Title of Invention

"A PROCESS FOR THE PREPARATION OF A PHARMACEUTICAL PRODUCT"

Abstract

In the present invention there is provided a process for the preparation of a pharmaceutical product useful for decrease of size or disappearance of a tumor upon administration to a patient having a tumor that employs Hedgehog/Smoothened signalling for inhibition of tumor cell differentiation and apoptosis comprising mixing an effective amount of cyclopamine as herein with a solvent, base cream, ointment, gel or liposomes

Full Text

This invention concerns the use of cyclopamine in vivo on basal cell carcinomas (BCC's) to achieve therapeutic effect by causing differentiation of the tumor cells and, at the same time, apoptotic death and removal of these tumor cells while preserving the normal tissue cells, including the undifferentiated cells of the normal epidermal basal layer and hair follicles. Causation of apoptosis by cyclopamine is by a non-genotoxic mechanism and thus unlike the radiation therapy and most of the currently used cancer chemotherapeutics which act by causing DNA-damage. These novel effects, previously unachieved by a cancer chemotherapeutic, make the use of cyclopamine highly desirable in cancer therapy, in the treatment of BCC's and other tumors that use the hedgehog/smoothened signal transduction pathway for proliferation and prevention of apoptosis. Basal cell carcinoma Is a common epithelial tumor. Its incidence Increases with increasing age. Current treatments for BCC's include the surgical excision of the tumor together with a margin of normal tissue and, when surgery is not feasible or desirable, destruction of the tumor cells by ionizing radiation or other means. Although scarring and disfigurement are potential side effects, surgical excisions that do not leave neoplastic cells behind can provide cure. Radiation therapy acts by causing irreparably high quantity of DNA-damage which, in turn, triggers apoptotic death of the tumor cells. This mode of action of radiation-therapy, i.e. apoptosis secondary to DNA-damage, is similar to those of many chemotherapeutic agents that are currently used in the treatment of cancers. However both radiation therapy and the cytotoxic cancer chemotherapeutics are capable of causing DNA-damage in the normal cells of patients in addition to the tumor cells. As a result their effectivity and usefulness in cancer therapy are seriously limited. A further dilemma with the use of radiation and genotoxic cancer chemotherapeutics is the disturbing fact that, even when cure of the primary tumor is achieved, patients have markedly increased risk of developing new cancers because of the DNA-damage and the resulting mutations they have undergone during the treatment of primary tumor. Induction of apoptosis selectively in tumor cells by non-genotoxic means would therefore be most desirable in the field of cancer therapy. BCC's frequently show inactivating mutations of the gene patched which encodes a transmembrane protein acting as a receptor for the hedgehog proteins identified first by their effect on the patterning of tissues during development When not liganded by hedgehog, the patched protein acts to Inhibit intracellular signal transduction by another transmembrane protein, smoothened. Binding of hedgehog to the patched causes relieving of this inhibition. Intracellular signal transduction by the relieved smootheened . then initiates a series of cellular events resulting ultimately in alterations of the expressions of the hedgehog target genes and of cellular behaviour. General features of this hedgehog/smoothened pathway of signal transduction, first Identified in Drosophila, are conserved in diverse living organisms from Drosophilato Human. However the pathway gets more complex in more advanced organisms (eg. presence in human of more than one genes that display significant similarity to the single patched gene of Drosophila). Inactivating mutations of the patched have been found to cause constitutive (ligand-free) signalling through the hedgehog/smooth&iedpathway. The hedgehog/smoothenedpsShNa/overada^, resulting from mutations of the patched and/or further downstream pathway elements, is found in all BCCs. The nevoid basal cell carcinoma syndrome (NBCCS) results from patched haploinsufficiency. Patients with the NBCCS, because of an already mutant patched in all cells, develop multiple BCC's as they grow older. STATEMENT OF THE INVENTION According to the present invention there is provided a process for the preparation of a pharmaceutical product useful for decrease of size or disappearance of a tumor that employs Hedgehog/Smoothened signalling for inhibition of tumor cell differentiation and apoptosis comprising mixing an effective amount of cyclopamine as herein with a solvent, base cream, ointment, gel or liposomes Cyclopamine, a steroid alkaloid, has the chemical formula shown below. (Formula Removed) It is found naturally in the lily Veratrum califomicum and can be obtained by purification from this.and other sources. Inhibition of the hedgehog/smoothened pathway by cyclopamine has been found in chicken embryos and in cultured cells of mouse. For topical applications, cyclopamine can be dissolved in ethanol or another suitable solvent and mixed with a suitable base cream, ointment or gel. Cyclopamine may also be entrapped in hydrogels or in other pharmaceutical forms enabling controlled release and may be adsorbed onto dermal patches. The effects shown in figures Fig 1Ato Fig 1D, Fig 2A to Fig 2F, Fig 3Ato Fig 3G and Fig 4Ato Fig 4D have been obtained by a cream preparation obtained by mixing a solution of cyclopamine in ethanol with a base cream so as to get a final concentration of 18 mM cyclopamine In cream. The base cream used is made predominantly of heavy paraffin oil( 10% w/w), vaseline (10% w/w), stearyl alcohol (8% w/w), polyoxylsteareth-40 (3% w/w) and water (68% w/w) but another suitably formulated base cream is also possible. Optimal concentration of cyclopamine in a pharmaceutical form as well as the optimal dosing and application schedules can obviously be affected by such factors as the particular pharmaceutical form, the localisation and characteristics of the skin containing the tumor (e.g. thickness of the epidermis) and the tumor size; however these can be determined by following well known published optimisation methods. The dosing and the application schedules followed for the tumors shown in Fig 1A (BCC on the nasolabial fold, - 4x5 mm on surface) and Fig 1C (BCC on the forehead, ~ 4x4 mm on surface) are as follows: 10 ± 2 µl cream applied directly onto the BCC's with the aid of a steel spatula four times per day starting ~ 9.00 a.m. with ~ 3% hours in between. Night-time applications, avoided in this schedule because of possible loss of cream from the patient skin to linens during sleep, can be performed by suitable dermal patches. Preservation of the undifferentiated cells in the normal epidermis and in hair follicles following exposure to cyclopamine, as described in this invention, provide information about the tolerable doses in other possible modes of administration as well; e.g. direct intratumoral injection of an aqueous solution or systemic administration of the same or of cyclopamine entrapped in liposomes. Fig 1 A, Fig 1B, Fig 1C and Fig 1D show rapid clinical regressions of the BCC's following exposure to cyclopamine. Besides the visual disappearance of several tumor areas within less than a week of cyclopamine exposure, there is a loss in the typically translucent appearance of the BCC's as seen by the comparison of Fig 1B to Fig 1A and of Fig 1D to Fig 1C. Fig 2A to Fig 2F show microscopic appearences of the tumor areas subjected to surgical excisions together with a margin of normal tissue on the fifth and sixth days of cyclopamine applications when the BCC's had lost most of their pre-treatment areas but still possessed few regions that, although markedly decreased in height, had not yet completely disappeared and therefore had residua] tumor cells for microscopic analyses. Fig 2A and Fig 2B show, on tissue sections, the skin areas corresponding to the visually disappeared tumor nodules. The tumors are seen to have disappeared to leave behind large cystic structures containing little material Inside and no detectable tumor cells. Fig 2C shows microscopic appearance of a skin area that contained still visible BCC in vivo. These regions are seen to contain residual BCC's displaying large cysts in the tumor center and smaller cystic structures of various sizes located among the residual BCC cells towards the periphery. Fig 20 and Fig 2E show 1000X magnified appearances from the interior and palisading peripheral regions of these residual BCC's and show the presence of massive apoptotic activity among the residual BCC ceils regardless of the tumor region. These high magnifications show greatly increased frequency of the BCC cells displaying apoptotic morphology and formation of the cystic structures by the apoptotic removal of cells as exemplified on Fig 20 by the imminent joining together of the three smaller cysts into a larger one upon removal of the apoptotic septal cells. Fig 2F shows that the BCCs treated with the placebo cream (i.e. the cream preparation identical to the cyclopamine cream except for the absence of cyclopamine in placebo) show, by contrast, the typical neoplastic BCC cells and no detectable apoptotic activity. Cells undergoing apoptosis are known to be removed by macrophages and by nearby cells in normal tissues and the quantitation of apoptotic activity by morphological criteria on hemaloxyiene-eosine stained sections is known to provide an underestimate. Despite these, the quantitative data shown in Table 1 show greatly increased apoptotic activity caused by cyclopamine among the residual BCC cells. The loss of translucency In the cyclopamine-treated BCC's raises the intriguing possibility of differentiation of BCC's under the influence of cyclopamine. This possibility, which can be tested by immunoNstochemical analyses of the BCC's, is found to be the case in this invention. In norma! epidermis, differentiation of basal layer cells to the upper layer cells is accompanied by a loss of labelling with the monoclonal antibody Ber-Ep4. Ber-Ep4 labels also the BCC celts and is a known marker for these neoplasms. Fig 3A. Fig 3B and the quantitative data on Table I show that, white Ber-Ep4 strongly labels all peripheral palisading cells and over 90% of the interior cells of the placebo-treated BCCs, none of the residual peripheral or interior cells of the cyclopamine-treated BCCs are labelled by Ber-Ep4. Differentiation of the BCCs under the influence of cyclopamine, hitherto unknown by any other means and highly unusual because of achievement of it in vivo and in all cells by immunohistochemical criteria, has independent value in the treatment of cancer. Another differentiation marker, Ulex Europaeus lectin type 1, normally does not label the BCCs or the basal layer cells of normal epidermis but labels the differentiated upper layer cells. Fig 3C, showing the heterogenous labelling of the residual cells of cyclopamine-treated BCCs with this lectin, shows differentiation of some of the BCC cells beyond the differentiation step detected by Ber-Ep4 all the way to the step detected by Ulex Europaeus lectin type 1. The p53 is a master regulator of the cellular response to DNA-damage. Amount of mis protein is known to increase in the cell nucleus following exposure of cells to genotoxic agents. When the DNA-damage is increased beyond a threshold, p53 serves for the apoptotic death of cells. Radiation therapy of cancer and the genotoxic cancer chemotherapeutics that are currently common, act largely by this mechanism, i.e. by causation of apoptosis secondary to the damaging of DNA. The monoclonal antibody DO-7 can bind both normal and missense mutant (i.e., nonfunctional) forms of p53 and Is known to be capable of detecting the increase of p53 in the cells following exposure to DNA-damaging agents. Fig 3D, Fig 3E and the quantitative data in Table I show that both the DO-7 labelling intensity and the frequency of labelled cells are markedly decreased in cyclopamine-treated BCCs in comparison to the placebo-treated BCCs. Thus cyclopamine causes, not an increase, but rather a decrease of p53 in the nuclei of cyclopamine-treated BCC cells. Since expression of p53 is known to decrease in epidermal cells upon differentiation, the decreased DO-7 labelling of the cyclopamine-treated BCCs is likely to be secondary to the cyclopamine-induced differentiation of the BCC cells. In any case, massive apoptotic activity in the cyclopamine-treated BCCs despite markedly decreased p53 expression means that the cyclopamine-induced apoptosis of these tumor cells is by a non-genotoxic mechanism. Arrest of the proliferation of BCCs is known to be associated with their retraction from stroma. Although retraction from stroma can also be caused artefactuaUy by improper fixation and processing of the tissues, adherence to published technical details ensures avoidance of such artefacts. As shown in Fig 3F and Fig 3G, cyctopamine-treated, but not placebo-treated, BCCs are consistently retracted from stroma. Exposure of BCCs to cyclopamine thus appears to be associated also with an arrest of proliferation. Fig 4A to Fig 4D show Ber-Ep4 labelling of the normal skin tissue found on and around the cyctopamine-treated BCCs. Different epidermal areas that were treated with cyclopamine are seen in Fig 4A, Fig 4B and Fig 4C to display normal pattern of labelling with Ber-Ep4, i.e. labelling of the basal layer cells. Similarly Fig 4D shows normal Ber-Ep4 labelling of a hair follicle exposed to cyclopamine. Thus the undifferentiated cells of normal epidermis and of hair follicles are preserved despite being exposed to the same schedule and doses of cyclopamine as the BCCs. Causation of highly efficient differentiation and apoptosis of the tumor cells in vivo by cyclopamine at doses that preserve the undifferentiated tissue cells are hitherto unknown achievements that, together with the non-genotoxic mode of action of cyclopamine, support the use of cyclopamine not only on BCCs but also on those internal tumors that utilize the hedgehog/smoothened pathway for proliferation and for prevention of apoptosis and/or differentiation. escription Of The Figures Fig 1A, 1B, 1C, 1D: Rapid regressions of the cydopamine-treated BCC's as indicated by disappeared turner regions (exemplified by arrows), markedly decreased height from skin surface and by a loss of translucency in less than a week. 1A: BCC, located on left nasolabial fold, prior to treatment 1B: Same BCC on the fifth day of topical cyctopamine treatment. 1C: BCC, located on forehead, prior to tretment. 1D: Same BCC on the sixth day of topical cyclopamine treatment Fig 2Ar 2B, 2C, 2D, 2E, 2F: Microscopic appearences of the cyclopamine- and placebo-treated BCC's, showing the cyclopamine-induced massive apoptotic death and removal of the tumor cells and the disappearance of tumor nodules to leave behind cystic spaces with no tumor cells. Skin areas corresponding to the pre-treatment positions of the BCCs were excised surgically on the fifth and sixth days of cyclopamine exposure with a margin of normal tissue and subjected to conventional fixation, sectioning and hematoxyfene-eosine staining for microscopic analyses. 2A: Large cyst in the dermis corresponding to the position of a disappeared tumor nodule showing no residual tumor cells. 2B: Similar cysts in another dermal area that contained BCC prior to, but not after, treatment with cyclopamine. 2C: Low power view of an area of the BCC shown on Fig 1D showing residual cells and formation of a large cyst by the joining together of the numerous smaller cysts in between these cells. 20: High power view from an interior region of the same residual BCC as in Fig 2C showing greatly increased frequency of ' the apoptotic cells and the formation as well as enlargement of the cysts by the apoptotic removal of the BCC celts. 2E: High power view from from a peripheral region of the same residual BCC as in Fig 2C also showing greatly increased frequency of the apoptotic celfs and the formation of cysts by the apoptotic removal of BCC cells. 2F: High power view from an internal area of a placebo-treated BCC showing typical neoplastic cells of this tumor and the absence of apoptosis. Original magnifications are 100X for2A, 2B, 2C and 100DXfor2D, 2E, 2F. Fig 3A, 3B, 3C, 3D, 3E, 3F, 3G: Immunohistochemical analyses of the cyclopamine- and placebo-treated BCC's showing differentiation of all residual BCC cells under the influence of cyclopamine and the decrease of p53 expression in BCC's following exposure to cyclopamina 3A and 3B: Absence of staining with the monodonal antibodyBer-Ep4 In all residual cells of cydopamine-treated BCC (3A) contrasted with the strong staining in placebo-treated BCC (3B) showing that all residual cells in the cydopamine-treated BCCs are differentiated to or beyond a step detected by Ber-Ep4. Ber-Ep4 is a known differentiation marker that stains the BCC cells as well as the undifferentiated cells of the normal epidermis basal layer and of hair follicles but not the differentiated upper layer cells of normal epidermis. 3C: Heterogenous labelling of the residual cells of a cydopamine-treated BCC with the Ulex Europaeus lectin type 1 showing differentiation of some of the BCC cells all the way to the step detected by this lectin which normally does not label the BCCs or the basal layer cells of the normal epidermis but labels the differentiated upper layer cells. 3Dand3E: Decreased expression of p53 as detected by the monoclonal antibody DO-7in cydopamine-treated BCCs (3D) in comparison to the placebo-treated BCCs (3E). Expression of p53 is known to decrease upon differentiation of the epidermal basal cells and upon differentiation of cultured keratinocytes. it is also well known that the amount of p53, detectable by DO-7, increases in cells when they are exposed to DMA-damaging agents. 3F and 3G: Consistent retraction of BCCs from stroma, which is a feature known to be associated with the arrest of tumor cell proliferation, seen in cydopamine-treated (3F, arrow shows the retraction space) but not in placebo-treated (3G) tumors (difference of the cydopamine- and placebo-treated BCCs in terms of retraction from stroma is seen also in 3D, 2C vs 3B, 3E). Original magnifications are 40QX for 3A, 3B, 3D, 3E, 100OX for 3C and 100X for 3F, 3G. All immunohistochemicat labellings are with peroxidase-conjugated streptavidin binding to biotinyiated secondary antibody; labelling is indicated by the brown-colored staining. Sections shown in 3F and 3G are stained with Periodic Atid-Schiff and Alcian blue. Rg4A 4B, 4C, 40: Normal pattern of labelling of the cydopamine-treated normal skin with Ber-Ep4 showing that the undifferentiated cells of normal epidermis and of hair fdlides are preserved despite being exposed to the same schedule and doses of cydopamine as the BCCs. 4A: Ber-Ep4 labelling of the basal layer cells of the epidermis treated with cydopamine. 48 and 4C: Higher power views from different areas of cydopamine-treated epidermis showing Ber-Ep4 labelling of the basal cells. 4D: High power view of a hair follicle treated with cydopamine yet showing normal labelling with Ber-Ep4. Original magnification is 400X for 4A and 1000X for 4B, 4C, 4D. Immunohistochemical detection procedure is the same as in Fig 3A, 3B; labelling is indicated by brown coloring. Table 1: Induction of the Differentiation and Apoptosis of Basal Celt Carcinoma Cells by Topical Cyclopamine (Table Removed) Means ± standart deviations from at least 16 randomly selected high-power (1000 X) fields of the tissue sections of each tumor group are shown, p Color prints of the same figures as on page 12 (Fig 1A, 1B, 1C, 1D, Fig 2A, 2B, 2C, 2D, 2E, 2F, Fig 3A, 3B, 3C, 3D, 3E, 3F, 3G, Fig AA, 4B, AC, AD), added as page 12a because the Immunohistochemical data and findings, due to their nature, can be conveyed best in color rather than in gray-scale; we respectfully request consideration of this fact by the Patent Authority and the keeping of page 12a as part of this patent application. However the page 12a may be removed from the patent application if it is deemed necessary by the Patent Authority. I CLAIM :- 1 A process for the preparation of a pharmaceutical product useful for decrease of size or disappearance of a tumor that employs Hedgehog/Smoothened signalling for inhibition of tumor cell differentiation and apoptosis comprising mixing an effective amount of cyclopamine as herein described with a solvent, base cream, ointment, gel or liposomes. 2. The process as claimed in claim 1, wherein the said pharmaceutical product is formulated for topical or systemic administration. 3 The process as claimed in claim 2, wherein the said product for systemic administration is in the form of an aqueous solution. 4 The process as claimed in any one of the preceding claims, wherein the said product is in a form enabling controlled release. 5. The process as claimed in any one of the preceding claims, wherein the product is capable of being adsorbed onto a dermal patch. 6. A process for preparation of a pharmaceutical product, substantially as hereinbefore described with reference to any of the accompanying drawings.

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