CN101904852A - The application of 11-Deoxojervine in treatment basal cell carcinoma and other tumor - Google Patents

Abstract

The present invention relates to the application of 11-Deoxojervine in treatment basal cell carcinoma and other tumor.The present invention relates to 11-Deoxojervine and treat the application of basal cell carcinoma in vivo, differentiation by causing tumor cell and apoptosis and these tumor cells are removed the realization therapeutic effect efficiently, and, comprise that the undifferentiated cell of normal epidermis basal layer and hair follicle does not have influence to normal histiocyte.11-Deoxojervine causes apoptosis by the non-genomic toxic mechanism.These effects make 11-Deoxojervine very desirable aspect treatment basal cell carcinoma and other cancer, and these cancers are utilized the hedgehog/smoothened signal transduction pathway to break up and prevented apoptosis.

Description

The application of 11-Deoxojervine in treatment basal cell carcinoma and other tumor

The application is to be 07 month 02 day calendar year 2001, application number the dividing an application for the patent application of " application of 11-Deoxojervine in treatment basal cell carcinoma and other tumor " that be 01823442.9 (international application no is PCT/TR01/00027), denomination of invention the applying date.

The present invention relates to 11-Deoxojervine (cyclopamine) and treat the application of basal cell carcinoma (BCC ' s) in vivo, break up by inducing tumor cell, and thereby apoptosis is removed the realization therapeutical effect with these tumor cells, and, comprise that the undifferentiated cell of normal epidermis basal layer and hair follicle does not have influence to normal histiocyte.11-Deoxojervine causes apoptosis by the non-genomic toxic mechanism, and is therefore different by the method that damage dna plays a role with most chemotherapeutics of radiotherapy and current use.These previous inaccessiable new effects of cancer chemotherapeutic drug make 11-Deoxojervine be suitable for treatment of cancer, are suitable for treating other tumors of BCC ' s, and described tumor is utilized the hedgehog/smoothened signal transduction pathway to breed and prevented apoptosis.

Basal cell carcinoma is a kind of common epithelial tumor.Its sickness rate raises with age growth.Current treatment to BCC ' s comprises tumor and edge normal structure excision thereof, when performing the operation or perform the operation when undesirable, takes radiotherapy or other means to destroy tumor cell.Although have the potential side effect that stays cicatrix and disfeature, tumor cell is removed completely excision it can be cured.Radiotherapy is worked by causing a large amount of expendable DNA damage, and this damages triggering tumor cell apoptosis subsequently.This binding mode of radiotherapy promptly is secondary to the apoptosis of DNA damage, and is similar to the mechanism of action of the many chemotherapeutics that use in the current treatment of cancer.Yet radiotherapy and cytotoxicity cancer chemotherapeutic drug also can cause damage to the Normocellular DNA beyond the tumor cell.Therefore, their effectiveness and serviceabilities in treatment of cancer are very limited.Also there are another awkward situation of bothering in radiotherapy and genotoxicity cancer chemotherapeutic drug, even promptly patient's primary carcinoma disease is cured, its danger of suffering from the New Development cancer increases significantly, because caused DNA damage and therefore produce sudden change in the treatment of primary carcinoma disease.Therefore, in field of cancer prospect is arranged by non-genomic toxicity mode selective induction apoptosis of tumor cells most.

BCC ' s often demonstrates the inactivation sudden change of gene patched, and this gene code is a kind of to be the transmembrane protein of the proteic receptor of hedgehog, and the proteic discovery first of these hedgehog comes from its effect aspect the tissue development pattern.When not combining with hedgehog, patched protein suppresses the intracellular signal transduction by another kind of transmembrane protein smoothened.Hedgehog and patched combination of proteins can be removed this inhibitory action.Then, the intracellular signal transduction by the smoothened that removes starts a series of cellular activities, finally causes the expression of hedgehog genes of interest and cell behavior to change.This hedgehog/smoothen signal transduction pathway at first is confirmed in fruit bat, and its general feature is guarded in the multiple organism from the fruit bat to people.Yet this path is complicated more (having remarkable similarity as a plurality of genes of the mankind and the single patched gene of fruit bat) in more high-grade organism.Find that the inactivation sudden change of patched gene can cause composing type (no part) signal transduction by the hedgehog/smoothened path.In all BCC ' s, all found to come from the overacfivity of the hedgehog/smoothened path that the sudden change of patched gene and/or downstream passages element causes.Nevoid basal cell carcinoma syn drome (NBCCS) comes from the haplo-insufficiency of patched gene.NBCCS patient is owing to formerly exist saltant patched gene in all cells, and they can develop into multiple BCC ' s in the future.

11-Deoxojervine is a kind of steroid alkaloid, has following structural formula.

Find 11-Deoxojervine natural existence in Bulbus Lilii Veratrum califomicum, can from wherein or other source extract and obtain.In Embryo Gallus domesticus and Mus cultured cell, found the inhibitory action of 11-Deoxojervine to the hedgehog/smoothened path.

When topical application, 11-Deoxojervine can be dissolved in ethanol or other the suitable solvent, mix with suitable alkaline substrate emulsifiable paste, ointment or gel again.Also can be fixed in 11-Deoxojervine on the hydrogel or make other dosage form reaching controlled release, and absorb on the transdermal patches.What Figure 1A～Fig. 1 D, Fig. 2 A～Fig. 2 F, Fig. 3 A～Fig. 3 G and Fig. 4 A～Fig. 4 D showed is the effect of cream preparation, and said preparation obtains the 11-Deoxojervine emulsifiable paste that final concentration is 18mM for the 11-Deoxojervine alcoholic solution is mixed with the substrate emulsifiable paste.The emulsifiable paste matrix that uses mainly is made up of scheelite wax oil (10%w/w), vaseline (10%w/w), octadecanol (8%w/w), polyoxylsteareth-40 (3%w/w) and water (68%w/w), yet, other suitably the emulsifiable paste matrix of preparation also can use.The optium concentration of 11-Deoxojervine preparation, best dosage and application scheme will obviously be subjected to concrete dosage form, comprise the position of skin of tumor and the factor affecting such as size of characteristics (as the thickness of epidermis) and tumor; Yet these factors can be determined through the following known ground optimization method of delivering.Figure 1A (is positioned at the BCC on the muffle pleat, surface area be 4 * 5mm) and Fig. 1 C (be positioned at the BCC of forehead, surface area is that the dosage and the therapeutic scheme of the tumor shown in 4 * 4mm) is as follows: with steel spatula 10 ± 2 μ l emulsifiable pastes directly are applied to BCC ' s, four times a day, approximately since 9:00 in the morning, medication interval 3.5 hours.Should avoid this scheme when using night, when sleeping soundly owing to the patient, emulsifiable paste easily run off to bedding from skin, and can use the suitable skin paster this moment.To the protection of undifferentiated cell in normal epidermis and the hair follicle, as described herein, provide behind the contact 11-Deoxojervine about information with the tolerance dose of other possible application method; Use as the whole body of directly in tumor, injecting its aqueous solution or aqueous solution, or 11-Deoxojervine is encapsulated in the liposome.

Figure 1A, Figure 1B, Fig. 1 C and Fig. 1 D have shown quick clinical the lapsing to that is exposed to BCC ' s behind the 11-Deoxojervine.In the time in one week, not only can see the disappearance of several places lump in less than, simultaneously, Figure 1B compares with Fig. 1 C with Figure 1A and Fig. 1 D, and the typical translucent appearance of BCC ' s disappears.

Fig. 2 A～Fig. 2 F has shown at application 11-Deoxojervine the 5th and after the 6th day, with the undergo surgery microscopical appearance of lump of excision of edge normal structure, most areas before BCC ' s treatment this moment disappear, a few regions is still arranged, although it highly obviously descends, but complete obiteration not yet, therefore, residual tumor cell is still found in microscopic analysis.

Shown in Fig. 2 A and Fig. 2 B corresponding to the skin area on the tissue slice of disappearance lump.Find that through observing tumor disappears and stays the cystic structures that comprises small amount of matter in a large number, does not detect tumor cell.

Fig. 2 C has shown the microscopical appearance that still comprises the skin area of visible BCC in the body.Can be observed residual BCC ' s in these zones, it demonstrates at a large amount of cysts of tumor center and is arranged in the less cystic structures of peripheral residual BCC cell all size.

Fig. 2 D and Fig. 2 E shown that these residual BCC ' s are inner and gone along with sb. to guard him 1000 times of outer peripheral areas and amplify outward appearances, is also shown among the residual BCC of tumor region to have a large amount of apoptosis activities.The picture that these high powers are amplified shows, apoptosis form BCC cell and increase greatly by the frequency that the apoptosis of cell is removed the cystic structures that forms, by can illustration shown in Fig. 2 D, in case the barrier film cell of apoptosis is eliminated, three less cysts link to each other and form a bigger cyst.

Fig. 2 F shows the treatment (promptly except that do not contain 11-Deoxojervine, with 11-Deoxojervine cream preparation identical emulsifiable paste as placebo) of BCC ' s with the placebo emulsifiable paste, with last different, demonstrates typical B CC cell and acellular apoptosis activity.

Known generation apoptotic cells is removed by the adjacent cells in macrophage and the normal structure, and the apoptosis activity that known morphological criteria of cutting into slices based on hematoxylin-eosin staining is carried out is quantitatively on the low side.However, the quantitative data in the table 1 shows, increases greatly through the apoptosis activity in the residual BCC cell after the 11-Deoxojervine treatment.

The disappearance of the translucence in the BCC ' s of 11-Deoxojervine treatment has improved the interesting probability of BCC ' s differentiation under the influence at 11-Deoxojervine.The present invention finds that this probability exists really, can measure through the immunohistochemical analysis of BCC ' s.In normal epidermis, the disappearance of monoclonal antibody Ber-Ep4 labelling characteristic is followed in the make progress differentiation of confluent monolayer cells of basal layer cell.Ber-Ep4 is labelling BCC cell also, is the known mark thing of these tumors.The quantitative data of Fig. 3 A, Fig. 3 B and table 1 shows, although Ber-Ep4 consumingly among labelling placebo group BCC ' s all peripheries go along with sb. to guard him the inside cell of cell and 90%, Ber-Ep4 is any residual peripheral cell or the inner cell of Marking ring rake Mingzhi treatment group BCC ' s not.Other similar report is not seen in BCC ' s differentiation under the influence of 11-Deoxojervine so far, because it reaches the extraordinary effect that is reached according to the SABC standard in vivo in all cells, has unique value on treatment for cancer.

Another differentiation marker thing, 1 type Ulex Europaeus lectin, the basal layer cell of labelling BCC ' s or normal epidermis not normally, but the last confluent monolayer cells of labelling differentiation.Fig. 3 C shown with the heterogeneous labelling of this lectin to 11-Deoxojervine treatment group BCC ' s ground residual cell, shown that the step 1 that can't detect from Ber-Ep4 is up to the differentiation with some BCC cells of the detectable step of 1 type Ulex Europaeus lectin.

P53 is that cell is to the main regulon of DNA damage.Knownly will increase when cellular exposure this albumen amount in nucleus behind the genotoxicity medicine.When DNA damage surpassed certain threshold value, p53 is cell death inducing just.The radiotherapy of cancer and current genotoxicity chemotherapeutics commonly used mainly work by this mechanism, promptly by inducing the apoptosis that is secondary to DNA damage to work.Monoclonal antibody DO-7 can combine with the p53 of normal and missense mutant (being non-functional) form, and can the increase of p53 detects in the cell to being exposed to behind the DNA damage medicine.

Quantitative data in Fig. 3 D, Fig. 3 E and the Table I shows, compares with the BCC ' s of placebo treatment, and the mark intensity of the DO-7 among 11-Deoxojervine treatment group BCC ' s and labelling frequency all significantly reduce.Thereby 11-Deoxojervine causes the p53 in the BCC nucleus after its treatment to reduce, rather than raises.The p53 of the epidermis cell of known differentiation will reduce, thereby the DO-7 labelling of 11-Deoxojervine treatment group BCC ' s reduces the differentiation that may be secondary to the inductive BCC cell of 11-Deoxojervine.In any case although the remarkable reduction that has p53 to express, a large amount of apoptosis activity that exists in the BCC ' s cell of 11-Deoxojervine treatment shows that the apoptosis of the inductive tumor cell of 11-Deoxojervine is undertaken by the non-genomic toxic mechanism.

Known BCC ' s inhibition of proliferation is relevant with its contraction in substrate.Although, can avoid these illusions according to disclosed technical scheme because the inappropriate fixing and processing of tissue can produce the illusion from the contraction of substrate.Shown in Fig. 3 F and Fig. 3 G, 11-Deoxojervine is handled, but not shrinking from substrate appears in the BCC ' s concordance of placebo treatment ground.As if thereby it is also relevant with inhibition of proliferation that BCC ' s is exposed to 11-Deoxojervine.

Fig. 4 A～Fig. 4 D has shown the BCC ' s of 11-Deoxojervine processing or near the Bcr-Ep4 labelling of the normal skin tissue it.Fig. 4 A, Fig. 4 B and Fig. 4 C show the skin factor of different zone of handling with 11-Deoxojervine, to show the normal mode of Ber-Ep4 labelling, the i.e. labelling of basal layer cell.Similarly, Fig. 4 D has shown the normal Ber-Ep4 labelling of the hair follicle that is exposed to 11-Deoxojervine.Thereby, although the undifferentiated cell of normal epidermis and hair follicle is exposed to 11-Deoxojervine with BCC ' s same scheme and dosage, but still keep intact.

When not influence undifferentiated histiocytic 11-Deoxojervine dosage, inducing tumor cell breaks up and inducing apoptosis of tumour cell efficiently in vivo, these achievements are also not known at present, the non-genomic toxic action pattern of these mechanism and 11-Deoxojervine, support is used for the purposes of BCC ' s and some other oncotherapys with 11-Deoxojervine, and described tumor is utilized the hedgehog/smoothened signal transduction pathway to breed and prevented apoptosis and/or differentiation.

Accompanying drawing is described

Figure 1A, 1B, 1C, 1D: shown that 11-Deoxojervine handles lapsing to fast of back BCC ' s, be embodied in lump disappearance (shown in the arrow), apart from the height of skin surface obviously reduce and transparent outward appearance in less than disappearance in one week.1A: the preceding BCC of treatment that is positioned at left side muffle pleat.1B: same BCC is in the 5th day situation of 11-Deoxojervine topical therapeutic.1C: the preceding situation of BCC treatment that is positioned at forehead.1D: same BCC is in the 6th day situation of 11-Deoxojervine topical therapeutic.

Fig. 2 A, 2B, 2C, 2D, 2E, 2F: the microscopical appearance of 11-Deoxojervine BCC ' s that handle and placebo treatment, show the removing of the inductive a large amount of apoptosis of 11-Deoxojervine, tumor cell and lump disappearance, stay a large amount of blister cavities that do not contain tumor cell.11-Deoxojervine the pretreated the 5th and the 6th day, will be corresponding to the skin area and the normal surrounding tissue of BCC ' s position before handling, the excision that undergos surgery is carried out conventional fixing, section and hematoxylin-eosin staining do microscopic analysis then.2A:, show the tumor cell of noresidue corresponding to the utriculi majores in the corium of lump disappearance position.2B: another place's similar cyst in corium district, this zone comprise BCC before treating with 11-Deoxojervine, but do not comprise after the non-treatment.2C: the low power field in the BCC zone that Fig. 1 D shows has shown the utriculi majores of the continuous formation of countless utriculi brevioreses between residual cell and these cells.2D: the high power field of same residual BCC interior zone among Fig. 2 C shows that apoptotic cell reaches and the apoptosis of BCC cell is removed the cyst formation that causes and the frequency that enlarges increases greatly.2E: the high power field of the outer peripheral areas of same residual BCC among Fig. 2 C shows apoptotic cell and removes the cystic structures that causes by the apoptosis of BCC cell to form frequency and increase greatly.2F: the high power field of the BCC interior zone of placebo treatment has shown typical tumor cell and acellular apoptosis.The initial amplification of 2A, 2B, 2C is 100 *, the initial amplification of 2D, 2E, 2F is 1000 *.

Fig. 3 A, 3B, 3C, 3D, 3E, 3F, 3G: the reduction that p53 expressed among BCC ' s after the BCC ' s immunohistochemical analysis of 11-Deoxojervine and placebo treatment was presented at the differentiation of all residual BCC cells under the influence of 11-Deoxojervine and is exposed to 11-Deoxojervine.3A and 3B: with the Ber-Ep4 of the BCC of placebo treatment dye by force (3B) form contrast, all residual cells of 11-Deoxojervine treatments B CC do not have monoclonal antibody Ber-Ep4 dyeing (3A), show all residual cells of handling through 11-Deoxojervine all break up to or surpass the step that can Ber-Ep4 detects.The known differentiation marker thing of Ber-Ep4, can dye BCC cell and undifferentiated normal epidermis basal layer cell and hair follicle, but the last confluent monolayer cells of the normal epidermis of can not labelling having broken up.3C: with the heterogeneous labelling of Ulex Europaeus lectin I to residual cell among the BCC ' s of 11-Deoxojervine processing, shown that some BCC cells break up to can be by the detected step of this agglutinin always, this lectin is the basal layer cell of labelling BCC ' s or normal epidermis not normally, but the last confluent monolayer cells of labelling differentiation.3D and 3E: compare with the BCC ' s (3E) of placebo treatment, BCC ' s (3D) that 11-Deoxojervine is handled detects the reduction that p53 expresses with monoclonal antibody DO-7.When epithelium basal cell or keratinocyte differentiation phase, known p53 expresses and will reduce.As everyone knows, after being exposed to the DNA damage medicine, can detect the p53 that detects with DO-7 in cell increases.3F and 3G:BCC ' s concordance in substrate is shunk, it is reported that it is the feature that the tumor cell proliferation inhibition is associated, be found in 11-Deoxojervine and handle tumor (3F, arrow has shown between the shrinking zone), and invisible in the tumor of placebo treatment group (3G) (BCC ' s of 11-Deoxojervine and placebo treatment also can be referring to 3D, 2C and 3B, 3E from difference that substrate is shunk).The initial amplification of 3A, 3B, 3D, 3E is 400 *, 3C is 1000 *, and 3F, 3G be 100 *.All SABC are labeled as the link coupled streptavidin of peroxidase, and this streptavidin is in conjunction with biotinylated secondary antibody; Be labeled as brown.Section shown in 3F and the 3G is with periodic acid-schiff's reaction and the blue dyeing of Ai Xi.

Fig. 4 A, 4B, 4C, 4D: the Ber-Ep4 labelling normal mode of the normal skin that 11-Deoxojervine is handled, this pattern show, although with the undifferentiated cell of normal epidermis and hair follicle to be exposed to 11-Deoxojervine with same scheme and the dosage of BCC ' s, still keep intact.4A: the Ber-Ep4 labelling of cyclopamine-treated epidermis basal layer cell.4B and 4C: the high power field of the zones of different of cyclopamine-treated epidermis shows the Ber-Ep4 labelling of basal cell.4D: the high power field of the hair follicle of handling with 11-Deoxojervine still shows the normal labeled of Ber-Ep4.The initial amplification of 4A is 400 *, 4B, 4C, 4D be 1000 *.Method for immunohistochemical detection is with Fig. 3 A, 3B; It is brown that labelling shows.

Table 1: the differentiation and the apoptosis of the inductive basal cell cancerous cell of 11-Deoxojervine Local treatment

Shown mean+SD comes from high power (1000 *) visual field of 16 every group of tumor tissue sections selecting at random at least.To going along with sb. to guard him parameters peripheral and non-(inside) tumor region of going along with sb. to guard him, placebo and 11-Deoxojervine processing tumor, p＜0.001.

Same accompanying drawing (Figure 1A, 1B, 1C, 1D on the 12nd page, Fig. 2 A, 2B, 2C, 2D, 2E, 2F, Fig. 3 A, 3B, 3C, 3D, 3E, 3F, 3G, Fig. 4 A, 4B, 4C, 4D) coloured picture, its page number is augmented at this and is 12a, based on the data of SABC method and the character of data, coloured picture is better than grey figure transmission information; We wish that sincerely Patent Office can consider that these facts are with the part of these several pages of reservations as present patent application.Yet,, also can from patent application, remove 12a if Patent Office considers there is no need.

Claims (12)

1. 11-Deoxojervine or the purposes of its officinal salt in the topical therapeutic of basal cell carcinoma.

2. 11-Deoxojervine or its officinal salt are in the purposes of the preparation of the pharmaceutical compositions that is used for the topical therapeutic basal cell carcinoma.

3. the approach injected in comprising with tumor with non local approach of 11-Deoxojervine or its officinal salt is used for the purposes of the treatment of basal cell carcinoma.

4. 11-Deoxojervine or its officinal salt are in the purposes of the pharmaceutical compositions preparation that is used for the topical therapeutic basal cell carcinoma, and this treatment comprises that with non local approach the injection approach realizes in the tumor.

5. 11-Deoxojervine or the purposes of its officinal salt or derivatives thereof in the topical therapeutic basal cell carcinoma.

6. 11-Deoxojervine or its officinal salt or derivatives thereof are in the purposes of the preparation of the pharmaceutical compositions that is used for the topical therapeutic basal cell carcinoma.

7. the approach injected in comprising with tumor with non local approach of 11-Deoxojervine or its officinal salt or derivatives thereof is used for the purposes of the treatment of basal cell carcinoma.

8. 11-Deoxojervine or its officinal salt or derivatives thereof are in the purposes of the pharmaceutical compositions preparation that is used for the topical therapeutic basal cell carcinoma, and this treatment comprises that with non local approach the injection approach realizes in the tumor.

9. 11-Deoxojervine or its officinal salt are being used for the purposes of oncotherapy, and such tumor is utilized the hedgehog/smoothened signal transduction pathway to breed and/or prevented apoptosis or cell differentiation.

10. 11-Deoxojervine or its officinal salt purposes in the pharmaceutical compositions preparation that is used for the treatment of tumor, such tumor are utilized the hedgehog/smoothened signal transduction pathway to breed and/or are prevented apoptosis or cell differentiation.

11. 11-Deoxojervine or its officinal salt or derivatives thereof are being used for the purposes of oncotherapy, such tumor is utilized the hedgehog/smoothened signal transduction pathway to breed and/or is prevented apoptosis or cell differentiation.

12. 11-Deoxojervine or its officinal salt or derivatives thereof purposes in the pharmaceutical compositions preparation that is used for the treatment of tumor, such tumor are utilized the hedgehog/smoothened signal transduction pathway to breed and/or are prevented apoptosis or cell differentiation.

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