JP2009143963A - Pharmaceutical composition for treating tumor using hedgehog/smoothened signal for inhibiting apoptosis of tumor cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for treating tumor, using hedgehog/smoothened signals for inhibiting apoptosis of tumor cells. <P>SOLUTION: The pharmaceutical composition contains a sufficient amount of cyclopamine, its pharmaceutically acceptable salt or another pharmaceutically acceptable compound specifically inhibiting hedgehog/smoothened signal transmission. By administeration of a sufficient dose of the pharmaceutical composition by intratumoral injection or the like, apoptosis of the tumor cells is induced, the size of the tumor is decreased or the tumor disappears, and tumors such as basal cell cancer are treated. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention treats by maintaining normal tissue cells, including normal epidermal basal layer and undifferentiated cells of hair follicles, while causing differentiation of tumor cells and concomitant apoptosis and removal of these tumor cells It relates to the use of cyclopamine for basal cell carcinoma (BCC) in vivo to achieve an effect. Inducing apoptosis by cyclopamine is due to a mechanism of non-genotoxicity and is therefore different from radiotherapy, which has the effect of causing damage to DNA, and most cancer chemotherapeutic drugs currently used. These novel effects not achieved by conventional cancer chemotherapeutic agents use cancer treatment, and also BCC and hedgehog / smoothed signaling pathways for proliferation and prevention of apoptosis In the treatment of other tumors, the use of cyclopamine has become highly desirable.

  Basal cell carcinoma is a common epithelial tumor. Incidence increases with age. Current treatments for BCC include surgical removal of tumors along with normal tissue edges and destruction of tumor cells by ionizing radiation or other means when surgery is not feasible or desirable. including. As a potential side effect, it may impair wounds and aesthetics, but surgical excision that does not leave tumor cells provides recovery. Radiation therapy works by causing irreparably large amounts of DNA damage and then causing apoptosis of tumor cells. The mode of action of radiation therapy, ie apoptosis following DNA damage, is similar to many chemotherapeutic agents currently used in cancer treatment. However, radiotherapy and cytotoxic cancer chemotherapy have the potential to cause DNA damage in normal cells of patients in addition to tumor cells. As a result, their effectiveness and utility in cancer treatment is significantly limited. In addition, as a dilemma associated with the use of radiation and genotoxic cancer chemotherapy, even if the initial tumor treatment could be realized, DNA damage and the resulting mutations during the initial tumor treatment could lead to new patient There is an anxious factor that the risk of developing cancer increases significantly. Therefore, selectively causing apoptosis in tumor cells by non-genotoxic means may be optimal in the field of cancer treatment.

  BCC often affects the patterning of tissues during development, thereby leading to the non-patch of a patched gene that encodes a transmembrane protein that acts as a receptor for the first identified hedgehog protein. Indicates an activity mutation. When not liganded by hedgehog, patched proteins act to suppress intracellular signaling by other transmembrane proteins smoothened. The combination of hedgehog and patched results in a relaxation of this suppression. Intracellular signaling, alleviated by smoothing, then initiates a series of intracellular events that ultimately result in the expression of hedgehog target genes and the degeneration of intracellular roles. A general form of information transmission in the hedgehog / smooth pathway has been identified in Drosophila and has been passed on to various biological tissues from Drosophila to humans. However, the pathway is more complex in more advanced tissues (in humans, there are one or more genes that are very similar to the Drosophila single patched gene, etc.). Patched inactive mutations were found to result in structural (ligandless) signaling via the hedgehog / smoothed pathway. Hedgehog / smoothed pathway overactivity is caused by mutations in patched and / or further downstream pathway elements and is found in all BCCs. Nevus basal cell carcinoma syndrome (NBCCS) is caused by patched haploinsufficiency. Patients with NBCCS develop multiple BCCs as they age because all cells are already mutated patched.

  Cyclopamine, which is a steroid alkaloid, has the chemical formula shown below.

Cyclopamine is found in the lily family Veratrum californicum and can be obtained by purification from veratram californicum and other sources. Inhibition of the hedgehog / smoothed pathway by cyclopamine was found in chicken embryos and cultured mouse cells.

In order to solve the above problems, the invention according to claim 1 provides a method of using cyclopamine or a medically acceptable cyclopamine salt in local treatment of basal cell carcinoma.
The invention of claim 2 is a method of using cyclopamine or a medically acceptable cyclopamine salt for producing cyclopamine or a medically acceptable compound for use in local treatment of basal cell carcinoma. I will provide a.

The invention of claim 3 provides a method of using cyclopamine or a medically acceptable cyclopamine salt in the treatment of basal cell carcinoma by non-local means, including intratumoral injection.
The invention of claim 4 is cyclopamine or a medically acceptable for producing a medically acceptable compound for use in the treatment of basal cell carcinoma by non-local means, including intratumoral injection Provide a possible use of cyclopamine salts.

The invention according to claim 5 provides a method of using cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative in the local treatment of basal cell carcinoma.
The invention according to claim 6 provides the use of cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative for the manufacture of a medically acceptable compound for use in the local treatment of basal cell carcinoma. Provide a method.

  The invention of claim 7 provides a method of using cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative in the treatment of basal cell carcinoma by non-local means, including intratumoral injection.

  The invention of claim 8 provides cyclopamine, a medically acceptable, for the manufacture of a medically acceptable compound for use in the treatment of basal cell carcinoma by non-local means, including intratumoral injection And a method of using a cyclopamine salt or a cyclopamine derivative.

  The invention of claim 9 is cyclopamine or medically acceptable in the treatment of tumors using hedgehog / smoothed signaling pathways for proliferation and / or prevention of apoptosis or cell differentiation A method for using a suitable cyclopamine salt is provided.

  The invention of claim 10 provides a medically acceptable compound in the treatment of tumors using the hedgehog / smoothed signaling pathway for proliferation and / or prevention of apoptosis or cell differentiation. Methods of using cyclopamine or a medically acceptable cyclopamine salt for manufacture are provided.

  The invention of claim 11 is a medically acceptable cyclopamine in the treatment of tumors using hedgehog / smoothed signaling pathways for proliferation and / or prevention of apoptosis or cell differentiation. Methods of using cyclopamine salts or cyclopamine derivatives are provided.

  The invention of claim 12 provides a medically acceptable compound in the treatment of tumors using the hedgehog / smoothed signaling pathway for proliferation and / or prevention of apoptosis or cell differentiation. Methods of using cyclopamine, medically acceptable cyclopamine salts, or cyclopamine derivatives for the manufacture are provided.

  For topical application, cyclopamine can be dissolved by ethanol or other suitable solvent and a suitable base (base) cream, ointment, or gel mixture. Cyclopamine can be taken up by hydrogels, or controlled-release medically applicable forms, and can be absorbed in skin patches. The effect shown in FIGS. 1A-1D, 2A-2F, 3A-3G, 4A-4D is achieved by using a solution of cyclopamine in ethanol to obtain a final concentration of 18 mM cyclopamine in the cream. It has been obtained with a cream formulation obtained by mixing with a base cream. The base cream used was mainly composed of heavy paraffin oil (10% by weight), petrolatum (10% by weight), stearyl alcohol (8% by weight), polyoxyl steareth-40 (3% by weight) and water (68% by weight). However, other suitably formulated base creams are further possible. The optimal concentration and schedule of administration of the medically applicable form of cyclopamine as the optimal dosage depends on the specific medically applicable form, localization, skin characteristics including the tumor (epidermal thickness, etc. ), And can be clearly influenced by factors such as tumor size. However, this can be determined according to well-known published optimization methods. The dosage and dosing schedule according to the tumor shown in FIG. 1A (BCC on the nasal lip, approximately 4 × 5 mm on the surface) and FIG. 1C (forehead BCC, approximately 4 × 4 mm on the surface) is as follows: It is. 10 ± 2 μl of cream was applied directly onto the BCC using an iron spatula, 4 times a day, starting at about 9 am and approximately 3 and a half hours apart. Nighttime application, avoided due to the possibility of losing cream from the patient's skin to the sheets during sleep, can be performed on the appropriate skin patches. The maintenance of undifferentiated cells of normal epithelial cells and hair follicle cells by exposure to cyclopamine described in the present invention includes injection of an aqueous solution directly into a tumor, systemic administration of cyclopamine incorporated in an aqueous solution or liposome, etc. Information on tolerable dosages is provided by other possible dosage forms.

  According to the present invention, a pharmaceutical composition for treating a tumor using hedgehog / smoothed signal to inhibit tumor cell apoptosis was provided. Furthermore, a method for inducing at least one of tumor cell differentiation and apoptosis in vitro, wherein hedgehog / smoothed signaling is used to inhibit the process of inducing at least one of tumor cell differentiation and apoptosis A method was provided.

BCC located in the left nasal lip before treatment. BCC with topical cyclopamine treatment identical to FIG. 1A on day 5. BCC located on the forehead before treatment. BCC with topical cyclopamine treatment identical to FIG. 1A on day 6. A large cyst in the dermis corresponding to the position where the tumor cell fistula indicating no residual tumor cells disappeared (magnification 100 times). Similar cysts in other epidermal regions that contained BCC before cyclopamine treatment but no longer contained BCC after treatment (100x magnification). Low power image (100x magnification) of the BCC region of FIG. 1D showing residual cells and the formation of large cysts due to the joining of many small cysts between the cells. As shown in FIG. 2C, which shows a marked increase in the frequency of apoptotic cells and the expansion and simultaneous formation of cysts due to the removal of apoptotic BCC cells, a high-power image of an internal region of the same residual BCC (magnification 1000) Times). As shown in FIG. 2C, which shows a marked increase in the frequency of apoptotic cells and the formation and simultaneous formation of cysts due to the removal of apoptotic BCC cells, a high-power image of a region around the same residual BCC (magnification 1000) Times). High power image (1000x magnification) of typical tumor cells of the tumor and the internal area of BCC after placebo treatment without apoptosis. Immunohistochemical analysis showing that monoclonal antibody Ber-Ep4 is not stained in all residual cells of BCC after cyclopamine treatment (400X magnification). In contrast to FIG. 3A, immunohistochemical analysis showing that monoclonal antibody Ber-Ep4 is very strongly stained in all residual cells of BCC after placebo treatment (400 × magnification). Uses Ulex Europaeus lectin type I, which normally does not label BCC cells, or normal skin matrix layer cells, but shows the same BCC cell differentiation until the step detected by this lectin that labels differentiated upper cells Immunohistochemical analysis (1000x magnification) showing heterogeneous labeling of residual cells in BCC after successful cyclopamine treatment. Immunohistochemical analysis showing reduced expression of p53 detected by monoclonal antibody DO-7 in BCC after cyclopamine treatment (100x magnification). Immunohistochemical analysis showing the expression of p53 detected by monoclonal antibody DO-7 in BCC after placebo treatment compared to FIG. 3D (100 × magnification). Immunohistochemical analysis showing consistent contraction of BCC from the substrate (arrows indicate contraction) (100x magnification), a form known for cessation of tumor cell growth observed more after cyclopamine treatment . Immunohistochemical analysis showing no contraction observed after placebo treatment (difference between BCC after cyclopamine treatment and BCC after placebo treatment was also observed in 3D, 2C vs. 3B, 3E with respect to contraction from the substrate (Magnification 100 times). Ber-Ep4 labeling of the epidermal basal layer cells after cyclopamine treatment (400 × magnification). High output image of the treated epidermal region showing Ber-Ep4 labeling of basal cells (1000x magnification). High output image of the treated epidermal region showing Ber-Ep4 labeling of basal cells (1000x magnification). High power image (1000x magnification) of hair follicles after cyclopamine treatment showing normal labeling using Ber-Ep4. A photomicrograph showing BCC before treatment that caused an ulcer in the upper nasal region of a 68 year old male. In the lower half, a micrograph showing the same BCC as in FIG. 5A at 54 hours when cyclopamine was applied. Section from half of BCC cyclopamine applied at 54 hours, photomicrograph at 400x magnification with hematoxylene eosin (H & E) staining. A micrograph of a section of the same BCC from a non-treated site, with hematoxylene eosin (H & E) staining at 400 × magnification. Sections from half of BCC cyclopamine applied at 54 hours, using immunohistochemical staining for Ki67 antigen, magnification 200 ×. A microphotograph at a magnification of 200 times, which is a section clearly showing proliferative activity in the tumor cell farthest from the cyclopamine-treated region and using immunohistochemical staining for Ki67 antigen. Photomicrograph showing follicular epithelioma before treatment of cheek of 82-year-old man. The micrograph which shows the same skin region as FIG. 6A 24 hours after a cyclopamine treatment. 6 is a section of the deleted skin region shown in FIG. 6B with residual tumor cells, H & E, micrograph at 400 × magnification. FIG. 6C is another region of the same tissue, in addition to many apoptotic cells and cyst structures formed by removal, the tumor is observed to be invaded by mononuclear cells, H & E, 200 × magnification Micrograph. Photomicrograph showing pre-treatment BCC pigmented on the lower pudendal region of a 59 year old male. FIG. 7B is a photomicrograph showing the same BCC as FIG. 7A on the third day of cyclopamine treatment. FIG. 7B is a section from the post-treatment region of BCC shown in FIG. 7B, H & E, 200 × magnification micrograph. Close up of the area of residual tumor cells in the section from the area after treatment of BCC, H & E, photomicrograph at 400x magnification. FIG. 7B is a section from a punch specimen material obtained from the BCC shown in FIG. FIG. 7B is a section containing a portion of the BCC blob indicated by the arrow in FIG. 7A, where the tissue was deleted after 3 days of treatment and followed for 6 days without treatment, H & E, 100 × magnification micrograph.

  1A, 1B, 1C and 1D show rapid clinical regression of BCC due to exposure to cyclopamine. In addition to the visual disappearance of several tumor areas within a week of cyclopamine exposure, the typical translucent appearance is lost, as seen by comparing FIGS. 1B and 1A, 1D and 1C. Is called.

  2A-2F show the microscopic appearance of the tumor area that was subject to surgical removal with normal tissue margins 5 and 6 days after cyclopamine administration, with BCC disappearing in most pretreatment areas However, in only a small area, there is a significant decrease in height, but it has not yet completely disappeared, and thus has residual tumor cells that are subject to microscopic observation.

  FIG. 2A and FIG. 2B show the skin area corresponding to the nodule of the tumor that disappeared visually on the tissue section. Tumors can be observed to disappear, leaving a cystic structure containing very small amounts of material inside and undetectable tumor cells.

  FIG. 2C shows the microscopic appearance of a skin area that still contains visible BCC in vivo. It is observed that these regions include residual BCC, which shows a huge cyst in the center of the tumor, and small cyst structures located between the residual BCC cells due to various sizes towards the periphery.

  2D and 2E show 1000 times the appearance of residual BCC in the inner and fenced surrounding areas, indicating the presence of massive apoptotic activity between residual BCC cells, regardless of the tumor area. ing. High magnification is due to the apoptotic morphology of the cells and, due to the removal of apoptotic septal cells, as demonstrated in FIG. It shows a greatly increased frequency of BCC cells showing the formation of cystic structures.

  FIG. 2F shows BCC after treatment with placebo cream (ie, a cream of the same formulation as cyclopamine cream except in the absence of cyclopamine in the placebo), in contrast to neoplastic BCC cells. It shows that apoptotic activity is not detected.

  Apoptotic cells are known to be removed by macrophages and neighboring cells in normal tissue, and quantification of apoptotic activity by morphological criteria on hematoxylin and eosin-stained sections provides an underestimation It is known to do. Nevertheless, the quantitative data shown in Table 1 shows a marked increase in apoptotic activity caused by cyclopamine between residual BCC cells.

  The loss of permeability within cyclopamine-treated BCC increases the potential for interesting BCC differentiation due to the effects of cyclopamine. This possibility, which can be examined by immunohistochemical analysis of BCC, can be found in the case of the present invention. In normal epidermis, differentiation from basal layer cells to epithelial layer cells is achieved concomitant with the loss of label using the Ber-Ep4 monoclonal antibody. In addition, Ber-Ep4 labels BCC cells and is known as a marker for their neoplasms. 3A, 3B, and Table 1 show that Ber-Ep4 strongly labels all peripheral palisade cells after placebo treatment and over 90% of BCC internal cells, but after cyclopamine treatment. It is shown that residual peripheral cells or internal cells are not labeled with Ber-Ep4. The differentiation of BCC due to the effects of cyclopamine has not been known until now, and since it is realized in vitro and in all cells according to immunohistochemical standards, it is a very exceptional and independent treatment of cancer. Have value.

  Another differentiation marker, Ulex Europaeus lectin type I, does not normally label basal cells of BCC or normal epidermis, but does label upper cells after differentiation. FIG. 3C shows the heterogeneous labeling of BCC residual cells after cyclopamine treatment using lectins, from the step of differentiation detected by Ber-Ep4 to the step detected by Ulex Europaeus lectin type I. A part of BCC cell differentiation is shown.

  P53 is a major regulator of cellular responses to DNA damage. This amount of protein is known to increase in the cell nucleus upon exposure of the cells to genotoxic agents. If DNA damage increases above the threshold, p53 causes apoptotic death in the cell. Currently generally known cancer radiotherapy and genotoxic cancer chemotherapy work largely by this mechanism, i.e. by causing apoptosis caused by DNA damage. Monoclonal antibody DO-7 is known to be capable of binding to both normal and missense mutant forms (non-functional) of p53 and has the ability to detect an increase in intracellular p53 exposed to DNA damaging agents. is there.

  The quantitative data in FIGS. 3D, 3E and Table 1 show that DO-7 labeling intensity and frequency of labeled cells are significantly reduced in BCC after cyclopamine treatment compared to BCC after placebo treatment. Show. Thus, cyclopamine not only does not increase p53 in the nucleus of BCC cells after cyclopamine treatment, but rather causes a decrease. Since p53 expression is known to be a decrease in epidermal cells due to differentiation, a decrease in BCC DO-7 labeling following cyclopamine treatment is likely to occur due to cyclopamine-induced differentiation of BCC cells. In any case, despite the significant decrease in p53 expression, the massive apoptotic activity of BCC after cyclopamine treatment means that cyclopamine-induced tumor cell apoptosis is due to non-genotoxicity.

  Stoppage of BCC growth is known to be associated with contraction from the substrate. Although shrinkage from the substrate can occur artificially due to improper fixation and tissue processing, adherence to known technical details ensures avoidance of such effects. As shown in FIGS. 3F and 3G, BCC after cyclopamine treatment is consistently contracted from the substrate, although not placebo treatment. Thus, it is clear that exposure of BCC to cyclopamine is further associated with growth arrest.

  4A-4D show Ber-Ep4 labeling of normal skin tissue found on and around BCC after cyclopamine treatment. Different epidermal regions treated with cyclopamine display the normal pattern labeled with Ber-Ep4, ie, the label of basal layer cells, as shown in FIGS. 4A, 4B, and 4C . Similarly, FIG. 4D shows a normal Ber-Ep4 label for hair follicles exposed to cyclopamine. Thus, undifferentiated cells of normal epidermis and hair follicles are maintained despite exposure to the same schedule and amount of cyclopamine, similar to BCC.

  In an amount that maintains undifferentiated tissue cells, cyclopamine efficiently differentiates in vivo tumor cells, and the occurrence of apoptosis is coupled with the effects of non-genotoxic cyclopamine, in addition to BCC, proliferation and apoptosis. And / or a previously unknown achievement supporting the use of cyclopamine for other internal tumors that utilize the hedgehog / smoothened pathway to suppress differentiation.

It is particularly contemplated that the molecule can be derived from cyclopamine and that the molecule can be synthesized by processing the structural form and exerting the same receptor binding properties and biological / therapeutic effects as cyclopamine. . In this disclosure, this molecule is called “cyclopamine derivative” and is defined as follows. The molecule contains an atomic population of cyclopamine molecules that require the binding of cyclopamine to a biological target, but the continued ability of novel cyclopamine derivatives to specifically bind to the same biological target allows the cyclopamine of the present disclosure to be Including modification of the parent cyclopamine molecule in such a way as to exert a biological effect. Such alterations of cyclopamine are processed to permit permissible substitution and removal of the cyclopamine molecule or to make the resulting molecule stable and in particular capable of specifically binding to the same biological target as cyclopamine. By adding a molecular population (especially a small molecular population such as the methanol family) to a cyclopamine molecule provided to exert a biological effect according to the present disclosure. Novel molecules derived from cyclopamine are readily realized by those of ordinary skill in the art, and the presence or absence of the biological action of cyclopamine within the novel derived molecule is easily determined by testing the biological effects of the present disclosure by those skilled in the art. Can be determined.
Further Examples FIG. 5A shows a pre-treatment BCC of a 68 year old male who had a giant ulcer on the upper nasal area. Cyclopamine cream (18 mM cyclopamine in the above base cream) was applied to the lower half of the BCC shown in FIG. 5A. Every 3 hours, about 20 μl of cream was applied directly to the lower half and the upper half was not treated. Thus, the topmost tumor cells (FIG. 5A) are unlikely to receive cyclopamine by diffusion from the directly applied area, and are exposed to relatively low concentrations of cyclopamine, if any. . FIG. 5B shows a 54 hour treatment tumor just prior to surgical removal for the study. Rapidly shrinking tumors occurred in the lower half where cyclopamine was applied, while relatively little change was seen in the tumor region farthest from the directly applied region (region to the upper right portion of the diagram in FIG. 5B). FIG. 5C shows a section of the lower part of the deleted tissue (after cyclopamine treatment) stained with hematoxylene eosin. Many apoptotic cells are shown with various cysts formed by the death and removal of tumor cells (FIG. 5C). In contrast, the untreated area of the same tumor that is furthest away from the area where cyclopamine was applied shows aggregated tumor tissue that shows division and no apoptotic cells are detected (FIG. 5D). FIG. 5E and FIG. 5F show tissue sections obtained by immunohistochemical staining for each of the post-cyclopamine treatment and non-treatment using KiS5 antibody (Dako A / S Glostrup Denmark) against Ki67 antigen. Ki67 antigen is a known marker of proliferating cells, and is not expressed in the region of tumor cells after cyclopamine treatment (FIG. 5E), but clearly shows proliferative activity in tumor cells farthest from the region treated with cyclopamine. (FIG. 5F). Tissue sections stained with antibodies against the Ki67 antigen show that the growth of tumor cells is further stopped by cyclopamine under the conditions described.

  Hair follicle epithelioma is another tumor related to genetic mutations that results in an increase in the Hedgehog-smoothened signal (Vorechovsky et al., (1997) Cancer Res 57, 4777-4681, Nilsson M. M) et al. (2000) Proceeding of the National Academy Science USA (Proc. Natl. Acad. Sci) 97, 3438-3443). FIG. 6A shows an untreated hair follicle epithelioma on the cheek of an 82 year old male, and FIG. 6B shows the same skin area after only 24 hours exposure to cyclopamine cream (18 mM in base cream). Cyclopamine, approximately 25 μl of cream applied to the tumor every 3 hours). Due to the rapid contraction, treatment was discontinued at 24 hours and all skin areas corresponding to the first tumor were removed for study. FIGS. 6C and 6D show tissue regions containing residual tumor cells for 24 hours, revealing significant apoptotic activity within those residual tumor cells. Similar to the mononuclear infiltration of the tumor (FIG. 6D), the cyst space (FIGS. 6C and 6D) produced by the apoptotic removal of the tumor cells is shown. Another notable discovery of the present invention is the reduction in bruise size and pigmentation located in the vicinity of the treated tumor with 24 hours of treatment (compare FIGS. 6B and 6A). Bruises (benign melanocytic tumors) are sensitive to relatively low concentrations of cyclopamine so that cyclopamine can be diffused from the applied proximity area.

  FIG. 7A shows a pigmented, pre-treatment BCC in the lower pubic area of a 59 year old male. Cyclopamine cream (18 mM cyclopamine in the base cream) was applied to all of the patient's nodules except for one indicated by the arrow. This blob can be exposed to a relatively low concentration of cyclopamine, provided that it could only receive diffuse cyclopamine from the adjacent post-treatment area. The pigmented nature of this tumor facilitates clinical follow-up, and treatment (applying about 20 μl cyclopamine cream every 4 hours) has resulted in significant regression of the tumor in the treatment area, but still including the visible region 3 On the day, it was interrupted (FIG. 7B). The tumor was then followed without treatment to study the potential for a lag effect. No further explicit regression was observed without treatment, and the area corresponding to the first tumor was deleted on the 6th day of follow-up (day 9 from the start of treatment). A hematoxylene eosin stained section of the tumor treatment area reveals a cyst space in which many tumor cells have disappeared (FIG. 7C). These cysts lacking epithelial lining (FIG. 7C) conform to the representation of the tissue region cysts previously occupied by tumor cells. At this point (day 6 followed without treatment), tissue sections representing a relatively small number of apoptotic cells (FIG. 7C) followed the known rapid elimination of apoptotic cells from living tissue. On the other hand, residual cells, particularly in the vicinity of the end of the cyst, show a markedly frequent differentiation of the cells into a protrusion shape (eg, the high area illustrated in the region for the lower left of FIG. 7C, another region of FIG. 7D). Observed more clearly at magnification). The same area of differentiation or cyst is not present in punch biopsies taken from the same tumor prior to the start of treatment (FIG. 7E). Tumor nodules that received relatively low concentrations of cyclopamine (shown by arrows in FIG. 7A) had large cyst centers on day 6 of follow-up (FIG. 7F). However, around the residual nodule, the frequency of differentiated forms of cells (eg, with increased and even eosinophilic cytoplasm) increased further, and small cysts were present inside this periphery. Continue to have a typical BCC configuration (FIG. 7F). Thus, tumors responding to optimal concentrations of cyclopamine are relatively rapid, and suboptimal concentrations of exposure leave tumor cells that survive during follow-up.

  These further examples demonstrate the efficiency of the described treatment to obtain rapid clinical contraction of BCC with other tumors (hair follicle epithelioma) that show an increased hedgehog-smoothened signal. Clinical contraction appears to be related to cyclopamine-induced differentiation and apoptosis of tumor cells. In addition to this, the growth of tumor cells is inhibited. The efficiency of patients with different genotypes relative to several independent tumors followed the general utility of the described treatment.

  Cyclopamine was discovered as a teratogenic element in the palm tree plant (Keeller R. F. (1969), cytochemistry 8 223-225). Inhibition of abdominal cell precursor differentiation in brain development has been reported (Incardona JP, et al. (1998), Development 125 3443-3562, Cooper Em. (Cooper M. K) et al. (1998), Science 280, 1603-1607). Inhibition of cell differentiation by cyclopamine is performed by differentiation from bone marrow cells to red blood cells (Detmer K, et al. (2000), Development Biology 222-242), urogenital sinus to prostate It has also been reported in other organizations, including the differentiation into (Berman DM, (2000), Journal of Urology (J.Urol) 163,204). However, the opposite has been found to be true in the present invention with tumor cells exposed to cyclopamine. Similar to cyclopamine-induced differentiation of tumor cells, further apoptosis of tumor cells is induced. Induction of tumor cell apoptosis by cyclopamine, not previously described, has shown high efficiency. Furthermore, the induction of apoptosis by cyclopamine has no subsequent genotoxic effect, and further tumor cells are differentiated and undergo apoptosis, while the outer root sheath cells of the hair follicles close to the tumor cells and normal epidermal basal cells are Has a remarkable specificity of being well maintained. The described treatment without adverse effects was confirmed by the presence of healthy skin that appeared clinically normal in the patient's cyclopamine application area (the longest follow-up for the subject was 15 months or more at this time, Long-term treatment safety was also demonstrated). The above described forms of the treatment described in the present invention are highly sought after in cancer treatment and provide a solution to the longstanding problem of cancer treatment.

1A-1D show that within a period of less than a week, cyclopamine as indicated by the disappeared tumor area (indicated by arrows), a significant decrease in height from the surface of the skin, and the loss of translucency. FIG. 5 is a microscopic appearance of a rapid regression of BCC after treatment.

  FIGS. 2A-2F show BCC after cyclopamine treatment and BCC after placebo treatment, showing massive cyclopamine-induced apoptosis and removal of tumor cells and disappearance of tumor nodules leaving cyst spaces without tumor cells. This is the appearance of the microscope. The skin area corresponding to the location of the pre-treatment of BCC, usually along with the edge of the tissue, is surgically removed on the 5th and 6th day of cyclopamine exposure and is subject to fixation by conventional methods, incision removal, Hematoxylin and eosin stained for microscopic analysis.

  3A-3G show the effects of cyclopamine associated with exposure to cyclopamine and cyclopamine treatment showing differentiation of all residual BCC cells due to decreased p53 expression in BCC, and immunohistochemistry of BCC after placebo treatment It is analysis. All immunohistochemical labels used streptavidin-conjugated peroxidase conjugated with a biotin-labeled secondary antibody.

  FIGS. 3A and 3B show that all residual cells in BCC after cyclopamine treatment are differentiated by Ber-Ep4 towards or beyond the detected process. Ber-Ep4 is a known differentiation marker that stains BCC cells, but does not stain upper layer cells after differentiation of normal epidermis, as with normal epidermal basal layer and undifferentiated cells of hair follicles.

  The expression of p53 in FIGS. 3D and 3E is known to reduce differentiation of epidermal basal cells and keratinocytes after culture. It is well known that the amount of p53 detectable with DO-7 increases intracellularly when cells are exposed to genotoxic agents.

The sections shown in FIGS. 3F and 3G were stained using periodate Schiff and Alcian Blue.
4A-4D show that normal epidermis and hair follicle undifferentiated cells are maintained despite exposure to the same schedule and dosage of cyclopamine, as in BCC. The procedure of immunohistochemical detection is the same as in FIGS. 3A and 3B, and the label is shown in brown.

  Color prints of the same figure as page 12 (FIGS. 1A, 1B, 1C, 1D, 2A, 2B), as immunohistochemistry data and knowledge can be transmitted more optimally in color than in grayscale. 2C, 2E, 2F, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 4A, 4B, 4C, and 4D) were added as page 12a. . The applicant requires that this fact be considered by the Patent Office and that page 12a be treated as part of the patent application. However, page 12a may be removed from this patent application if deemed necessary by the Patent Office.

  Because the color and color of immunohistochemistry can be transmitted more optimally than grayscale, the color print of the same figure on page 12b (FIGS. 5A, 5B, 5C, 5D, 5E, 6A 6B, 6C, 6D, 7A, 7B, 7C, 7D, 7E, and 7F) were added as 12c pages. Applicants require that this fact be considered by the Patent Office and that page 12c be treated as part of the patent application. However, page 12c may be removed from this patent application if deemed necessary by the Patent Office.

  Color prints of the same figure as page 12 (FIGS. 1A, 1B, 1C, 1D, 2A, 2B), as immunohistochemistry data and knowledge can be transmitted more optimally in color than in grayscale. 2C, 2E, 2F, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 4A, 4B, 4C, and 4D) were added as page 12a. . The applicant requires that this fact be considered by the Patent Office and that page 12a be treated as part of the patent application. However, page 12a may be removed from this patent application if deemed necessary by the Patent Office.

Claims (12)

Use of cyclopamine or a medically acceptable cyclopamine salt in the local treatment of basal cell carcinoma. Use of cyclopamine or a medically acceptable cyclopamine salt for the production of a compound that is cyclopamine or a medically acceptable compound for use in the local treatment of basal cell carcinoma. Use of cyclopamine or a medically acceptable cyclopamine salt in the treatment of basal cell carcinoma by non-local means, including intratumoral injection. Use of cyclopamine, or a medically acceptable cyclopamine salt, for the manufacture of a medically acceptable compound for use in the treatment of basal cell carcinoma by non-local means, including intratumoral injection. Use of cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative in the local treatment of basal cell carcinoma. Use of cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative for the manufacture of a medically acceptable compound for use in the local treatment of basal cell carcinoma. Use of cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative in the treatment of basal cell carcinoma by non-local means, including intratumoral injection. Use of cyclopamine, a medically acceptable cyclopamine salt, or a cyclopamine derivative for the manufacture of a medically acceptable compound for use in the treatment of basal cell carcinoma by non-local means, including intratumoral injection Method. Use of cyclopamine, or a medically acceptable cyclopamine salt, in the treatment of tumors using the hedgehog / smoothed signaling pathway for growth and / or prevention of apoptosis or cell differentiation. Cyclopamine, or medicine, for the production of medically acceptable compounds in the treatment of tumors using the hedgehog / smoothed signaling pathway for growth and / or for prevention of apoptosis or cell differentiation To use a chemically acceptable cyclopamine salt. Use of cyclopamine, medically acceptable cyclopamine salts, or cyclopamine derivatives in the treatment of tumors using the hedgehog / smoothed signaling pathway for proliferation and / or prevention of apoptosis or cell differentiation . Cyclopamine, medical, for the production of medically acceptable compounds in the treatment of tumors using the hedgehog / smoothed signaling pathway for proliferation and / or for the prevention of apoptosis or cell differentiation Of acceptable cyclopamine salts or cyclopamine derivatives.