WO2024259525A1 - Nanoparticules comprenant du n,n,n-triméthyl chitosane revêtu d'agonistes peptidiques de la kinine b1 ou b2 et/ou des antagonistes peptidiques de la kinine b1 pour l'administration transvasculaire de médicament à travers la barrière hémato-encéphalique - Google Patents

Nanoparticules comprenant du n,n,n-triméthyl chitosane revêtu d'agonistes peptidiques de la kinine b1 ou b2 et/ou des antagonistes peptidiques de la kinine b1 pour l'administration transvasculaire de médicament à travers la barrière hémato-encéphalique Download PDF

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WO2024259525A1
WO2024259525A1 PCT/CA2024/050823 CA2024050823W WO2024259525A1 WO 2024259525 A1 WO2024259525 A1 WO 2024259525A1 CA 2024050823 W CA2024050823 W CA 2024050823W WO 2024259525 A1 WO2024259525 A1 WO 2024259525A1
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pro
arg
gly
ser
lys
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Fernand Gobeil Jr.
Yves Dory
Martin SAVARD
Maxime GAGNON
Philippe Tremblay
Alexandre MOQUIN
Stéphane GAGNE
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Ovensa Innovations Inc
Societe de Commercialisation des Produits de la Recherche Appliquee SOCPRA
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Ovensa Innovations Inc
Societe de Commercialisation des Produits de la Recherche Appliquee SOCPRA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • AD Alzheimer's disease
  • extracellular plaques containing various forms of amyloid-p protein (Ap)
  • NFTs intracellular neurofibrillary tangles
  • the blood-brain barrier is a major obstacle to the development of effective therapeutics for Alzheimer’s disease (AD) and other central nervous system (CNS) disorders.
  • AD Alzheimer’s disease
  • CNS central nervous system
  • Antibody therapeutics have been particularly challenging due to their large size; however, their target specificity, reduced off-target effects and prolonged pharmacokinetics make them a very attractive treatment strategy.
  • many avenues have been explored to overcome the challenge offered by the BBB.
  • none of these approaches has demonstrated the adequate balance of robustness, safety, and desirable physicochemical properties necessary for an effective brain drug delivery system.
  • a nanoformulation comprising N,N,N-trimethyl chitosan (TMC); a peptide agonist of kinin B1 receptor, a peptide antagonist of kinin B1 receptor or a peptide agonist of kinin B2 receptor; and a payload to be delivered across the bloodbrain barrier (BBB).
  • TMC N,N,N-trimethyl chitosan
  • BBB bloodbrain barrier
  • the agonist of kinin B1 receptor, the antagonist of kinin B1 receptor or the agonist of kinin B2 receptor is coupled to the TMC.
  • the nanoformulation described herein comprises a spacer coupling the agonist of kinin B1 receptor, the antagonist of kinin B1 receptor or the agonist of kinin B2 receptor to the TMC.
  • the spacer is a Mpa, Mpa-pAla spacer, Mpa-Aca, Mpa- PEG2, Mpa-PEG8, or Mpa-Gly.
  • the nanoformulation encompassed herein comprises a ratio of 1 : 7 of TMC: B1 R agonist or antagonist.
  • the nanoformulation encompassed herein comprises a B1R agonist coupled to TMC and a non-cross-linked B2R agonist.
  • the nanoformulation encompassed herein comprises a non-cross-linked B1 R agonist and a non-cross-linked B2R agonist.
  • the nanoformulation encompassed herein comprises a B1R antagonist coupled to TMC and a non-cross-linked B2R agonist.
  • the nanoformulation encompassed herein comprises a non-cross-linked B1 R antagonist and a non-cross-linked B2R agonist.
  • the nanoformulation encompassed herein comprises at least one B1 R agonist selected from the group consisting of: pAla-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DPhe-OH; pAla-Lys-Arg-Pro-Hyp-Gly-lgl-Ser-Pro-DPhe-OH;
  • the nanoformulation encompassed herein comprises a B1 R consisting of pAla-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DPhe-OH; pAla- Lys-Arg-Pro-Hyp-Gly-lgl-Ser-Pro-DPhe-OH; Gly-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DPhe-OH; Gly-Lys-Arg-Pro-Hyp-Gly-lgl-Ser-Pro-DPhe-OH; Sar-Lys-Arg-Pro-Pro-Gly- Phe-Ser-Pro-DPhe-OH; Sar-Lys-Arg-Pro-Hyp-Gly-lgl-Ser-Pro-DPhe-OH; Aca-Lys-Arg- Pro-Pro-Gly-Phe-Ser-Pro-DPhe-OH; Aca-Lys-Arg-Pro-Hyp-Gly-lgl-S
  • the nanoformulation encompassed herein comprises at least one B2R agonist selected from the group consisting of:
  • the nanoformulation encompassed herein comprises a B2R consisting of Arg-Pro-Hyp-Gly-Thi-Ser- N Chg-Thi-Arg-OH; Lys-Arg-Pro-Hyp-Gly- Phe-Cys-Pro-Phe-Arg-OH; Lys-Arg-Pro-Hyp-Gly-Thi-Ser- N Chg-Thi-Arg-OH; pAla-Arg- Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH; or Aca-Arg-Pro-Hyp-Gly-Thi-Ser- N Chg-Thi-Arg- OH.
  • the nanoformulation encompassed herein comprises at least one B1 R antagonist selected from the group consisting of:
  • the nanoformulation encompassed herein comprises a B1 R antagonist consisting of AcOrn-Arg-Oic-Pro-Gly-(aMe)Phe-Ser-DpNal-lle-OH; pAla-Orn-Arg-Oic-Pro-Gly-(aMe)Phe-Ser-DpNal-lle-OH; pAla-Orn-Arg-Oic-Pro-Gly- (aMe)Phe-Ser-DpNal-lle-OH; AcLys-Lys-Arg-Oic-Pro-Gly-(aMe)Phe-Ser-DpNal-lle-OH; Aca-Orn-Arg-Oic-Pro-Gly-(aMe)Phe-Ser-DpNal-lle-OH; or Lys-Orn-Arg-Oic-Pro-Gly- (aMe)Phe-Ser-DpNal-lle-OH.
  • B1 R antagonist consist
  • the agonist or antagonist of kinin B1 receptor further comprises a chelating compound at the N-terminus.
  • the chelating compound is 1 ,4,7-triazacyclononane-1 ,4,7- triacetic acid (NOTA); 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOT A); methylhydroxamates derived from triaza- and tetraaza macrocycles (NOTHA2 and DOTHA2); 1 ,4,7-triazacyclononane-1-glutaric acid-4, 7-diacetic acid (NODAGA), or a derivative thereof.
  • the nanoformulation encompassed herein comprises at least one B1 R agonist, B2R agonist, or B1 R antagonist selected from the group consisting of:
  • the nanoformulation encompassed herein comprises a ratio of 2:1 of agonist or an antagonist B1R ligand: agonist of B2R.
  • the agonist of kinin B1 receptor, the antagonist of kinin B1 receptor or the agonist of kinin B2 receptor further comprises a radiolabeling agent at its N-terminus.
  • the radiolabeling agent is 64 Cu, 67 Cu, 68 Ga, 131 1, 111 In, 153 Sm, 89 Sr, 90 Y, 177 Lu, 225 Ac or 213 Bi.
  • the payload is siRNA, a shRNA, an antibody, a small molecule, an antisense, a polynucleotide, or an aptamer.
  • the antibody is a monoclonal antibody, a polyclonal antibody or a humanized antibody.
  • the nanoformulation encompassed herein is formulated for an intravenous (IV) administration or intranasal (IN) administration.
  • the nanoformulation encompassed herein is for the treatment of a central nervous system (CNS) disease.
  • CNS central nervous system
  • the nanoformulation encompassed herein is for measuring the biological effects of the payload to treat a CNS disorder.
  • nanoformulation encompassed herein for treating a CNS disorder.
  • nanoformulation described herein in the manufacture of a medication for treating a CNS disorder.
  • Fig. 1 illustrates the chemical synthesis of kinin agonists functionalized TRIOZANTM NPs.
  • Fig. 2 illustrates representative curves showing displacements of [ 3 H]- Kallidin-[DesArg 10 -Leu 9 ] (A, C) and [ 3 H]-BK (B) binding to human recombinant B1 R and B2R, respectively, by the mAbs-unloaded F4 (A, B) and F7 (C) formulations. Displacement curves were fitted using a one-site model.
  • FIG. 5 illustrates comparison of tissue biodistribution of free mAb (F1 ) and mAb loaded-TRIOZANTM NPs containing both B1 R/B2R agonists (F4 and F6) following intravenous and intranasal administrations in AD transgenic mice (males and females combined) at 24h post-delivery.
  • Data represent mean ⁇ SEM.
  • a nanoformulation comprising N,N,N-trimethyl chitosan (TMC), an agonist or an antagonist of kinin B2 or B1 receptor and a payload to be delivered across the blood-brain barrier (BBB).
  • TMC N,N,N-trimethyl chitosan
  • BBB blood-brain barrier
  • kinin-TRIOZANTM nanoparticles allow mediated targeted delivery of drugs for the treatment of CNS diseases, and for elucidating and optimizing biological effects of novel drug candidates to treat CNS disorders.
  • TMC hydrophilic quaternized biopolymer N,N,N-trimethyl chitosan
  • TMC has been shown to be readily soluble in water (over a wide range of pH), safe, biodegradable, biocompatible, and can be easily chemically conjugated for active targeting of the loaded therapeutic agent. Moreover, because of its good mucoadhesive properties, it has been used to develop nanocarriers for brain targeting via intranasal administration, additionally to standard intravenous injections.
  • Another strategy to enhance drug delivery to the brain involves the targeted permeabilization of the BBB at inflammatory sites through the activation of overexpressed G proteins-coupled receptors (GPCRs), and that includes the kinin B2 and B1 receptors (B2R and B1R).
  • GPCRs G proteins-coupled receptors
  • B1R and B1R kinin B2 and B1 receptors
  • metabolically resistant, B1 R or B2R selective kinin peptide agonists have been shown effective in improving the delivery of drugs into the CNS via transient opening of the BBB with no significant side effects such as brain edema.
  • a dual B1 R/B2R agonist has been used to modulate the BBB paracellular permeability (Cote et al., 2013, Cancer Biol Ther, 14: 806-811 ).
  • a new type of nanodelivery system based on kinin- B1 R/B2R agonists coated nanoparticles (NPs) to promote effective transvascular drug delivery across the pathological BBB, which leads to increased brain penetration and therapeutic potency of a drug or payload that needs to cross the BBB, such as a siRNA, a shRNA, an antibody, a small molecule, an antisense, a polynucleotide, or an aptamer.
  • the antibody is a monoclonal antibody, a polyclonal antibody or a humanized antibody.
  • kinin agonists functionalized TRIOZANTM NPs were accomplished in the following way (Fig. 1 ). First the agonist sequence was prepared on solid support using standard SPPS procedures (Fmoc strategy, step 1). In the same way the two-residues Mpa-pAla spacer was introduced (step 2). In a third step the full spaceragonist peptide was released off the resin as its free thiol. Finally (step 4), the free thiol was coupled at the 6-OH position of TRIOZANTM subunits that had been freshly activated as its corresponding tosylate (S N 2 reaction leading to thioethers), thus yielding the desired TRIOZANTM polymers. According to the number of equivalents of peptide free thiol used during step 4, the degree of conjugation varies.
  • the spacer can be e.g. Mpa-pAla, Mpa-Aca, Mpa- PEG2, Mpa-PEG8, or Mpa-Gly.
  • Table 1 Binding affinities of novel peptide conjugates on HEK293 cells stably overexpressing human kinin B1 and B2 receptors
  • Table 2 Binding affinities of novel peptide B1 R antagonist conjugates on HEK293 cells stably expressing human kinin B1 receptors
  • Table 3 Binding affinities of novel peptide B2R agonist conjugates on HEK293 cells stably expressing human kinin B2 receptors
  • Table 4 Binding affinities of kinin analogues linked to or complexed with TRIOZANTM NPs on HEK293 cells stably expressing hB1 or hB2R
  • B1 R agonist, B1 R antagonist or B2R agonist peptides are:
  • TRIOZANTM is a positively charged polymer owing to its numerous quaternary ammonium groups. It can be electrostatically cross-linked by adding negatively charged polytriphosphate (3PP) leading to the formation of stable NPs, that act as nanocontainers.
  • 3PP negatively charged polytriphosphate
  • a payload but not limited to, is a mAb, which is non- covalently retained inside the NPs until they reach the desired location for delivery. This selectivity is provided by B1 R and B2R ligands. Similar to the mAb, these ligands can be non-covalently trapped inside the NPs, from which they can slowly leach out to bind to the cognate receptors.
  • nanoformulation F6 for which the NPs contain twice as much B1 R ligand as B2R ligand. This was done to limit potential hypotensive effects of NPs containing NG291 agonists, mostly attributable to systemic vascular B2R activation.
  • the ligands can be covalently attached to the NPs by a spacer, meaning that binding between receptors and ligands can be somehow hindered by the bulk of the NPs.
  • ligands trapped inside the NPs are in no position to meet their receptors counterparts. Therefore, a loss of affinity is to be expected in that situation as indeed observed in nanoformulation F4, where the B1 R ligand only is covalently linked to the NP, whereas the B2R ligand is not.
  • Binding experiments with HEK-293 cells stably expressing human B1 R (Fig. 2, panel A) or B2R (Fig. 2, panel B) were also performed with new unloaded, peptide B1 R agonists functionalized TRIOZANTM-NPs mixed with B2R agonists. Results showed that the NPs, prepared using the specified starting material described in Table 1 (below referred to as F4 nanoformulation, see Table 5), retained their abilities to bind to human B1 R 186 and B2R, and being able to fully displace selective radioligands.
  • the mAb liquid solution (F1 ; 2.5 mg/mL) and the new TRIOZANTM-NP formulations loaded with mAb, F4 and F6, in lyophilized forms (in PBS/sucrose) were readily soluble in deionized water.
  • the chosen 2.5 mg/mL of F1 corresponds to a dose of 10 mg/kg by injecting a volume of 4 mL/kg (e.g. 80 L for a 20 g mouse).
  • a summary analysis of the characteristics of the mAb solution and the water-soluble NP formulations, is presented in Table 5.
  • the final concentrations of loaded mAb in F4 and F6 NPs were in the same range as the original mAb solution (2.5-3.3 mg/mL).
  • NPs The mean diameters of NPs were found to be less than 200 nm with polydispersity index (PDI) values below 0.3, indicating monodisperse NP suspensions. Due to their favorable characteristics (aqueous solubility, B1 R/B2R binding capacity, uniform and small sizes (i.e., between 130 and 165 nm)), the new NPs were considered appropriate for in vivo studies.
  • PDI polydispersity index
  • TMC-DS-TPP (mAb + B1 R ago + B2R ago) 2.97 ⁇ 0.11 132.4 ⁇ 2.3 0.144 ⁇ 0.006
  • F1 Test mAbs (2.5 mg/mL); F4 TMC/TMC-B1 R ago: dextran sulfate (DS): tripolyphosphate (TPP) (5.4/0.6: 1 : 0.5 in wt: wt: wt): mAb 2.5 mg/mL + B2R ago 45 pM; and F6: TMC: DS: TPP (6: 1 : 0.5): mAb 2.5 mg/mL + B1 R ago 90 pM + B2R ago 45 pM; F7: TMC/TMC-B1 R antago: DS: TPP (5.4/0.6: 1 : 0.5 in wt: wt: wt): mAb 2.5 mg/mL.
  • Biodistribution studies were then performed to characterize the relative effects of the two NP preparations on the accumulation of the tested mAb at specific regions of the brain such as, entorhinal cortex and hippocampus (regions responsible for learning and memory), most likely to be affected in AD. Comparison of biodistribution profiles of the free mAb and as NPs was evaluated at 24h post-injection. For the purpose of these experiments, a new, highly sensitive LC-MS/MS assay was developed for a direct quantification of the tested mAb in blood and tissue extracts (specifically, brain, liver, serum) from transgenic AD adult mice.
  • the intranasal (IN) administration route has recently gained increasing interest for delivering drugs to the CNS while bypassing the BBB. Therefore, for comparison purposes, the potential of the new drug-loaded nanoformulations was investigated to distribute into the brain after IN administration.
  • the naked mAb alone (F1 ) and the mAb loaded-NP formulations (F4 and F6) were administered by intranasal drops at the same mAb dosage of 10 mg/kg.
  • the nanoformulations were well- tolerated with no apparent adverse effects for up to 24h after their administration.
  • Fmoc groups were removed by treating the resin with 20% piperidine in DMF for 10 min (2x).
  • the peptides were cleaved from resin by HFIP/ DCM (7/3) for 1 h and deprotected using the cleavage cocktail of TFA: triisopropylsilane (TIPS): H2O (95:2.5:2.5/ v:v:v) at room temperature for 3h.
  • TFA triisopropylsilane
  • H2O 95:2.5:2.5/ v:v:v
  • the solution of the released peptides was filtered, concentrated, and precipitated in cold diethyl ether.
  • Hyp trans-4-hydroxy-L-proline
  • Thi a-(2-th ieny I )- L-alanine
  • Orn L-ornithine
  • Oic L-(2S,3aS,7aS)-octahydro-1H-indol-2-carboxylic acid
  • p-Nal 3-(2-naphthyl)-alanine
  • Igl 2-indanyl-glycine
  • NChg N-cyclohexyl-glycine
  • Sar N- methyl-glycine
  • (aMe)Phe a-methyl-phenylalanine
  • Mpa 3-mercaptopropanoyl.
  • 1 mL of TEA and 13 mg B1 R agonist peptide were dissolved into 15 mL of water in a 100 mL round-bottom flask under inert condition (7 equivalent peptide/Triozan; TMC-B1 R). The reaction mixture was kept under stirring at room temperature for 3 days.
  • TMC/TMC-B1 R ratio 9/1 was used instead of the free B1 R peptide agonist.
  • the TRIOZANTM (+mAbs/B1 R/B2R) solution and a solution of PBS (pH 7.4, 0.01 M, NaC1 137 mM) containing dextran sulfate (DS, Mw 500,000 Da: 1 mg/mL): tripolyphosphate (TPP: 0.5 mg/mL), were loaded in two separate 10 mL syringes.
  • Both syringes were placed in a NanoAssemblr® containing a sterile NanoAssemblr® NxGen microfluidic chip.
  • the flow rate was set at 12 mL/min with a final wt/wt ratio between TRIOZANTM and DS equal to 6:1.
  • the freshly prepared NPs were centrifuged at 42,000 g for 45 min at 4°C.
  • the pellets (clear with a yellowish tint) were resuspended in PBS using short bursts of sonication and filtered through 0.45 pm PVDF filters.
  • Sucrose final concentration: 50 mg/mL was finally added to NP suspensions before freeze-drying.
  • HEK293 cells stably expressing either human kinin B1 R or B2R were grown in 24-well plates and incubated with 4 nM [ 3 H]Lys[Leu 8 ]desArg 9 BK (for B1 R) or with 1 nM [ 3 H]BK/well (for B2R) in serum-free DMEM for 1-2h in the absence or presence of increasing concentrations of competitors (10 11 -10 ⁇ 5 M for peptides; 10’ 1 °-10’ 4 g/mL for peptides-coupled NPs).
  • the average sizes (nm) and polydispersity indices of TRIOZANTM based-NPs were determined by dynamic light scattering (DLS) using the UNCLE-Unchained Labs system (Unchained Labs, Pleasanton, CA, USA). Ten L of each antibody was loaded into the multi-micro cell array. The DLS measurement was recorded at 20°C. Ten readings were taken for each individual analysis, with outliers discarded, and the remaining data averaged. The Uncle software (version 2.0) correlation function was subsequently used to calculate DLS measurements (size distribution and polydispersity). Alternatively, DLS analyses of the mAbs loaded-TRIOZANTM NPs were performed at room temperature using a Zetasizer Nano ZS (Malvern® Instrument).
  • mice Male and female double transgenic mice (B6C3-Tg (APPswe/PSEN1dE9); 9- to 12-month-old; bodyweight 35-50 g) were used in this study. Mice were purchased from the Jackson Laboratory (MMRRC Strain #034829-JAX). Animals were maintained under standard diurnal conditions and were allowed access to food and water ad libitum. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Universite de Sherbrooke (protocol #2020-2508) and performed in accordance with the Canadian Council on Animal Care guidelines.
  • mAb formulations (dose: 10 mg/kg) were administered IV or IN in isoflurane anesthetized-mice (5% induction, 2% maintenance).
  • IV administration formulations were given as a bolus ( ⁇ 1 min) with a 50 pL saline flush via the caudal vein.
  • NP formulations were concentrated about 6- to 7-fold using 10 kDa MWCO ultrafiltration membranes (Amicon Ultra). A total volume of 5-6 pL was delivered into each nostril using a pipette over the course of 5 min.
  • mice were collected from mice anesthetized with ketamine/xylazine (intramuscular injection, 87/13 419 mg/kg). Serum was prepared from blood harvested from cardiac puncture. Prior to organ harvesting, mice were euthanized by transection of the right atrium followed by an injection of 20 ml_ cold saline in the left ventricle to flush the blood from the brain and organs. Harvested tissue and serum samples were flash frozen in liquid nitrogen and stored at -80°C until analysis.
  • Tissue samples were prepared according to Delcourt et al. (2018, Mol Cell Proteomics, 17(12): 2402-2411 ) and Dubois et al. (2020, Nat Commun, 11(1 ): 1306). Briefly, tissue samples were homogenized using a Mini Bead Mill Homogenizer with 2.8 mm ceramic beads for hard tissue homogenization in 500 pL Lysis Buffer (8 M Urea, 10 mM HEPES pH 8.0). Lysates were sonicated to reduce viscosity followed by a 10 min centrifugation at 16,000 g to discard debris and insoluble parts. The supernatant was transferred in a LoBind tube and protein content was assessed using BCA protein assay.
  • a total of 50 pg of protein in 50 pL of lysis buffer with 20 nM of the stable isotope peptide were reduced in 5 mM DTT, boiled 2 min at 95°C, rested 30 min at room temperature and alkylated in 7.5 mM 2 Choloroacetamide for 30 min in the dark at room temperature.
  • the urea concentration in the lysate was reduced to 2 M with the addition of 50 mM NH4HCO3 and the samples were subjected to overnight trypsin digest (T rypsin Gold, MS Grade, Promega Corporation).
  • a total of 10 L of peptide mixture was loaded and separated onto a nanoHPLC system (Dionex Ultimate 3000) with a constant flow of 4 pl_/min onto a trap column (Ac-claim PepMap100 C18 column, 0.3 mm id x 5 mm, Dionex Corporation). Peptides were then eluted off towards an analytical column heated to 40°C (PepMap C18 nano column (75 pm x 25 cm)) with a linear gradient of 5-45% of solvent B (80% ACN with 0.1% FA) over a 42 min gradient at a constant flow (450 nL/min).
  • the amount of the mAb protein was calculated using the light to heavy peptide ratio. Data are presented as mean ⁇ SEM. Data were compared using two-way ANOVA with Sidak's correction. Statistical significance was set at *p ⁇ 0.05. Statistical analysis was performed using GraphPad 9.3.1 (GraphPad Software, Inc).

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Abstract

L'invention concerne une nanoformulation comprenant du N,N,N-triméthyl chitosane (TMC), un agoniste ou un antagoniste du récepteur B2 ou B1 de la kinine et une charge utile à administrer à travers la barrière hémato-encéphalique (BHE). La nanoformulation peut être utilisée pour traiter le système nerveux central (SNC) par exemple par administration intraveineuse (IV) ou intranasale (IN).
PCT/CA2024/050823 2023-06-19 2024-06-18 Nanoparticules comprenant du n,n,n-triméthyl chitosane revêtu d'agonistes peptidiques de la kinine b1 ou b2 et/ou des antagonistes peptidiques de la kinine b1 pour l'administration transvasculaire de médicament à travers la barrière hémato-encéphalique Ceased WO2024259525A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007746A1 (fr) * 1996-08-19 1998-02-26 Universite De Sherbrooke Antagonistes du recepteur de la bradykinine b1 et leur utilisation
WO2006128293A1 (fr) * 2005-06-01 2006-12-07 UNIVERSITé DE SHERBROOKE Agonistes peptidiques du recepteur de la kinine b1 et leurs utilisations
WO2012171125A1 (fr) * 2011-06-13 2012-12-20 Rival, Société En Commandite Polymères de n,n,n-trialkyle, procédés permettant leur élaboration, et utilisations de ceux-ci
WO2016205941A1 (fr) * 2015-06-25 2016-12-29 Societe De Commercialisation Des Produits De La Recherche Appliquee Socpra Sciences Sante Et Humaines, S.E.C. Nouvelles sondes théranostiques à base de kinine pour cancers solides et leurs utilisations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007746A1 (fr) * 1996-08-19 1998-02-26 Universite De Sherbrooke Antagonistes du recepteur de la bradykinine b1 et leur utilisation
WO2006128293A1 (fr) * 2005-06-01 2006-12-07 UNIVERSITé DE SHERBROOKE Agonistes peptidiques du recepteur de la kinine b1 et leurs utilisations
WO2012171125A1 (fr) * 2011-06-13 2012-12-20 Rival, Société En Commandite Polymères de n,n,n-trialkyle, procédés permettant leur élaboration, et utilisations de ceux-ci
WO2016205941A1 (fr) * 2015-06-25 2016-12-29 Societe De Commercialisation Des Produits De La Recherche Appliquee Socpra Sciences Sante Et Humaines, S.E.C. Nouvelles sondes théranostiques à base de kinine pour cancers solides et leurs utilisations

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CAPRIFICO ANNA E., FOOT PETER J. S., POLYCARPOU ELENA, CALABRESE GIANPIERO: "Overcoming the Blood-Brain Barrier: Functionalised Chitosan Nanocarriers", PHARMACEUTICS, MDPI AG, SWITZERLAND, vol. 12, no. 11, Switzerland, pages 1013, XP093258559, ISSN: 1999-4923, DOI: 10.3390/pharmaceutics12111013 *
CÔTÉ JÉRÔME, BOVENZI VERONICA, SAVARD MARTIN, DUBUC CÉLÉNA, FORTIER AUDREY, NEUGEBAUER WITOLD, TREMBLAY LUC, MÜLLER-ESTERL WERNER,: "Induction of Selective Blood-Tumor Barrier Permeability and Macromolecular Transport by a Biostable Kinin B1 Receptor Agonist in a Glioma Rat Model", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 7, no. 5, US , pages e37485, XP093258555, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0037485 *
CÔTÉ JÉRÔME, SAVARD MARTIN, NEUGEBAUER WITOLD, FORTIN DAVID, LEPAGE MARTIN, GOBEIL FERNAND: "Dual kinin B1 and B2 receptor activation provides enhanced blood–brain barrier permeability and anticancer drug delivery into brain tumors", CANCER BIOLOGY & THERAPY, LANDES BIOSCIENCE, US, vol. 14, no. 9, 19 September 2013 (2013-09-19), US , pages 806 - 811, XP093258557, ISSN: 1538-4047, DOI: 10.4161/cbt.25327 *
DUARTE-GUTERMAN PAULA; LIEBLICH STEPHANIE E.; QIU WANSU; SPLINTER JARED E.J.; GO KIMBERLY A.; CASANUEVA-REIMON LAURA; GALEA LIISA : "Oxytocin has sex-specific effects on social behaviour and hypothalamic oxytocin immunoreactive cells but not hippocampal neurogenesis in adult rats", HORMONES AND BEHAVIOR, ACADEMIC PRESS, NEW YORK, NY, US, vol. 122, 21 March 2020 (2020-03-21), US , XP086169210, ISSN: 0018-506X, DOI: 10.1016/j.yhbeh.2020.104734 *
GAGNON MAXIME, SAVARD MARTIN, TRAN THI MINH HUE, VINCENT LAURENCE, MOQUIN ALEXANDRE, TREMBLAY PHILIPPE, ROUCOU XAVIER, DORY YVES, : "Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease", MOLECULES, MDPI AG, CH, vol. 28, no. 13, CH , pages 5206, XP093258560, ISSN: 1420-3049, DOI: 10.3390/molecules28135206 *
GAO ET AL.: "RVG-peptide-linked trimethylated chitosan for delivery of siRNA to the brain", BIOMACROMOLECULES, vol. 15, 2014, pages 1010 - 1018, XP055479945, DOI: 10.1021/bm401906p *
KULHARNI ET AL.: "N,N,N-trimethyl chitosan : An advanced polymer with myriad of opportunities in nanomedicine", CARBOHYDRATE POLYMERS, vol. 157, 2017, pages 875 - 902, XP029848652, DOI: 10.1016/j.carbpol.2016.10.041 *
KUMAR ET AL.: "Evaluation of neuropeptide-loaded trimethyl chitosan nanoparticles for nose to brain delivery", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, vol. 61, 2013, pages 189 - 195, XP028729244, DOI: 10.1016/j.ijbiomac.2013.06.041 *

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