(19)
(11)EP 2 861 235 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
28.08.2019 Bulletin 2019/35

(21)Application number: 13744833.8

(22)Date of filing:  13.06.2013
(51)Int. Cl.: 
A61K 31/593  (2006.01)
A61P 35/00  (2006.01)
A61K 31/606  (2006.01)
(86)International application number:
PCT/IB2013/054846
(87)International publication number:
WO 2013/186734 (19.12.2013 Gazette  2013/51)

(54)

CHEMOPREVENTION OF COLORECTAL CANCER

CHEMOPRÄVENTION VON KOLOREKTALKARZINOMEN

CHIMIO-PRÉVENTION DU CANCER COLORECTAL


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 15.06.2012 IT MI20121041

(43)Date of publication of application:
22.04.2015 Bulletin 2015/17

(73)Proprietor: Sofar SPA
20060 Trezzano Rosa (MI) (IT)

(72)Inventors:
  • GRANDE, Alexis
    I-41126 Modena (IT)
  • FERRARINI, Fabrizio
    I-41125 Modena (IT)
  • PARENTI, Sandra
    I-41122 Modena (IT)

(74)Representative: Biggi, Cristina et al
Bugnion S.p.A. Viale Lancetti 17
20158 Milano
20158 Milano (IT)


(56)References cited: : 
WO-A2-99/56697
US-A1- 2001 049 364
WO-A2-2006/125073
  
  • OH C J ET AL: "Treatment with anti-tumor necrosis factor alpha (TNF alpha) monoclonal antibody dramatically decreases the clinical activity of psoriasis lesions", JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY, MOSBY, INC, US, vol. 42, no. 5 part 1, 1 May 2000 (2000-05-01), page 829/830, XP000990581, ISSN: 0190-9622, DOI: 10.1067/MJD.2000.105948
  • JONES GLENVILLE: "Vitamin D analogs.", ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA JUN 2010, vol. 39, no. 2, June 2010 (2010-06), pages 447-472 , tab, ISSN: 1558-4410
  • DELUCA HECTOR F: "Overview of general physiologic features and functions of vitamin D.", THE AMERICAN JOURNAL OF CLINICAL NUTRITION DEC 2004, vol. 80, no. 6 Suppl, December 2004 (2004-12), pages 1689S-1696S, ISSN: 0002-9165
  • CHRISTAKOS SYLVIA ET AL: "Vitamin D: metabolism.", ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA JUN 2010, vol. 39, no. 2, June 2010 (2010-06), pages 243-253 , tab, ISSN: 1558-4410
  • JONES GLENVILLE: "Vitamin D Analogs", ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMER, W.B. SAUNDERS COMPANY, PHILADELPHIA, vol. 39, no. 2, 1 June 2010 (2010-06-01), pages 447-472, XI, XP009160069, ISSN: 0889-8529, DOI: 10.1016/J.ECL.2010.02.003
  • CHRISTAKOS SYLVIA ET AL: "Vitamin D: Metabolism", ENDOCRINOLOGY AND METABOLISM CLINICS OF NORTH AMERICA, vol. 39, no. 2, June 2010 (2010-06), pages 243-253,VII, ISSN: 0889-8529
 
Remarks:
The file contains technical information submitted after the application was filed and not included in this specification
 
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The present invention relates to a new combination of
  1. (i) 5-aminosalicylic acid '(S-ASA) or a pharmacologically acceptable salt thereof or a derivative thereof selected from the group consisting of: sulfasalazine, olsalazine and balsalazide ,
  2. (ii) a group D vitamin, and
  3. (iii) at least one physiologically acceptable excipient and to the use thereof in the prevention and/or treatment of colorectal cancer.


[0002] Another aspect of the invention relates to a pharmaceutical composition containing said combination and to said composition for use in the prevention and/or treatment of colorectal cancer.

PRIOR ART



[0003] Non Steroidal Anti-Inflammatory Drugs, NSAIDS, are characterized by a recognized chemopreventive activity against colorectal cancer (CRC) (O'Morain C et al., World J Gastroenterol, 2009;1(1):21-15). Unfortunately, the systemic and gastrointestinal toxicity of NSAIDs drastically limits the administration thereof in the area of protocols that require long-term treatment of the patients concerned (Guadagni F. et al., Anticancer Res, 2007;27:3147-3162). Different studies indicate that 5-ASA is a promising alternative for obtaining a comparable chemopreventive activity against CRC, at the same time avoiding the side effects induced by NSAIDs (Rubin DT et al., Inflamm Bowel Dis, 2008;14:65-274). In fact, despite the chemical similarity with aspirin, i.e. a typical NSAID, 5-ASA is characterized by weak inhibition of cyclooxygenase, and by negligible systemic absorption, which are properties that clearly explain the clinical safety of this therapeutic agent (Bergman R. et al., Aliment Pharmacol Ther, 2006;23:841-855). In this respect, it comes as no surprise that the anti- inflammatory effect of the 5-ASA is mediated by other mechanisms, amongst which an important role is probably played by the inhibition of transcription factors that promote the immune response, such as NFkB and the PPARs (Lyakhovich A. et al., Aliment Pharmacol Ther, 2010;31(2):202-9).

[0004] US 2001/049364 disclosing a composition comprising 5-ASA, vitamin D3 and calcium for the prevention of the initiation and progression of colorectal cancer.

[0005] WO 2006/125073 discloses the use of 5-ASA sulfasalazine, olsalazine and balsalazide in the treatment of colorectal cancer.

[0006] Recent studies have then shown that the 5-ASA interferes with the proliferation signaling pathway mediated by the β-catenin. This protein is a transcription factor that is constitutionally activated in almost all cases of colorectal cancer. It is known that the state of phosphorylation of the β-catenin indirectly influences its transcription function, considering that this protein is degraded in the cytoplasm when it is phosphorylated whereas it is translocated to the nucleus when it is dephosphorylated. Bos et al., Carcinogenesis 2006;27(12):2371-82 have demonstrated that 5-ASA is able to inhibit the enzyme activity of the phosphatase PP2A, normally responsible for dephosphorylation of the β--catenin, thus reducing the nuclear translocation thereof. Parenti S. et al., Aliment Pharmacol Ther; 31(1):108-19 and Losi L. et al., Hum Pathol have also demonstrated that treating CRC cancer cells with 5-ASA induces the expression of a membrane protein, known as µ-protocadherin, which belongs to the superfamily of the cadherins and is able to sequester the B-catenin on the plasma membrane, preventing the β--catenin from translocating to the nucleus and activating the transcription of the target genes thereof.

[0007] EP223958O has further demonstrated that the nuclear translocation of the β--catenin is inhibited after treatment with 5-ASA owing to the induction of a membrane protein belonging to the superfamily of the cadherins, said itprotocadherin. This cadherin bonds and sequestrates in membrane the B-catenin, preventing the nuclear translocation thereof and the transcription of the target genes thereof.

[0008] Numerous epidemiological studies thereof suggest that also administering vitamin D3 (VD3) significantly reduces the occurrence of CRC. In addition to regulating calcium homeostasis, in fact, VD3 is also characterised by important antiproliferative, pro-differentiating and pro-apoptotic activity performed on numerous cell types amongst which it is also possible to cite cancer enterocytes of the colon (Jimenez-Lara AM., Int" J Biochem Cell, Biol 20()7;39(4):672-7). For this purpose, Palmer et al., J Cell Biol 2001 ;154(2):369-87 have demonstrated that VD3 interferes with the signal pathway of the β--catenin through a mechanism similar to that already mentioned for the 5-ASA that consists of a sequestration of this transcription factor on the plasma membrane that is in this case mediated by the hyperexpression of the E-cadherin or of the main epithelial cadherin.

DESCRIPTION OF THE INVENTION



[0009] The object of the present invention consists of finding new and more effective therapies for the treatment of colorectal cancer. It was surprisingly found that the combination of (i) 5 -aminosalicylic acid (5-ASA) or a derivative thereof selected from the group consisting of: sulfasalazine, olsalazine and balsalazide, or a pharmacologically acceptable salt thereof, (ii) a group D vitamin and (iii) at least one physiologically acceptable excipient, enables the active principles to exert a synergic effect compared with the action of each single treatment on the transduction pathway of the proliferative signal of the β--catenin, the constitutive activation of which is observed in the progression of 90% of the colorectal cancers.

[0010] The present invention thus relates to a combination of (i) 5-aminosalicylic acid (5-ASA) or a derivative thereof selected from the group consisting of: sulfasalazine, olsalazine and balsalazide, or a pharmacologically acceptable salt thereof, (ii) a group D vitamin, and (iii) at least one physiologically acceptable excipient, to a pharmaceutical composition comprising this combination and to the use of said combination and/or pharmaceutical composition in the prevention and/or in the treatment of colorectal cancer.

[0011] The combination of the invention has the advantage of being able to reduce the therapeutic doses of both components with respect to the dosage that is usual in the prevention and/or in the treatment of colorectal cancer.

[0012] Preferably, the present invention relates to a combination of (i) 5-aminosalicylic acid (5-ASA) and (ii) a group D vitamin, a derivative thereof, a metabolite or analogue.

[0013] The derivative of the 5-aminosalicylic acid (5-ASA) is preferably chosen from the group comprising sulfasalazine (acid 6-oxo-3-((4-(pyridin-2-yl)sulfamoyl]phenyl)hydrazinylidene]cyclohexa-1,4-diene-1-carboxylic), olsalazine (acid 5-[(2Z)-2-(3-carboxy-4-oxo-1-cyclohexa-2,5-dienilidene)hydrazinyl]-2-hydroxy-benzoic) and balsalazide (acid 5-[4-(2-carboxyethylcarbamyl")phenyl]diazenyl-2-hydroxy-benzoic).

[0014] Preferably, the group D vitamin, or a derivative thereof, metabolite or similar is chosen from the group comprising vitamin D3 (or cholecalciferol, IUPAC name: (3S,9S,10R,13R,14R,17R)-17-((2R,5R,E)-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-01), vitamin D2 (or ergocalciferol, IUPAC name: (3S,9S,10R,13R,14R,17R)-17-((2R,5R,E)-5,6-dimethylheptan-3-en-2-yl)-10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol), 25-hydroxy-vitamin D3, (or calcifediol or calcidiol, IUPAC name: (6R)-6-[(1R,3aR,4E,7aR)-4-[(2Z)-2-[(5S)-5-hydroxy-2-methylidene-cyclohexylidene] ethylidene]-7a methyl-2,3,3a,5,6,7-hexahydra-1H-inden-1-yl]-2-methyl-heptan-2-ol) and 1alpha,25 dihydroxy-vitamin D3 (or calcitriol, IUPAC name: (1R,3S)-5-[2-[(1R,3aR,7aS)-1-[(2R)-6-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidene-cyclohexane-1,3-diol).

[0015] More preferably, the combination of the invention comprises vitamin D3. According to one aspect of the present invention, said combination of 5-aminosalicylic acid (5-ASA) and vitamin D3 does not comprise calcium. According to a further aspect of the present invention, said combination consists of 5-aminosalicylic acid (5-ASA) and vitamin D3.

[0016] According to one aspect, the present invention relates to a combination of 5-aminosalicylic acid (5-ASA) and vitamin D3, to a pharmaceutical composition comprising this combination in association with at least one physiologically acceptable excipient and to the use of said combination and/or pharmaceutical composition in the prevention and/or in the treatment of colorectal cancer. '

[0017] According to another aspect, the present invention relates to the use of the combination and/or of the pharmaceutical composition of the present invention for preventing colorectal cancer in persons with a different risk of developing neoplasms, such as, for such example: healthy/normal individuals; patients affected by chronic inflammatory diseases of the intestine (such as Crohn's disease and ulcerous rectocolitis); patients subjected to endoscopic removal of adenomas and/or adenocarcinomas of the colon-rectum; patients affected by genetic cancer syndromes (like Lynch syndrome and familial adenomatous polyposis).

[0018] Suitable pathways for administering the combination and/or pharmaceutical composition of the invention include oral, intramuscular, subcutaneous or rectal administration, preferably oral administration.

[0019] The pharmaceutical composition of the present invention can be formulated as a combined preparation, for simultaneous, sequential or separate administration of the components of the combination, as pharmaceutical compositions in which the two components are present in the same dosage unit or in separate dosage units.

[0020] Preferably, separate administration of the two components occurs in an interval of time comprised between 1 and 24 hours.

[0021] The pharmaceutical composition of the present invention can be formulated in the form of tablets, capsules, granules, pills, lozenges.

[0022] According to a preferred aspect, the pharmaceutical composition of the present invention comprises the two components of the combination in association with at least one physiologically acceptable excipient in the same dosage unit in compressed form for oral administration.

[0023] The use of the combination of the invention for preventing and/or treating colorectal cancer involves daily administration of a quantity of 5-aminosalicylic acid (5-ASA) or a derivative thereof, or a pharmacologically acceptable salt thereof comprised in the range 0.5 to 5 g, preferably of about 2.4 g, and a quantity of a group D vitamin, a derivative thereof, a metabolite or analogue comprised in the range 500 to 10,000 IU, preferably about 2000 IU.

[0024] The quantity of composition of the invention to be administered to the patient can vary according to various factors that are well known to the skilled persons, for example, the weight of the patient, the administration pathway and the seriousness of the illness.

[0025] According to a further aspect, the invention refers to a pharmaceutical composition that is suitable for the controlled release of the two components into distinct zones of the intestine, such as the small intestine for the group D vitamin, a derivative thereof, a metabolite or analogue, intended for systemic absorption in this place, and the colon for the 5-aminosalicylic acid (5-ASA) or a derivative thereof, or a pharmacologically acceptable salt thereof, where this compound has a topical effect, so that the corresponding concentrations of the two active components are optimum for the purposes of the object of the invention.

[0026] According to a preferred aspect, the combination and/or pharmaceutical composition of the present invention is administered to mammals, particularly to humans.

SHORT DESCRIPTION OF THE FIGURES



[0027] 

Figure 1. Growth curve of the cell line HT29 subjected to treatment with 5-ASA, VD3 and 5-ASA + VD3. On the x axis treatment times are shown, on the y axis the number of cell expansions is shown compared with the initial number of cells. The data have been shown as averages ± s.e.m obtained from three independent experiments.

Figure 2. QRT-PCR analysis of the RNA messengers of genes belonging to the signal pathway of the β chain in HT29 cells treated with 5-ASA, VD3 and 5-ASA + VD3. The analysed genes are shown on the x axis, whilst the quantitative variations of the levels of RNA messengers are shown on they axis. The data have been displayed as averages ± s.e.m obtained from three independent experiments.

Figure 3. Growth curve of the cell line CaCo2 subjected to treatment with 5-ASA and ASA. On the x axis, the analysed compounds and the concentrations thereof are shown, on the y axis, the number of cell expansions is shown compared with the initial number of cells. The data have been displayed as averages ± s.e.m obtained from three independent experiments. '

Figure 4. QRT-PCR analysis of the RNA messengers of the genes belonging to the7 13GI20E signal pathway of the [3 chain in CaCo2 cells treated with 5-ASA and ASA. The genes analyzed are shown on the x axis, whereas the quantitative variations of the levels of RNA messenger are shown on the y axis. The data have been displayed as averages ± s.e.m obtained from three independent experiments.


EXAMPLES


MATERIAL AND METHODS 1



[0028] The cell lines CaCo2 and HT29 of colorectal adenocarcinoma obtained from the ATCC (Rockville, MD, USA) were grown in a DMEM culture medium (Euroclone, Devon, UK), to which were added 10% fetal bovine serum (Lonza, Walkersville, MD, USA) inactivated at heat and 1mM of L-glutamine (Euroclone). The 5-aminosalicylic acid (5-ASA, SOFAR S.p.A., Milan, Italy) and acetylsalicylic acid (ASA, Sigma Aldrich, St Louise, MO, USA) were dissolved in a culture medium in a concentration of respectively 20 and 10 mM. The vitamin D3 (VD3) (1 alpha, 25 dihydroxyvitamin D3, calcitriol, Sigma Aldrich) was diluted in the culture medium at a concentration of 107 M. For the experiments conducted with the cell line HT29, 300,000 cells were sown for each sample and 5-ASA, vitamin D3 or the combination of 5-ASA and VD3 were added to each well. For the experiments conducted with the cell line CaCo2, on the other hand, 300,000 cells were again sown for each sample, but adding 5-ASA or ASA to each well. The treated cells were then subjected to a cell count every 24 hours to monitor the anti-proliferation effect of the different stimuli. After 96 hours of treatment all the cells were collected, resorbed and the RNA was extracted by means of the "Qiagen total RNA purification kit" following the manufacturer's instructions (Qiagen, Valencia, CA). The integrity and concentration of the RNA were tested using the Bio-Analyzer technique (Applied Biosystem, Foster City, CA). 100 ng total of RNA were then retrotranscribed using the High Capacity cDNA Archive Kit (Applied Biosystems) on the basis of the instructions provided by the manufacturer. The QRT-PCR was then carried out with an ABI PRISM 7900 (Applied Biosystems) sequence detecting system for quantifying the relative levels of mRNA measured in the various samples. The triggers and probes for amplifying the mRNA of the µ-protocadherin, E-cadherin, p21waf-1, KLF4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were devised by Applied Biosystems. Each sample of cDNA was processed in triplicate in 50 ul of reaction volume using the Taqman Universal PCR Master Mix (Applied Biosystems).

[0029] Thermal cyclisation was started with initial denaturation at 50°C for 2 minutes and at 90°C for 10 minutes followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.

[0030] The QRT-PCR signals were evacuated using the corresponding quantification method ΔΔCt (Livak KJ, Schmittgen TD. Relative gene expression data were analysed using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25(4):402-8). This procedure calculates the relative difference in gene expression of the target gene normalised on the endogen control (GAPDH) and compared with a calibrating sample. The values obtained were expressed as a relative quantity (RQ) of the variation in the messenger levels.

EXAMPLE 1


Analysis of the antiproliferal effects promoted by treatment with 5-ASA and VD3 of the adenocarcinoma cell line HT29 of the colon-rectum.



[0031] The colorectal adenocarcinoma cell line HT29, which was chosen for its responsiveness to the VD3 deriving from the corresponding VDR receptor, was subjected to "in vitro" treatment with 5-ASA 20 mM and VD3 10-7 M, used individually and in association, in order to quantify the antiproliferative effect determined by the compounds analysed on cancerous colonocytes. The plotting of the growth curves, based on cell-count values obtained from this experiment, has shown that the treatment combined with 5-ASA and VD3 determines an additional antiproliferative effect that is clearly superior to what has been observed with the single treatments. This observation is evident above all at 96 hours of stimulation, where 14 expansions were detected in the control, 8 times in the treatment with 5-ASA, 10 times in the treatment with VD3 and only 5 times in the combined treatment (Figure 1). These data confirm the antiproliferal effects achieved individually by treatment with 5-ASA and VD3 in colorectal cancer cells, further demonstrating the existence of an additional effect resulting from the pharmacological association thereof.

EXAMPLE 2


QRT-PCR analysis of genes that regulate proliferation, differentiation and apoptosis in colorectal adenocarcinoma cells HT29 of the colon-rectum subjected to treatment with 5-ASA and VD3.



[0032] In order to check at the molecular level the effects determined on the cell line HT29 by the association 5-ASA / VD3, "Real Time", quantitative RT-PCR was used to analyse the expression of a series of genes the protein products of which are involved in the regulation of the proliferation, differentiation and apoptosis of cancer colonocytes such as µ-protocadherin, E-cadherin, p21waf-1 and KLF4. This analysis was conducted on cell cultures subjected to 96 hours of treatment with the studied drugs, which were used in the previously disclosed concentrations. The choice of the listed genes derives from a series of observations explained below. M-protocadherin and E-cadherin are proteins belonging to the family of the cadherins that mediate intercellular adhesion and therefore, indirectly, have an antimetastatic activity. It has also been demonstrated that they regulate cell proliferation by sequestering, on the plasma membrane, the transcription factor β-catenin and preventing it from activating the transcription of the target genes. For the reasons set out, the expression of these cadherins is frequently down-regulated in epithelial tumors. The codifying gene for the protein p21waf1 is one of the main negative targets of the β-catenin and is an important regulator of the cell cycle that is able to determine, following a massive induction of the expression thereof, proliferative arrest following cell death by apoptosis. Lastly, the KLF4 is a codifying oncosuppressor gene for a transcription factor that plays a crucial role in regulating the proliferation and differentiation of the colonocytes, partly deriving from the ability thereof to contrast the proliferative activity mediated by the β-catenin, or through molecular mechanisms that are only partially characterised. The importance of the genes mentioned so far is also fully underlined by the fact that they are all involved in the occurrence and progression of colorectal cancers.

[0033] The data obtained have demonstrated that single treatment with 5-ASA determines an increase in the messenger expression that is 2.5 times for the µ-protocadherin, 2.2 times for E-cadherin, 2.9 times for p21 and 2.2 times for KLF-4. After treatment with VD3, these values are respectively 1, 1.7, 1.5 and 1.6, whereas following combined treatment, they are respectively 3.2, 3.1, 4.2 and 3.1, showing that the association of the drugs in questions determines an additive effect on the induction of the messenger expression of the examined genes (Figure 2).

[0034] These results clearly make the association 5-ASA/VD3 suitable for chemiopreventive use against colorectal cancers.

EXAMPLE 3


Comparative analysis of the antiproliferal and molecular effects promoted by treatment with 5-ASA and ASA in the colorectal adenocarcinoma cell line.



[0035] In order to characterise further the profile of efficacy and safety associated with the chemopreventive properties of 5-ASA, a series of experiments was conducted of the same type as the series explained previously, in which a comparative analysis was conducted of the biological effects determined on cancer colonocytes following treatment with 5-ASA and with aspirin (ASA), i.e. the main drug of the family of NSAIDs. These experiments were conducted in the colorectal adenocarcinoma cell line, owing to the elective responsive thereof to the 5-ASA emerging from the comparison with other cell lines of the same origin, exposing the culture to a treatment time of 96 hours.

[0036] The obtained results have demonstrated that although the ASA has a more pronounced antiproliferative effect than 5-ASA, this is associated withi a clear toxic effect that is created at a concentration of 20 mM of the analysed drugs at which all the cells treated with 5-ASA survive, despite the complete proliferative block indicated by the cellularity "fold change" value equal to 1, whereas the cells subjected to treatment with ASA 80% mortality, indicated by the cellularity "fold change" value equal to 0.2 (Figure 3).

[0037] An additional observation that arises from these results consists of the fact that a 10 mM concentration of ASA and 20 mM of 5-ASA can in fact be considered to be equivalents of the two compared drugs as they promote, on the cancer cells subjected to the treatment, an absolutely superimposable antiproliferative effect (Figure 3).

[0038] On the samples taken from these cell cultures and from other cultures used as a control, the levels of messenger RNA of the genes listed in the previous paragraph (see Figure 2) was determined by Quantitative Real Time RT-PCR. The obtained results have shown that the messenger expression of the µ-protocadherin, E-cadherin, p21waf1 and KLF4 genes were induced respectively 3.8, 2.4, 8.0 and 5.5 times after treatment with 5-ASA 20 mM (Figure 4), confirming the data previously observed on the cell line HT29. The treatment with the equivalent dose of ASA, equal to 10 mM, does not, on the other hand, induce any of the genes analysed with the sole exception of the gene p21waf1 whose expression shows an increase of 5.8 times that thus appears to be comparable with what was observed in the corresponding treatment with 5-ASA. This datum is not surprising, considering the redundancy and heterogeneity that characterize molecular regulation of the gene p21waf1, the expression of which is not only regulated by the proliferation signaling pathway of the β chain, explored in our experimental conditions through the evaluation of the codifying genes for µ-protocadherin, E-cadherin and KLF4, but also by numerous signalling pathways. The most plausible conclusion arising from this result consequently consists of the consideration that the ASA induces the expression of the p21waf1 through molecular mechanisms that are other than those that mediate the same effect through 5-ASA, thus demonstrating a specificity of action of the latter in relation to the compared drug. Overall, the data presented so far thus indicate that: 1) 5-ASA inhibits the signal pathway of the β-catenin and, consistently, the proliferative activity of cancer colonocytes, working in cooperation with the VD3, which makes this pharmacological association suitable for chemiopreventive use against colorectal cancer; 2) the 5-ASA is less toxic and thus safer than the ASA for the purposes of prolonged use such as that required for the purposes of obtaining chemiopreventive protection; 3) equivalent doses of ASA and 5-ASA act through distinct molecular mechanisms, which vindicates the specificity of action of the 5-ASA compared with the ASA.

Bibliography



[0039] 
  1. 1. Reinacher-Schick A, Schoeneck A, Graeven U, Schwarte-Waldhoff I, Schmiegel W. Mesalazine causes a mitotic arrest and induces caspase-dependent apoptosis in colon carcinoma cells. Carcinogenesis 2003;24(3):443-51.
  2. 2. Luciani MG, Campregher C, Fortune JM, Kunkel TA, Gasche C. 5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint. Gastroenterology 2007;132(1):221-35.
  3. 3. Stolfi C, Pellegrini R, Franze E, Pallone F, Monteleone G. Molecular basis of the potential of mesalazine to prevent colorectal cancer. World J Gastroenterol 2008;14(28):4434-9.
  4. 4. Lyakhovich A,.Gasche C. Systematic review: molecular chemoprevention of colorectal malignancy by mesalazine. Aliment Pharmacol Ther;31(2):202-9.
  5. 5. Bos CL, Diks SH, Hardwick JC, Walburg KV, Peppelenbosch MP, Richel DJ. Protein phosphatase 2A is required for mesalazine-dependent inhibition of Wnt/beta-catenin pathway activity. Carcinogenesis 2006;27(12):2371-82.
  6. 6. Parenti S, Ferrarini F, Zini R, Montanari M, Losi L, Canovi B, et al. Mesalazine inhibits the beta-catenin signalling pathway acting through the upregulation of mu-protocadherin gene in colo-rectal cancer cells. Aliment Pharmacol Ther;31(1):108-19.
  7. 7. Losi L, Parenti S, Ferrarini F, Rivasi F, Gavioli M, Natalini G, et al. Down-regulation of mu-protocadherin expression is a common event in colorectal carcinogenesis. Hum Pathol.
  8. 8. Grande AP, S. Ferrarini, F., inventor Determination of 5-ASA efficacy in CRC prevention and/or treatment by gene expression analysis. 2009.13 13GI20E
  9. 9. Tagliafico E, Tenedini E, Manfredini R, Grande A, Ferrari F, Roncaglia E, et al. Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia. Leukemia 2006;20(10):1751-8.
  10. 10. Jimenez-Lara AM. Colorectal cancer: potential therapeutic benefits of Vitamin D. Int J Biochem Cell Biol 2007;39(4):672-7.
  11. 11. Palmer HG, Gonzalez-Sancho JM, Espada J, Berciano MT, Puig I, Baulida J , et al. Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling. J Cell Biol 2001 ;154(2):369-87.
  12. 12. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001 ;25(4):402-8.



Claims

1. A combination of:

(i) 5-aminosalicylic acid, or a pharmacologically acceptable salt thereof, or a derivative thereof selected from the group consisting of: sulfasalazine, olsalazine and balsalazide, and

(ii) a group D vitamin, and

(iii) at least one physiologically acceptable excipient

or a pharmaceutical composition comprising said combination for use in the prevention and/or treatment of colorectal cancer in a subject.
 
2. The combination or the composition for use according to claim 1 characterized in that said D vitamin is selected from the group comprising: vitamin D3, vitamin D2, 25-hydroxy-vitamin D3 and 1alpha,25 dihydroxy-vitamin D3.
 
3. The combination or the composition for use according to any one of claims 1-2, comprising 5- aminosalicylic acid and vitamin D3.
 
4. The combination or the composition for use according to anyone of claims 1-3 wherein the composition comprising the combination does not comprise calcium.
 
5. The combination or the composition for use according to any one of claims 1-3 characterized in that said 5-aminosalicylic acid, or a pharmacologically acceptable salt thereof is administered in a daily dose comprised between 0.5 and 5 g, approximately 2.4 g preferably and said group D vitamin or an analogue thereof is administered in a daily dose comprised between 500 and 10.000 IU, preferably approximately 2000 IU.
 
6. The combination or the composition for use in the prevention of colorectal cancer according to any one of claims 1-5, wherein said subject is healthy/normal or is a patient affected by chronic inflammatory diseases of the intestine, preferably Crohn's diseases or ulcerous rectocolitis, or is a patient subjected to endoscopic removal of adenomas and/or adenocarcinomas of the colon-rectum, or is a patient affected by genetic cancer syndromes, preferably Lynch syndrome or Familiar Adenomatous Polyposis.
 
7. The combination or the composition for use according to any one of claims 1-6, characterized in that this subject is a mammal, preferably a human.
 
8. The combination or the composition for use according to any one of claims 1-7, wherein said combination or composition is for oral, intramuscular, subcutaneous or rectal administration.
 
9. The combination or the composition for use according to claim 8, wherein said combination or composition is in the form of tablets, capsules, granules, pills, lozenges.
 
10. The combination or the composition for use according to any one of claims 8-9, wherein said combination or composition is for controlled release.
 
11. The combination or the composition for use according to anyone of claims 8-10, wherein said combination or composition is a combined preparation, suitable for simultaneous, sequential or separate administration of the components of the combination.
 
12. The combination or the composition for use according to anyone of claims 8-11, wherein the treatment of colorectal cancer is due to antiproliferative effect on cancer cells of colon-rectum.
 
13. The combination or the composition for use according to claim 12, wherein said antiproliferative effect involves overexpression of µ-protocadherin, E-cadherin, p21 and KLF4.
 


Ansprüche

1. Kombination von:

(i) 5-Aminosalicylsäure oder einem pharmakologisch verträglichen Salz davon oder einem Derivat davon, ausgewählt aus der Gruppe bestehend aus: Sulfasalazin, Olsalazin und Balsalazid, und

(ii) einem Vitamin der Gruppe D und

(iii) mindestens einem physiologisch verträglichen Hilfsstoff

oder einer pharmazeutischen Zusammensetzung, umfassend die Kombination zur Verwendung bei der Prävention und/oder Behandlung von Kolorektalkarzinomen bei einem Subjekt.
 
2. Kombination oder Zusammensetzung zur Verwendung nach Anspruch 1, dadurch gekennzeichnet, dass das D-Vitamin ausgewählt ist aus der Gruppe, umfassend: Vitamin D3, Vitamin D2, 25-Hydroxy-Vitamin D3 und 1alpha, 25-Dihydroxy-Vitamin D3.
 
3. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 1-2, umfassend 5-Aminosalicylsäure und Vitamin D3.
 
4. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 1-3, wobei die Zusammensetzung, die die Kombination umfasst, kein Calcium umfasst.
 
5. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 1-3, dadurch gekennzeichnet, dass die 5-Aminosalicylsäure oder ein pharmakologisch verträgliches Salz davon in einer täglichen Dosis zwischen 0,5 und 5 g, vorzugsweise etwa 2,4 g, verabreicht wird und das Vitamin der Gruppe D oder ein Analogon davon in einer täglichen Dosis zwischen 500 und 10.000 IE, vorzugsweise etwa 2000 IE verabreicht wird.
 
6. Kombination oder Zusammensetzung zur Verwendung bei der Prävention von Kolorektalkarzinomen nach einem der Ansprüche 1-5, wobei das Subjekt gesund/normal ist oder es handelt sich um einen Patienten, der von chronischen entzündlichen Darmerkrankungen, vorzugsweise Morbus Crohn oder Rektokolitis ulcerosa, betroffen ist oder es handelt sich um einen Patienten, der einer endoskopischen Entfernung von Adenomen und/oder Adenokarzinomen des Dick-Enddarms unterzogen wurde, oder es handelt sich um einen Patienten, der von genetischen Krebssyndromen, vorzugsweise Lynch-Syndrom oder familiärer adenomatöser Polypose, betroffen ist.
 
7. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 1-6, dadurch gekennzeichnet, dass dieses Subjekt ein Säugetier, vorzugsweise ein Mensch ist.
 
8. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 1-7, wobei die Kombination oder Zusammensetzung zur oralen, intramuskulären, subkutanen oder rektalen Verabreichung ist.
 
9. Kombination oder Zusammensetzung zur Verwendung nach Anspruch 8, wobei die Kombination oder Zusammensetzung in Form von Tabletten, Kapseln, Granulaten, Pillen, Lutschtabletten vorliegt.
 
10. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 8-9, wobei die Kombination oder Zusammensetzung für kontrollierte Freisetzung ist.
 
11. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 8-10, wobei die Kombination oder Zusammensetzung eine kombinierte Zubereitung ist, die zur gleichzeitigen, sequentiellen oder getrennten Verabreichung der Komponenten der Kombination geeignet ist.
 
12. Kombination oder Zusammensetzung zur Verwendung nach einem der Ansprüche 8-11, wobei die Behandlung von Kolorektalkarzinom auf eine antiproliferative Wirkung auf Krebszellen des Dick-Enddarms zurückzuführen ist.
 
13. Kombination oder Zusammensetzung zur Verwendung nach Anspruch 12, wobei die antiproliferative Wirkung eine Überexpression von µ-Protocadherin, E-Cadherin, p21 und KLF4 beinhaltet.
 


Revendications

1. Combinaison de :

(i) acide 5-aminosalicylique ou son sel pharmaceutiquement acceptable ou son dérivé sélectionné à partir du groupe consistant en : sulfasalazine, olsalazine et balsalazide, et

(ii) une vitamine du groupe D, et

(iii) au moins un excipient physiologiquement acceptable

ou une composition pharmaceutique comprenant ladite combinaison à utiliser dans la prévention et/ou le traitement du cancer colorectal chez un sujet.
 
2. Combinaison ou composition à utiliser selon la revendication 1, caractérisée en ce que ladite vitamine D est choisie à partir du groupe comprenant : la vitamine D3, la vitamine D2, 25-hydroxy-vitamin D3 et 1alpha, 25 dihydroxy-vitamin D3.
 
3. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 1 à 2, comprenant de l'acide 5-aminosalicylique et de la vitamine D3.
 
4. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 1 à 3, dans laquelle la composition comprenant la combinaison ne comprend pas de calcium.
 
5. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 1 à 3, caractérisée en ce que ledit acide 5-aminosalicylique ou son sel pharmaceutiquement acceptable est administré en une dose quotidienne comprise entre 0,5 et 5 g, approximativement 2,4 g de préférence et ladite vitamine du groupe D ou son analogue est administrée en une dose quotidienne comprise entre 500 et 10 000 UI, de préférence approximativement 2 000 UI.
 
6. Combinaison ou composition à utiliser dans la prévention du cancer colorectal selon l'une quelconque des revendications de 1 à 5, dans laquelle ledit sujet est en bonne santé/normal ou est un patient atteint de maladies inflammatoires chroniques de l'intestin, de préférence les maladies de Crohn ou de colite ulcéreuse, ou est un patient confronté à des résections endoscopiques d'adénomes et/ou d'adénocarcinomes du colon-rectum, ou est un patient atteint de syndromes génétiques du cancer, de préférence du syndrome de Lynch ou d'une polypose adénomateuse familiale.
 
7. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 1 à 6, caractérisée en ce que ledit sujet est un mammifère, de préférence un humain.
 
8. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 1 à 7, dans laquelle ladite combinaison ou composition est destinée à une administration par voie orale, intramusculaire, sous-cutanée ou rectale.
 
9. Combinaison ou composition à utiliser selon la revendication 8, dans laquelle ladite combinaison ou composition se présente sous forme de comprimés, de capsules, de granules, de pilules ou de pastilles.
 
10. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 8 à 9, dans laquelle ladite combinaison ou composition est à libération contrôlée.
 
11. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 8 à 10, dans laquelle ladite combinaison ou composition est une préparation combinée adaptée pour une administration simultanée, séquentielle ou séparée des composants de la combinaison.
 
12. Combinaison ou composition à utiliser selon l'une quelconque des revendications de 8 à 11, dans laquelle le traitement du cancer colorectal est dû à un effet antiprolifératif sur les cellules cancéreuses du colon-rectum.
 
13. Combinaison ou composition à utiliser selon la revendication 12, dans laquelle ledit effet antiprolifératif implique la surexpression de µ-protocadhérine, E-cadhérine, p21 et KLF4.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description