N), at 37 °C and 5%> CO 2 . After 24 h, the medium is replaced with Luciferase-assay medium (DMEM with 0.5% FBS). Compounds are thawed and diluted in assay medium at 3:1000 (about 300-fold) resulting in a starting concentration of about 30 ⁇ M.
  • each 30 ⁇ M sample is added to the first wells (in triplicate).
  • the MTP samples are then diluted at 3 -fold dilutions to a total of seven wells, ultimately resulting in a regiment of seven dilutions in triplicate, for each compound.
  • the protein ligand OCT-SHH is diluted in Luciferase-assay medium and added to each well at a final concentration of 0.3 ⁇ g/ml. Plates are then returned to the incubator for further incubation O/N, at 37 °C and 5% CO 2 . After about 24 h, plates are removed from the incubator and the medium is aspirated/discarded.
  • RNA samples were cultured for 3 days in the presence of vehicle; jervine, a Icnown Patched pathway antagonist (i) used here as a positive control; or 1 ⁇ M of compound D.
  • Total ribonucleic acid (RNA) was isolated from the cells and used for reverse transcriptase-polymerase chain reaction (RT-PCR). Specific primers for the detection of mouse gli-1 mRNA were used in the PCR, and the actin gene was used to demonstrate that equivalent amounts of mRNA samples were compared in the experiment.
  • the gli-1 and actin mRNA samples were then loaded on 1.5%> agarose gel and were detected by staining with ethidium bromide. The same samples were analyzed by the quantitative real-time polymerase chain reaction method to quantify the levels of gli-1 mRNA.
  • Figure 33 A shows the results of a representative experiment. It shows gli-1 mRNA expression in cells treated with a vehicle control (Lane 1); 5 ⁇ M jervine, the positive control compound (Lane 2); and 1 ⁇ M D (Lane 3). Compared with vehicle, D and jervine significantly decreased the expression of gli-1 mRNA inj?tc-null cells. The levels of actin mRNA were equivalent in all conditions, indicating that equal quantities of RNA were analyzed in the experiment. This qualitative result was confirmed by the quantitative real-time PCR analysis (Figure 33B), which shows that D and jervine downregulated the gli-1 mRNA levels.
  • a novel cell culture assay was established to determine the effects of D on activation of the Patched pathway in skin.
  • activation of the Patched pathway results in increased expression of the ptc gene.
  • To monitor the activity of the Patched pathway in embryonic skin we cultured pieces of skin from transgenic Patched pathway reporter mice. These mice were genetically engineered to harbor a foreign gene (lacZ).
  • lacZ encodes a bacterial beta-galactosidase. The gene was inserted in the ptc locus but allowed for normal ptc function.
  • Ptc activation in response to Shh-induced Patched pathway activation can then be monitored by the production of the lacZ gene product, beta- galactosidase, which is detectable by the enzymatic conversion of the substrate X-gal into a blue-colored reaction product.
  • FIG. 35C shows that D completely blocked the formation of BCC-like structures without affecting the integrity of normal skin cells. This confirms that D can prevent the appearance of BCC-like structures produced by activating the Patched pathway, the same pathway that underlies the human disease.
  • Transgenic mouse-derived skin punches were treated with vehicle or D for 5 hours in the absence of Shh. After the pretreatment, the vehicle or D was removed.
  • the skin punches were washed twice and then cultured in the presence of Shh for
  • Embryonic Mouse Skin Punch Assay Long-term Treatment of Pre-existing BCC- Like Structures with D Methods Day 17.5 embryonic sldn punches from transgenic Patched pathway reporter mice were cultured in the presence of Shh protein for 7 days to allow for the development of BCC-like structures. The Shh protein was removed at the end of the 7 days. The cultures were then exposed to Shh protein plus either vehicle or D for 3 days. The cultures were analyzed histologically after 10 days to assess the formation of BCC-like structures that are indicative of activation of the Patched pathway. Results
  • TUNEL terminal deoxynucleotidyltransferase mediated d-UTP nick end-labeling
  • Embryonic Mouse Skin Punch Assay Short-term Treatment of Pre-existing BCC-like Structures with D Methods Day 17.5 embryonic skin punches from transgenic Patched pathway reporter mice were cultured in the presence of Shh protein for 7 days. The Shh protein was removed at the end of the 7 days. The skin punches were then exposed to vehicle or 1 or 5 ⁇ M D for 5 hours on days 7 and 9. After each exposure to vehicle or D, the vehicle or D was washed off and the skin punches were cultured again in the presence of Shh protein. Cultures were analyzed by X-gal staining after 10 days in vitro to assess the " activity of the Patched pathway. Results
  • Ptc heterozygous transgenic mice were irradiated 3 times weekly for 6 months, during which time many small, and often microscopic, BCC tumors developed.
  • Four-mm diameter skin punch explants presumably containing BCC structures, were cultured for 6 days in the presence of vehicle, the positive control (jervine), or 5 ⁇ M D.
  • the explants were analyzed by X- gal staining to detect the level of Patched pathway activity, by histology to determine the effect of treatment on the morphology of ultraviolet radiation-induced BCCs, and by quantifying the level of gli-1 mRNA expression to characterize the extent of pathway inhibition.
  • X-gal staining of the treated explants shows that skin punches cultured in the presence of vehicle alone developed intensely stained blue foci indicative of a focal upregulation of the Patched pathway and BCC structures (blue spots in Figure 39A).
  • 5 ⁇ M D like the positive control, decreased the number and size of established BCC structures.
  • Histological analysis of sectioned explants demonstrated that D induced the regression of ultraviolet radiation-induced BCC tumors, as compared with the vehicle control ( Figure 39B).
  • the levels of gli-1 mRNA were high because of the activation of ptc target genes.
  • D at concentrations of 1 and 5 ⁇ M also significantly inhibited the level of gli-1 mRNA levels compared with vehicle alone. Quantification of gli-1 mRNA levels shows the almost complete inhibition of target gene activation by D (Figure 39C). This inhibition did not appear to be caused by non-specific cytotoxicity, as statistical comparison of the levels of the housekeeping GAPDH enzyme between treated and vehicle conditions shows no significant difference among groups in general cellular metabolic activity. Thus, these results demonstrate that D inhibits the Patched pathway and induces the regression of ultraviolet radiation-induced, BCNS-like, BCC tumors in cultured skin explants. These data confirm the results of previous experiments and suggest that D might be effective in treating BCC.
  • Specimens obtained from surgical procedures were cultured on fresh, living, day 17 embryonic mouse dermis from which all epidermal cells have been removed by digestion using dispase. Since dispase treatment digests basement membrane components, Matrigel, a commercially available basement membrane preparation, was applied between the dermis and BCC. Cultures were assembled on top of a plastic grid and incubated for 3 days (with or without D at a concentration of 10 ⁇ M) in a medium suitable for the long-term culture of human skin. After culture, the samples were processed for routine histology and subjected to quantitative in situ hybridization.
  • Cyanogen bromide e.g., DCM, DMF, THF, toluene.
  • Base neutral solvents (e.g., DCM, toluene, THF).
  • H2NOH neutral solvents (e.g., DCM, DMF, THF, toluene).
  • neutral solvents e.g., DCM, DMF, THF, toluene
  • R2NHCH2CO2Me NaBH(OAc)3, CICH2CH2CI or THF 134)
  • RCO 2 H EDC or HBTU, HOBt, DIEA, CH 2 C1 2 or DMF.
  • Carbonyldiimidazole, neutral solvents e.g., DCM, DMF, THF, toluene.
  • neutral solvents e.g., DCM, DMF, THF, toluene.

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Abstract

L'invention concerne des procédés et des réactifs destinés à inhiber des états de croissance aberrante résultant d'une augmentation de la fonction hedgehog, d'une perte de la fonction ptc ou d'une augmentation de la fonction smoothened. Les procédés comprennent une mise en contact de la cellule avec un antagoniste de hedgehog, p. ex. petite molécule, en quantité suffisante pour inhiber la croissance aberrante, p. ex. afin de produire un effet agoniste sur une voie de signalisation de ptc normale, ou un effet antagoniste sur l'activité de smoothened ou de hedgehog.
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CN108588011B (zh) * 2018-05-08 2022-04-05 中国农业科学院北京畜牧兽医研究所 一种提高玻璃化冷冻卵母细胞体外受精能力的方法

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