Bifunctional compositions for the treatment of cancer.

Abstract

The present invention relates to the use of bifunctional compositions in methods for the treatment or prevention of cancer. The compositions comprise a DNA ligand and a protein ligand for the treatment or prevention of cancer, where the DNA ligand interferes with DNA metabolism and the protein ligand interferes with signaling pathways. In particular, the present invention relates to the use of pyronaridine in methods for the treatment or prevention of cancer. The invention also relates to compositions, compounds and medicaments comprising a DNA ligand and a protein ligand, for example, compositions, compounds and medicaments comprising pyronaridine.

Description

1 / 58 Bifunctional compositions for the treatment of cancer. Field of invention

[0001] The present invention relates to methods for the treatment or prevention of cancer. In particular, the invention contemplates the use of bifunctional compositions and compounds in the treatment or prevention of cancer. The invention relates more specifically to the use of compositions comprising a DNA ligand and a protein ligand for the treatment or prevention of cancer. The invention also relates to compositions, compounds and medicaments comprising a DNA intercalator and a protein inhibitor. Background of the invention

[0002] Any discussion of the state of the art throughout the report should in no way be considered an admission that such state of the art is widely known or forms part of the general knowledge in the field.

[0003] Cancer survival rates are often very closely linked to the frequency of early diagnoses, the aggressiveness of the cancer, the availability of effective anticancer therapies that are targeted at the cancer, the overall health of the individual undergoing treatment, and / or whether there are surgical or other options for removing tumors and cancerous cells.

[0004] All types of cancer are generally characterized by uncontrolled cell division through anomalous molecular signaling that allows cells to bypass cell cycle arrest / apoptosis. The persistence of cell proliferation is often attributed, at least in part, to the disabling of cellular mechanisms for programmed cell death. Petition 870200158404, dated 12 / 17 / 2020, page 9 / 142 2 / 58

[0005] However, it is common to many cancers that the indiscriminate targeting of therapeutic agents, and the toxicity of the agents, limits the effectiveness of anticancer therapies in their treatments.

[0006] In order to circumvent the problem of targeting, while also providing therapies that have broad applicability, it may be useful to target cellular processes that are significantly increased in cell proliferation associated with cancer, such as the transcription and replication of DNA required during cell division. The mode of action of such therapies is often related to subsequent DNA damage initiating apoptotic pathways that lead to cell death. Selective apoptosis of proliferating cancer cells is crucial for the effective treatment of cancer.

[0007] DNA-binding molecules can interfere with DNA synthesis by blocking interactions between DNA and, for example, transcription factors, polymerases, ligases, nucleases, topoisomerases, and helicases. In particular, DNA ligands that intercalate between the base pairs of DNA strands are especially effective at disrupting cellular machinery and complexes associated with, and moving along the length of, DNA during transcription and replication. However, while such DNA ligands can be targeted to cancerous cells, they can also impact cells undergoing normal cell division and affect the expression of non-dividing cells, leading to a potential range of side effects.

[0008] Furthermore, the action of a DNA ligand may not be sufficient because, despite the potential therapeutic effect of interfering with DNA synthesis in order to trigger apoptosis, the ability of cancer cells to evade cell death may negate the therapeutic benefit associated with causing DNA damage to cancer cells.

[0009] As such, it would be advantageous to combine the activity of a DNA ligand with a means to inhibit the signaling cascades that increase the Petition 870200158404, dated 12 / 17 / 2020, page 10 / 142 3 / 58 tumor progression and escape from cell death and the immune response that can normally be triggered, for example, by a DNA damage response.

[0010] One class of molecules involved in cell death escape and tumor promotion are protein kinases, many of which are prolific and promiscuous kinases with vast sets of substrates. Unfortunately, inhibition of a protein kinase involved in multiple pathways can often present serious side effects, which can be a barrier to the development and commercialization of successful drugs.

[0011] One strategy to accelerate the drug development route for new therapies is to repurpose (or reposition) existing licensed drugs for new medical indications. Repurposing is effectively the reuse of drugs with specific indications for new applications, while taking advantage of existing knowledge and safety data. Recent examples of promising drug repurposing for the treatment and prevention of cancer include the use of antiparasitic and antipsychotic drugs. There have also been in vitro studies of some antimalarial drugs that have shown effectiveness against resistant strains of malaria being repurposed for co-administration with chemotherapeutic drugs in order to improve treatment by counteracting or reversing resistance to chemotherapeutic drugs.Certainly, while drug repurposing may allow for the omission of some aspects of drug development with regard to safety data, it does not circumvent the need to demonstrate that the repurposed drug is indeed effective for the new application through in vivo studies and human trials, since in vitro data are often not representative of in vivo results.

[0012] For example, in US 20110300137, the use of the antimalarial drug lumefantrine in conjunction with chemotherapeutic drugs is described, where lumefantrine enhances cancer treatment by inhibiting autophagy. Autophagy is a known mechanism employed by cancer stem cells against chemotherapeutic drugs, which allows the cells to... Petition 870200158404, dated 12 / 17 / 2020, page 11 / 142 4 / 58 remain in a quiescent state before proliferating again once the chemotherapeutic drug has been removed. In the quiescent state, cancer cells are protected against chemotherapeutic drugs, which are generally targeted at proliferating cells, thus contributing to cancer resistance to the chemotherapeutic drug. By inhibiting autophagy, cancer cells cannot avoid chemotherapeutic drugs and resistance is reduced; however, it is still the activity of the chemotherapeutic drugs that treats the cancer.However, although it has shown anti-autophagy activity in in vitro studies in US 20110300137, neither lumefantrine nor any of the various antimalarial drugs cited (some of which, such as pyronaridin, share very little similarity with the other members of the group supposedly exhibiting the same activity) have been shown or suggested as exhibiting any antiproliferative or apoptotic activity to effectively treat or prevent dividing cancer cells separately, nor have there been any in vivo studies to validate these findings.

[0013] In another example, CN 1135977C describes the use of the antimalarial drug, pyronaridine, in the preparation of combination antineoplastic drugs for the treatment of multidrug-resistant tumors. Similarly, this report describes the co-administration or pre-administration of pyronaridine with Adriamycin (ADR: doxorubicin) for multidrug-resistant cell lines with specific resistance to ADR, in order to enhance the cytotoxic effects of ADR. Although it has been shown that apoptosis of MDR cell lines by ADR can be enhanced in the presence of pyronaridine in in vitro studies, it has not been demonstrated or suggested that PND could induce apoptosis in proliferating cells separately, nor that any purported activity of pyronaridine is transferable and replicable in actual tumors. Likewise, the invention claimed in CN1 135977C is merely for the use of pyronaridine in Petition 870200158404, dated 12 / 17 / 2020, page 12 / 142 5 / 58 combination with antitumor drugs for the reversal of MDR in resistant tumors in vitro.

[0014] It would be useful to be able to repurpose a known drug that exhibits both DNA and protein binding activity, such as an antimalarial drug with a long history of tolerated use, for new therapeutic applications in order to accelerate the drug development route for cancer treatments.

[0015] An objective of the present invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative. Summary of the invention

[0016] Surprisingly, it has been found that a molecule comprising a DNA ligand and a protein ligand is useful for the treatment and prevention of cancer. In particular, the present inventors have found that an antimalarial drug comprising a DNA ligand and a protein ligand, pyronaridin, exhibits previously known antiproliferative activities against dividing cancer cells, where the activity is selective, thus minimizing side effects. The present inventors are the first to demonstrate in in vivo studies that pyronaridin exhibits selective cytotoxicity and can be used for the treatment and / or prevention of cancer, as opposed to merely enhancing the effect of chemotherapeutic drugs in immortalized MDR cell lines.

[0017] Similarly, in a first aspect of the present invention, a method is provided for inducing apoptosis in proliferating cancer cells for the treatment or prevention of cancer in a mammal, the method comprising the step of administering to said mammal a composition comprising a therapeutically effective amount of pyronaridine (PND), or a pharmaceutically acceptable derivative or salt thereof.

[0018] In a second aspect of the present invention, a method is provided for inducing apoptosis in proliferating cancer cells in a tumor in a Petition 870200158404, dated 12 / 17 / 2020, page 13 / 142 6 / 58 mammal, the method comprising the step of administering to said mammal a composition comprising a therapeutically effective amount of pyronaridine (PND), or a pharmaceutically acceptable derivative or salt thereof.

[0019] In a third aspect of the present invention, a method is provided for inducing cell death in cancerous cells in a tumor in a mammal, the method comprising the step of administering to said mammal a composition comprising a therapeutically effective amount of pyronaridine (PND), or a pharmaceutically acceptable derivative or salt thereof.

[0020] In a fourth aspect of the present invention, a method is provided for inducing cell death in cancerous cells for the treatment or prevention of cancer in a mammal, the method comprising the step of administering to said mammal a composition comprising a therapeutically effective amount of pyronaridine (PND), or a pharmaceutically acceptable derivative or salt thereof.

[0021] In a fifth aspect of the present invention, a method is provided for reducing the size of a tumor in a mammal, the method comprising the step of administering to said mammal a composition comprising a therapeutically effective amount of pyronaridine (PND), or a pharmaceutically acceptable derivative or salt thereof, wherein the administration reduces the size of the tumor by inducing apoptosis in proliferating cancer cells in the tumor.

[0022] In a sixth aspect of the invention, a method is provided for the treatment or prevention of cancer, the method comprising the step of administering to an individual in need of such treatment a composition comprising a DNA binder and a protein binder.

[0023] A DNA ligand can be any molecule that can bind DNA and interfere with DNA metabolism. By DNA metabolism is meant any cellular process by which DNA is maintained or in which DNA is used and includes, for example, DNA synthesis and degradation reactions involved in DNA replication and repair, as well as transcription. Replication Petition 870200158404, dated 12 / 17 / 2020, page 14 / 142 7 / 58 refers to any process by which DNA sequences are copied. The linker can link DNA non-specifically through electrostatic interactions, through specific groove-linking interactions, and / or through base pair intercalation. In specific embodiments, the DNA linker is a DNA intercalator. The DNA intercalator can act by base pair intercalation through the major groove of the DNA or minor groove of the DNA and / or screw intercalation.

[0024] DNA intercalators may comprise a planar ring system that facilitates DNA intercalation by allowing insertion between two adjacent base pairs in the DNA double strands, thereby distorting the DNA double helix and / or preventing DNA replication and / or transcription by interfering with DNA uncoiling, elongation, and / or ligation. Interference with DNA synthesis in proliferating cells, such as cancer cells, can slow or halt DNA replication and cell proliferation, interfere with the cell cycle, reduce cell viability, and / or lead to cell death.

[0025] In particular cases, DNA intercalators may initiate apoptotic pathways by inhibiting topoisomerase activity. These DNA intercalators may be classified as topoisomerase inhibitors or topoisomerase poisons, and may be directed to topoisomerase I and / or topoisomerase II.These DNA intercalators can also bind topoisomerase proteins. Preferably, the DNA intercalator is also capable of binding and inhibiting the activity of topoisomerase II. Without wanting to get bogged down in theory, it is proposed that by forming a complex with DNA and topoisomerase II in a proliferating cell undergoing DNA synthesis, the DNA and topoisomerase II become trapped in the "cleavable complex". This prevents the rejoining of the double-stranded DNA that was initially cut by topoisomerase to facilitate uncoupling. The subsequent breakage of the endogenous DNA strand triggers the DNA damage response, leading to apoptosis. This mode of action may be facilitated, or contributed to, by the fact that... Petition 870200158404, dated 12 / 17 / 2020, page 15 / 142 8 / 58 DNA intercalator stabilizes double-strand breaks and / or prevents re-annealing of the two DNA strands.

[0026] In cases where the DNA ligand is a DNA intercalator, the protein ligand of the invention composition may also contribute to the initiation of the apoptotic pathway by interacting with DNA and / or topoisomerase.

[0027] The planar ring system of the DNA intercalator may comprise two or more fused rings. Preferably the rings are aromatic. The rings may be any 5- or 6-membered aromatic compounds and may be substituted and / or heterocyclic. A substituted aromatic ring is one in which a hydrogen atom in the ring is replaced by another atom or group. A heterocyclic aromatic compound is one in which the ring structure comprises carbon and at least one other atom, such as, for example, nitrogen, oxygen and / or sulfur. For example, the DNA intercalator may comprise a benzene ring and / or a 6-membered heterocyclic compound such as pyridine, pyrimidine, pyrazine, phosphinine, diazine and / or thiazine. In other examples, the DNA intercalator may comprise a 5-membered heterocyclic compound such as pyrrole, imidazole, furan, thiazole and / or thiophene.

[0028] In preferred embodiments, the planar ring system comprises at least one pyridine ring, although the ring system may comprise 2 or more pyridine rings. The planar ring system may comprise three fused aromatic rings. The three fused rings may be 5- or 6-membered rings. In one embodiment, the planar ring system comprises three fused 6-membered aromatic rings, wherein at least one, and preferably two, of the aromatic rings are heterocyclic. Each of the rings may be benzene, in which case the planar ring system may be called anthracene. One or more of the benzene rings may be halogenated, nitrated, sulfonated, alkylated, acylated, and / or substituted with sterically permissible combinations thereof. The DNA intercalator may be a heterocyclic derivative of anthracene, wherein at least one of the rings has a ring structure comprising carbon and at least nitrogen. Anthracene derivatives with a ring structure Petition 870200158404, dated 12 / 17 / 2020, p. 16 / 142 9 / 58 comprising carbon and one, two, and three nitrogens can be called monazeanthracenes, diazaanthracenes, and triazeanthracenes, respectively.

[0029] Non-limiting examples of anthracenes / anthracene groups that may be part of the DNA intercalator include 1,-monoazaanthracene, 2,monoazaanthracene, 5,-monoazaanthracene, 1,2-diazaanthracene, 1,3-diazaanthracene, 1,4-diazaanthracene, 1,5-diazaanthracene, 1,6-diazaanthracene, 1,7-diazaanthracene, 1,8-diazaanthracene, 1,9-diazaanthracene, 1,10-diazaanthracene, 2,3-diazaanthracene, 2,5-diazaanthracene, 2,7-diazaanthracene, 2,8-diazaanthracene and 5,10-diazaanthracene. In preferred embodiments of the invention, the flat ring system of the DNA intercalator is a 1,5-diazaanthracene (also known as benzo(b)-1,5-natyridine), or a derivative thereof.

[0030] In preferred embodiments of the invention, the DNA intercalator planar ring system is an anthracene, a diazaanthracene, or, more preferably, a 1,5-diaza-anthracene with one or more substituents selected from the group consisting of -O(-), -OH, -OR, -OC6H5, OCOCH3, -NH2, -NR2, -NHCOCH3, -R, -C6H5, -NO2, -NR(+), -PR(+), -SR(+), -SO3H, -SO2R, -CO2H, -CO2R, -CONH2, -CHO, -COR, -CN, -F, -Cl, -Br, -I, -CH2C1, -CH=CHNO2, where R is any alkyl group.

[0031] The protein ligand can be any molecule that binds to and interferes with the activity of a protein, and can prevent any signaling pathway involving said protein. It should be understood that communication within and between cells occurs through signal transmission via signaling pathways (also known as signal transduction). In preferred embodiments of the invention, the protein ligand binds to a protein that is involved in a signaling pathway that leads to, or enhances / enables, cancer progression. This signaling pathway can be any pathway that results in any cancer-associated phenotype(s), including but not limited to, cell proliferation, cellular diversion of apoptosis and / or necrosis, cellular diversion of the immune response, incorporation and propagation of DNA damage, increased cell motility, and increased cell metabolism. By blocking a pathway of Petition 870200158404, dated 12 / 17 / 2020, page 17 / 142 10 / 58 signaling that results in a cancer-associated phenotype, the protein ligand may assist in the treatment or prevention of cancer by reducing, minimizing, reversing, preventing, delaying, or ablating, or any effect of the cancer-associated phenotype.

[0032] The protein ligand can block a signaling pathway by binding to a protein and preventing a post-translational modification of the protein, such as specific cleavage reactions, the formation of disulfide bonds, the addition of low molecular weight groups, for example, through acetylation, amidation, biotinylation, cysteinylation, deamidation, formylation, glutathionylation, glycation, glycosylation, hydroxylation, methylation, oxidation, palmitoylation or phosphorylation and / or conjugations with other types of molecules. In other examples, a signaling pathway can be blocked by the binding of the protein ligand to a protein and preventing the removal of low molecular weight groups added by any of the post-translational processes mentioned above.

[0033] The qualified recipient would know that one of the most common cellular mechanisms of signal transduction is protein kinase phosphorylation and protein phosphatase dephosphorylation. Similarly, in preferred embodiments of the invention, the protein ligand blocks a signaling pathway that leads to, or enhances / enables, cancer progression by interfering with the phosphorylation state of a protein in the signaling pathway. By interference is meant that (a) the protein ligand can bind a protein in the signaling pathway and prevent or allow the phosphorylation or dephosphorylation of this protein; or (b) the protein ligand can bind a protein in the signaling pathway and prevent or allow the protein to phosphorylate or dephosphorylate other substrates in the signaling pathway.

[0034] A particularly common feature of cancer is the increased activity, upexpression, or loss of downregulation of protein kinases, which may be collectively termed protein kinase upregulation. In preferred embodiments of the invention, the protein ligand Petition 870200158404, dated 12 / 17 / 2020, page 18 / 142 11 / 58 binds a protein kinase, and more preferably, the protein ligand binds a protein kinase that is involved in a signaling pathway that leads to, or enhances / enables, cancer progression. The protein ligand can bind the protein kinase and prevent, disrupt, or reduce the protein kinase's ability to phosphorylate targets involved in the signaling pathway. The action of the protein kinase can be prevented, disrupted, or reduced by the binding of the protein ligand temporarily, reversibly, permanently, or in response to particular stimuli.The action of protein kinase can be prevented, disrupted, or reduced by the binding of a protein ligand, for example, by the protein ligand inducing conformational changes in the protein kinase, the protein ligand occupying binding sites on the protein kinase, the protein ligand blocking other receptor sites that would ordinarily trigger phosphorylation events, and / or the protein ligand creating steric obstruction to kinase activity. Similarly, the protein ligand can be called a protein kinase inhibitor.

[0035] The protein ligand can bind and inhibit any protein kinase that is upregulated in a signaling pathway that leads to or enhances / enables cancer progression. In certain embodiments of the invention, the protein kinase is a kinase involved in the MAP (mitogen-activated protein) kinase pathway (also known as the Ras / MAPK pathway or Ras / Raf / MAPK pathway or MAP / Erk pathway or Ras / Raf / Mek / Erk pathway), which is involved in cell proliferation, cell survival, and metastasis. In particular, the protein ligand can bind and inhibit a MEK (MAPK / Erk kinase), can bind and inhibit a Raf kinase, or can bind and inhibit an ERK (extracellular receptor kinase) or isoforms thereof.

[0036] In other embodiments, the protein ligand binds a protein kinase in the PI3K / Akt (phosphoinositide 3-kinase / Protein Kinase B) kinase pathway, which is a combination of many individual pathways involved in proliferation, metabolism, cell survival, and response to DNA damage. In particular, the Petition 870200158404, dated 12 / 17 / 2020, page 19 / 142 12 / 58 protein ligand can bind and inhibit PI3K, Akt, PDK1, GSK-3, Weel, Mytl, CHK1, CHK2, ATR, ATM, mTOR, CDK2 or PIP5K, or isoforms thereof.

[0037] In a further example, the protein ligand binds a protein kinase in the RIPK3 / RIPK1 (receptor-interacting serine / threonine kinase 3 / 1; also known as the RIP1 and / or RIP3 kinase) kinase pathway, which is involved in apoptosis and necrosis. In particular, the protein ligand can bind and inhibit RIPK3, RIPK1, TAKI, or FADD, or isoforms thereof. In other examples, the protein ligand binds and inhibits PKC (protein kinase C) or isoforms thereof, which is involved in cell proliferation, motility, and survival.

[0038] In embodiments of the present invention, the protein ligand is a protein kinase inhibitor, wherein the protein kinase is selected from the group consisting of MEK, Raf, ERK, PI3K, Akt, PDK1, GSK-3, Weel, Mytl, CHK1, CHK2, ATR, ATM, mTOR, CDK2, PIP5K, RI PK3, RIPK1, TAKI, FADD and PCK, and / or isoforms thereof.

[0039] The protein linker can bind more than one protein kinase. Similarly, in embodiments of the present invention, the protein linker binds and inhibits one or more protein kinases selected from the group consisting of MEK, Raf, ERK, PI3K, Akt, PDK1, GSK-3, Weel, Mytl, CHK1, CHK2, ATR, ATM, mTOR, CDK2, PIP5K, RIPK3, RIPK1, TAK1, FADD and PCK, and / or isoforms thereof.

[0040] In embodiments of the present invention, the protein ligand binds to the kinase, Glycogen Synthase Kinase-3 (GSK-3), and may be referred to as a GSK-3 ligand. In this case, the protein ligand may be any molecule that binds to GSK3a and / or GSK3P, where it should be understood that GSK3a and GSK3P are paralogs that have different and overlapping phosphorylation substrates and respond to different and overlapping molecular signals. For ease of reference, both molecules will be collectively referred to as GSK-3, although it should be understood that the GSK-3 ligand may preferably bind one of the paralogs, may bind the paralogs with different specificities and Petition 870200158404, dated 12 / 17 / 2020, page 20 / 142 13 / 58 affinities and / or may preferentially bind one of the paralogs, for example, at particular cellular sites, in response to different stimuli and / or when used in the treatment or prevention of different cancers. Alternatively, the GSK-3 ligand may bind each of the paralogs at the same site with similar specificity and affinity.

[0041] GSK-3 is a prolific and promiscuous kinase known to be involved in tumor promotion through increased cellular metabolism and the formation of anti-apoptotic protein complexes. GSK-3 is present throughout the cell, including the nucleus. Similarly, without wanting to be bound by theory, it is proposed that interference with GSK-3 signaling in proliferating cells, such as cancer cells, may disrupt many relevant cellular processes, thereby slowing or halting cell proliferation and / or encouraging cell death.

[0042] In one or more embodiments, the protein linker may comprise one or more 5- or 6-membered aromatic rings, wherein the ring(s) may be substituted and / or heterocyclic. In other embodiments, the protein linker may comprise 5- or 6-membered rings that are not aromatic, and the rings may be substituted at any of the atoms in the ring structure. In embodiments of the invention, the protein linker may comprise one or more benzene or phenyl rings, and these may be halogenated, nitrated, sulfonated, alkylated, acylated, and / or substituted with sterically permissible combinations thereof. In further embodiments, the protein linker may comprise one or more pyrrolidine rings.The protein linker may comprise one or more 6-membered aromatic rings and / or one or more 5-membered rings, with one or more substituents selected from the group consisting of -O(-), -OH, -OR, -OC6H5, -OCOCH3, -NH2, -NR2, -nhcoch3, -R, -C6H5, -NO2, -NR3(+), -pr3(+), -SR2(+), -SO3H, -SO2R, -CO2H, -CO2R, -CONH2, -CHO, -COR, -CN, -F, -Cl, -Br, -I. CH2C1, -CH=CHNO2, -C2H5, -CR2H, -CR3, where R is any alkyl group. In preferred embodiments, the protein linker may comprise a phenyl ring. Petition 870200158404, dated 12 / 17 / 2020, p. 21 / 142 14 / 58 and one or more pyrrolidine rings, the phenyl ring and / or the pyrrolidine ring may be substituted with one or more substituents selected from the group consisting of -O(-), -OH, -OR, -OC6H5, -OCOCH3, -NH2, -NR2, -NHCOCH3, -R, C6H5, -NO2, -NR(+), -PR(+), -SR2(+), -so3h, -SO2R, -CO2H, -CO2R, CONH2, -CHO, -COR, -CN, -F, -Cl, -Br, -I, -CH2C1, -CH=CHNO2, -C2H5, CR2H, -CR3, where R is any alkyl group.

[0043] The protein ligand can bind to or interact with the protein at particular residues. These residues may, for example, be part of one or more binding or phosphorylation sites on the protein. Alternatively, the residues may be crucial for protein attachment or phosphorylation of a protein substrate.

[0044] In embodiments of the invention, the protein linker can bind or interact with specific residues in GSK-3. For example, the GSK-3 linker can bind to an active site of GSK-3p via a hydrogen bond to Valine 135, Glutamine 185 and via van der Waals interactions to several additional amino acids.

[0045] In the methods of the present invention, cancer can be treated or prevented by administering a composition comprising a DNA ligand and a protein ligand, wherein the ligands are separate molecules. This would allow the composition to comprise different molar ratios of the two ligands to localize to different parts of the cell and / or to localize together. For example, the composition may comprise an excess of the DNA ligand (i.e., a molar ratio of DNA ligand:protein ligand of 1000:1 to 1.1:1) or an excess of the protein ligand (i.e., a molar ratio of DNA ligand:protein ligand of 1:1000 to 1:1.1) or equal molar amounts of DNA ligand and protein ligand.The ratio of DNA ligand to protein ligand can be modified and adjusted depending on factors such as the type of cancer being treated, the stage of the cancer, the individual's age and general health, and sensitivity to the effects of the DNA ligand and / or protein ligand. Petition 870200158404, dated 12 / 17 / 2020, page 22 / 142 15 / 58

[0046] In other embodiments, the DNA ligand and the protein ligand are separate molecules that are conjugated together, which would typically be a 1:1 ratio, although the ligands may be conjugated in such a way as to allow an excess of either the DNA ligand or the protein ligand. For example, 1-10 molecules of the DNA ligand may be conjugated with a single protein ligand, or 1-10 molecules of the protein ligand may be conjugated with a single DNA ligand. By conjugation is meant that the DNA ligand and the protein ligand are produced separately and linked by an appropriate and pharmaceutically acceptable spacer or crosslinker or by electrostatic or covalent interactions, for administration in a set ratio. The spacer or crosslinker may be cleaved under certain stimuli or the conjugation may be irreversible.

[0047] In preferred embodiments of the invention, the DNA ligand and the protein ligand are components of a single compound. The DNA ligand and the protein ligand can be joined by an appropriate ligand into a single compound. The ligand can be rigid or flexible and / or can be of any length that facilitates the DNA ligand being able to bind DNA and the protein ligand being able to bind protein. The ligand does not need to allow the simultaneous binding of DNA and protein, although this is preferred in some embodiments. The ligand may comprise an amino group. The compound may comprise other ligands and / or active sites.

[0048] In the case of the DNA ligand and the protein ligand being conjugated or part of a single compound, it should be understood that this would result in the colocalization of the DNA ligand and the protein ligand. At any given time, the conjugated compound or ligands may be located in such a way that the DNA ligand or only the protein ligand may be able to bind DNA or protein, respectively. Alternatively, the conjugated compound or ligands may be located in such a way that both the DNA ligand and the protein ligand are able to bind their respective target molecules simultaneously. The ability of the DNA ligand and the protein ligand in Petition 870200158404, dated 12 / 17 / 2020, p. 23 / 142 16 / 58 compound or conjugated ligands in binding DNA and protein, respectively, either simultaneously or sequentially, can fluctuate over time or in response to cellular activities.

[0049] In preferred embodiments, the DNA ligand and the protein ligand are in the form of pyronaridine (PND) or a pharmaceutically acceptable derivative or salt thereof. Pyronaridine has the chemical formula C29H32C1N5O2 and the chemical name 4-[(7-chloro-2-methoxy-1,5-dihydrobenzo[b][1,5]naphthyridin-10yl)imino]-2,6-bis(pyrrolidin-1-ylmethyl)cyclohexa-2,5-dien-1-one (previously, 2-methoxy-7-chloro-10-(3',5'-bis(pyrrolin-1-ylmethyl)-4'-hydroxyphenylamino)benzo(b)1,5-naphthyridine). Pyronaridine is also known as malaridin and benzonaphthyridine 7351.

[0050] In therapeutic applications, pyronaridine is preferably used as a phosphate salt or a pyronaridine tetraphosphate, which has the chemical formula C29H44C1N5O18P4 and the chemical name 4-[(7-chloro-2-methoxy-1,5-dihydrobenzo[b][1,5]naphthyridin-10-yl)imino]-2,6-bis(pyrrolidin-1-ylmethyl)cyclohexa-2,5-dien-1-one;phosphoric acid.

[0051] Pyronaridine tetraphosphate is a common antimalarial agent that has been approved and used effectively in the treatment of malaria for over 30 years. More recently, it has been found to enhance the antitumor activity of some common chemotherapeutic drugs when targeted at multidrug-resistant cancers, although it has never been used separately in the treatment or prevention of cancer in in vivo studies, nor has it been suggested as a stand-alone anticancer therapy that can target proliferating cells. Surprisingly, the present invention has found that pyronaridine is an effective anticancer agent with both a DNA binder and a protein binder. Despite having two therapeutic targets, the compound is generally well-tolerated and safe for use.Without wanting to get bogged down in theory, it is hypothesized that pyronaridin selectively targets proliferating cancer cells for apoptosis and interferes with the molecular mechanisms that would normally allow it. Petition 870200158404, dated 12 / 17 / 2020, page 24 / 142 17 / 58 that cancer cells escape cell death. It is important that pyronaridin is selective in targeting proliferating cells, as this reduces the side effects that are commonly associated with less selective cancer treatments.

[0052] Pyronaridine comprises a DNA-binding ligand that is an anthracene derivative of three fused benzene rings and a protein ligand comprising a 6-membered aromatic ring and two 5-membered rings. In pyronaridine, the DNA-binding ligand is a substituted 1,5-diaza-anthracene, although in embodiments of the invention, the method is based on a pyronaridine derivative that may contain alternative substituents at positions in the ring structures. In other pyronaridine derivatives, the DNA ligand is a heterocyclic anthracene derivative with nitrogen present at different positions in the ring structures of the fused aromatic rings. It is proposed that this structural aspect of pyronaridine (i.e., the planar nature of the three fused benzene rings) allows the pyronaridine DNA ligand to act as a DNA intercalator to contribute to the initiation of apoptosis.

[0053] This characteristic is in contrast to other antimalarial agents that have been suggested to increase the activity of chemotherapeutic agents against resistant cell lines, such as amodiaquine, mefloquine, naphthoquine, pamaquine, primaquine, piperaquine, tafenoquine, mepacrine, halofantrine, pyronaridin, nitazoxanide, and atovaquone. It is possible that the lack of structural similarity between pyronaridin and these agents is responsible for the different functionality of pyronaridin (i.e., only cancer treatment) when compared to the other antimalarial agents (i.e., amplifies cancer treatment in resistant cells by inhibiting autophagy).

[0054] In pyronaridine, the protein linker comprises a phenyl group, although in embodiments of the invention, the method is based on a pyronaridine derivative that may have alternative substituents in alternative positions on the benzene ring. In other pyronaridine derivatives, the linker of Petition 870200158404, dated 12 / 17 / 2020, page 25 / 142 18 / 58 protein comprises a 6-membered heterocyclic ring. In pyronaridin, the protein linker further comprises 2 pyrrolidine rings, although in embodiments of the invention the method is based on a pyronaridin derivative that may have one or more substituted pyrrolidine rings and / or may comprise 5-membered aromatic rings or 5-membered rings with molecules other than carbon and nitrogen in the ring structure.

[0055] In other preferred embodiments of the invention, the method comprises administering to an individual in need of such treatment a composition comprising pyronaridine tetraphosphate.

[0056] In the method of the invention, cancer can be any cancer that presents as a solid tumor or a blood (liquid) cancer, including but not limited to sarcomas, carcinomas, lymphomas, leukemia, myelomas, and circulating tumor cells (CTCs). For example, carcinoma can be of the pancreas, bladder, breast, colon, mesothelioma, kidneys, liver, lung, including small cell lung cancer, non-small cell lung cancer, head and neck, esophagus, gallbladder, ovary, stomach, cervix, thyroid, prostate, or skin. In general, cancer will be characterized by uncontrolled cell proliferation.

[0057] In other examples, lymphoma can be B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, Burkett lymphoma, or an extranodal lymphoma of the stomach, breast, or brain.

[0058] Sarcoma can, for example, be fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, leiomyosarcoma, mesothelial sarcoma, angiosarcoma, liposarcoma, bone tumors and tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas or other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctantoma, follicular thyroid cancer and Kaposi's sarcoma. Petition 870200158404, dated 12 / 17 / 2020, page 26 / 142 19 / 58

[0059] Myeloma can be, for example, plasma cell myeloma or Kahler's disease or multiple myeloma. In other examples, leukemia can be myelogenous leukemia, granulocytic leukemia, lymphatic leukemia, lymphocytic leukemia or lymphoblastic leukemia, polythemia vera or erythremia.

[0060] In other non-limiting examples, cancer may include, for example, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, HIV and AIDS-related cancers, primary CNS lymphoma, anal cancer, gastrointestinal carcinoid tumors, cerebral astrocytomas, atypical teratoid / rhabdoid tumors, basal cell carcinoma, bile duct cancer, Ewing's osteosarcoma, malignant fibrous histiocytoma, cerebral glioma, bronchial tumors, cardiac tumors, embryonal tumors, germ cell tumors, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, mycosis fungoides, Sézary syndrome, ductal carcinoma in situ (DCIS), uterine cancer, ependymoma, esthesioneuroblastoma, extragonadal germ cell tumors, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer,Gastric cancer, gastrointestinal stromal tumors, testicular cancer, hypopharyngeal cancer, lip, mouth and oral cavity cancer, male breast cancer, Merkel cell carcinoma, midline tract carcinoma with NUT gene alterations, multiple endocrine neoplasia syndromes, myelodysplastic syndromes, nasal cavity and sino-paranasal cancer, nasopharyngeal cancer, oropharyngeal cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumors, pleuropulmonary blastoma, primary peritoneal cancer, salivary gland cancer, vascular tumors, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer and Wilms' tumor.

[0061] The methods of the present invention can prevent, reduce or delay the development of cancers that, for example, can normally be Petition 870200158404, dated 12 / 17 / 2020, page 27 / 142 20 / 58 develop from metastasis of any of the cancers mentioned above. The methods can also prevent, reduce, or delay the recurrence of any of the cancers mentioned above after treatment.

[0062] Cancer can also be any cancer in which the proliferative capacity of cancer cells is modulated by protein in some way. In some embodiments, cancer can be any cancer in which cancer cells overexpress protein. It should be understood that by “overexpress” is meant that the cancer cell expresses more protein than a healthy cell of the same tissue or cell type. For example, a cancer cell present in a breast tumor would be considered to overexpress a protein if, when compared with a non-cancerous breast tissue cell, the level of protein expression in the cancer cell present in a breast tumor is higher. Protein expression levels in a cell can be determined by those skilled in the art using well-known techniques such as, but not limited to, mRNA quantification, immunofluorescence, and western blotting.

[0063] Although the composition of the invention can be used alone to effectively treat and prevent cancer, in some embodiments of the invention, the method comprises administering to an individual needing such treatment a composition comprising a DNA ligand and a protein ligand and an additional bioactive agent. The bioactive agent may be included in the composition comprising the DNA ligand and the protein ligand or the bioactive agent may be co-administered with the composition comprising the DNA ligand and the protein ligand. By co-administration is meant that the agent and the composition may be administered at the same time or the composition and the agent may be administered, for example, at alternate times or one before the other or one after the other or a combination thereof (i.e., pre-administration or post-administration is included in the term co-administration). For example, the bioactive agent may be administered before the composition and Petition 870200158404, dated 12 / 17 / 2020, p. 28 / 142 21 / 58 also administered at the same time or for the same duration as the composition. By “same time” is meant not limited to a real time, but a period or duration of time. For example, a bioactive agent may be administered to an individual at the same time as a composition of the invention, meaning that the bioactive agent is administered according to any suitable schedule for a particular period (days, weeks, months or years), while the individual is also receiving a composition of the invention on the same schedule or on a different schedule for the same particular period (i.e., the patient may be receiving a daily dose of the composition of the invention for three months, while during these three months the individual is receiving a weekly dose of the bioactive agent).

[0064] The bioactive agent can be any compound that exhibits biological activity with respect to cancer, including therapeutic activity. The bioactive agent may be capable of binding to or interacting with cancer cells. The bioactive agent can be any agent, drug, compound, or composition that can be used for the detection, prevention, and / or treatment of cancer. In embodiments of the present invention, the bioactive agent is a therapeutic agent and, more preferably, a chemotherapeutic agent. In embodiments of the invention, the DNA ligand and the protein ligand are in the form of pyronaridin (PND) or a pharmaceutically acceptable derivative or salt thereof, and the bioactive agent is included in the composition comprising pyronaridin, or the bioactive agent is co-administered with the composition comprising pyronaridin.

[0065] In particularly preferred embodiments of the invention, the bioactive agent is a chemotherapeutic agent selected from the group consisting of Abiraterone acetate, Albumin-bound paclitaxel (NAB), Alemtuzumab, Altretamine, Asparaginase, Bendamustine, Bevacizumab, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carmustine, Cetuximab, Chlorambucil, Cisplatin, Cladribine, Crizotinib, Cyclophosamide, Cytarabine (Ara-C), Dacarbazine, Dactinomycin, Dasatinib, Petition 870200158404, dated 12 / 17 / 2020, page 29 / 142 22 / 58 Daunorubicin, DaunoXome (liposomal daunorubicin), DepoCyt (liposomal cytarabine), Docetaxel, Doxil (liposomal doxorubicin), Doxorubicin, Eribulin mesylate, Erlotinib, Estramustine, Etoposide, Everolimus, Floxuridine, Fludarabine, Fluorouracil, Gefitinib, Gemcitabine, Gliadel wafers, Hydroxyurea, Ibritumomab, Ibritumomab, Idarubicin, Ifosfamide, Imatinib, Ipilimumab, Irinotecan, Ixabepilone, Lapatinib, Lenalidomide, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mioxantrone, MG132, Nilotinib, Oxaliplatin, Paclitaxel, Panitumumab, Pazopanib, Peginterferon alfa-2b, Pemetrexed, Pentostatin, Pralatrexate, Procarbazine, Rituximab, Romidepsin, Ruxolitinib, Sipuleucel-T, Sorafenib, Streptozocin, Sunitinib, Temozolomide, Temsirolimus, Teniposide, Thalidomide, Thioguanine, Thiotepa, Topotecan, Tositumomab, Trastuzumab, Valrubicin, Vandetanib, Vemurafenib, Vinblastine, Vincristine and Vinorelbine.Those skilled in the art will understand that this is by no means an exhaustive list and that other chemotherapeutic agents developed in the future may be suitable for use in the methods of the present invention.

[0066] In more preferred embodiments of the invention, the DNA ligand and the protein ligand are in the form of pyronaridine (PND), or a pharmaceutically acceptable derivative or salt thereof, and a bioactive agent is included in the composition comprising pyronaridine or a bioactive agent is administered in conjunction with the composition comprising pyronaridine, wherein the bioactive agent is selected from the group consisting of cisplatin, gemcitabine, bortezomib and MG 132.

[0067] In embodiments of the invention, the method comprises administering a composition comprising two or more bioactive agents.

[0068] In other preferred embodiments of the present invention, the DNA ligand, protein ligand and / or additional bioactive agent is a prodrug. A prodrug is a drug derived from an active drug that may be inert or have lower toxicity than the active drug, and which can be processed into the active drug in vivo. Processing can occur enzymatically or Petition 870200158404, dated 12 / 17 / 2020, p. 30 / 142 23 / 58 chemical and / or processing can be triggered by biological signals, such as changes in pH and / or in response to binding to a cellular component.

[0069] In another aspect of the invention, the use of a DNA ligand and a protein ligand in the preparation of a medicament for the treatment or prevention of cancer is provided. Compositions and medications, dosages and administration

[0070] The compositions, compounds and medicaments of the present invention can be administered by oral, topical or parenteral routes, including intravenous, intramuscular, intraperitoneal and subcutaneous. They can be administered by injection directly into a tumor. They can be administered to organs, tissues and cells ex vivo.

[0071] For intramuscular, intraperitoneal, subcutaneous, and intravenous use, the compositions and medicaments of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. The aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, and tragacanth gum, and a humectant such as lecithin. Suitable preservatives for aqueous suspensions include ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate.

[0072] For oral administration, the compositions of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension. Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium or calcium carbonate, sodium or calcium phosphate, and lactose. Corn starch and alginic acid are examples of suitable disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, will generally be magnesium stearate, acid Petition 870200158404, dated 12 / 17 / 2020, page 31 / 142 24 / 58 stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate in order to retard absorption in the gastrointestinal tract.

[0073] Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent and soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

[0074] For topical administration, the compositions of the invention will generally be provided in the form of a liquid, lotion, emulsion, mousse, paste, cream, ointment or gel and may be useful for the treatment or prevention of skin cancers or cancer near the skin surface, such that the DNA binder and the protein binder are able to diffuse into the affected cells. The compositions of the invention for topical administration also include preservatives, humectants, emollients, moisturizers, consistency factors, chelating agents, excipients, diluents and pharmaceutically acceptable colorants.

[0075] The effective doses of the compositions, compounds and medications used in the present invention can be determined by conventional methods and will generally be dependent on the therapeutic agent used. The specific dosage level required for any particular individual will depend on a number of factors, including the severity of the condition being treated, the route of administration and the individual's weight.

[0076] For example, 1 unit dose may include from about 0.1 mg to about 10,000 mg of a single compound comprising a DNA ligand and a protein ligand, or alternative form(s), derivative(s), or salt(s) thereof, or combinations thereof and / or source(s) thereof described in any aspect and / or example of the invention. In another example, the unit dose includes from about 50 mg to about 10,000 mg, from about 100 mg to about 10,000 mg, from about 200 mg to about 10,000 mg, from about 500 mg to about 10,000 mg, from about 1,000 mg to about 10,000 mg, from about 2,000 mg to about 10,000 mg, from about 4,000 mg to Petition 870200158404, dated 12 / 17 / 2020, p. 32 / 142 25 / 58 approximately 10000 mg, approximately 8000 mg to approximately 10000 mg, approximately 5 mg to approximately 1000 mg, approximately 10 mg to approximately 1000 mg, approximately 20 mg to approximately 1000 mg, approximately 50 mg to approximately 1000 mg, approximately 100 mg to approximately 1000 mg, approximately 200 mg to approximately 1000 mg, approximately 400 mg to approximately 1000 mg, approximately 800 mg to approximately 1000 mg, approximately 0.5 mg to approximately 100 mg, approximately 1 mg to approximately 100 mg, approximately 2 mg to approximately 100 mg, approximately 5 mg to approximately 100 mg, approximately 10 mg to approximately 100 mg, approximately 20 mg to approximately 100 mg, about 40 mg to about 100 mg, or about 80 mg to about 100 mg.Preferably, the unit dose includes about 25 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg of a single compound comprising a DNA ligand and a protein ligand, or alternative form(s), derivative(s), or salt(s) thereof, or combinations thereof and / or source(s) thereof described in any aspect and / or example of the invention.

[0077] In other examples, 1 unit dose may include from about 0.1 mg to about 10000 mg of a DNA ligand and from about 0.1 mg to about 10000 mg of a protein ligand, or alternative form(s), derivative(s), or salt(s) thereof, or combinations thereof and / or source(s) thereof described in any aspect and / or example of the invention. In another example, the unit dose includes approximately 50 mg to approximately 10000 mg, approximately 100 mg to approximately 10000 mg, approximately 200 mg to approximately 10000 mg, approximately 500 mg to approximately 10000 mg, approximately 1000 mg to approximately 10000 mg, approximately 2000 mg to approximately 10000 mg, approximately 4000 mg to approximately 10000 mg, approximately 8000 mg to approximately 10000 mg, approximately 5 mg to approximately 1000 mg, approximately 10 mg to approximately 1000 mg, approximately 20 mg to approximately 1000 mg, approximately 50 mg to approximately 1000 mg, approximately 100 mg to approximately 1000 mg, approximately 200 mg to approximately 1000 mg, about 400 mg to about 1000 mg, about 800 mg to about 1000 mg, about 0.5 mg to about 100 mg, about 1 mg to about 100 mg, about 2 mg to about 100 mg, about 5 mg to about 100 mg, about 10 to about 100 mg, about 20 mg to about 100 mg, about 40 mg to about 100 mg, or about 80 mg to about 100 mg of one. Petition 870200158404, dated 12 / 17 / 2020, page 33 / 142 26 / 58 DNA ligand, and approximately 50 mg to approximately 10,000 mg, approximately 100 mg to approximately 10,000 mg, approximately 200 mg to approximately 10,000 mg, approximately 500 mg to approximately 10,000 mg, approximately 1,000 mg to approximately 10,000 mg, approximately 2,000 mg to approximately 10,000 mg, approximately 4,000 mg to approximately 10,000 mg, approximately 8,000 mg to approximately 10,000 mg, approximately 5 mg to approximately 1,000 mg, approximately 10 mg to approximately 1,000 mg, approximately 20 mg to approximately 1,000 mg, approximately 50 mg to approximately 1,000 mg, approximately 100 mg to approximately 1,000 mg, approximately 200 mg to approximately from 1000 mg, about 400 mg to about 1000 mg, about 800 mg to about 1000 mg, about 0.5 mg to about 100 mg, about 1 mg to about 100 mg, about 2 mg to about 100 mg, about 5 mg to about 100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, about 40 mg to about 100 mg, or about 80 mg to about 100 mg of a protein binder.Preferably, the unit dose includes about 25 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg of a DNA ligand and about 25 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg of a protein ligand, or alternative form(s), derivative(s), or salt(s) thereof, or combinations thereof and / or source(s) thereof described in any aspect and / or example of the invention.

[0078] The unit dose can be administered once, twice, three, four or five times a day or it can be administered every second or third day or once a week, once every two months or once every four months.

[0079] In some embodiments, the compositions, compounds and medications may be administered to an individual alone or in combination with other additional bioactive agent(s). In such embodiments, administration may be simultaneous or sequential.

[0080] Typically, in treatment applications, compositions, compounds, and medications may be administered for the duration of the cancer. Furthermore, it will be clear to a person skilled in the art that the optimal quantity and spacing of Petition 870200158404, dated 12 / 17 / 2020, page 34 / 142 27 / 58 Individual dosages may be determined by the nature and extent of the disease or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Optimal dosages may be determined using conventional techniques.

[0081] Some embodiments of the present invention may involve administering the composition or medication in multiple separate doses. Similarly, the treatment and prevention methods described herein encompass administering multiple separate doses to an individual, for example, over a defined period of time.

[0082] The compositions and medicaments of this invention may also be useful in combination (administered together or separately) with one or more additional therapeutic agents such as radiation therapy and / or one or more additional therapeutic agents selected from the group consisting of different types of chemotherapeutic drugs, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, targeted therapies, differentiation agents, hormone therapy and immunotherapy. Definitions

[0083] Unless the context clearly requires otherwise, throughout the report and claims, the words “includes,” “comprising,” and similar terms should be considered in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, in the sense of “including, but not limited to.”

[0084] The term treatment, and similar terms, in the context of this report includes the relief of symptoms associated with a cancer, regression and / or remission of the cancer. In certain embodiments, a treatment will slow, delay or stop the proliferation of cancer cells, or metastasis of a cancer, slow, delay or stop the increase in size of a tumor that would normally occur from cell proliferation in the tumor, prevent the differentiation of a cell lineage, reduce the size of the tumor or reverse the progression of one or more tumors, at least temporarily. Treatment Petition 870200158404, dated 12 / 17 / 2020, page 35 / 142 28 / 58 can cure cancer, or delay morbidity. Thus, in the context of this invention, the word treatment or its derivatives, when used in relation to a therapeutic application, includes all aspects of a therapy, such as relief of pain associated with the cancer being treated, relief of the severity of the cancer being treated, improvement of one or more symptoms of the cancer being treated, improvement in the overall well-being of the individual being treated. The use of the word treatment or its derivatives will be understood to mean that the individual being treated may experience any one or more of the benefits mentioned above. In general, treatment may be related to the death of proliferating cells present in the cancer.

[0085] The term prevention, and similar terms, in the context of this report refers to the prevention of the recurrence of all or some of the symptoms associated with a cancer after remission of said cancer, as well as the prevention of the formation of one or more cancers due to, for example, metastasis of a cancer. Prevention may avoid morbidity due to one or more cancers, or delay morbidity due to one or more cancers. In general, prevention may relate to the death of proliferating cells that may cause a cancer or cause the spread or recurrence of a cancer.

[0086] In the context of this report, the term approximately will be understood as indicating the usual tolerances that a person skilled in the art would associate with the given value.

[0087] In the context of this report, where a range is indicated for a parameter, it should be understood that the parameter includes all values ​​within the indicated range, including the indicated end chicks of the range.

[0088] In the context of the present invention, the term “individual” refers to an animal, preferably a mammal, more preferably a human, that has experienced and / or exhibited at least one symptom associated with cancer. Typically, the individual is a person presenting with cancer and under the clinical care of a physician. The individual may be human or non-human, such that reference to an individual means a Petition 870200158404, dated 12 / 17 / 2020, page 36 / 142 29 / 58 human or non-human, such as an individual of any species of social, economic or research importance including, but not limited to, members of the ovine, bovine, equine, porcine, feline, canine, primate, rodent classifications, especially domesticated members of these classifications, such as sheep, cattle, horses and dogs. Furthermore, as used herein, an “individual in need of such treatment” may additionally be an individual who has not exhibited any symptoms of cancer but who has been considered by a physician, clinician or other medical professional to be at risk of developing cancer. For example, the individual may be considered at risk of developing cancer (and thus in need of preventative or preventive treatment) as a consequence of the individual’s medical history, including, but not limited to, family history, predisposition, coexisting / contributing cancer.

[0089] In the context of the present invention, "pharmaceutically acceptable excipient or diluent" means any excipient or diluent that is not biologically undesirable, that is, the material can be incorporated into a pharmaceutical composition of the present invention and administered to an individual without causing any undesirable or improper biological effects, including, but not limited to, toxicity, incompatibility, instability, irritation, allergic response and the like. In a preferred embodiment, the excipient or diluent is approved by a regulatory agency or body (the regulatory agency or body being, for example, a Federal or State Government agency), or listed in the US Pharmacopoeia or other recognized pharmacopoeia for use in individuals. Brief Description of the Figures

[0090] The embodiments of the invention will now be described only by way of examples, with reference to the accompanying figures, for which captions are provided below: Figure 1. The chemical structure of pyronaridine tetraphosphate (PND). Petition 870200158404, dated 12 / 17 / 2020, page 37 / 142 30 / 58 Figure 2. Transcriptome analysis of possible pathways activated by pyronaridine (PND). Subnetworks for most upregulated genes (marked squares) in response to PND treatment of HL-60 cells. Pearson correlation analysis of the PND transcriptional signature compared to signatures deposited in the LINCS project. The marked compounds are correlated with the effects of PND, and the darker the fill, the better the correlation. Figure 3. Transcriptome analysis of potential pyronaridine (PND)-activated pathways and target validation. (A) Subnetworks for most upregulated genes (tagged squares) in response to PND treatment of MDA-MB-231 cells. Pearson correlation analysis of the PND transcriptional signature compared to signatures deposited in the LINCS project. Tagged compounds are correlated with PND effects, and the darker the fill, the better the correlation. (B) SDS-PAGE analysis reveals that PND caused inhibition of topoisomerase II activity in a dose-dependent manner.Lane 1, catenate kinetoplast DNA (kDNA); Lane 2, decatenate kDNA; Lane 3, linear kDNA; Lane 4, kDNA plus topoisomerase II; Lane 5, kDNA plus topoisomerase plus 5 μM of PND; Lane 6, kDNA plus topoisomerase plus 50 pM of PND; Lane 7, kDNA plus topoisomerase plus 500 pM of PND; Lane 8, kDNA plus topoisomerase II plus 1 pM etoposide (Topo II inhibitor); Lane 9, kDNA plus topoisomerase II plus PBS; Lane 10, kDNA plus topoisomerase II plus DMSO. Figure 4. Studies of PND molecular docking. Predicted interaction of PND amino acid residues and quinacrine in Glycogen Synthase Kinase 3 β (A), and interaction of PND specifically within the predicted binding pocket of GSK3p (B). Figure 5. Cytotoxic Concentration of 50% (CC50) of PND and Selective Cytotoxicity Index (SCI) in a panel of human cell lines. Petition 870200158404, dated 12 / 17 / 2020, pp. 38 / 142 31 / 58 The SCI was calculated as follows: SCI = CC50 of non-cancerous cells / CC50 of cancerous cells. Figure 6. Representative dose-response curves of PND used to determine CC50 values. For these analyses, cells were exposed to PND for 72 h and their viability was determined using the DNS assay. As an example, MDA-MB-231 (A) and HL-60 (B) were treated with a PND concentration gradient as indicated on the x-axis; the y-axis shows the percentage of cytotoxicity (dead cells). In this series of experiments, several controls were incubated: the PND diluent, PBS, as contained in the experimental samples (0.5% v / v); as a negative control, untreated cells; and as a negative control for cytotoxicity, 1 mM H2O2. Each experimental point represents the average of four replicates, and error bars show their corresponding standard deviations. The cytotoxic concentration of 50% (CC50) in micromolar units (μM) is defined as the concentration of PND required to disrupt the plasma membrane of 50% of the cell population after 72 h of incubation. Figure 7. PND induced phosphatidylserine externalization in MDA-MB-231 (A) and HL-60 (B) cancer cells after 24 h of incubation. The cell death mechanism was studied after double labeling of cells with annexin V-FITC and PI and monitored using a cytometer. (AB) The total percentages of cells undergoing apoptosis (y-axis) are expressed as the sum of both the early and late stages of apoptosis (green bars); while cells labeled only with PI, being annexin V-FITC negative, were counted as a population of necrotic cells (black bars). The calculation of two paired two-tailed Student's t-tests of PND-treated cells compared to PBS-treated (*) and untreated ($) control cells provided consistent P < 0.001 values ​​in both circumstances. Each bar represents the triplicate mean and the error bars represent the standard deviation.PND exerted its cytotoxic effect by means of mitochondrial membrane depolarization in MDA-MB-231 (C) and HL- cells. Petition 870200158404, dated 12 / 17 / 2020, pp. 39 / 142 32 / 58 (D). Cells were treated with PND for 6 hours and changes in mitochondrial membrane potential (ΔΨηι) were monitored by labeling with JC-1; a lipophilic fluorochrome aggregate-forming reagent and examined by flow cytometry. The JC-1 reagent emits a green fluorescent signal after mitochondrial depolarization. (CD) The percentages of cells emitting a green fluorescent signal are shown, y-axis, versus different treatments, x-axis. As a positive control for disruptive mitochondrial membrane potential (ΔΨηι), 1 mM H2O2 was used. Each bar represents the mean of three replicates and the error bars represent the standard deviation. Furthermore, analysis of two paired two-tailed Student's t-tests of cells treated with PND, compared with control cells treated with PBS (*) and untreated ($), provided consistent values ​​of P < 0.01 and P < 0.001, respectively. Figure 8. PND disrupted the cell cycle profile of two cancer cells, MDA-MB-231 (AD) and HL-60 (EH), and also exhibited apoptosis-induced DNA fragmentation in a dose-dependent manner. After 72 h of PND treatment, cells were collected in 24-well culture plates, fixed, permeabilized, labeled with DAPI, and analyzed by flow cytometry. Percentages for each of the cell cycle phases are shown along the y-axis, while the different treatments are shown along the x-axis. For this series of experiments, the following controls were involved: 1 mM H2O2 was used as a cell cycle disrupting agent; 0.1% PBS as a control vehicle / solvent; and untreated cells. Each bar indicates an average of three replicates, and the error bars indicate the corresponding standard deviations.For obtaining assay data and analysis purposes, the FL 9 detector, a single-cell gate, and the Kaluza flow cytometry software (Beckman Coulter) were used. Figure 9. PND caused a dose-dependent delay in DNA migration. Three different concentrations of both PND and Petition 870200158404, dated 12 / 17 / 2020, pp. 40 / 142 33 / 58 quinacrine molecules were individually incubated with 100 ng of plasmid DNA, and the potential for complex formation was analyzed by agarose gel electrophoresis. The reaction products were separated by 1% (w / v) agarose gel electrophoresis in Tris / acetate / EDTA buffer and labeled with ethidium bromide. Both the PI and free plasmid DNA were used as positive and negative controls for DNA mobility, respectively. Charge wells are located at the top of the image, indicated by two blue-tipped arrows (upper left and right corners). The dashed yellow line indicates the maximum mobility of the free supermolded DNA used as a reference. Three DNA mobility switches are indicated by the blue lines and arrows (left side of the image). Also, the direction of DNA migration is indicated by an arrow (right side of the image); from the cathode (negative) to the anode (positive).A representative image is shown that is used to review the potential formation of DNA complexes. Figure 10. Calf thymus DNA caused hypochromic and bathochromic effects on the absorbance maxima of PND. UV-Visible spectrophotometric titration (300-650 nm) of PND (22.8 μM) in Tris / HCl buffer after successive additions of calf thymus DNA (10.1 mM). The arrow indicates changes in spectrum when DNA is added. (B) Summary of UV-Visible titration data of the interaction between PND and CT DNA. Figure 11. PND induced an increase in the intensity of the positive and negative bands of the circular dichroism spectrum of calf thymus DNA. Changes in the circular dichroism (CD) spectrum of calf thymus DNA (CT). CT DNA (150 pM) in Tris / HCl buffer was subjected to CD analysis after 30 min (A) or 20 h (B) of incubation with PND in molar ratios of 0.03, 0.06, 0.2, and 0.3. Arrows specify the CD spectrum changes of CT DNA under an increasing gradient of PND concentrations. Blanks in CD spectra with the same gradient of PND concentrations in the absence of CT DNA incubated for 20 h (C). Millidegrees = mdegs. Petition 870200158404, dated 12 / 17 / 2020, pp. 41 / 142 34 / 58 Figure 12. PND treatment of mice resulted in a reduction in tumor growth. Metastatic β breast cancer tumors grew larger on average in control mice (750 mm²) than in PND-treated β mice (430 mm²), and the difference was significant from Day 1 to Day 15, f-test p-values ​​of 0.01. In (A), Days 1-9 are individual bars (left to right) for each mouse. Figure 13. Treatment with PND increases the activity of low-dose chemotherapeutic drugs in MDA-MB-231 breast cancer cell lines. Example 1 - Transcriptome analysis

[0091] A transcriptome analysis was performed to compare the effects of PND with known drug signatures, using different cell lines.

[0092] HL-60 cells (400,000 cells / 2 ml / well in a 6-well plate) were treated with 5.6 μM, or control PBS solvent for 6 hours. RNA was extracted the following day and submitted to whole transcriptome analysis at the University of Texas Genetic Analysis Center facility at El Paso. A total of three biological replicates were conducted for each treatment.

[0093] MDA-MB-231 cells were treated with twice the CC50 concentration of PND, or control PBS solvent, for 6 hours. RNA was extracted the following day and subjected to whole transcriptome analysis.

[0094] In order to explore the possible Mechanism of Action (MoA) of PND, the transcriptome results for each cell line obtained were compared with gene expression data from the LINCS project (http: / / www.lincsproject.org), which contains gene expression profiles of various human cells treated with FDA-approved and withdrawn compounds, as well as different well-characterized chemical reagents. It was hypothesized that the expression profile of each compound should also induce a cellular gene expression consistent with a known MoA of drugs deposited in LINCS. By using this approach, the cell signature Petition 870200158404, dated 12 / 17 / 2020, pages 42 / 142 35 / 58 treated with PND was comparable to each of the 476,251 genomic signature profiles in LINCS. The results shown in Figures 2 and 3A suggest that PND may be a GSK inhibitor and a topoisomerase inhibitor, respectively. The activity of PND as a topoisomerase inhibitor was confirmed using a TopoGen Topoisomerase II Assay Kit, and the results can be seen in Figure 3B. Example 2 - GSK-3 Coupling

[0095] The crystal structure of GSK was obtained from the Protein Data Bank (50Y4) and used to model the interactions with PND and Quinacrine (see Figure 4A). It is shown that PND interacts specifically within the predicted binding pocket of GSK3P (see Figure 4B). The coupling experiments were performed using GLIDE 5.0 within the Schrodinger Software package. The molecular coupling studies revealed that PND can interact with GSK3P with a high coupling score (-9.4 kcal / mol) while SB 216763 (from Example 1; see Figure 2) shows a similar score (-9.2 kcal / mol). Quinacrine showed low binding scores of -7.6 kcal / mol, indicating that the PND side group likely provides additional contacts within the binding pocket (see Figure 4A), allowing it to bind better to GSK-3. Example 3 - Analysis of PND toxicity on human breast cancer and hematological cells

[0096] The drug pyronaridine (PND) is a benzonaphthyridine derivative initially synthesized in 1970 at the Institute of Chinese Parasitic Disease and has been used in China for over 30 years for the treatment of malaria. Previous reports indicated that PND inhibits β-hematin formation, promoting β-hematin-induced red blood cell lysis, based on in vitro studies of Plasmodium falciparum Kl. In addition, PND was previously tested in combination with doxorubicin (DOX) in multidrug-resistant K562 / A02 and MCF-7 / ADR cancer cells and was found to increase the sensitivity of the cells to Petition 870200158404, dated 12 / 17 / 2020, pp. 43 / 142 36 / 58 doxorubicin. However, it has never been determined or suggested that PND has had an effect on its own in the treatment of cancer. Materials and methods Preparation of pyronaridine tetraphosphate (PND)

[0097] A stock solution of pyronaridine tetraphosphate (PND; 2-methoxy-7chloro-10[3,5-bis(pyrrolidinyl-1-methyl-)-4-hydroxyphenyl]aminobenzyl-(b)-1,5naphthyridine; APExBIO, Houston, TX, USA) and dilutions of PND were prepared fresh using Dulbecco's Phosphate Buffered Saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) as solvent. Both the stock solutions and their dilutions were added directly to the wells containing cells in culture medium. The chemical structure of pyronaridine tetraphosphate is shown in Figure 1. Cell lines and culture conditions

[0098] In this study, 19 human cell lines were used: seven from breast cancer; MDA-MB-468, MCF-7, T47D, HCC1419, HCC70, MDAMB-231 (triple negative) and their metastatic lung (LM) derivatives MDAMB-231 LM2; four human leukemia / lymphoma cells, HL-60, Ramos, Jurkat and CEM, three human ovarian cancers, Ovcar 8, Ovcar 5 and Ovcar 3, one lung cancer, one melanoma and one pancreatic cell line, A549, A375, Panc-1, respectively. In addition, for selectivity / comparison purposes, the non-cancerous cell lines MCF-10A, HS-27 were also included. The culture medium used for MDA-MB-231, MDA-MB-231 LM2, MDA-MB-468, MCF-7, A549, A375, Panc-1 and HS-27 was DMEM (Hyclone, Logan UT), while for T47D, HCC1419, HCC-70, HL-60, Ramos, Jurkat, CEM, Ovcar 8 and Ovcar 5 it was RPMI-1640 (Hyclone, Logan UT).Consistently, both DMEM and RPMI culture media were supplemented with 10% heat-deactivated fetal bovine serum (FBS: Hyclone), 100 U / ml penicillin, and 100 pg / ml streptomycin (Thermo Fisher Scientific Inc., Rockford, IL). MCF-10A cells were grown in DMEM / F12 supplemented with 10% FBS and 10 pg / ml insulin. Petition 870200158404, dated 12 / 17 / 2020, pp. 44 / 142 37 / 58 recombinant human insulin (Sigma), 0.5 pg / ml hydrocortisone (Sigma), 20 ng / ml epidermal growth factor, 2.5 mM L-glutamine, 100 U / ml penicillin, and 100 pg / ml streptomycin. Also, a slight modification of the culture medium for the HL-60 and Ovcar 3 cell lines was the addition of 20% FBS (ATCC, Manassas, VA, USA). In addition, Ovcar 3 cells required 10 pg / ml recombinant human insulin (Sigma). Adherent cells, growing in the logarithmic growth phase at a confluence of 60-75%, were detached using HyQtase enzyme (Thermo Fisher Scientific Inc.), counted, and seeded in 96-well plates at a cell density of 10,000 in 100 pL of culture medium per well. Cells growing in suspension were processed similarly to that described above except for the addition of HyQtase. Typically, the incubation conditions for all cells were 37°C in a humidified atmosphere with 5% CO2. Differential Nuclear Labeling Assay to quantify cell death

[0099] In order to analyze the potential cytotoxic activity of PND, the Differential Nuclear Labeling (DNS) assay was used, which has been validated for high-throughput screening (HTS) using live-cell biological imaging. For this assay, cells were seeded at a density of 10,000 cells / well in a 96-well plate in 100 μL of culture medium, incubated overnight, and treated with gradient concentrations of PND for 72 h. Two hours before imaging, two fluorescent nucleic acid intercalators were added to each well, Hoechst 33342 and propidium iodide (PI; Invitrogen); at a final concentration of 1 pg / ml each. Due to its high permeability, Hoechst labels all cells (dead and live), while PI labels only dead or dying cells.2x2 image montages were captured directly from each individual well of the culture plates using an IN Cell 2000 multi-well plate reader analyzer, an HTS and high-content analysis (HCA) system (GE Healthcare Life Sciences, Pittsburgh, PA). Petition 870200158404, dated 12 / 17 / 2020, pp. 45 / 142 The following 38 / 58 controls were included in each single plate: PBS as a control solvent / vehicle, hydrogen peroxide as a positive control for cytotoxicity, and untreated cells to determine the background toxicity due to cell manipulation and intrinsic factors associated with the culture protocol. Each experimental data point, as well as controls, was evaluated in triplicate. The cytotoxic concentration of 50% (CC50) values ​​were calculated based on a linear interpolation equation. CC50 is defined as the concentration of PND required to disrupt the plasma membrane integrity of 50% of the cell population, compared to solvent-treated cells. Calculation of the selective cytotoxicity index

[0100] The selective cytotoxicity index (SCI) indicates the ability of a given experimental compound to kill cancer cells more efficiently while causing minimal toxicity to cells of non-cancerous origin. Thus, the SCI for PND was calculated as follows: SCI = CC50 of non-cancerous cells / CC50 of cancerous cells. Analysis of phosphatidylserine externalization by annexin V / Pl assay

[0101] In order to determine whether PND-induced cell death occurs via apoptosis or necrosis, MDA-MB-231 and HL-60 cells were labeled with annexin V-FITC and PI and monitored by flow cytometry. Cells were seeded in 24-well plates at a density of 100,000 for MDA-MB-231 cells and 200,000 adherent HL-60 cells in 1 ml of culture medium. After overnight incubation, the cells were treated with PND and incubated for a further 24 h. For MDA-MB-231 cells, unattached cells were collected in a chilled tube, while adherent cells were detached using HyQtase (Thermo Fisher) and incubated for approximately 5 min at 37°C. Both unfixed and detached cells collected from each individual well were washed with ice-cold PBS and centrifuged at 260 xg for 5 min.HL-60 cells were centrifuged directly after the incubation period as they grew in suspension. The cells were then labeled. Petition 870200158404, dated 12 / 17 / 2020, pp. 46 / 142 39 / 58 cells were treated with a mixture of annexin V-FITC and PI in 100 μL of binding buffer and incubated on ice in the dark for 15 minutes, following the manufacturer's instructions (Beckman Coulter). Finally, the cells were resuspended by adding 400 μL of ice-cold binding buffer and analyzed by flow cytometry (Cytomics FC500; Beckman Coulter). For this series of experiments, cells were treated with PBS as a control solvent; treated with H2O2 as a positive cytotoxicity control; and untreated cells were included and processed in parallel. For each sample, 10,000 events / cells were collected and analyzed using CXP software (Beckman Coulter). Both experimental samples and their controls were processed and evaluated in triplicate. The sum of the early and late stages of apoptosis was calculated to obtain the percentage of apoptotic cells. Polychromatic analysis of mitochondrial membrane potential

[0102] MDA-MB-231 and HL-60 cells were seeded as described in the previous section and treated with PND for 7 h. After treatment, the cells were collected and labeled with 2 μM of the fluorophore 5,5',6,6'-tetrachlorol,1,3,3'-tetraethylbenzimidazolyl carbocyanine (JC-1) iodide following the manufacturer's instructions (MitoProbe; Life Technologies, Grand Island, NY, USA). Cells with healthy polarized mitochondria favor the formation of JC-1 aggregates that emit a red signal. Cells with depolarized mitochondria exhibit a green signal due to dispersed JC-1 monomers. Controls similar to the above were analyzed concurrently. Data acquisition and analysis were performed using CXP software (Beckman Coulter). Each data point was analyzed in triplicate. Analysis of the transitions between the phases of the cell cycle.

[0103] Asynchronous cultures in the exponential growth phase of MDA-MB-231 and HL-60 cells in 24-well plates were treated with various doses of PND. After 72 h of incubation, the cells were centrifuged and treated as in the previous section, fixed, permeabilized, and labeled with a DNA-intercalating fluorophore, 4,6-diamidino-2-phenylindole (DAPI); these three steps were Petition 870200158404, dated 12 / 17 / 2020, pp. 47 / 142 40 / 58 cells were obtained by adding 200 μL of a single nuclear isolation medium (NIM)-DAPI solution (Beckman Coulter) to the cells. The cell suspension was then incubated for 3 min at room temperature in the dark. The controls included in the series of experiments were similar to those described for the experiments above. Approximately 20,000 events / cells were collected per sample using a flow cytometer equipped with a 405 nm solid-state laser (Gallios; Beckman Coulter). Obtaining and distributing cell subpopulations within each cell cycle was performed using Kaluza cytometry software (Beckman Coulter). Additionally, duplicates were effectively eliminated by including a single-cell gate in the cell cycle acquisition protocol. DNA mobility assay

[0104] Typically, an experimental compound that intercalates or binds to double-stranded DNA (ds) will increase the resulting molecular mass of the complex formation, reducing its electrophoretic mobility on an agarose gel, compared to untreated dsDNA. In order to explore the potential interaction between PND and dsDNA, a DNA mobility exchange assay was conducted. Each reaction mixture was 10 ml total volume in PBS pH 7.4. Both PND (Sigma-Aldrich) and quinacrine (Sigma-Aldrich) were tested at three individual concentrations of 1 μM, 0.5 μM, or 0.25 pM, respectively. To each reaction mixture, 100 ng of plasmid dsDNA (pCMV-dR8.91; Addgene, Cambridge, MA) were added, and the mixture was then incubated for 30 min at 37°C and stopped by the addition of 2 ml of 6X gel loading buffer and placed on ice.The potential binding interaction between both PND and quinacrine with dsDNA was analyzed using electrophoresis on a 1% (w / v) agarose gel dissolved in TAE buffer (0.04 M Tris base, 0.04 M acetate, and 0.001 M EDTA) pH 8.0. To label the dsDNA complexes, ethidium bromide was added to the agarose gel throughout the electrophoresis process at a concentration of 0.5 pg / ml. DNA migration was visualized using a gel documentation system. Petition 870200158404, dated 12 / 17 / 2020, pp. 48 / 142 41 / 58 photographs were captured using a trans-UV light illuminator (Alpha Innotech, San Leandro, CA). The well-known fluorescent DNA intercalating compound, propidium iodide (PI), was included as a positive control at 1 pg per reaction. As a control, untreated plasmid DNA was included to determine its normal electrophoretic mobility in the gel. Analysis of PND interaction with calf thymus DNA by UV-Visible spectroscopy

[0105] UV-Visible measurements were obtained on a Varian Cary 100 spectrophotometer. Calf thymus DNA (CT) and buffers were purchased from Sigma Aldrich. The binding constant of PND with CT DNA was determined by absorption titration at room temperature by the stepwise addition of a CT DNA solution (10.1 mM; 5 μL additions) in buffer (5 mM Tris / HCl, 50 mM NaCl, pH = 7.39) to a working solution of 2 mL of PND (22.8 μM) in the same buffer. Absorption spectra were recorded at 424 nm and the titration was terminated when saturation was reached. In order to determine the binding affinity, the data were fitted to the Scatchard equation r / Cf = K(nr) (McGhee and von Hippel plots), where r is the number of moles of PND bound to 1 mol of CT DNA, n is the number of equivalent binding sites, and K is the affinity of the complex for these binding sites.The concentrations of free (Cf) and bound (Cb) complexes were calculated from Cf = C(a) and Cb = C-Cf, respectively, where C is the total concentration of PND. The fraction of the bound complex (a) was calculated from α = (Af-A)Z(Af-Ab), where Af and Ab are the absorbances of the free and fully bound drug at the selected wavelengths and A is the absorbance at any given point during the titration. Plotting r / Cf vs. r gives the binding constant Kb as the curve in graph

[16] . All experiments were performed in triplicate and the Kb values ​​were averaged. Analysis of PND interaction with calf thymus DNA using circular dichroism spectroscopy. Petition 870200158404, dated 12 / 17 / 2020, pp. 49 / 142 42 / 58

[0106] Circular dichroism (CD) spectra measurements were obtained using a JASCO-1100 spectropolarimeter equipped with a xenon lamp (JASCO, Easton, MD). CT DNA and buffers were purchased from Sigma Aldrich. A stock solution of CT DNA was prepared in Tris / HCl buffer (5 mM Tris / HCl, 50 mM NaCl, pH = 7.39) and its concentration (4.325 mM) was determined by spectrophotometry using a molar excitation coefficient of 6600 mL cm⁻¹ at 260 nm. A 150 μM dilution in Tris / HCl buffer was prepared and used for the experiments. A 2.0 mM PND stock solution was freshly prepared in MQ water prior to use. The appropriate volume of this solution was added to 3 ml of 150 μM CT DNA working solutions to obtain molar ratios of 0.03, 0.06, 0.2, and 0.3 of PND / DNA. Samples were prepared in triplicate and incubated for 30 minutes and 20 hours.All DNA and DNA / PND CD spectra were recorded at 25°C over a range of 205-380 nm and finally corrected with a blank and noise reduction. The final data are the average of three experiments and are expressed in millidegrees (mdegs). Statistical Analysis

[0107] For each data point, triplicate means and their corresponding standard deviations are recorded. Statistical significance was determined using two-tailed paired Student's t-tests, and a P-value of < 0.05 was considered significant. Results PND exhibits potent and selective toxicity to cancer cell lines.

[0108] The cytotoxic effects of PND were analyzed using live-cell imaging via differential nuclear labeling (DNS) assay in eleven human cancer cell lines and non-cancerous control cell lines (MCF-10A; Figure 5). For each individual cell line, dose-dependent response curves were created, also using the DNS assay, to determine the CC50 of PND. Petition 870200158404, dated 12 / 17 / 2020, page 50 / 142 43 / 58 in these cell lines. In general, PND exhibited potent cytotoxicity on tested cells with consistent CC50 values ​​at low micromolar concentrations ranging from 1.6 μM to 9.4 μM (Figure 5). As shown in Figure 6A-B, the effects at various concentrations of PND on the triple-negative breast cancer cell line MDA-MB-231 and the acute promyelocytic leukemia cell line HL-60 revealed that PND exhibited CC50 values ​​of 1.6 pM and 1.9 pM, respectively (Figure 6A-B). In these and other assays, untreated cells were used to define baseline levels of cell death by cell manipulation and cell culture. Cells treated with solvent were also used as a control for non-specific cell death and for normalization purposes, and cells treated with H2O2 were used as a positive control for cytotoxicity (Figure 6).PND showed a significant selective cytotoxicity index (SCI) in four of six breast cancer cell lines tested: MDA-MB-231, MDA-MB-231 LM2, MDA-MB-468, and MCF-7, with SCI values ​​of 4.13, 2.54, 3.88, and 4.13, respectively, compared to non-cancerous breast cells MCF-10A (Figure 5). Interestingly, SCI values ​​(<1) were not favorable in T47D and HCC-70, with values ​​below 1 (Figure 5). In addition, the highest SCI value in leukemia / lymphoma cells tested corresponded to the HL-60 cell line, with an SCI value of 3.5 (Figure 5). Additionally, PND showed an SCI value of 3 and 3.3 for Ramos and Jurkat cells, respectively, but low selectivity was observed for the CEM cell line (Figure 5). Good selectivity was detected in a melanoma cell line (SCI = 3.2) and in two of the three ovarian cancer cell lines tested (SCI = 3.9).However, low selectivity (< 2.0) was detected in pancreatic and lung cancer cell lines (Figure 5). Since PND exhibited low CC50 values ​​and showed significant selectivity (SCI > 3) in both the MDAMB-231 and HL-60 cell lines, both were selected for further analysis. Petition 870200158404, dated 12 / 17 / 2020, pp. 51 / 142 44 / 58 PND triggers the externalization of phosphatidylserine in both MDA-MB231 and HL-60 cells.

[0109] In order to determine whether PND induces cytotoxicity through apoptosis or necrosis, cells were treated with two different concentrations of PND for 24 h, 34 μM and 68 μM for HL-60 and 11 pM and 22 pM for MDAMB-231. Subsequently, the cells were labeled with annexin V-FITC and PI and analyzed by flow cytometry. It was found that PND induces significant phosphatidylserine (PS) externalization in both cell lines compared to positive and negative controls (P < 0.001; Figure 7A-B). PND induced significant PS externalization in HL-60 cells in a dose-dependent manner, showing 14.8% and 30.2% apoptotic cells at 34 pM and 68 pM, respectively (P = 0.0033, Figure 7A). Additionally, PND induced a higher percentage of PS externalization in MDA-MB-231 cells than in HL-60 cells at both concentrations tested, with 67.2% and 71.1% annexin-positive cells at all pM and 22 pM, respectively (Figure 7B).As expected, both solvent-treated and untreated cells did not exhibit any significant increase in apoptotic or necrotic death (Figure 7A-B). Furthermore, H2O2 induced its cytotoxic effect through apoptosis and necrosis in MDA-MB-231 and HL-60 cells, respectively (Figure 7A-B). Thus, PND induced PS externalization in both MDA-MB-231 and HL-60 cells, which is a well-known early event in apoptosis activation. PND induces mitochondrial depolarization in cancer cells.

[0110] An early biochemical event that triggers the intrinsic apoptosis pathway is mitochondrial depolarization, which can be quantified using a polychromatic JC-1 reagent and flow cytometry. JC-1 emits a red or green fluorescent signal when mitochondria are polarized or depolarized, respectively. Consequently, both MDA-MB231 and HL-60 cells were incubated for 6 h with PND and the mitochondrial membrane potential status (ΔΨηι) was recorded. As expected, Petition 870200158404, dated 12 / 17 / 2020, pp. 52 / 142 Based on PS externalization data (45 / 58), both cancer cells treated with PND showed significant mitochondrial depolarization compared to untreated or solvent-treated cells (Figure 7C-D). These results indicate that PND is capable of inducing mitochondrial depolarization in both types of cancer cells, further indicating that PND induces cell death via the intrinsic apoptosis pathway. PND disrupts the cell cycle profile and shows DNA fragmentation in MDAMB-231 and HL-60 cells.

[0111] In order to examine how MDA-MB-231 and HL-60 cells proliferate in the presence of PND, the cell cycle distribution profile was examined using flow cytometry (Figure 8). In order to determine the effects of PND on cell cycle progression, a strategy was used to quantify the cellular DNA content that is dependent on the violet light-excited DNA intercalation fluorophore, D API (4',6diamidino-2-femlmdol). After treatment of MDA-MB-231 cells with PND, a significant reduction in the G0 / G1 cell subpopulation was observed; however, this effect was not observed in HL-60 cells (Figure 8B and F). PND reduced the S and G2 / M subpopulations in both MDA-MB-231 and HL-60 cells compared to PBS and untreated controls (Figure 8B-D and FH).Additionally, PND induced significant DNA fragmentation in a concentration-dependent manner in both cancer cell lines, as indicated by a significant increase in the sub-GO / Gl subpopulation (P < 0.0005; Figure 8A and E). The differences in the percentages of both cells at each cell cycle phase between PBS-treated and untreated cells were essentially indistinguishable. These experiments revealed that PND disrupted the cell cycle profile distribution and induced DNA fragmentation (sub-G0 / Gl population) in both cancer cell types. PND interacts directly with dsDNA. Petition 870200158404, dated 12 / 17 / 2020, page 53 / 142 46 / 58

[0112] The potential interaction between PND and dsDNA was examined using a DNA mobility exchange assay using plasmid DNA as a binding substrate, and compared with that of quinacrine, a compound with comparable structure, and a well-known DNA intercalator. When 1 mM PND was incubated with DNA, a marked reduction in the migration of PND-treated DNA was observed compared to free plasmid DNA (exchanges are indicated on the left side of Figure 9). Approximately half of the total incoming DNA was located in the loading well with minimal mobility in the agarose gel. Furthermore, when 0.5 and 0.25 mM PND were incubated with DNA, there was a clear reduction in mobility (observed on the left side of Figure 9). Additionally, treatment with PND did not result in DNA degradation based on the absence of DNA fragments smaller than free supercoiled DNA (Figure 9).Previous studies also found that quinacrine, with a chemical structure similar to PND, interacts with DNA by intercalation. As shown in Figure 9, quinacrine caused the maximum retardation of DNA mobility at the highest concentration tested (1 mM), as evidenced by a stain, indicative of complexes with supercoiled DNA. Quinacrine also caused a clear retardation of DNA mobility, similar to PND, when tested at 0.5 and 0.25 mM (Figure 9). As was the case with PND-treated DNA, quinacrine did not exhibit any DNA degradation activity (Figure 9). PI, which was used as a positive control for DNA binding, also caused a retardation of DNA mobility. Our results clearly indicate that PND can interact directly with DNA, causing a change in its mobility in agarose gels, and the quinacrine-like behavior may suggest that PND also has the ability to intercalate between the bases of dsDNA. PND intercalates with DNA as determined by UV-Visible spectrophotometry titration.

[0113] In order to further prove the interaction with PND intercalation with CT DNA, spectrophotometric titrations were performed on Petition 870200158404, dated 12 / 17 / 2020, pp. 54 / 142 47 / 58 Tris / HCl buffer. PND shows strong absorption bands in the 300-500 nm region, typical of transitions between electron energy levels of conjugated aromatic rings. In general, the hypochromic and bathochromic effects observed in the UV-Visible absorbance maxima can be taken as evidence of stacking interactions between conjugated aromatic systems that intercalate the nucleobases of DNA. Figure 10A shows the absorption spectra of PND in the region studied by consecutive additions of CT DNA. The absorbance maximum at 424 nm was studied for the diagnosis of the compound-DNA interaction. Our results show a significant hypochromic effect (39%) and a shift to red at 8 nm. In addition, the Scatchard equation was used to determine the binding affinity [8.5 ± 0.7 x 10⁵ Ml] of PND to CT DNA.All PND binding data with CT DNA, shown in Figure 10B, are comparable to those of well-known intercalating agents with similar structure and indicate that PND is also capable of intercalating between DNA bases, as suggested by the mobility exchange assay. PND stabilizes the B conformation of CT DNA as observed by circular dichroism spectroscopy.

[0114] In addition, detailed conformational changes in DNA were studied using circular dichroism spectroscopy in Tris / HCl buffer. A typical CD spectrum of CT DNA in its B form shows a positive band with a maximum of 275 nm due to base stacking and a negative band with a minimum of 248 nm due to right-hand helix. Thus, changes in CD signals can be associated with corresponding changes in the secondary structure of DNA. Figure 11, A and B, shows the CD spectrum of CT DNA, and the effect of treatment with increasing amounts of PND for 30 minutes and 20 hours, respectively. Our results show that PND was able to increase the intensity of both the negative and positive bands in a concentration-dependent manner, but without significant red shifts in either. Petition 870200158404, dated 12 / 17 / 2020, pp. 55 / 142 48 / 58 These results are comparable to previously reported similar compounds and suggest that PND is capable of stabilizing the right-facing B form of DNA without significant conformational changes. The fact that the same spectral changes were observed for 30-minute and 20-hour incubations suggests that the type of interaction occurs within a few minutes, confirming the intercalated mode of binding. In addition, a positive signal appeared in the 300-340 nm range, a region where DNA does not absorb light, suggesting that some asymmetric alteration is possibly being induced in PND by binding to CT DNA, since no signal for PND is observed in the absence of nucleic acid (see Figure 10, C). Discussion

[0115] In all cancer cell lines analyzed, treatment with PND was found to cause cell death at low micromolar concentrations (from 1.6 μM to 9.4 μM). Apart from being less toxic to non-cancerous cells (MCF-10A; CC50 of 6.6 pM), PND showed favorable selectivity in several breast cancer cells compared to non-cancerous cells, with an SCI value >2.5. PND also exhibited favorable SCI values ​​(from 1.43 to 3.47) in leukemia / lymphoma cells, showing the highest selectivity for HL-60 cancer cells.

[0116] The mode of action of PND was analyzed in both MDA-MB-231 and HL-60 cancer cell lines. After exposure to a toxic agent, cells can undergo two main pathways: necrosis or apoptosis. Phosphatidylserine (PS) is preferentially located on the inner side of the plasma membrane leaflet, facing the cytosol, and when cells initiate the apoptosis pathway, it is translocated to the outer leaflet of the plasma membrane, which is a biochemical marker of apoptosis. Flow cytometry identified this apoptotic facet when using propidium iodide and annexin V conjugated with FITC, which has a high affinity for PS. MDA-MB-231 and HL-60 cells treated with PND exhibited Petition 870200158404, dated 12 / 17 / 2020, pp. 56 / 142 49 / 58 consistently externalizing PS, suggesting that PND utilizes the apoptosis pathway to inflict its cytotoxicity in a dose-dependent manner.

[0117] Furthermore, apoptosis can be initiated through intrinsic or extrinsic biochemical pathways. A critical biochemical event that triggers the activation of the intrinsic pathway is through mitochondrial depolarization. Thus, MDA-MB-231 and HL-60 cells were exposed to PND and labeled with the polychromatic reagent JC-1 to investigate whether mitochondrial depolarization was involved in their mechanism for inducing cell death. Our data clearly indicate that both cancer cell lines exhibited significant mitochondrial depolarization after PND exposure, indicating that the intrinsic pathway of apoptosis is involved in their cytotoxic mechanism.

[0118] A successful strategy for studying cell cycle profiling is based on quantifying cellular DNA content using flow cytometry. When using this approach, it is possible to distinguish three phases of the cell cycle: G0 / G1, S, and G2 / M. In addition, regardless of which initiation signal is used to activate the apoptotic cascade, the death signal ultimately results in DNA fragmentation; a late biochemical marker of apoptosis. When performing cell cycle analysis using flow cytometry, cells undergoing DNA fragmentation are easily identified by the presence of a sub-GO / G1 subpopulation, a distinct peak located on the left side of the cell cycle histograms. Thus, the occurrence of apoptosis-induced DNA fragmentation, as well as the cell cycle, was analyzed concomitantly after incubating the cells with PND for 72 h.These assays revealed that PND was able to disrupt the progression of the cell cycle profile in MDA-MB-231 and HL-60 cells, detecting a dissimilar pattern in each cell lineage. In addition, PND caused consistent DNA fragmentation in both MDA-MB-231 and HL-60 cells in a dose-dependent manner, confirming previous results that PND induces apoptosis. Petition 870200158404, dated 12 / 17 / 2020, page 57 / 142 50 / 58

[0119] The interaction between DNA and drugs or proteins can be easily detected by the retardation of DNA migration during the mobility exchange assay by gel electrophoresis. In these DNA binding assays, experimental compound-DNA complexes migrate more slowly than free DNA (uncomplexed control DNA), which results from the fact that DNA has a higher molecular weight. Additionally, when incubating a chemical compound with plasmid DNA, it is possible to detect any deleterious effects resulting in DNA degradation or fragmentation. The results indicate that PND interacts directly with DNA since it causes significant DNA retardation in agarose gels, in a concentration-dependent manner, but does not cause DNA degradation. The intercalated mode of PND DNA binding was confirmed by UV-Visible spectrophotometric titration, as well as CT DNA circular dichroism.In addition, CD spectra provided evidence of stabilization of CT DNA in its B form without conformational changes detectable by interaction with PDN. Collectively, the mobility exchange assay, UV-Visible series, and CD experiments provide compelling evidence that PDN links DNA by intercalation with DNA nucleobases.

[0120] In this preclinical study, the findings indicate that PND shows potent cytotoxicity, with low micromolar CC50 values ​​and a favorable selective cytotoxicity index, in relation to a panel of human cancer cells, showing significant selectivity against triple-negative MDA-MB-231 breast cancer cells (a class of aggressive tumors) and HL-60 leukemia cells. PND and related compounds and derivatives exhibit activity as anticancer drugs, since PND markedly and consistently shows its cytotoxic effect by activating the apoptosis pathway, as evidenced by PS externalization, mitochondrial depolarization, DNA fragmentation, and cell cycle interference. Furthermore, PND was found to interact directly with DNA, causing DNA retardation during agarose gel electrophoresis and spectral alterations. Petition 870200158404, dated 12 / 17 / 2020, pp. 58 / 142 51 / 58 in the UV-Visible profile of PND, as well as in the CD spectra of DNA, suggesting an intercalated mode of binding. Example 4 - Analysis of PND activity in metastatic breast cancer in mice Materials and Methods

[0121] 12 mice comparable to metastatic breast cancer cells of the MDA-MB-231 LM2-LUC4 cell line were injected with either an aqueous solution of pyronaridine (PND) or with placebo (water) daily using a force-feeding technique. Treatment was initiated when the tumors reached 150–300 mm in size (i.e., Day 1).

[0122] In the PND-treated group (n = 6), all 6 mice received 160 mg / kg from Day 1 to Day 9. Mice treated with PND numbers 2, 4, and 5 received 160 mg / kg throughout the study, while mice treated with PND numbers 1, 3, and 6 had the largest tumors in the group on Day 9 and had their doses increased on Day 10 to 240 mg / kg and again on Day 13 to 320 mg / kg. Control mice (n = 6) received placebo throughout the study. The size of each tumor was measured daily. The endpoint of the protocol was when a tumor reached β 1500 mm in size, triggering the euthanasia of the mouse. For the analysis, the significance level was set at P < 0.05. Results

[0123] A comparison of tumor sizes from Day 1 to Day 9 showed that on average the tumors grew larger in control mice (750 mm²) than in PND-treated mice (430 mm²). The difference was significant, f-test p-value of 0.01 (see Figure 12A). A comparison of tumor sizes from Day 1 to Day 15 (when all 12 mice were alive) showed that, on average, the tumors grew larger in the placebo group than in the PND-treated group, and the difference was significant, f-test p-value of 0.0001 (see Figure 12B). Petition 870200158404, dated 12 / 17 / 2020, pp. 59 / 142 52 / 58

[0124] There was a significant difference in survival duration favoring the group treated with PND, although, after Day 15, comparisons became complicated due to increased doses in the 3 mice treated with PND and due to euthanasia of the placebo mice.

[0125] During the study, no negative adverse events were reported except for discomfort in walking after Day 15. All 6 mice treated with PND showed yellowing of the eyes, skin, ears, and paws around Day 20, likely due to PND being a strong yellow pigment. No weight loss, feeding disturbances, or hygiene problems were observed. The 6 PND-treated mice were dissected, and no signs of PND accumulation were observed anywhere, including the stomach, intestines, or liver.

[0126] The results clearly demonstrated that in mice treated with PND, metastatic breast cancer tumors grew less than in mice that received placebo. Example 5 - Analysis of PND activity in conjunction with chemotherapy. Materials and Methods.

[0127] PND was added to MDA-MB-231 breast cancer cell line cultures 24 hours after the cells were treated with a selection of known commercial chemotherapeutic drugs, before the cells were analyzed for cell death. Each of the drugs was known to have serious side effects at doses commonly administered for cancer treatment. The dose of the chemotherapeutic drugs was reduced to half, one-quarter, one-eighth, and one-sixteenth of the initial dose; while the PND dose was kept the same. The effect of these combination therapies was measured by the concentration of each compound required to be cytotoxic to at least 50 percent of the cancer cells (CC50). For cisplatin, gemcitabine, bortezomib, and MG132, the initial dose (CC50) was 95.42 μM, 0.44 Petition 870200158404, dated 12 / 17 / 2020, pp. 60 / 142 53 / 58 μM, 0.01 μM and 0.5 μM, respectively, while the PND dose was kept constant at 5.5 μM. Results

[0128] The results shown in Figure 12 demonstrate that co-administration of PND (where PND is administered after the chemotherapeutic drug) can greatly reduce the amount of drug required to effectively kill cancer cells. Example 6 - Treatment of late-stage canine cancer with PND Materials and Methods

[0129] An open-label study was conducted to evaluate the efficacy in increasing longevity and to assess the safety of oral pyronaridine tetraphosphate (PND) in five canine patients with terminal cancer.

[0130] Eligible patients had a malignancy that was metastatic or not surgically removable and for which standard curative or palliative measures either did not exist or were no longer effective. Due to ethical considerations regarding the treatment of terminally ill dogs, there was no placebo group.

[0131] PND was obtained from Mangalam Drugs and Organics, Ltd., and encapsulated in gelatin capsules for twice-daily oral administration.

[0132] The primary outcome assessed was the difference between the patient's expected survival duration at the time of the study and their actual survival. Secondary outcomes were: antitumor activity, relief of symptoms associated with malignancy, and the safety and tolerability of PND.

[0133] The study on cisco dogs was as follows:

[0134] Case 1, Mast cell carcinoma: Dog (Golden Retriever), female, 6 years old, 28.7 kg. Cancer was confirmed by histopathology report. In mid-May 2019, oral PND administration was initiated in the dog, after which the patient did not receive any other cancer medication. The veterinarian expected a survival duration of between 4 and 6 months at the time of study entry. The patient received 23 mg / kg / day of PND (325 mg in the morning and 325 mg at night). Petition 870200158404, dated 12 / 17 / 2020, pp. 61 / 142 54 / 58

[0135] Case 2, Osteosarcoma: Dog (Saint Bernard), male, 5 years old, 49.7 kg. Oral PND administration was initiated on May 17, 2019, after which the patient did not receive any other cancer medication. The veterinarian expected a survival duration of between 4 and 6 months at the time of study entry. The patient received 20 mg / kg / day of PND (325 mg in the morning and 650 mg at night).

[0136] Case 3, Sickle Cell Sarcoma: Dog (Border Collie), male, 9 years old, 28.5 kg. In 2014 the dog was diagnosed with low-grade sickle cell sarcoma and euthanasia was scheduled for February 11, 2019, with a life expectancy of 1-2 weeks. Instead, the veterinarian initiated oral PND administration on February 11, 2019, after which the patient did not receive any other cancer medication. The patient received 15 mg / kg / day of PND (215 mg in the morning and 215 mg at night).

[0137] Case 4, Non-Hodgkin lymphoma: Dog (Boxer), female, 27 kg, 11 years old. Cancer was confirmed by biopsy report. In December 2018, oral PND administration was initiated for the dog, after which the patient did not receive any other cancer medication. The patient received 15 mg / kg / day of PND (210 mg in the morning and 210 mg at night). However, PND administration was interrupted in February 2019 (due to shipping problems from the USA to Canada) and never resumed.

[0138] Case 5, mast cell tumor: Dog, male, 27.9 kg. Cancer was confirmed by histopathology report. A tumor was surgically removed from the tongue and there was no clean border. In December 2018, oral PND administration was initiated, after which the patient did not receive any other cancer medication. The patient received 15 mg / kg / day of PND (210 mg in the morning and 210 mg at night). Results

[0139] Case 1: There were no reports of adverse events in the study drug. The volumes of four tumors were measured at study entry and compared with follow-up measurements. Nodule Petition 870200158404, dated 12 / 17 / 2020, pp. 62 / 142 Right mandibular nodule (55 / 58) measuring 2.5 cm x 2 cm versus 2 cm x 1 cm; left mandibular nodule measuring 3 cm x 2 cm versus 2.2 cm x 1 cm; right popliteal nodule (back of the knee) measuring 0.5 cm x 0.5 cm versus 0.2 cm x 0.2 cm; and left popliteal nodule measuring 0.5 cm x 0.5 cm versus 0.2 cm x 0.2 cm. The dog was still alive, and the relief of the symptom associated with the malignancy allowed for daily activities starting in June 2019. In all cases, follow-up measurements showed a clinically significant reduction in tumor size.

[0140] Case 2: The only reported adverse events possibly related to the study drug were drooling, facial swelling, and vomiting, which resolved with diphenhydramine. The tumor size at study entry was 3.7 cm x 2.6 cm. As of June 2019, the dog was still alive and in good condition, as symptom relief associated with the malignancy allowed for daily activities.

[0141] Case 3: There were no reports of adverse events associated with the study drug except for initial vomiting, which was controlled. The volumes of three tumors were measured at study entry: 2 cm x 2.5 cm; 3 cm x 3.8 cm; and 3 cm x 4 cm. At follow-up, the volume of each tumor was reported to have reduced by approximately 20%. In mid-May 2019, the dog was euthanized; the dog's survival was extended by 3 months.

[0142] Case 4: There were no reports of any adverse events related to the study drug. The volume of a tumor in the patient's neck was measured at the time of study entry and compared with follow-up measurements. The tumor shrank by 70% by March 2019. On April 20, 2019, the dog was euthanized; the dog's survival was extended by 2.5 months.

[0143] Case 5: The patient presented with enlargement of the left mandible. The volume of a tumor was measured at the time of study entry and compared with follow-up measurements. The tumor remained the same size: 2 cm x 1 cm versus 2 cm x 1 cm. The dog was still alive and the relief of Petition 870200158404, dated 12 / 17 / 2020, pp. 63 / 142 56 / 58 symptom associated with malignancy allowed daily activities starting in June 2019. Example 7 - Treatment of late-stage cancer in humans with PND Materials and methods

[0144] An open-label, dose-escalation study was conducted to evaluate the longevity and safety of oral pyronaridine tetraphosphate (PND) in five terminally ill human patients with selected cancers. Eligible patients had a malignancy that was metastatic or not surgically removable and for which standard curative or palliative measures were no longer available or effective. Exclusion criteria included clinically significant active infection, uncontrolled intercurrent disease, neutropenia, and pregnancy.

[0145] PND was obtained from Mangalam Drugs and Organics, Ltd, and the potentiated PND was encapsulated in size “00” gelatin capsules for oral administration. Each capsule contained either 125 mg of PND and microcrystalline cellulose as an inert filler (MCC) or 200 mg of PND (without filler).

[0146] Before study entry and before each dose increase, the patient's complete blood count (CBC) and complete metabolic panel (CMP) laboratory results were checked to ensure they were within normal reference ranges.

[0147] Patients were instructed to increase their dose if there was no unacceptable toxicity. Study Group 1 started with a daily dose of 250 mg for 2 weeks, then 375 mg for 2 weeks, then 500 mg onwards. Study Group 2 started with a daily dose of 200 mg for 2 weeks, then 400 mg for 2 weeks, then 600 mg onwards. Due to ethical concerns regarding the treatment of terminally ill patients, there was no placebo group.

[0148] The primary outcomes assessed were the difference between the patient's estimated expected survival time at the time of admission to Petition 870200158404, dated 12 / 17 / 2020, pp. 64 / 142 57 / 58 study (estimated at 2-4 weeks for each patient) and their actual survival duration. Secondary outcomes were symptom relief associated with malignancy, antitumor activity, safety, and tolerability of PND.

[0149] The study on the five humans was as follows:

[0150] Case 1, Male, age 65, Klatskin hepatic biliary tract (or hilar cholangiocarcinoma) diagnosed in March 2018. On December 20, 2018, the patient began taking PND as per protocol, 2 capsules / day.

[0151] Case 2, Male, age 43, lung cancer, stage IV, diagnosed in May 2017. The patient received chemotherapy with the last cycle in December 2018 and on January 22, 2019, began taking PND, 2 capsules / day.

[0152] Case 3, Woman, age 55, ovarian cancer, stage IV, diagnosed in June 2017. The patient received chemotherapy with the last cycle in November 2018 and on January 15, 2019, began taking PND, 2 capsules / day.

[0153] Case 4, Woman, age 54, breast cancer, stage IV, diagnosed in September 2017. On January 20, 2019, the patient began taking PND, 2 capsules / day.

[00154] Case 5, Woman, age 40, small cell lung cancer, stage IV, diagnosed in April 2017. The patient received chemotherapy with the last cycle in December 2018 and on January 29, 2019, began taking PND, 2 capsules / day. Results

[0155] Case 1: There were no reports of any adverse events related to the study drug. In June 2019, the patient was still alive, his most recent BCC and PMC results were within normal reference ranges, and his relief of the symptom associated with the malignancy allowed him to resume daily activities. Petition 870200158404, dated 12 / 17 / 2020, pp. 65 / 142 58 / 58

[0156] Case 2: There were no reports of adverse events related to the study drug. The patient died on February 22, 2019, from lung cancer.

[0157] Case 3: There were no reports of adverse events related to the study drug. The patient died on January 29, 2019, from ovarian cancer.

[0158] Case 4: The patient died on February 15, 2019 from breast cancer.

[0159] Case 5: The only reported adverse event related to the study drug was skin irritation during the first week of treatment that resolved within a week, possibly related to the study drug. In 2019, the patient was still alive, her most recent BCC and PMC results were within normal reference ranges, and her relief of the symptom associated with the malignancy allowed her to resume daily activities.

[0160] PND has been shown to be well tolerated (the maximum tolerated dose was not reached for any patient) and, having extended the lives of 2 in 5 cancer patients, there is justification for adequate and well-controlled studies leading to an approved PND-based anticancer drug that increases survival rates without serious side effects. Petition 870200158404, dated 12 / 17 / 2020, pp. 66 / 142

Claims

1 / 3 CLAIMS 1. Use of a composition comprising pyronaridine (PND) or a pharmaceutically acceptable salt thereof as a DNA binder and protein binder, characterized in that it is used in the preparation of a medicament to induce apoptosis in proliferating cancer cells for the treatment or prevention of cancer in a mammal.

2. Use according to claim 1, characterized by being in the preparation of a medicament for the treatment or prevention of cancer.

3. Use according to claim 1 or claim 3, characterized in that PND, as a protein ligand, inhibits a protein kinase selected from the group consisting of MEK, Raf, ERK, PI3K, Akt, PDK1, GSK-3, Weel, Mytl, CHK1, CHK2, ATR, ATM, mTOR, CDK2, PIP5K, RIPK3, RIPK1, TAKI, FADD and PCK, and / or isoforms thereof.

4. Use in accordance with any of claims 1 to 3, characterized in that the cancer or tumor is a carcinoma selected from the group consisting of pancreatic, bladder, breast, colon, mesothelioma, kidney, liver, lung, including small cell lung cancer, non-small cell lung cancer, head and neck, esophagus, gallbladder, ovary, stomach, cervical, thyroid, prostate or skin carcinoma, or a sarcoma selected from the group consisting of fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, leiomyosarcoma, mesothelioma, angiosarcoma, liposarcoma, or a tumor of the central and peripheral nervous system selected from the group consisting of astrocytoma, neuroblastoma, glioma and schwannomas, or other tumors selected from the group consisting of melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctantoma, follicular thyroid cancer and sarcoma of Kaposi,or a liquid cancer (blood cancer) selected from the group consisting of B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, Burkett lymphoma or an extranodal lymphoma of the stomach, breast or brain, myeloma, and extranodal lymphomas of the stomach, plasma cell myeloma, Kahler's disease, multiple myeloma, myelogenous leukemia, granulocytic leukemia, lymphatic leukemia, lymphocytic leukemia and lymphoblastic leukemia. Petition 870260083311, dated 08 / 17 / 2026, p. 9 / 13 2 / 3, 5. Use according to claim 4, characterized in that the cancer is breast cancer.

6. Use according to claim 4, characterized by the fact that it is a serum lymphoma cancer.

7. Use according to any of the preceding claims, characterized in that the composition further comprises a therapeutically effective amount of a chemotherapeutic drug selected from the group consisting of abiraterone acetate, albumin-bound paclitaxel (nab), alemtuzumab, altretamine, asparaginase, bendamustine, bevacizumab, bleomycin, bortezomib, brentuximab vedotin, busulfan, cabazitaxel, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, crizotinib, cyclophosphamide, cytarabine (Ara-C), dacarbazine, dactinomycin, dasatinib, daunorubicin, daunoXome (liposomal daunorubicin), DepoCyt (liposomal cytarabine), docetaxel, doxil (liposomal doxorubicin), Doxorubicin, Eribulin mesylate, Erlotinib, Estramustine, Etoposide, Everolimus, Floxuridine, Fludarabine, Fluorouracil, Gefitinib, Gemcitabine, Gliadel wafers, Hydroxyurea, Ibritumomab, Ibritumomab, Idarubicin, Ifosfamide, Imatinib, Ipilimumab,Irinotecan, Ixabepilone, Lapatinib, Lenalidomide, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mioxantrone, MG132, Nilotinib, Oxaliplatin, Paclitaxel, Panitumumab, Pazopanib, Peginterferon alfa2b, Pemetrexed, Pentostatin, Pralatrexate, Procarbazine, Rituximab, Romidepsin, Ruxolitinib, Sipuleucel-T, Sorafenib, Streptozocin, Sunitinib, Temozolomide, Temsirolimus, Teniposide, Thalidomide, Thioguanine, Thiotepa, Topotecan, Tositumomab, Trastuzumab, Valrubicin, Vandetanib, Vemurafenib, Vinblastine, Vincristine, and Vinorelbine, or a combination thereof.

8. Use according to claim 7, characterized in that the chemotherapeutic drug is selected from the group consisting of bortezomib, cisplatin, gemcitabine and MG132.

9. Use according to claim 8, characterized in that the chemotherapeutic drug is Cisplatin.

10. Use according to claim 8, characterized by the fact that the chemotherapeutic drug is Gemcitabine. Petition 870260083311, dated 08 / 17 / 2026, page 10 / 13

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