Composition comprising oncolytic peptide and chitosan

Abstract

The present disclosure relates to compositions, kits, and uses thereof by treating a subject having a tumor, such as a patient, by administering such compositions to the subject.

Description

Technical Field

[0001] The present disclosure relates to compositions, kits, and uses thereof for treating a subject, such as a patient, having a tumor by administering such compositions to the subject. Background Art

[0002] Oncolytic peptides are efficient anticancer agents. These peptides not only have a direct lysis effect on tumor cells by disrupting and permeabilizing cell membranes, but they are also very effective in attacking organelles such as mitochondria and lysosomes, and can cause their cracking. This destruction of organelle membranes causes the release of reagents with effective immunostimulatory functions, and such reagents are commonly referred to as damage-associated molecular pattern molecules (DAMPs), and include ATP, cytochrome C, mitochondrial CpG DNA sequences, mitochondrial formyl peptides, cathepsins (from lysosomes) and HMGB1 (from nuclei). Therefore, these oncolytic peptides stimulate the immune system and trigger the immune response that produces long-term antitumor immunity. The immune stimulation that occurs when releasing DAMP and tumor antigens and the oncolytic peptides to the tumor by being injected into the tumor can enhance antigen presentation, thereby resulting in a kind of in situ " vaccination " for tumors.

[0003] Although oncolytic peptides are promising tools in cancer immunotherapy, there is still room for improvement in their application in cancer treatment. Summary of the Invention

[0004] In a first aspect, the present disclosure relates to a combination comprising: a composition (a) comprising an oncolytic peptide; and a composition (b) comprising chitosan and / or a chitosan derivative, for treating a subject having a tumor, or to a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative for treating a subject having a tumor.

[0005] In some embodiments, compositions (a) and (b) are used for treatment simultaneously. In some other embodiments, compositions (a) and (b) are used for treatment sequentially. In still other embodiments, compositions (a) and (b) are combined for treatment into a single composition, i.e., a composition comprising: i) an oncolytic peptide, and ii) chitosan and / or a chitosan derivative.

[0006] Therefore, in some embodiments, the first aspect of the present disclosure relates to a combination comprising: a composition (a) comprising an oncolytic peptide; and a composition (b) comprising chitosan and / or a chitosan derivative, wherein composition (a) and composition (b) are used simultaneously, sequentially or in combination in the treatment of a subject having a tumor, or the first aspect of the present disclosure relates to a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative, wherein composition (a) and composition (b) are used simultaneously, sequentially or in combination in the treatment of a subject having a tumor, for example, wherein composition (a) and composition (b) are administered to the subject simultaneously, sequentially or as a combined composition.

[0007] In an alternative first aspect, the present disclosure relates to a composition (a) comprising an oncolytic peptide for use in treating a subject suffering from a tumor by simultaneous, sequential or combined administration with a composition (b) comprising chitosan and / or a chitosan derivative.

[0008] In yet another alternative first aspect, the present disclosure relates to a method of treating a subject having a tumor, the method comprising administering to the subject simultaneously, sequentially or in combination a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative.

[0009] In another alternative first aspect, the present disclosure relates to the use of a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative for treating a subject suffering from a tumor. In some embodiments, composition (a) and composition (b) are used to treat the subject simultaneously, sequentially, or as a combined composition, for example, composition (a) and composition (b) are administered to the subject simultaneously, sequentially, or as a combined composition.

[0010] In some embodiments, the above composition comprises a physiologically acceptable carrier.In some embodiments, composition (a) is administered into the tumor and composition (b) is administered into the tumor and / or into the tissue immediately surrounding the tumor.

[0011] In some embodiments, the aforementioned composition is a pharmaceutical composition.

[0012] In some embodiments, a composition comprising an oncolytic peptide and a composition comprising chitosan and / or a chitosan derivative are combined into one composition.

[0013] Thus, in some embodiments, the present disclosure relates to a composition for treating a subject having a tumor, comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative. In some embodiments, the composition is administered to the subject, for example, to a tumor in the subject.

[0014] Alternatively, the present disclosure relates to a method of treating a subject having a tumor, comprising administering to the subject a composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative. In some embodiments, the composition is administered into the tumor.

[0015] In another alternative, the present disclosure relates to the use of i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative in the preparation of a medicament for treating a subject having a tumor. In some embodiments, the medicament is administered to the subject, for example, to a tumor in the subject.

[0016] In yet another alternative, the present disclosure relates to a composition for treating a subject having a tumor, comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative. In some embodiments, the composition is administered to the subject, for example, to a tumor in the subject.

[0017] In yet another alternative, the present disclosure relates to a medicament for treating a tumor in a subject by administering to the subject a medicament, wherein the medicament comprises i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative. In some embodiments, the medicament is administered into the tumor.

[0018] In some embodiments, the aforementioned composition comprises a physiologically acceptable carrier. In some embodiments, the aforementioned composition is a pharmaceutical composition.

[0019] In another aspect, the present disclosure relates to a composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative.

[0020] In some embodiments, the composition comprises a physiologically acceptable carrier.

[0021] In some embodiments, the composition is a pharmaceutical composition.

[0022] In yet another aspect, the present disclosure relates to a composition for use as a medicament comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative.

[0023] In some embodiments, the composition comprises a physiologically acceptable carrier.

[0024] In some embodiments, the composition is a pharmaceutical composition.

[0025] In yet another aspect, the present disclosure relates to a method of preparing a composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative.

[0026] In some embodiments, the composition comprises a physiologically acceptable carrier and / or is sealed in a container.

[0027] In some embodiments, the composition is a pharmaceutical composition.

[0028] In yet another aspect, the present disclosure relates to a kit comprising a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative, or comprising parts / components for preparing composition (a) and composition (b).

[0029] In yet another aspect, the present disclosure relates to a kit comprising parts / components for preparing a composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of Example 3 are shown, i.e., the therapeutic efficacy of a composition comprising LTX-401 and chitosan in tumor-bearing mice compared to a composition comprising LTX-401 alone, a composition comprising LTX-401 and HPMC, or a control group. Therapeutic efficacy is expressed as the percentage of mice with a tumor volume less than 1 mL / day from the start of treatment.

[0031] Figure 2 The results of Example 4 are shown, i.e., the therapeutic effect of a composition comprising LTX-401 and chitosan in tumor-bearing mice compared to a composition comprising LTX-401 alone, a composition comprising chitosan alone, or a control group. Therapeutic efficacy is expressed as the percentage of survival per day after tumor challenge. DETAILED DESCRIPTION

[0032] A "subject" is an animal, such as a mouse, or a human, preferably a human. A subject can be a patient, i.e., a person suffering from cancer who is in need of treatment for the cancer or who is in need of treatment to prevent the recurrence of the cancer. The terms "subject" and "individual" are used interchangeably herein.

[0033] As used herein, "cancer" refers to a broad range of diseases characterized by the uncontrolled growth of abnormal cells in the body, resulting in tumors. "Tumor" or "cancerous tissue" or "(cancerous) lesions" include primary tumors and metastases. Tumors include solid tumors and non-solid tumors.

[0034] As used herein, "treatment" is therapeutic treatment.

[0035] "Therapeutic treatment" is treatment administered to a subject who exhibits signs or symptoms of cancer with the goal of alleviating or eliminating those signs or symptoms or slowing or stopping the progression of the disease, e.g., stopping or delaying the growth of a tumor or its increase in malignancy or its spread.

[0036] As used herein, the term "oncolytic peptide" refers to a positively charged amphiphilic amino acid derivative, peptide, or peptidomimetic that has an oncolytic effect, i.e., a positively charged amphiphilic amino acid derivative, peptide, or peptidomimetic that is capable of lysing tumor cell membranes. Because they are amphiphilic in nature, they have one or more hydrophilic groups, i.e., cationic groups, and one or more lipophilic groups. Therefore, these molecules a) are attracted to the negative charge of the tumor cell membrane and b) are able to interact with the lipophilic tail of the phospholipids contained in the tumor cell membrane through their lipophilic groups, which tail is composed of fatty acid chains.

[0037] Oncolytic peptides

[0038] As described above, the oncolytic peptides used in the present disclosure refer to positively charged amphiphilic amino acid derivatives, peptides or peptidomimetics with oncolytic effects. Oncolytic peptides have been described in patent publications WO 2007 / 107748 A2, WO 2010 / 060497 A1, WO 2011 / 051692 A1, WO 2016 / 091487 A1 and WO 2016 / 091490 A1, the contents of which are incorporated herein in their entirety.

[0039] Nonapeptide

[0040] In some embodiments, the oncolytic peptide used in the present disclosure is a nonapeptide or a pharmaceutically acceptable salt thereof, wherein the nonapeptide consists of 9 linearly arranged amino acids, wherein 5 of the 9 amino acids are cationic amino acids and 4 are lipophilic amino acids, and among the 4 lipophilic amino acids, 3 lipophilic amino acids are tryptophan and 1 lipophilic amino acid is a non-genetically encoded amino acid.

[0041] In some embodiments, in the nonapeptide described above, the lipophilic amino acids and cationic amino acids are arranged so that no more than two amino acids of either type are adjacent to each other and / or the peptide comprises two adjacent pairs of cationic amino acids and one or two adjacent pairs of lipophilic amino acids.

[0042] The term "amino acid" as used to define nonapeptides includes alpha, beta and gamma amino acids, as well as N-substituted glycines.

[0043] "Non-genetically encoded amino acids" are amino acids that differ structurally, not just stereospecifically, from the genetically encoded L amino acids. As disclosed herein, D amino acids, while not strictly genetically encoded amino acids, are not therefore considered non-genetically encoded amino acids.

[0044] Cationic amino acids can be the same or different. In some embodiments, the cationic amino acid is lysine and / or arginine. In some other embodiments, the cationic amino acid is histidine and / or a positively charged non-genetically encoded amino acid at pH 7.0. Lysine and arginine are preferred cationic amino acids.

[0045] Non-genetically encoded cationic amino acids include derivatives of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, homoarginine, trimethyllysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamoylimidopiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0046] The non-genetically encoded lipophilic amino acids may be the same or different.

[0047] In some embodiments, the non-genetically encoded lipophilic amino acid has a lipophilic side chain having at least 7 non-hydrogen atoms, preferably at least 8 non-hydrogen atoms or 9 non-hydrogen atoms, more preferably at least 10 non-hydrogen atoms, preferably carbon atoms. In some embodiments, the lipophilic side chain contains no more than 30 non-hydrogen atoms, more preferably no more than 25 non-hydrogen atoms. In some embodiments, the lipophilic side chain comprises at least one, preferably two, cyclic groups, which may be fused or linked.

[0048] In some embodiments, the lipophilic side chain contains a heteroatom such as O, N or S, but generally no more than one heteroatom, preferably nitrogen. The lipophilic side chain will preferably have no more than two polar groups, more preferably no polar groups or one polar group, and most preferably no polar groups.

[0049] Preferred non-genetically encoded lipophilic amino acids include: 2-amino-3-(biphenyl-4-yl)propionic acid (biphenylalanine), 2-amino-3,3-diphenylpropionic acid (diphenylalanine), 2-amino-3-(anthracen-9-yl)propionic acid, 2-amino-3-(naphthalen-2-yl)propionic acid, 2-amino-3-(naphthalen-1-yl)propionic acid, 2-amino-3-[1,1':4',1"-terphenyl-4-yl]-propionic acid, 2-amino-3-(2,5,7-tri-tert-butyl-1H-indole-3- The present invention also provides a kind of non-genetically encoded lipophilic amino acid, which is preferably selected from the group consisting of 2-amino-3-[1,1':3',1"-terphenyl-4-yl]-propionic acid, 2-amino-3-[1,1':3',1"-terphenyl-4-yl]-propionic acid, 2-amino-3-[1,1':2',1"-terphenyl-4-yl]-propionic acid, 2-amino-3-(4-naphthalen-2-yl-phenyl)-propionic acid, 2-amino-3-(4'-butylbiphenyl-4-yl)propionic acid, 2-amino-3-[1,1':3',1"-terphenyl-5'-yl]-propionic acid, and 2-amino-3-(4-(2,2-diphenylethyl)phenyl)propionic acid. Diphenylalanine and biphenylalanine are particularly preferred non-genetically encoded lipophilic amino acids.

[0050] In some embodiments, the aforementioned peptides may have some or all of the amino acids therein present in the D form.

[0051] In a preferred embodiment, the nonapeptide is a compound of the following formula (I) to formula (V), wherein C represents a cationic amino acid as defined above and L represents a lipophilic amino acid as defined above. The amino acids are covalently linked via peptide bonds. The free amino terminus or the carboxyl terminus of these molecules may be modified, the carboxyl terminus preferably being modified to remove its negative charge, most preferably the carboxyl terminus being amidated, and the amide group may be substituted.

[0052] CCLLCCLLC(I) (SEQ ID NO: 1)

[0053] LCCLLCCLC(II) (SEQ ID NO: 2)

[0054] CLLCCLLCC(III) (SEQ ID NO: 3)

[0055] CCLLCLLCC(IV) (SEQ ID NO: 4)

[0056] CLCCLLCCL (V) (SEQ ID NO: 5)

[0057] As described above, the peptide comprises a non-genetically encoded lipophilic amino acid. When the amino acid is represented by L', the preferred peptide is represented by the following formula:

[0058] CCL'LCCLLC(I') (SEQ ID NO: 6)

[0059] CCLLCCLL'C(I”)(SEQ ID NO:7)

[0060] CCLL'CCLLC(I"')(SEQ ID NO: 8)

[0061] LCCLL'CCLC(II')(SEQ ID NO:9)

[0062] Particularly preferred are the nonapeptides of formula (I) and formula (II), among which the peptide of formula (I") is particularly preferred.

[0063] The nonapeptides listed in the table below are most preferred:

[0064]

[0065]

[0066]

[0067] in:

[0068] Standard single-letter codes are used to represent gene-encoded amino acids

[0069] Lowercase letters represent D amino acids

[0070] Dip stands for diphenylalanine

[0071] Bip is biphenylalanine

[0072] Orn is ornithine

[0073] Dap is 2,3-diaminopropionic acid

[0074] Dab is 2,4-diaminobutyric acid

[0075] 1-Nal is 1-naphthylalanine

[0076] 2-Nal is 2-naphthylalanine

[0077] Ath is 2-amino-3-(anthracen-9-yl)propionic acid

[0078] Phe(4,4'Bip) stands for 2-amino-3-[1,1':4',1"-terphenyl-4-yl]propanoic acid

[0079] Preferred nonapeptides are LTX-302, LTX-313, LTX-315, LTX-320 and LTX- 329. The preferred peptides are preferably in the form of a pharmaceutically acceptable salt, preferably in the form of an acetate salt.

[0080] For use in the present invention, all nonapeptides described herein may be in the form of pharmaceutically acceptable salts. These peptides preferably have a modified, particularly amidated, C-terminus. Suitable pharmaceutically acceptable salts are well known in the art and include salts of inorganic or organic acids, such as hydrochlorides, trifluoroacetates, and acetates. Acetate is most preferred.

[0081] Particularly preferred is the nonapeptide known as LTX-315, in particular in the form of a pharmaceutically acceptable salt, preferably in the form of its acetate salt, such as LTX-315 having the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt thereof, preferably its acetate salt.

[0082] The nonapeptides and pharmaceutically acceptable salts thereof disclosed herein can be synthesized in any convenient manner by peptide synthesis methods known in the art, preferably by synthesis on a solid phase support. Synthesis methods are described, for example, in WO 2010 / 060497A1, WO 2016 / 091487A1, and WO 2016 / 091490A1.

[0083] Compounds containing disubstituted beta amino acids

[0084] In some embodiments, the oncolytic peptides used in the present disclosure are peptides, peptidomimetics, or amino acid derivatives having a net positive charge of at least +2 and comprising disubstituted β-amino acids. Each substituent in the β-amino acid, which may be the same or different, contains at least 7 non-hydrogen atoms, is lipophilic, and has at least one cyclic group, and one or more cyclic groups within the substituent may be connected or fused to one or more cyclic groups within another substituent, and when the cyclic groups are fused in this manner, the total number of non-hydrogen atoms of the two substituents combined is at least 12. The two substituents on the β-amino acid are preferably the same.

[0085] The amino group of a beta amino acid is attached to the beta carbon atom; the amino acid encoded by the gene is an alpha amino acid, in which the amino group is attached to the alpha carbon atom. This structure allows the backbone of a peptide containing one or more beta amino acids to be extended by one atom at each beta amino acid. In this structure, the alpha carbon atom and / or the beta carbon atom can be substituted. Either the alpha carbon atom or the beta carbon atom can be disubstituted; when the alpha carbon atom is disubstituted, the resulting peptide is a beta carbon atom. 2,2 Amino acids, when the β carbon atom is disubstituted, generate β 3,3 Amino acids. If there is a substituent on each α carbon atom or each β carbon atom, a β 2,3 According to the present invention, β 2,2 and β 3,3 Disubstituted amino acids are preferably used, β 2,2 Disubstituted amino acids are particularly preferred.

[0086] The β-amino acid is substituted by two groups comprising at least 7 non-hydrogen atoms. Preferably, one of the two substituents, more preferably both of the two substituents, contains at least 8, more preferably at least 10 non-hydrogen atoms. These groups are lipophilic in nature and, although they may be different, are preferably the same. Each group contains at least one cyclic group, typically a 6-membered ring, which may be aliphatic or aromatic, preferably aromatic, and may be substituted, and the substituents may include heteroatoms such as oxygen, nitrogen, sulfur or halogen, especially fluorine or chlorine. Preferred substituents include C1-C4 alkyl (especially tert-butyl), methoxy, fluorine and fluoromethyl. The cyclic group may be homocyclic or heterocyclic, preferably homocyclic of carbon atoms. Preferred lipophilic substituents contain two or three cyclic groups, preferably two cyclic groups, which may be connected or fused, preferably fused. Particularly preferred substituents include naphthyl.

[0087] Further preferred lipophilic substituent groups have a single substituted or unsubstituted cyclic group, preferably phenyl or cyclohexyl.

[0088] The cyclic group is usually separated from the peptide backbone (i.e., from the α carbon atom or β carbon atom of the β amino acid) by a chain consisting of 1 to 4 atoms, preferably 1 to 3 atoms; these connecting atoms may include nitrogen and / or oxygen, but are usually carbon atoms, preferably these connecting atoms are not substituted. These spacers are of course part of the substituents as defined herein.

[0089] Each substituent portion of the disubstituted beta amino acid typically contains 7 to 20 non-hydrogen atoms, preferably 7 to 13 non-hydrogen atoms, more preferably 8 to 12 non-hydrogen atoms, and most preferably 9 to 11 non-hydrogen atoms.

[0090] The compounds comprising disubstituted β amino acids are preferably peptides or peptidomimetics of 1 or 2 to 12 amino acids or equivalent subunits in length. Unless the context clearly indicates otherwise, references to "amino acids" herein include equivalent subunits in peptidomimetics. Preferred compounds comprise 3 to 12 amino acids, more preferably 5 to 12 amino acids. In some embodiments, the compounds comprising disubstituted β amino acids consist of such disubstituted β amino acids, i.e., are amino acid derivatives (i.e., modified amino acids).

[0091] Compounds comprising disubstituted β-amino acids preferably include a modified C-terminus, where the C-terminal modification group typically results in a charge reversal, i.e., removal of the negative charge of the carboxyl group and addition of a positive charge, such as by the presence of an amino group. Assuming the N-terminus is unmodified, such modification alone will result in a net charge of +2 for the molecule as a whole. Regardless of whether the C-terminus is modified to produce a charge reversal or simply to remove the negative charge of the carboxyl group, in some embodiments where the compound is a peptide or peptidomimetic, such compounds also contain one or more cationic amino acids. Thus, the total charge of the compound can be +3, +4, or higher.

[0092] Suitable C-terminal groups, which are preferably cationic in nature, generally have a maximum dimension of no more than 15 non-hydrogen atoms. The C-terminus is preferably amidated, and the amide group may be further substituted to form N-alkyl or N,N-dialkylamides. Primary and secondary amide groups are preferred. Suitable groups to replace the amide group include aminoalkyl groups, such as aminoethyl or dimethylaminoethyl; the nitrogen atom of the amide group may be formed as part of a cyclic group, such as pyrazolidine, piperidine, imidazolidine, piperazine, etc., with piperazine being preferred; these cyclic groups may themselves be substituted, for example, with alkyl or aminoalkyl groups.

[0093] In some embodiments, the compound comprising a disubstituted β amino acid is a peptide comprising one or more cationic amino acids. Lysine, arginine, ornithine, and histidine are preferred, but any non-genetically encoded or modified amino acid that carries a positive charge at pH 7.0 may also be included.

[0094] Suitable non-genetically encoded cationic amino acids and modified cationic amino acids include analogs of lysine, arginine, and histidine, such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, and homoarginine, as well as trimethyllysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamoylimidopiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0095] In some embodiments, the compound comprising a disubstituted β amino acid is a dipeptide, which typically includes one cationic amino acid. Longer peptides typically include additional cationic amino acids, so a peptide of 4 or 5 amino acids may have 2 or 3 cationic amino acids, while a peptide of 6 to 9 amino acids may have 3 to 6 cationic amino acids.

[0096] In some embodiments, the compound comprising a disubstituted beta amino acid comprises a beta amino acid linked to a C-terminal L-arginine amide residue. 2,2 Disubstituted amino acids, and dipeptides having this structure are preferred.

[0097] Peptides having three or more amino acids typically have one or more additional lipophilic amino acids, i.e., amino acids having lipophilic R groups. Typically, the lipophilic R groups have at least one, preferably two, cyclic groups, which may be fused or linked. The lipophilic R groups may contain heteroatoms such as O, N, or S, but typically no more than one heteroatom, preferably nitrogen. The R groups will preferably have no more than two polar groups, more preferably no polar groups or one polar group, and most preferably no polar groups.

[0098] Tryptophan is the preferred lipophilic amino acid, and the peptide preferably contains 1 to 3 tryptophan residues. Genetically encoded lipophilic amino acids that may be included also include phenylalanine and tyrosine.

[0099] The lipophilic amino acid can be non-genetically encoded, including genetically encoded amino acids with modified R groups.

[0100] The typical characteristics of peptidomimetics are that they retain the polarity, three-dimensional size and functionality (biological activity) of their corresponding peptides, but the peptide bonds have been replaced by bonds that are generally more stable. "Stable" refers to being more resistant to enzymatic degradation by hydrolytic enzymes. Generally, the bonds that replace the amide bonds (amide bond substitutes) retain many properties of the amide bond, such as conformation, spatial volume, electrostatic properties, the possibility of forming hydrogen bonds, etc. The design and synthesis techniques of peptidomimetics are generally discussed in Chapter 14 of "Drug Design and Development", edited by Krogsgaard, Larsen, Liljefors and Madsen and published by Horwood Academic Publishers in 1996. In the present disclosure, when the oncolytic peptides / compounds containing disubstituted β amino acids interact with the tumor cell membrane, rather than acting on the specific active site of a certain enzyme, some of the previously described issues about accurately simulating affinity, efficacy or substrate function are no longer applicable. Therefore, peptidomimetics can be easily prepared according to a specific peptide structure or desired functional group pattern. Suitable amide bond substitutes include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46, 47), retro-inverse amide (Chorev, M and Goodman, M., Acc. Chem. Res., 1993, 26, 266), thioamide (Sherman DB and Spatola AF, J. Am. Chem. Soc., 1990, 112, 433), thioester, phosphonate, ketomethylene (Hoffman, RV and Kim, HOJ Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl (Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl, methyleneamino (Sasaki, Y and Abe, J., Chem. Pharm. Bull., 199745, 13), methylenethio (Spatola, AF, Methods Neurosci, 1993, 13, 19), alkane (Lavielle, S. et al., Int. J. Peptide Protein Res., 1993, 42, 270) and sulfonamide (Luisi, G. et al., Tetrahedron Lett., 1993, 34, 2391).

[0101] In some embodiments, the compound comprising a disubstituted β amino acid contains only this disubstituted β amino acid and one other amino acid, i.e., these compounds have two amino acids connected by an amide bond. Due to the amide bond, it can be considered that such molecules are dipeptides. However, the amide bond in these compounds is actually not cut due to the disubstituted structure of the β amino acid, so it can be considered that these compounds are peptidomimetics. For the purposes of this disclosure, due to the presence of amide bonds, such compounds (and larger molecules with more amino acids) are considered to be peptides rather than peptidomimetics. This makes the naming clear without the need to test whether or to what extent a given amide bond is cuttable. In other words, if all amino acids in a compound comprising a disubstituted β amino acid as described herein are connected by amide bonds, the compound is considered to be a peptide, even if one or more amide bonds are not easy to cut.

[0102] In some embodiments, the compound comprising a disubstituted β-amino group is a peptidomimetic, which generally has an identifiable subunit that is roughly equivalent in size and function to an amino acid. Peptidomimetics generally have groups that are equivalent to the R groups of amino acids, and the discussion herein of suitable R groups and N-terminal and C-terminal modification groups, after appropriate adjustments, also applies to peptidomimetic compounds.

[0103] As discussed in the textbooks referenced above, peptidomimetics can involve not only replacement of amide bonds, but also replacement of larger structural moieties with dipeptidyl or tripeptidyl structures. In this case, mimetic moieties involving peptide bonds, such as azole-derived mimetics, can be used as dipeptide replacements. However, preferred are those peptidomimetics that replace only the amide bond as described above, as well as peptidomimetic backbones.

[0104] Suitable peptidomimetics include reduced peptides in which the amide bond has been reduced to a methyleneamine by treatment with a reducing agent such as borane or a hydride reagent such as lithium aluminum hydride. Such reduction has the additional advantage of increasing the overall cationicity of the molecule.

[0105] Other peptidomimetics include, for example, those formed by the stepwise synthesis of amide-functionalized polyglycine. Some peptidomimetic backbones can be readily obtained from their peptide precursors, such as already fully methylated peptides. Suitable preparation methods are described by Ostresh, JM et al. in Proc. Natl. Acad. Sci. USA (1994) 91, 11138–11142. Strong alkaline conditions favor N-methylation over O-methylation, resulting in methylation of some or all of the nitrogen atoms in the peptide bonds as well as the N-terminal nitrogen atom.

[0106] Preferred peptide bond substitutes include esters, polyamines and their derivatives and substituted alkanes and alkenes, particularly aminomethyl and ketomethylene.The peptidomimetic will preferably have an N-terminus and a C-terminus, which may be modified as discussed herein.

[0107] In some embodiments, the compound comprising a disubstituted β-amino acid is a compound (ie, a peptide, peptidomimetic, or amino acid derivative) comprising a group of formula (VI):

[0108]

[0109] wherein X and N in formula (VI) have their normal valences and are therefore further substituted when attached to other parts of the compound, for example to other amino acids or N-terminal capping groups or C-terminal capping groups, and wherein any two of R1, R2, R3 and R4 are hydrogen atoms and the other two are substituents, the substituents being the same or different, the substituents comprising at least 7 non-hydrogen atoms, being lipophilic and comprising a cyclic group. The cyclic group is not directly attached to an α-carbon atom or a β-carbon atom but is optionally attached to or fused to a cyclic group in another substituent. When the cyclic groups are fused, the total number of non-hydrogen atoms in the two substituents combined is at least 12, and wherein X represents O, C, N or S.

[0110] It is understood that when the cyclic groups of each part are fused, R 1-4 The total number of non-hydrogen atoms in the two groups is a minimum of 12, which is obtained by adding the minimum number of unfused groups (7 + 7 = 14) and subtracting 2, because two non-hydrogen atoms in each group actually participate in the formation of the ring. Preferably, when the cyclic groups of each part are fused, R 1-4 The total number of non-hydrogen atoms in the two groups is 14. Complex fused and linked groups can be envisioned, where the atoms attached to the C α or C β The two groups of can contain more than one pair of fused cyclic groups, with or without additional connecting bonds between the substituents. However, the two substituents are preferably not fused or connected, as molecules in which these groups have the greatest flexibility to move are preferred.

[0111] The nitrogen atom in the group of formula (VI) is preferably not in contact with R 1-4 Any atom in the group is bonded, of course, through C β or C α Except for the case of indirect connection. The five atoms in the above skeleton (NC β -C α -CX) are preferably linked to one another only in a linear manner and not in a cyclic manner.

[0112] R 1-4 The substituents of R are generally lipophilic in nature, preferably uncharged, and more preferably have no more than two, more preferably no more than one polar group. 1-4 One or two substituents preferably contain at least 8 non-hydrogen atoms, more preferably at least 9 or 10 non-hydrogen atoms, for example 7 to 13, 7 to 12, 8 to 12 or 9 to 11 non-hydrogen atoms. These two substituents are preferably identical, and this is for the consideration of synthetic convenience. Preferably, these two substituents are respectively R1 and R2, or R3 and R4, wherein R3 and R4 are most preferred.

[0113] As mentioned above, R 1-4 The cyclic group in is not directly attached to the α-carbon atom or the β-carbon atom because it is separated from these carbon atoms by a chain of 1 to 4, preferably 1 to 3 atoms. These connecting atoms may include nitrogen atoms and / or oxygen atoms, but are generally carbon atoms, and preferably these connecting atoms are unsubstituted. Preferred spacer moieties are shown in the examples and constitute R as defined herein. 1-4 part of a substituent.

[0114] X can be substituted or unsubstituted, but is preferably an N atom and is preferably substituted. When X is N, it can form a part of an amide bond with other amino acid. Perhaps, this nitrogen-atoms can be substituted, for example, by aminoalkyl, such as aminoethyl or aminopropyl, or dimethylaminoethyl. In some embodiments, this nitrogen-atoms can be a part for cyclic groups such as piperazinyl, and this piperazinyl itself can be substituted by alkyl or aminoalkyl.

[0115] Compounds comprising a group of formula (VI) will preferably have a modified C-terminus, which is preferably amidated.

[0116] The above definitions of preferred substituents on β-amino acids also apply to R 1-4Two substituents. The compounds comprising the formula I group are a preferred subset of those compounds previously described in this application, so the compound characteristics defined previously, such as the length of the compound and the other amino acids it contains, also apply to these compounds defined by containing the formula (VI) group, and vice versa. Particularly preferred compounds are composed of 1 to 7 or 8 (e.g., 1 to 5) amino acids in length, more preferably 1, 2, 3 or 4 amino acids. The peptidomimetic molecule will include the same number of subunits, but these subunits will generally be connected by amide bond mimetics. Preferred modes of connection are as described above, including ester groups, aminomethyl groups and ketomethylene groups.

[0117] In some embodiments, the compound comprising a disubstituted β-amino acid is as described in WO 2011 / 051692 A1. Figure 1 In some other embodiments, the compound comprising a disubstituted β-amino acid is the amino acid derivatives 1-14 and amino acid derivatives 23-38 shown in Tables 2 and 4 to 7 in Example 2 and Figure 3 of WO 2011 / 051692 A1. In still other embodiments, the compound comprising a disubstituted β-amino acid is the heptapeptide 15-22 shown in Table 3 in Example 2 of WO 2011 / 051692 A1.

[0118] Compounds comprising disubstituted beta amino acids may be in the form of salts, may be cyclic or esterified, and may be amidated as described above.

[0119] Compounds comprising disubstituted beta amino acids may be synthesized in any convenient manner and such manners are known to those skilled in the art.

[0120] Compounds comprising disubstituted β-amino acids and their synthesis are described in detail in WO 2011 / 051692 A1, the contents of which are incorporated herein by reference.

[0121] In a preferred embodiment, the compound comprising a disubstituted β-amino acid is LTX-401, a compound of formula (VII),

[0122] It is preferably in the form of a pharmaceutically acceptable salt, more preferably in the form of its hydrochloride or acetate.

[0123] Chitosan and chitosan derivatives

[0124] Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4) linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units).

[0125] Chitosan is commercially produced by the deacetylation of chitin, a structural element in the exoskeletons of crustaceans such as crabs and shrimps, but can also be produced from certain types of fungi and algae. Its main characteristics are molecular weight and degree of acetylation (DA) or degree of deacetylation (DDA), which corresponds to the molar fraction of deacetylated units and acetylated units. The molecular weight range of most commercial chitosans is <50-2000 kDa, with an average DDA of 50-100%. Based on molecular weight, chitosan can be divided into very low molecular weight (usually <50 kDa), low molecular weight (usually 50 to <100 kDa), medium molecular weight (usually 100-1000 kDa) and high molecular weight (usually >1000 kDa). In some embodiments, chitosan used in the present disclosure has a DDA of about 50% to 99%, such as 70% to 99%, such as 80%, preferably about 70% and above, for example 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0126] The chitosan used in the present disclosure may be very low molecular weight chitosan, low molecular weight chitosan, medium molecular weight chitosan or high molecular weight chitosan, preferably very low molecular weight chitosan, low molecular weight chitosan or medium molecular weight chitosan.

[0127] Chitosan contains active functional groups that can undergo chemical reactions, and modification of chitosan by, for example, acylation, carboxylation, alkylation, and quaternization results in chitosan derivatives having properties different from chitosan, such as improved solubility or bioactivity, but retaining the unique original pharmacological properties of chitosan (see Wang et al., Int. J. Mol. Sci. 21(2), 2020, 487, doi: 10.3390 / ijms21020487).

[0128] The chitosan derivative used in the present disclosure may also be a salt of a chitosan derivative, such as a pharmaceutically acceptable salt of chitosan, such as hydrochloride. Hereinafter, the term "chitosan derivative" refers to a chitosan derivative and a pharmaceutically acceptable salt thereof.

[0129] Chitosan derivatives useful in the present disclosure include O- and N-acylated chitosans, O- and N-alkylated chitosans, chitosans modified by the introduction of hydrophilic groups such as carboxylic acid groups, quaternary ammonium groups, sulfonic acid groups, phosphoric acid groups, amino groups, ether linkages consisting of oxygen groups, hydroxyl groups, carboxylate groups, and block polyether groups.

[0130] In some embodiments, the chitosan derivative used in the present disclosure is saccharified chitosan, which is obtained by reacting the free amino groups of chitosan with the carbonyl groups of reducing monosaccharides and / or oligosaccharides. Examples of such monosaccharides include naturally occurring D-triose, D-tetraose, D-pentose, D-hexose, D-heptaose, and the like, such as D-glucose, D-galactose, D-fructose, D-mannose, D-allose, D-altrose, D-idose, D-talose, D-fucose, D-arabinose, D-gulose, D-hammelose, D-lyxose, D-ribose, D-rhamnose, D-threose, D-xylose, D-psicose, D-sorbose, D-tagatose, D-glyceraldehyde, dihydroxyacetone, D-erythrose, D-threose, D-erythrulose, D-mannoheptulose, D-sedoheptulose, and the like. Examples of such oligosaccharides include fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), mannooligosaccharides (MOS), and the like. An example of a saccharified chitosan is galactochitosan. Saccharified chitosan and its synthesis are described in, for example, US 5747475, WO 2002 / 040055 A2 and WO 2013 / 109732 A2, the contents of which are incorporated herein by reference.

[0131] In some embodiments, the saccharified chitosan used in the present invention has about 0.1% to about 90% of its originally free amino groups saccharified, for example, about 0.1% to about 30% or for example, about 2% to 15%, for example, about 12.5%. In some embodiments, the saccharified chitosan used in the present invention has a molecular weight of about 50kD to about 2000kD, such as about 50kD to about 1500kD, such as about 250kD or 300kD. In some embodiments, the saccharified chitosan used in the present invention has a DDA of about 50% to 99%, such as 70% to 99%, such as 80%. In some embodiments, the saccharified chitosan used in the present disclosure has a DDA of about 50% to 99%, for example, about 70% to 99%, for example, about 80%, and about 0.1% to about 90% of its originally free amino groups undergo a saccharification reaction, for example, about 0.1% to about 30%, or for example, about 2% to 15%, for example, about 12.5%, and the molecular weight of the saccharified chitosan is about 50 kD to about 2000 kD, for example, about 50 kD to about 1500 kD, for example, about 250 kD or about 300 kD.

[0132] Compositions comprising oncolytic peptides

[0133] The composition may comprise one oncolytic peptide or several different oncolytic peptides.

[0134] The composition comprising an oncolytic peptide is typically a composition comprising an oncolytic peptide and a physiologically acceptable carrier. In some embodiments, the composition is a pharmaceutical composition.

[0135] In some embodiments, the physiologically acceptable carrier is water, saline or aqueous buffer. In some embodiments, the physiologically acceptable carrier is water, such as water for injection (WFI). In some other embodiments, the physiologically acceptable carrier is the water comprising salt, such as sodium chloride (saline). In some other embodiments, the physiologically acceptable carrier is an aqueous buffer or a mixture of several different aqueous buffers. In some embodiments, the composition comprises a mixture of multiple different physiologically acceptable carriers.

[0136] A buffer may be used to maintain the pH of the composition within a desired range. Preferably, the pH of the composition is from 4.0 to 7.4, more preferably from 5.0 to 7.0, even more preferably from 5.0 to 6.5, such as 6.0.

[0137] The composition comprising the oncolytic peptide is preferably a solution of the oncolytic peptide in a physiologically acceptable carrier, more preferably a solution of the oncolytic peptide in water, saline or an aqueous buffer or a mixture of a plurality of different aqueous buffers.

[0138] In some embodiments, the composition comprises an oncolytic peptide at a concentration of 0.1 mg / ml to 30 mg / ml, such as 1 mg / ml to 5 mg / ml or 1 mg / ml to 30 mg / ml, preferably 4 mg / ml to 25 mg / ml, or more preferably 6 mg / ml to 20 mg / ml. Where applicable (i.e., when the oncolytic peptide is actually a peptide), the amount of the aforementioned peptide refers to the net peptide amount, and for an oncolytic peptide in the form of its salt as defined herein, refers to the amount of the free base.

[0139] The net peptide amount is the actual peptide content of the crude peptide after its synthesis, after correction for purity (determined by analytical methods such as HPLC) and analysis of the net peptide content (determined by analytical methods such as elemental analysis). The net peptide content is the fraction of the peptide in question relative to the counterion, residual water, and peptide impurities. The net peptide (in %) is calculated as follows:

[0140]

[0141] The results are given as a percentage of the neat peptide in the crude peptide.

[0142] In some embodiments, the composition comprises one or more additional pharmaceutically acceptable excipients, such as those well known in the art for use in pharmaceutical compositions / medical products comprising aqueous solutions of active ingredients.

[0143] In some embodiments, the composition comprising the oncolytic peptide can be a solution contained in a ready-to-use container (e.g., a sealed container). In other embodiments, the composition can be prepared before it is used / administered, for example, by reconstituting an oncolytic peptide provided in solid form (e.g., lyophilized form), for example, by dissolving it in a pharmaceutically acceptable carrier.

[0144] In some embodiments, the composition comprises LTX-401 and a pharmaceutically acceptable carrier. In some embodiments, the composition is a solution of LTX-401 in a pharmaceutically acceptable carrier contained in a sealed container ready for use.

[0145] Compositions comprising nonapeptides

[0146] For compositions comprising one or more nonapeptides, in some embodiments, the composition comprises one or more of the following aqueous buffers: acetic acid buffer, acetate buffer, aspartic acid buffer, sodium benzoate buffer, benzoic acid buffer, carbonate buffer, citric acid buffer, glucono-δ-lactone buffer, glycine buffer, glycine HCl buffer, histidine buffer, histidine HCl buffer, hydrobromic acid buffer, phosphate buffer (such as PBS), sodium succinate buffer, disodium succinate buffer, succinic acid buffer, sulfuric acid buffer, tartaric acid buffer and sodium tartrate buffer. Preferably, the aqueous buffer is acetic acid / acetate buffer, citric acid buffer and / or phosphate buffer.

[0147] For example, in some embodiments, the composition comprising the nonapeptide comprises one or more preservatives. The following table lists examples of preferred preservatives. Also listed are suitable concentration ranges for these agents if present in the composition:

[0148] preservative Concentration (w / w) Benzalkonium chloride 0.004% to 0.02% Benzyl alcohol 0.75% to 5% Chlorobutanol 0.25 to 0.5% m-Cresol 0.1 to 0.35% Methylparaben 0.01% to 0.2% phenol 0.15 to 0.5% Potassium sorbate 0.05% to 0.2% Propylparaben 0.005 to 0.035 Thimerosal 0.001% to 0.012%

[0149] Benzyl alcohol is a particularly preferred preservative, preferably present at 0.75% to 2% w / w, preferably 1% w / w. Another preferred preservative is phenol, preferably present at 0.15% to 0.5%.

[0150] In some embodiments, the composition comprising the nonapeptide comprises one or more chelating agents to chelate metal ions. The following table lists examples of preferred chelating agents. Also listed are suitable concentration ranges for these agents if present in the composition:

[0151]

[0152] EDTA = ethylenediaminetetraacetic acid, DTPA = diethylenetriaminepentaacetic acid

[0153] Preferably, the chelating agent is disodium EDTA and / or sodium EDTA.Preferably, the chelating agent such as disodium EDTA or sodium EDTA is present at about 0.1% w / w.

[0154] In some embodiments, the composition comprising the nonapeptide comprises one or more antioxidants. The following table lists examples of preferred antioxidants. Also listed are suitable concentration ranges for the antioxidants if present in the composition:

[0155] antioxidants Concentration (w / w) ascorbic acid 0.02 to 0.15% Ascorbyl palmitate 0.01 to 0.03% Citric acid 0.10 to 2.0% Erythorbic acid 0.02 to 0.1% Methionine 0.01 to 0.15% Monothioglycerol 0.1 to 1% Potassium metabisulfite 0.05% to 0.10% Sodium ascorbate 0.1 to 4.8% Sodium bisulfite 0.1 to 0.15% Sodium formaldehyde sulfoxylate 0.1 to 0.15% Sodium thiosulfate 0.1 to 0.2% Thiourea 0.002 to 0.008%

[0156] A preferred antioxidant is thiourea.

[0157] In some embodiments, the composition comprising the nonapeptide comprises one or more tonicity adjusting agents, such as dextrose, glycerol, lactose, mannitol, potassium chloride, sodium chloride and / or sorbitol, to obtain an isotonic composition that reduces the pain experienced by the patient administering the composition. Preferably, the tonicity adjusting agent is sodium chloride.

[0158] In some embodiments, the composition comprising the nonapeptide comprises one or more stabilizers that inhibit peptide aggregation. Such stabilizers include anti-stacking agents. Examples of suitable stabilizers are dimethyl sulfoxide, dimethylacetamide, ethanol, glycerol, mannitol, N-methyl-2-pyrrolidone, polyethylene glycol (PEG) 200, PEG 300, PEG 350, PEG 400, PEG 600, propylene glycol and sorbitol. PEG stabilizers, such as PEG 200, are particularly preferred. Such stabilizers, such as PEG stabilizers, can be present at 1% to 8% w / w, preferably 2% to 7% w / w, for example 3% to 6% w / w.

[0159] In some embodiments, the composition comprising the nonapeptide comprises one or more viscosity increasing agents, such as gum arabic, agar, cellulose derivatives, such as methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium, gelatin, hyaluronic acid, maltodextrin, pectin, poloxamer 407, PEG, polyethylene oxide, povidone (PVP), starches such as corn starch and tapioca starch, gum tragacanth, and / or β-cyclodextrin.

[0160] The increased viscosity of the composition containing the nonapeptide (compared to the viscosity of water) can effectively slow the diffusion of the peptide from the site of administration. If the composition is administered intratumorally, the increased viscosity can prolong the time the composition remains within the tumor without leaking out, slowing the diffusion of the peptide by the blood, and prolonging the time the peptide is in direct contact with tumor cells. In this way, the efficacy of the peptide can be increased and / or the toxicity can be reduced.

[0161] Thus, in some embodiments, the composition comprising the nonapeptide comprises one or more viscosity increasing agents and has a viscosity suitable for injection, for example, into a tumor. In some embodiments, the viscosity of the composition comprising the oncolytic peptide is from 1.5 cP, 2 cP, 2.5 cP or 3 cP to 300 cP, for example from 2.5 cP to 250 cP, for example 5 cP to 100 cP or 10 cP to 200 cP and 20 cP to 240 cP, preferably a viscosity of 40 cP to 180 cP, for example 45 cP to 150 cP or 30 cP to 100 cP, more preferably a viscosity of 1.5 cP to 10 cP, for example 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP or 10 cP or 10 cP to 15 cP or 15 cP to 20 cP or 20 cP to 35 cP or 35 cP to 50 cP or 50 cP to 100 cP.

[0162] The viscosity increasing agent is preferably present at 0.4% w / w to 1.2% w / w, such as 0.5% w / w to 1% w / w.

[0163] Preferably, the one or more viscosity increasing agents are selected from the group consisting of hydroxyethylcellulose, hydroxypropylcellulose, HPMC, poloxamer, PEG and PVP.

[0164] If the composition comprising the nonapeptide is prepared before administration, the aforementioned excipients such as preservatives, antioxidants or stabilizers may not be required, or if present in the reconstituted composition, the aforementioned excipients are preferably present in a pharmaceutically acceptable carrier. In some embodiments, if the composition is prepared before its use / administration, any of the aforementioned excipients are present in a pharmaceutically acceptable carrier.

[0165] In some embodiments, the composition comprises LTX-315 and a pharmaceutically acceptable carrier. Preferably, LTX-315 has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt thereof, more preferably an acetate salt thereof.

[0166] In some embodiments, such compositions are solutions of LTX-315 in a pharmaceutically acceptable carrier contained in a sealed container ready for use. In some other embodiments, such compositions are solutions of LTX-315 that are reconstituted prior to use / administration by dissolving such LTX-315 in solid form (e.g., as a lyophilized powder) in a pharmaceutically acceptable carrier.

[0167] Preparation of compositions containing oncolytic peptides

[0168] In some embodiments, the composition comprising the oncolytic peptide is a pharmaceutical grade product. In some embodiments, the composition comprising the oncolytic peptide is a sterile composition. In some embodiments, the composition comprising the oncolytic peptide is a pharmaceutical composition.

[0169] Compositions comprising oncolytic peptides can be prepared using any conventional method.

[0170] In some embodiments, the composition comprising an oncolytic peptide is a solution contained in a ready-to-use sealed container. In order to manufacture such a product, the oncolytic peptide in its solid form (e.g., (lyophilized) powder form) can be dissolved in a pharmaceutically acceptable carrier under aseptic manufacturing conditions, and the resulting composition is preferably aseptically filtered, then filled into a container and sealed. Alternatively, the oncolytic peptide can be dissolved in a pharmaceutically acceptable carrier, and the resulting composition is then filled into a container, sealed, and then the container is autoclaved to obtain a sterile composition. If there is an excipient in the composition, these excipients are mixed or dissolved in a pharmaceutically acceptable carrier or mixed or dissolved in a solution comprising an oncolytic peptide, depending on the nature and amount of the excipient.

[0171] In some other embodiments, the composition comprising the oncolytic peptide is prepared before it is used / administered, for example, by reconstituting an oncolytic peptide provided in solid form (e.g., in the form of a lyophilized powder), for example, by dissolving it in a sterile pharmaceutically acceptable carrier. To manufacture such a product, in some embodiments, the oncolytic peptide is dissolved, sterile filtered, and lyophilized. In some other embodiments, the oncolytic peptide is dissolved, lyophilized, and the lyophilized oncolytic peptide is sterilized by gamma irradiation.

[0172] Compositions comprising chitosan and / or chitosan derivatives

[0173] The composition may comprise one or more chitosans, such as a plurality of different chitosans, e.g. chitosans of different molecular weights and / or different DDAs, and / or may comprise one or more chitosan derivatives, such as a plurality of different chitosan derivatives, e.g. derivatives having the same modification but different degrees of modification, or derivatives having different types of modification.

[0174] The composition comprising chitosan and / or chitosan derivatives is typically a composition comprising chitosan and / or chitosan derivatives and a physiologically acceptable carrier. In some embodiments, the composition comprising chitosan and / or chitosan derivatives is a pharmaceutical composition.

[0175] In some embodiments, the composition comprising chitosan and / or a chitosan derivative is a dispersion, suspension or solution of chitosan and / or a chitosan derivative in a physiologically acceptable carrier.

[0176] In some embodiments, the physiologically acceptable carrier is water, saline or aqueous buffer. In some embodiments, the physiologically acceptable carrier is water, such as water for injection (WFI). In some other embodiments, the physiologically acceptable carrier is the water comprising salt, such as sodium chloride (saline) or other salts such as potassium chloride, calcium chloride or magnesium chloride, or the aqueous buffer comprising salt, such as sodium chloride or other salts such as potassium chloride, calcium chloride or magnesium chloride. In some embodiments, the composition comprises a mixture of multiple different physiologically acceptable carriers. In some other embodiments, the physiologically acceptable carrier is an aqueous buffer or a mixture of several different aqueous buffers.

[0177] Buffer can be used to maintain the pH of the composition within the desired range. Typically, the pH of the composition is 4.0 to 7.5. Preferably, the pH of the composition is 5.0 to 7.4, more preferably 5.0 to 7.0, such as 6.5 or 6.0.

[0178] In some embodiments, the composition comprising chitosan and / or a chitosan derivative has a viscosity suitable for injection, for example, a viscosity suitable for injection into a tumor in a subject. In some embodiments, the composition comprising chitosan and / or chitosan derivatives has a viscosity of 1.0 cP, 1.5 cP, 2 cP, 2.5 cP or 3 cP to 300 cP, for example 2.5 cP to 250 cP, for example 5 cP to 100 cP or 10 cP to 200 cP and 20 cP to 240 cP, preferably 40 cP to 180 cP, for example 45 cP to 150 cP or 30 cP to 100 cP, more preferably 1.5 cP to 10 cP, for example 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP or 10 cP or 10 cP to 15 cP or 15 cP to 20 cP or 20 cP to 35 cP or 35 cP to 50 cP or 50 cP to 100 cP. In some embodiments, the composition comprises saccharified chitosan and has a viscosity of 1 cP to 100 cP.

[0179] In some embodiments, the composition comprises 0.1% w / w to 5% w / w chitosan and / or chitosan derivatives, such as 0.1% w / w to 2.5% w / w, such as 0.5% w / w, 1% w / w, 1.5% w / w or 2% w / w.

[0180] In some compositions, the composition comprises one or more additional pharmaceutically acceptable excipients, such as those excipients well known in the art for use in medical products comprising aqueous dispersions, suspensions or solutions of active ingredients.

[0181] In some embodiments, the composition comprises saccharified chitosan. In some other embodiments, the composition comprises galactochitosan.

[0182] Preparation of compositions comprising chitosan and / or chitosan derivatives

[0183] In some embodiments, the composition comprising chitosan and / or chitosan derivatives is a pharmaceutical grade product. In some embodiments, the composition comprising chitosan and / or chitosan derivatives is a sterile composition. In some embodiments, the composition comprising chitosan and / or chitosan derivatives is a pharmaceutical composition.

[0184] The composition comprising chitosan and / or chitosan derivatives can be prepared using any conventional method. In some embodiments, chitosan and / or chitosan derivatives are dispersed, suspended or dissolved in a physiologically acceptable carrier and the resulting composition is aseptically filtered, then packed in a container and sealed. In some embodiments, chitosan and / or chitosan derivatives are dispersed, suspended or dissolved in a physiologically acceptable carrier, and the resulting composition is autoclaved. In some embodiments, the composition comprising chitosan and / or chitosan derivatives is prepared just before it is used / applied, for example, by redissolving the chitosan and / or chitosan derivatives provided in solid form (for example lyophilized form), for example, dissolving, dispersing or being suspended in a sterile pharmaceutically acceptable carrier. In order to manufacture such products, in some embodiments, chitosan and / or chitosan derivatives are dissolved, aseptically filtered and lyophilized. In some other embodiments, chitosan and / or chitosan derivatives are dissolved, lyophilized, and the lyophilized compound is sterilized by gamma radiation.

[0185] Kit for composition (a) and composition (b)

[0186] Composition (a) and composition (b) can be provided in the form of a kit comprising the two compositions or comprising parts / components for preparing composition (a) and composition (b). In some embodiments, the kit comprises composition (a) and composition (b) each in a (sealed) container.

[0187] The kit typically comprises instructions for using composition (a) and composition (b).

[0188] In some other embodiments, the kit comprises an oncolytic peptide and a physiologically acceptable carrier for preparing composition (a) and chitosan and / or chitosan derivatives and a physiologically acceptable carrier for preparing composition (b). In some embodiments, the physiologically acceptable carrier for preparing composition (a) is the same as the physiologically acceptable carrier for preparing composition (b). In such embodiments, the kit may include 3 containers or 3 compartments arranged in one or two containers, wherein one container or compartment comprises an oncolytic peptide, one container or compartment comprises chitosan and / or chitosan derivatives (e.g., both are in solid form), and another container or compartment comprises a physiologically acceptable carrier for preparing composition (a) and composition (b). In some other embodiments, the physiologically acceptable carriers for preparing composition (a) and composition (b) are different. In such embodiments, the kit may comprise four containers or four compartments contained in one, two or three containers, one container or compartment comprising the oncolytic peptide, one container or compartment comprising chitosan and / or a chitosan derivative (e.g., both in solid form), one container or compartment comprising a physiologically acceptable carrier for preparing composition (a), and one container or compartment comprising a physiologically acceptable carrier for preparing composition (b).

[0189] The kit typically comprises instructions for preparing compositions (a) and (b), such as reconstituting the oncolytic peptide and chitosan and / or chitosan derivative in a physiologically acceptable carrier prior to use, and instructions for use of compositions (a) and (b).

[0190] In yet other embodiments, the kit comprises composition (a), chitosan and / or chitosan derivatives (eg, in solid form), and a physiologically acceptable carrier for preparing composition (b).

[0191] The kit typically comprises instructions for preparing composition (b), such as instructions for reconstituting chitosan and / or chitosan derivatives in a physiologically acceptable carrier prior to use, and instructions for use of composition (a) and composition (b).

[0192] In yet other embodiments, the kit comprises composition (b), an oncolytic peptide (eg, in solid form), and a physiologically acceptable carrier for preparing composition (a).

[0193] The kit typically comprises instructions for preparing composition (a), such as instructions for reconstituting the oncolytic peptide in a physiologically acceptable carrier prior to use, and instructions for using composition (a) and composition (b).

[0194] Composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative

[0195] A composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative may also be referred to herein as a "combination composition."

[0196] The combination composition may comprise one oncolytic peptide or a plurality of different oncolytic peptides. In addition, the combination composition may comprise one or more chitosans, such as a plurality of different chitosans, such as chitosans of different molecular weights and / or different DDAs, and / or may comprise one or more chitosan derivatives, such as a plurality of different chitosan derivatives, such as derivatives comprising the same modification but different degrees of modification, or derivatives comprising different modifications.

[0197] The combination composition is typically a composition comprising i) and ii) and a physiologically acceptable carrier. In some embodiments, the combination composition is a pharmaceutical composition.

[0198] In some embodiments, the combination composition is a dispersion, suspension or solution, i.e., a dispersion, suspension or solution of i) and ii) in a physiologically acceptable carrier. In some embodiments, the oncolytic peptide is dissolved in a physiologically acceptable carrier, and the chitosan and / or chitosan derivative is dispersed or suspended in the dissolved oncolytic peptide.

[0199] In some embodiments, the physiologically acceptable carrier is water, saline or an aqueous buffer. In some embodiments, the physiologically acceptable carrier is water, such as water for injection (WFI). In some other embodiments, the physiologically acceptable carrier is water comprising salt, such as sodium chloride (saline) or other salts such as potassium chloride, calcium chloride or magnesium chloride, or an aqueous buffer comprising salt, such as sodium chloride or other salts such as potassium chloride, calcium chloride or magnesium chloride. In some other embodiments, the physiologically acceptable carrier is an aqueous buffer or a mixture of several different aqueous buffers.

[0200] A buffer may be used to maintain the pH of the combination composition within a desired range. Preferably, the pH of the combination composition is between 4.0 and 7.4, more preferably between 5.0 and 7.0, such as 6.0 or 6.5.

[0201] The combination composition comprises an oncolytic peptide at a concentration of 0.1 mg / ml to 30 mg / ml, for example 1 mg / ml to 5 mg / ml or 1 mg / ml to 30 mg / ml, preferably 4 mg / ml to 25 mg / ml, or more preferably 6 mg / ml to 20 mg / ml. Where applicable (i.e., when the oncolytic peptide is actually a peptide), the amount of the peptide mentioned above refers to the net peptide amount and the free base.

[0202] The combination composition comprises 0.1% w / w to 5% w / w chitosan and / or chitosan derivatives, such as 0.1% w / w to 2.5% w / w, such as 0.5% w / w to 1.5% w / w, for example 0.6% w / w to 1% w / w or 1% w / w to 1.5% w / w or 1.5% w / w to 2% w / w or 2% w / w to 2.5% w / w.

[0203] In some embodiments, the combination composition has a viscosity suitable for injection, such as a viscosity suitable for injection into a tumor in a subject. In some embodiments, the viscosity of the combination composition is from 1.0 cP, 1.5 cP, 2 cP, 2.5 cP or 3 cP to 300 cP, for example, from 2.5 cP to 250 cP, for example, from 5 cP to 100 cP or from 10 cP to 200 cP and from 20 cP to 240 cP, preferably, from 40 cP to 180 cP, for example, from 45 cP to 150 cP or from 30 cP to 100 cP, more preferably, from 1.5 cP to 10 cP, for example, from 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP or 10 cP, or from 10 cP to 15 cP or from 15 cP to 20 cP or from 20 cP to 35 cP or from 35 cP to 50 cP or from 50 cP to 100 cP.

[0204] For combination compositions comprising nonapeptides, such compositions may further comprise the excipients described in the corresponding section herein, ie, the "Compositions comprising nonapeptides" section.

[0205] In some embodiments, the combination composition comprises LTX-401. In some other embodiments, the combination composition comprises LTX-315. In still other embodiments, the combination composition comprises saccharified chitosan, such as galactochitosan. In still other embodiments, the combination composition comprises LTX-401 and saccharified chitosan, such as galactochitosan, or comprises LTX-315 and saccharified chitosan, such as galactochitosan.

[0206] Preparation of combined compositions

[0207] In some embodiments, the combination composition is a pharmaceutical grade product. In some embodiments, the combination composition is a sterile composition. In some embodiments, the combination composition is a pharmaceutical composition.

[0208] The combined composition can be prepared using any conventional method. In some embodiments, chitosan and / or chitosan derivatives are dispersed, suspended or dissolved in a physiologically acceptable carrier, and the oncolytic peptide is dissolved in a dispersion, suspension or solution. In some other embodiments, chitosan and / or chitosan derivatives are dispersed, suspended or dissolved in a solution of the oncolytic peptide in a physiologically acceptable carrier. The solution of the oncolytic peptide can be obtained by dissolving a solid form of the oncolytic peptide, such as a powdered oncolytic peptide, in a physiologically acceptable carrier. In some embodiments, the combined composition is sterile filtered, then loaded into a container and sealed, or loaded into a container and autoclaved.

[0209] Kits for combined compositions

[0210] The components / ingredients for preparing the combination composition may be provided in a kit, ie the combination composition may be prepared prior to use / administration.

[0211] In some embodiments, the kit comprises composition (a) and composition (b) as described herein and instructions for preparing the combined composition. The kit typically comprises instructions for use of the combined composition.

[0212] In some other embodiments, the kit comprises an oncolytic peptide, chitosan and / or a chitosan derivative (e.g., both in solid form) and a physiologically acceptable carrier for preparing a combination composition. In such embodiments, the kit may comprise three containers or three compartments comprised in one or two containers, one container or compartment comprising an oncolytic peptide, one container or compartment comprising chitosan and / or a chitosan derivative (e.g., both in solid form), one container or compartment comprising a physiologically acceptable carrier for preparing a combination composition, or may comprise two containers or two compartments comprised in one container, one container or compartment comprising a mixture of an oncolytic peptide and chitosan and / or a chitosan derivative, one container or compartment comprising a physiologically acceptable carrier for preparing a combination composition.

[0213] The kit typically contains instructions for preparing the combination composition, such as instructions for reconstituting the oncolytic peptide and chitosan and / or chitosan derivative or a mixture thereof in a physiologically acceptable carrier before use, and instructions for using the combination composition.

[0214] In yet other embodiments, the kit comprises composition (a) and chitosan and / or a chitosan derivative (eg, in solid form).

[0215] The kit typically comprises instructions for preparing the combination composition, such as instructions for reconstituting chitosan and / or chitosan derivatives in composition (a) prior to use, and instructions for using the combination composition.

[0216] In yet other embodiments, the kit comprises composition (b) and an oncolytic peptide (eg, in solid form).

[0217] The kit typically comprises instructions for preparing the combination composition, such as instructions for reconstituting the oncolytic peptide in composition (b) prior to use, and instructions for using the combination composition.

[0218] Methods of treating a subject having a tumor / compositions as disclosed herein for treating a subject having a tumor Use in methods

[0219] Composition (a) and composition (b) disclosed herein are used simultaneously, sequentially or as a combined composition (i.e., a combined composition as disclosed herein) in a method for treating a subject having a tumor, for example, administered simultaneously, sequentially or as a combined composition to the subject.

[0220] In some embodiments, composition (a) and composition (b) are administered simultaneously, i.e., administered at the same time. In this context, simultaneously refers to the same time (e.g., by the same or different modes of administration), or in a short period of time, for example, a few minutes, a few hours or longer, but these administration operations are completed on the same day. In some embodiments, composition (a) and composition (b) are administered simultaneously, wherein the composition (b) comprising chitosan and / or a chitosan derivative is first administered, and then the composition (a) comprising an oncolytic peptide is administered. In some other embodiments, composition (a) and composition (b) are administered simultaneously, wherein the composition (a) comprising an oncolytic peptide is first administered, and then the composition (b) comprising chitosan and / or a chitosan derivative is administered.

[0221] In some other embodiments, composition (a) and composition (b) are administered sequentially. In this context, sequentially refers to administration on different days, for example, one day / several days or one week / several weeks apart, for example, by the same or different modes of administration. In some embodiments, composition (a) and composition (b) are administered sequentially, wherein a composition (b) comprising chitosan and / or a chitosan derivative is first administered, followed by administration of a composition (a) comprising an oncolytic peptide. In some other embodiments, composition (a) and composition (b) are administered sequentially, wherein a composition (a) comprising an oncolytic peptide is first administered, followed by administration of a composition (b) comprising chitosan and / or a chitosan derivative.

[0222] In some embodiments, composition (a) and composition (b) are each administered only once, i.e., one dose is administered at one time point during the treatment. In some other embodiments, composition (a) and composition (b) are each administered repeatedly, i.e., several doses are administered at several time points during the treatment. In some other embodiments, one of composition (a) and composition (b) is administered repeatedly, while the other is administered only once.

[0223] In some embodiments, the composition is a combination composition. In some embodiments, such a combination composition is administered only once. In some other embodiments, such a combination composition is administered repeatedly during the course of treatment.

[0224] The combination composition and composition (a) are preferably administered to the tumor. In some embodiments, the composition is injected into the tumor. In some other embodiments, the composition is administered to the tumor by perfusion / infusion of a preferably isolated (including partially isolated) limb, body region, or organ containing the tumor. Any one or a combination of both (injection and perfusion / infusion) modes of administration that localize the composition to the tumor site is acceptable, as long as the delivery mechanism ensures sufficient concentration of the components of the composition in the tumor.

[0225] Composition (b) is preferably administered into the tumor and / or the tissue immediately surrounding the tumor. Composition (b) may be administered by injection, perfusion / infusion or a combination of the two, as described above for composition (a) and the combination composition.

[0226] In the therapeutic methods / uses of the compositions disclosed herein for treatment, the compositions are preferably administered in a therapeutically effective amount. Such an amount can be administered as a single dose, i.e., a single administration, or as a divided dose, i.e., repeated administration, i.e., in a series of doses, e.g., over the course of several days, weeks, or months.

[0227] The actual dosage to be administered may vary and will depend on the age, weight, sex, medical history, pre-existing conditions and general condition of the subject, the severity of the condition being treated and the judgment of the health care professional.

[0228] Use of the methods of treatment / compositions for treatment disclosed herein may continue as long as the clinician supervising the patient's care deems the method effective and treatment is indicated.

[0229] In some embodiments, the use of the methods / compositions disclosed herein for treatment is for treating a tumor selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, stomach cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, liver cancer, bile duct cancer, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease, and adrenocortical cancer. In some embodiments, the tumor is a carcinoma, adenocarcinoma, lymphoma, melanoma, blastoma, leukemia, sarcoma, and germ cell tumor.

[0230] Example

[0231] The foregoing written description is believed to be sufficient to enable those skilled in the art to practice the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0232] Embodiment 1: Preparation of the composition disclosed herein

[0233] The following composition was prepared as follows:

[0234] Approximately half of the aqueous acetic acid / acetate buffer or saline solution was added to a reaction vessel equipped with a magnetic stirrer and stirred. LTX-401 (prepared as described in WO 2011 / 051692 A1) was added as a dry powdered acetate salt and stirred continuously until dissolved. If chitosan or HPMC was present in the composition, this was added while stirring. Subsequently, the remaining buffer or saline solution was added, and the mixture was stirred vigorously until the polymer was completely dissolved. All compositions were autoclaved at 121°C for 15 minutes.

[0235]

[0236] *24 mg / ml is equivalent to 20 mg / ml of free base, while 12 mg / ml is equivalent to 10 mg / ml of free base.

[0237] NTD: Not Determined

[0238] CS LMW = Chitosan low molecular weight, 340 kDa, DAA 85.4% (manufacturer Chitinor, Norway)

[0239] CS MMW = chitosan medium molecular weight, 350-600 kDa, DAA 88% (manufacturer Chitinor, Norway)

[0240] HPMC LMW = Hydroxypropyl methylcellulose low molecular weight, pharmaceutical grade HPMC metolose 60SH (LMW) (Safic Alcan, Cedex, La Defense, Paris, France)

[0241] HPMC HMW = Hydroxypropyl methylcellulose high molecular weight, pharmaceutical grade HPMC metolose 60SH (HMW) (Safic Alcan, Cedex, La Defense, Paris, France)

[0242] Viscosity was measured using a Fungilab / Evo Expert viscometer equipped with an LCP spindle. The test was performed at 25°C with a sample volume of 20 mL, a rotation speed of 10 rpm, and a shear rate of 12.2 s -1 , pH value was determined according to Ph.Eur. method.

[0243] Embodiment 2: In vitro cytotoxic activity

[0244] Compositions 1 to 15 in the above table were tested for in vitro cytotoxicity (oncolytic) activity in three cell lines by determining their respective IC 50 The half-maximal inhibitory concentration (IC) was used to evaluate the 50 The values ​​represent the concentration required to kill 50% of the cells. Cell viability was determined using a WST-1 kit (Roche). RPMI 1640 medium without FCS (assay medium) and 1% v / v Triton X-100 in the assay medium were used as negative and positive controls, respectively. Three independent experiments were performed for each cell line. Stock solutions of compositions 1 to 15 containing 0.8 mg / ml LTX-401 were prepared and diluted to 2 to 200 μg / ml in the assay medium.

[0245] The three cell lines used in this example are as follows (all obtained from ATCC, USA):

[0246] 143B, human osteosarcoma cell line

[0247] K7M2, a mouse osteosarcoma cell line

[0248] B16F10, a mouse melanoma cell line.

[0249] The cells were seeded in 96-well flat-bottom microplates in 100 μl aliquots per well and cultured in RPMI medium with FCS at 37°C for 16 hours. The cells were washed once with RPMI medium without FCS, and 100 μl of diluted compositions 1 to 15, negative control, or positive control was added to each well. The culture plates were incubated at 37°C for 4 hours, after which 10 μl of WST-1 solution was added to each well. The culture plates were incubated for another 2 hours. After aspirating 70 μl of the solution in each well, 100 μl of acidic isopropanol was added. Varioskan TM The absorbance (OD) of each well was measured at a wavelength of 590 nm using a LUX multifunctional microplate reader (Thermo Scientific). Cell viability was measured using the WST-1 assay for compositions 1 to 8 and compositions 10 to 14, which did not contain LTX-401, to evaluate the effects of HPMC or chitosan alone on cell viability. None of these compositions affected cell viability.

[0250] According to their IC in three different cell lines 50 , LTX-401 concentration and viscosity, composition 10, composition 12, composition 13 and composition 15 were selected for Example 3.

[0251] Example 3: In vivo cytotoxic activity against osteosarcoma

[0252] The following Composition 10, Composition 12, Composition 13, and Composition 15 were selected for in vivo testing in a syngeneic osteosarcoma mouse model, and their administration effects were studied by administering the compositions to tumors:

[0253] 10: LTX-401, 10 mg / ml (free base), containing 0.5% w / w HPMC HMW

[0254] 12: LTX-401, 10 mg / ml (free base), containing 1% w / w chitosan MMW

[0255] 13: LTX-401, 10 mg / ml (free base), containing 1.2% w / w chitosan MMW

[0256] 15: LTX-401, 10 mg / ml (free base)

[0257] Control group: normal saline

[0258] Balb / c mice (Taconic Biosciences) were inoculated subcutaneously in the right flank on day 0 with 8 × 10 6K7M2 tumor cells. The mice were divided into 5 groups of 7 each. When the tumor grew to the desired size of about 5mm in diameter, treatment was started. Each group injected the corresponding composition into the tumor site (50μl) by injection for 3 consecutive days. During the study, the tumor size was measured regularly and the mice were weighed to monitor them. The mice were observed until they reached a maximum tumor load of 1.5ml volume or 20mm length, or until serious adverse events occurred (ie, wounds or ulcers formed at the tumor / injection site), at which time the mice were euthanized. Tumor size was measured using a caliper and used as a healthy control by weighing and physical examination.

[0259] The results are as follows Figure 1 As shown: Tumors in mice given the LTX-401 composition grew slower compared to tumor growth in mice that received saline alone. Essentially no difference in tumor growth was observed between the group of mice treated with LTX-401 alone and the group of mice treated with LTX-401 / 0.5% HPMC. Mice treated with the composition comprising LTX-401 and chitosan exhibited significantly slower tumor growth, and even after 150 days, one mouse in the group receiving LTX-401 / 1% chitosan and two mice in the group receiving LTX-401 / 1.2% chitosan were still alive and in remission (no visible or palpable tumors).

[0260] Example 4: In vivo cytotoxic activity against melanoma

[0261] The following compositions were all prepared in aqueous acetic acid / acetate buffer at pH 5.2 as described in Example 1. The following chitosan was used in this study: Chitosan medium molecular weight, 330 kDa, DAA 86% (manufacturer Chitinor, Norway).

[0262] 1: LTX-401, 5 mg / ml (free base)

[0263] 2: Chitosan, 1.2% (w / w)

[0264] 3: LTX-401, 5 mg / ml (free base) and chitosan, 1.2% (w / w)

[0265] 4: Control group: aqueous acetic acid / acetate buffer

[0266] On day 0, immunocompetent C57BL / 6NRj mice (Janvier Labs, France) were injected subcutaneously with 5×10 4B16F1 melanoma cells / 50 μl RPMI-1640 medium. The mice were divided into 4 groups, 10 mice in each group, and the tumors were palpable and grew to a diameter of about 5 mm (a volume of about 40 mm). 3 Up to 80mm 3 ) of the desired size. The corresponding compositions 1 to 4 were administered by injection (50 μl) into the tumor site on two consecutive days (days 11 and 12, or day 12 and 13, or day 13 and 14, depending on tumor growth). During the study, the tumor size was measured regularly and the mice were weighed to monitor them. The tumor volume was measured using an electronic caliper and calculated according to the formula V = (L×W×W) / 2, where W is the width of the tumor and L is the length of the tumor. The mice were observed until the diameter of the mouse tumor reached a maximum load of approximately 12 mm to 13 mm (maximum load of volume 860 mm). 3 Up to 1100mm 3 ), or the appearance of metastasis, or the occurrence of a serious adverse event (i.e., formation of wounds or ulcers at the tumor / injection site). If any of these endpoints is reached, the mice are euthanized. A scoring form is used to assess the overall health of the animals based on parameters such as body weight and physical examination.

[0267] The results are as follows Figure 2 Survival curves show that treatment with Composition 3 (a combination of LTX-401 and chitosan) resulted in complete tumor regression in all animals, with a 100% survival rate, compared to 60% for animals treated with LTX-401 alone and 10% for animals treated with chitosan alone. None of the animals in the control group survived.

[0268] Implementation Plan

[0269] 1. A combination of a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative for use in treating a subject suffering from a tumor.

[0270] 2. A composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative for use in treating a subject suffering from a tumor.

[0271] 3. Composition (a) is used for treating a subject suffering from a tumor by simultaneous, sequential or combined administration with composition (b), wherein composition (a) comprises an oncolytic peptide and composition (b) comprises chitosan and / or a chitosan derivative.

[0272] 4. A method of treating a subject having a tumor, comprising administering to the subject a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative.

[0273] 5. Use of a composition (a) comprising an oncolytic peptide and a composition (b) comprising chitosan and / or a chitosan derivative for treating a subject suffering from a tumor.

[0274] 6. The combination, composition for use, method or use according to any one of embodiments 1 to 5, wherein the composition (a) and composition (b) are administered to the subject, and wherein the administration is simultaneous, sequential or combined.

[0275] 7. The combination, composition for use, method or use according to any one of embodiments 1 to 6, wherein the composition (a) comprises one or more oncolytic peptides, and / or wherein the composition (b) comprises one or more different chitosans and / or chitosan derivatives.

[0276] 8. The combination, the composition for use, the method or the use according to any one of embodiments 1 to 7, wherein the composition (a) and / or the composition (b) comprises a physiologically acceptable carrier.

[0277] 9. The combination, composition for use, method or use according to embodiment 8, wherein the physiologically acceptable carrier is selected from the group consisting of water, such as water for injection (WFI), saline and aqueous buffer.

[0278] 10. The combination, composition for use, method or use according to any one of embodiments 8 to 9, wherein the composition (a) and / or the composition (b) comprises a mixture of different physiologically acceptable carriers.

[0279] 11. The combination, composition for use, method or use according to any one of embodiments 8 to 10, wherein the composition (a) and / or the composition (b) comprises water, such as water for injection, which comprises a salt selected from the group consisting of sodium chloride, potassium chloride, calcium chloride and magnesium chloride.

[0280] 12. The combination, composition for use, method or use according to any one of embodiments 1 to 11, wherein the oncolytic peptide is a nonapeptide or a pharmaceutically acceptable salt thereof, and the nonapeptide or a pharmaceutically acceptable salt thereof consists of 9 linearly arranged amino acids, wherein, among these 9 amino acids, 5 amino acids are cationic amino acids and 4 amino acids are lipophilic amino acids, and wherein, among these 4 lipophilic amino acids, 3 lipophilic amino acids are tryptophan and 1 lipophilic amino acid is a non-genetically encoded amino acid.

[0281] 13. The combination, composition for use, method or use of embodiment 12, wherein the lipophilic amino acids and cationic amino acids are arranged so that no more than two amino acids of either type are adjacent to each other and / or the peptide comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic amino acids.

[0282] 14. The combination, composition for use, method or use according to any one of embodiments 12 to 13, wherein the cationic amino acids are the same or different and are selected from the group consisting of lysine, arginine, histidine and non-genetically encoded amino acids that carry a positive charge at pH 7.0, preferably selected from the group consisting of lysine and arginine.

[0283] 15. The combination, composition for use, method or use according to any one of embodiments 12 to 14, wherein the non-genetically encoded cationic amino acid is selected from the group consisting of derivatives of lysine, arginine and histidine.

[0284] 16. The combination, composition for use, method or use of any one of embodiments 12 to 15, wherein the non-genetically encoded cationic amino acid is selected from the group consisting of homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, homoarginine, trimethyllysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamoylimidopiperidine-4-carboxylic acid and 4-guanidinophenylalanine.

[0285] 17. The combination, composition for use, method or use of any one of embodiments 12 to 16, wherein the non-genetically encoded lipophilic amino acid has a lipophilic side chain having at least 7, at least 8, at least 9 or at least 10 non-hydrogen atoms, preferably wherein the non-hydrogen atoms are carbon atoms.

[0286] 18. The combination, composition for use, method or use according to any one of embodiments 12 to 17, wherein the non-genetically encoded lipophilic amino acid has a lipophilic side chain containing no more than 30 or no more than 25 non-hydrogen atoms, and / or wherein such lipophilic side chain comprises at least one, preferably two, optionally fused or linked cyclic groups, and / or wherein the lipophilic side chain comprises heteroatoms such as O, N or S, preferably only one heteroatom, more preferably a nitrogen atom, and / or wherein the lipophilic side chain comprises 2 or less than 2 polar groups, preferably one and more preferably no polar groups.

[0287] 19. The combination, composition for use, method or use of any one of embodiments 12 to 18, wherein the non-genetically encoded lipophilic amino acid is selected from the group consisting of 2-amino-3-(biphenyl-4-yl)propionic acid (biphenylalanine), 2-amino-3,3-diphenylpropionic acid (diphenylalanine), 2-amino-3-(anthracen-9-yl)propionic acid, 2-amino-3-(naphthalen-2-yl)propionic acid, 2-amino-3-(naphthalen-1-yl)propionic acid, 2-amino-3-[1,1':4',1"-terphenyl-4-yl]propionic acid, 2-amino-3-(2,5,7-tri-tert-butyl- 1H-indol-3-yl)propionic acid, 2-amino-3-[1,1':3',1"-terphenyl-4-yl]propionic acid, 2-amino-3-[1,1':2',1"-terphenyl-4-yl]propionic acid, 2-amino-3-(4-naphthalen-2-yl-phenyl)propionic acid, 2-amino-3-(4'-butylbiphenyl-4-yl)propionic acid, 2-amino-3-[1,1':3',1"-terphenyl-5'-yl]propionic acid, and 2-amino-3-(4-(2,2-diphenylethyl)phenyl)propionic acid, more preferably, the lipophilic amino acid is selected from the group consisting of diphenylalanine and biphenylalanine.

[0288] 20. The combination, composition for use, method or use according to any one of embodiments 12 to 19, wherein the nonapeptide is a compound of formula (I) to (V), wherein C represents a cationic amino acid and L represents a lipophilic amino acid, and wherein the amino acids are covalently linked via peptide bonds and preferably wherein the carboxyl terminus is amidated:

[0289] CCLLCCLLC(I),

[0290] LCCLLCCLC(II),

[0291] CLLCCLLCC(III),

[0292] CCLLCLLCC(IV),

[0293] CLCCLLCCL(V).

[0294] 21. The combination, composition for use, method or use according to any one of embodiments 12 to 20, wherein the nonapeptide is a compound of formula (I'), formula (I"), formula (I'") or formula (II'), wherein C represents a cationic amino acid and L' represents a non-genetically encoded lipophilic amino acid, and wherein the amino acids are covalently linked by peptide bonds, and preferably wherein the carboxyl terminus is amidated:

[0295] CCL'LCCLLC(I'),

[0296] CCLLCCLL'C(I"),

[0297] CCLL'CCLLC(I"'),

[0298] LCCLL'CCLC(II').

[0299] 22. The combination, composition for use, method or use according to any one of embodiments 1 to 21, wherein the oncolytic peptide is selected from the group consisting of LTX-301, LTX-302, LTX-303, LTX-304, LTX-305, LTX-306, LTX-307, LTX-308, LTX-309, LTX-310, LTX-311, LTX-312, LTX-313, LTX-314, LTX-315, LTX-316, LTX-317, LTX-318, LTX-319, LTX-320, LTX-321, LTX-322, LTX-323, LTX-324, LTX-325, LTX-326, LTX-327, LTX-328, LTX-329, LTX-330, LTX-331, LTX-332, LTX-333, LTX-334, LTX-335, LTX-336, LTX-337, LTX-338, LTX-339, LTX-340, LTX-341, LTX-342, LTX-343, LTX-344, LTX-345, LTX-346, LTX-347, LTX-348, LTX-349, LTX-350, LTX-351, LTX-352, LTX-353, LTX-354, LTX-355, LTX-356, LTX-357, LTX-358, LTX-359, LTX-360, LTX-361 X-316, LTX-317, LTX-318, LTX-319, LTX-320, LTX-321, LTX-322, LTX-323, LTX-324, LTX-325, LTX-326, LTX-3 27. LTX-328, LTX-329, LTX-330, LTX-331, LTX-332, LTX-333, LTX-334, LTX-335, LTX-336, LTX-337 and LTX-338.

[0300] 23. The combination, composition for use, method or use according to any one of embodiments 1 to 22, wherein the oncolytic peptide is selected from the group consisting of LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329, preferably wherein the oncolytic peptide is LTX-315.

[0301] 24. The combination, composition for use, method or use according to any one of embodiments 1 to 23, wherein the oncolytic peptide is LTX-315 in the form of a pharmaceutically acceptable salt, preferably in the form of an acetate salt.

[0302] 25. The combination, composition for use, method or use according to any one of embodiments 1 to 24, wherein composition (a) further comprises one or more preservatives and / or one or more chelating agents and / or one or more antioxidants and / or one or more tension regulators and / or one or more stabilizers and / or one or more viscosity enhancers.

[0303] 26. The combination, composition for use, method or use of any one of embodiments 1 to 11, wherein the oncolytic peptide is a peptide, peptidomimetic or amino acid derivative having a net positive charge of at least +2 and comprising disubstituted β-amino acids, wherein each substituent in the β-amino acid contains at least 7 non-hydrogen atoms, is lipophilic and has at least one cyclic group, wherein one or more cyclic groups within a substituent are optionally fused to one or more cyclic groups within another substituent.

[0304] 27. The combination, composition for use, method or use of embodiment 26, wherein one or more cyclic groups within a substituent are fused to one or more cyclic groups within another substituent and the total number of non-hydrogen atoms in the two substituents combined is at least 12.

[0305] 28. The combination, composition for use, method or use according to any one of embodiments 26 to 27, wherein the oncolytic peptide is a compound comprising a group of formula (VI):

[0306]

[0307] wherein X and N have their normal valences and are attached to the rest of the compound, and wherein any two from R1, R2, R3 and R4 are hydrogen atoms and the other two are substituents, the substituents comprising at least 7 non-hydrogen atoms, being lipophilic and comprising a cyclic group, wherein the cyclic group is not directly attached to an α carbon atom or a β carbon atom and is optionally attached to or fused to a cyclic group in the other substituent, and wherein X represents O, C, N or S.

[0308] 29. The combination, composition for use, method or use according to embodiment 28, wherein the substituent groups are the same or different.

[0309] 30. The combination, composition for use, method or use of any one of embodiments 28 or 29, wherein the cyclic group is linked to or fused to a cyclic group in another substituent, and wherein the total number of non-hydrogen atoms of the two substituents combined is at least 12.

[0310] 31. The combination, composition for use, method or use according to any one of embodiments 28 or 30, wherein the nitrogen atom in the group of formula (VI) does not interact with the group R 1-4 Any atom bonded to, and / or wherein, the five atoms in the skeleton (NC β -C α -CX) are connected to each other in a linear manner, and / or wherein R 1-4It is lipophilic in nature, preferably uncharged, and preferably has no more than two, more preferably no more than one polar group, and / or wherein X is a substituted nitrogen atom.

[0311] 32. The combination, composition for use, method or use according to any one of embodiments 28 or 31, wherein the compound comprising a group of formula (VI) has an amidated C-terminus.

[0312] 33. The combination, composition for use, method or use according to any one of embodiments 1 to 11 and 26 to 32, wherein the oncolytic peptide is LTX-401, a compound of formula (VII):

[0313]

[0314] 34. The combination, composition for use, method or use according to embodiment 33, wherein LTX 401 is in the form of a pharmaceutically acceptable salt, preferably a hydrochloride or acetate salt.

[0315] 35. The combination, composition for use, method or use according to any of the preceding embodiments, wherein composition (a) comprises the oncolytic peptide at a concentration of 0.1 mg / ml to 30 mg / ml, for example 1 mg / ml to 5 mg / ml or 1 mg / ml to 30 mg / ml, preferably 4 mg / ml to 25 mg / ml, or more preferably 6 mg / ml to 20 mg / ml.

[0316] 36. The combination, composition for use, method or use according to any preceding embodiment, wherein the pH of composition (a) is from 4.0 to 7.4, more preferably from 5.0 to 7.0, and even more preferably from 5.0 to 6.5, such as 6.0.

[0317] 37. The combination, composition for use, method or use according to any one of the preceding embodiments, wherein the oncolytic peptide is in the form of a pharmaceutically acceptable salt, more preferably in the form of a hydrochloride or acetate salt.

[0318] 38. The combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) comprises chitosan.

[0319] 39. The combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) comprises a plurality of different chitosans.

[0320] 40. The combination, composition for use, method or use according to any one of embodiments 38 to 39, wherein the chitosan is very low molecular weight chitosan, low molecular weight chitosan and / or medium molecular weight chitosan.

[0321] 41. The combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) comprises a chitosan derivative.

[0322] 42. The combination, composition for use, method or use according to embodiment 41, wherein composition (b) comprises a plurality of different chitosan derivatives.

[0323] 43. The combination, composition for use, method or use according to any one of embodiments 41 to 42, wherein the chitosan derivative is a saccharified chitosan, preferably a saccharified chitosan having a degree of saccharification of about 0.1% to about 90% of its originally free amino groups and / or a saccharified chitosan having a molecular weight of about 50 kD to about 2000 kD.

[0324] 44. The combination, composition for use, method or use according to any preceding embodiment, wherein the chitosan and / or chitosan derivative has a degree of deacetylation (DDA) of about 50% to 99%, preferably a DDA of about 70% to about 99%.

[0325] 45. The combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) comprises 0.1% w / w to 5% w / w of chitosan and / or chitosan derivatives, such as 0.1% w / w to 2.5% w / w, such as 0.5% w / w, 1% w / w, 1.5% w / w or 2% w / w.

[0326] 46. ​​The combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) is a dispersion, suspension or solution of chitosan and / or a chitosan derivative in a physiologically acceptable carrier.

[0327] 47. The combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) has a pH of 4.0 to 7.5, preferably 5.0 to 7.4, more preferably 5.0 to 7.0, such as 6.5 or 6.0.

[0328] 48. The combination, composition for use, method or use according to any one of the preceding embodiments, wherein the viscosity of composition (a) and / or composition (b) is from 1.0 cP, 1.5 cP, 2 cP, 2.5 cP or 3 cP to 300 cP, such as from 2.5 cP to 250 cP, such as from 5 cP to 100 cP or from 10 cP to 200 cP and from 20 cP to 240 cP, preferably from 40 cP to 180 cP, for example 45 cP to 150 cP or 30 cP to 100 cP, and more preferably 1.5 cP to 10 cP, for example 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP or 10 cP, or 10 cP to 15 cP or 15 cP to 20 cP or 20 cP to 35 cP or 35 cP to 50 cP or 50 cP to 100 cP.

[0329] 49. The combination, composition for use, method or use according to any preceding embodiment, wherein composition (a) is administered into the tumor, for example by injection and / or by perfusion / infusion into the tumor.

[0330] 50. A combination, composition for use, method or use according to any preceding embodiment, wherein composition (b) is administered into the tumor, for example by injection and / or by perfusion / infusion into the tumor and / or into the tissue immediately surrounding the tumor.

[0331] 51. A combination, composition for use, method or use according to any preceding embodiment, wherein composition (a) and composition (b) are administered simultaneously, e.g., simultaneously (e.g., by the same or different modes of administration) or within a short period of time, e.g., within minutes or hours or longer, but on the same day.

[0332] 52. The combination, composition for use, method or use according to embodiment 51, wherein composition (a) is administered first and composition (b) is administered secondarily.

[0333] 53. The combination, composition for use, method or use according to embodiment 51, wherein composition (b) is administered first and then composition (a).

[0334] 54. The combination, composition for use, method or use according to any one of embodiments 1 to 50, wherein composition (a) and composition (b) are administered sequentially, e.g. on different days (e.g. by the same or different administration routes), such as one or more days apart or such as one or more weeks apart.

[0335] 55. The combination, composition for use, method or use according to embodiment 54, wherein composition (a) is administered first and composition (b) is administered secondarily.

[0336] 56. The combination, composition for use, method or use according to embodiment 54, wherein composition (b) is applied first and then composition (a).

[0337] 57. The combination, composition for use, method or use according to any preceding embodiment, wherein each of composition (a) and composition (b) is administered only once, for example into a tumor.

[0338] 58. The combination, composition for use, method or use according to any one of embodiments 1 to 56, wherein each of composition (a) and composition (b) is administered repeatedly, for example, into a tumor.

[0339] 59. The combination, composition for use, method or use of any one of embodiments 1 to 56, wherein one of composition (a) and composition (b) is administered only once, e.g., into a tumor, and the other composition is administered repeatedly, e.g., into a tumor.

[0340] 60. The combination, composition for use, method or use according to any of the preceding embodiments, wherein composition (a) and composition (b) are administered in a therapeutically effective amount, for example, into a tumor, and wherein the amount is administered in one or more administrations.

[0341] 61. The combination, composition for use, method or use of any of the preceding embodiments, wherein the tumor is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, liver cancer, bile duct cancer, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.

[0342] 62. The combination, composition for use, method or use according to any preceding embodiment, wherein the tumor is a tumor selected from the group consisting of carcinoma, adenocarcinoma, lymphoma, melanoma, blastoma, leukemia, sarcoma and germ cell tumor.

[0343] 63. The combination, composition for use, method or use according to any one of the preceding embodiments, wherein composition (a) and composition (b) are pharmaceutical compositions.

[0344] 64. A composition comprising i) an oncolytic peptide and ii) chitosan and / or a chitosan derivative.

[0345] 65. The composition according to embodiment 64, comprising one or more oncolytic peptides and / or one or more different chitosans and / or chitosan derivatives.

[0346] 66. The composition of any one of embodiments 64 to 65, wherein the composition comprises a physiologically acceptable carrier.

[0347] 67. A composition according to embodiment 66, wherein the physiologically acceptable carrier is selected from the group consisting of: water, such as water for injection (WFI), saline and aqueous buffer.

[0348] 68. The composition of any one of embodiments 66 to 67, wherein the composition comprises a mixture of different physiologically acceptable carriers.

[0349] 69. A composition according to any one of embodiments 66 to 68, wherein the composition comprises water, such as water for injection, including a salt selected from the group consisting of sodium chloride, potassium chloride, calcium chloride and magnesium chloride.

[0350] 70. A composition according to any one of embodiments 64 to 69, wherein the oncolytic peptide is a nonapeptide or a pharmaceutically acceptable salt thereof, and the nonapeptide or a pharmaceutically acceptable salt thereof consists of 9 linearly arranged amino acids, wherein, among these 9 amino acids, 5 amino acids are cationic amino acids, and 4 amino acids are lipophilic amino acids, and wherein, among these 4 lipophilic amino acids, 3 lipophilic amino acids are tryptophan and 1 lipophilic amino acid is a non-genetically encoded amino acid.

[0351] 71. A composition according to embodiment 70, wherein the lipophilic amino acids and cationic amino acids are arranged so that no more than two amino acids of either type are adjacent to each other and / or the peptide contains two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic amino acids.

[0352] 72. A composition according to any one of embodiments 70 to 71, wherein the cationic amino acids are the same or different and are selected from the group consisting of: lysine, arginine, histidine and non-genetically encoded amino acids that carry a positive charge at pH 7.0, preferably selected from the group consisting of: lysine and arginine.

[0353] 73. The composition of any one of embodiments 70 to 72, wherein the non-genetically encoded cationic amino acid is selected from the group consisting of derivatives of lysine, derivatives of arginine, and derivatives of histidine.

[0354] 74. The composition of any one of embodiments 70 to 73, wherein the non-genetically encoded cationic amino acid is selected from the group consisting of homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid, homoarginine, trimethyllysine, trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4-amino-1-carbamoylimidopiperidine-4-carboxylic acid, and 4-guanidinophenylalanine.

[0355] 75. A composition according to any one of embodiments 70 to 74, wherein the non-genetically encoded lipophilic amino acid has a lipophilic side chain, the lipophilic side chain has at least 7, at least 8, at least 9 or at least 10 non-hydrogen atoms, preferably wherein the non-hydrogen atoms are carbon atoms.

[0356] 76. A composition according to any one of embodiments 70 to 75, wherein the non-genetically encoded lipophilic amino acid has a lipophilic side chain containing no more than 30 or no more than 25 non-hydrogen atoms, and / or wherein such lipophilic side chain comprises at least one, preferably two, optionally fused or linked cyclic groups, and / or wherein the lipophilic side chain comprises heteroatoms such as O, N or S, preferably only one heteroatom, more preferably a nitrogen atom, and / or wherein the lipophilic side chain comprises 2 or less than 2 polar groups, preferably one and more preferably no polar groups.

[0357] 77. A composition according to any one of embodiments 70 to 76, wherein the non-genetically encoded lipophilic amino acid is selected from the group consisting of: 2-amino-3-(biphenyl-4-yl)propionic acid (biphenylalanine), 2-amino-3,3-diphenylpropionic acid (diphenylalanine), 2-amino-3-(anthracen-9-yl)propionic acid, 2-amino-3-(naphthalen-2-yl)propionic acid, 2-amino-3-(naphthalen-1-yl)propionic acid, 2-amino-3-[1,1':4',1"-terphenyl-4-yl]propionic acid, 2-amino-3-(2,5,7-tri-tert-butyl-1H-indole- 1':3',1"-terphenyl-5'-yl]propionic acid, and 2-amino-3-(4-(2,2-diphenylethyl)phenyl)propionic acid. More preferably, the lipophilic amino acid is selected from the group consisting of diphenylalanine and biphenylalanine.

[0358] 78. The composition according to any one of embodiments 70 to 77, wherein the nonapeptide is a compound of formula (I) to formula (V), wherein C represents a cationic amino acid and L represents a lipophilic amino acid, and wherein the amino acids are covalently linked via peptide bonds and preferably wherein the carboxyl terminus is amidated:

[0359] CCLLCCLLC(I),

[0360] LCCLLCCLC(II),

[0361] CLLCCLLCC(III),

[0362] CCLLCLLCC(IV),

[0363] CLCCLLCCL(V).

[0364] 79. The composition of any one of embodiments 70 to 78, wherein the nonapeptide is a compound of formula (I'), formula (I"), formula (I'") or formula (II'), wherein C represents a cationic amino acid and L' represents a non-genetically encoded lipophilic amino acid, and wherein the amino acids are covalently linked by peptide bonds, and preferably wherein the carboxyl terminus is amidated:

[0365] CCL'LCCLLC(I'),

[0366] CCLLCCLL'C(I"),

[0367] CCLL'CCLLC(I"'),

[0368] LCCLL'CCLC(II').

[0369] 80. The composition of any one of embodiments 70 to 79, wherein the oncolytic peptide is selected from the group consisting of LTX-301, LTX-302, LTX-303, LTX-304, LTX-305, LTX-306, LTX-307, LTX-308, LTX-309, LTX-310, LTX-310, LTX-311, LTX-312, LTX-313, LTX-314, LTX-315, LTX-316 , LTX-317, LTX-318, LTX-319, LTX-320, LTX-321, LTX-322, LTX-323, LTX-324, LTX-325, LTX-326, LTX-327 , LTX-328, LTX-329, LTX-330, LTX-331, LTX-332, LTX-333, LTX-334, LTX-335, LTX-336, LTX-337 and LTX-338.

[0370] 81. The composition of any one of embodiments 70 to 80, wherein the oncolytic peptide is selected from the group consisting of LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329, preferably wherein the oncolytic peptide is LTX-315.

[0371] 82. The composition according to any one of embodiments 70 to 81, wherein the oncolytic peptide is LTX-315 in the form of a pharmaceutically acceptable salt, preferably in the form of acetate.

[0372] 83. A composition according to any one of embodiments 70 to 82, wherein composition (a) further comprises one or more preservatives and / or one or more chelating agents and / or one or more antioxidants and / or one or more tonicity regulators and / or one or more stabilizers and / or one or more viscosity increasing agents.

[0373] 84. A composition according to any one of embodiments 61 to 63, wherein the oncolytic peptide is a peptide, peptidomimetic or amino acid derivative having a net positive charge of at least +2 and comprising disubstituted β-amino acids, wherein each substituent in the β-amino acid contains at least 7 non-hydrogen atoms, is lipophilic and has at least one cyclic group, wherein one or more cyclic groups within the substituent are optionally fused to one or more cyclic groups within another substituent.

[0374] 85. A composition according to embodiment 84, wherein one or more cyclic groups in a substituent are fused to one or more cyclic groups in another substituent, and the total number of non-hydrogen atoms in the two substituents combined is at least 12.

[0375] 86. The composition of any one of embodiments 84 to 85, wherein the oncolytic peptide is a compound comprising group (IV):

[0376]

[0377] wherein X and N have their normal valences and are attached to the rest of the compound, and wherein any two from R1, R2, R3 and R4 are hydrogen atoms and the other two are substituents, the substituents comprising at least 7 non-hydrogen atoms, being lipophilic and comprising a cyclic group, wherein the cyclic group is not directly attached to an α carbon atom or a β carbon atom and is optionally attached to or fused to a cyclic group in the other substituent, and wherein X represents O, C, N or S.

[0378] 87. A composition according to embodiment 86, wherein the substituents are the same or different.

[0379] 88. A composition according to any one of embodiments 86 to 87, wherein the cyclic group is connected or fused to a cyclic group in another substituent, and wherein the total number of non-hydrogen atoms in the two substituents combined is at least 12.

[0380] 89. A composition according to any one of embodiments 86 to 88, wherein the nitrogen atom in the group of formula (VI) is not in contact with the group R 1-4 Any atom bonded to, and / or wherein, the five atoms in the skeleton (NC β -C α -CX) are connected to each other in a linear manner, and / or wherein R 1-4 It is lipophilic in nature, preferably uncharged, and preferably has no more than two, more preferably no more than one polar group, and / or wherein X is a substituted nitrogen atom.

[0381] 90. A composition according to any one of embodiments 86 to 89, wherein the compound comprising the group of formula VI has an amidated C-terminus.

[0382] 91. The composition of any one of embodiments 64 to 90, wherein the oncolytic peptide is LTX-401, a compound of formula (VII):

[0383]

[0384] 92. The composition according to embodiment 91, wherein LTX 401 is in the form of a pharmaceutically acceptable salt, preferably in the form of a hydrochloride or acetate salt.

[0385] 93. The composition according to any one of embodiments 64 to 92, wherein the oncolytic peptide is present in a concentration of 0.1 mg / ml to 30 mg / ml, such as 1 mg / ml to 5 mg / ml or 1 mg / ml to 30 mg / ml, preferably 4 mg / ml to 25 mg / ml, or more preferably 6 mg / ml to 20 mg / ml.

[0386] 94. A composition according to any one of embodiments 64 to 93, wherein the oncolytic peptide is in the form of a pharmaceutically acceptable salt, more preferably in the form of a hydrochloride or acetate salt.

[0387] 95. The composition of any one of embodiments 64 to 94, wherein the composition comprises chitosan.

[0388] 96. The composition of any one of embodiments 64 to 95, wherein the composition comprises a plurality of different chitosans.

[0389] 97. The composition of any one of embodiments 64 to 96, wherein the chitosan is very low molecular weight chitosan, low molecular weight chitosan and / or medium molecular weight chitosan.

[0390] 98. The composition of any one of embodiments 64 to 97, wherein the composition comprises a chitosan derivative.

[0391] 99. The composition of embodiment 98, wherein the composition comprises a plurality of different chitosan derivatives.

[0392] 100. A composition according to any one of embodiments 64 to 99, wherein the chitosan derivative is a saccharified chitosan, preferably a saccharified chitosan having a saccharification degree of about 0.1% to about 90% of its originally free amino groups and / or a saccharified chitosan having a molecular weight of about 50 kD to about 2000 kD.

[0393] 101. The composition according to any one of embodiments 64 to 100, wherein the chitosan and / or chitosan derivative has a degree of deacetylation (DDA) of about 50% to 99%, preferably about 70% to about 99%.

[0394] 102. A composition according to any one of embodiments 64 to 101, wherein the composition comprises 0.1% w / w to 5% w / w chitosan and / or chitosan derivatives, such as 0.1% w / w to 2.5% w / w, such as 0.5% w / w, 1% w / w, 1.5% w / w or 2% w / w.

[0395] 103. The composition of any one of embodiments 64 to 102, wherein the composition is a dispersion, suspension or solution of i) and ii) in a physiologically acceptable carrier.

[0396] 104. The composition of any one of embodiments 64 to 103, wherein the pH of the composition is 4.0 to 7.4, more preferably 5.0 to 7.0, such as 6.0 or 6.5.

[0397] 105. A composition according to any one of embodiments 64 to 104, wherein the viscosity of the composition is 1.0 cP, 1.5 cP, 2 cP, 2.5 cP or 3 cP to 300 cP, for example, 2.5 cP to 250 cP, for example, 5 cP to 100 cP or 10 cP to 200 cP and 20 cP to 240 cP, preferably 40 cP to 180 cP, for example, 45 cP to 150 cP or 30 cP to 100 cP, more preferably 1.5 cP to 10 cP, for example, 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP or 10 cP, or 10 cP to 15 cP or 15 cP to 20 cP or 20 cP to 35 cP or 35 cP to 50 cP or 50 cP to 100 cP.

[0398] 106. The composition of any one of embodiments 64 to 105, wherein the composition is a pharmaceutical composition.

[0399] 107. The composition of any one of embodiments 64 to 106, contained in a sealed container.

[0400] 108. A composition as defined in any one of embodiments 64 to 107 for use as a medicament.

[0401] 109. A composition as defined in any one of embodiments 64 to 108 for use in a method of treating a tumor in a subject.

[0402] 110. The composition for use according to embodiment 109, wherein the composition is administered to a subject, for example, into a tumor.

[0403] 111. The composition for use according to embodiment 110, wherein the composition is administered only once, for example, into a tumor.

[0404] 112. The composition for use according to embodiment 110, wherein the composition is administered repeatedly, for example, into a tumor.

[0405] 113. The composition for use according to any one of embodiments 109 to 112, wherein the composition is administered in a therapeutically effective amount, for example, into a tumor, and wherein the administration is performed by a single administration or multiple administrations.

[0406] 114. A composition for use according to any one of embodiments 109 to 113, wherein the tumor is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, liver cancer, bile duct cancer, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.

[0407] 115. The composition for use according to any one of embodiments 109 to 114, wherein the tumor is a tumor selected from the group consisting of carcinoma, adenocarcinoma, lymphoma, melanoma, blastoma, leukemia, sarcoma and germ cell tumor.

[0408] 116. A method for preparing a composition as defined in any one of embodiments 64 to 106, wherein chitosan and / or a chitosan derivative is dispersed, suspended or dissolved in a physiologically acceptable carrier and the oncolytic peptide is dissolved in said dispersion, suspension or solution, or wherein chitosan and / or a chitosan derivative is dispersed, suspended or dissolved in a solution of the oncolytic peptide in a physiologically acceptable carrier.

[0409] 117. The method of embodiment 116, wherein the composition is sterile filtered and then filled into containers and sealed.

[0410] 118. The method of embodiment 116, wherein the composition is filled into containers, sealed and autoclaved.

[0411] 119. A kit comprising composition (a) and composition (b) as defined in any one of embodiments 1 to 63 or comprising means / components for preparing said composition (a) and said composition (b).

[0412] 120. The kit according to embodiment 119 comprises an oncolytic peptide and a physiologically acceptable carrier for preparing composition (a) and chitosan and / or a chitosan derivative and a physiologically acceptable carrier for preparing composition (b), and optionally comprises instructions for preparing composition (a) and composition (b).

[0413] 121. A kit according to embodiment 120, wherein the physiologically acceptable carrier used to prepare composition (a) is the same as the physiologically acceptable carrier used to prepare composition (b).

[0414] 122. A kit according to embodiment 119, comprising composition (a), chitosan and / or chitosan derivatives (e.g. in solid form) and a physiologically acceptable carrier for preparing composition (b), and optionally comprising instructions for preparing composition (b).

[0415] 123. A kit according to embodiment 119, comprising composition (b), an oncolytic peptide (e.g., in solid form) and a physiologically acceptable carrier for preparing composition (a), and optionally comprising instructions for preparing composition (a).

[0416] 124. The kit of any one of embodiments 119 to 123, further comprising instructions for use of composition (a) and composition (b).

[0417] 125. A kit according to embodiment 119, comprising composition (a) and composition (b) as defined in any one of embodiments 1 to 63 and instructions for preparing a composition as defined in any one of embodiments 64 to 106 by combining said composition (a) and said composition (b).

[0418] 126. The kit according to embodiment 125 further comprises instructions for using the combined composition.

[0419] 127. A kit comprising means / components for preparing a composition as defined in any one of embodiments 64 to 106.

[0420] 128. A kit according to embodiment 127, wherein the kit comprises an oncolytic peptide, chitosan and / or a chitosan derivative (for example, both are in solid form) and a physiologically acceptable carrier for preparing a combined composition.

[0421] 129. The kit according to embodiment 128, wherein the oncolytic peptide, chitosan and / or chitosan derivative are contained in one container.

[0422] 130. The kit according to embodiment 128, wherein the oncolytic peptide and chitosan and / or chitosan derivative are contained in separate containers.

[0423] 131. A kit according to embodiment 127, wherein the kit comprises composition (a), which comprises chitosan and / or chitosan derivatives (e.g., in solid form) as defined in any one of embodiments 1 to 63, and optionally comprises instructions for preparing the composition as defined in any one of embodiments 64 to 106.

[0424] 132. A kit according to embodiment 127, wherein the kit comprises a composition (b) as defined in any one of embodiments 1 to 63, wherein composition (b) comprises a physiologically acceptable carrier and an oncolytic peptide (e.g., in solid form), and optionally comprises instructions for preparing the composition as defined in any one of embodiments 64 to 106.

[0425] 133. The kit according to any one of embodiments 127 to 132, further comprising instructions for using the composition as defined in any one of embodiments 64 to 106.

Claims

1. A composition (a) for treating a subject suffering from a tumor by simultaneous, sequential or combined administration with a composition (b), wherein the composition (a) comprises an oncolytic peptide and the composition (b) comprises chitosan and / or a chitosan derivative.

2. The composition for use according to claim 1, wherein The composition (a) comprises one or more oncolytic peptides, and / or wherein the composition (b) comprises one or more different chitosans and / or chitosan derivatives.

3. A composition for use according to any one of claims 1 to 2, wherein The composition (a) and / or the composition (b) comprises a physiologically acceptable carrier.

4. A composition for use according to any one of claims 1 to 3, wherein The oncolytic peptide is a nonapeptide or a pharmaceutically acceptable salt thereof, and the nonapeptide or a pharmaceutically acceptable salt thereof is composed of 9 linearly arranged amino acids, wherein, among these 9 amino acids, 5 amino acids are cationic amino acids, 4 amino acids are lipophilic amino acids, and wherein, among these 4 lipophilic amino acids, 3 lipophilic amino acids are tryptophan, and 1 lipophilic amino acid is a non-genetically encoded amino acid.

5. A composition for use according to claim 4, wherein The cationic amino acid is lysine or arginine, and / or wherein, The non-genetically encoded amino acid is diphenylalanine or biphenylalanine.

6. A composition for use according to any one of claims 1 to 5, wherein The oncolytic peptide is selected from the group consisting of LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329.

7. A composition for use according to any one of claims 1 to 6, wherein The oncolytic peptide is LTX-315.

8. A composition for use according to any one of claims 1 to 7, wherein The composition (a) further comprises one or more preservatives and / or one or more chelating agents and / or one or more antioxidants and / or one or more tonicity regulators and / or one or more stabilizers and / or one or more viscosity increasing agents.

9. A composition for use according to any one of claims 1 to 3, wherein The oncolytic peptide is a peptide, peptidomimetic or amino acid derivative having a net positive charge of at least +2 and comprising a disubstituted β-amino acid, wherein each substituent in the β-amino acid contains at least 7 non-hydrogen atoms, is lipophilic and has at least one cyclic group, wherein one or more cyclic groups within the substituent are optionally fused to one or more cyclic groups within another substituent.

10. A composition for use according to claim 9, wherein The oncolytic peptide is a compound comprising a group of formula (VI): wherein X and N have their normal valences and are attached to the rest of the compound, and wherein any two from R1, R2, R3 and R4 are hydrogen atoms and the other two are substituents, said substituents comprising at least 7 non-hydrogen atoms, being lipophilic and comprising a cyclic group, wherein said cyclic group is not directly attached to an α carbon atom or a β carbon atom and is optionally attached to or fused to a cyclic group in said other substituent, and wherein X represents O, C, N or S.

11. A composition for use according to claim 10, wherein The compound comprising the group of formula (VI) has an amidated C-terminus.

12. A composition for use according to any one of claims 9 to 11, wherein The oncolytic peptide is LTX-401, a compound of formula (VII):

13. A composition for use according to any one of the preceding claims, wherein The oncolytic peptide is in the form of a pharmaceutically acceptable salt.

14. A composition for use according to any one of the preceding claims, wherein The composition (a) comprises the oncolytic peptide at a concentration of 0.1 mg / ml to 30 mg / ml.

15. A composition for use according to any one of the preceding claims, wherein The pH of the composition (a) is 4.0 to 7.

4.

16. A composition for use according to any one of the preceding claims, wherein The composition (b) comprises chitosan.

17. A composition for use according to any one of the preceding claims, wherein The composition (b) comprises a chitosan derivative.

18. A composition for use according to claim 15, wherein The chitosan derivative is saccharified chitosan.

19. A composition for use according to any one of the preceding claims, wherein The chitosan and / or chitosan derivative has a degree of deacetylation (DDA) of about 50% to 99%.

20. A composition for use according to any one of the preceding claims, wherein The composition (b) comprises 0.1% w / w to 5% w / w of the chitosan and / or the chitosan derivative.

21. A composition for use according to any one of the preceding claims, wherein The composition (b) comprises chitosan and / or chitosan derivatives of very low molecular weight, low molecular weight or medium molecular weight.

22. A composition for use according to any one of the preceding claims, wherein The composition (b) is a dispersion, suspension or solution of the chitosan and / or the chitosan derivative in a physiologically acceptable carrier.

23. A composition for use according to any one of the preceding claims, wherein The pH of the composition (b) is 4.0 to 7.

5.

24. A composition for use according to any one of the preceding claims, wherein The viscosity of the composition (a) and / or the composition (b) is 1.0 cP to 300 cP.

25. A composition for use according to any one of the preceding claims, wherein The composition (a) is administered into the tumor and the composition (b) is administered into the tumor and / or into the tissue immediately surrounding the tumor.

26. A composition for use according to any one of the preceding claims, wherein The tumor is a tumor selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, stomach cancer, skin cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer, liver cancer, bile duct cancer, brain cancer, cervical cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.

27. A composition comprising: i) an oncolytic peptide; and ii) chitosan and / or a chitosan derivative.

28. The composition according to claim 25, wherein The composition comprises one or more oncolytic peptides and / or comprises one or more different chitosans and / or chitosan derivatives.

29. The composition according to any one of claims 25 to 26, wherein The composition comprises a physiologically acceptable carrier. 30.

4. The composition according to any one of claims 25 to 27, wherein The oncolytic peptide is a nonapeptide or a pharmaceutically acceptable salt thereof, and the nonapeptide or a pharmaceutically acceptable salt thereof is composed of 9 linearly arranged amino acids, wherein, among these 9 amino acids, 5 amino acids are cationic amino acids, 4 amino acids are lipophilic amino acids, and wherein, among these 4 lipophilic amino acids, 3 lipophilic amino acids are tryptophan, and 1 lipophilic amino acid is a non-genetically encoded amino acid.

31. The composition according to claim 28, wherein The cationic amino acid is lysine or arginine, and / or wherein, The non-genetically encoded amino acid is diphenylalanine or biphenylalanine.

32. The composition according to any one of claims 25 to 29, wherein The oncolytic peptide is selected from the group consisting of LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329.

33. The composition according to any one of claims 25 to 30, wherein The oncolytic peptide is LTX-315.

34. A composition according to any one of claims 25 to 31, wherein The composition may further comprise one or more preservatives and / or one or more chelating agents and / or one or more antioxidants and / or one or more tonicity adjusting agents and / or one or more stabilizers and / or one or more viscosity increasing agents.

35. The composition according to any one of claims 25 to 27, wherein The oncolytic peptide is a peptide, peptidomimetic or amino acid derivative having a net positive charge of at least +2 and comprising a disubstituted β-amino acid, wherein each substituent in the β-amino acid contains at least 7 non-hydrogen atoms, is lipophilic and has at least one cyclic group, wherein one or more cyclic groups within the substituent are optionally fused to one or more cyclic groups within another substituent.

36. The composition according to claim 33, wherein The oncolytic peptide is a compound comprising a group of formula (VI): wherein X and N have their normal valences and are attached to the rest of the compound, and wherein any two from R1, R2, R3 and R4 are hydrogen atoms and the other two are substituents, said substituents comprising at least 7 non-hydrogen atoms, being lipophilic and comprising a cyclic group, wherein said cyclic group is not directly attached to an α carbon atom or a β carbon atom and is optionally attached to or fused to a cyclic group in said other substituent, and wherein X represents O, C, N or S.

37. The composition according to claim 34, wherein The compound comprising the group of formula (VI) has an amidated C-terminus.

38. A composition according to any one of claims 33 to 35, wherein The oncolytic peptide is LTX-401, a compound of formula (VII):

39. The composition according to any one of claims 25 to 36, wherein The oncolytic peptide is in the form of a pharmaceutically acceptable salt.

40. The composition according to any one of claims 25 to 37, wherein The composition comprises the oncolytic peptide at a concentration of 0.1 mg / ml to 30 mg / ml.

41. A composition according to any one of claims 25 to 38, wherein The composition comprises chitosan.

42. A composition according to any one of claims 25 to 39, wherein The composition comprises a chitosan derivative.

43. The composition according to claim 40, wherein The chitosan derivative is saccharified chitosan.

44. A composition according to any one of claims 25 to 41, wherein The chitosan and / or chitosan derivative has a degree of deacetylation (DDA) of about 50% to 99%.

45. The composition according to any one of claims 25 to 42, wherein The composition comprises 0.1% w / w to 5% w / w of the chitosan and / or the chitosan derivative.

46. ​​A composition according to any one of claims 25 to 43, wherein The composition comprises very low molecular weight, low molecular weight or medium molecular weight chitosan and / or chitosan derivatives.

47. A composition according to any one of claims 25 to 44, wherein The composition is a dispersion, suspension or solution of i) and ii) in a physiologically acceptable carrier.

48. A composition according to any one of claims 25 to 45, wherein The pH of the composition is from 4.0 to 7.

4.

49. The composition according to any one of claims 25 to 46, wherein The viscosity of the composition is 1.0 cP to 300 cP.

50. A composition as defined in any one of claims 25 to 47 for use as a medicament.

51. A composition as defined in any one of claims 25 to 47 for use in a method of treating a tumour in a subject.

52. A process for the preparation of a composition as defined in any one of claims 25 to 47, wherein Chitosan and / or chitosan derivatives are dispersed, suspended or dissolved in a physiologically acceptable carrier, and the oncolytic peptide is dissolved in the dispersion, suspension or solution; or chitosan and / or chitosan derivatives are dispersed, suspended or dissolved in a solution of the oncolytic peptide in the physiologically acceptable carrier.

53. A kit comprising composition (a) and composition (b) as defined in any one of claims 1 to 24, or comprising means / components for preparing said composition (a) and said composition (b).

54. The kit according to claim 51, wherein The kit comprises composition (a) and composition (b) as defined in any one of claims 1 to 24, and instructions for preparing a composition as defined in any one of claims 25 to 47 by combining composition (a) and composition (b).

55. A kit comprising means / components for preparing a composition as defined in any one of claims 25 to 47.

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