USE OF MODIFIED AGENTS OF TRANSCRPTOMY MORE CHEMOTHERAPY OR RADIOTHERAPY AGAINST CANCER
DETAILED DESCRIPTION OF THE INVENTION
Technical field The present invention relates to the use of transcriptome-modifying compounds to assist in the treatment of malignant diseases by preventing malignant cells from making the gene changes necessary to cope with cellular insult and surviving chemotherapy or radiotherapy, more specifically related to with the combined use of transcriptome-modifying agents that inhibit DNA mediation machinery and deacetylation of histones, and more specifically with the use in combination with a treatment kit of hydralazine and magnesium valproate to help with the treatment of cancer together with chemotherapy or radiotherapy. Background of the Invention Cancer or malignancies are classified as solid or hematopoietic. Examples of the former include cancer such as breast, head and neck, colon and rectum, among others. Examples of hematology include leukemias and lymphomas. The DNA in the cell nucleus exists ordered in chromatin, and has several levels of order. The constitutive unit of chromatin is the nucleosome that consists of an octamer of nuclear proteins called histones and on which the DNA is coiled. The ordering or packaging of DNA in nucleosomes plays an important role for gene regulation. Covalent modifications of histones. such as acetylation, plays a fundamental role for the regulation of chromatin and for gene expression (Cho KS, Elizondo LI, Boerkoel CF: A chance in chromatin remodeling and human disease.) Curr Opin Genel Dev 2004: 14: 308-15 ). Currently, cancer remains a significant health problem worldwide, according to the International Agency for Research on Cancer and the World Health Organization, the incidence of this disease is increasing.
dramatically, estimating that of 10 million new cases that were observed in the year 2000, in 20 more years there will be 15 million. (Mignogna M). Fcdele S. Russo LL. The World Cancer Report and the burden of oral cancer. Eur J Cancer Prev. 2004; 3: 39-42) On the other hand, the survival of patients with the most common cancers such as lung, prostate and breast has improved discretely in recent years. The 5-year survival was 50% in 1974 and increased to 63% in the period from 1992 to 1999. { .Jemal A, Murray T, Ward E. Samuels A, Tiwari RC, Ghafoor A, Feuer EJ. Thun MJ. Cancer statistics, 2005. CA Cancer.) Clin. 2005; 55: 10-30). While advances in the forms of treatment have allowed small gains in survival, the results are still far from optimal. Currently, chemotherapy along with surgery and radiotherapy continue to be the mainstay of treatment since the vast majority of cancer patients require this form of therapy. The vast knowledge generated on the molecular basis of cancer in recent years has allowed the design of new forms of therapies that are generally aimed at blocking the function of oncogenes or at reactivating the expression of suppressor genes. Representative examples of these efforts are the use of monoclonal antibodies against some oncogenic receptors such as EGFR, HER2, etc. In the case of suppressor genes, some therapeutic efforts are the use of recombinant adenoviruses that carry the gene for coding the functional product of p53 (Hermislon TW.Kirn DH.Genetically based therapeutics for cancer: similarities and conlrasls wilh traditional drug discovery and deve / opment, Mol Ther, 2005; 1 1: 496-507). Generally speaking, we can call this new form of cancer therapy as directed towards a single gene product or unigenic therapy. However, this approach has severe limitations because the genome of the malignant cell is tremendously plastic and because the nature of cancer is multi-steppedThus, there is no single genetic alteration responsible for the development of the malignant phenotype. This means that, even though blocking or restitution of a gene or its product can produce an important antitumor effect, it is not maintained since, with certainty, the malignant cell in a variable period will develop resistance against it. This therapy will increase or decrease the expression of compensatory genes of the effect caused by the therapy in question (Ross JS Schenkein DP, Pietrusko R. Rolfe M. Linetle GP Stec, Stagliano NE, Ginsburg
GS, Symmans WF, Pusztai L. Hortobagyi GN. Targeted Iherapies for cancer 2004. Am J Clin Pathol. 2004; 22: 598-609). At present it is well known that malignant cells have multiple defects, properly mutations, deletions, duplications, amplifications, as well as epigenetic changes, the latter are stable functional changes due mainly to chromatin modifications, the two most important changes are the DNA mediation and histone acetylation. The epigenetic changes must be in a certain state and act in perfect functional balance to maintain the "malignant homeostasis". This concept is very relevant since all the defects of the malignant cells are not simply summations, this is consistent with the fact that the proteins encoded by the genes play multiple roles in networks of complex and interactive functions that show positive and negative feedback controls . In addition, through the multistep process that occurs in the generation of tumors, the cell must maintain a stable state between the positive and negative signals of both the oncogenic pathways and the suppressor genes to ensure that the processes of proliferation and Cell death occurs according to the dynamics of the malignant state. { Weinstein IB. Cancer. A ddiction lo oncogenes-lhe Achilles heal of cancer. Science. 2002; 297 (5578): 63-4). Added to the inherent complexity of the global gene expression of malignant cells, the picture is much more complicated when it comes to regulating gene expression as a consequence of exogenous stimuli, particularly the effect of chemotherapy or radiotherapy. Chemotherapy and radiotherapy produce immediate changes in gene transcription, these changes occur not only in those genes that are primarily relevant to the carcinogenic process but also in genes that are not directly involved, such as those involved in metabolism, transport, etc. (A laoui-Jamali MA, Dupre I, Oicing, H.Prediction of drug sensitivity and drug resistance in cancer by transcriptional and proteomic profiling, Drug Resisl Updal, 2004; 7: 245-55). The most important aspect, however, is that only those cells capable of having a transcription response adequate to the noxious stimulus are the only ones that will survive the insult. Obviously, in order for this "adequate" response to survive to occur, it is essential that the epigenetic mechanisms that regulate transcription are intact. Consequently, if the malignant cell is under the influence of transcriptome-modifying compounds, the response
transcriptional necessary for survival will not occur and the cell could have irreversible functional changes or suffer apoptosis. The transcription of eukaryotic cells can be defined as their ability to express biologically active proteins. Transcription is therefore a highly regulated phenomenon. The process starts at the gene level and ends at the protein level and involves multiple events; thus the transcription has several levels of regulation between which they appear: 1) the structure of the chromatin that is the physical structure of the DNA that includes the level of compactación of the chromatin that determines the capacity of the regulating proteins to be united to the promotoras regions or regulatory genes, 2) control of the initiation of transcription, 3) transport of the transcript. 4) the processing and modification of the transcripts. 5) the stability of the transcript, 6) the initiation of the translation, 7) the posttranslational changes, and 8) the transport and stability of the protein. . { Archamba lt ./. Friesen JD. Genetics of ukaryolic RNA polymerases I, II and III. Microbe! Rev. 1993; 57: 703-24) Undoubtedly, the higher the action on the level of transcription regulation, the more important the transcriptional effects will be. In this way the transcriptome modifying agents, by acting at the higher level of regulation, will have an effect on global gene expression of a large magnitude. Covalent modifications of histones, such as acetylation, and DNA mediation play a major role in determining the degree of chromatin compaction and ultimately in determining global gene expression.; Because of this, agents that inhibit DNA mediation and deacetylation of histones have been shown to have the property of altering expression in a very significant way. The loss of mediation could reduce the number of complexes with proteins that bind to domains mediated at some locus leading to a decrease in histone deacetylase activity to which the histone deacetylase inhibitor would have to inhibit. Also, the loss of the transcription repressor complexes may favor the reassociation of the promoters of the genes with the transcription activating complexes possessing histone acetylase activity. It is known that the most abundant form of DNA methyltransferase (DNMT l) can bind directly to histone deacetylases and that the amino terminal end also has the ability to bind co-repressors (Nakao M. Epigenetics: inleraction of DNA
melhylation and chromatin. Gene. 2001; 278: 25-31; Robertson KD.DNA melhylation and chromatin - unraveling the tangled web. Oncogene. 2002: 21: 5361-79). Although hundreds of potential antitumor agents have been tried, the treatment of human cancer remains very challenging, with only partially effective antitumor treatments and with the potential to cause side effects to practically all systems. Thus, there is not only the need to have more effective therapeutic options but also some more specific ones that attack the malignant cells in a more selective way about the gene transcription. That is why one of the objectives of the present invention is to provide a composition to help with the treatment of cancer based on the alteration of the transcriptome through the use of transcriptional modifiers such as hydralazine and magnesium valproate, with which they will make the cell incapable of surviving the harmful stimulus triggered by chemotherapy or radiotherapy.
The antihypertensive hydralazine is an inhibitor of DNA mediation that has been used to hypomethylate the DNA of T cells in experimental systems which makes these cells self-reactive (Yung R. Chang S, Hemali N, Johnson K, Richardson B. Mechanisms of drug-induced lupus, IV, Comparison of procainamide and hydralazine with analogs in vitro and in vivo, Arthritis Rheum, 1997; 40: 1436-43). More recently it has been shown that hydralazine produces demethylation of the promoter region of suppressor genes and induces its reactivation in vitro and in vivo models; and that, in addition, the reactivated gene products are functional (Segura B, Trejo-Becerrtl C, Pérez E, Chavez A, Solazar AM Lizano M, Dueñas-González A. Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and the go potential use in cancer therapy, Clin Cancer Res. 2003; 9: 1596-603). Hydralazine has direct inhibitory effects on DNA methyltransferase and in an in silico model it has been shown that two nitrogen atoms of the molecule interact with the amino acids Lys l 62 and Arg240 of the active site of the enzyme, which explains its unmeasured and reactivating properties of the function of suppressor genes (Angeles EE, Vazquez-Valadez, VH, Vasquez-Valadez O, Velazquez-Sanchez AM, Ramírez A, Martínez L, Diaz-Barriga S, Romero-Rojas A, Cabrera G. Lopez-Castañares R, Duenas-Gonzalez A: Com putational studies of I '-hydrazinophthalazine (Hydralazine) as andneoplasic agent Docking studies on methyltransferase Letters Drug Design Discovery 2005; 4: 282-286 The magnesium valproate also known as VPA, 2-propylpentanoic acid is a drug that has been used as an anticonvulsant for many years with a profile of
security well demonstrated. (Perucca E: Pharmacological and therapeutic properties of valproale: a summary after 35 years of clinical experience CNS Drugs 2002, 16: 695-7 4) It has recently been shown that this drug is an inhibitor of histone deacetylases. The inhibited enzymes are those of the class I and II families with the exception of histone 6 and 10 deacetylase. Hyperacetylation of histones H3 and H4 observed in vitro and in vivo accompanies their enzymatic inhibitory effect on this family of enzymes. This action on histones produces an important effect on the induction of differentiation, induction of apoptosis and inhibition of cell proliferation. { Gurvich N, Tsygankova OM, Meinkoíh JL, Klein PS: Misione deacetylase is a targel of valproic acid-mediated cellular differentiation. Cancer Res 2004, 64: 1 79-1086). That is why another objective of the present invention is to provide a treatment kit composed of hydralazine and magnesium valproate that contributes to the usual therapy used against cancer. Brief description of the figures Figures 1A, I B, 1 C, I D, 1 E, 1 F, and 1 G, show the effect of cytotoxicity in different malignant cell lines treated with the transcriptome modifying composition of the present invention. Figures 2A, 2B and 2C show how the transcriptome modifying composition of the present invention produces an enhancement of the cytotoxic effect of representative chemotherapeutic agents (cisplatin, doxorubicin and gemcitabine) in a malignant cell line. Figure 3A shows the mediation frequencies of each gene under the concept of the present invention. Figure 3B show representative cases of methylation frequencies under the concept of the present invention. Figure 3C shows the correlation between the percentage of methylation and the dose of hydralazine under the concept of the present invention. Figures 4A, 4B and 4C show representative cases of the messenger gene expression in the pre- and post-treatment biopsy. Figures 5, 6 and 7 show data of histone deacetylase activity according to the concept of the present invention.
Figure 8 shows data of the inhibitory effect of histone descacetylases of the composition of the present invention. Figure 9 shows the growth-blocking effect of tumors in animals with and without treatment with the composition under the concept of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention provides the use of a composition and a treatment kit to assist in the treatment of malignant diseases by the use of transcriptome modifying compounds that complement the treatment with chemotherapy or radiotherapy. The composition of transcriptome modifying agents is the combination of hydralazine and magnesium valproate that contributes to chemotherapy that may be, but not limited to, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan thiotepa, carmustine, lomustine, altretamine, dacarbazine, and procarbazine, cisplatin, carboplastin and oxaliplatin, doxorubicin, daunorubicin, epirubicin, idarubicin, mithomycin C, bleomycin, dactinomycin, retinoids, hormonal agents, vincristine, vinblaslin, vindesine, vinorelbine, irinotecan, topotecan, etoposide, teniposide, paclitaxel, docetaxel, 5-fluorouracil, gemcitabine. metrotrexate, interleukins, interferons, monoclonal antibodies such as trastuzumab, ceruximab, rituxan, myelotarg, and small inhibitory molecules such as gefitinib, erlotinib, and imatinib. The composition and treatment kit of the present invention can be used against several tumor types including, but not limited to, breast, ovarian, matrix, skin, bone, prostate, liver, kidney, lung cancer. , brain, head and neck, gallbladder, pancreas, colon and rectum, parathyroid, thyroid, adrenal gland, stomach, kidney, pheochromocytoma, vvilms tumor, testicle, nueroblastoma, sarcoma, acute and chronic leukemias, Ijnfomas and myelodysplastic syndromes . The composition of the present invention can be administered orally or by any other route of administration in a formulation comprising 83 mg of hydralazine plus magnesium valproate at a dose of 3 μg / kg of weight if the individual is slow acetylator or a dose of 1 82mg of hydralazine plus valproate of magnesium at a dose of 30mg / g of weight if the individual is a fast acetylator. Both agents should be administered in a controlled release formulation starting with their administration 7 days before the
first dose of chemotherapy or first session of radiotherapy, to allow the modification of the transcriptome prior to the cytotoxic insult of these treatments. Example of use 1 To demonstrate that the modifying composition of the transcriptome -hydralazine and magnesium valproate have antitumor effects a variety of malignant cell lines from cervical cancer, breast, colon, upper respiratory tract and digestive tract, and sarcoma were used. planted in 96-well plates (Falcon Becton Dickinson, Franklin Lakes, NJ) at a density of 1.5-2.5 x 103 cells / well in 0. 1 ml of complete medium. The next day the cells were treated with hydralazine at 10 μ? and valproate of magnesium at 1 mM for 4 days. The next day, cell viability was measured using an MTT assay. Briefly, 50 μ? of the MTT reagent in buffered phosphate solution to each well. Viable cells with active mitochondria reduce MTT to a purple precipitable compound -formazan- that is solubilized with DMSO adding 150 μ? to each well. Then it is read spectrophotometrically in an ELISA reader. All tests were performed in triplicate. The cytotoxic effect of each treatment was expressed in the percentage of cell viability relative to the control without treatment (% of control) that is defined as. { To s7o m treated cells) / ^ 70 nm untreated cells)] x 100. Figures 1A, IB, 1 C, ID, 1 E, 1 F, and 1 G, show that in all HeLa treated cell lines, line of cervical carcinoma; HT1080 of sarcoma; MCF-7 breast cancer, KB and HEP2 epidermoid carcinomas of the larynx and oral cavity respectively; SW480 of colon carcinoma, KB squamous cell carcinoma of the oral cavity, HEP2 epidermoid carcinoma of the larynx; D54 the transcriptome modifying composition produced significant cytotoxicity that varies from a reduction of 12.7% to 43.4% in viability. Example of use 2 Once it was demonstrated that the transcriptome modifying composition had inhibitory effects on the growth of the malignant cell lines, it was investigated whether the composition increased the cytotoxic effect of chemotherapeutic agents. With this objective, three drugs that are representative of their class were selected: alkylating agents such as cisplatin, antibiotics such as doxorubicin. antimetabolite like gemcitabine. The cells were seeded in 96-well plates (Falcon Becton Dickinson, Franklin Lakes, NJ) at a density of 1.5-2.5 x 103 cells / well in 0. 1 ml of complete media. The next day the
cells were treated with the chemotherapy agent at the concentration indicated in Figures 2A, 2B and 2C plus hydralazine at 10 μ? and magnesium valproate at 1 mM. The next day the medium containing the drugs was removed by adding fresh hydralazine and valproate magnesium at the same concentration for 48 more hours. The next day (day 4) cell viability was measured using an MTT assay. Briefly, 50 μ? of the MTT reagent in buffered phosphate solution to each well. Viable cells with active mitochondria reduce MTT to a precipitable purple-formazan compound that is solubilized with DMSO by adding 150 μ? to each well. Then it is read spectrophotometrically in an ELISA reader. All the tests were performed by
1 tripled. The cytotoxic effect of each treatment was expressed in the percentage of cell viability relative to the control without treatment (% of control) which is defined as [(570 nm treated cells) /! 570 nm untreated cells)] x 100. Figures 2A, 2B, and 2C show that in all cases there was a greater cytotoxicity of the transcriptome modifying composition plus the chemotherapy agent. To these conditions, a concentration of
1 5 12μ? of cisplatin which is an inhibitory concentration 50, produced a 37% reduction in viability in HeLa cells when treated with the transcriptome modifying composition. A similar effect was also demonstrated for adriamycin and gemcitabine for respective reductions of 27% and 37% respectively. Example of use 3 20 To demonstrate that the demethylating and reactivating effect of transcription of suppressor genes is clinically achievable, a phase I clinical study was conducted to demonstrate at what dose hydralazine could have an undesirable effect and reactivate transcription in patients with Cancer. With this objective, hydralazine was administered to groups of 4 patients each at a dose of: 1) 50mg / day, 2) 75mg / day, 3) 100mg / day and 4) 15 () mg / day for 10
days. Biopsies and peripheral blood samples were taken before starting treatment and on day 1 1. The condition of pre and post-treatment mediation of the promoters of the following genes was analyzed: APC. MGMT; ER, GSTP I, DAJ'K, RAR / i FHIT as well as the state of expression of its messengers by RT-PCR. The state of mediation of the gene subject to parental inactivation H 19 and a genomic clone that is found was also evaluated.
normally mediated, as well as the overall content of cylindrins mediated in the genome. The toxicity to hydralazine was assessed using the scale of the National Cancer Institute
United States of America (CTC NCI). Hydralazine was well tolerated, only the following undesirable effects were registered: nausea, dizziness, fatigue, headache and palpitations. Regarding the genes, it was found that 70% of the samples analyzed (89 of 128) had at least one of the genes mediated in the pretreatment biopsy. 8 genes for each of the 16 patient-biopsies, and that all patients had at least one methylated gene in their tumors. In individual analysis for each gene showed the following frequencies of mediation: APC 94%, ER 25% FHIT 88%, GSTI 'I 88%, MGMT%, p6 19%, RARfi 62%, and DAPK 1 00%. In post-treatment biopsies, a variable frequency of demethylation was found in each gene, ranging from 1 5% 2 of 13 samples for GM to 67% for the p ¡6 2 of 3 gene. Figure 3A. Representative cases are presented in Figure 3B. The correlation between the percentage of demethylation and the dose of hydralazine was as follows. 50 mg 40%, 75 mg 52%, 100 mg 43%, 10 mg 32% as can be seen in Figure 3C. The analysis of gene expression showed that 90% (1 16 of 128) of the tumor samples expressed the messenger in the biopsy pre- and post-treatment regardless of the state of mediation of the gene and therefore were not informative. Of the 12 informative cases, it was found that 9 of them did not have expression of the pre-treatment gene being mid but post-treatment they demethylated and re-expressed the gene. The representative cases are shown in Figure 4A, 4B, and 4C and the total frequency of gene expression is summarized in Figure 4D. The above results show that hydralazine at a dose range between 50 and15 () mg is effective to alter gene expression in cancer patients (Zamkrano P, Segura-Pacheco B, Perez-Cardenas E. Celina L, Revilla-Vazquez A, Taja-Chayeb L, Chavez-Blanco A, Angeles E, Cabrera G. Sandoval K, Trejo-Becerril C. Chanona-Vilchis J, Duenas-Gonzalez A. A phase I study of hydralazine and dcmelhylate reactivate the expression of tumor suppressor genes, BMC Cancer 2005: 5: 44). Example of use 4 To prove that magnesium valproate induces histone hyperacetylation and inhibition of histone deacetylase activity in the tumor of cancer patients, another clinical study was conducted in which different doses of magnesium valproate were administered to patients with cervical cancer. Twelve patients with this cancer of recent diagnosis and without previous treatment received the following doses of magnesium valproate
in gaipos of 4 patients. Group 1, 20mg / kg, group 2, 30mg kg, group 3, 40mg g. A biopsy of the tumor and a blood sample were taken before the treatment and the next day (day 6) since the magnesium valproate was administered for 5 days in divided doses every 8 hours. The hyperacetylation of histone H3 and histone H4 in the tumor was analyzed in the samples by western blot as well as the activity of histone deacetylases in nuclear extracts of the tumor using a colorimetric assay, as well as the levels of valproic acid in serum. The level of expression of the p21 and CAR genes in post-treatment biopsies was also analyzed. The toxicity of the treatment was recorded at the end of the cycle. All the patients completed the treatment, the average dose was 1890 mg per day with the averages corresponding to the doses of 20, 30 and 40 mg / kg of 1245, 2000 and 2425 mg respectively. Grade 2 drowsiness was observed in nine of the 12 patients. After treatment, hyperacetylation of H3 and H4 was found in nine and seven patients respectively, six of them had hyperacetylation in both histones (positive and negative control HeLa cells treated or not with trichostatin respectively). The deacetylase activity of histones decreased by 8 while there were no changes in two which was statistically significant (two-tailed t-test p <0.0264). (Positive control and negative nuclear extracts of HeLa cells treated or not with trichostatin), Figures 5, 6 and 7. These data demonstrate that magnesium valproate at the doses used is effective and well tolerated as an inhibitor of histone deacetylases which reflects changes in the expression of genes such as p21 (Figure 8) which increases its expression. Example of use 5 Once the capacity of hydralazine and valproate of magnesium to alter gene expression in the imorres of patients was determined, we investigated whether the composition of these transcriptome modifying agents increased the antitumor effect of chemotherapy in a sarcoma model in immunodeficient mice. To this end, groups of 6 atomic female mice were studied and injected with 6 million cells of the HT 1080 sarcoma cell line. Once the tumors were formed, the animals were treated systemically with hydralazine and magnesium valproate at doses equivalent to those used in patients. The treatment groups were as follows: 1) control treated with saline, 2) treated weekly with adrianiicin, 3) treated with the composition of hydralazine and valproate for 7 days followed by the same treatment
weekly with adriamycin. The results show that at 5 weeks the rumors of the untreated animals reach a volume of between 2 and 3 cnr5, and treatment with adriamycin produces an almost complete antitumor effect after 3 weeks of treatment, however from that time the tumors grow back while in the animals treated with the composition, the tumor re-growth is blocked as can be seen in Figure 9. The above, suggests that the modifying combination of the transcriptome prevents the tumor cell from making the tTnscriptional changes necessary to recover the growth capacity. This phenomenon commonly occurs in the treatment of cancer in patients, where a total or almost total antitumor response is often observed and subsequently relapsed. The composition of the present invention could therefore induce prolonged or complete remissions of the tumors. The composition of the present invention can be integrated in a treatment kit to be administered orally or by any other route of administration in a formulation comprising 83 mg of hydralazine plus magnesium valproate at a dose of 30 mg / g of weight if the The individual is slow acetylator and at a dose of 1 82mg of hydralazine plus magnesium valproate at a dose of 30mg Kg of weight if the individual is a fast acetylator. Both agents should be administered in a controlled release formulation to avoid peaks in serum levels produced by hydralazine and magnesium valproate and decrease the side effects resulting from its rapid absorption. Since the effect of hydralazine on the inhibition of methylation starts at least 48 hours after its administration and that the effect of magnesium valproate on transcription could be higher on a background of demethylation, the treatment with the composition preferably it must be started seven days before the first dose of chemotherapy or radiotherapy to allow modification of the transcriptome prior to the cytotoxic insult of chemotherapy or radiotherapy.