Combination of chemotherapeutic agent and alpha-lactalbumin-oleic acid complex for the treatment of cancer

Abstract

To provide a combination chemotherapy exhibiting a large therapeutic effect on cancer, especially bladder cancer.SOLUTION: There is provided a first chemotherapeutic agent and a second chemotherapeutic agent for use in the treatment of cancer, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of a peptide of at least 10 amino acids comprising an alpha-helical structure and oleic acid or oleate in a ratio of at least 3 oleic acid or oleate molecules per peptide molecule. Provided are either a first chemotherapeutic agent or a second chemotherapeutic agent for use in cancer therapy, for use in conjunction with other chemotherapeutic agents, pharmaceutical compositions related thereto, and methods of treatment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to chemotherapeutic agents for use in combination chemotherapy in the treatment of cancer. The combination includes a first chemotherapeutic agent and a second chemotherapeutic agent, the second chemotherapeutic agent having an anti-tumor The biologically active complex includes a biologically active complex having an α-helix. Peptides of at least 10 amino acids containing the structure, and oleic acid or oleate at a ratio of at least 3 oleic acid or oleate molecules per peptide molecule. The present invention also relates to pharmaceutical compositions and methods related thereto. [Background technology]

[0002] Cancers constitute a highly heterogeneous and highly complex group of diseases, but they are more prevalent than healthy tissues. and disrupting essential physiological functions. The common features across various cancer types are succinctly defined: Targets with improved tumor specificity Many cancer treatments have been developed, but when others don't work, doctors turn to radiation, surgery, Or they resort to using highly toxic substances that kill cancer cells and healthy cells alike. Because the drugs are not selective enough, patients experience severe side effects and "patients are unable to tolerate the drugs while treating their cancer." will die."

[0003] Bladder cancer is one of the most common and costly cancers in the United States due to its high recurrence rate and lack of curative treatment. It is a highly prevalent form of cancer. Its prevalence is approximately 3.4 million people, with 430,000 new cases diagnosed each year. It is estimated that approximately 200,000 deaths occur annually. Survival is affected by the recurrence rate and risk of dedifferentiation. Invasive tumors may require cystectomy and systemic chemotherapy. Substantial papillary tumors are limited to the mucosa and have a good short-term prognosis. G), topical treatments such as mitomycin, thiotepa, or epirubicin have been shown to improve the quality of life in many patients. has been shown to provide long-term disease-free intervals but can cause serious side effects (Malmstroem ,PU,Expert Rev Anticancer Ther,4,1057- 1067(2004);Schenkman, E. & Lamm, D., SciWorld J 4,387-399(2004)).

[0004] More specifically, limitations of current bladder cancer treatments contribute to the high recurrence rate and more frequent recurrence in this patient population. This is reflected in the risk of progression to malignant invasive disease. The current standard of care in BC is transurethral resection of the bladder tumor. Intravesical chemotherapy is It is generally used as adjuvant therapy after resection in patients with low-risk NMIBC It has been shown to reduce recurrence but not disease progression (Kang, M et al. ,Oncotarget 7,45479(2016);Sylvester,RJ ,et al.,Eur Urol 69,231-244(2016)). Calmette Intravesical immunotherapy with Bacillus Guerin (BCG) is recommended after surgery and has been shown to reduce tumor recurrence. It is superior to intravesical chemotherapy for preventing bladder cancer (Kamat, AM, et al. ncet 388,2796-2810(2016)). Mitomycin C is a new diagnostic It is widely used for intravesical chemotherapy of superficial bladder cancer in Japan, and has been shown to reduce tumor recurrence and improve disease-free survival. prolonging the septum (Wilhelm, S., et al., Nat Rev Mater 1,16014 (2016)). These treatments are associated with serious side effects and a significant risk of tumor recurrence. There remains a need for less toxic, more specific treatments with enhanced tumor-killing properties. There is a huge need that has not been addressed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Malmstroem, PU, ​​Expert Rev Anticancer Ther, 4, 1057-1067 (2004) [Non-patent document 2] Schenkman, E. & Lamm, D., SciWorld J 4, 387-399 (2004) [Non-patent document 3] Kang,M et al.,Oncotarget 7,45479(2016) [Non-patent document 4] Sylvester,RJ,et al.,Eur Urol 69,231-244(2016) [Non-patent document 5] Kamat,AM,et al.Lancet 388,2796-2810(2016) [Non-patent document 6] Wilhelm, S., et al., Nat Rev Mater 1,16014(2016) Summary of the Invention [Problem to be solved by the invention]

[0006] This study provides a chemotherapy combination that shows significant therapeutic effects against cancer, particularly bladder cancer. do. [Means for solving the problem]

[0007] The present invention relates to the use of two chemotherapeutic agents for the treatment of cancer. and for use in combination therapy, i.e., they are both used in the course of treatment. These are intended to be administered to the same subject in separate doses or in the same composition. These agents may be administered substantially simultaneously, i.e., These may be simultaneous or during the same treatment session. The administration may be for administration at different times within the same course of treatment.

[0008] In particular, the present invention relates to biologically active conjugates having antitumor activity, or methods for producing such conjugates. For pharmaceuticals containing the biologically active complex, the biologically active complex comprises at least 10 α-helical structures. A peptide of 10 amino acids and oleic acid or oleate can be prepared by adding 1000 or more amino acids per peptide molecule. at least three oleic acid or oleate molecules, and the chemotherapeutic agent is other chemotherapeutic agent. It is intended for use in conjunction with

[0009] The inventors discovered that the combined effect of the two chemotherapeutic agents resulted in unexpectedly high antitumor activity. Without being bound by theory, the inventors have identified the biologically active compounds of the present invention. When administered in combination with other chemotherapeutic agents, the complex is biologically active in tumor cells. The mechanism of action of the complex is to suppress the chemotherapeutic effect of the other chemotherapeutic agent when used individually. It is believed that the effect of the drug is enhanced compared to that of chemotherapy. However, the biologically active complexes of the present invention disrupt tumor membrane integrity, and Increasing the uptake of administered chemotherapeutic agents into tumor cells and / or tumor cell nuclei and / or possibly alter the expression of molecules involved in cancer pathways. Through the effects of the biologically active conjugate, tumor cells are exposed to the downstream effects of other chemotherapeutic agents. It is further believed that this may reduce the ability of the body to resist the effects of the drug. is the increased precision of targeting tumor cells versus healthy cells.

[0010] In one aspect, the present invention provides a method for treating cancer comprising administering to a patient a first chemotherapeutic agent and a second chemotherapeutic agent. and a second chemotherapeutic agent, wherein the second chemotherapeutic agent is a biologically active compound having antitumor activity. The biologically active complex contains at least 10 amino acids containing an α-helical structure. and oleic acid or oleate, at least It consists of a ratio of three oleic acid or oleate molecules.

[0011] In another aspect, the present invention provides a first chemotherapeutic agent for use in treating cancer. This is in combination with a second chemotherapeutic agent comprising a biologically active conjugate having antitumor activity. The biologically active complexes are composed of at least 10 α-helical structures. From peptides of amino acids and oleic acid or oleate, there are less per peptide molecule. Both consist of a ratio of three oleic acid or oleate molecules.

[0012] In this, and optionally other, embodiments, the first chemotherapeutic agent is administered to the subject. The drug is intended for use in cancer treatment, and the subject to which the drug is administered is a patient receiving a second chemotherapy agent. Having received, or in the process of receiving, or to be receiving, a dose or course of treatment.

[0013] In a further aspect, the present invention provides a second chemotherapeutic agent for use in treating cancer. The present invention provides a method for treating cancer using a first chemotherapeutic agent, the second chemotherapeutic agent having antitumor activity, and a method for treating cancer using a second chemotherapeutic agent having antitumor activity. The biologically active complexes include those containing an α-helical structure. and oleic acid or oleate to form peptides of at least 10 amino acids, including The ratio of oleic acid or oleate molecules to each oleic acid or oleate molecule is at least 3.

[0014] In this, and optionally other, embodiments, the second chemotherapeutic agent is administered to the subject. The drug is intended for use in cancer treatment, and the subject to which the drug is administered is a first chemotherapeutic agent. Having received, being in the process of receiving, or will receive a dose or course of treatment.

[0015] Chemotherapeutic agents include drugs used in cancer therapy, i.e., the treatment and / or prevention of cancer. Prevention can include preventing cancer from occurring in the first place, and preventing cancer from recurring. By referring to a first chemotherapeutic agent and a second chemotherapeutic agent, it is meant that the chemotherapeutic agents are different from each other. The first chemotherapeutic agent may be, for example, an intravesical chemotherapeutic agent, a topical chemotherapeutic agent, or and / or DNA-interacting chemotherapeutic agents. The DNA-interacting chemotherapeutic agent may be DNA alkylating, DNA crosslinker, and DNA intercalator chemotherapeutic agents include.

[0016] The agents may be for administration in combination, e.g., as part of the same composition. The first chemotherapeutic agent and the second chemotherapeutic agent can be bonded, for example, by one or more covalent bonds. Typically, the first chemotherapeutic agent and the second chemotherapeutic agent are bonded together, although it is possible that they can be bonded together. The therapeutic agents are not chemically bonded to each other.

[0017] In one embodiment, the first chemotherapeutic agent is an intravesical chemotherapeutic agent. refers to chemotherapy agents that are used or available for the treatment of bladder cancer. The therapeutic agents are atezolizumab, avelumab, bacillus Calmette-Guérin, bevacizumab, and carboplatin. Zantinib, cephalexin, ciprofloxacin, cisplatin, doxorubicin hydrochloride Salt, durvalumab, eflornithine, epirubicin, erdafitinib, erlotinib , fenretinide, gemcitabine, gefitinib, lapatinib, mitomycin C, nivolumab lumab, pazobanib, pembrolizumab, rapamycin, selenium , sorafenib, thiotepa, urocidin, valrubicin, and bicamphetamine In a preferred embodiment, the first chemotherapeutic agent is thiotepa, mitomycin, or thiazol-1,2-dione. and preferably selected from the group consisting of methicillin C, bacillus Calmette-Guerin, or epirubicin. is mitomycin C or epirubicin. In one embodiment, it is mitomycin C. In another embodiment, it is epirubicin. Mitomycin C can also be administered in the form of a prodrug, such as apaziquone. The compound is a natural product containing mitomycin A, mitomycin B, and mitomycin C. The mitomycin family is typically used in chemotherapy. Generally, the term "mitomycin" as used herein is used to refer to The term is used to clarify that it refers to the family of mitomycin compounds. Unless otherwise specified, particularly in the figures and examples, this is a shorthand for mitomycin.

[0018] The second chemotherapeutic agent comprises a biologically active conjugate having anti-tumor activity. In this method, the second chemotherapeutic agent consists of a biologically active conjugate having antitumor activity. "Anti-tumor activity" means any activity that stops, attenuates, slows, or inhibits the growth of any tumor cells. Antitumor activity refers to the ability of a conjugate to exhibit other deleterious effects. Antitumor activity is described in more detail below. Easily screened by any of a number of established techniques, including Preferably, the complex is a peptide molecule and oleic acid or an oleate. It is a non-covalent complex with a molecule.

[0019] The biologically active complexes consist of a peptide of at least 10 amino acids containing an α-helical structure. and oleic acid or oleate, with at least three olefins per peptide molecule. Many such biologically active complexes are , have been identified (e.g., WO 2010 / 079362, WO 2010 / 079362, No. 2012 / 052310, WO 2018 / 116165, and International Application No. P See CT / EP2019 / 066409). The peptides may be grown in their natural host (e.g., yeast, Expression, recombinant expression, or synthesis in mammals (humans, non-human primates, cattle, sheep, goats, horses, etc.) Peptides can be obtained from any suitable source, including chemical synthesis. The sequences of the wild-type peptides of non-human primates, cattle, sheep, goats, horses, etc. may contain or have, or include fragments and / or variant sequences thereof or a fragment and / or variant sequence thereof. wherein the peptide sequence is of eukaryotic origin, preferably of mammalian or yeast origin. Preferably, the mammalian-derived peptides are human sequences or fragments of human sequences and / or or a variant thereof. Preferably, the yeast-derived peptide is derived from Saccharomyces cerevisiae. Saccharomyces cerevisiae sequences or fragments and / or variants of Saccharomyces cerevisiae sequences be.

[0020] In one embodiment, the peptide in the biologically active complex has membrane-perturbing activity. The protein is a protein that is expressed as a nucleotide sequence (see, for example, WO 2018 / 116165).

[0021] The peptide in the biologically active conjugate is the peptide that, when present in the conjugate, is more potent than the unconjugated peptide. 1H NMR peak width of at least a portion of the complex compared to the corresponding peak width of the 1H NMR peak of the tinctorial As shown by the increased peak width of the H NMR peaks, This may increase the structural fluidity in the three-dimensional structure. The significance of this increase in activity has been described elsewhere (International Application No. PCT / EP2019 / 06 In summary, the inventors have demonstrated that peptides with diverse sequences have antitumor properties. It was found that oleates can form complexes with oleates, which share certain structural characteristics that confer their activity. This is due to the fact that it has an α-helical structure and is capable of binding oleic acid or oleate. and increasing the structural fluidity of the three-dimensional structure upon bonding. A marker of increased activity is the unconjugated peptide (i.e., when not conjugated with oleate). ) the width of at least a portion of the 1H NMR peak of the complex compared to the corresponding width of the 1H NMR peak of In one embodiment, the unconjugated peptide at least one 1H NMR peak of the complex compared to the corresponding width of the 1H NMR peak of the There is an increase in peak width in the NMR peaks. In one embodiment, the peptide At least one tryptophan residue is present, and at least one tryptophan residue in the complex is present. The 1H NMR peaks of the indole protons correspond to the 1H NMR peaks of the peptide alone. Another indicator of increased fluidity is the increase in the width of the peptide alone. 1H NMR peaks in the 1H NMR of the complex compared to the corresponding 1H NMR peaks The reduction in the chemical shift dispersion of the nuclei is

[0022] The peptide in the biologically active conjugate may have no cysteine ​​residues. Cysteine ​​residues typically contain disulfide bonds that provide rigidity to the 3D conformation of the peptide. By having a peptide without cysteine ​​residues, this dimer can be formed. Prevents sulfide bond formation, which increases the structural flexibility of peptides and enhances their biological activity. Promotes complex formation and improves antitumor effects.

[0023] The peptide in the biologically active complex is the peptide fragment that is biologically active. As long as the complex shows antitumor activity, it is possible to use longer protein fragments. In an embodiment, the peptide in the biologically active complex is 50 10 amino acids or less, i.e., it is a sequence having a maximum length of 50 amino acids. In particular, it can be less than 45, 40, or 35 amino acids in length. This is preferably at least 10, 12, 15, 20, or 25 meshes in length. Early studies used the full-length protein, but more recent studies have shown that it has antitumor efficacy. It has been shown that this can be achieved by using appropriate protein fragments or variants thereof. The shorter amino acid chains of such fragments make it easier, faster, and more reproducible. This allows for a reliable manufacturing process that complies with Good Manufacturing Practices (GMP). This is especially important when considering the manufacturing process that requires can be efficiently chemically synthesized.

[0024] In a preferred embodiment, the peptide in the biologically active complex is α-lacto Albumin or SAR-1, or a variant or fragment thereof, preferably the N-terminus thereof By N-terminal fragment, we generally mean the N-terminal fragment of the wild-type protein. It refers to a fragment containing the N-terminal amino acid (or an amino acid variant of the N-terminal amino acid).

[0025] Biologically active conjugates in which the peptide is α-lactalbumin are e.g., HAMLE T (tumor cell lethal human α-lactalbumin) has been previously described. Many peptides derived from α-lactalbumin have also been shown to have therapeutic effects in their own right. It has been found that the full-length α-lactoprotein Examples of albumin sequences are SEQ ID NOs: 5 and 6. KQFTKCELSQ LLKDIDGYGG IALPELICTM FHTSGY DTQA IVENNESTEY GLFQISNKLW CKSSQVPQSR NI CDISCDKF LDDDITDDIM CAKKILDIKG IDYWLAHKA L CTEKLEQWLC EKL (SEQ ID NO: 5) KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGY DTQA IVENNESTEY GLFQISNKLW AKSSQVPQSR NI ADISADKF LDDDITDDIM AAKKILDIKG IDYWLAHKA L ATEKLEQWLA EKL (SEQ ID NO: 6)

[0026] Biologically active conjugates in which the peptide is SAR1 or a fragment thereof have also been reported. (e.g., WO 2018 / 116165 and International Application No. PCT / (See EP2019 / 066409). Preferably, the SAR1 is derived from yeast or is a yeast-derived SAR1. It is a variant or fragment of the parent sequence. An example of a full-length SAR1 sequence is SEQ ID NO:7. MAGWDIFGWF RDVLASLGLW NKHGKLLFLG LDNAGK TTLL HMLKNDRLAT LQPTWHPTSE ELAIGNIKFT TF DLGGHIQA RRLWKDYFPE VNGIVFLVDA ADPERFDEA R VELDALFNIA ELKDVPFVIL GNKIDAPNAV SEAEL RSALG LLNTTGSQRI EGQRPVEVFM CSVVMRNGYL E AFQWLSQYI (SEQ ID NO: 7)

[0027] The term "variant" refers to a variant that has a similar biological function but whose amino acid sequence differs from that of the sequence. The amino acid sequence is different in that one or more amino acids in the sequence are substituted with other amino acids. refers to a protein or polypeptide that differs from the base sequence from which it is derived. A substitution is considered "conservative," in which one amino acid is replaced by another amino acid with roughly similar properties. A non-conservative substitution is one in which an amino acid is replaced with an amino acid of another type. is.

[0028] By "conservative substitution" is meant the substitution of an amino acid with another amino acid of the same class, Class is defined below.

[0029] Examples of Amino Acid Classes Non-polar A, V, L, I, P, M, F, W Polar uncharged G, S, T, C, Y, N, Q Acidic D, E Basic K, R, H

[0030] As is well known to those skilled in the art, altering the primary structure of a peptide by conservative substitutions can result in The activity of the peptide is not significantly altered by the addition of The side chain of the amino acid forms similar bonds and contacts to the side chain of the amino acid it replaces. Even if the substitution is in a region that is important for determining the peptide structure, That's how it is.

[0031] This is possible provided that the non-conservative substitutions do not interfere with the function of the peptide. A smaller number of non-conservative substitutions are possible without altering the biological activity of the polypeptide. .

[0032] Determining the effect of any substitution (and indeed any amino acid deletion or insertion) is entirely It is easy to determine whether the variant polypeptide retains the basic properties and activity of the base protein. It is within the ordinary skill of a person skilled in the art to determine whether or not a polypeptide has been modified. When determining whether a variant falls within the scope of the present invention, one skilled in the art should consider the composition of the compound containing the variant. Biological activity of the complexes formed with unfolded forms of the native protein The polypeptide may be used to determine whether it has a protective effect on the immune system (e.g., tumor cell death). At least 60%, preferably at least 70%, more preferably at least 60% of natural protein. is at least 80%, and even more preferably 90%, 95%, 96%, 97%, 98%, It has 99% or 100% activity.

[0033] Variants of polypeptides may be derived from naturally occurring protein sequences such as alpha-lactalbumin or SAR1. For sequences, or for active fragments of such natural protein sequences, at least about 70% identity, e.g., at least 75%, 80%, 85%, 90%, 91%, 92% , 93%, 94%, 96%, 97%, 98%, or 99% identity It may comprise or consist essentially of:

[0034] The level of sequence identity was determined using BLASTP with the native protein sequence as the base sequence. This is suitably determined using a computer program. This means that the sequence identity percentage is determined. The software is available at http: / / blast.ncbi.nlm.nih.gov / Blast. cgi (accessible as of March 12, 2009).

[0035] In one embodiment, the variant is one that does not have any cysteine ​​residues.

[0036] If the peptide is a fragment, preferably the peptide is a fragment of up to 40 amino acids in length. amino acids, for example up to 30 amino acids in length, or up to 25 amino acids in length. The peptides can be 10 to 40 amino acids in length, e.g., 11, 12, or 16 amino acids in length. , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or In one embodiment, it can be 39 amino acids in length. In others, it is 23 amino acids in length, and in others, it is 3 In particular, the peptide is a protein with an N-terminal α-helical domain. It can be an N-terminal fragment of a protein.

[0037] In a particularly preferred embodiment, the peptide in the biologically active complex has SEQ ID NO: Nos. 1 to 4, or functional variants or fragments thereof, preferably SEQ ID NO: Containing or consisting of 1. [Table 1]

[0038] In a preferred embodiment, the peptide in the biologically active complex is SEQ ID NO: 1 or Or rather.

[0039] In one embodiment, the peptide in the biologically active conjugate is up to 19 amino acids in length. and truncated forms of SEQ ID NO: 1 or SEQ ID NO: 2, which are amino acids of the amino acid sequence shown.

[0040] In one embodiment, the first agent is mitomycin C and the second agent is SEQ ID NO: 1 is.

[0041] In one embodiment, the first agent is mitomycin C and the second agent is SEQ ID NO:2 is.

[0042] In one embodiment, the first agent is mitomycin C and the second agent is SEQ ID NO:3 is.

[0043] In one embodiment, the first agent is mitomycin C and the second agent is SEQ ID NO:4 is.

[0044] In one embodiment, the first agent is epirubicin and the second agent is SEQ ID NO: 1. do.

[0045] In one embodiment, the first agent is epirubicin and the second agent is SEQ ID NO:2. do.

[0046] In one embodiment, the first agent is epirubicin and the second agent is SEQ ID NO: 3. do.

[0047] In one embodiment, the first agent is epirubicin and the second agent is SEQ ID NO: 4. do.

[0048] Optional combinations of chemotherapeutic agents are shown in Table 2. The combinations shown in Table 2 include: The combination includes functional equivalents of the listed agents. Possible combinations include peptides with sequences that are longer than the sequences shown. The present invention further includes a peptide having the sequence shown, or a functional fragment or variant of the peptide. nothing. [Table 2] JPEG2026001029000003.jpg230157JPEG2026001029000004.jpg228155JPEG2026001029000005.jpg229155JPEG2026001029000006.jpg68154

[0049] In one embodiment, the use is in the treatment or prevention of carcinoma, lymphoma, or brain tumors, preferably or gastrointestinal cancer, mucosal cancer, bladder cancer, kidney cancer, lung cancer, glioblastoma, and skin papilloma Preferably, the method is used in the treatment or prevention of bladder cancer. The cancer is a human cancer. In one embodiment, this is the treatment of bladder cancer. In some cases, this is bladder cancer prevention.

[0050] The first chemotherapeutic agent is selected to produce a therapeutic effect when administered without the second chemotherapeutic agent. may be administered or intended to be administered at a dose less than half the dose required for The first chemotherapeutic agent is administered in an amount of at least 0.001, 0.01, 0.1, 0. 5, 1, or 1.25 mg / kg, or at least 0.001, 0.0 administered or administered in amounts of 1, 0.1, 0.5, 1, or 1.25 μg / kg In certain embodiments, the first chemotherapeutic agent may be the most effective by weight. 1000, 500, 100, 50, 10, 4, 2, and 1.5 mg / kg or body weight at doses up to 1000, 500, 50, 10, 4, 2, or 1.5 μg / kg In certain embodiments, the second chemotherapy The drug should be administered in doses of at least 0.001, 0.01, 0.1, 0.5, 1, 2, 4, or or 8 mg / kg, or at least 0.001, 0.01, 0.1, 1 , 5, 10, 20, 40, or 45 μg / kg, or for administration. The second chemotherapeutic agent may be of a maximum of 5000, 1000, 500, or , 100, 50, 25, or 10 mg / kg, or up to 10,000, 5 mg / kg body weight or administered in amounts of 000, 100, 500, 75, or 50 μg / kg. Preferably, the first chemotherapeutic agent is administered in an amount of 0.5 to 2 mg by body weight. / kg of body weight, and / or the second chemotherapeutic agent is administered in the range of 25 to 75 mg / kg of body weight. Administer in the kg range.

[0051] The first chemotherapeutic agent and the second chemotherapeutic agent are generally administered together. and the second chemotherapeutic agent can be administered in combination, together, or sequentially. as part of the same composition, as separate compositions but simultaneously, or in the same treatment program or regimen. This may mean that they are administered as part of a regimen.

[0052] In another aspect, the present invention provides a method for treating a rheumatoid arthritis comprising administering to a patient a first chemotherapeutic agent, a second chemotherapeutic agent, and a pharmaceutically acceptable carrier. and a second chemical compound. Chemotherapy agents include biologically active conjugates with antitumor activity, and biologically active conjugates The body produces peptides of at least 10 amino acids containing an alpha helix structure, and oleic acid or oleate, at least three oleic acids or oleic acids per peptide molecule The first chemotherapeutic agent and / or the second chemotherapeutic agent of the pharmaceutical composition are the same or different from the first chemotherapeutic agent and / or the second chemotherapeutic agent of the present invention. It may further include any of the features described in any other aspect herein.

[0053] In a further aspect, the present invention provides a compound according to the fourth aspect of the invention for use in therapy. The present invention provides a pharmaceutical composition for

[0054] In a further aspect, the present invention provides a method of treating or preventing cancer, the method comprising: A subject in need of treatment or prevention is administered a first chemotherapeutic agent in conjunction with a second chemotherapeutic agent. wherein the second chemotherapeutic agent comprises a biologically active conjugate having antitumor activity; The biologically active complexes consist of peptides of at least 10 amino acids containing an α-helical structure. and oleic acid or oleate, with at least three oleic acids per peptide molecule. The first compound in the method for treating or preventing cancer is The first, second and third aspects of the invention may be Any of the features described above may be further included.

[0055] The compositions according to this aspect of the invention are preferably in the form of, for example, a cream, ointment, gel or is a pharmaceutical composition in a form suitable for topical use as an aqueous or oily solution or suspension This includes commonly known, pharmaceutically acceptable carriers, fillers, and / or adjuvants. The topical solution or cream is preferably prepared with a diluent or cream base. The composition can be delivered by any route. For example, the composition can be delivered by any route, particularly by a suction device. The composition can be administered orally to target the gastrointestinal tract. Further routes of delivery are, for example, intravenous or intramuscular. When targeting the bladder, the composition is typically administered via urinary infusion. It can be administered intravesically by a bladder catheter.

[0056] Typically, the preparation of biologically active conjugates of the present invention is carried out as described elsewhere. (See, for example, WO 2018 / 116156). In this case, the peptide can be simply combined with oleic acid or a salt thereof in a solution, such as an aqueous solution. The preparation can be carried out by mixing together the oleate:peptide mixture. The ratio is preferably in the range of 20:1 to 3:1, for example about 5:1 oleate:peptide. The mixing can be carried out at a temperature of 0 to 50°C, conveniently at ambient temperature and pressure. This simple preparation method is particularly advantageous for the use of such peptides in conjugates. This method can be performed at the site when needed for treatment. Kits can be provided containing the peptides and salts for pre-mixing.

[0057] In the detailed description and claims of this specification, the terms "comprise" and "contain" The word "comprises" and variations of that term, such as "including" and "including," mean "including but not limited to" and "including but not limited to." "Excluding" means "not including" and does not exclude other ingredients, components or steps. Furthermore, unless the context requires otherwise, the singular includes the plural and, in particular, the indefinite article is used. Where appropriate, the specification shall contemplate the plural as well as the singular, unless the context otherwise requires. It needs to be understood as such.

[0058] Preferred features of each aspect of the invention are as described with respect to any of the other aspects. Within the scope of this application, the preceding paragraphs, claims, and / or Various aspects, embodiments, examples, and other aspects, particularly those described in the detailed description below and in the drawings. It is clear that the individual features may be used independently or in any combination. That is, all embodiments and / or features of any embodiment are intended to be included in the scope of such features. The features may be combined in any manner and / or in any combination unless the features are inconsistent.

[0059] One or more embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0060] [Figure 1] Figure 1 shows a schematic diagram of a mouse bladder tumor model for measuring the dose-dependent therapeutic effect of α1-oleate. Bladder cancer was induced in C57BL / 6 female mice by intravesical instillation of MB49 cells (2 × 105 in 100 μl of PBS). Mice were treated by intravesical instillation of α1-oleate (1.7 mM, 8.5 mM, or 17 mM) on days 3, 5, 7, 9, and 11 and sacrificed on day 12. Sham-treated mice received PBS. [Figure 2] FIG. 2 shows the dose-dependent effect of α1-oleate as estimated from macroscopic examination of evident bladder lesions. [Figure 3] Figure 3 shows a comparison of bladder weight, bladder size, and tumor area, with data shown as the mean ± standard error of two experiments (n = 6 + 5 mice, *P < 0.05, **P < 0.01, and ***P < 0.001 compared to sham-treated mice). [Figure 4] Figure 4 shows the accumulation of α1-oleate in tumor tissue. Alexa-Fluor 568-labeled α1-oleate was used to challenge tumor-bearing mice and track the molecule in the tumor tissue. (A) Scheme of intravesical Alexa-α1-oleate challenge. Intravesical instillation was performed on day 11, and tissue was collected 6 hours later. (B) Detection of Alexa-Fluor 568-labeled α1-oleate in tumor-bearing mice by confocal imaging of frozen tissue sections. Healthy mice challenged with labeled α1-oleate served as controls. (C) Quantification of the fluorescence intensity in (B). Mean ± standard error of five images per dose is shown. [Figure 5]Figure 5 shows dose-dependent changes in gene expression in tumor-bearing mice. (A) Heatmap showing the dose-dependent reduction in the number of regulated genes in treated mice. Red (left) indicates upregulated genes, and blue (right) indicates downregulated genes, with a cutoff fold change of >2.0 compared to healthy bladders. (B) Dose-dependent reduction in molecular mechanisms in cancer pathway genes. (C) Individual regulated genes in this pathway are shown. (D) Principal component analysis of mRNA profiles in whole bladder tissue. Increasing doses of α1-oleate shifted the transcriptome profile from a sham-treated cluster of tumor-bearing to a cluster of healthy bladder tissue. (E) Probe scatter plot comparing the transcriptome profiles of tumor-bearing and healthy bladders (log2 signal intensity values). (F) Venn diagram showing genes significantly regulated in sham-treated and α1-oleate-treated mice compared to healthy bladders. [Figure 6] Figure 6 shows a schematic diagram of the mouse bladder cancer model. Bladder cancer was induced in C57BL / 6 female mice by intravesical instillation of MB49 cells (2 x 10 cells in 100 μl of PBS). Mice were treated by intravesical instillation of mitomycin C (25 μg, *25 μg is half the therapeutic dose for mice; intraperitoneal administration of 50 μg results in systemic toxicity) or α1-oleate (8.5 mM) on days 3, 5, 7, 9, and 11 and sacrificed on day 12. Sham-treated mice received PBS. [Figure 7] Figure 7 shows bladder size (and any effect of tumor size on gross bladder appearance) after death of mice in the model of Figure 1, where mice were sham-treated, treated with 8.5 mM α1-oleate, or 25 μg mitomycin, as well as healthy controls (all shown at the same magnification). [Figure 8] FIG. 8 shows the schematic diagram of FIG. 1 modified to examine the therapeutic effect of the combination of α1-oleate and mitomycin C. [Figure 9]Figure 9 shows the bladder size (and any effect of tumor size on gross bladder appearance) of mice in the model of Figure 3 after death, treated with sham, 1.7 mM α1-oleate, 25 μg mitomycin, and 1.7 mM α1-oleate + 25 μg mitomycin (all shown at the same magnification). [Figure 10] Figure 10 shows a study comparing post-mortem bladder size (and any effect of tumor size on gross bladder appearance) in the 1.7 mM α1-oleate + 25 μg mitomycin study of Figure 4 with bladder size (and any effect of tumor size on gross bladder appearance) in mice from the same model but treated with 17 mM α1-oleate (all shown at the same magnification). [Figure 11] Figure 11 shows a schematic diagram of a mouse bladder tumor model for measuring the therapeutic effects of α1-oleate, mitomycin C (MMC), and the combination of the two. Bladder cancer was induced in C57BL / 6 female mice by intravesical instillation of MB49 cells (2 × 105 in 100 μl of PBS). Mice were treated by intravesical instillation of α1-oleate (1.7 mM), MMC (25 μg), and α1-oleate (1.7 mM) and MMC (25 μg) on ​​days 3, 5, 7, 9, and 11 and sacrificed on day 12. Sham-treated mice received PBS. [Figure 12] FIG. 12 shows the effects of α1-oleate, MMC, and α1-oleate plus MMC as estimated from macroscopic examination of apparent bladder lesions. [Figure 13] FIG. 13 shows the effects of α1-oleate, MMC, and α1-oleate plus MMC as estimated from bladder histology. [Figure 14]Figure 14 shows a schematic diagram of a mouse bladder tumor model for measuring the dose-dependent therapeutic effects of α1-oleate, mitomycin C (MMC), and the combination of the two. Bladder cancer was induced in C57BL / 6 female mice by intravesical instillation of MB49 cells (2 × 10 cells in 100 μl of PBS). Mice were treated by intravesical instillation of α1-oleate (1.7 mM and 8.5 mM), MMC (25 μg), and α1-oleate (1.7 mM and 8.5 mM) and MMC (25 μg) on ​​days 3, 5, 7, 9, and 11. The sham group was sacrificed on day 12, and the treatment groups were sacrificed at 4 or 8 weeks. Sham-treated mice were given PBS. [Figure 15] FIG. 15 shows the dose-dependent effects of α1-oleate, the effect of MMC, and the dose-dependent effects of α1-oleate and MMC, as estimated from macroscopic examination of apparent bladder lesions. [Figure 16] FIG. 16 shows the dose-dependent effects of α1-oleate, the effect of MMC, and the dose-dependent effects of α1-oleate and MMC, as estimated from bladder histology. [Figure 17] Figure 17 shows a schematic diagram of a mouse bladder tumor model for measuring the therapeutic effects of α1-oleate, mitomycin C (MMC), epirubicin (Epi), a combination of α1-oleate and MMC, and a combination of α1-oleate and Epi. Bladder cancer was induced in C57BL / 6 female mice by intravesical instillation of MB49 cells (2 × 10 cells in 100 μl of PBS). Mice were treated by intravesical instillation of α1-oleate (1.7 mM), MMC (25 μg), Epi (25 μg), α1-oleate (1.7 mM) and MMC (25 μg), and α1-oleate (1.7 mM) and Epi (25 μg) on ​​days 3, 5, 7, 9, and 11 and sacrificed on day 12. Sham-treated mice received PBS. [Figure 18] FIG. 18 shows the effects of α1-oleate, Epi, and α1-oleate plus Epi as estimated from macroscopic examination of apparent bladder lesions. [Figure 19] FIG. 19 shows a schematic diagram similar to that shown in FIG. [Figure 20]FIG. 20 shows the effects of α1-oleate, MMC, Epi, α1-oleate and MMC, and α1-oleate and Epi, as estimated from macroscopic examination of evident bladder lesions. [Figure 21] Figure 21 shows the therapeutic efficacy of α1-oleate and mitomycin C in a mouse bladder cancer model. (a) A schematic diagram of the treatment model is shown. Bladder cancer was induced in C57BL / 6J female mice by intravesical instillation of MB49 cells (2 × 10 cells in 50 μl of PBS). Treatment groups received α1-oleate (1.7 mM or 8.5 mM) alone or MMC (25 μg / mL), or their combination, on days 3, 5, 7, 9, and 11. Sham-treated mice received PBS, and all mice were sacrificed on day 12. (b) The effect of treatment estimated from macroscopic examination of the bladder is shown. (c, d, e) Pathology scores, bladder weight, bladder size, and tumor area are shown (see also Figure 20). Data are presented as the mean ± standard error of two experiments (n = 5 + 6 mice per group, analyzed by one-way ANOVA). [Figure 22] Figure 22 shows the reduction in tumor size by α1-oleate and mitomycin C, alone or in combination. Tumor area was compared between sham-treated mice and mice given 1.7 mM α1-oleate, 25 μg MMC, or the combination. (a) Sham-treated mice showed large tumors filling the bladder lumen (dotted line). (b-c) Mice treated with α1-oleate (1.7 mM) and MMC showed reduced tumor size. (d) Further reduction in tumor size in mice treated with a combination of α1-oleate (1.7 mM) and MMC (25 μg / mL) is shown. (e) A healthy bladder used as a negative control is shown. Representative images are shown (n = 5 + 6 mice per group). (n = 5 + 6 mice per group, analyzed by one-way ANOVA). Tumor area was determined in H&E-stained whole bladder sections and quantified using ImageJ. [Figure 23]Figure 23 shows the therapeutic efficacy of α1-oleate and epirubicin in a mouse bladder cancer model. (a) A schematic diagram of the treatment model is shown. Treatment groups received α1-oleate alone (α1-oleate 1.7 mM or 8.5 mM), epirubicin (25 μg / mL), or their combination by intravesical instillation on days 3, 5, 7, 9, and 11. Sham-treated mice received PBS, and all mice were sacrificed on day 12. (b) Visible bladder lesions as determined by macroscopic examination are shown. (c, d, e) Pathology score, bladder weight, bladder size, and tumor area are shown (see also Figure 22). Data are presented as the mean ± standard error of two experiments (n = 5 + 5 mice per group, analyzed by one-way ANOVA). [Figure 24] Figure 24 shows the reduction in tumor size with α1-oleate and epirubicin treatment, alone or in combination. (a) Sham-treated mice showed large tumors filling the bladder lumen (dotted line). (b-c) Mice treated with α1-oleate (1.7 mM or 8.5 mM) and epirubicin showed a reduction in tumor size. (d, e) Further reduction in tumor size is shown in mice given a combination of α1-oleate (1.7 mM or 8.5 mM) and epirubicin. Tumor area was determined in H&E-stained whole bladder sections and quantified using ImageJ. [Figure 25] Figure 25 shows the inhibition of tumor recurrence by the combination of α1-oleate and mitomycin C. The combination of α1 and mitomycin C provided long-term protection, inhibiting tumor recurrence for 4 weeks. (a) The effect of α1-oleate and MMC, alone or in combination, as determined by macroscopic examination of the bladder is shown (see also the schematic diagram in Figure 19a). (c, d, e) Reduction in pathology score, bladder weight, and bladder size is shown (p<0.001, see also Figure 6). Data are shown as the mean ± standard error of two experiments (n=5 + 5 mice). [Figure 26]Figure 26 shows tumor parameters in long-term follow-up of mice treated with α1-oleate or mitomycin C. (a) Quantification of pathology score, bladder size, bladder weight, and tumor area is shown. (b-e) Tumor area was compared after 4 weeks between mice receiving 1.7 mM and 8 mM α1-oleate and 25 μg MMC. (b, c) Evidence of tumor recurrence in mice receiving α1-oleate (1.7 mM) alone and MMC (25 μg) is shown. (d-e) The combination of α1-oleate 1.7 mM or 8.5 mM with mitomycin C shows the inhibition of tumor recurrence. No tumors were detected in H&E-stained whole bladder sections. Representative images are shown (n = 5 + 5 mice per group). [Figure 27] Figure 27 shows tumor parameters over time in mice treated with α1-oleate or epirubicin. (a) Quantification of pathology score, bladder size, bladder weight, and tumor area is shown. (b-e) Tumor area was compared after 4 weeks between mice receiving 1.7 mM and 8 mM α1-oleate and 25 μg epirubicin. (b, c) Evidence of tumor recurrence in mice receiving α1-oleate (1.7 mM) alone and epirubicin (25 μg) is shown. (d-e) The combination of α1-oleate 1.7 mM or 8.5 mM with epirubicin inhibits tumor recurrence. No tumors were detected in H&E-stained whole bladder sections. Representative images are shown (n = 5 + 5 mice per group). DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention provides a novel method for targeting and killing tumor cells with high efficacy and precision using combination therapy. The inventors have demonstrated that the biological activity of the present invention in tumor cells is The effect of the highly active complex is greater than the direct antitumor properties of the complex alone. The conjugate also makes tumor cells more susceptible to the effects of other chemotherapy drugs. Furthermore, the complex of the present invention was not found to affect healthy cells (see Example 2). ), suggesting that any enhanced efficacy of additional chemotherapy agents is restricted to tumor cells. It is further noted that this enhancement can be achieved, for example, by disrupting the cell wall (by directly disrupting the lipid bilayer). by disrupting contacts or by disrupting membrane-bound proteins, including ion channels. and / or gene expression, especially genes involved in the molecular mechanisms of cancer pathways This may occur through regulation of the α-glucan-1-phosphate dehydrogenase (α-glucan-1-phosphate dehydrogenase) (see Example 3). Data collected on the conjugate and other chemotherapeutic agents will be used in combination with their individual use (Example 4). (Example 5) shows a clear synergistic enhancement in therapeutic effect. [Example]

[0062] Example 1 - Dose Escalation Study of α1-Olate in Tumor-Bearing Mice A dose-escalation study was conducted in a murine MB49 bladder cancer model (inoculation on day 0, Figure 1). Mice in the treatment group received five bladder injections of α1-oleate on days 3, 5, 7, 9, and 11. Intravesical instillation was performed and sham-treated mice received PBS instillation at these time points. The macroscopic appearance of the bladder in mice was significantly improved compared to controls that did not receive tumor cells. The patient developed a palpable tumor that rapidly grew from the mucosa and gradually expanded into the bladder. The bladder lumen was filled to replace functional bladder tissue.

[0063] Treated mice received increasing concentrations of α1-oleate (1.7, 8 in 100 μg). (0.5 or 17 mM, 5-6 mice per group, 2 experiments per dose). On day 1, the bladder was collected and evaluated macroscopically (Figure 2), and bladder size, weight, and tumor area were recorded (Figure 3). 3) After treatment with 1.7 mM α1-oleate, tumor growth was suppressed, and bladder weight and bladder size were increased. The urinary tract infection and tumor size were reduced (P<0.001 compared to sham-treated mice, Figure 3). Further dose-dependent reductions in bladder weight, bladder size, and tumor size were observed at 8.5 m The highest concentration was recorded in mice given α1-oleate (P<0.001). At 17 mM, no visible tumor tissue remained. Bladder size and weight in the rats were not different from those in healthy control mice.

[0064] The results show increasing therapeutic efficacy with increasing doses of the compound until most of the tumor is eliminated. This paper identifies α1-oleate as a tumoricidal complex having

[0065] Example 2 - Accumulation of α1-oleate in tumor tissue To examine whether α1-oleate reaches tumor tissue, tumor-bearing mice were treated with Al exa-Fluor568-labeled α1-oleate was inoculated (day 11, n=2). Six hours after instillation, frozen tissue sections were harvested and subjected to confocal imaging. The Fluorescent-labeled conjugate showed a dose-dependent accumulation in tumor tissue (1.7 vs. 8 0.5 mM, 8.5 mM vs. 17 mM) (Figure 4A-4C), Alexa-Fluor 568 α1-oleate was not detected in healthy mice subjected to the same procedure (Figure 4B and Figure 4C). These results were compared using anti-α1 antibodies to compare the tumor-bearing cells treated with α1-oleate. Accumulation of α1-oleate in tumor tissues was confirmed by dot blot analysis in mice. This was confirmed by staining frozen tissue sections with anti-α1 antibody. Tissues from healthy mice were negative.

[0066] Example 3 - Dose-dependent inhibition of gene expression in tumor-bearing mice To further characterize tumor response to α1-oleate, whole-genome analysis was performed on total bladder RNA. Transcriptome profiling was performed. Gene expression was analyzed in the sham-treated group and in the dose-increasing group. The results were compared between healthy mice and control mice fed with α1-oleate supplemented with α1-oleate. It was used as.

[0067] Transcriptome data revealed a significant difference between sham-treated and α1-oleate-treated mice. Significant differences in gene expression were revealed in the number of differentially expressed genes. A dose-dependent reduction in tumor size was observed, consistent with a significant reduction in tumor size in treated mice. (Fold change compared to healthy controls, see heatmap in Figure 5A). The molecular mechanisms involved in the pathways and genes involved were identified by Ingenuity Pathway Analysis (IPA). As shown in Fig. 5B and Fig. 5C, the activation of ATP was dose-dependent (Fig. 5B). (Figure 5D) showed that tumor-bearing sham-treated mice were significantly more sensitive than treated mice and healthy controls. The transcriptome profile clearly differed from that of the control group. The 9.3% variability was dominated by the difference between sham-treated and healthy bladders. As the expression profile of the treated mice increased, the expression profile of the treated mice shifted to a healthy one. The variability (% variability) distinguished the untreated group from the α1-oleate-treated mice. Corroborated by a scatter plot analysis of lobe signal intensity (Figure 5E), healthy control and treated mice revealed a dose-dependent decrease in the number of differentially expressed transcripts between the The results are also shown in a Venn diagram (Figure 5F).

[0068] Example 4 - Examination of the individual therapeutic effects of α1-oleate and mitomycin C Another study was conducted in a murine MB49 bladder cancer model, with mice in treatment groups receiving 3, 5, 7, and 9 , and on day 11, 8.5 mM α1-oleate or 25 μg mitomycin C Five intravesical instillations were performed, and sham-treated mice received PBS instillations at these time points. (Figure 6).

[0069] Bladders were harvested on day 12 and evaluated (Figure 7). After treatment with mitomycin, tumor growth was suppressed to a similar extent compared with sham-treated mice. Bladder weight, bladder size, and tumor size were reduced.

[0070] Example 5 - Examination of the therapeutic effect of the combination of α1-oleate and mitomycin A modified version of the previous study was performed in which mice in the treatment group received 1.7 mM α1-oleate, 25 μg mitomycin, or a combination of 1.7 mM α1-oleate + 25 μg mitomycin Five intravesical instillations were performed on each group, and sham-treated mice received PBS instillations at these time points. (Figure 8).

[0071] This result was obtained with the combination of 1.7 mM α1-oleate + 25 μg mitomycin. Treatment consisted of either 1.7 mM α1-oleate or 25 μg mitomycin alone. Unlike other treatments, this treatment produces bladders that are equivalent or nearly equivalent to healthy bladders (Figure 9). ). Furthermore, Figure 10 shows that the combination treatment was superior to the 10-fold concentration of α-1-oleate alone (i.e. It produces effects in tumors that are perceived to be equivalent to those of 17 mM α1-oleate. Indicates that.

[0072] Example 6 - Examination of the therapeutic effect of the combination of α1-oleate and mitomycin A modified version of the previous study was performed in which mice in the treatment group received 1.7 mM α1-oleate, 25 μg mitomycin, or a combination of 1.7 mM α1-oleate + 25 μg mitomycin Five intravesical instillations were performed on each group, and sham-treated mice received PBS instillations at these time points. (Figure 11).

[0073] This result was obtained with the combination of 1.7 mM α1-oleate + 25 μg mitomycin. Treatment consisted of either 1.7 mM α1-oleate or 25 μg mitomycin alone. Unlike other treatments, this procedure produces bladders that are equivalent or nearly equivalent to healthy bladders (Figure 1). 2) Furthermore, Figure 13 shows that the combined treatments were comparable or superior to those seen in healthy bladders. shows that the two produce nearly identical bladder histology images.

[0074] Example 7 - Examination of the therapeutic effect of the combination of α1-oleate and mitomycin A modification of the previous study was performed in which treatment groups of mice received either 1.7 mM or 8.5 mM α1- oleate, 25 μg mitomycin, or 1.7 mM or 8.5 mM α1-oleate Five intravesical instillations of a combination of acetaminophen and 25 μg of mitomycin were administered to sham-treated mice. , PBS instillation was performed at these time points (Figure 14).

[0075] This result was obtained with the combination of 1.7 mM α1-oleate + 25 μg mitomycin. Treatment consisted of either 1.7 mM α1-oleate or 25 μg mitomycin alone. Unlike other treatments, this procedure produces bladders that are equivalent or nearly equivalent to healthy bladders (Figure 1). 5). Furthermore, Figure 15 shows that the combination treatment was superior to the 5-fold concentration of α-1-oleate alone (i.e. produces effects in tumors that are perceived to be equivalent to those of 8.5 mM alpha-1-oleate Figure 16 shows that the combination treatments resulted in bladder swelling comparable to or greater than that seen in healthy bladders. 15 and 16 show that the bladder tissue images were almost the same as those of the control group. The results show that the effects are maintained over a long period of 1 week and 8 weeks.

[0076] Example 8 - Therapeutic Effect of Combination of α1-Olate and Mitomycin or Epirubicin Consider A modified version of the previous study was performed in which mice in the treatment group received 1.7 mM α1-oleate, 25 μg mitomycin, 25 μg epirubicin, 1.7 mM α1-oleate + 25 μg The combination of mitomycin or 1.7 mM α1-oleate + 25 μg epirubicin Five intravesical instillations of the combination were performed, and sham-treated mice received PBS instillations at these time points. This was carried out (Figure 17).

[0077] This result was consistent with the treatment with a combination of 1.7 mM α1-oleate and 25 μg epirubicin. The conditions were 1.7 mM α1-oleate, 25 μg mitomycin, or 25 μg epirubicin. Treatment with 1.7 mM α1-oleate + 25 μg myosin alone did not significantly affect the efficacy of the treatment with either myosin or α1-oleate alone. and from mice treated with the combination of cyclosporin and cyclosporin, and similar or nearly identical to healthy bladders. Furthermore, Figure 18 shows that the α1-oleate and epi- Treatment with rubicin in combination with α-1-oleate was five times more effective than treatment with α-1-oleate alone (i.e., 8.5 The results show that α-1-oleate produces effects in tumors that are comparable to those of α-1-oleate (1 mM).

[0078] Materials and Methods Chemicals and Antibodies Oleic acid (Croda, Lot No.: 0001120439), poly-L-lysine solution Solution (Sigma, Cat. No. RNBF4239), Alexa-Fluor 568 Tan Protein labeling kit (Thermo Scientific, catalog number A10238) , ECL Plus detection reagent (GE healthcare, Cat. No. RPN213 2) Richard-Allan Scientific Signature Series Hema Toxiclin and Eosin-Y (Thermo Scientific, Cat. No. #7 211 and 7111), DAPI (Sigma, Cat. No. D9542), anti-α-lactate Albumin (Mybiosource, Cat. No. MBS175270), monoclonal Neutral mouse anti-β-actin (Sigma-Aldrich, Cat. No. A2228), Reclonal rabbit anti-mouse IgG-HRP (Dako, Cat. No. P0260), Reclonal goat anti-rabbit IgG-HRP (Cell Signaling, catalog no. No. 7074), rabbit polyclonal anti-VEGF (Abcam, Cat. No. ab461 54), mouse monoclonal anti-Ki-67 (BD Biosciences, catalog No. 556003), rabbit monoclonal anti-cyclin D1 (Thermo Fish er, Catalog No. SC8396), Goat anti-Rabbit IgG Alexa-Fluor 48 8 (Thermo Fisher, Cat. No. A-11034), goat anti-mouse IgG Alexa568 (Thermo Fisher, Cat. No. A-11004), D RAQ5 (Abcam, catalog number ab108410).

[0079] Peptide synthesis and conjugate preparation α1 was synthesized using Fmoc solid phase chemistry (Mimotopes). The α10 ... The α1 sequence was Ac-KQFTKAELSQ LLKDIDGY GG IALPELIA™ FHTSGYDTQ-OH.

[0080] Bladder cancer model C57BL / 6 female mice were bred at the Department of Laboratory Medicine, Lund University, and aged from 7 weeks to 12 weeks. For the procedure, mice were administered ketamine (1.48 mcg in 100 μl of NaCl). g, Intervet) and xylazine (0.22 mg in 100 μl NaCl, Vet On day 0, the bladder was emptied and the rats were anesthetized by intraperitoneal injection of a mixture of acetaminophen and methylprednisolone. , via a 0.61 mm outer diameter soft polyethylene catheter (Clay Adams). Preconcentration was achieved by intravesical instillation of 100 μl of poly-L-lysine solution (0.1 mg / ml). After 30 minutes of conditioning, MB49 mouse bladder cancer cells (2 μg / mL in 100 μl of PBS) were added. x10 5 On days 3, 5, 7, 9, and 11, 100 μl of α1-oleate was instilled. (α1: 1.7mM, 8.5mM, or 17mM, oleic acid: 8.5mM , 42.5 mM, or 85 mM) or PBS (sham-treated control). Us left the catheter in place to prolong tumor exposure to the peptide-oleate conjugate. The animals were kept under anesthesia for approximately 1 hour on a preheated block in a dark place. Groups of 5-6 mice were sacrificed after 12 days and their bladders were imaged and histologically examined. Two independent experiments were performed.

[0081] statistical analysis Results are presented as mean ± standard error and groups are compared by one-way ANOVA. P values ​​are reported. , GraphPad Prism version 7 (GraphPad Software Student's t-test and one-way analysis of variance followed by Bonn- fitzpatrick's method were used. The results were calculated using the Erroni post-hoc test. P<0.05 was considered statistically significant. I met. *P<0.05;**P<0.01;***P<0.001.

[0082] Example 9 - Further investigation of combination therapy Materials and Methods Chemicals and Antibodies Oleic acid (Croda, Lot No.: 0001120439), poly-L-lysine solution Solution (Sigma, Cat. No. RNBF4239), Richard-Allan Sc ientific Signature Series Hematoxylin and Eosin-Y (Therm Scientific, Catalog Numbers #7211 and 7111), epirubicin (S igma-Aldrich, Cat. No. E9406) and mitomycin (Sigma -Aldrich, Cat. No. M0440).

[0083] Peptide synthesis and conjugate preparation The N-terminal α-helical domain of α-lactalbumin was identified as the tumoricidal component. It forms a complex with oleic acid (Ho J, Rydstrom A, Manimek alai MSS,Svanborg C,Grueber G.Low resolu tion solution structure of HAMLET and th e importance of its alpha-domains in tum oricidal activity.PLoS One 2012;7: e5305 1) For this study, 39 nucleotides were synthesized using Fmoc solid-phase chemistry with purity exceeding 95%. The amino acid peptide (aa1-39, Ac-KQFTKAELSQLLKDIDGYGG Polypeptide (IALPELIATMFHTSGYDTQ-OH) was synthesized. group, France). α1 peptide is used to form the α1-oleate complex. The stock solution was mixed with sodium oleate at a molar ratio of 1:5. The appropriate concentration for each experiment was adjusted. The solution was further diluted in PBS to obtain a final concentration.

[0084] Bladder cancer model MB49 (RRID:CVCL_7076) cells were cultured by Sara M, Uppsala University, Sweden. MB49 bladder cancer was established as previously described. (Mossberg AK, Hou Y, Svensson M, Holmqvis t B,Svanborg C.HAMLET treatment delays b ladder cancer development.The Journal of Urology 2010;183: 1590-7). C57BL / 6 female mice were They were bred at the Department of Laboratory Medicine, Lund University, and used at 7 to 12 weeks of age. Mice were given ketamine (1.48 mg in 100 μl of 0.9% NaCl solution, Interv et) and xylazine (0.22 mg in 100 μl of 0.9% NaCl solution, Vetme On day 0, the bladder was emptied and the rats were anesthetized by intraperitoneal injection of a mixture of 0.5 mL of HCl and 0.5 mL of HCl. 10 mm via a 0.61 mm outer diameter soft polyethylene catheter (Clay Adams) Preconditioning was performed by intravesical instillation of 0.01 μl of poly-L-lysine solution (0.1 mg / ml). After 30 min, MB49 mouse bladder cancer cells (2 × 10 in 50 μl medium) were added. 5 Thin On days 3, 5, 7, 9, and 11, the mice were instilled with α1-oleate (1.7 mM or 8.5 mM), chemotherapy drugs (MMC 25 μg, epirubicin 25 μg), α Patients were randomly assigned to receive either a combination of 1-oleate and chemotherapy or a PBS sham treatment group. The catheter was left in place for approximately 1 minute, but this was not necessary as the mouse was kept under anesthesia. The time until the quality disappeared (retention time) was set to 2 to 3 hours. A group of 5-6 mice was sacrificed after 12 days. A group of 5-8 mice was observed for 4 weeks. The bladders were imaged and processed for histology or RNA extraction. Two independent experiments were performed for each condition. All experiments were performed with mycoplasma-free cells. It was done with cells.

[0085] Histological examination The bladders were embedded in OCT compound (VWR) and serial 5 μm sections were taken through the center of each bladder. and placed on a positively charged microscope slide (Superfrost / Plus; T Hermo Fisher Scientific). Hematoxylin and eosin (H& E) For staining, use Richard-Allan Scientific Signature Use Leeds' Hematoxylin 7211, followed by Eosin-Y7111 for counterstaining. Images were acquired using an AX10 microscope (Carl Zeiss). For product analysis, tumor circumference was measured using ImageJ software.

[0086] Gene expression analysis Frozen bladder tissue was pulverized using liquid nitrogen. (Tran T. Hien1,A mbite, I., Lam Yim Wan, Butler, D., Tran T. H. iep, Hoeglund, U., Babjuk, M. and Svanborg, C. Bladder cancer treatment without toxic ity - A dose-escalation study of alpha1- oleate.International Journal of Cancer.i n press) total RNA was extracted (RNeasy Mini kit, Qiagen ) Using the GeneChip 3´IVT Express kit, 100ng of total The RNA was amplified and then fragmented. The labeled aRNA was then used to Hybridization was performed on 430PM array strips (Affymetrix) at 45°C for 16 hours. The cells were then lysed, washed, stained, and then analyzed (Applied Biosystems, Ther moFisher Scientific), GeneAtlas system (Af All samples were scanned using the MRI scanner (Fymetrix). The samples passed internal quality control tests (signal strength based on signal-to-noise ratio, hybridization time, etc.). (Sample quality according to GAPDH signal and 3'-5' ratio <3). Riptome Analysis Console software (v.4.0.1.3 6, Applied Biosystems, ThermoFisher Scient The transcriptome data were analyzed using a robust multi-average method implemented in The data were normalized. Tumor-bearing bladders or treated healthy bladders were compared with untreated healthy bladders. Fold changes were calculated by comparing with control tissues. Relative expression levels were analyzed and absolute values ​​were Genes with a fold change >2.0 were considered differentially expressed. Heat maps were constructed using Ingenuity Pathway Analysis (I Differentially expressed genes were functionally characterized using Qiagen (Qiagen) software. I added it.

[0087] statistical analysis Results are presented as mean ± standard error. P values ​​were calculated using Prism version 7 (GraphP Ad Software Inc.) to perform Student's t-test or one-way analysis. Calculated by ANOVA followed by Bonferroni's post-hoc test. p<0.0 5 was accepted as statistically significant. *p<0.05; **p<0.01; ***p<0. 001.

[0088] result Effect of combination therapy with α1-oleate and mitomycin The effects of α1-oleate and mitomycin C on the murine MB49 bladder cancer model Bladder cancer was investigated by intravesical instillation of MB49 cells (see Figure 19A for study design). Treatment groups were administered α1-oleate (1.7 mM) or α1-ol ... or 8.5 mM), MMC (0.1 mL, 25 μg / dose), or α1-oleate A mixture of acetaminophen (1.7 mM or 8.5 mM) and MMC (0.1 mL, 25 μg / dose) The combination was administered to sham-treated mice on days 3, 5, 7, 9, and 11. The bladders were collected at the time of death on day 12, and tumor formation was assessed by measuring bladder weight, bladder size, and The tumor area was quantified as tumor size, tumor size, and pathological score (Fig. 21c to Fig. 21f). , and quantified in H&E stained whole bladder tissue sections (Figure 22).

[0089] Sham-treated mice developed large tumors filling the bladder lumen after 12 days (12 / 12 Mice, Figure 21b). The therapeutic efficacy of α1-oleate and mitomycin C was compared with a sham-treated group. Tumor progression was significantly increased in a dose-dependent manner with α1-oleate alone (p<0.01, Figure 19c-c). 19f) and mitomycin C alone (0.1 mL, 25 μg / dose) This resulted in a delay (Figures 21 and 22).

[0090] The combined effect of mitomycin and α1-oleate was investigated by first administering α1-oleate to mice. Two hours later, the mice were inoculated with mitogen-activated ATP (1.7 or 8.5 μM). A second inoculation of Ishin was performed (see Figure 21a), and this procedure was repeated on days 3, 5, 7, 9, and 11. The treatment was repeated once a day in the 12th day of chemotherapy. The therapeutic effect was detected between the α1-oleate and α1-oleate (Fig. 21b). In mice given the combination with alpha-amycin C (p=0.03), a higher dose of alpha-amycin This was most pronounced in mice given 1-oleate (8.5 mM). No tissue was detected in treated mice at the time of death by visual inspection, and bladder weight and bladder size was not different from that of healthy bladders of control mice (p=0.99, Figure 21 The reduction in tumor area was confirmed by microscopic observation after H&E staining. No tumors were detected in any of the placement groups (p<0.001) (Figure 20). Since α1-oleate at M doses is effective, further enhancing the therapeutic effect of MMC combination therapy No further increase was detected at day 12 (p=0.99).

[0091] Effect of combination therapy with α1-oleate and epirubicin The mechanism of action of epirubicin is similar to that of mitomycin, involving DNA intercalation. These two compounds have similar toxicity profiles. Therefore, the following experiments were carried out to investigate the effects of α1-oleate and epirubicin alone and in combination. The therapeutic efficacy of the combination of epirubicin and α1-oleate was investigated (Figure 23a). The effect of α1-oleate was first examined by inoculating mice with α1-oleate (1.7 or 8.5 μM). Two hours later, the mice received a second inoculation of epirubicin (see Figure 23a). The technique was repeated once a day on the 3rd, 5th, 7th, 9th, and 11th days, and then the victim died on the 12th day. made him do so.

[0092] Tumor formation was determined by bladder weight, bladder size, tumor size, and pathology score. As shown, epirubicin delayed the progression of leukemia (p<0.001 compared to sham-treated controls). ) (Fig. 22c-22f). Reduction of tumor area was confirmed by H&E staining of whole bladder tissue sections. The therapeutic effect of epirubicin alone was comparable to that of α1-oleate (1.7 At higher doses (8.5 mM), α1-oleate was more effective. The results were significant (Figure 24, p=0.004).

[0093] A strong synergistic effect was detected between epirubicin and α1-oleate. Examination revealed no urinary bladder mass in treated mice at the time of death, and bladder weight and size were significantly higher than in control mice. The bladder tissue samples were not different from those of healthy bladders of control mice (Figs. 23c-23e and 21c-21d). The reduction in tumor area was confirmed by H&E staining, and no tumor tissue was detected (Figure 21e). Figure 24). The 8.5 mM dose was more effective than 1.7 mM as a standalone treatment. (p=0.001), but did not significantly increase the therapeutic effect of the MMC combination therapy after 12 days. (p=0.62).

[0094] This result suggests that α1-oleate treatment inhibits the chemotherapeutic agent mitochondria to the point where tumor formation is suppressed. This suggests that it enhances the effects of cyclosporine and epirubicin.

[0095] Long-term follow-up The duration of the therapeutic effect was assessed by following the mice for a total of 4 weeks (Figure 28). Treatment groups were not followed beyond 12 days when they were sacrificed due to rapid tumor growth. However, long-term protection was observed in the treatment group. After 4 weeks, low doses of α1-oleate Mice given methicone (1.7 mM), MMC, or epirubicin showed increased tumor growth. However, in the combination therapy group, mice maintained protection with no evidence of tumor recurrence (Figure 1). 25 and 26). The combination of low doses of α1-oleate (1.7 mM) with MMC or epirubicin ( The effect of combining MMC with epirubicin (25 μg / dose) was greater than that of MMC or epirubicin alone. was potent (p<0.001 compared with MMC or epirubicin), but at higher doses The effect was comparable to that of α1-oleate (8.5 μM) and showed no significant difference. Further protection was observed in mice treated with a combination of α-oleate and epirubicin. (Figure 27).

Claims

1. A first chemotherapeutic agent and a second chemotherapeutic agent for use in combination cancer treatment. The second chemotherapeutic agent comprises a biologically active conjugate having antitumor activity, The biologically active complex is a peptide of at least 10 amino acids containing an alpha helix structure, and From oleic acid or oleate, at least three oleic acids or oleates per peptide molecule a first chemotherapeutic agent and a second chemotherapeutic agent, wherein: a is an oleate molecule;

2. A first chemotherapeutic agent for use in the treatment of cancer, said first chemotherapeutic agent comprising to be administered to a subject who is to be administered, has been administered, or is currently administering a second chemotherapeutic agent. wherein the second chemotherapeutic agent is a biologically active compound having antitumor activity. and the biologically active complex comprises: a peptide of at least 10 amino acids containing an alpha helix structure, and From oleic acid or oleate, at least three oleic acids or oleates per peptide molecule and a ratio of oleate molecules to the first chemotherapeutic agent.

3. A second chemotherapeutic agent for use in the treatment of cancer, said second chemotherapeutic agent comprising to be administered to, or has been administered to, or is currently administering a first chemotherapeutic agent. wherein the second chemotherapeutic agent is a biologically active compound having antitumor activity. and the biologically active complex comprises: a peptide of at least 10 amino acids containing an alpha helix structure, and From oleic acid or oleate, at least three oleic acids or oleates per peptide molecule and a second chemotherapeutic agent consisting of a ratio of oleate molecules.

4. The first chemotherapeutic agent may be an intravesical chemotherapeutic agent, a topical chemotherapeutic agent, a DNA-interacting chemotherapeutic agent, or a chemotherapeutic agents, DNA alkylating chemotherapeutic agents, and / or DNA crosslinking chemotherapeutic agents. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of claims 1 to 3.

5. The first chemotherapeutic agent is atezolizumab, avelumab, bacillus Calmette-Guerin, venom Basizumab, carbozantinib, cephalexin, ciprofloxacin, cisplatin, Doxorubicin hydrochloride, durvalumab, eflornithine, epirubicin, erdaphycitin nib, erlotinib, fenretinide, gemcitabine, gefitinib, lapatinib, myeloid leukemia Tomycin C, nivolumab, pazobanib, pembrolizumab, Pamycin, selenium, sorafenib, thiotepa, urocidin, bal Rubicin, and Vicinium, preferably Thiotepa, Mitomycin C, Calmette-Guérin or epirubicin, more preferably mitomycin C or epirubicin. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of claims 1 to 4, chemotherapy agents.

6. The peptide in the biologically active complex is a protein having membrane-perturbing activity. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of claims 1 to 5, Therapeutic agents.

7. The peptide in the biologically active complex, when present in the complex, is non- of complex peptides １ The width of the complex is smaller than the corresponding width in the H NMR peak. At least some １ The non-H NMR peaks were significantly increased in width as indicated by the increased peak width. Claims 1 to 6, wherein the structural fluidity in the three-dimensional structure is increased compared to the peptide of the complex. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of the above.

8. The peptide in the biologically active complex has no cysteine ​​residues. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of claims 1 to 7.

9. The peptide in the biologically active complex may be up to 50 amino acids in length. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of claims 1 to 8, 。

10. The peptide in the biologically active complex is α-lactalbumin or SA R-1, or a variant or fragment thereof, preferably an N-terminal fragment thereof. The first chemotherapeutic agent and / or the second chemotherapeutic agent according to any one of claims 1 to 9.

11. The peptide in the biologically active complex is any one of SEQ ID NOs: 1 to 4, or any one of claims 1 to 10, comprising or consisting of a variant or fragment thereof The first chemotherapeutic agent and / or the second chemotherapeutic agent according to KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGY DTQ (SEQ ID NO: 1) MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO: 2) KQFTKAELSQ LLKDI (SEQ ID NO: 3) MAGWDIFGWF RDVLA (SEQ ID NO: 4).

12. The peptide in the biologically active complex is any one of SEQ ID NOs: 1 to 4, preferably The first chemotherapeutic agent according to any one of claims 1 to 11, preferably consisting of SEQ ID NO: 1; and / or a second chemotherapeutic agent.

13. The use may be for the treatment or prevention of carcinoma, lymphoma, or brain tumors, preferably gastrointestinal cancer, mucosal cancer, Treatment or prevention of membranous cancer, bladder cancer, kidney cancer, lung cancer, glioblastoma, and skin papilloma, more preferably The first chemical compound according to any one of claims 1 to 12, preferably for the treatment or prevention of bladder cancer. Therapeutic agent and / or second chemotherapeutic agent.

14. a first chemotherapeutic agent, a second chemotherapeutic agent, and a pharmaceutically acceptable carrier, excipient, and and / or an adjuvant, wherein the second chemotherapeutic agent has antitumor activity. and a biologically active complex having a peptide of at least 10 amino acids containing an alpha helix structure, and From oleic acid or oleate, at least three oleic acids or oleates per peptide molecule A pharmaceutical composition comprising:

15. 15. The pharmaceutical composition of claim 14 for use in cancer treatment.

16. The first chemotherapeutic agent is mitomycin, preferably mitomycin C. Item 16. The pharmaceutical composition according to item 14 or 15.

17. 16. The pharmaceutical composition of claim 14 or 15, wherein the first chemotherapeutic agent is epirubicin. thing.

18. The second chemotherapeutic agent comprises a biologically active conjugate having antitumor activity, The biologically active complex is A peptide having or comprising the following peptide sequence: KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGY DTQ From oleic acid or oleate, at least three oleic acids or oleates per peptide molecule The pharmaceutical composition according to any one of claims 14 to 17, wherein the ratio of oleate molecules is:

19. A method of treating or preventing cancer, comprising administering to a subject in need of treatment or prevention a second chemical administering a first chemotherapeutic agent in conjunction with a therapeutic agent, wherein said second chemotherapeutic agent is an anti-tumor and a biologically active complex having an activity, the biologically active complex comprising: a peptide of at least 10 amino acids containing an alpha helix structure, and From oleic acid or oleate, at least three oleic acids or oleates per peptide molecule is the ratio of oleate molecules.

Images