Medicament for treatment of tumors wherein hedgehog/smoothened signaling is employed for inhibition of apoptosis of the tumor cells
This invention concerns the use of cyclopamine in vivo on basal cell carcinomas (BCC's) to achieve therapeutic effect by causing differentiation of the tumor cells and, at the same time, apoptotic death and removal of these tumor cells while preserving the normal tissue cells, including the undifferentiated cells of the normal epidermal basal layer and hair follicles. Causation of apoptosis by cyclopamine is by a non-genotoxic mechanism and thus unlike the radiation therapy and most of the currently used cancer chemotherapeutics which act by causing DNA-damage. These novel effects, previously unachieved by a cancer chemotherapeutic, make the use of cyclopamine highly desirable in cancer therapy, in the treatment of BCC's and other tumors that use the hedgehog/smoothened signal transduction pathway for proliferation and prevention of apoptosis.
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This application is a continuation of U.S. application Ser. No. 10/682,584, filed on 9 Oct. 2003 which is a continuation-in-part of PCT/TR01/00027, filed on 2 Jul. 2001 designating the United States, and a continuation-in-part of PCT/TR02/00017, filed on 19 Apr. 2001 designating the United States. U.S. application Ser. No. 10/682,584, PCT/TR01/00027 and PCT/TR02/00017 are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTIONBasal cell carcinoma (BCC) is a common epithelial tumor. Its incidence increases with increasing age. Current treatments for BCC's include the surgical excision of the tumor together with a margin of normal tissue and, when surgery is not feasible or desirable, destruction of the tumor cells by ionizing radiation or other means. Although scarring and disfigurement are potential side effects, surgical excisions that do not leave neoplastic cells behind can provide cure. Radiation therapy acts by causing irreparably high quantity of DNA-damage which, in turn, triggers apoptotic death of the tumor cells. This mode of action of radiation-therapy, i.e. apoptosis secondary to DNA-damage, is similar to those of many chemotherapeutic agents that are currently used in the treatment of cancers. However, both radiation therapy and the cytotoxic cancer chemotherapeutics are capable of causing DNA-damage in the normal cells of patients in addition to the tumor cells. As a result, their effectivity and usefulness in cancer therapy are seriously limited. A further dilemma with the use of radiation and genotoxic cancer chemotherapeutics is the disturbing fact that, even when cure of the primary tumor is achieved, patients have markedly increased risk of developing new cancers because of the DNA-damage and the resulting mutations they have undergone during the treatment of primary tumor. Induction of apoptosis selectively in tumor cells by non-genotoxic means would therefore be most desirable in the field of cancer therapy.
BCC's frequently show inactivating mutations of the gene patched which encodes a transmembrane protein acting as a receptor for the hedgehog proteins identified first by their effect on the patterning of tissues during development. When not liganded by hedgehog, the patched protein acts to inhibit intracellular signal transduction by another transmembrane protein, smoothened. Binding of hedgehog to the patched causes relieving of this inhibition. Intracellular signal transduction by the relieved smoothened then initiates a series of cellular events resulting ultimately in alterations of the expressions of the hedgehog target genes and of cellular behaviour. General features of this hedgehog/smoothened pathway of signal transduction, first identified in Drosophila, are conserved in diverse living organisms from Drosophila to Human. However, the pathway gets more complex in more advanced organisms (e.g. presence in human of more than one genes that display significant similarity to the single patched gene of Drosophila). Inactivating mutations of the patched have been found to cause constitutive (ligand-free) signalling through the hedgehog/smoothened pathway. The hedgehog/smoothened pathway overactivity, resulting from mutations of the patched and/or further downstream pathway elements, is found in all BCC's. The nevoid basal cell carcinoma syndrome (NBCCS) results from patched haploinsufficiency. Patients with the NBCCS, because of an already mutant patched in all cells, develop multiple BCC's as they grow older. Hedgehog/smoothened signalling is known to be employed for normal functions in several normal tissues and for the maintenance of normal epithelial stem cells (Zhang Y et al (2001) Nature 410:599-604).
Cyclopamine, a steroid alkaloid, has the chemical formula shown below.
It is found naturally in the lily Veratrum californicum and can be obtained by purification from this and other sources. Inhibition of the hedgehog/smoothened pathway by cyclopamine has been found in chicken embryos and in cultured cells of mice. Cyclopamine has been found to inhibit the differentiation of neuronal precursor cells in developing brain (Incardona J P et al (1998) Development 125:3553-3562; Cooper M K et al (1998) Science 280:1603-1607). Studies with other differentiating cell types have also reported an inhibitory action of cyclopamine on cellular differentiation. Differentiation of bone marrow cells to erythroid cells (Detmer K. et al (2000) Dev. Biol. 222:242) and the differentiation of urogenital sinus to prostate (Berman D M et al (2000) J. Urol. 163:204) have been found to be inhibited by cyclopamine. Inhibition of hedgehog/smoothened signalling by cyclopamine has been reported to exert no significant effect on the viability of cells (Taipale J. et al (2000) Nature 406;1005-1009).
SUMMARY OF THE INVENTIONThis invention concerns the use of cyclopamine in vivo on basal cell carcinomas (BCC's) to achieve therapeutic effect by causing differentiation of the tumor cells and, at the same time, apoptotic death and removal of these tumor cells while preserving the normal tissue cells, including the undifferentiated cells of the normal epidermal basal layer and hair follicles. Causation of apoptosis by cyclopamine is by a non-genotoxic mechanism and thus unlike the radiation therapy and most of the currently used cancer chemotherapeutics which act by causing DNA-damage. These novel effects, previously unachieved by a cancer chemotherapeutic, make the use of cyclopamine highly desirable in cancer therapy, in the treatment of BCC's and other tumors that use the hedgehog/smoothened signal transduction pathway for proliferation and prevention of apoptosis.
In one aspect, the present invention is directed to the use of cyclopamine or a pharmaceutically acceptable salt or a derivative of cyclopamine in the topical treatment of basal cell carcinomas, particularly for the manufacture of a pharmaceutical compound for use in the topical treatment of basal cell carcinomas.
In a further aspect, the invention is directed to the use of cyclopamine or a pharmaceutically acceptable salt of cyclopamine or a derivative thereof in the treatment of basal cell carcinomas by non-topical means, including by intratumoral injections, or for the manufacture of a pharmaceutical compound for use in such a treatment.
In a further aspect, the invention is directed to the use of cyclopamine or a pharmaceutically acceptable salt of cyclopamine or a derivative of cyclopamine in the treatment of tumors that use the hedgehog/smoothened signal transduction pathway for proliferation and/or for the prevention of apoptosis or cellular differentiation, or for the manufacture of a pharmaceutical compound for use in such treatment.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Heterogenous labelling of the residual cells of a cyclopamine-treated BCC with the Ulex Europaeus lectin type 1 showing differentiation of some of the BCC cells all the way to the step detected by this lectin which normally does not label the BCC's or the basal layer cells of the normal epidermis but labels the differentiated upper layer cells. 3D and 3E: Decreased expression of p53 as detected by the monoclonal antibody DO-7 in cyclopamine-treated BCC's (3) in comparison to the placebo-treated BCC's (3E). Expression of p53 is known to decrease upon differentiation of the epidermal basal cells and upon differentiation of cultured keratinocytes. It is also well known that the amount of of p53, detectable by DO-7, increases in cells when they are exposed to DNA-damaging agents. 3F and 3G: Consistent retraction of BCC's from stroma, which is a feature known to be associated with the arrest of tumor cell proliferation, seen in cyclopamine-treated (3F, arrow shows the retraction space) but not in placebo-treated (3G) tumors (difference of the cyclopamine-and placebo-treated BCC's in terms of retraction from stroma is seen also in 3D, 2C vs 3B, 3E). Original magnifications are 400× for 3A, 3B, 3D, 3E, 1000× for 3C and 100× for 3F, 3G. All immunohistochemical labellings are with peroxidase-conjugated streptavidin binding to biotinylated secondary antibody; labelling is indicated by the brown-coloured staining. Sections shown in 3F and 3G are stained with Periodic Acid-Schiff and Alcian blue.
Color prints of the same figures as on pages 1/2 (
Cyclopamine was discovered as a teratogenic compound of Veratrum plants (Keeler R. F. (1969) Phytochemistry 8:223-225). It has been reported to inhibit differentiation of the precursors of the ventral cells in the developing brain (Incardona J. P. et al (1998) Development 125:3553-3562; Cooper M. K. et al. (1998) Science 280:1603-1607). Inhibition of cellular differentiation by cyclopamine has been reported in other systems as well, including the differentiation of bone marrow cells to erythroid cells (Detmer K. et al (2000) Dev. Biol. 222-242) and the differentiation of urogenital sinus to prostate (Berman D. M. et al (2000) J. Urol. 163-204). However, the opposite was found to be true in this invention with the tumor cells exposed to cyclopamine. Along with the cyclopamine-induced differentiation of tumor cells, apoptosis of tumor cells was also induced. Induction of tumor cell apoptosis by cyclopamine, again previously undescribed, is shown to be highly efficient. Furthermore, induction of apoptosis by cyclopamine was not secondary to a genotoxic effect and had extreme specificity; even the outer root sheath cells of hair follicles and normal epidermis basal cells that were adjacent to the tumor cells were well preserved while the tumor cells had differentiated and were undergoing apoptosis. Lack of adverse effects of the described treatment is confirmed also by the presence of clinically normal-looking healthy skin and hair at the sites of cyclopamine application in patients (longest duration of follow-up of a human subject is over 31 months at the time of writing and shows safety of the treatment also in the long term). Above summarised features of the treatment described in this invention make it highly desirable in cancer therapy and provide solutions to the long-standing problems of cancer therapy.
It is specifically contemplated that molecules can be derived from cyclopamine or synthesised in such a way that they possess structural features to exert similar receptor binding properties and biological/therapeutic effects as cyclopamine. Such a molecule is called here a “derivative of cyclopamine” and defined as follows: A molecule that contains the group of atoms of the cyclopamine molecule required for the binding of cyclopamine to its biological target but contains also modifications of the parent cyclopamine molecule in such ways that the newly derived molecule continues to be able to bind specifically to the same biological target to exert the biological effects of cyclopamine disclosed herein. Such modifications of cyclopamine may include one or more permissible replacement of or a deletion of a molecular group in the cyclopamine molecule or addition of a molecular group (particularly a small molecular group such as the methyl group) to the cyclopamine molecule, provided that the resultant molecule is stable and possesses the capability of specific binding to the same biological target as cyclopamine to exert the biological effects described herein. Derivation of such new molecules from cyclopamine can be readily achieved by those skilled in the art and the possession or lack of the biological effects of cyclopamine in the newly derived molecule can also be readily determined by those skilled in the art by testing for the biological effects disclosed herein.
For topical applications, cyclopamine can be dissolved in ethanol or another suitable solvent and mixed with a suitable base cream, ointment or gel. Cyclopamine may also be entrapped in hydrogels or in other pharmaceutical forms enabling controlled release and may be adsorbed onto dermal patches. In a pharmaceutical composition for topical administration, the cyclopamine or a salt or derivative thereof should be present in a concentration of 0.001 mM to 100 mM, preferably 12 to 24 mM. The effects shown in
Cells undergoing apoptosis are known to be removed by macrophages and by nearby cells in normal tissues and the quantification of apoptotic activity by morphological criteria on hematoxylene-eosine stained sections is known to provide an underestimate. Despite these, the quantitative data shown in Table 1 show greatly increased apoptotic activity caused by cyclopamine among the residual BCC cells.
The loss of translucency in the cyclopamine-treated BCC's raises the intriguing possibility of differentiation of BCC's under the influence of cyclopamine. This possibility, which can be tested by immunohistochemical analyses of the BCC's, is found to be the case in this invention. In normal, epidermis, differentiation of basal layer cells to the upper layer cells is accompanied by a loss of labelling with the monoclonal antibody Ber-Ep4. Ber-Ep4 labels also the BCC cells and is a known marker for these neoplasms.
Another differentiation marker, Ulex Europeaus lectin type 1, normally does not label the BCC's or the basal layer cells of normal epidermis but labels the differentiated upper layer cells.
The p53 is a master regulator of the cellular response to DNA-damage. Amount of this protein is known to increase in the cell nucleus following exposure of cells to genotoxic agents. When the DNA-damage is increased beyond a threshold, p53 serves for the apoptotic death of cells. Radiation therapy of cancer and the genotoxic cancer chemotherapeutics that are currently common, act largely by this mechanism, i.e. by causation of apoptosis secondary to the damaging of DNA. The monoclonal antibody DO-7 can bind both normal and missense mutant (i.e. non-functional) forms of p53 and is known to be capable of detecting the increase of p53 in the cells following exposure to DNA-damaging agents.
Arrest of the proliferation of BCC's is known to be associated with their retraction from stroma. Although retraction from stroma can also be caused artefactually by improper fixation and processing of the tissues, adherence to published technical details ensures avoidance of such artefacts. As shown in
Causation of highly efficient differentiation and apoptosis of the tumor cells in vivo by cyclopamine at doses that preserve the undifferentiated tissue cells are hitherto unknown achievements that, together with the non-genotoxic mode of action of cyclopamine, support the use of cyclopamine not only on BCC's but also on those internal tumors that utilize the hedgehog/smoothened pathway for proliferation and for prevention of apoptosis and/or differentiation.
Trichoepithelioma is another tumor associated with genetic changes that cause increased hedgehog-smoothened signalling (Vorechovsky L. et al. (1997) Cancer Res. 57:4677-4681; Nilsson M. et al. (2000) Proc. Natl. Acad. Sci. USA 97:3438-3443).
The tumor nodule (marked by arrow in
These examples illustrate effectiveness of the described treatment in the causations of tumor cell differentiation and apoptosis and in obtaining rapid clinical regression of the tumors displaying hedgehog/smoothened signalling. Effectiveness on several independent tumors in unrelated patients with differing genotypes is consistent with the general utility of the described treatment.
Of the numerous substances known in the art to display inhibitory activity on tumor cell proliferation, only a small minority prove to be usable or effective in the treatment of tumors in patients. A major reason for this is the causation of harm also to the normal cells (particularly to the progenitor and stem cells) and the development of intolerable adverse effects. As hedgehog/smoothened signalling is well known to be employed by several normal cell types and for the maintenance of stem cells (Zhang Y et al (2001) Nature 410:599-604), use of cyclopamine on tumors of patients would have been anticipated to lead to adverse effects, especially on the normal tissues around tumors that are exposed to the same schedule and doses of cyclopamine as the tumors. However, treatment with cyclopamine under the described conditions has not revealed undue adverse effects on normal tissue components (including the putative stem cells) by histological/immunohistochemical criteria. Moreover, former skin sites of cyclopamine application that have been followed up more than 31 months at the time of this writing continue to display healthy-looking normal skin and hair, suggesting functional preservation as well of the stem cells and long-term safety. Our finding that a transient exposure to cyclopamine can suffice for the causations of tumor cell differentiation and apoptosis is further surprising and facilitates treatment of internal tumors as well. The term transient administration of cyclopamine for treatment as used here means administration of cyclopamine for a period that is short enough so that causation of the apoptosis and/or differentiation of the normal tissue cells do not happen to such an extend to lead to intolerable adverse effects. We describe in this invention that tumor cells can be caused to undergo apoptosis and/or differentiation in vivo much faster than normal tissue cells so that during the same period of exposure to cyclopamine relatively much smaller proportion or no normal tissue cells undergo cyclopamine-induced apoptosis and/or differentiation, making thereby the clinically detectable or intolerable adverse effects minimal or nonexistent. It is also clear that the therapeutic effectiveness described herein and the rapid disappearance of treated tumors could not be possible without the causation of tumor cell apoptosis since merely inhibiting or slowing the tumor cell proliferation by cyclopamine would, at best, help one only to keep the tumor at its pre-treatment size.)
Claims
1-24. (canceled)
25. A medicament for causing decrease of size or disappearance of a tumor in a tumor-bearing subject,
- wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of the tumor cells,
- wherein the medicament comprises cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling,
- wherein the medicament has labeling that instructs administration to the subject in a dose that suffices for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor.
26. A medicament for causing decrease of size or disappearance of a tumor in a human,
- wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of the tumor cells,
- wherein the medicament comprises cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling,
- wherein the medicament has labeling that instructs administration to said human in a dose that suffices for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor.
27. A medicament for causing decrease of size or disappearance of a tumor in a human,
- wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of the tumor cells,
- wherein the medicament comprises cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling,
- wherein the medicament has labeling that instructs administration to said human in a dose that suffices for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor, and
- wherein administration as instructed in the labeling induces apoptosis of the tumor cells and results in decrease of size or disappearance of the tumor.
28. A medicament for causing decrease of size or disappearance of a tumor in a human,
- wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of the tumor cells,
- wherein the medicament comprises cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling,
- wherein the medicament has labeling that instructs administration to said human in a dose that suffices for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor,
- wherein said administration causes decrease of size or disappearance of the tumor by inducing apoptosis of the tumor cells while sparing normal cells of said human that are labeled by an antibody that marks both the tumor cells and normal cells having stem cell features.
29. A medicament for treatment of a tumor-bearing human,
- wherein the tumor is one wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of tumor cells,
- wherein the medicament comprises cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling,
- wherein the medicament has instructions of administration effective for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor,
- wherein administration as instructed to said human induces apoptosis of the tumor cells and results in decrease of size or disappearance of the tumor.
30. A medicament comprising cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling,
- wherein the medicament has labeling that instructs administration to a tumor-bearing human,
- wherein the tumor is one wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of the tumor cells,
- wherein the instructions comprises administration in a dose that is sufficient for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor,
- wherein said administration causes decrease of size or disappearance of the tumor by inducing apoptosis of the tumor cells while sparing normal cells of said human that are labeled by an antibody that marks both the tumor cells and normal cells having stem cell features.
31. A medicament according to claim 25, wherein the medicament is formulated for topical or systemic administration.
32. A medicament according to claim 25, wherein the medicament is formulated for intratumoral injection.
33. A medicament according to claim 25, wherein the medicament is an aqueous solution or liposomal preparation.
34. A medicament according to claim 25, wherein the medicament is in a pharmaceutical form enabling controlled release.
35. A medicament according to claim 25, wherein the medicament is manufactured in the form of a cream or ointment or gel or hydrogel or the medicament is adsorbed onto a dermal patch.
36. A kit or labeled container comprising
- (i) cyclopamine or a pharmaceutically acceptable salt thereof or another compound that selectively inhibits Hedgehog/Smoothened signaling and
- (ii) instructions secured to the container or associated with the kit or container and giving directions for administration of cyclopamine or of said salt or of said another compound to a tumor-bearing subject,
- wherein the tumor is one wherein Hedgehog/Smoothened signaling is utilized for inhibition of apoptosis of tumor cells,
- wherein the instructions comprises administration of cyclopamine or of said salt or of said another compound in a dose that is sufficient for induction of apoptosis of the tumor cells and for decrease of size or disappearance of the tumor, and
- wherein administration as instructed induces apoptosis of the tumor cells and results in decrease of size or disappearance of the tumor.
37. A medicament or kit or labeled container according to claim 25, wherein said non-cyclopamine compound inhibits Hedgehog/Smoothened signaling by binding to the same biological target to which cyclopamine binds to exert inhibition of Hedgehog/Smoothened signaling.
38. A medicament or kit or labeled container according to claim 36, wherein said non-cyclopamine compound inhibits Hedgehog/Smoothened signaling by binding to the same biological target to which cyclopamine binds to exert inhibition of Hedgehog/Smoothened signaling.
Type: Application
Filed: Jan 13, 2011
Publication Date: May 5, 2011
Applicant: (Bor)
Inventors: Sinan Tas (Bor), Oktay Avci (Izmir)
Application Number: 12/930,677
International Classification: A61K 9/127 (20060101); A61P 35/00 (20060101); C07D 491/107 (20060101); A61K 31/438 (20060101); A61K 9/14 (20060101);