Methods of producing polymeric particles

Abstract

The present invention relates to a process for the preparation of particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer, wherein said process comprises the polymerisation of an oil-in-water emulsion, wherein said emulsion comprises (i) an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid, and (ii) an aqueous phase, wherein the oil soluble acid has an octanol-water partition coefficient (logP) above -0.5. The present invention also provides the above-mentioned process for the preparation of particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer further comprising preparing the oil phase. Particles produced according to said processes, pharmaceutical compositions comprising said particles and therapeutic methods are also provided.

Description

[0001] 171870 / 02

[0002] Methods of producing polymeric particles

[0003] The present invention relates to methods of preparing particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer comprising a therapeutically or diagnostically active agent and particles produced by such methods and their uses in medicine.

[0004] Background of the Invention

[0005] The use of nanotechnology in medicine offers many exciting possibilities with potential in a number of medicinal applications envisaged. In particular, nanomedicine is expected to lead to big improvements in the treatment of complex diseases. Two areas in which the use of nanoparticles has begun to demonstrate particular value are drug delivery and molecular imaging.

[0006] Particles, e.g. nanoparticles, for the delivery of therapeutic agents have the potential to circumvent many challenges associated with traditional delivery approaches, including lack of patient compliance to prescribed therapy, adverse side effects and poor clinical efficacy due to lack of targeted delivery. Important technological advantages of particles for drug delivery include the ability to deliver water-insoluble and unstable drugs, incorporation of both hydrophobic and hydrophilic therapeutic agents and the ability to utilise various routes of administration. Particle, e.g. nanoparticle, delivery systems may also facilitate targeted drug delivery and controlled release applications, enhancing drug bioavailability at the site of action, reducing dosing frequency and overall dosage size, thereby minimising side effects.

[0007] Polymeric nanoparticles (NPs) have received a great deal of attention in the field of medicine, in particular those comprising biodegradable polymers such as poly(lactic acid), poly(glycolic acid) and poly(alkyl cyanoacrylate) (PACA). Many methods for preparing nanoparticles are known, such as emulsion (including miniemulsion) polymerisation, self-assembly and nanoprecipitation. Anionic emulsion polymerisation is described in, for example, US 2008 / 0138418.

[0008] Miniemulsion processes are known for the production of nanoparticles with average sizes typically in the range 1-1000 nm, most typically 50-500 nm asdisclosed e.g. in Landfester in Macromol. Rapid Comm. 2001, 22, 896-936 and Landfester et al in Macromolecules 1999, 32, 5222-5228. The method was first described in 1972 by Ugelstad and Vanderhoff. The miniemulsion technique for the preparation of polymeric nanoparticles is a technology by which a dispersion is prepared, by converting a stable nanoemulsion of a dispersed phase in a continuous phase into a nanoparticle dispersion by polymerisation reactions. The technology involves mixing various components in the dispersed phase before emulsification with the continuous phase takes place, resulting in the production of an emulsion in which each droplet has an identical composition of active agent and monomers. All types of polymerisation reactions may be applied in these droplet nanoreactors.

[0009] The particles formed are typically identical or almost identical to the droplets from which they are prepared, in terms of size and size distribution, resulting in high reproducibility of the process.

[0010] Emulsions are usually stabilised by a surfactant and a co-stabiliser. The costabiliser contributes to the osmotic stabilisation of the emulsion by increasing the osmotic pressure, which counteracts the capillary or Kelvin pressure due to surface tension of the droplets and reduces Ostwald ripening by minimising diffusion of the monomer from small to large droplets.

[0011] Effective methods of preparing PACA NPs include single step anionic polymerisation of an oil in water miniemulsion, as described in WO 2014 / 191502. For example, effective drug delivery has been shown using a PACA based NP prepared in this way which contains encapsulated cabazitaxel (WO 2019 / 185685 and WO 2020 / 192950).

[0012] However, many active agents (APIs) are difficult to encapsulate due to poor solubility in alkyl cyanoacrylate monomers and / or due to instability of the monomer plus API formulation.

[0013] Alkyl cyanoacrylate (ACA) monomers are very reactive and polymerise very easily in the presence of radical and / or anionic initiators. A side effect of the reactivity of alkyl cyanoacrylate monomers is that they are vulnerable to premature polymerisation (either anionic or radically initiated) caused by structural features of the API. The structural features include for instance M-NH3 (wherein M is a metal, i.e. a metal-ammonia complex), R-NH2, R1R2-NH, R1R2R3N, RI=NR2, R-OH, R-CI, R-Br, R-l, R-SH, RIN=NR2, R-NHOH, structurally associated H2O and enols (whereR, Ri, R2 and R3, following convention, refer to the rest of the molecule, moieties attached to the named atom(s), which may be the same or different).

[0014] This premature polymerisation severely limits the range of active ingredients that can be dissolved into ACA monomers prior to polymerisation and has significantly reduced the potential of PACA particles. To avoid premature polymerisation of the alkyl cyanoacrylate during a miniemulsion polymerisation it is known to add an anionic or radical inhibitor, such as methane sulphonic acid (MSA, anionic inhibitor) and butylated hydroxytoluene (BHT, radical inhibitor). MSA prevents polymerisation of the ACA by adjusting the pH of the polymer droplet and preventing the formation of anions that trigger polymerisation. This is achieved either by neutralisation of an anion (via donation of a proton) to a neutral molecule, or formation of a cation (via donation of a proton) thus preventing nucleophilic attack by a lone pair of electrons (e.g. on nitrogen). These inhibitors do however have various disadvantages. For instance, MSA is a strong acid that may decompose acid labile compounds (for instance active pharmaceutical ingredients, i.e. API’s) while neither MSA nor BHT are suitable for use in a clinical setting due to their toxicity.

[0015] A newer approach to inhibit premature polymerisation is disclosed in WO 2020 / 039081, in which compounds such as vanillin act as a combined anionic and radical inhibitor. These inhibitors act as electrophiles but not acids (i.e. do not donate a proton). However, this system is still not able to produce nanoparticles with all drugs of interest, in particular some hydrophilic drugs are hard to formulate with this approach.

[0016] There remains a need for new ways to produce PACA particles with hard to encapsulate drugs, in particular drugs with reactive nucleophilic groups. It is also an objective, where necessary, to enhance solubility of the API and / or add greater flexibility to the method of manufacture by allowing for the oil phase of an emulsion to be sufficiently stable to mean it need not be used immediately in the emulsion forming step.

[0017] Summary of the invention

[0018] The present inventors have devised a convenient method whereby difficult to formulate APIs can surprisingly be formulated into PACA particles through controlling / lowering the pH in the oil phase of the emulsion formed during particle production.The present invention provides a process for the preparation of particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer, wherein said process comprises the polymerisation of an emulsion, wherein said emulsion comprises:

[0019] (i) an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid; and

[0020] (ii) an aqueous phase.

[0021] The particles are preferably 50 nm to 10000 nm in diameter and may be termed “nanoparticles”.

[0022] In one aspect, the process described above further comprises preparing the oil phase through mixing the at least one species of alkyl cyanoacrylate monomer, active agent and oil soluble acid.

[0023] Thus, the present invention further provides a process for the preparation of particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer which comprise an active agent, wherein said process comprises preparing an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid and forming an emulsion between said oil phase and an aqueous phase, followed by polymerisation of said emulsion to form said particles.

[0024] In preferred embodiments, the emulsion is an oil-in-water emulsion.

[0025] In an embodiment, the oil soluble acid is present in an amount of 0.01 to 10 wt%, 0.01 to 5 wt% or 0.01 to 2 wt%.

[0026] The nucleophilic group(s) on the API may be what is known in the art as weak nucleophiles, for example metal complexes. In some embodiments, the active agent has one or more nucleophilic groups selected from the group consisting of an amine, a hydroxyl, a sulfhydryl, a carboxyl, a phosphate ester and a phosphonate or phosphinate species.

[0027] In some embodiments, the oil soluble acid is present in an amount of 0.001 to 15 wt% of the oil phase of the emulsion. The oil phase comprises the non-aqueous components of the emulsion, including the at least one alkyl cyanoacrylate monomer.

[0028] In some embodiments, the oil soluble acid has an octanol-water partition coefficient (logP) in the range of -0.5-3.0, preferably in the range of 0.0-3.0, e.g. in the range of 1.0-2.0.In an embodiment, the active agent is a therapeutic agent or a diagnostic, e.g. an imaging agent. In further embodiments, the active agent may be selected from the group comprising aroma compounds, flavours, fragrances, dyes, nutraceuticals, herbicides and pesticides.

[0029] The emulsion formed in the process is preferably a miniemulsion as described herein and known in the art.

[0030] In embodiments the oil phase and / or the aqueous phase, preferably the aqueous phase, further comprises a surfactant. Suitable surfactants include polyalkylene glycols such as polyethylene glycols (PEG), polypropylene glycols (PPG) and mixtures thereof. More than one surfactant, e.g. two surfactants, may be present.

[0031] The polymerisation is preferably anionic polymerisation and in some embodiments the surfactant initiates the anionic polymerisation reaction. Thus in preferred embodiments, the processes of the present invention employ anionic polymerisation of an oil-in-water emulsion, preferably an oil-in-water miniemulsion.

[0032] Thus, in some embodiments, the emulsion may comprise an anionic polymerisation initiator, i.e. an initiator able to start the anionic polymerization of the at least one alkyl cyanoacrylate monomer. Preferably, the anionic polymerisation reaction is initiated by the at least one surfactant.

[0033] In some embodiments, when the process includes preparing the oil phase (e.g. through mixing the at least one species of alkyl cyanoacrylate monomer, active agent and oil soluble acid), said preparation step (or mixing step), including the time prior to formation of the emulsion, is at least 15 minutes, preferably 30 minutes to 24 hours, more preferably 30 minutes to 6 hours.

[0034] In a further aspect, the present invention provides a formulation comprising at least one species of an alkyl cyanoacrylate monomer, an active agent having one or more nucleophilic groups and an oil soluble acid. Other aspects of the invention apply mutatis mutandis to this aspect. Said formulation preferably has the active agent dispersed throughout and is preferably a homogenous mixture. Said formulation may comprise and be used for the “oil phase” in the emulsions described elsewhere herein.

[0035] Thus in a further aspect, the invention provides an emulsion, preferably an oil-in-water emulsion, wherein said emulsion comprises:(i) an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid; and

[0036] (ii) an aqueous phase.

[0037] Particles produced by the processes of the invention as described herein constitute a further aspect of the invention.

[0038] In a further aspect, the present invention provides a pharmaceutical composition comprising a particle according to the invention, or a particle obtained by the process according to the invention, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0039] In a further aspect, the present invention provides a particle according to the invention, or a particle obtained by the process according to the invention, for use in medicine, preferably for use in drug delivery or molecular imaging.

[0040] The present invention further provides a particle of a poly(alkyl cyanoacrylate) homopolymer or copolymer as described herein which comprises a sulfonate, carboxylate or phosphate salt of an active agent, preferably a docusate, 4-Dodecylbenzenesulfonate acetate, decanoate, dodecanoate or D-a-Tocopherol succinate salt of an active agent as described herein.

[0041] Emulsion

[0042] An emulsion is a mixture of two or more immiscible liquids. Typically, in accordance with the present invention, the emulsion is stabilised by one or more surfactants.

[0043] Emulsions may be direct (oil-in-water) or inverse (water-in-oil). In the emulsions of the invention, oil-in-water emulsions are preferred and therefore the aqueous phase preferably forms the continuous phase.

[0044] In the processes described herein, the active agent is preferably encapsulated in the particle (e.g. nanoparticle).

[0045] In preferred embodiments, the emulsion is a miniemulsion. A miniemulsion may be prepared by any known method in the art, such as that described in US 2009 / 0297613. Processes typically involve forming the oil and water phases, mixing these and subjecting the mixture to high shear forces, e.g. ultrasonication or homogenisation, to form a stable (nano)emulsion of oil droplets containing the monomer with a stabiliser / surfactant on the surface. In other embodiments, anemulsion may be formed by simple mixing, without use of energy intensive methods. Polymerisation of the monomer droplets then occurs by initiation at the droplet interface to form a dispersion of polymeric nanoparticles which have the same size as the droplets did before polymerisation. Miniemulsion processes are known for the production of nanoparticles with average sizes typically in the range 1-1000 nm, most typically 50-500 nm. As polymerisation occurs within the original monomer droplets during miniemulsion processes, this advantageously allows good control over particle size and composition.

[0046] Alternatives to so-called ‘miniemulsion’ processes include those described in WO 2012 / 131018 (see Example 1 therein describing aqueous emulsion polymerisation to form loaded PACA nanoparticles). For example, a surfactant may be used in an aqueous phase to produce micelles and then the oil phase is added (e.g. drop wise) and the oil phase associates with the micelles and polymerisation takes place to form the particles.

[0047] Surfactant

[0048] The emulsions of the invention may comprise at least one surfactant. The emulsion may comprise the surfactant in the oil phase or in the aqueous phase, preferably in the aqueous phase. Thus, in preferred embodiments, the emulsions do not comprise a surfactant in the oil phase.

[0049] Any typical surfactant known in the art may be used, suitable surfactants include fatty acids of glycerols, sorbitol and other multifunctional alcohols, poloxamers, poloxamines, polysorbates, polyoxyethylene ethers and polyoxyethylene esters, ethoxylated triglycerides, ethoxylated phenols and diphenols, polysaccharides (e.g. hyaluronic acid and sialic acid), proteins, metal salts of fatty acids, metal salts of fatty alcohol sulfates, sodium lauryl sulfate, metal salts of sulfosuccinates and mixtures thereof. Non-ionic surfactants are preferred.

[0050] The surfactant preferably comprises 0.05 to 5 wt% of the aqueous phase of the emulsion, e.g. miniemulsion, more preferably 0.1 to 2 wt%.

[0051] In addition to these components, the emulsion may further comprise a costabiliser in the oil phase. The co-stabiliser is typically highly water insoluble, i.e. has a solubility of less than 5x10'5mol / L, more preferably less than 5x10'6mol / L and still more preferably less than 5x10'7mol / L and may be any substance which iscompatible with the polymerisable monomer(s), such as a hydrocarbon, silane, organosilane, fatty acid ester, oil (e.g. plant oil), hydrophobic dye or lipid. Examples of suitable co-stabilisers include hexadecane, cetyl alcohol, miglyol and olive oil. Particularly preferred co-stabilisers include miglyols and plant oils.

[0052] The co-stabiliser preferably comprises 0.5 to 5 wt% of the oil phase of the emulsion, more preferably 1 to 3 wt%.

[0053] The oil phase content of an emulsion, of the invention may be up to 50%, but is typically up to 15-25 wt%, preferably up to 15 wt% or in the range of 5-15 wt%.

[0054] One preferred class of surfactants for use in the present invention are polyalkylene glycols. The emulsions of the present invention may comprise at least one polyalkylene glycol selected from the group consisting of polyethylene glycols (PEG) and polypropylene glycols (PPG) or mixtures thereof. At least one of these polyalkylene glycols may initiate the anionic polymerisation reaction.

[0055] The polyalkylene glycols are usually added to the continuous phase of the emulsion, i.e. the aqueous phase. One of the polyalkylene glycols may in some instances be covalently attached to a targeting moiety. It is especially preferred if the polyalkylene glycols are so water-soluble that a homogeneous solution may be prepared.

[0056] By the term "polyethylene glycol" (PEG) we mean any polymer containing mostly ethylene oxide repeating units, i.e. -CH2-CH2-O- units. Typical polyethylene glycols have a molecular mass less than 20000 g / mol, preferably less than 10000 g / mol. By the term "polypropylene glycol" (PPG) we mean any polymer containing mostly propylene oxide repeating units, i.e. -CH2-CH2-CH2-O- units.

[0057] The polyalkylene glycols may have a hydroxy or amino end group, or a mixture thereof. The polyalkylene glycols are water soluble. By water soluble we mean that they must have a solubility in water which is high enough to enable the formation of a homogenous solution in water, which may then be added to the emulsion, i.e. a solubility of more than 10 g / L at room temperature and pressure (RTP).

[0058] Examples of suitable polyalkylene glycols include polyethylene glycol and polypropylene glycol homopolymers and copolymers thereof (e.g. poloxamers). It should be noted that the term "polyethylene glycol" is intended to cover polysorbates (e.g. polysorbate 80). Most importantly, the copolymers may be block copolymers. Examples of suitable copolymers include polypropylene glycol)-poly(ethylene glycol) block copolymers, polyalkylamine - polyalkylene glycol blockcopolymers, lipid - polyalkylene glycol block copolymers and polylysine -polyalkylene glycol block copolymers.

[0059] The length of the blocks of each polymer may be varied so as to alter the properties of the copolymer, with the proviso that the copolymer remains water soluble. Increasing PPG content for example, reduces water solubility. In one embodiment, there is preferably a hydroxyl or amino end group directly attached to the PPG. Preferably the end group is an amino end group.

[0060] Preferably, the ratio of polyethylene glycol units to PPG units is in the range 1 :5 to 5: 1 , such as 1:1. Each block may contain 2 - 40 monomer units. It is further preferred however if polyethylene glycol units are in excess.

[0061] Typical molecular weights for the block copolymers are lower than 20000 g / mol, preferably lower than 10000 g / mol, such as 1000 to 8000.

[0062] A polyethylene glycol may further comprise a hydrophobic component so as to optimise properties and enable efficient hydrophobic interaction with the monomers in the oil phase. Preferably the hydrophobic component is attached covalently to the polyethylene glycol, most preferably between the amino or hydroxyl end group and the rest of the polyethylene glycol moiety.

[0063] The hydrophobic component is typically an alkyl chain, polyether or a lipid. A particularly preferred hydrophobic component is polypropylene oxide (PPG) thus forming a polyethylene glycol / polypropylene glycol block copolymer. It should be understood that PPG is equivalent to PPO.

[0064] Preferably, the amount of polyalkylene glycol(s) (in total) is 0.05-5 wt%, more preferably 0.1-2 wt% of the water phase of the emulsion.

[0065] The Examples show use of Kolliphore surfactants.

[0066] Organic solvents are typically organic compounds capable of dissolving one or more other substances. Preferably, the emulsions of the invention do not comprise organic solvents, for example do not comprise water-soluble organic solvents in the aqueous phase.

[0067] Polymerisable monomers

[0068] The polymerisable monomers of use in the present invention comprise at least one alkyl cyanoacrylate monomer. These are monomers whose use in the preparation of particles, e.g. nanoparticles, has been widely reported. The alkyl cyanoacrylate may be a monofunctional or difunctional acrylate i.e. containingsingle or multiple acrylate functionalities. Any straight or branched chain alkyl cyanoacrylate monomer or derivative thereof may be used, however preferred monomers are those of C1-C10 alkyl cyanoacrylates, more preferably C2-C8 alkyl cyanoacrylates. A single monomer may be used or mixtures of different alkyl cyanoacrylates may be used. Preferred alkyl cyanoacrylates include ethyl cyanoacrylate, butyl (n-butyl) cyanoacrylate, 2-ethylbutyl cyanoacrylate, 2-ethylhexyl cyanoacrylate, n-octyl cyanoacrylate, 3-methylbutyl cyanoacrylate, 1-heptyl cyanoacrylate, 2-heptyl cyanoacrylate, 3-heptyl cyanoacrylate, 2-phenyl ethyl cyanoacrylate, neopentyl cyanoacrylate, 1 -pentyl cyanoacrylate, 2-pentyl cyanoacrylate, 3-pentyl cyanoacrylate, 3,3-dimethyl-1 butyl cyanoacrylate and derivatives and mixtures thereof. 2-ethylbutyl cyanoacrylate, 2-ethylhexyl cyanoacrylate, 3-methylbutyl cyanoacrylate, 1 -heptyl cyanoacrylate and 2-heptyl cyanoacrylate are particularly preferred.

[0069] In some embodiments, a cyanoacrylate homopolymer is used, i.e. formed from a single monomer species. Alternatively, a mixture of alkyl cyanoacrylates of differing chain length, e.g. one with a short alkyl chain and one with a long alkyl chain such as butyl cyanoacrylate mixed with 2-ethylbutyl cyanoacrylate or n-octyl cyanoacrylate may be used.

[0070] The alkyl cyanoacrylate monomers are preferably present in an amount of 1 to 100 wt%, more preferably 75-100 wt%, even more preferably 95-100 wt% of the total amount of monomers.

[0071] In addition to the alkyl cyanoacrylate monomers, other co-monomers may also be present. It is preferable if these co-monomers are also biocompatible or biodegradable. Suitable co-monomers include, but are not limited to acrylates, vinyl esters, vinyl ethers, vinyl epoxides, cyclic siloxanes and lactones.

[0072] The polymerisable monomers preferably comprise 25 to 99.5 wt% of the oil phase of the emulsion, e.g. miniemulsion, more preferably 30 to 70 wt%.

[0073] Active Agent

[0074] The active agents of use in the present invention comprise one or more nucleophilic groups. As is well understood in the art, the term “nucleophilic group” refers to a group which is able to donate a pair of electrons to form a new covalent bond.The nucleophilic group(s) on the API may be what is known in the art as weak nucleophiles, for example metal complexes. In some embodiments, the active agent has one or more nucleophilic groups selected from the group consisting of an amine, a hydroxyl, a sulfhydryl, a carboxyl, a phosphate ester and a phosphonate or phosphinate species.

[0075] Active agents may be hydrophilic in their natural state, i.e. when first added to the oil phase. Through formation of a complex with the oil soluble acid, the active agent may be rendered hydrophobic and thus exhibit improved solubility in the oil phase. Likewise, the character of the nucleophilic group(s) possessed by an active agent in its standard state, pre-formulation, may be altered during the processes of the present invention, e.g. through association with components present in the emulsion, in particular the oil soluble acid.

[0076] In some embodiments, the active agent and the oil soluble acid are mixed before they are added to the alkyl cyanoacrylate monomer(s). This may improve solubility of the active agent in the monomer.

[0077] The active agent may be any agent which has a medicinal application, e.g. therapeutic agents or diagnostic agents such as imaging agents, as well as any other compound of interest for nanoencapsulation, such as, but not limited to, aroma compounds, flavours, fragrances, dyes, nutraceuticals, herbicides and pesticides. In a preferred embodiment, the active agent comprises 1 to 75 wt% of the oil-phase of the emulsion, e.g. miniemulsion, more preferably 10-30 wt%.

[0078] Exemplary therapeutic agents include chemotherapeutic agents, cytostatic drugs, antineoplastic agents, prophylactic agents, nutraceutical agents, antibiotics, antiviral agents, antiinflammatory agents, small molecule kinase inhibitors, nucleic acids, proteins, peptides, lipids, carbohydrates, hormones, metals, ceramics, vaccines, immunological agents, and mixtures thereof. Preferred agents include anticancer drugs such as cytostatic drugs or imaging agents such as dyes, e.g. fluorescent dyes. The active agent is preferably not a preservative (e.g. antimicrobial) agent, and particularly preferably not hydroquinone. Active agents of particular interest are NR668, DIR, Trametinib, Regorafenib, Topotecan, Monomethyl Auristatin E, Pirtobrutinib and SNX281.

[0079] The present invention provides a method of formulating active agents featuring functional groups that may initiate premature polymerisation of alkyl cyanoacrylate monomers in the emulsion.

[0080]

[0081] Preferably, polymerisation according to the invention is an anionic polymerisation and / or the polymerisation is performed in a single step. Thus, in a particularly preferred embodiment the polymerisation is an anionic polymerisation and is performed in a single step (e.g. a single step anionic polymerisation).

[0082] Anionic emulsion polymerisation is described in, for example, US 2008 / 0138418.

[0083] An emulsion is typically prepared by adding an oil phase containing the alkyl cyanoacrylate monomer(s), the active agent and an oil soluble acid to an aqueous solution optionally comprising a surfactant and subjecting this to high shear forces, e.g. by ultrasonication, to form oil droplets containing the monomer(s) in water. In other processes, high shear forces may not be required to form an emulsion on mixing. The presence of the oil soluble acid prevents a premature polymerisation of the monomer caused by. e.g. a reactive active agent.

[0084] Poly(alkyl cyanoacrylate) nanoparticles may be synthesized by the following method, which is also used in the Examples hereto:

[0085] 1) The substance to be encapsulated (e.g. an active agent or a dye) is mixed with an oil soluble acid and optionally a co-stabilizer (e.g. Miglyol), and solubilized in an alkyl cyanoacrylate monomer, forming the oil phase.

[0086] 2) An acidic water phase (e.g. an aqueous phase) is prepared, consisting of e.g. 0.1 M HCI and amphiphilic stabilizers (e.g. non-ionic PEG-based stabilizers / surfactants such as Kolliphore HS15 and Kolliphore P338).

[0087] Optionally, the surfactant can be added to the oil phase.

[0088] 3) An oil-in-water pre-emulsion is formed by mixing the oil phase and the aqueous phase.

[0089] 4) An emulsion is then formed by subjecting the mixture to high shear forces, e.g. by ultrasonication.

[0090] 5) Nanoparticles are formed by the in-situ polymerization of the oil droplets, initiated by nucleophilic moieties (anionic polymerization) and / or radicals (radical polymerization) present in the water phase or oil phase (or both). The reaction is completed within 12-48 hrs on stirring at room temperature.Such processes are well known in the art and hence the mechanisms involved will be well known to the skilled person.

[0091] Thus the processes of the invention may employ anionic or radical polymerisation, preferably anionic polymerisation.

[0092] The aqueous phase is preferably acidic, e.g. with a pH less than 6, 5, 4, 3 or 2.

[0093] Oil soluble acid

[0094] The following discussion describes the acids of use in the invention in terms of their lipophilicity, how oil soluble they are, and also how acidic they are.

[0095] As well as selecting the innate characteristics of the acids, the inventors have also determined that the molar amount of the acid (acidic / proton-donating groups) relative to the amount of the active agent (nucleophilic groups therein) impacts the success of the processes of the invention. Thus, oil soluble acids for use in the present invention have a molar amount of acidic groups that is equal to or in excess (preferably in excess) of the molar amount of nucleophilic groups in the active agent in the oil phase, preferably at least 1.5 to 5 (e.g. 2-5) molar equivalents in excess. Without wishing to be bound by theory, it is believed that an excess of protons in the oil phase causes the nucleophilic groups on the API to be unreactive, i.e. inhibits their ability to act as initiators of polymerisation.

[0096] Preferred oil soluble acids for use in the present invention have a pKa of at least -2.0, such as between -2.0 and 6, such as between -1.8 and 6, e.g. 0 and 6. For a polyprotic acid, all pKas are preferably at least -2.0, such as between -2.0 and 6, such as between -1.8 and 6, e.g. 0 and 6.

[0097] pKa can be determined by several methods, such as the in silico pKa prediction tool MoKa (https: / / www.moldiscovery.com / software / moka / ) as described in Milletti F, et al., Eur J Med Chem. 2010 Sep;45(9):4270-9.

[0098] The oil soluble acids for use in the present invention preferably have an octanol-water partition coefficient (logP) above -0.5, such as in the range of -0.5-3.0, preferably in the range of 0.0-3.0. These logP values ensure that the acid used in the processes according to the invention have an optimal distribution throughout the emulsion, i.e. sufficient inhibitor is present in the oil phase.

[0099] LogP can be determined by several methods, including shake flask, pH-Metric, reverse phase HPLC and electrochemical, or calculated by use ofappropriate software. A preferred method of measuring LogP is using an in silica LogP prediction tool such as ACD / Labs partition coefficients LogP prediction software (https: / / www.acdlabs.com / products / percepta-platform / physchem- suite / logp / ). Further standard approaches of measuring LogP are the shake flask method (as described in OECD GUIDELINE FOR THE TESTING OF CHEMICALS, Test Guideline No. 107, adopted 27.07.95, Partition Coefficient (n-octanol / water): Shake Flask Method) or the HPLC method (as described in OECD GUIDELINE FOR THE TESTING OF CHEMICALS, Test Guideline No. 117, adopted 13.04.14, Partition Coefficient (n-octanol / water): High Performance Liquid Chromatography (HPLC) Method).

[0100] The acids of use in the invention are added to the oil phase of the emulsion. Preferably the aqueous phase of the emulsion does not comprise an organic acid, e.g. an oil soluble acid of use in the invention.

[0101] Preferred oil soluble acids include organic acids, preferably a carboxylic, sulfonic or phosphoric acid, preferably a carboxylic or sulfonic acid. 4-Dodecylbenzenesulfonic acid, decanoic acid, dodecanoic acid, D-a-Tocopherol succinate, docusic acid, acetic acid, 2-Naphthalene sulfonic acid, Hexadecylphosphoric acid, Pamoic acid, Sodium decanesulfonic acid, Sodium dodecyl benzenesulfonic acid, Sodium dodecyl sulfonic acid and Sodium tetradecyl sulfonic acid are particularly preferred, docusic and acetic acid are especially preferred.

[0102] In some embodiments, the oil soluble acid is an organic acid with three or more (at least three) carbon atoms, for example between 3 and 40 carbon atoms, preferably between 10 and 35 carbon atoms.

[0103] In some embodiments, the oil soluble acid is not acetic acid.

[0104] Particles

[0105] Particles according to the present invention may have a largest dimension, preferably diameter, of equal to or less than 10000 nM, preferably from 50 nm to 10000nm, such as from 50 nm to 5000 nm, 70 nm to 3000 nm, 70 nm to 1000 nm, 50 nm to 750 nm or 50 nm to 500 nm, more preferably from 70 to 500 nm, e.g. 90-500nm.

[0106] In preferred embodiments, the particles according to the present invention are nanoparticles. As used herein, the term “NP” means nanoparticle. As such, the term “PACA NPs” means poly(alkyl cyanoacrylate) nanoparticles. As used herein,the term “nanoparticle” means any particle having a largest dimension, preferably diameter, of less than 1000 nm, such as from 1 nm to 1000 nm. Preferably, the term “nanoparticle” means any particle having a largest dimension of from 70 to 500 nm.

[0107] The particle size is influenced by a number of factors, including the amount of surfactant present, the viscosity of the system as a whole and the shear rate used to produce the droplets. Typical particle size distribution curves (measured using, for example, dynamic light scattering) for miniemulsions are Gaussian in shape and are relatively narrow.

[0108] The particles of the present invention preferably have a polydispersity index (PDI) of 0.3 or less, more preferably 0.2 or less, such as about 0.1.

[0109] The polydispersity index is defined in context of measurement of particle size by dynamic light scattering as given by Malvern Panalytics (ref.

[0110] https: / / www.rnalvernpanalytical.com / en / learn / knowledqe- center / whitepapers / WP111214DLSTermsDefined.html), and is the number calculated from a 2-parameter fit to the correlation data (cumulants analysis).

[0111] Calculations for this are defined in ISO standard documents ISO 13321:1996 E, and ISO 22412:2008.

[0112] Nanoparticles made by emulsion interfacial polymerisation are typically vesicular systems, which contain the active agent in the core of the nanoparticle, surrounded by a polymer shell.

[0113] The present invention provides a particle obtained by the process according to the invention, e.g. a particle of a poly(alkyl cyanoacrylate) homopolymer or copolymer obtained by polymerisation of an emulsion, wherein said emulsion comprises

[0114] (i) an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid; and

[0115] (ii) an aqueous phase.

[0116] The present invention further provides a nanoparticle of a poly(alkyl cyanoacrylate) homopolymer or copolymer which comprise a sulfonate, carboxylate or phosphate salt of an active agent, preferably a docusate salt, a 4-Dodecylbenzenesulfonate salt, an acetate salt, a decanoate salt, a dodecanoate salt or a D-a-Tocopherol succinate salt of an active agent.The processes of the present invention may comprise a further step wherein the particles are isolated, e.g. from the aqueous phase. This may be carried out by any known method in the art.

[0117] The particles, e.g. nanoparticles, provided by, and produced in accordance with, the current invention may be formulated as a pharmaceutical composition comprising the particles together with one or more pharmaceutically acceptable carriers, diluents or excipients. Such carriers, diluents and excipients are well known in the art. The pharmaceutical compositions may also comprise additional active agents. Importantly, the particles according to the invention are free of toxic inhibitors, such as MSA and BHT, which would render them unsuitable for clinical / pharmaceutical use.

[0118] Uses

[0119] The particles, e.g. nanoparticles, and compositions thereof may be used in medicine, in particular in drug delivery and imaging applications. In addition, the particles may also be highly advantageous in nanoencapsulation of various materials / compounds, such as aroma compounds, fragrances, flavours, dyes, nutraceuticals, herbicides and pesticides. Hence, the present invention relates to particles, e.g. nanoparticles, according to the present invention for use in medicine and for use in nanoencapsulation of various materials / compounds, such as aroma compounds, fragrances, flavours, dyes, nutraceuticals, herbicides and pesticides.

[0120] The nanoparticles and compositions thereof may be used in medicine, in particular in drug delivery and imaging applications. Hence, the present invention relates to particles, e.g. nanoparticles, according to the present invention for use in medicine. In a further embodiment, the present invention relates to the particles, e.g. nanoparticles, according to the current invention for use in the treatment or prevention, or the diagnosis of particular disorders and diseases. Examples of disorders or diseases which can be treated or prevented in accordance with the present invention include infections, inflammatory diseases, neurodegenerative diseases, auto-immune diseases, cardiovascular diseases, and cancer, such as lung cancer, breast cancer, prostate cancer, brain cancer, head and neck cancer, ovarian cancer, skin cancer, testicular cancer, pancreatic cancer, colorectal cancer, kidney cancer, cervical cancer, gastrointestinal cancer, bladder cancer andcombinations thereof. In some embodiments the purpose is not to treat or prevent a microbial infection.

[0121] In an alternative embodiment, the invention provides a method for treating a disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of the particles, e.g. nanoparticles, and compositions thereof. Exemplary diseases or disorders are described elsewhere herein.

[0122] In an alternative embodiment, the invention provides the use of the particles, e.g. nanoparticles, or compositions thereof in the manufacture of a medicament for use in therapy, e.g. for use in the treatment of disease or disorder. Exemplary diseases or disorders are described elsewhere herein.

[0123] The particles or compositions thereof are preferably administered in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount of the particles necessary to treat or prevent the particular disease or disorder. Any route of administration may be used to deliver the particles to the subject. Suitable administration routes include intramuscular injection, transdermal administration, inhalation, topical application, oral administration, rectal or vaginal administration, intratumural administration and parenteral administration (e.g. intravenous, peritoneal, intra-arterial or subcutaneous). The preferable route of administration is injection.

[0124] The exact dosage and frequency of administration depends on the particular particles, active agent and optional targeting agents used, the particular condition being treated, the severity of the condition being treated, the age, weight, sex, extent of disorder and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the particles according to the instant invention.

[0125] In the following non-limiting Examples, preparation of particles using the processes according to the present invention are described.Examples

[0126] To achieve a reduction in pH in the oil phase of the emulsion, an organic acid was added, in a molar excess to the molar equivalent of reactive groups in the (active pharmaceutical ingredient) API, to the alkyl cyanoacrylate (ACA) monomer before the introduction of the API to be encapsulated.

[0127] Method of producing nanoparticles containing a fluorescent dye (DIR)

[0128] Method

[0129] The alkyl cyanoacrylate (“oil”) phase was prepared by adding docusic acid (0.21 g, 0.50 mmol) and Miglyol 812n (0.06 ml) to an alkyl cyanoacrylate monomer, 2-ethylbutyl cyanoacrylate (2-EBCA) (0.60 ml) and then combining with 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DIR, 10% in Miglyol 0.013 ml, 0.0013 mmol) (the API).

[0130] The same procedure was followed in parallel but omitting the docusic acid. The aqueous phase was prepared by dissolving Kolliphor HS15 (0.034 g) and Kolliphore P338 (0.068 g) in 0.1 M HCL (4.2 ml).

[0131] The aqueous phase was placed on ice and an ultrasonic tip sonicator was placed therein.

[0132] The alkyl cyanoacrylate phase was added to the aqueous phase and sonication was initiated (50% amplitude, 6x30 s with 10 s pause) to form a nanoemulsion.

[0133] The suspension was left on a carousel for 48h at room temperature before storing it in refrigerator. Nanoparticles formed through in situ polymerisation of the oil droplets.

[0134] The quality of the nanoparticles was measured by diluting the sample 1:100 in 0.1 mmol phosphate buffer and analysing it by dynamic light scattering to determine the polydispersity index (PDI).

[0135] The polydispersity index is defined in context of measurement of particle size by dynamic light scattering as given by Malvern Panalytics (ref.

[0136] https: / / www.rnalvernpanalytical.com / en / learn / knowledge- center / whitepapers / WP111214DLSTermsDefined.html), and is the number calculated from a 2-parameter fit to the correlation data (cumulants analysis).Calculations for this are defined in ISO standard documents ISO 13321:1996 E, and ISO 22412:2008.

[0137] Results

[0138] Without the addition of docusic acid, the suspension was inhomogeneous, with the addition of docusic acid we generated nanoparticles with a size of 140 nm and PDI of 0.11.

[0139] Further Examples

[0140] The above protocol was also followed to prepare the nanoparticles as set out in Table 1.

[0141] For Monomethyl Auristatin E preparation #1, vanillin (0.005 g) (Merck) and chloroform (0.246 ml) (Merck) were also added to the oil phase. Chloroform was evaporated before polymerization.

[0142] For Monomethyl Auristatin E preparation #2, benzyl alcohol (0.05 ml) (Merck) and chloroform (0.247 ml) (Merck) were also added to the oil phase. Chloroform was evaporated before polymerization.

[0143] For Monomethyl Auristatin E preparation #3, chloroform (0.247 ml) (Merck) was added to the oil phase. Chloroform was evaporated before polymerization.

[0144] Table 1

[0145]

[0146]

[0147] Additional Examples

[0148] The following further formulations of use in the methods of the invention were also investigated, the formulations (oil phase) are as set out in Table 2. These tests primarily investigated if reactivity of the drugs could be reduced, and / or the stability of the drug could be increased, so that it is feasible to use them for encapsulation in nanoparticles. Tests were performed in different monomers and using different acids.

[0149] Table 2

[0150]

[0151] Trametinib was tested for reactivity with docusic acid and acetic acid, no reactivity was observed, indicating its compatibility with these acids. The formulation comprising Trametinib and docusic acid was also tested to see if polymerisation of the ACA monomer occurred overnight. There was no polymerisation observed overnight, indicating that premature polymerisation of the ACA monomer is prevented. Regorafenib did not react with docusic acid indicating compatibility. Topotecan was stable with acetic acid, indicating its suitability for encapsulation using the method of the invention.

Claims

Claims1. A process for the preparation of particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer, wherein said process comprises the polymerisation of an emulsion, wherein said emulsion comprises:(i) an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid; and (ii) an aqueous phase,wherein the oil soluble acid has an octanol-water partition coefficient (logP) above -0.5.

2. A process for the preparation of particles of a poly(alkyl cyanoacrylate) homopolymer or copolymer which comprise an active agent, wherein said process comprises preparing an oil phase comprising at least one species of alkyl cyanoacrylate monomer, an active agent with one or more nucleophilic groups and an oil soluble acid and forming an emulsion between said oil phase and an aqueous phase, followed by polymerisation of said emulsion to form said particles.

3. The process according to claim 2 wherein the step of preparing the oil phase, including the time prior to formation of the emulsion, is at least 15 minutes, preferably 30 minutes to 24 hours, more preferably 30 minutes to 6 hours.

4. The process according to any preceding claim wherein the one or more nucleophilic groups are selected from the group consisting of an amine, a hydroxyl, a sulfhydryl, a carboxyl, a phosphate ester and a phosphonate or phosphinate species.

5. The process according to any preceding claim wherein the polymerisation is an anionic polymerisation.

6. The process according to any preceding claim wherein said polymerisation is performed as a single step.

7. The process according to any preceding claim wherein the emulsion is a miniemulsion.

8. The process according to any preceding claim wherein the molar amount of acidic groups in the oil soluble acid in the oil phase is equal to or in excess of the molar amount of nucleophilic groups in the active agent in said oil phase, preferably 1.5 to 5 molar equivalents in excess.

9. The process according to any preceding claim wherein the oil soluble acid has a pKa between 0 and 6, or for a polyprotic acid all pKas are between 0 and 6.

10. The process according to any preceding claim, wherein the oil soluble acid has an octanol-water partition coefficient (logP) in the range of -0.5-3.0, preferably in the range of 0.0-3.0.

11. The process according to any preceding claim wherein the oil soluble acid is an organic acid, preferably a carboxylic, sulfonic or phosphoric acid, more preferably a carboxylic or sulfonic acid, most preferably docusic acid, 4-dodecylbenzenesulfonic acid, decanoic acid, dodecanoic acid, D-a-Tocopherol succinate, 2-Naphthalene sulfonic acid or acetic acid.

12. The process according to any preceding claim, wherein the oil phase or the aqueous phase, preferably the aqueous phase, comprises a surfactant.

13. The process according to any preceding claim wherein the particle is 50 -10000 nm, preferably 50 - 5000 nm, more preferably 70-3000 nm, most preferably 90 - 500 nm, in diameter.

14. The process according to any preceding claim wherein the active agent is a therapeutic or diagnostic agent, preferably a cytostatic drug or imaging agent.

15. The process according to any preceding claim wherein the at least one species of alkyl cyanoacrylate monomer is selected from the group consisting of 3-methylbutyl cyanoacrylate, 2-ethylbutyl cyanoacrylate, 1 -heptyl cyanoacrylate, 2-heptyl cyanoacrylate and 2-ethylhexyl cyanoacrylate.

16. The process according to any preceding claim wherein the active agent and the oil soluble acid are pre-mixed before addition of the alkyl cyanoacrylate monomer.

17. The process according to any preceding claim wherein the emulsion is an oil-in-water emulsion.

18. A particle produced by a process according to any of the preceding claims.

19. A particle of a poly(alkyl cyanoacrylate) homopolymer or copolymer which comprises an active agent with one or more nucleophilic groups and further comprises an oil soluble acid.

20. A particle of a poly(alkyl cyanoacrylate) homopolymer or copolymer which comprises a sulfonate, carboxylate or phosphate salt of an active agent, preferably a docusate, 4-dodecylbenzenesulfonate, decanoate, dodecanoate or D-a-Tocopherol succinate salt thereof.

21. A pharmaceutical composition comprising a particle as defined in any one of claims 18 to 20 and one or more pharmaceutically acceptable carriers, diluents or excipients.

22. The particle of any one of claims 18 to 20 or the pharmaceutical composition of claim 21 for use in medicine.

23. A formulation comprising at least one species of an alkyl cyanoacrylate monomer, an active agent having one or more nucleophilic groups and an oil soluble acid, said monomer, active agent and acid being as defined in any preceding claim.

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