Compositions comprising acetylated derivatives of l-cysteine and 5-methyl-1-phenyl-2(1H)-pyridone in nanoparticles and uses thereof

Abstract

The present invention discloses pharmaceutical compositions comprising acetylated derivatives of L-cysteine and 5-methyl-1-phenyl-2(1H)-pyridone formulated into nanoparticles, which can be used for treating an altered physiological condition in a subject in need, such as for the treatment or prophylaxis of a condition such as hepatocellular carcinoma (HCC) at any of its stages, hepatic fibrogenic process, neoplastic lesions, modulation of the oxidising process and epigenetic alterations, as well as tumour reduction.

Description

COMPOSITIONS INCLUDING DERIVATIVES ACETYLATES OF L-CYSTEINE AND 5-METHYL-1-PHENYL-2(1 H)-PYRIDONE IN NANOPARTICLES AND THEIR USES I Field of Invention

[0001] The present invention relates to the field of pharmacology, and more particularly to compositions comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-pyridone formulated in nanoparticles, as well as their uses in therapies for a patient in need thereof. Background of the Invention

[0002] Worldwide, primary liver cancer is the sixth most diagnosed cancer and the third leading cause of liver-related death. In 2020, 906,000 new cases were diagnosed, and 830,000 deaths were recorded. Primary liver cancer includes hepatocellular carcinoma (HCC), which accounts for 75%–85% of cases; 10%–15% of cases are related to intrahepatic cholangiocarcinoma and other rare types.

[0003] In Mexico, HCC is the ninth most common malignant neoplasm, with a similar distribution between both sexes, and represents the third highest rate of mortality from malignancy. In the United States, HCC is a highly lethal malignancy, with mortality rates increasing significantly in recent decades.

[0004] Despite advances in diagnosis and therapeutic development, statistical projections indicate that the incidence rate will continue to increase by 2030.

[0005] HCC is an epithelial hepatic neoplasm of heterogeneous onset and cause. Like liver cirrhosis, it represents one of the terminal stages of fibrotic liver disease and is associated with chronic inflammation. The main risk factors for developing this disease include hepatitis B (HBV) and C (HCV) virus infections, autoimmune hepatitis, chronic alcohol and tobacco use, nonalcoholic steatohepatitis (NASH), obesity, and diabetes mellitus.

[0006] HCC can arise from both mature hepatocytes and progenitor cells; however, the molecular basis of HCC depends on various factors and mechanisms.

[0007] Hepatocytes are differentiated cells that remain quiescent in the G0 phase. When they receive mitogenic signals, they enter the cell cycle and eventually divide and proliferate. The cell cycle is controlled by the activity of cyclins and cyclin-dependent kinases (Cdk). Cyclin D1 plays a critical role since its de novo expression, mediated by cytokines and growth factors, allows the hepatocyte to enter the cell cycle. Cell cycle progression is key to HCC; however, it is strictly controlled by members of the INK family, which are Cdk inhibitors and of which p15 / p16 / p18 / p19 are known. Another important family of proteins that block cell cycle entry is Cip / Kip: p21 / p27 / p57. The occurrence of events that can decrease the expression of the different proteins that regulate the cell cycle eventually cause tumorigenesis.On the contrary, the increase in the expression of these proteins results in the arrest of the cell cycle, especially the increase in p27 that allows to preserve the quiescent state of the hepatocytes, restricting the activity of the Cdks. In a quiescent state, hepatocytes express basal levels of membrane receptors, which can be activated by cytokines (IL-8, TNFa, TGF[3) or growth factors (EGF, HGF) inducing the expression of early response genes, and re-entry into the cell cycle through the expression of cyclin D1.

[0008] Apoptosis contributes to liver tissue homeostasis through cell elimination without affecting the biological functionality of the tissue. The pathways most affected in HCC are p53, TGF-[3 and mechanisms involving death receptors. Mutations in the tumor suppressor gene p53 are alterations with greater relevance to the development of HCC, these mutations are related to endogenous processes and environmental carcinogens. For example, exposure to aflatoxin B1 (AFB1) leads to a somatic mutation in the third base at codon 249 in the TP53 gene. On the other hand, oxidative stress, HBV and HCV infections induce damage and mutations in tumor suppressor genes including TP53. Because of this, mutations occurring in TP53 may play an important role in both the initiation and progression of HCC.

[0009] The transforming growth factor-beta (TGF-[3) pathway plays an important role in multiple mechanisms, but is dependent on the etiological context; it has been shown to stop tumor growth by inhibiting the cell cycle through the increase of CDK inhibitors: p15, p21, p27 and p57. On the other hand, TGF-[3 induces apoptosis by negative activation of Bcl2 through SMAD3 and the embryonic liver fold adaptor protein (ELF), and therefore reduces the risk of developing HCC. However, in most patients with HCC the TGF-[3 pathway is deregulated and can compromise apoptosis.

[0010] The epidermal growth factor receptor (EGFR) is required in HCC to activate anti-apoptotic signals. In HCC cells, EGFR is activated by TGF-[3] which, in turn, requires the participation of TACE / ADAM17 to activate TNF-[a] which enhances EGF function and evasion of apoptosis. Some functions of TGF-[3] that have been characterized in HCC are pro-tumorigenic such as promotion, differentiation, proliferation and evasion of the immune system. Death receptor-mediated apoptosis is another altered mechanism in HCC, generally compromising the Fas pathway. Alterations in the Fas pathway are due to Fas dysregulation and the concomitant decrease of downstream molecules such as FADD or FLICE or by the increased expression of molecules that counteract the pro-apoptotic effect of Fas, such as NF-κB, Bcl-2 and Bcl-X.Similarly, the caspase 8 inhibitor FLICE is constitutively expressed in HCC, impairing apoptosis. In HCC, on the other hand, it impairs death. Fas-induced can also be suppressed by regulation of hepatocyte growth factor (HGF) and activation of the PI3K / AKT pathway.

[0011] Furthermore, numerous studies indicate that chronic inflammation leads to carcinogenesis. Regardless of the different etiological factors involved in HCC, the common denominator that causes malignant transformation of liver cells is the perpetuation of the wound healing response, resulting in an inflammatory cascade. The six main mechanisms that trigger inflammation are regulated by cytokines, chemokines, transcription factors, and proteins that are part of the inflammatory signaling cascade. Among the best-studied cytokines involved in the inflammatory process are interleukin 1α (IL-1α), IL-1β3, IL-6, IL-8, and TNF-α, of which IL-6 is believed to be the most important interleukin.In this sense, when liver tissue suffers continuous damage, Kupffer cells increase IL-6 production and local inflammation, inducing a compensatory response of liver cell proliferation and eventual malignant transformation. In various liver diseases, including alcoholic hepatitis, NASH, and HBV and HCV infections, elevated levels of IL-6 have been found in patient serum. Recent studies have postulated IL-6 as an indicator of tumor size, developmental stage, and aggressiveness measured through invasion and metastasis. In experimental models of HCC using diethylnitrosamine (DEN) as a damage inducer, IL-6 has been shown to be essential in the early stages of HCC.Another interleukin that has an important role in the early stages of HCC is IL-1 [3] since it activates the transdifferentiation of hepatic stellate cells, promotes the production of C-reactive protein, facilitates the invasion of hepatic cells through JNK and the evasion of cell death mediated by TGF- [3. Although interleukins have a central role in the development of HCC mediated by inflammation, it has been proposed that the regulatory nucleus of this event is NF-KB, which is a transcription factor formed by two subunits RelA (p65) and RelB (p50). Under physiological conditions these isoforms are dimenezized and inactive in the cytoplasm; however, in response to inflammatory stimuli, NF-KB is translocated to the nucleus by binding to sequences. DNA-specific target genes involved in inflammation mechanisms such as IL-1 , IL-2, IL-6, IL-8, IL-12 and TNF-α; proliferation, Bcl-2L1 , PAI12 and cyclin; anti-apoptotic factors, Fas, BCL-2, c-FLIP and Caspase. Similarly, NF-κB activation in HCC promotes proliferation and maintains the inflammatory environment, mainly through IL-6, which activates the STAT3 signaling pathway, which positively feedbacks to NF-κB; STAT3 favors the tumor environment, activating the secretion of the cytokines mentioned above.

[0012] The liver performs multiple metabolic and detoxification functions, and is therefore constantly exposed to countless antigens. To prevent autoimmune damage due to antigenic overstimulation, there are mechanisms aimed at preventing this damage, which are listed below: 1. Constitutive secretion of IL-10 by Kupffer cells. 2. Secretion of TGF-[3 by sinusoidal endothelial cells (LSECs) and Kupffer cells. 3. Decreased antigen-presenting capacity of LSECs due to decreased expression of B7-1 / CD80 and B7-2 / CD86. 4. Constitutive expression of PD-L1 and PD-L2 by hepatocytes, hepatic stellate cells, LSECs and Kupffer cells.

[0013] All these mechanisms contribute to the tolerance of the immune system to antigens associated with tumor cells and to the progression of HCC. Furthermore, an increase has been shown during stages of chronic inflammation, especially during chronic infections by Hepatitis B and C viruses, showing an increase in the expression of PD-1 , CTLA-4 and Tim3, as well as overexpression of PD-L1 and PD-L2 by Kupffer cells, LSECs and other intrahepatic leukocytes, these profiles are closely related to the degree of inflammation.

[0014] Failure of antigen presentation by APCs as a result of decreased HLA I expression and deficiencies in antigen processing by tumor cells contributes to tumor evasion by HCC. Furthermore, an increase in T lymphocytes has been found. regulators, type I NKT cells (iNKT), monocyte-like myeloid suppressor cells (MDSC), tumor-associated macrophages (TAMs); as well as a decrease in the response of CD4 + Helper T lymphocytes. The increase in different cytokines has been associated with a characteristic innate immune response that provides the tumor microenvironment with capacities to promote the development of HCC and its metastases. This profile is given by the increase of anti-inflammatory cytokines such as: IL-4, IL-5, IL-8 and IL-10; as well as the decrease of proinflammatory cytokines such as IL-1, tumor necrosis factor (TNF) and Interferon gamma (IFNy). High levels of IL-10 in patients with HCC have been linked to unfavorable prognoses and immune dysfunction, for example, low activity of interleukins-activated NK (LAK).

[0015] The multiple mechanisms that drive the development of HCC are generally associated with distinct genetic and epigenetic alterations. Indeed, malignant phenotypes are governed by these mechanisms, which alter the regulation of the expression of tumor suppressor genes and oncogenes. The best-studied genetic alterations in HCC include translocations, deletions, and single-nucleotide variations, amplification of long chromosome fragments, and loss of small DNA fragments. Among the chromosomes amplified in HCC is chromosome 1q, where the 1q21 region is amplified in at least 50% of HCC patients; the oncogene CHD1 L, which promotes p53 degradation, is located in this region of the chromosome. The p53 gene is located on chromosome 17, in the 17p13.1 region, a region frequently lost or hypermethylated in HCC.Another altered chromosome is 8q, specifically the 8q24 region. This region houses the oncogenes c-Myc and PTK2, which have been well characterized in HCC and have been identified as the main regulators of malignant transformation in the early stages of the disease. Another genetic alteration reported in most cancers, including HCC, are mutations, which contribute to tumor initiation and progression. In particular, somatic mutations are present in neoplastic tissue and accumulate during cancer progression. Mutations accumulated in the open reading frame can lead to both loss of function and gain of oncogenic functions. Mutations in the non-coding region can change transcription, translation, or the stability of the gene product. The genes most frequently mutated are shown in Table 1. Table 1 Genes with the highest mutation frequency

[0016] On the other hand, epigenetic modifications involve changes in gene function and expression without modifying nucleotide sequences. Among the best-studied epigenetic alterations are DNA methylation, histone modification (acetylation and methylation), and regulation by non-coding RNAs (ncRNAs). The enzymes involved in epigenetic mechanisms have been grouped as writers, readers, and erasers. The enzymes responsible for writing epigenetic marks are DNA methyltransferases (DNMTs), histone acetyltransferases (HATs), and histone methyltransferases (HMTs). The group of enzymes that erase these marks includes DNA demethylases, histone deacetylases (HDACs), and histone demethylases (HDMS). While enzymes that erase epigenetic marks include CpG-binding proteins, chromodomain-containing enzymes, and bromodomain-containing enzymes.

[0017] Treatment options for HCC are divided into surgical and non-surgical therapies.

[0018] Current surgical therapies include liver resection, liver transplantation, and ablation, all of which have 5-year survival rates of 70–90%. However, the choice of surgical treatment for HCC requires careful analysis of various factors such as tumor size, involvement of major vascular structures, liver function, and the patient's clinical status, so not all patients may be candidates for this type of treatment.

[0019] On the other hand, nonsurgical therapies, in addition to broadening the criteria, are currently being applied to treat patients with HCC who are not candidates for surgical treatment. Nonsurgical therapies, also known as downstaging therapies, are defined as therapies that reduce viable tumor burden and include locoregional therapies (LRT) and systemic therapies.

[0020] Locoregional therapies include transarterial chemoembolization (TACE), transarterial radioembolization (TARE), and stereotactic body radiation therapy (SBRT), which have been used for the past few decades to downsize tumors.

[0021] For more than a decade, sorafenib was the only clinically effective systemic treatment for advanced HCC. However, in recent years, rapid advances in molecularly targeted therapies, immunotherapies, and combination therapies have revolutionized the treatment of advanced HCC, and a range of drugs have been developed to inhibit specific molecular abnormalities associated with cancer progression. Some of these clinically approved drugs include levantinib, regorafenib, cabozantinib, and ramucirumab.

[0022] The above therapies are still under development and / or have serious adverse effects such as myocardial infarction / ischemia, gastrointestinal perforation, drug-induced hepatitis, hemorrhage, and hypertension / hypertensive crisis, as well as adverse reactions including diarrhea, fatigue, alopecia, infection, hand-foot skin reaction (MedDRA palmar-plantar ehtrodysesthesia syndrome) and rash.

[0023] Specifically, for non-surgical therapies, some international clinical practice guidelines for the treatment of HCC propose radioembolization with yttrium-90 (90Y)-labeled glass microspheres, which has been shown to be effective in inducing necrosis in HCC with a good safety profile, but has not been shown to improve cell survival efficacy.

[0024] Likewise, most international guidelines recommend the use of other drugs such as sorafenib in patients who meet certain criteria, such as patients with Child A cirrhosis (preserved liver function), advanced BCLC stage (metastasis), and patients who are not candidates for transplant, resection, and local ablative therapy.

[0025] Patients with Child C cirrhosis should only be offered palliative care unless they are candidates for liver transplantation. Alternatively, percutaneous ethanol injection may be recommended in cases of very early (BCLC 0) and early (BCLC A) cirrhosis of HCC. Downstaging for liver transplantation may be considered in a suitable candidate. Stereotactic external beam radiotherapy (EBRT) and external beam radiotherapy (EBRT) are also considered as options, either alone or in combination with transarterial chemoembolization (TACE) for residual or metastatic HCC disease.

[0026] Recent studies have demonstrated the effect of substances contained in the diet as possible treatments in the early stages of HCC. Furthermore, it has been shown that w-3 polyunsaturated fatty acids act as ligands for PPARy and RXR, triggering epigenetic mechanisms: inhibiting DNMT1 and HDAC1, and modulating genetic transcription and inducing antitumor effects. This suggests the ability of synthetic or natural molecules to regulate antioxidant and anti-inflammatory mechanisms, particularly through regulation of epigenetic mechanisms, which could be an adjuvant strategy for HCC therapy.

[0027] Cysteine ​​derivatives such as mecysteine, N-acetylcysteine, carbocisteine, fudosteine, its analogue erdosteine ​​and L-lysine N-acetylcysteinate (“Nacystelyn” or NAL) are considered compounds that restore the viscoelastic properties of mucus; they are also attributed with anti-inflammatory and antioxidant properties.

[0028] N-acetylcysteine ​​(NAC) is an acetylated derivative of the amino acid L-cysteine, which has been widely used clinically as an antidote for acetaminophen poisoning and has also been used since 1960 as an effective mucolytic in patients with pulmonary fibrosis.

[0029] The pharmacodynamic mechanisms of NAC, and of acetylated L-cysteine ​​derivatives in general, are related to their antioxidant capacity per se, reducing reactive oxygen species (ROS) that can cause oxidative damage to lipids, proteins, and DNA, leading to cell damage and death. These mechanisms are also implicated in various diseases, including liver disease.

[0030] NAC has also been observed to exert anticancer responses, specifically by inducing Notch2 degradation through the lysosomal pathway, preventing proliferation, migration, and invasion responses, and inducing apoptosis in glioblastoma multiforme. Importantly, Notch proteins are conserved transmembrane receptors that control crucial steps in development, cell growth, and differentiation, and dysregulation of Notch signaling is linked to the genesis of many human cancers.

[0031] Additionally, NAC possesses anti-inflammatory activity where nuclear factor-κB (NF-κB) plays an important role, specifically, NAC can decrease NF-κB activity by suppressing the ubiquitination and degradation of IκB, a repressor of NF-κB, preventing nuclear translocation and activation of this nuclear factor and finally preventing the expression of several genes related to the proinflammatory response such as interleukin-1 [3 (IL-113), IL-6, TNF-α [Qiu, 2013] and TGF-β.

[0032] On the other hand, pirfenidone (PFD) or 5-methyl-1-phenyl-2-(1 H)-pirfenidone is another molecule with antioxidant properties, with the ability to directly inhibit the production of ROS, possibly through a mechanism mediated by the transcriptional factor Nrf2 (antioxidant gene regulator). Currently, PFD is used as an antifibrotic drug that acts on multiple fibrogenic pathways to reduce fibrosis in idiopathic pulmonary fibrosis, downregulates the production of growth factors, decreases fibroblast proliferation and influences the differentiation of fibroblasts into myofibroblasts mediated by transforming growth factor beta (TGF-[3).

[0033] The use of 6-oxo-7-phenyl-6,7-dihydro-1 H-pyrrolo[2,3-b]pyridine derivatives has also been explored for the treatment of inflammatory and fibrotic disorders. Overall, the potential mechanism of PFD derivatives as antifibrotic agents has been investigated, where studies suggest the possible mechanism of these compounds as antifibrotic agents was through the p38 MAPK pathway.

[0034] Due to their effects, NAC and PFD have been widely studied for different types of treatments. For example, document MX 322609 B protects a composition comprising galatomannan and NAC for treating or preventing a skin disease or condition resulting from the production of ROS in a subject's skin, including some modalities for its use in burns, wounds, ulcers and health problems associated with exposure to UV radiation, including different types of skin cancer. However, the types of skin cancer linked to sunlight exposure described include, in order of increasing severity, basal cell cancer, squamous cell cancer and malignant melanoma, which together with the described composition differs from the subject matter to be protected in the present patent application and does not solve or suggest a solution for the treatment of HHC, which is far from the etiology of skin cancer.

[0035] Likewise, WO 201 1 / 097273 A1 describes a method and composition for treating non-alcoholic fatty liver disease (NADFLD) with docosahexaenoic acid (DHA) and NAC, however, it does not propose a solution to HCC, nor does it suggest its combination with other active ingredients.

[0036] Document MX 290751 B protects a microemulsifiable composition of Pirfenidone, which offers advantages over other pharmaceutical forms of oral administration known in the state of the art, such as tablets, capsules, suspensions and solutions for use in the restoration of tissues with fibrotic lesions and for their prevention. However, the document does not explore other modalities in which PFD can be used in compositions with other active ingredients, nor does it offer a specific treatment for HCC.

[0037] WO 2018 / 088886 A1 describes the use of a pharmaceutical composition in the form of prolonged-release tablets containing pirfenidone for the treatment of alcoholic and non-alcoholic steatohepatitis (NAFLD / NASH) and advanced liver fibrosis, by reducing serum cholesterol and triglyceride levels, as well as reducing the content of accumulated fat in liver tissue in the form of macrosteatosis and microsteatosis. However, it does not explore the treatment of HCC or the combination with other active ingredients.

[0038] On the other hand, the use of nanotechnology is one of the most widely used technological advances in drug formulation. Targeted delivery of active ingredients combines knowledge of pharmacology, pharmaceutical development, polymer science, conjugate chemistry, and molecular biology, with the goal of improving the pharmacokinetics of active ingredients, reducing their toxicity, immunogenicity, and biorecognition.

[0039] The use of nanocarriers or nanoparticles delays the degradation and inactivation of the active ingredient in the bloodstream; it also helps maintain the structure and stability of the drug(s), protecting them from changes in pH, temperature, protease activity, etc.

[0040] Other advantages offered by the use of nanoparticles include the entry and transport of poorly soluble or hydrophobic drugs, either singly or in combination, and drug delivery to more precise sites, such as an organ, a cell, a cellular compartment, or even an organelle.

[0041] Nanoparticles can be organic polymers composed of phospholipids and cholesterol, whose characteristics include biodegradability, biocompatibility, non-immunogenicity, amphiphilic properties, lipid modification capabilities, and drug delivery. Liposomes, on the other hand, are spherical vesicles composed of phospholipids. Amphiphilic phospholipids and cholesterol. Amphiphilic phospholipids form a closed bilayer that encapsulates the components of the aqueous medium and is in contact with the hydrophilic heads of the membrane phospholipids.

[0042] Metal oxide nanoparticles have also shown potential in drug delivery. Among these nanoparticles, silver oxide nanoparticles and cerium oxide (CeO2) nanoparticles stand out; cerium oxide in particular has antioxidant and cytotoxic properties toward cancer cells, thus offering them anticancer activity.

[0043] In this regard, document WO 2010 / 065329 A2 describes a parenteral pharmaceutical composition comprising an isothiocyanate (ITC) formulated in pharmaceutically acceptable nanoparticles that solubilize and stabilize the ITC; where in turn, one of the embodiments of the invention, the ITC is a phenethyl isothiocyanate N-acetylcysteine ​​conjugate (PEITC-NAC) for the treatment of liver cancer. However, this technology does not explore the possibility of including metallic nanoparticles and does not offer a solution for the treatment of liver cancer using drugs with an epigenetic effect.

[0044] Likewise, document US 11,517,588 B2 describes a nanoparticle comprising a core composed of at least one nitric oxide donor, which is encapsulated in a polymer and a lipid by means of a simple oil-in-water emulsion to form a nanoparticle; one of the embodiments described in this document details that the nitric oxide donor is a dinitrosyl iron complex (DNIC), the polymer is poly D,L-lactide-co-glycolic acid (PLGA) and the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), wherein the nanoparticle is designed to treat liver cancer. However, it also does not consider the use of other types of nanoparticles, such as those of metallic origin proposed in this patent application, nor does it suggest the incorporation of compounds other than nitric oxide donors.

[0045] As a result of the above, we have sought to eliminate the drawbacks presented by the compositions of drugs formulated in nanoparticles to treat cancer that are currently used, developing Compositions comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-pyhdone formulated in nanoparticles and uses thereof as an effective therapy for HCC that, in addition to providing a stable nanoparticle system, allows the delivery of drugs effectively and safely to their target site. Objects of the invention

[0046] Taking into account the defects of the prior art, it is an object of the present invention to provide a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-phosphate formulated in nanoparticles.

[0047] Another object of the present invention is to provide a pharmaceutically acceptable and therapeutically effective composition for an altered physiological condition in a subject in need thereof.

[0048] Another object of the present invention is to provide a pharmaceutically acceptable and therapeutically effective composition for the treatment of HCC.

[0049] Another object of the present invention is to provide a pharmaceutically acceptable and therapeutically effective composition for slowing the development of HCC.

[0050] Another object of the present invention is to provide a pharmaceutically acceptable and therapeutically effective composition for the intervention in the hepatic fibrogenic process.

[0051] Another object of the present invention is to provide a pharmaceutically acceptable and therapeutically effective composition for preventing the development of neoplastic lesions.

[0052] Another object of the present invention is to provide a pharmaceutically acceptable and therapeutically effective composition for modulating the oxidative process and modulating epigenetic alterations.

[0053] These and other objects are achieved by compositions comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated into nanoparticles and uses thereof in accordance with the present invention. Brief description of the invention

[0054] For this purpose, a pharmaceutical composition has been invented that comprises acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles.

[0055] Other aspects of the invention consider the use of a pharmaceutical composition for the manufacture of a medicament for the treatment or prophylaxis of a condition such as hepatocellular carcinoma (HCC), wherein the treatment or prophylaxis of HCC includes at least one of the reduction of the hepatic fibrogenic process, reduction of neoplastic lesions, modulation of the oxidative process, reduction of tumors and reduction of epigenetic alterations. Brief description of the figures

[0056] The novel aspects considered characteristic of the present invention will be set forth with particularity in the appended claims. However, certain embodiments, features, objects, and advantages thereof will be better understood from the detailed description when read in conjunction with the accompanying drawings, in which: Fig.lA

[0057] [Fig. 1 A] FIG. 1A corresponds to representative photographs of experimental groups of rats, the white asterisks show the development of neoplastic lesions in the group treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles in accordance with an embodiment of the present invention. NT indicates the tissues of the untreated group; HCC indicates the tissues of the group subjected to HCC induction and development treatment; HCC-NAC / PFD- Nano designates the tissues of the group subjected to induced HCC development treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles in accordance with an embodiment of the present invention. Fig.lB

[0058] [Fig.l B] FIG. 1 B shows the results of the analysis of liver weight variation in experimental groups of rats. NT indicates the tissues of the untreated group; HCC indicates the tissues of the group subjected to HCC induction and development treatment; HCC-NAC / PFD-Nano indicates the tissues of the group subjected to induced HCC development treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated into nanoparticles according to an embodiment of the present invention. Fig.lC

[0059] [Fig.l C] FIG. 1 C shows the results of the analysis of the variation in relative liver weight with respect to total body weight of the experimental groups of rats. NT indicates the tissues of the untreated group; HCC indicates the tissues of the group subjected to HCC induction and development treatment; HCC-NAC / PFD-Nano indicates the tissues of the group subjected to induced HCC development treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pinedone formulated in nanoparticles according to an embodiment of the present invention. Fig.lD

[0060] [Fig.l D] FIG. 1 D shows the results of serum y-GTP enzyme analysis in the liver of experimental groups of rats. NT indicates the tissues of the untreated group; HCC indicates the tissues of the group subjected to HCC induction and development treatment; HCC-NAC / PFD-Nano indicates the tissues of the group subjected to induced HCC development treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated into nanoparticles according to an embodiment of the present invention. Fig.l E

[0061] [Fig. 1 E] FIG. 1 E corresponds to the results of serum ALT enzyme analysis in the liver of experimental groups of rats. NT indicates the tissues of the untreated group; HCC indicates the tissues of the group subjected to HCC induction and development treatment; HCC-NAC / PFD-Nano indicates the tissues of the group subjected to induced HCC development treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated into nanoparticles in accordance with an embodiment of the present invention. Fig. 2A

[0062] [Fig.2A] FIG. 2A shows photographs of hematoxylin and eosin stained liver tissue from all experimental groups. Fig. 2B

[0063] [Fig.2B] FIG. 2B shows the results of the variation analysis of atypical hepatocytes displayed in hematoxylin and eosin (H&E) staining. Fig. 2C

[0064] [Fig.2C] FIG. 2C corresponds to photographs of liver tissue with Masson's Trichrome staining, to mark extracellular matrix fibers. Fig.2D

[0065] [Fig.2D] FIG. 2D shows the results of the quantification of the extracellular matrix area detected in trichrome staining. Fig. 2E

[0066] [Fig.2E] FIG. 2E corresponds to the results of the immunohistochemical test using the hepatocellular carcinoma marker Glipican 3. Fig. 2F

[0067] [Fig.2F] FIG. 2F shows the results of quantification of the Glipican 3 positive area in the immunohistochemical test (IHC) in the liver tissue of all experimental groups. Fig. 3A

[0068] [Fig.3A] FIG. 3A is the protein expression profile of the tumor marker [3-catenin] obtained by western blot of cytoplasmic extracts from all experimental groups. Fig. 3B

[0069] [Fig.3B] FIG 3B is the protein expression profile of tumor markers [3-catenin and c-Myc obtained by western blot of nuclear extracts from all experimental groups. Fig. 3C

[0070] [Fig.3C] FIG. 3C shows the results of the relative quantification of the protein expression profile of the tumor marker [3-catenin cytoplasmic extracts, normalized with [3-Actin] of all experimental groups. Fig.3D

[0071] [Fig.3D] FIG. 3D corresponds to the results of the relative quantification of the protein expression profile of the tumor marker [3-catenin nuclear extracts, normalized with [3-Actin of all experimental groups. Fig. 3E

[0072] [Fig.3E] FIG. 3E shows the results of the relative quantification of the protein expression profile of the tumor marker c-Myc in nuclear extracts, normalized with [3-Actin] of all experimental groups. Fig. 4A

[0073] [Fig.4A] FIG. 4A is the protein expression profile of PPARa, PPARy and PPARy2 obtained by western blot of cytoplasmic extracts from all experimental groups. Fig. 4B

[0074] [Fig.4B] FIG. 4B corresponds to the results of the relative quantification of the protein expression profile of PPARa in cytoplasmic extracts, normalized with [3-Actin of all experimental groups. Fig.4C

[0075] [Fig.4C] FIG. 4C shows the results of the relative quantification of the PPARγ protein expression profile in cytoplasmic extracts, normalized with p-Actin from all experimental groups. Fig. 4D

[0076] [Fig.4D] FIG. 4D corresponds to the results of the relative quantification of the protein expression profile of PPARy2 in cytoplasmic extracts, normalized with [3-Actin of all experimental groups. Fig. 4E

[0077] [Fig.4E] FIG. 4E is a protein expression profile of PPARa, PPARy and PPARy2 by western blot of nuclear extracts from all experimental groups. Fig. 4F

[0078] [Fig.4F] FIG. 4F shows the results of the relative quantification of the PPARa protein expression profile in nuclear extracts, normalized with Lamin-B1 from all experimental groups. Fig.4G

[0079] [Fig.4G] FIG. 4G corresponds to the results of the relative quantification of the protein expression profile of PPARy in nuclear extracts, normalized with Lamin-B1 of all experimental groups. Fig. 4H

[0080] [Fig.4H] ​​FIG. 4H shows the results of the relative quantification of the PPARy2 protein expression profile in nuclear extracts, normalized with Lamin-B1 from all experimental groups. Fig. 4l

[0081] [Fig.4l] FIG. 4I is the protein expression profile of SREBP and Psrebp-1C (Ser372) by western blot of cytoplasmic extracts from all experimental groups. Fig.4J

[0082] [Fig.4J] FIG. 4J corresponds to the results of the relative quantification of the protein expression profile of SREBP, normalized with [3-Actin of all experimental groups. Fig.4K

[0083] [Fig. 4K] FIG. 4K shows the results of the relative quantification of the protein expression profile of Psrebp-1 C with respect to the expression of SREBP of all experimental groups. Fig. 5A

[0084] [Fig. 5A] FIG. 5A corresponds to the results of the immunohistochemical test to detect the expression and localization of the GSTP1 enzyme (brown color), in all experimental groups. Fig. 5B

[0085] [Fig.5B] FIG. 5B shows the results of GSTP1 protein expression by western blot of total extracts. Fig. 5C

[0086] [Fig. 5C] FIG. 5C corresponds to the results of the quantification of the GSTP1-positive area in the immunohistochemical test (IHC) of the liver tissue of all experimental groups. Fig. 5D

[0087] [Fig.5D] FIG. 5D shows the results of the relative quantification of the protein expression profile of GSTP1 in total extracts, normalized with [3- Actin in all experimental groups. Fig. 6A

[0088] [Fig.6A] FIG. 6A is the protein expression profile of the antioxidant enzymes Nrf2, CAT and SOD, by western blot in total extracts. Fig.6B

[0089] [Fig.6B] FIG. 6B shows the results of the relative quantification of the protein expression profile of the Nrf2 enzyme, normalized with [3-Actin of all experimental groups. Fig.6C

[0090] [Fig.6C] FIG. 6C corresponds to the results of the relative quantification of the protein expression profile of the CAT enzyme, normalized with [3-Actin of all experimental groups. Fig.6D

[0091] [Fig.6D] FIG. 6D shows the results of the relative quantification of the protein expression profile of the SOD enzyme, normalized with [3-Actin of all experimental groups. Fig.7A

[0092] [Fig.7A] FIG. 7A is the protein expression profile of DNMT1 , DNMTI Ac, DNMT3a, DNMT3b, UHRF1 , and PCNA enzymes in the liver tissue of all experimental groups. Fig.7B

[0093] [Fig.7B] FIG. 7B shows the results of the relative quantification of the protein expression profile of the enzymes DNMT1, DNMTI Ac, DNMT3a, DNMT3b, UHRF1 and PCNA, normalized with Lamin-B1 of all experimental groups. Fig.7C

[0094] [Fig. 7C] FIG. 7C corresponds to the results of double-label immunofluorescence analysis directed against DNMT1 and 5-meC modification in liver tissue from all experimental groups. Fig.7D

[0095] [Fig.7D] FIG. 7D shows the results of the representative Dot blot assay of the global DNA methylation profile of all experimental groups. Fig.7E

[0096] [Fig.7E] FIG. 7E corresponds to the results of the relative quantification of the global methylation level by Dot blot of all experimental groups. Fig.7F

[0097] [Fig.7F] FIG. 7F shows the results of quantification of the degree of global DNA methylation by ELISA analysis of all experimental groups. Fig.8A

[0098] [Fig.8A] FIG. 8A is the protein expression profile of DNMT1 , DNMTIAc, DNMT3a, DNMT3b, UHRF1 , and PCNA enzymes in hepatoblastoma cells in cell culture (HepG2) treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-pyridone formulated into nanoparticles according to an embodiment of the present invention in a 5-Aza-2'-deoxycytidine (5-Aza) DNA methylation inhibition model. Fig.8B

[0099] [Fig.8B] FIG. 8B corresponds to the results of double-label immunofluorescence analysis directed against DNMT1 and the 5mC modification in hepatoblastoma cells in cell culture (HepG2) treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-pyridone formulated in nanoparticles according to an embodiment of the present invention in a 5-Aza-2'-deoxycytidine (5-Aza) DNA methylation inhibition model. Fig.8C

[0100] [Fig.8C] FIG. 8C shows the results of the representative Dot blot assay of the global DNA methylation profile of HepG2 cells with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-pyridone formulated in nanoparticles according to an embodiment of the present invention in a DNA methylation inhibition model with 5-Aza-2'-deoxycytidine (5-Aza). Fig.8D

[0101] [Fig.8D] FIG. 8D corresponds to the results of the relative quantification of global methylation by Dot blot in liver tissue treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-pyridone formulated in nanoparticles in accordance with an embodiment of the present invention in a model of inhibition of DNA methylation with 5-Aza-2'-deoxycytidine (5-Aza). Description of some way of carrying out the invention

[0102] The present invention provides a pharmaceutical composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles as an alternative to current therapies and pharmaceutical compositions intended to treat HCC, which have limited effectiveness depending on the stage of the patient or degree of severity of the disease.For this purpose, a pharmaceutical composition has been developed comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles, which is therapeutically effective for an altered physiological condition in a subject who needs it, such as a subject who needs treatment or prophylaxis for HCC, a patient who needs to slow down the development of HCC, a patient who needs therapy to intervene in the hepatic fibrogenic process, to prevent the development of neoplastic lesions or to modulate the oxidative process and epigenetic alterations.

[0103] On the other hand, the invention additionally considers the use of a pharmaceutical composition for the manufacture of a medicament for the treatment or prophylaxis of a condition such as hepatocellular carcinoma (HCC), hepatic fibrogenic process, neoplastic lesions, modulation of the oxidative process and epigenetic alterations, as well as the reduction of tumors in subjects with HCC.

[0104] For this purpose, a pharmaceutical composition has been invented to treat an altered physiological condition in a subject who needs it, which comprises acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles.

[0105] Thus, in one aspect of the invention, a pharmaceutical composition is described for treating an altered physiological condition in a subject in need thereof, comprising at least one acetylated derivative of L-cysteine ​​and at least one derivative of 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles.

[0106] In a non-limiting embodiment of the present invention, the acetylated derivative of L-cysteine ​​is N-acetylcysteine ​​(NAC).

[0107] In a non-limiting embodiment of the present invention, the 5-methyl-1-phen I-2 (1 H)-pyridone derivative is pirfenidone (PFD).

[0108] In another non-limiting embodiment of the present invention, the nanoparticles are nanoparticles of organic nature, are transition metal oxides or are bioactive molecules.

[0109] Regarding organic nanoparticles, these are selected from the group consisting of liposomes, micelles, proteins, polymers, and dendrimers. In a more preferred embodiment of the present invention, the organic nanoparticles are liposomes.

[0110] As for the nanoparticles that are transition metal oxides, in a non-limiting embodiment, the transition metal oxides are selected from the group consisting of aluminum oxide, magnesium oxide, titanium dioxide, zinc oxide, iron oxide, zene oxide, and silver oxide. In a more preferred embodiment of the present invention, the transition metal oxide is zene oxide.

[0111] The composition of the present invention may further comprise additional components such as pharmaceutically acceptable excipients.

[0112] In another aspect of the invention, a pharmaceutical composition is described for use in the treatment or prophylaxis of a condition.

[0113] In a preferred embodiment of the present invention, the pharmaceutical composition is for use in the treatment or prophylaxis of hepatocellular carcinoma (HCC), hepatic fibrogenic process, neoplastic lesions, modulation of the oxidative process and epigenetic alterations.

[0114] In a more preferred embodiment of the present invention, the pharmaceutical composition is for use in tumor reduction in subjects with HCC.

[0115] In another aspect of the invention, the use of a pharmaceutical composition for the manufacture of a medicament for the treatment or prophylaxis of a condition is described.

[0116] In a preferred embodiment of the present invention, the use of a pharmaceutical composition for the treatment of hepatocellular carcinoma (HCC), hepatic fibrogenic process, neoplastic lesions, modulation of the oxidative process and epigenetic alterations is described.

[0117] In a more preferred embodiment of the present invention, the use of a pharmaceutical composition for tumor reduction in subjects with HCC is described.

[0118] Referring now to FIG. 1A, representative photographs of the dissected liver tissues at the end of the experimental protocol are shown, recording their weight and physical characteristics for each of the experimental groups of rats. The tissues of the untreated group (NT) present a normal liver morphology, red-brown in color, with a smooth surface and soft consistency, without the presence of neoplastic lesions. The livers of the HCC group presented a pale color, abnormal architecture and multiple nodules (neoplastic lesions marked with white asterisks); however, the group treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1H)-phosphate formulated in nanoparticles in accordance with an embodiment of the present invention (also referred to as NAC / PFD-Nano), retained the color and liver structure, and the number and size of the nodules were significantly smaller.

[0119] On the other hand, FIG. 1 B and FIG. 1 C show the results of the analysis of the variation in liver weight of the experimental groups of rats, where both the variation in individual liver weight per group (FIG. 1 B) and the weight of the liver relative to the entire body of the rat (FIG. 1C), reveals an increase in the weight of the livers in the carcinogenic damage group (HCC group) compared to the group that received treatment with PFD (p<0.05), observing a condition of hepatomegaly by increasing around 4.5% compared to the group treated with a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles in accordance with a modality of the present invention (p<0.0001).

[0120] Furthermore, to evaluate the effect of a composition comprising acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-pyridone formulated in nanoparticles in accordance with an embodiment of the present invention, two markers of liver damage were determined. As a first marker of damage, FIG. 1 D shows the levels of the enzyme y-GTP in protein extract from the liver of the experimental groups of rats. Whereas, as a second marker of damage, FIG. 1 E shows the serum levels of the enzyme ALT in the liver of the experimental groups of rats. For both FIG. 1 D and FIG. 1 E, the levels of ALT and GGTP were significantly increased (pO.0001) in the HCC group, while the NAC / PFD-Nano group was effective in preventing the increase of these markers (p<0.05).

[0121] To determine the histological effect of treatment with a NAC / PFD-Nano composition, H&E and Masson's Trichrome staining were performed. FIG. 2A shows representative images of H&E staining of liver tissue from all experimental groups, while FIG. 2C shows representative images of Masson's Trichrome staining to label extracellular matrix fibers in liver tissue from all experimental groups. For both FIG. 2A and FIG. 2C, a lobular architecture and portal spaces surrounded by vascular structures, bile ducts and canicles, and a well-defined portal triad are observed in the NT group. The liver lobules have a characteristic polygonal shape with well-defined boundaries. In this structure, parenchymal cords consisting of polygonal hepatocytes with a round nucleus and surrounded by vascular sinusoids are observed.Histopathological analysis of the HCC group revealed severe damage and loss of liver structure, mostly poorly differentiated tumor hepatocytes arranged in rows of two or more cells with nuclei of variable size and shape, surrounded by bile canaliculi. Nuclear membrane irregularities were also observed. increasing the nucleus-cytoplasm ratio. While the HCC group treated with NAC / PFD-Nano presented a structural appearance similar to the untreated group (NT), with hepatic lobes and portal triads remaining well defined and delimited, where single-hepatocyte cords were observed and the liver cells appeared well differentiated and slightly atypical; that is, giant, binucleated cells, without cell membrane and with chromatin condensation.

[0122] Through quantitative histological analysis depicted in FIG. 2B and FIG. 2D, an increase in atypical hepatocytes and extracellular matrix area was observed in the HCC group compared to the NT group. While a significant decrease in the number of atypical hepatocytes and extracellular matrix area was also observed in the HCC group treated with NAC / PFD-Nano compared to the HCC group (pcO.0001).

[0123] Similarly, FIG. 2E shows representative images of the immunohistochemical test using the hepatocellular carcinoma marker Glipican 3 in tissues from the different experimental groups; while FIG. 2F shows the quantification of the positive areas of the immunohistochemical test. The transmembrane protein Glipican-3 (GPC3) is a serological and histochemical marker of hepatocellular carcinoma, which is overexpressed in a high percentage in HCC (70-100%), has a higher detection sensitivity in poorly differentiated HCC, and its presence in early and intermediate stages means poor prognosis for patients. In the tissues of the control group in FIG. 2E, no positive areas for GPC3 were found.However, the group subjected to chemically induced HCC showed positive staining with cytoplasmic localization, with an intense granular pattern and intense granular staining with perinuclear distribution, which has a statistically significant difference (p<0.0001 ) compared to the NT group (FIG. 2F). On the other hand, treatment with NAC / PFD-Nano was able to prevent the expression of GPC3, since in this group the positive areas are observed to a lesser extent than the HCC group (FIG. 2E) and statistically lower (FIG. 2F; pcO.0001 ).

[0124] On the other hand, FIG. 3A shows the protein expression profile of the tumor marker [3-catenin by western blot of cytoplasmic extracts from all experimental groups, while FIG. 3B shows the protein expression profile of the tumor marker [3-catenin]. protein expression of tumor markers [3-catenin and c-Myc by western blot of nuclear extracts from all experimental groups. The transcription factors [3-Catenin and c-Myc are a therapeutic target since they are aberrantly expressed in different human cancers and are involved in the tumor transformation of liver cells. For this reason, it was determined whether the combination of NAC / PFD-Nano is capable of regulating the expression of both proteins in the experimental model, where FIG. 3C shows the relative quantification of the protein expression profile of the tumor marker [3-catenin cytoplasmic extracts, normalized with [3-Actin from all experimental groups, FIG. 3D shows the relative quantification of the protein expression profile of the tumor marker [3-catenin nuclear extracts, normalized with [3-Actin from all experimental groups and FIG.3E shows the relative quantification of the protein expression profile of the tumor marker c-Myc in nuclear extracts, normalized with [3-Actin] from all experimental groups, from which it is observed that HCC induction increases the nuclear expression of both c-Myc (p<0.0001) and [3-Catenin (pO.001). While treatment with NAC / PFD-Nano significantly decreases the expression of both proteins in the nucleus.

[0125] Likewise, FIG. 4A and FIG. 4E show the variation in the protein expression profile of PPARa, PPARy and PPARy2 by western blot of cytoplasmic and nuclear extracts (respectively) from all experimental groups. Peroxisome proliferator-activated receptors (PPARs) play an important role in the regulation of multiple proteins involved in lipid metabolism, energy homeostasis, cell differentiation and tumorigenesis, making it relevant to evaluate the expression and cytoplasmic and nuclear localization of PPARa, PPARy and PPARy2.

[0126] Quantitatively, for cytoplasmic extracts from all experimental groups, FIG. 4B shows the relative quantification of the protein expression profile of PPARα normalized with [3-Actin], FIG. 4C shows the relative quantification of the protein expression profile of PPARγ normalized with [3-Actin] and FIG. 4D shows the relative quantification of the protein expression profile of PPARγ2 normalized with [3-Actin]. Compared to the NT group, Carcinogenic damage significantly increased the cytoplasmic expression of PPARa (p<0.0001 ), PPARy (p<0.05) and PPARy2 (pcO.0001 ). However, the group that received treatment with NAC / PFD-Nano presented a statistically significant overexpression with respect to the damage group (HCC) of the isoforms PPARa (p<0.01 ) and PPARy (p<0.01 ). While treatment with NAC / PFD-Nano prevents the expression of PPARy2.

[0127] For nuclear extracts from all experimental groups, FIG. 4F is the relative quantification of the PPARα protein expression profile normalized with Lamin-B1 , FIG. 4G shows the relative quantification of the PPARγ protein expression profile normalized with Lamin-B1 and FIG. 4H shows the relative quantification of the PPARγ2 protein expression profile normalized with Lamin-B1. Given their role as transcription factors, these isoforms carry out their function at the nuclear level, where PPARα expression decreased significantly during induced damage (HCC; pcO.0001 ) compared to the NT group, but the expression of PPARγ (pcO.0001 ), PPARγ2 (pcO.0001 ) and in the induced damage group (HCC) significantly increased compared to the NT group. However, treatment with NAC / PFD-Nano reduces the overexpression and localization of PPARy (pcO.0001) and PPARy2 (pcO.0001) compared to the induced damage group (FIG. 4G and FIG. 4H respectively).

[0128] FIG. 4I shows the protein expression profile of SREBP and SREBP phosphorylated at Ser372 obtained by western blot of cytoplasmic extracts from all experimental groups. The transcriptional factors PPARγ and SREBP regulate each other to control fatty acid and triglyceride metabolism, so it is important to evaluate the expression of SREBP and its active form phosphorylated at Ser372.

[0129] Quantitatively, FIG. 4J shows the protein expression profile of SREBP, normalized with [3-Actin] of all experimental groups, while FIG. 4K shows the relative quantification of the protein expression profile of Psrebp-1 C with respect to the expression of SREBP of all experimental groups. Where the HCC group shows a statistically significant increase in the phosphorylated form of SREBP (p<0.0001) in compared with the NT and NAC / PFD-Nano groups. That is, treatment with NAC / PFD-Nano reduces SREBP expression and phosphorylation (FIG. 4K).

[0130] Oxidant stress can induce the accumulation of genetic and epigenetic alterations, allowing upregulation of genes related to proinflammatories, oncosuppressors and oncopromoters involved in the development of HCC, in addition, an excess of ROS can damage lipids, proteins and DNA, and in this way, several cellular processes are altered such as gene expression, cell adhesion, cell metabolism, cell cycle and cell death. In addition, ROS are related to the activation of cellular signaling pathways such as mitogen-activated protein kinase (MAPK), NF-KB, phosphatidylinositol 3-kinase (PI3K), p53, [3-catenin / Wnt and angiogenesis. On the other hand, GSH plays an important role during HCC.GSH and glutathione peroxidase (GPx) enzyme activity have been shown to be increased in HCC tissues compared with adjacent normal liver; furthermore, preoperative plasma levels of GSH and oxidized glutathione (GSSG) were low, but post-resection GSH levels increased, suggesting that GSH is able to modulate oxidative damage during carcinogenesis. Finally, as an additional effective mechanism induced by NAC administration, it was found that pretreatment with this antioxidant drug abolished the induction of autophagy by ionizing radiation or hyperthermia, two treatments commonly used in HCC patients.

[0131] In order to determine the effect of NAC / PFD-Nano on proteins regulated by oxidative stress, IHC, Western Blot, and enzyme recycling techniques were performed using the Griffith method for enzymes involved in the antioxidant response mechanism. FIG. 5A shows the immunohistochemical test to detect the expression and localization of the GSTP1 enzyme in all experimental groups, where the presence of this GSTP1 marker is not detected in the NT and HCC groups, while in the group treated with NAC / PFD-Nano there is a significant increase in positive areas with cytoplasmic localization compared to the HCC group (pcO.0001). These results are also confirmed by observing FIG. 5B, which shows the protein expression levels of GSTP1 by western blot of total extracts, which revealed a significant increase in GSTP1. in the NAC / PFD-Nano treated group compared to the HCC group (p<0.01) in the quantitative analysis of FIG. 5C for the GSTP1 positive area in the immunohistochemical test (IHC) and FIG. 5D for the relative quantification of the protein expression profile of GSTP1 in total extracts normalized with [3- Actin in all experimental groups.

[0132] Similarly, FIG. 6A shows the protein expression profile of the antioxidant enzymes Nrf2, CAT and SOD, by western blot in total extracts. And in particular, FIG. 6B shows the relative quantification of the protein expression profile of the Nrf2 enzyme, normalized with [3-Actin, FIG. 6C shows the relative quantification of the protein expression profile of the CAT enzyme normalized with [3-Actin and FIG. 6D shows the relative quantification of the protein expression profile of the SOD enzyme, normalized with [3-Actin; where it can be seen that the HCC group has a statistically significant increase in the expression of Nrf2, compared to the NT group (pcO.001); while the group treated with NAC / PFD-Nano shows a statistically significant reduction in the expression of Nrf2, compared to the HCC group (FIG. 6B; pcO.001).While CAT expression was statistically higher in the NAC / PFD-Nano group compared to the HCC group (FIG. 6C p<0.05), SOD expression did not show significant differences between groups, as the expression of this protein was similar in all three experimental groups (FIG. 6D).

[0133] DNA methylation is the best understood and widely studied epigenetic mechanism. The enzymes responsible for methylation are DNA methyltransferases and are classified into two categories: de novo, including DNMT3A (chromosome 2) and DNMT3B (chromosome 20), and maintenance, DNMT1 (chromosome 19). These enzymes are involved in preserving chromosome stability and genome integrity, embryonic development, cell differentiation, and organismal growth. The main function of DNMT1 is to maintain the methylation state after DNA synthesis. It has a high affinity for hemimethylated DNA. DNMT3A and DNMT3B are mainly active during embryonic development and identify non-motylated cytosines and establish new methylation patterns. The N-terminal domain of DNMT allows nuclear localization. In DNMT1, there is a binding region consisting of repeated dipeptide lysine-glycine regions, which connects the N-domain with the C-domain. The C-region is the domain that carries out the methyltransferase function by binding to various substrates such as cytosine. DNMT3A and 3B are structurally similar, containing the TRX-DNMT3-DNMT3L (ADD) region, which interacts with unmodified regions. The PWW domain allows the specific binding of these enzymes to DNA. All isoforms contain a nuclear antigen-binding domain (PBD). Specifically, motif IV carries out the methylation reaction in the C-terminal domain. DNA methylation occurs at cytosine bases found in CpG islands. Methylation of these islands in promoter regions is generally linked to the inactivation of gene expression.Methylated CpG islands can promote chromatin condensation by directly inhibiting the interaction of DNA-binding proteins with target sites and can also provide recognition signals for the recruitment of methyl-CpG-binding domain-containing proteins.

[0134] FIG. 7A shows the protein expression profile by western blot of methylation enzymes and accessory proteins such as DNMT1 , DNMTIAc, DNMT3a, DNMT3b, UHRF1 , and PCNA in the liver tissue of all experimental groups. DNA methylation is a stable but reversible epigenetic mark that regulates gene expression, so it is relevant to evaluate the expression of the different isoforms of these enzymes that participate in this process, that is, DNMTs and accessory proteins, where it is observed that the induction of HCC reduces the expression of DNMT1 , while treatment with NAC / PFD-Nano increases its expression. This effect is corroborated by performing the statistical analysis shown in FIG. 7B, where these significant changes are appreciated (p <0.001 ). The methyltransferase function of this enzyme is regulated at the post-translational level through the specific acetylation of lysine residues.Acetylation of residues K1127, K1129, K1131 and K1133 in the catalytic domain of DNMT1 were affected in HCC, NAC / PFD- Nano significantly increased DNMT1 acetylation (FIG. 7A and FIG. 7B; p<0.0001).

[0135] Likewise, the results obtained also demonstrate that treatment with NAC / PFD-Nano affects the expression of DNMT3a since there is an increase statistically significant compared to the NT and HCC groups (pcO.001). However, DNMT3b expression did not show significant differences between groups (FIG. 7A and FIG. 7B).

[0136] For DNMTs to perform their function, they must be recruited to the DNA replication fork, forming a replication machinery with multiple associated proteins. In particular, the proliferating nuclear antigen (PCNA), which functions as a DNA clamp and interacts with DNA-modifying enzymes, such as DNMT1, which increase its activity and affinity for DNA. This response is due to the N-terminal domain of DNMT1, which harbors a PCNA-binding domain, called the PBD. However, DNMT1 can also be recruited by PCNA to DNA repair sites, to restore methylation patterns after repair. Another DNMT1-associated protein is UHRF1 , which plays an important role in DNA methylation, since it recognizes hemimethylated DNA generated in replication and recruits DNMT1 to guarantee methylation patterns.

[0137] HCC induction reduces UHRF1 expression, whereas NAC / PFD-Nano treatment significantly increases its expression (FIG. 7A and FIG. 7B, pcO.001). Notably, DNMT1 has a PCNA binding domain, which allows both to interact to regulate cell replication. In the HCC group, statistically significant elevated levels of PCNA were determined (FIG. 7A and FIG. 7B, pcO.001), which were prevented by NAC / PFD-Nano treatment, since this study group demonstrated protein expression levels similar to the NT group (FIG. 7A).

[0138] FIG. 7C shows the double-label immunofluorescence analysis directed against DNMT1 and the modification in 5-meC in the liver tissue of all experimental groups in order to confirm the localization and activation of DNMT1, where the DNMT1-positive nuclei (green channel) were immuno-labeled and quantified, as well as the 5-methylcytosine methylations of the DNA (5mC, red channel), it can be observed in the NT group a large number of nuclei that are expressing this protein as well as a greater 5mC label, contrary to the HCC group there is a decrease in the intensity of both labels, particularly in the neoplastic nodules, while the treated with NAC / PFD-Nano, more DNMT1-positive nuclei were detected in the neoplastic nodules as indicated in FIG. 7C.

[0139] Once protein expression was confirmed, we assessed whether DNMT1 function was modulated by NAC / PFD-Nano treatment. FIG. 7D shows the representative dot blot assay of the global DNA methylation profile of all experimental groups using the 5mC label.

[0140] Quantitatively, FIG. 7E shows the global methylation levels by Dot blot, while FIG. 7F shows the degree of global DNA methylation by ELISA analysis, where the induction of carcinogenic damage decreases DNA methylation compared to the NT group (pcO.001 ). Treatment with NAC / PFD-Nano restored global DNA methylation as shown in FIG. 7D and FIG. 7E (pcO.001 ). In the carcinogenic damage group (HCC), a trend of decreased DNA methylation is observed; however, treatment with NAC / PFD-Nano restores the percentage of methylation, even above normal values ​​(FIG. 7F, p<0.001 ).

[0141] Similarly to the studies carried out in vivo, in vitro analyses were also carried out considering a compound that induces methylation inhibition; where FIG. 8A shows the protein expression profile of the enzymes DNMT1, DNMTI Ac, DNMT3a, DNMT3b, UHRF1, and PCNA treated with a composition of NAC and PFD formulated in nanoparticles in accordance with an embodiment of the present invention in a model of DNA methylation inhibition with 5-Aza-2'-deoxycytidine (5-Aza). Treatment with 5-Aza preferentially affects the expression of DNMT1 (p<0.00001) and its active form DNMTI ac (p0.0001), the expression of the isoforms DNMT3a and DNMT3b is not significantly affected. On the other hand, NAC / PFD-Nano treatment significantly increases the expression of DNMT1 (pcO.0001 ) and its activation in the form of DNMTI ac (pcO.0001 ) and even with prior inhibition with 5-Aza.It is noteworthy that in both the HCC model and the HepG2 cell line, treatment with NAC / PFD-Nano did not affect DNMT3b expression.

[0142] Likewise, treatment with the DNMT inhibitor (5-Aza) did not show significant effects on UHRF1 expression; however, it increased PCNA expression (FIG. 8A; p<0.0001 ). After treatment with 5-Aza and subsequent treatment with NAC / PFD-Nano, a significant increase in UHRF1 expression (pcO.0001 ) and a decrease in PCNA (p<0.01 ) was observed.

[0143] FIG. 8B shows dual-label immunofluorescence analysis targeting DNMT1 and 5mC modification in liver tissue treated with a composition of NAC and PFD formulated into nanoparticles according to an embodiment of the present invention in a 5-Aza-2'-deoxycytidine (5-Aza) DNA methylation inhibition model to confirm DNMT1 localization and activation in the in vitro system.Where the DNMT1-positive nuclei (green channel) were immuno-labeled and quantified, as well as 5-methylcytosine DNA methylation (5mC, red channel), a large number of nuclei expressing this protein as well as a greater 5mC mark can be observed in the NT group, on the contrary, in cells treated with 5-Aza there is a decrease in the intensity of both marks, particularly in the cell nuclei, while in the group treated with NAC / PFD-Nano more positive nuclei are detected for both DNMT1 and DNA methylation (5mc). These data were quantified, it can be concluded that NAC / PFD-Nano treatment after DNMT inhibition significantly restores global DNA methylation.

[0144] FIG. 8C shows the representative dot blot assay of the global DNA methylation profile treated with a composition of NAC and PFD formulated in nanoparticles according to an embodiment of the present invention in a DNA methylation inhibition model with 5-Aza-2'-deoxycytidine (5-Aza) to confirm the effect of NAC / PFD-Nano in regulating the expression of enzymes involved in DNA methylation.

[0145] Quantitatively, FIG. 8D shows the global methylation level by Dot blot in liver tissue treated with a composition of NAC and PFD formulated in nanoparticles according to an embodiment of the present invention in a DNA methylation inhibition model with 5-Aza-2'-deoxycytidine (5-Aza). Where it is observed that the NAC / PFD-Nano treatment significantly restores methylation even after treatment with 5-Aza (pcO.0001), confirming what was observed visually (FIG. 8C).

[0146] Altered expression and function of DNMTs leads to dysregulated methylation patterns and contributes to cancer development. Both hypermethylation and hypomethylation of genomic DNA have been detected in HCC at different stages of development. Hypermethylation occurs mainly in CpG islands in promoter regions and in cis-regulatory regions of transcription. Generally, hypermethylation of promoter regions promotes the repression of gene expression, and frequently occurs in tumor suppressor genes including GATA4, CDKL2, CDKN1A, CDH1 , NEFH34, NOTCH3, P15, P16 and Retinoblastoma 1. Nishida and colleagues have characterized hypermethylated and inactive tumor suppressor genes in early stages of HCC, including: HIC1 , GSTP1 , SOCS1 , RASSF1 , CDKN2A, APC, RUNX3 and PRDM2. In advanced stages of HCC, hypermethylation of the RASSF2, MINT1, MINT2, RPRM, SFRP2, CDH1 and DCC genes has been reported.On the other hand, hypomethylation occurs at specific loci, in promoter regions, 5'UTR regions and intergenic regions. The changes produced by hypomethylation are linked to genomic instability, mutations and genomic rearrangements that occur in inactive chromatin regions. In an analysis of global hypomethylation, different hypomethylated genes were determined in HCC; CEBPp, CCL20, PGK1 and PDHK1 , which have gained relevance in recent years as potential therapeutic targets. Other genes that have also been reported as hypomethylated in HCC are MAD2L1 and CDC20, these are highly expressed in high-grade dysplastic nodules compared to low-grade ones and have a close relationship with tumor aggressiveness.

[0147] Two mechanisms of action have been described through which NAC exerts its effects. The first is related to its mucolytic action, which is due to the rupture of disulfide bonds in the glycoprotein matrix of reticulated mucus, decreasing its viscosity; and the second, its antioxidant mechanism, is due to its ability to act as a precursor to reduced glutathione (GSH), increasing its levels at the cytosolic level.

[0148] During excessive ROS production, NAC exerts its antioxidant response through the sulfhydryl (SH) group present in its structure, acting as an effective free radical scavenger. Another protective response carried out by NAC is its ability to increase GSH levels. GSH is composed of three amino acids: glutamate, glycine, and cysteine. NAC provides the limiting amino acid, cysteine, necessary to replenish GSH levels. It is also important to mention that NAC is an effective mucolytic agent, whose response is related to its SH group and its ability to interact with disulfide bonds in mucoproteins, breaking them down into smaller, less viscous units.

[0149] Clinical studies have demonstrated the safety and tolerability of NAC in human patients; specifically, data from four prospective clinical trials, conducted in 331 patients treated with NAC (oral or intravenous), have shown minor side effects such as nausea, vomiting, and other gastrointestinal discomfort. In rare cases, NAC may cause rash, fever, headache, drowsiness, and low blood pressure.

[0150] The invention also provides pharmaceutical compositions comprising one or more compounds of the invention, in combination with one or more pharmaceutically acceptable carriers or excipients. Such excipients include, but are not limited to, fillers, binding agents, lubricants, preservatives, water, buffers, and disintegrants. The compositions may be in the form of combined solids or liquids for oral administration (e.g., as tablets, lozenges, hard or soft capsules, extended-release capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups, or elixirs) or for parenteral administration, such as solutions or suspensions suitable for parenteral administration (e.g., a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, or intraperitoneal dosing, or as a suppository for rectal dosing).Pharmaceutically acceptable carriers and excipients are those compounds, solutions, substances, or materials that can be used to produce formulations of the compounds of the present invention suitable for administration to a subject. In particular, the carriers and excipients of the present invention are those useful in the... preparation of pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and that may exhibit favorable pharmacological profiles, and include carriers and excipients that are acceptable for veterinary use, as well as pharmaceutical use in humans. Suitable pharmaceutically acceptable carriers and excipients are well known in the art and can be determined by those skilled in the art as the clinical situation requires. Suitable carriers and excipients are set forth, for example, in the Pharmacopoeia of the United Mexican States (FEUM, 13 § Ed.) and its supplements.

[0151] The person skilled in the art will understand that diluents used for parenteral or oral administration are included within the scope of the terms carriers and excipients. Examples of suitable carriers and excipients include saline, buffered saline, dextrose, water, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween™-80), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g., Cremophor™ EL), poloxamer 407 and 188, a cyclodextrin or cyclodextrin derivative (including HPCD ((2-hydroxypropyl)cyclodextrin) and (2-hydroxyethyl)cyclodextrin; see, e.g., U.S. patent application publication 20060194717), hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholides, polymeric matrices, biocompatible polymers, lipospheres, vesicles, particles and liposomes.The excipients, carriers, and diluents included in a formulation have different purposes depending on, for example, the nature of the drug, the mode of administration, and the purpose for which the formulation is intended. Examples of generally used excipients include, but are not limited to: stabilizing agents, solubilizing agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, antibacterials, antioxidants, chelating agents, sweeteners, perfuming agents, flavoring agents, coloring agents, administration aids, and combinations thereof.

[0152] The compositions may further contain common carriers and excipients such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium and sodium starch glycolate.

[0153] Pharmaceutically acceptable excipients also include tonicity adjusting agents that render the composition isotonic with blood; these are particularly desirable in injectable formulations. Suitable tonicity adjusting agents include, but are not limited to, monosaccharides, disaccharides, trisaccharides, alditols, and mixtures thereof. Preferred agents are sucrose, dextrose, trehalose, mannitol, lactose, glycerol, and sorbitol.

[0154] NAC can be administered enterally, intravenously, and by inhalation; it is well tolerated and safe at high doses. When administered orally, it undergoes intestinal absorption and hepatic metabolism, which provides and directs the released cysteine ​​for GSH synthesis. Its Cmax is reached within 1 to 2 hours after oral administration.

[0155] The bioavailability of NAC after enteral administration is <10%, and only a small portion of the molecule reaches plasma and tissues intact. After complete hepatic biotransformation, the metabolites cysteine, cystine, organic sulfate, and glutathione are the main excretable products.

[0156] NAC, when administered intravenously, bypasses the first-pass intestinal-hepatic effect, allowing it to reach high plasma concentrations rapidly. This route of administration is the one most commonly used to treat acetaminophen poisoning. After intravenous administration of 150 mg / kg for 15 minutes, NAC Cmax was 554 mg / L, and Vd ranged from 0.33 to 0.47 L / kg. Regarding its excretion, its clearance ranges from 0.190 to 0.21 L / h / Ke, with an estimated half-life of 2.15 h. NAC is undetectable in plasma 12 hours after dosing.

[0157] Pirfenidone (PFD) or 5-methyl-1-phenyl-2-(1 H)-pyridone is a molecule with a molecular weight of 185.23 g / mol, partially soluble in water, methanol, alcohol and chloroform, which can diffuse through cell membranes without requiring the participation of a receptor. Among its properties Pharmacokinetics, pirfenidone is easily absorbed from the gastrointestinal tract, reaching most tissues and crossing the blood-brain barrier; it reaches its maximum blood levels after 1-2 hours and is almost completely eliminated in the urine after 6 hours. With respect to its biosafety, no significant toxicity attributable to the drug has been reported at doses of 2500 mg / day; reported side effects include nausea, photosensitivity, and gastrointestinal problems. Among its main reported properties are: It has been established that PFD activates the translocation of Nrf2 to the nucleus where it activates the transcription of the GCLC, GCLM and HMOX1 genes in CEH. On the other hand, its antiproliferative and anti-inflammatory effect is carried out through the decrease of the cytokines IL6, MCP1 and TNFa; and antifibrogenic by decreasing the expression of COL1A1, TGF-1 [3 and TIMP1.In 2006, Armendáriz-Borunda et al. reported that PFD modulates mechanisms related to necroinflammation, steatosis and, interestingly, reverses advanced liver fibrosis in patients with HCV. In 2002, it was reported that PFD decreases the expression levels of collagen types I, II and IV and TGF-1 [3; while through an in silico analysis carried out by Silva Gómez et al. in 2021, it was possible to determine that PFD acts as a PPARy ligand-agonist. Furthermore, PFD induces nuclear overexpression of PPARy, postulating that this mechanism could slow the development of HCC (Silva Gómez JA, et al. 2021).

[0158] This science seeks to minimize drug degradation and loss, prevent the onset of side effects, and increase the bioavailability of the active ingredients at the sites of action. A drug's therapeutic potential depends not only on its physicochemical properties but also on processes related to its interaction within a living organism, which leads to adjustments in its half-life, its elimination via various pathways, and its biotransformation.

[0159] The size of drug nanocarriers varies between 3 and 200 nm, and can have different natures depending on the intended purpose, for example, proteins (albumin), lipids (liposomes), polymers (micelles, nanoparticles or dendrimers), viruses (viral nanocarriers), metals (metallic nanocarriers), among others.

[0160] The use of liposomes reduces the nonspecific toxicity of the drug itself, which can be especially useful when releasing drugs such as highly toxic anticancer drugs. Their size ranges between 50 and 100 nm to ensure the drug's release mechanism; this size allows them to cross tissues and bind to the target sites. Once the target sites are reached, the release of the active ingredients depends on the lipid nature of the bilayer, the size of the active ingredient molecule, and its interaction with the lipid membrane. Liposomes can adhere to the cell membrane, where, through the enzymatic action of lipases or mechanical forces, the active ingredient(s) are released into the extracellular space, where they diffuse to the cell membrane and subsequently to the cytoplasm.When the drug is hydrophilic, the release occurs directly into the cytoplasm, due to the fusion of the liposomal membrane with the cell membrane.

[0161] NAC / PFD-loaded liposomes were generated based on our previous experience with ocular liposomal formulations. The ideal formulation for the release and absorption of the active ingredients, according to their pH, osmolality, viscosity, stability, and encapsulation efficiency, is related to the following composition: Pirfenidone (0.2 mg), NAC (200 nM), kolifor HS 15 (50 mg), Polyethylene glycol (100 mg), ethyl alcohol (14 pL), anhydrous citric acid (0.8 mg), sodium citrate (4.675 mg), benzalkonium chloride (0.1 mg), purified water grade 2 (Q;S; 1.0 mL). The self-formed and thermodynamically stable liposomal formulation (PL) loaded with NAC / PFD was formed as previously described [Altamirano Vallejo JC, et al. 2018; Navarro-Partida J, et al.

[2020] Briefly, PFD (Tecoland Corporation, Irvine, CA, USA) and NAC, Sigma-Aldrich (St. Louis, MO, USA) were first added to a lipid mixture containing polyethylene glycol (PEG-12, Sigma-Aldrich, St. Louis, MO, USA).UU.), glyceryl dimyristate and ethyl alcohol (Sigma-Aldrich, St. . Louis, MO, USA). An aqueous mixture containing grade 2 purified water, polyethylene glycol (15)-hydroxystearate (Kolliphor HS 15, Sigma-Aldrich, St. Louis, MO, USA), anhydrous citric acid (Sigma-Aldrich, St. Louis, MO, USA), dehydrated sodium citrate (Sigma-Aldrich, St. Louis, MO, USA) and benzalkonium chloride (Sigma-Aldrich, St. Louis, MO, USA) was mixed in a. flask and were set aside for compound preparation at room temperature. The water mixture was gently added to the lipid mixture to obtain the final formulation. The PL was filtered through 0.22 mm pore size membranes (Merck Millipore, Billerica, MA, USA) under aseptic conditions.

[0162] Characterization of NAC / PFD loaded liposome formulation

[0163] The physicochemical and microscopic characterization of the NAC / PFD formulation was performed. First, the morphology of PFD and NAC in aqueous solution, in ethanolic solution, and in liposomes was explored by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Aqueous and ethanolic solutions of NAC / PFD were prepared by adding ultrapure water or ethanol to the required amount of NAC / PFD crystals to achieve concentrations of 0.1%. A TESCAN MIRA3 LMU FE-SEM (Tescan Orsay Holding, as, Brno-Kohutovice, Czech Republic) was used for SEM analysis, while a JEOL JEM-1010 scanning electron microscope (Jeol USA, Peabody, MA, USA) was used for TEM studies. SEM samples were kept at -4 °C before being mounted in pieces and coated with gold using a Denton Vacuum Desk II sputter coater (SPI Supplies, West Chester, PA, USA).v / v ) and were deposited on FF 300 square mesh copper grids (Electron Microscopy Sciences, Fort Washington, PA, USA) for observation. Manual particle counting and measurement were performed using SEM micrographs in a 63.6 mm field of view to calculate the size and distribution of PLs.

[0164] Finally, the physicochemical properties, size distribution, and zeta potential of different PL formulations and diluted samples were determined at 37 °C, which is normal body temperature [Purslow C, et al. 2005]. The osmolality of 10 pL of sample was measured at room temperature using a Vapro 5600 vapor pressure osmometer (ELITechGroup, Paris, France). The viscosity of the PL formulations was measured at a shear rate of 100 s -1 using a stress-controlled AR-G2 rheometer (TA Instruments, New Castle, DE, USA) with a 60 mm cone and plate geometry of 2 o. The pH was controlled with an Orion Star A210 (Thermo Fisher Scientific, Waltham, MA, USA). Intensity size distributions, Polydispersity index (Pdl) and zeta potential values ​​of liposomal formulations diluted in double distilled water (ddH2O) or 1 mM PBS buffer (pH 7.4) were determined by dynamic light scattering (DLS) in a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). DLS measurements were performed using a dispersant refractive index of 1.33 and an absorption index of 0.01 . Zeta potential values ​​were obtained using the same diluted samples in a disposable capillary cell (DTS1070). All experiments were performed in triplicate.

[0165] The present invention will be better understood from the following examples, which are presented solely for illustrative purposes to allow a full understanding of the preferred embodiments of the present invention, without implying that there are no other non-illustrated embodiments that can be put into practice based on the detailed description given above. Examples

[0166] Example 1. Preparation of cerium oxide nanoparticles

[0167] A preparation of cene oxide nanoparticles as a NAC / PFD transport system was carried out.

[0168] The synthesis of metal nanoparticles of cene oxide coupled with N-acetyl cysteine ​​and pirfenidone is briefly described below. 1. In 200 mL of water at 80 S C, 3.2 g of sucrose and 0.4 g of pva (polyvinyl alcohol) were dissolved until a completely clear solution was obtained. 2. 13 g of nitrate hexahydrate, 10 g of NAC and 10 g of PFD were added. 3. Continuous stirring was carried out until all the solution evaporated. 4. At the end of evaporation, a semi-brown foam was obtained due to the burnt sucrose. 5. In the same glass, the foam was heated to a temperature of 220 S C for 4 h. 6. Once cold, it was heated to 450 S C for 4 hours in a muffle furnace, in a sintered alumina container (to avoid contamination). 7. It was cooled and cerium oxide coupled to NAC and PFD (NAC / PFD- Nano) was obtained. 8. This process yields approximately 10 g of NAC / PFD-nano, which must be suspended in an injectable solution.

[0169] Example 2.

[0170] Biochemical analyses consisted of determining alanine aminotransferase (ALT) and γ-glutamyl transpeptidase (GGT) enzyme activity in serum; reduced glutathione (GSH) and malondialdehyde (MDA) levels were quantified in liver tissue. For biochemical analysis of ALT and GGT, blood was drawn by cardiac puncture, followed by centrifugation at 4000 rpm / 15 min to obtain serum; 0.5 g of liver from an experimental animal model was used to quantify GSH and MDA levels.

[0171] Experimental Animal Model.

[0172] Male Fischer-344 rats were purchased through CIRCULO ADNSA de CV and maintained at the Bioteho of the University Center for Health Sciences (CUCS) of the University of Guadalajara (UdeG). All experiments were conducted in accordance with the guidelines approved by the Ethics, Research and Biosecurity Committees of CUCS with approval number CI-03020, and in accordance with the specifications of the Mexican Official Standard NOM-062-ZOO-1999, which indicates all the technical specifications for the production, care, and use of laboratory animals. A total of 18 male Fischer-344 rats were distributed into 3 groups. The animals had ad libitum access to water and food and were maintained at a temperature of 25 ± 2 °C with 12-hour light / dark cycles. Experimental interventions began when the rats reached approximately 180 grams in weight for the development of HCC.As reported by Castro-Gill et al. in 2021, damage is induced. by weekly intraperitoneal administration of diethylnitrosamine (DEN; 50 mg / kg) and three days later 2-acetylaminofluorene (2-AAF; 25 mg / kg) was administered intragastrically. The HCC-NAC / PFD-Nano group received an intravenous bolus (500 pL) of NAC-PFD nano (0.5 mg / mL of the active ingredients) 3 times a week for 12 weeks of treatment. At the end of each treatment, all animals were euthanized by the administration of isoflurane by inhalation.

[0173] ALT determination

[0174] The activity of the ALT enzyme was determined by the method implemented by Reitman and Frankl, in which the formation of the pyruvate complex and 2,4-dinitrophenylhydrazine is measured, the color obtained is absorbed at a wavelength of 515 nm. The determination is briefly described below in test tubes are placed 0.25 mL of the substrate solution and 0.05 ml of test serum except for the blank tubes, these are mixed and incubated at 37 ° C for 60 min. After this time, the chromogenic reagent or serum is added as appropriate, it was incubated again for 15 min at 37 ° C and then the reaction is stopped with 2.5 mL of 0.4 N NaOH. The samples are read at a wavelength of 515 nm.

[0175] The determination of the activity of the y-GTP enzyme is based on the fact that the substrate glutamyl-p-nitroanilide, in the presence of the y-GTP enzyme and the y-1 -glutamyl receptor, produces y-1 -glutamylglycine and p-nitroaniline; which can be quantified at a wavelength of 410 nm. In test tubes, 200 pL of serum, 400 pL of Tris-HCI, 200 mM / pH 8.2, 100 pL of MgCl2, 200 mM, 100 pL of Glycyl-glycine, 40 mM, pH 8.2 and 200 pL of Gamma-glutamyl-p-nitroanilide, 10 mM, are added; this mixture is incubated for 10 min / 37 °C. Finally, the reaction is stopped with 2 mL of 1.5 M acetic acid and read at 410 nm. Enzyme activity is determined by interpolation of the sample value onto a p-nitroaniline standard curve and is reported in pmol of substrate hydrolyzed per liter of plasma per minute.

[0176] Determination of reduced glutathione (GSH) and malondialdehyde (MDA)

[0177] The determination of GSH is based on the use of Ellman's reagent, which reacts with sulfhydryl groups to produce a colored product, and thus reduced cisterns are measured. To quantify this tripeptide, 0.5 g of liver was homogenized in 1.2 mL of precipitating solution (5 mM EDTA in 5% TCA), followed by centrifugation for 20 minutes at 12,000 rpm. 0.1 mL of supernatant was taken (in duplicate) and 2.1 mL of phosphate solution (0.3 M Na2HPO4) and 0.25 mL of Ellman reagent (prepared with 40 mg of 5,5'-dithiobisnitrobenzoic acid in 100 mL of 1% sodium citrate) were added. The mixture was stirred, and the absorbance at 412 nm was read.

[0178] On the other hand, for the determination of MDA, one of the final products of oxidative damage at the lipid level, we relied on the thiobarbital acid method, of oxidized lipid nature. 0.5 g of liver were weighed and homogenized in 5 ml of water. 300 pL of the homogenate were taken, then 700 pL of Tris-HCI (150 mM) and 2 mL of 0.375% TBA dissolved in 15% TCA were added, and the mixture was kept boiling for 45 min. Finally, the samples were cooled and centrifuged at 3000 rpm / 15 min, and the supernatant was read at a wavelength of 532 nm.

[0179] Example 3.

[0180] An in vivo histological analysis was performed in the liver of an animal experimental model and in vitro to determine the effect of a composition with NAC / PFD-Nano.

[0181] Resection and analysis of liver tissue

[0182] The abdomens of rats from each experimental group were exposed for subsequent liver tissue removal. Photographic documentation was obtained and small fractions were dissected and fixed in 4% paraformaldehyde for histological analysis. The remaining liver tissue was stored at -80°C until use.

[0183] Hematoxylin and Eosin Staining

[0184] The color differentiation of hematoxylin and eosin staining is based on the basophilic nature of the nuclei, which makes them affine to hematoxylin, while the cytoplasm is acidophilic with a high affinity for eosin. The process is briefly described below: 1) Deparaffinization and hydration: before starting the staining, it is necessary to deparaffinize the tissue; to do this, it is left for 24 hours / 55 ° C. It is then subjected to a hydration in solutions with increasing concentrations of alcohol from lowest to highest concentration. 2) Once the tissues have been hydrated, the following procedure is followed for staining: a) The sections are placed in Harris Hematoxylin for 40 s, b) rinsed with distilled water until the color is gone, c) bathed in 1% acid alcohol, d) washed gently with hot water to turn / 30 s, e) washed gently with cold water / 30 s. f) They are placed in Eosin / 15 s, and g) rinsed gently with water.

[0185] Masson's trichrome stain

[0186] This staining is based on the selective detection of tissue collagen through the use of acid aniline dyes (aniline blue, acid fuchsin). The most commonly used acid is picnic acid, which acidifies the medium and provides contrast color for muscle and cytoplasm. The procedure is briefly described below. 1) Deparaffinize and hydrate the tissue. 2) Once the tissues are hydrated, follow the following procedure for staining: 3) incubate the samples with Bouin's fluid for 1 h at 56 °C or overnight. 4) allow to cool for 10 min. 5) wash with running water until the sections become clear. 6) stain with hematoxylin stock solution. Weigert's ferric (freshly prepared) for 10 min, 7) wash with running water for 10 min and rinse with distilled water, 8) incubate with Briebñch's acid-scarlet fuchsin solution for 15 min, then rinse with distilled water, 9) incubate in phosphotungstic-phosphomolybdic acid solution for 10 min, 10) add aniline blue for 10 min and rinse with distilled water, 11) transfer to 1% acetic acid / 3 min, to differentiate, 12) dehydrate, rinse with alcohol baths from lowest to highest concentration, 13) mount with resin.

[0187] Histological analysis by immunofluorescence.

[0188] Immunofluorescence (IF) allows the localization of proteins of interest to be visualized in both cells and tissues. It is achieved by the specificity and affinity of antibodies to recognize their target molecules, and by the combination of specific secondary antibodies labeled with fluorophores. The procedure is briefly described below. 1) Deparaffinize the tissues in an oven for 24 h / 55 ° C. 2) Subsequently, the Slides were dehydrated with alcohols from highest to lowest concentration. 3) To restore the molecular structure and immunoreactivity, each sample was treated with citrate pH regulator / 40 min / 90 °C. 4) All slides were carefully placed on a humid chamber and incubated with the primary antibody diluted in PBS-Thton (0.2%)-BSA (5%) for 24 h. 5) For incubation with the secondary antibody, excess primary antibody was carefully removed with PBS. They were then incubated in the humid chamber at room temperature (2 h / darkness). 6) Hoechst staining (1:1000 / 5 min) will allow staining of the nuclei. 7) Mounting medium was placed on the tissues until they were completely covered, and finally, a coverslip was placed over the samples and sealed with varnish. 8) Finally, the tissues were analyzed by confocal microscopy using a ZEISS LSM 800 laser scanning microscope.

[0189] Cultivation and treatment of HCC cell lines

[0190] The HepG2 cell line was cultured in standard plates (100x20mm), with Dulbecco's Modified Eagle's Medium (DMEM, Corning), 37 °CZ 5% CO2, added with 10% fetal bovine serum (FBS, GIBCO) and 1% Antibiotic-Antimycotic 100x (Anti-Anti, Gibco). The cells were grown until they were ~80-85% confluent; subsequently, they were seeded in multi-well plates (p6), and starved for 8 h (DMEM without FBS). The cells were subjected to different treatments for 24 h; pirfenidone (500 pm) / NAC 300 pm and 5-Azacitidine (5-Aza; 20 pm)

[0191] Example 4.

[0192] An analysis of protein expression of interest was performed in in vivo models in the liver of an animal experimental model and in vitro to determine the effect of a NAC / PFD-Nano composition.

[0193] Extraction and quantification of proteins from cell cultures and liver tissue.

[0194] The cytoplasmic and nuclear fractions were obtained with the pH regulators used in previous work. To extract the cytoplasmic fraction, 200 pL of pH regulator B1 was used. The tissues / cells were disrupted (Polytron PT1200E / Vortex / 5 min / 4 °C) and centrifuged (17,000 rpm / 10 min / 4 °C) to recover the supernatant (cytoplasmic proteins), and the nuclear pellet was preserved. The nuclei were resuspended in 100 pL of pH regulator B2 and the same procedure was followed to obtain nuclear proteins. The mini-Bradford technique was used to quantify protein, using bovine albumin (BSA) as a standard protein to perform the calibration curve; the reading was made at 595 nm in the Multiskan GO microplate reader.

[0195] Protein analysis by SDS-PAGE-Western Blot (WB)

[0196] SDS-PAGE-WB is the most widely used semiquantitative method for protein analysis and describes a phenomenon in which charged particles move toward the opposite electrode under the influence of an electric field. In the presence of SDS, electrophoretic mobility is primarily based on the weight of the molecule; on the other hand, the WB technique consists of several steps, described below.

[0197] Polyacrylamide gel electrophoresis

[0198] For the analysis of proteins of interest, polyachlamide gels were prepared at different concentrations, depending on the molecular weight of the protein of interest. All samples were denatured by chemical agents contained in the pH regulator Laemmli Sample (2X) supplemented with 10% of [3-Mercaptoethanol, and physical (100 °C / 10 min). 30 pg of each sample were loaded into an electrophoresis chamber with running pH regulator, and a voltage of 80 Volts was applied for 20 min, after that time the voltage was increased to 100 Volts for 2 h, respectively. Finally, the remnants of running pH regulator were removed from the polyachlamide gel, to proceed with the transfer of the proteins to a PVDF membrane.

[0199] Wet transfer

[0200] For protein transfer, the wet transfer method was followed. The proteins were electro-transferred to a PVDF membrane, which was activated in methanol (5 min). The material was then moistened with pH transfer regulator to assemble the cassette, applying a milliamp of 220 mA for 3 h in cold conditions (4 °C). To verify the transfer, the PVDF membrane is stained with Ponceau red dye which has a high affinity for proteins.

[0201] Membrane blockage

[0202] To prevent both primary and secondary antibodies from binding non-specifically to the PVDF membrane (areas without the protein of interest), 10% milk (Svelty 0% fat) dissolved in PBS / Tween 0.2% (PBS / t) was used as a blocking agent. It was blocked overnight at 4 °C / shaking. Finally, 3 wash cycles with PBS / t were performed to remove excess milk. Incubation with antibodies

[0203] To determine the protein of interest, the membranes were incubated with the corresponding primary antibody in BSA (1%)-PBS / t, overnight / 4°C. A loading control was used for each protein; for proteins of the cytoplasmic fraction, [3-Actin (Mouse IgG, Santa Cruz Biotechnology) was used, and for nuclear proteins, Lamin-[31 / H¡stone-H3 (Mouse IgG, Santa Cruz Biotechnology). Subsequently, the membranes were washed with PBS / t, and incubated with the secondary antibody.

[0204] Immunofluorescence of tissue and HepG2 cells

[0205] The tissue and cells on slides previously fixed in Paraformaldehyde (PFA 4% and 2% respectively), were carefully washed to remove excess PFA, and preserved in 1X PBS, permeabilized for 10 min (PBS / Thton (2%), blocked for 1 h with PBS / BSA (5%) / Triton (0.2%). Incubated with the primary antibody, removing excess blocking solution, for this, the cells were washed with PBS. The primary antibody was prepared in PBS (1x) / BSA (5%) and incubated for 24 h / 4 ° C in a humid chamber. Then, the cells or tissue are carefully washed with PBS, and incubated with the secondary antibody 1 h / 4 ° C / in darkness, staining is carried out with Hoechst (1:2000), the cells or tissue are washed with PBS and left with Hoechst / 10 min. Finally, excess Hoechst is removed with PBS washes, and a volume of Vecta-Shield is placed over the cells or tissue, covered with a coverslip, and sealed with varnish.The samples were stored to avoid contact with light.

[0206] Example 5.

[0207] DNA profiling analysis was performed to evaluate the action of DNA methyltransferases.

[0208] Global DNA methylation analysis: DOT-BLOT method

[0209] Dot blotting is a technique that allows a known amount of sample (DNA) to be transferred to an inherent support, such as a nitrocellulose membrane. This procedure is essential for determining antigen concentration. The procedure is described below.

[0210] Genomic DNA was purified using a commercial DNA extraction kit (QIAGEN). DNA from all samples was quantified using the Nanodrop2000, and 1 pg aliquots were made from each sample. The DNA was then denatured and neutralized by incubating at 95 °C / 10 min, and adding NH4OAc, respectively. Subsequently, the samples were placed on a PVDF membrane and incubated at 80 °C / 30 min, in order to fix the DNA to the membrane. The membrane was blocked using 5% BSA in TBS (1x)-Tween 20 (0.1%), with moderate shaking, at room temperature, for 1 hour. To remove excess blocking medium, washes were performed with TBS, and the membrane was incubated with the primary antibody 5meC (1:10000; Santa Cruz Biotechnology) and Isotype IgG (1:1000; Santa Cruz Biotechnology) overnight at 4 °C (The same amount of DNA stained with methylene blue was used as a loading control).After incubation, a series of washes with TBS are performed, followed by incubation of the membrane with the secondary antibody for 1 h at RT. Finally, the membrane is washed with TBS and visualized by chemiluminescence using the CHEMIDOC BioRaD instrument.

[0211] Analysis of DNA methylation percentage

[0212] For the determination of the methylation percentage, a colonometric technique was used, for which the MethylFIash ™ Global DNA Methylation Kit (5-mC) was used. The procedure is briefly described. To determine the percentage of DNA methylation of the test samples, 100 pL of SU and 100 ng of DNA from the test samples (2-4 pL) were added, the solution was mixed by gently tilting from side to side or gently shaking the plate several times to ensure that the solution covers the bottom of the well. evenly. The strip plate was covered with a plate seal or Parafilm and incubated at 37°C / 60 minutes. During the last 10 minutes of sample incubation, the 5-mC Detection Complex Solution was prepared: in each 1 mL of diluted wash buffer (BL), 1 pL of the 5-MeC antibody was added, mixed, and then 1 pL of identification solution (SI) and 0.5 pL of enhancing solution (SP) were added. 50 pL of the 5-mC detection complex solution was added to each well, then incubated at room temperature for 50 minutes. 100 pL of developer solution (DS) was added to each well in a column, not a row, simultaneously vertically using a multichannel pipette so that the replicates develop at the same time. The plate was gently shaken against a flat surface for 5 to 10 seconds and incubated at room temperature for 3 to 4 minutes.Color development in the sample and control wells was monitored. After a few minutes, the DS turned blue in the presence of sufficient methylated DNA. The color in the NC wells generally remained unchanged. The enzymatic reaction was stopped when the color of the 5% CP wells turned deep blue. 100 μL of stop solution (SP) was added to each well in a column, not a row, simultaneously and vertically using a multichannel pipette so that the replicates stopped at the same time. The solution was mixed by gently shaking the plate against a flat surface and waiting 1 to 2 minutes to allow the color reaction to completely stop. The color changed to yellow after adding SP, and the absorbance was measured in a microplate reader at 450 nm within 2 to 15 minutes.

[0213] Therefore, the present invention should not be considered as restricted except as required by the prior art and by the scope of the appended claims.

Claims

Claims

1. A pharmaceutical composition, characterized in that it comprises acetylated derivatives of L-cysteine ​​and 5-methyl-1-phenyl-2(1 H)-phosphate formulated in nanoparticles.

2. The pharmaceutical composition according to claim 1, further characterized in that the acetylated derivative of L-cysteine ​​is N-acetylcysteine ​​(NAC).

3. The pharmaceutical composition according to claim 1, further characterized in that the 5-methyl-1-phenyl-2(1 H)-pyridone derivative is pirfenidone (PFD).

4. The pharmaceutical composition according to claim 1, further characterized in that the nanoparticles are nanoparticles of organic nature.

5. The pharmaceutical composition according to claim 4, further characterized in that the nanoparticles of organic nature are selected from the group consisting of liposomes, micelles, proteins, polymers and dendrimers.

6. The pharmaceutical composition according to claim 1, further characterized in that the nanoparticles are transition metal oxides.

7. The pharmaceutical composition according to claim 6, further characterized in that the transition metal oxides are selected from the group consisting of aluminum oxide, magnesium oxide, titanium dioxide, zinc oxide, iron oxide, cene oxide and silver oxide.

8. The pharmaceutical composition according to claim 1, further characterized in that the nanoparticles are bioactive molecules.

9. The pharmaceutical composition according to any one of claims 1 to 8, further characterized in that additionally comprises at least one pharmaceutically acceptable excipient.

10. A pharmaceutical composition as claimed in any one of claims 1 to 9 for use in the treatment or prophylaxis of a condition.

11. The pharmaceutical composition for use according to claim 10, wherein the condition is hepatocellular carcinoma (HCC).

12. The pharmaceutical composition for use according to claim 11, wherein the treatment or prophylaxis of hepatocellular carcinoma includes at least one of the reduction of the hepatic fibrogenic process, reduction of neoplastic lesions, modulation of the oxidative process, reduction of tumors and reduction of epigenetic alterations.

13. The use of a pharmaceutical composition as claimed in any one of claims 1 to 9 in the manufacture of a medicament for the treatment or prophylaxis of a condition.

14. The use according to claim 13, wherein the condition is hepatocellular carcinoma (HCC).

15. The use according to claim 14, wherein the treatment or prophylaxis of hepatocellular carcinoma includes at least one of decreasing the hepatic fibrogenic process, decreasing neoplastic lesions, modulating the oxidative process, reducing tumors and decreasing epigenetic alterations.

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