RU2308948C2 - Using cyclopamine in treatment of basal cell epithelioma (carcinoma) and other tumors - Google Patents

Abstract

FIELD: medicine, oncology, biology, pharmacy.

SUBSTANCE: the claimed invention involves using inhibitors. Invention relates to using cyclopamine and its pharmaceutically acceptable salts for preparing a pharmaceutical agent for induction of apoptosis of tumor cells that use transfer of Hedgehog/Smoothened signal for prophylaxis of apoptosis. Cyclopamine causes apoptosis by not genotoxic mechanism and therefore it distinguishes from radiation therapy and most of the modern anticancer chemotherapeutic agent that effect inducing DNA damages. These new effects early not achieved in cancer chemotherapy provide using cyclopamine as to be very desirable in cancer therapy, in treatment of BCC (basal cell carcinoma) and other tumors that use way of signal transduction of hedgehog/smoothened protein for proliferation and prophylaxis of apoptosis.

EFFECT: valuable medicinal properties of cyclopamine.

6 cl, 1 tbl, dwg 7

Description

This invention relates to the use of in vivo - to achieve a therapeutic effect against basal cell epithelioma (carcinoma, BCC) - cyclopamine, which causes the differentiation of tumor cells and at the same time apoptotic death and removal of these tumor cells while maintaining normal tissue cells, including undifferentiated cells of the normal epidermal basal layer and hair follicles. Cyclopamine does not induce apoptosis by the genotoxic mechanism and is therefore different from radiation therapy and most modern anti-cancer chemotherapeutic agents that act by damaging DNA. These new effects, previously unattainable in cancer chemotherapy, make cyclopamine use in cancer therapy, in the treatment of SCD and other tumors that use the hedgehog / smoothened signal transduction pathway to proliferate and prevent apoptosis, highly desirable.

Basal cell epithelium (carcinoma) is a common epithelial tumor. With age, it occurs more often. Current methods of treatment of SCD include surgical excision of the tumor along with border normal tissue and, when surgery is not possible or undesirable, the destruction of tumor cells by ionizing radiation or other methods. Although possible side effects are scarring and an unaesthetic appearance, surgery that does not leave tumor cells can lead to a cure. Radiation therapy causes significant permanent damage to DNA, which, in turn, includes the mechanism of apoptotic death of tumor cells. This mode of action of radiation therapy, i.e. apoptosis is secondary to DNA damage, similar to the methods of action of many chemotherapeutic agents currently used to treat cancer. However, both radiation therapy and cytotoxic anticancer chemotherapeutic agents can cause DNA damage in normal patient cells in addition to tumor cells. As a result, the effectiveness and suitability of these methods in cancer therapy is greatly limited. An additional difficulty arising from the use of radiation and genotoxic anticancer chemotherapeutic substances is the alarming fact that even when the cure of the primary tumor is achieved, the risk of developing new cases of cancer due to DNA damage and mutations resulting from the treatment of the primary tumor is markedly increased. Therefore, the selective induction of apoptosis in tumor cells by a non-genotoxic method is most desirable in the field of cancer therapy.

In BCC, inactivated mutations of the patched gene, which encodes a transmembrane protein that acts as a receptor for hedgehog proteins, are identified primarily by their effect on tissue structuring during development. Without being bound as a ligand to the hedgehog protein, patched acts by inhibiting intracellular signal transduction by the mechanism of another transmembrane protein, smoothened. The binding of hedgehog to patched causes a weakening of this inhibition. Then, intracellular signal transduction along the attenuated smoothened protein pathway initiates a series of cellular events, ultimately leading to changes in the expression of target hedgehog genes and cell behavior. The general features of the hedgehog / smoothened signal transduction pathway, originally identified in Drosophila (in Drosophila), persist in a variety of living organisms from Drosophila to Man. However, the pathway becomes more complex in progressive organisms (for example, the presence in the human body of more than one gene, showing significant similarity to a single patched Drosophila gene). Inactivated mutations in patched were found to induce constitutive (without ligand) signaling along the hedgehog / smoothened pathway. The overactivity of the hedgehog / smoothened pathway resulting from patched mutations and / or additional elements of the 5'-3'path is found in all SCDs. Nevoid Basal Cell Epithelioma Syndrome (NBCCS) is the result of patched haplo-insufficiency. In patients with NBCCS, due to the presence of an already mutant patched in all cells, multiple SCD develops with age.

Cyclopamine, a steroidal alkaloid, has the chemical formula shown below:

It is found in nature in the lily Veratrum californicum and is obtained by purification from it and other sources. Inhibition of the hedgehog / smoothened pathway by cyclopamine was found in chick embryos and in cultured mouse cells.

WO 01 / 27135A2 discloses the use of hedgehog / smoothened signaling inhibitors (Hh / Smo), in particular cyclopamine, to inhibit cell proliferation (including tumor cell proliferation). In contrast to the specified analogue in the present invention, cyclopamine or its pharmaceutically acceptable salts are used for apoptosis of tumor cells in vivo, which allows to achieve a rapid reduction and disappearance of the treated tumor.

For topical use, cyclopamine can be dissolved in ethanol or in another suitable solvent and mixed with a suitable cream, ointment or gel base. Cyclopamine can also be formulated in hydrogels or other pharmaceutical forms that allow controlled release, and can be absorbed onto skin patches. The results depicted in Figures 1A-1D, Figs. 2A-2F, Figs. 3A-3G, and Figs. 4A-4D, are obtained by using a preparation obtained by mixing a solution of cyclopamine in ethanol with a cream base in such a way as to achieve a final concentration 18 mM cyclopamine in cream. The cream base used mainly consists of heavy paraffin oil (10% by weight), petroleum jelly (10% by weight), stearyl alcohol (8% by weight), polyoxystearate-40 (3% by weight) and water (69% by weight) .%), but a cream base prepared in another suitable way is also possible. The optimal concentration of cyclopamine in the pharmaceutical form, as well as the optimal doses and regimens, can obviously be influenced by factors such as the specific pharmaceutical form, location and characteristics of the skin containing the tumor (for example, epidermis thickness) and tumor size; however, they can be determined by the following well-known published optimization methods. Dosage and application regimens for the treatment of tumors depicted in Fig. 1A (BCC on the nasolabial fold, area ~ 4 × 5 mm) and Fig. 1C (BCC on the forehead, area ~ 4 × 4 mm), the following: 10 ± 2 μl of cream applied directly to the BCC with a steel spatula four times a day, starting from -9.00 in the morning, at intervals of ~ 3.5 hours. At night, instead of the applications avoided according to this scheme because of the possible loss of the cream due to getting it from the skin on the bedding, use suitable skin patches. Preservation of undifferentiated cells in the normal epidermis and in the hair follicles after exposure to cyclopatin, as shown in this description, also provides information on tolerated doses for other methods of use; for example, by direct injection of an aqueous solution into the tumor or by systemic use of the same cyclopamine placed in liposomes.

On figa, figv, figs and fig.1D shows a rapid clinical regression of SCD after exposure with cyclopamine. In addition to the visual disappearance of several areas (regions) of the tumor during the exposure with cyclopamine for less than a week, the usually transparent appearance of SCD is lost, which is seen when comparing Figv with Fig.1A and Fig.1D with Fig.1C.

FIGS. 2A-2F show the microscopic appearance of areas of the tumor undergoing surgical excision with borderline normal tissue on the fifth or sixth day of application of cyclopamine applications, when BCC has lost the bulk of the pre-treated areas, but still contains several areas, although it has decreased markedly in weight, but not completely disappeared and, therefore, having residual tumor cells for analysis under a microscope.

On figa and 2B shown on sections of tissue areas of the skin corresponding to visually disappearing nodules of the tumor. It can be seen that the tumors disappear, leaving behind extensive cystic structures containing a little material inside and not containing distinguishable tumor cells.

FIG. 2C shows a microscopic view of a skin region containing still visible BCC in vivo. It can be seen that these areas contain large cysts detecting residual SCD in the center of the tumor and smaller cystic structures of various sizes, localized among the residual SCD cells, towards the periphery.

2D and 2E show, with a magnification of 1000x, an inside view and peripheral palisade sections of these residual SCDs and a powerful apoptotic activity in the residual SCD cells is shown regardless of the tumor area. Such a large increase allows us to see a significantly increased frequency of BCC cells revealing aloptotic morphology and the formation of cystic structures during apoptotic cell removal, as shown in Fig. 2D, by combining three smaller cysts into one larger cyst when removing apoptotic septal cells.

FIG. 2F shows that in BCC treated with a placebo cream (i.e., a cream preparation identical to cyclopamine cream, except that there is no cyclopamine in placebo), on the contrary, typical neoplastic BCC cells are observed and there is no noticeable apoptotic activity.

It is known that cells undergoing apoptosis in normal tissues are removed using macrophages and nearby cells and that quantitative determination of apoptotic activity using morphological criteria on hematoxylene-eosin stained sections gives underestimated results (underestimation). Despite this, the quantitative data presented in Table I show increased apoptotic activity stimulated by cyclopamine in residual SCD cells.

The loss of transparency in cyclopamine-treated BCC indicates an interesting possibility of differentiation of BCC under the influence of cyclopamine. It was found that this possibility, which can be checked using immunochemistry assays of BCC, is carried out in this invention. In the normal epidermis, the differentiation of the cells of the basal layer into cells of the higher layers is accompanied by the loss of labeling with the monoclonal antibody Ber-Ep4. Ber4-Ep4 also marks BCC cells and is a known marker for these neoplasms. Fig. 3A, Fig. 3B and the quantitative data in Table I show that although Ber-Ep4 clearly labels all peripheral elongated cells of the palisade tissue and more than 90% of the internal cells of the BCC placebo-treated, none of the residual peripheral or internal cells of the BCC-treated cyclopamine tagged with Ber-Ep4. Differentiation of SCD under the influence of cyclopamine, unknown until now with the use of any other methods and is highly unusual, since it is achieved in vivo and in all cells, judging by immunohistochemical parameters, has independent significance in the treatment of cancer.

Another differentiation marker, Ulex Europaeus type 1 lectin, usually does not label BCC or basal layer cells of the normal epidermis, but it does differentiate cells of the upper layers. FIG. 3C, showing heterogeneous labeling of residual cells with BCC cyclopamine treated with this pectin, shows the differentiation of some BCC cells at the differentiation stage detected with Ber-Ep4 to the stage detected with Ulex Europaeus type 1.

p53 is the main protein - a regulator of the cellular response to DNA damage. It is known that the amount of this protein increases in the cell nucleus after exposure to a genotoxic agent. When DNA damage exceeds the limit, p53 contributes to apoptotic cell death. Radiation therapy for cancer and genotoxic chemotherapeutic anticancer drugs, currently common, mainly act on this mechanism, i.e. causing apoptosis secondary to DNA damage. The DO-7 monoclonal antibody can bind both normal and missense mutant (i.e. non-functional) forms of p53, and DO-7 is known to detect an increase in p53 in cells after exposure to DNA damaging agents.

Fig. 3D, Fig. 3E and the quantitative data shown in Table I show that both the labeling intensity of DO-7 and the frequency (occurrence) of labeled cells are markedly reduced in BCC treated with cyclopamine compared to BCC treated with placebo. Therefore, cyclopamine is not an increase, but rather a decrease in p53 in the nuclei of BCC cells treated with cyclopamine. As is known, the level of p53 expression decreases in epidermal cells upon differentiation; therefore, the reduced labeling of cyclopamine-treated BCC with DO-7 is apparently secondary to cyclopamine-induced differentiation of BCC cells. In any case, the potent apoptotic activity in cyclopamine-treated BCC despite a markedly reduced p53 expression means that the cyclopamine-induced apoptosis of these tumor cells is due to a non-toxic mechanism.

It is known that inhibition of SCD proliferation is associated with their retraction from the stroma. Although retraction from stroma can also be caused as an artifact (as a result of human activity) due to improper fixation and processing of tissues, the accuracy of compliance with published technical conditions ensures the possibility of avoiding such artifacts. As shown in FIG. 3R and 3O treated with cyclopamine but not placebo, BCC is constantly being drawn from the stroma. Therefore, exposure to SCD with cyclopamine is also likely to be associated with suppression of proliferation.

4A-4D show Ber-Ep4 labeling of normal skin tissue located on and around BCC treated with cyclopamine. The various epidermal sites that were treated with cyclopamine, as can be seen in Figs. 4A, 4B and 4C, give a normal imprint of labeling with Ber-Ep4, i.e. labeling of cells of the basal layer. Similarly, FIG. 4B shows normal labeling with Ber-Ep4 hair follicles exposed with cyclopamine. Consequently, undifferentiated cells of the normal epidermis and hair follicles persist despite exposure according to the same scheme and with the same doses of cyclopamine as SCD.

The fact that cyclopamine induces highly effective differentiation and apoptosis of tumor cells in vivo at doses at which undifferentiated tissue cells are preserved is an achievement not known so far, which, along with the non-genotoxic effect of cyclopamine, confirms the possibility of using cyclopamine not only for SCD, but also for such internal tumors that use the hedgehog / smoothened pathway for proliferation and for the prevention of apoptosis and / or differentiated.

Specifically, those cases are considered when molecules can be formed from cyclopamine or synthesized in such a way that they have structural features that make it possible to exhibit similar binding properties to cyclopamine and biological / therapeutic properties. Such molecules are referred to herein as “cyclopamine derivatives” and are defined as follows. A molecule that contains a group of atoms of the cyclopamine molecule required to bind cyclopamine to its biological target, but also includes such modifications in the molecule of the original cyclopamine that the newly formed molecule retains the ability to specifically bind to the same biological target, exhibiting the biological properties of cyclopamine presented in this description. Such modifications of cyclopamine may include one or more permissible substitutions or deletions of the molecular group in the cyclopamine molecule or additions of a molecular group (in particular a small molecular group such as a methyl group) to the cyclopamine molecule, provided that the resulting molecule is stable and has the ability to specifically binding to the same biological target as cyclopamine, exhibiting the biological effect described herein. Derivatization of such new cyclopamine molecules can be easily carried out by those skilled in the art, and the presence or absence of the biological properties of cyclopamine in a newly obtained molecule can also be easily determined by those skilled in the art by checking for the biological effect presented herein.

Additional examples

On figa shows a large ulcerated SCD in the upper nose of a 68-year-old man before treatment. The cyclopamine cream (18 mM cyclopamine in the cream base described above) is applied to the lower half of the BCC shown in Fig. 5A. Every three hours - 20 μl of the cream is applied directly to the lower half, and the upper half is left untouched. Thus, the upper part of the tumor cells (Fig. 5A), which is unlikely, receive cyclopamine due to possible diffusion from the area onto which cyclopamine is applied directly, and are exhibited with significantly lower cyclopamine concentrations, if any. FIG. 5B shows a tumor after 54 hours of treatment before surgical excision for examination. While rapid regression of the tumor in the lower half that Cyclopamine is applied to is obvious, it is clear that the region of the tumor farthest from the half of the direct application remained relatively unchanged (Figure 5B; region closer to the upper corner of the figure). Figure 5C shows a hematoxylene-eosin stained section of the lower (cyclopamine treated) portion of the excised tissue. Numerous apoptotic cells with cysts of various sizes are visible, which are formed as a result of the death and removal of tumor cells (Fig. 5C). In contrast, in the untreated area of the same tumor, farthest from the half-treated with cyclopamine, solid tumor tissue with mitotic figures is visible and there are no noticeable apoptotic cells (Figure 5B). Figures 5E and 5F show immunohistochemically stained sections of tissue, respectively, of the treated and untreated regions of the tumor, obtained with the Ki-S5 monoclonal antibody (Dako A / S, Glostrup, Denmark) against the Ki-67 antigen. The Ki-67 antigen, a known marker of proliferating cells, no longer expresses in the cyclopamine-treated region of the tumor (Figure 5E), while the tumor cells farthest from the region directly coated with cyclopamine clearly show proliferative activity (Figure 5F). Thus, staining of tissue sections with an anti-Ki-67 antigen again shows inhibition of proliferation of tumor cells by cyclopamine under the described conditions.

Trichoepithelioma is another tumor associated with genetic changes that cause increased hedgehog / smoothened signal transmission (Vorechovsky I. et al. (1997) Cancer Res. 57: 4677-4681; Nilsson M. et al. (2000) Proc. Natl. Acad. Sci. USA 97: 3438-3443). Fig. 6A shows trichoepithelioma on the cheek of an 82-year-old man before treatment, and Fig. BB shows the same skin area after only 24 hours of exposure with cyclopamine cream (18 mM cream-based cyclopamine; - 25 μl of the cream is applied every three hours directly to the tumor area). Due to rapid regression, treatment is stopped after 24 hours and the entire skin area corresponding to the original tumor is excised for examination. On figs and fig.6D shows areas of the skin that contain residual tumor cells after 24 hours and show noticeable aloptotic activity among these residual tumor cells. Visible cystic areas resulting from apoptotic removal of tumor cells (Fig.6C, 6B), as well as tumor infiltration by mononuclear cells (Fig.6D). Another noteworthy patient in this patient is the reduction in size and pigmentation of the nevus adjacent to the treated tumor after 24 hours of treatment (FIG. 6B compared to FIG. 6A). Since cyclopamine may diffuse from the surrounding area onto which it was applied, the nevus (a benign amelanotic tumor) appears to be sensitive to relatively low concentrations of cyclopamine.

7A shows a pigmented SCD on the lower eyelid of a 59-year-old man before treatment. Cyclopamine cream (18 mM cream-based cyclopamine) is applied to all nodes except one marked by an arrow. This nodule, which, apparently, received cyclopamine only by diffusion from the adjacent treated area, is exposed with cyclopamine in a relatively low concentration. Since the pigmented nature of this tumor facilitates observation of the clinical course, treatment (application of 20 μl cyclopamine cream every four hours) is interrupted on the third day, when the tumor regresses significantly in the treated area, but still contains visible parts (Fig. 7B). The tumor is then observed without treatment in order to study possible late effects. A clear further clinical regression in the absence of treatment is not observed, and the skin region corresponding to the initial tumor is excised on the sixth day of medical observation (ninth day from the start of treatment). In hematoxylene-eosin stained sections of the treated area of the tumor, there are many cystic areas in which there are no tumor cells (Fig. 7C). The absence of the epithelium smoothing these cysts (Fig. 7C) is consistent with the idea that these cysts are tissue sites that previously occupied tumor cells. At the same time (the sixth day of subsequent medical observation without treatment), tissue sections show a relatively small number of apoptotic cells (Fig. 7C), which is consistent with the known speed of cleaning of living tissues from apoptotic cells. On the other hand, residual tumor cells, especially near the edges of the cyst, show an unusually high frequency of cells exhibiting spinous differentiation properties (for example, the area to the left down in Fig. 7C; it is more clearly visible at higher magnification, as seen in the example of another Fig.7D). Similar areas of differentiation or cysts are absent in the material obtained by excision using a dermatome from the same tumor before treatment (Fig. 7E). A tumor node that receives a relatively low concentration of cyclopamine (indicated by an arrow in FIG. 7A) contains a large cystic center on the sixth day of subsequent medical follow-up (FIG. 7F). The remaining periphery of the node, however, continues to contain cells with a typical BCC morphology, although the frequency of cells with signs of differentiation (for example, with an increased and more eosinophilic cytoplasm) again increases and there are smaller cysts in this peripheral region (Fig. 7F). Thus, while the response of the tumor to the optimal concentration of cyclopamine is relatively quick, when exposed to concentrations below the optimal, tumor cells remain, which persistently persist during subsequent observation.

These additional examples demonstrate the effectiveness of the described treatment to achieve rapid clinical regression of SCD and other tumors that have enhanced hedgehog / smoothened signal transmission. It is seen that clinical regression is associated with cyclopamine-induced differentiation and apoptosis of tumor cells. In addition, proliferation of tumor cells is also prevented. Efficacy against several independent tumors in non-relative patients with different genotypes corresponds to the general nature of the usefulness of the described treatment.

Cyclopamine was discovered as a teratogenic component of Veratrum plants (Keeler R.F. (1969) Phyto chemistry 8: 223-225). It has been reported that it inhibits the differentiation of ventral cell precursors in brain development (Incardona J.P. et al. (1998) Development 125: 3553-3562; Cooper M.K. et al. (1998) Science 280: 1603-1607). Cyclopamine has also been reported to block cell differentiation in other systems, including also the differentiation of bone marrow cells into erythroid cells (Detmer K. et al. (2000) Dev. Biol. 222: 242) and the differentiation of the urogenital sinus into the prostate (Berman DM et al . (2000) J. Urol. 163: 204). However, in the present invention, it was found that in the case of exposure of tumor cells with cyclopamine, the opposite is true. Along with cyclopamine-induced tumor cell differentiation, tumor cell apoptosis is also stimulated. It was also shown that the previously undescribed induction of tumor cell apoptosis by cyclopamine is highly effective. In addition, the induction of apoptosis by cyclopamine is not accompanied by a genotoxic effect and is extremely specific; even the cells of the outer shell of the roots of the hair follicles and the normal basal cells of the epidermis adjacent to the tumor cells are preserved, while the tumor cells differentiate and undergo aloptosis. The absence of a harmful side effect of the described treatment is also confirmed by the presence of clinically normal looking healthy skin in the patient at the places of cyclopamine application (the maximum duration of subsequent medical observation of a sick person is more than 15 months at the time of writing this work and shows the safety of treatment also for a long time). The summarized above characteristic features of the treatment method described in this invention make it highly desirable in cancer therapy and provide a solution to the problems long ago facing cancer therapy.

Description of Shapes

Figa, 1B, 1C, 1D: Rapid regression of cyclopamine-treated BCC, as seen by the disappearing areas of the tumor (shown by arrows), a markedly reduced weight of the skin surface and the loss of transparency in less than a week. 1A: BCC, located on the left nasolabial fold, before treatment. 1B: The same SCD on the fifth day of topical application of cyclopamine. 1C: BCC, located on the forehead, before treatment. 1D: The same SCD on the sixth day of topical application of cyclopamine.

Fig. 2A, 2B, 2C, 2D, 2E, 2F: Microscopic view of the BCC treated with cyclopamine and placebo, clearly showing cyclopamine-induced aloptotic death and removal of tumor cells and the disappearance of tumor nodes leaving cystic regions without tumor cells . The skin areas corresponding to the provisions of the SCD before treatment are excised surgically on the fifth and sixth days of exposure with cyclopamine together with normal border tissue and subjected to normal fixation, sections are made, stained with hematoxylene-eosin and examined under a microscope. 2A: Large cyst in the dermis, corresponding to the position of the disappeared tumor node, no residual tumor cells. 2B: A similar cyst in another area of the skin that contained BCC before, but not after, treatment with cyclopamine. 2C: Small enlargement view of the BCC region shown in FIG. 1D showing residual cells and the formation of a large cyst by connecting multiple smaller cysts between these cells. 2D: View with a large increase in the internal region of the same residual SCD as in FIG. 2C, showing a significantly increased frequency of apoptotic cells and formation, as well as an increase in cysts during aloptotic removal of SCD cells. 2E: View with a large increase from the peripheral region of the same residual SCD as in FIG. 2C, also showing a significantly increased frequency of apoptotic cells and cyst formation due to apoptotic removal of SCD cells. 2F: View with a large magnification on the side of the internal BCC region treated with a placebo, showing typical neoplastic cells of this tumor and the absence of apoptosis. 100X magnification for 2A, 2B, 2C and 1000X for 2D, 2E, 2F.

3A, 3B, 3C, 3D, 3E, 3F, 3G: Immunohistochemical analysis of BCC treated with cyclopamine and placebo showing the differentiation of all residual BCC cells under the influence of cyclopamine and a decrease in p53 expression in BCC after exposure to cyclopamine. 3A and 3B: The absence of Ber-Ep4 monoclonal antibody staining in all residual cells treated with BCC cyclopamine (3A) contrasts with the intense staining in BCC treated with placebo (3B), which shows that all residual cells in BCC treated with cyclopamine are differentiated to or higher than detectable with Ber-Ep4. Ber-Ep4 is a well-known differentiation marker that stains BCC cells, as well as undifferentiated cells of the basal layer of the normal epidermis and hair follicles, but not differentiated cells of the upper layer of the normal epidermis. 3C: Heterogeneous labeling of residual BCC cells treated with cyclopamine with Ulex Europaeus type 1 lectin, showing the differentiation of some BCC cells to the stage detected by this lectin, which usually does not mark BCC cells or basal layer cells of the normal epidermis, but marks differentiated cells top layers. 3D and 3E: Reduced p53 expression detected by DO-7 monoclonal antibody in BCC treated with cyclopamine (3D) compared to BCC treated with placebo (3E). It is known that p53 expression decreases the differentiation of epidermal basal cells and the differentiation of cultured keratinocytes. It is also known that the amount of p53 detected by DO-7 increases in cells when they are exposed with DNA damaging agents. 3F and 3G: Continuous BCC retraction from the stroma, which is known to be associated with inhibition of tumor cell proliferation observed in cyclopamine-treated tumors (3F, arrow shows the place of retraction) but not placebo (3G) (difference between BCC treated with cyclopamine and placebo, from the point of view of retraction from the stroma is also seen in 3D, 2C in comparison with 3B, 3E). 400X magnification for 3A, 3B, 3D, 3E, 1000X for 3C and 100X for 3F, 3G. All immunohistochemical labeling operations are performed with streptavidin conjugated to peroxidase that binds to a biotinylated secondary antibody; labeling is indicated by brown staining. The sections shown in FIGS. 3F and 3G are stained with periodic acid — Schiff's base and Alcian blue.

4A, 4B, 4C, 4C: Normal imprint of labeling of cyclopamine-treated normal skin with Ber-Ep4, showing that undifferentiated cells of the normal epidermis and hair follicles persist despite the fact that they are exposed according to the same scheme and with the same doses cyclopamine, like BCC. 4A: Introduction of the Ber-Ep4 label into cells of the cyclopamine-treated basal layer of the epidermis. 4B and 4C: View at a larger magnification from various regions of the epidermis treated with cyclopamine, showing labeling of basal cells with Ber-Ep4. 4D: View with a large increase in hair follicles treated with cyclopamine but still showing normal labeling with Ber-Ep4. 400X magnification for 4A and 100X for 4B, 4C, 4D. The method of immunohistochemical detection is the same as in FIGS. 3A, 3B; tagging is shown in brown.

5A shows ulcerated SCD in the upper nasal region of a 68-year-old man before treatment.

Fig. 5B shows the same BCC as in Fig. 5A after 54 hours of cyclopamine application to its lower half.

Fig. 5C shows a slice of half of the BCC on which cyclopamine was applied after 54 hours. Hematoxylene-eosin staining (H&E), magnification 400X.

5B shows a slice of an untreated area of the same BCC. H&E, magnification 400X.

Figure 5E shows a slice of half of the BCC on which cyclopamine was applied after 54 hours; immunohistochemical staining for Ki-67 antigen. Magnification 200X.

On figa shows trichoepithelium on the cheek of an 82-year-old man before treatment.

Fig. 6B shows the same skin site as in Fig. 6A after 24 hours of cyclopamine treatment.

FIG. 6C shows a section of the excised skin of FIG. 6B with residual tumor cells. H&E, magnification 400X.

On fig.6D shows another area of the same tissue as on figs. In addition to numerous aloptotic cells and the formation of cystic structures, tumor infiltration by mononuclear cells is seen during their removal. H&E, magnification 200X.

7A shows pigmented SCD on the lower eyelid of a 59-year-old man before treatment.

Fig. 7B shows the same BCC as in Fig. 7A, on the third day of cyclopamine treatment.

FIG. 7C shows a slice of the treated BCC region depicted in FIG. 7B. H&E, magnification 200X.

On Fig.7D close-up shows the region of residual tumor cells in a slice of the treated area of SCD, depicted in Fig 7B. H&E, magnification 400X.

Fig. 7E shows a section of material using a dermatome obtained from the BCC depicted in Fig. 7A before treatment. H&E, magnification 400X.

FIG. 7P shows a slice containing a portion of the BCC assembly, marked with an arrow in FIG. 7A. Tissue excision is performed after 3 days of treatment and 6 days of subsequent medical observation without treatment. H&E, magnification 100X.

Table 1: Induction of differentiation and apoptosis of basal cell epithelial cells using topical cyclopamine.

Peripheral elongated BCC palisade tissue cells treated BCC non-palisade tissue cells treated Placebo Cyclops Placebo Cyclops amine amine % Cells showing ≥2 morphological features apoptosis in H&E stained sections 0 ± 0 20 ± 8 0.2 ± 0.4 18 ± 11 tissue % Cells labeled with Ber-Ep4 100 ± 0 0 ± 0 91 ± 8 0 ± 0 % DO-7-labeled cells 58 ± 27 1b ± 11 67 ± 22 5 ± 3

Shown is the mean ± standard deviation from at least 16 randomly selected sections of tissue sections of each tumor group with a large increase (1000X), p <0.001 for placebo compared with treated cyclopamine for all parameters, both for peripheral cells of palisade tissue and non-palisade (internal) regions of the tumor.

Claims (6)

1. The use of cyclopamine or its pharmaceutically acceptable salt for the manufacture of a pharmaceutical agent for the induction of apoptosis of tumor cells that use Hedgehog / Smoothened signaling to prevent apoptosis. 2. The use according to claim 1 for the manufacture of pharmaceuticals for antitumor therapy. 3. The use according to claim 1 for the manufacture of pharmaceuticals in the form of an aqueous solution or embedded in liposomes. 4. The use according to claim 1 for the manufacture of pharmaceutical preparations of prolonged action. 5. The use according to claim 1 for the manufacture of pharmaceutical products for local use. 6. The use according to claim 4 for the manufacture of a pharmaceutical agent in the form of a cream, ointment, gel or hydrogel.

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