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WO2011072133A1 - Compositions thérapeutiques et procédés pour la distribution d'agents actifs liés de manière clivable à des nanoparticules - Google Patents

Compositions thérapeutiques et procédés pour la distribution d'agents actifs liés de manière clivable à des nanoparticules Download PDF

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Publication number
WO2011072133A1
WO2011072133A1 PCT/US2010/059704 US2010059704W WO2011072133A1 WO 2011072133 A1 WO2011072133 A1 WO 2011072133A1 US 2010059704 W US2010059704 W US 2010059704W WO 2011072133 A1 WO2011072133 A1 WO 2011072133A1
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Prior art keywords
therapeutic composition
nanoparticle
active agent
paclitaxel
cleavable linker
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Eugene Zubarev
Jacob Gibson
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William Marsh Rice University
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William Marsh Rice University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic 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
    • A61K47/69—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
    • A61K47/6925—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 microcapsule, nanocapsule, microbubble or nanobubble
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • active agents have been linked to numerous carriers, such as nanoparticles.
  • carriers may not be able to effectively deliver and/or release the active agents at desired sites. This problem can be farther amplified by the lack of solubility of many active agents and/or carriers. Accordingly, there is currently a need to design more effective approaches for the delivery of active agents to desired sites for treating various diseases and conditions (including cancer).
  • the present disclosure pertains to therapeutic compositions that comprise an active agent and a nanopartiele, where the active agent is covalently linked to the nanopartiele through a cleavable linker.
  • the active agent is an anticancer drug, such as paciitaxei.
  • multiple active agents may be covalently linked to the nanopartiele through cleavable linkers.
  • the active agent is substantially inactive when it is covalently linked to the nanopartiele,
  • the cleavable linker is a chemically cleavable linker, such as a linker that comprises a hydrazone species that can release the active agent(s) from the nanopartiele in an acidic environment (e.g., a tumor site).
  • the nanoparticle is a gold nanoparticle (AuNP), such as a gold nanoparticle with a diameter of about 2 nm.
  • the nanoparticle is functionalized with one or more functional groups and/or polymers, such as polyethylene oxide (PEO).
  • the therapeutic composition may also comprise a targeting agent, such as an antibody, for directing the therapeutic composition to a desired site.
  • Additional embodiments of the present disclosure pertain to methods of treating a condition in a subject by administering the therapeutic compositions of the present disclosure to the subject.
  • the subject is a human being, and the condition to be treated is cancer.
  • the cleavable linker in the therapeutic composition may be cleaved at or near a tumor site in the subject to release the active agent at that site.
  • the active agent is substantially inactive when it is covalently linked to the nanoparticle. In such embodiments, the cleaving and subsequent release of the active agent results in its activation.
  • compositions of the present invention allow for more effective and specific approaches for treating many diseases with reduced side effects, streamlined treatment formulations, and improved patient outcomes.
  • FIGURE 1 depicts various aspects of a paclitaxel-linked gold nanoparticle as a specific embodiment of a therapeutic composition.
  • FIG. 1A depicts a structural view of the paclitaxel-linked gold nanoparticle, showing multiple paclitaxel molecules covalently linked to a gold nanoparticle through cleavable linkers that contain hydrazone species.
  • the hydrazone species in the linkers can be degraded in an acidic environment (e.g., pH ⁇ 5-6) to release the paclitaxel from the gold nanoparticles.
  • an acidic environment e.g., pH ⁇ 5-6
  • the hydrazone species remain intact, thereby retaining the paclitaxel molecules on the gold nanoparticles.
  • FIG. IB shows the chemical structure of paclitaxel.
  • the 2' site is labeled (paclitaxel becomes substantially inactive if the 2' site is blocked)
  • FIG. 1C illustrates how a hydrazone species can be degraded in an acidic environment.
  • FIGURE 2 schematically illustrates how the paclitaxel-linked gold nanoparticle shown in FIG. 1A can be synthesized in some embodiments.
  • FIG. 2A shows the esterification of paclitaxel at the 2' site (the esterification at the 2' site renders the paclitaxel substantially inactive).
  • FIG. 2B shows the formation of a linker with a hydrazine group at one of its ends.
  • FIG. 2C shows the coupling of the linker in FIG. 2B to the esterified paclitaxel in FIG. 2A to form a paclitaxel-linker compound, where the paclitaxel becomes coupled to the linker at the 2' site to form a hydrazone species (shown in dotted square).
  • FIG. 2D shows the coupling of the paclitaxel-linker compound in FIG. 2C to phenol- terminated gold nanoparticles to form the paclitaxel-linked gold nanoparticle shown in FIG. 1 A.
  • FIGURE 3 shows experimental results obtained from in vitro studies that tested the in vitro efficacy of the paclitaxel-linked gold nanoparticles (labeled as "Au(Tax2'hydrazoneP4sux)") in MCF-7 breast carcinoma cells.
  • paclitaxel a potent inhibitor of cell proliferation that is widely used in the treatment of various forms of cancer.
  • paclitaxel acts non- specifically against both cancer cells and normal cells.
  • the non-specificity of active agents can cause unnecessary toxicity to healthy cells and tissues. As a result, such non-specificity can lead to undesired side effects in a subject (e.g., nausea, hair loss, reduced immune response, etc.). Such non-specificity can also reduce an active agent's therapeutic efficacy.
  • active agents have been linked to carriers, such as nanoparticles.
  • carriers such as nanoparticles.
  • many carriers may not be able to effectively deliver and/or release the active agents at a desired site.
  • hydrophobic nature of many active agents and nanoparticles may render them insoluble.
  • various embodiments of the present disclosure address the above-mentioned problems.
  • various embodiments of the present disclosure pertain to therapeutic compositions that comprise: (1) one or more active agents; (2) a cleavable linker; and (3) a nanoparticle that is covalently linked to the active agent(s) through the cleavable linker.
  • the active agent is substantially inactive when it is covalently linked to the nanoparticle.
  • the therapeutic compositions of the present disclosure may also be associated with one or more targeting agents.
  • the cleavable linker is specifically cleaved at a desired treatment site (i.e., tumor site), thereby releasing the active agent at that site.
  • FIG. 1A A specific and non-limiting example of a therapeutic composition in accordance with the present disclosure is shown in FIG. 1A.
  • the active agent is paclitaxel (shown in FIG. IB, also known as Taxol® when contained in a commercial excipient, Cremaphor EL), a potent but non-specific inhibitor of cell proliferation that is used in the treatment of various forms of cancer.
  • the nanoparticle is a gold nanoparticle that is about 2 nm in diameter.
  • multiple paclitaxel molecules are covalently linked to the gold nanoparticle in this example through cleavable linkers that are coupled to the paclitaxel molecules at the 2' site through hydrazone species.
  • the blocking of the 2' site renders the paclitaxel molecule substantially inactive.
  • Active agents of the present disclosure generally refer to biologically active compounds, such as compounds that can be used to treat one or more conditions in a subject (e.g., a human being).
  • active agents of the present disclosure may refer to anti-cancer drugs, antibiotics, chemotherapeutics, antioxidants, and/or anti-inflammatory drugs.
  • the active agents of the present disclosure may also be derived from various compounds.
  • the active agents of the present disclosure can be small molecules, proteins, aptamers, DNA, anti-sense oligo nucleotides, miR A, siR A, and the like.
  • the active agent is an anti-cancer agent, such as paclitaxel, docetaxel, doxorubicin, and the like.
  • the active agent is paclitaxel.
  • the active agent is substantially inactive when it is covalently linked to the nanoparticle. However, the active agent may become active once it is cleaved from the nanoparticle.
  • substantially inactive refers to a state of an active agent that exhibits a therapeutic activity that is less than the normal or average therapeutic activity of the active agent.
  • the therapeutic activity of an active agent can be determined by the measurement of various parameters, as known by persons of ordinary skill in the art. Such parameters include, without limitation, target binding activities (e.g., as measured by Kj values), inhibition activities (e.g., as measured by IC 50 values), changes in a dose response curve, changes in survival rates of subjects, and the like.
  • target binding activities e.g., as measured by Kj values
  • inhibition activities e.g., as measured by IC 50 values
  • changes in a dose response curve changes in survival rates of subjects, and the like.
  • substantially inactive may refer to a state of an active agent that exhibits little or no therapeutic activity.
  • paclitaxel is virtually inactive if the 2' site is blocked. Therefore, in some embodiments, paclitaxel may be covalently linked to the nanoparticle through the 2' site. Applicants envision that the inactivation of the active agent in covalently linked form can help prevent any non-specific or undesired effects of the active agent against various non-targeted cells or tissues.
  • the cleavable linkers of the present disclosure may have numerous lengths and components.
  • the cleavable linkers of the present disclosure may contain one or more polymers, such as polyethylene oxides (PEO), polyesters, polyethylene glycols, polystyrenes, polypropylenes, polyamides, and the like.
  • the polymers may consist of two or more monomeric units. In more specific embodiments, the polymers may consist of about four to five monomeric units.
  • the polymers may be homopolymers or heteropolymers.
  • the polymers of the present disclosure may be linked to an active agent at one end and a nanoparticle at another end.
  • the cleavable linkers of the present disclosure contain one or more moieties or sites that can be cleaved and/or degraded under various conditions. The cleavage and/or degradation thereby results in the release of the active agent from the nanoparticle.
  • various cleavable linkers may be used in various embodiments of the present disclosure (e.g., without limitation, chemically cleavable linkers, photo-cleavable linkers, and enzymatically cleavable linkers).
  • the cleavable linker is a chemically cleavable linker.
  • one or more moieties and/or sites in the linker may be cleaved and/or degraded by various chemicals and/or reaction conditions (e.g., change in pH).
  • the cleavable linker is cleaved in an acidic environment (e.g., pH ⁇ 5-6).
  • the cleavable linker comprises one or more hydrazone species.
  • hydrazone species refer to a class of organic compounds with the structure . This structure is illustrated below:
  • Hydrazone species are usually formed by the reaction of hydrazine with ketones or aldehydes. Furthermore, as illustrated in FIG. 1C, hydrazone species are readily degraded under acidic conditions (i.e., pH -5-6). Advantageously, many tumor sites have an acidic pH level. Thus, in some embodiments, therapeutic compositions of the present disclosure with cleavable linkers containing hydrazone species may be selectively cleaved at or near tumor sites to selectively release active agents from nanoparticles at those sites.
  • Ri and/or R 2 may be derived from alkyl, acyl, benzophenone, methyl, ethly, ester, ether and other similar functional groups.
  • the hydrazone species can include, without limitation, acyl hydrazones, benzophenone hydrazones, acetone hydrazones, ⁇ , ⁇ -dialkylhydrazones, and the like.
  • the cleavable linker is a photo-cleavable linker.
  • one or more moieties in the linker may be cleaved and/or degraded by photolysis.
  • the photolysis may be initiated by any photon with sufficient energy to affect the chemical bonds of the linker.
  • photons include visible light, uv light, x-rays, and gamma rays.
  • the cleavable linker is an enzymatically cleavable linker.
  • one or more moieties in the linker may be cleaved and/or degraded by various enzymes.
  • the enzyme that cleaves and/or degrades the cleavable linker may only be expressed in diseased cells, such as cancer cells.
  • Nanoparticles suitable for use in the present disclosure generally refer to particles that are capable of associating with an active agent, desirably through covalent bonds. Nanoparticles in the present disclosure also refer to particles that are capable of delivering an active agent to a targeted area. In some embodiments, the nanoparticles of the present disclosure are soluble in water.
  • the nanoparticles of the present disclosure may be derived from one or more carbon nanotubes (CNTs), including, without limitation, single-walled nanotubes (SWNTs), oxidized SWNTs, multi-walled nanotubes (MWNTs), and oxidized MWNTs.
  • CNTs carbon nanotubes
  • SWNTs single-walled nanotubes
  • MWNTs multi-walled nanotubes
  • the nanoparticles of the present disclosure may be derived from graphene, graphene nanoribbons, graphite, graphite oxide nanoribbons, carbon black, oxidized carbon black, and other nanoparticles.
  • the nanoparticles of the present disclosure may be derived from liposomes.
  • nanoparticles of the present disclosure are gold nanoparticles (AuNPs).
  • AuNPs gold nanoparticles
  • EPR enhanced permeation and retention effect
  • EPR refers to a property where various active agents and macromolecular particles tend to accumulate in tumor tissue more than they accumulate in normal tissue.
  • mice bearing tumors and injected with AuNPs retained about eight times more of the AuNPs in the tumors than in the normal cells, which cleared the AuNPs twice as fast as the tumor cells.
  • AuNPs gold nanoparticles in the therapeutic compositions of the present disclosure can provide an additional advantage in enhancing the selectivity and efficacy of active agents in treating cancer (and other similar conditions).
  • the nanoparticles of the present disclosure may be functionalized with one or more molecules, polymers, chemical moieties, and/or functional groups.
  • the nanoparticles of the present disclosure may be functionalized with hydroxyl groups and/or polyethylene oxides (PEO).
  • PEO polyethylene oxides
  • gold nanoparticles may be functionalized with PEO.
  • the functionalization occurs on the surfaces of the nanoparticles.
  • nanoparticles are functionalized with one or more molecules, polymers, chemical moieties, and/or functional groups, they should be allowed to circulate for longer periods of time in a living body.
  • the functionalization would increase the chance of nanoparticles accumulating at or near a desired site (e.g., a tumor site).
  • the nanoparticles of the present disclosure may also have various sizes. For instance, in some embodiments, the nanoparticles of the present disclosure may be from about 1 nm to about 500 nm in diameter. In other embodiments, the nanoparticles may be from about 1 nm to about 50 nm in diameter. In more specific and preferred embodiments, the nanoparticles of the present disclosure may be about 2 nm in diameter (e.g., -1.9 nm).
  • nanoparticles of the present disclosure may also be associated with one or more targeting agents.
  • therapeutic compositions of the present disclosure may also be associated with one or more targeting agents.
  • the targeting agent may be coupled directly to the nanoparticles in the therapeutic composition.
  • the targeting agent may be coupled to the cleavable linker of the therapeutic composition. In various embodiments, such coupling may occur through covalent and/or non- covalent bonds.
  • Targeting agents of the present disclosure generally refer to compounds that target a particular cell, organ, and/or tissue for which treatment is desired.
  • the targeting agent may be compounds such as antibodies, RNA, DNA, aptamers, small molecules, dendrimers, and/or proteins.
  • the targeting agent can be a monoclonal or polyclonal antibody.
  • the antibody may be a chimeric antibody or an antibody fragment (e.g., Fab fragment of a monoclonal antibody).
  • the targeting agent may be an antibody that specifically targets epidermal growth factor receptors (e.g., Cetuximab).
  • epidermal growth factor receptors e.g., Cetuximab
  • EGFRs epidermal growth factor receptors
  • anti- EGFR antibodies and other EGFR inhibitors may be used to deliver anti-cancer agents to cancer cell lines in some embodiments.
  • Other suitable targeting agents can also be envisioned by a person of ordinary skill in the art.
  • targeting agents in the therapeutic compositions of the present disclosure can help facilitate their efficacy by directing the active agent to a desired site.
  • various therapeutic compositions of the present disclosure may not contain any targeting agents.
  • Additional aspects of the present disclosure pertain to methods of treating a condition in a subject by administering a therapeutic composition of the present disclosure to the subject.
  • the cleavable linker in the therapeutic composition may be cleaved in an acidic environment after administration, such as a tumor site.
  • the active agent is substantially inactive when it is covalently linked to the nanoparticle, the cleaving and subsequent release may result in the activation of the active agent.
  • the therapeutic compositions of the present disclosure can be used to treat numerous conditions in different subjects through various modes of administration.
  • the methods of the present disclosure can be used to treat various conditions.
  • the methods of the present disclosure may be used to treat various types of cancer (e.g., breast cancer, head and neck cancer, colorectal cancer, lymphatic cancer, etc.).
  • the active agent is an anti-cancer drug (e.g., paclitaxel).
  • the targeting agent if used may be a compound that recognizes one or more markers on a cancer cell.
  • the targeting agent may be an antibody that recognizes EGFRs.
  • the cancer-treating methods of the present disclosure may utilize a therapeutic composition where the nanoparticle is a gold nanoparticle, the active agent is paclitaxel, and the cleavable linker contains a hydrazone species (e.g., the therapeutic composition shown in FIG. 1A).
  • the methods of the present disclosure can be used to treat microbial infections, such as bacterial, viral, and/or fungal infections.
  • the active agent can be an antibiotic
  • the targeting agent if used can be an antibody that recognizes one or more markers on the pathogenic microbes and/or infected cells.
  • the methods of the present disclosure can be used to treat other conditions, such as inflammation.
  • a person of ordinary skill in the art will also recognize that the methods of the present disclosure can be used to treat other conditions that have not been specifically described above.
  • various subjects may be treated by the methods and therapeutic compositions of the present disclosure.
  • the methods and compositions of the present disclosure may be used to treat human beings.
  • the subjects being treated may be in need of such treatment for one or more conditions.
  • the subjects may be non- human animals, such as mice, rats, other rodents, or larger mammals, such as dogs, monkeys, pigs, cattle and horses.
  • the therapeutic compositions of the present disclosure can be administered to subjects by various methods known to persons of ordinary skill in the art.
  • the therapeutic compositions of the present disclosure can be administered by oral administration, inhalation, subcutaneous administration (sub-q), intravenous administration (I.V.), intraperitoneal administration (LP.), intramuscular administration (I.M.), and/or intrathecal injection.
  • the therapeutic compositions of the present disclosure can be administered by topical application (e.g, transderm, ointments, creams, salves, eye drops, and the like). Additional modes of administration can also be envisioned by persons of ordinary skill in the art.
  • Additional embodiments of the present disclosure also pertain to methods of making therapeutic compositions of the present disclosure. Such methods generally comprise covalently linking a nanoparticle to an active agent through a cleavable linker. A person of ordinary skill in the art will recognize that various methods may be used to make the therapeutic compositions of the present disclosure.
  • FIG. 2 A specific example of a method of making a therapeutic composition is shown in FIG. 2.
  • paclitaxel is esterified at the 2' site to render it inactive (FIG. 2A).
  • a linker is also formed that contains a hydrazine group at one of its ends (FIG. 2B).
  • the linker is covalently coupled to the esterified paclitaxel to form a paclitaxel-linker compound (FIG. 2C).
  • the paclitaxel is coupled to the linker at the 2' site through a hydrazone (shown in dotted square).
  • compositions of the present disclosure can also be formulated in conventional manners known to persons of ordinary skill in the art.
  • the formulation may also utilize one or more physiologically acceptable carriers or excipients.
  • compositions can also comprise formulation materials for modifying, maintaining, or preserving various conditions, including pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, and/or adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to: amino acids (e.g., glycine); antimicrobials; antioxidants (e.g., ascorbic acid); buffers (e.g., Tris- HC1); bulking agents (e.g., mannitol and glycine); chelating agents (e.g., EDTA); complexing agents (e.g., hydroxypropyl-beta-cyclodextrin); and the like.
  • amino acids e.g., glycine
  • antimicrobials e.g., ascorbic acid
  • buffers e.g., Tris- HC1
  • bulking agents e.g., mannitol and glycine
  • chelating agents e.g., EDTA
  • complexing agents e.g., hydroxypropyl-beta-cyclodextrin
  • a therapeutic composition may comprise a plurality of the same active agents.
  • a therapeutic composition may comprise a plurality of different active agents.
  • therapeutic compositions of the present disclosure may comprise one active agent per nanoparticle, about 2-10 active agents per nanoparticle, or about 50-100 active agents per nanoparticle.
  • the therapeutic compositions of the present disclosure may comprise about 70-75 active agents per nanoparticle.
  • a therapeutic composition may comprise about 70-75 paclitaxel molecules covalently linked to a single gold nanoparticle.
  • Figure 2 illustrates the convergent synthetic strategy employed in the manufacturing of the new PTX-AuNP delivery system beginning with the aforementioned conversion of paclitaxel with pyruvic acid under standard DIPC/DPTS conditions (FIG. 2A).
  • FIG. 2A commercially available aminotetraethylene glycol t-boc-hydrazide was coupled with glutaric anhydride in order to introduce a terminal carboxyl group that can be coupled to the surface of mercaptophenol-functionalized AuNPs.
  • FIG. 2B simply dissolving both agents in an equimolar ratio resulted in quantitative conversion to the desired product in the absence of base or catalyst.
  • Paclitaxel 2' pyruvate (FIG. 2A). To a solution of 100 mg of paclitaxel in 4 mL of CH 2 CI 2 were added 60 mg (1.5 equiv) of DPTS and 13 mg (1.3 equiv) of pyruvic acid. Under vigorous stirring, 75 mg (5 equiv) of the coupling agent DIPC were added to catalyze ester formation. Upon addition of DIPC, the colorless solution immediately became yellow in color and progressed to a dark orange color as the reaction continued. Near complete consumption of starting PTX was observed after 2-3 hours, as evidenced by TLC. The reaction was quenched upon removal of DPTS via three water extractions.
  • Paclitaxel-hydrazone linker compound (PTX-L-COOH) (FIG. 2C).
  • the t-Boc protected hydrazide linker was first treated with a solution of 20% TFA in CH 2 CI 2 . After 30 minutes, TFA and residual solvent were removed under vacuum pressure and the resulting colorless oil was dissolved in 1.5 mL of THF. Following the liberation of the t-Boc group, 50 mg of paclitaxel pyruvate was dissolved in 1 mL of THF and added to the hydrazide linker solution. Hydrazone formation was then catalyzed by the addition of 1-2 drops of TFA.
  • PTX-AuNPs Formation of PTX-AuNPs. Preactivation of the carboxyl terminated intermediate was achieved upon the addition of 20 mg of DIPC to a 3 mL solution of methylene chloride containing 15 mg of Paclitaxel-hydrazone linker compound (PTX-L-COOH) and 25 mg of DPTS. Immediately following the addition of DIPC, a solution of 5 mg of mercaptophenol- functionalized AuNPs in 0.30 mL of DMF was added to the above mixture. The reaction was monitored by GPC and additional amounts of PTX-L-COOH were added in 3-5 mg aliquots until saturation of nanoparticle was complete based on the sharpness and quality of the product signal in GPC.
  • DIPC Paclitaxel-hydrazone linker compound
  • the reaction was quenched upon DPTS removal via three extractions with DI water followed by drying of the crude solution over Na 2 S0 4 .
  • Methylene chloride was removed under vacuum, and the residual product was then diluted in DMF and placed on regenerated cellulose membrane filters (Millipore MWCO 30 kDa) and subjected to 3 rounds of centrifugation at 40 minutes each before being analyzed for purity by GPC.
  • the formed AuNP(PTX) n was diluted with CH 2 C1 2 and DMF was removed via water extractions. The product solution was again dried over Na 2 S0 4 and the final product precipitated from hexanes and dried under high vacuum leaving 14 mg of a light brown powder.
  • PTX- AuNPs The antiproliferative capabilities of PTX- AuNPs from Example 1 were first investigated in vitro. To this end, cellular assays were performed to compare the ability of the PTX-AuNPs to inhibit cell growth relative to the parent drug. Briefly, MCF-7 breast cancer cells were treated over a range of concentrations (based on equivalent PTX content) for all samples and relative cell survival was measured 72 hours post treatment following the MTT protocol. Additionally, the paclitaxel pyruvate compound was also analyzed, as it represented the agent released from the gold surface upon degradation of the hydrazone linker. Results of these experiments are shown in FIG. 3.
  • MCF-7 cells were plated in 6 well plates and allowed to proliferate until approximately 80-90% confluent. Once optimal confluency was reached, increasing concentrations of PTX- AuNPs were added to each well in order to ensure sufficient uptake for imaging (up to 100 ⁇ / ⁇ .). Following 12 hours of incubation with PTX-AuNPs, the cells were fixed with a solution of gluteraldehyde and imaged by transmission electron microscopy (TEM). MCF-7 breast cancer cells were plated in 96-well plates at a concentration of 4,000 cells/well (100 ⁇ ) and allowed to attach for 24 hours.
  • TEM transmission electron microscopy
  • paclitaxel exhibits high potency towards inhibiting cell growth, as evidenced by an IC 50 value of approximately 4 nM.
  • This potent inhibitory quality of paclitaxel pyruvate confirms the usefulness of a hydrazone strategy towards effecting intracellular drug delivery based on a AuNP carrier.
  • the released drug is capable of passive diffusion through the endosomal membrane in a manner analogous to that of commercially available paclitaxel diffusing through the external cell membrane.

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Abstract

La présente invention, conformément à différents modes de réalisation, porte sur des compositions thérapeutiques qui comprennent : (1) un agent actif (par exemple, le paclitaxel); et (2) une nanoparticule (par exemple, une nanoparticule d'or). Dans de tels modes de réalisation, l'agent actif est lié par liaison covalente à la nanoparticule par l'intermédiaire d'un coupleur clivable (par exemple, un coupleur contenant une espèce d'hydrazone). D'autres modes de réalisation de la présente invention portent sur des procédés de traitement d'un état pathologique chez un sujet par administration des compositions thérapeutiques décrites ci-dessus au sujet.
PCT/US2010/059704 2009-12-09 2010-12-09 Compositions thérapeutiques et procédés pour la distribution d'agents actifs liés de manière clivable à des nanoparticules Ceased WO2011072133A1 (fr)

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