Peptide water-soluble delivery system for anti-cancer drugs

Abstract

The present invention relates to micelles of SN-38 peptide conjugates loaded with one or more free therapeutically active agents, such as free SN-38 lactones, having anticancer activity, methods of preparing the micelles, pharmaceutical compositions comprising the micelles, and therapeutic indications of the micelles as anticancer drugs.

Description

[0001] This application claims the benefit of European patent application EP22382287 filed on March 28, 2022. Technical Field

[0002] The present invention relates to the field of delivery systems including anticancer drugs, methods for their preparation and therapeutic indications thereof. Background Art

[0003] Camptothecins 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 stability at acidic pH. The lactone ring opens to carboxylation at neutral or alkaline pH. This conversion is non-enzymatic and reversibly converts to the lactone upon pH acidification. The carboxylates are 100-1000 times less potent than the lactones.

[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 and is formed by hydrolysis of irinotecan (its water-soluble prodrug) by liver carboxylesterase and metabolized by glucuronidation by UGT1A1. It has the following formula.

[0005]

[0006] SN-38 is 1000-fold more active than irinotecan itself and can be used to treat the same types of cancer as irinotecan. In vitro cytotoxicity assays have shown that the potency of SN-38 relative to irinotecan varies between 2-fold and 2000-fold.

[0007] SN-38 is quite restrictive at the chemical, pharmacological and toxicity levels. At the chemical level, unlike irinotecan, which is soluble in water at acidic pH, SN-38 is almost insoluble in water at acidic or neutral pH and in most solvents and oils, so it is not feasible to administer SN-38 to patients to maintain the active (lactone) form of the molecule. Many solvents have been tested, and only dimethyl sulfoxide, formic acid and HP and 0.5% SN-38 was dissolved with NaOH 0.1M, but the alkaline pH of this NaOH 0.1M aqueous solution opened the lactone ring and thus inactivated SN-38. Therefore, the high lipophilicity of SN-38 hinders the administration of this drug to humans in a clinically acceptable vehicle. At neutral or alkaline pH, the equilibrium shifts to the less active species due to the opening of the lactone ring, while at more acidic pH, the formation of lactones with greater cancer inhibitory power is favored. The drug also includes an asymmetric carbon at position 20, wherein the S form is the pharmacologically active configuration.

[0008] In summary, the two forms of SN-38 are in equilibrium in water, namely, the lipophilic lactone (SN-38 lactone, which has strong anticancer activity) and the hydrophilic carboxylate (SN-38 carboxylate, which has no anticancer activity). SN-38 lactone is mainly in acidic pH conditions, and it is almost 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), while SN-38 carboxylate is mainly in neutral pH and alkaline pH, and it is easily soluble in water. Both SN-38 forms are in equilibrium, and when a solution of SN-38 carboxylate (basic pH) is poured into an acidic pH solution, the carboxylate form is converted to the SN-38 lactone, which precipitates and forms thorny crystals even in the presence of nonionic surfactants such as This is also the case in the presence of F68 (see EP2644191B1). This precipitation process is associated with strong intermolecular interactions between free SN-38 lactone molecules.

[0009] Like many other lipophilic small molecule drugs, camptothecin self-assembles in water and forms 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).

[0010] In view of the unfavorable characteristics of the drug, several studies have led to chemical conjugation of SN-38 with solubilizing chemical groups to produce SN-38 prodrugs. Several conjugation strategies have been applied to SN-38 to release the drug instead of using free drug SN-38.

[0011] Some of these strategies are based on releasing SN-38 from soluble conjugates. For example, US8299089B2 proposes the use of multi-arm PEG conjugated to SN-38 through appropriate linkers to improve solubility and release SN-38 due to ester hydrolysis. However, these conjugates must be administered at high concentrations in order to be effective in vitro / in vivo.

[0012] Likewise, WO2015 / 051307A1 discloses several conjugates that release SN-38 from 4-arm polyethylene glycol at a slow rate via a β-elimination reaction mechanism, thereby enabling low-dose and long-term exposure regimens. Other conjugates are based on, for example, the use of peptide conjugates to bypass liver activation and reduce gastrointestinal toxicity and inter-patient variability compared to irinotecan. Thus, for example, 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, No. 7, pp. 2145-2153, proposed conjugating SN-38 to a cationic peptide (Vectocell) via an esterase-cleavable linker to deliver significantly higher levels of SN-38 than irinotecan without irinotecan-related toxicity, thereby increasing the therapeutic window of DTS-108 in preclinical models.

[0013] Other strategies for dissolving free SN-38 lactone in aqueous solution are also disclosed in the prior art, and the other strategies cover the use of amphiphilic polymers, which can self-assemble into core-shell structures due to aggregation of hydrophobic parts when in an aqueous environment. EP3753966A1 discloses an amphiphilic block copolymer, which includes a hydrophilic segment, a hydrophobic segment, and a linker for connecting the hydrophilic segment to the hydrophobic segment. The linker contains an unsaturated structure to enhance the interaction between the poorly soluble drug (SN-38) and the copolymer.

[0014] Also disclosed are polymer structures that can self-assemble into micelles that include encapsulated SN-38 in their structures. For example, 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(2), pp. 1314-1329, disclose amphiphilic chitosan (CS)-g-poly(methyl methacrylate)-poly(acrylic acid) copolymers loaded with SN-38 polymer nanoparticles that are surface-modified with peptide shuttles to improve transport across the blood-brain barrier (BBB).

[0015] CN102060991A1 describes an amphiphilic prodrug of 7-ethyl-10-hydroxycamptothecin, the OH at position 10 or position 20 of which is linked to a hydrophilic group such as PEG-200-2000 and which can form micelles.

[0016] 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, disclosed polymer micelles that release SN-38, which are constructed by covalently linking SN-38 to a block copolymer PEG-pGlu and then self-assembling the amphiphilic block copolymer in an aqueous medium.

[0017] CN110124052A discloses a conjugate of E-selectin peptide ligand coupled with polyethylene glycol monomethyl ether and an anti-tumor drug such as camptothecin, hydroxycamptothecin, SN-38, paclitaxel, docetaxel, dasatinib, gemcitabine, doxorubicin or podophyllotoxin, which can self-assemble into nanoparticles in aqueous solution.

[0018] Finally, Lei, Fan et al. disclosed cross-linked micelles in "Nanoscale platform for delivery of active IRINOX to combat pancreatic cancer", Journal of Controlled Release, 2021, Vol. 330, pp. 1229-1243, which were prepared using an amphiphilic PEG-b-poly(L-glutamic acid) / SN-38 conjugate and subsequently loaded with dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt).

[0019] Despite the efforts made on the delivery systems of SN-38 to allow for adequate administration and bioavailability of SN-38 for the treatment of cancer, there is still an unmet medical need in finding improved systems for the effective administration of SN-38, especially considering that neither irinotecan nor SN-38 can cross the BBB in significant amounts, and therefore, they are not effective against brain tumors with an intact blood-brain barrier (BBB), such as diffuse intrinsic pontine glioma (DIPG) and high-grade glioma (HGG), including its pediatric form (pHGG). Summary of the invention

[0020] The present inventors have discovered that certain SN-38 peptide conjugates form micelles by spontaneous self-assembly in aqueous media and are capable of loading free drugs into their cores.

[0021] These micelles are particularly advantageous when loaded with SN-38 lactone and / or another anticancer drug. Unexpectedly, by forming micelles with these SN-38 peptide conjugates and free SN-38 lactone, the apparent solubility of free SN-38 lactone in water at acidic pH was increased by at least 500 times, which is much higher than the prior art.

[0022] The SN-38 lactone form is extremely insoluble in water at acidic pH. However, an acidic pH is required to preserve SN-38 in its lactone (active) form, which is reversibly and pH-dependently converted to SN-38 carboxylate (an inactive form that is readily soluble in water due to the opening of the lactone ring at neutral or alkaline pH). The intrinsic water solubility of the SN-38 lactone at pH 3 was determined to be 8 μg / mL (own data). The reported solubility of SN-38 in water, pH 3.5 buffer, and pH 7.4 buffer is 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).

[0023] In contrast, 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 times, while the SN-38 liposomes disclosed by Zhang et al. achieve an apparent solubility of 0.111 mg / mL of SN-38. This is achieved without the need for pharmaceutical excipients or lipid components, and the solubility of the SN-38 liposomes exceeds the best liposome formulation of SN-38 reported in the prior art by more than 40 times.

[0024] Since SN-38 lactone is insoluble, in clinical practice, SN-38 is administered like irinotecan (its water-soluble prodrug), which is approved for several cancer indications. After administration in the bloodstream, irinotecan releases SN-38 lactone as its active metabolite due to the action of carboxylesterase. However, due to the limited expression of these enzymes, the conversion rate of irinotecan to SN-38 is low in humans. This would explain why irinotecan is very effective in animal models rich in carboxylesterase (mice) and less effective in humans (low carboxylesterase). Advantageously, the present invention is superior in terms of the need for carboxylesterase because it carries free SN-38 lactone.

[0025] Finally, unlike irinotecan and SN-38, the micelles of the present invention cross the blood-brain barrier (BBB) ​​and achieve therapeutic concentrations of SN-38 in the brain and cerebrospinal fluid (CSF), thereby causing therapeutic activity in DIPG and pHGG xenografts. Thus, a water-soluble brain-permeable delivery system for anticancer drugs that can be used to treat brain cancer is provided.

[0026] SN-38 peptide conjugates are formed from some peptides disclosed in WO2015 / 001015A1 and specific linkers of a specific size larger than the linkers disclosed in the referenced literature. These conjugates themselves provide in vitro antitumor activity that is up to 100 times better than the in vitro activity of irinotecan. They have shown high water solubility and their own activity is much higher than the in vitro activity of irinotecan. The SN-38-loaded micelles of the present invention show antitumor activity against several cell lines from brain and extracranial cancers and are more active than the peptide conjugates alone.

[0027] The micelles are prepared by spontaneous micellization by dissolving the SN-38 peptide conjugate in water at a pH <7, preferably <3. Such micellar acidic solutions can neutralize alkaline solutions containing free SN-38 carboxylate at a concentration of up to 25 mg / mL. After acidification at alkaline pH, the precipitation of free SN-38 lactone due to the pH change causes intermolecular interactions between free SN-38 lactone molecules and conjugated SN-38 molecules in the SN-38 peptide conjugate. Micellar dispersions of SN-38 peptide conjugates containing free SN-38 lactone can be easily filtered through 0.22 μm and 0.45 μm pore filters without significant loss of free SN-38 lactone. The final system consists of a drug-drug co-crystal consisting of two forms of SN-38 lactone, namely the free form and the conjugated form. Under these conditions, the free SN-38 lactone did not form crystallites or larger solid structures and remained significantly soluble at concentrations as high as 4 mg / ml.

[0028] These micellar dispersions of SN-38-binding peptide conjugates containing free SN-38 lactone are nanometer-sized. They can be used to load free SN-38 into the micelle core. Thus, for example, as shown in Example 15, in this example, the loading was increased from the current 21.5% SN-38 loading (w / w) (already included in the conjugated product) to 35% w / w SN-38 in the final micelle product, which includes free SN-38 lactone loaded in the micelle. The size of this micelle is about 40 nm at a concentration of 11 mM (20 mg / mL) in the absence of free SN-38 lactone, and about 100 nm after encapsulation of free SN-38 lactone, which is attributed to the effect of loading the micelle with free SN-38 lactone.

[0029] Thus, a first aspect of the present invention relates to micelles comprising a SN-38 peptide conjugate and one or more free therapeutically active agents having anticancer activity, wherein: the micelle is a core-shell structure comprising an inner core and an outer shell, wherein the free therapeutically active agent is loaded 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,

[0030] (Z)-(L)-P-(W) s –(Y)

[0031] (I)

[0032] Wherein: Z is a free 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 connected to the linker L through only one of the two hydroxyl groups (a) or (b) of the pharmaceutically active ingredient;

[0033]

[0034] L is a linker, which is a diradical composed of 2 to 8 diradicals L' and has the following formula: -L' a -(L' b ) n -L' c ;

[0035] L a ' is a diradical selected from the group consisting of: -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-;

[0036]

[0037]

[0038] L8-L 11 The substitution in any of may be at any position of the ring;

[0039]

[0040] L b ' is a diradical independently selected from the group consisting of: -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 -S-; L1; L2; L3; L4; and combinations thereof;

[0041] L c ' is a diradical selected from the group consisting of: -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;

[0042]

[0043]

[0044] P is a diradical of a peptide selected from the group consisting of: (a) a peptide comprising an amino acid sequence X1KAPETALX2, wherein the amino acid sequence has an intrapeptide bond between X1 and X2, wherein the intrapeptide bond is an amide bond; wherein X1 is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutyric acid); and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid); i.e.

[0045]

[0046] For the amino acid Dap, the codes Dap and Dpr are used equivalently herein;

[0047] (b) a peptide having a length of 12-20 amino acid residues, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond or a diselenide bond, and the peptide comprising the following amino acid sequence: X3KAPETALX4AAA; the amino acid sequence having at least one intrapeptide disulfide bond or a diselenide bond between X3 and X4, wherein X3 and X4 are identical and are selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine); i.e.

[0048]

[0049] (c) a peptide of 9-11 amino acid residues in length, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond or a diselenide bond, and the peptide consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6, X5KAPETALX6A and X5KAPETALX6AA, the amino acid sequence having at least one intrapeptide disulfide bond or a diselenide bond between X5 and X6; wherein X5 and X6 are identical and selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine); i.e.

[0050]

[0051] (d) having 16 amino acid residues and comprising the amino acid sequence X7NX8KAPETALX9AAAX 10 H peptide, the amino acid sequence is between X7 and X9 and between X8 and X 10 There is an intrapeptide disulfide bond or a diselenide bond between them; wherein X7-X 10 independently selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine); provided that X7 and X9 are identical and X8-X 10 are equivalent; that is

[0052] and (e) a peptide comprising an amino acid sequence X1KAPETALX2 (SEQ ID NO: 7), wherein 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), the peptide being a linear peptide;

[0053] W is a diradical selected from the group consisting of: -NH-(CH2) r -C(=O)- and -NH-CH((CH2) rNH2)-C(=O)-; Y is a free 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; and R3 is a free radical selected from the group consisting of (C1-C6)-alkyl;

[0054] L a ' is connected to the free radical Z via a bond selected from the group consisting of an ester bond, an ether bond, a urethane bond, a silyl ether bond, a sulfonate bond, a phosphate bond, a ketal bond, a hemiketal bond, a carbonate bond and a urethane bond, the bond being in the L drawn a 'The C=O, SO2, Si, P, CH or CH2 group on the left side of the formula is formed with one of the hydroxyl groups of the SN-38;

[0055] When n = 0, L a ' connected to the free radical L through a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond c ', the key is drawn in the L a The functional group on the right side of the formula is c 'Formed between the functional groups on the left side of the formula;

[0056] When n = 1, L a ' connected to the free radical L through a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond b ', the key is drawn in the L a The functional group on the right side of the formula is b ' is formed between the functional groups on the left side of the formula; and L b ' connected to the free radical L through a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond c ', the key is drawn in the L b The functional groups on the right side of the formula are similar to the L c 'Formed between the functional groups on the left side of the formula;

[0057] When n is greater than 1, L b ' are identical or different and are connected by a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond; one L b 'Connected to L at the terminal end via a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond, and a thioester bonda ', the key is drawn in the L a The functional groups on the right side of the formula are similar to the L b 'formed between the functional groups on the left side of the formula; and another L b 'Connected to L at the terminal end via a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond, and a thioester bond c ', the key is drawn in the L b The functional groups on the right side of the formula are similar to the L c 'Formed between the functional groups on the left side of the formula;

[0058] L c 'By using the L drawn c 'The carbonyl group on the right side of the formula and the amino group of the first amino acid of the peptide sequence P form an amide bond to connect to the diradical P;

[0059] When s=0, P is directly linked to Y via an amide bond, a carboxylic acid bond or an ester bond, the bond being formed between the C=O at the C-terminus of the last amino acid of the sequence P and the free radical Y, the free radical Y being -NH2, -OH, -OR3 or -NHR3; and

[0060] When s=1, P is linked to the free radical W via an amide bond formed with the C=O at the C-terminus of the last amino acid of the sequence P, the bond being formed between the functional group on the left side of the drawn formula of W and the functional group (C=O) at the C-terminus of the last amino acid of the sequence P on the right side of the drawn sequence; and W is linked to Y as follows: -C(=O)-NH-(CH2) r -C(=O)-Y or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y.

[0061] A line between two amino acids of a sequence above or below represents an intrapeptide bond between the side chains of the two amino acids. In a specific embodiment, a line between two amino acids of a sequence above or below represents an intrapeptide bond between the side chains of the two amino acids.

[0062] A second aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of micelles as defined above and an appropriate amount of a pharmaceutically acceptable carrier or excipient.

[0063] A third aspect of the invention relates to the use of a micelle as defined above as a medicament.

[0064] A fourth aspect of the invention relates to the use of a micelle as defined above for treating cancer in a mammal, including a human.

[0065] A fifth aspect of the present invention relates to a micelle as defined above for use in the treatment of cancer, wherein the compound of formula (I) is used in combination therapy in combination with a chemotherapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Schematic diagram showing the manufacturing process of the novel water-soluble SN-38 lactone micelle product.

[0067] Figure 2 The photographs were taken immediately after preparing the product G2B-002-20-9 or a product consisting of only vehicle or 20 mg / mL irinotecan in vehicle in the absence of G2B-001 micelles containing 2 mg / mL loading of free SN-38 lactone. The product G2B-002-20-9 was not turbid, while the products made with vehicle or 20 mg / mL irinotecan in vehicle showed turbidity corresponding to insoluble SN-38 lactone crystals. Left: vehicle containing 2 mg / mL SN-38 lactone; center: G2B-002-20-9; right: 20 mg / mL irinotecan in vehicle containing 2 mg / mL SN-38 lactone.

[0068] Figure 3 The photographs were taken immediately after preparing the product G2B-006-20-9 or a product consisting of only the vehicle in the absence of G2B-003 micelles containing a 2 mg / mL loading of free SN-38 lactone. The product G2B-006-20-9 is not turbid, while the product made with the vehicle shows turbidity corresponding to insoluble SN-38 lactone crystals. Left: G2B-006-20-9; Right: vehicle.

[0069] Figure 4 The photographs of the product G2B-002-20-9 obtained at different times. The product was stored at 4°C for 24 weeks without turbidity. Left: G2B-002-20-9, immediately after preparation; Center: G2B-002-20-9, one week after preparation; Right: G2B-002-20-9, 24 weeks after preparation.

[0070] Figure 5 The photographs of the product G2B-006-20-9 obtained at different times. The product was stored at 4°C for 24 weeks without turbidity. Left: G2B-006-20-9, immediately after preparation; Center: G2B-002-20-9, six weeks after preparation; Right: G2B-006-20-9, 24 weeks after preparation.

[0071] Figure 6Photographs of the products detailed in Table 4 containing camptothecin (CPT) in the concentration range of 1 mg / mL to 0.25 mg / mL.

[0072] Figure 7 Photographs 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).

[0073] Figure 8 Comparison of the antiproliferative activities of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line HSJD-DIPG-007. The values ​​of the dots represent the mean and SD of three replicates at the compound concentration.

[0074] Fig. 9 Comparison of the antiproliferative activities of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line HSJD-DMG-001. The values ​​of the dots represent the mean and SD of three replicates at the compound concentration.

[0075] Fig.10 Comparison of the antiproliferative activities of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line HSJD-GBM-001. The values ​​of the dots represent the mean and SD of three replicates at the compound concentration.

[0076] Fig.11 Comparison of the antiproliferative activities of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line RH4. The values ​​of the dots represent the mean and SD of three replicates at the compound concentration.

[0077] Fig.12 Comparison of the antiproliferative activities of G2B-002-20-9, free SN-38, and irinotecan against the cancer cell line A673. The values ​​of the dots represent the mean and SD of three replicates at the compound concentration.

[0078] Fig.13 Kaplan-Meier survival curves obtained in each of the groups of mice carrying HSJD-DIPG-007 are shown.

[0079] Fig.14 Kaplan-Meier survival curves obtained in each of the groups of mice bearing HSJD-GBM-001 are shown.

[0080] Fig.15Shown are tumor growth (% tumor volume on treatment day 1) of subcutaneous PDX-bearing mice treated with saline control, 10 mg / kg irinotecan, and G2B-002 versions at doses of 1 mg / kg and 10 mg / kg SN-38 lactone. Each dot represents tumor growth of an individual PDX model.

[0081] Fig.16 The concentration-time data of SN-38 lactone in the mouse retina after intravenous administration of G2B-002-20-9 or 10 mg / kg irinotecan at doses of 10 mg / kg and 1 mg / kg SN-38 lactone are shown. The dots are individual data, and the lines connect the data mean values ​​for each time point.

[0082] Fig.17 The transport of different micelles formed by Compound Ia and SN-38 compared to SN-38 and SN-38 peptide conjugate (G2B-001) in the human in vitro BBB model is shown. DETAILED DESCRIPTION

[0083] In this application, unless otherwise specified, all terms used herein should be understood as their common meanings known in the art. Other more specific definitions of certain terms used in this application are set forth below and are intended to be applied throughout the specification and claims.

[0084] Unless otherwise indicated, amino acids referenced herein are L-amino acids. 1-letter codes and 3-letter codes have been used ambiguously. For the following amino acids, the following abbreviations have been used: diaminopropionic acid (Dap), diaminobutyric acid (Dab), selenocysteine ​​(Sec) and penicillamine (Pen). In the context of the present invention, penicillamine includes only D-penicillamine.

[0085] The term "apparent solubility" is the water solubility of the free SN-38 lactone measured after inclusion in a micellar aqueous dispersion of the SN-38 peptide conjugate.

[0086] When the term "core" is applied to the micelles of the present invention, it refers to the center of the micelle formed by the SN-38 peptide conjugate.

[0087] When the term "shell" is applied to the micelles of the present invention, it refers to the layer formed by the SN-38 peptide conjugate.

[0088] As used herein, the terms "drug" and "therapeutic agent" are used interchangeably.

[0089] The terms "drug-loaded" and "encapsulated" are used interchangeably. According to the present invention, a "drug-loaded" micelle refers to a micelle in which the drug or therapeutic agent is located within the core of the micelle.

[0090] In the case of using a numerical interval in the present invention, the numerical interval includes the extreme values ​​of the interval. Specifically, as used herein, the term "included between" refers to a range of values, including the endpoint values ​​of the range.

[0091] Unless otherwise indicated, all percentages mentioned herein are expressed in units of weight relative to the total weight of the product, provided that the sum of the amounts of the components equals 100%.

[0092] Compound (Ia) is also named G2B-001. Compound (Ib) is also named linear G2B-001. Compound (Ic) is also named G2B-003. Compound (Id) is also named G2B-004. Compound (Ie) is also named G2B-005.

[0093] As an example of the nomenclature used, in 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 the final concentration of G2B-001 is 20 mg / mL (see also Example 15).

[0094] For the purpose of the present invention, the word "comprising" encompasses the case of "consisting of."

[0095] As mentioned above, micelles of peptide conjugates binding to SN-38 loaded with at least one drug having anticancer activity are part of the present invention. In a specific embodiment, the micelles of peptide conjugates binding to SN-38 are loaded with camptothecin. In another specific embodiment, the camptothecin is selected from the group consisting of SN-38, camptothecin (CPT) or topotecan. In a specific embodiment, the drug in the inner core is SN-38 lactone.

[0096] The SN-38 peptide conjugates used to prepare the micelles of the present invention mentioned above can be in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" as used herein encompasses any salt formed by a pharmaceutically acceptable non-toxic acid or base (including an inorganic or organic acid or base). Regarding salts, there are no restrictions, except that if used for therapeutic purposes, they must be pharmaceutically acceptable. Because some of the compounds of formula (I) are alkaline compounds, salts can be prepared from pharmaceutically acceptable non-toxic acids (including inorganic acids 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.

[0097] In another specific embodiment, the micelles are those in which: in the SN-38 peptide conjugate of formula (I), Z is linked to the linker L via the hydroxyl group (b) of the pharmaceutically active ingredient. In another specific embodiment, the micelles are those in which: in the SN-38 peptide conjugate of formula (I), Z is linked to the linker L via the hydroxyl group (a) of the pharmaceutically active ingredient.

[0098] In another specific embodiment, the micelles are those in which: In the SN-38 peptide conjugate of formula (I), P is a diradical of a peptide selected from the group consisting of:

[0099] (a) a peptide comprising an amino acid sequence DapKAPETALD, wherein the amino acid sequence has an intrapeptide bond between Dap and D, wherein the intrapeptide bond is an amide bond, and the amino acid sequence is

[0100] (b) a peptide having a length of 9 to 20 amino acid residues, wherein the peptide has at least one intrapeptide bond, wherein the intrapeptide bond is a disulfide bond, and wherein the peptide comprises the following amino acid sequence: CKAPETALCAAA, wherein the amino acid sequence has at least one intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, wherein the amino acid sequence is

[0101] (c) a peptide of 9-11 amino acid residues in length, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond, and the peptide consisting of an amino acid sequence selected from the group consisting of CKAPETALC, CKAPETALCA and CKAPETALCAA, the amino acid sequence having at least one intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, the amino acid sequence being:

[0102]

[0103] (d) a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH, which has intrapeptide disulfide bonds between the first and third cysteine ​​residues, which are cysteine ​​1 and cysteine ​​11, and between the second and fourth cysteine ​​residues, which are cysteine ​​3 and cysteine ​​15, the amino acid sequence being:

[0104] and

[0105] (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14), ie, a linear peptide.

[0106] In the sequences SEQ ID NO:8 to SEQ ID NO:13, specific amino acids have been selected for X1-X 10 , specific intra-peptide bonds are drawn in the sequence.

[0107] In another specific embodiment, the peptides of the present invention are those having one intrapeptide bond. In another specific embodiment, the peptides of the present invention are those having two intrapeptide bonds.

[0108] In another specific embodiment, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), P is a diradical of a peptide selected from the group consisting of: (a) a peptide having an amino acid sequence DapKAPETALD (SEQ ID NO: 8) having an intrapeptide bond between Dap and D, wherein the intrapeptide bond is an amide bond; (b) a peptide having an amino acid sequence CKAPETALC (SEQ ID NO: 10) having at least one intrapeptide disulfide bond between cysteines in positions 1 and 9; (c) a peptide having an amino acid sequence DapKAPETALD (SEQ ID NO: 14), i.e., a linear peptide. In another specific embodiment, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), P is a diradical of a peptide DapKAPETALD (SEQ ID NO: 8) having an intrapeptide bond between Dap and D, wherein the intrapeptide bond is an amide bond.

[0109] In another specific embodiment, the micelles are those in which: in the SN-38 peptide conjugate of formula (I), L a ' is a diradical selected from the group consisting of: -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 .

[0110] In another specific embodiment, in conjunction with any of the specific embodiments above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L is a linker, the linker is a biradical consisting of 3 to 8 biradicals, and n is an integer from 1 to 6. In another specific embodiment, in conjunction with any of the specific embodiments above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L is a linker, the linker is a biradical consisting of 5 to 8 biradicals. In another specific embodiment, in conjunction with any of the specific embodiments above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L is a linker, the linker is a biradical consisting of 6 to 8 biradicals. In another specific embodiment, in conjunction with any of the specific embodiments above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L is a linker, the linker is a biradical consisting of 6 to 7 biradicals. In another specific embodiment, in combination with any of the above or below specific embodiments, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L is a linker that is a diradical composed of 6 diradicals.

[0111] In another specific embodiment, the micelles are those in which: in the SN-38 peptide conjugate of formula (I), L a ' is L3 of the following formula, and L c ' is -C(=O)-(CH2) r -C(=O)-.

[0112]

[0113] In another specific embodiment, the micelles are those in which: in the SN-38 peptide conjugate of formula (I), L b 'Selected from -NH-(CH2) r -O-, -(CH2) r -O- and -(CH2) r In another specific embodiment, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L a ' is L3 of the above formula, L b 'Selected from -NH-(CH2) r -O-, -(CH2) r -O- and -(CH2) r -NH- and their combination, and L c ' is -C(=O)-(CH2) r -C(=O)-.

[0114] In another specific embodiment, in conjunction with any of the specific embodiments above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L is a linker, the linker is a biradical composed of 2 biradicals, and n=0. In another specific embodiment, in conjunction with any of the specific embodiments above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L a 'For L 12 And L c 'For L 13 In another specific embodiment, in combination with any of the above or below specific embodiments, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L a ' is -C(=O)-NH-(CH2) r -C(=O)- and L c 'For L 15 .

[0115] In another specific embodiment, in combination with any of the above or below specific embodiments, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L a ' is connected to the radical Z through a bond, the bond being L drawn with a The C=O on the left side of the formula forms an ester bond and is connected to the free radical L through a chemically feasible bond. b ', the chemically feasible bond is selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond, and a thioester bond, and is formed with the functional group on the right side of the drawn formula. In another specific embodiment, in combination with any of the above or below specific embodiments, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L a ' is connected to the radical Z through a bond, the bond being L drawn with a The C=O on the left side of the formula forms an ester bond and is connected to the free radical L through a chemically feasible bond. b ', the chemically feasible bond is an amide bond, which is formed with the functional group on the right side of the drawn formula. In another specific embodiment, in combination with any specific embodiment above or below, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L a ' is connected to the radical Z through a bond, the bond being L drawn with a The C=O on the left side of the formula forms a carbonate or carbamate bond and is connected to the free radical L through a chemically feasible bond. b ', the chemically feasible bond is an amide bond (NH-CO or CO-NH), which is formed with the functional group on the right side of the drawn formula.

[0116] In another specific embodiment, in combination with any of the above or below specific embodiments, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L b 'With free radical L a ' and the drawn L b The functional groups on the left side of the formula together form a chemically feasible bond; and L b 'Connected to the radical L through a chemically feasible bond c ', the chemically feasible bond is selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond, and is drawn with L b 'The functional group on the right side of the formula is formed; wherein, when n is greater than 1, L b ' are identical or different and are connected by a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond; one L b 'Connect to L at the end a 'And another L b 'Connect to L at the end c '.

[0117] In another specific embodiment, in combination with any of the above or below specific embodiments, the micelles are those micelles as follows: In the SN-38 peptide conjugate of formula (I), L c ' is connected to the diradical P via an amide bond, the amide bond being drawn with L c The carbonyl group on the right side of the formula is formed by the amino group of the first amino acid in the peptide sequence P and is connected to the free radical L through a chemically feasible bond. b ', the chemically feasible bond is selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond, and is formed with the functional group on the left side of the drawn formula.

[0118] In another specific embodiment, the micelles are those micelles as follows: wherein the SN-38 peptide conjugate of formula (I) is a compound selected from the group consisting of: Compound of formula (Ia): Also named G2B-001 or SN38-Linker A-MiniAp4. MiniAp4 is DapKAPETALD, wherein Dap is 2,3-diaminopropionic acid. These two names are used interchangeably. These names are used equivalently herein. The compound of the following formula (Ia) or a pharmaceutically acceptable salt thereof is a compound of formula (I), wherein P is DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=0, the linker is the linker A below, and SN-38 is connected to the linker via a hydroxyl group (b).

[0119]

[0120] In another specific embodiment, the compound of formula (I) is a compound of the following formula (Ib) (also named linear G2B001) or a pharmaceutically acceptable salt thereof, wherein, in formula (I), P is a linear DapKAPETALD having no intrapeptide bond between Dap and D, Y=CONH2, W=0, the linker is the linker A described below, and SN-38 is connected to the linker via a hydroxyl group (b).

[0121]

[0122] In another specific embodiment, the compound of formula (I) is a compound of formula (Ic) (also named G2B003, shown below) or a pharmaceutically acceptable salt thereof, wherein, in formula (I), P is DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=0, the linker is linker A described below, and SN-38 is linked to the linker via a hydroxyl group (a).

[0123]

[0124] Joint A is formed by: La': L3, where r=4 on the right and r=3 on the left:

[0125]

[0126] Lb': 1 unit of diradical -NH-(CH2)3-O-, 2 units of diradical -(CH2)2-O- and 1 unit of diradical -(CH2)3-NH-; and Lc': -C(=O)-(CH2)2-C(=O)-; and the diradicals are connected corresponding to the linker A as shown below:

[0127]

[0128] In another specific embodiment, the compound of formula (I) is a compound of formula (Id) (also named G2B-004, shown below) or a pharmaceutically acceptable salt thereof, wherein, in formula (I), P is DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=0, the linker is linker C below, and SN-38 is connected to the linker via hydroxyl group (a). Linker C is formed by: La': L 12 and Lc':L 13 , and n=0.

[0129]

[0130] In another specific embodiment, the compound of formula (I) is a compound of the following formula (Ie) or a pharmaceutically acceptable salt thereof, wherein, in formula (I), P is DapKAPETALD having an intrapeptide bond between Dap and D, Y=CONH2, W=0, the linker is the linker D described below, and SN-38 is connected to the linker via a hydroxyl group (a).

[0131]

[0132] This compound is also named G2B-005; the linker D is formed by: La': -C(=O)-NH-(CH2) r -C(=O)-, where r=1, Lb': L 15 , and n=0.

[0133] The micelles of the present invention are formed in an aqueous medium. Therefore, the micelles of the present invention may be in the form of an aqueous micellar dispersion.

[0134] Micelles of the SN-38 peptide conjugate as defined above containing free therapeutic agent can be obtained by the following operations: a) spontaneously self-assembling the SN-38 peptide conjugate as defined above in water at pH <7; b) contacting the micellar acidic solution with a basic solution containing free SN-38 carboxylate at a concentration of up to 25 mg / ml; and c) optionally lyophilizing the micellar acidic solution.

[0135] In step b), the initial pH of the mixture is an acidic pH of <7, and the SN-38 carboxylate is converted into an SN-38 lactone, which forms an intermolecular interaction with the SN-38 molecules conjugated in the SN-38 peptide conjugate. In a specific embodiment, the spontaneous self-assembly of the SN-38 peptide conjugate as defined above is carried out in water at a pH of <5. In a specific embodiment, the spontaneous self-assembly of the SN-38 peptide conjugate as defined above is carried out in water at a pH of <3. In a specific embodiment, the alkaline solution containing free SN-38 carboxylate has a concentration of 2 mg / ml-25 mg / ml. In a specific embodiment, the alkaline solution containing free SN-38 carboxylate has a concentration of 2 mg / ml-12 mg / ml.

[0136] In a particular embodiment, the micelles according to the invention are those micelles in which each individual micelle in the micellar aqueous dispersion comprises only one type of therapeutic agent useful for treating cancer. In another particular embodiment, the micellar aqueous dispersion is a micellar aqueous dispersion in which the free therapeutic agent is SN-38 lactone. In another particular embodiment, the micellar aqueous dispersion is a micellar aqueous dispersion in which each individual micelle comprises two or more therapeutic agents useful for treating cancer.

[0137] The SN-38 peptide conjugate of formula (I) can be generated 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 liquid phase synthesis or preferably by solid phase peptide synthesis, for which there are many published procedures (see M. Amblard et al., "Methods and protocols of modern solid-phase peptide synthesis", Molecular Biotechnology 2006, Vol. 33, pp. 239-254). The compound of formula (I) can also be prepared by any combination of liquid phase synthesis and / or solid phase synthesis. For example, the main body of peptide P is synthesized by solid phase synthesis, followed by removal of the protecting groups in solution. The combination of SN-38 with linker L and peptide P can be performed in solid phase or in solution. The construction of linker L can also be prepared by any combination of liquid phase synthesis and / or solid phase synthesis.

[0138] The peptide of the present invention can also be obtained by generating a DNA template and subcloning into an expression vector (see JH Lee et al., Eur. J. 30 Biochem. 2001, Vol. 268, pp. 2004-2012).

[0139] Compounds of formula (Ia) may be prepared by a process comprising reacting a compound of formula (III) with a compound of formula (IV) as described above to produce a compound of formula (Ia).

[0140]

[0141] The preparation of pharmaceutically acceptable salts of compounds of formula (I) can be carried out by methods known in the art. For example, they can be prepared by conventional chemical methods from parent compounds containing basic or acidic moieties. In general, such salts are prepared, for example, by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture thereof.

[0142] A method of preparing micelles from these SN-38 peptide conjugates and SN-38 lactones by spontaneous micellization by dissolving them in water at pH <7, specifically pH <3 is also part of the present invention. Such micellar acidic solutions can neutralize alkaline solutions containing free SN-38 carboxylate at a concentration of up to 25 mg / ml. Upon acidification at alkaline pH, the appearance of free SN-38 lactone due to the pH change causes intermolecular interactions between free SN-38 lactone molecules and SN-38 molecules conjugated in the SN-38 peptide conjugates. In general, the micelles can be formed with gentle mixing. In general, they are formed in less than 30 minutes. In general, the micelles are formed at room temperature.

[0143] The system produces nanometer-sized drug-drug co-crystals composed of two forms of SN-38 lactone, namely the free form and the conjugated form. Under such conditions, the free SN-38 lactone does not form microcrystals or larger solid structures and remains significantly soluble at concentrations up to 4 mg / ml.

[0144] Such micellar aqueous dispersions of SN-38 peptide conjugates containing free SN-38 lactone can be easily filtered through 0.22 μm and / or 0.45 μm pore filters without losing any free SN-38 lactone. For example, filtration can be performed using a 0.45 or 0.22 polypropylene syringe filter.

[0145] The aqueous micellar dispersion of the present invention can also be freeze-dried. After reconstitution with water, it retains the same amount of soluble SN-38 lactone. Freeze-drying can be performed, for example, using a Telstar freeze dryer.

[0146] In a particular embodiment, the concentration of the peptide conjugate in the micelle in the micelle aqueous dispersion is at most 50mg / ml. In another particular embodiment, the concentration of the peptide conjugate in the micelle in the micelle aqueous dispersion is 1mg / ml to 50mg / ml. In another particular embodiment, the concentration of the peptide conjugate in the micelle in the micelle aqueous dispersion is 1mg / ml to 20mg / ml. In another particular embodiment, the concentration of the peptide in the micelle aqueous dispersion is 5mg / ml to 20mg / ml. In another particular embodiment, the concentration of the peptide conjugate in the micelle aqueous dispersion is 10mg / ml to 20mg / ml. In general, the concentration of the peptide conjugate of the micelle of the present invention in the micelle aqueous dispersion is selected from 20mg / mL, 10mg / mL and 5mg / mL.

[0147] In another specific embodiment, the proportion of free SN-38 lactone in the micellar aqueous dispersion is up to 25 mg / mL. In another specific embodiment, the proportion of free SN-38 lactone in the micellar aqueous dispersion is 2 mg / ml-25 mg / ml. In another specific embodiment, the proportion of free SN-38 lactone in the micellar aqueous dispersion is 2 mg / ml-12 mg / ml. In another specific embodiment, the proportion of free SN-38 lactone ranges from 1 mg / ml to 8 mg / ml, specifically 8 mg / mL, 4 mg / mL, 2 mg / mL, 1 mg / mL.

[0148] In a specific embodiment, the micelle of the present invention is selected from the group consisting of: a) micelles of a peptide conjugate of formula (Ia), wherein the inner core is loaded with SN-38, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of SN-38 is 16 mg / ml;

[0149] b) micelles of the peptide conjugate of formula (Ia), wherein the inner core is loaded with SN-38, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of SN-38 is 9 mg / ml; c) micelles of the peptide conjugate of formula (Ia), wherein the inner core is loaded with SN-38, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 10 mg / ml, and the concentration of SN-38 is 9 mg / ml; and d) micelles of the peptide conjugate of formula (Ic), wherein the inner core is loaded with SN-38, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of SN-38 is 9 mg / ml.

[0150] In another specific embodiment, the micelle of the present invention is e) a micelle of a peptide conjugate of formula (Ia), wherein the inner core is loaded with camptothecin lactone, the concentration of the SN-38 peptide conjugate in the micelle aqueous dispersion is 20 mg / ml, and the concentration of camptothecin lactone is 0.25 mg / ml-4 mg / ml, specifically 0.25 mg / ml-1 mg / ml. In another specific embodiment, the micelle of the present invention is f) a micelle of a peptide conjugate of formula (Ia), wherein the inner core is loaded with camptothecin lactone and SN-38, the concentration of the SN-38 peptide conjugate in the micelle aqueous dispersion is 20 mg / ml, the concentration of camptothecin lactone is 0.5 mg / ml-1 mg / ml, and the concentration of SN-38 is 0.5 mg / ml-1 mg / ml.

[0151] Pharmaceutical compositions comprising a therapeutically effective amount of micelles as defined above and a suitable amount of a pharmaceutically acceptable carrier or excipient are also part of the invention.

[0152] The term "therapeutically effective amount" as used herein refers to the amount of the compound (according to the micelle of the present invention) sufficient to prevent or alleviate to some extent the development of one or more symptoms of the disease being treated when administered. The specific dosage of the compound administered according to the present invention will of course be determined by the specific circumstances surrounding the case, including the compound administered, the route of administration, the specific condition being treated, and similar considerations.

[0153] The term "pharmaceutical composition" refers to a mixture of the compounds described herein with other chemical components, such as diluents or carriers. Pharmaceutical compositions facilitate administration of the compounds to an organism. The term "pharmaceutically acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. Each component must also be suitable for contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with the benefit / risk ratio.

[0154] The compositions of the present invention may be administered in a parenteral form suitable for injection, such as intravenous bolus injection, intravenous infusion, implantation, orally, intrathecally, or intranasally.

[0155] An important feature of the micelles of the present invention is their biological activity of inhibiting the cell growth of the tested tumor cell lines. As shown 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 aggregation of tumor cells, which may cause death in animals and humans. It is also one of the main causes of disease death in children and adolescents. Although significant progress has been made in the treatment of several types of cancer, particularly childhood cancer, in the past fifty years, progress has been limited for other types. The annual incidence of childhood tumors is between 100-160 cases per million. One in every 500 children under the age of 15 is at risk each year. In industrialized countries, this incidence is slightly lower. Brain and spinal cord tumors account for 25% of all neoplasms (40%-50% of all pediatric solid tumors). Despite the progress that has been made, the survival data for these patients remains low, about 55%. This contrasts with the considerable increase in survival seen in recent years in other types of patients, such as those affected by leukemia or extracranial tumors.

[0156] Conventional cancer treatment methods include surgical treatment, administration of chemotherapeutic agents and more recently, immune response-based therapies involving administration of antibodies or antibody fragments that may be conjugated to a therapeutic moiety. However, to date, the success of such treatments has been limited.

[0157] Camptothecins, one of the four major structural classes of anticancer compounds derived from plants, are cytotoxic alkaloids consisting of a five-ring structure containing a pyrrole (3,4β) quinoline moiety, an S-configured lactone form, and a carboxylate form. Irinotecan is made from natural camptothecin found in the Chinese ornamental tree Camptotheca acuminata. Several types of cancer have been treated with irinotecan at some point, among them glioblastoma multiforme (GBM) in adults and diffuse intrinsic pontine glioma (DIPG), pediatric glioblastoma (pGBM), neuroblastoma, rhabdomyosarcoma, Ewing sarcoma, and retinoblastoma in children.

[0158] Gliomas are a group of tumors that start in glial cells in the brain or spine, accounting for about 30% of all brain tumors and central nervous system tumors and 80% of all malignant brain tumors. Typically, brain gliomas are treated with a combination of surgery, radiotherapy, and chemotherapy. DIPG mainly affects children, usually between the ages of 5 and 7. Unfortunately, this is the majority of brain stem tumors, accounting for 60%-70%, and it is one of the tumors with the worst prognosis of all tumors. Treatment has not been shown to be effective, and the average survival period is 9 months. Radiation and steroid administration are the only methods that have a palliative effect and very cautiously increase survival rates. To date, there is no useful chemotherapy, and multiple clinical trials have been tested, but no favorable results. Pediatric glioblastoma is another group of tumors, accounting for one-third of hemispheric tumors. Its peak incidence is between 8 and 12 years old. Glioblastoma also affects adult patients, with about 2-3 cases per 100,000 people.

[0159] Ewing sarcoma is the second leading cause of malignant bone tumors in children and adolescents. The annual incidence is 0.6 per million inhabitants. It rarely appears before the age of 5 years, and the peak incidence is between 10 and 15 years of age, affecting more boys than girls, but this gender relationship varies with the age range. Its most common starting site is the pelvic (hip) bone, the chest wall (such as the ribs or shoulder blades), or the middle of the leg bones. In the absence of bone lesions, Ewing sarcoma can also present as an extraskeletal lesion. In this variant, there is a high risk of lymphatic spread, and treatment is generally similar to that of rhabdomyosarcoma.

[0160] Soft tissue sarcomas are divided into rhabdomyosarcomas and non-rhabdomyosarcomas. Rhabdomyosarcomas account for 50% of all childhood soft tissue sarcomas. It is the third most frequent extracranial solid tumor after neuroblastoma and Wilms' tumor. The age peak is bimodal, with the first peak between 2 and 5 years of age and the second peak in adolescence, between 15 and 19 years of age. Although most adult sarcomas appear in the limbs, childhood sarcomas can originate from anywhere in the body, including skeletal muscle and soft tissue. The most affected areas for children are the head and neck and the urogenital tract. The limbs of 20% of patients are affected. The overall survival rate is poor unless the tumor is present in a location where it can be completely removed, which sometimes requires amputation or excretion. The survival rate varies between 7%-70%, depending on the location.

[0161] Neuroblastoma is the most common extracranial solid tumor in children. Due to its embryonic origin, neuroblastoma can be present in almost any part of the sympathetic nervous system, but localized disease is most often located on the adrenal glands (44%). Children between 1.5 and 6 years old at diagnosis can still be cured with conventional treatment, but when they are diagnosed with metastatic disease (stage 4 neuroblastoma), their chances of survival decrease. About 50% of newly diagnosed patients already present with bone (60%), bone marrow (50%), lymph node (42%) and / or liver (15%) metastases and require intensive chemotherapy treatment, surgery and radiotherapy, but their survival rate remains poor and there has been little progress in the past few decades.

[0162] Finally, retinoblastoma is the most common cause of childhood eye tumors, with a global incidence of 1 in 20,000 live births. It usually presents in the first 2 years of life. 30%-40% are bilateral, in these cases there is always a positive family history. Of the unilateral, 10% have a germline mutation in the Rb gene located on chromosome 13. If detected early, they have a 95% survival rate. In certain areas of the world, when the diagnosis is late, the survival rate drops dramatically, to less than 20%. Treatment depends on the tumor control achieved. When tumor regression cannot be controlled by chemotherapy and brachytherapy, enucleation is recommended. Even if enucleation is performed, in some cases there is invasion of the optic nerve by the tumor, which will necessitate additional chemotherapy treatment.

[0163] Micelles as defined above for use as a medicament are part of the present invention. Micelles as defined above for use in the treatment of cancer in mammals (including humans) are also part of the present invention, as they are active in all types of cancer that have been tested. This aspect can also be systematically described as the use of micelles as defined above in 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 for treating a mammal (including humans) suffering from or susceptible to cancer, the method comprising administering to the patient a therapeutically effective amount of micelles as defined above and a pharmaceutically acceptable excipient or carrier.

[0164] In a specific embodiment, the micelle is used for the purposes as defined above, wherein the cancer is located in the brain. In another specific embodiment, the micelle or the micellar aqueous dispersion is used for the purposes as defined above, wherein the micelle or the micellar aqueous dispersion crosses the blood-brain barrier and releases free therapeutic agents in the brain parenchyma and cerebrospinal fluid. In a specific embodiment, the micelle of the present invention is used for the purposes as defined above, wherein cancer treatment includes treating tumors selected from the group consisting of extracranial solid tumors, eye tumors and CNS tumors. In another specific embodiment, the micelle of the present invention is used for the purposes as defined above, wherein the cancer is selected from the group consisting of: adult glioma, pediatric glioma, retinoblastoma, Ewing sarcoma, DIPG, neuroblastoma, medulloblastoma, ependymoma, atypical teratoid-rhabdoid tumor (ATRT) and rhabdomyosarcoma. In another specific embodiment, the micelle of the present invention is used for the purposes as defined above, wherein the cancer is a pediatric brain tumor. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the pediatric glioma is selected from the group consisting of diffuse intrinsic pontine glioma (DIPG) and pediatric high-grade glioma. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the cancer is diffuse intrinsic pontine glioma. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the cancer is pediatric high-grade glioma. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the cancer is retinoblastoma. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the cancer is Ewing sarcoma. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the cancer is neuroblastoma. In another specific embodiment, the micelle is used for the purpose as defined above, wherein the cancer is rhabdomyosarcoma.

[0165] In another specific embodiment, the micelles are used for the purposes as defined above, wherein the cancer is medulloblastoma. In another specific embodiment, the micelles are used for the purposes as defined above, wherein the cancer is ependymoma. In another specific embodiment, the micelles are used for the purposes as defined above, wherein the cancer is atypical teratoid-rhabdoid tumor (ATRT). In another specific embodiment, the micelles are used for the purposes as defined above, wherein the cancer is adult glioma. In another specific embodiment, the micelles are used for the purposes as defined above, wherein they are active against cancer cell lines and patient-derived xenografts of Ewing sarcoma, DIPG, pHGG, glioblastoma multiforme, rhabdomyosarcoma, retinoblastoma, medulloblastoma, ependymoma, atypical teratoid-rhabdoid tumor (ATRT) and neuroblastoma.

[0166] In another specific embodiment, the micelles are for the use as defined above, wherein they are active against the cancer cell lines used in these experiments, including pediatric gliomas, including cancer types of diffuse intrinsic pontine glioma (HSJD-DIPG-007), pediatric diffuse midline glioma (HSJD-DMG-001) and pediatric high-grade glioma (HSJD-GBM-001), and cell lines of other pediatric solid tumors, such as cell lines of Ewing sarcoma (A673) and cell lines of rhabdomyosarcoma (RH4).

[0167] The micelles of the present invention can be used in the same manner as other known chemotherapeutic agents, i.e., simultaneously or sequentially in combination with other treatments, depending on the condition to be treated. The micelles of the present invention can be used alone or in combination with other suitable biologically active compounds. Thus, the micelles of the present invention are used in combination therapy with chemotherapeutic agents to treat cancer in mammals (including humans).

[0168] In a specific embodiment, the dose of free SN-38 is at least 1 mg / kg and up to 12 mg / kg (preclinical studies in mice). In a specific embodiment, the micelles are administered in combination with another chemotherapeutic agent. In another specific embodiment, the micelles are administered simultaneously with another chemotherapeutic agent. In another specific embodiment, the micelles are administered separately in any order within a therapeutically effective interval.

[0169] Throughout the specification and claims, the word "comprise" and variations of the word are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprise" covers the case of "consisting of ...". Additional objects, advantages and features of the present invention will become apparent to those skilled in the art after investigating this specification, or can be learned by the practice of the present invention. The following examples and drawings are provided by way of example, and they are not intended to limit the present invention. The figure marks associated with the drawings and placed in brackets in the claims are intended only to increase the understandability of the claims and should not be interpreted as limiting the scope of the claims. In addition, the present invention covers all possible combinations of the specific and preferred embodiments described herein.

[0170] Example

[0171] Protected amino acids, handles, and resins were supplied by Luxembourg Industries (Tel Aviv, Israel), Neosystem (Strasbourg, France), CalbiochemNovabiochem AG (Leufelfingen, Switzerland), Bachem AG (Bubendorf, Switzerland), or Iris Biotech (Marktredwitz, Germany). Other reagents and solvents used are summarized in Table 1 middle.

[0172] Table 1 Commercial suppliers and reagents used. DCM was passed through an Al2O3 column. DMF was stored at molecular sieves and bubbling with nitrogen to eliminate volatile reagents.

[0173]

[0174]

[0175] General method for preparing SN-38 peptide conjugates:

[0176] General considerations for manual synthesis: Solid phase peptide extension and other solid phase manipulations were performed manually in a polypropylene syringe equipped with a polyethylene porous disk. Solvents and soluble reagents were removed by aspiration. Dimethylformamide (DMF) (5 × 30 seconds) and dichloromethane (DCM) (5 × 30 seconds) washings were performed between different synthesis steps, using 10 mL of solvent per gram of resin each time.

[0177] General considerations regarding microwave-assisted synthesis:Microwave-assisted solid phase peptide synthesis was performed on a Liberty Blue automated microwave peptide synthesizer using H-Rink amide Protide resin (loading: 0.56 mmols / g). Linear peptides were synthesized on a 0.5 mmol scale using 5 excess Fmoc-amino acids (0.2 M) relative to the resin.

[0178] Logo test: The tests used to identify and control the synthesis are 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).

[0179] Protocol used during the manual synthesis of the compounds : The compounds were synthesized at 100 μmol scale using the following method and protocol: The resin for manual synthesis was selected based on the Y group: If Y is OH, the terminal will be COOH and 2-chlorotrityl chloride resin will be selected from the available resins. If Y is NH2, the terminal will be CONH2 and Rink amide MBHA resin will be selected from the available resins.

[0180] Initial resin conditioning: 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).

[0181] Removal of Fmoc group: 20% (v / v) piperidine in DMF was used with one 30 second treatment followed by two 10 minute treatments to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Two more DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) treatments (2 x 5 minutes) were performed to ensure removal of the Fmoc group from the secondary amine (proline).

[0182] Coupling method described for 100 μmol scale:

[0183] Coupling method 1: The protected amino acid (4 eq., 400 μmol), TBTU (4 eq., 400 μmol, 128 mg) dissolved in DMF (1 mL-3 mL per gram of resin) were added to the resin in sequence, followed by DIEA (8 eq., 800 μmol, 136 μl). The mixture was reacted for 1 hour with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 seconds) and DCM (5×30 seconds). The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed using a 30 second treatment and two 10 minute treatments with 20% (v / v) piperidine in DMF. If the amino acid to be deprotected is proline, an additional treatment (2×5 minutes) of DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0184] Coupling method 2: The protected amino acid (4 eq., 400 μmol), PyBOP (4 eq., 400 μmol, 208 mg), HOAt (12 eq., 1.2 mmol, 163 mg) dissolved in DMF (1 mL-3 mL per gram of resin) were added to the resin in sequence, followed by DIEA (12 eq., 1.2 mmol, 204 μL). The mixture was reacted for 1 hour with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 seconds) and DCM (5×30 seconds). The coupling reaction was carried out twice under the same conditions. The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with a 20% (v / v) piperidine solution in DMF using one 30 second treatment and two 10 minute treatments. If the amino acid to be deprotected is proline, DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) treatment is applied once more (2 x 5 min) to ensure removal of the Fmoc group.

[0185] Coupling method 3:The protected amino acid (4 eq., 400 μmol), PyBOP (4 eq., 400 μmol, 208 mg), HOBt (12 eq., 1.2 mmol, 162 mg) dissolved in DMF (1 mL-3 mL per gram of resin) were added to the resin in sequence, followed by DIEA (12 eq., 1.2 mmol, 204 μL). The mixture was reacted for 1 hour with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 seconds) and DCM (5×30 seconds). The coupling reaction was performed twice under the same conditions. The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with a 20% (v / v) piperidine solution in DMF using one 30 second treatment and two 10 minute treatments. If the amino acid to be deprotected is proline, DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) treatment is applied once more (2 x 5 min) to ensure removal of the Fmoc group.

[0186] Coupling method 4, 100 μmol scale: Protected amino acid (3 eq., 300 μmol), DIC (3 eq., 300 μmol, 46 μL) and Oxyma (3 eq., 300 μmol, 43 mg) in DCM / DMF (1:1). The mixture was reacted for 45 min with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with 20% (v / v) piperidine in DMF using one 30 sec treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2×5 min) of DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0187] Coupling method 5, 100 μmol scale: Protected amino acid (3 eq., 300 μmol), DIC (3 eq., 300 μmol, 46 μL) and HOBt (3 eq., 300 μmol, 41 mg) in DCM / DMF (1:1). The mixture was reacted for 45 min with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed using a 30 sec treatment and two 10 min treatments with 20% (v / v) piperidine in DMF. If the amino acid to be deprotected was proline, an additional treatment (2×5 min) of DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0188] Protocol used during microwave-assisted automated synthesis: The compounds were synthesized at 500 μmol scale using the following method and protocol: The resin for microwave-assisted automated synthesis was selected based on the Y group: If Y is OH, the terminal will be COOH, and Cl-TCP(Cl)ProTide resin will be selected from the available resins. If Y is NH2, the terminal will be CONH2, and Rink amide ProTide resin will be selected from the available resins.

[0189] Initial resin treatment: The resin was treated 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).

[0190] Coupling and deprotection conditions for microwave-assisted automated peptide synthesis:

[0191] Coupling conditions:

[0192]

[0193]

[0194] Deprotection conditions:

[0195] CEM Preferences Deprotection mixture Microwave method 1 10% (v / v) piperazine in 10:90 (EtOH / NMP) standard 2 20% (v / v) piperidine in DMF or NMP standard

[0196] method Ramp-up time Total time Maximum temperature Standard deprotection 20-30 seconds 1:05 90℃

[0197] Method for cyclizing a peptide sequence P:

[0198] Cyclization method 1: disulfide bond 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 in 100 μM concentration of aqueous ammonium bicarbonate buffer at 10 mM and pH 8.0. The solution was stirred vigorously at room temperature for 24 hours. Thereafter, the product was acidified to pH 2-3 with TFA, frozen and lyophilized.

[0199] Cyclization method 2: Amide bond:Cyclization is performed on the resin. With 20% (v / v) piperidine solution in DMF, the Fmoc group is removed using a 30-second treatment and two 10-minute treatments. Use Boc2O (3 equivalents, 1000 μmol, 56 mg) and DIEA (30 equivalents, 3000 μmol, 240 μL) to protect the N-terminal amine with a Boc protecting group. First, OAl and Alloc groups are deprotected (3 × 15 minutes) by being added in tetrakis (triphenylphosphine) palladium (0) (0.1 equivalent, 10 μM, 12 mg), phenylsilane (10 equivalents, 1000 μmol, 123 mg) in DCM. 0.02M sodium diethylcarbamate washing resin (3 × 5 minutes) is used in DCM. Then, coupling of the amino group of Dap with the carboxylate group of aspartic acid was achieved by adding PyBOP (4 eq., 400 μmol, 208 mg), HOAt (12 eq., 1.2 mmol, 163 mg), DMF (1 mL-3 mL per gram of resin) and DIEA (12 eq., 1.2 mmol, 204 μL). The coupling was maintained for 1.5 hours and repeated overnight.

[0200] Cyclization method 3: Amide bond: Cyclization is performed on resin.With 20% (v / v) piperidine solution in DMF, use 30 seconds of processing and twice 10 minutes of processing to remove Fmoc group.Use Boc2O (3 equivalents, 1000 μmol, 56mg) and DIEA (30 equivalents, 3000 μmol, 240 μL) to protect N-terminal amine with Boc protecting group.First, by being added in tetrakis (triphenylphosphine) palladium (0) (0.1 equivalent, 10 μM, 12mg), phenylsilane (10 equivalents, 1000 μmol, 123mg) in DCM, OAl and Alloc groups are deprotected (3 × 15 minutes).Wash resin (3 × 5 minutes) for 0.02M sodium diethyldithiocarbamate in DCM. Then, coupling of the amino group of Dap with the carboxylate group of aspartic acid was achieved by two 30 min cycles of 4 equivalents of Oxyma (400 μmol, 57 mg) and 4 equivalents of N,N'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL).

[0201] Cyclization method 4: Amide bond:Cyclization is performed on resin.With 20% (v / v) piperidine solution in DMF, use 30 seconds of processing and twice 10 minutes of processing to remove Fmoc group.Use Boc2O (3 equivalents, 1000 μmol, 56mg) and DIEA (30 equivalents, 3000 μmol, 240 μL) to protect N-terminal amine with Boc protecting group.First, by being added in tetrakis (triphenylphosphine) palladium (0) (0.1 equivalent, 10 μM, 12mg), phenylsilane (10 equivalents, 1000 μmol, 123mg) in DCM, OAl and Alloc groups are deprotected (3 × 15 minutes).Wash resin (3 × 5 minutes) for 0.02M sodium diethyldithiocarbamate in DCM. Then, coupling of the amino group of Dap and the carboxylate group of aspartic acid was achieved by two 1-hour cycles of 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg).

[0202] General method for constructing linker L:

[0203] General method for forming disulfide bonds: A disulfide bond can be achieved by reacting two thiols. A 100 μM concentration of thiol was dissolved in 10 mM aqueous ammonium bicarbonate buffer at pH 8.0, and the solution was stirred vigorously at room temperature for 24 hours. Thereafter, the solution was acidified to pH 2-3 with TFA, frozen and lyophilized.

[0204] General method for the formation of thioethers: The thioether bond is achieved by reacting the N-terminal bromoacetyl group with a cysteine ​​thiol as described in PL Barker et al., J. Med. Chem. 1992, Vol. 35, pp. 2040-2048.

[0205] General method for ether formation: Ether formation can be achieved by reacting the hydroxyl group with a haloalkyl compound, preferably under basic conditions, as described in Greene, Protective Groups in Organic Synthesis, 5th edition, Peter GM Wuts. 2014 John Wiley & Sons, Inc., pp. 26-29.

[0206] General method for ester formation:Ester formation can be achieved by reacting the hydroxyl group with a carboxylic acid using typical esterification conditions, such as Fischer esterification in the presence of acid catalyst, or reacting the hydroxyl group with the corresponding acid chloride, as described in Greene, Protective Groups in Organic Synthesis, 5th edition, Peter GM Wuts. 2014 John Wiley & Sons, Inc., pp. 271-279.

[0207] General method for the formation of thioesters: The thioester bond is achieved by reacting a thiol with a carboxylic acid as described in M. Kazemi et al., Journal of Sulfur Chemistry, 2015, Vol. 36:6, pp. 613-623.

[0208] General Method for Formation of Ethyl Carbamates: The hydroxyl group can be reacted with an isocyanate to produce the corresponding urethane as described herein: MT Nguyen et al., J. Org. Chem. 1998, 63, Vol. 20, pp. 6878-6885.

[0209] General method for the formation of silyl ethers: The hydroxyl group is reacted with a halotrialkylsilyl group to produce the corresponding silyl ether. An acid scavenger is usually required, as described in Greene, Protective Groups in Organic Synthesis, 5th edition, Peter GM Wuts. 2014 John Wiley & Sons, Inc., pp. 456-463.

[0210] General Method for Formation of Sulfonates: The hydroxyl group is reacted with an alkyl or aryl sulfonyl halide to produce the corresponding sulfonate ester as described in F. David et al., Org. Process Res. Dev. 2010, 14, 4, pp. 999-1007.

[0211] General method for forming phosphate esters: The hydroxyl group can be reacted with a dialkyl or diaryl phosphate having one hydroxyl group under dehydrating conditions or using Mitsunobu reaction conditions to form the corresponding phosphate ester, wherein one of the substituents is a chain attached to the hydroxyl group.

[0212] General method for ketal formation:Ketals can be formed by reacting the hydroxyl group with a halomethyleneoxyalkyl compound, or by adding the hydroxyl group to a substituted dihydropyran or dihydrofuran under acidic conditions, as described in Greene, Protective Groups in Organic Synthesis, 5th edition, Peter GM Wuts. 2014 John Wiley & Sons, Inc., pp. 69-77.

[0213] General method for the formation of hemiketals: The hydroxyl group can be contacted with an aldehyde to provide 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.

[0214] General Method for Formation of Carbamates: Carbamates can be formed by reacting a hydroxy group with a haloformate or isocyanate as described in Greene, Protective Groups in Organic Synthesis, 5th edition, Peter GM Wuts. 2014 John Wiley & Sons, Inc., pp. 371-374.

[0215] General method for forming carbonates: Carbonates can be formed by reacting SN-38 with PNPC as described in Eur J Pharm Biopharm, 2017, 115, 149-158, or with triphosgene as described in J. Med. Chem., 2008, 51, 21, 6916-6926.

[0216] Coupling of Fmoc-TTDS-OH: The coupling of Fmoc-TTDS-OH (2 eq.) was carried out by two 30 min cycles of 4 eq. Oxyma and 4 eq. N,N'-diisopropylcarbodiimide (DIC) in DMF. or This was achieved with 4 eq. DIC and 4 eq. HOBt in DCM for 2 h. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed using 20% ​​(v / v) piperidine in DMF using one 30 sec treatment followed by two 10 min treatments.

[0217] Coupling of 5-hexynoic acid: Coupling of 5-hexynoic acid (2 eq, 200 μmol, 23 mg) was achieved by 2 30 min cycles of 4 eq Oxyma (400 μmol, 57 mg) and 4 eq N,N′-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL) in DMF:DCM (1:1) or 4 eq DIC (400 μmol, 61 μL) and 4 eq HOBt (400 μmol, 54 mg) in DMF:DCM 1:1 in DMF for 4 h or 2 eq PyBOP (400 μmol, 208 mg) in DMF:DCM 1:1, 6 eq HOAt (600 μmol, 81.5 mg) and 6 eq DIEA (600 μmol, 102 μL) in DMF for 1.5 h. The solvent was removed by suction and the resin was washed with DMF (5 x 30 sec) and DCM (5 x 30 sec). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0218] Coupling of diglycolic anhydride: The coupling of diglycolic anhydride (10 equivalents, 1000 μmol, 116 mg) was achieved by two 60-minute cycles of 10 equivalents of DIEA (1000 μmol, 174 μL) in DMF. The solvent was removed by aspiration and the resin was washed with DMF (5×30 seconds) and DCM (5×30 seconds). The coupling was repeated under the same conditions. The degree of coupling was monitored using the Kaiser colorimetric assay.

[0219] General method for cleavage from resin: Final cleavage of the resin and side chain deprotection: This was performed by treating the resin (2 hours) with TFA (95%), H2O (2.5%) and TIS (2.5%). Tert-butyl methyl ether was added to the obtained product and the mixture was centrifuged (3×8 minutes). The supernatant was discarded and the pellet was resuspended in a mixture of H2O, MeCN and TFA (1000:1000:1). The product was filtered off and frozen.

[0220] General methods used to characterize the compounds:The 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, and using MeCN (0.036% TFA) and H2O (0.045% TFA) 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) coupled to an electrospray ion source ESI-MS Micromass ZQ) and using MassLynx 4.1 software (Waters, Milford, MA). BEH C18 column (50×2.1 mm×1.7 μm, Waters). The flow rate was 0.6 mL / min, and MeCN (0.07% formic acid) and H2O (0.1% formic acid) were used as solvents. The samples were analyzed with positive ionization: the ion spray voltage was 30 V, and the capillary temperature was 1 kV). Accurate mass was obtained by mass spectrometer: LTQ-FT Ultra (Thermo Scientific), and the samples were introduced in direct infusion (automated nano-electrospray). NanoMate (Advion BioSciences, Elm City, New York, USA) drew samples from 384-well plates (protein Lobind) with disposable conductive pipette tips and injected the samples into the mass spectrometer through a nano-electrospray ionization chip (nanoESI Chip) (consisting of 400 nozzles in a 20×20 array). The spray voltage was 1.70 kV, and the delivery pressure was 0.50 psi; ionization was nano-electrospray ionization, positive ionization.

[0221] NMR experiments were performed on a Bruker Avance III 600MHz spectrometer equipped with a TCI cryoprobe. Samples were prepared by dissolving the compound in 3mM-4mM 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 modeling. Residue-specific assignments were obtained from 2D total correlation spectroscopy (TOCSY) and correlation spectroscopy (COSY) experiments, while 2D nuclear Overhauser effect spectroscopy (NOESY) allowed sequence-specific assignments. 13C resonances were assigned by 2D 1H13C HSQC spectra. All experiments were performed at 298K, except for the NOESY spectra obtained at 278K. The amide proton temperature coefficient was determined by a series of one-dimensional spectra obtained between 278K and 308K. TOCSY and NOESY mixing times were 70 milliseconds and 250 milliseconds, respectively.

[0222] Example 1: Preparation of (S)-(4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrrolidone tert-Butyl pyro[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate

[0223] 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 anhydrous pyridine (9 mL) were added to 150 ml of anhydrous DCM. The mixture was stirred at room temperature overnight. The reaction mixture was then washed with HCl (0.5 N) × 3, saturated NaHCO3 × 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: Linear gradient from 0% to 100% MeCN in H2O in 2 minutes, using an Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, and using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.89 minutes. Yield: 96%, [M+H] exp +:493.5Da. 1H NMR (400MHz, chloroform-d) δ7.81(d,J=9.2Hz,1H),7.45(d,J=2.5Hz,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 = 7.7Hz, 2H), 1.60 (s, 1H), 1.57-1.41 (m, 3H), 1, 21 (s, 10H), 0.99 (t, J = 7.7Hz, 3H), 0.61 (t, J = 7.3Hz, 3H).

[0224] 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

[0225] In a round-bottom flask, 400 mg of (S)-(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)carbonic acid tert-butyl ester (Example 1), 1.6 equivalents of 5-azido-pentanoic acid (193 mg) were purged with N2 and then dissolved in anhydrous DCM. The mixture was cooled at 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 hour and at room temperature overnight. The mixture was washed with saturated NaHCO3×3, HCl (0.1N)×2, and brine. The organic layer was dried over MgSO4 and removed under vacuum. The compound was used without further purification. [M+H] exp + :618.36Da.

[0226] Example 3: Preparation of (S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran and [3', 4': 6, 7] indolizino [1, 2-b] quinolin-4-yl 5-azidopentanoate (as a result of Example 1-2, with azido Nitride modified SN-38)

[0227] 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 using (DCM / MeOH (10%)) with a purity of more than 95%. Overall yield of Example 1: 8%, [M+H] +: 518.58 Da. Reversed phase UPLC-PDA: linear gradient from 0% to 100% MeCN in H2O in 2 minutes, using an Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, and using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.819 minutes. [M+H] exp + :518.58Da. 1 H-NMR (400MHz, chloroform-d) δ1.00(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).

[0228] Alkyne-Azide Cycloaddition Method: 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. This reaction was performed without microwaves following the procedure described by Lumiprobe (https: / / www.lumiprobe.com / protocols / click-chemistry-dna-labeling). However, this reaction took about 2 days to complete. Cu was used with the ligand THTPA. Because SN-38-N3 is not soluble in H2O, only DMF was used for the reaction instead of buffer and DMSO, because it is not soluble in water or even in the mixture (water / DMSO). Alternatively, the alkyne-azide cycloaddition (click reaction) of alkyne-TTDS-DapKAPETALD with SN-38-N3 was performed using microwaves: alkyne-TTDS-DapKAPETALD (1 eq.), CuTHTPA (0.15 eq.) and sodium ascorbate (0.3 eq.) were added to a microwave vial with 10 ml of SN-38-N3 (1.5 eq.) and dissolved in 3 ml of DMF. During all reactions, the mixture was stirred at 30° C. with the aid of MW (Discover SP MW) for 2 to 4 hours. The crude mixture was purified by semi-preparative HPLC (C18).

[0229] Preparation of reagents for alkyne-azide cycloaddition (Click chemistry):100 mM copper(II)-THPTA stock solution in 55% DMSO: 50 mg copper(II) sulfate pentahydrate was dissolved in 1 mL distilled water and 116 mg tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) ligand was dissolved in 1.1 mL DMSO. The two solutions were then mixed.

[0230] 5 mM ascorbic acid stock solution: Dissolve 18 mg ascorbic acid in 20 mL distilled water.

[0231] General methods used for purification and characterization of products: The crude material was purified by RP-HPLC on a 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, and using MeCN (0.036% TFA) and H2O (0.045% TFA) 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) coupled to an electrospray ionization source ESI-MS Micromass ZQ and using MassLynx 4.1 software (Waters, Milford, MA). BEH C18 column (50×2.1 mm×1.7 μm, Waters). The flow rate was 0.6 mL / min, and MeCN (0.07% formic acid) and H2O (0.1% formic acid) were used as solvents. The samples were analyzed with positive ionization: ion spray voltage was 30 V, and capillary temperature was 1 kV). Exact mass was obtained by mass spectrometry: LTQ-FT Ultra (Thermo Scientific), and the samples were introduced in direct infusion (automated nano-electrospray). NanoMate (Advion BioSciences, Elm City, New York, USA) drew samples from a 384-well plate (protein Lobind) with a disposable conductive pipette tip and passed through a nanoliter electrospray ionization chip (nanoESI The samples were injected into the mass spectrometer using a 400 nozzle chip (consisting of 400 nozzles in a 20×20 array). The spray voltage was 1.70 kV and the delivery pressure was 0.50 psi; ionization was nanoelectrospray ionization, positive ionization. All peptides were obtained with a purity higher than 95%.

[0232] Example 4: Preparation of hexynoic acid-TTDS- having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (hexynoic acid-TTDS - SEQ ID NO: 7).

[0233] To manually couple 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:

[0234]

[0235] 46 μL DIC and 43 mg Oxyma in DMF / DCM (1:1) were used. The mixture was reacted for 45 minutes with intermittent manual stirring. After each coupling, 20% (v / v) piperidine in DMF was used to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group using one 30 second treatment followed by two 10 minute treatments. Two more DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) treatments (2×5 minutes) were performed to ensure removal of the Fmoc group from the secondary amine (proline). Cyclization was performed on the resin following Cyclization Method 2: With 20% (v / v) piperidine in DMF, the Fmoc group was removed using one 30 second treatment and two 10 minute 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). First, the OAl and Alloc groups were deprotected by adding 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). Then, the coupling of the amino group of Dap with the carboxylate group of aspartic acid was achieved by adding PyBOP (4 eq., 400 μmol, 208 mg), HOAt (12 eq., 1.2 mmol, 163 mg), DMF (1 mL-3 mL per gram of resin) and DIEA (12 eq., 1.2 mmol, 204 μL). The coupling was maintained for 1.5 hours and repeated overnight.

[0236] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 eq., 200 μmol, 108.53 mg) was achieved by 4 eq. DIC (400 μmol, 61 μL) and 4 eq. HOBt (400 μmol, 54 mg) in DCM for 2 h. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed using 20% ​​(v / v) piperidine in DMF, using one 30 sec treatment followed by two 10 min treatments.

[0237] Coupling of 5-hexynoic acid: To couple 5-hexynoic acid with the peptide anchored to the resin, the following protocol was used: Hexynoic acid (4 eq., 400 μmol, 45 mg), PyBOP (4 eq., 400 μmol, 208 mg) and HOAt (12 eq., 1.2 mmol, 163 mg) in DMF (1 mL-3 mL per gram of resin) were added sequentially to the resin, followed by 12 eq. of DIEA (1.2 mmol, 204 μL). The mixture was reacted for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The coupling was repeated under the same conditions. The degree of coupling was monitored using the Kaiser colorimetric assay.

[0238] The peptide was then cleaved and lyophilized. Product characterization. Reversed phase UPLC: Linear gradient from 20% to 60% MeCN in H2O in 2 min, using an Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, and 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%.

[0239] Example 5: Preparation of the compound of formula (Ia), namely G2B-001

[0240] Starting with hexynoic acid-TTDS-DapKAPETALD prepared as in Example 4 and using SN-38-N3 prepared as in Example 3, the following protocol was followed to obtain the compound of formula (Ia): 10 ml of 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 to a microwave vial and dissolved in 3 ml of DMF. During all reactions, the mixture was stirred at 30° C. for 2 to 4 hours with the assistance of MW (CEM DiscoverSP MW). The crude material was purified on a semi-preparative scale by RP-HPLC. This afforded the compound with a purity greater than 95%. Product characterization: Reversed phase UPLC-PDA: Linear gradient from 0% to 100% MeCN in H2O in 2 minutes using an Acquity BEH C18 (50×2 mm×1.7 μm) column at 0.61 mL / min and using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.462 minutes. UPLC-MS [M+H] exp + : 1824.76 Da; Yield (synthesis and purification): 30%.

[0241] Example 6: Preparation of hexynoic acid-TTDS- without an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Linear Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (hexynoic acid-TTDS-linear SEQ ID NO: 14). DapKAPETALD (SEQ ID NO: 14), a linear peptide.

[0242] To manually couple 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:

[0243]

[0244] 46 μL DIC and 43 mg Oxyma in DMF / DCM (1:1) were used. The mixture was reacted for 45 minutes with intermittent manual stirring. After each coupling, 20% (v / v) piperidine in DMF was used, using one 30 second treatment followed by two 10 minute treatments to remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Two more DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) treatments (2×5 minutes) were performed to ensure removal of the Fmoc group from the secondary amine (proline).

[0245] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 eq., 200 μmol, 108.53 mg) was achieved with 4 eq. DIC (400 μmol, 61 μL) and 4 eq. HOBt (400 μmol, 54 mg) in DCM for 2 h. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed using 20% ​​(v / v) piperidine in DMF, using one 30 sec treatment followed by two 10 min treatments.

[0246] Coupling of 5-hexynoic acid: To couple 5-hexynoic acid with the peptide anchored to the resin, the following protocol was used: Hexynoic acid (4 eq., 400 μmol, 45 mg), PyBOP (4 eq., 400 μmol, 208 mg) and HOAt (12 eq., 1.2 mmol, 163 mg) in DMF (1 mL-3 mL per gram of resin) were added sequentially to the resin, followed by 12 eq. of DIEA (1.2 mmol, 204 μL). The mixture was reacted for 1.5 h with intermittent manual stirring. The solvent was removed by aspiration and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The coupling was repeated under the same conditions. The degree of coupling was monitored using the Kaiser colorimetric assay.

[0247] Deprotection of OAl and Alloc groups was performed by 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). The peptide was then cleaved and lyophilized. Product characterization. Reverse phase HPLC: linear gradient from 10% to 60% MeCN in H2O in 30 min using an Xbridge 25 cm 3.5 μm column, 1 mL / min, and MeCN (0.1% TFA) and H2O (0.1% TFA) were used as solvents; retention time: 11.526 min. Yield (synthesis and purification): 9%.

[0248] Example 7: Preparation of a compound of formula (Ib) having no amide between the Dap side chain amino group and the Asp side chain carboxylic acid Bonded Straight Chain G2B-001

[0249] Starting with hexynoic acid-TTDS-linear DapKAPETALD prepared as in Example 6 and using SN-38-N3 prepared as in Example 3, the following protocol was followed to obtain the compound of formula (Ib), also named linear G2B-001: 10 ml of 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-linear DapKAPETALD, 0.15 equivalents of CuTHPTA and 0.3 equivalents of sodium ascorbate were added to a microwave vial and dissolved in 3 ml of DMF. During all reactions, the mixture was stirred at 30° C. for 2 to 4 hours with the assistance of MW (CEM Discover SP MW). The crude material was purified by RP-HPLC on a semi-preparative scale. This afforded the compound with a purity greater than 95%. Product characterization: Reverse phase HPLC-MS: Linear gradient from 0% to 80% MeCN in H2O over 7 min using a Luna 3 μm C18(2)100A 50×2.1 mm column, 0.85 mL / min, and MeCN (0.1% formic acid) and H2O (0.1% formic acid) as solvents; retention time: 5.08 min. UPLC-MS [M+H] exp + : 1845.10 Da; Yield (synthesis and purification): 22%.

[0250] Example 8: Preparation of N3-Pen-SN-38

[0251]

[0252] In a two-necked round-bottom flask, 1 equivalent of SN-38 (600 mg) was added and dissolved in anhydrous DCM (60 ml) at 100 ml / g. A turbid solution was observed. This reaction mixture was cooled to 0°C-2°C, and 6 equivalents of DIEA (2 ml) were added and stirred for 15 minutes. After 15 minutes, 3 equivalents of N3-Pen-Cl (700 mg) [dissolved in 500 μL anhydrous DCM] were added portionwise. It was cooled for 15 minutes and then continued to stir at room temperature. The progress of the reaction was checked by HPLC. When the starting material was less than 3%, the reactants were post-processed. The initial volume of the reaction mixture was ~45 ml, which was further supplemented to 300 ml using anhydrous DCM. It was extracted three times with distilled water (20% volume of the final volume of the reaction mixture), 3×3 minutes×60 ml. The DCM layer was dried for 30 minutes using sodium sulfate (Na2SO4) and evaporated on a rotary evaporator. The solid obtained was treated twice with 80:20 hexane:ether and kept in a desiccator to dry. The yield after workup was 730 mg. Product characterization. Reverse phase HPLC: linear gradient from 10% to 90% MeCN in H2O in 30 minutes, using a SS column 250×4.6 mm filled with C-18 silica gel for chromatography R (5 μm), 1 mL / min, and MeCN (0.1% TFA) and H2O (0.1% TFA) were used as solvents; retention time: 19.730 minutes. Observed mass = 518.20 Da, calculated mass = 517.19 Da.

[0253] Example 9: Preparation of the compound of formula (Ic), namely G2B-003

[0254] Starting with hexynoic acid-TTDS-DapKAPETALD prepared as in Example 4 and using N3-Pen-SN-38 prepared as in Example 8, the following protocol was followed to obtain the compound of formula (Ic), which was also named G2B-003. 10 ml of 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 to a vial and dissolved in 3 ml of DMF. During all reactions, the mixture was stirred at 30 ° C for 2 to 4 hours with the assistance of a microwave (CEM Discover SP MW). The crude material was purified on a semi-preparative scale by RP-HPLC. This obtained the compound with a purity higher than 95%. Product characterization: Reverse phase HPLC: linear gradient from 20% to 70% MeCN in H2O in 30 min using Xbridge 25 cm 3.5 μm column, 1 mL / min, and MeCN (0.1% TFA) and H2O (0.1% TFA) as solvents; retention time: 12.00 min. UPLC-MS [M+H] exp + : 1826.15 Da; Yield (synthesis and purification): 17%.

[0255] Example 10: Preparation of SN-38-O-CO-NH-Gly-COOH

[0256] A solution of glycine-COOBut (1 equivalent) and DMAP (2 equivalents) in anhydrous DCM (15 mL) was added dropwise to a solution of bis(4-nitrophenyl) carbonate (1.3 equivalents) in anhydrous DCM (15 mL), and the resulting solution was stirred at 50 ° C overnight. Then, the reaction mixture was diluted in DCM (150 mL) and washed with 0.5N HCl (100 mL). The aqueous layer 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 equivalent) was reacted with SN-38 (1.2 equivalents) in the presence of DMAP (2 equivalents) in anhydrous DCM. The reactant was stirred at 50 ° C overnight. The solvent was evaporated under reduced pressure, and the residue was purified by flash chromatography. The tert-butyl ester group was removed by treating SN-38-O-CO-NH-Gly-COOBut with TFA:DCM:TIS (40:40:20) during 6 hours. Then, the solvent was evaporated. Toluene was added twice to remove traces of TFA. No further purification was performed.

[0257] Example 11: Preparation of Boc-Val-Cit-PAB-SN-38.

[0258] 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 anhydrous DMF at room temperature for 16 hours. Then, the reaction mixture was diluted with AcOEt and washed with HCl (0.5M) (×3) and brine (×4). Then, it was dried with 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 with a yield of 52% and a purity of more than 90%. Yield 52%. Reversed phase UPLC-PDA: linear gradient from 0% to 100% MeCN in H2O in 2 min, using an Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, and using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.42 min. [M+H] exp + :898.4Da.

[0259] Example 12: Preparation of H2N-Val-Cit.PAB-SN-38

[0260] In a round bottom flask, 1 equivalent of Boc-Val-Cit-PAB-SN-38 prepared as in Example 11 was stirred in a mixture of DCM:TFA:H20 (44.75:49.75:0.5) for 5 minutes. Then, the solvent was evaporated. Toluene was added twice to remove traces of TFA. No further purification was performed.

[0261] Example 13: Preparation of G2B-004 (Id).

[0262] G2B-004 was prepared starting with diglycolic acid-DapKAPETALD (prepared using the standard method described above, i.e., coupling method 4) and 2 equivalents of H2N-Val-Cit-PAB-SN-38 (prepared as in Example 12) using PyBOP (4 equivalents), HOAt (12 equivalents) dissolved in DMF (1 mL-3 mL per gram of resin), followed by the addition of DIEA (12 equivalents). The mixture was reacted for 1 hour with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 seconds) and DCM (5×30 seconds). The coupling reaction was carried out twice (1 hour and overnight). G2B-004 was then cleaved and lyophilized. Product characterization. Reversed phase UPLC: linear gradient from 0% to 100% MeCN in H2O in 2 min, using an Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, and using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.17 min. UPLC-MS [M+H] exp + : 1807.84 Da. Yield (synthesis and purification): 21%.

[0263] Example 14: Preparation of G2B-005 (Ie)

[0264] Nval-Pro-Gly-DapKAPETAL1D was prepared using the standard method described above (Coupling Method 4). G2B-005 was prepared starting with Nval-Pro-Gly-DapKAPETALD and 2 equivalents of SN-38-O-CO-NH-Gly-OH prepared as in Example 10 using PyBOP (4 equivalents), HOAt (12 equivalents) dissolved in DMF (1 mL-3 mL per gram of resin), followed by the addition of DIEA (12 equivalents). The mixture was reacted for 1 hour with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 seconds) and DCM (5×30 seconds). The coupling reaction was carried out twice (1 hour and overnight). G2B-005 was then cleaved and lyophilized. Product Characterization. Reversed phase UPLC: linear gradient from 0% to 100% MeCN in H2O in 2 minutes, using an Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, and using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents; retention time: 1.51 minutes. UPLC-MS [M+H] exp + : 1640.77 Da. Yield (synthesis and purification): 18%.

[0265] Example 15: Preparation of Several Micellar Aqueous Solutions Containing SN-38 Lactone Encapsulated in Micelles of SN-38 Peptide Conjugates Dispersion.

[0266] All formulations were simple mixtures of an alkaline aqueous solution of free SN-38 carboxylate (0.05 N NaOH 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 the two components in the alkaline and acidic solutions could be varied. Mixing equal volumes of alkaline and acidic solutions resulted in an acidic solution containing SN-38 lactone encapsulated in G2B-001 micelles that had a pH <7 (experimental values ​​were 2.3-2.5), was completely translucent over time, and was physically stable (no precipitate or turbidity after storage at room temperature for several weeks). A diagram of this process is shown in Figure 1 middle.

[0267] Example 15a: Preparation of G2B-001 micelles at a concentration of 20 mg / mL and containing 16% w / w free SN-38 lactone. This formulation was designated G2B-002-20-16.

[0268] In this formulation, the weight / weight percentage (% w / w) of free SN-38 in the final formulation was 16% w / w, i.e., free SN-38 / (SN-38+G2B-001)×100=16%. The final concentration of free SN-38 in the aqueous formulation was 4 mg / mL and the final concentration of G2B-001 was 20 mg / mL, and the final pH was <3, ensuring that the free SN-38 was primarily in the form of the active lactone. The particle size was 310 nm (Z-average), and the polydispersity index (PDI) was 0.55 (measured by dynamic light scattering).

[0269] Example 15b: Preparation of G2B-001 micelles at a concentration of 20 mg / mL and containing 9% w / w free SN-38 lactone. One preparation is referred to as G2B-002-20-9.

[0270] In this formulation, the weight / weight percentage (% w / w) of free SN-38 in the final formulation was 9% w / w, i.e., free SN-38 / (SN-38+G2B-001)×100=9%. The final concentration of free SN-38 in the aqueous formulation was 2 mg / mL and the final concentration of G2B-001 was 20 mg / mL, and the final pH was <3, ensuring that the free SN-38 was primarily in the form of the active lactone. The particle size was 103 nm (Z-average), and the polydispersity index (PDI) was 0.50 (measured by dynamic light scattering). The solution was transparent.

[0271] Example 15c: Preparation of G2B-001 micelles at a concentration of 10 mg / mL and containing 5% w / w free SN-38 lactone. One preparation is called G2B-002-10-5 .

[0272] In this formulation, the weight / weight percentage (% w / w) of free SN-38 in the final formulation was 5% w / w, i.e., free SN-38 / (SN-38+G2B-001)×100=5%. The final concentration of free SN-38 in the aqueous formulation was 0.5 mg / mL and the final concentration of G2B-001 was 10 mg / mL, and the final pH was <3, ensuring that the free SN-38 was primarily in the form of the active lactone. The particle size was 40 nm (Z-average), and the polydispersity index (PDI) was 0.40 (measured by dynamic light scattering).

[0273] The following table shows the amount of SN38 in the peptide and the amount of free SN-38 lactone:

[0274]

[0275] Example 15d: Preparation of G2B-003 micelles at a concentration of 20 mg / mL and containing 5% w / w free SN-38 lactone. One preparation is referred to as G2B-006-20-9.

[0276] It was prepared as G2B-002-20-9, but with a peptide conjugate of formula (Ic) instead of a peptide conjugate of formula (Ia). In this formulation, the weight / weight percentage (% w / w) of free SN-38 in the final formulation was 9% w / w, i.e., free SN-38 / (SN-38+G2B-003)×100=9%.

[0277] Example 16: Loading of soluble SN-38 lactone in micellar system

[0278] In order to study the loading efficiency of soluble SN-38 lactone in the system of the present invention, G2B-001 or G2B-003 micelles at a concentration of 40 mg / mL in an acidic solution (0.5 mL) were added to SN-38 carboxylate at a concentration of 4 mg / mL or 8 mg / mL in an alkaline solution (0.5 mL) in a plastic vial (1.5 mL). After gentle mixing, the sample was kept at room temperature (18°C-22°C) for 0.5 hours. The resulting solution was then filtered through a 0.45 μm polypropylene syringe filter to remove insoluble SN-38 lactone crystals. The concentration of soluble SN-38 lactone in the filtered solution was determined using a high performance liquid chromatograph (Shimazdu) with a fluorescence detector. The loading efficiency (LE) of soluble SN-38 lactone was calculated according to the following formula:

[0279] LE=C / L×100

[0280] 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 product is Table 2The products named G2B-002-20-9 and G2B-006-20-9 obtained soluble SN-38 lactone at a final concentration of 2 mg / mL after filtration, and the loading efficiency was close to 100% in both cases. (Table 2) The same process in the absence of G2B-001 or G2B-003 micelles produced insoluble SN-38 lactone in the presence of vehicle alone. (Table 2) Similarly, the same process produced insoluble SN-38 lactone in the presence of irinotecan at a concentration of 20 mg / mL in acidic solution. (Table 2) .

[0281] Table 2. SN-38 lactone solubilization parameters for G2B-001 or G2B-003 solutions with a final pH of 2.3-2.6

[0282]

[0283] When comparing it with the solubility data of SN-38 liposomes or micelles disclosed in the prior art, it is also seen that the solubility data of the micelles of the present invention is also higher.

[0284] Example 17: Visual inspection of turbidity of micellar system

[0285] To investigate the physical stability of the products of the present invention, G2B-002-20-9 and G2B-006-20-9, both of which carried a final concentration of 2 mg / mL of soluble SN-38 lactone, the present inventors performed a visual inspection of the products. Table 3 Turbidity data after visual inspection of the products are summarized. For comparison, the inventors used products obtained in the absence of G2B-001 or G2B-003 micelles, consisting of 20 mg / mL SN-38 in linear G2B-001, SN-38 alone in vehicle, or 20 mg / mL irinotecan and SN-38 in vehicle, as described in the Example “Loading of soluble SN-38 lactone in micellar system”.

[0286] Table 3. Visual inspection of the turbidity of the micelle system loaded with SN-38 lactone of G2B-001 or G2B-003 solution containing 2 mg / mL theoretical loading amount of SN-38 lactone

[0287]

[0288] nd: Not tested.

[0289] Figure 2-Figure 5Photographs of products G2B-002-20-9 and G2B-006-20-9 or alternative products obtained in the absence of G2B-001 micelles consisting of SN-38 alone in vehicle or 20 mg / mL irinotecan and SN-38 in vehicle, as described in Example “Loading of soluble SN-38 lactone in micellar system” are shown. Products G2B-002-20-9 and G2B-006-20-9 are not turbid, while products including SN-38 alone in vehicle as well as irinotecan and SN-38 show turbidity corresponding to insoluble SN-38 lactone crystals. Immediately after preparation ( Figure 2-Figure 3 ) or up to 6 months after preparation ( Figure 4-Figure 5 ) Photographed. At 1 week to 4 weeks, the product of SN-38 included in the linear G2B-001 was not turbid.

[0290] Example 18: Visual inspection of turbidity of micellar system carrying water-insoluble camptothecin

[0291] In order to study whether the system of the present invention can carry products other than SN-38, the inventors selected camptothecin of the following formula:

[0292]

[0293] Like SN-38, camptothecin is extremely insoluble as a lactone at acidic pH but is readily soluble as a carboxylate at basic pH.

[0294] In a plastic vial (1.5 mL), G2B-001 micelles at a concentration of 40 mg / mL in an acidic solution (0.5 mL) were added to camptothecin carboxylates at concentrations of 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL in an alkaline solution (0.5 mL). After gentle mixing, the sample was kept at room temperature (18° C.-22° C.). The final pH of the preparation was acidic (pH=1.5) to ensure conversion to camptothecin lactone. Immediately after preparation and after 24 hours, the inventors performed a visual inspection of the product. Table 4 summarizes the turbidity data after visual inspection of the product. For comparison, the inventors used the product obtained in the absence of G2B-001 micelles, which was composed only of camptothecin lactone in the vehicle.

[0295] Table 4. Visual inspection of the turbidity of the micellar system of G2B-001 containing camptothecin lactone (CPT) with a theoretical loading (CPT concentration) of 0.25 mg / mL to 1 mg / mL

[0296]

[0297] Figure 6Photographs of the products detailed in Table 4 are shown. Products G2B-CPT-20-5, G2B-CPT-20-2, and G2B-CPT-20-1 were not turbid, whereas the product including only CPT in the vehicle showed turbidity corresponding to insoluble CPT crystals. The photographs were taken immediately after preparation.

[0298] Example 19: Visual inspection of turbidity of micellar system carrying SN-38 lactone and water-insoluble camptothecin

[0299] In order to investigate whether the system of the present invention can carry SN-38 and camptothecin simultaneously, the present inventors prepared G2B-001 micelles at a concentration of 40 mg / mL in an acidic solution (0.5 mL) in a plastic vial (1.5 mL), to which camptothecin carboxylate at a concentration of 2 mg / mL or 4 mg / mL in an alkaline solution (0.25 mL) and SN-38 carboxylate at a concentration of 2 mg / mL or 4 mg / mL in an alkaline solution (0.25 mL) were added.

[0300] After gentle mixing, the sample was kept at room temperature (18°C-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, the inventors performed a visual inspection of the product. Table 5 summarizes the turbidity data after visual inspection of the product. For comparison, the inventors used the product obtained in the absence of G2B-001 micelles, which was composed only of SN-38 and camptothecin lactone in the vehicle.

[0301] Table 5. Visual inspection of the turbidity of micellar systems of G2B-001 containing SN-38 and camptothecin lactone (CPT) at theoretical loadings (SN-38 and CPT concentrations) of 0.5 mg / mL and 1.0 mg / mL

[0302]

[0303] Figure 7 Photographs of products formulated with G2B-001 or obtained in the absence of G2B-001 micelles detailed in Table 5, which are composed solely of SN-38 and CPT in the vehicle, are shown. Products G2B-SN38-CPT-20-5-5 and G2B-SN38-CPT-20-2-2 are not turbid, whereas products including only SN-38 and CPT in the vehicle show turbidity corresponding to insoluble SN-38 and CPT crystals. The photographs were taken immediately after preparation.

[0304] Example 20: Stability of the lyophilized product G2B-002-20-9.

[0305] In order to study 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 soluble SN-38 lactone) was frozen in liquid nitrogen and lyophilized in a Telstar device (LyoQuest). After lyophilization, the dried sample was suspended in the same volume of water (280 μL). The resulting solution did not show turbidity. The soluble SN-38 lactone was analyzed by HPLC, indicating that the soluble SN-38 lactone had a recovery rate of 100.6±7.8% (average and standard deviation of 4 samples prepared independently). Therefore, the product G2B-002-20-9 of the present invention is stable after lyophilization and reconstitution in water.

[0306] Example 21: In vitro activity of product G2B-002-20-9

[0307] The activity of the product G2B-002-20-9 carrying soluble SN-38 lactone was compared with that of 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 gliomas, including diffuse intrinsic pontine glioma (HSJD-DIPG-007), pediatric diffuse midline glioma (HSJD-DMG-001), and pediatric high-grade glioma (HSJD-GBM-001) cancer types, and cell lines of other pediatric solid tumors, such as Ewing sarcoma cell lines (A673) and rhabdomyosarcoma cell lines (RH4). Briefly, between 3000 and 30,000 cancer cells were cultured in 96-well plates, and 24 hours later, the cells were exposed to the compounds G2B-002-20-9 (soluble SN-38 lactone at concentrations ranging from 10 μM to 0.0000001 μM), SN-38 lactone (at concentrations ranging from 10 μM to 0.0000001 μM), or irinotecan (at concentrations ranging from 100 μM to 0.000001 μM). To add the drugs to the cells in culture, G2B-002-20-9, irinotecan, and SN-38 were prepared from stock solutions in culture medium. The cell viability after 72 hours of incubation with drugs was determined using a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS assay; Promega, Fitchburg, WI). The drug concentration required to cause a 50% decrease in cell proliferation (IC50 and 95% confidence interval) was calculated using Graphpad Prism 8 software (La Jolla, CA). The results are expressed as a percentage of cell viability compared to untreated control wells. The activity of G2B-002-20-9 carrying the SN-38 lactone was comparable to that of the free SN-38 lactone and superior to that of irinotecan in all cell lines tested. Figure 8 Results for HSJD-DIPG-007 cells are shown. Fig. 9 Results for HSJD-DMG-001 cells are shown. Fig.10 Results for HSJD-GBM-001 cells are shown. Fig.11 Results for RH4 cells are shown. Fig.12 Results for A673 cells are shown.

[0308] Example 22: Antitumor efficacy of the products of the invention in DIPG and pHGG xenografts

[0309] The product G2B-002-20-9, administered intravenously in mice bearing intracranial human brain tumors, was compared to the activity of the closely related drug irinotecan. The inventors established brain tumor xenografts in immunodeficient athymic nude mice by intracranial injection of half a million cells in 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 with mutations H3.3-K27M and ACVR1-R206H. Xenograft HSJD-GBM-001 is a pHGG-related model with wild-type H3.3 and mutation p.G245S in gene TP53. The goal of this experiment is to study whether the activity of the new compound G2B-002-20-9, which carries soluble SN-38 lactone and penetrates the blood-brain barrier, is better than the activity of irinotecan, which is metabolized to SN-38 lactone by carboxylesterase after administration to the mouse blood and does not significantly penetrate 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 through the tail vein. Treatment was started 31 days after inoculation of HSJD-DIPG-007 cells and 8 days after inoculation of HSJD-GBM-001 cells. For mice bearing HSJD-DIPG-007, G2B-002-20-9 was treated on days 1, 2, 3, 4, 5, 8, 9, 10, 11, and 12, whereas for mice bearing HSJD-GBM-001, treatment days were 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 22, 23, 24, 25, and 26. For mice bearing HSJD-DIPG-007, irinotecan was treated on days 1, 2, 3, 4, and 5, whereas for mice bearing HSJD-GBM-001, treatment days were 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 22, 23, 24, 25, and 26. For mice bearing HSJD-DIPG-007 tumors, treatment groups received 10 mg / kg G2B-002-20-9 (soluble SN-38 lactone dose), 40 mg / kg irinotecan, or control (saline solution). A minimum of eight mice received each treatment. For mice bearing HSJD-GBM-001 tumors, treatment groups received 10 mg / kg G2B-002-20-9 (soluble SN-38 lactone dose), 10 mg / kg irinotecan, or saline control (saline solution). Seven mice received each treatment. G2B-002-20-9 was safely administered in experiments with mice bearing HSJD-DIPG-007.During the treatment period, after 10 doses of G2B-002-20-9, the body weight loss was -7.3±3.5% (mean and standard deviation), and there was no toxic death. Treatment with 40 mg / kg irinotecan caused one toxic death and -16.4±9.4% body weight loss. After stopping treatment, the survival curve obtained in the study is shown in. Fig.13 The survival time of mice treated with G2B-002-20-9 (median survival = 73 days) was significantly longer than that of control mice (median survival = 67 days; P = 0.0344) and irinotecan (median survival = 65.5 days; P = 0.0009), while irinotecan did not improve animal survival compared with controls (P = 0.5851). In the experiment with mice carrying HSJD-GBM-001, all mice tolerated the treatment and did not lose significant weight. After stopping treatment, the survival curves obtained in the study are shown in Fig.14 The survival time of mice treated with G2B-002-20-9 (median survival = 50 days) was significantly longer than that of 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 with controls (P = 0.0020). Overall, these experiments demonstrate that G2B-002-20-9 is superior to irinotecan in treating mice bearing intracranial DIPG and pHGG.

[0310] Example 23: Anti-tumor efficacy of the product of the invention in subcutaneous xenografts derived from patients

[0311] The inventors compared the activity of the novel compounds G2B-002-10-5, G2B-002-20-9 and G2B-002-20-16 administered intravenously to mice bearing subcutaneous human tumors with that of the closely related drug irinotecan. Patient-derived xenograft (PDX) tissue was obtained from immunodeficient athymic nude mice at Hospital Sant Joan de Deu (Barcelona, ​​Spain). PDX were established from biopsies of pediatric patients with Ewing sarcoma, neuroblastoma, osteosarcoma and rhabdomyosarcoma as detailed in one publication (1). PDX are identified in Table 6 middle.

[0312] Table 6 Identification of PDX models to include in the study of anti-tumor efficacy of novel compounds

[0313]

[0314] The goal of this experiment was to investigate whether the activity of the novel product carrying SN-38 lactone is comparable to that of irinotecan 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. When the volume of the subcutaneous tumor was 100 mm 3 -300mm 3 Treatment was started when the tumor was within the range of 10 mg / kg. Treatment days were 1, 2, 3, 4, 5, 8, 9, 10, 11 and 12. We included seven treatment groups in the study: 10 mg / kg G2B-002-20-16, 1 mg / kg G2B-002-20-16, 10 mg / kg G2B-002-20-9, 1 mg / kg G2B-002-20-9, 1 mg / kg G2B-002-10-5, 10 mg / kg irinotecan and saline control (normal saline solution). One mouse received each treatment. All mice tolerated the treatment and had no significant weight loss. On day 15, the inventors 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 had significantly reduced volumes compared with control tumors treated with saline (P = 0.0001; paired one-way ANOVA with Dunnett's multiple comparison test) ( Fig.15 ). Overall, these experiments demonstrated that G2B-002-20-16, G2B-002-20-9, and G2B-002-10-5 were similar to irinotecan in treating mice bearing extracranial Ewing sarcoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma.

[0315] Example 24: Expression of SN-38 lactone in the mouse retina following intravenous administration of G2B-002-20-9 and irinotecan Distribution in

[0316] The inventors evaluated the distribution of SN-38 lactone in the retina after intravenous administration of G2B-002-20-9 or 10 mg / kg irinotecan at a dose sufficient to provide 10 mg / kg and 1 mg / kg free SN-38 lactone. The inventors intravenously administered G2B-002-20-9 in an amount sufficient to provide 10 mg / kg or 1 mg / kg free SN-38 lactone at each dose level in 18 athymic nude mice, and intravenously administered 10 mg / kg irinotecan in another 12 mice. At 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours and 8 hours in the group receiving G2B-002-20-9 and at 0.25 hours, 0.5 hours, 1 hour and 2 hours in the group receiving irinotecan, they killed the animals by decapitation (three at each time point), dissected the retinas and cryopreserved the samples until analysis using published methods.

[0317] Following administration of a dose of G2B-002-20-9 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), which was achieved at the 0.5 hour time point. For this dose level, the concentration of SN-38 lactone remained above 100 ng / g for at least 4 hours ( Fig.16 ).

[0318] Following administration of a dose of G2B-002-20-9 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), which was achieved at the 0.5 hour time point. For this dose level, the concentration of SN-38 lactone remained above 100 ng / g for at least 0.5 hours ( Fig.16 ).

[0319] After administration of 10 mg / kg irinotecan, the concentration 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 ng / g-72 ng / g), which was achieved at the 0.25 hour time point. After 2 hours, SN-38 lactone in this group was below the detection limit (5 ng / g) ( Fig.16 ).

[0320] Overall, these experiments demonstrate that the novel invention improves the retinal distribution of soluble SN-38 lactone.

[0321] Example 25: In vitro G2B-002-20-9 micelle transport across the human BBB model.

[0322] It is well described that irinotecan and SN-38 cannot cross the BBB in vivo. (Pharmaceutics 2020, 12 399 and Cancer Res 1993, 53(12) 2823-9.) All compounds were evaluated at 100 μM in Ringer Hepes using an in vitro BBB human model. The time assay was 2 hours at 37°C. Receptors and time 0 were analyzed by UPLC. All compounds were evaluated by triplicate and 25 μM fluorescein yellow (LY). As mentioned in the article ACIE, 2015, 3967, when Papp permeability is higher than that of natural ligands such as Tf 0.953 10 -6 When the Papp permeability is 1 / 2, then transport is considered relevant. Often times, the Paap of BBB shuttle-cargo conjugates is lower than the Paap of the BBB shuttle alone because they are transporting cargo. Results are shown in Fig.17 middle.

[0323] Example 26: Expression of SN-38 lactone in the brain and cerebrum of mice following intravenous administration of G2B-002-20-9 and irinotecan Distribution in cerebrospinal fluid

[0324] The inventors evaluated the distribution of SN-38 lactone in the brain and cerebrospinal fluid (CSF) one hour after intravenous administration of G2B-002-20-9 or 40 mg / kg irinotecan at a dose sufficient to provide 10 mg / kg free SN-38 lactone. The inventors intravenously administered G2B-002-20-9 in an amount sufficient to provide 10 mg / kg free SN-38 lactone in 4 athymic nude mice and intravenously administered 40 mg / kg irinotecan in another 4 mice. One hour later, they anesthetized the mice and obtained 5 μL of CSF using a glass capillary. They then killed the animals by decapitation, dissected the brains, and stored the samples frozen until analysis using published methods. In the brain, SN-38 lactone could not be detected one hour after administration of 40 mg / kg irinotecan, i.e., its concentration was below the quantification limit of 5 ng / g. After administration of G2B-002-20-9, the average SN-38 concentration in the brain was 33 ng / g (range 13 ng / g-60 ng / g). In CSF, after administration of 40 mg / kg irinotecan, the average concentration of SN-38 lactone reached 0.5 ng / mL (range 0.2 ng / mL-0.8 ng / mL). After administration of G2B-002-20-9, the median concentration of SN-38 lactone reached 61 ng / mL (range 33 ng / mL-97 ng / mL). Overall, these experiments show that the new invention improves the CNS distribution of soluble SN-38 lactone. In the case of CSF, the distribution of SN-38 lactone increased by about 100 times.

[0325] Citation list

[0326] Patent Literature

[0327] -EP2644191B1

[0328] -US8299089B2

[0329] - WO2015 / 051307A1

[0330] -CN10206099A1

[0331] -CN110124052A

[0332] Non-patent literature

[0333] -Sosnik, Drug self-assembly: A phenomenon at the nanometer scale with major impactin the structure-biological properties relationship and the treatment of disease, Prog Mater Sci, 2016, Vol. 82, pp. 39-82

[0334] - F Meyer-Losic et al., "DTS-108, A novel Peptidic prodrug of SN-38: In vivo Efficacy and Toxicokinetic Studies", Clinical Cancer Research, 2008, Vol. 14, No. 7, pp. 2145-2153

[0335] -A. Buckin et al., "Amphiphilic Polymeric Nanoparticles Modified with a Protease-Resistant Peptide Shuttle for the Delivery of SN-38in Diffuse Intrinsic Pontine Glioma", ACS Applied Nano Materials, Vol. 4, No. 2, 2021, pp. 1314-1329

[0336] -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

[0337] -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

[0338] - 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

[0339] - MJ Hatfield and PM Potter, Exp. Opin. Ther. Pat., 2011, Vol. 21(8), pp. 1159-1171

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[0342] - Greene, Protective Groups in Organic Synthesis, 5th edition, Peter GMWuts. 2014 John Wiley & Sons, Inc., published by John Wiley & Sons, Inc. in 2014, pp. 26-29, 271-279, 456-463, 69-77 and 371-374

[0343] - M. Kazemi and L. Shiri, Journal of Sulfur Chemistry, 2015, Vol. 36:6, pp. 613-623.

[0344] -MT Nguyen et al., J. Org. Chem., 1998, 63, Vol. 20, pp. 6878-6885.

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[0347] - JH Lee et al., Eur. J. 30 Biochem., 2001, Vol. 268, pp. 2004-2012

[0348] - M. Amblard et al., "Methods and protocols of modern solid-phase peptide synthesis", Molecular Biotechnology, 2006, Vol. 33, pp. 239-254

[0349] -Journal of Medicinal Chemistry (J. Med. Chem.), 2008, 51, 21, 6916-6926.

Claims

1. A micelle comprising a SN-38 peptide conjugate and one or more free therapeutically active agents having anticancer activity, wherein: The micelle is a core-shell structure comprising an inner core and an outer shell, wherein the free therapeutically active agent is loaded 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, (Z)-(L)-P-(W) s –(Y) (I) in: Z is a free 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 connected to the linker L through only one of the two hydroxyl groups (a) or (b) of the pharmaceutically active ingredient; L is a linker, which is a diradical composed of 2 to 8 diradicals L' and has the following formula: -L' a -(L' b ) n -L' c- ; L a ' is a diradical selected from the group consisting of: -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-; L8-L 11 The substitution in any of may be at any position of the ring; L b ' is a diradical independently selected from the group consisting of: -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 -S-; and combinations thereof; L c ' is a diradical selected from the group consisting of: -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)-; P is a diradical of a peptide selected from the group consisting of: (a) a peptide comprising an amino acid sequence X1KAPETALX2, wherein the amino acid sequence has an intrapeptide bond between X1 and X2, wherein the intrapeptide bond is an amide bond; wherein 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); that is, SEQ ID NO:1: (b) a peptide having a length of 12-20 amino acid residues, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond or a diselenide bond, and the peptide comprising the following amino acid sequence: X3KAPETALX4AAA; the amino acid sequence having at least one intrapeptide disulfide bond or a diselenide bond between X3 and X4, wherein X3 and X4 are identical and are selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine); SEQ ID NO:2: (c) a peptide of 9-11 amino acid residues in length, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond or a diselenide bond, and the peptide consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6, X5KAPETALX6A and X5KAPETALX6AA, the amino acid sequence having at least one intrapeptide disulfide bond or a diselenide bond between X5 and X6; wherein X5 and X6 are identical and selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine); i.e. SEQ ID NO:3: SEQ ID NO:4: SEQ ID NO:5: (d) having 16 amino acid residues and comprising the amino acid sequence X7NX8KAPETALX9AAAX 10 H peptide, the amino acid sequence is between X7 and X9 and between X8 and X 10 There is an intrapeptide disulfide bond or a diselenide bond between them; wherein X7-X 10 independently selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine); provided that X7 and X9 are identical and X8-X 10 is equivalent, that is SEQ ID NO:6: and (e) a peptide comprising an amino acid sequence X1KAPETALX2 (SEQ ID NO: 7), wherein 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), the peptide being a linear peptide; W is a diradical selected from the group consisting of: -NH-(CH2) r -C(=O)- and -NH-CH((CH2) r NH2)-C(=O)-; Y is a free 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 connected to the free radical Z via a bond selected from the group consisting of an ester bond, an ether bond, a urethane bond, a silyl ether bond, a sulfonate bond, a phosphate bond, a ketal bond, a hemiketal bond, a carbonate bond and a urethane bond, the bond being in the L drawn a 'The C=O, SO2, Si, P, CH or CH2 group on the left side of the formula is formed with one of the hydroxyl groups of the SN-38; When n = 0, L a ' connected to the free radical L through a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond c ', the key is drawn in the L a The functional group on the right side of the formula is c 'Formed between the functional groups on the left side of the formula; When n = 1, L a ' connected to the free radical L through a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide ester bond and a thioester bond b ', the key is drawn in the L a The functional group on the right side of the formula is b ' is formed between the functional groups on the left side of the formula; and L b ' connected to the free radical L through a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond c ', the key is drawn in the L b The functional groups on the right side of the formula are similar to the L c 'Formed between the functional groups on the left side of the formula; When n is greater than 1, L b ' are identical or different and are connected by a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond and a thioester bond; one L b 'Connected to L at the terminal end via a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond, and a thioester bond a ', the key is drawn in the L a The functional groups on the right side of the formula are similar to the L b 'formed between the functional groups on the left side of the formula; and another L b 'Connected to L at the terminal end via a chemically feasible bond selected from the group consisting of an amine bond, an amide bond, an ether bond, a thioether bond, a disulfide bond, an ester bond, and a thioester bond c ', the key is drawn in the L b The functional groups on the right side of the formula are similar to the L c 'Formed between the functional groups on the left side of the formula; L c 'By using the L drawn c 'The carbonyl group on the right side of the formula and the amino group of the first amino acid of the peptide sequence P form an amide bond to connect to the diradical P; When s=0, P is directly linked to Y via an amide bond, a carboxylic acid bond or an ester bond, the bond being formed between the C=O at the C-terminus of the last amino acid of the sequence P and the free radical Y, the free radical Y being -NH2, -OH, -OR3 or -NHR3; and When s=1, P is linked to the free radical W via an amide bond formed with the C=O at the C-terminus of the last amino acid of the sequence P, the bond being formed between the functional group on the left side of the drawn formula of W and the functional group (C=O) at the C-terminus of the last amino acid of the sequence P on the right side of the drawn sequence; and W is linked to Y as follows: -C(=O)-NH-(CH2) r -C(=O)-Y or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y.

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

3. The micelle according to any one of claims 1 to 2, wherein In the SN-38 peptide conjugate of formula (I), P is a diradical of a peptide selected from the group consisting of: (a) a peptide comprising an amino acid sequence DapKAPETALD, wherein the amino acid sequence has an intrapeptide bond between Dap and D, wherein the intrapeptide bond is an amide bond, and the amino acid sequence is SEQ ID NO: 8: (b) a peptide having a length of 9 to 20 amino acid residues, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond, and the peptide comprising the following amino acid sequence: CKAPETALCAAA, the amino acid sequence having at least one intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, the amino acid sequence being SEQ ID NO: 9: (c) a peptide of 9-11 amino acid residues in length, the peptide having at least one intrapeptide bond, the intrapeptide bond being a disulfide bond, and the peptide consisting of an amino acid sequence selected from the group consisting of CKAPETALC, CKAPETALCA and CKAPETALCAA, the amino acid sequence having at least one intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, the amino acid sequence being SEQ ID NO: 10: SEQ ID NO:11 SEQ ID NO:12) (d) a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH, which has intrapeptide disulfide bonds between the first and third cysteine ​​residues, which are cysteine ​​1 and cysteine ​​11, and between the second and fourth cysteine ​​residues, which are cysteine ​​3 and cysteine ​​15, the amino acid sequence being: SEQ ID NO:13: and (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

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

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

6. The micelle according to any one of claims 1 to 5, wherein In the SN-38 peptide conjugate of formula (I), L a ' is a diradical selected from the group consisting of: -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 .

7. The micelle according to any one of claims 1 to 6, wherein In the SN-38 peptide conjugate of formula (I), L is a linker selected from the group consisting of: a)L a ' is L3, L b 'Selected from -NH-(CH2) r -O-, -(CH2) r -O- and -(CH2) r -NH- and their combination, and L c ' is -C(=O)-(CH2) r -C(=O)-, b) A diradical composed of two diradicals, n=0, L a 'For L 12 , and L c 'For L 13 ;and c) A diradical composed of two diradicals, n=0, L a ' is -C(=O)-NH-(CH2) r -C(=O)-, and L c 'For L 15 .

8. The micelle according to claim 7, wherein The SN-38 peptide conjugate of formula (I) is a compound selected from the group consisting of: Compounds of formula (Ia): Compounds of formula (Ib): Compounds of formula (Ic): Compound of formula (Id): Compounds of formula (Ie):

9. The micelle according to any one of claims 1 to 8, wherein The micelles are in the form of an aqueous dispersion of micelles according to any one of claims 1 to 8, the free therapeutic anti-cancer drugs being the same or different.

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

11. The micelle according to claim 10, wherein The micelle is selected from the group consisting of; a) micelles of a peptide conjugate of formula (Ia), wherein the inner core is loaded with SN-38 lactone, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of SN-38 lactone is 4 mg / ml; b) micelles of a peptide conjugate of formula (Ia), wherein the inner core is loaded with SN-38 lactone, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of SN-38 lactone is 2 mg / ml; c) micelles of a peptide conjugate of formula (Ia), wherein the inner core is loaded with SN-38 lactone, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 10 mg / ml, and the concentration of SN-38 lactone is 1 mg / ml; d) micelles of a peptide conjugate of formula (Ic), wherein the inner core is loaded with SN-38 lactone, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of SN-38 lactone is 2 mg / ml; e) micelles of the peptide conjugate of formula (Ia), wherein the inner core is loaded with camptothecin lactone, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, and the concentration of camptothecin lactone is 0.25 mg / ml-4 mg / ml; and f) micelles of the peptide conjugate of formula (Ia), wherein the inner core is loaded with camptothecin lactone and SN-38, the concentration of the SN-38 peptide conjugate in the micellar aqueous dispersion is 20 mg / ml, the concentration of the camptothecin lactone is 0.5 mg / ml-1 mg / ml, and the concentration of the SN-38 is 0.5 mg / ml-1 mg / ml.

12. The micelle according to any one of claims 1 to 10, wherein The micelles are freeze-dried micelles.

13. The micelle according to any one of claims 1 to 12, wherein The micelles can be obtained by the following operation: a) the SN-38 peptide conjugate according to any one of claims 1 to 12 spontaneously self-assembles in water at pH <7; b) contacting the micellar acidic solution with a basic solution containing free SN-38 carboxylate at a concentration of 1 mg / ml-12 mg / ml, which forms intermolecular interactions between free SN-38 lactone molecules and SN-38 molecules conjugated in the SN-38 peptide conjugate; and c) Optionally, the micellar solution of step b) is freeze-dried.

14. A pharmaceutical composition, wherein The pharmaceutical composition comprises a therapeutically effective amount of the micelle according to any one of claims 1 to 13 and an appropriate amount of a pharmaceutically acceptable carrier or excipient.

15. Use of the micelle according to any one of claims 1 to 13 as a medicament.

16. Use of the micelle according to any one of claims 1 to 13 in treating cancer in a mammal, including a human, wherein: The cancer is selected from the group consisting of: a) a tumor selected from the group consisting of extracranial solid tumors, eye tumors and CNS tumors; b) a cancer selected from the group consisting of adult glioma, pediatric glioma, retinoblastoma, Ewing sarcoma, DIPG, neuroblastoma, medulloblastoma, ependymoma, atypical teratoid-rhabdoid tumor (ATRT), and rhabdomyosarcoma; c) Pediatric brain tumors; d) Pediatric high-grade glioma; e) diffuse intrinsic pontine glioma (DIPG) tumors; and f) Diffuse midline glioma.

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