TWI721202B - Compounds for enhancing bax/bcl-2 expression and activity and therapeutic use thereof - Google Patents

Abstract

The present invention relates to a novel type of BAX/BCL-2 modulator having a pyrimido[5,4-d ]pyrimidine main structure. The BAX/BCL-2 modulator can enhance the expression and activity of BAX/BCL-2 in cells. The present invention also relates to a pharmaceutical composition comprising the BAX/BCL-2 modulator of the invention encapsulated in a pharmaceutically acceptable carrier so that it can be directly delivered into cells. The present invention thus provides a method of preventing or treating BAX/BCL-2-related disorders or conditions comprising administering to a subject in need thereof a therapeutically effective amount of the BAX/BCL-2 modulator of the invention or the pharmaceutical composition of the invention.

Description

Compounds for enhancing the performance and activity of BAX/BCL-2 and their therapeutic uses

The present invention provides methods for enhancing the performance and activity of BAX/BCL-2 and related therapeutic uses.

The BCL-2 protein family determines the promise of cell apoptosis (an ancient cell suicide program necessary for development, tissue stability, and immunity). These proteins manage the mitochondrial outer membrane permeability (MOMP) and can be pro-apoptotic (especially Bax, BAD, Bak and Bok) or anti-apoptotic (among various other proteins, especially including intrinsic Bcl-2 , Bcl-xL and Bcl-w). There are a total of 25 genes in the Bcl-2 family known to date. It has been found that human BCL-2 is a gene located near the junction of abnormal junctions of chromosomes 18 and 14 (t14; 18) in tumor cells of patients with follicular lymphoma. This chromosomal translocation causes misregulation of the normal BCL-2 expression pattern and leads to cancer (Tsujimoto et al., 1985; Nunez et al., 1989). Various physiological death signals and pathological cell damage trigger the genetically programmed pathway of apoptosis (Vaux and Korsmeyer 1999). Apoptosis is reflected in the two main execution programs downstream of the death signal: apoptotic protease pathway and organelle dysfunction, of which mitochondrial dysfunction is the most obvious feature (for reviews, see Green and Reed 1998; Thornberry and Lazebnik 1998). Since members of the BCL-2 family reside upstream of irreversible cell damage and many of their efforts focus on the content of mitochondria, they play a key role in determining whether cells will survive or die. Recent studies on the involvement of mitochondria in cancer have revealed that when comparing metastatic mitochondria and mitochondria that are untransformed cells, there are a lot of differences in the structure and function of these organelles. In particular, modern research largely supports the observations of Warburg and his successors' metabolism, and at the same time refines and greatly expands the knowledge of the mitochondrial state and the mechanism of its function in tumor development. The comprehensive mechanism of the Warburg effect has not been isolated; however, multiple intertwined etiologies and response mechanisms have been described recently. This recognition of the indicative characteristics of cancer cell metabolism is also directly applied to current clinical care. It uses glucose analogues to identify cancerous lesions characterized by high glucose uptake through the increasingly widely adopted positron emission tomography (PET) imaging ( BioMed Research International Volume 2013 (2013), Article ID 612369). Apoptosis can be initiated through two pathways (ie, external and internal pathways). However, the external pathway is initiated by the stimulation of cell surface death receptors, and the internal pathway is activated by many different intracellular pressures and is caused by most chemotherapeutic agents. The BCL-2 family proteins are mainly involved in the intrinsic pathway, in which they regulate the mitochondrial outer membrane permeability (MOMP). MOMP causes apoptosis triggering factors (such as cytochrome c) to be released from the mitochondrial membrane space into the cytoplasm, where the apoptosis triggering factors activate the cascade of apoptotic proteases that perform common proteolytic events, leading to cell death (3). MOMP marks the promise of death and is usually regarded as the irreversible point of the cell. Here, the rational and preliminary results of recent efforts to induce MOMP by using BCL-2 family proteins as anti-cancer therapy are reviewed and other related targets for drug development are discussed (Clin Cancer Res; 21(12), June 2015 15th). Subsequently, the over-expression of Bcl-2 in prostate cancer, breast cancer, colon cancer, and glioblastoma is described. Overexpression of Mcl-1 (another anti-apoptotic Bcl-2 related protein) has been identified in relapsed acute myeloid leukemia and is associated with a poor prognosis. Other changes in the expression of Bcl-2 related proteins identified in cancer cells include different mutations in the Bax gene and changes in the ratio of pro-apoptotic and anti-apoptotic Bcl-2 proteins (Bettaieb et al., 2003; and Oncogene (2004) 23 , 2934-2949). The overexpression of Bcl-2 and related anti-apoptotic proteins has been shown to inhibit cell death induced by many stimuli including growth factor deprivation, hypoxia and oxidative stress. However, the ability of anti-apoptotic Bcl-2 family proteins to suppress cell death induced by cytotoxic anticancer drugs makes these proteins particularly interesting as potential targets for cancer drug discovery. Regardless of the main mode of action, whether it is a single-stranded or double-stranded DNA break, whether microtubules are depolymerized or aggregated, whether nuclear hormone receptor activation (glucocorticoid receptor) or inhibition (estrogen and androgen receptor), Basically all traditional anti-cancer drugs seem to rely to a large extent on the Bcl-2/Bax-dependent mechanism to kill cancer cells (reviewed by Debatin et al., 2002; Reed, 2008). Therefore, Bcl-2 operates at a remote point in the conservative cell death pathway used by most anti-cancer drugs. This composition is different from the previously identified mechanisms involving drug excretion, drug metabolism, drug inactivation, and related mechanisms. Intrinsic form of chemical resistance. This observation probably explains why the performance of various Bcl-2 family proteins has shown prognostic significance for many types of cancers and leukemias treated by chemotherapy. Defects in the expression of pro-apoptotic members of the BCL-2 family are also found in cancer, which leads to the loss of tumor suppressor function of these killer genes. The best recorded is BAX, where homozygous deletion or inactivation mutations have been specifically identified in cancers that accompany microsatellite instability due to defective DNA mismatch repair. In this regard, the human BAX gene contains a homopolymer extension of eight guanosine residues in the sense strand, and this extension is the target of frame shift mutations. The defective performance of pro-apoptotic BCL-2 family genes was also found in the background of loss of p53 function. Among them, the direct targets of p53 transcription factors are BAX, BID, PUMA, and NOXA, which confirms that there is a strong relationship between the genome supervision of p53 and the cell death genes of the BCL-2 family. Recently, it has been observed that p53 protein interacts with the cytoplasm of pro-apoptotic and anti-apoptotic Bcl-2 family proteins, which directly regulate the biological activities of p21-Bax and p26-Bcl-2 proteins, and indicate that p53 is involved in transcription and transcription. The latter two levels regulate the Bcl-2 family. The activities of other pro-apoptotic members of the Bcl-2 family are also suppressed by post-translational modifications. For example, the pro-apoptotic protein BAD is phosphorylated by Akt (PKB) and other protein kinases known to be highly active in cancer, which leads to its isolation by 14-3-3 (Oncogene (2008) 27, 6398-6406 ). The overexpression of Bcl-2 prevents apoptosis induced by most chemotherapeutic drugs (including alkylating agents and topoisomerase inhibitors). Moreover, fibroblasts defective in both Bax and Bak are resistant to apoptosis induced by various agents that induce mitochondrial stress. However, fibroblasts with a single defect of Bax or Bak do not have a significant defect in apoptosis induced by these agents, which indicates that both Bax and Bak proteins can be functionally redundant. This apparent redundancy can be cell type dependent, because HCT116 cells lacking Bax are resistant to apoptosis induced by staurosporine (Theodorakis et al., 2002). Many anti-cancer agents (such as arsenic and lonidamine) can directly permeate the outer mitochondrial membrane at least in vitro, and the overexpression of Bcl-2 prevents mitochondrial permeation and apoptosis induced by arsenic and lonidamine . Based on the role of Bcl-2 protein in regulating mitochondrial membrane permeation and its usually modified performance in human cancers, Bcl-2 protein is a reasonable target for inducing apoptosis by itself or in combination with other anti-cancer drugs (Anti-apoptotic member) or prototype (pro-apoptotic member). However, molecular markers should be used when using Bcl-2 targeted therapy. This is because not all anti-apoptotic cells overexpress Bcl-2, as confirmed in breast cancer cell lines screened by NCI cells (Nieves-Neira And Pommier, 1999; and Oncogene (2004) 23, 2934-2949). The Bcl-2 protein is an inhibitor of programmed cell death. It homodimerizes with itself and forms a heterodimer with the homologous protein Bax (a promoter of cell death). The Bax/Bcl-2 ratio can be used as a rheostat that determines the sensitivity of cells to apoptosis. The secondary cell location of some members of the Bcl-2 family also seems to regulate their functions. A small part of Bcl-2 and Bcl-xL can be found on the outer mitochondrial membrane. In contrast, Bax mainly exists in the cytoplasm before apoptosis is induced. During the early stage of apoptosis, Bax translocates from the cytoplasm to the mitochondria, where it participates in mitochondrial division and the release of cytochrome c. Regulating the insertion step can regulate cell apoptosis. The lower level of this ratio can lead to resistance of human cancer cells to apoptosis. Therefore, the Bax/Bcl-2 ratio can affect tumor progression and aggressiveness (Iran Biomed J. 2015 April; 19(2): 69-75). As in the extrinsic pathways, vehicles that target the intrinsic pathways involved in both tumorigenesis and chemoresistance are used in therapeutic pathways. These anti-cancer strategies attempt to develop drug-designed inhibitors of anti-apoptotic proteins (eg, Bcl-2, Bcl-xL, and IAP) that are commonly expressed in cancer cells. Efforts to target the Bcl-2 protein involve the development of agents that destroy the Bcl-2 complex. The BH3 mimic binds to the hydrophobic groove of the anti-apoptotic protein, which mimics the role of the BH3 only protein in binding to the pro-survival protein, resulting in the release of the BH3 only protein from the complex and activates BAX and BAK. So far, nearly twelve BH3 mimics have been studied as Bcl-2 inhibitors in different stages of human clinical trials, such as AT-101 (R-(−)-Gossypol), ABT-199 (Vitamin Venetoclax), ABT-737, ABT-263 (navitoclax, an orally available derivative of ABT-737), GX15-070 (obatoclax) and TW37. The field of Bcl-2 family member inhibitors is under continuous development, which emphasizes the importance of these molecules as potent anti-cancer agents. Moreover, targeting the specific BH4 domain of Bcl-2 is also emerging as a novel strategy for anti-cancer therapy. Therefore, Bcl-2, through its BH4 domain, cooperates with several proteins that regulate different cellular pathways (such as hypoxia and angiogenesis) involved in tumor progression and chemoresistance. Recently, it was discovered that a small molecule (ie, BDA-366) can act as a powerful and effective BH4 domain antagonist, which exhibits significant anti-cancer activity in vitro and in vivo, thus providing a proof of concept for this approach. Another innovative approach to inhibit Bcl-2 stems from the following evidence: the human bcl-2 gene contains a GC-rich sequence located in its promoter that has the possibility of forming a G four-stranded structure and functions as a transcriptional repressor element. Therefore, the G four-strand specific ligand can regulate the transcription of bcl-2 with the help of the stabilization of the four-strand structure (AGING, April 2016, Vol. 8 Issue 4). Bcl-XL is the major anti-apoptotic protein in the Bcl-2 family. Its overexpression is more widely observed in human lung cancer cells than Bcl-2, which indicates that Bcl-XL is more biologically related and therefore more important than lung cancer. The best therapeutic target. Here, the UCSF DOCK 6.1 program kit and NCI chemical library database are used to screen small molecules that selectively target the BH3 domain (aa 90-98) of Bcl-XL. Two new Bcl-XL inhibitors (BXI-61 and BXI-72) that exhibit selective toxicity against lung cancer cells are comparable to normal human bronchial epithelial cells. Fluorescence polarization analysis revealed that BXI-61 and BXI-72 preferentially bind to Bcl-XL protein in vitro with high binding affinity instead of Bcl2, Bcl-w, Bfl-1/A1 or Mcl-1. Treatment of cells with BXI-72 results in the destruction of Bcl-XL/Bak or Bcl-XL/Bax interactions, so that Bak is oligomerized and cytochrome c is released from mitochondria. Importantly, BXI-61 and BXI-72 show more potent anti-human lung cancer efficacy than ABT-737, and have less thrombocytopenia in vivo. BXI-72 overcomes the acquired radiation resistance of lung cancer. Based on these findings, the development of BXI as a new class of anticancer agents is guaranteed and represents a new strategy for improving lung cancer outcomes (Cancer Res. 2013, September 1, 73(17):5485-5496). Bax (a regulator of cell death) is needed in the decision-making stage of apoptosis. Recent studies have identified Bax's serine 184 (S184) as a key functional switch that controls its pro-apoptotic activity. Here, the structure bag surrounding S184 is used as a docking site to screen the NCI library of small molecules using the UCSF-DOCK program kit. Three compounds (ie, the small molecule Bax agonists SMBA1, SMBA2, and SMBA3) induce configuration changes in Bax by blocking S184 phosphorylation, which promotes the insertion of Bax into the mitochondrial membrane and the formation of Bax oligomers. The latter causes the release of cytochrome c and apoptosis in human lung cancer cells, which occurs in a Bax rather than Bak-dependent manner. SMBA1 strongly inhibits lung tumor growth by selectively activating Bax in vivo through apoptosis without significant normal tissue toxicity. Develop Bax agonists as a new class of anti-cancer drugs to provide strategies for the treatment of lung cancer and other malignant tumors with Bax (NATURE COMMUNICATIONS DOI: 10.1038/ncomms5935). BID (BH3 Interaction Domain Death Receptor Agonist) protein is located outside the death receptor and mitochondria in apoptotic signaling, and acts as an inducer of permeabilization of the outer mitochondrial membrane in type II cells. The content of BID is critical to the viability of various cells, because its silence makes cells resist apoptosis induced by death receptor ligands (such as TNF-related apoptosis-inducing ligand (TRAIL)). Moreover, it has been confirmed that the content of BID is lower than the functional dose in cells of several cell lines because it can be sensitive to TRAIL through the overexpression of BID. For the above reasons, BID has been considered for therapeutic agent development. However, in order to define the way to invest in BID, a number of major issues should be resolved. The main problem is to control the amount of BID delivered to the cells. Although full-length BID has been shown to participate in apoptosis signal transduction, effective activation of apoptosis requires specific cleavage of BID by apoptotic protease 8 and the production of an active truncated form (tBID). TBID expressed in cells directly induces apoptosis. Therefore, in order to take advantage of the selectivity of some anti-cancer agents against cancer cells, it is a better strategy to sensitize cells by full-length BID. To sensitize cells, an overexpression system based on adenovirus or pcDNA vector is usually used. However, it does not provide strict control of the expression level of BID in cells. Therefore, the content of BID in transfected cells exceeds the content of endogenous protein several times and in some cases direct activation of apoptosis is observed rather than sensitization of cells to apoptotic stimuli. According to the above, tBID appears in cells treated with adenovirus vectors expressing full-length BID (BMC Cancer201414:771 DOI: 10.1186/1471-2407-14-771). Rapamycin and its derivatives are promising therapeutic agents with both immunosuppressant and anti-tumor properties. The effects of these rapamycins are mediated by specific inhibition of mTOR protein kinase. It is known that mTOR acts as part of an evolutionarily conserved signaling pathway that controls the cell cycle in response to changes in nutrient content. The mTOR signaling network contains many tumor suppressor genes (including PTEN, LKB1, TSC1, and TSC2) and many proto-oncogenes (including PI3K, Akt, and eIF4E), and mTOR signaling is constitutively activated in many tumor types. These observations indicate that mTOR is an ideal target for anticancer agents and that rapamycin is such an agent. In fact, early preclinical and clinical studies indicate that rapamycin derivatives as anti-tumor agents have efficacy alone and in combination with other treatment modalities. Rapamycin appears to inhibit tumor growth by stopping tumor cell proliferation, inducing tumor cell apoptosis, and inhibiting tumor angiogenesis. The effect of rapamycin immunosuppressant is caused by inhibiting the proliferation of T and B cells by the same mechanism that rapamycin blocks the proliferation of cancer cells. Therefore, it may be considered that the immunosuppression induced by rapamycin will be harmful to the use of rapamycin as an anticancer agent. In contrast, when rapamycin is combined with the widely used immunosuppressant cyclosporine, compared with the tumor incidence observed when cyclosporine is used alone, in organ transplantation trials The frequency of tumor formation that occurs is reduced. Existing evidence indicates that with regard to tumor growth, the anticancer activity of rapamycin significantly exceeds the immunosuppressive effect of rapamycin. In recent years, rapamycin and its analogues (such as CCI-779 or temsirolimus, RAD001 or everolimus, sirolimus, FK-50 and AP23576) have been clinically used. It is used to treat various cancers, including kidney cancer, mantle cell lymphoma and metastatic breast cancer. Dipyridamole (DPM) (such as aspirin) inhibits platelet adhesion and therefore tends to prevent vascular embolization of heart attacks and strokes. There is a report on the European Stroke Prevention Study in Lancet (page 1, 371-4) issued on December 12, 1987. The introduction of this report commented that the use of aspirin to treat patients who survived a small stroke TIA (transient ischemic attack) lacks the indicated benefit. In this trial, 300 mg/day of dipyridamole was added to the treatment with aspirin and the results were significant. After two years, stroke deaths were reduced by 50%, deaths due to myocardial infarction were reduced by 38%, and deaths due to cancer were reduced by 25%. The anticancer effect of dipyridamole indicated above can only be due to its prevention of metastasis; however, Eva Bestida of the University of Barcelona and others have been published in Cancer Research, September 1985 (pages 4,048-4,062) It is reported that the growth of certain human cancer cells is inhibited by dipyridamole. It inhibits more than 80% of adenosine, thymidine and uridine. These substances are needed for the prosperity of cancer cells. This can indicate the anti-cancer effect of dipyridamole in addition to preventing metastasis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as an attractive cytokine to selectively target cancer cells; however, its efficacy is challenged by many resistance mechanisms. Therefore, current research is investigating the possibility of dipyridamole enhancing the efficacy of TRAIL and the possible underlying mechanism. Dipyridamole significantly sensitized the following p53 mutant human cancer cell lines to the anti-tumor activity of TRAIL: SW480, MG63, and DU145, as evidenced by the ability of TRAIL to effectively cleave initiator and performer apoptotic proteases. Although dipyridamole upregulates both DR4 and DR5 and increases its cell surface expression, RNA interference reveals a preferential dependence on DR5. Moreover, dipyridamole inhibits survivin performance and its important results have been confirmed by small interfering RNA. Mechanistically, dipyridamole induces the transcriptional shutdown expressed by survivin, accompanied by G1 block characterized by D-type cyclin and cdk6 downregulation. In addition, the transcription mechanism driven by CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) induction is responsible for the up-regulation of DR5 by dipyridamole. Importantly, dipyridamole-induced enhancement of TRAIL efficacy and changes in protein performance do not depend on protein kinase A or protein kinase G. In summary, the findings of this study illustrate the novel mechanism of dipyridamole action and highlight its promising use as a potential enhancer of TRAIL efficacy (Oncogene (2008) 27, 3435-3445). Preliminary observations indicate that dipyridamole (DPM) can increase the sensitivity of human ovarian cancer cells to cisplatin. In another study, Jekunen et al. showed that DPM enhanced the cytotoxicity of cisplatin in cisplatin-sensitive 2008 human ovarian cancer cells to 4.7 times in a synergistic manner, and in the cisplatin-resistant 2008/C13*5.25 subline Increased to 5.8 times. It has been found that DPM increases the cellular uptake of cisplatin in a concentration-dependent manner without increasing trypan blue or propidium iodide uptake or changing cell size. It is believed that the cumulative increase in cisplatin induced by DPM is not related to the non-specific increase in membrane permeability. In a nude mouse model with human bladder cancer, Keane et al. found that the tumor size was reduced by 20% when cisplatin was combined with DPM. Using human testicular cancer in the same model, it achieved complete tumor regression. Barberi-Heyob et al. found that DPM increased the growth inhibitory activity of cisplatin in MCF-7 human breast cancer cells in a synergistic manner. Janice R. Perussi et al. reported that the cytotoxicity of cisplatin in cisplatin-sensitive MDA/S human breast cancer cells was enhanced by DPM, indicating that there is a strong correlation between Pt accumulation and enhanced cisplatin cytotoxicity, but in cisplatin In platinum-resistant MDA/R cells, the synergistic interaction between cisplatin and DPM does not involve an increase in cisplatin cell accumulation (Quím. Nova Vol. 26, No. 3, São Paulo, May/June 2003). Gemcitabine (2',2'-difluorodeoxycytidine) is a pyrimidine nucleoside analogue. It exerts its cytotoxic effect in cells and has activity against many different solid tumors (including pancreatic cancer, breast cancer, lung cancer and bladder cancer). Because gemcitabine has strong hydrophilicity, passive diffusion through the lipid bilayer of the hydrophobic cytoplasmic membrane is relatively slow. In order to enter cells efficiently, gemcitabine requires a physiological nucleoside transporter to cross the serosal membrane. These transporters are divided into two types, namely balanced transporters and concentrated transporters. The bidirectional human balanced nucleoside transporter (hENT) is found in most cell types, and both hENT1 and hENT2 can mediate gemcitabine uptake in the direction of a concentration gradient. hENT protein is a transmembrane glycoprotein located in the plasma membrane. Its ability is inhibited by nitrobenzyl mercaptopurine ribonucleoside (NBMPR) (specific hENT1 inhibitor at low nanomolar concentrations) and dipyridamole (hENT1/2 inhibitor). In the upper division, the sensitivity to gemcitabine is reduced to 1/39 and 1/1,800 (The Oncologist 2008; 13:261-276). Fluorouracil (5-FU) (especially sold under the trade name Adrucil) is a medicine used to treat cancer. By injecting it into a vein, it is used for colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and cervical cancer. As a cream, it is used for solar keratosis and basal cell carcinoma, and as eye drops for the treatment of ocular surface squamous cell tumors. The defect of apoptosis has involved the chemoresistance of cancer cells. Studies have found that the combination of high levels of anti-apoptotic Bcl-2 and low levels of Bax is associated with high 5-FU resistance in various cancers, including human breast cancer, pancreatic cancer, human head and neck cancer, and colon tumor cells ( Int J Cancer. April 1, 2002; 98(4):498-504; and Br J Cancer. November 2000; 83(10): 1380-1386). Dipyridamole has been shown to enhance the cytotoxicity of 5-FU and other fluoropyrimidines in vitro. This effect may be related to the increase in the intracellular concentration of FdUMP and the inhibition of extracellular nucleoside uptake through the "salvage" pathway. In vitro studies have shown that dipyridamole may increase many cytotoxic agents (including etoposide, doxorubicin, and vinblastine) by changing the cellular uptake and retention of cytotoxic agents. ) And mitoxantrone (mitoxantrone)) (Investigational New Drugs 12: 283-287, 1994). Multidrug resistance (MDR) of cancer cells is the simultaneous development of resistance to various anti-cancer drugs that seem to be structurally and mechanistically unrelated. One type of MDR is characterized by a decrease in the accumulation of hydrophobic natural product drugs. In some multi-drug resistant cells, drug excretion is mediated by an adenosine triphosphate (ATP)-dependent membrane transporter called P-glycoprotein (Pgp) (product of the MDR1 gene) (Juliano and Ling, 1976). Pgp acts as an active transport mechanism for various molecules (including certain chemotherapeutic drugs). As data on the role of Pgp in drug resistance accumulates, it is clear that other transporters can confer resistance to cytotoxic agents. The MDR protein 1 (MRP1) gene was cloned from a multi-drug resistant lung cancer cell line, and it was found that, like Pgp, it is a member of the ATP-binding cassette (ABC) superfamily of the transporter gene (Cole et al., 1992). Transfection studies indicate that, similar to MDR1, MRP1 overexpression is sufficient to confer resistance to various lipophilic natural product antitumor drugs. MRP1 is the first identified member of the gene family encoding the multispecific organic anion transporter (MOAT) protein (Borst et al., 1999). The other two homologs of MRP1, cMOAT/MRP2 and MRP3, encode proteins that mediate MDR when transfected into drug-sensitive cells (Borst et al., 1999). All of these membrane-embedded proteins act as drug discharge pumps, preventing cytotoxic agents from reaching lethal levels in cells. P-glycoprotein (P-gp) is a key role in the multi-drug resistance phenotype in cancer. The protein confers resistance by mediating the ATP-dependent excretion of a surprising series of anticancer drugs. Its wide specificity has been the subject of many attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy is to develop compounds that compete with anticancer drugs for transmission or act as direct inhibitors of P-gp. Although quite successful in vitro, there are currently no compounds that can be used to "block" P-gp-mediated resistance in the clinic. The failure can be attributed to toxicity, adverse drug interactions, and several pharmacokinetic issues. In addition, multi-drug resistance-related protein 1 (MRP1) transmits a wide range of therapeutic agents and various physiological substrates, and can develop drug resistance in certain cancers (including lung cancer, breast cancer and prostate cancer) and childhood neuroblastoma Play a role. Several studies above have shown that in vitro, dipyridamole can significantly increase the cytotoxicity and anti-tumor activity of various chemotherapeutic agents. The underlying mechanism here is to prevent nucleoside and nucleobase salvage, and to increase the intracellular accumulation of toxic metabolites via inhibited P-glycoprotein and MRP1 (Clin Pharmacol Ther 2003; 73: 51-60. Drug Metab Dispos. 2014 April; 42(4): 623-631. Oncogene (2003) 22, 7340-7358). Studies have investigated the role of dipyridamole as a single agent in the prevention of tumor occurrence and metastasis in multiple models of triple-negative (estrogen and progesterone receptor negative, Her-2 normal) breast cancer (a subtype with little effective therapy) The potential role. These findings provide evidence that intraperitoneal administration of dipyridamole impairs the growth and metastasis of primary tumors in a breast cancer xenograft animal model. Moreover, the data identified a new mechanism of action of dipyridamole, which was shown to inhibit ERK1/2-MAPK, NF-kB and Wnt signaling pathways, and prevent the accumulation of inflammatory cells in the tumor microenvironment. In addition, dipyridamole is the most potent BCRP inhibitor among the tested compounds, with an IC50 value of 6.4 +/- 0.9 mM. Therefore, dipyridamole has the potential to treat multidrug resistance in cancer (Cancer Prev Res (Phila). May 2013; 6(5):. Doi:10.1158/1940-6207.CAPR-12-0345). However, clinical administration of DPM did not improve the anticancer activity of 5-FU or cisplatin in patients with advanced colorectal cancer, metastatic breast cancer, advanced non-small cell lung cancer, or advanced measurable pancreatic cancer. The increased dose intensity of 5-FU or cisplatin observed for DPM is not clinically relevant. The path for nanoparticle drug carriers to enter cells is different from conventional drugs. Conventional drugs enter cells through dose-dependent diffusion. In other words, the higher the concentration of the drug in the blood, the higher the concentration of the drug in the cells, and the drug can only enter the cytoplasm. Nanoparticle drug carriers are absorbed by cells through endocytosis and enter the cells to form lysosomes. In the initial stage after injection, the concentration of the nanoparticle drug carrier increases in a time-dependent manner. Endocytosis is the process of incorporating extracellular material into a cell. This process can be classified into three types, namely endocytosis, pinocytosis and receptor-mediated endocytosis. Cytophagy occurs only in specialized cells. These cells proliferate and aggregate when stimulated by extracellular substances and engulf the substances in the lysosomes of the cells for decomposition. This process occurs in the macrophages and neutrophils of the immune system. The pinocytosis system internalizes the extracellular fluids and molecules in the cell membrane to form a pocket by means of the recess of the cell membrane, and then pinches it off into the cell to form a vesicle. The vesicles then travel into the cytoplasm and fuse with other vesicles, such as endosomes and lysosomes. Depending on the structure of the carrier, pinocytosis can be divided into two types, namely fluid-phase pinocytosis and adsorptive pinocytosis. If the carrier does not have a functional group that interacts with the cell, the cell will engulf the drug carrier through fluid phase pinocytosis. This process is slow and depends on the concentration of the carrier around the cell membrane. When the carrier has a hydrophobic group or is positively charged, adsorptive pinocytosis will occur. These carriers will be physically adsorbed by the cell membrane and increase the engulfing capacity of the cell. The above two types of endocytosis are non-specific processes and are not suitable for delivering drugs to their targets. Targeting can only be achieved by enhanced penetration and retention (EPR) in certain cancer tissues. Receptor-mediated endocytosis is a process in which cells absorb molecules (endocytosis) by sprouting inwardly of serosal vesicles containing proteins with receptor sites specific to the uptake molecules. After the drug carrier binds to the receptor on the cell, the intrinsic signal will trigger the cell membrane to form a coated pit. The surface area of the coated pit is equivalent to 1% to 2% of the cell membrane. The coated follicles will separate from the cell membrane and enter the cell to form coated vesicles in the cell, then form endosomes and move in a jumping motion inside the cell. The intranuclear system includes a complex structure of microtubules and vesicles. Vesicles can be fused with Golgi. Due to the proton pump (ATPase), endosomes usually become acidic. Endosomes will then fuse with lysosomes to form secondary lysosomes. The cell membrane therapeutic agent is effectively delivered to the barrier to be overcome at the target site in the mitochondria, cytoplasm or nucleus. Hydrophobic phospholipids are the main components of cell membranes that hinder the delivery of therapeutic agents. Therefore, various delivery systems (such as liposomes, nanoparticles, and viral vectors) have been developed to transfer small molecules, peptides, proteins, and oligonucleotides across membranes. This method of drug delivery is referred to herein as a cell penetrating drug delivery system. Many drug carrier systems (liposomes, cell penetrating peptides, cationic polymer conjugates and polymeric nanoparticles) have been developed for intracellular delivery of therapeutic agents. It needs to be suitable for crossing a series of membrane barriers to reach the site of drug action in the cell. During this process, an important part of the drug molecule will be lost at each continuous barrier. These barriers include cell association of drug carriers and internalization by endocytosis; intracellular transport and release of drugs or drug carriers into the cytoplasm; translocation of drugs or drug carriers from the cytoplasm to the nucleus or any other organelle ; And nucleus/organelle uptake. Cells contain several intracellular organelles with specific functions. The intracellular targeting of these specific organelles of therapeutic agents is expected to not only significantly enhance the therapeutic efficacy, but also reduce non-specific effects and therefore toxicity. Therefore, there is a great interest in using different carrier systems to achieve intracellular target-specific delivery of therapeutic agents. Carriers that promote endocytosis of drugs include nano-sized polymer carriers and liposomes. Depending on the nature of the drug and the manufacturing process, nano-sized drug carriers can be classified into nanoparticle, nanoliposome, nanosuspended particle, solid lipid nanoparticle, magnetic nanocarrier and the like. In addition to the above-mentioned carriers, cell penetrating peptides (CPP), biodegradable nanoparticles, and viral vectors can also be used as delivery systems to enhance the penetration of drugs into cells. Since cell membranes constitute the main barrier for intracellular delivery of large-sized hydrophilic proteins, peptides and oligonucleotides, cell penetrating peptides (CPP) have been developed to overcome this barrier. These CPPs can ferry the molecule or colloidal drug carrier system labeled with them across the cell membrane, enter the cytoplasm and reach the nucleus. The characteristics of CPP are attributed to the existence of a segment of 9-16 cationic amino acid residues; the most commonly studied CPP includes HIV-1 transactivated transcription activator (TAT) peptide, HSV VP-22 (herpes simplex) Virus type 1 transcription factor) peptide and penetratin. Several theories have been proposed to determine the exact mechanism by which these CPP enter cells. For example, the penetration of TAT through cell membranes has been shown to be independent of receptors and transporters, and it has been suggested to enter cells by destabilizing the phospholipid bilayer to form antimicrobial cells. The main benefit of TAT coupling is that together with the effective delivery of the molecule, the biological activity of the coupled molecule is retained, and the size of the delivered molecule is not a rate limiting factor. TAT has been proposed to enhance not only intracellular delivery but also nuclear delivery, and therefore has been studied for nucleic acid delivery. TAT peptides coupled to antisense oligonucleotides have been shown to deliver the oligonucleotides to the nucleus. After internalization, it was also found that the TAT peptide and BODIPY-ceramide (which is a marker of the Golgi body) co-localized inside the Golgi body. Therefore, it is likely to be directly transported from the early endosomes to the Golgi without entering the late endosomes. There may be a secretory pathway in which the peptide enters the cytoplasm from the endoplasmic reticulum. Gene therapy has proven to have great potential in the treatment of hereditary, acquired and neurodegenerative disorders. Among non-viral gene delivery methods, various drug delivery systems and polymers are being studied, such as liposomes, cationic lipid-DNA, and polymer complexes. To overcome the relatively inefficient cellular uptake of non-viral gene expression vectors, it has been explored to couple TAT peptides to the vector. Kleeman et al. have used polyethyleneimine (PEI) covalently coupled to TAT via a polyethylene glycol (PEG) spacer to confirm gene expression in alveolar basal epithelial cells. The transfection efficiency in lung is higher than that of unconjugated PEG complex in vivo. In a similar study by Rudolph et al., solid lipid particles coupled to dimeric HIV-1 TAT have demonstrated enhanced gene delivery to the lung. The amino acid composition of CPP usually contains positively charged amino acids with high relative abundance (such as lysine or arginine) or alternates between polar/charged amino acids and non-polar, hydrophobic amino acids. The sequence of patterns. The two types of structures are called polycationic or amphiphilic, respectively. The third type of CPP is a hydrophobic peptide that contains only non-polar residues, has a low net charge, or has a hydrophobic amino acid group that is essential for cellular uptake. Among the cell penetrating peptides, arginine-rich cell penetrating peptides have been studied most extensively. Examples include the TAT peptide from HIV transactivator protein TAT, Penetratin, the 16 amino acid domain of Antennapedia protein from Drosophila, and the coat of flock house virus (FHV) Peptide (sequence 35-49) and oligoarginine. A dynamic process of intracellular delivery system mediated by biodegradable nanoparticles; involving endocytosis, exocytosis and sorting into different intracellular compartments. It appears that the NP surface and its interaction with the cell surface controls the uptake and intracellular transport of the biodegradable nanoparticles and therefore the encapsulated therapeutic agent. Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Therefore, viral vector systems are suitable options for cell-penetrating drug delivery systems. Cell penetrating peptides and biodegradable nanoparticles are not only used to modify drugs, but also coupled to carriers to enhance transmembrane effects. Dipyridamole is a balanced nucleoside transporter (ENT) inhibitor. Nucleoside transporter (NT) plays an important role in the transport of nucleosides across cell membranes. Dipyridamole blocks the balanced nucleoside transporter (ENT), which promotes the diffusion of adenosine across the membrane. Dipyridamole will increase the concentration of extracellular endogenous adenosine mainly in situations where the extracellular formation of adenosine is increased (for example, occurring during hypoxia or inflammation). However, the extracellular endogenous adenosine concentration induced by dipyridamole causes vasodilation, which contributes to the metabolic control of organ perfusion. Dipyridamole pressure myocardial imaging is a widely used and successful technique for diagnosing and evaluating coronary artery disease. Coronary artery dilation with IV dipyridamole is associated with a significant reduction in blood flow to the collateral-dependent myocardium, which is consistent with coronary blood stealing in patients with CAD. In addition, there are other studies that have discovered the vasoconstriction and vasodilation effects of dipyridamole in many organs (including kidney, lung, pancreas, brain, etc.). Dipyridamole not only causes vasoconstriction in some organs, but can also cause hypotension and subsequent side effects, such as dizziness and palpitations due to the dilation of cardiovascular blood vessels. The effect of lowering blood pressure makes dipyridamole unsuitable for the treatment of physiologically unstable patients, such as those suffering from (but not limited to) sepsis, ischemic stroke, hemorrhagic stroke, acute lung injury, acute liver injury, Patients with myocardial infarction and cardiorenal syndrome. Moreover, the blood flow restriction effect of dipyridamole limits its application in the treatment of diseases involving organs rich in blood vessels. Since the pharmacological effects of dipyridamole are mainly on the cell membrane, a delivery system designed for membrane penetration to avoid binding to the balanced nucleoside transporter on the membrane while enhancing intracellular signal transduction and PPARγ regulation can prevent the increase The effect of tissue hypoperfusion caused by cardiovascular expansion and local blood flow restriction. Therefore, the limitation of dipyridamole in clinical application due to the decrease of blood pressure in acute and severe patients can be lifted. Dipyridamole is also a non-selective phosphodiesterase inhibitor. The increase in intracellular drug delivery will enhance the inhibition of intracellular phosphodiesterase (PDE) by dipyridamole. The members of the PDE family have a unique cell and tissue specific distribution. Depending on the distribution curve of PDE on the cell membrane or in the cytoplasm in different tissues, dipyridamole can be used as an anti-inflammatory, antioxidant, and smooth muscle relaxant for the treatment of diseases related to PDE regulation. The unique cell and tissue specific distribution of PDE is shown in the following table (see US 2012/0065165): Increasing the ability of dipyridamole to penetrate the membrane can promote the inhibition of PDE3, PDE5 and PDE8 in specific tissues and endow dipyridamole with therapeutic effects in diseases related to PDE3, PDE5 and PDE8. In this case, dipyridamole can be used to treat lower urinary tract dysfunction and erectile dysfunction like other PDE5 inhibitors. Moreover, since dipyridamole is a non-selective PDE inhibitor, it can be used to treat PDE-related diseases when it is delivered by a transmembrane drug delivery system. There are several molecules that act on mitochondria that are currently in use or are being tested in clinical trials. Certain clinically approved anticancer drugs (e.g. paclitaxel and VP-16 (etoposide) and vinorelbine) and an increasing number of experimental anticancer drugs (e.g. ceramide, MKT077, CD437, cloni Daming and betulinic acid) have been found to act directly on mitochondria to trigger apoptosis. CD437 can induce apoptosis in various human cancer cells in vitro and in vivo. In intact cells, CD437-dependent apoptotic protease activates the release of cytochrome C from the mitochondria before activation. Moreover, when added to isolated mitochondria, CD437 caused membrane permeabilization. This effect is prevented by inhibitors of the mitochondrial permeability transition pore complex (mPTPC), such as cyclosporine A. Therefore, CD437 represents a low molecular weight compound that exerts its cytotoxic effect (ie, by directly acting on the surface or inside of mitochondria) through mPTPC. Similarly, arsenic trioxide used to treat acute promyelogenous leukemia has multiple effects on mitochondria. It is known that via its action on the voltage-dependent anion channel VDAC leads to the induction of mPTPC formation. Arsenic trioxide is also known to act on the respiratory chain and inhibit the activity of the respiratory chain. The apoptotic factors that play a major role in cell apoptosis include Bcl-2 and Bcl-Xl. Compounds that act by binding to these proteins have been identified and studied for their efficacy; some examples include chromene derivatives and gossypol, which have recently been shown to act on proteins of the Bcl-2 family. In fact, there are so many different and structurally different compounds, so it is suggested to collectively refer to them as mitochondria-targeted anticancer drugs (mitocans) to reflect their mitochondrial-mediated anticancer effects. The selective accumulation pathway targeting tumor mitochondria requires two levels of specific accumulation; drug accumulation in tumors and then drug accumulation in cancer cell mitochondria. Generally speaking, drug treatment can be adjusted by subtle modification of the chemical structure to change its known physical-chemical properties that determine its accumulation in each compartment. Of course, these modifications must be implemented without adversely affecting the molecular target. The second approach involves coupling ligands larger than simple organic functional groups to alter the biodistribution of active molecules. Again, as long as the coupling does not adversely affect the desired pharmacological activity of the molecule, this approach will work. Using ligands that are known to have affinity for target tissues, these approaches have been extremely effective in changing the distribution of drugs in the body and achieving higher accumulation in target tissues. There are ligands that have been shown to mediate the tumor-specific accumulation of drugs, and there are ligands that are known to be mitochondrial. However, it is still unclear whether there are ligands with two properties to the extent that will allow a high desired accumulation level. Therefore, it can be said with certainty that so far, the dual strategy is the most feasible way. This dual strategy will require the use of a targeted delivery route to achieve high tumor accumulation, followed by a second route to ensure that the drug then accumulates in the mitochondria where it will exert its effect. Although there are many tissue-specific delivery studies aimed at increasing the weight content of anticancer drugs, studies aimed at subcellular delivery have only received more attention. Nonetheless, there are some interesting mitochondrial delivery routes suggesting the promise of improved cancer therapy. Pharmaceutical nanocarriers (such as liposomes, micelles, and solid nanoparticles) provide a way that can be regarded as a non-chemical way to change the disposal of drug molecules. All chemical processes can be performed on the components of the nanocarrier system, which can then be loaded with drugs to provide targeted delivery. Most pharmaceutical nanocarriers can be modified to target specific tissues and even specific cell types. Long-circulating liposomes and nanoparticles can passively target leaky vascular regions with enhanced penetration and retention (EPR) effects, and can also be modified with antibodies or other targeting ligands to provide cell-specific recognition. If nano-carriers that affect both the tumor-specific accumulation of drugs and mediate the mitochondrial-specific accumulation in tumor cells can be developed for clinical therapy, it can be the ultimate tool in the mitochondrial targeted anti-cancer pathway. The first step in this direction has been taken in recent years. The current nano-carrier technology has reached the extent that the demand for subcellular delivery can be actually met by using nano-carriers specifically designed for these purposes. In the route for delivering the mPTPC-inducing drug wasptoparan (mastoparan) into cells, liposomes are modified with both transferrin and the gene fusion peptide Chol-GALA. The transferrin modification enhances the uptake of liposomes into cells via endocytosis, after which the peptide promotes release from endosomes into the cytosolic fluid. Therefore, by only increasing the intracellular content of the drug, the delivery route achieves a higher concentration of the drug that can potentially interact with subcellular targets. Micelles have also been suggested for the delivery of hydrophobic drugs to various subcellular organelles, including mitochondria. It is found that the luciferin-labeled microcell lines used in this study are distributed through several cytoplasmic organelles (including most of them associated with mitochondria). The uptake of these micelles is not limited to a single cell type. Moreover, the cellular internalization cargo (cargo) is incorporated into the micelles to a higher degree than the free cargo itself. There are currently several examples of nanocarriers specifically designed to accumulate in mitochondria. Possibly, the earliest such systems are called DQAsome. Prepared from the mitochondrial molecule dequalinium chloride. These vesicle nano-carriers have been developed for mitochondrial-specific DNA delivery, but have also been shown to change the subcellular distribution of paclitaxel to increase the drug mitochondria In the accumulation. Mitochondrial-specific delivery results in improved apoptotic activity at paclitaxel concentrations where the free drug does not have a significant cytotoxic effect. DQAsome loaded with paclitaxel has also been tested for its ability to inhibit the growth of human colon cancer tumors in nude mice, and the data strongly suggests that encapsulation of paclitaxel in DQAsome leads to improved efficacy. The anti-tumor efficiency of paclitaxel encapsulated by DQAsomal is further improved by modifying the surface of DQAsomal with folic acid (FA). Folic acid is a high-affinity folate membrane-bound protein, which has been expressed in a variety of human tumors. FA conjugates are internalized by receptor-mediated endocytosis in a tumor cell-specific manner, leading to increased toxicity of the corresponding drugs. Another approach for mitochondrial specific nanocarriers is to modify existing nanocarriers with mitochondrial ligands. In this regard, TPP once again acts as a mitochondrial ligand in liposome and polymer-based nanocarriers. Liposomes have been well characterized as delivery systems and are a popular choice due to their biocompatibility, ease of surface modification, and ability to encapsulate hydrophilic or hydrophobic drugs. It is the first to indicate that liposomes can be modified by using mitochondrial residues to make the mitochondrial system derived from reports on the so-called proteoliposomes. The proteolipid system incorporates the crude mitochondrial membrane part in the preimplanted embryo Prepared in liposomes coexisting with endogenous mitochondria. Further research on the concept of using ligands to alter the subcellular distribution of liposomes is based on stearyl triphenyl phosphonium (STPP). The stearyl residues of STPP act as lipid anchors to use TPP residues to modify the surface of liposomes and produce liposome formulations that have a clear predisposition to mitochondria. STPP-liposomes have been shown to effectively guide the accumulation of rhodamine-labeled phosphatidylethanolamine (Rh-PE) to mitochondria in living cells. Based on flow cytometry, STPP-liposomes exhibit the same level of cell association as liposomes with the same cationic charge. However, the subsequent subcellular localization by confocal microscopy analysis is significantly different, indicating that the mitochondrial specific association of the nanocarrier is determined by the mitochondrial ligand rather than the surface charge. It was also found that TPP ligand does not change the in vivo distribution of long-circulating pegylated liposomes and tumor accumulation. However, STPP-liposomes do improve both the in vitro and in vivo efficacy of Ceramide. In summary, these data indicate that the tendency of long-circulating liposomes to passively accumulate in solid tumors (via the EPR effect) can be combined with the organelle-specific tropism conferred by appropriate ligand modification to enhance the effect of encapsulated anti-tumor agents. Alternative approaches to the development of mitochondrial-specific liposomes focused on the concept that liposomes that tend to selectively fuse with mitochondrial membranes are more likely to associate with mitochondria when they enter cells. These liposomes, called MITO-Porter, utilize octaarginine residues to be surface-modified to facilitate entry into cells as intact vesicles (via giant pinocytosis). The lipid composition is selected based on the high level of fusion with the mitochondrial membrane in living cells and the release of its cargo to the inner mitochondrial compartment. Based on confocal microscopy data, MITO-porter liposomes have been used to deliver green fluorescent protein and propidium iodide to mitochondria, indicating that it can be used to deliver macromolecules and small molecules to mitochondria. The development of mitochondrial-specific nanocarriers is not limited to lipid-based carriers, and also includes the use of mitochondrial residues to generate polymer systems capable of mitochondrial-specific intracellular delivery of biologically active molecules. TPP has been modified to produce nanoparticles based on mitochondrial mitochondrial N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Interestingly, early studies indicated that although the characterized polymer exhibits association with isolated mitochondria, experiments with ovarian cancer cells revealed significant lysosomal association of the polymer. However, in recent studies, microinjection and incubation experiments using fluorescently labeled constructs based on microscopic analysis have shown the ability of mitochondria to target. Subsequently, the photosensitizer mesochlorin e 6 (Mce 6) was used to synthesize the HPMA copolymer-drug conjugate. The mitochondrial targeting of HPMA copolymer-bound Mce 6 enhances cytotoxicity compared with non-targeting HPMA copolymer-Mce 6 conjugate. The authors indicate that "small modifications may be required to suit the current design and allow tumor site-specific mitochondrial targeting of other therapeutic agents." Therefore, these systems can theoretically be applied to mitochondria to specifically deliver a series of pro-apoptotic substances for cancer therapy. Inorganic nanoparticles have also been shown to be capable of mitochondrial specific delivery. In a recent study, hypocrellin A (a photodynamic drug) was encapsulated in a water-soluble amorphous silica nanocage (HANC). These drug-loaded nanocages are reported to specifically accumulate in the mitochondria of cancer cells and improve the photosensitizing effect of Hypocrellin A. However, it is unclear which mediates the specific accumulation of mitochondria in the nanocage system. However, all in all, the various studies described so far strongly support that nanocarriers can indeed control the subcellular accumulation of bioactive molecules and therefore represent a useful tool in the development of mitochondrial targeted anti-cancer strategies (GGM D'Souza et al., / Biochimica et Biophysica Acta 1807 (2011) 689-696). Extracellular adenosine disrupts the mitochondrial membrane potential in HuH-7 cells (Fas-deficient human liver cancer cell line), and this effect is inhibited by the adenosine transporter inhibitor dipyridamole or by overexpression of Bcl-XL. Adenosine down-regulates the expression of mRNA and protein of Bcl-XL and apoptosis protein inhibitor 2 (IAP2) to directly inhibit apoptotic proteases-3, -7 and -9, but it up-regulates DIABLO (an inhibitor of IAP) in other ways ) MRNA and protein performance. These adenosine effects are attenuated by dipyridamole (Cell Biol Toxicol (2010) 26:319-330). Extracellular adenosine induces apoptosis in various cancer cells through internal and external pathways. In the former pathway, adenosine uptake in cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Dipyridamole, which inhibits the cellular uptake of adenosine, significantly reverses the growth suppression induced by adenosine.

(I) 其中R₁ 、R₂ 、R₃ 及R₄ 中之每一者獨立地選自由雜環基及二(羥烷基)胺基組成之群， 或其醫藥上可接受之鹽。 本發明亦係關於式I化合物或其醫藥上可接受之鹽之用途，其用於製造用於預防或治療BAX/BCL-2相關病症或病況之醫藥。在較佳實施例中，醫藥包含囊封於醫藥上可接受之載劑中之式I化合物或其醫藥上可接受之鹽。 本發明進一步係關於用於預防或治療BAX/BCL-2相關疾病之醫藥組合物，其包含治療有效量之囊封於醫藥上可接受之載劑中之式I化合物或其醫藥上可接受之鹽。在較佳實施例中，化合物係雙嘧達莫且載劑係脂質體。 本發明更詳細闡述於以下部分中。本發明之其他特徵、目的及優點可容易地在本發明之具體實施方式及申請專利範圍中發現。It has been found in the present invention that certain substituted pyrimido[5,4- d ]pyrimidine compounds (such as dipyridamole) can enhance the performance and activity of BAX/BCL-2. Therefore, the present invention provides a novel type of BAX/BCL-2 modulator with the main structure of pyrimido[5,4-d ]pyrimidine and the use of the BAX/BCL-2 modulator to prevent or treat BAX/BCL-2 related diseases Or methods of disease conditions, such as cancer, myeloproliferative disorders, prostatic epithelial dysplasia, lymphangiomyocytosis, Kimura disease, and keloids. The present invention also relates to methods for increasing the performance and activity of BAX/BCL-2. Although studies have confirmed that dipyridamole exhibits anti-cancer activity, its effect is not significant at low doses. Dipyridamole is an ENT-1 inhibitor known to block the uptake of adenosine into cells. Since adenosine can promote the apoptosis of cancer cells, the blockade reduces the anti-cancer efficacy of dipyridamole. In the present invention, dipyridamole can be encapsulated in a carrier to promote cell membrane penetration and accumulation in the cell, and thereby activate the cell's endogenous apoptosis mechanism. The enhanced apoptosis-promoting effect of dipyridamole in cells can be achieved by increasing the formation of BAX/BCL-2 heterodimers, increasing the formation of BAX homodimers and reducing the formation of BCL-2 homodimers . In the present invention, it was also found that dipyridamole can reduce the performance of BCL-XL, reduce the inhibition of ENT-1 and regulate the expression of apoptotic proteins of cancer cells, thereby increasing its anti-cancer activity. In a preferred embodiment, dipyridamole is encapsulated in a cell penetrating vehicle to increase the concentration of dipyridamole inside the cell and reduce the inhibition of ENT-1. In the embodiment of the treatment of cancer, the increase of BCL-XL and the decrease of cytoplasmic adenosine can be avoided and therefore the anticancer efficacy is decreased. In an embodiment, the present invention relates to a method for preventing or treating BAX/BCL-2 related disorders or conditions, which comprises administering a therapeutically effective amount of a compound of formula (I) to an individual in need thereof:

(I) wherein each of R ₁ , R ₂ , R ₃ and R ₄ is independently selected from the group consisting of a heterocyclic group and a di(hydroxyalkyl)amino group, or a pharmaceutically acceptable salt thereof. The present invention also relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of medicines for the prevention or treatment of BAX/BCL-2 related diseases or conditions. In a preferred embodiment, the medicine comprises a compound of formula I or a pharmaceutically acceptable salt thereof encapsulated in a pharmaceutically acceptable carrier. The present invention further relates to a pharmaceutical composition for preventing or treating BAX/BCL-2 related diseases, which comprises a therapeutically effective amount of a compound of formula I encapsulated in a pharmaceutically acceptable carrier or a pharmaceutically acceptable compound thereof salt. In a preferred embodiment, the compound is dipyridamole and the carrier is a liposome. The invention is explained in more detail in the following sections. Other features, objectives and advantages of the present invention can be easily found in the specific embodiments of the present invention and the scope of the patent application.

(I) 其中R₁ 、R₂ 、R₃ 及R₄ 中之每一者獨立地選自由雜環基及二(羥烷基)胺基組成之群， 或其醫藥上可接受之鹽。 在實施例中，R₁ 及R₃ 係雜環基、較佳六氫吡啶基，且R₂ 及R₄ 係二(羥烷基)胺基、較佳N,N-二(羥乙基)胺基。 在較佳實施例中，化合物係雙嘧達莫。 在另一實施例中，化合物囊封於載劑中，例如非離子體、聚合體囊胞、奈米粒子、脂質體、奈米懸浮粒子、固體脂質奈米粒子、磁性奈米載劑、微胞、巨分子偶聯物或微粒藥物載劑。 在較佳實施例中，載劑係脂質體。在另一實施例中，脂質體之直徑在約50-700 nm、較佳約60-530 nm、更佳80-350 nm且最佳約130-230 nm之範圍內。 此項技術中已知當雙嘧達莫以游離形式投與時，其結合至細胞膜上之ENT受體並活化阻斷腺苷進入細胞中之信號傳導路徑。發明者發現，雙嘧達莫可調控細胞凋亡/抗細胞凋亡蛋白之表現且藉助細胞穿透藥物遞送系統進入細胞中引起細胞凋亡。癌細胞細胞凋亡路徑之活化可促進已知與異常細胞凋亡/抗細胞凋亡蛋白表現相關聯之癌症的治療。 圖1a顯示若雙嘧達莫以游離形式投與，則其主要在細胞外部起作用且將促進腺苷之累積，此將導致減少之細胞凋亡。圖1b顯示在細胞內部之雙嘧達莫可活化細胞凋亡/抗細胞凋亡蛋白之表現。在此情形中，可避免雙嘧達莫在細胞外部之不合意作用。在下文之實例中，本發明證實藉由將雙嘧達莫直接遞送至細胞中，可避免結合至細胞膜上之受體以便減少副作用，例如氧化壓力及由腺苷累積造成之血管收縮。 在實施例中，本發明之式(I)化合物囊封於載劑中以遞送至細胞中。在較佳實施例中，載體係非離子體、聚合體囊胞、奈米粒子、脂質體、奈米懸浮粒子、固體脂質奈米粒子、磁性奈米載劑、微胞、巨分子偶聯物或微粒藥物載劑。較佳地，載體係脂質體。適用於本發明之脂質體具有在約10-300 nm、較佳約80-280 nm、更佳約120-270 nm範圍內之直徑。 在本發明之另一實施例中，載劑可為直徑小於1 mm之非離子體、聚合體囊胞或聚合物。可基於表面電位、親水性/疏水性、大小、形態、形狀及/或表面曲率進行修飾。 本發明之脂質體調配物可包含各種性質(例如，單層或多層)、組成、大小及特性之囊泡，包括不同組合、pH及滲透強度之水性介質在內。在較佳實施例中，脂質體脂質層膜之主要成分係選自由天然或合成磷脂組成之群，例如以下所列示之彼等： - 1,2-二月桂醯基-sn-甘油基-3-磷酸膽鹼(DLPC) - 1,2-二肉豆蔻醯基-sn-甘油基-3-磷酸膽鹼(DMPC) - 1,2-二棕櫚醯基-sn-甘油基-3-磷酸膽鹼(DPPC) - 1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC) - 1,2-二油醯基-sn-甘油基-3-磷酸膽鹼(DOPC) - 1,2-二肉豆蔻醯基-sn-甘油基-3-磷酸乙醇胺(DMPE) - 1,2-二棕櫚醯基-sn-甘油基-3-磷酸乙醇胺(DPPE) - 1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺(DSPE) - 1,2-二油醯基-sn-甘油基-3-磷酸乙醇胺(DOPE) - 1-肉豆蔻醯基-2-棕櫚醯基-sn-甘油基-3-磷酸膽鹼(MPPC) - 1-棕櫚醯基-2-肉豆蔻醯基-sn-甘油基-3-磷酸膽鹼(PMPC) - 1-硬脂醯基-2-棕櫚醯基-sn-甘油基-3-磷酸膽鹼(SPPC) - 1-棕櫚醯基-2-硬脂醯基-sn-甘油基-3-磷酸膽鹼(PSPC) - 1,2-二肉豆蔻醯基-sn-甘油基-3-[磷酸-rac-(1-甘油)] (DMPG) - 1,2-二棕櫚醯基-sn-甘油基-3-[磷酸-rac-(1-甘油)] (DPPG) - 1,2-二硬脂醯基-sn-甘油基-3-[磷酸-rac-(1-甘油)] (DSPG) - 1,2-二油醯基-sn-甘油基-3-[磷酸-rac-(1-甘油)] (DOPG) - 1,2-二肉豆蔻醯基-sn-甘油基-3-磷酸鹽(DMPA) - 1,2-二棕櫚醯基-sn-甘油基-3-磷酸鹽(DPPA) - 1,2-二棕櫚醯基-sn-甘油基-3-[磷酸-L-絲胺酸] (DPPS) - 磷脂醯絲胺酸(PS)，及 - 天然L-a-磷脂醯膽鹼(來自雞蛋，EPC；或來自大豆、SPC；及HSPC)。 較佳磷脂係長飽和磷脂，例如彼等具有多於12、較佳多於14、更佳多於16、最佳多於18個碳原子之烷基鏈者。 用於本發明之較佳脂質體組合物較佳係彼等其中脂質體係單層及/或多層者，且包含： (i) 1至100、較佳40至70 mol%較佳選自由以下組成之群之生理上可接受之磷脂：DLPC、DMPC、DPPC、DSPC、DOPC、DMPE、DPPE、DSPE、DOPE、MPPC、PMPC、SPPC、PSPC、DMPG、DPPG、DSPG、DOPG、DMPA、DPPA、DPPS、PS、EPC、SPC及HSPC； (ii) 1至100、較佳40至70 mol%神經鞘脂質、較佳神經鞘磷脂； (iii) 1至100、較佳40至70 mol%表面活性劑，其較佳以疏水性烷基醚(例如，Brij)、烷基酯、聚山梨醇酯、山梨糖醇酯及/或烷基醯胺為特徵； (iv) 5至100、較佳50至100 mol%兩親性聚合物及/或共聚物，較佳包含至少一個親水性聚合物或共聚物之嵌段(例如聚乙二醇)及至少一個疏水性聚合物或共聚物之嵌段(例如聚(丙交酯)、聚(己內酯)、聚(環氧丁烷)、聚(氧化苯乙烯)、聚(苯乙烯)、聚(乙基乙烯)或聚二甲基矽氧烷)之嵌段共聚物， (v) 0至60 mol%、較佳20至50 mol% 毒素滯留增強化合物、較佳固醇衍生物、較佳膽固醇；或 (vi) 0至30 mol%、較佳1至5 mol%空間穩定劑、較佳聚乙二醇化化合物、較佳聚乙二醇化脂質、更佳DSPE-PEG。 在較佳實施例中，脂質體樣囊泡係自聚合物製得且出於脂質在形式上並不視為脂質體而是稱為聚合體囊胞之原因，不包含脂質。然而，出於本發明之目的，聚合體囊胞意欲由如定義本發明及申請專利範圍所用之術語脂質體涵蓋。 類似地，自合成表面活性劑製得且不包含脂質之脂質體樣囊泡稱為非離子體。然而，出於本發明之目的，非離子體意欲由如定義本發明及申請專利範圍所用之術語脂質體涵蓋。 在本發明之實施例中，可使用不同高分子聚合物之聚合，其包含呈三嵌段共聚物形式之彼等(例如ABA及BAB)及呈嵌段共聚物形式之彼等(例如PLLA-PEG、PLGA-PEG、PLA-PEG、PLLA-mPEG、PLGA-mPEG及PLA-mPEG)。可設計各種形狀(例如星形及L形式)，其包括PEG-(PLGA)₈ 、PEG-(PLLA)₈ 及PEG-(PDLA)₈ 星形嵌段共聚物。聚乙二醇化修飾可用於修飾任何媒劑(例如聚合物媒劑及脂質體)以達成降低血漿蛋白之結合速率之效應(參見Park, J.等人，(2009) 「PEGylated PLGA nanoparticles for the improved delivery of doxorubicin. Nanomedicine.」 5(4):410-418.；Lück, M.等人，(1998) 「Plasma protein adsorption on biodegradable microspheres consisting of poly(D,L-lactide-co-glycolide), poly(L-lactide) or ABA triblock copolymers containing poly(oxyethylene). Influence of production method and polymer composition.」 J. Control Release. 55(2-3):107-20.；及Sempf, K.等人，(2013) 「Adsorption of plasma proteins on uncoated PLGA nanoparticles.」 Eur. J. Pharm. Biopharm. 85(1):53-60)。 動物劑量不應藉由基於體重之簡單轉換外推至人類等效劑量(HED)。食品暨藥物管理局(Food and Drug Administration)已提出動物劑量至人類劑量之外推僅藉助正規化之BSA (其通常以mg/m2表示)正確執行。人類劑量當量可藉由使用下式更適當地計算：HED (mg/kg) = 動物劑量(mg/kg)乘以動物Km/人類Km。為將小鼠中所用之劑量轉換為基於表面積之人類劑量，將22.4 mg/kg (鮑爾小鼠(Baur’s mouse)劑量)乘以小鼠之Km因子(3)且然後除以人類之Km因子(37) (參見下表)。 基於來自FDA指南草案之數據的值 為將以mg/kg表示之劑量轉換為以表示mg/m² 之劑量，乘以K_m 值。根據本發明，脂質體-雙嘧達莫在小鼠中之有效劑量係10 mg/kg-100 mg/kg，在倉鼠中6-60 mg/kg，在大鼠中5-50 mg/kg，在天竺鼠中3.75-37.5 mg/kg，在兔子中2.5-25 mg/kg，在猴中2.5-25 mg/kg，在狗中1.5-15 mg/kg，在貓中2.4-24 mg/kg，在狒狒中1.5-15 mg/kg，在兒童中1.2-12 mg/kg，且在成年人中0.81-8.1 mg/kg。考慮到物種間之藥物敏感性差異，不限制物種之最寬劑量範圍係0.4-160 mg/kg、較佳0.6-120 mg/kg、更佳0.8 mg/kg-100 mg/kg。 在實施例中，雙嘧達莫脂質體可用於治療癌症。在實施例中，癌症係耐藥性或非耐藥性的。在實施例中，癌症係乳癌及肝癌。 在實施例中，雙嘧達莫脂質體可與其他抗癌藥物組合投與，例如烷基化劑、抗代謝藥、抗生素、激素、免疫調節劑、有絲分裂抑制劑、靶標治療劑及鉑藥物。在實施例中，抗癌藥物包含化學治療藥物及靶標藥物。在實施例中，化學治療藥物包含烷基化劑(例如環磷醯胺、甲基二(氯乙基)胺(Mechlorethamine)及美法侖(Melphalan))、抗有絲分裂劑(長春花鹼、長春新鹼(Vincristine)及紫杉醇(Taxol))、DNA嵌入劑(道諾黴素(Daunorubicin)及多柔比星)、DNA斷裂劑、抗代謝劑(例如卡培他濱(Capecitabine)、克拉屈濱(Cladribine)、阿糖胞苷(Cytarabine)、磷酸氟達拉濱(Fludarabine phosphate)、5-氟尿嘧啶(5-Fluorouracil)、吉西他濱(Gemcitabine)、6-巰嘌呤、胺甲喋呤(Methotrexate) (胺甲喋呤(Amethopterin)，MTX)、米托蒽醌(Mitoxantron)、培美曲塞二鈉(Pemetrexed disodium) (七水合物)及替加氟(Tegafur) (FT-207))及拓樸異構酶抑制劑(例如喜樹鹼(Camptothecin)、拓撲替康(Topotecan)、伊立替康(Irinotecan)、鬼臼毒素(Podophyllotoxin)及依託泊苷)。在實施例中，靶標藥物包含曲妥珠單抗(Trastuzumab)、拉帕替尼(Lapatinib)、吉非替尼(Genfitinib)、埃羅替尼(Erlotinib)、西妥昔單抗(Cetuximab)、貝伐珠單抗(Bevacizumab)、利妥昔單抗(Rituximab)、硼替佐米(Bortizomib)、伊馬替尼(Imatinib)、舒尼替尼(Sunitinib)、雷帕黴素、西羅莫司、替西羅莫司、依維莫司、地磷莫司(Ridaforolimus) (德福羅莫司(deforolimus))及索拉非尼(Sorafenib)。在實施例中，其他抗癌藥物係5-氟尿嘧啶(5-FU)。 現已一般性闡述本發明，藉助參考以下實例可更容易地理解本發明，該等實例提供用於產生本發明醫藥組合物及其在治療癌症中之用途的例示性方案。提供實例僅出於說明性目的，且並不意欲以任何方式限制本發明之範圍。已努力確保所用數字(例如，量、溫度等)之準確性，但當然將允許一些實驗誤差及偏差。實例 實例 1 ：雙嘧達莫脂質體之製備 脂質體係利用含有正電荷及中性電荷之磷脂及膽固醇製備，其中膽固醇之莫耳百分比為5%至75%。製備小的單層囊泡。將經乾燥脂質膜利用硫酸銨水合且隨後擠出穿過一系列聚碳酸酯膜過濾器。經由跨膜pH梯度或去水-再水合將雙嘧達莫囊封於脂質體中，且經由以上製造製程，所擠出脂質體之直徑在50-602 nm之範圍內(溶於葡萄糖溶液中)。脂質體-雙嘧達莫之直徑為約50至602 nm，如表1中所示。 表1實例 2 ： BCL-2 蛋白家族之細胞存活力 (MTT 分析 ) 及西方墨點分析 癌細胞系培養 用於人類乳癌之培養基係L-15培養基。為獲得完成生長培養基，將以下組分添加至基礎培養基：胎牛血清至10%之最終濃度。將培養物在37℃無CO₂ 下培育。所測試癌細胞系包括MDA-MB-231、PANC-1、BXPC-3、HCT-116、HT29、A375及MeWo。 MTT (溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-四唑鎓鹽, Sigma, St. Louis)之儲積液係藉由將5 mg MTT/ml溶於磷酸鹽緩衝鹽水(pH 7.5)中製備並藉助0.22 mm過濾器過濾。所用MTT分析基本上類似於Mosmann (1983)初始所闡述者。簡言之，將100 mL MTT溶液(0.5 mg/mL)添加至所處理癌細胞系之單層並將微板於37℃下培育3小時。將100 mL DMSO添加至各孔並充分混合以溶解藍黑色晶體。在microELISA讀取器上使用570 nm之測試波長及630 nm之參考波長讀取板。 處理後，將細胞用150 mL RIPA細胞溶解緩衝液(10mM Hepes pH=7.9、1.5mM MgCl2、10mM KCl、1.0mM DTT、0.1% Triton-X 100)洗滌，並於1200 g在4℃下離心10分鐘。然後收集含有癌細胞總蛋白之上清液並藉由西方墨點進行分析。西方墨點方法係如下執行：使用Bradford分析量測蛋白質濃度；將6X試樣緩衝液(0.8 mM Tris-HCl、10 mM EDTA、10% SDS、60%甘油、0.6 M β-巰基乙醇、0.06%溴酚藍，pH 6.8)添加至50 μg全細胞蛋白中；並將等體積之溶解緩衝液添加至試樣。於95℃下加熱10分鐘以使蛋白質變性之後，立即將試樣於冰上冷卻。 然後將試樣藉由12% SDS-PAGE電泳(100 V)分離並藉由濕墨點自SDS-PAGE凝膠轉移至PVDF膜。然後將PVDF膜用5%脫脂奶在室溫下處理60分鐘以阻斷非特異性結合。將膜與初級抗體一起於4℃下培育過夜並用PBST洗滌三次。將膜與二級抗體一起於室溫下培育60分鐘並用PBST洗滌三次。然後將膜用PBS再洗滌一次並與增強之化學發光(ECL)受質一起培育用於檢測。使用自動化化學發光及螢光成像系統(UVP Biospectrum)獲得影像照片。實例 2.1 ： 在用雙嘧達莫脂質體處理之 MCF-7 細胞中之細胞存活力及 Bax/BCL-2 比率 分析中所用之細胞系係人類乳癌細胞MCF-7。將細胞用雙嘧達莫脂質體(10 mM及20 mM)或雙嘧達莫游離形式(10 mM及20 mM)處理。結果顯示於圖4a及4b中，且正規化於圖5a及5b及表2中。 表2實例 3 ： 在用雙嘧達莫脂質體與雷帕黴素之組合處理之 MDA-MB-231 細胞中之細胞存活力及 Bax/BCL-2 比率 分析中所用之細胞系係人類乳癌細胞MDA-MB-231。將細胞用雷帕黴素(100 mM)、雙嘧達莫脂質體(3.125 mM及25 mM)或雙嘧達莫游離形式(3.125 mM及25 mM)與雷帕黴素(100 mM)之組合處理。結果顯示於表3中。 表3實例 4 ： 在用雙嘧達莫脂質體與雷帕黴素之組合處理之 PANC-1 細胞中之細胞存活力及 Bax/BCL-2 比率 分析中所用之細胞系係人類乳癌細胞PANC-1。將細胞用雷帕黴素(10 mM)、雙嘧達莫脂質體(3.125 mM及25 mM)或雙嘧達莫游離形式(3.125 mM及25 mM)與雷帕黴素(10 mM)之組合處理。結果顯示於表4中。 表4實例 5 ： 在用雙嘧達莫脂質體與 5-Fu 之組合處理之 PANC-1 、 BxPC-3 、 HCT-116 、 HT29 、 A375 及 MeWo 細胞中之細胞存活力及 Bax/BCL-2 比率 將細胞用5-Fu (10 mM及100 mM)、雙嘧達莫脂質體(3.125 mM及25 mM)或雙嘧達莫游離形式(25 mM)與5-Fu (10 mM)之組合處理。結果顯示於表5中。 表5 此實例中之結果證實藉由使用雙嘧達莫脂質體與5-Fu之組合，該等藥物對胰臟癌、肝癌、乳癌及皮膚癌細胞之細胞毒性效應藉助平衡Bcl-2與Bax比率而增加。亦可減少該等藥物之劑量。舉例而言，5-Fu之劑量可減少至少8倍。 而且，藉助統計分析，發現在所測試之8種癌細胞系中，Bax/Bcl-2比率因雙嘧達莫脂質體處理之增加與癌細胞存活率負相關(R=-0.789,p ＜ 0.01)。此結果證明，雙嘧達莫與5-Fu在抑制各種類型癌細胞系之生長方面產生協同效應。而且，包含雙嘧達莫及5-Fu之醫藥組合物可增強5-Fu在癌細胞、尤其對5-Fu治療具有抗性之癌症中之細胞毒性效應(圖2)。實例 6 ： 用雙嘧達莫脂質體與吉西他濱之組合處理之 A375 及 MeWo 細胞的細胞存活力 在A375及MeWo癌細胞系中，發現雙嘧達莫脂質體展現膜穿透效應。藉由此膜穿透效應，雙嘧達莫對吉西他濱之拮抗有效降低20-100%。此結果證明雙嘧達莫脂質體增加藥物穿透進入細胞中。結果顯示於表6中。細胞外雙嘧達莫阻斷ENT-1且阻止吉西他濱進入癌細胞中以產生其細胞毒性效應。此實例中之結果證實雙嘧達莫脂質體使此現象降低20-40%，此指示雙嘧達莫脂質體增強進入細胞之藥物的比率。 表6 其他化學治療藥物(例如圖3中所示之雷帕黴素類似物(rapamycin analogs, Rapalogs))可與本發明本發明之雙嘧達莫脂質體組合使用。 自以上數據，已發現本發明之雙嘧達莫脂質體當與化學治療藥物一起投與時在抑制癌細胞增殖方面產生協同效應，其中10-40%以上的細胞死亡(圖4)。癌細胞死亡之協同效應並非簡單地起因於增加Bax或降低Bcl-2，而是增加Bax/Bcl-2比率(圖5及6)。根據實例，雙嘧達莫在進入細胞中後使Bax/Bcl-2比率增加25-200%，且對癌細胞增殖之抑制增加20-50%。當與5-Fu組合時，雙嘧達莫藉助進入細胞中使5-50%之癌細胞增殖受到抑制，且使Bax/Bcl-2比率增加15-50%。雙嘧達莫與雷帕黴素之組合藉助進入細胞中使10-30%之癌細胞增殖受到抑制，且使Bax/Bcl-2比率增加10-200%。 對於熟悉此項技術者而言，預計將存在以上說明性實例中所闡述本發明之各種修改及變化。因此，僅將隨附申請專利範圍中出現之該等限制應用於本發明。Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those familiar with the art. The meaning and scope of the terms should be clear; however, in the case of any potential ambiguity, the definitions provided in this article take precedence over any dictionary or external definitions. As utilized in accordance with the present invention, unless otherwise indicated, the following terms should be understood to have the following meanings. The term "BAX/BCL-2 modulator" as used herein refers to an agent that can modulate the performance or activity of the BAX/BCL-2 homodimer. The term "alkyl" as used herein refers to a saturated linear or branched hydrocarbon group having 1 to 6 carbon atoms, especially 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 1-methyl Base ethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3- Methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl , 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl Base, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1, 2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl. C ₁ -C ₄ -Alkyl means (for example) methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1- Dimethyl ethyl. The term "heterocyclyl" as used herein refers to a monocyclic group having 5 to 8 ring members, wherein in each case 1, 2, 3, or 4 of the ring members are independently selected from each other Heteroatoms of the group consisting of oxygen, nitrogen or sulfur. The term "preventing, prevention" as used herein refers to delaying the onset of symptoms in susceptible individuals or reducing the occurrence of diseases. The term "treating, treatment" as used herein means reducing and/or improving the symptoms of susceptible individuals or increasing the survival rate of individuals suffering from certain fatal diseases or conditions. The term "BAX/BCL-2 related disorder or condition" as used herein means a disorder or condition in which the regulation of BAX/BCL-2 is beneficial. For example, these disorders or conditions include cancer, myeloproliferative disorders, prostatic epithelial dysplasia, lymphangiomyocytosis, Kimura disease, and keloids. The term "individual" as used herein means an animal, especially a mammal. In a preferred embodiment, the term "individual" means a human being. In another preferred embodiment, the term "individual" refers to a companion animal, such as a cat or a dog. The term "therapeutically effective amount" as used herein refers to the amount of the active ingredient used alone or in combination with other treatments/medicines for the treatment of PPARγ-related disorders or conditions to show therapeutic efficacy. The term "carrier" or "pharmaceutically acceptable carrier" refers to particles that can encapsulate active pharmaceutical ingredients. Examples of carriers suitable for the present invention include non-ionic bodies (niosome), polymeric vesicles, nanoparticles, liposomes, nano-suspended particles, solid lipid nanoparticles, magnetic nano-carriers, micelles, and macromolecules Conjugates, particulate drug carriers, and the like. Unless the context requires otherwise, singular terms shall include plural forms and plural terms shall include the singular. The inventors of the present invention surprisingly found that compounds with a pyrimido[5,4-d ]pyrimidine structure can enhance the performance and activity of BAX/BCL-2 homodimers, and therefore can serve as a novel type of BAX/BCL-2 Modifier. In a preferred embodiment, the pyrimido[5,4- d ]pyrimidine compound is dipyridamole. Therefore, the present invention provides a method for preventing or treating BAX/BCL-2 related disorders or conditions, which comprises administering to an individual in need thereof a therapeutically effective amount of a compound of formula (I):

(I) wherein each of R ₁ , R ₂ , R ₃ and R ₄ is independently selected from the group consisting of a heterocyclic group and a di(hydroxyalkyl)amino group, or a pharmaceutically acceptable salt thereof. In the embodiment, R ₁ and R ₃ are heterocyclic groups, preferably hexahydropyridyl, and R ₂ and R ₄ are bis(hydroxyalkyl)amino groups, preferably N,N-bis(hydroxyethyl) Amine group. In a preferred embodiment, the compound is dipyridamole. In another embodiment, the compound is encapsulated in a carrier, such as nonionic body, polymeric vesicle, nanoparticle, liposome, nanosuspended particle, solid lipid nanoparticle, magnetic nanocarrier, micro Cells, macromolecular conjugates or microparticle drug carriers. In a preferred embodiment, the carrier is a liposome. In another embodiment, the diameter of the liposome is in the range of about 50-700 nm, preferably about 60-530 nm, more preferably 80-350 nm, and most preferably about 130-230 nm. It is known in the art that when dipyridamole is administered in a free form, it binds to the ENT receptor on the cell membrane and activates the signal transduction pathway that blocks the entry of adenosine into the cell. The inventors found that dipyridamole can regulate the expression of apoptosis/anti-apoptotic proteins and enter the cell via a cell penetrating drug delivery system to cause apoptosis. Activation of the apoptotic pathway of cancer cells can promote the treatment of cancers that are known to be associated with abnormal apoptosis/anti-apoptotic protein expression. Figure 1a shows that if dipyridamole is administered in a free form, it mainly acts on the outside of the cell and will promote the accumulation of adenosine, which will result in reduced apoptosis. Figure 1b shows the expression of dipyridamole activating apoptosis/anti-apoptotic protein inside the cell. In this case, the undesirable effect of dipyridamole on the outside of the cell can be avoided. In the following examples, the present invention demonstrates that by directly delivering dipyridamole to cells, binding to receptors on the cell membrane can be avoided to reduce side effects such as oxidative stress and vasoconstriction caused by adenosine accumulation. In the examples, the compound of formula (I) of the present invention is encapsulated in a carrier for delivery to cells. In a preferred embodiment, the carrier system is nonionic, polymeric vesicles, nanoparticles, liposomes, nano-suspended particles, solid lipid nanoparticles, magnetic nano-carriers, micelles, macromolecular conjugates Or particulate drug carriers. Preferably, the carrier system liposomes. Liposomes suitable for use in the present invention have a diameter in the range of about 10-300 nm, preferably about 80-280 nm, and more preferably about 120-270 nm. In another embodiment of the present invention, the carrier may be a non-ionic body, polymeric vesicle or polymer with a diameter of less than 1 mm. It can be modified based on surface potential, hydrophilicity/hydrophobicity, size, morphology, shape, and/or surface curvature. The liposome formulation of the present invention may contain vesicles of various properties (for example, unilamellar or multilamellar), composition, size and characteristics, including aqueous media of different combinations, pH and osmotic strength. In a preferred embodiment, the main component of the liposome lipid layer membrane is selected from the group consisting of natural or synthetic phospholipids, such as those listed below:-1,2-Dilauroyl-sn-glyceryl- Choline 3-phosphoric acid (DLPC)-1,2-Dimyristyl-sn-glyceryl-3-phosphocholine (DMPC)-1,2-Dipalmitoyl-sn-glyceryl-3-phosphate Choline (DPPC)-1,2-Distearyl-sn-glyceryl-3-phosphocholine (DSPC)-1,2-Dioleyl-sn-glyceryl-3-phosphocholine ( DOPC)-1,2-Dimyristyl-sn-glyceryl-3-phosphoethanolamine (DMPE)-1,2-Dipalmitoyl-sn-glyceryl-3-phosphoethanolamine (DPPE)-1, 2-Distearyl-sn-glyceryl-3-phosphoethanolamine (DSPE)-1,2-Dioleyl-sn-glyceryl-3-phosphoethanolamine (DOPE)-1-myristyl- 2-palmitoyl-sn-glyceryl-3-phosphocholine (MPPC)-1-palmitoyl-2-myristyl-sn-glyceryl-3-phosphocholine (PMPC)-1-hard Tallow-2-palmitoyl-sn-glyceryl-3-phosphocholine (SPPC)-1-palmitoyl-2-stearyl-sn-glyceryl-3-phosphocholine (PSPC) -1,2-Dimyristyl-sn-glyceryl-3-[phosphate-rac-(1-glyceryl)] (DMPG)-1,2-Dipalmitoyl-sn-glyceryl-3-[ Phosphoric acid-rac-(1-glycerol)] (DPPG)-1,2-Distearyl-sn-glyceryl-3-[phosphate-rac-(1-glycerol)] (DSPG)-1,2- Dioleyl-sn-glyceryl-3-[phosphate-rac-(1-glyceryl)] (DOPG)-1,2-Dimyristyl-sn-glyceryl-3-phosphate (DMPA)- 1,2-Dipalmitoyl-sn-glyceryl-3-phosphate (DPPA)-1,2-Dipalmitoyl-sn-glyceryl-3-[phospho-L-serine] (DPPS) -Phospholipid serine (PS), and-Natural La-phospholipid choline (from eggs, EPC; or from soybeans, SPC; and HSPC). Preferred phospholipids are long saturated phospholipids, for example, those having alkyl chains of more than 12, preferably more than 14, more preferably more than 16, and most preferably more than 18 carbon atoms. The preferred liposome compositions used in the present invention are preferably those in which the lipid system is unilamellar and/or multilamellar, and contains: (i) 1 to 100, preferably 40 to 70 mol%, preferably selected from the following composition Physiologically acceptable phospholipids of the group: DLPC, DMPC, DPPC, DSPC, DOPC, DMPE, DPPE, DSPE, DOPE, MPPC, PMPC, SPPC, PSPC, DMPG, DPPG, DSPG, DOPG, DMPA, DPPA, DPPS, PS, EPC, SPC and HSPC; (ii) 1 to 100, preferably 40 to 70 mol% sphingolipid, preferably sphingomyelin; (iii) 1 to 100, preferably 40 to 70 mol% surfactant, It is preferably characterized by hydrophobic alkyl ethers (for example, Brij), alkyl esters, polysorbates, sorbitol esters and/or alkyl amides; (iv) 5 to 100, preferably 50 to 100 mol% amphiphilic polymer and/or copolymer, preferably comprising at least one block of hydrophilic polymer or copolymer (e.g. polyethylene glycol) and at least one block of hydrophobic polymer or copolymer (e.g. Poly(lactide), poly(caprolactone), poly(butylene oxide), poly(styrene oxide), poly(styrene), poly(ethylethylene) or polydimethylsiloxane) (V) 0 to 60 mol%, preferably 20 to 50 mol%, a toxin retention enhancing compound, preferably a sterol derivative, preferably cholesterol; or (vi) 0 to 30 mol%, preferably 1 to 5 mol% steric stabilizer, preferably pegylated compound, preferably pegylated lipid, more preferably DSPE-PEG. In a preferred embodiment, liposome-like vesicles are made from polymers and do not contain lipids for the reason that lipids are not considered liposomes in form but are called polymeric vesicles. However, for the purposes of the present invention, polymeric vesicles are intended to be covered by the term liposome as used to define the scope of the present invention and patent applications. Similarly, liposome-like vesicles made from synthetic surfactants that do not contain lipids are called nonionic bodies. However, for the purposes of the present invention, nonionic bodies are intended to be covered by the term liposomes as used to define the scope of the present invention and patent applications. In the embodiment of the present invention, the polymerization of different high molecular polymers can be used, including those in the form of triblock copolymers (such as ABA and BAB) and those in the form of block copolymers (such as PLLA- PEG, PLGA-PEG, PLA-PEG, PLLA-mPEG, PLGA-mPEG and PLA-mPEG). Various shapes (such as star and L forms) can be designed, including PEG-(PLGA) ₈ , PEG-(PLLA) ₈ and PEG-(PDLA) ₈ star block copolymers. PEGylation modification can be used to modify any vehicle (such as polymer vehicles and liposomes) to achieve the effect of reducing the binding rate of plasma proteins (see Park, J. et al., (2009) "PEGylated PLGA nanoparticles for the improved" delivery of doxorubicin. Nanomedicine."5(4):410-418.; Lück, M. et al., (1998) "Plasma protein adsorption on biodegradable microspheres consisting of poly(D,L-lactide-co-glycolide), poly (L-lactide) or ABA triblock copolymers containing poly(oxyethylene). Influence of production method and polymer composition." J. Control Release. 55(2-3):107-20.; and Sempf, K. et al., ( 2013) "Adsorption of plasma proteins on uncoated PLGA nanoparticles." Eur. J. Pharm. Biopharm. 85(1):53-60). Animal doses should not be extrapolated to human equivalent doses (HED) by simple conversion based on body weight. The Food and Drug Administration (Food and Drug Administration) has proposed that the extrapolation of animal doses to human doses can only be carried out correctly with the help of normalized BSA (which is usually expressed in mg/m2). The human dose equivalent can be calculated more appropriately by using the following formula: HED (mg/kg) = animal dose (mg/kg) multiplied by animal Km/human Km. To convert the dose used in mice to a human dose based on surface area, 22.4 mg/kg (Baur's mouse dose) was multiplied by the mouse Km factor (3) and then divided by the human Km factor (37) (See the table below). The value based on the data from the draft FDA guideline is to convert the dose expressed in mg/kg to the dose expressed in mg/m ² and multiply it by the K _m value. According to the present invention, the effective dose of liposome-dipyridamole is 10 mg/kg-100 mg/kg in mice, 6-60 mg/kg in hamsters, and 5-50 mg/kg in rats. 3.75-37.5 mg/kg in guinea pigs, 2.5-25 mg/kg in rabbits, 2.5-25 mg/kg in monkeys, 1.5-15 mg/kg in dogs, 2.4-24 mg/kg in cats, 1.5-15 mg/kg in baboons, 1.2-12 mg/kg in children, and 0.81-8.1 mg/kg in adults. Taking into account the differences in drug sensitivity between species, the widest dose range without limiting species is 0.4-160 mg/kg, preferably 0.6-120 mg/kg, more preferably 0.8 mg/kg-100 mg/kg. In an embodiment, dipyridamole liposomes can be used to treat cancer. In an embodiment, the cancer is drug-resistant or non-drug-resistant. In the examples, the cancer is breast cancer and liver cancer. In an embodiment, dipyridamole liposomes can be administered in combination with other anticancer drugs, such as alkylating agents, antimetabolites, antibiotics, hormones, immunomodulators, mitotic inhibitors, target therapeutic agents, and platinum drugs. In an embodiment, the anti-cancer drugs include chemotherapeutic drugs and target drugs. In an embodiment, the chemotherapeutic drugs include alkylating agents (such as cyclophosphamide, methyl bis(chloroethyl) amine (Mechlorethamine) and Melphalan), anti-mitotic agents (vinblastine, vinblastine) Vincristine and Taxol), DNA intercalators (Daunorubicin and Doxorubicin), DNA fragmentation agents, antimetabolites (e.g. Capecitabine, Cladribine) (Cladribine), Cytarabine, Fludarabine phosphate, 5-Fluorouracil, Gemcitabine, 6-mercaptopurine, Methotrexate (amine Methotrexate (Amethopterin, MTX), Mitoxantron, Pemetrexed disodium (heptahydrate) and Tegafur (FT-207)) and Topology Structure enzyme inhibitors (such as Camptothecin, Topotecan, Irinotecan, Podophyllotoxin and Etoposide). In an embodiment, the target drug includes Trastuzumab (Trastuzumab), Lapatinib (Lapatinib), Gefitinib (Genfitinib), Erlotinib (Erlotinib), Cetuximab (Cetuximab), Bevacizumab (Bevacizumab), Rituximab (Rituximab), Bortizomib (Bortizomib), Imatinib (Imatinib), Sunitinib (Sunitinib), Rapamycin, Sirolimus, Tesirolimus, Everolimus, Ridaforolimus (deforolimus) and Sorafenib. In the embodiment, the other anticancer drug is 5-fluorouracil (5-FU). Now that the present invention has been described in general, the present invention can be more easily understood by referring to the following examples, which provide exemplary solutions for producing the pharmaceutical composition of the present invention and its use in the treatment of cancer. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure the accuracy of the numbers used (for example, quantity, temperature, etc.), but of course some experimental errors and deviations will be allowed. Examples Example 1 : Preparation of dipyridamole liposomes The lipid system is prepared using phospholipids and cholesterol containing positive and neutral charges, wherein the molar percentage of cholesterol is 5% to 75%. Prepare small unilamellar vesicles. The dried lipid membrane was hydrated with ammonium sulfate and then extruded through a series of polycarbonate membrane filters. Dipyridamole is encapsulated in liposomes through transmembrane pH gradient or dehydration-rehydration, and through the above manufacturing process, the diameter of the extruded liposomes is in the range of 50-602 nm (dissolved in glucose solution) ). The diameter of liposome-dipyridamole is about 50 to 602 nm, as shown in Table 1. Table 1 Example 2 : Cell viability of the BCL-2 protein family (MTT analysis ) and Western blot analysis The culture medium used for human breast cancer is L-15 medium. To obtain a complete growth medium, the following components are added to the basal medium: Fetal Bovine Serum to a final concentration of 10%. The culture was incubated at 37°C without CO ₂ . The cancer cell lines tested included MDA-MB-231, PANC-1, BXPC-3, HCT-116, HT29, A375 and MeWo. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Sigma, St.Louis) storage solution is obtained by adding 5 mg MTT /ml was prepared in phosphate buffered saline (pH 7.5) and filtered through a 0.22 mm filter. The MTT analysis used is basically similar to that originally described by Mosmann (1983). Briefly, 100 mL of MTT solution (0.5 mg/mL) was added to the monolayer of the treated cancer cell line and the microplate was incubated at 37°C for 3 hours. Add 100 mL of DMSO to each well and mix well to dissolve the blue-black crystals. Use the test wavelength of 570 nm and the reference wavelength of 630 nm to read the plate on the microELISA reader. After treatment, the cells were washed with 150 mL RIPA cell lysis buffer (10mM Hepes pH=7.9, 1.5mM MgCl2, 10mM KCl, 1.0mM DTT, 0.1% Triton-X 100), and centrifuged at 1200 g at 4°C for 10 minute. Then the supernatant containing the total protein of cancer cells was collected and analyzed by western blotting. The Western blot method is implemented as follows: use Bradford analysis to measure protein concentration; mix 6X sample buffer (0.8 mM Tris-HCl, 10 mM EDTA, 10% SDS, 60% glycerol, 0.6 M β-mercaptoethanol, 0.06% Bromophenol blue, pH 6.8) was added to 50 μg of whole cell protein; and an equal volume of lysis buffer was added to the sample. After heating at 95°C for 10 minutes to denature the protein, immediately cool the sample on ice. The sample was then separated by 12% SDS-PAGE electrophoresis (100 V) and transferred from the SDS-PAGE gel to the PVDF membrane by wet ink dots. The PVDF membrane was then treated with 5% skim milk at room temperature for 60 minutes to block non-specific binding. The membrane was incubated with the primary antibody overnight at 4°C and washed three times with PBST. The membrane was incubated with the secondary antibody for 60 minutes at room temperature and washed three times with PBST. The membrane was then washed again with PBS and incubated with enhanced chemiluminescence (ECL) substrate for detection. Use automated chemiluminescence and fluorescence imaging system (UVP Biospectrum) to obtain image photos. Example 2.1 : The cell line used in the analysis of cell viability and Bax/BCL-2 ratio in MCF-7 cells treated with dipyridamole liposomes is the human breast cancer cell line MCF-7. The cells were treated with dipyridamole liposomes (10 mM and 20 mM) or the free form of dipyridamole (10 mM and 20 mM). The results are shown in Figures 4a and 4b, and normalized in Figures 5a and 5b and Table 2. Table 2 Example 3 : Cell line human breast cancer cells used in the analysis of cell viability and Bax/BCL-2 ratio in MDA-MB-231 cells treated with the combination of dipyridamole liposome and rapamycin MDA-MB-231. Combine cells with rapamycin (100 mM), liposomes of dipyridamole (3.125 mM and 25 mM) or the free form of dipyridamole (3.125 mM and 25 mM) and rapamycin (100 mM) deal with. The results are shown in Table 3. Table 3 Example 4 : The cell line used in the analysis of cell viability and Bax/BCL-2 ratio in PANC-1 cells treated with the combination of dipyridamole liposome and rapamycin, human breast cancer cell line PANC- 1. Combine cells with rapamycin (10 mM), liposomes of dipyridamole (3.125 mM and 25 mM) or the free form of dipyridamole (3.125 mM and 25 mM) and rapamycin (10 mM) deal with. The results are shown in Table 4. Table 4 Example 5 : Cell viability and Bax/BCL-2 in PANC-1 , BxPC-3 , HCT-116 , HT29 , A375 and MeWo cells treated with the combination of dipyridamole liposomes and 5-Fu the ratio of cells with 5-Fu (10 mM and 100 mM), dipyridamole liposomes (3.125 mM and 25 mM) in free form or dipyridamole (25 mM) and 5-Fu (10 mM) of combined treatment . The results are shown in Table 5. Table 5 The results in this example confirm that by using the combination of dipyridamole liposomes and 5-Fu, the cytotoxic effects of these drugs on pancreatic cancer, liver cancer, breast cancer and skin cancer cells balance Bcl-2 and Bax The ratio increases. The dosage of these drugs can also be reduced. For example, the dose of 5-Fu can be reduced by at least 8 times. Moreover, with the help of statistical analysis, it was found that among the 8 cancer cell lines tested, the increase in the ratio of Bax/Bcl-2 due to dipyridamole liposome treatment was negatively correlated with the survival rate of cancer cells (R=-0.789, p <0.01 ). This result proves that dipyridamole and 5-Fu have a synergistic effect in inhibiting the growth of various types of cancer cell lines. Moreover, the pharmaceutical composition containing dipyridamole and 5-Fu can enhance the cytotoxic effect of 5-Fu in cancer cells, especially cancers resistant to 5-Fu treatment (Figure 2). Example 6 : Cell viability of A375 and MeWo cells treated with a combination of dipyridamole liposomes and gemcitabine In A375 and MeWo cancer cell lines, it was found that dipyridamole liposomes exhibited a membrane penetration effect. With this membrane penetration effect, the antagonism of dipyridamole to gemcitabine is effectively reduced by 20-100%. This result proves that dipyridamole liposomes increase drug penetration into cells. The results are shown in Table 6. Extracellular dipyridamole blocks ENT-1 and prevents gemcitabine from entering cancer cells to produce its cytotoxic effect. The results in this example confirm that dipyridamole liposomes reduce this phenomenon by 20-40%, which indicates that dipyridamole liposomes enhance the rate of drug entering cells. Table 6 Other chemotherapeutic drugs (such as rapamycin analogs (Rapalogs) shown in Figure 3) can be used in combination with the dipyridamole liposomes of the present invention. From the above data, it has been found that the dipyridamole liposome of the present invention produces a synergistic effect in inhibiting the proliferation of cancer cells when administered with chemotherapeutic drugs, in which more than 10-40% of the cells die (Figure 4). The synergistic effect of cancer cell death is not simply caused by increasing Bax or decreasing Bcl-2, but increasing the Bax/Bcl-2 ratio (Figures 5 and 6). According to an example, dipyridamole increases the Bax/Bcl-2 ratio by 25-200% after entering the cell, and increases the inhibition of cancer cell proliferation by 20-50%. When combined with 5-Fu, Dipyridamole inhibits the proliferation of 5-50% of cancer cells by entering the cell and increases the Bax/Bcl-2 ratio by 15-50%. The combination of dipyridamole and rapamycin inhibits the proliferation of 10-30% of cancer cells by entering the cell and increases the ratio of Bax/Bcl-2 by 10-200%. For those familiar with the art, it is expected that there will be various modifications and changes of the present invention described in the above illustrative examples. Therefore, only the limitations appearing in the scope of the attached patent application are applied to the present invention.

Figures 1a and 1b are schematic diagrams showing the different effects of dipyridamole (a) outside the cell and (b) inside the cell on the apoptosis of cancer cells. Figure 2 confirms the relationship between cell viability and Bax/Bcl-2 ratio (Y-axis: cell viability; X-axis: Bax/Bcl-2 ratio). Figure 3 shows the structure of representative rapamycin analogs (Rapalogs). Figures 4a and 4b show the cell viability of the triple-negative breast cancer cell line MDA-MB-231 treated with dipyridamole with or without 5-FU. Figures 5a and 5b show the performance of Bax, Bcl-2 and Bcl-xL in cancer cells treated with dipyridamole with or without 5-FU. Figures 6a and 6b show the ratio of Bax/Bcl-2 in cells treated with dipyridamole with or without 5-FU.

Claims (9)

Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof,

It is used to prepare an agent that increases the ratio of BAX/BCL-2 in an individual, wherein each of R ₁ , R ₂ , R ₃ and R ₄ is independently selected from heterocyclic groups and di(hydroxyalkyl) amines BAX/BCL-2 performance and activity are related to breast cancer, pancreatic cancer or skin cancer, wherein the compound is encapsulated in liposomes or solid lipid nanoparticles, and wherein the agent and Rapa Rapamycin or 5-Fluorouracil is administered in combination. Such as the use of claim 1, wherein R ₁ and R ₃ are heterocyclic groups, and R ₂ and R ₄ are bis(hydroxyalkyl)amino groups. The use of claim 1, wherein the heterocyclic group is hexahydropyridyl. The use according to claim 1, wherein the bis(hydroxyalkyl)amino group is N,N-bis(hydroxyethyl)amino. Such as the use of claim 1, wherein the compound is dipyridamole. The use of claim 1, wherein the liposome has a diameter in the range of about 50-602 nm. Such as the use of claim 1, wherein the system is human or non-human mammal. Such as the use of claim 7, wherein the non-human mammal is a cat or a dog. Such as the use of claim 1, wherein the cancer is a drug-resistant or non-drug-resistant cancer.

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