WO2024254448A2 - Nanoformulations de promédicament de boronate dans des traitements de maladies - Google Patents

Nanoformulations de promédicament de boronate dans des traitements de maladies Download PDF

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WO2024254448A2
WO2024254448A2 PCT/US2024/033004 US2024033004W WO2024254448A2 WO 2024254448 A2 WO2024254448 A2 WO 2024254448A2 US 2024033004 W US2024033004 W US 2024033004W WO 2024254448 A2 WO2024254448 A2 WO 2024254448A2
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amb
peg
cmt
cur
moiety
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WO2024254448A3 (fr
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Dandan Guo
Juntao Luo
Changying SHI
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Research Foundation of the State University of New York
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/025—Boronic and borinic acid compounds
    • 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
    • A61K47/54—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 compound
    • A61K47/541—Organic ions forming an ion pair complex with the pharmacologically or therapeutically active agent
    • 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
    • A61K47/62—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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
    • 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
    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6907—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 colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle

Definitions

  • the immunocompromised patients suffering dialysis, organ transplant, catheterization, HIV infection, chemotherapy or under intensive care medication are at higher risk for lethal fungal infection, commonly by Candida, Aspergillus, Cryptococcus, and Pneumocystis.
  • the challenge of fungal infections and the development of drug resistance demand highly efficacious antifungal therapeutics.
  • Polyenes and azoles play the critical role for invasive fungal infections treatment of immunocompromised patients.
  • Amphotericin B (AmB) is the gold standard for the treatment of life-threatening systemic fungal infections for over sixty- years in the clinic due to its broad activity spectrum, low resistant rate, and outstanding pharmacological and clinical efficacy.
  • the antifungal activity of AmB is attributed with its high binding affinity with ergosterol in fungal membrane, creating an ion permeable transmembrane channel and causing cell destruction.
  • the clinical use of AmB is associated with severe nephrotoxicity and infusion-related complication syndrome due to the proinflammatory cytokine production and hyperkalemia, causing cardiac arrhythmias and even death.
  • the conventional AmB sodium deoxycholate formulation i.e. Fungizone®
  • Lipid-based formulations of AmB such as AmBisome®, ABELCET®, and Amphotin LIP® have been developed to significantly lower the incidence of infusion-related reactions and increase the renal tolerance.
  • Lipid based AmB has been reported to cause idiosyncratic reactions such as chest pain, flushing, and abdomen pain due to liposome formulation other than AmB itself.
  • prohibitive cost and limited manufacturer of Lipid AmB drastically compromise its clinic application. Much effort have been made to develop cost-effective formulations with reduced toxicity.
  • Embodiments disclosed herein are directed to prodrug compounds having the following form ula:PEG-D(X)-(L-BA) m -d n , wherein PEG is a polyethylene glycol moiety, and has an average molecular weight of about 44 to about 40,000 Dalton; D(X) is a dendritic polymer moiety having one or more branched monomer units (X); L is an optional linker group; BA is at least one boronic acid moiety; d is at least one 1 , 2- or 1 , 3- cis-diol or catechol containing therapeutic compound, or an a- or p- hydroxyl ketone containing therapeutic compound; and subscript m is an integer from 2 to 16 and subscript n is any number that greater than 0 and less than m.
  • prodrug compounds wherein the polyethylene glycol moiety has an average molecular weight of about 5000 Da.
  • the prodrug compounds have branched monomer unit X that is a natural or synthetic amino acids with more than one amine group.
  • each branched monomer unit X is a lysine moiety.
  • the prodrug compounds comprise a boric acid that can be an aryl boronic acid or an aliphatic boronic acid and according to embodiments that boric acid component is a phenylboronic acid or a phenylboronic acid comprising a substitute group on the phenyl ring.
  • embodiments include a prodrug compound having a nonlinear dendritic structure, and according to an embodiment the X unit is a lysine moiety.
  • the therapeutic compound of the prodrug is covalently bonded to the boric acid BA via a reversible boronate ester bond.
  • the prodrug compounds include at least one boronic acid moiety is selected from the group consisting of phenyl boronic acid, nitrophenyl boronic acid, methoxyl phenylboronic acid, benzyl boronic acid, methylene boronic acid, ethylene boronic acid, and proplene boronic acid.
  • the therapeutic drug molecule of the prodrug compound include curcumin, doxorubicin, daunorubicin, amphotericin B, nystatin, anidulafungin; tetracyclines, aminoglycosides, nicotinamide mononucleotide (NMN), and nicotinamide adenine dinucleotide (NAD/NADH).
  • therapeutic compounds of the prodrug include tetracycline HCI, doxycycline, minocycline, tigecycline, eravacycline, sarecycline, omadacycline, chlortetracycline, oxytetracycline, and chemically modified tetracycline 3 (CMT-3).
  • therapeutic compounds of the prodrug include gentamycin, tobramycin, and kanamycine.
  • the therapeutic compound is amphotericin B (AmB).
  • the linker (L) of the prodrug compound can be polyethylene glycol moiety, amino acid, polyamino acids, polyserine moiety, enzyme cleavable peptide moiety, disulfide bond moiety and acid labile moiety, polyglycine moiety, poly(serine-glycine) moiety, aliphatic amino acid moieties, 6-amino hexanoic acid moiety, 5-amino pentanoic acid moiety, 4-amino butanoic acid moiety, and beta-alanine moiety.
  • linker (L) groups include lysine, arginine, aspartic acid, glutamic acid, linear oligo-lysine, linear oligo-aspartic acid, linear oligo-arginine, and linear oligo-glutamic acid.
  • the prodrug compound comprises selfassemble monodispersed micelles, and the micelles have a particle size of about 10- 20nm.
  • the prodrug reduce toxic side effects, immunogenicity and improved efficacy of drug molecules when administered in therapeutically effective amounts to mammal in need thereof.
  • FIG. 1 presents a design of AmB conjugated telodendrimer:
  • FIG. 1A presents model reaction of AmB and PBA conjugates by bivalent or trivalent boronate ester bond formation.
  • FIG. 1 B presents a schematic illustration for the architectures of PBA containing TDs: PEG 5k BAi, PEG 5k BA 2 , PEG 5k BA 4 , and PEG 5k BA 8 .
  • FIG. 1C presents a schematic illustration for AmB conjugation and release on PBA-containing TDs via reversible boronate ester bond.
  • FIG. 2 presents the characterization of AmB-TDs prodrug formulations:
  • FIG. 2A presents the particle size distribution of AmB-PEG 5k BAi , AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BA8.
  • GIG. 2B presents TEM images of blank PEG 5k BA 4 , PEG 5k BA 8 , AmB-PEG 5k BA 4 and AmB-PEG 5k BA 8 .
  • FIG. 2A presents the particle size distribution of AmB-PEG 5k BAi , AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BA8.
  • GIG. 2B presents TEM images of blank PEG 5k BA 4 , PEG 5k BA 8 , AmB-PEG 5k BA 4 and AmB-PEG 5k BA 8 .
  • FIG. 2C presents the UV-Vis absorbance spectrum of AmB formulations including: Free AmB in DMSO, Fungizone, Ambisome, AmB-PEG 5k BAi, AmB-PEG 5k BA2, AmB-PEG 5k BA 4 and AmB- PEG 5k BA 8 .
  • FIG. 2D presents an in vitro drug release profile of Fungizone, Ambisome, AmB-PEG 5k BAi , AmB-PEG 5k BA 2 , AmB-PEG 5k BA 4 and AmB-PEG 5k BA 8 in PBS at 37 °C.
  • FIG. 2E presents an in vitro drug release profile of AmB-PEG 5k BA4 and AmB- PEG 5k BAa in neutral, acidic pH and with glucose at 37 °C.
  • FIG. 3 presents the antifungal activity, cytotoxicity and biocompatibility of Am B-TD formulations:
  • FIG. 3A presents an MIC of AmB formulations for C. albicans inhibition.
  • FIG. 3B presents Candida albicans at invasion phase growth inhibition by Free AmB in DMSO, Fungizone, Ambisome, AmB-PEG 5k BAi, AmB-PEG 5k BA2, AmB- PEG 5k BA4 and AmB-PEG 5k BA8 at AmB concentration of 2.5 pg/mL.
  • FIG. 3 presents the antifungal activity, cytotoxicity and biocompatibility of Am B-TD formulations:
  • FIG. 3A presents an MIC of AmB formulations for C. albicans inhibition.
  • FIG. 3B presents Candida albicans at invasion phase growth inhibition by Free AmB in DMSO, Fungizone, Ambisome, AmB-PEG 5k BAi, AmB-PEG 5k BA2, AmB- PEG 5k BA4 and AmB-P
  • FIG. 3C presents cell viability of Free AmB in DMSO, Fungizone, Ambisome, AmB-PEG 5k BAi, AmB- PEG 5k BA 2 , AmB-PEG 5k BA 4 and AmB-PEG 5k BA 8 in 293T, RAW 264.7, and CHO cells.
  • FIG. 3D the hemolytic property of Free AmB in DMSO, Fungizone, Ambisome, AmB- PEG 5k BAi, AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BA 8 for overnight incubation at 37 °C.
  • FIG.3E presets the TNF-a production level of RAW 264.7 cells with Fungizone, Ambisome, AmB-PEG 5k BAi, AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BA8 for 2h incubation at 37 °C.
  • FIG. 4 presents the PK and biodistribution of AmB-TD formulations:
  • FIG. 4A presents the PK of Fungizone, Ambisome, AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BA 8 at AmB dose of 1 mg/kg by i.v. injection.
  • FIG. 4B presents the PK of Ambisome, AmB-PEG 5k BA4 and AmB-PEG 5k BA 8 at AmB dose of 10 mg/kg by i.v. injection.
  • FIG. 4C presents a summary of PK analysis of AmB formulations.
  • FIG. 4D presents representative ex vivo images of major organs taken out at 24 h after i.v.
  • FIG. 4E presents an ex vivo semi-quatitative fluorescent intensity of major organs at 24 h post-injection.
  • FIG. 5 the MTD, blood chemistry and pathology profiles of mice treated by AmB formulations:
  • FIG. 5A presents bodyweight changes of healthy mice administer intravenously with three consecutive Fungizone, Ambisome, AmB- PEG 5k BA 4 and AmB-PEG 5k BA 8 and blank PEG 5k BA 4 .
  • FIGs. 5B-5F [present the BUN, CRE, K + , ALT and AST and TNF-a levels of 24 h after last dose of AmB formulations.
  • FIG. 5G presents the histological examinations of the kidneys by H&E staining from animals treated with AmB formulations.
  • FIG. 6 presents the antifungal efficacy of AmB-PEG5kBA4 in C. albicans infected immunocompetent mice.
  • FIG. 6A presents a schematic illustration of C. albicans infected immunocompetent mouse model and treatment.
  • FIG. 6B presents the cumulative mice survival
  • FIG. 6C presents the bodyweight change
  • FIG. 6D presents the body temperature changes of mice was monitored.
  • FIG. 6E presents the colony count of C. albicans in major organ of mice treated by AmB formulations.
  • FIG. 6F presents the TNF-a and IL-6 level in plasma of mice treated by AmB formulations.
  • FIG. 6G presents GMS staining for C. albicans in the kidney of mice treated by AmB formulations.
  • FIG. 6H presents the histological examinations of the kidneys by H&E staining of mice treated by AmB formulations.
  • FIG. 7 presents the antifungal efficacy of AmB-PEG5kBA4 in C. albicans infected immunocompromised mice.
  • FIG. 7A presents a schematic illustration of C. albicans infected immunocompromised mouse model and treatment.
  • FIG. 7 presents FIG. 7B presents the cumulative mice survival,
  • FIG. 7C presents the bodyweight change, of mice was monitored.
  • FIG. 7D presents the colony count of C. albicans in major organ of mice treated by AmB formulations.
  • FIG. 7E presents the TNF-a and IL-6 level in plasma of mice treated by AmB formulations.
  • FIG. 7F presents the GMS staining for C. albicans in the kidney of mice treated by AmB formulations.
  • FIG. 7G presents the histological examinations of the kidneys by H&E staining of mice treated by AmB formulations.
  • FIG. 8 presents a scheme of the chemical structures and synthesis route of PEG 5k BAi, PEG 5k BA2, PEG 5k BA4 and PEG 5k BA 8 .
  • FIG. 8A presnts the chemical structures of PEG 5k BAi , PEG 5k BA2, PEG 5k BA4 and PEG 5k BA8.
  • FIG. 8B presents the synthesis route of PEG 5k BAi .
  • FIG. 8C presents the synthesis route of PEG 5k BA2.
  • FIG. 8D presents the synthesis route of PEG 5k BA4.
  • FIG. 8E presents the synthesis route of PEG 5k BA 8 .
  • FIG. 9 Chemical structure of clinical medicine and compounds with 1 ,2- and 1 ,3 Cis-diol group that can be conjugated on BA containing TDs to form nanoprodrug formulations.
  • FIG. 10 Chemical structure of clinical medicine and compounds with a- hydroxy ketone group that can be conjugated on BA containing TDs to form nanoprodrug formulations.
  • FIG. 11 Chemical structure of clinical medicine and compounds with enolatable dione group that can be conjugated on BA containing TDs to form nanoprodrug formulations.
  • FIG. 12 Chemical structure of PEG 2k BA 4 , PEG 2k NBA 4 , PEG 5k BA 4 , PEG 5k NBA 4 , PEG 2k Arg 4 BA 4 , PEG 2k Glu 4 BA 4 and PEG 2k Glu 8 BA 4 .
  • the TDs are prepared with both PEG 5k and PEG 2k .
  • the number of BA moieties in the TD are 1 , 2, 4, and 8.
  • PEG linker, C6 linker, Arginine, and glutamate are used as linker to increase flexibility of BA moieties on TD.
  • FIG. 13 BA-containing TD forTCL, DCL, and CMT-3 prodrug conjugation.
  • TCL Tetracycline
  • DCL doxycycline
  • CMT-3 with 1 ,2- and 1 ,3-diol groups can be conjugated onto the boronic acid group containing TDs to form prodrug nanoformulation via boronate ester bond, which is labile and responsive to acidic pH and ROS.
  • FIG.14 MALDI-TOF of CMT-3 react with (A) PBA and (B) NBA small molecules.
  • MALDI-TOF result indicates CMT-3 can be reacted with PBA or NBA small molecules via boronate ester bonds in 1 :1 or 1 :2 molar ratio.
  • FIG. 15 Particle size of CMT-3-PEG2kBA4 and CMT-3-PEG2kNBA4 with and without TEA during drug loading process.
  • CMT-3 can be efficiently conjugated on both PEG2kBA4 and PEG2kNBA4 with TEA as catalyst and form homogeneous nanoparticles with particle size of 275 nm and 10 nm, respectively.
  • CMT-3 can also efficiently be conjugated on PEG2kNBA4 without TEA at 1 :2.5 mass ratio.
  • PEG2kNBA4 exhibits significantly higher efficiency for CMT-3 conjugation.
  • FIG. 16 MALDI-TOF of CMT-3-PEG2kBA4 and CMT-3-PEG2kNBA4 formulations before and after CMT-3 conjugation.
  • MW of TDs increased after CMT-3 conjugation.
  • significant lower signal or non-signal of free CMT-3 were observed in the CMT-3-TD conjugated nanoprodrug formulation.
  • the CMT-3 conjugation efficiency was calculated.
  • the conjugation efficiency of CMT-3-PEG2kBA4 and CMT-3-PEG2kNBA4 are 84% and over 90%, respectively.
  • FIG. 17A UV spectrum of free CMT-3, CMT-3-PEG2kBA4, CMT-3- PEG2kNBA4, and CMT-3 PEG5kCA4VE4 formulations.
  • FIG. 17B Fluorescent intensity of free CMT-3 in DMSO, CMT-3-PEG2kBA4, CMT-3-PEG2kNBA4, and CMT- 3 PEG5kCA4VE4 in PBS.
  • FIG. 17 C Fluorescent intensity of CMT-3-PEG2kBA4 and CMT-3-PEG2kNBA4 in PBS, pH 5.5 and pH 2.0 buffer.
  • FIG. 18 In vitro CMT-3 release profile CMT-3-PEG2kBA4, CMT-3- PEG2kNBA4, and CMT-3 PEG5kCA4VE4 at pH 7.4 and pH 5.5. CMT-3 release percentage at 8h. As shown in Fig.18, both CMT-3-PEG2kBA4 and CMT-3- PEG2kNBA4 exhibited significant prolonged drug release in comparison with CMT-3 encapsulated PEG5kCA4VE4 formulation at neutral pH. Acidic pH 5.5 accelerated the CMT-3 release in CMT-3-PEG2kBA4, while has slightly effect on release profile of CMT-3-PEG2kNBA4. In comparison with PEG2kNBA4, PEG2kBA4 exhibited higher sensitivity to acidic pH.
  • FIG. 19 MTS of CMT-3 and nanoformulations:
  • FIG. 19A Cell viability of THP-1 , HEK, and RAW 264.7 cells incubated with free CMT-3, CMT-3-PEG2kBA4, CMT-3-PEG2kNBA4, and CMT-3 PEG5kCA4VE4 for 72 h.
  • FIG. 19B Cell viability of THP-1 , HEK, and RAW 264.7 cells incubated with blank polymer PEG2kBA4 and PEG2kNBA4 for 72 h.
  • FIG. 20 Cellular uptake of CMT-3: (FIG. 20A) Fluorescence intensity of RAW 264.7 and THP-1 cells incubated with free CMT-3, CMT-3-PEG2kBA4, CMT-3- PEG2kNBA4, and CMT-3 PEG5kCA4VE4 at 4h and 8h. (FIG. 20B) Fluorescent images of RAW 264.7 cells incubated with CMT-3, CMT-3-PEG2kBA4, CMT-3- PEG2kNBA4 at 4h.
  • both CMT-3-PEG2kBA4 and CMT-3- PEG2kNBA4 exhibited significantly lower cellular uptake in comparison with random diffusion of free CMT-3 in THP-1 and RAW cells at 4 and 8 h incubation.
  • FIG. 21 Hemolytic toxicity of (FIG. 21 A) CMT-3-PEG2kBA4 and CMT-3- PEG2kNBA4, and (FIG. 21 B) blank TD PEG2kBA4 and PEG2kNBA4 at 30 min, 4h, and overnight incubation.
  • Nonhemolytic toxicity has been found in both CMT-3- PEG2kBA4 and CMT-3-PEG2kNBA4, and blank TDs at 30 min, 4 h, and overnight incubation.
  • FIG. 22 A Particle size of CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4.
  • FIG. 22B Fluorescent spectrum and intensity of free CMT-3, CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4.
  • FIG. 22C UV spectrum of free CMT-3, CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4.
  • CMT-3 can be effectively conjugated on both PEG5kBA4 and PEG5kNBA4 with homogeneous particle size of 315 nm and 11 nm, respectively.
  • fluorescent intensity of CMT-3 significantly increased after conjugation in PEG5kBA4 and PEG5kNBA4. Red shift of UV spectrum was observed in both CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4, indicating efficient conjugation of CMT-3 on BA containing TDs.
  • FIG. 23 MTS of CMT-3 and nanoformulations FIG. 23A Cell viability of THP-1 , HEK, and RAW 264.7 cells incubated with free CMT-3, CMT-3-PEG5kBA4, CMT-3-PEG5kNBA4 for72 h.
  • FIG. 23B Cell viability of THP-1 , HEK, and RAW 264.7 cells incubated with blank polymer PEG2kBA4 and PEG2kNBA4 for 72 h.
  • FIG. 23C IC50 of free CMT-3, CMT-3-PEG2kBA4, CMT-3-PEG2kNBA4 in THP-1 , HEK, and RAW 264.7 cells. As shown in FIG.
  • FIG. 24 A Cell viability of RAW 264.7 cells incubated with free CMT-3, CMT-3-PEG5kBA4, CMT-3-PEG5kNBA4 for overnight with or without LPS.
  • FIG. 24B LPS induced proinflam matory cytokine IL-6 and TNF-a production in RAW 264.7 cells treated by free CMT-3, CMT-3-PEG5kBA4, CMT-3-PEG5kNBA4 for overnight incubation.
  • RAW cells incubated with CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4 exhibited significant higher cell viability than that of free CMT-3.
  • FIG 25A LTA or LPS induced TLR activation in HEK TLR-2, HEK TLR-4 and THP-1 Blue cells treated by free CMT-3, CMT-3-PEG5kBA4, CMT-3-PEG5kNBA4 for overnight incubation.
  • FIG 25B Cell viability of HEK TLR-4 and THP-1 Blue cells incubated with free CMT-3, CMT-3-PEG5kBA4, CMT-3-PEG5kNBA4 for overnight with LPS.
  • Free CMT-3 exhibited dose-dependent inhibition of TLR signaling pathway activation by LTA/LPS in HEK TLR2, HEK TLR4, and THP-1 blue cells due to its cytotoxicity.
  • CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4 showed dose-dependent inhibition of TLR signaling pathway activation by LPS in HEK TLR4, and THP-1 blue cells, rather than HEK TLR2 cells.
  • CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4 significantly reduced cytotoxicity of CMT-3 in HEK TLR and THP-1 blue cells during overnight incubation.
  • FIG. 26A Growth curve of gram-positive and gram-negative bacteria treated with different concentration of free CMT-3, CMT-3-PEG5kBA4, CMT-3- PEG5kNBA4 for overnight incubation.
  • FIG. 26B MIC of free CMT-3, CMT-3- PEG5kBA4, CMT-3-PEG5kNBA4 in gram-positive and gram-negative bacteria.
  • CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4 kept similar potency of antibacterial activity or slightly increased MIC in E.Coli. (K12), E.Coli (DH5a), Staph, aureus, Strep.
  • FIG. 27A CMT-3 adsorption on hydroxyapatite.
  • FIG. 27B CMT-3 degradation by UV.
  • FIG. 27C Cell viability of RAW 264.7 cells incubated with UV degraded of free CMT-3, CMT-3-PEG5kBA4, CMT-3-PEG5kNBA4 for 72 h.
  • CMT-3-PEG5kBA4 and CMT-3-PEG5kNBA4 exhibited less hydroxyapatite adsorption in comparison with free CMT-3, demonstrating CMT-3- PEG5kBA4 and CMT-3-PEG5kNBA4 may reduce the damage of bone tissue. Less UV degradation of CMT-3-PEG5kNBA4 has been found.
  • FIG. 28 PK and biodistribution of CMT-3-PEG5kBA4 via i.v. injection.
  • FIG. 28A Body weight change of the mice treated by 10 mg/kg CMT-3-PEG5kBA4.
  • FIG. 28B Pharmacokinetics of CMT-3 in mice treated by CMT-3-PEG5kBA4 via i.v. injection.
  • FIG. 28C Biodistribution of CMT-3 in mice treated by CMT-3-PEG5kBA4 via i.v. injection after 24h. Less than 2% body weight loss shown in the mice treated by 10 mg/kg CMT-3-PEG5kBA4 via i.v. injection, indicating minimized toxicity of nanoprodrug formulation.
  • CMT-3-PEG5kBA4 significantly prolong the blood circulation time of CMT-3 with over 57-fold AUC in comparison with CMT-3 encapsulated PEG5kCA4VE4 formulation. After 24 h of the injection, majority of CMT- 3 were distributed in the liver and spleen.
  • FIG. 29 Particle size of TCL-PEG2kBA4, TCL-PEG2kNBA4 and TCL- PEG5kBA4, TCL-PEG5kNBA4.
  • TCL can be effectively conjugated on PEG2kBA4, PEG2kNBA4, PEG5kBA4 and PEG5kNBA4 with homogeneous particle size of 269 nm, 9 nm, 318 nm and 11 nm, respectively.
  • FIG.30A UV spectrum (FIG.30B) Fluorescent spectrum and intensity of free TCL, TCL-PEG2kBA4, TCL-PEG2kNBA4 and TCL-PEG5kBA4, TCL- PEG5kNBA4.
  • FIG.30C In vitro TCL release profile of free TCL, TCL-PEG2kBA4, TCL-PEG2kNBA4 and TCL-PEG5kBA4, TCL-PEG5kNBA4 at pH 7.4 and pH 5.5.
  • TCL-PEG2kBA4, TCL- PEG2kNBA4 and TCL-PEG5kBA4, TCL-PEG5kNBA4 significantly prolonged drug release profile in comparison with free TCL.
  • Acidic pH 5.5 accelerated the TCL release in TCL-PEG2kBA4, TCL-PEG5kBA4, and TCL-PEG2kNBA4 while has slightly effect on release profile of TCL-PEG5kNBA4.
  • PEG5kBA4 exhibited higher sensitivity to acidic pH.
  • FIG. 31A Fluorescent intensity of TCL in free TCL, TCL-PEG2kBA4, TCL-PEG2kNBA4 mixed with Ca2+ at different concentration for 10 min and FIG. 31 B 5 h. Fluorescent intensity of free TCL significantly increased by interaction with Ca2+ in a dose-dependent manner. Only slightly fluorescent intensity increase has been shown in both TCL-PEG2kBA4 and TCL-PEG2kNBA4 at highest Ca2+ concentration, indicating nanoprodrug nanoformulation TCL-PEG2kBA4 and TCL-PEG2kNBA4 efficiently prevent TCL interacting with Ca2+, which may further reduce the damage of bone tissue.
  • FIG. 32 Cytotoxicity of free TCL, TCL-PEG2kBA4, TCL-PEG2kNBA4.
  • FIG. 32 A Cell viability of RAW 264.7, TH P-1 and HEK cells incubated with free TCL, TCL-PEG2kBA4, TCL-PEG2kNBA4 for 72 h.
  • FIG. 32 B Hemolytic toxicity of free TCL, TCL-PEG2kBA4, TCL-PEG2kNBA4 for 0.5 h, 4 h and overnight incubation.
  • both TCL-PEG2kBA4 and TCL-PEG2kNBA4 show nearly 100% cell viability up to 100 pg/mL, demonstrating reduced cytotoxicity of TCL in nanoprodrug formulation.
  • Nonhemolytic toxicity has been found in both TCL- PEG2kBA4, TCL-PEG2kNBA4, TCL-PEG5kBA4 and TCL-PEG5kNBA4at 30 min, 4 h, and overnight incubation.
  • FIG. 33 Particle size of DCL-PEG2kBA4, DCL-PEG2kNBA4 and DCL- PEG5kBA4, DCL-PEG5kNBA4.
  • TCL can be effectively conjugated on PEG2kBA4, PEG2kNBA4, PEG5kBA4 and PEG5kNBA4 with homogeneous particle size of 181 nm, 192 nm, 261 nm and 11 nm, respectively.
  • FIG. 34A UV spectrum
  • FIG. 34B Fluorescent spectrum and intensity of free DCL, DCL-PEG2kBA4, DCL-PEG2kNBA4 and DCL-PEG5kBA4, DCL- PEG5kNBA4.
  • FIG. 34C In vitro DCL release profile of free DCL, DCL-PEG2kBA4, DCL-PEG2kNBA4 and DCL-PEG5kBA4, DCL-PEG5kNBA4 at pH 7.4 and pH 5.5.
  • Acidic pH 5.5 accelerated the DCL release in DCL-PEG2kBA4 and DCL-PEG5kBA4, while has slightly effect on release profile of DCL-PEG5kNBA4 and DCL-PEG2kNBA4.
  • PEGnkBA4 exhibited higher sensitivity to acidic pH.
  • FIG. 35 Cytotoxicity of free DCL, DCL-PEG2kBA4, DCL-PEG2kNBA4.
  • FIG. 35 A Cell viability of RAW 264.7, THP-1 and HEK cells incubated with free DCL, TCL-PEG2kBA4, DCL-PEG2kNBA4 for 72 h.
  • FIG. 35 B Hemolytic toxicity of free DCL, DCL-PEG2kBA4, DCL-PEG2kNBA4 for 0.5 h, 4 h and overnight incubation.
  • FIG. 35 C Fluorescence intensity of RAW 264.7 cells incubated with free DCL, TCL- PEG2kBA4, DCL-PEG2kNBA4 for 4 and 8h.
  • both DCL- PEG2kBA4 and DCL-PEG2kNBA4 show nearly 100% cell viability up to 100 pg/mL, demonstrating reduced cytotoxicity of DCL in nanoprodrug formulation.
  • Nonhemolytic toxicity has been found in both DCL-PEG2kBA4, DCL-PEG2kNBA4, DCL-PEG5kBA4 and DCL-PEG5kNBA4at 30 min, 4 h, and overnight incubation.
  • Both DCL-PEG2kBA4 and DCL-PEG2kNBA4 exhibited significantly lower cellular uptake in comparison with random diffusion of free DCL in RAW cells at 4 and 8 h incubation.
  • FIG. 36 Anti-bacterial activity of DCL and its nanoprodrug formulations.
  • FIG. 36A Growth curve of gram-positive and gram-negative bacteria treated with different concentration of free DCL, DCL-PEG2kBA4, DCL-PEG2kNBA4 for overnight incubation.
  • FIG. 36B MIC of free DCL, DCL-PEG2kBA4, DCL-PEG2kNBA4 in grampositive and gram-negative bacteria.
  • DCL-PEG2kBA4 and DCL-PEG2kNBA4 kept similar potency of antibacterial activity or slightly increased MIC in E.Coli. (K12), Pseudo. Aeruginosa, Staph, aureus, Strep. Pneumoniae and Listeria, indicating efficient drug release of CMT-3 in nanoprodrug formulations.
  • FIG. 38 Particle size of CUR-PEG2kBA4, CUR-PEG2kNBA4.
  • CUR can be effectively conjugated on PEG2kBA4, PEG2kNBA4, with homogeneous particle size of 13 nm and 11 nm, respectively.
  • FIG. 39A Free CUR detection in CUR-PEG2kBA4 and CUR- PEG2kNBA4 formulations.
  • FIG. 39B MALDI-TOF of CUR-3-PEG2kBA4 and CUR-3- PEG2kNBA4 formulations before and after CMT-3 conjugation.
  • the MW of PEG2kBA4 and PEG2kNBA4 before and after CUR conjugation were characterized by MALDI-TOF.
  • MW of TDs increased after CUR conjugation.
  • significant lower signal or non-signal of free CUR were observed in the CUR-TD conjugated nanoprodrug formulation. Based on the peak intensity ratio of free CUR to CHCA matrix, the CUR conjugation efficiency was calculated.
  • the conjugation efficiency of CUR-PEG2kBA4 and CUR-PEG2kNBA4 are over 95%.
  • FIG. 40A UV spectrum
  • FIG. 40B Fluorescent spectrum and intensity of free CUR, CUR-PEG2kBA4, CUR-PEG2kNBA4 and CUR PEG5kBA4, CUR - PEG5kNBA4.
  • FIG. 40C In vitro CUR release profile of free CUR, CUR-PEG2kBA4, CUR-PEG2kNBA4 and CUR-PEG5kBA4, CUR-PEG5kNBA4 at pH 7.4, pH 8.5, pH 5.5 and pH 1 .6. Red shift of UV spectrum was observed in CUR-PEG2kBA4 and CUR- PEG2kNBA4, indicating efficient conjugation of CUR on BA containing TDs.
  • FIG. 41 MTS of CUR and nanoformulations.
  • FIG. 41A Cell viability of THP-1 , HEK, and RAW 264.7 cells incubated with free CUR, CUR-PEG5kBA4, CUR- PEG5kNBA4 for 72 h.
  • FIG. 41 B Cell viability of THP-1 , HEK, and RAW 264.7 cells incubated with blank polymer PEG2kBA4 and PEG2kNBA4 for 72 h.
  • FIG. 41 C IC50 of free CUR, CUR-PEG2kBA4, CUR-PEG2kNBA4 in THP-1 , HEK, and RAW 264.7 cells. As shown in FIG.
  • FIG. 42A LTA or LPS induced TLR activation in HEK TLR-2, HEK TLR- 4 and THP-1 Blue cells treated by free CUR, CUR-PEG5kBA4, CUR-PEG5kNBA4 for overnight incubation.
  • FIG. 42B Cell viability of HEK TLR-4 and THP-1 Blue cells incubated with free CUR, CUR-PEG5kBA4, CUR-PEG5kNBA4 for overnight with LPS.
  • Free CUR, CUR-PEG5kBA4 and CUR-PEG5kNBA4 exhibited dose-dependent inhibition of TLR signaling pathway activation by LTA/LPS in HEK TLR2, HEK TLR4, and THP-1 blue cells due to their cytotoxicity.
  • FIG. 43A Cell viability of RAW 264.7 cells incubated with free CUR, CUR-PEG5kBA4, CUR-PEG5kNBA4 for overnight with or without LPS.
  • Free CUR, CUR-PEG5kBA4 and CUR-PEG5kNBA4 significantly reduced TNF-a and IL-6 production induced by LPS in a dose-dependent manner due to their cytotoxicity.
  • Free CUR exhibited moderate antibacterial activity at 100 pg/mL in both gram-positive and gram-negative bacteria.
  • CUR-PEG5kBA4 and CUR-PEG5kNBA4 exhibited less antibacterial activity, indicating the effective conjugation of CUR on the BA containing TDs.
  • FIG. 44 Treatment of LPS induced sepsis mouse model.
  • FIG.44A Body temperature and body weight change of LPS-induced sepsis mice without treatment and treated by CUR-PEG5kBA4 and CMT-3-PEG5kBA4.
  • FIG. 44B Proinflammatory cytokine TNF-a and IL-6 in serum of LPS-induced sepsis mice without treatment and treated by CUR-PEG5kBA4 and CMT-3-PEG5kBA4.
  • CUR-PEG5kBA4 and CMT-3- PEG5kBA4 significantly improve the body temperature of the LPS-induced sepsis mice at 24 h.
  • the body weight change showed no significant difference between treated and untreated group. In comparison with untreated group, CMT-3-PEG5kBA4 significantly reduced TNF-a production at 1 h, while CUR-PEG5kBA4 significantly reduced IL-6 production at 24 h.
  • FIG. 45 Particle size of PEG2kArg4BA4, PEG2kGlu4BA4, PEG2kGlu8BA4, CUR- PEG2kArg4BA4, CUR-PEG2kGlu4BA4, and CUR- PEG2kGlu8BA4.
  • CUR can be effectively conjugated on PEG2kArg4BA4 and PEG2kGlu4BA4 with homogeneous particle size of 19 nm and 13 nm, respectively. Multiple peaks were observed in CUR-PEG2kGlu8BA4.
  • FIG. 46A Free CUR detection in CUR-PEG2kArg4BA4, CUR- PEG2kGlu4BA4 and CUR-PEG2kGlu8BA4 formulations.
  • FIG. 46B MALDI-TOF of CUR-PEG2kArg4BA4, CUR-PEG2kGlu4BA4 and CUR-PEG2kGlu8BA4 formulations before and after CUR conjugation.
  • the MW of PEG2kArg4BA4 and PEG2kGlu4BA4 before and after CUR conjugation were characterized by MALDI-TOF. As shown in Figure 35, MW of TDs increased after CUR conjugation.
  • FIG. 47B Fluorescent spectrum and intensity of CUR-PEG2kArg4BA4, CUR-PEG2kGlu4BA4 and CUR-PEG2kGlu8BA4. Red shift of UV spectrum was observed in CUR-PEG2kArg4BA4, CUR-PEG2kGlu4BA4, indicating efficient conjugation of CUR on BA containing TDs. In comparison with free CUR, fluorescent intensity of CUR significantly reduced after conjugation in nanoprodrug formulations.
  • FIG. 48 Cell viability of RAW 264.7 incubated with CUR-PEG2kArg4BA4,
  • blank TDs exhibited noncytotoxicity in RAW cells up to 1 mg/mL.
  • CUR-PEG2kArg4BA4 exhibited 4-5-fold higher IC50 than PEG2kGlu4BA4 and PEG2kGlu8BA4 in RAW cells. All the CUR- PEG2kArg4BA4, CUR-PEG2kGlu4BA4 and CUR-PEG2kGlu8BA4significantly reduced the cytotoxicity of CUR.
  • FIG. 49A Cell viability of RAW 264.7 and THP-1 cells incubated with CUR-PEG2kArg4BA4, CUR-PEG2kGlu4BA4 and CUR-PEG2kGlu8BA4, PEG2kArg4BA4, PEG2kGlu4BA4 and PEG2kGlu8BA4 for overnight with LPS.
  • FIG. 49B LPS-induced proinflam matory cytokine TNF-a production in RAW 264.7 and THP-1 cells incubated with CUR-PEG2kArg4BA4, CUR-PEG2kGlu4BA4 and CUR- PEG2kGlu8BA4, PEG2kArg4BA4, PEG2kGlu4BA4 and PEG2kGlu8BA4.
  • FIG. 50 Particle size of CMT-3-PEG2kArg4BA4, CMT-3- PEG2kGlu4BA4, and CMT-3-PEG2kGlu8BA4.
  • CMT-3 can be effectively conjugated on PEG2kArg4BA4 and PEG2kGlu4BA4 with homogeneous particle size of 17 nm and 107 nm, respectively. Precipitation was observed in CUR-PEG2kGlu8BA4 on the second day.
  • FIG. 51 A Free CMT-3 detection in CMT-3-PEG2kArg4BA4, CMT-3- PEG2kGlu4BA4 and CMT-3-PEG2kGlu8BA4formulations. (FIG.
  • FIG. 53 Particle size of NMN-PEG2kBA4, NMN-PEG2kArg4BA4, NMN- PEG2kGlu4BA4, and NMN-PEG2kGlu8BA4.
  • NMN can be effectively conjugated on PEG2kBA4, PEG2kArg4BA4 and PEG2kGlu4BA4 with homogeneous particle size of 223 nm, 14 nm, and 253 nm, respectively. Multiple peaks were observed in NMN- PEG2kGlu8BA4 on the second day.
  • FIG. 54A Free NMN detection in NMN-PEG2kBA4, NMN- PEG2kArg4BA4, NMN-PEG2kGlu4BA4 and NMN-PEG2kGlu8BA4 formulations.
  • FIG. 54B MALDI-TOF of NMN-3-PEG2kBA4, NMN-3-PEG2kArg4BA4, NMN- PEG2kGlu4BA4 and NMN-PEG2kGlu8BA4formulations before and after CUR conjugation.
  • the MW of PEG2kBA4, PEG2kArg4BA4 and PEG2kGlu4BA4, PEG2kGlu8BA4 before and after CMT-3 conjugation were characterized by MALDI- TOF.
  • FIG. 55A Cell viability of RAW 264.7 and THP-1 cells incubated with NMN-PEG2kBA4, NMN-PEG2kArg4BA4, NMN-PEG2kGlu4BA4 and NMN- PEG2kGlu8BA4 for overnight with LPS.
  • FIG. 55B LPS-induced proinflammatory cytokine TNF-o production in RAW 264.7 and THP-1 cells incubated with NMN-3- PEG2kBA4, NMN-3-PEG2kArg4BA4, NMN-PEG2kGlu4BA4 and NMN- PEG2kGlu8BA4.
  • FIG. 55B LPS-induced proinflammatory cytokine TNF-o production in RAW 264.7 and THP-1 cells incubated with NMN-3- PEG2kBA4, NMN-3-PEG2kArg4BA4, NMN-PEG2kGlu4BA4 and NMN- PEG2kGlu8BA4.
  • FIG. S1 presents MALDI-TOF MS characterization of TD intermediates and TDs.
  • FS1A presents an MS spectrum detected by MALDI-TOF MS of TD intermediates: PEG 5k NH 2 , PEG 5k Lysi(NH 2 ) 2 , PEG 5k Lys 3 (NH 2 ) 4 , and PEG 5k Lys 7 (NH 2 ) 8 .
  • FIG. S1 B presents an MS specturm detected by MALDI-TOF MS of TDs: PEG 5k BAi, PEG 5k BA 2 , PEG 5k BA 4 and PEG 5k BA 8 .
  • FIG. S2 presenst a scheme and MS spectrum of AmB-PBA conjugation in AmB and PBA model reaction.
  • FIG. S4 presents the characterization of AmB and PBA-containing TDs conjugation.
  • FIG. S4A presents characterization of conjugation between AmB and TDs-PEG 5k BA 2 , PEG 5k BA 4 and PEG 5k BAs by TLC.
  • FIG. S4B presents fluorescent intensity of Fungizone, AmB-PEG 5k BAi , AmB-PEG 5k BA 2 , AmB-PEG 5k BA 4 , and AmB- PEG 5k BA 8 at neutral and acidic pH.
  • FIG. S5 presents the characterization of AmB conjugation on TDs by MALDI-TOF MS.
  • FIG. S5A presents the MS spectra of AmB-PEG 5k BAi at neutral and acidic pH.
  • FIG. S5B presents the MS spectra of AmB-PEG 5k BA 2 at neutral and acidic pH.
  • FIG. S5C presents the MS spectra of AmB-PEG 5k BA 4 at neutral and acidic pH.
  • FIG. S5D presents the MS spectra of AmB-PEG 5k BAs at neutral and acidic pH.
  • FIG. S6 presents particle size distributions and zeta potentials measured by DLS of blank TDs and AmB-TDs.
  • FIG. S6 presents particle size distributions and zeta potentials measured by DLS of blank TDs and AmB-TDs.
  • S6A presents particle size distributions of PEG 5k BAi, PEG 5k BA 2 , PEG 5k BA 4 and PEG 5k BA 8 .
  • FIG. S7 presents the particle size distribution by TEM and CMC of blank TDs and AmB-TDs.
  • FIG. S7A presents the particle size distributions of PEG 5k BA 4 , PEG 5k BA 8 , AmB-PEG 5k BA 4 , and AmB-PEG 5k BA 8 from TEM images.
  • FIG. S7B presents the CMC of PEG 5k BAi , PEG 5k BA 2 , PEG 5k BA 4 and PEG 5k BAs.
  • FIG. S7C presents the CMC of AmB-PEG 5k BAi, AmB-PEG 5k BA2, AmB-PEG 5k BA 4 , and AmB- PEG 5k BA 8 .
  • FIG. S8 presents in vitro cytotoxicity of blank TDs-PEG 5k BAi , PEG 5k BA 2 , PEG 5k BA 4 and PEG 5k BAs in 293T, RAW 264.7, and CHO cells by MTS assay.
  • FIG. S9 presents hemolytic toxicity in vitro of AmB formulations and blank TDs.
  • FIG. S9A presents hemolysis of Free AmB, Fungizone, AmBisome, AmB- PEG 5k BAi, AmB-PEG 5k BA 2 , AmB-PEG 5k BA 4 , and AmB-PEG 5k BA 8 for 30 min and 4h at 37 °C.
  • FIG. S9B presents hemolysis of blank TDs-PEG 5k BAi , PEG 5k BA 2 , PEG 5k BA 4 and PEG 5k BA 8 for 30 min, 4h, and overnight incubation at 37 °C.
  • FIG. S10 presents the fluorescent intensity of Doxil incubated with Fungizone, AmBisome, AmB-PEG 5k BAi, AmB-PEG 5k BA2, AmB-PEG 5k BA 4 , and AmB- PEG 5k BA 8 at 1 :1 mass ratio for 30 min and overnight.
  • FIG. S11 presents the TNF-a production level in RAW 264.7 cells induced by Fungizone, AmBisome, AmB-PEG 5k BAi , AmB-PEG 5k BA2, AmB-PEG 5k BA 4 , and AmB-PEG 5k BA 8 with concentration of AmB at 5p g/mL for 1 h incubation.
  • FIG. S12 present the MTD, blood chemistry of mice treated by AmB formulations.
  • FIG. S12A present the bodyweight changes of healthy mice administer intravenously with seven consecutive AmBisome, and AmB-PEG 5k BA 4 at 15 mg/kg dose daily.
  • FIG. S13 presents the histological examinations of the damaged livers by H&E staining from animals treated with Fungizone (1.5 mg/kgx3), AmB-PEG 5k BA4 (15 mg/kgx7), and AmBisome (15 mg/kgx/).
  • FIG S14 presents the histological examinations of the damaged hearts by H&E staining from animals treated with Fungizone (1.5 mg/kgx3), AmB-PEG 5k BA4 (15 mg/kgx7), and AmBisome (15 mg/kgx7).
  • FIG. S15 presents the antifungal efficacy of AmB-PEG 5k BA4 in C. albicans infected immunocompetent mice.
  • FIG. S15A presents a schematic illustration of C. albicans infected immunocompetent mouse model and treatment extension.
  • FIG. S15B presents the cumulative mice survival.
  • FIG. S15C presents the bodyweight change.
  • FIG. S15D presents the monitored body temperature changes of the infected mice.
  • FIG. S15E presents the colony count of C. albicans in major organ and blood of mice treated by AmB formulations.
  • FIG. S15F presents GMS staining for C. albicans in the heart of mice treated by AmB formulations.
  • FIG. S15G presents the histological examinations of the hearts by H&E staining of mice treated by AmB formulations.
  • FIG. S16 presents the characterization of immunocompromised mice induced by CY.
  • FIG. S16A presents the WBC counting change of mice treated by 200 mg/kg CY via i.p. injection.
  • FIG. S16B presents the bodyweight change of mice treated by CY.
  • FIG. S16C presents C the body temperature change of mice treated by CY.
  • FIG. S17 presents the antifungal efficacy of AmB-PEG 5k BA4 in C. albicans infected immunocompromised mice.
  • FIG. S17A presents the body temperature change of mice was monitored.
  • FIG. S17B presents the colony count of C. albicans in major organs and blood of mice treated by AmB formulations.
  • FIG. S C presents the GMS staining for C. albicans in the heart of mice treated by AmB formulations.
  • FIG. S17D presents the histological examinations of the hearts by H&E staining of mice treated by AmB formulations.
  • FIG. S18 presents the antifungal efficacy of AmB-PEG 5k BA4 in C. albicans infected immunocompromised mice for kept 1-2 months.
  • FIG. S18A presents the body weight change of mice was monitored.
  • FIG. S18B presents the GMS staining for C. albicans in the kidny of mice treated by AmB-PEG 5k BA4.
  • FIG. S18C presents the histological examinations of the hearts by H&E staining of mice treated by AmB- PEG 5k BA4.
  • FIG. S18D presents the histological examinations of the kidney by H&E staining of mice treated by AmB-PEG 5k BA4.
  • FIG. S18E presents the histological examinations of the livers by H&E staining of mice treated by AmB-PEG 5k BA4.
  • FIG. S19 presents an illustrative graphic for the prodrug design of AmMB for in vivo fungal infection control with preferred release in acidic extracellular and intracellular environments.
  • Embodiments disclosed herein are directed to biocompatible well- defined AmB-conjugated TD prodrug nanoformulations that have been rationally designed and prepared efficiently via reversible boronate ester chemistry for AmB systemic delivery.
  • the AmB-PEG 5k BA4 and AmB-PEG 5k BA8 prodrug nanoformulations have stable and small particle sizes with zeta potentials close to neutral, which are desired for improving macrophage targeting for systemic drug delivery.
  • the burst release was significantly minimized by increasing density of phenylboronic add (PBA) moieties of TDs.
  • PBA phenylboronic add
  • Embodiments herein utilize the sustained drug release profiles of AmB-PEG 5k BA4 and AmB-PEG 5k BA8 that can reduce systemic toxic effects of Fungizone®.
  • AmB-PEG 5k BAs efficient drug release and higher efficacy for invasive phase C. albicans inhibition were shown in AmB- PEG 5k BA4. Therefore, AmB-PEG 5k BA4 is promising to further development in systemic delivery of AmB in severe fungal infections.
  • Embodiment herein provide AmB-PEG 5k BA4 having exhibited prolonged blood circulation time, comparable MTD with AmBisome®, reduced nephrotoxicity, and significantly improved antifungal treatment efficacy and survival rate than Fungizone® and AmBisome® in immunocompromised mouse model.
  • Embodiments herein provide stable and efficient reversible AmB conjugation in the small-sized TD nanocarriers with optimized release profile is crucial for optimizing systemic drug delivery, minimizing nephrotoxicity, administration-related toxicity and improving treatment efficacy in vivo. Therefore, cost-effective AmB-PEG 5k BA4 prodrug formulation significantly enhanced systemic antifungal treatment efficacy than Fungizone® and AmBisome®, which holds a great potential for clinical translation.
  • Embodiments disclosed herein are directed to a novel pH sensitive prodrug strategy to reversibly conjugate 1 , 2 or 1 , 3 cis-diol containing drug molecules in a telodendrimer via boroate chemistry to improve the drug solubility, targeted drug delivery and controlled release for improved drug availability, reduced toxicity, and enhance drug efficacy.
  • Embodiments can be applied broadly to different kinds of drugs, e.g. antibiotics, antifungal drug, anticancer drugs etc.
  • Polymeric micelles and nanoparticles have been widely applied for hydrophobic drug delivery, including AmB, via non-covalent physical interactions.
  • chitosan, gelatin, poly glycolic acid (PGA), and poly lactide-co-glycolide acid (PLGA) have been studied for AmB encapsulation.
  • the AmB encapsulated PLGA formulation with reduced renal toxicity and high oral bioavailability has potential for AmB oral delivery.
  • Prodrug conjugation of AmB to protein or polymers via amide or imine Schiff base linkages significantly reduce the hemolysis and cytotoxicity, however, also significantly compromise the antifungal potency of AmB.
  • the 1 ,3-cis-diols has lower reactivity than the 1 ,2-cis-diol for boronic acid coupling, thus the increased valency of both cis-diols and boronic acids in the coupling pairs may increase the chance for efficient coupling.
  • AmB has three pairs of 1 ,3-cis-diols at the polar side of molecule for pore formation in fungal plasma membrane.
  • a multivalent and flexible boronic acid scaffold can maximize the multivalence for AmB conjugation via boronate ester.
  • the reversible boronate chemistry enables the drug release at slightly acidic environment, or upon the competition by the diol-containing glucose.
  • the well-defined linear-dendritic telodendrimer nanoplatform can be decorated precisely with functional moiety for efficient drug interactions in nanocarrier.
  • telodendrimer nanoplatform for boronic acid anti-cancer drug bortezomib delivery via reversible boronate chemistry for ovarian cancer treatment.
  • the optimal constructs of boronate moieties in TD enable effective AmB prodrug conjugation and encapsulation in a solvent-free and catalyst-free manner, which ease the formulation process for clinical translation.
  • the optimized AmB-TD nanoformulations improve the bioavailability, reduce its nephrotoxicity and administration related side effects, while sustaining the antifungal activity of AmB both in vitro and in vivo.
  • Amphotericin B is the gold standard treatment of life-threatening systemic fungal infections. The severe nephrotoxicity and the infusion side effects hinder its clinical application.
  • PBA phenylboronic acid
  • TD linear dendritic telodendrimer
  • PA phenylboronic acid
  • Optimized AmB-TDs prodrug self-assemble into monodispersed micelles with small particle size from 14-16 nm with neutral surface zeta potential.
  • the optimized AmB prodrugs sustain the drug release with approximately 30% drug released after 8 h dialysis in a sink condition, whereas burst release was observed in the conventional Fungizone and suboptimal AmB prodrug formulations.
  • AmB-TD prodrugs respond to acidic pH for accelerated drug release, but not sensitive to glucose competition. UV-vis spectrum indicates that AmB prodrugs reduce the aggregation status of AmB, indicating the reduced cytotoxicity and hemolytic activity, in comparison to both Fungizone and AmBisome. All AmB-TDs prodrugs exhibit comparable antifungal activity to Fungizone and more effective than Ambisome.
  • AmB-TD prodrug demonstrates a comparable maximum tolerate dose (MTD) with AmBisome and an over-20 fold MTD enhancement than Fungizone.
  • MTD maximum tolerate dose
  • a high single dose of AmB- PEG 5k BA4 were effective in both immunocompetent and immunocompromised mice for treatment of systemic fungal infections caused by Candida albicans. Therefore, AmB-PEG 5k BA4 prodrug nanoformulation seems to be very promising as an alternative for the treatment of systemic fungal infections.
  • Amphotericin B was purchased from Gold Biotechnology, Inc. (St Louis, MO). Fungizone (Amphotericin B solubilized) was purchased from Sigma-Aldrich (St. Louis, MO). Ambisome was received from Pharmacy of SUNY Upstate Medical University. Monomethyl-term inated polyethylene glycol) monoamine hydrochloride (MeO-PEG-NHs HCI, /W w 5 kDa) was purchased from Biopharma PEG Scientific Inc. (Watertown, MA). (Fmoc)Lys(Fmoc)- OH were purchased from Chem-lmpex International Inc. (Wood Dale, IL).
  • CellTiter 96® AQueous MTS reagent powder was purchased from Promega (Madison, Wl, USA).
  • TNF-a and IL-6 mouse uncoated ELISA kits were obtained from ThermoFisher Scientific. VetScan Critical care plus rotors were purchased from Zoetis (United States). Pointe Liquid ALT (SGPT), AST (SGPT), Creatinine, and Urea Nitrogen (BUN) Reagent Set were obtained from MedTextDX (Canton Ml).
  • TD named PEG 5k BA4 means that the molecular weight of PEG is 5 kDa with four phenylboronic acid (PBA) moieties conjugated on the periphery amino groups at both a and £ position of lysine in the TD.
  • PBA phenylboronic acid
  • TD Synthesis General preparation for TD synthesis has been reported in our previous publications ⁇ Id), and is described mainly as following: the TDs were synthesized by solution-phase peptide chemistry beginning from MeO-PEG 5k - NH2 HCI. /V-terminal-protected lysine was used to synthesize the branched scaffold of TD.
  • TAA triethylamine
  • DIC N,N’-Diisopropylcarbodiimide
  • HOBt N- hydroxybenzotriazole
  • the ice-chilled ether was added to the reaction solution to precipitate PEGylated intermediates, and then washed by chilled ether for three times. Then Fmoc-protection groups were removed by the treatment with 20% 4-methyl- piperidine in DMF for 30 min. PEGylated intermediate was precipitated and washed with chilled ether for three times. Then 4-carboxyphenylboronic acid was conjugated was conjugated to generate PEG 5k BA2 (FIG. 8C).
  • the branched scaffold was further branched by another step of Fmoc-Lys (Fmoc)-OH coupling and then 4-carboxyphenylboronic acid was coupled to generate PEG 5k BA4 (FIG.
  • NPs and AmB-NPs were measured by Malvern Nano-Zetasizer (Malvern Panalytical Ltd, UK) at room temperature in PBS and H2O, respectively.
  • the morphology and particle size were further measued by transmission electron microscopy (TEM) (JEOL JEM-1400 instrument at 80 kV) with negative staining by UranyLess.
  • TEM transmission electron microscopy
  • CMC Critical Micelle Concentration
  • Fluorescent emission intensity was measured by microplate reader (BioTek Synergy H1 ) at the wavelength of 620 nm with the excitation at 543 nm.
  • CMC of TDs were determined at the intersection of the tangents to the two linear fitting of the curve of fluorescence intensity as a function of the log concentrations of the TDs and AmB-TDs.
  • Murine macrophage cell line Raw 264.7, Chinese hamster ovary cells CHO, and human embryonic kidney cell line HEK293 were purchased from American Type Culture Collections (Manassas, VA, USA). Raw 264.7 and HEK293 cells were cultured in DMEM medium, while THP-1 was cultured in RPMI 1640 medium, supplemented with 10% FBS, 100 U/mL penicillin G and 100 pg/mL streptomycin at 37 °C using a humidified 5% CO2 incubator.
  • Candida albicans C. albicans
  • ATCC Manassas, VA, USA).
  • Antifungal Susceptibility Testing Antifungal susceptibility tests for AmB formulations were performed using the broth microdilution method according to study. The AmB formulations were prepared as stock solutions at 1 mg/mL concentration of AmB in water or 100% DMSO for free AmB. The final concentrations of AmB were 0.0125 to 0.2 pg/mL. Total volume of 200 pL of Candida albicans in culture medium with different AmB concentration of different AmB formulations were conducted in a 96-well plate at 28 °C. The fungi growth was monitored at CD of 530 nm using a microplate reader (BioTek Synergy H1 ).
  • Minimum inhibitory concentration (MIC) of AmB was defined as the lowest concentration resulting in at least 90% growth inhibition after overnight incubation.
  • T 0 further evaluate the antifungal activity of AmB formulations of Candida albicans at invasive phase. Total volume of 200 pL of Candida albicans in culture medium without AmB were conducted in a 96-well plate at 28 °C for 12 h. Then AmB of different formulations were added into 96-well plate with Candida albicans growth for 12 h with the final concentration of AmB at 2.5 pg/mL. The fungi growth was monitored at OD of 530 nm using a microplate reader (BioTek Synergy H1 ) after overnight incubation.
  • Hemolytic Assays Fresh blood from a healthy human volunteer was collected into PBS solution with 20 mM EDTA. Red bold cells (RBCs) were separated by centrifugation at 1000 rpm for 10 min. Then the RBCs were washed by PBS for three times and were re-suspended in PBS. AmB formulations or blank TDs were added into 200 pL RBC solutions with the concentration range of AmB at 10, 50 and 100 pg/mL, and 100, 500 and 1000 pg/mL of blank TDs followed by gentle mix and incubation at 37 °C for 0.5 h, 4 h and overnight, respectively. The samples were centrifuged at 1000 rpm for 5 min.
  • the hemoglobin in supernatant was measured by the UV absorbance at 540 nm using a NanoDrop spectrophotometer. PBS and Triton X-100 (2%) were also incubated with RBCs for a negative and positive control, respectively.
  • the blank PEG5kBA4 was injected intravenously at 500 mg/kg by three doses.
  • the mice were euthanized to collect blood plasma and critical organs e.g. the heart and kidney, and liver for blood chemistry and histology studies.
  • the blood chemistry was conducted by VetScan Critical care plus rotors via Vetscan VS2 (Zoetis) or by reagent set (MedTextDX).
  • mice survival or physical conditions e.g. body weight changes were monitored daily until the mice euthanized.
  • the mouse serum was collected and stored at -80 °C for cytokine analysis by mouse TNF-a and IL-6 ELISA assays.
  • the MTD was defined as the allowance of 15% loss of median body weight and causing neither death due to toxic effects nor significant changes in the general signs within 24 h after the last dose.
  • IVIS In vivo Imaging System
  • Animal Imaging The fluorescent nanoformulations for in vivo near infrared fluorescence (NIRF) optical imaging were prepared by loading of a hydrophobic near-infrared dye DiD into AmB TD formulations with a ratio of 0.2:1 :10 (w/w/w, DiD/AmB/TD). Then the fluorescent nanoformulations were filtered with a 0.22 pm sterile filter. Healthy 6-week-old C57BL/6J mice with or without Candida albicans inoculation via tail vein injection were administered with DiD- encapsulated AmB-TDs formulations. The mice were anaesthetized and imaged by IVIS 50 at different time points.
  • NIRF near infrared fluorescence
  • mice were euthanized, all the major organs were excised for ex vivo imaging.
  • the associated fluorescent intensities were analyzed by Living Image software (Caliper Life Sciences) using operator-defined regions of interest (ROI) measurements.
  • tissue and blood samples were made in sterile Sabouraud’s broth and plated on Sabouraud-dextrose agar. Gentamicin (40 pg/mL) and chloramphenicol (200 pg/mL) were added into Sabouraud-dextrose agar for fungi selective growth. Colonies were counted after incubation at room temperature for 48 h. The plasma was collected and stored at - 80 °C for cytokine analysis by mouse TNF-a and IL-6 ELISA assays.
  • Candida albicans Infected Immunocompromised Mouse Model Healthy 6-week-old C57BL/6J mice were treated with immunosuppressive agent cyclophosphamide (CY) at a dosage of 200 mg/kg by I.P. injection. Mice were monitored daily post-treatment to evaluate blood leucocyte count and by body weight. Three days after the CY treatment, the Candida albicans with cell concentration at 7X10 6 was injected intravenously through tail veins of the mice.
  • CY immunosuppressive agent cyclophosphamide
  • tissue and blood samples were made in sterile Sabouraud’s broth and plated on Sabouraaud-dextrose agar. Gentamicin (40 pg/mL) and chloramphenicol (200 pg/mL) were added into Sabouraud- dextrose agar for fungi selective growth 32 . Colonies were counted after incubation at room temperature for 48 h. The plasma was collected and stored at -80 °C for cytokine analysis by mouse TNF-a and IL-6 ELISA assays.
  • the TDs were synthesized using solution peptide chemistry with the structural scaffold shown in FIG. 1 B, TDs were synthesized from PEG 5k -NH2, and the stepwise synthetic routes are illustrated in detail in FIG. 8B-8E.
  • the dendritic oligolysine scafold was synthesized by the coupling of the lysine protected with Fmoc groups.
  • the efficient amide bond formation allows the precise and controlled construction of TD scaffold with precise number of primary amino groups for PBA conjugation.
  • the molecular weight of all the intermediates in TD synthesis have been confirmed by MALDI-TOF MS (FIG. S1 ), revealing the accurate mass increase accordingly throughout the TD synthesis step by step.
  • PBAs were conjugated on TD through amide bond formation.
  • the TDs bearing different number of PBA are denoted as PEG 5k BAi, PEG 5k BA2, PEG 5k BA4 and PEG 5k BAs (chemical structures are shown in FIG. 8A).
  • Table S1 MW of TDs detected by MALDI-TOF MS
  • AmB-TD prodrug conjugation The presence of boronic acid (BA) groups on the designed TDs provide efficient anchors for AmB conjugation via reversible boronate ester bond.
  • BA boronic acid
  • the detected molar ratios AmB/TD are around 1 :1.5 for PEG 5k BAi, PEG 5k BA2, and PEG 5k BA4 TDs, and 1 :1.04 was determined for AmB-PEG 5k BAa TD conjugate, indicating excessive PBA in TDs for AmB conjugation via the formation of multivalent boronate linkages.
  • the detected molar ratio of AmB to TD showed less molar of TDs are required to conjugate with AmB, indicating partial of the TDs in the polymeric micelles are without AmB conjugation, which may explain two populations of TD molecular weight in AmB-PEG 5k BAi MALDI-TOF MS spectra shown in FIG.
  • Table 1 MW characterization of blank TDs, AmB-TDs prodrug formulations.
  • AmB-TD prodrug nanocarrier Characterization The self-assembly behavior of TDs was characterized by dynamic light scattering (DLS) and critical micelle concentration (CMC) measurements. As shown in Table 2, the particle size of AmB-TDs was significantly increased after AmB conjugation as compared with blank TDs. As expected, AmB-PEG 5k BA4 and AmB-PEG 5k BAs self-assembled into monodispersed micelles with small particle size and homogenous distribution ranging from 14-16 nm and exhibit great stability with particle size of 13 nm of both formulations for storage over a month with at 4 °C, as evident by both DLS and TEM studies (FIG. 2A, FIG. 2B and FIG. S6D).
  • DLS dynamic light scattering
  • CMC critical micelle concentration
  • the relatively aggregation particles observed from blank PEG 5k BA4 and PEG 5k BAa of TEM images may be due to their high CMCs and strong hydrogen bond formation between high density of PBA moieties on TDs during the TEM grid dryness process (FIG. S7A).
  • FOG. S7A TEM grid dryness process
  • the well dispersed spherical structure was observed in both AmB-TDs, suggesting lower CMC and disruption of strong hydrogen bond among PBA moieties.
  • PBA with relatively high-water solubility were hardly able to form hydrophobic core of micelles, as shown in Table 2 and Fig. S7B, neither blank PEG 5k BAi nor PEG 5k BA2 were able to form micelles up to 5 mg/mL.
  • the comparable CMC after AmB conjugation may be caused by their similar AmB-TD conjugation molar ratio of 1 :1.47 and 1 : 1.04 for AmB-PEG 5k BA4 and AmB-PEG 5k BAs, respectively.
  • CMC of AmB-PEG 5k BAi and AmB-PEG 5k BA2 were not detectable due to less stable micellar formation, which is supported by their particle size measurement in DLS.
  • Blank PEG 5k BAi , PEG 5k BA2, and PEG 5k BA4 showed relatively negative zeta potential ranging from -8 ⁇ -17 mv, while zeta potential of PEG 5k BAs was close to neutral.
  • Blank PEG 5k BAi, PEG 5k BA2 and their AmB conjugated formulations were not able to form stable micelles, therefore, the negatively charged PBA moieties significantly affect their zeta potential measurement.
  • Negatively charged PBA moieties in blank PEG 5k BA4 with relatively high CMC may form micelle with loosed core and reduce its zeta potential.
  • zeta potentials of AmB- PEG 5k BA4 and AmB-PEG 5k BAs are close to neutral due to stable micelles formation.
  • Fungizone® show highly aggregated state of AmB in aqueous solution with significantly increased absorbance at 326 nm and reduced monomer signal at 420 nm.
  • AmB also dimerizes and aggregates in the lipid environments due to its physicochemical properties. Therefore, strong absorbance of oligomer at 324 nm and quite weak signal at 416 were observed in AmBisome UV-Vis spectra. Similar spectra pattern was shown in AmB-PEG 5k BAi prodrug, demonstrating PEG 5k BAi fail to reduce the aggregation state of AmB after conjugation due to low density of PBA moieties and less stable micellar formation, as evident by both DLS and CMC studies.
  • spectra of AmB-PEG 5k BA2, AmB conjugated PEG 5k BA4 and PEG 5k BA8 show red-shift of oligomer aggregation absorbance from 324-326 nm towards 354-368 nm region and merged partially with monodispersed absorbance at 372 nm as well as increased intensity of monodispersed absorbance at 422 nm, confirming reduced AmB oligo-aggregation and formation of monomer or partial dimeraggregation.
  • the ratio of oligomer and monomer absorbance intensity was used to evaluate the degree of AmB aggregation.
  • the ratios of Fungizone® and AmB-PEG 5k BAi prodrug were reach to 9.79 and 7.87, respectively, while the ratios of AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BAs remarkably decreased to 0.88, 0.69 and 0.54.
  • the dramatically reduced ratios indicate AmB- PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BAs nanoformulations significantly reduced the aggregation status of AmB, with the density of PBA increased, less aggregation status of AmB formed, while PEG 5k BAi showed no effect.
  • Main peak ratio (MPR, ODdimer/ODmono) can estimate the ratio between the population size of the loose aggregate and monoers 33 .
  • MPR ODdimer/ODmono
  • the mpr result indicates dimer aggregation was mainly observed in Fungizone, Ambisome and AmB-PEG 5k BAi.
  • the UV-Vis spectra of AmB formulations suggest the aggregation state of is highly dependent on the density of PBA moieties of TDs, which serve as anchor to conjugate AmB in its monomeric or dimeric state by boronate easter bond in the micelle nanoformulations.
  • the remarkable reduction in aggregation state of AmB in the AmB-TDs prodrug micelles is potentially beneficial for reducing the toxicity of AmB.
  • dimeric/loose aggregates show increase the toxicity than that of the tight/oligomer aggregates due to the faster release, these formulations were limited to non-covalent lipid-based AmB-encapsulated formulations.
  • the AmB were covalently bind to the nanocarriers, therefore, AmB can be released in monomeric forms other than aggregated forms to lowered toxicity for systemic application.
  • Table 2 Characterization of blank TDs, AmB-TDs prodrug formulations.
  • AmB- PEG 5k BA4 exhibited a relatively slow release of less than 30% in first 8 h and an effective AmB release of nearly 20% during 8-24 h, which may reduce the toxicity of AmB as AmBisome during the systemic administration as well as retain the potency and availability of the drug even with a low dose application.
  • the differences in release profiles from four AmB-TDs formulations is attributed to the density of PBA functionalities for AmB conjugation via boronate ester bond. Increased number of PBA moieties on TDs exhibits higher capability AmB conjugation and superior stability of the micelles, leading to reduced burst release and slower sustained release.
  • the higher branched structure in PEG 5k BA4 and PEG 5k BAs create additional barrier to prevent cleaved AmB diffusion.
  • the boronate ester bond is sensitive to both acidic pH and cis-diol containing sugar, e.g. glucose.
  • the AmB release from PEG 5k BA4 and PEG 5k BAs conjugates were found to be efficient responsive to acidic pH at 5.5. Over 60% and 42% AmB was released from PEG 5k BA4 and PEG 5k BAs in 24 h at pH 5.5, which was faster than AmB release of two formulations at neutral pH with 50% and 33%, respectively.
  • the moderate pH responsive AmB release may be because the primary amino moiety of AmB can be protonated at acidic condition and further neutralize the pH locally in the micelle.
  • AmB-PEG 5k BA2 significantly reduced cytotoxicity in RAW 264.7 cells and slightly increased IC50 in CHO cells.
  • AmB-PEG 5k BA4 and AmB-PEG 5k BAs exhibited comparable or even superior biocompatibility as compared with AmBisome.
  • Hemolysis is another general parameter for determining the biocompatibility of AmB formulations.
  • Free AmB with AmB concentration ranging from 10 -100 pg/mL triggered over 100% of hemolysis within 30 min, as considered hemolysis of 2% triton X-100 is 100%.
  • free AmB, Fungizone, AmB-PEG 5k BAi and AmB-PEG 5k BA2 delayed the onset of hemolysis to 4h and exhibited dose-dependent hemolysis property. After incubation for overnight, as shown in FIG. 3D, Fungizone with AmB concentration ranging from 10-100 pg/mL reached to over 100% of hemolysis.
  • AmB-PEG 5k BAi and AmB-PEG 5k BA2 resulted in nearly 90% of hemolysis at 50 and 100 pg/mL and over 50% hemolysis at 10 pg/mL of AmB after overnight incubation.
  • non-hemolysis was observed in AmBisome, AmB-PEG 5k BA4 and AmB-PEG 5k BAs treated RBCs at all the concentration of AmB during the different incubation time of 30 min, 4h and overnight (FIG. 3D and FIG. S9).
  • Non-hemolytic toxicity was found in all the blank TDs with their concentration up to 1000 pg/mL during the incubation.
  • AmB-PEG 5k BAi and AmB-PEG 5k BA2 with less effective micellar formation (none-detectable CMC), relatively fast drug release and high aggregated AmB in PEG 5k BAi slightly reduced the cytotoxicity and hemolysis.
  • AmB- PEG 5k BA4 and AmB-PEG 5k BAs exhibited non-hemolytic toxicity, comparable or even less cytotoxicity than AmBisome.
  • the favorable biocompatibility of AmB-PEG 5k BA4 and AmB-PEG 5k BA8 mainly attribute to their high stability micelles, significantly reduced aggregation degree and optimized drug release profiles.
  • Innate immune cell stimulation by AmB and TNF-a production A “sepsis-like syndrome” infusion-related toxicity of AmB distinguished by inflammatory cytokine release has been reported up to 70% of patients at 1-3 h starting to receive AmB.
  • AmB as a microbial product can stimulate immune cells through Toll-like receptors (TLR)2 and TLR4 with CD14.
  • TLR Toll-like receptors
  • AmB initiates production of inflammatory cytokines e.g. TNF-a, IL-6, and IL-i p, chemokines, e.g. IL-8 and MCP-1 , and nitric oxide (NO) in murine and human innate immune cells in vitro.
  • inflammatory cytokines e.g. TNF-a, IL-6, and IL-i p
  • chemokines e.g. IL-8 and MCP-1
  • NO nitric oxide
  • the stimulation of RAW cells by AmB-TDs formulations may attribute to the concentration applied to the Raw cells was much lower than the CMCs of AmB- TD conjugates prodrugs, inducing less stability of micelles and rapid release of AmB. Still with higher density of PBA, the AmB-PEG 5k BA4 and AmB-PEG 5k BAs significantly reduced TNF-a production from innate immune cells.
  • PK Pharmacokinetics
  • AmB-TDs biodistribution of AmB-TDs
  • the PK profiles of AmB-PEG 5k BA2, AmB-PEG 5k BA4 and AmB-PEG 5k BA8 were investigated in C57BL/6J mice after intravenous administration in comparison with Fungizone and AmBisome at the dose of AmB of 1 mg/kg, respectively. As shown in FIG.
  • Fungizone and AmB-PEG 5k BA2 administration at 1 mg/kg has a rapid clearance from blood circulation with area under the curve (AUG) of 15.1 and 9.3 pg/mL/h and half-life (ti/2a) of 0.004 h and 0.003 h, respectively, analyzed by UV-Vis of AmB in plasma at different time points.
  • AmBisome, AmB-PEG 5k BA4 and AmB- PEG 5k BA8 at 1 mg/kg exhibit significantly prolonged blood circulation time as compared with Fungizone.
  • AmB-PEG 5k BA4 exhibited comparable AUG of PK profile with AmBisome, which contributes to over 3-fold increase of AUG relative to Fungizone.
  • AmB-PEG 5k BAs performed the longest blood circulation time among various AmB formulations with ti/2a of 1.5 h and over 12-fold higher AUG than that of Fungizone.
  • the prolonged circulation time of AmB-PEG 5k BA4 and AmB- PEG 5k BAs at 1 mg/kg can be attributed to high density of PBA in TD for AmB conjugation to minimize initial burst drug release and the existing of AmB- unconjugated PBA moieties for alleviating the competition by glucose as demonstrated in FIG. 2E.
  • the concentration of AmB-TDs was still above their CMCs through rapid dilution by the blood at 10 mg/kg dose, forming stable micelles during the blood circulation, whereas AmB-TDs concentrations were close to their CMCs after I.V. injection at 1 mg/kg resulting in less stability of micelles.
  • the optimal particle sizes and prolonged blood circulation time allow the AmB-TDs conjugates accumulate and penetrate to the targeted tissues.
  • the signal of DiD in the lung was less compared with that in the liver but was still higher than the fluorescent signals from other critical organs, e.g. the heart, brain, kidney, colony, and intestine, indicating possibility of lung fungal infection treatment.
  • Both AmB-PEG 5k BA4 and AmB-PEG 5k BAs showed dramatically lower levels of fluorescent signals to 25% and 40% of those from the liver.
  • the fluorescent signal from AmB-PEG 5k BA4 in the kidney further reduced kidney accumulation than that from AmB-PEG 5k BAs, demonstrating minimized nephrotoxicity during the systemic administration.
  • MTD Maximum tolerance dose
  • AmB has been reported to produce significant cardiac toxicity with ventricular arrhythmias and nephrotoxicity with acute renal failure in the patients overdoses or even administered in conventional dosages and infusion rates. Therefore, we evaluated MTD of AmB-TDs conjugates by dose escalation in C57BL/6J mice using a single dose intravenous injection or a schedule of multiple doses with consecutive I.V. injection for three or seven days. According to the treatment dose of AmBisome applied in the clinic, we tested the MTD of various AmB formulations up to 10 mg/kg at single dose and triple-dose treatment studies.
  • mice in the triple-dose treatments we test the bold enzyme levels of mice in the triple-dose treatments.
  • FIG. 5B one out of three mice in three doses Fungizone of 1 mg/kg and 1.5 mg/kg groups showed 1.8 and 3.2-fold increased level of BUN as compared with that of control group, indicating kidney dysfunction.
  • 1.7-fold of creatinine level (FIG. 5C) higher than control group was observed the serum from the identical mice with high BUN in 1 mg/kg Fungizone group, which further confirmed the incidence of damage of kidney by nephrotoxicity.
  • the serum potassium level of 8.0 ⁇ 0.85 mmol/L was noted as the upper limit of a normal reference range.
  • the liver function evaluation typically includes alanine transaminase (ALT) and aspartate transaminase (AST). As shown in FIG.
  • mice treated by triple doses of AmB-PEG 5k BA4 at 1 mg/kg and PEG 5k BA4 at 500 mg/kg showed similar levels of ALT and AST with those in control group.
  • Slightly higher serum level of ALT (17-77 U/L) and AST (54-298 U/L) than their normal ranges were observed in one mouse of each Fungizone treated group, one mouse treated by AmB-PEG 5k BA4 at 10 mg/kg, and two mice injected by AmBisome at 10 mg/kg, which may indicate possible liver toxicity due to the higher accumulation of nanoformulations in the liver observed from biodistribution studies.
  • Necrosis in the liver was only found in the mice treated by triple doses of Fungizone at 1.5 mg/kg from pathology studies (FIG. S13). Seven consecutive doses of 15 mg/kg AmBisome and AmB-PEG 5k BA4 were further evaluated in C57BL/6J mice. As shown in FIG. S12A, the bodyweight changes of both groups were less than 10%. In addition, seven doses i.v. injections at 15 mg/kg of AmBisome and AmB-PEG 5k BA4 were well tolerated without noticeable changes in their blood chemistry evaluation including BUN, CRE, ALT and AST shown in FIG. S12B - FIG. S12E.
  • mice were sacrificed, and the heart, liver, kidney were harvested for histology analysis on 24 h after the last dose.
  • the pathology results of the kidney treated by various AmB- formulations demonstrated the remarkably superior biocompatibility and less nephrotoxicity of AmB-PEG 5k BA4 than Fungizone and comparable safety with AmBisome for i.v. injection in vivo.
  • FIG. S13 and FIG. S14 marginally necrosis in the liver and heart was found in the mice treated by triple doses of Fungizone at 1.5 mg/kg and seven doses of AmBisome and AmB-PEG 5k BA4 at 15 mg/kg.
  • the slightly increased ALT and AST might only indicate the transient alteration in the liver function since magnitude of change in both parameters has no relationship to the prognosis or severity of liver damage.
  • AmB-PEG 5k BA4 showed comparable MTD level (15 mg/kgx7) of AmBisome and reached to over 20-fold higher than the MTD (1 .5 mg/kgx3) of Fungizone.
  • mice treated by AmBisome and AmB-PEG 5k BA4 at 10 mg/kg showed non-colony in their major organs, while small number of colonies were observed in the kidney, lung, liver and spleen of one or two mice treated by Fungizone (FIG. 6E and FIG. S15E).
  • the dead mouse in untreated group exhibited extremely high level of TNF-a and IL-6 in serum, revealing the fungal infection leaded to cytokine storm and caused mice death.
  • the AmB formulation treatment and functional immune system efficiently eliminated the fungal pathogens, therefore, prevented the development of cytokine storm and saved the mice.
  • Table S7 provides a summary of Candida organisms presence and inflammation percentage with tissue injury in the hearts from the immunocompromised mice treated by AmB formulations:
  • CY induced immunosuppression in mice increased the mortality rate elicited by C. albicans.
  • the untreated control of immunocompromised mice exhibited 100% mortality within a 5-day period.
  • the treatment of Fungizone at 0.5 mg/kg *2 in one day, AmBisome and AmB-PEG 5k BA4 at 10 mg/kg of one dose was administered to immunocompromised mice 24 h after C. albicans infection.
  • the AmB formulation treatment postponed the incidence of death from 29 h to over 40 h.
  • the survival percentage was significantly increase to 20%, 37.5% and 62.5% by the treatment of Fungizone, AmBisome, and AmB-PEG 5k BA4, respectively, as compared with untreated group.
  • Bodyweight of mice shown in FIG. 7C started recovery on Day 4 in AmBisome and AmB-PEG 5k BA4 treatment groups, while bodyweight continuously lost over 20% was observed in untreated and Fungizone treated group, which may be due to toxicity of Fungizone despite the low dose. All the survived mice were euthanized on Day 6 or 8 to collect the major organ as well as organs of dead mice for evaluating antifungal treatment efficacy via colony counting. As shown in FIG.
  • FIG. 7F Macroscopic observation of the kidneys from dead mice infected with C. albicans in untreated and Fungizone treated groups shown in FIG. 7F revealed abundant microabscessess with over 10% and 5% presence of Candida organisms, respectively, which upon microscopic observation demonstrated hyphae and yeasts.
  • the kidneys from dead mice treated by both AmBisome and AmB-PEG 5k BA4 showed 2% Candida organisms presence with most budding-yeasts. No Candida organisms were found in the kidney from survived mice treated by AmBisome and AmB- PEG 5k BA4, while 1 % budding-yeasting Candida organisms were detected in survived mouse receiving Fungizone. Similar results were also shown in the hearts from dead and survived mice in each group (FIG.
  • FIG. 7E This GMS staining results are well corelated with the results of colony culture and counting from major organs shown in FIG. 7E.
  • the pathology analysis of kidney and heart were also conducted in survived and dead mice of each group. As we expected, 50% necrotic proximal tubules and 10-15% inflammation were observed in the kidneys (FIG. 7F) from dead mice treated by AmBisome and AmB-PEG 5k BA4, indicating the cytokine storm with high level of proinflammatory cytokines in serum induced severe damage of kidney and heart and finally caused mice death.
  • mice dead in untreated and Fungizone group may be due to severe infection status of C. albicans other than the tissue damage by the proinflammatory cytokines.
  • the survived mice receiving Fungizone exhibited the most severe tissue damage level with 20% proximal tubules necrosis in the kidney and 15% inflammation in the heart among the survived mice treated by AmBisome and AmB- PEG 5k BA4.
  • Table S8 provides a summary of tissue damage and Candida organism presence of the survived immunocompromised mice treated by AmB-PEG 5k BA4:
  • biocompatible well-defined AmB-conjugated TD prodrug nanoformulations have been rationally designed and prepared efficiently via reversible boronate ester chemistry for AmB systemic delivery.
  • the AmB-PEG 5k BA4 and AmB-PEG 5k BAs prodrug nanoformulations have stable and small particle sizes with zeta potentials close to neutral, which are desired for improving macrophage targeting for systemic drug delivery.
  • the burst release was significantly minimized by increasing density of PBA moieties of TDs.
  • the sustained drug release profiles of AmB-PEG 5k BA4 and AmB-PEG 5k BAs can reduce systemic toxic effects of Fungizone®.
  • AmB-PEG 5k BA4 In comparison with AmB-PEG 5k BA8, efficient drug release and higher efficacy for invasive phase C. albicans inhibition were shown in AmB-PEG 5k BA4. Therefore AmB- PEG 5k BA4 is promising to further development in systemic delivery of AmB in severe, fungal infections. As a result, AmB-PEG 5k BA4 exhibited prolonged blood circulation time, comparable MTD with AmBisome®, reduced nephrotoxicity, and significantly improved antifungal treatment efficacy and survival rate than Fungizone® and AmBisome® in immunocompromised mouse model.
  • Stable and efficient reversible AmB conjugation in the small-sized TD nanocarriers with optimized release profile is crucial for optimizing systemic drug delivery, minimizing nephrotoxicity, administration- related toxicity and improving treatment efficacy in vivo. Therefore, cost-effective AmB-PEG 5k BA4 prodrug formulation significantly enhanced systemic antifungal treatment efficacy than Fungizone® and AmBisome®, which hods a great potential for clinical translation.
  • Multifunctional Telodendrimer Nanocarriers Restore Synergy of Bortezomib and Doxorubicin in Ovarian Cancer TreatmentSynergized BTZ/DOX Combination Codelivered by Nanocarriers. Cancer research 2017, 77 (12), 3293-3305; Guo, D.; Ji, X.; Luo, J. Rational nanocarrier design towards clinical translation of cancer nanotherapy. Biomedical Materials 2021 , 16 (3), 032005. DOI: 10.1088/1748-605x/abe35a; Guo, D.; Shi, C.; Wang, X.; Wang, L.; Zhang, S.; Luo, J.
  • Riboflavin-containing telodendrimer nanocarriers for efficient doxorubicin delivery High loading capacity, increased stability, and improved anticancer efficacy.
  • a drug-specific nanocarrier design for efficient anticancer therapy is provided.
  • Amphotericin B release rate is the link between drug status in the liposomal bilayer and toxicity.
  • Intravenous administration of amphotericin B entrapped in liposomes Induction of high serum levels of TNFa. Annals of Oncology 1991 , 2 (2), 141-144; Traslavina, R. P.; King, E. J.; Loar, A. S.; Riedel, E. R.; Garvey, M. S.; Ricart-Arbona, R.; Wolf, F. R.; Couto, S. S. Euthanasia by CO2 inhalation affects potassium levels in mice. Journal of the American Association for Laboratory Animal Science 2010, 49 (3), 316-322; Udensi, U. K.; Tchounwou, P. B. Potassium homeostasis, oxidative stress, and human disease.

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Abstract

L'amphotéricine B (AmB) est la référence standard pour le traitement d'infections fongiques systémiques potentiellement mortelles. Pour développer des formulations d'AmB économiques à toxicité réduite, des modes de réalisation divulgués introduisent un nombre contrôlé de fractions d'acide phénylboronique (PBA) dans une nanoplateforme de télodendrimère (TD) pour optimiser la conjugaison AmB via une stratégie d'ester de boronate réversible. Cette nanoplateforme de TD de boronate flexible multivalent peut également être appliquée dans le développement de promédicaments pour différentes molécules de médicament contenant du cis-diol ou molécules de médicament contenant de l'hydroxyle cétone α- ou β-.
PCT/US2024/033004 2023-06-07 2024-06-07 Nanoformulations de promédicament de boronate dans des traitements de maladies Ceased WO2024254448A2 (fr)

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