WO2023242766A1 - Nanoconjugués d'or - Google Patents

Nanoconjugués d'or Download PDF

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Publication number
WO2023242766A1
WO2023242766A1 PCT/IB2023/056148 IB2023056148W WO2023242766A1 WO 2023242766 A1 WO2023242766 A1 WO 2023242766A1 IB 2023056148 W IB2023056148 W IB 2023056148W WO 2023242766 A1 WO2023242766 A1 WO 2023242766A1
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composition
nanoconjugate
aunp
compound
gemcitabine
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PCT/IB2023/056148
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English (en)
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Raghuraman Kannan
Sai Giridhar Sarma KANDANUR
Abilash Gangula
Agasthya Suresh BABU
Tilak CHHETRI
Dhananjay SURESH
Anandhi UPENDRAN
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Alembic Pharmaceuticals Limited
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Publication of WO2023242766A1 publication Critical patent/WO2023242766A1/fr

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    • 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
    • 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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/6923—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 an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Gemcitabine is a well-known broad-spectrum anticancer drug used alone or in combination therapy for treatment of several cancer types including pancreatic, bladder, non-small cell lung cancer, ovarian, breast, head, neck, thyroid, and bone cancers.
  • the therapeutic efficacy of Gem is compromised primarily by two of its inherent structural limitations that result in poor delivery of administered drug to the tumor cells.
  • gem is rapidly deaminated into its therapeutically inactive form of 2',2'-difluorodeoxyuridine (dfdu) by the enzyme cytidine deaminase (CD A) which is abundant in blood and liver.
  • Gem is administered at high doses (1000 mg/m 2 for 30 min intravenous infusion) that would cause toxic side effects and development of resistance.
  • the hydrophilicity of Gem hinders its passive diffusion across the plasma membrane thus necessitating high tumor expression of nucleoside transporters to cause efficient uptake and therapeutic effect.
  • the low tumor expression of nucleoside transporters leads to therapeutic inefficacy and resistance to Gem.
  • poor plasma stability and membrane permeability of Gem are the major roadblocks in realizing its therapeutic potential.
  • the present disclosure provides, among other things, gemcitabine-containing compounds having improved stability and tumor uptake.
  • such compounds are of Formula I:
  • the present disclosure also provides nanoconjugates (e.g., comprising gold nanoparticles) comprising provided compounds, and optionally comprising a thioctic acid terminated peptide.
  • the present disclosure provides a gold nanoparticle (AuNP) comprising a compound of the following structure:
  • each « is independently a point of attachment of the compound to hydrogen or the AuNP gold surface.
  • AuNP gold nanoparticle
  • the present disclosure further provides pharmaceutical compositions, methods of treating cancer, methods for enhancing the cytotoxicity of gemcitabine, and processes for the preparation of provided compounds, compositions, and nanoconjugates.
  • Figure 3 depicts TEM images of Au-[DTGT],
  • Figure 5 depicts TEM images of P 4 cMET-Au-[DTGT],
  • Figure 6 depicts UV-Visible spectra of Au-[DTGT], P 4 BN-Au[DTGT], and
  • Figure 7 depicts HPLC spectra: a) HPLC spectrum of DTGT in 1.5 M NaCN, where the peak at 3.8 min corresponds to Gem that is released on digestion of DTGT with NaCN. b) HPLC-based standard curve of DTGT in 1.5 M NaCN.
  • Figure 8 depicts standard curves: a) HPLC-based standard curve of P4CMET in 1.5 M NaCN, b) HPLC-based standard curve of P4BN in 1.5 M NaCN.
  • Figure 9 depicts standard curves: a) Gemcitabine and b) Theoretical Gemcitabine equivalence in DTGT.
  • Standards of drug in DPBS were processed similar to that of constructs in CDA stability study.
  • Figure 10 depicts a stability profile of Gemcitabine and modified Gemcitabine formulations in the presence of cytidine deaminase (CDA).
  • CDA cytidine deaminase
  • Figure 11 depicts MTT assay plots for growth inhibitory analysis of Gemcitabine and its modified analogs in lung cancer (A549 (a), H23 (b)) and pancreatic cancer (PANC-1 (c), BxPC3 (d)) cell lines after 72 hours of treatment.
  • Figure 12 depicts SRB assay plots for growth inhibitory analysis of Gemcitabine and its modified analogs in pancreatic cancer cell lines after 72 hours of treatment: MCF-7 (a), MBA-MB-468 (b), MDA-MB-231.
  • a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • precancerous e.g., benign
  • malignant pre-metastatic
  • metastatic metastatic
  • non-metastatic e.g., metastatic
  • present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant.
  • a relevant cancer may be characterized by a solid tumor.
  • a relevant cancer may be characterized by a hematologic tumor.
  • Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
  • an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc).
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance.
  • Such natural amino acids include the nonpolar, or hydrophobic amino acids, glycine, alanine, valine, leucine isoleucine, methionine, phenylalanine, tryptophan, and proline. Cysteine is sometimes classified as nonpolar or hydrophobic and other times as polar. Natural amino acids also include polar, or hydrophilic amino acids, such as tyrosine, serine, threonine, aspartic acid (also known as aspartate, when charged), glutamic acid (also known as glutamate, when charged), asparagine, and glutamine. Certain polar, or hydrophilic, amino acids have charged side-chains. Such charged amino acids include lysine, arginine, and histidine.
  • protection of a polar or hydrophilic amino acid side-chain can render that amino acid nonpolar.
  • a suitably protected tyrosine hydroxyl group can render that tyroine nonpolar and hydrophobic by virtue of protecting the hydroxyl group.
  • a patient refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals including but not limited to humans).
  • a patient is a human.
  • a patient is suffering from or susceptible to one or more disorders or conditions.
  • a patient displays one or more symptoms of a disorder or condition.
  • a patient has been diagnosed with one or more disorders or conditions.
  • the disorder or condition is or includes cancer, or presence of one or more tumors.
  • the patient is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
  • protecting group refers to temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, thiols, and acetals and ketals of aldehydes and ketones, respectively.
  • Protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3 rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.
  • Reference As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
  • Stable when applied to compositions herein, means that the compositions maintain one or more aspects of their physical structure and/or activity over a period of time under a designated set of conditions.
  • the period of time is at least about one hour; in some embodiments the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or longer.
  • the period of time is within the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc.
  • the designated conditions are ambient conditions (e.g., at room temperature and ambient pressure).
  • the designated conditions are physiologic conditions (e.g., in vivo or at about 37 °C for example in serum or in phosphate buffered saline).
  • the designated conditions are under cold storage (e.g., at or below about 4 °C, -20 °C, or -70 °C).
  • the designated conditions are in the dark.
  • therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen.
  • a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc.
  • the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition.
  • a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.
  • treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.
  • Unnatural amino acid refers to amino acids not included in the list of 20 amino acids naturally occurring in proteins, as described above. Such amino acids include the D-isomer of any of the 20 naturally occurring amino acids. Unnatural amino acids also include homoserine, ornithine, norleucine, and thyroxine. Other unnatural amino acids side-chains are well known to one of ordinary skill in the art and include unnatural aliphatic side chains. Other unnatural amino acids include modified amino acids, including those that are N-alkylated, cyclized, phosphorylated, acetylated, amidated, azidylated, labelled, and the like.
  • an unnatural amino acid is a D-isomer. In some embodiments, an unnatural amino acid is a L-isomer.
  • the present disclosure encompasses the recognition that a combined strategy that incorporates both chemical modification and nanocarrier delivery of Gem would result in superior overall therapeutic performance in clinical practice. Indeed, the present disclosure validates a synergistic effect by demonstrating that certain chemical modifications of Gem result in improved plasma stability which is further enhanced by conjugating the Gemcitabine modification to a gold nanocarrier.
  • Gem is a deoxy cytidine analog and therefore subject to deamination by cytidine deaminase, a process that transforms Gem and other deoxy cytidines (e.g., cytarabine and decitabine) into inactive metabolites.
  • the present disclosure provides insight for improving the enzymatic stability of Gem by synthesizing a novel prodrug of Gem (DTGT) and conjugating it with biocompatible gold nanoparticles to form an Au-DTGT conjugate.
  • DTGT novel prodrug of Gem
  • the present disclosure modifies Gem at the 4-(N) position with a threonine moiety to generate a metabolically stable threonine derivative of gemcitabine (GT).
  • nanoparticles are pegylated to further prevent enzymatic degradation, reduce phagocytic clearance, and prolong the circulation time.
  • the present disclosure provides superior tumor uptake of Gem by conjugating it to nanoparticles that promote EPR-based tumor accumulation and endocytic internalization, thereby circumventing the need of transporters for membrane permeability.
  • the smaller size (e.g., ⁇ 30 nm) of such nanoparticles facilitates efficient penetration of dense tumor extracellular matrix to deliver the drug to the tumor core.
  • provided nanoparticles can be functionalized with receptor-specific peptides for actively targeting the tumor cells.
  • the present disclosure provides compounds of Formula A:
  • L is a multivalent linker moiety having one or more thiol functional groups; each AA is independently a naturally or unnaturally occurring L or D amino acid; each Drug is independently a therapeutic entity capable of being deaminated by cytidine deaminase (CD A); each n is independently 0 or 1 ; x is 1, 2, or 3; and each y is independently 0 or 1 ; wherein n and y cannot both be 0; and wherein all linkages between Drug-AA, Drug-L, and AA- L when present comprise amide bonds.
  • CD A cytidine deaminase
  • each Drug independently is or comprises a cytidine or deoxy cytidine that is capable of being deaminated by CD A.
  • each Drug is preferably Gem.
  • L is a multivalent moiety (e.g., linker) having one or more thiol functional groups; each AA is independently a naturally or unnaturally occurring L or D amino acid; GEM is gemcitabine; each n is independently 0 or 1 ; x is 1, 2, or 3; and each y is independently 0 or 1 ; wherein n and y cannot both be 0; and wherein all linkages between GEM-AA, GEM-L, and AA-L when present comprise amide bonds.
  • linker e.g., linker
  • L is a multivalent linker moiety having one or more thiol functional groups; each AA is independently a naturally or unnaturally occurring L or D amino acid;
  • GEM is gemcitabine; each n is independently 0 or 1 ; x is 1, 2, or 3; and each y is independently 0 or 1 ; wherein n and y cannot both be 0; and wherein all linkages between GEM-AA, GEM-L, and AA-L when present comprise amide bonds.
  • L is a multivalent linker moiety having one or more thiol functional groups (e.g., a linker capable of forming covalent Au-S bonds with a gold NP).
  • L is or comprises a heterofunctional crosslinker containing one or more thiol functional groups and one or more amine reactive groups (e.g., a reactive group capable of forming an amide bond with GEM or AA).
  • L contains one or more amine reactive groups selected from the group consisting of isothiocyanates, isocyanates, sulfonyl chlorides, aldehydes, carbodiimides, acyl azides, anhydrides, fluorobenzenes, carbonates, NHS esters, imidoesters, epoxides, fluorophenyl esters, and combinations thereof.
  • L is or comprises a polyaminocarboxylate (e.g., aminopolycarboxylic acid).
  • L is a thiol-functionalized derivative of NTA, EDTA, DTP A, EGTA, BAPTA, NOTA, DOTA, mcotianamine, EDDHA, or EDDS.
  • L is a thiol-functionalized derivative of DTPA.
  • L comprises one thiol functional group.
  • L comprises two thiol functional groups.
  • L is preferably dithiolated diethylenetriamine pentaacetic acid (DTD TP A):
  • each AA is independently a naturally or unnaturally occurring amino acid. In some embodiments, each AA is independently a naturally occurring amino acid. In some embodiments, each AA is independently an unnaturally occurring amino acid. In some embodiments, AA is preferably threonine (Thr). In some embodiments, AA is preferably L-Thr.
  • gemcitabine is attached to AA or L via a functional group (e.g., amine) capable of covalently linking gemcitabine directly or indirectly to AA or L, and wherein the linkage comprises an amide bond.
  • a functional group e.g., amine
  • gemcitabine is covalently linked to AA or L via the primary amine group (4-(N)) of gemcitabine.
  • the primary amine group of Gem is connected to AA via an amide bond.
  • the primary amine group of Gem is connected to L via an amide bond.
  • x is 3. In some embodiments, x is 2. In some embodiments, x is 1.
  • n is i. In some embodiments, n is 0.
  • x is 3 and each n is i. In some embodiments for a given occurrence of [(GEM) n (AA) y ], n is 1. In some embodiments for a given occurrence of [(GEM)n(AA) y ], n is 0. In some embodiments where x is 3, n is 1 for two occurrences of [(GEM)n(AA) y ], and n is 0 for the other occurrence of [(GEM) n (AA) y ].
  • n is 0 for two occurrences of [(GEM) n (AA) y ], and n is 1 for the other occurrence of [(GEM)n(AA) y ], In some embodiments where x is 3, n is 0 for each occurrence of [(GEM)n(AA)y],
  • n is 0 for one occurrence of [(GEM) n (AA) y ], and n is 1 for the other occurrence of [(GEM) n (AA) y ] .
  • n is 0 for each occurrence of [(GEM) n (AA) y ]
  • x is 1 and n is 1. In some embodiments, x is 1 and n is 0.
  • each y is 0. In some embodiments, each y is 1.
  • y is 1. In some embodiments for a given occurrence of [(GEM) n (AA) y ], y is 0.
  • a compound of Formula I or II has the structure:
  • NP nanoparticles
  • delivery vehicles for Gem include polymeric NP, lipid NP, silica NP, magnetic NP, liposomes, and micellar NP. All these formulations involve physical entrapment of Gem within the NP and suffer from two major drawbacks: i) The encapsulation strategies for loading of drug in a nanoparticle are usually inefficient resulting in very low levels of drug loading ( ⁇ 10%).
  • a bifunctional crosslinker enables high loading of Gem by covalent methods.
  • a single molecule of DT has three carboxylic acid moieties that serve as chemical handles for covalent conjugation of three Gem analogs.
  • DT has two sulfhydryl groups that can be readily conjugated to a gold NP.
  • a single nanoparticle of ⁇ 10 nm size can accommodate a monolayer containing around 150 molecules of DT, each of which has up to 3 molecules of Gem.
  • Such nanoconjugates can achieve relatively high drug loading of Gem (20-30%) in Au-DTGT bv covalent methods.
  • the metabolic stability of Gem is significantly improved by provided nanoconjugates by i) protection from CDA degradation by chemical modification at 4-(N) position which is susceptible to CDA, and ii) conjugating with pegylated AuNP.
  • Another aspect of the present disclosure is the recognition that the use of gold NPs as delivery vehicles offer several synthetic advantages over other NPs being used as delivery vehicles of Gem.
  • Polymeric NPs are susceptible to aggregation and cause toxicity.
  • the practical use of lipid and liposomal NP is limited by low drug loading capacities and poor biodistribution due to high NP uptake by liver and spleen.
  • the inorganic NP such as iron and silica suffer from drawbacks such as low solubility and concerns of toxicity.
  • the synthetic techniques of polymeric, lipid, silica, magnetic and micellar NP involve nanoprecipitation, desolvation, homogenization, ionic gelation, emulsification, sol-gel process, pyrolysis, selfassembly, and co-precipitation. These techniques suffer from limitations such as complexity, lack of reproducibility, use of high temperatures and pressure.
  • the present disclosure provides, among other things, processes for the synthesis of gold nanoparticles which are extremely facile, rapid (15 min), and reproducible. Unlike other inorganic NP vehicles such as silica and iron, the provided gold-based NP are highly water soluble (e.g., up to concentrations of 50 mg/mL).
  • gold NP are versatile and adaptable in comparison to other NPs as they can be easily tuned to several sizes, shapes, and surface functionalities.
  • the unique optical properties of gold NP enable them to act as contrast/imaging agent and catalyst for photothermal and photodynamic therapy while simultaneously serving as delivery vehicles. This attribute sets gold NP apart from other NP that just serve as vehicles as it opens opportunities for a clinician to track the drug in- vivo as well as execute multi-modal treatment options, all using a single NP platform.
  • the present disclosure also recognizes, for the first time, that unlike any other NP systems, gold NP possess a unique ability to sensitize tumors to Gem treatment.
  • the present disclosure provides nanoconjugates comprising a provided compound covalently linked to a gold nanoparticle (AuNP) via at least one Au-S bond.
  • a provided compound is compound covalently linked to an AuNP via one Au-S bond.
  • a provided compound is compound covalently linked to an AuNP via two Au-S bonds.
  • an AuNP is PEGylated.
  • an AuNP is PEGylated prior to conjugation with a provided compound.
  • a provided nanoconjugate comprises a single layer of compound surrounding the AuNP.
  • Targeting peptides can provide for or accentuate accumulation of NP at tumor sites.
  • a provided nanoconjugate further comprises a targeting peptide.
  • a variety of chemistries are known to the skilled artisan for linking a peptide to an AuNP, by way of nonlimiting example the use of sulfur moieties (e.g., thiols, thioctic acid, disulfides) on the peptide that can form a covalent bond with the gold surface.
  • a peptide is conjugated to a nanoconjugate via a thioctic acid terminal group on the peptide.
  • a nanoconjugate comprises a thioctic acid terminated peptide covalently linked to AuNP via at least one Au-S bond.
  • a thioctic acid terminated peptide is thioctic acid terminated bombesin, thioctic acid terminated cMET or thioctic acid terminated GE11.
  • nanoconjugates are provided as a plurality of individual nanoconjugates within a composition.
  • Nanoconjugate compositions may be characterized by various parameters, (e.g., average size, drug loading, peptide loading, conjugation efficiency, etc.).
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size of less than about 40 nm.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size of less than about 35 nm, about 30 nm, about 25 nm, about 20 nm, about 15 nm, or about 10 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 5 nm to about 25 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 5 nm to about 35 nm.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 8 nm to about 22 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 5 nm to about 15 nm. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging from about 15 nm to about 25 nm.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a hydrodynamic size ranging of about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a zeta potential of about -15 mV, about -16 mV, about -17 mV, about -18 mV, about -19 mV, about -20 mV, about -21 mV, about -22 mV, about -23 mV, about -24 mV, about -25 mV, about -26 mV, about -27 mV, about -28 mV, about -29 mV, or about -30 mV.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a Drug (e.g., Gem) loading of about 5% to about 60%. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a Drug (e.g., Gem) loading of about 10% to about 50%, about 15% to about 50%, about 25% to about 35%, or about 15% to about 30%.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of at least about 40 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of at least about 45 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of at least about 50 mg/mL.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of about 40 mg/mL to about 75 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of about 40 mg/mL to about 60 mg/mL. In some embodiments, a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have an aqueous solubility of about 40 mg/mL to about 55 mg/mL.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a targeting peptide (e.g., a thioctic acid terminated peptide) loading of about 5% to about 60%.
  • a provided nanoconjugate composition is characterized in that, on average in the composition the nanoconjugates have a targeting peptide (e.g., a thioctic acid terminated peptide) loading of about 5% to about 10%, about 5% to about 15%, about 15% to about 60%, about 25% to about 60%, about 35% to about 60%, or about 45% to about 60%.
  • the present disclosure provides compositions for use in therapy.
  • the present disclosure provides a method of treating a cancer in a patient in need of such of treatment, comprising administering to the patient a provided pharmaceutical composition.
  • a patient exhibits one or more reduced side effects compared to a patient treated with an equivalent amount of Drug (e.g., gemcitabine) alone.
  • the present disclosure provides an improved method of treating cancer in a patient in need of such of treatment, the improvement comprising administering to the patient a provided pharmaceutical composition.
  • a cancer treated in accordance with the present disclosure is breast, ovarian, non-small cell lung, bladder, testicular, or pancreatic cancer.
  • a provided compound or nanoconjugate composition provides methods for killing or inhibiting the growth of a cancer cell, comprising contacting the cell with a provided compound or nanoconjugate composition.
  • the present disclosure further provides methods for enhancing the cytotoxicity or cytostaticity of gemcitabine in a cancer cell, comprising contacting the cancer cell with a provided compound or nanoconjugate composition.
  • a provided compound or nanoconjugate exhibits an IC50 toward the cancer cell at least 10-fold lower compared to gemcitabine alone.
  • the present disclosure further provides methods for delivering or introducing a Drug (e.g., Gem) into a cancer cell, comprising contacting the cell with a provided compound or nanoconjugate composition.
  • a Drug e.g., Gem
  • the present disclosure provides an improved method of delivering gemcitabine into a cancer cell, the improvement comprising contacting the cell with a provided compound or nanoconjugate composition.
  • gemcitabine is delivered to the cell independent of nucleoside transporters.
  • compositions described herein may be made as described in the Exemplification below, as well as by other methods known by one skilled in the art.
  • the present disclosure provides a process for preparing a compound of Formula I or II, comprising steps of i) covalently linking Gem to AA via an amide bond, and ii) covalently linking DTDTPA to AA via an amide bonds.
  • the exact composition of each AA can be varied using various amino acids and peptide coupling chemistries known in the art (e.g., Hong, S. et al. Molecules 2018, 23, 2608).
  • the reaction mixture was then stirred in an oil bath at 55 °C for 17 hours, cooled to room temperature, and quenched by adding brine (15 mL).
  • the mixture was then extracted using ethyl acetate (2 50 mL) and the combined organic layer was washed with 100 mL of 20% LiCl solution, 100 mL of saturated NaHCCh aqueous solution, 100 mL of brine solution, dried over MgSO4, and concentrated under reduced pressure to afford the crude intermediate GT-N-Boc.
  • the crude product was purified by silica gel column chromatography (1-2% MeOH/DCM as a solvent system) to afford the desired product GT-Ol(GT-N-Boc) as an off white solid (960 mg, 62%).
  • the GT-N-Boc obtained above is further deprotected using the following procedure to obtain GT.
  • 960 mg (4.01 mmol) of GT-N-Boc and 40 mL of anhydrous DCM were charged under N2 atmosphere at RT.
  • 40 mL of 4N HC1 in dioxane was charged and the reaction mixture was stirred overnight ( ⁇ 14 hours) under N2 atmosphere at RT. After 14 hours, the solvent was evaporated under reduced pressure, and the residue was triturated with hexane to obtain the desired product GT as a white solid (427 mg, 57%).
  • Protocol for the synthesis of S-trityl-DT _T o a 250 mL two neck round bottom flask fitted with a magnetic stir bar, 3 g of DTDTPA (DT) and 45 mL of dry DMF were charged under N2 atmosphere at RT. To this, 3.26 g of trityl chloride was charged under N2 atmosphere at RT. The reaction mixture was stirred for two days under N2 atmosphere at RT. After 2 days, the reaction was quenched by the addition of 240 mL of 10% NaOAc solution to produce a white precipitate. The contents were continued to stir for 30 min and the precipitate was filtered using sintered funnel.
  • Protocol for the conjugation of GT to S-trityl-DT To a 250 mL two neck round bottom flask fitted with a magnetic stir bar, 1 g of S-trityl-DT and 60 mL of dry DMF were charged under N2 atmosphere at RT. To this, 425 mg of DIPEA was charged under
  • Protocol for the deprotection of sulfur The S-trityl-DTGT obtained above is further deprotected using the following procedure to obtain DTGT.
  • DTGT To a 50 mL two neck round bottom flask fitted with a magnetic stir bar, 60 mg of S-trityl-DTGT, 3 mL of 10% TFA in DCM, and 3 mL of 10% TES in DCM were consecutively charged and the reaction mixture was stirred for 2.5 hours under N2 atmosphere at RT. After 2.5 hours, the reaction was quenched by the addition of 0.3 mL of 10% pyridine in MeOH and the reaction mixture was flushed with N2 to minimize the amount of DCM.
  • aqueous solution of m-PEG-SH, 2000 daltons (18 mg in 2 mL of H2O) was added to the nanoparticle suspension dropwise at RT under vigorous stirring (1000 rpm).
  • the reaction mixture was continued to stir for 16 hours at 1000 rpm followed by washing with water several times using a 10 kDa (molecular weight cut off) centrifugal filter.
  • the final suspension of gold nanoparticles (AuNP) was concentrated to 1.5 mL.
  • 0.39 mL of concentrated solution of Au NP taken in a 5 mL glass vial fitted with a magnetic stir bar 0.61 mL of water was charged.
  • Protocol for the synthesis of P4BN A thioctic-bombesin peptide was synthesized following the traditional solid-phase peptide synthesis (SPPS) procedure employing Fmoc chemistry methodology and the final peptides were purified by HPLC. A 4- hydroxymethylphenoxyacetyl- 4'-methylbenzyhydrylamine resin was used as the solid support for the synthesis. Fmoc-protected amino acids were activated using one equivalent of 0.45 M HBTU/HOBt solutions and two equivalents of N, N-di isopropyl ethylamine. The amino acids were Fmoc deprotected using piperidine and coupled using NMM.HBTU. Following the coupling of all of the amino acids in the appropriate sequence, thioctic acid (lipoic acid) was coupled using DIC.HOBt.
  • Protocol for the MTT Assay To conduct the MTT assay 1x106 cells (at 70% confluency; p+2) were seeded onto 96-well plates overnight (triplicates per dose per construct). Drugs or nanoparticle-constructs at specific concentrations were then prepared in serum-free RPMI media to test the toxicity profile at various concentrations for a period of 72 hours. Drugs or nanoparticle-constructs at specific concentrations were also added to cell-free wells (duplicates) as a background control.
  • Protocol for the SRB Assay NCI-60 Screening Methodology was followed to evaluate the in vitro efficacy of the constructs. Briefly, cells were seeded in 96-well tissue culture plates at a density appropriate for the cell line. The next day, a control plate was processed as described below to determine the density at TO (zero time). The remaining plates were treated with constructs and controls over a 7 log ug/mL concentration range. The plates were then incubated for 72 hours following which they were fixed with TCA (4 °C; 1 hour; final cone. 10%), dried and stained with sulphorhodamine B (0.4% w/v in 1% acetic acid; 100 uL per well) for 10 minutes.
  • TCA 4 °C; 1 hour; final cone. 10%

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Abstract

La présente invention concerne des composés, des nanoconjugués et des compositions de ceux-ci pour le traitement du cancer.
PCT/IB2023/056148 2022-06-15 2023-06-14 Nanoconjugués d'or WO2023242766A1 (fr)

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