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WO2016101068A1 - Chitosanes conjugués et procédé d'utilisation et de fabrication de ces derniers - Google Patents

Chitosanes conjugués et procédé d'utilisation et de fabrication de ces derniers Download PDF

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WO2016101068A1
WO2016101068A1 PCT/CA2015/051356 CA2015051356W WO2016101068A1 WO 2016101068 A1 WO2016101068 A1 WO 2016101068A1 CA 2015051356 W CA2015051356 W CA 2015051356W WO 2016101068 A1 WO2016101068 A1 WO 2016101068A1
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Prior art keywords
chitosan
alkyl
linear
conjugate
ramified
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Vincent D. PICKENHAHN
Marc Lavertu
Michael D. Buschmann
Gregory De Crescenzo
Vincent DARRAS
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Polyvalor SC
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Polyvalor SC
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/3311—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group
    • C08G65/3318—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing a hydroxy group heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J11/00—Recovery or working-up of waste materials
    • C08J11/04—Recovery or working-up of waste materials of polymers
    • C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • This application generally relates to the field of conjugated chitosans, methods of use, and methods of making same.
  • Chitosan a linear and cationic polysaccharide is derived from chitin by deacetylation. This non-toxic cationic polysaccharide holds great interest due to its biocompatibility, biodegradability and mucoadhesive properties. Rinaudo, Progress in Polymer Science 2006, 31 (7), 603-632. Chitosan and its derivatives have been proposed for applications including gene and drug delivery, tissue repair, water purification and cosmetics. Two general approaches have been explored to chemically modify chitosan - lateral "graft" and "block” modifications. The former involves conjugation to chitosan lateral functional groups (amine or hydroxyl) while the latter relies on conjugation to chitosan end groups.
  • Chitosan lateral grafting can potentially compromise the ability of chitosan to bind nucleic acid and thus limit the stability and efficiency of chitosan/nucleic acid complexes for gene delivery applications. Indeed, lateral grafting can impede the ability of chitosan to electrostatically bind to negatively charged species by reducing its effective charge density and by potentially creating steric hindrance with bulky moieties. Casettari et al., Progress in Polymer Science 2012, 57 (5), 659-685.
  • Chitosan block modification strategies have been recently proposed as a means to modify the chitosan properties without compromising its ability to bind oppositely charged macro-ions such as nucleic acids.
  • Two different chitosan attachment sites have been explored to date: the first site is formed after chitosan depolymerization by nitrous acid (HONO) where a 2,5-anhydro-D- mannose unit (M-Unit) is formed at the reducing end of the cleaved polymer (Allan et al., Carbohydrate research 1995, 277 (2), 257-272), while the second site is available on the open- chain form, present in trace amounts, of the chitosan reducing extremity (either GlcNH 2 or GlcNHAc units) and allows mutarotation between the alpha and beta anomers.
  • HONO nitrous acid
  • M-Unit 2,5-anhydro-D- mannose unit
  • 2012/0238735 describes polyethylene glycol (PEG)-chitosan conjugates prepared by an oxime-click chemistry reaction of a single PEG moiety with an aldehyde on a single chitosan backbone. While this reaction affords a control over the ratio PEG/chitosan, the presence of the aldehyde on the chitosan depends on a mutarotation equilibrium in which the amount of non-reactive cyclic form at equilibrium is very high compared to the reactive opened- ring intermediate one (e.g., only 0.0051% of aldehyde opened form for D-Glucose at 52°C, pH 5).
  • PEG polyethylene glycol
  • the present disclosure relates to a chitosan conjugate of formula (I):
  • CS represents a chitosan residue
  • X comprises a Zl group, the Zl being a linear thioacetal or forming an n-membered thioacetal ring, n being from 4 to 9, the Zl optionally being ramified and/or substituted
  • POLY represents a ligand, or a pharmaceutically acceptable salt thereof.
  • CS represents a chitosan residue
  • X comprises a thioacetal forming an n-membered ring, n being from 4 to 9, the ring optionally being ramified and/or substituents, and the ring further comprising a free SH, or a pharmaceutically acceptable salt thereof.
  • the present disclosure relates to a process for manufacturing depolymerized chitosan residues, comprising: depolymerization of chitosan with nitrous acid (HONO) to obtain a depolymerized chitosan residue salt, and drying the depolymerized chitosan residue salt under acidic conditions, the acidic conditions comprising a pH ⁇ 4, preferably pH ⁇ 3.5, more preferably pH ⁇ 3.
  • HONO nitrous acid
  • the present disclosure relates to a process for manufacturing a conjugate of chitosan residues, comprising: depolymerization of chitosan with nitrous acid (HONO) to obtain a depolymerized chitosan residue salt, drying the depolymerized chitosan residue salt under acidic conditions, the acidic conditions comprising a pH ⁇ 4, preferably pH ⁇ 3.5, more preferably pH ⁇ 3, rehydration of the salt in an aqueous solvent to obtain a solution, and incorporating a thiol molecule in the solution thus producing a reaction medium for obtaining said chitosan conjugate, said reaction medium being at a pH of about 1.
  • HONO nitrous acid
  • the above drying step comprises thermal vacuum drying. [15] In another non-limiting embodiment, the above drying step comprises freeze-drying.
  • the present disclosure relates to a process for manufacturing a conjugate of chitosan residues, comprising: providing a nitrous acid (HONO) depolymerized chitosan residue salt previously dried under acidic conditions, said acidic conditions comprising a pH ⁇ 4, preferably pH ⁇ 3.5, more preferably pH ⁇ 3, rehydration of the salt in an aqueous solvent to obtain a solution, and incorporating a thiol molecule in the solution thus producing a reaction medium for obtaining said chitosan conjugate, said reaction medium being at a pH of about 1.
  • HONO nitrous acid
  • the herein described reaction for obtaining said chitosan conjugate can be performed at a temperature selected from the range of about 5 °C to about 90 °C. In a particular practical embodiment, the temperature is selected from the range of about 20 °C and 50 °C.
  • the herein described reaction for obtaining said chitosan conjugate can be performed substantially instantly (e.g., in case where there is a direct freeze-drying of the reaction medium) or can be performed for a given amount of time, for example up to several weeks. In a particular practical embodiment, the reaction can be performed for a time period ranging from about 24h to about 72h.
  • the present disclosure relates to particles comprising the chitosan conjugate of formula (I) as described previously, wherein the ligand comprise a polyethylene glycol (PEG) molecule, and wherein the particles have a substantially spherical form and have a reduced zeta potential than a comparative particle being prepared in the same conditions to said particle except for comprising chitosan residues instead of chitosan-PEG conjugates.
  • the ligand comprise a polyethylene glycol (PEG) molecule
  • PEG polyethylene glycol
  • Figure 1 is a non-limiting schematic representation of a chitosan conjugate and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 2 is a non-limiting schematic representation of another chitosan conjugate and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 3 is a non-limiting schematic representation of other chitosan conjugates and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 4 is a non-limiting schematic representation of a prior art grafted chitosan residues on a surface ("standard pathway”) and of a grafted conjugate on a surface in accordance with a non-limiting embodiment of the present disclosure (“BCL pathway”), as well as (bottom) a non-limiting schematic representation of a reaction scheme to obtain the grafted conjugate on a surface in accordance with the non-limiting embodiment of the present disclosure (“BCL pathway”) shown in (top).
  • Figure 5 is a non-limiting schematic representation of another chitosan conjugate and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 6 is a non-limiting schematic representation of another chitosan conjugate and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure in which the ligand includes a siRNA.
  • Figure 7 is a non-limiting flowchart of an experimental design for (A) mechanistic studies and (B) reactivity studies performed in the present disclosure.
  • Figure 8 is a non-limiting schematic representation of chitosan conjugates in accordance with a non-limiting embodiment of the present disclosure for quantitation of chitosan residues derivatization efficiency: functionalization degree (F) calculations.
  • Figure 10 is a non-limiting schematic representation of potential reaction scheme occurring during conjugation of 2,5-anhydro-D-mannose (M-Unit) and 2 thiol-bearing molecules (3- mercaptopropionic acid and ⁇ -mercaptoethanol, MPA and BME respectively) giving the following expected products:
  • Product A is the hemithioacetal intermediate that is in equilibrium with its corresponding oxonium, whereas products B and C correspond to the oxathiolane (for BME reactions only) and thioacetal, respectively.
  • Molecule D represents the ⁇ , ⁇ -unsaturated sulfide.
  • Figure 13 is a non-limiting Diffusion ordered spectroscopy experiments (DOSY) spectrum of the CS HC1 salt M-Unit conjugated to MPA. 32 gradients between 1 1.2 and 358.4 gauss.cm-1 with a gradient pulse ( ⁇ ) of 1 ms, a diffusion time ( ⁇ ) of 60 ms. Both CS and MPA have the same translational diffusion coefficient at 25°C in 2% DCl in D 2 0 indicating that they are conjugated given the large difference between their molar masses.
  • DOSY Diffusion ordered spectroscopy experiments
  • Figure 14 is a non-limiting schematic representation of potential reaction scheme for thiol addition to the aldehyde group of the M-Unit CS HCl salt under acidic aqueous conditions.
  • a & B x80000 and xl60000, respectively
  • polyplexes formed with CS 92-10 are heterogeneous in size and present various morphologies (globular, rod-like and toroidal).
  • Pictures C and D (x80000 and xl60000, respectively): polyplexes formed with CS-b- PEG 2 (CS 92-10 and mPEG-SH 2kDa), are substantially uniformly spherical.
  • Figure 17 is a non-limiting schematic representation of proposed model of reaction scheme for thiol addition to the aldehyde group of the M-Unit CS HCl salt under acidic aqueous conditions in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 18 is a non-limiting graph of 13 C solid state NMR (CP-MAS) of an extra-dried CS 99-1 salt.
  • Figure 19 is a non-limiting graphical representation of M-Unit chitosan 92-2 (deacetylation degree of 92%; 2-3 kDa) / Thiol-hook models (Ethanedithiol and propanedithiol, EDT and PDT, respectively) conjugation efficiencies determined by ! H NMR (N>3 +SD).
  • Figure 20 is a non-limiting schematic representation of proposed model of a reaction scheme for a triskelion linker synthesis that corresponds to a two-steps process taking place in organic conditions in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 21 is a non-limiting schematic representation of a proposed model of reaction scheme for a 2-step process for the conjugation of the acetyl-protected triskelion to the M-Unit aldehyde in accordance with a non-limiting embodiment of the present disclosure.
  • Figure 22 is a non-limiting schematic (left) and graphical representation (right) of the relative proportion of the M-Unit aldehyde / Triskelion linker conjugation products determined by liquid chromatography-mass spectrometry (LCMS).
  • Products A & B correspond to the desired products obtained by intramolecular cyclization (A: M-Unit-triskelion conjugate; B: M-Unit- triskelion conjugates linked by disulfide bond through the third remaining thiol moiety).
  • Products C & D correspond to the side-products obtained by intermolecular thioacetylation (D: M-Unit- (triskelion) 2 conjugate; E: Oxidized M-Unit-(triskelion) 2 conjugates. (N>3 + SD).
  • Figure 23 is a non-limiting graphical representation of the M-Unit chitosan (deacetylation degree of 92%; 2-3 kDa and lOkDa*) / Triskelion linker conjugation efficiencies determined by ⁇ NMR (N>3 ⁇ SD).
  • chitosan residue generally refers to a chitosan residue having a deacetylation degree (%DDA) from about 50% to about 100% and/or a molecular weight (Mn) of from about 0.2 to about 200 kDa.
  • %DDA deacetylation degree
  • Mn molecular weight
  • chitosan residue may also generally refer to any modified chitosan residue where the modification(s) is either on the chitosan lateral amines and/or on the chitosan hydroxyl groups. The person of skill will readily envision the types of modifications which can be suitable for this purpose.
  • M-Unit generally refers to a 2,5-anhydro-D-mannose linked at the reducing end of a chitosan residue via a glycosidic bond.
  • Triskelion generally refers to a mercapto compound having a structure where at least two S atoms are on one end and at least one S atom is on an opposite end of the structure, for example as per the following formula:
  • the Triskelion molecule may include a compound as described for example, but without being limited thereto, in EP0528590 (e.g., Zl includes two free S atoms, LI is -S-CH 2 -CH 2 - and ⁇ includes one free S atom).
  • linear alkyl generally refers to an alkyl chain having any length within CJ-CJOO, for example but without being limited thereto, Cj-Cgo, Q-Cso, C2-C90, C 2 - C 8 o, Ci-C 50) C]-C 40> C!-C 3 o ; C1 -C25, C!-C 2 o, C!-C 15; C!-C 10 and the like, optionally ramified and/or substituted.
  • thioacetal generally refers to the chemical structure RCH(SR') 2 where R is not H, and where R' is an alkyl chain C1 -C100 if a linear thioacetal.
  • ligand also referred herein as "POLY" generally refers to any desired moiety and/or surface, for example but without being limited thereto, a nucleic acid molecule (e.g., linear DNA, mRNA, shRNA, or siRNA), a polypeptide, a non-peptidic polymer (e.g., a poly(alkylene oxide) such as a PEG), another chitosan conjugate (of identical structure or not), a planar or particulate surface, and the like.
  • the ligand may further include one or more functional group(s) in protected or unprotected form, optionally attached via a linker group.
  • the functional group can be, for example, one or more of a passive or active targeting moiety (fusogenic peptide, folate TAG, Galactose, and the like), dyes/fluorophores, polymers, and the like.
  • the chitosan conjugate can be represented according to the following formula:
  • alkyl chain C C ⁇ o, and/or alkyl n-membered ring, n being from 4 to 9, optionally ramified
  • Optional linker and/or substituted optionally including at least one S atom such as -S-CH 2 -CH 2 -; Cleavable or uncleavable covalent linkage; and the like
  • alkyl chain Ci-C ]0 o, and/or alkyl n-membered ring, n being from 4 to 9, optionally ramified
  • Optional linker or spacer and/or substituted optionally including at least one S atom such as -S-CH 2 -CH 2 -; Cleavable or uncleavable covalent linkage; and the like
  • polypeptide polypeptide, nucleic acid, planar or particulate
  • a method of making such chitosan conjugates which makes use of a thiol-based chemistry.
  • the proposed thiol- based chemistry may advantageously overcome at least some of the limitations of the hereinbefore discussed oxime-click pathway.
  • thiol moieties are highly reactive towards double bonds as well as towards carbonyl groups in aqueous conditions at pH as low as 1 where CS amines are present only in the ionized and non-reactive form (Lienhard, G. E.; Jencks, W. P., Thiol Addition to the Carbonyl Group. Equilibria and Kinetics 1. Journal of the American Chemical Society 1966, 88 (17), 3982-3995).
  • the reactive species is the dehydrated carbonyl compound so that dehydration and hemithioacetal formation represent the rate limiting steps of this pH dependent process (Lienhard, G. E.; Jencks, W. P., Thiol Addition to the Carbonyl Group. Equilibria and Kineticsl . Journal of the American Chemical Society 1966, 88 (17), 3982-3995; Schubert, M. P., Compounds Of Thiol Acids With Aldehydes. Journal of Biological Chemistry 1936, 114 (1), 341- 350).
  • Hemithioacetals can be stabilized by thioacetal formation via a second thiol nucleophilic attack (intra- or inter-molecular) associated with the release of water (Campaigne, E., Chapter 14 - Addition Of Thiols Or Hydrogen Sulfide To Carbonyl Compounds. In Organic Sulfur Compounds, Kharasch, N., Ed. Pergamon: 1961 ; pp 134-145; Fournier, L.; Lamaty, G.; Natat, A.; Roque, J. P., Addition des thiols sur les cetones-III: Reinvestigation duate de I'addition du mercapto-2/ethanol. Tetrahedron 1975, 31 (8), 1025-1029), as depicted in Scheme 3.
  • the process for manufacturing a conjugate of chitosan residues described in the present disclosure can have at least one of the following benefits over the oxime click method developed previously: it can be used for CS derivatization without interfering with amine groups that are fully protonated and thus unreactive; it is efficient in aqueous media; and there is no need for an additional chemical treatment to stabilize the products.
  • CS-thiol adducts were unexpectedly produced with at least 50% coupling rates within acidic aqueous solvent, for example, using linear thioacetal the inventors unexpectedly obtained CS-thiol adducts with about 55%) to about 70% coupling rates (intermolecular pathway in aqueous conditions) and using thiol hook/triskelion the inventors unexpectedly obtained CS-thiol adducts with about 70%» to about 90%) conjugation efficiencies (intramolecular pathway in aqueous conditions with a co-solvent addition to solubilize the linkers).
  • the herein described reaction medium can also include a co-solvent such as any suitable polar protic and/or aprotic co-solvent.
  • a co-solvent such as any suitable polar protic and/or aprotic co-solvent.
  • the suitable polar protic and/or aprotic co-solvent can be selected from, but without being limited thereto, methanol, ethanol, 2-propanol, butanol, isobutanol, tert-butanol, tetrahydrofuran, dioxane, dichloromethane, and any combination thereof.
  • the person of skill will readily be able to select a suitable co-solvent without undue effort.
  • the above co-solvent is present in the reaction medium at a proportion selected from the range of > 0 v/v % to about 95 v/v %>.
  • the person of skill may wish to use a lower proportion in water.
  • the person of skill will readily be able to select a suitable proportion of co-solvent depending on the particular properties of the co- solvent without undue effort.
  • the presence of such co-solvent in the reaction medium can be advantageous for example for solubilisation of the herein described triskelion linker prior to conjugation with M-Unit and/or M-Unit chitosans.
  • FIG. 1 represents a non-limiting schematic representation of a chitosan conjugate and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • This chitosan conjugate includes a chitosan residue having a single M-Unit obtained via depolymerization by nitrous acid (HONO), the M-Unit being linked to a thioacetal (Zl) and each S atom in the thioacetal being linked to a respective ligand, which in this case is represented with a polyethylene glycol (PEG) molecule.
  • HONO nitrous acid
  • Zl thioacetal
  • PEG polyethylene glycol
  • the ligand bearing a thiol moiety e.g., PEG-SH
  • PEG-SH a thiol moiety
  • the reaction scheme of Figure 1 provides a conjugate with two ligands per conjugate molecule.
  • FIG. 2 is a non-limiting schematic representation of another chitosan conjugate and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • Zl thioacetal
  • Z2 a peptide bond
  • the structure stabilization by the second POLY-SH attack may be sterically hindered by the presence of the first POLY-SH attached to the M-Unit and that a reaction according to the scheme depicted in Figure 2, where the ligand includes a "thiol hook" ((SH-CH 2 ) 2 CHR) may provide higher coupling rates.
  • the ligand is functionalized with the thiol hook prior to conjugation with the chitosan residue.
  • the functionalized ligand is represented with a functionalized PEG (PEG-NH-thiol hook).
  • the reaction scheme of Figure 2 provides a conjugate with a single ligand per conjugate molecule.
  • FIG. 3 is a non-limiting schematic representation of other chitosan conjugates and a reaction scheme to make same in accordance with a non-limiting embodiment of the present disclosure.
  • a cleavable linkage e.g., a disulphide linkage
  • a chemical trigger e.g., exposure to a reductive environment, such as upon entry in an endosome
  • an uncleavable covalent linkage e.g., S-CH 2 -S0 2 -
  • a physical trigger e.g., exposure to light, such as a laser
  • the cleavable or uncleavable covalent linkage links the peptide bond to the ligand, which in this particular case is represented with a PEG molecule.
  • the cleavable or uncleavable covalent linkage links the peptide bond to the ligand, which in this case is represented with a functionalized surface, for example a nanoparticle surface or a planar surface.
  • the cleavable linkage links the peptide bond to a second chitosan residue which may have identical characteristics as those of the first chitosan residue.
  • the cleavable linkage (a disulphide linkage) links the peptide bond to a siR A. While these non-limiting embodiments all include a peptide bond, the person of skill will readily understand that this bond is optional. The person of skill will also be able to envision other chitosan conjugates having alternate structures based on the herein teachings.
  • Figure 3 also represents a schematic representation of the reaction scheme for making block- copolymers in accordance with a non-limiting embodiment of the present disclosure.
  • This reaction scheme will be referred hereinafter as the "Triskelion" reaction scheme.
  • this reaction scheme has an increased coupling rate relatively to the reaction scheme of Figure 1 , since it involves in a first step coupling a small linker (small linkers are more reactive than long ligand chains and they can be used at much higher concentration), and coupling the ligand (POLY) in a second step having smoother conditions than the first step, where for example the first step includes a reaction at a pH value of about 1.
  • this reaction scheme also allows the introduction of a cleavable or an uncleavable covalent linkage in the chitosan conjugate.
  • the "Triskelion” strategy involves a two-step process: Firstly, a Triskelion molecule bearing 3 or more thiol groups (preferably 3 thiol moieties) is conjugated to the CS M-Unit aldehyde. This conjugation takes place at about pH 1, and it results in a functionalized CS chain (CS-b-Triskelion) that bears a highly reactive chemical moiety (the 3 rd unreacted thiol group) on its end group. Secondly, once the CS-b-Triskelion has been synthesized, further conjugations with POLY can be performed in milder conditions (pH 4-6).
  • the Triskelion reaction scheme of Figure 3 includes providing as starting materials, a "Triskelion" molecule and a chitosan residue having a single M-Unit including a reactive aldehyde.
  • the Triskelion molecule includes a thiol hook linked to an -CH 2 -CH 2 -SH moiety (L2) via a peptidic bond (72).
  • L2 -CH 2 -CH 2 -SH moiety
  • the CS-b-Triskelion intermediate is then reacted, as further explained in more detail later in this text, via a step (b) either with a ligand functionalized to include a cleavable linkage (Z3) thus forming a CS-b-SS-ligand conjugate (in this case the ligand is represented by PEG) or via a step (c) with a ligand functionalized to include an uncleavable covalent linkage (Z3) thus forming a CS-b-ligand conjugate (in this case the ligand is also represented by PEG).
  • a step (b) either with a ligand functionalized to include a cleavable linkage (Z3) thus forming a CS-b-SS-ligand conjugate (in this case the ligand is represented by PEG) or via a step (c) with a ligand functionalized to include an uncleavable covalent linkage (Z3) thus forming a CS-b-ligand conjugate (in this
  • Figure 4 is a schematic representation of a prior art grafted chitosan residues on a surface, for example a planar surface, a nanoparticle surface, and the like.
  • Conventional techniques for grafting chitosan residues to surfaces usually result in chitosan residues which are grafted, adsorbed or electrostatically bound in a mostly longitudinal configuration (parallel to the surface).
  • the Triskelion reaction scheme described here affords chitosan residues which are grafted in a mostly transverse configuration (perpendicular to the surface).
  • Figure 4 is a schematic representation of a chitosan conjugate having a cleavable linkage S-S or an uncleavable covalent linkage S-CH 2 -S0 2 - which is grafted in a mostly transverse configuration onto a surface.
  • the reaction scheme to obtain this conjugate includes reacting a CS-b-Triskelion intermediate with a functionalized surface, which includes a functional group comprising the cleavable or the uncleavable covalent linkage.
  • a chitosan conjugate grafted in a mostly transverse configuration as described here may have its amines and other chitosan residue functional group(s) free to interact with their environment and may therefore provide novel properties in terms of bioactivity relatively to those chitosan conjugates of the prior art bound in a mostly longitudinal configuration.
  • Figure 5 represents a non-limiting example of a double chitosan residue conjugate and a reaction scheme for making same.
  • the reaction scheme to obtain this conjugate is based on the previously described Triskelion reaction scheme.
  • two CS-b-Triskelion intermediates each having a molecular weight of n kDa, are reacted with each other (oxidation) so as to obtain a chitosan conjugate having a molecular weight of 2n kDa, which includes two chitosan residues.
  • a chitosan conjugate according to this embodiment can be particularly advantageous for cellular delivery of nucleic acid molecules since the the disulfide linkage between the two chitosan residues can be cleaved within the endosome (reductive environment), favoring the release of the nucleic acid molecules.
  • Figure 6 represents a non-limiting example of a chitosan conjugate which includes a nucleic acid molecule, represented in this case with a siRNA molecule.
  • the reaction scheme to obtain this conjugate is based on the previously described Triskelion reaction scheme.
  • the starting materials include the CS-b- Triskelion intermediate and a nucleic acid molecule modified to include a linker carrying a thiol group or another disulfide linkage (which in this case is represented with an siRNA having a 2- disulfanepyridine - C 5 H 5 NS 2 ).
  • the chitosan residue may have any molecular weight from 0.2 to 5 kDa so long as the chitosan residues is soluble in water when neutralized and can be attached this way without any electrostatic interactions between the chitosan residue and the nucleic acid.
  • siRNA any other suitable nucleic acid molecule can be used, for example, an shRNA, mRNA, oligonucleotide, linear DNA, and the like.
  • chitosan conjugate may be used, for example, for administration to a subject, such as by subcutaneous injection.
  • Figure 7 represents a non-limiting of a flowchart of an experimental design for (A) mechanistic studies and (B) Chitosan M-Unit reactivity studies, which are further discussed later in the Examples:
  • Glucosamine (GlcNH 2 ) was treated with nitrous acid (HONO) to form the 2,5-anhydro-D- mannose (M-Unit).
  • M-Unit was reacted with 2 thiol-bearing molecules ( ⁇ -mercaptoethanol and 3-mercaptopropionic acid, BME and MP A, respectively).
  • the reaction products were treated using one of 3 methods: a. Method I: Direct LC-MS analyses to determine to which extent thioacetal formation occurs in situ; b. Method II: Freeze-drying (FD) + LC-MS analyses to assess the effect of FD on the thioacetal proportion and to ascertain that no by-products appear post FD; and c.
  • Method III Acetate buffer pH 4 + FD + LC-MS analyses to determine the effect of an increase in pH prior to FD (this pH increase was included here to prevent any CS acid hydrolysis that could occur when Method II, i.e. FD at pH 1, would be transposed to the polymer).
  • CS 92-200 was depolymerized with nitrous acid (HONO) to produce CS 92-2 HCl salt bearing the M-Unit at the cleaved end of the polymer.
  • M-Unit CS 92-2 HCl salt were reacted with MP A and BME and the reaction products treated with one of 3 workups: a. Workup I: Dialysis vs. HCl ImM solution + FD to remove all thiol model excess and to determine the in situ thioacetal formation rate; b. Workup II: FD + Dialysis vs. HCl ImM solution + FD to determine the effect of FD on the functionalization rate; c.
  • mPEG-SH 2 kDa and the plasmid DNA (pDNA) pEGFPLuc were purchased from JenKem Technology USA and from Clontech Laboratories, respectively.
  • the eluents consisted of 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B).
  • the initial mobile phase contained 1% eluent B and was held for 3 min.
  • Eluent B content was increased from 1 % to 20 % from 3 to 5 min then from 20 % to 80 % from 5 to 7 min.
  • the system returned to the initial conditions at 7.2 min and was held constant for up to 15 min to allow column equilibration.
  • the injection volume was 1-3 ⁇ .
  • a needle wash solution containing methanol: water (60:40 v/v) was used after each injection to reduce carry-over. Mass spectra were acquired for m/z ranging from 50 to 1200.
  • the deacetylation degree (%DDA) of chitosan was determined by 1H NMR spectroscopy as previously described using a Bruker Avance 500 spectrometer equipped with a Bruker 5 mm BBFO probe. Lavertu et al., Journal of Pharmaceutical and Biomedical Analysis, 2003, 32 (6), 1 149-1 158.
  • Modified CS depolymerized CS and thiol coupled CSs
  • SB-806M HQ and SB-803 HQ columns SB-806M HQ and SB-803 HQ that are more suitable for the analysis of low molecular weight chitosans.
  • Method III was included to prevent any CS acid hydrolysis that could occur when Method II, i.e. FD at pH 1, would be transposed to the polymer.
  • the CS hydrochloride salt carrying the 2,5-anhydro-D-mannose unit (M-Unit), was allowed to react at pH 1 with the two thiol models: 3-mercaptopropionic acid (MP A) and ⁇ -mercaptoethanol (BME). Each reaction was allowed to stir for 72h, at two different temperatures (25 and 50°C) under Ar atmosphere and were treated using three different workups: Workup I) Dialysis vs. HCl ImM solution followed by freeze-dry (FD) to remove all thiol-bearing molecule excess and to determine the in situ thioacetal functionalization degree); Workup II) Direct FD, dialysis vs.
  • MP A 3-mercaptopropionic acid
  • BME ⁇ -mercaptoethanol
  • H mol peaks refers to the well-defined proton peaks of the thiol-bearing molecule conjugated to CS and H M _ Unil peaks corresponds to the well-defined M-Unit characteristic proton peaks.
  • Both integrations in Equation 1 are normalized to the number of protons used for the calculation, namely a and ⁇ for the thiol-bearing molecule and M-Unit, respectively.
  • Equation 1 Equation 1
  • Equation 2 Equation 3
  • protons used for integration are defined in Figure 8, for purified BME and MPA chitosan adducts.
  • Both CS-b-PEG 2 /pDNA and CS/pDNA polyplexes were prepared at room temperature, by adding 100 of the diluted polymer solution to 100 of the pDNA solution followed by immediate mixing by pipetting up and down.
  • the polyplexes were analyzed for their size and morphology by dynamic light scattering (DLS) and environmental scanning electron microscopy (ESEM) lh after their formation.
  • DLS dynamic light scattering
  • ESEM environmental scanning electron microscopy
  • the expected products of all conjugations implemented with thiol-bearing molecules include hemithioacetal, thioacetal, oxathiolane and ⁇ , ⁇ -unsaturated sulfide intermediate ( Figure 10).
  • the first thiol attack on the aldehyde forms a hemithioacetal intermediate (A), which is in equilibrium with its corresponding protonated hemimercaptal form (oxonium) via a proton transfer.
  • This structure may react in several ways: it could be stabilized with a second nucleophilic attack forming the corresponding thioacetal (Q after water removal.
  • Method I refers to direct LC-MS analysis of the reaction medium;
  • Method II corresponds to the direct freeze-drying (FD) of the reaction medium before analysis;
  • Method III corresponds to an increase in pH with acetate buffer pH 4 followed by FD.
  • FD direct freeze-drying
  • Method III corresponds to an increase in pH with acetate buffer pH 4 followed by FD.
  • CS depolymerization concentrations are possible (typically up to 2% w/v for CS with Mn of a few hundreds of kg.mol "1 ) but limited by the high viscosity of CS solutions, which may compromise stirring efficiency and homogeneity of the depolymerization medium.
  • the depolymerized (i.e. less viscous) CS hydrochloride salt was freeze-dried, with all CS amines protonated, thus avoiding Schiff base formation and subsequent HMF formation upon rehydration.
  • the Workup I was proposed to evaluate the in situ thioacetal formation by removing all unreacted thiol moieties prior to FD, whereas the Workup II was implemented to assess the role of freeze-drying in hemithioacetal intermediate stabilization.
  • Workup III was initially suggested to protect M-Unit CS HCl salt from acid hydrolysis during FD at pH 1 in Workup II, however SEC-MALLS analyses of Workup II polymers did not reveal any glycosidic linkage hydrolysis at pH 1.
  • Other SEC-MALLS analyses involving different CS salts (Mn of 2, 4 and 10 kg.moi 1 ) that were freeze-dried at low pH also did not reveal any alteration of the polymer (data not shown).
  • F below corresponds to the functionalization degree, considering 2 thiol molecules per potential aldehyde and calculated using Equation 2 for BME and Equation 3 for MPA with N>3 ( ⁇ SD). F was also calculated using Equation 5, considering only the relative proportion of the remaining gem-dio ⁇ per M-Unit. (*) corresponds to the results of the conjugations implemented with 20 equivalents (instead of 5) of thiol-bearing molecule per end unit.
  • the gem-diol signal should decrease concomitantly with the conjugation of thiols onto the M-Unit of chitosan (one gem-diol consumed for two conjugated thiols).
  • the calculated conjugation efficiencies obtained with either Equation 2 (BME) or 3 (MP A) and the following equation should therefore be the same if two thiols react regioselectively onto the terminal aldehyde function of chitosan:
  • CS-b-PEG 2 block-copolymer/pDNA polyplexes are homogeneously spherical
  • Such particles having a size of less than a micron and which include chitosan conjugates as described herein where the ligand is a PEG molecule may be used as a cell delivery system.
  • such particles when used as a delivery system in a host blood vessels such particles have a prolongation of their circulation time within the host blood vessels relatively to known particles having a size of less than a micron, at least because these particles (i) have a net neutral charge, (ii) have less aggregation, (iii) have less protein interaction than the known particles, or (iv) any combinations thereof, which reduces recognition by the host immune system.
  • FIG. 17 illustrates a reaction scheme which summarizes a proposed mechanistic model for thiol-based end-group derivatization of chitosans described here.
  • CS nitrous acid (HONO) depolymerization induces the formation of M-Unit that carries an aldehyde moiety at the end of the cleaved polymer (1).
  • the equilibrium between the M-Unit aldehyde and its hydrated form (gew-diol) is strongly displaced towards the latter (2). If the CS depolymerization medium is freeze-dried at pH well below the CS pKa (i.e.
  • the stabilization of the latter into its thioacetal form (6) can occur at least either by increasing the amount of thiol-bearing reactants in the medium (in situ stabilization), or by freeze-drying the reaction medium when low amounts of thiol are engaged.
  • any suitable PEG entity may be used so long as it produces a conjugate having desired properties.
  • Protocol 2,5-anhydro-D-mannose (M-Unit) conjugation with thiol-hook models.
  • Thiol-hook models (Ethanedithiol, EDT and Propanedithiol, PDT) were used to assess the intramolecular thioacetylation process, where both thiol attacks occur simultaneously on the M-Unit aldehyde forming instantaneously the stable thioacetal conjugate.
  • the synthesized 2,5-anhydro-D- mannose M-Unit (0.1 mmol, 16.2 mg) was dissolved in 5mL degassed 30 or 40 %v/v 2-propanol in ddH 2 0 for EDT or PDT coupling, respectively.
  • the pH of the solution was adjusted to 1 with 3M HC1 solution prior to the addition of the thiol-bearing molecule (0.5 mmol, 41.9 ⁇ ⁇ for EDT and 50.2 ⁇ , for PDT).
  • the reaction mixture was stirred for 72h at 50°C, under Ar atmosphere and covered with aluminum foil.
  • the reaction mixture turned clear pink-orange after 72h and was split into 2 parts (Methods I and II): the first was dedicated to the direct LC-MS analysis of the reaction medium in order to determine the thioacetal proportion in resulting conjugates that formed in situ; whereas the second one was immediately flash-frozen and then freeze-dried prior to LC-MS analyses to assess the effect of FD on the thioacetal proportion in resulting conjugates and to ascertain that no by-products appear post FD.
  • PDT propanedithiol
  • the freeze-drying step post-reaction may favor the conjugation as observed for PDT 4-5, PDT 24-5 and PDT 72-5. Nevertheless, with 72h reaction duration, the freeze-drying step seems to increase the conjugation to a lower extent than with shorter reaction durations.
  • the proposed triskelion linker synthesis is depicted in Figure 20 and corresponds to a two- steps process that takes place in organic conditions: 1) the triol (1,2,6-hexanetriol) was treated with mesylate chloride (methanesulfonyl chloride) to transform all triol hydroxyls into leaving groups. 2) The leaving groups were displaced by potassium thioacetate (CH 3 COSK) to give the triskelion under its protected form.
  • mesylate chloride methanesulfonyl chloride
  • CH 3 COSK potassium thioacetate
  • DCM dichloromethane
  • THF Tetrahydrofurane
  • Step 1 Triskelion linker deprotection.
  • the reaction medium stirred for 10 min at room temperature and under inert atmosphere.
  • the isolated organic layer was carefully concentrated under reduced pressure to give a clear yellowish oil confirmed to be the pure product (95% yield) by 1H NMR and was stored under inert atmosphere until conjugation reaction.
  • Step 2 Reaction 1 Triskelion linker conjugation (30% THF). The following description corresponds to the conjugation performed in a degassed mixture of 30% v/v THF in ddH 2 O.
  • Step 2 Reaction 2 Triskelion linker conjugation (90% MeOH). The following description corresponds to the conjugation performed in a degassed mixture of 90% v/v Methanol in ddH 2 0.
  • Reaction media were treated according to the following methods prior to LCMS analysis: Method I where the reaction medium was concentrated by rotavap followed by extractions vs. Method II where extractions were performed without preliminary concentration of the medium by rotavap.
  • Results The relative proportion of the M-Unit aldehyde / Triskelion linker conjugation products determined by LCMS is depicted in Figure 22.
  • Products A & B correspond to the desired products obtained by intramolecular cyclization (A: M-Unit-triskelion conjugate; B: M-Unit- triskelion conjugates linked by disulfide bond through the third remaining thiol moiety).
  • A M-Unit-triskelion conjugate
  • B M-Unit- triskelion conjugates linked by disulfide bond through the third remaining thiol moiety.
  • the thioacetylation process between the M-Unit aldehyde and the triskelion thiol hook forms a 5- membered ring.
  • Products C & D correspond to the side-products obtained by intermolecular thioacetylation (D: M-Unit-(triskelion) 2 conjugate; E: Oxidized M-Unit-(triskelion)2 conjugates.
  • D M-Unit-(triskelion) 2 conjugate
  • E Oxidized M-Unit-(triskelion)2 conjugates.
  • the thioacetylation process between the M-Unit aldehyde and the triskelion' s third thiol moiety forms a linear thioacetal.
  • the major compounds observed correspond to the intramolecular cyclization product (5-membered ring thioacetal), meaning that the intramolecular thioacetylation is favored vs. its intermolecular counterpart.
  • Step 1 Triskelion linker deprotection was performed as described above. The amount of triskelion used in the following examples corresponds to 20 equivalents per chitosan's M-Unit aldehyde.
  • Step 2 Reaction 2 (2 kDa CS. 85% MeOH. pH 1 , 24, 48 and 72h.
  • the reaction media corresponding to the time-points (24h, 48h and 72h) were treated with Workup II (concentration to dryness under reduced pressure prior unreacted thiol molecule models removal).
  • Step 3 Purificalion and analyses: All reaction media were treated with IN sodium hydroxide solution (pH of the solutions was increased up to 10) in order to remove some potential hemithioacetal intermediates (even if they were not observed by LCMS in the conditions implemented therein), ensuring that only the stables thioacetals conjugates will be detected by ⁇ NMR. After acidification of the solutions for chitosan solubilization, unreacted triskelion was discarded by 5 successive reprecipitations in fresh THF. The remaining precipitates were dissolved in 5 mL ddH 2 0 and these solutions were flash-frozen and freeze-dried. Conjugation efficiencies were determined by ⁇ NMR, using both the herein described Equation 1 and Equation 5.
  • the freeze-drying step post-reaction may increase the conjugation degree as observed for THF 1-72, MeOH 1-72 and MeOH 1-72* samples. All reactions performed at pH 4 are less efficient than their pH 1 counterpart; this observation is in good agreement with previous results since the thioacetylation process is pH dependent.

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Abstract

L'invention concerne un conjugué de chitosane de formule (I) : dans laquelle CS représente un résidu chitosane ; X comprend un groupe Zl, le groupe Zl étant un thioacétal linéaire ou formant un cycle thioacétal à n chaînons, n étant de 4 à 9, le groupe Zl étant éventuellement ramifié et/ou substitué ; et POLY représente un ligand, ou un sel pharmaceutiquement acceptable de ce dernier. L'invention concerne également des utilisations d'un tel conjugué, ainsi que des procédés de fabrication de ce dernier.
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CN111870739A (zh) * 2020-06-12 2020-11-03 广州暨南大学医药生物技术研究开发中心有限公司 一种多功能改性壳聚糖自愈合水凝胶的制备方法及应用
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CN111484568A (zh) * 2019-01-25 2020-08-04 中国科学院理化技术研究所 一种壳聚糖-抗菌性多肽接枝聚合物及其制备方法和应用
CN111484568B (zh) * 2019-01-25 2021-12-14 中国科学院理化技术研究所 一种壳聚糖-抗菌性多肽接枝聚合物及其制备方法和应用
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CN112851832A (zh) * 2021-01-21 2021-05-28 浙江工商大学 N,o-硫醚壳寡糖衍生物及其制备方法和应用

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