Patent Application
20170319505
The present invention relates to nanoparticles encapsulated within polymeric microparticles, particularly for use in medicine, and includes methods for treatment of disorders. Pharmaceutical compositions, processes for production of the microparticles and methods for their use are also disclosed.
The present invention is directed at compositions and products, and methods of making and administering such compositions and products, including for the treatment of mammals and particularly humans.
Drug delivery poses several significant challenges, particularly with regard to the site and duration of action. In the case of treatment of certain tumours, for example, there remains a need for delivery systems that are able to target anti-cancer drugs to the tumour site and which are able to sustain an efficacious drug concentration over an extended period ideally while minimising the need for frequent repeat drug administration.
WO2012/042273 and WO2012/042274 describe apparatus and process for the preparation of solid beads that encapsulate a bioactive agents and which are suitable for use in sustained release, e.g., via depot injection.
WO2013/042125 describes double nano-encapsulation for protecting and controlling the release of active agents, either hydrophobic or hydrophilic, from stable nanoparticles of opposite characteristics.
WO2011/154711 describes glycated gold nanoparticles that act as carriers for delivery of peptides such as insulin.
WO 2011/156711 and WO2012/170828 describe therapeutic or bioaffecting film delivery systems in which nanoparticles having actives bound to or associated therewith are incorporated within a polymeric film matrix.
WO2014/125256 describes nanoparticle delivery systems for use in targeting biologically active agents to the central nervous system (CNS), e.g., for treatment of CNS disorders.
There remains an unmet need for further nanoparticle delivery systems and for methods of delivering such bioactive agents to a subject at the appropriate location and/or for the appropriate duration. The present invention addresses these and other needs.
Broadly, the present invention relates to nanoparticle-drug conjugates encapsulated in micron-scale beads. The present inventors have surprisingly found that it is possible to encapsulate metal- (e.g. gold-) core nanoparticles within biocompatible polymeric microparticles with high entrapment efficiency.
Accordingly, in a first aspect the present invention provides a microparticle comprising at least one biocompatible polymer, the microparticle encapsulating at least one nanoparticle, the nanoparticle comprising:
In accordance with this and other aspects of the present invention, the at least one nanoparticle preferably further comprises at least one biologically active agent (e.g. a pharmaceutical active as a “payload”) and/or a detectable label (e.g. a fluorophore). However, it has been found that certain nanoparticles exhibit biological activity even in the absence of an additional active agent. Therefore, it is specifically contemplated herein that the nanoparticle need not necessarily have any added active agent. When the nanoparticle does comprise said biologically active agent the agent is generally associated with the core and/or corona of the nanoparticle. Conveniently this association may be in the form of a covalently link, e.g., via a linker group attached to the nanoparticle core or to one or more of the nanoparticle ligands. For example, an alkyl and/or glycol linker may connect an agent such as a chemotherapeutic agent to the nanoparticle core. In certain cases the active agent may be non-covalently bound to said corona and/or said core. For example, the active agent may comprise a peptide or polypeptide bound non-covalently (e.g. via electrostatic interaction or Van der Waals forces or otherwise) to the ligands that make up the corona of the nanoparticle.
In certain cases the at least one biologically active agent may comprise a chemotherapeutic agent and/or may be covalently linked to said core via a linker.
In certain cases the at least one biologically active agent may be covalently linked to said core via a linker comprising C2-C15 alkyl and/or C2-C15 glycol.
In certain cases the biologically active agent comprises a chemotherapeutic or “anti-cancer” agent, which may preferably be selected from the group consisting of: temozolomide, irinotecan, chlorotoxin, carmustine, platinum(IV), platinum(II), camptothecin, doxorubicin, docetaxel, Maytansine, Maytansinoids, monomethyl auristatin E (MMAE) and histone deacetylase (HDAC) inhibitors such as Panobinostat, Vorinostat, Romidepsin and Chidamide.
In certain cases the nanoparticle further comprises a cell, tissue or tumour targeting moiety. Owing to the advantageous avidity effects of the multiple ligands presented by the nanoparticle, it has been found that a number of targeting moieties are capable of targeting the nanoparticles described herein to certain sites of interest, e.g., tumour or diseased tissue sites, organ types or particular cell types. In certain cases, the targeting moiety may be selected from the group consisting of: folic acid, lactose, albumin, glutamine, a cell surface receptor binding ligand (e.g. an EGFR ligand such as Gefitinib) and an antibody, particularly an antibody that binds selectively to a tumour associated antigen.
In some cases the at least one biologically active agent comprises at least one peptide or polypeptide that is non-covalently bound to the corona. For example, the peptide or polypeptide may be selected from the group consisting of: insulin, GLP-1, amylin, exenatide, octreotide, teriparatide, glucagon, a cytokine, and an antibody.
In some cases the corona of the nanoparticle comprises at least 5, 10, 20 or at least 50 ligands per nanoparticle core. The corona may, in certain cases, be a mixed corona. For example, the corona may comprise two or more different species of ligand (e.g., carbohydrate and non-carbohydrate, such as alpha-galactose and PEGamine; glutathione and carbohydrate; two different saccharides such as glucose and galactose or N-acetyl glucosamine and lactose). The ratio of the first species of ligand to the second species of ligand may be in the range 1:100 to 100:1, preferably 80:20 to 20:80, yet more preferably 60:40 to 40:60).
In some cases the number of molecules of said biologically active agent per nanoparticle core is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.
In some cases the diameter of the core of the nanoparticle is in the range 1 nm to 5 nm. The diameter of the nanoparticle including its ligands may in some cases be in the range 2 nm to 50 nm, or 3 nm to 30 nm, or 4 nm to 20 nm, or 5 nm to 15 nm.
In some cases the core of the nanoparticle comprises a metal selected from the group consisting of: Au, Ag, Cu, Pt, Pd, Fe, Co, Gd, Eu and Zn, or any combination thereof. In certain cases the nanoparticle has a core comprising or consisting of gold atoms.
The microparticle of this and other aspects of the present invention typically has more than one, e.g., more than 10, 100, 1000, 10000, 100000, 106, 107 or more than 108 individual nanoparticles encapsulated therein.
In certain cases the microparticle comprises a plurality of said nanoparticles, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the nanoparticles in said microparticle have at least one biologically active agent conjugated or bound thereto.
In certain cases the microparticle comprises at least two different species of biologically active agent. Preferably, the at least two different species of biologically active agent work in concert to potentiate or otherwise enhance the biological or therapeutic activity in comparison to either one species alone. Examples specifically contemplated herein are insulin and GLP-1, two or more chemotherapeutic agents, a chemotherapeutic agent in combination with an inhibitor of anti-cancer drug resistance.
In certain cases the at least two difference species of biologically active agent comprise a first and a second agent. In some cases the first biologically active agent is conjugated or bound to a first nanoparticle and a second of said at least two different species of biologically active agent is conjugated or bound to a second nanoparticle. The two or more nanoparticles may then advantageously be present within a single microparticle (i.e. two different nanoparticles). Alternatively or additionally, the at least one nanoparticle may have at least two different species of biologically active agent conjugated or bound thereto (i.e. a combination payload nanoparticle).
In some cases the microparticle has a diameter along its longest dimension that is within the range 1 μm to 900 μm, 5 μm to 500 μm, 10 μm to 100 μm or 25 μm to 75 μm. In some cases the morphology of the microparticle is substantially spherical. Without wishing to be held to any particular theory, the present inventors believe that the spherical shape affords greater structural resistance, e.g., resistance to pressure differentials. In one particularly contemplated form the microparticle is spherical and comprises nanoparticles conjugated to a chemotherapeutic such as temozolomide or carmustine. Such microparticles may find use in the treatment of brain tumours, including secondary tumours, in which the microparticles are implanted following or at the time of surgical resection, e.g. to prevent or inhibit cancer recurrence. Owing to their structurally more robust shape, the spherical microparticles are expected to overcome shortcomings that have been seen with GLIADEL Wafer in this therapeutic context. However, other non-spherical shapes are specifically contemplated herein. In certain cases the microparticle of the present invention may be relatively flattened or disc-shaped, e.g., the microparticle morphology may be lenticular.
In certain cases the surface porosity of the microparticle of the present invention may be characterised by an average surface pore size of <20 nm, <100 nm, <1 μm or <10 μm. As described in WO2012/042274 (incorporated herein by reference in its entirety), certain process conditions such as the temperature of the second liquid and/or the pH of the second liquid (e.g. pH 3 to 9).
In accordance with this and other aspects of the present invention the at least one biocompatible polymer may be any polymer disclosed in WO2012/042274 (incorporated herein by reference in its entirety —see, in particular, pages 9 and 10 thereof). In certain cases the polymer may be selected from the group consisting of: a polylactide, a polycaprolactone, a polyanhydride and a co-polymer of lactic acid and glycolic acid (“PLGA”). The weight average molecular weight (MW) of the polymer may be from 4 to 700 kDaltons, preferably 4 to 120 kDaltons, more preferably from 4 to 15 kDaltons. Polymer end groups and inherent viscosities may, in certain cases, be as set forth in WO2012/042274 (incorporated herein by reference in its entirety—see, in particular, pages 9 and 10 thereof). In certain cases the polymer comprises at least one end group or terminal functional group selected from the group consisting of: carboxyl, hydroxyl and ester.
Without wishing to be bound by any particular theory, the present inventors believe that the encapsulation efficiency of nanoparticles within the microparticle and/or the rate or extent of nanoparticle release from the microparticle, e.g. in use, may be influenced (increased or decreased) by virtue of interactions between the nanoparticle corona and the polymeric matrix, e.g., through the selection of a polymer end-group that binds to a functional group present on one or more of the ligands making up the nanoparticle corona. One example of this contemplated herein is a microparticle wherein the at least one biocompatible polymer comprises a co-polymer of lactic acid and glycolic acid (“PLGA”) having a carboxylate end group and said nanoparticle corona comprises at least one amine group. In particular, the nanoparticle may comprise a corona of PEGamine ligands, such as 1-amino-17-mercapto-3,6,9,12,15,-pentaoxa-heptadecanol, and alpha-galactose ligands, such as 2-thio-ethyl-α-D-galactoside. Accordingly, in certain cases of the present invention the polymer or co-polymer comprises at least one end group or terminal functional group that binds to a functional group present on the corona of said nanoparticle.
In certain cases in accordance with the present invention the at least one biocompatible polymer may have a ratio of lactide to glycolide (“L/G ratio”) in the range 1:1 to 1:0, optionally having a ratio of lactide to glycolide in the range 70:30 to 90:10. The L/G ratio is thought to influence the rate of microparticle breakdown in vivo and therefore to influence the duration of release of nanoparticles and/or actives from the microparticle
In certain cases the nanoparticles are distributed substantially homogeneously throughout the microparticle. For example, the density of nanoparticles measured in terms of number of nanoparticles per unit volume of the microparticle may, in certain cases, not vary by more than 50%, more than 20% or more than 10% about the mean. Distribution of nanoparticles within the microparticle may, for example, be determined by electron microscopy. Nanoparticles may be visualised and counted and, if necessary, statistical analyses applied to compute a measure of nanoparticle distribution homogeneity.
In certain cases the nanoparticles have a distribution within the microparticle that is biased towards the surface of the microparticle. For example, more than 50%, more than 80% or more than 90% of the nanoparticles may be located closer to the surface of the microparticle than they are to the centre of the microparticle. As above, the distribution of nanoparticles within the microparticle may, for example, be determined by electron microscopy. Nanoparticles may be visualised and counted and, if necessary, statistical analyses applied to compute a measure of nanoparticle distribution heterogeneity.
In certain cases, the average concentration of nanoparticles expressed in terms of % weight of nanoparticle per weight of microparticle polymer (w/w) is within the range 0.01% to 25% w/w, optionally in the range 0.05% to 10% w/w.
In certain cases, the average concentration of biologically active agent, where present, expressed in terms of % weight of agent per weight of microparticle polymer is within the range 0.005% to 20% w/w, e.g., 0.01% to 10% w/w.
As described herein, microparticles of the present invention advantageously provide sustained release in clinical use. In certain cases the in vivo release profile (i.e. release of nanoparticles, particularly nanoparticles having a pharmaceutical payload, from the microparticle) of the microparticle following depot injection of the microparticle in a mammalian subject is in the range 1 week to 6 months, optionally in the range 3 weeks to 3 months. Release profile can be determined according to in vivo experimental techniques (e.g. animal models) known to those skilled in the art and/or can be modelled using one or more in vitro assays of drug release. Preferably the release profile is such that the rate of release of the nanoparticles into the body (e.g. cellular or extracellular space) is substantially slower than the rate of release of nanoparticles in the absence of encapsulation (e.g. a suspension of nanoparticles injected directly). In preferred cases, substantially slower in this context means at least 10 times, 20 times, 50 times or 100 times slower.
In a second aspect the present invention provides a pharmaceutical composition comprising a plurality of microparticles of the first aspect of the invention and a pharmaceutically acceptable carrier or excipient.
In some cases the pharmaceutical composition comprises an anti-agglomerating agent. The anti-agglomerating agent may take the form of a coating applied to the microparticles, e.g., mannitol.
In some cases the carrier is an aqueous or organic liquid, such as a biocompatible oil. In some cases the pharmaceutical composition takes the form of a lyophilised powder of microparticles suitable for reconstitution, e.g., prior to use.
In certain cases the pharmaceutical composition may be formulated for delivery via an injectable route, such as subcutaneous or intramuscular depot injection, or formulated for implantation during surgery.
In a third aspect the present invention provides a microparticle of the first aspect of the invention or a pharmaceutical composition of the second aspect of the invention for use in medicine.
In a fourth aspect the present invention provides a microparticle of the first aspect or a pharmaceutical composition of the second aspect for use in a method of treatment of a cancer, an endocrine disorder, a central nervous system (CNS) disorder or an ocular disorder in a mammalian subject.
In some cases the microparticle comprises an chemotherapeutic agent and the microparticle or composition is for treatment of a cancer selected from the group consisting of: a brain cancer, such as a glioblastoma, glioma or astrocytoma; an ovarian cancer; a liver cancer; and a skin cancer. In some cases the chemotherapeutic agent is selected from the group consisting of: temozolomide, irinotecan, chlorotoxin, carmustine, platinum(IV), platinum(II), camptothecin, doxorubicin, docetaxel, Maytansine, Maytansinoids, monomethyl auristatin E (MMAE) and histone deacetylase (HDAC) inhibitors such as Panobinostat, Vorinostat, Romidepsin and Chidamide.
In a fifth aspect the present invention provides a method of treatment of a cancer, an endocrine disorder or central nervous system (CNS) disorder or an ocular disorder in a mammalian subject, comprising administering a therapeutically effective amount of a microparticle of the first aspect of the invention or a pharmaceutical composition of the second aspect of the invention to the subject in need of said treatment.
In some cases the method is a treatment of a cancer selected from the group consisting of: a brain cancer, optionally glioblastoma, glioma or astrocytoma; an ovarian cancer; a liver cancer; and a skin cancer. In some cases the chemotherapeutic agent is selected from the group consisting of: temozolomide, irinotecan, chlorotoxin, carmustine, platinum(IV), platinum(II), camptothecin, doxorubicin, docetaxel, Maytansine, Maytansinoids, monomethyl auristatin E (MMAE) and histone deacetylase (HDAC) inhibitors such as Panobinostat, Vorinostat, Romidepsin and Chidamide.
In accordance with the microparticle or pharmaceutical composition for use of the fourth aspect of the invention or the method of the fifth aspect of the invention, the time interval between administration of doses may be at least 1 week, such as at least 2, 3, or 4 weeks, and preferably at least 1, 2, 3, 4, 5 or 6 months.
In a sixth aspect the present invention provides an article of manufacture comprising:
In certain cases the insert and/or label provides instructions, dosage and/or administration information relating to the use of the microparticle or pharmaceutical composition in a cancer, an endocrine disorder or central nervous system (CNS) disorder or an ocular disorder in a mammalian subject.
In a seventh aspect the present invention provides a process for producing a microparticle of the first aspect of the invention, the process comprising:
In certain cases the nanoparticles are provided in an organic solvent:water mixed phase, optionally a DMSO:water mixed phase, and further optionally a DMSO:water mixed phase having a DMSO:water ratio within the range 80:20 to 95:5. Biocompatible nanoparticles as described herein are typically provided in the form of an aqueous suspension. The present inventors have surprisingly found that it is possible to prepare nanoparticles, such as gold core glutathione or glycated nanoparticles, in a DMSO-mixed water phase, e.g., 90:10 DMSO:water. The DMSO:water mixed phase provides certain advantageous compatibility with the microparticle production process, e.g., as described in WO2012/042274. In particular, this addresses the problem that polymer such as PLGA is insoluble in water. Therefore, use of high concentrations of nanoparticles provided in a mixed phase has particular suitability for use in the process of this aspect of the present invention.
In certain cases the first liquid comprises DMSO, poly-lactide-co-glycolide co-polymer (“PLGA”) and nanoparticles, the nanoparticles being as defined in connection with the first aspect of the present invention.
In certain cases the second liquid may be as defined in WO2012/042274 (the contents of which is expressly incorporated herein by reference—see, e.g., pages 13 to 15 thereof). In certain cases the second liquid comprises water. The second liquid may comprise a water-alcohol mix (e.g. having a water:alcohol ration in the range 95:5 to 75:25). In particular cases the second liquid may comprise a mixture of water and tert-butanol (e.g. 85:15 water:tert-butanol).
In accordance with this aspect of the present invention the second liquid may have a temperature as described in WO2012/042274 (the contents of which is expressly incorporated herein by reference). In certain cases, the temperature of the second liquid may be within the range 1° C. to 10° C., such as 4° C. to 8° C.
In some cases the concentration of polymer in the first liquid may be at least 20%, at least 30% or at least 40% w/v.
In some cases the process of this aspect of the present invention employs an apparatus as described in WO2012/042274 (the contents of which is expressly incorporated herein by reference—see, e.g., pages 17 to 23 thereof). In some cases the process of this aspect of the present invention employs an apparatus as described in WO2012/042273 (the contents of which is expressly incorporated herein by reference—see, e.g., claims 1 to 47 thereof).
In some cases the process further comprises collecting the solid microparticles by separating the solid microparticles from the second liquid, optionally under vacuum. In certain cases, the step of collecting the microparticles employs a bead collection device as described in WO2013/014466 (the contents of which is expressly incorporated herein by reference—see, e.g., claims 1 to 30 thereof). The method of separating the solid microparticles may be as described in WO2013/014466 (the contents of which is expressly incorporated herein by reference—see, e.g., claims 32 to 35 thereof).
In some cases the process further comprises collecting the solid microparticles and formulating or packaging the microparticles into a pharmaceutical composition or delivery form. This may, in certain cases, comprise freeze-drying the microparticles and/or subjecting the microparticles to a sterilization step. In some cases the microparticles may be formulated into a liquid for delivery by depot injection.
In some cases the process of this aspect of the invention further comprises a preceding stage in which the nanoparticles are produced, the preceding stage comprising:
In certain cases the preceding stage of producing the nanoparticles further comprises conjugating or binding said biologically active agent to the nanoparticle. Where conjugation involves a covalently attached linker that conjugates the active to the nanoparticle this is advantageously achieve during the self-assembly process. In particular, the preceding stage of producing the nanoparticles may comprise providing a sulphur derivatised linker (e.g. thioalkyl, thioglycol or thioalkylpolyglycol) covalently attached to the agent and combining:
In some cases the sulphur derivatised linker covalently attached to the agent comprises thio alkyl and/or thio glycol linker covalently linked to the agent.
In some cases, particularly where the biologically active agent comprises a peptide or polypeptide, the nanoparticle may be produced first and brought into contact with a solution comprising the peptide or polypeptide and the peptide or polypeptide allowed to bind non-covalently to the nanoparticle corona.
In accordance with all aspects of the present invention the microparticles and methods for their production may be sterile. Additionally or alternatively, the nanoparticles and methods for their production may be sterile. However, it is specifically contemplated herein that the production of the nanoparticles and/or the production of the microparticles includes one or more sterilization steps.
In an eighth aspect the present invention provides a method for extending the in vivo half-life of a biologically active agent when administered to a mammalian subject, the method comprising:
In accordance with the present invention, particularly the fourth and fifth aspects thereof, the subject may be a human, a companion animal (e.g. a dog or cat), a laboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate), a domestic or farm animal (e.g. a pig, cow, horse or sheep). Preferably, the subject is a human.
The present invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided. These and further aspects and embodiments of the invention are described in further detail below and with reference to the accompanying examples and figures.
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
As used herein, “nanoparticle” refers to a particle having a nanomeric scale, and is not intended to convey any specific shape limitation. In particular, “nanoparticle” encompasses nanospheres, nanotubes, nanoboxes, nanoclusters, nanorods and the like. In certain embodiments the nanoparticles and/or nanoparticle cores contemplated herein have a generally polyhedral or spherical geometry.
Nanoparticles comprising a plurality of carbohydrate-containing ligands have been described in, for example, WO 2002/032404, WO 2004/108165, WO 2005/116226, WO 2006/037979, WO 2007/015105, WO 2007/122388, WO 2005/091704 (the entire contents of each of which is expressly incorporated herein by reference) and such nanoparticles may find use in accordance with the present invention.
As used herein, “corona” refers to a layer or coating, which may partially or completely cover the exposed surface of the nanoparticle core. The corona includes a plurality of ligands which generally include at least one carbohydrate moiety, one surfactant moiety and/or one glutathione moiety. Thus, the corona may be considered to be an organic layer that surrounds or partially surrounds the metallic core. In certain embodiments the corona provides and/or participates in passivating the core of the nanoparticle. Thus, in certain cases the corona may include a sufficiently complete coating layer substantially to stabilise the semiconductor or metal-containing core. However, it is specifically contemplated herein that certain nanoparticles having cores, e.g., that include a metal oxide-containing inner core coated with a noble metal may include a corona that only partially coats the core surface. In certain cases the corona facilitates solubility, such as water solubility, of the nanoparticles of the present invention.
Nanoparticles are small particles, e.g. clusters of metal or semiconductor atoms, that can be used as a substrate for immobilising ligands.
Preferably, the nanoparticles have cores having mean diameters between 0.5 and 50 nm, more preferably between 0.5 and 10 nm, more preferably between 0.5 and 5 nm, more preferably between 0.5 and 3 nm and still more preferably between 0.5 and 2.5 nm. When the ligands are considered in addition to the cores, preferably the overall mean diameter of the particles is between 2.0 and 20 nm, more preferably between 3 and 10 nm and most preferably between 4 and 5 nm. The mean diameter can be measured using techniques well known in the art such as transmission electron microscopy.
The core material can be a metal or semiconductor (said semiconductor optionally comprising metal atoms or being an organic semiconductor) and may be formed of more than one type of atom. Preferably, the core material is a metal selected from Au, Fe or Cu. Nanoparticle cores may also be formed from alloys including Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and may be used in the present invention. Preferred core materials are Au and Fe, with the most preferred material being Au. The cores of the nanoparticles preferably comprise between about 100 and 500 atoms (e.g. gold atoms) to provide core diameters in the nanometre range. Other particularly useful core materials are doped with one or more atoms that are NMR active, allowing the nanoparticles to be detected using NMR, both in vitro and in vivo. Examples of NMR active atoms include Mn+2, Gd+3, Eu+2, Cu+2, V+2, Co+2, Ni+2, Fe+2, Fe+3 and lanthanides+3, or the quantum dots described elsewhere in this application.
Nanoparticle cores comprising semiconductor compounds can be detected as nanometre scale semiconductor crystals are capable of acting as quantum dots, that is they can absorb light thereby exciting electrons in the materials to higher energy levels, subsequently releasing photons of light at frequencies characteristic of the material. An example of a semiconductor core material is cadmium selenide, cadmium sulphide, cadmium tellurium. Also included are the zinc compounds such as zinc sulphide.
In some embodiments, the nanoparticle or its ligand comprises a detectable label. The label may be an element of the core of the nanoparticle or the ligand. The label may be detectable because of an intrinsic property of that element of the nanoparticle or by being linked, conjugated or associated with a further moiety that is detectable. Preferred examples of labels include a label which is a fluorescent group, a radionuclide, a magnetic label or a dye. Fluorescent groups include fluorescein, rhodamine or tetramethyl rhodamine, Texas-Red, Cy3, Cy5, etc., and may be detected by excitation of the fluorescent label and detection of the emitted light using Raman scattering spectroscopy (Y. C. Cao, R. Jin, C. A. Mirkin, Science 2002, 297: 1536-1539). In some cases, the detectable label may comprise fluorescein isothiocyanate (FITC). In certain cases, the detectable label (e.g. FITC) may be covalently linked to the core of the nanoparticle, e.g. via a linker.
In some embodiments, the nanoparticles may comprise a radionuclide for use in detecting the nanoparticle using the radioactivity emitted by the radionuclide, e.g. by using PET, SPECT, or for therapy, i.e. for killing target cells. Examples of radionuclides commonly used in the art that could be readily adapted for use in the present invention include 99mTc, which exists in a variety of oxidation states although the most stable is TcO4−; 32P or 33P; 57Co; 59Fe; 67Cu which is often used as Cu2+ salts; 67Ga which is commonly used a Ga3+ salt, e.g. gallium citrate; 68Ge; 82Sr; 99Mo; 103Pd; 111In which is generally used as In3+ salts; 125I or 131I which is generally used as sodium iodide; 137Cs; 153Gd; 153Sm; 158Au; 186Re; 201Tl generally used as a Tl+ salt such as thallium chloride; 39Y3+; 71Lu3+; and 24Cr2+. The general use of radionuclides as labels and tracers is well known in the art and could readily be adapted by the skilled person for use in the aspects of the present invention. The radionuclides may be employed most easily by doping the cores of the nanoparticles or including them as labels present as part of ligands immobilised on the nanoparticles.
As used herein the term “biologically active agent” is intended to encompass drugs and pro-drugs that exert an effect on a biological system, preferably a therapeutic effect. Class of active agent contemplated herein include small molecule organic compounds, peptides, polypeptides and nucleic acids. Particular examples include: chemotherapeutic agents (e.g. temozolomide, irinotecan, chlorotoxin, carmustine, platinum(IV), platinum(II), camptothecin, doxorubicin, docetaxel Maytansine, Maytansinoids, monomethyl auristatin E (MMAE) and/or histone deacetylase (HDAC) inhibitors such as Panobinostat, Vorinostat, Romidepsin and Chidamide); peptides or polypeptides (e.g. insulin, GLP-1, amylin, exenatide, octreotide, teriparatide, glucagon, a cytokine, and/or an antibody); DNA or RNA (including, e.g. siRNA).
Microparticles in accordance with the present invention may be in the form of solid beads. As used herein in connection with microparticles or beads, solid is intended to encompass a gel. Microparticles as used herein specifically include any polymeric particle or bead of micron scale (typically up to 999 μm in diameter). Microparticles contemplated herein advantageously include the monodisperse polymeric beads obtainable by the process described in WO 2012/042274 (the entire contents of which are expressly incorporated herein by reference—see, e.g., claims 1 to 44 thereof).
Process for Encapsulating Nanoparticles within Microparticles
In certain cases the microparticles are produced by the Q Sphera® process as described in WO2012/042274 (the entire contents of which are expressly incorporated herein by reference—see, e.g., claims 1 to 44 thereof).
The microparticles and compositions of the invention may be administered to patients by any number of different routes, including enteral or parenteral routes. Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol), and rectal systemic routes.
Administration be performed e.g. by injection, especially depot injection.
The microparticles of the invention may be formulated as pharmaceutical compositions that may be in the forms of solid or liquid compositions. Such compositions will generally comprise a carrier of some sort, for example a solid carrier or a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0.1 wt % of the compound.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution or liquid which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.
In addition to one or more of the compounds, optionally in combination with other active ingredient, the compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, isotonicising agent, preservative or anti-oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g., intramuscular injection.
Preferably, the pharmaceutically compositions are given to an individual in a prophylactically effective amount or a therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful activity providing benefit to the individual. The actual amount of the compounds administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA); Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994. By way of example, and the compositions are preferably administered to patients in dosages of between about 0.01 and 100 mg of active compound per kg of body weight, and more preferably between about 0.5 and 10 mg/kg of body weight.
The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.
Gold nanoparticles having a corona of carbohydrate ligands or glutathione ligands were synthesised essentially as described previously (WO 2011/154711; and Lund et al., 2011, Biomaterials Vol. 32 pp. 9776-9784, the entire contents of which are expressly incorporated herein by reference).
To a suspension of galactose (3 g, 16.65 mmol) in 2-bromoethanol (30 ml), acid resin Amberlite 120-H is added to reach pH 2. The reaction is stirred for 16 hours at 50-60° C. The reaction mixture is filtered and washed with MeOH. Triethylamine is added to reach pH 8. The crude of the reaction is concentrated and co evaporated 3 times with toluene. The reaction mixture is dissolved pyridine (75 mL) and Ac2O (35 mL) and a catalytic amount of DMAP are added at 0° C. and stirred for 3 h at rt. The mixture is diluted with AcOEt and washed with 1.H2O; 2.HCl (10%) 3. NaHCO3 dis 4. H2O. The organic layer is collected and dried over anhydrous Na2SO4. TLC (Hexane:AcOEt 3:1, 2 elutions) shows a major product (desired) and a lower Rf minority. The product is purified by flash chromatography using the mixture hexane:ethyl acetate 6:1 as eluent and the 2-bromoethyl-alpha-galactoside (2) is obtained.
The product of the previous reaction, 2 is dissolved in 27 ml of 2-butanone. To this solution, a catalytic amount of tetrabutylammonium iodide and 4 equivalents of potassium thioacetate are added. The resulting suspension is stirred for 2 hours at room temperature. Throughout this period the reaction is tested by TLC (hexane-AcOEt 2:1, 2 elutions) for the disappearance of the starting material. The mixture is diluted with 20 ml of AcOEt and washed with a saturated NaCl solution. The organic phase is dried, filtered and evaporated under vacuum. The product is purified in hexane/AcOEt 2:1→1:1 to obtain the acetylthio-alpha-galactoside 3.
The new product of the reaction, 3 is dissolved in a mixture dichloromethane-methanol 2:1. To this mixture a solution of 1N sodium methoxide (1 equivalent) is added and stirred for 1 hour at room temperature. Amberlite IR-120H resin is added to achieve pH 5-6. The resulting mixture is then filtered and concentrated to dryness to obtain the final product (α-galactose C2SH).
To a solution of PPh3 (3 g, 11.4 mmol) in 20 ml dry THF, DIAC (2.3 g, 11.4 mmol) is added. The mixture is allowed to stir at 0° C. 15 min until the appearance of a white product. To this mixture a solution of hexaethyleneglycol (1.45 mL, 5.7 mmol) and HSAc (610 μl, 8.55 mmol) in dry THF (20 mL) is added dropwise (addition funnel). After 15 min the products begin to appear on TLC at Rf 0.2. The solution is concentrated in an evaporator. The crude of the reaction is dissolved in 50 ml of dichloromethane and washed with a solution of K2CO3 10%. The organic phase is dried over anhydrous Na2SO4, filtered and concentrated under vacuum. Flash chromatography of the crude using AcOEt:Hexane 1:1, AcOEt and finally DCM:MeOH 4:1 as eluyent gave the acetyl-thio-hexaethyleneglycol derivative.
The reaction product is dissolved in 5 ml of DMF and PPh3 (2.25 g, 8.55 mmol), NaN3 (0.741 g, 11.4 mmol) and BrCl3C (0,845 ml, 8.55 mmol) are added and the solution subsequently stirred for 40 min at room temperature. The resulting product has a higher Rf than the starting product when performing TLC (DCM:MeOH 25:1). The reaction mixture is diluted with 100 ml of diethylether and washed three times with H2O. The organic phase is dried over anhydrous Na2SO4, filtered and evaporated under vacuum. The product is purified by flash chromatography using the mixture of eluyents DMC/MeOH 200:1 and DCM/MeOH 40:1 to obtain the azido-acetylthio-hexaethyleneglycol derivative.
To remove the triphenyl phosphine oxide, the reaction product is dissolved in 10 ml of THF and 0.5 g of MgCl2 is added to this solution. The reaction is stirred for 2 h at 80° C. until a white precipitate appears and then is filtered through celite. The product is dissolved in a mixture of ethanol:H2O 3:1 and added Zn dust (0.45 g, 6.84 mmol) and NH4Cl (0.6 g, 11.4 mmol). The reaction was stirred at reflux for 1 h until the presence of starting material is no longer detectable by TLC (DCM/MeOH 25:1). The reaction is filtered through celite and the solvent is evaporated. The crude de reaction is diluted with AcOEt and extract with 5 ml H2O. The aqueous phase is evaporated to dryness to obtain the amino-thiol-hexaethylenglycol product.
Alpha-galactose C2 derivative 3 and hexaethyleneglycol amine linker 6 were taken from Midatech Biogune stock. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl), HAuCl4, NaBH4 were purchased from Sigma-Aldrich Chemical Company. Imidazole-4-acetic acid monohydrochloride was purchased from Alfa Aesar. Company High quality MeOH and Nanopure water (18.1 mΩ) were used for all experiments and solutions.
α-GalC2 (alpha)
Preparation of AL/α-Gal NPs: To a mix of amine-mercapto hexaethylenglycol linker 6 and alpha-galactose ligand 3 in a ratio 1:1 (0.58 mmol, 3 eq.) in MeOH (49 mL) was added an aqueous solution of gold salt (7.86 mL, 0.19 mmol, 0.025M). The reaction was stirred for 30 seconds and then, an aqueous solution of NaBH4 (1N) was added in several portions (4.32 mL, 4.32 mmol). The reaction was shaken for 100 minutes at 900 rpm. After this time, the suspension was centrifuged 1 minute at 14000 rpm. The supernatant is removed and the precipitated was dissolved in 2 mL of water. Then, 2 mL of the suspension were introduced in two filters (AMICON, 10 KDa, 4 mL) and were centrifuged 5 minutes at 4500 g. The residue in the filter was washed twice more with water. The final residue was dissolved in 80 mL of water.
For the preparation of gold NPs manufacture was under laminar flow cabinet. All glass and plastic material (such as eppendorfs, vials and bottles) and solvent (water, HAc) were first sterilized in an autoclave. All other disposables (such as tips and filters) came pre-sterilized.
Oxidized ligand, glutathione (Fluka 49741) was dissolved in 9:1 methanol:water and gold III chloride (Sigma-Aldrich, Poole, UK) added. The organic ligand was used at a fourfold molar excess relative to the gold. The solution was then mixed for 5 min gently on a flat-bed shaker. The nanoparticles were produced by reduction following the rapid addition of a 20 fold molar excess relative to the gold, of freshly made 1 M sodium borohydride (Sigma-Aldrich, Poole, UK) under vigorous vortexing. The samples were vortexed for a total of 30 s followed by a further 1 h gentle mixing on the flat bed shaker. As the nanoparticles are not soluble in methanol/water solvent, initial purification was by bench centrifugation, supernatant removal and dispersion of the nanoparticle pellet in water. Further purification was achieved by 4 water washes in 10 kDa vivaspin centrifugation devices (GE Healthcare). The gold concentration of all nanoparticle preparations was determined by a simple colorimetric assay. Briefly 10 μl of nanoparticle sample or 12 mg/ml gold standard (Fluka (Sigma-Aldrich, Poole, UK)) and blanks were digested with 30 μl of 50:50 water:aqua regia in an ELISA plate for 1 min, this was followed by addition of 150 μl of 2 M NaBr, the 405 nm absorbance was then measured immediately, the assay having excellent linearity over the 0-10 μg range.
Colloidal gold nanoparticles were prepared in-house using standard techniques.
Encapsulation of gold nanoparticles (GNPs) in PLGA microparticles (solid beads) was performed using the following three GNPs solutions:
As PLGA is insoluble in water, it is difficult to produce a stable solution of PLGA, DMSO and water. As a maximum, a mixture containing up to ˜10% water can be generated.
The present inventors therefore sought to obtain gold nanoparticles (GNPs) prepared in a DMSO-water mixed phase. Highly concentrated solution of GNPs are contemplated for use herein.
To prepare a solution of PLGA and GNPs, first a highly concentrated solution of PLGA in DMSO (60-80% w/v) was prepared. This was a linear PLGA (L/G 75:25) with a carboxylic acid terminus, molecular weight ˜10 kDaltons. This solution was then diluted by 33%-50% with one of the above GNP solutions, to yield a final polymer concentration of 40% w/v.
A solution of antisolvent (85:15 Water/tert-Butanol) was produced and cooled to ˜4° C. The solution was then placed in a piezoelectric droplet generating device (see WO2012/042273) and a stable droplet stream was produced. Droplets were allowed to fall into and react with the above antisolvent solution at a temperature of between 5-8° C. Rapid extraction reaction took place, producing solid microparticles of diameter ˜53 μm.
The solid microspheres were dried in vacuo using a Bead Harvester device (see WO2013/014466) and stored at 4° C.
In order to quantify the encapsulation efficiency, a spectrophotometric assay was undertaken using NP3 (which has a visible absorption at ˜520 nm). A serial dilution was prepared in acetonitrile. An accurate mass of GNP-containing microspheres was then dissolved in acetonitrile and the absorption of the solution quantified. Using this method, we determined that NP3 was successfully encapsulated with an entrapment efficiency of ˜96%.
Using UV-Vis spectrophotometry, we also determined that encapsulated samples of NP1 and NP2 had not aggregated during the process, i.e. no UV-Vis absorption bands were detected after dissolution of microspheres in acetonitrile. The present inventors contemplate loading & release studies making use of inductively coupled plasma mass spectrometry (ICPMS) techniques for quantification.
A quantity (3.5 mg) of PLGA microparticles encapsulating NP2 (i.e. glutathione nanoparticles) was treated with 200 μl 50% aqua regia and the microparticles solubilised visually to >50% (some particles could still be seen). The material was centrifuged and the supernatant tested for gold content using an in-house colorimetric assay.
30 μl supernatant gave 0.21 μg Au, which equates to 1.4 μg Au/3.5 mg PLGA-NP microparticle. Based on a theoretical maximum of 1.94 μg gold of NP2 if encapsulation efficiency had been 100%, these results represent 72% recovery. The present inventors conclude that a high level of nanoparticle incorporation into PLGA microspheres was demonstrated.
Further examples of nanoparticles have been encapsulated and characterised essentially as described in Example 2.
40% w/v RG752H (Resomer® RG 752 H, Poly(D,L-lactide-co-glycolide) acid terminated, lactide:glycolide 75:25, Mw 4,000-15,000) in DMSO with 0.25 mg/mL C2-glucose GNPs (9:1 DMSO:Water) successfully produced microspheres using a piezoelectric droplet generator device. The antisolvent was 15% v/v tertiary butanol at room temperature. An electron micrograph showing the microparticles can be seen in
The average diameter of the microparticles was 53.49 μm; Standard deviation 4.34 μm; CV 8.12%.
40% w/v RG752H in DMSO with 0.5 mg/mL C2-glucose GNPs (9:1 DMSO:Water) successfully produced microspheres using the piezoelectric droplet generator device. Antisolvent—15% v/v tertiary butanol at room temperature.
Average diameter—46.67 μm Standard deviation—2.11 μm CV—4.53%.
40% w/v RG752H in DMSO with 0.18 mg/mL Colloidal Gold (9:1 DMSO:Water) successfully produced microspheres using the piezoelectric droplet generator device. Antisolvent—15% v/v tertiary butanol at room temperature.
Average diameter—44.82 μm Standard deviation—3.35 μm CV—7.47%.
As colloid gold is detectable by UV spectrophotometry at 520 nm the concentration within the microspheres was analysed. It produced a loading of 0.174 mg/mL which produced an encapsulation efficiency of 96.4%.
FITC-PEG-SH-1kda GNPs c(Au) 0.587 g/1 were centrifuged and resuspended in 250 μL DMSO. Transferred to 33% w/v RG752H in DMSO formulation. Successfully manufactured with the piezoelectric droplet generator using 15% v/v tertiary butanol as the antisolvent.
Average diameter—59.4 μm Standard deviation—3 μm CV—5.1%
In 1 mg of GNP microspheres there were 2.18 μg/mL of the FITC-PEG-SH-1kda detected.
The successful encapsulation of nanoparticles coupled to a detectable label illustrates the provision of encapsulated, payload-coupled nanoparticles according to the present invention.
Glutathione-conjugated GNPs c(Au) 6.52 mg/mL were centrifuged for 30 minutes in amicon to remove the majority of water then the GNPs were resuspended in 200 μL of DMSO. 200 μL was then added to a 30% w/v RG752H formulation in DMSO. This was then used to manufacture microspheres using the piezoelectric droplet generator. 15% v/v tertiary butanol was used as the antisolvent.
Average diameter—49.36 μm Standard deviation—4.48 μm CV—9.07%
Nanoparticles were dissolved in aqua regia and a colorimetric assay used to measure gold concentration. The assay was used to produce a standard curve using the gold AAS standard solution read at 382 nm. A linear trend was shown between a HLQ of 600 μg/mL and an LLQ of 10 μg/mL (see
The microspheres produced a gold concentration of 0.8279 mg/mL. The formulation theoretical maximum—1.304 mg/mL gold concentration, which gives an encapsulation efficiency of 63.49%.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1420080.2 | Nov 2014 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/076364 | 11/11/2015 | WO | 00 |
