Compositions comprising cytidine analogs and uses and methods thereof

Abstract

The present invention provides a composition comprising a cytidine analogue, in particular decitabine or azacitidine. In particular, the present invention provides compositions comprising cytidine analogs, in particular decitabine or azacitidine, which are useful for topical administration. The invention also provides the use of such a composition for keratinized / dermatological and non-keratinized / dermatological medical conditions, such as for the treatment of human papilloma virus (HPV)-related precancerous conditions.

Description

[0001] The present invention relates to the field of compositions comprising cytidine analogs, in particular decitabine or azacitidine. In particular, the present invention relates to compositions comprising cytidine analogs, in particular decitabine or azacitidine, and packages comprising the compositions, which are useful for topical administration. The present invention also relates to the use of such compositions for the treatment of precancerous conditions associated with human papillomavirus (HPV).

[0002] Oncogenic types of human papillomavirus (HPV) can cause a variety of precancerous symptoms in the anogenital region as well as invasive tumors in the anogenital region and the head and neck region. The development and maintenance of the malignant phenotype in HPV-related tumors is essentially dependent on the sustained expression of HPV E6 and HPV E7 oncoproteins (von Knebel Doeberitz M, Oltersdorf T, Schwarz E, Gissmann L. Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells. Cancer Res. 1988 Jul 1; 48(13): 3780-6; reviewed in zur Hausen H. Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancers. Cancer Res. 1989 Sep 1; 49(17): 4677-81). Previous studies have shown that certain epigenetic modifications of the HPV genome itself, particularly hypermethylation within the upstream regulatory region (URR) of the virus, can trigger dysregulated expression of the HPV E6 / HPV E7 oncoprotein (Vinokurova S, von Knebel Doeberitz M. Differential methylation of the HPV 16 upstream regulatory region during epithelial differentiation and neoplastic transformation. PLoS One. 2011; 6(9): e24451. doi: 10.1371 / journal.pone.0024451). Based on this, it has been hypothesized that treatment of HPV-transformed cells with demethylating agents such as decitabine (5-aza-2'-deoxycytidine, DAC) can eliminate the abnormal methylation pattern in the affected cells and subsequently downregulate the expression of the HPV E6 / HPV E7 oncoprotein to regress the malignant phenotype.This hypothesis has been confirmed in vitro using a variety of HPV-transformed cell lines (Stich M, Ganss L, Puschhof J, Prigge ES, Reuschenbach M, Guiterrez A, Vinokurova S, von Knebel Doeberitz M. 5-aza-2'-deoxycytidine (DAC) treatment downregulates the HPV E6 and E7 oncogene expression and blocks neoplastic growth of HPV-associated cancer cells. Oncotarget. 2016 Jul 16; 8(32): 52104-52117).

[0003] In the prior art, medicines comprising decitabine are usually developed for injection or infusion, particularly for intravenous administration. For example, US2003 / 229047 A1 describes a liquid preparation comprising decitabine, wherein decitabine is dissolved in a solvent comprising glycerol, propylene glycol, polyethylene glycol or a combination thereof. However, the composition in US2003 / 229047A1 is not only a liquid, but it also seems to be unstable. Similarly, the preparation disclosed in WO 2006 / 071491A1 obviously does not meet the stability requirements of decitabine. In addition, it comprises at least 60% water, and is therefore also a liquid. Although WO 2014 / 064717 A1 has paid attention to the problem of stability, the decitabine composition disclosed therein is also formulated for intravenous administration, and is therefore a liquid preparation. Similarly, the lipid nanoparticles described as carriers in WO 2012 / 073125 are developed for intravenous administration, particularly for the treatment of thyroid cancer.

[0004] However, systemic administration of drugs containing demethylating agents such as decitabine or azacitidine carries the risk of causing systemic side effects because the delivery and uptake of the drug are not restricted to a specific target site.

[0005] In view of the above, the object of the present invention is to overcome the shortcomings of the prior art and provide a composition comprising a cytidine analog, specifically decitabine or azacitidine, which is useful for local administration and / or local efficacy. Local administration is conducive to achieving local (local / topical) effects, such as local effects on transformed cells of HPV-related precancerous symptoms, while minimizing the risk of systemic drug uptake and related side effects. However, the decitabine preparations of the above-mentioned prior art are mainly developed for injection or infusion, in particular for intravenous administration. Therefore, the decitabine preparations of the prior art are generally liquid compositions, which are conducive to intravenous administration, but are not conducive to local administration (local / topical administration).

[0006] In addition, it is well known that dissolved decitabine exhibits significant instability under a variety of conditions, including oxidative or hydrolytic degradation in alkaline and acidic pH ranges (Rogstad DK, Herring JL, Theruvathu JA, Burdzy A, Perry CC, Neidigh JW, Sowers LC. Chemical decomposition of 5-aza-2'-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry. Chem Res Toxicol. 2009 Jun; 22 (6): 1194-204). In addition, HPV-related precancerous symptoms mainly occur in keratinized or non-keratinized epithelium in the anogenital area and are often associated with local irritation such as itching and pain. In view of this, the present invention also provides a composition comprising a cytidine analog, particularly decitabine or azacitidine, wherein the cytidine analog, particularly decitabine or azacitidine, is stably formulated. Furthermore, it is an object of the present invention to provide a composition comprising a cytidine analog, in particular decitabine or azacitidine, which is formulated so that the cytidine analog, in particular decitabine or azacitidine, can penetrate into the lower layers of the epithelium after topical application. Furthermore, it is an object of the present invention to provide a composition comprising a cytidine analog, in particular decitabine or azacitidine, and a pharmaceutically acceptable carrier or vehicle or diluent, which enables the stable preparation of the active agent, i.e. the cytidine analog, in particular decitabine or azacitidine, over a wide range of final concentrations. It is also an object of the present invention to provide a composition comprising a cytidine analog, in particular decitabine or azacitidine, which exhibits good consistency for topical application, in particular a consistency that allows the product to be applied evenly for comfortable and non-irritating application to epithelial lesions.

[0007] These objects are achieved by what is described below and in the appended claims.

[0008] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as they may vary. It should also be understood that the terms used herein are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art.

[0009] Hereinafter, the key element of the present invention will be described.These key elements are listed with specific embodiments, but it should be understood that they can be combined in any manner and any number, thereby creating additional embodiments.The embodiments and preferred embodiments with multiple descriptions should not be construed as limiting the present invention to only the embodiments clearly described.This description should be understood as supporting and comprising the embodiments that the embodiments clearly described are combined with any number of open elements and / or preferred elements.In addition, any arrangement and any combination of all key elements described in this application should be considered as disclosed by the description of this application, unless the context otherwise indicates.

[0010] Throughout this specification and the claims that follow, unless the context requires otherwise, the terms "comprises", "comprising" and variations thereof will be understood to imply the inclusion of the stated components, integers or steps, but not the exclusion of any other unstated components, integers or steps. The term "consisting of" is a specific embodiment of the term "comprising", in which any other unstated components, integers or steps are excluded. In the context of the present invention, the term "comprising" encompasses the term "consisting of". Thus, the term "comprising" encompasses "containing" and "consisting of", for example, a composition "comprising" X may consist of only X, or may include some additional components such as X+Y.

[0011] In the context of describing the present invention (particularly in the context of the claims), the articles used are understood to cover the singular and the plural, unless otherwise specified herein or the context is clearly contradictory. The enumeration of numerical ranges herein is intended only as a shorthand method of referring to each individual value falling within the range individually. Unless otherwise specified herein, each individual value is incorporated into the specification as if it were quoted separately in this article. Any language in the specification should not be understood to indicate that any unclaimed element is essential to the implementation of the present invention.

[0012] The word "substantially" does not exclude "completely", for example a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0013] The term "about" in relation to a numerical value x refers to x ± 10%.

[0014] Composition

[0015] In a first aspect, the present invention provides a pharmaceutical composition comprising:

[0016] - decitabine or azacitidine, or a pharmaceutically acceptable salt thereof;

[0017] - polyethylene glycol (PEG); and

[0018] -Dimethyl sulfoxide (DMSO).

[0019] The pharmaceutical composition is suitable for topical application. For this reason, the pharmaceutical composition is usually semisolid at room temperature, such as about 25°C. Therefore, the composition can be easily and evenly applied topically to a specific target site, such as a lesion, and it is substantially retained in the area where it is applied (rather than flowing down). In addition, for topical application, the composition is preferably deformable and / or easy to apply at room temperature, such as about 25°C. Therefore, it can be easily and comfortably applied, and easily removed from the tube / primary packaging, while avoiding stimulation or pain during application. Therefore, preferably, the composition is not a liquid composition (such as a composition developed for injection or intravenous administration, etc.). On the contrary, compared with a liquid composition (such as a liquid composition for injection), the composition of the present invention for topical application has a higher viscosity, which is to ensure specific targeting, easy application (such as with fingers) and uniform application to the affected area during topical application. Preferably, the composition is semisolid at room temperature.

[0020] Therefore, on the one hand, the viscosity of the composition should be high enough to substantially prevent the composition from flowing from the applied area to or accidentally smearing other areas. On the other hand, the viscosity of the composition should be low enough to enable the patient to easily take out the desired dose of the composition from the container (e.g., tube, etc.) in which the composition is available, so as to evenly apply the composition to the affected area, thereby ensuring uniform application of the active component and avoiding or minimizing friction on the application site, which can cause local irritation and / or local pain. In order to obtain a viscosity suitable for topical application (especially the semi-solid state of the composition), a thixotropic agent as a viscosity increasing agent is added so that the composition is generally in the form of an ointment. The thixotropic agent has the advantage of being easy to apply, and when standing, such as after application, the viscosity increases, so that the composition generally does not flow from the affected area of ​​the skin to which it is applied or accidentally smeared to the unaffected area. In the composition of the present invention, polyethylene glycol (PEG) is used as a thixotropic agent. It should be understood that for pharmaceutical compositions, the PEG (and any other components) contained in the composition are pharmaceutical grade compounds, i.e., they have a chemical purity acceptable for pharmaceutical use.

[0021] In the composition of the present invention, the pharmaceutically active ingredient is typically decitabine or azacitidine (or its salt). In some embodiments, the composition does not contain other active ingredients, i.e., active ingredients other than decitabine or azacitidine (or its salt). In other embodiments, the composition may contain additional active ingredients, i.e., active ingredients other than decitabine or azacitidine (or its salt). However, PEG, DMSO and other excipients, solvents, diluents or carriers are generally not considered to be active pharmaceutical ingredients.

[0022] Decitabine and azacitidine are chemical analogs of the nucleoside cytidine (cytidine analogs). Decitabine is the deoxy derivative of azacitidine.

[0023] Decitabine, also known as 5-aza-2'-deoxycytidine, is represented by the following formula (1):

[0024]

[0025] Azacitidine, also known as 5-azacytidine, radakamycin or 4-amino-1-β-D-ribofuranosyl-s-triazine-2(1H)-one, is shown in the following formula (2):

[0026]

[0027] Decitabine or azacitidine can be in the form of a (preferably pharmaceutically acceptable) salt. Examples thereof include, but are not limited to, pharmaceutically acceptable salts prepared from inorganic or organic acids. Examples of such inorganic acids include, but are not limited to, hydrochloric acid and hydrobromic acid. Examples of such organic acids include, but are not limited to, aliphatic organic acids, alicyclic organic acids, aromatic organic acids, aromatic aliphatic organic acids, heterocyclic organic acids, carboxylic acids and sulfonic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, maleic acid, pamoic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, pantothenic acid, benzenesulfonic acid, toluenesulfonic acid, sulfanilic acid, methanesulfonic acid, cyclohexylaminosulfonic acid, stearic acid, alginic acid, [β]-hydroxybutyric acid, malonic acid, galactonic acid (galactic acid) and galacturonic acid. In some embodiments, the acid is selected from hydrochloric acid, L-lactic acid, acetic acid, phosphoric acid, (+)-L-tartaric acid, citric acid, propionic acid, butyric acid, caproic acid, L-aspartic acid, L-glutamic acid, succinic acid, EDTA, maleic acid, methanesulfonic acid, hydrochloric acid, hydrobromic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, phosphorous acid, perchloric acid, chloric acid, chlorous acid, carboxylic acid, sulfonic acid, ascorbic acid, carbonic acid and fumaric acid. The sulfonic acid can be selected from ethanesulfonic acid, 2-hydroxyethanesulfonic acid and toluenesulfonic acid.

[0028] Preferably, the pharmaceutical composition comprises decitabine or azacitidine (or its salt) at a concentration of 0.005mM to 100mM, more preferably at a concentration of 0.01mM to 50mM, even more preferably at a concentration of 0.05mM to 25mM, and still more preferably at a concentration of 0.1mM to 10mM (0.0228mg / g to 2.28mg / g). As shown in the attached examples, in an HPV-transformed precancerous tumor model, topical administration of the composition of the present invention containing this dose of decitabine or azacitidine resulted in dose-dependent tumor cell degradation.

[0029] In another aspect, the present invention also provides a pharmaceutical composition for medical use comprising 0.1 mM to 10 mM (0.0228 mg / g to 2.28 mg / g) of decitabine or azacitidine or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is topically administered.

[0030] Such pharmaceutical compositions for use as described herein preferably comprise polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO).Thus, the description of the pharmaceutical composition provided herein also applies to the pharmaceutical composition for use, and vice versa.

[0031] As described above, for topical application, on the one hand, the viscosity of the composition should be high enough to substantially prevent the composition from flowing from the area to which it is applied or being inadvertently smeared onto other areas, while on the other hand, the viscosity of the composition should be low enough to enable the patient to easily take out the desired dose of the composition from the container (e.g., tube, etc.) in which the composition is available to apply the composition evenly to the affected area, thereby ensuring uniform application of the active ingredient and avoiding or minimizing friction of the application site, such as the anogenital area, which may cause local irritation and / or local pain. The viscosity of the product is characterized by its complex viscosity η*, which combines the G' (elastic modulus) and G" (viscous modulus) of the composition. Therefore, the composition of the present invention described herein has a complex viscosity of preferably from about 1000 Pa.s to about 25000 Pa.s at 25°C, preferably from about 2000 Pa.s to about 15000 Pa.s at 25°C, and even more preferably from about 3000 Pa.s to about 11000 Pa.s at 25°C. The complex viscosity is usually measured under oscillation. In particular, the complex viscosity can be suitably measured at room temperature (about 25°C) using a rheometer (e.g. Anton Paar), in particular using a cone-plate geometry with a gap of 400 nm, at a predetermined strain amplitude fixed to 5 Pa and an oscillating ramp set to a fixed frequency of 1 Hz. The complex viscosity value can be the average of 10 values ​​collected within 60 seconds. A specific example of determining the complex viscosity is provided in method C of Example 2.

[0032] In order to obtain the desired complex viscosity, PEG is used as a thixotropic agent (thickening agent) in the composition of the present invention. PEG is usually prepared by the polymerization of ethylene oxide, and it is commercially available in a wide range of molecular weights from 100 g / mol to 10,000,000 g / mol. Typically, the numbers contained in the PEG name represent their average molecular weight (e.g., n=9 PEG has an average molecular weight of about 400 g / mol and will be labeled as PEG 400; PEG 300 has a molecular weight of about 300 g / mol, PEG 1000 has a molecular weight of about 1000 g / mol, PEG 1500 has a molecular weight of about 1500 g / mol, etc.). PEGs of different molecular weights are referred to herein as different PEG components. The PEG component of the composition of the present invention allows a semi-solid ointment base of the composition to be provided for adjusting viscosity within a desired range.

[0033] The amount of PEG in the composition of the present invention may be at least 30% (by weight of the finished product), at least 35% (by weight of the finished product), at least 40% (by weight of the finished product), at least 45% (by weight of the finished product), at least 50% (by weight of the finished product), at least 55% (by weight of the finished product), at least 60% (by weight of the finished product), at least 65% (by weight of the finished product) or even higher than 65%. Preferably, the amount of PEG in the composition of the present invention is at least 70% (by weight of the finished product), preferably at least 75% (by weight of the finished product), more preferably at least 80% (by weight of the finished product), even more preferably at least 85% (by weight of the finished product), still more preferably at least 90% (by weight of the finished product), particularly preferably at least 91% or at least 92% (by weight of the finished product), most preferably at least 93% or at least 94% (by weight of the finished product). In some embodiments, the amount of PEG in the composition of the present invention may be 94% to 95% (by weight of the finished product).

[0034] In some embodiments, a single PEG component can be used, particularly to provide a composition in a semi-solid form. For this reason, PEG 800 to PEG 1500 can be used as a single component PEG, such as PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400, PEG 1500. In some embodiments, a single PEG component can be combined with a higher amount of DMSO to provide a composition that is more fluid compared to a single PEG component alone. Therefore, the higher the molecular weight of PEG (i.e., the more "solid" the PEG is), the higher the amount of DMSO that can be used. For example, if PEG 1500 is used as a single PEG component, its usage amount can be 40% to 45% (by weight of the finished product), and the usage amount of DMSO in such a composition can be used at 53% to 58% (by weight of the finished product). For other PEGs with lower molecular weights (used as single PEG components), the amount of DMSO can be reduced (compared to the example of PEG 1500 described above).

[0035] Preferably, the composition of the present invention comprises at least two different PEG components. For example, the composition of the present invention may comprise 2, 3, 4, 5, 6, 7, 8, 9 or more than 9 (different) PEG components. Preferably, the composition of the present invention comprises (exactly) two or three different PEG components, even more preferably, the composition of the present invention comprises (exactly) two different PEG components.

[0036] The first PEG component preferably has a solid physical state at 25°C and the second PEG component included in the composition preferably has a liquid physical state at 25°C, ie, solid PEG is preferably combined with liquid PEG.

[0037] Without being bound by any theory, the inventors speculate that in this combination, the short-chain liquid PEG components such as PEG200 to PEG 600 are arranged in parallel crystal layers (or folded layers) of solid PEG components such as PEG 1000 to PEG 8000, especially in parallel crystal layers (or folded layers) of terminal OH-groups, thereby causing swelling. Without being bound by any theory, it is speculated that this arrangement leads to favorable plastic deformation characteristics.

[0038] Preferably, the first PEG component is selected from PEG 1000 to PEG 8000, such as PEG 1000, PEG 1500, PEG 3000, PEG 3350, PEG 4000, PEG 6000 or PEG 8000; and the second PEG component contained in the composition is selected from PEG 200 to PEG 600, such as PEG 200, PEG 300, PEG 400, PEG 500 or PEG 600.

[0039] For example, PEG 300 and PEG 1500, PEG 400 and PEG 3350, or PEG 400 and PEG 4000 can be combined. Even more preferably, the composition comprises (exactly) two different PEG components, wherein the first PEG component is PEG 1500 and the second PEG component is PEG 300.

[0040] In the composition of the present invention, the ratio (w / w) between (i) the first PEG component, in particular a PEG component having a solid physical state at 25°C, for example, a PEG selected from PEG 1000 to PEG 8000, and (2) the second PEG component, in particular a PEG component having a liquid physical state at 25°C, for example, a PEG selected from PEG 200 to PEG 600, can be 1:10 to 10:1, preferably 1:3 to 3:1, more preferably 1:2 to 2:1, even more preferably 1:1.75 to 1.75:1, and still more preferably 1:1.5 to 1.5:1.

[0041] In some embodiments, the amount (weight) of the first PEG component, in particular the amount (weight) of the PEG component having a solid physical state at 25° C., such as the amount (weight) of the PEG selected from PEG 1000 to PEG 8000, is about the same as the amount (weight) of the second PEG component, in particular the amount (weight) of the PEG component having a liquid physical state at 25° C., such as the amount (weight) of the PEG selected from PEG 200 to PEG 600. For example, the ratio (w / w) between (i) the first PEG component, in particular the PEG component having a solid physical state at 25° C., such as the PEG selected from PEG 1000 to PEG 8000, and (2) the second PEG component, in particular the PEG component having a liquid physical state at 25° C., such as the PEG selected from PEG 200 to PEG 600, can be 1:1.35 to 1.35:1, for example 1:1.

[0042] In some embodiments, the amount (weight) of the first PEG component, in particular the amount (weight) of the PEG component having a solid physical state at 25° C., e.g. the amount (weight) of PEG selected from PEG 1000 to PEG 8000, is higher than the amount (weight) of the second PEG component, in particular the amount (weight) of the PEG component having a liquid physical state at 25° C., e.g. the amount (weight) of PEG selected from PEG 200 to PEG 600. For example, the ratio (w / w) between (i) the first PEG component, in particular the PEG component having a solid physical state at 25° C., e.g. PEG selected from PEG 1000 to PEG 8000, and (2) the second PEG component, in particular the PEG component having a liquid physical state at 25° C., e.g. PEG selected from PEG 200 to PEG 600, is preferably 1.1:1 to 3:1, more preferably 1.1:1 to 2:1, even more preferably 1.1:1 to 1.75:1, and still more preferably 1.1:1 to 1.5:1.

[0043] Preferably, the amount (weight) of the first PEG component, in particular the amount (weight) of the PEG component having a solid physical state at 25°C, such as the amount (weight) of PEG selected from PEG 1000 to PEG 8000, is lower than the amount (weight) of the second PEG component, in particular the amount (weight) of the PEG component having a liquid physical state at 25°C, such as the amount (weight) of PEG selected from PEG 200 to PEG 600. Therefore, preferably, the ratio (w / w) between (i) the first PEG component, in particular a PEG component having a solid physical state at 25°C, such as PEG selected from PEG 1000 to PEG 8000, and (2) the second PEG component, in particular a PEG component having a liquid physical state at 25°C, such as PEG selected from PEG 200 to PEG 600, is preferably 1:3 to 1:1.1, more preferably 1:2 to 1:1.1, even more preferably 1:1.75 to 1:1.1, still more preferably 1:1.5 to 1:1.1, and particularly preferably about 1:1.3575.

[0044] In some embodiments, PEG 1500 and PEG 300 can be combined in a ratio of 1:1 to 1:2, preferably 1:1 to 1:1.5, such as 1:1.3575 (PEG 1500:PEG 300). In another example, PEG 3350 and PEG 400 can be combined in a ratio of 1:1 to 1:2, preferably 1:1 to 1:1.5, such as 1:1.3575 (PEG 3350:PEG 400). In another example, PEG 4000 and PEG 400 can be combined in a ratio of 1:1 to 1:2, preferably 1:1 to 1:1.5, such as 1:1.3575 (PEG 4000:PEG 400). Particularly preferably, PEG 1500 and PEG 300 are combined in a ratio of 1:1 to 1:1.5, such as 1:1.3575 (PEG 1500:PEG 300).

[0045] In addition, the compositions of the present invention typically include DMSO. The inventors have found that aqueous solvents do not provide sufficient stability for decitabine or azacitidine. Among different non-aqueous solvents, only DMSO exhibits sufficient solubility for the corresponding dosage strength. Therefore, DMSO is advantageously used as a non-aqueous solvent for decitabine or azacitidine. In some embodiments, DMSO can be combined with other non-aqueous, polar solvents, particularly with propylene glycol, for example, in a 1:1 ratio. In other embodiments, the composition does not include solvents other than DMSO (and PEG).

[0046] Typically, the composition may advantageously include enough DMSO to provide solubility at the expected decitabine or azacitidine concentration, while (substantially) excessive DMSO should be avoided. Thus, the final concentration of DMSO may depend on the concentration of decitabine or azacitidine (or its salt) in the composition. The DMSO concentration may be determined based on the expected concentration of decitabine or azacitidine (or its salt) in the composition. The solubility of decitabine in DMSO is approximately 46 mg / ml.

[0047] In some embodiments, the composition comprises DMSO at a concentration (weight to weight (w / w)) of 1% to 60%, such as 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, or 1% to 20%. Preferably, the composition comprises DMSO at a concentration of 1% to 15%, more preferably 2% to 10%, even more preferably 4% to 7%, still more preferably 5% to 6%, particularly preferably about 5.7%. Although the inventors have found that this concentration of DMSO is particularly advantageous for topical formulations (compositions) of the present invention comprising about 0.1 mM to 10 mM decitabine or azacitidine (or its salt), for topical formulations (compositions) of the present invention comprising higher concentrations of decitabine or azacitidine (or its salt), i.e., more than 10 mM decitabine or azacitidine (or its salt), it may be necessary to increase the concentration of DMSO in the final composition due to the solubility of decitabine in DMSO (approximately 46 mg / ml). In such cases where the DMSO concentration is higher, the concentration of the liquid PEG component in the composition can be reduced to compensate for the increased fluidity of the drug formulation with a higher DMSO concentration in the final composition.

[0048] Preferably, the composition of the present invention is substantially free of water. For example, the composition may contain less than 10% (w / w) water, preferably less than 5% water, more preferably less than 3% or less than 4% water, more preferably less than 2% water, even more preferably less than 1% water, and still more preferably less than 0.5% water. Generally, the less water included in the composition, the more preferred. Ideally, the composition does not contain water or does not contain any moisture. As shown in the attached examples, aqueous compositions have a negative impact on the stability of decitabine in the composition.

[0049] Therefore, aqueous solvents are preferably avoided. In some embodiments, the composition may contain less than 10% (w / w) aqueous solvent, preferably less than 5% aqueous solvent, more preferably less than 3% or less than 4% aqueous solvent, more preferably less than 2% aqueous solvent, even more preferably less than 1% aqueous solvent, and still more preferably less than 0.5% aqueous solvent. Generally, the less aqueous solvent included in the composition, the more preferred. Ideally, the composition does not contain an aqueous solvent.

[0050] In some embodiments, polar protic solvents are preferably avoided. Protic solvents are solvents containing labile H +Solvents that are proton-free, i.e., they can easily release protons. Protic solvents include, for example, solvents with hydrogen atoms (such as hydroxyl groups) bonded to oxygen atoms, hydrogen atoms (such as amine groups) bonded to nitrogen atoms, or hydrogen atoms (such as hydrogen fluoride) bonded to fluorine atoms. Although the most common polar protic solvent is water, other common polar protic solvents that can be avoided, non-limiting examples of which include formic acid, n-butanol, isopropanol, nitromethane, ethanol, methanol, and acetic acid. In some embodiments, the composition may include less than 10% (w / w) of a polar protic solvent, preferably less than 5% of a polar protic solvent, more preferably less than 3% or less than 4% of a polar protic solvent, more preferably less than 2% of a polar protic solvent, even more preferably less than 1% of a polar protic solvent, and still more preferably less than 0.5% of a polar protic solvent. Typically, the fewer polar protic solvents included in the composition, the more preferred. Ideally, the composition does not include a polar protic solvent. For example, the composition may be substantially free of ethanol. In some embodiments, the composition may contain less than 10% (w / w) ethanol, preferably less than 5% ethanol, more preferably less than 3% or less than 4% ethanol, more preferably less than 2% ethanol, even more preferably less than 1% ethanol, and still more preferably less than 0.5% ethanol. Generally, the less ethanol contained in the composition, the more preferred. Ideally, the composition does not contain ethanol.

[0051] In some embodiments, the compositions of the present invention are substantially free of glycerol. For example, the composition may contain less than 10% (w / w) glycerol, preferably less than 5% glycerol, more preferably less than 3% or less than 4% glycerol, more preferably less than 2% glycerol, even more preferably less than 1% glycerol, and still more preferably less than 0.5% glycerol. Generally, the less glycerol included in the composition, the more preferred. Ideally, the composition does not contain glycerol.

[0052] Preferably, the composition is substantially homogeneous. For example, the PEG included in the composition is preferably not in the form of PEGylated liposomes. In some embodiments, the composition is (substantially) hormone-free. In particular, the composition does not include thyroid stimulating hormone (TSH).

[0053] In some embodiments, the composition of the present invention does not correspond to a composition comprising glycerol. In some embodiments, the composition of the present invention does not correspond to a composition comprising polypropylene glycol. In some embodiments, the composition of the present invention does not correspond to a composition comprising glycerol and polypropylene glycol. In some embodiments, the composition of the present invention does not correspond to a composition comprising a combination of glycerol, polypropylene glycol and PEG.

[0054] In some embodiments, the compositions of the present invention do not include an acidifying agent.

[0055] In some embodiments, the composition of the invention does not correspond to a composition comprising any of the following components selected from stabilizers (other than PEG), acidifying agents, buffer salts, pH adjusting agents, desiccants and antioxidants, or mixtures thereof.

[0056] In some embodiments, the compositions of the present invention do not contain any of the following components: stabilizers other than PEG, acidulants, buffer salts, pH adjusters, desiccants, antioxidants, glycerol and polypropylene glycol or mixtures thereof.

[0057] In some embodiments, the composition of the present invention consists (essentially) of decitabine or azacitidine (or its salt), PEG and DMSO. In other words, preferably, the composition does not contain any components other than decitabine or azacitidine (or its salt), PEG and DMSO. The inventors have found that no other components are required to formulate the composition, which can be easily applied topically (particularly topically to the target area), which is effective for treating precancerous symptoms associated with human papillomavirus (HPV) and provides sufficient stability of the active ingredient (decitabine or azacitidine (or its salt)).

[0058] In some embodiments, the composition of the present invention comprises decitabine or azacitidine (or its salt), PEG 1500, PEG 300 and DMSO. In particular, the composition of the present invention may consist of decitabine or azacitidine (or its salt), PEG 1500, PEG 300 and DMSO.

[0059] In some embodiments, the composition comprises (or consists of):

[0060] - 0.1 mM to 10 mM (0.0228 mg / g to 2.28 mg / g) of decitabine (or azacitidine),

[0061] - about 5.7% (w / w) DMSO,

[0062] - about 40% (w / w) PEG 1500, and

[0063] - about 54% to 55% (w / w) PEG 300.

[0064] Preferably, the composition of the present invention can show the stability of its active ingredient decitabine or azacitidine over an extended period of time. The composition of the present invention prevents the degradation of the active ingredient, so that more than 90% or more than 93% or more than 94% of the initially formulated active ingredient can preferably be retained for at least 3 months, especially under storage conditions of 5°C ± 3°C and / or 25°C ± 2°C. Stability can be determined by, for example, HPLC.

[0065] Medical treatment and medical uses

[0066] The composition of the present invention can be used in medicine, preferably wherein the pharmaceutical composition is topically applied. Non-limiting examples of medical symptoms that can be treated with the composition of the present invention include medical symptoms or medical conditions in keratinized epithelium / skin and non-keratinized epithelium / skin (e.g., epithelium / skin that can be treated topically). Thus, the composition can be topically applied to non-keratinized epithelium or keratinized epithelium or to the transition zone from non-keratinized epithelium to keratinized epithelium. Preferably, the composition of the present invention is used to treat papillomavirus (PV)-related precancerous symptoms, preferably wherein the pharmaceutical composition is topically applied, for example, to the lesions and / or genital area (of an animal or human patient). For example, the composition of the present invention can be used to treat animals infected with species-specific types of (human) papillomavirus, such as horse sarcoma in horses that may be caused by bovine papillomavirus. Preferably, the composition of the present invention is used to treat human papillomavirus (HPV)-related precancerous symptoms, preferably wherein the pharmaceutical composition is topically applied, for example, to the lesions and / or genital area (of a human patient).

[0067] Thus, the present invention also provides a method for treating medical symptoms in keratinized epithelium / skin, such as papillomavirus (PV)-related medical symptoms in keratinized epithelium / skin and non-keratinized epithelium / skin, or a method for reducing the risk of cancer, the method comprising administering (an effective amount of) a composition of the present invention as described above to a subject in need thereof. In some embodiments, the method is used to treat precancerous symptoms associated with human papillomavirus (HPV). In some embodiments, the composition is administered topically, such as topically to a lesion and / or genital area (e.g., a lesion and / or genital area of ​​an animal or human patient).

[0068] The present invention also provides the use of the composition of the present invention as described above in the preparation of a medicament for treating medical symptoms in keratinized epithelium / skin and non-keratinized epithelium / skin, for example, in the preparation of a medicament for treating papillomavirus (PV)-related medical symptoms such as human papillomavirus (HPV)-related precancerous symptoms in keratinized epithelium / skin and non-keratinized epithelium / skin.

[0069] The term "disease" as used herein is generally intended to be synonymous with, and used interchangeably with, the terms "disorder" and "symptom" (e.g., medical symptoms) because these words all reflect an abnormal state of the human or animal body or a part thereof that impairs normal function, often manifests itself in various signs and symptoms, and results in a reduction in the life span or quality of life of the human or animal.

[0070] As used herein, reference to "treatment" of a subject or patient is intended to include prevention, prophylaxis, alleviation, relief, and therapy. The terms "subject" or "patient" are used interchangeably herein to refer to all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. Preferably, the subject or patient is a human.

[0071] Human papillomavirus (HPV), particularly high-risk HPV, such as HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, and potentially HPV 66 and HPV 82, can cause cancer. HPV-related cancers (and corresponding precancerous symptoms) usually occur in parts of the body where HPV infects epithelial cells, such as the cervix, oropharynx, anus, penis, vagina, and vulva. Therefore, HPV-related precancerous symptoms include cervical precancerous symptoms (which can develop into cervical cancer), oropharyngeal precancerous symptoms (which can develop into oropharyngeal cancer), anal precancerous symptoms (which can develop into anal cancer), penile precancerous symptoms (which can develop into penile cancer), vaginal precancerous symptoms (which can develop into vaginal cancer), and vulvar precancerous symptoms (which can develop into vulvar cancer). Typically, HPV infects squamous epithelial cells, so most HPV-related precancers and HPV-related cancers are squamous cell precancers and squamous cell carcinomas, respectively. However, some HPV-related (especially cervical) precancers and HPV-related cancers (especially cervical cancer) can be adenocarcinomas or corresponding precancers.

[0072] As used herein, the term "precancer" (also referred to as "precancer") refers to a symptom that can (or may) develop into cancer. Treatment of precancerous symptoms generally includes reducing the risk of precancerous symptoms developing into cancer. HPV-related precancerous symptoms can be identified, for example, by cytology-based screening such as Pap smears (particularly for cervical cancer screening). There are different stages of precancerous disease, which can be classified according to their respective healthcare systems. For example, the Bethesda system divides squamous cell abnormalities into the following categories, from the mildest to the most severe: atypical squamous cells (ASC), which have two subgroups, ASC-US (unclear significance) and ASC-H (high-grade squamous intraepithelial lesions cannot be excluded); low-grade squamous intraepithelial lesions (LSILs); high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma. In the Bethesda system, glandular cell abnormalities are divided into the following categories: atypical glandular cells (AGC), adenocarcinoma in situ of the cervix (AIS), and adenocarcinoma. Of these stages, squamous cell carcinoma and adenocarcinoma are cancers, while the other (earlier) stages are precancerous conditions.

[0073] As described above, the compositions of the present invention are preferably topically applied to a target area, such as a precancerous lesion or an area that appears to be affected by or suspected of being affected by a precancerous symptom. The target area may be the anogenital area or the genital area or any other body part affected by HPV-related precancerous symptoms, particularly the vulva. Examples of body parts to which the compositions of the present invention may be topically applied include all body parts where HPV-infected cells are located, such as the cervix, oropharynx, anal canal, penis, vagina, and vulva. Thus, the compositions may be applied to a body surface that is exposed to air or to a surface that is not exposed to air (i.e., a body cavity).

[0074] In some embodiments, the medical condition or medical disorder to be treated with the composition of the present invention refers to precancerous lesions of the vulva, more specifically to vulvar intraepithelial neoplasia grade 2 / 3.

[0075] In some embodiments, precancerous conditions can be identified during a colposcopy and a composition of the invention can be administered to, for example, the cervical area, during, for example, a colposcopy.

[0076] In a specific embodiment, the composition of the invention can be used to treat HPV-induced precancerous lesions. Thus, the treatment of HPV-related cancers is preferably excluded.

[0077] Advantageously, the treatment methods according to the present invention do not cause epithelial disruption or epithelial ulceration of normal tissue surrounding the target lesion / disease tissue being treated.

[0078] Application can be done with a gloved finger or with the aid of a medical device, such as an applicator, spatula, or cervical cap.

[0079] The administration scheme can be designed as follows: within the treatment cycle, the composition is administered at least 5 times, for example, 5 to 15 times. For example, for each treatment cycle, administration can be performed once a day or twice a day, or once every two days or once every three days. The total treatment period can include at least 2 treatment cycles, for example, 2 to 5 treatment cycles are included in a period of at least 2 weeks, such as 2 to 8 weeks. Preferably, the composition comprises 1 mM to 1 M, preferably 1 mM to 50 mM of active ingredient. At each administration, 5 mg to 500 mg, preferably 50 mg to 250 mg of the preparation is applied to the affected tissue / lesion.

[0080] Packaging of the composition

[0081] Another aspect of the present invention is the packaging for compositions of the present invention. In embodiments, compositions of the present invention are packaged in primary containers comprising a volume of 0.05ml to 50ml. Preferably, the primary packaging of preparation allows to repeatedly take out / extract preparation from container (such as pipe or any other suitable applicator or container), thereby being convenient to the patient and conducive to transportation and storage. Primary container can preferably be designed to pipe, which can be deformable or non-deformable. Pipe can be made of plastic material or metal material or alloy material, for example, made of aluminum. Preferably, deformable (aluminum) pipe is used to prevent moist air from being sucked into pipe. More specifically, at least in use, the outlet of pipe can be equipped with an applicator to allow the preparation of a limited amount to be taken out according to the length of extrudate.

[0082] In another embodiment, the formulation may be present in a disposable primary packaging container (eg, a small sealed bag).

[0083] Preferably, the primary packaging (eg when a tube is used) may comprise a protective inner lacquer or other protective inner layer.

[0084] Alternatively, the composition may be provided as a capsule comprising the composition of the invention. Preferably, the capsule may consist of a self-dissolving material, delivering the composition of the invention upon administration, for example upon insertion into the anogenital region. Other embodiments allowing packaging of the composition of the invention refer to (bioadhesive) patches or tampons. The composition of the invention, preferably as an ointment, may be applied to such patches or tampons.

[0085] Method for preparing the composition

[0086] The present invention also provides a method for preparing the pharmaceutical composition according to any one of the preceding claims, the method comprising the following steps:

[0087] (1) preparing a DMSO solution of decitabine or azacitidine (or its salt);

[0088] (2) melting the PEG component to obtain a homogeneous composition; and

[0089] (3) Mixing the decitabine (or azacitidine) solution of step (1) with the melted PEG component of step (2) to obtain a homogenous composition.

[0090] Although step (3) is after step (1) and step (2), step (1) and step (2) may be performed in any order, for example, step (1) is before step (2), or step (2) is before step (1), or step (1) and step (2) may be performed substantially in parallel (simultaneously). Advantageously, decitabine or azacitidine is initially dissolved in DMSO and therefore not dissolved in PEG. Thus, decitabine or azacitidine is contacted with PEG only after being previously dissolved in DMSO.

[0091] In some embodiments, step (1) is performed at 20°C to 30°C, preferably at 22°C to 28°C, more preferably at 23°C to 27°C, even more preferably at about 25°C.

[0092] In some embodiments, in step (1), a stock solution can be used, and an appropriate amount of the stock solution for the relevant decitabine concentration can be added to the remaining weight of DMSO in a suitable container and mixed to obtain a homogenous solution. For example, the following concentrations can be used to prepare a decitabine stock solution dissolved in DMSO (depending on the final dosage strength):

[0093]

[0094]

[0095] In other embodiments, a DMSO solution of decitabine or azacitidine (or a salt thereof) is prepared in step (1) without using a stock solution.

[0096] If more than one polyethylene glycol (PEG) component is used, the different PEG components can be combined and melted in step (2), for example in a suitable container at a moderate product temperature, for example at 50° C. to 60° C., preferably at 51° C. to 59° C., more preferably at 52° C. to 58° C., even more preferably at 65° C. ± 2° C. In some embodiments, the melting of the PEG components is carried out under (continuous) stirring. As an example, the PEG components PEG 1500 and PEG 300 can be weighed as follows: 40% (w / w) PEG 1500 and 30% (w / w) to 55% (w / w) PEG 300. In step (2), the different PEG components to be combined can be provided in the same container. The PEG component may be melted in step (2), in particular under stirring, for example at 50 to 60°C, preferably at 51 to 59°C, more preferably at 52 to 58°C, even more preferably at 65°C±2°C (e.g. in a water bath).

[0097] Thereafter, the PEG component may be cooled (actively or passively), for example to 30°C to 45°C, preferably 32°C to 40°C, more preferably 36°C±2°C, before step (3) begins.

[0098] In step (3), the decitabine (or azacitidine) solution of step (1) is combined with the molten PEG component of step (2) to obtain a homogeneous composition. To this end, the decitabine (or azacitidine) solution of step (1) can be transferred (added) to the molten polyethylene glycol mixture of step (2). In order to obtain a homogeneous mixture, it can be stirred for an appropriate time or mixed; for example, on a laboratory test tube oscillator (vortexer). Thereafter (when a homogeneous composition is obtained), the composition can be cooled (actively or passively), for example, to room temperature (about 25° C.), for example, while stirring. In some embodiments, it can optionally be mixed again afterwards, for example using a drug mixing system.

[0099] After step (3), the resulting composition may be packaged, for example, filled into a suitable container such as an ointment tube. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] The following is a brief description of the accompanying drawings. The accompanying drawings are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the present invention in any way.

[0101] Figure 1 , showing (A) stability test of decitabine in aqueous buffer (about 25° C., two independent samples) and (B) degradation products of decitabine in aqueous buffer (about 25° C.) in Example 1.

[0102] Figure 2 , showing the stability of decitabine dissolved in DMSO at 2°C to 8°C in Example 1, as shown in the left (lower) figure, and the stability of decitabine dissolved in DMSO at room temperature (about 25°C), as shown in the right (upper) figure.

[0103] Figure 3 , showing the stability of decitabine dissolved in DMSO in Example 1 in a polyethylene glycol-based formulation (1 mM) at 2°C to 8°C, as shown in the left (lower) figure, and the stability of decitabine dissolved in DMSO in a glycerol / carbopol-based formulation (1 mM) at 2°C to 8°C, as shown in the right (upper) figure.

[0104] Figure 4, showing the stability of decitabine dissolved in DMSO in a polyethylene glycol-based formulation (1 mM) at different temperatures in Example 1. "Starting value" = concentration on the day after preparation; "Room temperature": about 25°C; Quantitative value: 1099.4 μM / 1110.8 μM / 1048.9 μM.

[0105] Figure 5 , shows the stability of an exemplary decitabine formulation containing 2.28 mg / g (10 mM, nominal concentration) decitabine in Example 1 stored at different specified temperatures for three months. The percentage value represents the actual decitabine content relative to the nominal decitabine content.

[0106] Figure 6 , shows an overlay of the flow profiles of the PEG-based placebo formulation and the PEG-based 10 mM decitabine formulation in Example 2.

[0107] Figure 7 , showing the PEG-based placebo formulation in Example 2 at a constant rate of 1.0 seconds -1 The viscosity of the solution changes as a function of time (t).

[0108] Figure 8 , shows the LVER overlay of the PEG-based placebo formulation and the PEG-based 10 mM decitabine formulation in Example 2.

[0109] Fig. 9 , shows the results of a 2-week topical treatment experiment on organotypic cultures (OTCs) in Example 3, which consist of keratinocytes and HPV-transformed SiHa cells grown on dermal equivalents and treated with decitabine-containing polyethylene glycol (PEG)-based formulations of different intensities [0.1 mM, 1 mM or 10 mM] or placebo formulations as controls. Representative areas of tumor nests and adjacent normal epithelium are shown at 10x magnification (A1-D1) and 40x magnification (A2-D2), which are from full-section scans of hematoxylin and eosin (HE)-stained tissue sections of one biological replicate under each treatment condition. The black outline indicates the tumor nest area composed of HPV-transformed SiHa cells, the dotted gray outline indicates the stratified normal epithelial cells, and the black rectangles in images A1-D1 indicate more detailed tissue areas displayed at 40x magnification in the corresponding images A2-D2.

[0110] Fig.10 , Fig.10 The whole-slice scan of p16 in Example 5 at 20 times magnification is shown. INK4aImmunohistochemical staining results from a 2-week (A, B) and 4-week (C, D) topical treatment experiment of HPV-induced precancerous lesions from the vulva cultured in vitro with a PEG-based formulation containing decitabine [1 mM] / a corresponding placebo formulation. Example

[0111] Hereinafter, specific examples illustrating various embodiments and various aspects of the present invention are presented. However, the scope of the present invention should not be limited by the specific embodiments described herein. The preparation and examples given below are to enable those skilled in the art to more clearly understand and implement the present invention. However, the scope of the present invention is not limited by the exemplary embodiments, which are only intended to illustrate various aspects of the present invention, and functionally equivalent methods are also within the scope of the present invention. In fact, various modifications to the present invention beyond the description herein will become apparent to those skilled in the art, according to the above, the accompanying drawings, and the examples below. All such modifications are within the scope of the appended claims.

[0112] Example 1: Stability of decitabine in different formulations

[0113] To evaluate the stability of decitabine in different formulations, HPLC analysis was performed as follows:

[0114] The HPLC method was based on the method previously published by Rogstad, D. et al.: Chemical decomposition of 5-aza-2'-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry. Chem Res Toxicol 2009; 22(6): 1194-204. The parameters / values ​​used are shown in Table 1 below.

[0115]

[0116] (Table 1)

[0117] step:

[0118] 1. Mix 200 μL of sample solution with 444 μL of mobile phase

[0119] 2. Inject immediately

[0120] 3. Quantification using internal standard and a separately recorded internal standard calibration curve (constant internal standard concentration and increasing decitabine concentration)

[0121] 4. Peak identification was as described in Rogstad, D. et al., Chemical decomposition of 5-aza-2'-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry. Chem Res Toxicol 2009; 22(6): 1194-204.

[0122] A. Stability of Decitabine in Aqueous Media

[0123] To test the stability of β-decitabine in aqueous media, two stock solutions of β-decitabine in DMSO were prepared and mixed with HPLC mobile phase (20 mM ammonium acetate pH 6.8) at a ratio of 1:2. The samples were stored at room temperature (approximately 25°C) and their content of β-decitabine, which is considered to represent the active anomer of decitabine, was measured for up to 14 days.

[0124] The results are as follows Figure 1 shown. Figure 1 A shows the decrease of β-decitabine over the observation period, which is well described by a first-order decay function. One of the samples was also analyzed for the presence of a known degradation product of β-decitabine. The results can be seen in Figure 1 B. These results clearly indicate that aqueous media cannot be used due to the observed instability of decitabine in aqueous media.

[0125] In summary, these data suggest that aqueous formulations are not suitable for decitabine formulation because decitabine degrades rapidly in an aqueous environment.

[0126] B. Solubility of Decitabine in Different Non-aqueous Media

[0127] Due to the unsuitability of aqueous media, different non-aqueous media were tested for the solubility of decitabine in non-aqueous media. Since decitabine is a nucleoside, several polar anhydrous solvents that are well suited for further formulation experiments were selected to determine the solubility of decitabine. However, among the four tested solvents - glycerol, PEG 300, anhydrous ethanol and DMSO, only DMSO showed sufficient solubility for the relevant dose strengths. Therefore, the DMSO stock solution was selected as a candidate for the next step of stability testing and formulation testing.

[0128] C. Long-term stability of decitabine in DMSO

[0129] A 3 mM stock solution of decitabine in DMSO was prepared and aliquots were stored at room temperature and at 2° C. to 8° C. The β-decitabine concentration was determined by HPLC for approximately three months.

[0130] The results are as follows Figure 2 These tests showed little change in the concentration of β-decitabine and, although the sample size for these preliminary tests was too small to achieve statistical significance, adequate long-term stability was observed, especially when compared to (in)stability in aqueous buffer.

[0131] D. Long-term stability of candidate formulations

[0132] Previous testing of formulations without decitabine resulted in the selection of two formulations that exhibited viscosities suitable for patient handling and administration and were also compatible with the DMSO concentrations required for relevant dose strengths of β-decitabine:

[0133] (1) Formulations based on polyethylene glycol mixtures; and

[0134] (2) Glycerin-based gels.

[0135] Therefore, the first batch of these formulations were prepared for preliminary in vitro testing as follows:

[0136] (1) Polyethylene glycol (PEG) preparations

[0137] In brief, the following steps were performed:

[0138] 1. Mix and melt the polyethylene glycol (PEG) component in a suitable container with continuous stirring at moderate product temperature (50°C to 60°C).

[0139] 2. Prepare a stock solution of decitabine in DMSO. Add an appropriate amount of the stock solution for the relative decitabine concentration to the remaining weight of DMSO in a suitable container and mix to obtain a homogenous solution.

[0140] 3. Transfer the DMSO solution of decitabine into the molten polyethylene glycol mixture in step 1 and stir for an appropriate time to obtain a homogenous mixture.

[0141] In more detail, the exemplary formulation is obtained by the following steps:

[0142] 1. Prepare a stock solution of decitabine in DMSO according to the final dose strength:

[0143]

[0144] 2. Weigh the polyethylene glycol compounds in a 50 mL centrifuge tube: 40% (m / m) PEG 1500 60-x% (m / m) PEG 300 (x% (m / m) = DMSO stock solution (approximately 5.7% for a dose strength of 10 mM, see below)

[0145] 3. Melt in a water bath (50°C to 60°C)

[0146] 4. Add an appropriate amount of DMSO stock solution (see above) to the melted PEG component

[0147] 5. Mix on a laboratory tube oscillator (vortexer)

[0148] 6. Transfer the melted batch into the Topitec tank

[0149] 7. Allow it to cool

[0150] 8. Mix with Topitec (level 1, 60 seconds)

[0151] 9. Fill into ointment tube

[0152] 10. Closed packaging (closed tube and shrink wrapped)

[0153] Thus, the following compositions were obtained (Table 2):

[0154]

[0155] (2) Glycerin gel preparation

[0156] 1. Prepare a stock solution of decitabine in DMSO.

[0157]

[0158] 2. Pipette glycerol into the Topitec tank

[0159] 3. Add decitabine stock solution (5.8% m / m of final mass)

[0160] 4. Join 974P (0.5% m / m of glycerol mass)

[0161] 5. Mix immediately with Topitec

[0162] 6. Fill into ointment tube

[0163] Thus, the following compositions were obtained (Table 3):

[0164]

[0165] Note: For dose strengths >10 mM, higher concentration.

[0166] Aliquots of these batches were stored at 2°C to 8°C to assess their long-term stability.

[0167] The results are as follows Figure 3 shown. Figure 3 The β-decitabine content of samples quantified up to 112 days (PEG) and up to 76 days (glycerol) are shown. Again, although the sample sizes for these preliminary tests were too small to achieve statistical significance, adequate long-term stability was observed.

[0168] E. Stability test at different temperatures

[0169] The stability of the PEG-based formulations described above was then tested at different temperatures.

[0170] Preliminary tests such as Figure 4 shown. Figure 4 The β-decitabine content of three aliquots of the same production batch of the PEG-based formulation is shown: the bar on the left is the content on the day after preparation, the bar in the middle is the content after about one month at 2°C to 8°C, and the bar on the right shows an aliquot that was not stored under controlled conditions after use in the in vitro experiments but was placed at room temperature and then quantified also after about one month. As can be seen, even under these uncontrolled conditions, no significant degradation occurred, indicating that the final developed drug product can be stored at room temperature.

[0171] Subsequently, production batches of different strengths [0.1 mM, 1 mM and 10 mM], essentially as described in Table 2, were stored under controlled conditions, i.e. at -80°C, -20°C, 5°C and 25°C. The decitabine content was determined by HPLC after production (T0) and after storage for one month (T1), two months (T2) and three months (T3) under the corresponding conditions.

[0172] The results are as follows Figure 5 In these preliminary analyses, essentially no changes in the measured values ​​were observed for storage at -80°C, -20°C, 5°C, or 25°C. Therefore, the formulation is stable even for long-term storage at room temperature (about 25°C).

[0173] In summary, the data obtained in Example 1 indicate that it is necessary to use a stock solution of decitabine dissolved in DMSO for preparation due to lack of solubility in other available media. The long-term stability data of decitabine in dimethyl sulfoxide (DMSO) looked promising, and two potential formulations were developed based on decitabine dissolved in DMSO - a glycerol-based gel and a polyethylene glycol-based ointment. In preliminary directed testing, both showed good long-term stability, and since the in vitro test results are not described here, the polyethylene glycol-based formulation was selected for further development.

[0174] Example 2: Evaluation of formulation viscosity

[0175] For topical application, on the one hand, the viscosity of the composition should be high enough to substantially prevent the composition from flowing from the area to which it is applied or from being inadvertently smeared on other areas. On the other hand, the viscosity of the composition should be low enough to allow the patient to easily take out the desired dose of the composition from the container (e.g., tube, etc.) in which the composition is available, apply the composition evenly on the affected area to ensure uniform application of the active ingredient, and avoid or minimize friction on the application site, which may cause local irritation and / or local pain.

[0176] Hereinafter, examples for determining the viscosity of exemplary compositions are provided.

[0177] A: Flow profile measurement

[0178] The flow profiles of the PEG-based placebo formulation and the PEG-based 10 mM decitabine formulation were determined. The PEG-based 10 mM decitabine formulation was prepared as follows:

[0179] (1) preparing a DMSO solution of decitabine or azacitidine (or its salt) at room temperature (about 25° C.);

[0180] (2) melting the PEG component, for example, stirring at 65°C ± 2°C to obtain a homogenous composition, and then cooling to 36°C ± 2°C;

[0181] (3) mixing the decitabine (or azacitidine) solution of step (1) with the melted PEG component of step (2) to obtain a homogenous composition; and

[0182] (4) Cool to room temperature (about 25°C) while stirring.

[0183] The PEG-based placebo formulation was prepared as follows:

[0184] (1) melting the PEG component, for example, stirring at 65°C ± 2°C to obtain a homogeneous composition; then cooling to 36°C ± 2°C;

[0185] (2) adding the melted PEG component of step (1) to DMSO to obtain a homogenous composition; and

[0186] (3) Cool to room temperature (about 25°C) while stirring.

[0187] Each sample was loaded into a rheometer (Anton Paar: Model: MCR 302) at room temperature (25°C); constant temperature bath (Viscotherm VT2 from Anton Paar), gap 398 nm (cone-plate geometry CP4-40 mm).

[0188] The samples were subjected to logarithmic increasing stress sweep and logarithmic decreasing stress sweep. The conditions are shown in Table 4 below.

[0189]

[0190] (Table 4)

[0191] The results are as follows Figure 6 Table 5 below shows the 1.0 second -1 The viscosity value (in Pa.s) extracted from the starting point of the increasing curve and the yield point value (in Pa) extrapolated from the maximum value of the curve at the beginning of the increasing curve:

[0192]

[0193] (Table 5)

[0194] Figure 6 The shape of the flow curves in Figure 2 indicates that both samples exhibit shear thinning properties. The term "shear thinning" as used herein refers to a common characteristic of non-Newtonian fluids in which the viscosity of the fluid decreases with increasing shear rate or shear stress. Shear thinning can be observed in suspensions, emulsions, polymer solutions, and gels. Due to the shear thinning property, the viscosity of the ointment can be reduced by increasing the shear rate. Basically, the "shear thinning" property is a measure of the ability of the ointment structure to temporarily deform during its application by rubbing on the skin as a result of the decrease in viscosity when the shear rate increases.

[0195] exist Figure 6 At the beginning of the flow curve shown, the shear stress increases rapidly to a maximum value corresponding to the yield point. From this value onwards, the sample begins to flow. As shown in the figure, the increasing and decreasing curves are not the same, which reflects the thixotropic behavior.

[0196] In conclusion, the results showed that the PEG-based 10 mM decitabine formulation was more viscous and had a higher yield point compared to the PEG-based placebo formulation.

[0197] B: Viscosity at a constant rate

[0198] The placebo sample was loaded into a rheometer (Anton Paar: Model: MCR 302) at room temperature (25°C); constant temperature bath (Viscotherm VT2 from Anton Paar) gap 398 nm (cone-plate geometry CP4-40 mm).

[0199] A linear stress sweep is applied to the sample until the stress is reached within 1 second. -1 The viscosity was measured after 300 seconds of pre-shearing.

[0200] The results are as follows Figure 7 As shown in Table 6 below:

[0201]

[0202] (Table 6)

[0203] At the beginning of the measurement, the viscosity dropped rapidly and never stabilized during the measurement. At the end of the pre-shear at a constant rate, the viscosity increased rapidly from 102 Pa.s to 131 Pa.s. This observation confirms the thixotropic nature of the sample, which quickly recovers some of its viscosity once the constraint stops. This means that the sample will not leak out of the tube after being subjected to pressure, nor will it leak from the affected area of ​​the skin to the unaffected area when the friction on the skin stops.

[0204] C: Determination of the linear viscoelastic range (LVER) and complex viscosity

[0205] Oscillatory measurements are useful for studying processes involving changes in the sample's structure. The strain must be kept small enough not to interfere with ongoing physical or chemical processes. Stress sweep tests (i.e., measuring the elastic modulus and loss modulus as a function of stress at a fixed frequency) were performed to determine the shear-thinning properties of the sample.

[0206] Each sample (as described in A) was loaded into a rheometer (Anton Paar: Model MCR302) at room temperature (25°C); constant temperature bath (Viscotherm VT2 from Anton Paar), gap 398 nm (cone-plate geometry CP4-40 mm).

[0207] Step 1: Determination of the linear viscoelastic region (LVER)

[0208] model Instrument Setup Step Duration Sample loading and rest time Temperature: 25℃(±0.5℃) 300 seconds Incremental logarithmic scan 0.01Pa to 1000Pa 602 seconds

[0209] (Table 7)

[0210] The results are as follows Figure 8 As shown in Table 8 below:

[0211]

[0212] (Table 8)

[0213] The term "elastic modulus" (also known as "Young's modulus" or storage modulus (G')) is defined herein as the change in stress in a material with applied strain (i.e., the ratio of shear stress (force per unit area) to shear strain (proportional deformation)). Essentially, the elastic modulus is a quantitative measure of the stiffness of an elastic material, which measures the ability of the subject material to recover its original shape and original dimensions.

[0214] The term “loss modulus” (also called viscous modulus (G”)) characterizes the viscous part of the viscoelastic properties, which can be considered as the liquid-like properties of the sample. It characterizes the deformation energy lost (dissipated) through internal friction when flowing. Viscoelastic solids with G′>G″ have an elastic modulus higher than the loss modulus.

[0215] The elastic modulus and loss modulus measurements are reported as force per unit area (N / m 2 ) or Pascal (Pa), where one Pascal is equivalent to the force applied to one square meter (1m 2 ) is a force of one newton (1 N) on an area of ​​​​1. The unit Pascal is a term recognized in the art and is commonly used to define units of pressure, tensile strength, stress, and elasticity.

[0216] For both samples (PEG-based placebo formulation and PEG-based 10 mM decitabine formulation), the linear viscoelastic region is within the oscillation constraint of 1 Pa to about 15 Pa. Above 15 Pa, both moduli G' and G" continue to decrease. Shear thinning behavior (i.e., a decrease in elastic modulus G' as a function of applied stress) is observed.

[0217] The term "loss tangent tan δ" or "tan δ" refers herein to the tangent of the phase angle, which is the ratio of the viscous modulus (G") to the elastic modulus (G'), and is a useful quantitative indicator of the presence and degree of elasticity in a fluid. Tan δ values ​​less than 1 indicate elasticity-dominated (i.e., solid-like) behavior, while values ​​greater than 1 indicate viscosity-dominated (i.e., liquid-like) behavior.

[0218] The tan δ of both samples is less than 1, which confirms the elasticity-dominated behavior.

[0219] Step 2: Determination of complex viscosity

[0220] Complex viscosity measurements were performed within the linear viscoelastic range of deformation at a predetermined strain amplitude fixed at 5 Pa and a frequency fixed at 1 Hz in order not to disturb or limit structural changes.

[0221] model Instrument Setup Step Duration Sample loading and rest time Temperature: 25℃(±0.5℃) 300 seconds Pre-cut 5.0Pa 60 seconds Measurement 5.0Pa 60 seconds

[0222] (Table 9)

[0223] The results are shown in Table 10 below:

[0224]

[0225] (Table 10; RSD: relative standard deviation)

[0226] The complex viscosity of the PEG-based 10 mM decitabine formulation was higher than its corresponding placebo. Despite the rather high complex viscosity at rest, oscillatory measurements showed that both samples were able to flow under shear constraints, which is compatible with transdermal administration. This means that both samples can be easily spread on the skin by friction.

[0227] In summary, complex viscosity measurements indicate that for topical application, the complex viscosity should be from about 1000 Pa.s to about 25000 Pa.s at 25°C, preferably from about 2000 Pa.s to about 15000 Pa.s at 25°C, and even more preferably from about 3000 Pa.s to about 11000 Pa.s at 25°C. The complex viscosity can be suitably measured at room temperature (about 25°C) using a rheometer (e.g., Anton Paar), particularly using a cone-plate geometry with a gap of 400 nm, at a predetermined strain amplitude fixed to 5 Pa and an oscillating ramp set at a fixed frequency of 1 Hz. The complex viscosity value is the average of 10 values ​​collected over 60 seconds.

[0228] Example 3: Decitabine formulation according to the present invention in organotypic culture (OTC) of HPV-induced precancerous tumors Application of the model

[0229] In order to demonstrate that the drug substance decitabine penetrates into the epithelial layer and induces a beneficial therapeutic effect on HPV-transformed cells upon topical application of the developed polyethylene glycol (PEG)-based decitabine-containing formulation, an organotypic culture (OTC) model was treated with different strengths of the drug, as shown in Table 11. The OTC used consisted of keratinocytes co-cultured on dermal equivalents (DE) and HPV-transformed SiHa cells, thus representing a three-dimensional (3D) in vitro model of HPV-transformed precancerous lesions, which allows the application of topical formulations to surfaces exposed to air.

[0230] In order to be able to apply the drug topically to the surface exposed to air of the OTC model in a standardized manner, a small plate was made with a sealing film material in the laboratory to allow the entire surface exposed to air of the OTC to be covered. The plate was coated with the drug, carefully placed on the OTC, and removed after two hours of incubation (Table 11). The OTC model was topically treated with a decitabine-containing preparation based on PEG of different strengths [0.1mM, 1mM or 10mM] or a corresponding placebo preparation as a control for more than two weeks (5 times per week, according to Table 11). The experiment was repeated three times biologically under each condition. The OTC model was harvested on the 15th day after the start of treatment, and then fixed in formalin and embedded in paraffin.

[0231]

[0232] (Table 11)

[0233] Tissue architecture and tumor cell morphology were evaluated based on formalin-fixed paraffin-embedded (FFPE) OTC sections stained with hematoxylin and eosin (HE). The therapeutic effects of PEG-based decitabine-containing formulations on tissue architecture and tumor cell morphology under topical treatment were compared with the corresponding placebo formulations. Fig. 9 As shown. Dose-dependent degradation of tumor cells was observed, such as the loss of intact nuclei and the accumulation of cell debris within tumor nests (A→D). It is worth noting that after a 2-week treatment period, epithelial stratification still existed in the OTC model treated with placebo or different strengths of decitabine, and no separation of normal epithelium from the underlying dermis equivalent (DE) was observed in either placebo or decitabine-treated samples. In summary, the data show that after topical application to the OTC model of HPV-transformed precancerous tumors, treatment with the PEG-based decitabine-containing formulation of the present invention results in decitabine penetration into the epithelium and preferential degradation of HPV-transformed cells.

[0234] Through the data set of Example 3, It is proved that the preparation of the present invention can achieve the desired treatment for HPV transformed cells effects (such as degradation of tumor cells) while maintaining epithelial integrity. The experiment is based on an innovative organotypic culture (OTC) model that morphologically mimics HPV-induced precancerous lesions. In this model, HPV-transformed cells are co-cultured with normal human keratinocytes, resulting in the formation of tumor cell nests surrounded by normal stratified epithelial cells. This precancerous model setup allows same The therapeutic effects on HPV-transformed tumor cells and normal keratinocytes were evaluated. and Especially allowing topical Application. In a group of experiments containing decitabine formulations, the results showed A sufficiently high dose of decitabine from the combination of the present invention is released from the body and delivered to target cells. thereby Inducing the desired therapeutic effect in HPV-transformed cells, while relatively No damage to surrounding normal epithelial cells This finding demonstrates the real clinical potential of the present invention for HPV-induced precancerous lesions.

[0235] Example 4 Measurement of Stability and pH of Decitabine under Controlled Storage Conditions

[0236] The stability of decitabine (0.228 mg / g, corresponding to 1 mM decitabine) in a formulation as described in Table 2 (polyethylene glycol-based formulation; ointment) was measured (at the beginning of the experiment (0), and after one month (1) and three months (3) under controlled storage conditions (i) 5°C + / - 3°C (Table 12) and (ii) 25°C + / - 2°C (60% RH + / - 5%; RH: relative humidity)) (Table 13). At these time points, in addition to the stability of decitabine, the pH of the formulation was also measured. The stability of decitabine was measured by HPLC as described in Example 1.

[0237]

[0238] (Table 12)

[0239]

[0240] (Table 13)

[0241] The stability of decitabine is defined as "% LC": "% of labelled amount" defines the amount of decitabine as active ingredient in the tested formulation provided as % based on the initial amount of decitabine as a reference value (100%: 0.228 mg / g). The results clearly show that a very high percentage of decitabine remains stable for up to 3 months, regardless of the storage conditions applied. The pH of the formulation is essentially constant during the observation period. Thus, an unexpectedly high level of stability of the decitabine formulation is achieved without the addition of any components other than PEG and DMSO.

[0242] Example 5 Effect of decitabine preparation on HPV-induced precancerous tumors cultured in vitro

[0243] Example 5 investigated the effect of decitabine formulations on HPV-induced precancerous tumors cultured in vitro following their topical administration.

[0244] 1. Sample preparation:

[0245] Biopsies obtained from patients with precancerous lesions of the vulva (ie, high-grade vulvar intraepithelial neoplasia, HG-VIN) were cut into individual pieces using a scalpel. A portion of each individual biopsy was fixed in formalin and embedded in paraffin.

[0246] Corresponding formalin-fixed paraffin-embedded (FFPE) tissue from this reference biopsy sample was tested for the presence of HPV DNA, genotyped for high-risk (HR) HPV types, and analyzed by immunohistochemistry for the cellular protein p16 INK4a Overexpression of HR-HPV DNA and p16 INK4a Combined positivity of expression indicates HPV-induced transformation.

[0247] In addition, fresh (i.e., unfixed) biopsy sections were carefully placed on top of different dermis equivalents (DE; separated from the ex vivo cultured lesions by a dashed outline in the images) and cultured ex vivo for 3 to 5 days to allow lesional HPV-transformed epithelial cells to adhere and grow on the surface of the DE. Subsequently, over a period of more than two weeks ( Fig.10 A. Fig.10 B) or four weeks ( Fig.10 C. Fig.10 D) at a strength of 1 mM using a PEG-based decitabine formulation as described in Table 2 ( Fig.10 B. Fig.10 D), or a corresponding placebo preparation as a control ( Fig.10 A. Fig.10 C), the respective cultures were treated ex vivo topically 5 times per week. Standardized topical application was achieved by carefully placing small plates made of laboratory sealing film loaded with the preparation on the surface of the ex vivo culture exposed to air to allow the preparation to cover the entire epithelial surface. After the treatment period, the cultures were fixed in formalin and embedded in paraffin.

[0248] Tissue sections from the resulting FFPE blocks were subjected to p16 INK4a Immunohistochemical staining was performed and whole-slice scanning was performed. The tissue architecture and tumor cell morphology as well as p16 expression were evaluated on the scanned images. INK4a The expression of proteins was compared with that of PEG-based decitabine formulations [1 mM] ( Fig.10 B. Fig.10 D) or the corresponding placebo preparation ( Fig.10 A. Fig.10 C) Processed biopsy section from the same specimen.

[0249] 2. Results

[0250] Fig.10 In vitro culture and treatment with PEG-based decitabine formulations ([1 mM], Fig.10 B and Fig.10 D) or the corresponding placebo preparation ( Fig.10 A and Fig.10C) Images of two HG-VIN biopsies processed more than two weeks or four weeks later. In the analysis of the respective reference biopsy samples, both lesions were confirmed to be HPV-transformed (i.e., HR-HPV DNA positive and p16 INK4a HPV-transformed HG-VIN ex vivo cultures treated with placebo formulations for more than two weeks (A) or four weeks (C) showed a multi-stratified epithelium with strong and diffuse nuclear and cytoplasmic p16 INK4a Expression (brown staining in the original color image, dark grey staining in the submitted black and white image and highlighted with arrows) indicates actively HPV-transformed cells.

[0251] In contrast, ex vivo cultured HG-VIN biopsies from the same lesions treated with a PEG-based decitabine formulation [1 mM] for more than two weeks (B) or four weeks (D) did not show strong and diffuse nuclear and cytoplasmic p16 INK4a In contrast, evidence of tumor cell degradation was seen, including the loss of intact nuclei and accumulation of cellular debris above the DE.

[0252] The results obtained show that topical treatment of HPV-transformed precancerous lesions cultured in vitro with a PEG-based decitabine-containing formulation according to the present invention results in penetration of decitabine into target cells, reversal of the HPV-transformed phenotype, and substantially complete degradation of the corresponding tumor cells. These effects were demonstrated by using the experimental method of Example 5 as a real disease model based on patient samples and by adopting an administration route intended for use in a clinical setting (i.e., topical administration).

Claims

1. A pharmaceutical composition comprising: - decitabine or azacitidine, or a pharmaceutically acceptable salt thereof; - polyethylene glycol (PEG); and -Dimethyl sulfoxide (DMSO).

2. The pharmaceutical composition according to claim 1, wherein the composition comprises decitabine (or its salt) at a concentration of 0.005 mM to 100 mM, preferably 0.01 mM to 50 mM, more preferably 0.1 mM to 10 mM (0.0228 mg / g to 2.28 mg / g).

3. A pharmaceutical composition comprising 0.1 mM to 10 mM (0.0228 mg / g to 2.28 mg / g) of decitabine (or a pharmaceutically acceptable salt thereof) for medical use, wherein the pharmaceutical composition is topically administered.

4. The pharmaceutical composition according to any one of the preceding claims, having a complex viscosity of about 1000 Pa.s to about 25000 Pa.s at 25°C, preferably about 2000 Pa.s to about 15000 Pa.s at 25°C, even more preferably about 3000 Pa.s to about 11000 Pa.s at 25°C.

5. The pharmaceutical composition according to any one of the preceding claims, comprising at least two different PEG components, wherein the first PEG component has a solid physical state at 25°C and the second PEG component has a liquid physical state at 25°C.

6. The pharmaceutical composition according to any one of the preceding claims, comprising at least two different PEG components, wherein the first PEG component is PEG 1000 to PEG 8000 and the second PEG component is PEG 200 to PEG 600.

7. A pharmaceutical composition according to any one of the preceding claims, comprising two different PEG components, wherein the first PEG component is PEG 1500 and the second PEG component is PEG 300.

8. A pharmaceutical composition according to any one of the preceding claims, comprising DMSO in a concentration (weight to weight (w / w)) of 1% to 15%, preferably 2% to 10%, more preferably 4% to 7%, even more preferably 5% to 6%, still more preferably about 5.7%.

9. The pharmaceutical composition according to any one of the preceding claims, wherein the composition is a non-liquid, semi-solid composition at 25°C.

10. A pharmaceutical composition according to any one of the preceding claims, wherein the composition does not comprise water.

11. The pharmaceutical composition according to any one of the preceding claims, wherein the composition does not comprise (any other) polar protic solvents, such as ethanol.

12. A pharmaceutical composition according to any one of the preceding claims, wherein the composition does not contain any of the following components: stabilizers other than PEG, acidulants, buffer salts, pH adjusters, desiccants, antioxidants, glycerol and polypropylene glycol or mixtures thereof.

13. A pharmaceutical composition according to any one of the preceding claims, wherein the composition does not comprise any components other than decitabine or azacitidine (or a salt thereof), PEG and DMSO.

14. A pharmaceutical composition according to any one of the preceding claims comprising decitabine or azacitidine (or a salt thereof), PEG 1500, PEG 300 and DMSO.

15. A pharmaceutical composition according to any one of the preceding claims, comprising or consisting of 0.1 mM to 10 mM (0.0228 mg / g to 2.28 mg / g) decitabine (or azacitidine), 5.7% (w / w) DMSO, 40% (w / w) PEG1500 and 54% (w / w) to 55% (w / w) PEG 300.

16. A pharmaceutical composition according to any one of the preceding claims for use in medicine.

17. The pharmaceutical composition according to any one of claims 1 to 15 for use according to claim 16, wherein the pharmaceutical composition is administered topically.

18. A pharmaceutical composition according to any one of claims 1 to 15 for use according to claim 16 or 17, wherein the composition is for treating a medical symptom or medical condition of keratinized epithelium / skin and non-keratinized epithelium / skin.

19. A pharmaceutical composition according to any one of the preceding claims for use in the treatment of human papillomavirus (HPV) related precancerous conditions, wherein the pharmaceutical composition is administered topically.

20. The pharmaceutical composition according to any one of claims 1 to 15 for use according to any one of claims 16 to 19, wherein the composition is applied to the anogenital area, in particular the vulva.

21. A method for preparing a pharmaceutical composition according to any one of the preceding claims, comprising the steps of: (1) preparing a DMSO solution of decitabine or azacitidine (or its salt); (2) melting the PEG component to obtain a homogeneous composition; and (3) Mixing the decitabine (or azacitidine) solution of step (1) with the melted PEG component of step (2) to obtain a homogenous composition.

22. A package comprising a pharmaceutical composition according to any one of claims 1 to 15.

23. The package according to claim 22, wherein the package is designed as a deformable tube.

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