Methods and compositions for synthesizing therapeutic nanoparticles

Abstract

Improved methods and reactants are provided for the chemical synthesis of therapeutic nanoparticles. The nanoparticles comprise a polymer core to which one or more homing molecules and one or more therapeutic agents are attached. Improved speed, yield, and purity are achieved using the methods disclosed herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e)(1) of U.S. Provisional Patent Application No. 62 / 943,594, filed December 4, 2019, and U.S. Provisional Patent Application No. 63 / 110,182, filed November 5, 2020, the disclosures of both of which are incorporated by reference herein in their entireties for all purposes.

[0002] (Statement regarding Federal Government Support) Not applicable. [Technical field]

[0003] The present disclosure is in the field of nanoparticles for treating various cancers, said nanoparticles comprising a polymer core and further comprising one or more homing molecules (i.e., targeting molecules) and / or one or more therapeutic molecules. Improved methods for synthesizing such nanoparticles are provided herein. [Background technology]

[0004] Treatment of many brain diseases, such as brain tumors and brain metastases, requires the delivery of therapeutic molecules to the brain. Direct delivery of therapeutics to the brain poses serious risks to the subject (e.g., skull fracture) and is impractical for continuous administration, as is required for most chemotherapy treatments. However, systemic delivery (e.g., via the bloodstream) cannot efficiently deliver molecules to the brain due to the presence of the blood-brain barrier (BBB), a tightly bound layer of endothelial cells lining the blood vessels of the brain. A similar permeability barrier, known as the blood-tumor barrier (BTB), exists in certain solid tumors.

[0005] Breast cancer often metastasizes to the brain, and these brain metastases could be treated with chemotherapy molecules used in breast cancer therapy if the therapeutics could be delivered to the brain in sufficient concentrations. One such chemotherapy drug is camptothecin, an alkaloid that inhibits DNA topoisomerase I, thereby preventing cell division. Camptothecin has solubility issues in aqueous environments such as the cytoplasm, so more soluble derivatives of camptothecin have been developed. One such derivative is 7-ethyl-10-hydroxy-camptothecin (SN38), which was originally discovered as a metabolite of the camptothecin analog irinotecan.

[0006] To address the problem of delivering therapeutic agents across the BBB and BTB, nanoparticles have been developed that contain chemotherapeutic molecules and homing (i.e., targeting) molecules that bind to transferrin receptors present on the surface of brain endothelial cells (and endothelial cells of tumor vasculature), thereby allowing the nanoparticles to pass through the endothelial cells by transcytosis. See, for example, U.S. Patent Nos. 9,468,681, 10,166,291, and 10,182,986. Each of these patents is incorporated herein by reference for all purposes, or at least for the teachings regarding the properties of nanoparticles, how to make and use them, and how they are manipulated.

[0007] Specific steps in the synthesis of such nanoparticles include (1) converting mucic acid to a polymerizable reactive derivative (i.e., a "mucic acid monomer" or MAM), (2) polymerizing the mucic acid monomer to form a mucic acid polymer (MAP), (3) converting a therapeutic molecule to a selective reactive derivative, and (4) attaching one or more of the derivatized therapeutic molecules to the MAP.

[0008] Nanoparticles containing camptothecin (CPT) that can cross the blood-brain barrier and blood-tumor barrier have been described. See, for example, U.S. Patent Nos. 9,446,149; 9,468,681; 10,166,291 and U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019), each of which is incorporated by reference herein for all purposes, or at least for the description of nanoparticles. However, existing methods for preparing such nanoparticles are reagent and time consuming, require high temperatures, and have low yields. Furthermore, as discussed above, derivatives of CPT that are more soluble under aqueous conditions would be preferred. Thus, there is a need for improved methods for synthesizing nanoparticles containing chemotherapeutic drugs such as CPT and its derivatives, such as SN38, that are rapid, economical in terms of reactants, and have high yields.

[0009] Additionally, the treatment of non-CNS malignancies, such as non-CNS tumors, and hematological malignancies would benefit from improved vehicles for the delivery of chemotherapeutic agents. Summary of the Invention

[0010] Provided herein are improved methods for the synthesis of therapeutic nanoparticles comprising mucic acid-polyethylene glycol (PEG) polymers and chemotherapeutic drugs such as camptothecin (CPT) and its derivatives and metabolites, e.g., 7-ethyl-10-hydroxycamptothecin (SN38). The improvements include, among others, new intermediates, the use of more stable reactants, the formation of more stable intermediates, faster reaction times, higher yields of intermediates and products, and new linkers for conjugating targeting (homing) molecules and therapeutic molecules (e.g., macromolecular therapeutics, e.g., antibodies) to the nanoparticles. Derivatized nanoparticles (e.g., containing conjugated CPT or SN38 molecules) for use in the methods described herein are also provided, as are mucic acid polymer (MAP) conjugates (e.g., MAP conjugated to CPT, SN38, transferrin and / or trastuzumab) for use in the assembly of therapeutic nanoparticles.

[0011] Thus, provided herein are, inter alia, the following embodiments:

[0012] 1. A method for the synthesis of mucic acid di(aspartylamine) ditrifluoroacetate from mucic acid diaminochloride, comprising: (a) the structure [ka] mucic acid diamino chloride having the following structure: [ka] and converting the compound into di(aspartyl(Ot-butyl)-Boc) mucic acid having the formula: (b) Di(aspartyl(Ot-butyl)-Boc) mucic acid, [ka] and converting the resulting mixture to mucic acid di(aspartylamine) di-trifluoroacetate having the formula: A method comprising:

[0013] 2. The method according to embodiment 1, wherein in step (b): (a) Mucinic acid di(aspartyl(Ot-butyl)-Boc) has the following structure: [ka] is converted to a neutral species, di(aspartylamine) of mucic acid having the formula (b) A method wherein mucic acid di(aspartylamine) neutral species is converted to mucic acid di(aspartylamine) ditrifluoroacetate.

[0014] 3. A method for synthesizing mucic acid di(aspartylamine) ditrifluoroacetate, comprising the steps of: [ka] A neutral species of mucic acid di(aspartylamine) having The following structure: [ka] to mucic acid di(aspartylamine) ditrifluoroacetate having the formula:

[0015] 4. The following structure [ka] Mucin di(aspartylamine) neutral species having

[0016] 5. A method for synthesizing a 10-TBDPS derivative of SN38, comprising heating a mixture of SN38 and tert-butyl(chloro)diphenylsilane (TBDPSCl) in a base (e.g., an amine base, e.g., a trialkylamine, e.g., triethylamine) and a solvent (e.g., dichloromethane (DCM)), wherein the product has the structure: [ka] The method comprising:

[0017] In certain embodiments, the mixture is heated at a temperature ranging from 15° C. to 90° C. for a time ranging from 5 minutes to 48 hours. In further embodiments, upon completion of the reaction, the reaction mixture is washed successively with dilute acid (e.g., 0.2N HCl), weak base (e.g., saturated NaHCO3), and brine. In further embodiments, the neutralized solution is dried (e.g., with MgSO4). In further embodiments, the dried solution is evaporated under vacuum to provide a solid. In further embodiments, the solid is recrystallized (e.g., by dissolving the residue (e.g., in DCM) and precipitating the product (e.g., with hexanes)).

[0018] In additional embodiments, a derivative or analog of SN38 or CPT having a 20-OH group and / or a 10-OH group is used as the starting material in place of SN38.

[0019] Combinations of the above embodiments are also contemplated as additional embodiments of the invention(s) disclosed herein.

[0020] 6. A method for synthesizing 20-Boc-aminoacyl, 10-TBDPS derivatives of SN38, comprising combining 10-TBDPS-SN38 with a Boc-amino acid-OH in the presence of a solvent, a base, and a coupling agent. In certain embodiments, the reactants are combined in a solvent (e.g., DCM) and / or a coupling agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl)) and / or a base (e.g., 4-dimethylaminopyridine (DMAP)). In certain embodiments, the reaction is carried out at a temperature between 0° C. and 20° C., or any integer or fractional value therebetween.

[0021] 7. The method of embodiment 6, wherein said amino acid is selected from the group consisting of glycine (Gly), valine (Val), gamma-aminobutyric acid (GABA) and hexanoic acid (Hex).

[0022] 8. The following structure [ka] 20-(Boc-Gly)-10-TBDPS-SN38.

[0023] 9. The following structure [ka] 20-(Boc-GABA)-10-TBDPS-SN38.

[0024] 10. The following structure [ka] 20-(Boc-Hex)-10-TBDPS-SN38.

[0025] 11. The following structure [ka] 20-(Boc-Val)-10-TBDPS-SN38 having the formula:

[0026] 12. A method for synthesizing a 10-OBoc derivative of SN38 (10-OBoc-SN38), comprising the steps of combining SN38 with di-tert-butyl dicarbonate in the presence of a base (e.g., pyridine) and a solvent (e.g., DCM), wherein 10-OBoc-SN38 has the following structure: [ka] The method comprising:

[0027] 13. A method for synthesizing 20-(Boc-aminoacyl), 10-OBoc derivative of SN38, comprising the steps of combining 10-OBoc-SN38 with Boc-amino acid-OH in the presence of a solvent (e.g., DCM), a base (e.g., DMAP) and a coupling agent (e.g., EDC.HCl) at low temperature, wherein 20-(Boc-aminoacyl)-10-OBoc-SN38 has the following structure: [ka] wherein R is an α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. In certain embodiments, the reaction is carried out at a temperature between 0° C. and 20° C. or any integer or fractional value therebetween.

[0028] 14. The method of embodiment 12, wherein the amino acid functional groups are selected from the functional groups of glycine (Gly), alanine (Ala), β-alanine (β-Ala), valine (Val) and leucine (Leu).

[0029] 15. The following structure [ka] 20-(Boc-Gly)-10-OBoc-SN38.

[0030] 16. The following structure [ka] 20-(Boc-Ala)-10-OBoc-SN38 having the formula:

[0031] 17. The following structure [ka] 20-(Boc-β-Ala)-10-OBoc-SN38.

[0032] 18. The following structure [ka] 20-(Boc-Val)-10-OBoc-SN38 having the formula:

[0033] 19. The following structure [ka] 20-(Boc-Leu)-10-OBoc-SN38.

[0034] 20. The following structure [ka] 20-(HCl.Gly)-SN38.

[0035] 21. The following structure [ka] 20-(TFA.GABA)-SN38.

[0036] 22. The following structure [ka] 20-(TFA.Hex)-SN38 having the formula:

[0037] 23. The following structure [ka] 20-(HCl.Val)-SN38.

[0038] 24. The following structure [ka] 20-(TFA.Gly)-SN38.

[0039] 25. The following structure [ka] 20-(TFA.Ala)-SN38.

[0040] 26. The following structure [ka] 20-(TFA.β-Ala)-SN38.

[0041] 27. The following structure [ka] 20-(TFA.Val)-SN38.

[0042] 28. The following structure [ka] 20-(TFA.Leu)-SN38.

[0043] 29. The following structure [ka] Mucinous acid polymer-SN38 conjugate (MAP-Gly-SN38).

[0044] In certain embodiments, x in compound 16 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0045] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0046] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0047] 30. The following structure [ka] A mucic acid polymer-SN38 conjugate (MAP-GABA-SN38).

[0048] In certain embodiments, x in compound 17 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0049] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0050] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0051] 31. The following structure [ka] Mucinous polymer-SN38 conjugate (MAP-Hex-SN38),

[0052] In certain embodiments, x in compound 18 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0053] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0054] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0055] 32. The following structure [ka] Mucinous acid polymer-SN38 conjugate (MAP-Val-SN38) having the formula:

[0056] In certain embodiments, x in compound 19 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0057] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0058] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0059] 33. The following structure [ka] Mucinous acid polymer-SN38 conjugate (MAP-Ala-SN38) having the formula:

[0060] In certain embodiments, x in compound 37 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0061] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0062] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0063] 34. The following structure [ka] Mucinous acid polymer-SN38 conjugate (MAP-β-Ala-SN38),

[0064] In certain embodiments, x in compound 38 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0065] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0066] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0067] 35. The following structure [ka] Mucinous acid polymer-SN38 conjugate (MAP-Leu-SN38).

[0068] In certain embodiments, x in compound 39 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0069] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0070] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0071] 36. Nanoparticles comprising a MAP-SN38 conjugate according to any of embodiments 29 to 35.

[0072] 37. The following structure [ka] 20-(Boc-Gly)-CPT.

[0073] 38. The following structure [ka] 20-(Boc-Val)-CPT having the formula:

[0074] 39. The following structure [ka] 20-(Boc-Ala)-CPT having the formula:

[0075] 40. The following structure [ka] 20-(Boc-β-Ala)-CPT having the formula:

[0076] 41. The following structure [ka] 20-(Boc-GABA)-CPT.

[0077] 42. The following structure [ka] 20-(Boc-Phe-Gly)-CPT having the formula:

[0078] 43. The following structure [ka] 20-(TFA.Gly)-CPT.

[0079] 44. The following structure [ka] 20-(TFA.Ala)-CPT.

[0080] 45. The following structure [ka] 20-(TFA.β-Ala)-CPT.

[0081] 46. ​​The following structure [ka] 20-(TFA.Val)-CPT.

[0082] 47. The following structure [ka] 20-(TFA.GABA)-CPT.

[0083] 48. The following structure [ka] 20-(TFA.Phe-Gly)-CPT having the formula:

[0084] 49.Structure below [ka] 1. A method for synthesizing a nitrophenylboronic acid-polyethylene glycol conjugate (NPBA-PEG-AA) having the formula: (a) suspending NH2-PEG-acetic acid (AA) in a base (e.g., N,N'-diisopropylethylamine, DIPEA) and a solvent (e.g., DCM); (b) dissolving 3-carboxy-5-nitrophenylboronic acid in a solvent mixture (e.g., DCM and dimethylformamide (DMF)); (c) adding a coupling agent (e.g., N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, EEDQ) to the solution of (b); (d) mixing the solutions (a) and (c); (e) acidifying the mixture; Including, The method, wherein n is a number ranging from 2 to 2,000 or any integer value therebetween, for example, 100 to 300, 20 to 300, 120 to 180, and / or 140 to 160.

[0085] In certain embodiments, the reaction is carried out at low temperature, for example, a temperature ranging from 0° C. to 20° C. or any integer or decimal value therebetween, and then allowed to warm to ambient temperature prior to acidification and purification. In certain embodiments, the mixture is acidified to a pH of about 4. In additional embodiments, the product is purified by extraction with a solvent (e.g., isopropyl acetate, iPrOAc).

[0086] In certain embodiments, the value of n is such that the weight average molecular weight of the PEG (i.e., -CH2-CH2-O-) portion of the compound is in the range of about 2 to about 15 kDa, e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0087] 50. The following structure [ka] The compound (NPBA-PEG-AA-PFP) has the formula:

[0088] In certain embodiments, n is a number ranging from 2 to 2,000 or any integer value therebetween, such as a number ranging from 100-300, 20-300, 120-180, and / or 140-160.

[0089] In certain embodiments, the value of n is such that the weight average molecular weight of the PEG (i.e., -CH2-CH2-O-) portion of the compound is in the range of about 2 to about 15 kDa, e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0090] 51. A method for producing the compound of embodiment 50, comprising: Combining the NPBA-PEG-AA of embodiment 49 with bis(pentafluorophenyl)carbonate and a base catalyst (e.g., N-methylmorpholine). A method comprising:

[0091] 52. A method for synthesizing an NPBA-PEG-transferrin conjugate, comprising: Mixing the NPBA-PEG-AA-PFP of embodiment 50 with holo-transferrin (i.e., iron-bound transferrin). A method comprising:

[0092] 53. A method for synthesizing an NPBA-PEG-trastuzumab conjugate, comprising: Mixing the NPBA-PEG-AA-PFP of embodiment 50 with trastuzumab. A method comprising:

[0093] 54. A method for synthesizing an NPBA-PEG-therapeutic polypeptide conjugate, comprising: Mixing the NPBA-PEG-AA-PFP of embodiment 50 with a therapeutic polypeptide. A method comprising:

[0094] 55. The following structure [ka] 20-Boc-aminoacyl, 10-TBDPS derivative of SN38 (20-(Boc-aminoacyl)-10-TBDPS-SN38), having the formula: where R is the α-carbon atom of one or more amino acids optionally attached to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker can include more than one amino acid, i.e., the linker can be a dipeptide, tripeptide, or oligopeptide.

[0095] 56. The 20-Boc-aminoacyl, 10-TBDPS SN38 derivative according to embodiment 55, wherein the amino acid functional group is selected from the functional groups of glycine, valine, γ-aminobutyric acid (GABA) and hexanoic acid.

[0096] 57. The following structure [ka] 20-Boc-aminoacyl, 10-OBoc derivative of SN38 (20-(Boc-aminoacyl)-10-OBoc-SN38), having the formula: where R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker can include more than one amino acid, i.e., the linker can be a dipeptide, tripeptide or oligopeptide.

[0097] 58. The 20-Boc-aminoacyl, 10-OBoc SN38 derivative according to embodiment 57, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine and leucine.

[0098] 59. The following structure [ka] where R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker can include more than one amino acid, i.e., the linker can be a dipeptide, tripeptide, or oligopeptide.

[0099] 60. The HCl salt according to embodiment 59, wherein the amino acid functional group is selected from the functional groups of glycine and valine.

[0100] 61. The following structure [ka] where R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 portion and the MAP / PEG portion of the structure) represents an amino acid. "One or more" reflects the fact that the linker can include multiple amino acids, i.e., the linker can be a dipeptide, tripeptide, or oligopeptide.

[0101] 62. The TFA salt according to embodiment 61, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine, GABA, hexanoic acid and leucine.

[0102] 63. The following structure [ka] where R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 moiety and the MAP / PEG moiety of the structure) represents an amino acid. "One or more" reflects the fact that the linker may include multiple amino acids, i.e., the linker may be a dipeptide, tripeptide, or oligopeptide.

[0103] In certain embodiments, x in the MAP-amino acid-SN38 conjugate is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0104] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0105] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0106] 64. The MAP-amino acid-SN38 conjugate according to embodiment 63, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine, GABA, hexanoic acid and leucine.

[0107] 65. Nanoparticles comprising a MAP-amino acid-SN38 conjugate according to any of embodiments 63 or 64.

[0108] 66. The following structure [ka] 20-Boc-aminoacyl derivative of camptothecin (20-(Boc-aminoacyl)-CPT), having the formula: where R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 moiety and the MAP / PEG moiety of the structure) represents an amino acid. "One or more" reflects the fact that the linker may comprise multiple amino acids, i.e., the linker may be a dipeptide, tripeptide, or oligopeptide.

[0109] 67. The 20-(Boc-aminoacyl)CPT derivative according to embodiment 66, wherein the amino acid functional group is selected from the functional groups of glycine, valine, alanine, β-alanine, GABA and the dipeptide phenylalanine-glycine.

[0110] 68. The following structure [ka] The trifluoroacetic acid (TFA) salt of the 20-aminoacyl derivative of CPT (20-(TFA.aminoacyl)-CPT), wherein R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 moiety and the MAP / PEG moiety of the structure) represents an amino acid. "One or more" reflects the fact that the linker can include multiple amino acids, i.e., the linker can be a dipeptide, tripeptide, or oligopeptide.

[0111] 69. The TFA salt according to embodiment 68, wherein the amino acid functional group is selected from the functional groups of glycine, alanine, β-alanine, valine, GABA and the dipeptide phenylalanine-glycine.

[0112] 70. The following structure [ka] where R is the α-carbon atom of one or more amino acids optionally bonded to an amino acid functional group. The term "R is the α-carbon atom of one or more amino acids" reflects the fact that -NH-R-CO2- in the above structure (i.e., the linker between the SN38 moiety and the MAP / PEG moiety of the structure) represents an amino acid. "One or more" reflects the fact that the linker may include multiple amino acids, i.e., the linker may be a dipeptide, tripeptide, or oligopeptide. In certain embodiments, the amino acid functional group is selected from the functional groups of alanine, β-alanine, valine, GABA, and the dipeptide phenylalanine-glycine.

[0113] In certain embodiments, x in the MAP-amino acid-CPT conjugate is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion of the polymer (i.e., -O-CH2-CH2-) is in the range of about 500 Da to about 50,000 Da.

[0114] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0115] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0116] 71. Nanoparticles comprising a MAP-amino acid-CPT conjugate according to embodiment 70. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0117] The present disclosure may be more readily understood by reference to the following description in conjunction with the accompanying examples, all of which form a part of this disclosure. It will be understood that the present disclosure is not limited to the specific products, methods, conditions, or parameters described or shown herein, and that the terms used herein are intended to describe specific embodiments by way of example only, and are not intended to limit the claimed invention. Similarly, unless specifically stated otherwise, any explanation of a possible mechanism or mode of action or reason for improvement is intended to be illustrative only, and the disclosure herein is not intended to be bound by the correctness or incorrectness of such suggested mechanism or mode of action or reason for improvement. It will be recognized that throughout this text, the explanations refer to compositions and methods of making and using said compositions. That is, when the present disclosure describes or claims features or embodiments related to compositions or methods of making or using the compositions, it will be understood that such explanations or claims are intended to extend these features or embodiments to embodiments in each of these contexts (i.e., compositions, methods of making, and methods of using).

[0118] In this disclosure, the singular forms "a," "an," and "the" include plural references, and a reference to a particular value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, a reference to "a material" is a reference to at least one of such materials and equivalents thereof known to one of ordinary skill in the art.

[0119] When values ​​are expressed as approximations using the descriptor "about", it will be understood that the particular value forms another embodiment. In general, the use of the term "about" indicates an approximation that may vary depending on the desired properties sought to be obtained by the disclosed subject matter, interpreted in the particular context in which it is used based on its function. A person skilled in the art would be able to interpret this as normal. In some cases, the number of significant figures used for a particular value may be one non-limiting way of determining the range of the word "about". In other cases, the gradations used in a series of values ​​may be used to determine the intended range available to the term "about" for each value. When present, all ranges are inclusive and combinable. That is, reference to values ​​set forth in a range includes all values ​​within that range.

[0120] For clarity, it will be understood that certain features of the present disclosure that are described herein in the context of separate embodiments can also be provided in combination in a single embodiment. That is, unless expressly incompatible or expressly excluded, each embodiment is deemed combinable with any other embodiment(s), and such combinations are deemed to be separate embodiments. Conversely, various features of the present disclosure that are described for brevity in the context of a single embodiment can also be provided separately or in any subcombination. Finally, while an embodiment may be described as part of a series of steps or as part of a more general structure, each said step can also be considered to be an independent embodiment in itself and combinable with others.

[0121] The transitional phrases "comprising," "consisting essentially of," and "consisting of" are intended to imply their generally accepted meaning in patent language, i.e., (i) "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps; (ii) "consisting of" excludes elements, steps, or ingredients not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to specified materials or steps that do not "materially affect" the basic and novel characteristic(s) of the claimed invention. Also, embodiments described in terms of the phrase "comprising" (or its equivalent) provide as embodiments those described independently in terms of "consisting of" and "consisting essentially of." For embodiments defined in terms of "consisting essentially of," the basic and novel characteristic(s) are the methods of preparing and using the materials of the invention (and the systems used in such methods and compositions resulting therefrom) and the ease of operation of the materials themselves, which are capable of achieving the emphasized characteristics using only the elements specified in the claims. That is, although other materials may be present in the compositions of the present invention, the presence of these additional materials is not necessary to provide the described benefits of those compositions (i.e., the effects may be additive) and / or these additional materials do not detract from the performance of the product composition. Similarly, while additional steps may also be employed in the method, their presence is not necessary to achieve the described effects or advantages and / or they do not detract from the described effects or advantages.

[0122] When a list is presented, unless specifically stated otherwise, each individual element of the list and every combination of that list is understood to be a separate embodiment. For example, a list of embodiments presented as "A, B, or C" is interpreted to include the embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C." Similarly, C 1-3Terms such as alkyl include, in alternative embodiments, C alkyl, C alkyl, C alkyl, C 1-2 Alkyl and C 2-3 It also includes alkyl.

[0123] Throughout this specification, words will have their ordinary meaning as understood by those of ordinary skill in the relevant art, except that, to avoid doubt, the meaning of certain terms will be specifically defined or clarified.

[0124] Reference to an alcohol, aldehyde, amine, carboxylic acid, ketone, or other similarly reactive functional group also includes protected analogs thereof. For example, a reference to a hydroxy or an alcohol also includes substituents in which the hydroxy is protected by acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers (the most common of which include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), ethoxyethyl ether (EE). References to amines also include substituents in which the amine is protected with a BOC glycine, carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts) group or sulfonamide (Nosyl and Nps) group. Substituents containing a carbonyl group also include those in which the carbonyl is protected with an acetal or ketal, acylal or diacetane group. References to substituents containing a carboxylic acid or carboxylate group also include substituents in which the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert-butyl ester, ester of 2,6-disubstituted phenol (e.g., 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), silyl ester, ortho ester or oxazoline.

[0125] (abbreviation) AA: Acetic acid ACN: Acetonitrile Ala: Alanine, Alanyl ARS: Alizarin Red S Asp: aspartate, aspartic acid β-Ala: β-alanine, β-alanyl BBB: Blood-brain barrier Boc: tert-butoxycarbonyl BTB: Blood-tumor barrier CPME: Cyclopentyl methyl ether CPT: Camptothecin CV: column volume DCM: dichloromethane DIC: N,N'-diisopropylcarbodiimide DIPEA: N,N'-diisopropylethylamine DiSPA-PEG: Di(succinimidyl propionate)-PEG DMA: Dimethylacetamide DMAP: 4-dimethylaminopyridine DMF: Dimethylformamide DMSO: Dimethyl sulfoxide EDC.HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline EtOAc: ethyl acetate GABA: gamma-aminobutyric acid, gamma-aminobutyryl Gly: glycine, glycyl GPC: Gel Permeation Chromatography HBSS: Hanks' balanced salt solution HCl: Hydrochloric acid Hex: 6-aminohexanoic acid HIC: Hydrophobic interaction chromatography HOPO: Hydroxypyridine N-oxide IPA: Isopropyl alcohol iPrOAc: isopropyl acetate MAM: Polymerizable mucic acid monomer MAP: Mucinous acid polymer MeOH: Methanol MTBE: Methyl tert-butyl ether NHS: N-hydroxysuccinimide NP (or P): Nanoparticle NPBA: 3-carboxy-5-nitrophenylboronic acid Mono-NPBA-PEG-Tf: mono-PEGylated fraction of transferrin Mono-NPBA-PEG-Tras: mono-PEGylated fraction of trastuzumab PEO: Polyethylene oxide PBS: Phosphate-buffered saline PEG: Polyethylene glycol PES: Polyethersulfone PFP: Pentafluorophenyl PyAOP: (7-azabenzotriazol-1-yloxy)trispyrrolizinophosphonium hexafluorophosphate SN38: 7-Ethyl-10-hydroxy-camptothecin SpP: strands per particle TBDPS: tert-butyldiphenylsilane; tert-butyldiphenylsilyl TBDPSCl: tert-Butryl(chloro)diphenylsilane TEA: Triethylamine Tf: Holo-transferrin TFA: Trifluoroacetate, trifluoroacetic acid THF: tetrahydrofuran Tras: trastuzumab UF / DF: Ultrafiltration and diafiltration Val: Valin, Baril

[0126] (Reaction components) The procedures described herein include standard solvents and catalysts known in the art. Although specific compounds (e.g., acids, bases, coupling agents) are described in this disclosure, it will be apparent to one of skill in the art that different reagents can be used.

[0127] Thus, exemplary solvents include, but are not limited to, chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-dichloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-methyltetrahydrofuran), alcohols (e.g., methanol, ethanol, isopropanol, tert-butanol), alkanes (e.g., pentane, hexane, heptane), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethylsulfoxide, dimethylformamide, acetone, N-methyl-2-pyrrolidone), and polar protic solvents (e.g., water, ethanol, acetic acid, propionic acid).

[0128] Exemplary acids include, but are not limited to, mineral acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).

[0129] Exemplary bases include, but are not limited to, amino bases (e.g., 1,4-diazabicyclo[2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert-butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammonium hydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butylmagnesium chloride), pyridines (e.g., 4-dimethylaminopyridine, 2,6-lutidine, pyridine), carbonates (e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and hydrides (e.g., sodium hydride, calcium hydride, potassium hydride).

[0130] Exemplary coupling agents include carbodiimide reagents (e.g., N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1-hydroxy-7-azabenzotriazole, 6-chloro-1-hydroxybenzotriazole, N-hydroxysuccinimide, 1-hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2-(hydroxyimino ) acetate salts), anhydride systems or forming reagents (e.g., ditert-butyl carbonate, acetic anhydride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ethyl chloroformate), acyl azoles (e.g., carbonyldiimidazole), acid halide generating reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriphenylphosphonium dihydrogen trifluoride), phosphonium salt coupling reagents (e.g., benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), tetramethylamino Uranium reagents (e.g., 2-(2-oxo-1(2H)-pyridyl-1,1,3,3-tetramethyluronium tetrafluoroborate), aminium reagents (e.g., 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate), oxymauranium salts (e.g., 1-((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), uranium antimonate salts (e.g., benzotriazol-1-yloxy-N,N-dimethyl-methaniminium hexachloroaluminum), These include, but are not limited to, organophosphoric reagents (e.g., diethyl cyanophosphonate, 1-oxo-chlorophosphorane, 2-propanephosphonic anhydride), triazine-based reagents (e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine), organosulfur reagents (e.g., pentafluorophenyl-4-nitrobenzenesulfonate), pyridinium reagents (e.g., 2-chloro-1-methylpyridinium iodide), polymer-bound reagents (e.g., polymer-supported N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline).

[0131] (Synthesis of polymerizable mucic acid monomers (MAM)) Provided herein, inter alia, is a method for synthesizing polymerizable derivatives of mucic acid ("mucic acid monomers" or MAMs). In the first step, mucic acid is reacted with methanol (MeOH) in the presence of sulfuric acid to form the 1,6-dimethyl diester of mucic acid (compound 1). This diester is then reacted with N-Boc-ethylenediamine in the presence of triethylamine (TEA) and MeOH to form the N-Boc protected mucic acid ethylenediamine (compound 2).

[0132] An alternative preparative scale method for the preparation of N-Boc protected mucic acid ethylenediamine involves reacting mucic acid with MeOH in the presence of sulfuric acid to produce a methoxylated mucic acid derivative, which is then reacted with N-(2-aminoethyl)(tert-butoxy)carboxamide in TEA and MeOH to produce the N-Boc protected mucic acid diamine. See Example 24.

[0133] In conventional methods, this first step (i.e., conversion of mucic acid to N-Boc-protected mucic acid ethylenediamine) is carried out in two separate reactions. First, a diester is formed, the diester is purified, and then the isolated diester is converted to N-Boc-protected mucic acid ethylenediamine. See, for example, U.S. Pat. No. 10,166,291 and U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019). In the methods described herein, conversion of mucic acid to N-Boc-protected mucic acid ethylenediamine is accomplished in a single reaction. See Example 1.

[0134] Also, any and all combinations of two or more of the foregoing embodiments are contemplated as additional embodiments of the inventions disclosed herein.

[0135] In the next step, the Boc protecting group is removed from the N-Boc protected mucic acid ethylenediamine by reaction with MeOH and HCl. This converts the N-Boc protected mucic acid ethylenediamine to mucic acid ethylenediamine chloride (compound 3). See Example 2. An alternative preparative scale method for preparing mucic acid ethylenediamine chloride (mucic acid diamino chloride) by reaction with MeOH and HCl is provided in Example 25.

[0136] In the next step, mucic acid diamino chloride is reacted with Boc-L-aspartic acid 4-tert-butyl ester in the presence of hydroxypyridine N-oxide (HOPO), N,N'-diisopropylcarbodiimide (DIC) and acetonitrile (ACN) to convert mucic acid ethylenediamine chloride to mucic acid di(aspartyl(Ot-butyl)-Boc) (compound 4). See Example 3. The reaction time can range from 5 minutes to 48 hours at temperatures ranging from 15°C to 90°C. In the conventional method, the reaction of mucic acid ethylenediamine with di(aspartyl(O-benzyl)-Boc) in the presence of ACN and pyridine resulted in the addition of a di(aspartyl(O-benzyl)-Boc) protecting group (e.g., U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019)) rather than the di(aspartyl(Ot-butyl)-Boc) protecting group used in the method disclosed herein. Thus, the method described herein utilizes new reagents (HOPO and DIC instead of pyridine) and a different protecting group (Ot-butyl instead of O-benzyl). The advantage of the t-butyl protecting group used in the present method compared to the benzyl group used in the conventional method is that the t-butyl protecting group can be removed with HCl or trifluoroacetic acid (TFA). Thus, in the next synthetic step, both protecting groups (t-butyl and Boc) can be removed with one reagent (e.g., TFA or HCl).

[0137] An alternative method for the synthesis of mucic acid di(aspartyl(Ot-butyl)-Boc) on a preparative scale is the reaction of Boc-L-aspartic acid 4-tert-butyl ester with ethyl cyanohydroxyiminoacetate (Oxima) in dichloromethane (DCM) proceeding with the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl). This mixture is mixed with mucic acid diaminochloride (compound 3) in an aqueous solution of sodium carbonate. The crude product is concentrated and crystallized by the addition of ACN while distilling off the aqueous phase. See Example 26.

[0138] Also, any and all combinations of two or more of the foregoing embodiments are contemplated as additional embodiments of the inventions disclosed herein.

[0139] In the final step of the synthesis of mucic acid polymerizable monomer, mucic acid di(aspartyl(Ot-butyl)-Boc) is converted to mucic acid di(aspartylamine) (compound 5) by reacting with TFA in the presence of DCM. See Example 4. Reaction times can range from 5 minutes to 96 hours at temperatures ranging from 15° C. to 90° C. Previous methods (see, for example, U.S. Patent Application Publication No. 2019 / 0381188 (December 19, 2019)) required two steps for this conversion, first converting mucic acid di(Asp(O-benzyl)-Boc) to mucic acid di(Asp(O-benzyl)-amine, and then converting mucic acid di(Asp(O-benzyl)-amine to mucic acid di(Asp-amine). The use of t-butyl and Boc protecting groups in the methods described herein (instead of the O-benzyl and Boc protecting groups previously described) allows for the removal of the two protecting groups in one step in aqueous or organic solution, avoiding the need for homogeneous or heterogeneous hydrogenation.

[0140] In preparative scale applications, mucic acid di(aspartyl(Ot-butyl)-Boc) is converted to mucic acid di(aspartylamine) neutral species (compound 20), which is converted to mucic acid di(aspartylamine) di-TFA salt (compound 5). In these embodiments, mucic acid di(aspartyl(Ot-butyl)-Boc) can be reacted with TFA and triisopropylsilane in DCM and water, and optionally further recrystallized using tetrahydrofuran (THF) to form mucic acid monomer neutral species (20). See Example 27. The neutral mucic acid monomer can then be reacted with TFA in DCM and water and combined with a solution of seed crystals of compound 5 in ether to precipitate MAM-di-TFA salt (5). See Example 28.

[0141] Also, any and all combinations of two or more of the foregoing embodiments are contemplated as additional embodiments of the inventions disclosed herein.

[0142] (Synthesis of mucic acid polymer (MAP)) Provided herein, inter alia, are methods for synthesizing polymers of mucic acid ("mucic acid polymers" or MAPs). In certain embodiments, di-TFA-mucic acid di(aspartylamine) (MAM, compound 5) is reacted with di(succinimidylpropionate)-PEG (diSPA-PEG) (e.g., diSPA-PEG) in a solvent (e.g., dimethylsulfoxide, DMSO) to form a polymer of mucic acid (e.g., di-succinimidylpropionate)-PEG (diSPA-PEG) (e.g., di-SPA-PEG 3.5k ), add a base (e.g., N,N'-diisopropylethylamine, DIPEA), and react at a temperature ranging from 15°C to 70°C (e.g., 35°C) for a time ranging from 1 hour to 96 hours (e.g., 66 hours). After the desired polymer length (generally in the range of 10 kDa to 120 kDa) is obtained, the reaction mixture is dialyzed against DMSO, dialyzed against water, and filtered to obtain MAP (compound 6). [ka]

[0143] The molecular weight of the polymer is controlled by adjusting the molar ratio of MAM to diSPA-PEG, with the highest molecular weight being obtained at a 1:1 molar ratio of reactants. Molecular weights of the resulting polymer decrease when the molar ratio of MAM to diSPA-PEG is greater or less than 1. In our examples, a further decrease in molecular weight correlates with increasing ratios of MAM to diSPA-PEG. See Example 29.

[0144] In certain embodiments, x in compound 6 is a number in the range of 20 to 200 and y is a number in the range of 5 to 200 (or any integer value therebetween, e.g., 10 to 150, 20 to 120, 50 to 100, or 10 to 25). In certain embodiments, x is selected such that the number average molecular weight of the PEG portion (i.e., -O-CH2-CH2-) of the polymer is in the range of about 500 Da to about 50,000 Da.

[0145] In certain embodiments, the weight average molecular weight of the MAP ranges from 5 to 150 kDa or any integer value therebetween, such as 20 to 120 kDa, and the values ​​of x and y are selected accordingly.

[0146] In additional embodiments, the values ​​of x and y are selected such that after assembly of the MAP or drug conjugate MAP into nanoparticles, the size of the nanoparticles is in the range of 10-900 nm or any integer value therebetween, for example, 100-800 nm, 200-500 nm, 400-700 nm, or 20-100 nm.

[0147] In some embodiments, the methods described herein are practiced with targeted nanoparticles comprising a nanoparticle core described herein conjugated to any one of the targeting agents described herein and any one of the chemotherapeutic agents described herein, wherein the total size of the targeted nanoparticle is about 20 nm to about 100 nm. In some embodiments, the size of the targeted nanoparticle is about 40 nm to about 100 nm. In some embodiments, the size of the targeted nanoparticle is about 40 nm to about 90 nm. In some embodiments, the size of the targeted nanoparticle is about 40 nm to about 80 nm. In some embodiments, the size of the targeted nanoparticle is about 40 nm to about 70 nm. In some embodiments, the size of the targeted nanoparticle is about 40 nm to about 60 nm. In some embodiments, the size of the targeted nanoparticle is about 40 nm to about 50 nm. In some embodiments, the size of the targeted nanoparticle is about 50 nm to about 100 nm. In some embodiments, the size of the targeted nanoparticle is about 50 nm to about 90 nm. In some embodiments, the size of the targeted nanoparticle is about 60 nm to about 80 nm. In some embodiments, the size of the targeted nanoparticles is between about 50 nm and about 70 nm, In some embodiments, the size of the targeted nanoparticles is between about 70 nm and about 100 nm.

[0148] In certain embodiments, the solvent is, for example, dimethylsulfoxide (DMSO), dimethylformamide (DMF), or dimethylacetamide (DMA).

[0149] In certain embodiments, the elevated temperature is any temperature in the range of 20°C to 40°C, i.e., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0150] Dialysis can be performed through membranes of various porosities, for example membranes with molecular weight cut-offs of 1 kDa, 3 kDa, 5 kDa, 10 kDa, 30 kDa or 50 kDa.

[0151] Also, any and all combinations of two or more of the foregoing embodiments are contemplated as additional embodiments of the inventions disclosed herein.

[0152] (Measurement of polymer molecular weight) Molecular weight measurements of MAPs are performed by gel filtration chromatography (e.g., a Malvern OMNISEC gel permeation chromatography (GPC) system) using polyethylene oxide (PEO) standards. The chromatography equipment can include, for example, a solvent delivery pump, a degasser, an automatic sampling injector, a column compartment, a refractive index detector, and a light scattering detector.

[0153] Samples are prepared, for example, by dissolving MAP in phosphate buffered saline (PBS) containing 0.02% sodium azide at a concentration ranging from 1 to 5 mg / mL, and then filtering the sample, for example, through a 0.45 micron (pore size 0.45 μm) filter. Injection of 100 μL of the filtered sample, along with PEO molecular weight standards, is performed on a gel filtration or gel permeation column (e.g., 2×Agilent PL Aquagel-OH 40 columns, 300 mm long×7.5 mm diameter, 8 μm particle size) with a mobile phase of PBS+0.02% sodium azide at a flow rate of, for example, 0.7 mL / min and a column temperature of, for example, 30° C. Peak fractions are detected by refractive index and / or light scattering and analyzed, for example, using Malvern OMNISEC software.

[0154] (Camptothecin (CPT) and its derivatives, metabolites and analogues) In particular, the present disclosure provides methods and compositions for preparing selectively reactive derivatives of CPT and its derivatives, metabolites and analogs. In general, these molecules contain five fused aromatic rings, one or more of which are heteroaromatic and contain one or more pendant hydroxyl groups, as shown in the diagram below showing the structure of CPT. [ka]

[0155] Exemplary molecules in these categories include topotecan, irinotecan, ciratan, cositecan, exatecan, raltotecan, gimatecan, belotecan, rubitecan, SN38, diflomotecan, karenitecan, namitecan, elomotecan, delimotecan, chimitecan, ZBH-1205, DRF-1042 and FL118.

[0156] Binding of derivatized 7-ethyl-10-hydroxy-camptothecin (SN38) to MAP Nanoparticles for the treatment of brain tumors and brain metastases have been described in the past that utilize CPT as a therapeutic molecule. However, as mentioned above, more soluble derivatives of CPT, such as SN38, provide a higher therapeutic index and lower toxicity. Therefore, the present disclosure provides a method for synthesizing nanoparticles with a polymeric mucic acid backbone to which multiple SN38 molecules are attached. To this end, the present disclosure provides methods and compositions for synthesizing multiple derivatized variants of SN38 that can be covalently attached to the above-mentioned MAPs.

[0157] Thus, provided herein is a method for synthesizing reactive derivatives of, among other things, the chemotherapy molecule SN38. The method involves attaching a number of different amino acid-based linker molecules to the SN38 molecule via the SN38 hydroxyl group. SN38 and other CPT derivatives have two hydroxyl groups, the 10 hydroxyl and the 20 hydroxyl. Conjugation at the 20 hydroxyl stabilizes the active lactone form of these molecules and reduces or prevents conversion to the less active carboxylate form after administration to a subject. However, the 10 hydroxyl is also reactive.

[0158] In the methods described herein, the reactivity of the 10-hydroxyl of SN38 is first blocked (i.e., protected) and then a linker is added to the 20-hydroxy. Exemplary amino acid linkers include glycine, alanine, β-alanine, γ-aminobutyric acid (GABA), valine, leucine, isoleucine, pentanoic acid, hexanoic acid, arginine, histidine, lysine aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, tyrosine, tryptophan, and dipeptides (e.g., phenylalanine-glycine), tripeptides, and oligopeptides.

[0159] Methods are provided herein for attaching a number of different linkers to the 20-hydroxyl group of SN38. Each of the various linkers exemplified herein (such as glycine, alanine, β-alanine, leucine valine, γ-aminobutyric acid and 6-aminohexanoic acid, as well as other dipeptides such as phenylalanine-glycine) can be conjugated to the 20-hydroxyl of SN38 and then reacted with the carboxyl group of MAP (via an amidation reaction between the amine group of the linker and the carboxyl group of the polymer) to covalently attach the SN38 molecule to the MAP. Different linkers have different stabilities, which affect the properties (e.g., size, density) of the resulting nanoparticles, which in particular affect the rate at which SN38 is released from the nanoparticles after crossing the blood-brain barrier (BBB) ​​or blood-tumor barrier (BTB).

[0160] (Synthesis of 10-hydroxy-protected SN38 (10-TBDPS-SN38)) In the first step of the synthesis of derivatized SN38, a tert-butyldiphenylsilyl (TBDPS) protecting group is added to the 10-hydroxyl group of SN38 (to block reactivity at that site) by mixing SN38 with tert-butyl(chloro)diphenylsilane (TBDPSCl) in DCM and triethylamine. The mixture is stirred and refluxed (e.g., 45° C.) for about 16 hours, washed with HCl (e.g., 0.2 N), saturated NaHCO3, and brine, and finally dried over MgSO4 and evaporated in vacuo. The product (10-TBDPS-SN38, compound 7) is dissolved in DCM, precipitated with hexane, and the solid is dried.

[0161] The TBDPS protecting group is selective for the 10-hydroxy group of SN38 (the 20-OH group is free to react in the subsequent step), and the resulting compounds are less susceptible to spontaneous loss of the protecting group during storage, providing more stable intermediates.

[0162] An added advantage of using TBDPS to protect the 10-OH group of SN38 is that it can be removed using the same conditions (1M to 12M HCl or 1:1 to 1:4 v / v water:TFA) used to remove the Boc protecting group from the linker attached to the 20-OH group.

[0163] Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxyglycine linker A protected glycine (Gly) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by mixing 10-TBDPS-SN38 with Boc-Gly-OH in DCM at 0° C. EDC.HCl and 4-dimethylaminopyridine (DMAP) are added and the mixture is stirred at 0° C. Stirring can be performed for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval in between. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1 N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to give 20-(Boc-Gly)-10-TBDPS-SN38 (compound 8).

[0164] (Synthesis of 20-(HCl.Gly)-SN38) 20-(Boc-Gly)-10-TBDPS-SN38 is deprotected at both the 10-hydroxy and 20-hydroxy groups by treatment with HCl (concentrations ranging from 1N to 12N) in a hexane bath at room temperature, which restores the 10-hydroxy group and produces an SN38 derivative in which a glycine linker is covalently attached to the 20-hydroxy group (20-(HCl.Gly)-SN38, compound 12).

[0165] Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxy GABA linker A protected γ-aminobutyric acid (GABA) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by mixing 10-TBDPS-SN38 with Boc-GABA-OH in DCM at 0° C. EDC.HCl and DMAP are added and the mixture is stirred at 0° C. Stirring can be performed for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval in between. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1 N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to give 20-(Boc-GABA)-10-TBDPS-SN38 (compound 9).

[0166] (Synthesis of 20-(TFA.GABA)-SN38) 20-(Boc-GABA)-10-TBDPS-SN38 is deprotected at both the 10-hydroxy and 20-hydroxy groups by treatment with TFA at concentrations ranging from 1:1 to 4:1 v / v in water in a hexane phase at room temperature, which restores the 10-hydroxy group and produces an SN38 derivative in which the GABA linker is covalently attached to the 20-hydroxy group (20-(TFA.GABA)-SN38, compound 13).

[0167] Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxyhexanoic acid linker A protected 6-aminohexanoic acid (Hex) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by mixing 10-TBDPS-SN38 with Boc-Hex-OH in DCM at 0° C. EDC.HCl and DMAP are added and the mixture is stirred at 0° C. Stirring can be performed for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval in between. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1 N HCl, and once with brine. The solution is dried (e.g., with MgSO4) and evaporated under vacuum to give 20-(Boc-Hex)-10-TBDPS-SN38 (compound 10).

[0168] (Synthesis of 20-(TFA.Hex)-SN38) 20-(Boc-Hex)-10-TBDPS-SN38 is deprotected at both the 10-hydroxy and 20-hydroxy groups by treatment with TFA at concentrations ranging from 1:1 to 4:1 v / v in water in a hexane phase at room temperature, which restores the 10-hydroxy group and produces an SN38 derivative in which a Hex linker is covalently attached to the 20-hydroxy group (20-(TFA.Hex)-SN38, compound 14).

[0169] (Synthesis of 10-hydroxy-protected SN38 with a protected 20-hydroxyvaline linker) A protected valine (Val) linker is added to the 20-hydroxy position of 10-TBDPS-SN38 by mixing 10-TBDPS-SN38 with Boc-Val-OH in DCM at 0° C. EDC.HCl and DMAP are added and the mixture is stirred at 0° C. Stirring can be performed for 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, or any interval in between. After stirring, the mixture is washed twice with 0.5% NaHCO3, once with water, twice with 0.1 N HCl, and once with brine. The solution can be dried (e.g., with MgSO4) and evaporated under vacuum to provide 20-(Boc-Val)-10-TBDPS-SN38 (compound 11).

[0170] (Synthesis of 20-(HCl.Val)-SN38) 20-(Boc-Val)-10-TBDPS-SN38 is deprotected at both the 10-hydroxy and 20-hydroxy groups by treatment with HCl (concentrations ranging from 1N to 12N) in a hexane bath at room temperature, which restores the 10-hydroxy group and produces an SN38 derivative in which a valine linker is covalently attached to the 20-hydroxy group (20-(HCl.Val)-SN38, compound 15).

[0171] (Use of the same protecting group on both the 10- and 20-OH groups of SN38) An alternative route to synthesize reactive derivatives of SN38 with amino acid linkers is to place a Boc-O protecting group on the 10-OH group and a Boc-amino acid protecting group on the 20-OH. Reacting this compound (20-(Boc-aminoacyl)-10-OBoc-SN38) with TFA restores the 10-OH group and adds the TFA salt of the amino acid linker to the 20-OH. Using this method, 20-Boc-glycyl, -alanyl, -β-alanyl, -leucyl, and -valyl derivatives of SN38 have been synthesized. See Examples 33-35 below. Other amino acids that can be used in this manner include GABA, isoleucine, and hexanoic acid, as well as dipeptides such as phenylalanine-glycine. [ka]

[0172] Synthesis of Polymer-Drug Conjugates In particular, methods are provided herein for forming polymer-drug conjugates (i.e., MAP-linker-SN38 nanoparticles) by covalently attaching derivatized SN38 to MAP. Many different conjugates are provided, differing in the nature (i.e., size and chemistry) of the linker between the polymer and the drug. A variety of conjugates are made using SN38 derivatized with various types of linkers. In one embodiment, the linker is a glycyl (Gly) moiety. In one embodiment, the linker is a valyl (Val) moiety. In one embodiment, the linker is a gamma-aminobutyric acid (GABA) moiety. In one embodiment, the linker is a 6-aminohexanoic acid (Hex) moiety. In one embodiment, the linker is an alanyl (Ala) moiety. In one embodiment, the linker is a beta-alanyl (beta-Ala) moiety. In one embodiment, the linker is a leucyl (Leu) moiety.

[0173] To form the polymer-drug conjugate, MAP is dissolved in DMSO, then EDC.HCl and N-hydroxysuccinimide (NHS) are added. After the addition of EDC.HCl and NHS, the derivatized SN38 and DIPEA are added, and the mixture is stirred at room temperature for about 18 hours. The derivatized SN38 can be, for example, 20-(HCl.Gly)-SN38, 20-(HCl.Val)-SN38, 20-(TFA.GABA)-SN38, 20-(TFA.Hex)-SN38, 20-(TFA.Gly)-SN38, 20-(TFA.Ala)-SN38, 20-(TFA.β-Ala)-SN38, 20-(TFA.Val)-SN38, or 20-(TFA.Leu)-SN38. The reaction mixture is then dialyzed against DMSO, then against water at pH 4, using a 10 kDa membrane. The dialyzed product is filtered (e.g., through a 0.22 μm filter), frozen and lyophilized. Depending on the derivatized SN38 used, the product is MAP-Gly-SN38 (compound 16), MAP-GABA-SN38 (compound 17), MAP-Hex-SN38 (compound 18), MAP-Val-SN38 (compound 19), MAP-Ala-SN38 (compound 37), MAP-β-Ala-SN38 (compound 38) or MAP-Leu-SN38 (compound 39).

[0174] (Nanoparticle formation) To convert the polymer-drug conjugate (i.e., any one of compounds 16, 17, 18 or 19, 37, 38 or 39) into nanoparticles, dissolve the lyophilized polymer-drug conjugate in water at pH 4 at a concentration of 1-10 mg / mL (e.g., 4 mg / mL).

[0175] The size of the nanoparticles ranges from about 20 nm to about 60 nm in diameter. Exemplary nanoparticle diameters are 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm or more.

[0176] (SN38 release from nanoparticles in vitro) When 0.1 mg of nanoparticles at a concentration of SN38 / mL were incubated in PBS at pH 6.5, 7.0 or 7.4 in a humidified oven at 37 °C, the release of SN38 from MAP-Gly-SN38, MAP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-SN38, MAP-β-Ala-SN38 and MAP-Leu-SN38 nanoparticles all showed first-order kinetics. The release rate was observed to be strongly dependent on pH. As the pH increased from 6.5 to 7.4, the release half-life decreased for all nanoparticle formulations, suggesting that hydrolysis plays an important role in the release of SN38. Longer half-lives were observed for nanoparticles with more hydrophobic and sterically hindered linkers.

[0177] (Derivatized CPT for linking to MAP) In certain embodiments, the present disclosure provides nanoparticles comprising CPT as a therapeutic agent. Derivatives of CPT conjugated to amino acids (e.g., 20-(TFA.Gly)-CPT) are used in the synthesis of said nanoparticles. Exemplary amino acid linkers include glycine, alanine, β-alanine, γ-aminobutyric acid (GABA), valine, leucine, isoleucine, pentanoic acid, hexanoic acid, and dipeptides (e.g., phenylalanine-glycine), tripeptides, and oligopeptides.

[0178] 20-(TFA.Gly)-CPT is synthesized in two steps. In the first step, CPT is mixed with Boc-Gly-OH in DMAP under an inert atmosphere (e.g., under argon) and DCM is added to slurry the reaction. DIC is then added dropwise to the reaction mixture over a few minutes, and the mixture is then stirred at room temperature for several hours (e.g., 3 hours). Approximately half of the solvent is then removed in vacuum, and cold MeOH is added to precipitate the product (20-(Boc-Gly)-CPT, compound 21), which is obtained by filtration. The solid is washed with cold MeOH and cold methyl tert-butyl ether (MTBE) and dried in vacuum. See Example 30.

[0179] In the second step, TFA is slowly added to a stirred suspension of 20-(Boc-Gly)-CPT in DCM and the mixture is stirred for several hours (e.g., 2 hours). The product (20-(TFA.Gly)-CPT, compound 22) is then precipitated with MTBE, filtered, washed, and dried under vacuum. See Example 31.

[0180] Additional CPT derivatives can be produced by using other Boc-protected amino acids (or peptides) instead of Boc-Gly-OH. For example, using Boc-Ala-OH as the starting material gives 20-(Boc-Ala)-CPT (compound 41) and 20-(TFA.Ala)-CPT (compound 45). Using Boc-β-Ala-OH as the starting material gives 20-(Boc-β-Ala)-CPT (compound 42) and 20-(TFA.β-Ala)-CPT (compound 46). Using Boc-Val-OH as the starting material gives 20-(Boc-Val)-CPT (compound 40) and 20-(TFA.Val)-CPT (compound 47). Using Boc-GABA-OH as the starting material gives 20-(Boc-GABA)-CPT (compound 43) and 20-(TFA.GABA)-CPT (compound 48). When Boc-Phe-Gly-OH is used as the starting material, 20-(Boc-Phe-Gly)-CPT (compound 44) ​​and 20-(TFA.Phe-Gly)-CPT (compound 49) are obtained.

[0181] (Synthesis of CPT-polymer conjugates) Conjugates of CPT and MAP are provided in certain embodiments. To synthesize these conjugates, MAP and 20-(TFA.aminoacyl)-CPT (see previous section for exemplary amino acid and peptide derivatives of CPT) are dissolved in DMSO in an inert atmosphere (e.g., under argon). In a separate container, (7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate (PyAOP) is dissolved in DMSO. The PyAOP solution is then added to the MAP / 20-(TFA.aminoacyl)-CPT solution and stirred for a few minutes (e.g., 2 minutes), after which DIPEA is added and the reaction mixture is stirred in the dark at room temperature for 12-24 hours (e.g., 18 hours). The product is precipitated with cold (0-4° C.) ethyl acetate (EtOAc) (optionally in several batches) and washed with cold EtOAc. The product can be further isolated by lyophilization. See Example 32.

[0182] (Nitrophenylboronic acid (NPBA)-polyethylene glycol (PEG) conjugate) In certain embodiments, macromolecules, such as targeting (homing) molecules or macromolecular therapeutics (e.g., antibodies), are linked to MAPs via nitrophenylboronic acid-polyethylene glycol (NPBA-PEG) linkers. Such NPBA-PEG linkers are amine derivatives of PEG (e.g., PEG 3.5k , PEG 5k ) with a carboxyl derivative of NPBA to form an NPBA-PEG-acetate (AA) conjugate linked by an amide bond. In certain embodiments, the PEG derivative contains an amino group at one end (for reaction with NPBA) and a carboxyl group at the opposite end (which can be activated in a subsequent reaction to react with a macromolecule such as a polypeptide). See Example 36. [ka]

[0183] In certain embodiments, n is a number ranging from 2 to 2,000 or any integer value therebetween, for example, 100-300, 20-300, 120-180, or 140-160.

[0184] In certain embodiments, the value of n is such that the weight average molecular weight of the PEG (i.e., -CH2-CH2-O-) portion of the compound is in the range of about 2 to about 15 kDa, e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0185] The NPBA-PEG-AA conjugate is activated for reaction with a polypeptide (such as, for example, transferrin) by forming a pentafluorophenyl (PFP) derivative. In these embodiments, bis(pentafluorophenyl)carbonate is combined with the NPBA-PEG-AA conjugate to form the NPBA-PEG-pentafluorophenyl ester (e.g., NPBA-PEG 5k -AA-PFP). See Example 37. [ka]

[0186] In certain embodiments, n is a number ranging from 2 to 2,000 or any integer value therebetween, such as a number ranging from 100-300, 20-300, 120-180, and / or 140-160.

[0187] In certain embodiments, the value of n is such that the weight average molecular weight of the PEG (i.e., -CH2-CH2-O-) portion of the compound is in the range of about 2 to about 15 kDa, e.g., about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa.

[0188] Polymer conjugates containing homing (targeting) molecules In certain embodiments, the MAPs described herein include a homing (or targeting) molecule instead of, or in addition to, a small molecule therapeutic, such as CPT or SN38. Targeting molecules are known in the art and include, for example, small molecules (e.g., vitamins, e.g., folic acid), sugars (e.g., mannose, allose, altrose, glucose, gulose, idose, galactose, talose, disaccharides, trisaccharides, oligosaccharides), peptides (e.g., RGD), polypeptides (e.g., antibodies, proteins that bind to cell surface receptors such as transferrin), nucleic acids, peptoids, and peptide nucleic acids (PNAs).

[0189] For example, MAPs (and nanoparticles made from MAPs) can include transferrin, a homing molecule that targets the polymer (or nanoparticles formed from the polymer) to endothelial cells of the blood-brain barrier or blood-tumor barrier. In these embodiments, the homing molecule is attached to the NPBA-PEG polymer by reaction with the PFP active ester described above. Conjugation of the ester to holo-transferrin is achieved by conjugating holo-transferrin to the NPBA-PEG-pentafluorophenyl ester since primary amine groups of the protein react with the PFP active ester. The progress of the reaction is monitored by HPLC to determine the mono-PEGylated transferrin product (e.g., mono-NPBA-PEG 5k -Tf) is purified by hydrophobic interaction chromatography (HIC) based on the differential binding of mono-PEGylated transferrin compared to more highly PEGylated fractions (e.g., di- and tri-PEGylated fractions). See Examples 38 and 39. This method prevents the loss of iron from transferrin during purification, as occurs using conventional methods, thereby maintaining the holo-transferrin structure.

[0190] (Polymer conjugates containing macromolecular therapeutic agents) In certain embodiments, the MAPs described herein include macromolecular therapeutic agents instead of, or in addition to, small molecule therapeutic agents such as CPT or SN38. Macromolecular therapeutic agents are known in the art and include, for example, therapeutic polypeptides (e.g., antibodies, enzymes, and biologically active proteins) and nucleic acids.

[0191] Exemplary therapeutic polypeptides include, but are not limited to, antibodies such as trastuzumab (anti-Her2), anti-Her3, anti-Trop2, anti-PSMA, anti-LIV-1, anti-FOLR1, anti-DLL3, anti-PDGF, anti-FRα, anti-PTK7, anti-mesothelin, anti-c-MET, anti-MUC1, anti-CD70, anti-CD74, anti-CD30, anti-CD33, anti-FLT3, anti-CD22, anti-CD20, and anti-CD19.

[0192] Certain polypeptide therapeutics, such as antibodies directed against tumor markers as exemplified in the previous paragraph, also find use as targeting (homing) molecules.

[0193] For example, MAPs (and nanoparticles made from MAPs) can include the anti-Her2 antibody trastuzumab. In these embodiments, the antibody is attached to the NPBA-PEG polymer by reaction with the PFP active ester described above.

[0194] Since the primary amine groups of proteins react with the PFP active ester, the conjugation of trastuzumab to the NPBA-PEG-pentafluorophenyl ester is achieved by conjugating the ester with trastuzumab. The progress of the reaction was monitored by HPLC, and the mono-PEGylated trastuzumab product (e.g., mono-NPBA-PEG) was obtained. 5k -Tras) is purified by HIC based on the difference in binding of monoPEGylated trastuzumab compared to more highly PEGylated fractions (e.g., di- and tri-PEGylated fractions). See Examples 41 and 42.

[0195] (Measurement of the number of polymer strands per nanoparticle) To determine the number of polymer strands per nanoparticle, the average molecular weight of the nanoparticles is divided by the molecular weight of the corresponding polymer-drug (ie, MAP-SN38) conjugate.

[0196] The molecular weight of the nanoparticles is determined by gel permeation chromatography (GPC) using PEO standards of nominal molecular weight 100 kDa and 200 kDa. The molecular weight of the polymer-drug conjugate is calculated from the molecular weight of the polymer (i.e., MAP) used in the synthesis of the conjugate and adjusted for drug loading as follows: MW コンジュゲート =MW ポリマー / (1-T) (In the formula, MW ポリマー is the molecular weight of the MAP used in the synthesis of the conjugate, and T is the percentage loading of the therapeutic agent, expressed as a decimal.

[0197] As used herein, strands per particle (SpP) is the number of mucic acid polymer ("MAP") therapeutic agent conjugate molecules present in a particle or nanoparticle. In measuring SpP, a particle or nanoparticle refers to an entity that has at least one MAP-therapeutic agent-protein conjugate molecule that behaves as a single unit in any aqueous solution, such as water at neutral pH, PBS at pH 7.4, or any formulation administered to a patient, at a concentration suitable for administration to humans. In calculating strands per particle, a MAP-therapeutic agent conjugate molecule is a single MAP polymer with a covalently attached therapeutic agent.

[0198] The methods disclosed herein provide for the evaluation of particles, said particles comprising one or more MAP-therapeutic agent conjugate molecules. In general, the methods require providing a sample comprising a plurality of said particles and determining a value for the number of MAP-therapeutic agent conjugates per particle in the sample, thereby evaluating a preparation of particles. The value for the particle sample is a function of the values ​​obtained for a plurality of particles.

[0199] As stated above, SpP is defined as the number of MAP-therapeutic agent conjugate molecules that self-assemble into a particle or nanoparticle, and thus SpP = [MAP-therapeutic drug conjugate molecule] / P (or NP) where [MAP-therapeutic agent conjugate molecules] is the number of MAP-therapeutic agent conjugate molecules and P (or NP) is a single particle (or nanoparticle).

[0200] In certain embodiments, the method further comprises comparing the measured value to a reference value. The comparison can be used in many ways. As an example, depending on the comparison or measurement made in the method, a decision or step can be taken, e.g., a manufacturing parameter in the process for making the particles is changed, the sample is sorted, selected, accepted or discarded, released or withheld, processed into a pharmaceutical product, shipped, transferred to another location, combined with another substance, e.g., combined with an excipient, labeled, packaged, commercially offered, or sold or offered for sale. For example, based on the result of the measurement or based on the comparison with a reference standard, the batch from which the sample was taken can be processed, e.g., as described above.

[0201] To calculate the number of strands per particle, the size of the particle, e.g., molecular weight by light scattering of the self-assembled particle, the size of the individual polymer, e.g., molecular weight by light scattering of the individual polymer, and the loading of the therapeutic agent, e.g., wt %, are determined. Using these values, SpP is calculated as follows: SpP=MW 粒子 / (MW コンジュゲート ) (In the formula, MW 粒子 is the molecular weight of the particle, and MW コンジュゲート is the molecular weight of the MAP-therapeutic drug conjugate molecule and is calculated as follows: MW コンジュゲート =MW ポリマー / (1-T % ) (In the formula, MW ポリマー is the molecular weight of MAP, and T %is the drug loading rate expressed as a decimal, e.g., T at 10% loading % =0.1.))

[0202] Measurement of SpP has been demonstrated with MAPs with various linkers and drugs. MAPs of the same molecular weight with CPT and SN38 result in approximately 1.5 to 2.5 strands per particle. Different linkers result in varying strands per particle and thus particle density, as demonstrated with SN38. Linkers of glycine, gamma-aminobutyric acid, hexanoic acid, alanine, beta-alanine, leucine, and valine result in varying strands per particle from approximately 1.3 to approximately 4.6.

[0203] Polymer molecular weight distribution and particle dispersity: MAPs are synthesized with a range of molecular weights. Different molecular weights result in different particle sizes and numbers of strands per particle. Particles may be formed with MAPs (referred to herein as strands) that are smaller or larger than average. Strands may also combine to a maximum size that can be limited by shear.

[0204] Particle Shape: Particle shape is assumed to be approximately spherical. Self-assembly is assumed to be driven by hydrophobic regions created by the therapeutic agent of the MAP-therapeutic agent conjugate molecule.

[0205] (Formulations, kits and routes of administration) Therapeutic compositions comprising the nanoparticles as disclosed herein are also provided. Such compositions typically comprise the nanoparticles and a pharma- ceutically acceptable carrier. Supplementary active compounds can also be incorporated into the nanoparticle compositions.

[0206] The therapeutic compositions disclosed herein are particularly useful for treating cancer, cancer metastasis, and other disorders of the brain and central nervous system. Thus, a "therapeutically effective amount" of a composition comprising nanoparticles is any amount that alleviates symptoms or stimulates, for example, tumor regression. For example, the dosage of nanoparticles is about 0.1-1.0 mg / kg body weight, or about 0.5-2.0 mg / kg body weight, or about 1-5 mg / kg body weight, or about 1 mg / kg body weight to about 10 mg / kg body weight or more (or any integer value therebetween), and the frequency of administration is, for example, hourly, twice daily, once daily, twice weekly, once weekly, twice monthly, or once monthly, depending on, for example, body weight, route of administration, severity of disease, etc. Thus, a therapeutically effective amount may consist of multiple administrations of the same or different amounts of nanoparticles. In certain embodiments, a single administration of nanoparticles is a therapeutically effective amount.

[0207] In certain embodiments, the nanoparticles are administered at a dose of 1-20 mg of nanoparticles per square meter of the subject's body surface area (or any integer or subvalue therebetween), as is typical for chemotherapy drug doses.

[0208] Various pharmaceutical compositions and techniques for preparing and using them are known to those skilled in the art in light of the present disclosure. For detailed listings of suitable pharmaceutical compositions and techniques for their administration, reference can be made to texts such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0209] The nanoparticles described herein can be suspended in a physiologically compatible carrier for administration. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with the nanoparticles and any other components of the formulation and is not harmful to the recipient. Those skilled in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include water (e.g., water at pH 4), phosphate buffered saline, Hanks' balanced salt solution + / - glucose (HBSS), and multiple electrolyte solutions such as, for example, Plasma-Lyte™ A (Baxter).

[0210] The amount of nanoparticle suspension administered to a subject varies depending on the site of administration, the therapeutic goal, and the number of nanoparticles in the solution. Typically, the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, "therapeutically effective amount" or "effective amount" refers to the number of nanoparticles administered necessary to effectively treat a particular disorder; i.e., to cause a reduction in the amount and / or an improvement in the severity of symptoms associated with the disorder. For example, in the case of cancer metastasis to the brain, administration of a therapeutically effective amount of nanoparticles will result in a reduction and / or reversal of symptoms of metastasis; e.g., regression of metastatic tumors. The therapeutically effective amount will vary depending on the type and extent of brain injury, and may also vary depending on the overall condition of the subject.

[0211] The disclosed therapeutic compositions may also include pharma- ceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, i.e., carriers. These carriers can, for example, stabilize the nanoparticles and / or promote retention of the nanoparticles in the body. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials which can function as pharma- ceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; polyols such as glycerin, sorbitol, and mannitol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic, compatible substances used in pharmaceutical preparations. Wetting agents, emulsifying agents and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening agents, flavorings and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0212] The nanoparticles can be administered to a subject by any suitable route, including, but not limited to, inhalation, topical, nasal, oral, parenteral (e.g., intravenous, intraperitoneal, intravesical or intrathecal), or rectal, in a vehicle containing one or more pharma- ceutically acceptable carriers (the proportion of which is determined by the solubility and chemical properties of the compound, the selected route of administration, and standard practice). Administration of the compounds described herein can be performed using any method known in the art. For example, administration can be transdermal, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intraventricular, intrathecal, intranasal, aerosol, suppository, or by oral administration. Pharmaceutical compositions of the nanoparticles described herein can be for administration by injection, or for oral, pulmonary, nasal, transdermal, or ocular administration.

[0213] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intraarterial, intraocular, intracranial, subthecal or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-releasing capsules (active agent incorporated within a biocompatible coating designed for sustained release); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations, such as inhalants, aerosols and sprays. The dosage of the compositions of the present disclosure will vary depending on the extent and severity of the need for treatment, the activity of the composition being administered, the general health of the subject, and other considerations well known to those skilled in the art.

[0214] In additional embodiments, the compositions described herein are delivered intracranially at or near the site of brain injury or metastasis. Such local delivery allows for non-systemic delivery of the composition, thereby reducing the physical burden of the composition compared to systemic delivery. Local delivery can be achieved, for example, by intracranial injection, or by the use of various medical implanted devices, including but not limited to stents and catheters, or by inhalation, bloodletting, or surgery. Methods for coating, implanting, embedding, and otherwise binding desired agents to medical devices such as stents and catheters are established in the art and are contemplated herein.

[0215] Another aspect of the disclosure relates to a kit for administering nanoparticles, optionally in combination with another therapeutic agent, to a subject. In one embodiment, the kit includes a composition of nanoparticles formulated in a pharmaceutical carrier suitable for administration, e.g., by injection. EXAMPLES

[0216] The following examples are provided to illustrate some of the concepts described within this disclosure. Each example is considered to provide a particular individual embodiment of the compositions, methods of preparation, and methods of use, but none of the examples should be considered to limit the more general embodiments described herein.

[0217] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Temperatures are in degrees Celsius and pressures are at or near atmospheric unless otherwise specified. References to molecular weight refer to number average molecular weight unless otherwise specified.

[0218] Examples 1-4 describe the conversion of mucic acid to polymerizable mucic acid monomers (MAMs). Examples 5 and 6 describe the polymerization of MAMs to form mucic acid polymers (MAPs). Examples 7-15 describe the derivatization of 7-ethyl-10-hydroxy-camptothecin (SN38) with four different amino acid-based linkers to a form capable of reacting with MAPs to form covalently linked MAP-SN38 conjugates. Examples 16-20 describe the reactions used to synthesize MAP-SN38 conjugates linked by amino acid-based linkers. Example 21 describes a method for converting MAP-SN38 conjugates to nanoparticles. Examples 22 and 23 describe the characterization of MAP-SN38 nanoparticles. Examples 24-29 describe routes for the preparative scale synthesis of MAPs. Examples 30-32 describe the derivatization of camptothecin (CPT) and its conjugation to MAPs. Examples 33-35 describe the derivatization of SN38 for conjugation to MAP. Examples 36 and 37 describe the synthesis of nitrophenylboronic acid-polyethylene glycol (NPBA-PEG) polymers with reactive termini suitable for protein conjugation. Examples 38-40 describe the conjugation of targeting (homing) molecules to NPBA-PEG polymers and characterization of the products. Examples 41-43 describe the conjugation of macromolecular therapeutics to NPBA-PEG polymers and characterization of the products.

[0219] Example 1. Synthesis of N-Boc protected mucilaginous diamine (compound 2) A 1 L round bottom flask containing 18 g (86 mmol) of mucilaginous acid and a magnetic stir bar was charged with 400 mL of methanol (MeOH) and 1.4 mL of concentrated sulfuric acid. The mixture was heated to reflux (85° C.) overnight (approximately 18 hours) with constant stirring. After approximately 18 hours, 32 mL of triethylamine (TEA) was added to the flask and the mixture was stirred at reflux (85° C.) for 60 minutes. To the reaction mixture was then added 30 g (187 mmol) of N-Boc-ethylenediamine dissolved in 50 mL of MeOH. The reaction was stirred at reflux (85° C.) overnight (approximately 18 hours). The reaction mixture was cooled to room temperature, filtered, and washed with MeOH. The collected solid was then taken up in 450 mL of MeOH and refluxed for 1 hour with constant stirring. The reaction mixture was cooled to room temperature, vacuum filtered, and washed with MeOH. The collected solid was dried under vacuum overnight to give 26.1 g (52.8 mmol, 62% yield) of N-Boc protected mucilaginous diamine (2). [ka]

[0220] Example 2. Synthesis of mucic acid diamino chloride (compound 3) To a 500 mL round bottom flask containing 25.9 g (52 mmol) of N-Boc protected mucilaginous diamine (2) and a magnetic stir bar, 466 mL of 3 M hydrochloric acid (HCl) in MeOH was added slowly with constant stirring. After the slow addition of HCl, the reaction flask was transferred to an orbital shaker and shaken at room temperature overnight. The reaction slurry was filtered and the solid was washed three times (150 mL) with MeOH. The collected solid was dried at 35° C. overnight and under vacuum for several hours to give 18.0 g (49 mmol, 94% yield) of mucilaginous diamino chloride (3). [ka]

[0221] Example 3. Synthesis of mucic acid di(Asp(OtBu)-Boc) (Compound 4) To a 250 mL round bottom flask containing 14.9 g (51 mmol) of Boc-L-aspartic acid 4-tert-butyl ester, 6 g (54 mmol) of hydroxypyridine N-oxide (HOPO) and a magnetic stir bar, 140 mL of acetonitrile (ACN) and 8.5 mL (54 mmol) of N,N'-diisopropylcarbodiimide (DIC) were added. The reaction mixture was stirred for 10 min. Next, a suspension of 6.3 g (17 mmol) of mucic acid diamino chloride (3) in a mixture of water (55 mL) and TEA (5.4 mL) was added to the reaction flask. The reaction mixture was heated (80° C.) for approximately 24 h with constant magnetic stirring. After heating, the ACN was removed from the reaction mixture using vacuum. Additional water (150 mL) was added to the concentrated reaction, and the mixture was heated (85° C.) for 1 h. After cooling, the reaction mixture was collected by vacuum filtration and washed three times with water (100 mL). The washed solid was placed back into the round bottom flask with ACN (150 mL). The reaction mixture was heated to reflux with constant stirring until dissolved. After cooling, the reaction mixture was collected by filtration and washed three times with cold ACN (100 mL). The collected solid was placed back into the round bottom flask with ACN (150 mL) and the reaction mixture was heated to reflux with constant stirring for about 1 h. After cooling, the reaction mixture was collected by filtration and washed three times with cold ACN (100 mL). The collected solid was dried under vacuum overnight to give 5.9 g (7 mmol, 41% yield) of mucilaginous acid di(Asp(OtBu)-Boc) (4). [ka]

[0222] Example 4. Synthesis of mucic acid monomer (MAM) (compound 5) To a 40 mL vial containing 1 g (1 mmol) of mucic acid di(Asp(OtBu)-Boc) (4) and a magnetic stir bar was added dichloromethane (DCM, 7.5 mL). Trifluoroacetic acid (TFA, 7.5 mL, 98 mmol) was slowly added to the reaction mixture with constant stirring. After stirring for 1 h at room temperature, methyl tert-butyl ether (MTBE, 100 mL) was slowly added to precipitate the reaction mixture. The precipitated mixture was centrifuged and subsequently washed once with MTBE (100 mL). The collected solid was dried under vacuum and then dissolved in water (30 mL). The dissolved product was passed through a 0.22 μm filter, frozen, and lyophilized to give 0.2767 g (0.384 mmol, 32% yield) of MAM (5). [ka]

[0223] Example 5. Synthesis of mucic acid polymer (MAP) (compound 6) 224 mg (0.30 mmol) of MAM (5), 1 g of di(succinimidyl propionate)-PEG (diSPA-PEG 3.5k A 10 mL round bottom flask containing MAP (0.27 mmol) and a magnetic stir bar was sealed and the two solids were dried under vacuum at room temperature for 4 h. Then, 4.1 mL of anhydrous dimethyl sulfoxide (DMSO) was added to the reaction flask and the mixture was heated (35 °C). After solubilization, 208 μL of anhydrous N,N'-diisopropylethylamine (DIPEA, 1.19 mmol) was added to the reaction mixture. The reaction mixture was heated (35 °C) for 66 h with constant magnetic stirring. The reaction mixture was dialyzed against DMSO and then against water using a 10 kDa membrane. The dialyzed product was then passed through a 0.22 μm filter, frozen, and lyophilized to give 1.03 g (quantitative yield) of MAP (6). [ka]

[0224] For compound 6 synthesized by this method, x was about 80 and y was about 16. Thus, the average molecular weight of compound 6, determined as the arithmetic mean of the number average molecular weight and the weight average molecular weight, was about 65 kDa. These values ​​were comparable to those of different starting materials (e.g., diSPA-PEG). 2k ) is used (e.g., for a 2 kDa PEG, x is approximately 46).

[0225] Example 6. Measurement of molecular weight of MAP The molecular weight of MAP(6) was measured on a Malvern Omnisec gel permeation chromatography (GPC) system, which included a solvent delivery pump, a degasser, an automatic sampling injector, a column compartment, a refractive index detector, and a light scattering detector. Samples were prepared for analysis by dissolving known concentrations of MAP(6) in the range of 1-5 mg / mL in phosphate buffered saline (PBS) + 0.02% sodium azide. After complete dissolution, the samples were passed through a 0.45 μm filter. 100 μL of the filtered sample was injected onto a 2×PL Aquagel-OH 40 column (Agilent) at a flow rate of 0.7 mL / min to measure the absolute molecular weight. The analysis was performed at 30 °C. The resulting polymer peaks were analyzed using Malvern OMNISEC software.

[0226] Example 7.10-Synthesis of TBDPS-SN38 (Compound 7) A 250 mL round bottom flask containing 2 g (5.10 mmol) of SN38 and a magnetic stir bar was charged with 100 mL of anhydrous DCM and 4.2 mL (30.58 mmol) of TEA. To this reaction mixture was then added 7.9 mL (30.58 mmol) of tert-butyl(chloro)diphenylsilane (TBDPSCl). The mixture was heated to reflux (50 °C) overnight (~16 h) with constant stirring and then washed with 0.2 N HCl (2 x 50 mL), saturated NaHCO3 (100 mL) and brine (100 mL). The organic solution was dried over MgSO4 and evaporated in vacuum. The residue was dissolved in anhydrous DCM and precipitated with hexane. This precipitation was repeated to further remove excess TBDPSCl. The solid was dried in vacuum to give 1.2 g (1.9 mmol, 37% yield) of 10-TBDPS-SN38 (7). [ka]

[0227] Example 8. Synthesis of 20-(Boc-Gly)-10-TBDPS-SN38 (Compound 8) To a 40 mL vial containing 0.5 g (0.8 mmol) of 10-TBDPS-SN38 (7), 8.9 mL of anhydrous DCM and a magnetic stir bar were added. After solvent addition, the reaction was cooled to 0 °C. To another vial was added 0.35 g of Boc-Gly-OH (2.0 mmol), 0.39 g (2.0 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl), 0.1 g (0.8 mmol) of 4-dimethylaminopyridine (DMAP), and 3.8 mL of anhydrous DCM. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0 °C for 3 h, the mixture was washed with 0.5% NaHCO3 (2 x 50 mL), water (50 mL), 0.1 N HCl (2 x 50 mL), and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to give 0.54 g of crude 20-(Boc-Gly)-10-TBDPS-SN38 (8) (0.7 mmol, 86% yield). [ka]

[0228] Example 9. Synthesis of 20-(Boc-GABA)-10-TBDPS-SN38 (Compound 9) To a 40 mL vial containing 1 g (1.6 mmol) of 10-TBDPS-SN38 (7), 17.7 mL of anhydrous DCM and a magnetic stir bar were added. After solvent addition, the reaction was cooled to 0 °C. To a separate vial, 0.82 g (4.0 mmol) of Boc-GABA-OH, 0.77 g (4.0 mmol) of EDC.HCl, 0.20 g (1.6 mmol) of DMAP, and 7.6 mL of anhydrous DCM were added. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0 °C for 3 h, the mixture was washed with 0.5% NaHCO3 (2 x 50 mL), water (50 mL), 0.1 N HCl (2 x 50 mL), and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to give 1.05 g of crude 20-(Boc-GABA)-10-TBDPS-SN38 (9) (1.28 mmol, quantitative yield). [ka]

[0229] Example 10. Synthesis of 20-(Boc-Hex)-10-TBDPS-SN38 (Compound 10) To a 40 mL vial containing 0.6 g (0.95 mmol) of 10-TBDPS-SN38 (7), 10.4 mL of anhydrous DCM and a magnetic stir bar were added. After solvent addition, the reaction was cooled to 0 °C. To a separate vial, 0.55 g of Boc-Hex-OH (2.4 mmol), 0.45 g (2.4 mmol) of EDC.HCl, 0.12 g (0.98 mmol) of DMAP, and 4.5 mL of anhydrous DCM were added. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0 °C for 3 h, the mixture was washed with 0.5% NaHCO3 (2 x 50 mL), water (50 mL), 0.1 N HCl (2 x 50 mL), and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to give 0.88 g of crude 20-(Boc-Hex)-10-TBDPS-SN38 (10) (1.0 mmol, 111% yield). [ka]

[0230] Example 11. Synthesis of 20-(Boc-Val)-10-TBDPS-SN38 (Compound 11) To a 100 mL round bottom flask containing 1.0 g (1.6 mmol) of 10-TBDPS-SN38 (7), 17.7 mL of anhydrous DCM and a magnetic stir bar were added. After solvent addition, the reaction was cooled to 0 °C. To a separate vial, 0.87 g of Boc-Val-OH (4.0 mmol), 0.77 g (4.0 mmol) of EDC.HCl, 0.20 g (1.6 mmol) of DMAP, and 7.6 mL of anhydrous DCM were added. The contents of the second vial were then transferred to the flask containing 7. After stirring at 0 °C for 3 h, the mixture was washed with 0.5% NaHCO3 (2 x 50 mL), water (50 mL), 0.1 N HCl (2 x 50 mL), and brine. The organic solution was dried over MgSO4 and evaporated in vacuo to give 1.24 g of crude 20-(Boc-Val)-10-TBDPS-SN38 (11) (1.5 mmol, 93% yield). [ka]

[0231] Example 12. Synthesis of 20-(HCl.Gly)-SN38 (Compound 12) To a 100 mL round bottom flask containing 1.2 g (1.5 mmol) of 20-(Boc-Gly)-10-TBDPS-SN38 (8) and a magnetic stir bar, 75 mL of 12 N HCl was added slowly with constant stirring. Hexane (20 mL) was added on top of the HCl solution. After stirring overnight (~16 h) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed three times with hexane (3 x 20 mL). After that, the HCl was removed from the reaction mixture using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.44 g (0.91 mmol, 60%) of 20-(HCl.Gly)-SN38 (12). [ka]

[0232] Example 13.20-Synthesis of (TFA.GABA)-SN38 (Compound 13) To a 50 mL round bottom flask containing 0.37 g (0.45 mmol) of 20-(Boc-GABA)-10-TBDPS-SN38 (9), 8.6 mL of water, and a magnetic stir bar, 8.6 mL of TFA was added slowly with constant stirring. Hexane (20 mL) was added over the TFA solution. After stirring overnight (~16 h) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed three times with hexane (3 x 20 mL). The reaction mixture was then concentrated using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.16 g (0.27 mmol, 60%) of 20-(TFA.GABA)-SN38 (13). [ka]

[0233] Example 14.20-Synthesis of (TFA.Hex)-SN38 (Compound 14) To a 50 mL round bottom flask containing 0.37 g (0.43 mmol) of 20-(Boc-Hex)-10-TBDPS-SN38 (10), 8.3 mL of water, and a magnetic stir bar, 8.3 mL of TFA was added slowly with constant stirring. Hexane (20 mL) was added over the TFA solution. After stirring overnight (~16 h) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed three times with hexane (3 x 20 mL). The reaction mixture was then concentrated using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.16 g (0.26 mmol, 60%) of 20-(TFA.Hex)-SN38 (14). [ka]

[0234] Example 15. Synthesis of 20-(HCl.Val)-SN38 (Compound 15) To a 100 mL round bottom flask containing 1.2 g (1.4 mmol) of 20-(Boc-Val)-10-TBDPS-SN38 (11) and a magnetic stir bar, 75 mL of 12 N HCl was added slowly with constant stirring. Hexane (20 mL) was added on top of the HCl solution. After stirring overnight (~16 h) at room temperature, the hexane layer was removed from the reaction. The reaction was then washed three times with hexane (3 x 20 mL). After that, the HCl was removed from the reaction mixture using vacuum. The residue was dissolved in MeOH and precipitated with MTBE. The solid was dried under vacuum to give 0.46 g (0.86 mmol, 60% yield) of 20-(HCl.Val)-SN38 (15). [ka]

[0235] Example 16. Synthesis of MAP-Gly-SN38 (Compound 16) A 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was charged with 8 mL of anhydrous DMSO. To this reaction mixture was then added 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of N-hydroxysuccinimide (NHS). Following the addition of EDC.HCl and NHS, 105.2 mg (0.22 mmol) of 20-(HCl.Gly)-SN38 (12) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred overnight (approximately 18 hours) at room temperature. The reaction mixture was dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized to give 98 mg (quantitative yield) of MAP-Gly-SN38 (16). [ka]

[0236] As noted in Example 5, for compounds 6 and 16, x was approximately 80 and y was approximately 16.

[0237] Example 17. Synthesis of MAP-GABA-SN38 (Compound 17) To a 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was added 8 mL of anhydrous DMSO. To this reaction mixture was then added 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS. Following the addition of EDC.HCl and NHS, 128.3 mg (0.22 mmol) of 20-(TFA.GABA)-SN38 (13) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred overnight (approximately 18 hours) at room temperature. The reaction mixture was dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized to give 91 mg (quantitative yield) of MAP-GABA-SN38 (17). [ka]

[0238] As noted in Example 5, for compounds 6 and 17, x was approximately 80 and y was approximately 16.

[0239] Example 18. Synthesis of MAP-Hex-SN38 (Compound 18) To a 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was added 8 mL of anhydrous DMSO. To this reaction mixture was then added 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS. Following the addition of EDC.HCl and NHS, 134.4 mg (0.22 mmol) of 20-(TFA.Hex)-SN38 (14) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred overnight (approximately 18 hours) at room temperature. The reaction mixture was dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized to give 97 mg (quantitative yield) of MAP-Hex-SN38 (18). [ka]

[0240] As noted in Example 5, for compounds 6 and 18, x was approximately 80 and y was approximately 16.

[0241] Example 19. Synthesis of MAP-Val-SN38 (Compound 19) To a 10 mL round bottom flask containing 100 mg of MAP (6) and a magnetic stir bar was added 8 mL of anhydrous DMSO. To this reaction mixture was then added 51.9 mg (0.27 mmol) of EDC.HCl and 24.9 mg (0.22 mmol) of NHS. Following the addition of EDC.HCl and NHS, 114.3 mg (0.22 mmol) of 20-(HCl.Val)-SN38 (15) and anhydrous DIPEA (0.22 mmol) were added to the reaction mixture. The mixture was stirred overnight (approximately 18 hours) at room temperature. The reaction mixture was dialyzed against DMSO and then against water at pH 4 using a 10 kDa membrane. The dialyzed product was passed through a 0.22 μm filter, frozen, and lyophilized to give 96 mg (quantitative yield) of MAP-Val-SN38 (19). [ka]

[0242] As noted in Example 5, for compounds 6 and 19, x was approximately 80 and y was approximately 16.

[0243] Example 20. Characterization of Drug Loading The loading of SN38 onto the MAP was measured on an Agilent 1100 HPLC system using a reversed-phase column (Synergi 4 μm Hydro-RP 80 Å, Phenomenex) coupled to a fluorescence detector set at 375 / 536 nm (ex / em). ACN / 10 mM potassium phosphate buffer, pH 4 (1:1 v / v) was used as the eluent at a flow rate of 0.5 mL / min.

[0244] For analysis, the polymer-drug conjugates (compounds 16-19) were dissolved at a concentration of 1 mg / mL in PBS at pH 7.4. The amount of unconjugated SN38 was measured by first mixing 10 μL of sample with 10 μL of 0.1 N HCl and incubating at room temperature for 30 min. 6.7 μL of water was added, followed by 73.3 μL of ACN, and incubated at room temperature for 3 h. The mixture was centrifuged at 1,4000 g for 10 min at 4 °C, and the supernatant was passed through a 0.45 μm filter. 10 μL of the filtered sample was injected and the SN38 concentration was measured. The resulting peak area of ​​eluted SN38 was compared to the peak area of ​​SN38 of known concentration.

[0245] To measure the total amount of SN38, 10 μL of sample was mixed with 6.7 μL of 0.1 N NaOH. The solution was incubated at room temperature for 3.5 hours to release SN38 from the parent polymer. Then, 10 μL of 0.1 N HCl was added (to convert the carboxylate SN38 form to the lactone form) and the mixture was incubated for 45 minutes. Then, 73.3 μL of ACN was added and the mixture was incubated at room temperature for 3 hours. The samples were then centrifuged and processed as above. Polymer-bound SN38 was determined from the difference between the total SN38 concentration and the unconjugated SN38 concentration.

[0246] Example 21. Formation of MAP-Linker-SN38 Nanoparticles The polymer-drug conjugates (compounds 16-19) were dissolved in water at pH 4 at a concentration of 4 mg / mL to form MAP-linker-SN38 nanoparticles. The solution was passed through a 0.22 μm filter and frozen for subsequent analysis. The nanoparticles were diluted to 2 mg / mL in water at pH 4 and the hydrodynamic diameter was measured by dynamic light scattering (DLS) using a ZetaPALS (Brookhaven Instruments Corporation) instrument. Five runs were performed for 1 min each for the nanoparticles. The diameter of all nanoparticles was approximately 25 nm, ranging from 19 to 32 nm.

[0247] Example 22. In Vitro Release Test Experiments were performed to evaluate the release of SN38 from MAP-Gly-SN38, MAP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-SN38, MAP-β-Ala-SN38 and MAP-Leu-SN38 nanoparticles. See Examples 33-35 below for the synthesis of MAP-Ala-SN38, MAP-β-Ala-SN38 and MAP-Leu-SN38 nanoparticles. These studies were performed at 0.1 mg SN38 / mL in PBS at pH values ​​of 6.5, 7.0 or 7.4.

[0248] PBS medium was pipetted into the cuvette and incubated at 37° C. for 2 hours in a humidified oven to equilibrate. Nanoparticle formulations were mixed into the relevant medium and returned to the oven. Samples were removed at pre-determined time points and flash frozen at −80° C. until time of analysis. The amount of unconjugated SN38 and total SN38 were measured as described above in Example 20. Polymer-bound SN38 concentration was determined from the difference between total SN38 and unconjugated SN38 concentrations.

[0249] The results showed that the release of SN38 from all seven nanoparticles: MAP-Gly-SN38, MAP-GABA-SN38, MAP-Hex-SN38, MAP-Val-SN38, MAP-Ala-SN38, MAP-β-Ala-SN38 and MAP-Leu-SN38, showed first-order kinetics. The release rate was observed to be strongly dependent on pH. As the pH increased from 6.5 to 7.4, the release half-life decreased for all nanoparticle formulations, suggesting that hydrolysis plays an important role in the release of SN38. The more hydrophobic and sterically hindered the linker was, the longer the nanoparticle half-life was, ranging from about 30 h to more than 168 h.

[0250] Example 23. Determination of the number of strands per particle The number of strands per nanoparticle (SpP) was determined by calculating the ratio of the molecular weight of the particle to that of the MAP. Polyethylene oxide (PEO) standards of nominal molecular weight 100 kDa and 200 kDa as well as samples of interest were prepared for GPC analysis (Omnisec GPC, Malvern) by dissolving at 1-5 mg / mL in water or PBS + 0.02% sodium azide at pH 3. Concentrations were chosen to approximate those expected after particle formation. After dissolution, samples were passed through a 0.45 μm filter and analyzed on a Zenix SEC300 or Zenix-C SEC300 column (Sepax, 3 μm) at 0.35 mL / min at 30 °C. Light scattering and differential refractive index information was used with Malvern OMNISEC software or similar molecular weight analysis software to determine the molecular weight of the particles.

[0251] The strands per particle were determined by dividing the molecular weight of the particle by the molecular weight of the MAP-SN38 conjugate molecule, which was calculated from the molecular weight of the polymer adjusted for drug loading according to the following formula: MW コンジュゲート =MW ポリマー / (1-T % ) (In the formula, MW ポリマー is the molecular weight of MAP, and T % is the drug loading rate expressed as a decimal, e.g., T at 10% loading % =0.1.)

[0252] Measurement of SpP was demonstrated with MAPs with various linkers and drugs. MAPs of the same molecular weight conjugated with CPT or SN38 produce nanoparticles with approximately 1.5 to 2.5 strands per particle. The use of different linkers for derivatized SN38 alters the number of strands per particle and, consequently, the density of the particles. The number of strands per particle varied from approximately 1.3 to approximately 4.6, depending on the linker (glycine, γ-aminobutyric acid, hexanoic acid, alanine, β-alanine, leucine, or valine).

[0253] Example 24. Preparative Scale Synthesis of N-Boc Protected Mucor Acid Diamine (Compound 2) To a mechanically stirred 15 liter reaction vessel under nitrogen was added 450 g (2.1 mol) of mucilaginous acid and MeOH (5.75 L). To the reaction vessel was added a solution of MeOH (1 L) and concentrated sulfuric acid (34.2 mL, 0.64 mol). The mixture was heated to an internal temperature of 65° C. for 48 hours. The contents of the reaction vessel were cooled to 22° C. at which point 0.81 L (5.78 mol) of TEA was added over 20 minutes. The reaction was stirred for 1 hour at which point a solution of 0.76 kg (4.71 mol) of N-(2-aminoethyl)(tert-butoxy)carboxamide in 0.9 L of MeOH was added over 20 minutes. The reaction was heated to an internal temperature of 63° C. and after 60 minutes 1.8 L of MeOH was added to aid stirring. After a total of 2 hours at 63° C., the reaction vessel was cooled at 1° C. per minute and held at 20° C. overnight. The reaction slurry was filtered and the cake was washed with MeOH (3×0.5 L). The solid was dried on the vacuum filter overnight to completely remove the liquid. The isolated solid was returned to the reaction vessel with MeOH (9.0 L) and stirred at 63° C. for 1 h. The reaction vessel was set to cool at 1° C. per minute and held at 20° C. for approximately 60 h. The slurry was filtered, washed with MeOH (2×1.0 L) and the solid was dried overnight in a vacuum oven at 45° C. to give 963 g of N-Boc protected mucilaginous diamine (2) (91% yield as solvate with MeOH, 82% yield as anhydrous). [ka]

[0254] Example 25. Preparative Scale Synthesis of Mucic Acid Diaminochloride (Compound 3) To a mechanically stirred 15 L reaction vessel was added 890 g of N-Boc protected mucilaginous diamine (2) (1.80 mmol), 3.6 L of water, and 7.1 L of MeOH. To this slurry was added 12 M HCl (3.6 L) over 2 hours while maintaining the temperature in the range of 20° C. to 30° C. The reaction was stirred overnight at which point the slurry was filtered and washed with 1.8 L of MeOH. The isolated solid was placed in a tray and dried in a vacuum oven at 45° C. The isolated solid was charged to a 15 L reaction vessel under nitrogen along with water (5.5 L) and stirred at 45° C. 5.5 L of MeOH was added over 30 minutes resulting in crystallization. The reaction vessel was cooled and the reaction contents were allowed to cool to room temperature. The solid was isolated by filtration and washed with MeOH (2×1.0 L). The solid was collected and dried overnight in a vacuum oven at 40° C. to give 504 g (1.37 mol, 76% yield) of mucilaginous diamino chloride (3). [ka]

[0255] Example 26. Preparative-scale synthesis of mucic acid di(Asp(OtBu)-Boc) (Compound 4) To a mechanically stirred 15 L reactor was added 1.27 kg Boc-L-aspartic acid 4-tert-butyl ester (4.39 mol), 0.62 kg Oxima(ethyl cyanohydroxyiminoacetate) (4.39 mol) and 3.5 L DCM. The reaction mixture was cooled to 5° C. and stirred for 30 min. To the reaction mixture was added 0.84 kg EDC.HCl (4.40 mol) as a solid over 10 min. EDC.HCl was rinsed into the reaction with an additional 0.5 L DCM and the reaction was allowed to stir for approximately 30 min and then cooled to 10° C. In a separate 12 L flask was charged 1.02 kg sodium carbonate (9.61 mol) and 5.0 L water. The components were dissolved and then cooled to room temperature. 504 g (1.37 mol) of 3 was added to the sodium carbonate solution over 10 min. This mixture was allowed to stir for 15 minutes at which point it was added to the Boc-L-aspartic acid 4-tert-butyl ester in the 15 L reaction flask over approximately 3 minutes. The mixture reaction was exothermic, but the cooling jacket was set at 10° C. for 30 minutes, then 15° C. for an additional 45 minutes. The reaction jacket was then set to 33° C., and after 1 hour, 8.06 L of water was added and stirred for 5 minutes. Stirring was then discontinued, and after 15 minutes the reaction phases were separated. The DCM (bottom layer) was removed, and an additional 1.0 L of DCM was added to the reactor. The mixture was stirred for 10 minutes, then allowed to settle. The DCM layer was collected, combined with the first collected organic layer, and concentrated to a total volume of approximately 3 L. 1 L of water was added to the organic mixture to facilitate further distillative removal of DCM. The organic reaction mixture was then added to a 15 L reactor containing 11.1 L of water heated to 40° C. The stirred mixture was allowed to return to 20° C., forming a slurry. The water was removed, leaving the solids in the reactor. 10.1 L of water was added to the wet solids. The reaction mixture was heated to 37° C. to facilitate stirring. The reaction was then cooled to 3° C. overnight and stirred. The solids were filtered and then added back to the reactor with an additional 10 L of water. The slurry was stirred at room temperature for 3 hours. The solids were filtered again, washed with 1.0 L of water, and dried to yield 815 g of material. 790 g of crude 4 was added to a 5 L round bottom flask along with 2.4 L of ACN and heated to 60° C. Water was continuously distilled off by azeotropic distillation under partial vacuum and continued addition of ACN to a volume of about 1.6 L.To this was added 3.1 L of ACN to induce crystallization. The slurry was briefly heated to reflux and then cooled to 5° C. and held there for 30 min. After cooling, the slurry was filtered and washed with ACN (4×0.5 L, 5° C.). The solid was vacuum heated to 50° C. to remove the solvent. 6.3 L of DCM was added to the reaction mixture in two portions at which point the slurry was concentrated and the ACN was distilled off. The solid was then vacuum heated to 80° C. to remove the DCM and give a free-flowing powder. The solid was then placed in a vacuum oven and dried at 80° C. under reduced pressure to give 310 g (0.35 mol, 26%) of mucic acid di(Asp(OtBu)-Boc) (4) as a 1:1 molar solvate with ACN. [ka]

[0256] Example 27. Preparative-scale synthesis of mucic acid monomer neutral species (compound 20) A 5 L jacketed round bottom flask equipped with mechanical stirring was charged with 300 g of 4 (0.34 mol), 1.2 L of DCM, 0.11 L of water (6.13 mol) and 0.279 L of triisopropylsilane (1.36 mol). The reaction mixture was cooled to 5° C. and then a slurry was formed. 1.2 L of TFA, previously cooled in an ice bath for 1 h, was added to the stirred slurry. The reaction was held at an internal temperature of 4° C. overnight. An additional 0.30 L of TFA was added and stirring was continued at 4° C. overnight. 0.90 L of hexane was added at which point the reaction was stirred at 5° C. for 10 min. The reaction mixture was then poured into a separatory funnel and the lower phase was separated into a 5 L round bottom funnel. The reaction material was washed with additional DCM (300 mL) and combined with the first collected fraction. The combined collected fractions were concentrated on a rotary evaporator at <30° C. An additional 0.90 L of DCM was added to the flask and then removed using a rotary evaporator at 25° C. Then 0.60 L of toluene was added and concentrated on a rotary evaporator to azeotropically remove water and TFA to give an oil. This oil was filtered through a plug of Celite and washed 4 times with DCM (0.40 L). DCM was removed on a rotary evaporator at 35° C. The resulting biphasic mixture was added to a clean 5 L reaction vessel and heated to 23° C. 3.0 L of cyclopentyl methyl ether (CPME) was added over 20 minutes, forming an oily solid. The vessel was cooled to 22° C. and stirred overnight to give sticky crystals. 1.5 L of DCM was distilled off at 35° C. using a short path distillation column. The resulting slurry was filtered and washed with CPME (2×0.45 L). The solid was then dried on a rotary evaporator in a round bottom flask and vacuum dried at room temperature overnight to give 261 g of mucic acid monomer neutral species (208.8 g, 82% yield considering titer).

[0257] For further recrystallization, 0.24 kg of mucic acid monomer neutral seeds were added to a 1 L Erlenmeyer flask along with water (0.20 L). The mixture was stirred at room temperature for 1 hour to obtain a solution. The solution was filtered through a filter funnel with additional water (50 mL) and transferred to a 5 L round bottom flask. 2.2 L of tetrahydrofuran (THF) was added to the flask over 30 minutes with stirring. The mixture was stirred for a total of 60 minutes at which point it was concentrated on a rotary evaporator at 35° C. to remove 1 L of distillate. Additional THF (0.98 L) was added over 10 minutes. The slurry was cooled to room temperature and stirred for 20 minutes. The cooled slurry was filtered and washed with THF (2×0.25 L), followed by drying in a vacuum oven at room temperature for 2.5 days to obtain 119 g (0.23 mol, 66% yield) of the neutral form of mucic acid monomer (20). [ka]

[0258] Example 28. Preparative-scale synthesis of MAM (Compound 5) A 3 L round bottom flask containing 120 g (0.23 mol) of 20 and DCM (1.2 L) was stirred for 10 min at which point water (0.06 L) was added. Stirring was continued for 15 min at which point TFA (66.0 mL, 0.86 mol) was added over 5 min. The solids were dissolved and the mixture was stirred at room temperature for 1 h. The mixture was concentrated on a rotary evaporator at 30° C. To a 5 L round bottom flask equipped with mechanical stirring was added ether (4.0 L) and MAM seed crystals (5, 12 g). The 20 / TFA / water solution was added slowly to the ether with stirring. After 1 h, the ether was decanted off and fresh additional ether was added (3.6 L). The mixture was stirred at room temperature until the MAM-diTFA salt precipitated. The solids were filtered and washed once with ether (0.2 L). The solid was dried under vacuum at room temperature and heated at 30° C. for 1 h to give 122 g (75% yield) of MAM (5). [ka]

[0259] Example 29. Preparative-scale synthesis of mucic acid polymer (compound 6) MAP (6) was synthesized by polymerization of mucic acid monomer (MAM) (5) and di-SPA-PEG 3.5k It was synthesized by step-growth polymerization of

[0260] 108.52 mg (0.158 mmol) of MAM (5) containing approximately 1.5 equivalents of TFA, 579.54 mg of diSPA-PEG 3.5k A 20 mL glass vial containing (0.157 mmol) and a magnetic stir bar was sealed and the two solids were dried under vacuum at room temperature for 1 hour. To the reaction flask was added 3.43 mL of anhydrous DMSO under argon. The reactants were solubilized at room temperature. After solubilization, 126 μL of anhydrous DIPEA (0.72 mmol) was added to the reaction mixture under argon. The reaction mixture was stirred at room temperature with constant magnetic stirring for 3 hours.

[0261] The reaction mixture was precipitated in cold isopropyl alcohol (IPA, ×10 vol, 0-4 °C). The reactor was washed with DMSO (3 × 1 mL) and then precipitated in cold IPA. The resulting white suspension was stirred at 0-4 °C for 30 min and centrifuged at 4 °C for 12 min. The white fluffy polymer that precipitated at the bottom was isolated by decanting the IPA. The polymer was washed twice with cold IPA (×5 vol) and the procedure was repeated. The product was vacuum dried overnight at room temperature in a Schlenk line to remove the IPA and then dissolved in Milli-Q water (8 mL). The solution in water was frozen and lyophilized to give 612.5 mg (quantitative yield) of MAP (6).

[0262] The molecular weight of the polymer was controlled by incorporating a stoichiometric distortion between the two monomers. At 1:1, the molecular weight of MAP(6) was the highest, and the molecular weight decreased as the molar ratio moved away from 1:1. Exemplary stoichiometric distortions and experimental molecular weights obtained by GPC are shown in Table 1. [Table 1]

[0263] Example 30. Synthesis of 20-(Boc-Gly)-CPT (Compound 21) 200.4 mg (0.58 mmol) of CPT, 302.7 mg (1.73 mmol) of Boc-Gly-OH, and 140.6 mg (1.15 mmol) of DMAP were added to a 10 mL round bottom flask equipped with a magnetic stir bar and a rubber septum. The vessel and its contents were purged with argon for 10 minutes, at which point anhydrous DCM (1 mL) was added to slurry the material. 280 μL (1.81 mmol) of DIC was added dropwise via syringe to the reaction mixture over approximately 3 minutes. The reaction was stirred at room temperature for 3 hours, at which point approximately 50% of the solvent was removed in vacuo. To the concentrated slurry was added 4 mL of cold MeOH. The reaction was stirred for several minutes to promote further precipitation and then filtered into a Buchner funnel. The solid was washed with additional cold MeOH and then cold MTBE. The isolated solid was dried under high vacuum to give 110.8 mg (0.22 mmol, 38%) of 20-(Boc-Gly)-CPT (21).

[0264] In the above procedure, Boc-Gly-OH can be replaced with other Boc-protected amino acids and dipeptides. By replacing Boc-Ala-OH, Boc-β-Ala-OH, Boc-Val-OH, Boc-GABA-OH and Boc-Phe-Gly-OH, 20-(Boc-Ala)-CPT (compound 41), 20-(Boc-β-Ala)-CPT (compound 42), 20-(Boc-Val)-CPT (compound 40), 20-(Boc-GABA)-CPT (compound 43) and 20-(Boc-Phe-Gly)-CPT (compound 44) ​​were obtained by the same procedure, respectively. [ka]

[0265] Example 31. Synthesis of 20-(TFA.Gly)-CPT (Compound 22) To a scintillation vial equipped with a magnetic stir bar and vent needle was added 97.1 mg (0.20 mmol) of 20-(Boc-Gly)-CPT (21). A stirred suspension was produced by the addition of DCM (0.2 mL). With stirring, TFA (0.2 mL) was added slowly. The reaction mixture was stirred for 2 h. After 2 h, the product was precipitated with MTBE, filtered, and washed. The isolated sample was dried under high vacuum to give 82.5 mg (0.16 mmol, 83% yield) of 20-(TFA.Gly)-CPT (22). [ka]

[0266] Substituting 20-(Boc-Gly)-CPT with 20-(Boc-Ala)-CPT, 20-(Boc-β-Ala)-CPT, 20-(Boc-Val)-CPT, 20-(Boc-GABA)-CPT, and 20-(Boc-Phe-Gly)-CPT in the above method gives 20-(TFA.Ala)-CPT (compound 45), 20-(TFA.β-Ala)-CPT (compound 46), 20-(TFA.Val)-CPT (compound 47), 20-(TFA.GABA)-CPT (compound 48), and 20-(TFA.Phe-Gly)-CPT (compound 49), respectively.

[0267] Example 32. Synthesis of MAP-Gly-CPT (Compound 23) 149.2 mg (0.07 mmol based on COH) of MAP (6) and 21.5 mg of 20-(TFA.Gly)-CPT (22) (0.04 mmol) were added to a 20 mL scintillation vial containing a magnetic stir bar. The headspace of the vessel was purged with argon for approximately 30 minutes and then sealed under argon. Anhydrous DMSO (approximately 9 mL, approximately 85% of the total reaction volume) was added and the vial contents were allowed to dissolve with stirring. An 8 mL scintillation vial containing 166.3 mg of (7-azabenzotriazol-1-yloxy)trispyrrolizinophosphonium hexafluorophosphate (PyAOP, 0.32 mmol, 4.4 equiv.) and a magnetic stir bar was purged under argon. The contents of the vessel were allowed to dissolve in anhydrous DMSO (approximately 3.2 mL) with stirring. Once all solids in both vessels had dissolved, 1.6 mL of the PyAOP solution (83.2 mg PyAOP, 0.16 mmol PyAOP, approximately 15% of the total reaction volume) was transferred to the polymer-containing vessel. This reaction was stirred for approximately 2 minutes, at which point approximately 45 μL (0.26 mmol) of anhydrous DIPEA (target 31.3 μL, 0.18 mmol) was added. The reaction vessel was stirred in the dark at room temperature for approximately 18 hours. After 18 hours, the reaction vessel was removed from the argon atmosphere and approximately 1 / 3 of the reaction contents were transferred to a 50 mL glass centrifuge tube. The reaction was precipitated by slow addition of cold ethyl acetate (EtOAc) (0-4 °C). This suspension was stirred for approximately 30 minutes at 0-4 °C and then centrifuged. The EtOAc layer was decanted off and the next 1 / 3 of the reaction medium was added to the same vessel. This procedure was repeated until all of the reaction medium had been precipitated in the same tube. The precipitated solid was washed a total of three times with cold EtOAc (×5 volumes) while vortexing to resuspend the solid. After the last wash cycle, the last of the EtOAc was decanted off and the solid was frozen in the centrifuge tube using liquid nitrogen. The frozen centrifuge tube was exposed to high vacuum for approximately 3 hours to dry the solid, yielding 115.2 mg (quantitative yield) of MAP-Gly-CPT (23). [ka]

[0268] The same method was also used to produce MAP-CPT conjugates using 20-(TFA.Ala)-CPT, 20-(TFA.β-Ala)-CPT, 20-(TFA.Val)-CPT, 20-(TFA.GABA)-CPT or 20-(TFA.Phe-Gly)-CPT as the starting material instead of 20-(TFA.Gly)-CPT to produce MAP-Ala-CPT (compound 50), MAP-β-Ala-CPT (compound 51), MAP-Val-CPT (compound 52), MAP-GABA-CPT (compound 53) and MAP-Phe-Gly-CPT (compound 54). [ka] [ka] [ka]

[0269] As described in Example 5, x was about 80 and y was about 16 for compounds 6, 23 and 50-54.

[0270] Example 33.10-Synthesis of OBoc-SN38 (Compound 24) Under an argon atmosphere, a reaction vessel was charged with 2.0 g (5.10 mmol) of SN38. 204 mL of anhydrous DCM was added to the reaction vessel, followed by 12.2 mL (151 mmol) of anhydrous pyridine and 1.5 mL (6.53 mmol) of di-tert-butyl dicarbonate. The reaction was stirred at room temperature for approximately 16 hours, at which point the reaction was filtered through Celite 545 (filter aid) and washed with 0.5 N HCl and saturated aqueous sodium bicarbonate. The organic phase was dried over magnesium sulfate and concentrated in vacuo. The reaction mixture was diluted in DCM and then precipitated into hexanes. The solid was filtered and dried in vacuo to give 2.2 g (4.47 mmol, 88% yield) of 10-OBoc-SN38 (24). [ka]

[0271] Example 34. Synthesis of 20-(Boc-Gly)-10-OBoc-SN38 (Compound 25) A reaction vessel cooled to 0 °C under argon was charged with 502 mg (1.02 mmol) of 10-OBoc-SN38 (24) and 8.8 mL of DCM. A cooled solution containing 3.8 mL of DCM, 447 mg (2.55 mmol) of Boc-Gly-OH, 489 mg (2.55 mmol) of EDC.HCl, and 125 mg (1.02 mmol) of DMAP was added to the reaction vessel. The reaction was stirred at 0 °C for 3 h. The reaction was washed with 0.5% aqueous sodium bicarbonate, water, 0.1 N HCl, and brine. The organic phase was dried over magnesium sulfate and evaporated in vacuo to give 639 mg (0.98 mmol, 97% yield) of 20-(Boc-Gly)-10-OBoc-SN38 (25).

[0272] In the above procedure, Boc-Gly-OH can be replaced with other Boc-protected amino acids. By replacing Boc-Ala-OH, Boc-β-Ala-OH, Boc-Val-OH and Boc-Leu-OH as starting materials, 20-(Boc-Ala)-10-OBoc-SN38 (compound 29), 20-(Boc-β-Ala)-10-OBoc-SN38 (compound 30), 20-(Boc-Val)-OBoc-SN38 (compound 31) and 20-(Boc-Leu)-10-OBoc-SN38 (compound 35) were obtained by the same procedure, respectively. [ka]

[0273] Example 35. Synthesis of 20-(TFA.Gly)-SN38 (Compound 26) To a reaction vessel containing 250 mg (0.38 mmol) of 20-(Boc-Gly)-10-OBoc-SN38 (25) and 1.88 mL of DCM was added 0.47 mL (6.14 mmol) of TFA. The reaction was stirred at room temperature for 2.5 h. The reaction mixture was precipitated into cold (4 °C) MTBE, isolated by centrifugation, washed again with MTBE, and dried in vacuum to give 133 mg (0.24 mmol, 61% yield) of 20-(TFA.Gly)-SN38 (26). [ka]

[0274] Substituting 20-(Boc-Gly)-10-OBoc-SN38 with 20-(Boc-Ala)-10-OBoc-SN38, 20-(Boc-β-Ala)-10-OBoc-SN38, 20-(Boc-Val)-10-OBoc-SN38 and 20-(Boc-Leu)-10-OBoc-SN38 as starting material gave 20-(TFA.Ala)-SN38 (compound 32), 20-(TFA.β-Ala)-SN38 (compound 33), 20-(TFA.Val)-SN38 (compound 34) and 20-(TFA.Leu)-SN38 (compound 36), respectively.

[0275] The TFA salts of the aforementioned amino acid-linked SN38 derivatives can be used to form MAP-SN38 conjugates as described in Examples 17 and 18.

[0276] Example 36 NPBA-PEG 5k Synthesis of -AA (Compound 27) 20g (4mmol) of NH2-PEG 5kA 250 mL round bottom flask containing AA hydrochloride, DIPEA (2.1 mL, 12 mmol) was charged with anhydrous DCM (40 mL) and flushed with argon. The solution was cooled in an ice / water bath. In a 20 mL vial containing a magnetic stir bar, 934 mg (5.5 mmol) of 3-carboxy-5-nitrophenylboronic acid (NPBA) was dissolved in anhydrous DCM / dimethylformamide (DMF) (8 mL, 4:1 v / v). 1038 mg (5.25 mmol) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was added to the solution of NPBA. The activated acid was stirred for 15 min and then added dropwise to the pre-cooled solution of PEG-amine in DCM. The reaction mixture was stirred at 0° C. for 20 min. Deionized water (100 mL) was added to the reaction mixture, stirred vigorously and warmed to room temperature. At this stage, the pH of the reaction mixture was 9. Monosodium phosphate (NaH2PO4, 2.5 g) was then added to bring the pH to 7. The pH of the reaction mixture was further reduced to 4 by adding 1 M HCl (12 mL). The reaction mixture was transferred to a separatory funnel and isopropyl acetate (iPrOAc, 100 mL) was added. The mixture was shaken well and allowed to settle to obtain separate phases. The organic phase was discarded and the extraction was repeated with iPrOAc (3 x 100 mL). The aqueous phase was transferred to a 250 mL round bottom flask and concentrated on a rotary evaporator at room temperature at 20 Torr for 30 min. The aqueous solution was lyophilized to obtain a powder. The lyophilized powder was dissolved in DMSO / water (40 mL, 3:1 v / v). This solution was purified by hydrophobic interaction chromatography on a C18Aq Teledyne ISCO column (475 g) pre-equilibrated with 12-15 mL injections of 95% mobile phase A (water + 0.05% TFA) and 5% mobile phase B (ACN + 0.05% TFA). A gradient from 5% B to 40% B was run before washing the column and subsequent injections. The purification was repeated on the remaining product solution. Fractions containing product were analyzed by HPLC. Fractions with purity >99.5% were combined and lyophilized to give 14.2 g (2.71 mmol, 68% yield) of NPBA-PEG. 5k -AA(27) was obtained. [ka]

[0277] For compound 27 synthesized by this method, n was approximately 114. These values ​​were consistent with the results for different starting materials (e.g., NH2-PEG 3.5k -AA) is used (e.g., for a 3.5 kDa PEG, n is about 80).

[0278] Example 37 NPBA-PEG 5k Synthesis of -AA-PFP (Compound 28) In an 8 mL reaction vessel, 301.8 mg (0.06 mmol) of NPBA-PEG was added. 5k -AA (27) was added along with a magnetic stir bar. The reaction vessel was purged with three successive vacuum and argon cycles. The solid was dissolved in anhydrous DCM (1.2 mL) and then 35 μL of anhydrous N-methylmorpholine (0.35 mmol) was added via syringe. The reaction vessel was briefly opened and 45.3 mg of bis(pentafluorophenyl)carbonate (0.12 mmol) was added. The reaction headspace was purged with argon and the reaction was stirred at room temperature for approximately 2 h. The reaction solution was added dropwise to a stirred volume of MTBE (25 mL) to promote precipitation. The mixture was stirred under an argon atmosphere for 30 min and then centrifuged at 2600 g. The liquid layer was decanted at which point heptane (20 mL) was added to the centrifuge tube. The sample was mechanically stirred before being centrifuged a second time at 2600 g (8 min). The liquid was decanted from the vessel, leaving behind the remaining wet solid. The vessel was briefly cooled with liquid nitrogen, then the solvent was removed by pumping using a Schlenk line to obtain 300 mg (0.055 mmol, 96% yield) of NPBA-PEG. 5k -AA-PFP (28) was obtained. [ka]

[0279] As described in Example 36, in compounds 27 and 28, n was approximately 114.

[0280] Example 38 NPBA-PEG 5k -Synthesis of Tf crude mixture 35 g (0.440 mmol) of holotransferrin (Tf) was placed in a reaction vessel, and 1.75 L of 50 mM HEPES (pH 7.8) was slowly added to dissolve Tf at a concentration of 20 g / L. After solubilization, 4.77 g (0.881 mmol) of NPBA-PEG was dissolved in 44 mL of DMSO. 5k -AA-PFP (28) was added slowly to the reaction mixture and mixed well. The reaction mixture was incubated at room temperature for 2 h. The crude reaction mixture was analyzed on an Agilent 1290 HPLC system equipped with a reversed phase (RP) column (Zorbax 300SB-CN 4.6 mm ID x 150 cm) with mobile phase A (water + 0.1% TFA) and mobile phase B (ACN / IPA (4:1 v / v) + 0.1% TFA) at a flow rate of 0.7 mL / min. The column was connected to a spectrophotometric detector set at a wavelength of 280 nm and a reference wavelength of 360 nm. The crude reaction mixture was further analyzed on an Agilent 1290 HPLC system equipped with a hydrophobic interaction chromatography (HIC) column (TSK gel Butyl-NPR 4.6 mm IDx3.5 cm) with mobile phase A (20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0) and mobile phase B (20 mM sodium phosphate, pH 7.0 / IPA (4:1 v / v)) at a flow rate of 0.8 mL / min. The column was connected to an absorbance detector set at a wavelength signal of 280 nm and a reference of 360 nm. After 2 h of incubation, the crude reaction mixture was ready for HIC purification.

[0281] Example 39. Mono-NPBA-PEG 5k -Isolation of Tf NPBA-PEG 5k1.75 L of the -Tf crude mixture (20 g / L) was added to the reaction vessel. 7 L of mobile phase A (1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 7.4) was added to the crude mixture to give a concentration of 1.2 M ammonium sulfate, 16 mM sodium phosphate, pH 7.4. After mixing, the sample mixture was loaded onto an AxioChrom 140 column (1.7 L) packed with GE Sepharose Phenyl High Performance HIC resin at a pump flow rate of 200 mL / min. A residence time of approximately 10 min was allowed for the sample mixture to be loaded onto the HIC column. IPA / 10 mM sodium phosphate, pH 7.4 (1:4 v / v, mobile phase B) was used as the eluent. Before the elution step, 2 column volumes (CV) of 20% mobile phase B were washed to remove unbound species. Purified NPBA-PEG 5k -Tf was eluted with a gradient of 20 to 100% mobile phase B over 15 CV. 5k The fractions of mono-NPBA-PEG (-Tf) were collected and combined for ultrafiltration and diafiltration (UF / DF). 5k -Tf UF / DF. mono-NPBA-PEG 5k The -Tf conjugate was concentrated to 50 g / L by diafiltration with 7 volumes of PBS (pH 7.4). 5k -Tf was sterile filtered through a 0.22 μm polyethersulfone (PES) membrane.

[0282] Example 40. Determination of the number of NPBA per Tf Mono NPBA-PEG 5k5k The number of NPBA per Tf in -Tf is mono-NPBA-PEG 5k -TfNPBA-PEG 5k The ratio of the molar concentration of NPBA to the molar concentration of Tf was calculated using the Alizarin Red S (ARS) detection assay of NPBA. 5kSamples of -AA (27) were prepared at various concentrations (0-0.0098 mM) dissolved in PBS (pH 7.2). ARS was prepared in water at 0.049 mM. Samples were then mixed with ARS and fluorescence was measured (excitation wavelength: 468 nm, emission wavelength: 572 nm) using a Varioskan LUX spectrophotometer (ThermoFisher) and SkanIt (ThermoFisher) or similar fluorescence analysis software. Linear regression of these data (fluorescence vs. concentration) yielded a calibration curve for the determination of NPBA in samples of interest.

[0283] Samples of interest (e.g., Tf and mono-NPBA-PEG 5k -Tf) was dissolved in PBS (pH 7.2) at 0.007 mM. 5k -TfNPBA-PEG 5k The molar concentration of mono-NPBA-PEG 5k The mean fluorescence values ​​of -Tf samples and various known concentrations of NPBA-PEG 5k The fluorescence value was calculated from the calibration curve of the -AA standard (0 to 0.0098 mM) using the following formula. Fluorescence = ε*C N +b (where ε = slope of the linear regression of the calibration curve, b = intercept of the linear regression of the calibration curve, C N = concentration of NBPA-PEG (mM). Therefore, C N (mM)=(fluorescence-b) / ε.)

[0284] Then, according to the following formula, C N Divide by the known Tf concentration to get the number of NPBAs per Tf (N Tf ) was calculated. N Tf =(NPBA-PEG 5k (mmol / L) / (Tf mmol / L)

[0285] Mono-NPBA-PEG using ARS detection assay for NPBA 5k The determined number of NPBAs in -Tf is mono-NPBA-PEG 5kThe reaction with ARS, and therefore the fluorescent signal, requires an available NPBA functional group for reaction. Some molecules (e.g., citrate) form complexes with NPBA, decreasing the fluorescent signal and mono-NPBA-PEG 5k In other cases, excess unreacted NPBA-PEG conjugates are present in the crude sample, increasing the fluorescent signal and mono-NPBA-PEG 5k - The number of NPBAs determined per Tf may vary. Exemplary experimental results are shown in Table 2. [Table 2]

[0286] Example 41 NPBA-PEG 5k Synthesis of -Tras crude mixture 5.5 g (0.038 mmol) of trastuzumab was buffer exchanged into 50 mM HEPES (pH 7.8) and the final protein concentration was adjusted to 10 g / L. After solubilization, NPBA-PEG was dissolved in 3.78 mL of DMSO. 5k 409.07 mg (0.076 mmol) of -AA-PFP (28) was slowly added to the reaction mixture and mixed well. The reaction mixture was incubated at room temperature for 2 h. The crude reaction mixture was analyzed on an Agilent 1290 HPLC system equipped with a reversed phase (RP) column (Zorbax 300SB-CN 4.6 mm ID x 150 cm) with mobile phase A (water + 0.1% TFA) and mobile phase B (ACN / IPA (4:1 v / v) + 0.1% TFA) at a flow rate of 0.7 mL / min. The column was connected to an absorbance detector set at a signal of 280 nm and a reference of 360 nm. After 2 h of incubation, the crude reaction mixture was ready for HIC purification.

[0287] Example 42 Mono-NPBA-PEG 5k -Isolation of Tras 0.55L of NPBA-PEG 5kThe -Tras crude mixture (10 g / L) was added to the reaction vessel. 1.1 L of mobile phase A (1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 7.4) was added to the crude mixture to give a concentration of 1 M ammonium sulfate, 13.3 mM sodium phosphate, pH 7.4. After mixing, the sample mixture was loaded onto an AxioChrom 140 column (1.7 L) packed with GE Sepharose Phenyl High Performance HIC resin at a flow rate of 200 mL / min. A residence time of approximately 10 min was allowed for the sample mixture to be loaded onto the HIC column. IPA / 10 mM sodium phosphate, pH 7.4 (1:4 v / v, mobile phase B) was used as the eluent. Before the elution step, a 2 CV wash with 40% mobile phase B was used to remove unbound species. Purified NPBA-PEG 5k -Tras was eluted with a gradient of 40 to 100% mobile phase B over 15 CV. 5k The fractions of mono-NPBA-PEG (-Tras) were collected and combined for UF / DF. 5k Used for UF / DF of -Tras. mono-NPBA-PEG 5k The -Tras conjugate was concentrated to 50 g / L by diafiltration with 7 volumes of PBS (pH 7.4). 5k -Tras was sterile filtered through a 0.22 μm PES membrane.

[0288] Example 43. Determination of the number of NPBA per Tras Mono NPBA-PEG 5k5k -The number of NPBA per Tras is mono-NPBA-PEG 5k -Tras' NPBA-PEG 5k The ratio of the molar concentration of Tras to the molar concentration of NPBA was calculated using the ARS detection assay of NPBA. 5kSamples of -AA (27) were prepared at various concentrations (0–0.0098 mM) in PBS (pH 7.2). ARS was prepared in water at 0.049 mM. Samples and ARS were then mixed and fluorescence was measured (excitation wavelength: 468 nm, emission wavelength: 572 nm) using a Varioskan LUX spectrophotometer (ThermoFisher) and SkanIt (ThermoFisher) or similar fluorescence analysis software. Linear regression of these data (fluorescence vs. concentration) yielded a calibration curve for the determination of NPBA in samples of interest.

[0289] Samples of interest (e.g., Tras and mono-NPBA-PEG 5k Mono-NPBA-PEG (Tras) was dissolved in PBS (pH 7.2) at 0.007 mM. 5k The molar concentration of -Tras NPBA-PEG is mono-NPBA-PEG 5k The mean fluorescence values ​​of -Tras samples and various known concentrations of NPBA-PEG 5k The fluorescence value was calculated from the calibration curve of the -AA standard (0 to 0.0098 mM) using the following formula. Fluorescence = ε*C N +b (where ε = slope of the linear regression of the calibration curve, b = intercept of the linear regression of the calibration curve, C N = concentration of NBPA-PEG (mM). Therefore, C N (mM)=(fluorescence-b) / ε.)

[0290] Then, according to the following formula, C N Divide by the known Tras concentration to obtain the number of NPBAs per Tras (N Tras ) was calculated. N Tras =(NPBA-PEG mmol / L) / (Tras mmol / L)

[0291] Mono-NPBA-PEG using ARS detection assay for NPBA 5k Determine the number of -Trans NPBA mono-NPBA-PEG 5kAs described in Example 40, mono-NPBA-PEG 5k The number of NPBA per -Tras can vary due to unwanted conjugation of the molecule with NPBA, which reduces the fluorescent signal, or due to the presence of excess unreacted NPBA-PEG conjugates in the crude sample, which increases the fluorescent signal. Exemplary experimental results are shown in Table 3. [Table 3]

[0292] As will be appreciated by those skilled in the art, numerous modifications and variations of the present disclosure are possible in light of these teachings, all of which are contemplated herein. For example, in addition to the embodiments described herein, the present disclosure contemplates and claims inventions resulting from combinations of features of the disclosures cited herein with features of the cited prior art documents that are complementary to the features of the present disclosure. Similarly, it will be understood that any described material, feature, or article can be used in combination with any other material, feature, or article, and that such combinations are considered to be within the scope of the present disclosure.

[0293] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated by reference in their entirety for all purposes, or at least as relevant to the context in which they are cited. In addition to the documents already mentioned, this disclosure contains subject matter related to U.S. Patent Application No. 12 / 540,319, filed Aug. 12, 2009, now U.S. Patent No. 8,557,292, U.S. Patent Application No. 13 / 782,458, filed Mar. 1, 2013, U.S. Patent Application No. 13 / 782,486, filed Mar. 1, 2013, U.S. Patent Application No. 13 / 852,303, filed Mar. 28, 2013, U.S. Patent Application No. 15 / 180,201, filed Jun. 13, 2016, now U.S. Patent No. 10,287,401, and International Application No. PCT / US2009 / 053620, filed Aug. 12, 2009, the contents of which are incorporated by reference for all purposes.

Claims

1. The following structure: 【Chemistry 1】 (Wherein, R is —CH(CH ３ ) -, -CH ２ -CH ２ -, -CH(i-Pr)-, -CH ２ -CH ２ -CH ２ --, --CH ２ -CH ２ -CH ２ -CH ２ -CH ２ - or -CH(CH 2 i-Pr)-; x is 20 to 200; and y is 5-200.

2. A nanoparticle comprising the MAP-SN38 conjugate of claim 1.

3. The following structure: 【Chemistry 2】 (Wherein, R is —CH(CH ３ ) -, -CH ２ -CH ２ -, -CH(i-Pr)-, -CH ２ -CH ２ -CH ２ - or -CH(CH2Ph)-C(O)-NH-CH ２ - and x is 20 to 200; mucic acid polymer (MAP)-CPT conjugates having the moiety y=5-200.

4. The MAP-CPT conjugate of claim 3, wherein x is 114.

5. The MAP-CPT conjugate of claim 3, wherein x is 80.

6. The MAP-CPT conjugate of claim 3, wherein x is 46.

7. The MAP-CPT conjugate of claim 3, wherein y is 16.

8. The MAP-CPT conjugate of claim 3, which is conjugated to transferrin.

9. The MAP-CPT conjugate of claim 3, which is conjugated with an antibody.

10. The MAP-CPT conjugate of claim 9, wherein the antibody is trastuzumab.

11. (a) transferrin, and (b) Antibody The MAP-CPT conjugate of claim 3, wherein

12. The MAP-CPT conjugate of claim 11, wherein the antibody is trastuzumab.

13. A nanoparticle comprising the MAP-CPT conjugate according to any one of claims 3 to 12.

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