Water-soluble peptide delivery system for anticancer drugs

Abstract

Micelles of peptidic conjugates of SN-38 loaded with one or more free therapeutically active agents having anti-cancer activity, such as free SN-38 lactone, processes for their preparation, pharmaceutical compositions containing them, and their therapeutic applications as anti-cancer agents.

Description

[Technical field]

[0001] This application claims the benefit of European Patent Application No. 22382287, filed March 28, 2022.

[0002] The present invention relates to the field of delivery systems containing anticancer drugs, processes for their preparation and their therapeutic applications. [Background technology]

[0003] Campthotecins are a family of topoisomerase I inhibitors with anticancer properties. They are chemically characterized by the presence of a lactone ring that confers anticancer activity and is stable at acidic pH. The lactone ring opens to a carboxylate at neutral or acidic pH. Such conversion is nonenzymatic, and it can be converted back to the lactone by acidification of the pH. The carboxylate is 100-1000 times less potent than the lactone.

[0004] Irinotecan is a camptothecin approved for the treatment of several types of cancer. SN-38 (7-ethyl-10-hydroxycamptothecin) is the active metabolite of irinotecan, formed via hydrolysis of its water-soluble prodrug irinotecan by hepatic carboxylesterases and metabolized via glucuronidation by UGT1A1. It has the formula: [ka]

[0005] SN-38 has 1000 times greater activity than irinotecan itself and may be used more than irinotecan to treat the same types of cancer. In vitro cytotoxicity assays show that the potency of SN-38 varies from 2 to 2000 times compared to irinotecan.

[0006] SN-38 has significant limitations at the chemical, pharmacological, and toxicity levels. At the chemical level, unlike irinotecan, which is water-soluble at acidic pH, SN-38 is practically water-insoluble at acidic or neutral pH, making it impractical to administer SN-38 to patients while maintaining the active (lactone) form of the molecule in most solvents and oils. Many solvents have been tested, including dimethyl sulfoxide, formic acid, and Transcutol® HP, as well as NaOH (0.1M), which is capable of solubilizing only 0.5% of SN-38, but the basic pH of this 0.1M aqueous NaOH solution opens the lactone ring, thus inactivating SN-38. Thus, the highly lipophilic character of SN-38 prevents the administration of this drug to humans in a clinically acceptable vehicle. At neutral or basic pH, the balance shifts in favor of less active species due to lactone ring opening, whereas at more acidic pH the formation of the lactone with high cancer inhibitory power is favored.The drug also has an asymmetric carbon at position 20, with the S-form being the pharmacologically active configuration.

[0007] In summary, two forms of SN-38, lipophilic lactone (SN-38 lactone with strong anticancer activity) and hydrophilic carboxylate (SN-38 carboxylate without anticancer activity), are in equilibrium in water. SN-38 lactone predominates under acidic pH conditions, which is practically insoluble in water (<40 μg / ml according to JA: Zhang et al., Development and characterization of a novel liposome-based formulation of SN-38. International Journal of Pharmaceutics (2004), vol. 270(1-2), pp. 93-107), whereas SN-38 carboxylate predominates at neutral and basic pH, which is freely soluble in water. Both SN-38 forms are in equilibrium, and when an SN-38 carboxylate solution (basic pH) is poured into an acidic pH solution, the carboxylate form precipitates and forms needle-like crystals, even in the presence of non-ionic surfactants such as Pluronic® F68 (see EP 2644191). This precipitation process is associated with strong intermolecular interactions between the free SN-38 lactone molecules.

[0008] Like many other lipophilic small molecule drugs, camptothecin self-assembles in water to form insoluble aggregates (see A. Sosnik, Drug self-assembly: A phenomenon at the nanometer scale with major impact in the structure-biological properties relationship and the treatment of disease, Prog Mater Sci 2016, vol. 82, pp. 39-82).

[0009] Considering the unfavorable characteristics of this drug, some studies have led to chemical conjugation of SN-38 with solubilizing chemical groups to generate SN-38 prodrugs. Several conjugation strategies have been applied to SN-38 to release the drug without using the free drug SN-38.

[0010] Some of these are based on the release of SN-38 from soluble conjugates. For example, US Patent No. 8,299,089 proposes the use of multi-arm PEG to conjugate SN-38 via a suitable linker to improve solubility and release SN-38 by ester hydrolysis. However, these conjugates must be administered at high concentrations to be effective in vitro / in vivo.

[0011] Similarly, International Patent Publication No. 2015 / 051307 discloses several conjugates that release SN-38 from a 4-arm polyethylene glycol via a slow β-elimination mechanism to allow for low dose and long exposure regimens. Other conjugates are based on the use of peptidic conjugates to bypass hepatic activation and reduce gastrointestinal toxicity and interpatient variability, as compared to, for example, irinotecan. Thus, for example, in "DTS-108, A novel peptidic prodrug of SN-38: In vivo efficacy and toxicokinetic studies", Clinical Cancer Research 2008, vol.14, issue 7, pp. 2145-2153, F Meyer-Losic et al. propose conjugating SN-38 to a cationic peptide (Vectocell) via an esterase-cleavable linker to deliver significantly higher levels of SN-38 than irinotecan, without the associated irinotecan toxicity, resulting in an increased therapeutic window of DTS-108 in preclinical models.

[0012] Other strategies to solubilize free SN-38 lactone in aqueous solution have also been disclosed in the state of the art, including the use of amphiphilic polymers that can self-assemble into a core-shell structure by aggregation of hydrophobic moieties when in an aqueous environment. EP 3753966 discloses an amphiphilic block copolymer that includes a hydrophilic chain segment, a hydrophobic chain segment, and a linker for connecting the hydrophilic chain segment to the hydrophobic chain segment. The linker includes an unsaturated structure to enhance the interaction between the poorly soluble drug (SN-38) and the copolymer.

[0013] Polymeric structures capable of self-assembling into micelles containing SN-38 encapsulated in their structures have also been disclosed. For example, in “Amphiphilic Polymeric Nanoparticles Modified with a Protease-Resistant Peptide Shuttle for the Delivery of SN-38 in Diffuse Intrinsic Pontine Glioma” ACS Applied Nano Materials 2021, vol. 4 (2), pp. 1314-1329, A. Buckin et al. disclose SN-38-loaded polymeric nanoparticles of amphiphilic chitosan (CS)-g-poly(methyl methacrylate)-poly(acrylic acid) copolymers whose surfaces are modified with a peptide shuttle that improves transport across the BBB.

[0014] China Patent Publication No. 102060991 describes an amphiphilic drug precursor of 7-ethyl-10-hydroxycamptothecin in which the 10- or 20-OH is linked to a hydrophilic group (e.g., PEG-200-2000) and can form micelles.

[0015] Ri, Masaki et al., "A phase I / II study for dose-finding, and to investigate the safety, pharmacokinetics and preliminary efficacy of NK012, an SN-38-incorporating macromolecular polymeric micelle, in patients with multiple myeloma," Internal Medicine (Tokyo, Japan) (2018), vol. 57(7), pp. 939-946, discloses SN-38-releasing polymeric micelles that are covalently bound to a block copolymer PEG-pGlu and then constructed by self-assembly of the amphiphilic block copolymer in an aqueous medium.

[0016] China Patent Publication No. 110124052 discloses conjugates of E-selectin peptide ligands bound to polyethylene glycol monomethyl ether and antitumor drugs such as camptothecin, hydroxycamptothecin, SN-38, paclitaxel, docetaxel, dasatinib, gemcitabine, doxorubicin, or podophyllotoxin, which can self-assemble into nanoparticles in aqueous solution.

[0017] Finally, Lei, Fan et al., Nanoscale platform for delivery of active IRINOX to combat pancreatic cancer Journal of Controlled Release 2021, vol. 330, pp.1229-1243, disclose crosslinked micelles prepared using amphiphilic PEG-b-poly(L-glutamic acid) / SN-38 conjugates and then loaded with dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt).

[0018] Despite efforts regarding the delivery system of SN-38 to enable proper administration and bioavailability of SN-38 for the treatment of cancer, there remains an unmet medical need for the discovery of an improved system for the efficient administration of SN-38, especially considering that neither irinotecan nor SN-38 can significantly cross the BBB. Thus, they have no efficacy in brain tumors with an intact blood-brain barrier (BBB), such as diffuse intrinsic pontine glioma (DIPG) and high-grade glioma (HGG), including pediatric type (pHGG). Summary of the Invention

[0019] The present inventors have found that certain peptide conjugates of SN-38 can form micelles by spontaneous self-assembly in aqueous media and can load free drug into their cores.

[0020] These micelles are particularly advantageous when loaded with SN-38 lactone and / or another anticancer drug. Unexpectedly, the formation of micelles with these peptide conjugates of SN-38 and free SN-38 lactone increases the apparent solubility of free SN-38 lactone in water at acidic pH by at least 500-fold, which is well above the state of the art.

[0021] The lactone form of SN-38 is highly water insoluble at acidic pH. However, acidic pH is necessary to keep SN-38 in its lactone (active) form, which undergoes a reversible and pH-dependent conversion to SN-38 carboxylate (a readily water-soluble inactive form by opening of the lactone ring at neutral or basic pH). The intrinsic aqueous solubility of SN-38 lactone at pH 3 has been determined to be 8 μg / mL (own data). The reported solubilities of SN-38 in water, pH 3.5 and 7.4 buffers are 11-38 μg / ml, 7.2 μg / ml, and 36 μg / ml, respectively (see Zhang et al., "Development and characterization of a novel liposome-based formulation of SN-38" International Journal of Pharmaceutics, 2004, vol. 270, pp. 93-107).

[0022] Conversely, in the present invention, the apparent solubility of free SN-38 lactone in water at acidic pH is at least 4 mg / ml, thus increasing the solubility by at least 500-fold, while the SN-38 liposomes disclosed by Zhang et al. achieve an apparent solubility of 0.111 mg / mL of SN-38, without the need for pharmaceutical excipients or lipid components, which exceeds by more than 40-fold the solubility of the best liposomal formulation of SN-38 reported in the previous state of the art.

[0023] Due to the insolubility of SN-38 lactone, in clinical practice SN-38 is administered as its water-soluble prodrug, irinotecan, which is approved for several cancer indications. After administration into the bloodstream, irinotecan releases SN-38 lactone as its active metabolite by the action of carboxylesterase enzymes. However, due to limited expression of these enzymes, conversion of irinotecan to SN-38 is low in humans. This explains why irinotecan is highly potent in animal models (mice) that are rich in carboxylesterases, but is less effective in humans (which have fewer carboxylesterases). Advantageously, the present invention has an advantage over the need for carboxylesterases, since it retains the SN-38 lactone in a free form.

[0024] Finally, unlike irinotecan and SN-38, the micelles of the present invention cross the blood-brain barrier (BBB) ​​to achieve therapeutic concentrations of SN-38 in the brain and cerebrospinal fluid (CSF) and provide therapeutic activity in DIPG and PHGG xenografts, thus providing a water-soluble, brain-penetrating delivery system for anticancer drugs useful for the treatment of brain cancer.

[0025] SN-38 peptide conjugates are formed from some peptides disclosed in International Patent Publication No. 2015 / 001015 and specific linkers of a specific size that are larger than those disclosed in the referenced documents. These conjugates alone provide antitumor activity in vitro up to 100 times better than the in vitro activity of irinotecan. They already exhibit high water solubility and activity alone that is higher than the activity of irinotecan in vitro. The SN-38-loaded micelles of the present invention show antitumor activity against several cell lines derived from brain and extracranial cancers, and are more active than the peptide conjugates alone.

[0026] Micelles are prepared from peptide conjugates of SN-38 by their dissolution in water at pH <7, preferably <3, by spontaneous micellization. An acidic solution of such micelles can neutralize a basic solution containing free SN-38 carboxylate at a concentration of up to 25 mg / mL. After acidification to basic pH, precipitation of free SN-38 lactone due to pH change leads to intermolecular interactions between the free SN-38 lactone molecules and the SN-38 molecules conjugated to the peptide conjugates of SN-38. Micellar dispersions of peptide conjugates of SN-38 containing free SN-38 lactone can be easily filtered through filters with pores of 0.22 and 0.45 μm without significant loss of free SN-38 lactone. The final system is composed of a drug-drug cocrystal composed of two forms of SN-38 lactone, the free form and the conjugated form. Under these conditions, free SN-38 lactone does not form microcrystals or large solid structures and remains apparently soluble at concentrations up to 4 mg / ml.

[0027] The micellar dispersions of these SN-38 binding peptide conjugates containing free SN-38 lactone are in the nanometer size scale. They can be used to load free SN-38 into their micelle cores. Thus, as illustrated for example in Example 15, the loading increases in this example from the current 21.5 (w / w)% SN-38 loading already contained in the conjugated product to up to 35 (w / w)% SN-38 in the final micelle product with free SN-38 lactone loaded into the micelles. The size of the micelles is about 40 nm at a concentration of 11 mM (20 mg / mL) without free SN-38 lactone and about 100 nm after encapsulation of free SN-38 lactone due to the effect of loading the micelles with free SN-38 lactone.

[0028] Thus, a first aspect of the present invention is a micelle comprising a peptide conjugate of SN-38 and one or more free therapeutically active agents having anti-cancer activity, the micelle is a core-shell structure comprising an inner core and an outer shell, the free therapeutically active agent is loaded into the inner core, and the peptide conjugate of SN-38 forms the outer shell; The peptide conjugate of SN-38 is a compound of formula (I) or a pharma- ceutically acceptable salt thereof: [ka] Z is the pharma- ceutical active ingredient SN-38 or a pharma- ceutical acceptable salt thereof, said pharma-ceutical active ingredient SN-38 having formula (II), and Z is attached to a linker, L, independently by only one of the two hydroxyl groups (a) or (b) of said pharma-ceutical active ingredient; [ka] L is a biradical consisting of 2 to 8 biradicals L', and has the formula: -L' a -(L' b ) n -L' c -, L a ' is -C(=O)-(CH2) r -C(=O)-;-C(=O)-(CH2) r -NH-;-C(=O)-(CH2) r -S-;-C(=O)-(CH2) r -O-;-C(=O)-NH-(CH2) r -C(=O)-;-C(=O)-NH-(CH2) r -NH-;-C(=O)-NH-(CH2) r -S-;-C(=O)-NH-(CH2) r -O-;-(CH2) r -C(=O)-;-(CH2) r -NH-;-(CH2) r -S-;-(CH2) r -O-;-Si(R1)(R2)-(CH2) r -NH-;-Si(R1)(R2)-(CH2) r -C(=O)-;-Si(R1)(R2)-(CH2) r-O-;-Si(R1)(R2)-(CH2) r -S-;-SO2-(CH2) r -NH-;-SO2-(CH2) r -C(=O)-;-SO2-(CH2) r -O-;-SO2-(CH2) r -S-;-P(=O)(OR1)-O-(CH2) r -NH-;-P(=O)(OR1)-O-(CH2) r -C(=O)-;-P(=O)(OR1)-O-(CH2) r -O-;-P(=O)(OR1)-O-(CH2) r -S-;-CH(OH)-(CH2) r -NH-;-CH(OH)-(CH2) r -C(=O)-;-CH(OH)-(CH2) r -O-;-CH(OH)-(CH2) r -S-; [ka] is a biradical selected from the group consisting of L8-L 11 The substituents on any of the rings: [ka] Located in one of the L b ' is independently -NH-(CH2) r -C(=O)-;C(=O)-(CH2) r -C(=O)-;-S-(CH2) r -C(=O)-;-O-(CH2) r -C(=O)-;-NH-(CH2) r -;-C(=O)-(CH2) r -;-S-(CH2) r -;-O-(CH2) r -;-NH-CH-((CH2) r NH2)-C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-(CH2) r -C(=O)-;-(CH2) r-O-;-(CH2) r -NH-;-(CH2) r -S-;-C(=O)-(CH2) r -NH-;-C(=O)-(CH2) r -O-;-C(=O)-(CH2) r -S-;-NH-(CH2) r -O-;-NH-(CH2) r -NH-;-NH-(CH2) r is a biradical selected from the group consisting of -S-, L1; L2; L3; L4; and combinations thereof; L c ' is:-NH-(CH2) r -C(=O)-;-NH-CH-((CH2) r -NH2)-C(=O)-;-C(=O)-(CH2) r -C(=O)-;-S-(CH2) r -C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-O-(CH2) r -C(=O)-, -(CH2) r -C(=O)-;L1, L2, L3, L4 [ka] is a biradical selected from the group consisting of P is (a) a peptide comprising the amino acid sequence X1KAPETALX2, where X1 is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutanoic acid) and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid), with an intrapeptide bond between X1 and X2 being an amide bond, i.e. [ka] (For the amino acid Dap, the symbols Dap and Dpr are used equally herein) (b) a peptide having at least one intrapeptide bond that is a disulfide bond or a diselenide bond, the peptide having an amino acid sequence of X3KAPETALX4AAA, the peptide having a length of 12 to 20 amino acid residues and having at least one intrapeptide disulfide bond or diselenide bond between X3 and X4 (X3 and X4 are equal and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine)); i.e. [ka] (c) a peptide having at least one intrapeptide bond which is a disulfide bond or a diselenide bond, the peptide having an amino acid sequence selected from the group consisting of X5KAPETALX6; X5KAPETALX6A; and X5KAPETALX6AA, the peptide having a length of 9 to 11 amino acid residues and having at least one intrapeptide disulfide bond or diselenide bond between X5 and X6 (X5 and X6 are equal and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine)), i.e. [ka] (d) having 16 amino acid residues, between X7 and X9, and between X8 and X 10 Between (X7~X 10 are independently selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), with the proviso that X7 and X9 are equal and X8 to X 10 The amino acid sequence X7NX8KAPETALX9AAAX has an intrapeptide disulfide bond or diselenide bond. 10 Peptides containing H, i.e. [ka] And (e) a peptide (SEQ ID NO: 7) comprising the amino acid sequence X1KAPETALX2 (X1 is selected from the group consisting of Dap and Dab, and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid)), which is a linear peptide. is a biradical of a peptide selected from the group consisting of W is -NH-(CH2) r -C(=O)-, and -NH-CH((CH2) r is a biradical selected from the group consisting of: —NH2)—C(═O)—; Y is a radical selected from the group consisting of -NH2, -OH, -OR3, and -NHR3; s is an integer independently selected from 0 to 1; n is an integer from 0 to 6, r is an integer independently selected from 1 to 5; k is an integer from 5 to 8; R1 and R2 are independently selected from (C1-C6)-alkyl; R3 is a radical selected from the group consisting of (C1-C6)-alkyl, L a ' is linked to the radical Z through a bond selected from the group consisting of ester, ether, urethane, silyl ether, sulfonate, phosphate, ketal, hemiketal, carbonate and carbamate bonds, said bond being formed between a C=O, SO2, Si, P, CH or CH2 group on the left side of the formula of draw La` and one of the hydroxyl groups of SN-38, If n=0, L a ' is connected to the radical L through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester. c ', and the bond is drawn L a The functional group on the right side of the formula of ` and L c ' is formed between the functional group on the left side of the formula, If n=1, L a' is connected to the radical L through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester. b ', and the bond is drawn L a The functional group on the right side of the formula of ` and L b The functional group on the left side of the formula is formed between L b ' is connected to the radical L through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester. c ', and the bond is drawn L b Draw the functional group on the right side of the formula of L' c ' is formed between the functional group on the left side of the formula, If n is greater than 1, L b ' are equal or different and are linked between them via a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters; L b one end of the ' is bonded to L via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester. a ', and the bond is drawn by drawing the functional group on the right side of the formula of drawing La` and drawing L b ' is formed between the functional group on the left side of the formula and another L b The terminus is connected to the L through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester. c ', and the bond is drawn L b Draw the functional group on the right side of the formula of L' c ' is formed between the functional group on the left side of the formula, L c 'Drawing L c ' is attached to the biradical P via an amide bond formed between the carbonyl group on the right side of the formula and the amino group of the first amino acid of the peptide sequence P, when s=0, P is directly linked to Y via an amide, carboxylic acid or ester bond, said bond being formed between the C=O of the C-terminus of the last amino acid of the sequence P and a radical Y which is -NH2, -OH, -OR3 or -NHR3; When s=1, P is linked to a radical W through an amide bond formed with the C=O of the C-terminus of the last amino acid of sequence P, said bond being formed between the functional group on the left side of the formula of drawing W and the functional group (C=O) of the C-terminus of the last amino acid of sequence P on the right side of the drawing, W being: -C(=O)-NH-(CH2) r -C(=O)-Y, or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y, Concerning micelles.

[0029] The line between two amino acids in the above or following sequences represents an intrapeptide bond between the side chains of the two amino acids. In certain embodiments, the line between two amino acids in the above or following sequences represents an intrapeptide bond between the side chains of the two amino acids.

[0030] A second aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a micelle as defined above together with a suitable amount of a pharma- ceutically acceptable carrier or excipient.

[0031] A third aspect of the invention relates to a micelle as defined above for use as a medicament.

[0032] A fourth aspect of the invention relates to a micelle as defined above for use in the treatment of cancer in a mammal, including a human.

[0033] A fifth aspect of the invention relates to a micelle as defined above for use in the treatment of cancer, wherein the compound of formula (I) is for use in combination therapy with a chemotherapeutic agent. [Brief description of the drawings]

[0034] [Figure 1]FIG. 1 shows a schematic of the manufacturing process for the novel water-soluble SN-38 lactone micelle product. [Diagram 2] Figure 2 is a photograph taken immediately after preparation of product G2B-002-20-9 or products consisting of vehicle alone or 20 mg / mL irinotecan in vehicle, with a loading of 2 mg / mL of free SN-38 lactone in the absence of G2B-001 micelles. Product G2B-002-20-9 was not turbid, whereas products made with vehicle or 20 mg / mL irinotecan in vehicle showed turbidity corresponding to insoluble SN-38 lactone crystals. Left: vehicle with 2 mg / mL SN-38 lactone; center: G2B-002-20-9; right: 20 mg / mL irinotecan in vehicle with 2 mg / mL SN-38 lactone. [Diagram 3] Figure 3 shows photographs taken immediately after preparation of product G2B-006-20-9 or a product consisting of vehicle only, containing a loading of 2 mg / mL of free SN-38 lactone without G2B-003 micelles. Product G2B-006-20-9 was not turbid, whereas the product made with vehicle showed turbidity corresponding to insoluble SN-38 lactone crystals. Left: G2B-006-20-9; Right: vehicle. [Figure 4] Figure 4 shows photographs of the product G2B-002-20-9 obtained at different times. The product remained turbidity-free during 24 weeks of storage at 4°C. Left: G2B-002-20-9 immediately after preparation; Center: G2B-002-20-9 1 week after preparation; Right: G2B-002-20-9 24 weeks after preparation. [Diagram 5] Figure 5 shows photographs of the product G2B-006-20-9 obtained at different times. The product remained turbidity-free during 24 weeks of storage at 4°C. Left: G2B-006-20-9 immediately after preparation; Center: G2B-006-20-9 6 weeks after preparation; Right: G2B-006-20-9 24 weeks after preparation. [Figure 6] FIG. 6 is a photograph of the products detailed in Table 4 containing camptothecin (CPT) in the concentration range of 1 to 0.25 mg / mL. [Figure 7]FIG. 7 is a photograph of the products detailed in Table 5 containing a mixture of SN-38 lactone and camptothecin (CPT) at concentrations of 1 mg / mL and 0.5 mg / mL in the presence or absence of G2B-001 (vehicle). [Figure 8] Figure 8 is a comparison of the antiproliferative activity of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line HSJD-DIPG-007. Dotted values ​​represent the mean and SD from triplicate experiments at compound concentrations. [Figure 9] Figure 9 is a comparison of the antiproliferative activity of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line HSJD-DMG-001. Dotted values ​​represent the mean and SD from triplicate experiments of compound concentrations. [Figure 10] Figure 10 is a comparison of the antiproliferative activity of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line HSJD-GBM-001. Dot values ​​represent the mean and SD from triplicate experiments at compound concentrations. [Figure 11] Figure 11 is a comparison of the antiproliferative activity of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line RH4. Dotted values ​​represent the mean and SD from triplicate experiments at compound concentrations. [Figure 12] Figure 12 is a comparison of the antiproliferative activity of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line A673. Dot values ​​represent the mean and SD from triplicate experiments at compound concentrations. [Figure 13] Figure 13 shows the Kaplan-Meier survival curves obtained for each of the groups of mice bearing HSJD-DIPG-007. [Figure 14] Figure 14 shows the Kaplan-Meier survival curves obtained for each of the groups of mice bearing HSJD-GBM-001. [Figure 15]FIG. 15 shows tumor growth (% of tumor volume on day 1 of treatment) of mice bearing subcutaneous PDX treated with saline control, irinotecan 10 mg / kg, and G2B-002 version with SN-38 lactone at doses 1 and 10 mg / kg. Each dot represents the tumor growth of one individual PDX model. [Figure 16] Figure 16 shows concentration-time data of SN-38 lactone in mouse retina after intravenous administration of G2B-002-20-9 at doses of 10 mg / kg and 1 mg / kg SN-38 lactone or 10 mg / kg irinotecan. The dots represent individual data points and the lines connect the average data points at each time point. [Figure 17] FIG. 17 shows the transport of different micelles formed by compound Ia and SN-38 in comparison to SN-38 and a SN-37 peptide conjugate (G2B-001) in a human in vitro BBB model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] All terms used herein in this application should be understood in their ordinary sense known in the art unless otherwise stated. Other more specific definitions for certain terms used in this application are as set forth below and are intended to apply throughout the specification and claims.

[0036] Unless otherwise specified, the amino acids referred to herein are L-amino acids. One-letter and three-letter symbols are used without ambiguity. The following abbreviations are used for the following amino acids: diaminopropionic acid (Dap), diaminobutiric (Dab), selenocysteine ​​(Sec), and penicillamine (Pen). In the context of the present invention, penicillamine includes only D-penicillamine.

[0037] The term "apparent solubility" is the aqueous solubility of free SN-38 lactone measured after inclusion in an aqueous micellar dispersion of a peptide conjugate of SN-38.

[0038] The term "inner core" as applied to the micelles of the present invention refers to the center of the micelle formed by the peptide conjugate of SN-38.

[0039] The term "external shell" as applied to the micelles of the present invention refers to the layer formed by the peptide conjugates of SN-38.

[0040] As used herein, the terms "drug" and "therapeutic agent" are used interchangeably.

[0041] The terms "drug-loaded" and "encapsulated" are used interchangeably. In the present invention, a "drug-loaded" micelle refers to a micelle that has a drug or therapeutic agent located in the core of the micelle.

[0042] In the present invention, when a numerical interval is used, this includes the extreme values ​​of the interval. In particular, as used herein, "included between" denotes a range of values ​​including the endpoints of the range.

[0043] Unless otherwise stated, all percentages referred to herein are expressed by weight, based on the total weight of the product, where the sum of the amounts of the ingredients equals 100%.

[0044] Compound (Ia) is also referred to as G2B-001. Compound (Ib) is also referred to as G2B-001 linear. Compound (Ic) is also referred to as G2B-003. Compound (Id) is also referred to as G2B-004. Compound (Ie) is also referred to as G2B-005.

[0045] As an example of the nomenclature used, for G2B-002-20-9, the weight / weight percentage (w / w(%)) of free SN-38 in the final formulation is 9(w / w)%, i.e., free SN-38 / (SN-38+G2B-001)*100=9%, and for G2B-001, the final concentration is 20 mg / mL (see also Example 15).

[0046] The word "comprise" for purposes of the present invention includes "consisting of."

[0047] As described above, the micelles of SN-38-binding peptide conjugates loaded with at least one drug having anticancer activity are part of the present invention. In a particular embodiment, the micelles of SN-38-binding peptide conjugates are loaded with camptothecin. In another particular embodiment, the camptothecin is selected from the group consisting of SN-38, camptothecin (CPT), or topotecan. In a particular embodiment, the drug in the inner core is SN-38 lactone.

[0048] The peptide conjugate of SN-38 used to prepare the micelles of the present invention described above may be in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" as used herein includes any salt formed from a pharmaceutically acceptable non-toxic acid or base, including inorganic or organic acids or bases. There are no limitations on the salt, except that it must be pharmaceutically acceptable when used for therapeutic purposes. When a part of the compound of formula (I) is a basic compound, the salt can be prepared from a pharmaceutically acceptable non-toxic acid, including inorganic and organic acids. Such acids include, for example, hydrochloric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, etc.

[0049] In another particular embodiment, the micelle is a peptidic conjugate of SN-38 of formula (I) in which Z is linked to the linker L through a hydroxyl group (b) of the pharma- ceutical active ingredient. In another particular embodiment, the micelle is a peptidic conjugate of SN-38 of formula (I) in which Z is linked to the linker L through a hydroxyl group (a) of the pharma-ceutical active ingredient.

[0050] In another particular embodiment, the micelle comprises a peptidic conjugate of SN-38 of formula (I), wherein P is (a) SEQ ID NO:8: [ka] a peptide comprising the amino acid sequence DapKAPETALD, with an intrapeptide bond between Dap and D being an amide bond, (b) SEQ ID NO:9: [ka] a peptide having a length of 9 to 20 amino acid residues, comprising an amino acid sequence of CKAPETALCAAA, having at least one intrapeptide bond which is a disulfide bond, and having at least one intrapeptide disulfide bond between cysteines 1 and 9; (c) [ka] and a peptide having a length of 9 to 11 amino acid residues, the peptide consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA, having at least one intrapeptide bond which is a disulfide bond and having at least one intrapeptide disulfide bond between cysteines 1 and 9, (d) [ka] a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH with intrapeptide disulfide bonds between the first and third cysteines, which are cysteines 1 and 11, and between the second and fourth cysteines, which are cysteines 3 and 15; (e) a peptide containing the amino acid sequence DapKAPETALD (SEQ ID NO: 14), i.e., a linear peptide A micelle that is a biradical of a peptide selected from the group consisting of:

[0051] X1-X 10 In the sequences of SEQ ID NOs: 8-10, where specific amino acids have been selected for, specific intrapeptide bonds are depicted in the sequence.

[0052] In another particular embodiment, the peptide of the invention is a peptide with one intrapeptide bond. In another particular embodiment, the peptide of the invention is a peptide with two intrapeptide bonds.

[0053] In another particular embodiment, the micelle comprises a peptidic conjugate of SN-38 of formula (I), in which P is (a) a peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 8) with an intrapeptide bond between Dap and D being an amide bond; (b) a peptide having the amino acid sequence CKAPETALC (SEQ ID NO: 10) with at least one intrapeptide disulfide bond between the cysteines at positions 1 and 9; (c) a peptide having the amino acid sequence DapKAPETALD, i.e., a linear peptide (SEQ ID NO: 14) In another particular embodiment, the micelle is a biradical of a peptide selected from the group consisting of: In another particular embodiment, the micelle is a peptidic conjugate of SN-38 of formula (I), in which P is a biradical of a peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 8) with an intrapeptide bond between Dap and D being an amide bond.

[0054] In another particular embodiment, the micelle comprises a peptidic conjugate of SN-38 of formula (I) in which La′ is C(═O)—(CH2) r -C(=O)-, -C(=O)-(CH2) r -NH-, -C(=O)-(CH2) r -S-;-C(=O)-(CH2) r -O-;-C(=O)-NH-(CH2) r -C(=O)-;L1, L2, L3, L4, L5, L6, L7, and L 12 A micelle that is a biradical selected from the group consisting of:

[0055] In another specific embodiment in combination with any of the above or below specific embodiments, the micelle is a micelle in which, in the peptidic conjugate of SN-38 of formula (I), L is a linker that is a biradical composed of 3 to 8 biradicals, and n is an integer of 1 to 6. In another specific embodiment in combination with any of the above or below specific embodiments, the micelle is a micelle in which, in the peptidic conjugate of SN-38 of formula (I), L is a linker that is a biradical composed of 5 to 8 biradicals. In another specific embodiment in combination with any of the above or below specific embodiments, the micelle is a micelle in which, in the peptidic conjugate of SN-38 of formula (I), L is a linker that is a biradical composed of 6 to 8 biradicals. In another specific embodiment in combination with any of the above or below specific embodiments, the micelle is a micelle in which, in the peptidic conjugate of SN-38 of formula (I), L is a linker that is a biradical composed of 6 to 7 biradicals. In another particular embodiment in combination with any of the above or below particular embodiments, the micelle is a peptidic conjugate of SN-38 of formula (I), in which L is a linker that is a biradical composed of 6 biradicals.

[0056] In another particular embodiment, the micelle comprises a peptidic conjugate of SN-38 of formula (I), wherein La′ is L3 of the following formula:c ' is C(=O)-(CH2) r It is a micelle where -C(=O)-. [ka]

[0057] In another particular embodiment, the micelle comprises a peptidic conjugate of SN-38 of formula (I) b ' is -NH-(CH2) r -O-, -(CH2) r -O-; and -(CH2) r In another particular embodiment, the micelle is a micelle selected from the group consisting of L, -NH-, and combinations thereof. a ' is L3 in the above formula, and L b ' is -NH-(CH2) r -O-, -(CH2) r -O-; and -(CH2) r -NH-, and combinations thereof; L c ' is C(=O)-(CH2) r It is a micelle where -C(=O)-.

[0058] In another specific embodiment in combination with any of the above or below specific embodiments, the micelle is a micelle of a peptidic conjugate of SN-38 of formula (I) in which L is a linker that is a biradical composed of two biradicals, and n = 0. In another specific embodiment in combination with any of the above or below specific embodiments, the micelle is a micelle of a peptidic conjugate of SN-38 of formula (I) in which L is a linker that is a biradical composed of two biradicals, and n = 0. a ' is L 12 And L c ' is L 13 In another particular embodiment in combination with any of the above or below particular embodiments, the micelle is a peptidic conjugate of SN-38 of formula (I), a ' is -C(=O)-NH-(CH2) r-C(=O)-, L c ' is L 15 are micelles.

[0059] In another particular embodiment in combination with any of the above or below particular embodiments, the micelle comprises a peptidic conjugate of SN-38 of formula (I), L a ' is an ester bond formed with the C=O group on the left side of the formula of drawing La' to radical Z through a bond, and a functional group on the right side of the formula of drawing to radical L through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester. b In another particular embodiment in combination with any of the above or below particular embodiments, the micelle is a peptidic conjugate of SN-38 of formula (I) in which L a 'Drawing L a 'The C=O group on the left side of the formula is formed with an ester bond through a bond with the radical Z, and the functional group on the right side of the formula is formed with an amide bond through a feasible bond with the radical L b In another particular embodiment in combination with any of the above or below particular embodiments, the micelle is a peptidic conjugate of SN-38 of formula (I) in which L a is linked to radical Z through a bond which is a carbonate or carbamate bond formed with the C=O group on the left side of the formula of the drawing La', and to radical Lb' through a chemically feasible bond which is an amide bond (NH-CO or CO-NH) formed with the functional group on the right side of the formula of the drawing.

[0060] In another particular embodiment in combination with any of the above or below particular embodiments, the micelle is a peptidic conjugate of SN-38 of formula (I), in which Lb' forms a chemically feasible bond with a radical La' having a functional group on the left side of the depicted formula Lb', Lb' is bonded to a radical Lc' through a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters formed with a functional group on the right side of the depicted formula Lb', and when n is greater than 1, Lb' are equal or different and bonded therebetween through a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfide esters, and thioesters, and one end of Lb' is bonded to La' and the other end of Lb is bonded to Lc'.

[0061] In another particular embodiment in combination with any of the above or below particular embodiments, the micelle is a peptidic conjugate of SN-38 of formula (I) in which Lc' is bound to a biradical P through an amide bond formed with the carbonyl group on the right side of the formula of the drawn Lc' and the amino group of the first amino acid of the peptide sequence P, and to a radical Lb' through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester formed with a functional group on the left side of the drawn formula.

[0062] In another particular embodiment, the micelle is a compound in which the peptidic conjugate of SN-38 of formula (I) is selected from the group consisting of compounds of formula (Ia) (also referred to as G2B-001 or SN38-Linker A-MiniAp4). MiniAp4 is DapKAPETALD, where Dap is 2,3-diaminopropionic acid. Both names are used interchangeably. These names are used equally in this specification. The compound of formula (Ia) below or a pharma- ceutically acceptable salt thereof is a compound of formula (I) in which P is DapKAPETALD with an intrapeptide bond between Dap and D, Y=CONH2, W=0, and the linker is the following linker A, and SN-38 is attached to the linker by hydroxyl (b). [ka]

[0063] In another particular embodiment, the compound of formula (I) is a compound of the following formula (Ib), also referred to as G2B-001 linear, or a pharma- ceutically acceptable salt thereof, in which in formula (I), P is a linear DapKAPETALD with no peptide bond between Dap and D, Y=CONH2, W=0, and the linker is the following linker A, and SN-38 is attached to the linker by the hydroxyl (b). [ka]

[0064] In another particular embodiment, the compound of formula (I) is a compound of formula (Ic) as shown below, also referred to as G2B003, or a pharma- ceutically acceptable salt thereof, wherein in formula (I), P is DapKAPETALD with a peptide bond between Dap and D, Y=CONH2, W=0, and the linker is linker A as shown below, and SN-38 is attached to the linker by the hydroxyl (a). [ka]

[0065] The linker A is formed as follows: a ':L3:r = 4 on the right and 3 on the left: [ka] Lb': one unit of biradical -NH-(CH2)3-O-, two units of biradical -(CH2)2-O-, and one unit of biradical -(CH2)3-NH-; and Lc': -C(=O)-(CH2)2-C(=O)-; and the biradicals are linked as shown in the following diagram corresponding to linker A: [ka]

[0066] In another particular embodiment, the compound of formula (I) is a compound of formula (Id) shown below, also referred to as G2B-004, or a pharma- ceutically acceptable salt thereof, in which P is DapKAPETALD with a peptide bond between Dap and D, Y=CONH2, W=0, and the linker is the linker C shown below, and SN-38 is attached to the linker by the hydroxyl (a). a ':L 12 and L c ':L 13 and n=0. [ka]

[0067] In another particular embodiment, the compound of formula (I) is a compound of the following formula (Ie) or a pharma- ceutically acceptable salt thereof, wherein in formula (I), P is DapKAPETALD with a peptide bond between Dap and D, Y=CONH2, W=0, and the linker is the following linker D, and SN-38 is attached to the linker by the hydroxyl (a). [ka]

[0068] This compound is also called G2B-005, and the linker D is La'-C(=O)-NH-(CH2) r It is formed by -C(=O)-(r=1), Lb':L15 and n=0.

[0069] The micelles of the present invention are formed in an aqueous medium, and thus may be in the form of an aqueous dispersion of micelles.

[0070] A micelle-peptide conjugate of SN-38 containing a free therapeutic agent as defined above can be obtained by a) spontaneous self-assembly of a peptide conjugate of SN-38 as defined above in water at pH<7, b) contacting an acidic solution of micelles with a basic solution containing free SN-38 carboxylate at a concentration of up to 25 mg / ml, and c) optionally lyophilizing the acidic solution of micelles.

[0071] In step b), the initial pH of the mixture is an acidic pH<7, and the SN-38 carboxylate converts to SN-38 lactone and forms an intermolecular interaction with the SN-38 molecule conjugated in the SN-38 peptide conjugate. In a particular embodiment, the spontaneous self-assembly of the SN-38 peptide conjugate defined above is carried out in water with a pH<5. In a particular embodiment, the spontaneous self-assembly of the SN-38 peptide conjugate defined above is carried out in water with a pH<3. In a particular embodiment, the basic solution containing free SN-38 carboxylate has a concentration of 2-25 mg / ml. In a particular embodiment, the basic solution containing free SN-38 carboxylate has a concentration of 2-12 mg / ml.

[0072] In a particular embodiment, the micelles of the present invention are micelles in which each individual micelle in the aqueous dispersion of micelles contains only one therapeutic agent useful for the treatment of cancer. In another particular embodiment, the aqueous dispersion of micelles are micelles in which the free therapeutic agent is SN-38 lactone. In another particular embodiment, the aqueous dispersion of micelles are micelles in which each individual micelle contains two or more therapeutic agents useful for the treatment of cancer.

[0073] The peptide conjugate of SN-38 of formula (I) can be made in whole or in part by chemical synthesis. The amino acids required for the preparation of the compound of formula (I) are commercially available. The compound of formula (I) can be easily prepared, for example, by synthesis in solution phase, of which many methods have been published (see M. Amblard, et al., "Methods and protocols of modern solid-phase peptide synthesis. Molecular Biotechnology 2006, Vol. 33, p. 239-254), or preferably by solid-phase peptide synthesis. The compound of formula (I) can also be prepared by any combination of solution phase synthesis and / or solid phase synthesis, for example by synthesizing the body of peptide P via solid phase synthesis and then removing the protecting groups in solution. The linker L and the binding of SN-38 to peptide P can be performed in solid phase or in solution. The construction of linker L can also be prepared by any combination of solution phase synthesis and / or solid phase synthesis.

[0074] The peptides of the present invention can also be obtained by preparing DNA templates and subcloning into expression vectors (see JH Lee et al. Eur. J. 30 Biochem. 2001. Vol. 268. pp. 2004-2012).

[0075] The compound of formula (Ia) may be prepared by a process comprising reacting a compound of formula (III) with a compound of (IV) as described above to obtain a compound of formula (Ia). [ka]

[0076] The preparation of pharma- ceutically acceptable salts of the compounds of formula (I) can be carried out by methods known in the art. For example, they can be prepared from parent compounds that contain a basic or acidic moiety by conventional chemical methods. In general, such salts are prepared, for example, by reacting the free acid or base form of these compounds with an aqueous or organic solvent or mixtures thereof that contain a chemically equivalent amount of a suitable pharma- ceutically acceptable base or acid.

[0077] A process for preparing micelles from SN-38 and these peptide conjugates of SN-38 lactone by spontaneous micellization upon dissolution in water at pH<7, especially pH<3, is also part of the present invention. An acidic solution of such micelles can neutralize a basic solution containing free SN-38 carboxylate at a concentration of up to 25 mg / ml. After acidification to basic pH, the appearance of free SN-38 lactone due to the change in pH leads to intermolecular interactions between the free SN-38 lactone molecules and the SN-38 molecules conjugated to the peptide conjugate of SN-38. Generally, micelles can be formed with gentle mixing. Generally, they are formed within less than 30 minutes. Generally, micelles are formed at room temperature.

[0078] This system produces drug-drug cocrystals on the nanometer size scale that contain two forms of SN-38 lactone, free and conjugated. Under these conditions, free SN-38 lactone does not form microcrystals or larger solid structures and remains apparently soluble at concentrations up to 4 mg / ml.

[0079] Such aqueous dispersions of micelles of peptide conjugates of SN-38 containing free SN-38 lactone can be easily filtered through 0.22 μm and / or 0.45 μm pore filters without any loss of free SN-38 lactone. For example, filtration can be performed with syringe 0.45 or 0.22 polypropylene filters.

[0080] The aqueous dispersion of the micelles of the present invention can also be freeze-dried. After reconstitution in water, it retains the same amount of soluble SN-38 lactone. Freeze-drying can be carried out, for example, in a Telstar freeze dryer.

[0081] In a particular embodiment, the concentration of the micellar peptide conjugate in the aqueous micellar dispersion is up to 50 mg / ml. In another particular embodiment, the concentration of the micellar peptide conjugate in the aqueous micellar dispersion is 1-50 mg / ml. In another particular embodiment, the concentration of the micellar peptide conjugate in the aqueous micellar dispersion is 1-20 mg / ml. In another particular embodiment, the concentration of the micellar peptide conjugate in the aqueous micellar dispersion is 5-20 mg / ml. In another particular embodiment, the concentration of the micellar peptide conjugate in the aqueous micellar dispersion is 10-20 mg / ml. In general, the concentrations of the micellar peptide conjugates of the present invention in the aqueous micellar dispersion are 20, 10, and 5 mg / ml.

[0082] In another particular embodiment, the proportion of free SN-38 lactone in the aqueous dispersion of micelles is up to 25 mg / ml. In another particular embodiment, the proportion of free SN-38 lactone in the aqueous dispersion of micelles is 2 to 25 mg / ml. In another particular embodiment, the proportion of free SN-38 lactone in the aqueous dispersion of micelles is 2 to 12 mg / ml. In another particular embodiment, the proportion of free SN-38 lactone in the aqueous dispersion of micelles is 1 to 8 mg / ml, in particular 8, 4, 2, 1 mg / ml.

[0083] In certain embodiments, the micelles of the present invention comprise a) micelles of peptide conjugate of formula (Ia) with an inner core loaded with SN-38, the concentration of peptide conjugate of SN-38 in an aqueous dispersion of the micelles being 20 mg / ml and the concentration of SN-38 being 16 mg / ml; b) micelles of peptide conjugate of formula (Ia) in which the inner core is loaded with SN-38, the concentration of peptide conjugate of SN-38 in the aqueous dispersion of the micelles is 20 mg / ml and the concentration of SN-38 is 9 mg / ml; c) micelles of peptide conjugate of formula (Ia), the inner core of which is loaded with SN-38, the concentration of peptide conjugate of SN-38 in an aqueous dispersion of the micelles being 10 mg / ml and the concentration of SN-38 being 9 mg / ml; and d) Micelles of peptide conjugate of formula (Ia), the inner core of which is loaded with SN-38, the concentration of peptide conjugate of SN-38 in the aqueous dispersion of the micelles being 20 mg / ml and the concentration of SN-38 being 9 mg / ml. is selected from the group consisting of:

[0084] In another particular embodiment, the micelles of the invention are e) micelles of the peptide conjugate of formula (Ia) whose inner core is loaded with camptothecin lactone, and the concentration of the peptide conjugate of SN-38 in the aqueous dispersion of the micelles is 20 mg / ml, and the concentration of camptothecin lactone is 0.25-4 mg / ml, in particular 0.25-1 mg / ml. In another particular embodiment, the micelles of the invention are f) micelles of the peptide conjugate of formula (Ia) whose inner core is loaded with camptothecin lactone and lactone, and the concentration of the peptide conjugate of SN-38 in the aqueous dispersion of the micelles is 20 mg / ml, the concentration of camptothecin lactone is 0.5-1 mg / ml, and the concentration of SN-38 is 0.5-1 mg / ml.

[0085] Pharmaceutical compositions containing a therapeutically effective amount of micelles as defined above together with an appropriate amount of a pharma- ceutically acceptable carrier or excipient are also part of the invention.

[0086] The term "therapeutically effective amount" as used herein refers to an amount of a compound (a micelle according to the present invention) sufficient when administered to prevent the onset of the disease presented or to alleviate to some extent one or more symptoms of said disease. The particular dose of a compound administered according to the present invention will, of course, be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations.

[0087] The term "pharmaceutical composition" refers to a mixture of a compound described herein with other chemical components, such as a diluent or carrier. A pharmaceutical composition facilitates administration of a compound to an organism. The term "pharmaceutical acceptable excipient or carrier" refers to a pharmaceutical acceptable substance, composition, or vehicle. Each component must be pharmaceutical acceptable in the sense of being compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication commensurate with the benefit-risk ratio.

[0088] The compositions of the present invention may be administered by parenteral forms suitable for injection, such as intravenous bolus injection, intravenous infusion, implantation into the body, orally, intrathecally, or intranasally.

[0089] An important property of the micelles of the present invention is their biological activity in inhibiting cell growth of the tumor cell lines tested. As illustrated in the examples, the compounds of the present invention show antitumor properties in several cancer cell lines. Cancer is a heterogeneous disease characterized by the accumulation of tumor cells that can result in death in both animals and humans. It is also one of the leading causes of death from disease among children and adults. While there has been substantial progress in the treatment of some types of cancer over the past 50 years, there has been limited progress against other types, especially childhood cancers. The annual incidence of tumors in children is 100-160 cases per million cases. Under the age of 15, the annual risk is 1 in 500 children. This incidence is slightly lower in developed countries. Tumors of the brain and spinal cord account for 25% of all neoplasms (40-50% of all pediatric solid tumors). Despite progression, survival data for these patients remains low at approximately 55%. This is in contrast to the substantial increases in survival seen in recent years in other types of patients, such as those with leukemia or extracranial tumors.

[0090] Traditional methods of treating cancer include surgical procedures, administration of chemotherapeutic agents, and more recently, immune response-based therapies, including administration of antibodies or antibody fragments that can be conjugated to therapeutic moieties. However, to date, such treatments have met with limited success.

[0091] Camptothecin is one of the four major structural classes of plant-derived anticancer compounds, a cytotoxic alkaloid consisting of a five-membered ring structure containing a pyrrole (3,4β) quinoline moiety in the S-configured lactone form and in the carboxylate form. Irinotecan is made from the natural camptothecin found in the Chinese ornamental tree Camptotheca acuminata. Several types of cancer, particularly glioblastoma multiforme (GBM) in adults, and diffuse intrinsic pontine glioma (DIPG), paediatric glioblastoma (pGBM), neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma, and retinoblastoma in children, have been treated with irinotecan at some point.

[0092] Gliomas are a group of tumors that begin in the glial cells of the brain or spine, and comprise approximately 30% of all brain and central nervous system tumors, as well as 80% of all malignant brain tumors. Treatment of brain gliomas is often a combined approach using surgery, radiation therapy, and chemotherapy. DIPG primarily affects children, usually between 5 and 7 years of age. Unfortunately, it is the majority of brain stem tumors, accounting for 60-70%, and has one of the poorest prognoses of all. No treatment has been shown to be effective, and the median survival time is 9 months. Radiation and administration of steroids are the only treatments that have a palliative effect and extend survival only slightly. So far, no chemotherapy is useful, and several clinical trials have been tested, but without favorable results. Pediatric glioblastoma is another group of tumors that includes one-third of hemispheric tumors. It has a peak age of onset between 8 and 12 years of age. Glioblastoma also affects adults with approximately 2-3 cases per 100,000 population.

[0093] Ewing's sarcoma is the second leading cause of malignant bone tumors in children and adolescents. The annual incidence is 0.6 per million inhabitants. It is rare under the age of 5, with the highest age of onset being 10-15 years, affecting more boys than girls, but this gender relationship varies with age range. The most common body locations for it to develop are the pelvic bones (hip bone), chest wall (such as ribs or scapula), or mid-leg bones. Ewing's sarcoma can also present as an extraosseous lesion in the absence of bone lesions. In this variant, there is a high risk of lymphatic spread, and treatment is usually similar to that for rhabdomyosarcoma.

[0094] Soft tissue sarcomas are divided into rhabdomyosarcoma and non-rhabdomyosarcoma. Rhabdomyosarcoma accounts for 50% of all soft tissue sarcomas in children. It is the third most common solid extracranial tumor in incidence, behind neuroblastoma and Wilms' tumor. The peak age is bimodal, with the first peak occurring at 2-5 years of age and the second peak occurring during adolescence at 15-19 years of age. Adolescent sarcomas are mostly located in the extremities, but in children, they can occur anywhere in the body, both in skeletal muscle and soft tissue. The most commonly affected areas in children are the head and neck and genitourinary tract. In 20% of patients, the extremities are affected. Overall survival is poor unless the tumor is found in a location that allows complete resection, sometimes requiring amputation of a limb or urination. Survival varies from 7-70% depending on location.

[0095] Neuroblastoma is the most common extracranial solid tumor in children. Due to its embryonic origin, neuroblastoma can be found virtually in any part of the sympathetic nervous system, but the most common location of locoregional disease is the adrenal gland (44%). Children aged 1.5–6 years at diagnosis can be cured with conventional treatment, but if malignant disease (stage 4 neuroblastoma) is diagnosed, the probability of survival decreases. Approximately 50% of newly diagnosed patients already present with metastases to bone (60%), bone marrow (50%), lymph nodes (42%), and / or liver (15%), requiring intensive chemotherapy treatments, surgery, and radiation therapy, but their survival remains poor and has made little progress over the past decade.

[0096] Finally, retinoblastoma is the most common cause of childhood eye tumors, with a worldwide incidence of 1 case per 20,000 live births. It typically presents within the first 2 years of life. Of these, 30-40% are bilateral, and these cases always have a positive family history. In unilateral cases, 10% have a germline mutation in the Rb gene located on chromosome 13. If detected early, these have a 95% survival rate, but in certain parts of the world, if the diagnosis is late, the survival rate drops significantly to less than 20%. Treatment depends on the tumor control achieved. If tumor regression is not controlled by chemotherapy and brachytherapy, eye removal is recommended. Even if eye removal is performed, some cases with tumor invasion into the optic nerve require further chemotherapy treatment.

[0097] The micelles as defined above for use as a medicament are part of the present invention.The micelles as defined above for use in the treatment of cancer in mammals, including humans, which are active in all types of cancer tested, are also part of the present invention.This embodiment may also be framed as the use of the micelles as defined above for the preparation of a medicament for the treatment and / or prevention of cancer in mammals, including humans.The present invention also relates to a method of treatment of cancer in mammals, including humans, suffering from or susceptible to cancer, comprising administering to said patient a therapeutically effective amount of the micelles as defined above together with a pharma- ceutically acceptable excipient or carrier.

[0098] In a particular embodiment, the micelle is for use as defined above, and the cancer is located in the brain.In another particular embodiment, the micelle or aqueous micelle dispersion is for use as defined above, and the micelle or aqueous micelle dispersion crosses the blood-brain barrier and releases free therapeutic agent in the brain parenchyma and cerebrospinal fluid.In a particular embodiment, the micelle of the present invention is for use as defined above, and the treatment of cancer comprises the treatment of tumors selected from the group consisting of extracranial solid tumors, eye tumors, and CNS tumors.In another particular embodiment, any micelle of the present invention is for use as defined above, and the cancer is selected from the group consisting of adult glioma, pediatric glioma, retinoblastoma, Ewing's sarcoma, DIPG, neuroblastoma, medulloblastoma, ependymoma, atypical teratocarcinoma rhabdoid tumor (ATRT), and rhabdomyosarcoma.In another particular embodiment, the micelle of the present invention is for use as defined above, and the cancer is pediatric brain tumor. In another particular embodiment, the micelles are for the use as defined above, and the pediatric glioma is selected from the group consisting of diffuse intrinsic pontine glioma (DIPG) and pediatric high-grade glioma. In another particular embodiment, the micelles are for the use as defined above, and the cancer is diffuse intrinsic pontine glioma. In another particular embodiment, the micelles are for the use as defined above, and the cancer is pediatric high-grade glioma. In another particular embodiment, the micelles are for the use as defined above, and the cancer is retinoblastoma. In another particular embodiment, the micelles are for the use as defined above, and the cancer is Ewing's sarcoma. In another particular embodiment, the micelles are for the use as defined above, and the cancer is neuroblastoma. In another particular embodiment, the micelles are for the use as defined above, and the cancer is rhabdomyosarcoma.

[0099] In another particular embodiment, the micelle is for use as defined above, and the cancer is medulloblastoma. In another particular embodiment, the micelle is for use as defined above, and the cancer is ependymoma. In another particular embodiment, the micelle is for use as defined above, and the cancer is atypical teratocarcinoma rhabdoid tumor (ATRT). In another particular embodiment, the micelle is for use as defined above, and the cancer is adult glioma. In another particular embodiment, the micelle is for use as defined above, and they are active against Ewing's sarcoma, DIPG, pHGG, glioblastoma multiforme, rhabdomyosarcoma, retinoblastoma, medulloblastoma, ependymoma, atypical teratocarcinoma rhabdoid tumor (ATRT), and neuroblastoma cancer cell lines and patient-derived xenografts.

[0100] In another particular embodiment, the micelles are for the use defined above, and they are active against the cancer cell lines used in these experiments, including pediatric glioma cancer types, including diffuse intrinsic pontine glioma (HSJD-DIPG-007), pediatric diffuse midline glioma (HSJD-DMG-001) and pediatric high grade glioma (HSJD-GBM-001), as well as cell lines of other solid tumors, such as Ewing's sarcoma (A673) and rhabdomyosarcoma (RH4).

[0101] The micelles of the present invention can be used in the same manner as other known chemotherapeutic agents, i.e., in combination with other treatments, either simultaneously or sequentially depending on the condition being treated. They can be used alone or in combination with other suitable bioactive compounds. Thus, the micelles of the present invention are intended for use in the treatment of cancer in mammals, including humans, in combination therapy with chemotherapeutic agents.

[0102] In certain embodiments, the dose of free SN-38 is at least 1 mg / kg and up to 12 mg / kg (preclinical studies in mice). In certain embodiments, the micelles are administered in combination with another chemotherapeutic agent. In another particular embodiment, the micelles are administered simultaneously with another chemotherapeutic agent. In another particular embodiment, the micelles are administered separately in any order at a therapeutically effective interval.

[0103] Throughout the specification and claims, the word "comprise" and variations of this word are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprise" includes the instance of "consisting of". Further objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the invention. The following examples and figures are provided for illustration purposes and are not intended to limit the invention. Reference signs in parentheses in the claims in conjunction with the figures are merely an attempt to increase the intelligibility of the claims and should not be construed as limiting the scope of the claims. Furthermore, the present invention encompasses all possible combinations of the specific preferred embodiments described herein.

[0104] Working Example Protected amino acids, handles, and resins were supplied by Luxembourg Industries (Tel-Aviv, Israel), Neosystem (Strasbourg, France), CalbiochemNovabiochem AG (Laufelfingen, Switzerland), Bachem AG (Bubendorf, Switzerland), or Iris Biotech (Marktredwitz, Germany). Other reagents and solvents used are summarized in Table 1. [Table 1]

[0105] General Method for Preparation of Peptide Conjugates of SN-38 General considerations for manual synthesis: Solid-phase peptide elongation and other solid-phase manipulations were performed manually in polypropylene syringes fitted with polyethylene porous disks. Solvents and soluble reagents were removed by aspiration. Washing between the different synthesis steps was performed with dimethylformamide (DMF) (5×30 s) and dichloromethane (DCM) (5×30 s) using 10 ml of solvent / g resin each time.

[0106] General considerations for microwave synthesis: Microwave solid-phase peptide synthesis was performed on a Liberty Blue automated microwave peptide synthesizer using H-Rink amide Protide resin (loading: 0.56 mmol / g). Linear peptides were synthesized on a 0.5 mmol scale using a 5-fold excess of Fmoc-amino acids (0.2 M) relative to resin.

[0107] Identification tests: The tests and synthetic controls used for identification were as follows: A) Kaiser colorimetric assay for detection of solid-phase bound primary amines (E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598); B) p-nitrophenyl ester test for solid-phase bound secondary amines (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791).

[0108] Protocols used during manual synthesis of compounds: Compounds were synthesized on a 100 μmol scale using the following methods and protocols: The resin for manual synthesis was chosen depending on the group Y: if Y is OH, the terminus is COOH, 2-chlorotrityl chloride resin is chosen among others available; if Y is NH2, the terminus is HCONH2, Rink amide MBHA resin is chosen among others available.

[0109] Initial conditioning of the resin: The resin was conditioned by washing with MeOH (5×30 sec), DMF (5×30 sec), DCM (5×30 sec), 1% TFA in DCM (1×30 sec and 2×10 min), DCM (5×30 sec), DMF (5×30 sec), DCM (5×30 sec), 5% DIEA in DCM (1×30 sec and 2×10 min), DCM (5×30 sec), DMF (5×30 sec).

[0110] Removal of the Fmoc group: Removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was carried out with 20 (v / v)% piperidine in DMF using a 30 s treatment followed by two treatments of 10 min each. Two further treatments (2 x 5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure removal of the Fmoc group from the secondary amine (proline).

[0111] Coupling method described on 100 μmol scale Coupling method 1: Protected amino acid (4 equiv., 400 μmol), TBTU (4 equiv., 400 μmol, 128 mg) dissolved in DMF (1-3 mL / g resin) were added successively to the resin, followed by DIEA (8 equiv., 800 μmol, 136 μl). The mixture was reacted with intermittent manual stirring for 1 h. The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The extent of coupling was confirmed by Kaiser colorimetric assay. The Fmoc group was removed with a 20 (v / v)% piperidine solution in DMF using a 30 s treatment and two 10 min treatments. When the amino acid to be deprotected was proline, further treatments (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were applied to ensure removal of the Fmoc group.

[0112] Coupling method 2: Protected amino acid (4 equiv., 400 μmol), PyBOP (4 equiv., 400 μmol, 208 mg), HOAt (12 equiv., 1.2 mmol, 163 mg) dissolved in DMF (1-3 mL / g resin) were added successively to the resin, followed by DIEA (12 equiv., 1.2 mmol, 204 μL). The mixture was reacted with intermittent manual stirring for 1 h. The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling reaction was carried out twice under the same conditions. The extent of coupling was confirmed by Kaiser colorimetric assay. The Fmoc group was removed with a 20 (v / v)% piperidine solution in DMF using a 3 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, further treatments (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were applied to ensure removal of the Fmoc group.

[0113] Coupling method 3: Protected amino acid (4 equiv., 400 μmol), PyBOP (4 equiv., 400 μmol, 208 mg), HOBt (12 equiv., 1.2 mmol, 162 mg) dissolved in DMF (1-3 mL / g resin) were added successively to the resin, followed by DIEA (12 equiv., 1.2 mmol, 204 μL). The mixture was reacted with intermittent manual stirring for 1 h. The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling reaction was carried out twice under the same conditions. The extent of coupling was confirmed by Kaiser colorimetric assay. The Fmoc group was removed with a 20 (v / v)% piperidine solution in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, further treatments (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were applied to ensure removal of the Fmoc group.

[0114] Coupling method 4, scale 100 μmol: protected amino acid (3 equiv., 300 μmol), DIC (3 equiv., 300 μmol, 46 μL), and Oxyma (3 equiv., 300 μmol, 43 mg) in DCM / DMF (1:1). The mixture was reacted with intermittent manual stirring for 45 min. The solvent was removed by aspiration and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The extent of coupling was confirmed by Kaiser colorimetric assay. The Fmoc group was removed with a 20 (v / v)% piperidine solution in DMF using a 30 sec treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0115] Coupling method 5, scale 100 μmol: protected amino acid (3 equiv., 300 μmol), DIC (3 equiv., 300 μmol, 46 μL), and HOBt (3 equiv., 300 μmol, 41 mg) in DCM / DMF (1:1). The mixture was reacted with intermittent manual stirring for 45 min. The solvent was removed by aspiration and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The extent of coupling was confirmed by Kaiser colorimetric assay. The Fmoc group was removed with a 20 (v / v)% piperidine solution in DMF using a 30 sec treatment and two 10 min treatments. When the amino acid to be deprotected was proline, an additional treatment (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0116] Protocols used during microwave automated synthesis: Compounds were synthesized on a 500 μmol scale using the following methods and protocols. Resins for microwave automated synthesis were selected depending on the group Y: if Y is OH, the terminus is COOH and Cl-TCP(Cl)ProTide resin is selected among others available. If Y is NH2, the terminus is CONH2 and Rink amide ProTide resin is selected among others available.

[0117] Initial conditioning of the resin: The resin was conditioned by washing with MeOH (5×30 sec), DMF (5×30 sec), DCM (5×30 sec), 1% TFA in DCM (1×30 sec and 2×10 min), DCM (5×30 sec), DMF (5×30 sec), DCM (5×30 sec), 5% DIEA in DCM (1×30 sec, 2×10 min), DCM (5×30 sec), DMF (5×30 sec).

[0118] Coupling and deprotection conditions for microwave automated peptide synthesis. Coupling Conditions [Table 2]

[0119] Method for cyclization of peptide sequence P Cyclization method 1: Disulfide or diselenide bond: Cyclization was performed in solution after cleavage from the resin or on the resin after selective deprotection of Cys, Sec, or Pen residues. The peptide was dissolved at a concentration of 100 μM in aqueous ammonium bicarbonate buffer at 10 mM and pH 8.0. The solution was stirred vigorously for 24 hours at room temperature. After this, the product was acidified with TFA to pH 2-3, frozen and dried.

[0120] Cyclization method 2: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with 20 (v / v)% piperidine solution in DMF using a 30 s treatment and two 10 min treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 eq., 1000 μmol, 56 mg) and DIEA (30 eq., 3000 μmol, 240 μL). The OAI and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 μM, 12 mg), phenylsilane (10 eq., 1000 μmol, 123 mg) in DCM (3×15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3×5 min). Coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by addition of PyBOP (4 equiv., 400 μmol, 208 mg), HOAt (12 equiv., 1.2 mmol, 163 mg), DMF (1-3 mL / g of resin), and DIEA (12 equiv., 1.2 mmol, 204 μL). The coupling was left for 1.5 h and repeated overnight.

[0121] Cyclization method 3: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with 20 (v / v)% piperidine solution in DMF using a 30 s treatment and two 10 min treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 eq., 1000 μmol, 56 mg) and DIEA (30 eq., 3000 μmol, 240 μL). The OAI and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 μM, 12 mg), phenylsilane (10 eq., 1000 μmol, 123 mg) in DCM (3×15 min). The resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3×5 min). Coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by two cycles of 4 equivalents of Oxyma (400 μmol, 57 mg) and 4 equivalents of N,N′-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL) for 30 min.

[0122] Cyclization method 4: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with 20 (v / v)% piperidine solution in DMF using a 30 s treatment and two 10 min treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 eq., 1000 μmol, 56 mg) and DIEA (30 eq., 3000 μmol, 240 μL). The OAI and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 μM, 12 mg), phenylsilane (10 eq., 1000 μmol, 123 mg) in DCM (3×15 min). The resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3×5 min). After this, coupling of the amino group of Dap and the carboxylate group of aspartic acid was achieved by two cycles of 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg) for 1 h.

[0123] General Method for Construction of Linker L General method for the formation of disulfides: Disulfide bonds can be achieved by the reaction of two thiols. The thiols are dissolved at a concentration of 100 μM in aqueous ammonium carbonate buffer at 10 mM and pH 8.0, and the solution is stirred thoroughly for 24 hours at room temperature. The solution is then acidified to pH 2-3 with THF, frozen and lyophilized.

[0124] General method for formation of thioethers: Thioether bonds are achieved by reaction of cysteine ​​thiols with the N-terminal bromoacetyl group as described in PL Barker et al. J. Med. Chem., 1992. vol 35, pp. 2040-2048.

[0125] General methods for the formation of ethers: The formation of ethers can be achieved by reaction of a haloalkyl compound with a hydroxyl group, preferably under basic conditions as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GM Wuts. 2014 John Wiley & Sons, Inc. pp. 26-29.

[0126] General methods for the formation of esters: The formation of esters can be achieved by the reaction of a hydroxyl group and a carboxylic acid using typical esterification conditions such as the Fischer ester synthesis reaction in the presence of an acid catalyst, or alternatively by the reaction of a hydroxyl group with the corresponding acid chloride as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GM Wuts. 2014 John Wiley & Sons, Inc. pp. 271-279.

[0127] General method for the formation of thioesters: Thioester bonds are achieved by reaction of carboxilic acids with thiols as described in M. Kazemi et al., Journal of Sulfur Chemistry, 2015, vol. 36:6, pp. 613-623.

[0128] General method for the formation of urethanes: The reaction of isocyanates with hydroxyl groups can lead to the corresponding urethanes as described in MT Nguyen et al., J. Org. Chem. 1998, 63, vol. 20, pp. 6878-6885.

[0129] General method for the formation of silyl ethers: The reaction of a halotrialkylsilyl with a hydroxyl group leads to the corresponding silyl ether. An acid scavengers is usually required, as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GM Wuts. 2014 John Wiley & Sons, Inc. pp. 456-463.

[0130] General method for the formation of sulfonate salts: reaction of an alkyl or aryl sulfonyl halide with a hydroxyl group leads to the corresponding sulfophosphonate ester as described in F. David et al., Org. Process Res. Dev. 2010, 14, 4, pp. 999-1007.

[0131] General methods for the formation of phosphates: The reaction of a dialkyl or diaryl phosphate ester having one hydroxyl group with a hydroxyl group under dehydrating conditions or using Mitsunobu reaction conditions can form the corresponding phosphate ester in which one of the substituents is a chain attached to the hydroxyl group.

[0132] General methods for the formation of ketals: Ketals can be formed by the reaction of a hydroxyl group with a halomethylenoxyalkyl compound or the addition of a hydroxyl group to a substituted dihydropyran or dihydrofuran under acidic conditions, as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GM Wuts. 2014 John Wiley & Sons, Inc. pp. 69-77.

[0133] General method for the formation of hemiketals: Contact of an aldehyde with a hydroxyl group can result in the formation of the corresponding hemiketal as described here: https: / / www.cliffsnotes.com / study-guides / chemistry / organic-chemistry-ii / aldehydes-and-ketones / reactions-of-aldehydes-and-ketones.

[0134] General Methods for the Formation of Carbamates: Carbamates can be formed by reaction of haloformates or isocyanates with hydroxyl groups as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GM Wuts. 2014 John Wiley & Sons, Inc.. pp. 371-374.

[0135] General methods for the formation of carbonates: Carbonates can be formed by reaction of PNPC as described in Eur J Pharm Biopharm, 2017, 115, 149-158 or triphorhodin with SN-38 as described in J. Med. Chem. 2008, 51, 21, 6916-6926.

[0136] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equiv.) was achieved by removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group with 20 (v / v)% piperidine in DMF using two cycles of 30 min for 2 h, followed by 30 s treatment, then two treatments of 10 min each, of either 4 equiv. of oxyma and 4 equiv. of N,N'-diisopropylcarbodiimide (DIC) in DMF or 2 equiv. of DIC and 4 equiv. of HOBt in DCM.

[0137] Coupling of 5-hexynoic acid: Coupling of 5-hexynoic acid (2 equiv., 200 μmol, 23 mg) was achieved by two 30 min cycles of either 4 equiv. Oxyma (400 μmol, 57 mg) and 4 equiv. N,N'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL) in DMF:DCM (1:1) for 4 h, or 4 equiv. DIC (400 μmol, 61 μL) and 4 equiv. OBt (400 μmol, 54 mg) in DMF:DCM 1:1 for 4 h, or 2 equiv. PyBOP (400 μmol, 208 mg) in DMF for 1.5 h in DMF:DCM 1:1, 6 equiv. HOAt (600 μmol, 81.5 mg) and 6 equiv. DIEA (600 μmol, 102 μL). The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0138] Coupling of diglycolic anhydride: Coupling of diglycolic anhydride (10 equiv., 1000 μmol, 116 mg) was achieved by two cycles of 10 equiv. DIEA (1000 μmol, 174 μL) in DMF for 60 min. The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0139] General method for cleavage from resin: Final cleavage of resin and deprotection of side chains: This was performed by treating the resin with TFA (95%), HO (2.5%), and TIS (2.5%) (2 h). Tert-butyl methyl ether was added to the resulting product and the mixture was centrifuged (3 x 8 min). The supernatant was discarded and the pellet was resuspended in a mixture of HO, MeCN, and TFA (1000:1000:1). The product was filtered and frozen.

[0140] General methodology for compound characterization: Compounds were characterized by UPLC (Acquity high-class system (PDA detector, sample manager FNT and Quaternary solvent manager, Acquity BEH C18 (50 × 2 mm × 1.7 μm) column, 0.61 mL / min, MeCN (0.036% TFA) and H2O (0.045% TFA) were used as solvents. In all cases, a 2 min linear gradient was used and UPLC-MS spectroscopy (Waters high class (PDA detector, sample manager FNT and Quaternary solvent manager) was used with MassLynx 4.1 software (Waters, Milford, MA) connected to an electrospray ion source ESI-MS Micromass ZQ. BEH C18 column (50 × 2.1 mm × 1.7 μm, A 1000-milliliter (1000-milliliter) HPLC-MS was used. The flow rate was 0.6 mL / min, MeCN (0.07% formic acid), and H2O (0.1% formic acid) were used as solvents. Samples were analyzed in positive mode ionization: ion spray voltage was 30 V, and capillary temperature was 1 kV). Accurate masses were obtained by a mass spectrometer: LTQ-FT Ultra (Thermo Scientific) with sample flow in direct injection (automated nanoelectrospray). A NanoMate (Advion BioSciences, Ithaca, NY, USA) aspirated samples from a 384-well plate (protein Lobind) using disposable conductive pipette tips and injected the samples through a nanoESI Chip (consisting of 400 nozzles in a 20 × 20 array) into the mass spectrometer. The spray voltage was 1.70 kV, the delivery pressure was 0.50 psi, and ionization was NanoESI positive mode ionization.

[0141] NMR experiments were performed on a Bruker Avance III 600 MHz mass spectrometer equipped with a TCI cryoprobe. Samples were prepared by dissolving compounds at 3–4 mM in 90% H2O / 10% D2O and adjusting the pH to 2–3. Chemical shifts were referenced to internal sodium-3-(trimethylsilyl)propanesulfonate (DSS). Suppression of water signals was achieved by excitation sculpting (ES). Residue-specific assignments were obtained from TOCSY (Total correlation spectroscopy) and COSY (correlation spectroscopy) experiments, allowing sequence-specific assignments by NOESY (2D nuclear Overhauser effect spectroscopy). 13C resonances were assigned from 2D 1H13C HSQC spectra. All experiments were performed at 298 K except for the NOESY spectra, which were acquired at 278 K. Amide proton temperature coefficients were determined from a series of one-dimensional spectra acquired from 278 to 308 K. Mixing times for TOCSY and NOESY were 70 ms and 250 ms, respectively.

[0142] Example 1: Preparation of (S)-tert-butyl (4,11-diethyl 4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate In a 250 ml flask, 1.5 g of 7-ethyl-10-hydroxy-camptothecin (SN-38), 1.3 equivalents of di-tert-butyl dicarbonate (1.15 mL), and excess of dry pyridine (9 mL) were added in 150 ml of dry DCM. The mixture was stirred at room temperature overnight. The reaction mixture was then washed with HCl (0.5N) x 3, saturated NaHCO3 x 1, and brine. The organic layer was dried over MgSO4 and the solvent was removed under vacuum. No further purification was performed. Reverse-phase UPLC-PDA: Acquity BEH C18 (50 × 2 mm × 1.7 μm) column, linear gradient of 0 to 100% MeCN in HO in 2 min using 0.61 mL / min, and MeCN (0.036% TFA), and HO (0.045% TFA) were used as solvents; retention time: 1.89 min. Yield:96%, [M+H]exp+: 493.5 Da. 1H NMR (400MHz, choroform-d) δ 7.81 (d, J= 9.2 Hz, 1H), 7.45 (d, J= 2.5 Hz, 1H), 7.25-7.20 (m, 1H), 6.91- 6.85 (m, 1H), 5.32 (d, J= 16.3, 1H), 4.86 (d, J= 1Hz, 6H), 3.85 (s, 1H), 2.73 (q, J= 7.7 Hz, 2H), 1.60 (s, 1H), 1.57-1.41 (m, 3H), 1,21 (s, 10H), 0.99 (t, J =7.7 Hz, 3H), 0.61 (t, J= 7.3 Hz, 3H).

[0143] Example 2: Preparation of (S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 5-azidopentanoate In a round bottom flask, 400 mg of (S)-tert-butyl (4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl) carbonate (Example 1), 1.6 equivalents of 5-azido-pentanoic acid (193 mg) were dissolved in dry DCM after purging with N2. The mixture was cooled to 0°C. Then, 1.4 equivalents of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (218 mg) were added and the mixture was stirred at 0°C for 1 h and at room temperature overnight. The mixture was washed with NaHCO3 (sat) x 3, HCl (0,1N) x 2, and brine. The organic layer was then dried over MgSO4 and removed under vacuum. The compound was used without purification. [M+H]exp+: 618.36 Da

[0144] Example 3: Preparation of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 5-azidopentanoate (modification of SN-38 with azide as a result of Examples 1-2) In a round bottom flask, 1.5 g of (S)-9-((tert-butoxycarbonyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 5-azidopentanoate (Example 2) was stirred in 50 ml of HCl (4N in dioxane) at room temperature for 2 hours. The solvent was then removed under vacuum and the crude mixture was purified on silica with a purity of >95% using (DCM / MeOH (10%)). Overall yield from Example 1: 8%. [M+H]+: 518,58 Da. Reverse-phase UPLC-PDA: Acquity BEH C18 (50 × 2 mm × 1.7 μm) column, linear gradient of 0 to 100% MeCN in HO in 2 min using 0.61 mL / min, and MeCN (0.036% TFA) and HO (0.045% TFA) were used as solvents; retention time: 1.819 min. [M+H]exp+: 518.58 Da. 1H-NMR (400MHz, chloroform-d) δ 1.00 (t, 3H), 1.38 (t, 3H), 1.65 (m, 2H), 1.69 (m, 2H), 2.20 (m, 2H), 2.56 (m, 2H), 3.13 (q, 3H), 3.27 (td, 2H), 5.20 (s, 2H), 5.39-5.72 (dd, 2H), 7.42 (d, 1H), 7.48 (s, 1H), 7.62 (dd, 1H), 8.45 (d, 1H).

[0145] Methods for alkyne-azide cycloaddition: Alkyne-azide cycloaddition (click reaction) coupling was performed in solution using the protocol described in SFM van Dongen et al.; Bioconjugate Chem. 2009, vol. 20, pp. 20-23. The reaction was performed without the aid of microwaves using the procedure described in lumiprobe (https: / / www.lumiprobe.com / protocols / click-chemistry-dna-labeling). However, the reaction took approximately 2 days to complete. Cu was used along with the ligand (THTPA). As SN-38-N3 is insoluble in H2O, which is insoluble in water or even in mixtures (water / DMSO), the reaction was performed using only DMF instead of buffer and DMSO. Alternatively, the alkyne-azide cycloaddition of SN-38-N3 with alkyne-TTDS-DapKAPETALD was performed using microwaves. To a 10ml microwave vial of SN-38-N3 (1.5 eq.), alkyne-TTDS-DapKAPETALD (1 eq.), CuTHTPA (0.15 eq.), and sodium ascorbate (0.3 eq.) were added and dissolved in 3mL of DMF. The mixture was stirred for 2-4 hours with MW (discover SP MW) assistance at 30°C for the entire reaction. The crude mixture was purified by semi-preparative HPLC (C18).

[0146] Preparation of reagents for alkyne-azide cycloaddition (click reaction): 100 mM copper(II)-THPTA stock in 55% DMSO: 50 mg of copper(II) sulfate pentahydrate was dissolved in 1 mL of distilled water, and 116 mg of tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand was dissolved in 1.1 mL of DMSO. The two solutions were then mixed.

[0147] 5 mM ascorbic acid stock solution: 18 mg of ascorbic acid was dissolved in 20 ml of distilled water.

[0148] General methods for purification and characterization of products: Crude materials were purified by RP-HPLC in semi-preparative scale and characterized by UPLC (Acquity high-class system (PDA detector, sample manager FNT and Quaternary solvent manager, Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, MeCN (0.036% TFA) and HO (0.045% TFA) were used as solvents. In all cases, a linear gradient of 2 min was used) connected to an electrospray ion source ESI-MS Micromass ZQ and UPLC-MS spectroscopy (Waters high class (PDA detector, sample manager FNT and Quaternary solvent manager). BEH C18 column (50×2.1 mm×1.7 μm, A 100% ethanol chromatograph (Waters) was used. The flow rate was 0.6 mL / min, MeCN (0.07% formic acid), and H2O (0.1% formic acid) were used as solvents. Samples were analyzed in positive mode ionization: ion spray voltage was 30 V, and capillary temperature was 1 kV). Accurate masses were obtained by a mass spectrometer: LTQ-FT Ultra (Thermo Scientific) with direct infusion sample flow (automated nanoelectrospray). A NanoMate (Advion BioSciences, Ithaca, NY, USA) aspirated samples from a 384-well plate (protein Lobind) using disposable conductive pipette tips and injected the samples through a nanoESI Chip (consisting of 400 nozzles in a 20x20 array) into the mass spectrometer. The spray voltage was 1.70 kV, the delivery pressure was 0.50 psi, and ionization was NanoESI positive mode ionization. All peptides were obtained with purity above 95%.

[0149] Example 4: Preparation of hexynoic acid-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (hexynoic acid-TTDS-SEQ ID NO: 7) with an amide bond between the amino group of the Dap side chain and the carboxylic acid of the Asp side chain For the manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-Asp(OAl)-OH (118.5 mg). Subsequent amino acids were coupled sequentially using coupling method 4 as follows: [Table 3]

[0150] DMF / DCM (1:1) containing 46 μL DIC and 43 mg Oxyma was used. The mixture was reacted with intermittent manual stirring for 45 min. After each coupling, removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was performed with 20 (v / v)% piperidine in DMF using a 30 s treatment followed by two treatments of 10 min each. Two further treatments with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed (2×5 min) to ensure removal of the Fmoc group from the secondary amine (proline). Cyclization was performed on the resin by cyclization method 2. The Fmoc group was removed with a solution of 20 (v / v)% piperidine in DMF using a 30 s treatment and two treatments of 10 min. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 equiv., 1000 μmol, 56 mg) and DIEA (30 equiv., 3000 μmol, 240 μL). The OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg), phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3×5 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3×15 min). Coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by addition of PyBOP (4 equiv., 400 μmol, 208 mg), HOAt (12 equiv., 1.2 mmol, 163 mg), DMF (1-3 mL / g resin), and DIEA (12 equiv., 1.2 mmol, 204 μL). The coupling was left for 1.5 h and repeated overnight.

[0151] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equiv., 200 μmol, 108.53 mg) was achieved with 4 equiv. DIC (400 μmol, 61 μL) and 4 equiv. HOBt (400 μmol, 54 mg) in DCM for 2 h, followed by removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group with 20 (v / v)% piperidine in DMF using a 30 s treatment followed by two treatments of 10 min each.

[0152] Coupling of 5-hexynoic acids: The following protocol was used to couple 5-hexynoic acids to peptides immobilized on resin. Hexynoic acids (4 equiv., 400 μmol, 45 mg), PyBOP (4 equiv., 400 μmol, 208 mg), and HOAt (12 equiv., 1.2 mmol, 163 mg) in DMF (1-3 mL / g resin) were added successively to the resin, followed by 12 equiv. of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0153] The peptide was then cleaved and lyophilized. Product characterization: Reverse-phase UPLC: linear gradient of 20-60% MeCN in H2O in 2 min using an Acquity BEH C18 (50 x 2 mm x 1.7 μm) column, 0.61 mL / min, MeCN (0.036% TFA), and H2O (0.045% TFA) were used as solvents; retention time: 0.939 min. UPLC-MS [M + H]exp+: 1308.15 Da; yield (synthesis and purification): 7.5%

[0154] Example 5: Preparation of compound of formula (Ia), G2B-001 Starting with hexynoic acid-TTDS-DapKAPETALD prepared in Example 4 and using SN-38-N3 prepared in Example 3, the compound of formula (Ia) was achieved according to the following protocol: In a 10 ml microwave vial, 1.5 equivalents of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 5-azidopentanoate, 1 equivalent of alkyne-TTDS-DapKAPETALD, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate were added and dissolved in 3 mL of DMF. During all reactions, the mixture was stirred at 30°C for 2-4 hours utilizing MW (CEM discover SP MW). The crude was purified by RP-HPLC in a semi-preparative scale. This gave the compound with a purity of >95%. Product characterization. Reversed phase UPLC-PDA: linear gradient of 0-100% MeCN in H2O in 2 min using Acquity BEH C18 (50 x 2 mm x 1.7 μm) column, 0.61 mL / min, MeCN (0.036% TFA) and H2O (0.045% TFA) were used as solvents; retention time: 1.462 min. UPLC-MS [M + H]exp+: 1824.76 Da; yield (synthesis and purification): 30%

[0155] Example 6: Preparation of linear hexynoic acid-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (hexynoic acid-TTDS-SEQ ID NO: 14) which does not contain an amide bond between the amino group of the Dap side chain and the carboxylic acid of the Asp side chain. DapKAPETALD (SEQ ID NO: 14) i.e. linear peptide For manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-Asp(OAl)-OH (118.5 mg). Subsequent amino acids were coupled sequentially using coupling method 4 as follows: [Table 4]

[0156] DMF / DCM (1:1) containing 46 μL DIC and 43 mg Oxyma was used. The mixture was reacted with intermittent manual stirring for 45 min. After each coupling, removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was performed with 20 (v / v)% piperidine in DMF using a 30 s treatment followed by two treatments of 10 min each. Two further treatments (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure removal of the Fmoc group from the secondary amine (proline).

[0157] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equiv., 200 μmol, 108.53 mg) was achieved with 4 equiv. DIC (400 μmol, 61 μL) and 4 equiv. HOBt (400 μmol, 54 mg) in DCM for 2 h. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was then removed with 20 (v / v)% piperidine in DMF using a 30 s treatment followed by two treatments of 10 min each.

[0158] Coupling of 5-hexynoic acids: The following protocol was used to couple 5-hexynoic acids to peptides immobilized on resin. Hexynoic acids (4 equiv., 400 μmol, 45 mg), PyBOP (4 equiv., 400 μmol, 208 mg), and HOAt (12 equiv., 1.2 mmol, 163 mg) in DMF (1-3 mL / g resin) were added successively to the resin, followed by 12 equiv. of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0159] The OAI and Alloc groups were deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg), phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3×15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3×5 min). The peptide was then cleaved and lyophilized. Product characterization: A linear gradient of 10-60% MeCN in H2O in 30 min using an Xbridge 25 cm 3.5 μm column, 1 mL / min, MeCN (0.1% TFA), and H2O (0.1% TFA) were used as solvents; retention time: 11.526 min. Yield (synthesis and purification): 9%

[0160] Example 7: Preparation of G2B-001m, a compound of formula (Ib) that does not contain an amide bond between the amino group in the side chain of Dap and the carboxylic acid in the side chain of Asp Starting with hexynoic acid-TTDS-DapKAPETALD linear prepared in Example 6 and using SN-38-N3 prepared in Example 3, the compound of formula (Ib), also referred to as G2B-001 linear, was achieved according to the following protocol: In a 10 ml microwave vial, 1.5 equivalents of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl 5-azidopentanoate, 1 equivalent of alkyne-TTDS-DapKAPETALD linear, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate were added and dissolved in 3 mL of DMF. During all reactions, the mixture was stirred at 30 °C for 2-4 hours utilizing MW (CEM discover SP MW). The crude was purified by RP-HPLC on a semi-preparative scale. This gave the compound in >95% purity. Product characterization: Reverse-phase HPLC-MS: Linear gradient of 0-80% MeCN in H2O in 7 min using a Luna 3 μm C18(2) 100A 50×2.1 mm column, MeCN (0.1% formic acid), and H2O (0.1% formic acid) were used as solvents; retention time: 5.08 min. UPLC-MS [M + H]exp+: 1845.10 Da; Yield (synthesis and purification): 22%.

[0161] Example 8: Preparation of N3-Pen-SN-38 [ka]

[0162] In a two-necked round bottom flask, add 1 equivalent of SN-38 (600 mg) dissolved in 100 ml per gram of dry DCM (60 ml). A cloudy solution is observed. Cool the reaction mixture to 0-2°C, add 6 equivalents of DIEA (2 ml) and stir for 15 minutes. After 15 minutes, add 3 equivalents of N3-Pen-Cl (700 mg) (dissolved in 500 μl of dry DCM) dropwise. Allow it to cool for 15 minutes and then continue stirring at room temperature. Check the progress of the reaction by HPLC. If the starting material is less than 3%, take the reaction for workup. The initial volume of the reaction mixture is about 45 mL and is made up to 300 ml using dry DCM. Extract 3 times with distilled water (volume of 20% of the final volume of the reaction mixture), 3 min × 60 ml. Dry the DCM layer with sodium sulfate (Na2SO4) for 30 minutes and evaporate on a rotary evaporator. Treat the solid twice with 80:20 Hex:Ether and keep drying in a desiccator. Yield after workup, 730 mg, characterization of product. Reverse phase HPLC: SS column 250 x 4.6 mm packed with C-18 silica gel (5 μm) for chromatography R, linear gradient of 10-90% MeCN in H2O in 30 min using 1 mL / min, MeCN (0.1% TFA) and H2O (0.1% TFA) were used as solvents; retention time: 19.730 min, observed mass = 518.20 Da. calculated mass = 517.19 Da.

[0163] Example 9: Preparation of compound of formula (Ic), G2B-003 Starting with hexynoic acid-TTDS-DapKAPETALD prepared in Example 4 and using N3-Pen-SN-38 prepared in Example 8, the compound of formula (Ic), also referred to as G2B-003, was achieved according to the following protocol: In a 10 ml vial, 1.5 equivalents of N3-Pen-SN-38, 1 equivalent of hexynoic acid-TTDS-DapKAPETALD, 0.15 equivalents of CuTHPTA, and 0.3 equivalents of sodium ascorbate were added and dissolved in 3 mL of DMF. During all reactions, the mixture was stirred at 30° C. for 2-4 hours using microwave (CEM discover SP MW). The crude was purified by RP-HPLC on a semi-preparative scale. This gave the compound in a purity of more than 95%. Product characterization: Reverse phase HPLC: Xbridge 25 cm 3.5 μm column, linear gradient of 20-70% MeCN in HO in 30 min using 1 mL / min, MeCN (0.1% TFA) and HO (0.1% TFA) were used as solvents; retention time: 12.00 min. UPLC-MS [M + H]exp+: 1826.15 Da; yield (synthesis and purification): 17%

[0164] Example 10: Preparation of SN-38-O-CO-NH-Gly-COOH A solution of Glycine-COOBut (1 eq.) and DMAP (2 eq.) in dry DCM (15 mL) was added dropwise to a solution of bis(4-nitrophenyl)carbonate (1.3 eq.) in dry DCM and the resulting solution was stirred at 50° C. overnight. The reaction mixture was then diluted with DCM (150 mL) and washed with 0.5 N HCl (100 mL). The aqueous phase was washed with DCM (5×100 mL) and all organic fractions were collected, dried over MgSO4 and filtered. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (Hexane:DCM:Et2O; 5:4:1). The resulting PNP-Gly-COOBut (1 eq.) was reacted with SN-38 (1.2 eq.) in the presence of dry DCM containing DMAP (2 eq.). The reaction was stirred at 50° C. overnight. The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography. The t-butyl ester group was removed by treating SN-38-O-CO-NH-Gly-COOBut with TFA:DCM:TIS (40:40:20) for 6 hours. The solvent was then evaporated. Toluene was added twice to remove traces of TFA. No further purification was performed.

[0165] Example 11: Preparation of Boc-Val-Cit-PAB-SN-38 In a round bottom flask, 1 equivalent of 7-ethyl-10-hydroxy-camptothecin (SN-38), 1 equivalent of Boc-Val-Cit-PAB-PNP (from iris biotech), 2 equivalents of DIEA, and catalytic DMAP were stirred in dry DMF at room temperature for 16 hours. The reaction mixture was then diluted with AcOEt and washed with HCl (0.5M) (x3), and brine (x4). It was then dried over MgSO4, the volatiles were evaporated, and the crude mixture was purified by silica chromatography (AcOEt:MeOH; 20:1; 15:1; 9:1). The desired compound was obtained in 52% yield with a purity of more than 90%. Yield 52%. Reverse phase UPLC-PDA: linear gradient of 0-100% MeCN in HO in 2 min using Acquity BEH C18 (50 x 2 mm x 1.7 μm) column, 0.61 mL / min; MeCN (0.036% TFA) and HO (0.045% TFA) were used as solvents; retention time: 1.42 min. [M+H]exp+: 898.4 Da.

[0166] Example 12: Preparation of H2N-Val-Cit.PAB-SN-38 In a round bottom flask, 1 equivalent of Boc-Val-Cit-PAB-SN-38 prepared in Example 11 was stirred in a mixture of DCM:TFA:H2O (44.75:49.75:0.5) for 5 minutes. The solvent was then evaporated. Toluene was added twice to remove traces of TFA. No further purification was performed.

[0167] Example 13: Preparation of G2B-004(Id) G2B-004 was prepared starting from diglycolic acid-DapKAPETALD (prepared using coupling method 4 of the standard method described above) and 2 equivalents of H2N-Val-Cit-PAB-SN-38 (prepared in Example 12) using PyBOP (4 equivalents), HOAt (12 equivalents) was dissolved in DMF (1-3 mL / g resin), and then DIEA (12 equivalents) was added. The mixture was allowed to react with intermittent manual stirring for 1 hour. The solvent was removed by aspiration and the resin was washed with DMF (5 x 30 seconds) and DCM (5 x 30 seconds). The coupling reaction was carried out twice (1 hour and overnight). G2B-004 was then cleaved and lyophilized. Product characterization. Reverse phase UPLC: Acquity BEH C18 (50 x 2 mm x 1.7 μm) column, linear gradient of 0-100% MeCN in H2O in 2 min using 0.61 mL / min, and MeCN (0.036% TFA) and H2O (0.045% TFA) were used as solvents; retention time: 1.17 min. UPLC-MS [M + H]exp+: 1807.84 Da; yield (synthesis and purification): 21%.

[0168] Example 14: Preparation of G2B-005(Ie) Nval-Pro-Gly-DapKAPETALlD was prepared using the standard method described above (coupling method 4). G2B-005 was prepared starting from Nval-Pro-Gly-DapKAPETALD and 2 equivalents of SN-38-O-CO-NH-Gly-OH prepared in Example 10 using PyBOP (4 equivalents), HOAt (12 equivalents) was dissolved in DMF (1-3 mL / g resin) and then DIEA (12 equivalents) was added. The mixture was reacted with intermittent manual stirring for 1 hour. The solvent was removed by aspiration and the resin was washed with DMF (5 x 30 seconds) and DCM (5 x 30 seconds). The coupling reaction was carried out twice (1 hour and overnight). G2B-005 was then cleaved and lyophilized. Characterization of the product. Reverse phase UPLC: Acquity BEH C18 (50 x 2 mm x 1.7 μm) column, linear gradient of 0-100% MeCN in HO in 2 min using 0.61 mL / min, and MeCN (0.036% TFA) and HO (0.045% TFA) were used as solvents; retention time: 1.51 min. UPLC-MS [M + H]exp+: 1640.77 Da; yield (synthesis and purification): 18%.

[0169] Example 15: Preparation of several aqueous micellar dispersions containing SN-38 lactone encapsulated within micelles of SN-38 conjugates All formulations are simple mixtures of a basic aqueous solution of free SN-38 carboxylate (NaOH, 0.05 N in water) and an acidic aqueous solution of G2B-001 or other SN-38 peptide conjugates (forming micelles) (tartaric acid, 20 mg / mL (134 mM) in water), where the concentrations and volumes of both components in the basic and acid solutions can be altered. The result of mixing equal volumes of basic and acidic solutions is an acidic solution with a pH < 7 (experimental values ​​are 2.3-2.5), completely translucent, physically stable over time (no appearance of precipitate or turbidity after several weeks of storage at room temperature), and containing SN-38 lactone encapsulated in G2B-001 micelles. A scheme of the process is shown in Figure 1.

[0170] Example 15a: Preparation of G2B-001 micelles at a concentration of 20 mg / ml containing 16% (w / w) of free SN-38 lactone. This formulation is known as G2B-002-20-16. In this formulation, the weight / weight percentage ((w / w)%) of free SN-38 in the final formulation is 16(w / w)%, i.e., free SN-38 / (SN-38+G2B-001)*100=16%. The final concentrations in the aqueous formulation are 4 mg / mL for free SN-38 and 20 mg / mL for G2B-001, with a final pH of less than 3, ensuring that the majority of the free SN-38 is the active lactone. The particle size is 310 nm (Z-average) and the polydispersity index (PDI) is 0.55 (measured by dynamic light scattering).

[0171] Example 15b: Preparation of G2B-001 micelles at a concentration of 20 mg / mL containing 9% (w / w) of free SN-38 lactone. This formulation is known as G2B-002-20-9. In this formulation, the weight / weight percentage ((w / w)%) of free SN-38 in the final formulation is 9(w / w)%, i.e., free SN-38 / (SN-38+G2B-001)*100=9%. The final concentrations in the aqueous formulation are 2 mg / mL for free SN-38 and 20 mg / mL for G2B-001, and the final pH is less than 3, ensuring that the majority of the free SN-38 is the active lactone. The particle size is 103 nm (Z-average) and the polydispersity index (PDI) is 0.50 (measured by dynamic light scattering). The solution is clear.

[0172] Example 15c: Preparation of G2B-001 micelles at a concentration of 10 mg / mL containing 5% (w / w) of free SN-38 lactone. This formulation is known as G2B-002-10-5. In this formulation, the weight / weight percentage ((w / w)%) of free SN-38 in the final formulation is 5(w / w)%, i.e., free SN-38 / (SN-38+G2B-001)*100=5%. The final concentrations in the aqueous formulation are 0.5 mg / mL for free SN-38 and 10 mg / mL for G2B-001, with a final pH of less than 3, ensuring that the majority of the free SN-38 is the active lactone. The particle size is 40 nm (Z-average) and the polydispersity index (PDI) is 0.40 (measured by dynamic light scattering).

[0173] The table below shows the amount of SN38 and the amount of free SN-38 lactone in the peptides. [Table 5]

[0174] Example 15d: Preparation of G2B-003 micelles at a concentration of 20 mg / mL containing 5% (w / w) of free SN-38 lactone. This formulation is known as G2B-006-20-9.

[0175] It was prepared as G2B-002-20-9 using the peptide conjugate of formula (Ic) instead of the peptide conjugate of formula (Ia), in which the weight / weight percentage ((w / w)%) of free SN-38 in the final formulation is 9(w / w)%, i.e., free SN-38 / (SN-38+G2B-003)*100=9%.

[0176] Example 16: Loading of soluble SN-38 lactone in micellar systems To test the loading efficiency of soluble SN-38 lactone in the present system, micelles of G2B-001 or G2B-003 at a concentration of 40 mg / mL in acidic solution (0.5 mL) were added to SN-38 carboxylate at a concentration of 4 or 8 mg / mL in basic solution (0.5 mL) in a plastic vial (1.5 mL). After gentle mixing, the samples were stored at room temperature (18-22 °C) for 0.5 h. The resulting solution was then filtered through a 0.45 μm polypropylene syringe filter to remove the insoluble SN-38 lactone crystals. The concentration of soluble SN-38 lactone in the filtered solution was determined with a high performance liquid chromatography apparatus (Shimazdu) with a fluorescence detector. The loading efficiency (LE) of soluble SN-38 lactone was determined by the formula: LE=C / L*100 (C is the concentration of SN-38 lactone in the filtered micellar system, and L is the theoretical loading of SN-38 lactone in the final product before filtration). The LE of the products is presented in Table 2. The products designated G2B-002-20-9 and G2B-006-20-9 achieved a final concentration of soluble SN-38 lactone of 2 mg / mL after filtration, where the loading efficiency was nearly 100% in both cases (Table 2). In the absence of G2B-001 or G2B-003 micelles, the same process resulted in insoluble SN-38 lactone in the presence of vehicle alone (Table 2). Similarly, the same process in the presence of irinotecan at a concentration of 20 mg / mL in acid solution resulted in insoluble SN-38 lactone (Table 2).

[0177] [Table 6]

[0178] It is also observed that the solubility data of the micelles of the present invention is higher when compared to the solubility data of SN-38 liposomes or micelles disclosed in the prior art.

[0179] Example 17: Visual inspection of turbidity in micellar systems To test the physical stability of the products of the invention G2B-002-20-9 and G2B-006-20-9, both with a final concentration of soluble SN-38 lactone of 2 mg / ml, we carried out visual inspection of these products. Table 3 summarizes the turbidity data from visual inspection of the products. For comparison, we used products obtained in the absence of G2B-001 or G2B-003 and consisting of G2B-001 linear SN-38 at 20 mg / mL, SN-38 only in vehicle, or irinotecan 20 mg / mL and SN-38 in vehicle, as described in the example "Loading of soluble SN-38 lactone in micellar systems".

[0180] [Table 7]

[0181] Figures 2-5 show photographs of products G2B-002-20-9 and G2B-006-20-9, or alternatively products consisting of only SN-38 in vehicle or irinotecan 20 mg / mL and SN-38 in vehicle, obtained in the absence of G2B-001, as described in the example "Loading of soluble SN-38 lactone in micellar systems". Products G2B-002-20-9 and G2B-006-20-9 were not turbid, while products containing only SN-38 in vehicle, as well as products containing irinotecan and SN-38, showed turbidity corresponding to insoluble SN-38 lactone crystals. Photographs were taken immediately after preparation (Figures 2-3) or up to 6 months after preparation (Figures 4-5). Products containing SN-38 in the G2B-001 linear form did not become turbid in 1-4 weeks.

[0182] Example 18: Visual examination of turbidity of micellar systems carrying water-insoluble camptothecin To test whether the system of the present invention possesses products other than SN-38, the inventors chose camptothecin of the following formula: [ka]

[0183] Like SN-38, camptothecin is highly insoluble in the lactone at acidic pH and soluble in the free state as the carboxylate at basic pH.

[0184] G2B-001 micelles at a concentration of 40 mg / mL in acid solution (0.5 mL) were added to camptothecin carboxylate at concentrations of 0.25, 0.5, and 1 mg / mL in basic solution (0.5 mL) in plastic vials (1.5 mL). After gentle mixing, the samples were kept at room temperature (18-22 °C). The final pH of the formulation was acidic (pH = 1.5) to ensure conversion to camptothecin lactone. Immediately after preparation and after 24 hours, we performed visual inspection of the products. Table 4 summarizes the turbidity data from visual inspection of the products. For comparison, we used the product obtained in the absence of G2B-001 micelles and consisting of camptothecin lactone in the vehicle.

[0185] [Table 8]

[0186] Figure 6 shows photographs of the products detailed in Table 4. Products G2B-CPT-20-5, G2B-CPT-20-2 and G2B-CPT-20-1 were not turbid, while the product containing only CPT in the vehicle showed turbidity corresponding to insoluble CPT crystals. Photographs were taken immediately after preparation.

[0187] Example 19: Visual examination of turbidity of micellar systems carrying SN-38 lactone and water-insoluble camptothecin To test whether the system of the present invention can simultaneously carry SN-38 and camptothecin, we formulated G2B-001 micelles at a concentration of 40 mg / mL in acidic solution (0.5 mL) in plastic vials (1.5 mL) and added camptothecin carboxylate at a concentration of 2 or 4 mg / mL in basic solution (0.25 mL) and SN-38 carboxylate at a concentration of 2 or 4 mg / mL in basic solution (0.25 mL).

[0188] After gentle mixing, the samples were kept at room temperature (18-22 °C). The final pH of the formulation was acidic (pH = 1.5) to ensure conversion to lactone. Immediately after preparation and after 24 hours, we performed visual inspection of the products. Table 5 summarizes the turbidity data from visual inspection of the products. For comparison, we used a product obtained in the absence of G2B-001 micelles and consisting of only SN-38 and camptothecin lactone in the vehicle.

[0189] [Table 9]

[0190] Figure 7 shows photographs of the products formulated with G2B-001 detailed in Table 5, or alternative products obtained in the absence of G2B-001 micelles and consisting only of SN-38 and CPT in the vehicle. Products G2BSN-38-CPT-20-5-5 and G2B-SN38-CPT-20-2-2 were not turbid, whereas products containing only SN-38 and CPT in the vehicle showed turbidity corresponding to insoluble SN-38 and CPT crystals. Photographs were taken immediately after preparation.

[0191] Example 20: Stability of product G2B-002-20-9 after lyophilization To test whether the soluble SN-38 lactone carried by the product G2B-002-20-9 of the present invention is stable after lyophilization, G2B-002-20-9 (280 μL in a 1.5 mL plastic vial, containing 2 mg / mL of soluble SN-38 lactone) was frozen in liquid nitrogen and lyophilized in a Telstar apparatus (LyoQuest). After lyophilization, the dried sample was suspended in an equal amount of water (280 μL). The resulting solution showed no turbidity. Analysis of the soluble SN-38 lactone by HPLC showed a recovery of 100.6 ± 7.8% of soluble SN-38 lactone (average and standard deviation of four samples prepared independently). Thus, the product G2B-002-20-9 of the present invention was stable after lyophilization and reconstitution with water.

[0192] Example 21: In vitro activity of product G2B-002-20-9 The activity of the product G2B-002-20-9, which possesses soluble SN-38 lactone, was compared to one of the free SN-38 lactone and irinotecan. Cancer cell lines were obtained from a repository maintained at Hospital Sant Joan de Deu (Barcelona, ​​Spain). Cancer cell lines used in these experiments included pediatric glioma cancer types, including diffuse intrinsic pontine glioma (HSJD-DIPG-007), pediatric diffuse midline glioma (HSJD-DMG-001), and pediatric high-grade glioma (HSJD-GBM-001), as well as cell lines of other pediatric solid tumors, such as Ewing's sarcoma (A673) and rhabdomyosarcoma (RH4). Briefly, 3000-30,000 cancer cells were cultured in 96-well plates, and after 24 hours, the cells were exposed to compounds G2B-002-20-9 (concentration range of soluble SN-38 lactone ranging from 10-0.0000001 μM), SN-38 lactone (concentration range of 10-0.0000001 μM), or irinotecan (concentration range of 100-0.000001 μM). To add drugs to cells in culture, G2B-002-20-9, irinotecan, and SN-38 were prepared in culture medium from stock solutions. The tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetratetrazolium] (MTSassay; Promega, Fitchburg, WI) was used to determine cell viability after 72 hours of incubation with the drug. The concentration of drug required to produce a 50% reduction in cell proliferation (IC50 and 95% confidence interval) was calculated with Graphpad Prism 8 software (La Jolla, CA). Results are expressed as a percentage of cell viability compared to untreated control wells. The activity of G2B-002-20-9 carrying SN-38 lactone was equivalent to that of free SN-38 lactone and superior to irinotecan in all tested cell lines. Figure 8 shows the results with HSJD-DIPG-007 cells. Figure 9 shows the results for HSJD-DMG-001 cells, Figure 10 shows the results for HSJD-GBM-001 cells, and Figure 11 shows the results for RH4 cells.FIG. 12 shows the results with A673 cells.

[0193] Example 22: Antitumor efficacy of the products of the invention in DIPG and pHGG xenografts We compared the activity of the product G2B-002-20-9 and the closely related drug irinotecan administered intravenously to mice bearing intracranial human brain tumors. We established brain tumor xenografts in immunodeficient athymic nude mice by intracranial injection of 500,000 cells into the fourth ventricle of anesthetized mice using a stereotaxic apparatus. Two different xenografts were used. Xenograft HSJD-DIPG-007 is a clinically relevant model of DIPG and carries the mutations H3.3-K27M and ACVR1-R206H. Xenograft HSJD-GBM-001 is a relevant model of pHGG and carries wild-type H3.3 and the mutation p.G245S in the gene. The aim of this experiment was to test whether the activity of the novel compound G2B-002-20-9, which possesses soluble SN-38 lactone and crosses the blood-brain barrier, is superior to one of the irinotecans, which are metabolized to SN-38 lactone by carboxylesterase after administration to the blood of mice and do not significantly cross the blood-brain barrier. HSJD-DIPG-007 cells were injected into 25 mice and HSJD-GBM-001 cells were injected into 21 mice. Treatment consisted of intravenous injection via the tail vein. Treatment was started 31 days after seeding of HSJD-DIPG-007 cells and 8 days after seeding of HSJD-GBM-001 cells. Days of treatment with G2B-002-20-9 were 1, 2, 3, 4, 5, 8, 9, 10, 11, and 12 for mice carrying HSJD-DIPG-007 and 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 22, 23, 24, 25, and 26 for mice carrying HSJD-GBM-001. Days of treatment with irinotecan were 1, 2, 3, 4, and 5 for mice carrying HSJD-DIPG-007 and 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 22, 23, 24, 25, and 26 for mice carrying HSJD-GBM-001. In mice bearing HSJD-DIPG-007 tumors, treatment groups received 10 mg / kg G2B-002-20-9 (a dose of soluble SN-38 lactone), 40 mg / kg irinotecan, or control (saline). A minimum of eight mice received each treatment.In mice bearing HSJD-GBM-001 tumors, treatment groups received G2B-002-20-9 10 mg / kg (a dose of soluble SN-38 lactone), irinotecan 10 mg / kg, or control (saline). Seven mice received each treatment. In experiments with mice bearing HSJD-DIPG-007, G2B-002-20-9 was administered safely. During treatment, the weight loss after 10 doses of G2B-002-20-9 was -7.3 ± 3.5% (mean and standard deviation), with no toxic deaths. Irinotecan treatment at 40 mg / kg resulted in one toxic death and a weight loss of -16.4 ± 9.4%. After cessation of treatment, the survival curves obtained in the study are shown in Figure 13. Mice treated with G2B-002-20-9 survived significantly longer (median survival = 73 days) than control mice (median survival = 67 days; P = 0.0344) and irinotecan (median survival = 65.5 days; P = 0.0009), but irinotecan did not improve the survival of the animals compared to the control (P = 0.5851). In experiments with mice bearing HSJD-GBM-001, all mice tolerated the treatment without significant weight loss. After cessation of treatment, the survival curves obtained in the study are shown in Figure 14. Mice treated with G2B-002-20-9 survived significantly longer than control mice (median survival = 35 days; P = 0.0003) and irinotecan (median survival = 40 days; P = 0.008). Irinotecan improved animal survival by 5 days compared to controls (P=0.0020). Collectively, these studies demonstrate that G2B-002-20-9 is superior to irinotecan in treating mice bearing intracranial DIPG and pHGG.

[0194] Example 23: Antitumor efficacy of products of the invention in subcutaneous patient-derived xenografts We compared the activity of novel compounds G2B-002-10-5, G2B-002-20-9, and G2B-002-20-16 with one of the closely related drugs irinotecan, which were administered intravenously to mice bearing subcutaneous human tumors. Patient-derived xenograft (PDX) tissues were obtained from immunodeficient athymic nude mice at Hospital Sant Joan de Deu (Barcelona, ​​Spain). PDXs were established from biopsies of pediatric patients with Ewing's sarcoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma, as detailed in one publication (1). Identification of PDXs is shown in Table 6.

[0195] [Table 10]

[0196] The aim of this experiment was to test whether the activity of the novel product bearing SN-38 lactone is comparable to that of the one of irinotecan, which is metabolized to SN-38 lactone by carboxylesterase after administration into the blood of mice. Each PDX was inserted subcutaneously into 7-8 mice. Treatment consisted of intravenous injection via the tail vein. Treatment was started when the subcutaneous tumor volume was in the range of 100-300 mm3. Treatment days were 1, 2, 3, 4, 5, 8, 9, 10, 11, and 12. We included seven treatment groups in the study: G2B-002-20-16 at 10mg / kg, G2B-002-20-16 at 1mg / kg, G2B-002-20-9 at 1mg / kg, G2B-002-20-9 at 1mg / kg, G2B-002-10-5 at 1mg / kg, irinotecan at 10mg / kg, and saline control (saline solution). One mouse was administered each treatment. All mice tolerated the treatment without significant weight loss. On day 15, we measured the tumor volume and calculated the ratio between the final volume after treatment and the initial volume on day 1. All tumors treated with irinotecan and G2B-002 products were significantly reduced in volume compared to saline-treated control tumors (P=0.0001; paired one-way ANOVA with Dunnett's multiple comparison test) (Figure 15). Collectively, these experiments show that G2B-002-20-16, G2B-002-20-9 and G2B-002-10-5 are similar to irinotecan in treating mice bearing extracranial Ewing's sarcoma, neuroblastoma, osteosarcoma and rhabdomyosarcoma.

[0197] Example 24: Distribution of SN-38 lactone in mouse retina following intravenous administration of G2B-002-20-9 and irinotecan We evaluated the distribution of SN-38 lactone in the retina following intravenous administration of G2B-002-20-9 at a dose sufficient to provide 10 mg / kg and 1 mg / kg of free SN-38 lactone, or 10 mg / kg of irinotecan. We administered sufficient G2B-002-20-9 intravenously to 18 athymic nude mice at each dose level to provide 10 mg / kg and 1 mg / kg free SN-38 lactone, and 12 mice at 10 mg / kg irinotecan. The animals were sacrificed by decapitation (3 per time point) at 0.25, 0.5, 1, 2, 4, and 8 hours for the G2B-002-20-9 group, and 0.25, 0.5, 1, and 2 hours for the irinotecan group, and the retinas were dissected and samples were frozen and stored until analysis using published methods.

[0198] Following administration of G2B-002-20-9 at a dose sufficient to provide 10 mg / kg of free SN-38 lactone, the mean maximum concentration of SN-38 lactone in the retina was 2878 ng / g (range 2330-3593), achieved at 0.5 hours. At this dose level, the concentration of SN-38 lactone remained above 100 ng / g for at least 4 hours (Figure 16).

[0199] Following administration of G2B-002-20-9 at a dose sufficient to provide 1 mg / kg of free SN-38 lactone, the mean maximum concentration of SN-38 lactone in the retina was 867 ng / g (range 254-1844) and was achieved at 0.5 hours. At this dose level, the concentration of SN-38 lactone remained above 100 ng / g for at least 0.5 hours (Figure 16).

[0200] After administration of 10 mg / kg irinotecan, concentrations of SN-38 lactone remained below 100 ng / g at all time points. The mean maximum concentration of SN-38 lactone in the retina was 57 ng / g (range 48-72 ng / g), achieved at 0.25 hours. After 2 hours, SN-38 lactone was below the detection limit (5 ng / g) in this group (Figure 16).

[0201] Taken together, these experiments demonstrate that the novel invention improves retinal distribution of soluble SN-38 lactone.

[0202] Example 25: Transport of G2B-002-20-9 micelles through a human BBB model in vitro The inability of irinotecan and SN-38 to cross the BBB in vivo is well documented (Pharmaceutics 2020, 12 399 and Cancer Res 1993, 53(12) 2823-9. Using an in vitro BBB human model, all compounds were evaluated at 100 μM in Ringer Hepes. The time assay was 2 hours at 37°C. Acceptor and 0 hours were analyzed by UPLC. All were evaluated in triplicate and with Lucifer Yellow (LY) at 25 μM. As mentioned in the paper ACIE, 2015,3967, the permeability of Papp was estimated at Tf 0.953 10 -6 Transport is considered adequate if the Paap is higher than that of the natural ligand such as . Many times, the BBB shuttle-cargo conjugates have a lower Paap than the BBB shuttle alone because they are shuttling the cargo. The results are shown in Figure 17.

[0203] Example 26: Distribution of SN-38 lactone in the brain and cerebrospinal fluid of mice following intravenous administration of G2B-002-20-9 and irinotecan We evaluated the distribution of SN-38 lactone in the brain and cerebrospinal fluid 1 hour after intravenous administration of G2B-002-20-9 at a dose sufficient to provide 10 mg / kg of free SN-38 lactone, or 20 mg / kg of irinotecan. We administered G2B-002-20-9 at a dose sufficient to provide 10 mg / kg of free SN-38 lactone intravenously to four athymic nude mice, and 40 mg / kg of irinotecan intravenously to another four mice. After 1 hour, the mice were anesthetized and 5 μL of CSF was obtained using a glass capillary. The animals were then sacrificed by decapitation, the brains dissected, and the samples were frozen and stored until analysis using published methods. In the brain, SN-38 lactone was not detectable 1 hour after administration of 40 mg / kg of irinotecan, i.e., the concentration was below the limit of quantification of 5 ng / g. After administration of G2B-002-20-9, the mean concentration of SN-38 in the brain was 33 ng / g (range 13-60 ng / g). In the CSF, SN-38 lactone achieved a mean concentration of 0.5 ng / mL (range 0.2-0.8 ng / mL) after administration of 40 mg / kg irinotecan. After administration of G2B-002-20-9, SN-38 lactone achieved a median concentration of 61 ng / mL (range 33-97 ng / mL). Taken together, these experiments show that the novel invention improves CNS distribution of soluble SN-38 lactone. In the CSF, the increase in SN-38 lactone distribution is approximately 100-fold.

[0204] Citation List Patent Literature - European Patent No. 2644191 - U.S. Patent No. 8,299,089 - International Patent Publication No. 2015 / 051307 - China Patent Publication No. 10206099 - China Patent Publication No. 110124052 Non-patent literature - Sosnik, Drug self-assembly: A phenomenon at the nanometer scale with major impact in the structure-biological properties relationship and the treatment of disease, Prog Mater Sci, 2016, vol. 82, pp.39-82 - F Meyer-Losic et al in ”DTS-108, A novel Peptidic prodrug of SN-38: In vivo Efficacy and Toxicokinetic Studies”, Clinical Cancer Research, 2008, vol.14, issue 7, pp. 2145-2153 - A. Buckin et al., in ”Amphiphilic Polymeric Nanoparticles Modified with a Protease-Resistant Peptide Shuttle for the Delivery of SN-38 in Diffuse Intrinsic Pontine Glioma” ACS Applied Nano Materials, 2021, vol. 4, issue 2, pp.1314-1329 - Ri, Masaki et al.; in ”A phase I / II study for dose-finding, and to investigate the safety, pharmacokinetics and preliminary efficacy of NK012, an SN-38-incorporating macromolecular polymeric micelle, in patients with multiple myeloma ”Internal Medicine (Tokyo, Japan) (2018), vol. 57(7), pp. 939-946 - Lei, Fan et al:, Nanoscale platform for delivery of active IRINOX to combat pancreatic cancer Journal of Controlled Release (2021), vol. 330, pp. 1229-1243 - Zhang et al., ”Development and characterization of a novel liposome -based formulation of SN-38” International Journal of Pharmaceutics, 2004, vol. 270, pp. 93-107 - M. J. Hatfield and P. M. Potter, Exp. Opin. Ther. Pat. 2011, vol. 21(8), pp. 1159-1171 - A. Madder et al., Eur. J. Org. Chem,. 1999, pp. 2787-2791 - Barker et al. J. Med. Chem., 1992, vol. 35, pp. 2040-2048. - Greene’s Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc. pp.26-29, 271-279, 456-463, 69-77, 371-374 - M. Kazemi & L. Shiri, Journal of Sulfur Chemistry, 2015, vol. 36:6, pp.613-623. - M. T. Nguyen et al., J. Org. Chem. 1998, 63, vol. 20, pp. 6878-6885. - F. David et al., Org. Process Res. Dev. 2010, vol. 14, 4, pp. 999-1007. - S.F.M. van Dongen et al.; Bioconjugate Chem., 2009, vol. 20, pp. 20-23. - J.H. Lee et al. Eur. J. 30 Biochem., 2001, vol. 268. pp. 2004-2012 - M. Amblard, et al., ”Methods and protocols of modern solid-phase peptide synthesis. Molecular Biotechnology 2006, vol. 33, pp. 239-254 - J. Med. Chem. 2008, 51, 21,6916-6926.

Claims

1. A micelle comprising an SN-38 peptide conjugate and one or more free therapeutic agents having anticancer activity, The micelle has a core-shell structure including an inner core and an outer shell, the free therapeutic activator is filled in the inner core, and the SN-38 peptide conjugate forms the outer shell. The SN-38 peptide conjugate is a compound of formula (I) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 Z is a radical of the pharmaceutically active ingredient SN-38 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically active ingredient SN-38 has formula (II), and Z is independently bonded to linker L by only one of the two hydroxyl groups (a) or (b) of the pharmaceutically active ingredient. 【Chemistry 2】 L is a biradical composed of 2 to 8 biradicals L', and its formula is: -L' ａ - (L' ｂ ) ｎ -L' ｃ - is a linker having L ａ 'が、-C(=O)-(CH ２ ) ｒ -C(=O)-;-C(=O)-(CH ２ ) ｒ -NH-;-C(=O)-(CH ２ ) ｒ -S-;-C(=O)-(CH ２ ) ｒ -O-;-C(=O)-NH-(CH ２ ) ｒ -C(=O)-;-C(=O)-NH-(CH ２ ) ｒ -NH-;-C(=O)-NH-(CH ２ ) ｒ -S-;-C(=O)-NH-(CH ２ ) ｒ -O-;-(CH ２ ) ｒ -C(=O)-;-(CH ２ ) ｒ -NH-;-(CH ２ ) ｒ -S-;-(CH ２ ) ｒ -O-;-Si(R １ )(R ２ )-(CH ２ ) ｒ -NH-;-Si(R １ )(R ２ )-(CH ２ ) ｒ -C(=O)-;-Si(R １ )(R ２ )-(CH ２ ) ｒ -O-;-Si(R １ )(R ２ )-(CH ２ ) ｒ -S-;-SO ２ -(CH ２ ) ｒ -NH-;-SO ２ -(CH ２ ) ｒ -C(=O)-;-SO ２ -(CH ２ ) ｒ -O-;-SO ２ -(CH ２ ) ｒ -S-;-P(=O)(OR １ )-O-(CH ２ ) ｒ -NH-;-P(=O)(OR １ )-O-(CH ２ ) ｒ -C(=O)-;-P(=O)(OR １ )-O-(CH ２ ) ｒ -O-;-P(=O)(OR １ )-O-(CH ２ ) ｒ -S-;-CH(OH)-(CH ２ ) ｒ -NH-;-CH(OH)-(CH ２ ) ｒ -C(=O)-;-CH(OH)-(CH ２ ) ｒ -O-;-CH(OH)-(CH ２ ) ｒ -S-; 【Transformation 3】 A biradical selected from the group consisting of, L ８ -L １１ A substituent in any of the rings 【Chemistry 4】 It could be in any of the following positions, L ｂ ’ is independently, -NH-(CH ２ ) ｒ -C(=O)-; C(=O)-(CH ２ ) ｒ -C(=O)-; -S-(CH ２ ) ｒ -C(=O)-; -O-(CH ２ ) ｒ -C(=O)-; -NH-(CH ２ ) ｒ -; -C(=O)-(CH ２ ) ｒ -; -S-(CH ２ ) ｒ -; -O-(CH ２ ) ｒ -; -NH-CH-((CH ２ ) ｒ NH ２ )-C(=O)-; -S-CH ２ -CH(NH ２ )-C(=O)-; -(CH ２ ) ｒ -C(=O)-; -(CH ２ ) ｒ -O-; -(CH ２ ) ｒ -NH-; -(CH ２ ) ｒ -S-; -C(=O)-(CH ２ ) ｒ -NH-; -C(=O)-(CH ２ ) ｒ -O-; -C(=O)-(CH ２ ) ｒ -S-; -NH-(CH ２ ) ｒ -O-; -NH-(CH ２ ) ｒ -NH-; -NH-(CH ２ ) ｒ -S-, and combinations thereof; 【Transformation 5】 A biradical selected from the group consisting of, L ｃ 'が、:-NH-(CH ２ ) ｒ -C(=O)-;-NHH-CH-((CH ２ ) ｒ NH ２ )-C(=O)-;-C(=O)-(CH ２ ) ｒ -C(=O)-;-S-(CH ２ ) ｒ -C(=O)-;-S-CH ２ -CH(NH) ２ )-C(=O)-;-O-(CH ２ ) ｒ -C(=O)-、-(CH ２ ) ｒ -C(=O)-; 【Transformation 6】 A biradical selected from the group consisting of, P, (a) X is an amide bond １ and X ２ Amino acid sequence X having an intrapeptide bond between them １ KAPETALX ２ (X １ X is selected from the group consisting of Dap and Dab. ２ This refers to a peptide containing D (aspartic acid) and E (glutamic acid), i.e. 【Transformation 7】 (b) Having at least one peptide internal bond which is a disulfide bond or a diselenide bond, X ３ KAPETALX ４ It contains an amino acid sequence that is AAA, X ３ and X ４ (X ３ and X ４ A peptide having a length of 12 to 20 amino acid residues, wherein each of these is equally selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), and has at least one intrapeptide disulfide bond or diselenide bond between them; 【Transformation 8】 (c) Having at least one peptide internal bond which is a disulfide bond or a diselenide bond, X ５ KAPETALX ６ ;X ５ KAPETALX ６ A; and X ５ KAPETALX ６ It consists of an amino acid sequence selected from the group consisting of AA, X ５ and X ６ (X ５ and X ６ Peptides having a length of 9 to 11 amino acid residues, wherein each of these is equally selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), and each of these has at least one intrapeptide disulfide bond or diselenide bond between them, i.e. 【Chemistry 9】 (d) Having 16 amino acid residues, X ７ and X ９ Between and X ８ and X １０ Between (X ７ ~X １０ It is independently selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), except X ７ and X ９ are equal to X ８ ~X １０ (is equal to) an amino acid sequence X having an intrapeptide disulfide bond or diselenide bond. ７ NX ８ KAPETALX ９ AAAX １０ H-containing peptides, i.e. 【Chemistry 10】 Furthermore (e) Amino acid sequence X １ KAPETALX ２ (X １ X is selected from the group consisting of Dap and Dab. ２ (Selected from the group consisting of D (aspartic acid) and E (glutamic acid)), the peptide is a linear peptide (SEQ ID NO: 7) A peptide biradical selected from the group consisting of, W is -NH-(CH ２ ) ｒ -C(=O)-, and -NH-CH((CH ２ ) ｒ NH ２ It is a biradical selected from the group consisting of )-C(=O)-, Y is -NH ２ -OH, -OR ３ , and -NHR ３ A radical selected from the group consisting of, s is an integer independently selected from 0 to 1, n is an integer from 0 to 6, r is an integer independently selected from 1 to 5. k is an integer between 5 and 8. R １ and R ２ However, independently, (C １ -C ６ ) - Selected from alkyl groups, R ３ However, (C １ -C ６ A radical selected from the group consisting of )-alkyl groups, L ａ ' is bonded to radical Z via a bond selected from the group consisting of ester, ether, urethane, silyl ether, sulfonate, phosphate, ketal, hemiketal, carbonate, and carbamate bonds, wherein the bond is C=O,SO on the left side of the formula 'drawn La'. ２ Si, P, CH, or CH ２ It is formed between the group of and one of the hydroxyl groups of SN-38, If n = 0, L ａ ' is formed by a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters, which forms a radical L ｃ It is coupled to ', and the coupling is drawn L ａ The functional group on the right side of the formula and L ｃ Formed between the functional group on the left side of the formula, If n=1, L ａ ' is formed by a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfide esters, and thioesters, which forms a radical L ｂ It is coupled to ', and the coupling is drawn L ａ The functional group on the right side of the formula and L ｂ Formed between the functional group on the left side of the formula, L ｂ ' is formed by a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters, which forms a radical L ｃ It is coupled to ', and the coupling is drawn L ｂ The functional group on the right side of the formula and drawing L ｃ Formed between the functional group on the left side of the formula, If n is greater than 1, L ｂ ' are equal or different, and are bonded to each other via chemically feasible bonds selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters, L ｂ One end of ' is connected to L via a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters. ａ It is bonded to the functional group on the right side of the expression La' and L ｂ Formed between the functional group on the left side of the formula and another L ｂ The terminal is L via a chemically feasible bond selected from the group consisting of amines, amides, ethers, thioethers, disulfides, esters, and thioesters. ｃ It is coupled to ', and the coupling is drawn L ｂ The functional group on the right side of the formula and drawing L ｃ Formed between the functional group on the left side of the formula, L ｃ 'but, drawing L ｃ The biradical P is bound via an amide bond formed by the carbonyl group on the right side of the formula and the amino group of the first amino acid in peptide sequence P. When s = 0, P is directly bonded to Y via an amide, carboxylic acid, or ester bond, and the bond is between the C=O at the C-terminus of the last amino acid of sequence P and -NH ２ -OH, -OR ３ , or -NHR ３ It is formed between the radical Y, When s = 1, P is bonded to radical W via an amide bond formed at the C=O at the C-terminus of the last amino acid of sequence P, and this bond is formed between the functional group on the left side of the equation of drawn W and the functional group (C=O) at the C-terminus of the last amino acid of sequence P on the right side of the drawn sequence, and W is as follows: -C(=O)-NH-(CH ２ ) ｒ -C(=O)-Y, or -C(=O)-NH-CH((CH ２ ) ｒ NH ２ ) - C (= O) - Y, Micelle.

2. The micelle according to claim 1, wherein the drug in the internal core is SN-38 lactone.

3. In the SN-38 peptide conjugate of formula (I), P is (a) Sequence ID 8: 【Chemistry 11】 A peptide comprising the amino acid sequence DapKAPETALD, which has an intrapeptide bond between Dap and D, which is an amide bond. (b) Sequence ID 9: 【Chemistry 12】 A peptide having a length of 9 to 20 amino acid residues, comprising the amino acid sequence CKAPETALCAAAA, which has at least one intrapeptide bond that is a disulfide bond, and at least one intrapeptide disulfide bond between cysteine ​​1 and 9, (c) 【Chemistry 13】 A peptide having a length of 9 to 11 amino acid residues, comprising an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA, having at least one intrapeptide bond which is a disulfide bond, and having at least one intrapeptide disulfide bond between cysteine ​​1 and 9, and (d) 【Chemistry 14】 A peptide comprising the amino acid sequence CNCKAPETALCAAAACH, having 16 amino acid residues, with intrapeptide disulfide bonds between the first cysteine ​​(cysteine ​​1 and 11) and the third cysteine, and between the second cysteine ​​(cysteine ​​3 and 15) and the fourth cysteine, (e) Peptide containing the amino acid sequence DapKAPETALD (SEQ ID NO: 14) The micelle according to claim 1, which is a peptide biradical selected from the group consisting of the following.

4. In the SN-38 peptide conjugate of formula (I), P is (a) A peptide having the amino acid sequence DapKAPETALD, which involves an intrapeptide bond between Dap and D, which is an amide bond (SEQ ID NO: 8); (b) A peptide having the amino acid sequence CKAPETALC having at least one intrapeptide disulfide bond between the cysteine ​​at positions 1 and 9 (SEQ ID NO: 10), and (c) Peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 14) The micelle according to claim 3, which is a peptide biradical selected from the group consisting of the following.

5. The micelle according to claim 4, wherein in the peptide conjugate of formula (I) SN-38, P is a biradical of peptide DapKAPETALD (SEQ ID NO: 8) having an intrapeptide bond between Dap and D, where P is an amide bond.

6. In the peptide conjugate of SN-38 of formula (I), L ａ 'But, -C(=O)-(CH ２ ) ｒ -C(=O)-, -C(=O)-(CH ２ ) ｒ -NH-, -C(=O)-(CH ２ ) ｒ -S-;-C(=O)-(CH ２ ) ｒ -O-;-C(=O)-NH-(CH ２ ) ｒ -C(=O)-;L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ７ , and L １２ A micelle according to claim 1, which is a biradical selected from the group consisting of the following.

7. The micelle according to claim 1, wherein L is a linker selected from the group consisting of the following: a) L ａ 'However, L in the following equation ３ And L ｂ 'But, -NH-(CH ２ ) ｒ -O-, -(CH ２ ) ｒ -O-; and -(CH ２ ) ｒ Selected from the group consisting of -NH- and combinations thereof, L ｃ 'But, -C(=O)-(CH ２ ) ｒ -C (=O)-, 【Chemistry 15】 b) A biradical composed of two biradicals, n=0, L ａ ' is L １２ And L ｃ ' is L １３ And, c) A biradical composed of two biradicals, n=0, L ａ ' is -C(=O)-NH-(CH ２ ) ｒ -C (=O)- and L ｃ ' is L １５ That is the case.

8. The peptide conjugate of SN-38 in formula (I) 【Chemistry 16】 【change】 The micelle according to claim 7, which is a compound selected from the group consisting of the above.

9. The micelle according to claim 1, wherein the therapeutic anticancer agent is the same or different, and is in the form of an aqueous dispersion of the micelles as defined in claim 1.

10. The micelle according to claim 9, wherein the concentration of the SN-38 peptide conjugate in the aqueous dispersion of micelles is up to 50 mg / ml, the free therapeutic anticancer agent is SN-38, and the concentration of free SN-38 is up to 25 mg / ml.

11. a) Micelles of the peptide conjugate of formula (Ia), in which the concentration of the SN-38 peptide conjugate in an aqueous dispersion of micelles is 20 mg / ml and the concentration of the SN-38 lactone is 4 mg / ml, with the internal core filled with SN-38 lactone; b) Micelles of the peptide conjugate of formula (Ia) with an internal core filled with SN-38 lactone, wherein the concentration of the SN-38 peptide conjugate in the aqueous dispersion of micelles is 20 mg / ml and the concentration of SN-38 lactone is 2 mg / ml; c) Micelles of the peptide conjugate of formula (Ia), in which the concentration of the SN-38 peptide conjugate in an aqueous dispersion of micelles is 10 mg / ml and the concentration of the SN-38 lactone is 1 mg / ml, with the internal core filled with SN-38 lactone; d) Micelles of the peptide conjugate of formula (Ic) with an internal core filled with SN-38 lactone, wherein the concentration of the SN-38 peptide conjugate in the aqueous dispersion of micelles is 20 mg / ml and the concentration of SN-38 lactone is 2 mg / ml; e) Micelles of the peptide conjugate of formula (Ia) with an internal core filled with camptothesin lactone, wherein the concentration of the SN-38 peptide conjugate in the aqueous dispersion of micelles is 20 mg / ml and the concentration of camptothesin lactone is 0.25–4 mg / ml; and f) Micelles of the peptide conjugate of formula (Ia), in which the concentration of the SN-38 peptide conjugate in the aqueous dispersion of micelles is 20 mg / ml, the concentration of camptothesin lactone is 0.5–1 mg / ml, and the concentration of SN-38 is 0.5–1 mg / ml, with the internal core filled with camptothesin lactone and SN-38. A micelle according to claim 1, selected from the group consisting of the following.

12. The micelle according to claim 1, which is a freeze-dried micelle.

13. a) Spontaneous self-assembly of the SN-38 peptide conjugate as defined in any of claims 1 to 12 in water with pH < 7; b) Contacting a basic solution containing free SN-38 carboxylate at a concentration of 1-12 mg / ml with an acidic solution of micelles to form intermolecular interactions between free SN-38 lactone molecules and SN-38 molecules conjugated to SN-38 peptide conjugates, and c) Optionally, freeze-dry the micelle solution from step b). The micelle according to claim 1, which can be obtained by the means described herein.

14. A pharmaceutical composition comprising a therapeutically effective amount of the micelles described in claim 1, together with an appropriate amount of pharmaceutically acceptable carrier or excipient.

15. A method for treating cancer, comprising the step of administering the micelles described in claim 1 to a mammal, including a human, that requires them, The aforementioned cancer, a) Tumors selected from the group consisting of extracranial solid tumors, ocular tumors, and CNS tumors; b) Cancers selected from the group consisting of adult glioma, pediatric glioma, retinoblastoma, Ewing's sarcoma, DIPG, neuroblastoma, medulloblastoma, ependymoma, atypical teratoma rhabdoid tumor (ATRT), and rhabdomyosarcoma; c) Pediatric brain tumors; d) Pediatric high-grade glioma; e) Pediatric brainstem glioma (DIPG) tumors; and f) Diffuse median glioma A method selected from the group consisting of the following.

16. In the SN-38 peptide conjugate of formula (I), P is (a) Sequence ID 8: 【Chemistry 17】 A peptide comprising the amino acid sequence DapKAPETALD, which has an intrapeptide bond between Dap and D, which is an amide bond. (b) Sequence ID 9: [Chemistry 18] A peptide having a length of 9 to 20 amino acid residues, comprising an amino acid sequence CKAPETALCAAAA having at least one peptide bond which is a disulfide bond, and at least one intrapeptide disulfide bond between cysteine ​​1 and 9, (c) 【Chemistry 19】 A peptide having a length of 9 to 11 amino acid residues, comprising an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA, having at least one intrapeptide bond which is a disulfide bond, and having at least one intrapeptide disulfide bond between cysteine ​​1 and 9, and (d) 【Chemistry 20】 A peptide comprising the amino acid sequence CNCKAPETALCAAAACH, having 16 amino acid residues, with intrapeptide disulfide bonds between the first cysteine ​​(cysteine ​​1 and 11) and the third cysteine, and between the second cysteine ​​(cysteine ​​3 and 15) and the fourth cysteine, (e) Peptide containing the amino acid sequence DapKAPETALD (SEQ ID NO: 14) The micelle according to claim 2, which is a peptide biradical selected from the group consisting of the following.

17. The micelles according to claim 8, wherein the free therapeutic anticancer agents are the same or different, and are in the form of an aqueous dispersion of micelles as defined in claim 1.

18. The micelle according to claim 10, which is freeze-dried.

19. A pharmaceutical composition comprising a therapeutically effective amount of the micelles according to claim 13, together with an appropriate amount of pharmaceutically acceptable carrier or excipient.

Images