POLYMER-LlPIB HYBRID ¾11CR0P RTICLES

TECHNICAL FIELD

[0001 ] The present invention relates to a novel composition for the delivery of an active substance (eg a pharmaceutical agent such as a drug or othe biologically active molecule including proteins and peptides) to a subject.

PRIORITY DOCUMENT j 0002] The present -application: claims priority from Australian Provisional Patent Application o 2 15900861 titled "Novel drug dciivery compositiQii" filed on 11 March 2015, the content of which, is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[00031 The following publications; are referred to in the present specification and their contents, are hereby incorporated by reference in their entirety:

Rao JP and KE Ge keler, Polymer nanoparticles: Preparation techniques and size control. Prog Pofy Sci 36:887-913 (201 i}[37]; and

Makadia H and SI Siegei, Poly Lacti.c-eo-Glycolic Acid (PLGA) as Biodegradable Controlled Drug Delivery Carrier. Polymers 3:1.377-1397 (201J)[38L

BACKGROUND

10004] A. major challenge facing naaoteelinology research is: the development of smarter and more owerful carriers for the effective delivery of pharaiaceutieal agents. In parti etiiar, formulating com lex p arnmeeutical agents suc as proteins, peptides and lipophilic molecules, with nanocarrters is often required so as to overcome physicoeheniieal limitations and thereby attain the full pharmaceutical potential of the molecules 11 ). To this end, lipid-based compositions (eg liposomes and solid lipid nanoparticies) which encapsulate pharmaceutical agents have been the most widely investigated and, indeed, some compositions of this type have been adopted in certain drug delivery applications (eg oral delivery of poorly water soluble drugs, anti-cancer formulations and vaccinations). However, even though lipid-based compositions can offer high levels of bioeompatibtlity, favourable p annacokinetie profiles and a relatively simple ^" manufacture, to date tire clinical use of such compositions has been somewhat; limited due to instability, insufficient drag loading and/or characteristic ' ^' burst" release profiles of the encapsulated .pharmaceutical agents (2-6J.

[0005 ] An alternati ve approach to the formulation of pharmaceutical agents with nanoearriers involve the use of polymeric nanoparticles:. Such natioparticles have been investigated for use in the encapsulation of phannaeeutieal agents with poor solubility and permeability, and have been found to provide higher levels of stability in biological fluid (ie, relative to lipid-based systems) while offering the possibility ^' of a controlled rate of release of the pharmace tical agent in a manner enabling, effective dreg delivery.

However, the bioeonipatibility of many polymeric nanopartieles is not as high as lipid systems [7-9] and, consequently, considerable research effort has been devoted to engineering novel nanostruetured earner systems which might combine the advan tages of lipid-based systems with those of polymeric

nanoparticles _. , whilst minunismg the physicochemieal and biological limitations of the two nanoearriers. j 0006 ] A wide range of "hybrid" polymer-iipid n nocornpo sites have been recentl reported which aim to address multi-faceted drug delivery "challenges [10-13, 45], The most extensively fabricated type of hybrid particle consists of a lipid shell-polymer core architecture commonly assembled via a two-step method whereby anionic poly(D,L-lactide-co-gl ¾ _' 'colide) (PLC A) nanoparticles are mixed with cationic liposomes at a desired, ratio [1, 14, .15]. However., single-step methods also exist which minimise batch- to-batch, variation of physicochemieal: properties. Such methods utilise phospholipids as enlnlsifi ers in the nanoparticle synthesis, resulting in the self-assembly of lipkl-eoated. polymer nanoparticles [11, 16 ^' }. PLGA is an FDA-approved biodegradable polymer that has received the most extensive attention in the field of polymer-lipid hybrids due to its biocompatibility [17]. Lipid shell-polymer core morphology of PLGA-lipid hybrids have demonstrated several potential advantages over conventional delivery systems such as controllable particle size for high uptake, surface functionality for targeted delivery,, high drug loading, entrapment of multiple pharmaceutical .agents for combination therapies and "tuneable" drug, release profiles [ 12]. However, limitations are still present in such systems in. regard to the stability of the lipid component and the burst release profiles from agents encapsulated within the lipid shell.

10007] Previously, the present applicant developed a novel rranostruetured lipid carrier system which consists of lipid encapsulated within a three-d imensional porous silica matrix or coacervate;- providing, silica-lipid hybrid (SLH) mieroparticles [18-201. These SLH mieroparticles can be prepared by spray drying a silica-stabilised emulsion, with the water removal process ind ucing the aggregation of silica particles into a sponge-like matrix, whereby oil droplets are attached by lipophilic negatively or positively charged surfactants |21], The surfactant char e ^' impacts the nanostruciure of the dr SLH mieroparticles due to the enhanced stabilising effect of nanoparticles when a charge neutralisation, mechanism is operative in a Pickering emulsion j 18]. The oral absorption of ^' a number of lipophilic drugs has been shown to increase as a result of increased solubilit i SLH mieroparticles, due to the enhanced and controlled digestion of l ipid adsorbed in the ihree-d iensioiMl silica matrix: by die- digestive enzyme* lipase 122-24], The increased interracial surface area o f lipi d,, bi nding support of hydropMlic silica and reduced interference effect of digestion products have been shown to enhance lipase adsorption and action in SLH mieroparticies [25], However, the exact effect of the surface chemistry of the solid matrix support on. the digestibility of the encapsulate lipid .is not well understood,

[0008] The ability to form hybrid n krostructwes wi th porous three-dimensional, matrices whereby lipid is: adsorbed, may be controlled initially by the ability of solid nanoparticles to form a stable emulsion with medium chain, triglycerides (MCT). PLGA iianoparticles with a slight negative surface charge, due to- the use of PVA as a stabili ser, have shown the ability to impart kinetic stability to a range of non-polar oils by forming weak interactions win* the oil-water interface [26], Herein, the present applicant investigated whether the stabilising and controlled delivery characteristics of polymeric nanoparticles (such as PLGA nanoparticles) might be usefully combined with the soiubiiising effect of lipid droplets to form dry- polymer (nanopaiticle) -lipid hybrid (PLH) micropartieles with a novel polymerie nanoparticle shell-lipid core archi tecture through the process of spray drying. Further, the present applicant investigated the use of cfyoprotectant (such as mannitol) in the spray drying step, and identified an additional form of dry PLH niicroparticies including the eryoproteetam (ie. polymer nanopaiticie)-iipid-cryoprotectant hybrid (PLCfi) microparricies) with a novel architecture consistin of a tht¾e-dimensionai matrix (or, in other words, coacervate) of the polymeric nanoparticles, lipid droplets and cryoprotectant.

SUMMARY

[0009] hi a first aspect, the present invention provides a dr composition comprising three-dimensional porous rnieropar itles, wherein said imeroparticles comprise; (i) an active substance, (ii) polymeric nanoparticles, (til) lipid droplets, (iv) a nanoparticle stabilising agent, and optionally, (v) a cryoproieetant; _: wherein said active substance is carried by said nanoparticles and/or lipid droplets,

[0010] Typically, the active substance is a pharmaceutical agent such as a drug (particularly, a poorly water soluble drug) or other biologically active molecule: (eg a protein such as an antibody or antibody fragment).

[0 1 1] The polymeric nanoparticles preferabl comprise a biocompatible and/or biodegradable polymer such as a PLGA polymer,

[00121 The lipid droplets preferably comprise a medium chain triglyceride (MCT),

[0013] The nanoparticle stabilising agent is preferably selected from poly vinyl, alcohol (PVA) and didodecytdimetivyi ammonium bromide (DMAB). 1001 ] The: optional cryoprotect nt ma be selected from mannitol, maitodextrin, lactose, trehalose, sucrose, glucose, fructose and sorbitol.

[00 ,15 ] The microparticles of the composition preferably do not consist of a lipid shell-polymer

nanoparticie core architecture like that seen in previously described hybrid polynier-lipid oanocoraposites [L 14, 15].

[0016 ] Preferably;, the composition of the present invention is produced b a method comprising spray drying an oil in. water (o.-'w) emulsion comprisin lipid drople ts and polymeric nanoparticles i the aqueous phase.

[0017] In a second aspect, the present invention provides a method for administering an -active substance to a subject, wherein said method comprises administering to said subject, a composition. according to the first aspect.

[0018] The composition may be formulated into a medicament for oral, nasal, pulmonary, iiitra-niuscular or subcutaneous admimstra.ri.on to the subject.

[001 ] In a third aspect, the present invention provides a metilod for producing a composition accordin to the first aspect, wherein said method comprises spray dryin an emulsion comprising lipid droplets and polymeric nanoparticles in the aqueous phase.

[0020 ] In an embodi ment of the method of the third aspect, the method comprises providing the emulsion comprising lipid droplets and polymeric- nanoparticles in an aqueous phase, and thereafter removing the aqueous phase b spray -.drying.

BRIEF DESCRIPTION OF THE FIGURES

[0021] Figure 1 provides a schematic representation of a two-step fvbrication method of polymer-lipid hybrid (PLH) mieropartieles according to tire present invention. (A) Oil. phase: MCT (10 wt%) with dissolved anionic emulsifier, lecithin (0.6 wt%) and PLGA nanoparticles (80 wt% relative to oii) prepared by emulsk i-dif¾s).o:n-evaporati:on are dispersed in water. (B) PLGA-stahi sed emulsion is formed (nb. positivel charged PLGA nanoparticles coat negatively charged lipid droplets and negatively charged PLGA nanoparticles weakl cover lipid droplets due to electrostatics). (C) Manniioi solution. (20 wt% relative to PLGA nanoparticles) is. dispersed in the emttlsion, (D) LGA-raannitol stabilised emulsion is formed; coating of negatively charged PLGA nanoparticles on emulsion interface ^' increases due to increase in zeta potential . (E) The emulsion is spray dried to .form dry PLH and PLCH mtcropartieies in the absence and presence of mannitol, respectively; 10022 J Figure 2 provides graphical results showing: (A) Zeta potential as a -function of H for (0) PLGA-1 nanopartieles, (Δ) PI.GA-2 nanopartieles and (▲) a submicron emulsion stabilised with lecithin, on the addition of 10 mM NaQ; and (B) Zeta potential as a function of pH for (■} PLMH-l, ( ^" ·) PLH-1 , (o) PLMH-2 and {□) PLH-2 inieropartieie dtspmions, on the addition of 10 mM NaQ;

[0023] Figure 3 provides eonf cal. laser scanning microscopy (C.LSM) cross section images and scanning electron micrograph (SEM) images demonstrating the differences in surface morphology and aggregation properties for PLH-1 (A and B) and PLMH- l TOicroparticles. The SEM images show: (A) A large aggregate of individual PLH mieropartieles, (B) up -close image demonstrating the surface morphol ogy of P LH mieroparticles, (C) minimal aggregation of PLCH micropartic ies, and ( D) up-close imag demonstrating the surface morphology of PLCH microparticies;

[0024] Figure 4 provides graphical results illustrating the reeonsrittition properties of (·) PLH-1, (o) PLH-2, (■) PLMH-l and (□) PLMH-2 mieroparticles, with mixing in digestion media over a 60-minute time period;

[0025] Figure 5 provides graphical results showing the effect of (A) PJLGA-l. nanopartieles and (B) PLGA-2 nanopartieles on the digestion profiles of submicron MCI emulsions when PLGA nanopartieles were added to the aqueous phase at (Δ) 0 wt%, (X) 10 wt and (*) 50 wt%, relative to the lipid content in the emulsion systems. Inset: particle aggregation demonstrated for PLG A nanopartieles (50 ¾!%) and emulsion droplets by an increase in average particle, size over a 60-mintite: period;

[0026] Figure 6 provides graphical results showing the lipasc-mediated digestion kinetics for MCT in, (□) PLH-1 mieroparticks, (o) PLH-2 microparticies, (■) PLMH-l microparticies, {·) PLMH-2 microparticies: and ( A ) a snbmteron emulsion, under fasted state conditions over a 3-hour digestion period-. Inset; the qual ities of pseudo-first order fit; 0027] Figure 7 provides graphical results showin the kinetics of lipolysis of microparticies according, to the present invention as a function of the percent particle redtspersion after 30 minutes agitation in digestion medium: extent of lipid hydroiysis after 30 rata (circles, left, axis) and the pseudo-first order rate constant, k (squares, right axis);

[00281 Figu re 8 provides CLSM cros section images and schematic representations of PLH and P LCH micropartieles at 0, 5 and 60 minutes redtspersion in digestion media. The degree of redispersion is clearly demonstrated to he greater in PLCH mieroparticles, compared to PLH microparticies, as the particle size and lipid content decreases significantly ove time. Schematic representations: PLGA -· mid grey, lipid = light grey, uniform circular droplet shape, mannitol = dark grey dots. Scale bars = 1 um: [0029 ] ^' Figure 9 provides graphical results for the release of the poorly water soluble drug, cinnarizine (CJfN), as a function, of time for: (A) (■) negati vely charged PLCH mieropartieks, ( ^A ) a swhmi ^' cron. emulsion, (X) negatively charged PLGA-i nanoparfieles, and with dashed line) pure ON drug, in 0.1% SLS solution; and (B) a two-step dissolution for (■) negatively charged PLCH micropartieies, (*) negatively charged ^' PLGA, nanoparticl.es, and {♦) pure drug, in simulated gastric conditions (< 60 mm.) and simulated intestinal conditions {≥ 60 mm);

{00.30] Figure 10 provides further graphical results tor the release of O into ft.1% SLS solution, showing the effect of charge on PLR PLCH microparticlc compositions as well as the presence of the eryoproteetant, nianmrol, in the PLCH micropartieies: (■) negatively charged PLCH micropartieies, ( ^' ·± ^■ ) positively charged PLCH micropartieies, (·) negatively charged PLH micropartieies and { ^' ♦) positively charged PLH micropartieies; and

[003-1 J Figure 11 provides graphical results of the release of ON into -0.1% SLS solution, which sho the effect of substituting high molecular weight PLGA with low molecular weight PLGA. The

compositions used in these studies were negati vely charged PLCH micropartieies with (■) high

molecular weight PLGA (MW ^» 30000-60000) and ( ) low molecular weight PLGA (MW = 7000- 17000);

[0032 ] Figure 1.2 provides the results of a CIN release study into 0.1% SLS solution, showing the effect of substituting medium chain lengt triglycerides (MCT) in the compositions with long chain, length triglycerides (LCT).. The compositions used in these studies were negatively charged PLCH

micropartieies formulated, with .{■) MCT and ( * ) LCT; and

[0033 ) Figu re 13 provides the pharmacokinetic profiles of CIN from a PLH microparticle-based composition against comparative silica-containing lipid compositions: PLH (■), siliea-lipid hybrid microparticlc composition (·), and siliea-stabilised eubosomes (▼ ) over 24 h. Doses were normalised to 10 mg kg, and plotted as mean plasma concentration ± SEM, n=4.

DETAILED DESCRIPTION

[0034] Novel ieropartielcs comprisin polymer nanopartieles and lipid were prepared by spray drying polymer (eg PLGA) nanopartieie-stabilised lipid emulsions. The resultant hybrid micropartieies demonstrated three-dimensional porous properties (ie including internal proposity) enabling their use in, for example, compositions for the deliver of an active substance (eg a pharmaceutical agent such as a drug or other biologically active molecule) to a subject. By including a cryoprotectant (eg .mannttol) in the- emulsion prior to spray drying, the ^■ three-dimensional porous properties were able to be increased inasmuch as the: internal porosity of the- m-icroparticles was increased. [0035] in a first aspect, the: present inveation. provides a dry composition comprising three-dimensional pOTOis raicropartieles. wherein said mieropartieies comprise: (i) an active substance, (it) polymeric nanoparo ^' cles, (iii) lipid droplets, (iv) a nanoparticle stabilising agent, and optionally, (v) a cryoprotectoit; wherein said active substance is carried by said nanoparticles and or lipid droplets.

[0036] The composition may comprise a dry substance comprising loose, aggregated and/or partially aggregated mieroparticles. By "dry composition", it s; to be understood that the composition comprises less: than about 10 weight percent (wt%) of water, more preferably less than about 5 wi%. As .such, the composition will typically consist of a free flowing po wder with no requirement for an anti-cakin agent (eg calcium carbonate and powdered cellulose).

[0037] lit the absence of a cryoprotectant, the niieropartieies will typically comprise an average diameter size i the range of 1 to 10 pm. However, preferably, the mieroparticles will comprise an average diameter size i the range of 2 to 5,5 μτη. Using scanning electron microscopy (SEM), the individual mieroparticles typically have a smooth spherical morphology, while confoeal laser scanning microscopy (CLSM) indicates that the mieropartieies consist of lipid droplets encapsulated within, a solid outer shell o ftanopartioles (see Figure .1 ). These mieropartieies may be present in the composition in the Form of large aggregates (eg with an average maximum dimension i the range of, for example, 20 to 100 μηχ). Hereinafter, mieroparticles lacking a cryoprotectant are referred to as polymer (nanoparticle)-lipid hybrid ( Pi I f) iniciOparticies.

[0038 J In the presence of a cryoprotectant, the mieropartieies will also typically comprise an. average diameter size in the range of 1 to 10 p.m. However, i this ease, the mieropartieies will preferabl comprise an average diameter size in the range of 2 to 6: . Using scanning electron microscopy (S EM), the individual mieroparticles typically haw a rough spherical morphology, while confoeal laser scanning microscopy (CLSM) indicates that the mieropartieies consist of a three-dimensional matrix (or, in other words, a eoacervate) of the polymer nanoparticles, lipid droplets and cryoprotectant (ie. with no polymer nanoparticle sheil-iipid core architecture like that observed in the PLH niieropartieies). These

mieroparticles: may be present i the -composition largely in the form of individual mieroparticles (ie. with minimal aggregation). Hereinafter, mieropartieies including a cryoprotectant are referred, to as polymer (nanopartie1e)-lipid-cn _r -'oprotectant hybrid (PLCH) mieroparticles. The redisperstbility of PLCH micropartklcs (ie. to nanoparticles) appears to be significantly greater than that of PLH mieroparticles,

[0039 J Therefore, mieroparticles according to the presen t invention do not consist of a l ipid shell - polymer nanoparticle core architecture (ie. as seen in previously described hybrid polymer- ipid nanoeoraposites [1, 14, 15]).. 10040] The: micropartici.es are porous, with an average pore size- typically in the range of 25-500 nm. The internal porosit of.PLCH mlcropartic.les appears to be more extensive than that of PLH mieroparticles.

[0041 ] The raicroparticles may be positively or negatively charged, or otherwise be neutral

[0042 ] The active substance is carried in the composition by the polymeric nanoparticles and or lipid droplets. By "carried", it is to be understood that the active substance may be dissol ed within the polymeric nanoparticles and/or lipid droplets, and/or associated in anothe manner (eg the active substance may be wholly or partiall adsorbed to the surface of the nanoparticles); such that the active substance may be released from the nanoparticles and/or lipid droplets (eg following degradation and/or diffusion).

[0043] The active substance ma be selected .from, for example, iiutriceutical substances, cosmetic substances (including sunscreens and liV-absorhing .molecules), pesticide compounds, agrochemicals and foodstuffs. However, more typically, the active substance is a pharmaceutical agent such as a drug or other biological ty active molecule (e protein such, as an antibody or antibody fragment, peptide such as an antigenic peptide of vaccine significance _* or a nucleic ac id molecule such as a antisense

oligonucleotide or small interfering RNA (siRMA)).

[0044] The composition of the present invention may be partic olarly suitabie for th e de livery of a poorly water soluble (ie. lipopliilic) drug to a subject Poorly water soluble drugs are understood by those skilled in the art as compounds whereby low aqueous dissolution presents the major barrier to drug absorption across the gastrointestinal tract (GIT) into the blood. Consequently, the drug's oral bioavailability is limited.

[0045 ) The acti ve substance may be a poorly sol ub le drug compound such as those of the group consisting of:

anti-inflammatory agents including celecoxib (4-[5-( ^' 4-mefl»ylphenyl)-3-(iriflaoromet[iyl) yrazoM-yjj benzenesulfonamiclc), tttdomethacin (l-(4-clii.om

vald.eeo.xib (4-(5-methy!-3-phenylisoxa2ol-4-yi) beiizenesuifonainide), n eloxicam ((8£)-S- hydroxy-[(5- methyl - 1 ,3 -thiazol-2-yl)amino [methyl idene ) -9-methyi - 10,10-dioxo- 10i ^(> -thia-9 -azabie lo 14.4.0] dee a- 1 ,3,5-ή'ίεη-7-οηβ), rofecoxib (4-(4-meihylsulfonylp.heny1)-3-phenyi- diclofenac (2-(2-

(2,6-clichlorop¾enyiaminQ)plienyl)acetic acid), naproxen ((+)~(¾-2-(6-methoxyiiapfc

acid) and combinations thereof;

anti-cancer agents such as paclitaxel, 7-Ethyi-l 0-hydroxy-camptothecin (SN-38), etoposide, taxotere, docetaxel, temozolomide and combinations thereof;

anti-emetic agents such, as cmnarizine {(.E)-!-(Dipheny1fflcth.yi)-4-(3-phenylprop-2-eny

vitamins and derivatives thereof includin vitamin B. vitamin D, retinol (vitamin A) and retiuoic acid; antibiotic agents including tetracycline, rifarnpaoin, claritferomycia, erythromycin and combinations thereof; anti-psychoti drugs including sriprasidone, aripriprazole, etc.; and

cardiovascular drugs such as statins, etc.

[0046 ] The active substance, and particularly those that are poorly water soiubie drugs, may be present i the composition within (and/or at the surface of) the polymeric nanoparticies and/or the lipid droplets. As a consequence, the composition enables the possible two phase release of a drug (e the drug may be initially released from the polymeric nanoparticies in a first phase and thereafter released from the lipid droplets: in a second phase, o vice versa), or the release of two different drugs (eg in a combination: therapy). In one exa ple of a composition for use in a combination therapy, the first active substance may be eeieeoxib present within (and/or at the surface of) the polymeric nanoparticies, and the second active substance is an anti-cancer agent (such as taxotere, docetaxel and temozolomide) dissolved within the lipid droplets,

} 0047 J Folymeric aanopatticies suitable for use in the composition of the present invention may be selected from those well known to those skilled in the art. Examples include those comprising:

a polyester (eg poiy(lactic) acid (PLA), poly(methyl methactylate) (P MA), polyaerylic acid (PAA ^' poly inyl alcohol (PVA) and the like); a polyamide (eg poly-paraphenylene terephthaiamide and poly t imino ( 1 , 6-clioxoh examethylene) iminohexamethylene,); a co-polyn er (eg poiy(3ae tide)-bloek- poly ethylene oxide)-biock-poiy(!actide) (PEO-PL.A); and mixtures thereof. Other suitable polymeric nanoparticies, and their methods of preparation, are described by Rao and Geckeler [37] such: as poiyioxyetiiyiene glycol.) polymer (POP), poly(ethylene glycol )-pol (lac ide) (PEG-PLA),

polyeaproiacione (PCL), and polystyrene (PS) copolymer; the entire disclosure of Rao and Geckel er j 37 j is incorporated herein by reference. j0048] Preferably, the polymeric nanoparticies will be biocompatible and/or biodegradable. B the term, "biocompatible", those skilled in the art will understand thai the nanoparticies, when administered to a subject, will not produce any substantial adverse effect. :[4 i]. By the terra, "biodegradable", those skilled in the art will, understand that the nanoparticies, when administered to a subject, are broken down (ie.

degraded) by hydrolysis and/or enzymatic processes within the body j 41 ]. As such, the polymeric nanoparticies may be preferabl selected from PLA, PLG, PLGA, PCL, chitosan, chitin, gelatin, poly- cyanoacrylare (PC ) and poly-alkyl-cyanoacrylate (P ACA) polymers. However, most preferably, the polymeric nanoparticies comprise a PLGA polymer, j 0049 ) Due to its bioeompatibility and Modegradahility, PLGA polymers have been widely used to pr duce materials for introduction into a subject, such as drug delivery device and tissue engineering scaffolds 138].. PLGA also has "tuneable mechanical properties' ^! (eg by controlling relevant parameters such as polymer molecular weight, laefide to glycoli.de ratio, and drug concentration, ¾ is possible to control drug dosage and release profile from a PLGA carrier) and, as mentioned above, is ED A approved

[38]; the PLGA polyasers: being biodegradable into biocompatible degradation products. In particular, PLGA is degraded into lactic and- glycolie acids by hydrolysis (and possibly some enzymatic action \M\). in turn, the lactic acids enter the tricarboxylic acid cycle and are metabolised and eventually eliminated from the body as carbon dioxide and water [39], while glycolie acid is believed to be excreted either unchanged in the kidney or, like the lactic acids, via the tricarboxylic acid cycle where it is metabolised and eventually eliminated from the body as carbon dioxide and water. Bi degradable PLG polymers, suitable for the pro duction of polymeric nanoparticles for use in the present invention, are reviewed b Makadia and. Siege! [38], along with, the methods of their synthesis and fabrication, into, inter alia, PLGA polymeric nanoparticles; the entire disclosure of Makadia and Siegel [38] is incorporated herei b reference. Some particular examples include PLGA polymers with a. poly lactic acid (PLA)/poly glycolie acid- (PGA) ratio of 50:50, 65:35, 75:25 and 85:3.5. In addition, PLGA copolymers may also be suitable. For example, di-block PLGA/PEG co-polymers (PLGA-PEG) and tri-bloek PLGA/PEG PLGA copolymers may be suitable and may provide an added benefit of increased shelf stabilit [38 |.

[0050] With regard, to PLG A polymers, typically, the higher the content of poly giycolie acid in the PLGA, the faster is the rate of degradation (eg PLGA 50:50 degrades faster than 65:35, which in turn degrades faster than 75:25 cte.)[38], as the hydrophilieity of the PLGA is mereased with increasing PG content (thereby resulting in quicker degradation by hydrolysis). Also, with the increasing- hydrophiiieit (and decreasing hydrophobicity; , degradation of the lipid droplet content of the composition through lipolysis will reduce d ue to the inhibition of lipase. On the other hand, the use of a biodegradable polymer with higher molecular weight will generall exhibit a lower rate of degradation. The polymers used in the present invention, and particularly where- a PLGA polymer is used, may have a molecular weight (MW) in the range of, for example,, 5-100 kDa, more preferably 25-75 M a, and most preferably, 30-60 kPa. The ability to select PLG polymers with different MW and/or PL AJ PGA ratios enables "tuning" of the release profile of the acti ve substance from the composition of the present invention, in addition, the use of a PLGA with a higher content of PGA may also assist in raising the glass transition temperature (tg), which can make ^' the: production of the composition of the present i nvention more amenabl e to a spra drying method [ 381; although as discussed below, the inclusion of a eryoprotectant can enable the use of PLGA polymers with lower amounts of PGA (eg PLGA 75:25 and PLGA 85: 15).

[0051 ] Most preferably, me polymeric nanoparticles of the composition of the present invention comprise a PLGA 50:50 polymer with a MW of 30-60 kDa. π

10052 J The polymeric nanoparticles preferably haw an average diameter in the range of 2 .- 500 nm, more preferably 5 - 200 nm, and. most preferably about 150 nm,

[0053 ] The composition of the present in vention .comprises lipi d droplets, preferably droplets of a medium chain triglyceride (MCT), although droplets of a long chain triglyceride (LOT) can also be suitable. Those skilled in the art will understand that MCTs contain 6-12 carbon fatty acid esters of glycerol. Suitable examples of MCTs include eaproic acid (Co: ) _: , capiryh© acid (C 8:0), caprie acid (CT0;0) and lauric acid {C12.:0), and mixtures thereof. A specific suitable example is Mig!yoPP 812 (Ci emer GmbH & Co, Cincinnati, OH, United States of America) which consists of a mixture of caprylic triglyceride and caprie triglyceride. Suitable LCTs include soybean oil and safflo er oil. The lipid droplets- may optionally comprise a standard emulsifier, preferably an anionic surfactant such as,, for example, lecithin and sodium deoxycholate. Anionic surfactants induce a negative charge into the droplets.

[0054] Th nanoparticle stabilising agent may be selected from any of those well known to those skilled in the art including sodium do-decyl sulphate (SDS), polyoxyethylene-polyoxypropylene block copolymer surfactant (.Pturonie F-68; Signia-Aldrieh Co. LLC, S .Louis, MO, United States of ^' America),

P!uronic® F-127 surfactant (Sigma-Aldrich Co. LLC), poloxamine, polyethylene glycol (PEG), poly tactic acid (PLA), polyethylene glycol sorbitan monooleate (T een® 80; Sigma-Aldrich Co. LLC) and ^' Vitamin E TP-GS (d-alpha tocopheryl polyethylene glycol 1000 succinate; Antares Health Products Inc., Batavia, IL, United States of America). However, preferred stabilising agents include poly vinyl alcohol (PVA.) and didodecyldimeth i ammonium bromide (DMAB). The stabilisin agent is preferabl present during the synthesis of the polymeric nanopartieies, in which case, the stabilising agent may confer a. charge on the -nanoparticle- surface. For example, the presence of PVA during the synthesis of PLGA nanoparticles produces a negati ve charge whereas the presence of DMAB during the synthesis of PLGA nanoparticles produces a positive charge that coats the nanoparticle. Whe used in the composition with negatively charged lipid droplets (eg lecithins-stabilised lipid droplets), such positively charged PLGA nanoparticle will be caused to electrostatically coat the lipid droplet surface and thereby increase the stability of the toee-dimensional structure of the porous micro particles in the dry composition.

}(K.)551 The optional eryoproteciant niay be selected from an of those well known to those skilled in the art. However, preferred eryoprotectants include manmtoi, maltGdex n, lactose, trehalose, sucrose, glucose, fructose and sorbitol.

[00561 The composition of the present invention may comprise; 0.01 to 20 wt of the active substance (preferably, 1-5 wt%), 10 to 60 wt¾ of the polymeric n ioparticles (prelerabiy, 25-50 wt%), 5 to 50 wt of lipid droplets (preferably, 25-50 wt%), 1 to 40 wt% of the nanoparticle s tabilising agent (preferably. 10-25 wt%), and 0 to 17,5 wt% of: the cryoprotectant (preferably, 0- 1 wt%); wherein the wr% amounts are based on the total weight of the composition.

[0057 ] Where an e ulsifier is used to stabilis the lipid droplets, the emulsifier may be present in an, amount of 0.01 to 2.5 wt% based on: the total weight of the composition.

[0058] While the active substance may be present in the composition in an amount in the range of 0.01 to 20 wt%, it will be understood fay those skilled in the art that the actual amount present may vary considerably depending upon, for example, the particular components of the composition, the solubility of the particular active substance (which can o ften be increased by the presence of an emu isi fter) and the mariner of release of the active substance that is desired.

[0059] With an increasing amount of polymer nanopartieks, the rate of degradation of the lipid droplets through, lipolysis b lipase will decrease.

[0060] The composition of the present in vention may be produced by, for example, spra drying, freeze drying and fhridised bed procedures.

[0061 ] Preferably, the composition of the present invention is produced by a method comprising spra drying an oil in .water (p/w) emulsion comprising lipid emulsion droplets and. polymeric: nanopartieks in the aqueous phase. The polymeric nanopartieles in the aqueons phase may have a stabilising effect on the lipid droplets. The spra drying will preferably be conducted at a temperature less than the t of the biocompatible polymer (eg less than 60°C for PLGA). At temperatures greater than the tg of the polymer,, the polymeric nanopartieles will disintegrate and agglomerate during the spray drying process. Other spray dryin parameters, such as emulsion, flow rate and air flow rate, are preferably set, to provide a high level or optimal level of removal of residual moisture (eg an emulsion flow rate I L/min, such as for example, a flow rate in the range of 0.25 to 0,75 mL/min, and an air flow rate < 1 m^ min, such as for example, a flow rate in the range of 0.25 to 0.75 nr'/min). With PLGA nanopartieles, the emulsion flow rate may preferably be about 0.5 m Vmin, and the air flow rate ma preferably be about 0.6 m'/min.

[0062 ] More preferably, the composi tion of the present invention is produced by a two-step method of providing an w' emulsion comprising lipid droplets stabilised by polymeric nanopartieles in an aqueous phase, and thereafter removing the aqueous phase by spray drying. The emulsion may be produced by homogenising a mixture comprising lipid in an aqueous dispersion of polymeric nanopartieles. The active substance · is: preferably present in the mixture. The acti ve substance ^' ma be: incl uded in the polymer preparation for nanoparticic production (ie. such that the active substance is carried by the nanopartieles). 10063 J The: emulsion ma comprise a standard emiiisifier, preferably an anionic surfactant such as lecithin. The eraulsifiet ma be present in n amount of 0.1. to 5 vt%, preferably about 0.5 to 2 t%, of the wei gh t o f the l ipid droplets. The amount of polymeric nanopar deles present in the emulsion may be up to 80 wt % relative to the weight, of the lipid emulsion, droplets. The emulsion may optionall include a eryoproteetanf (eg mannitol). The cr oproteeta may h present in an amouat in the range o abosii 1 to 35 wt , preferably 10 to 20 \vt%, .relative to the weight of the polymeric nanoparticles .

J0O6 ] The average diameter of the individual micropartieks of the composition will typically be in th range of 1-20 pat, preferably 2-10 pin, and most preferably <5 urn (for example, in the order of I to 4 pm or 2,5-3,5 μηι). Micropartieks of this size are suitable for a wide range of uses. In., terms of therapeutic uses, raicroparticles of this size are particularly suitable for administration to the lung. That is, studies have shown that mieropartieks <5 pm, particularly in the order of 2.5-3.5 urn have the best level of penetration and retention in the lung, even when the subject is experiencing airflow obstruction (eg associated with mild to moderate asthma)[40j, fn contrast raicroparticles 5 μιη get have a tendency to ge t "stuck at the back of the throat" or, in other words, the oropharynx, while sub-micron particles, (ie particles < 1 pm) are not readily retained in the lun (ie they exit upon .exhalation).

[0065] The composi tion, of the present invention may be formulated into, for example, a medicament for the treatment _. aftd/or prevention of vari us; diseases or disorders (eg human or veterinary therapeutics). The medicament may be suitable for, for example, oral administration, delivery to the mucous membranes (eg nasal and/or pulmonary administration) or su be utaneous administration. For oral .administration, the medicament may be in the form of any suitable oral dosage form, i ncluding tablets, caplets, capsules, liquid emulsions and suspensions and elixirs. For nasal and/or pulmonary delivery , the medicament may be provided in the form of a dry powder for a dry powder inhaler device (eg a device well known t thos skilled in the art which, typically, produce a drug aerosol by directin turbulent air through loose powder). For subcutaneous administration, the composition may be formulated into a solid medicament, suitable for implantation into the body by, for example, surgery. Alternatively, the composition may be formulated into a depot-forming composition that may be subcutaneously injected into the subject. Such solid and depot-ferraing implantable medicaments ma be particularly suitable for long term

requirements (eg where a sustained release of the active substance is desired).

[0066] in a second aspect, the present invention provides a method for administering an active .substance to a subject, wherein said method comprises administering to said subject a composition accordin to the first aspect.

[0067] The eoinposition may be formulated into a medicament fo oral, nasal, pulmonary, intramuscular or subcutaneous .administration to the subject:. [0068 ] Generally, the subject will be a human, typically an adult. However, the present invention may also be applicable to ηαη-human subjects such as., for example, li vestock (eg cattle, sheep and horses), exotic animals (eg tigers, lions, elephants and the like) and companion animals (such as dogs and eats).

}0069 ) I a third aspect, the present invention provides a method for producing a composition according to the first aspect, wherein said method comprtses spray drying an oil in water (o w) emiilsion comprising lipid droplets and polymeric nanoparticie in the aqueous phase.

[0070] The polymeric nanopariieles in the aqueous phase may have a stabilising effect on the lipid droplets.

[0071 ] In an embodiment of the method of the third aspec t, the method comprises providing the o/w emulsion comprising lipid droplets and polymeric nanoparticies in an aqueous phase, and thereafter removing the aqueous phase by spray drying.. 0072] Again, the polymeric nanoparticies in the aqueous phase may have stabilising effect on the lipid droplets.

[0073] The spray drying will preferably be conducted at a temperature less than the tg of the biocompatible polymer (eg less than 60"C for PLGA).

[0074] The: emulsion may he produced by homogenising mixture comprising lipid in a aqueous dispersion of polymeric nanopartieles. The active substance is preferably present in the mixture. The active substance may be included in the polymer preparation for nanoparticie production (ie, such that the active substance is carried by the aanopartieks). The emulsion may be formed from an oil in water (o/w) emulsion comprising 1-70% (w/w) lipid in water, preferably, .1-25% (w/w), more preferably 5-20% (w/w), and most preferably about 10% (w/w) lipid in water. The o/w emulsion may comprise a suitable amount of a standard emuisifier, preferably an anionic surfactant such, as lecithin, suck as 0.1 to 5 t% (relative to the amount of the lipid) or, more preferably, about 0.5-2wt %.. The amount of polymeric nanoparticies present in the emulsion may be up to 80 wt% relative to the weight of the lipid droplets. The emulsion may optionally include a cryopiOtectaftt (eg mannitol). The cryoproteetant may be present in an amount in. the range of about 1 to 35 wt%, preferably 1 to 20 wf%, relative to the weight of the polymeric nanoparticies.

[0075] In a further aspect, the present in vention provides an aqueous preparation for producing a composition according to the first: aspect, wherein said preparation comprises: (!) an active substance, (j.i) polymeric nanoparticies _: , (Hi) lipid, (iv) a nanoparticie stabilising agent, and optionally, (v) a

ciyoprotectant 10076 ] Such an aqueous preparation may be provided as, for example, a component of ^' kit, wherein said kit ma further comprise instructions ^' for spray dry ing the preparation to produce a composition according to the first aspect, in preparation for the spray drying, the aqueous preparation may be mixed (preferably homogenised) to form an emu si n of lipid droplets stabilised by the polymeric nanoparticies.

[0077] In a still further aspect, the present invention provides a spray-dried composition comprising three-dimensional porous mieroparticles.. wherein said imcropar tides comprise: (i) an active substance, (ii) polymeric nanoparticl.es, (hi} lipid, droplets, (iv) a nanopartick stabilisin agent, and optionally, (y) a cryoproteetant-; wherein said active substance is carried by said nanoparticies and/or lipid droplets,

[00781 The invention is hereinafter described with reference to the following non-limiting exampk'Cs) and accompanying figures.

EXAMPLE(S)

Example 1 Bioacth e hybrid particles from PLGA nanoparticfc-stahilised lipid droplets

Materials and Methods [0079] Materials

PolyiD -lactide-co-glyeoiidc) (PLGA; 50:50, MW - 30000-60000 Da), didodeeyldimethyl ammonium bromide (DMAB) and. polyvinyl alcohol (FVA; MW = 30000-70000 Da) were purchased from Sigma- Aldrich Pry Ltd (Castle Hill, NSW, Australia). Medium chain triglyceride (MCT; MiglyolD 812) was obtained from Hamilton Laboratories ( Adelaide, SA, Australia), and soybean lecithin (containing >94% phosphatidycholine and <2% triglycerides) from.BDH Merck (Sydney; NSW, Australia). Ethyl Acetate (AH Grade) was obtained from Sigma Aldrieh (Australia). Materials used for the lipoiysis study, including sodium taurodeoxychoiatc (NaTDC) 99%, tiiztm maleate, type X-E L- - lecithin

(approximately 60% pure phosphatidylcholine, from dried egg yolk), porcine paiicreatm extract (activity equivalent to 8 x DSP specification), calcium chloride dehydrate and sodium hydroxide pellets, were all purchased from Sigma- Aldrieh (Australia).

[008 1 Prepa ation of PLGA nanoparticies

Nanoparticies were prepared b a modified emulsion-diftusion-evaporanon method developed- by

Hariharan et L [27]. The average diameter of die nanoparticies was about 180 nm, S00 nig PLGA (50:50) was dissolved in 25 mL ethyl, acetate at room temperature for 2 hours. The organic phase was then added to 50 mL of an aqueous phase containing DMAB (PLGA-1 nanoparticies) or PV A (PLGA-2 nanopartteks) as a stabiliser (250 g in 50 mL, 0,5%, w/V}. The resulting primary emulsion was stirred at 1 00 rpm for 3 hours: and subsequently homogenised at; 15000 rpm for 5 minutes using a high-pressure honiogeniser (AvestinsSi Em«!siFles-C5 Momogeniser), ^' Water was added wit constant stirring to this nanoeraulsion to i eilitate diffusion and finally, evaporation of ethyl acetate, leading to the

nanopreeipH ion of nanaparticles.

[008 i ] Preparation of PLGA-lipid hybrid microcapsules

PLH rnieroparticles were prepared using a two-step method (ie. homogenisation followed by spray drying of PLC A nanoparttele-stabilised emulsions) de veloped by Tan el etl [19] and shown in Figure .1. The initial o/w emulsions were prepared by diss lving 0,6% (w w) leeitMa in 10% (w/w) oil (Miglyoi® 812) and MISli-Q -water was added as the continuous phase, PLGA nanoparticies were dispersed in Milli-Q water, which contained 80% (wt relative to oil content) of nanopartieles. The coarse emulsion was tumbled for 12 hours after the addition of PLGA nanopartieles prior to being homogenised (Avestirri ^; EmulsjFlex-C5 Bomogenizer; Avestiii Inc., Ottawa, ON, Canada) under a pressure of 1 00 bar for 5 cycles. The PLGA nanopartiele-stabitised emulsion was then spra dried (Mini Spray Dryer B-290; BlICHl Labortechn!k AG, Flawil, Switzerland) to form PLH. micropartieles under the conditions given in

Table 1.

[0082] Table !

[0083] Preparation of PLCH micropartieles

PLCH rnieroparticles were prepared in a similar maime to that described above for PLH ieroparhelcs ₄ although in this ease a mannitol dispersion, containing 20% (wt relative -to PLGA nanopartieles) niannitol, was added to the PLGA nanopaxtiele dispersion. This mixture was then added to the emulsion before homogenisaiion and spray dried following the method described above.

[0084] Physicocbemical characterisation of PLGA-lipid- hybrid micropartides

[00851 Scanning electro microscop (SEM) - The particle size- and surf ce morphology of PL H mierocapsules was examined by high .resolution analytical scanning electron microscopy, SEM (Quanta 450: FE1, Hills boro, OR , United States of America). Each sample was mounted on. double-sided .adhesive tape and sputter coated with a platinum layer prior to imaging.

[0086] Lipid loading content - The lipid loading content of PL microcapsules was determined by fherraogravimetric analysis (TO A). The particles were heated at a scanning rate of iO°C min from 20- 550°€ under nitrogen purging; the lipid completel decomposed by SOO^C. The amount of lipid loaded within, the mierocapsuies could be determined ^' b the weight loss within this temperature range, minus that corresponding to water moisture.

[0087] Di spersi hiiity study - The reconstitution properties of PLHs were assessed based on changes in droplet size over ape riod of time as characterised by laser diffraction (DLS) usin a Malvern Mastersizer and dynamic light scattering using a Malvern, Zetasizer Nano, respectively (Malvern Instruments inc., Malvern, United Kingdom), Each composition (5 mg/mi powder) was redispersed in lipid digestion medium following the method of Jang et al. [28],

[| ^' )881 In vitro lipoiysis studies

[1)0891 Preparation of lipid digestion medium - The lipid digestion medium was prepared according to the method adapted from Sek et at. [29] . The fasted state mixed micelles (i _. e. phos holipid/hife salt (1.25 »M PC/5MM NaTDQ), were prepared in the following sequence: egg lecithin was dissol ved in

ch!orofomi (4 mL) followed by evaporation of c oroform under vacuum (Ro ta vapor RE, BlJCHl Labortechmk, Switzerland) to form a thin film of lecithin around the bottom of a 50 mL round-bottom fla.sk; a D ^" and digestion buffer [ ^' 50 mM Trizma maleate (pH 7.5), 150 mM aO, and 5 mM

CaCl2.2HiO] was added and the mixture was -stirred for ~ 12 h to produce a transparent (light yellow) micelkr solution. Pancreatin extracts (containing pancreatic lipase, coltpase and other non-specific lipolytic enzymes such as phospbolipase A?) were freshly prepared each da b stirring Ig of porcine pancreatin powder in 5 mL of digestion buffer for 15mm, followed by centrifugatkm (at -5000 rpm, 4 ^Q C) for 20 nrin. The supernatant phase was collected and stored on ice until use,

[0090] Lipid digesti on kinetics studies - The progress of lipid digestion was monitored for 1.80 min by usin a pH-stat titration unit (ΤΪΜ854 Titration Manager, Radiometer, Copenhagen, Denmark) according to the lipolysis protocol as described b Sek et at [29]. Briefly, a known quantity of sample composition (equivalent to -200 mg lipid) was dispersed in. 18 niL of buffered micellar solution b stirriiig continuously for 1.0 min in a glass reaction vessel with thermostat (37 ^s C). The pH of the digestion medium was re-adjusted with 0.1 M NaOE-or HO to 7.50 ± 0,01 . Lipolysis was initiated by the additio of 2 mL of pancreatin extract (eontainiiig - 2000 TBU of pancreatic lipase acti vity) into the digestio medium. Free -fatty acids (FFA) produced in the reaction vessel were immediately titrated with NaOH via an auto-burette to maintain a constant pH in the digestion medium at the pre-set value of 7.50 ± 0.01 throughout the experiment A solution of 0.6 M NaOH was used for long-chain lipids as per the established experiment, protocol ( 29),

Remits mid Discussion

[0091] Formation of hybrid particles from PLGA nanoparticle stabilised lipid droplets

[0092 J anoeomposite nncrcfarticles consi sting of PLGA nanoparticles and MCT droplets were prepared via a two-step fabrication method, whereby the oil-phase containing the anionic surfactant, lecithin* was homogenised with an aqueous dispersion of either positively or negatively charged PLGA nanoparticles, The consequent nanopartiele-stabillsed emulsions were then spray dried to form dry PLGA lipid hybrid (PLH) inieropartieles of 1-5 μηι in size with a three-dimensional microstmcture controlled by the interfaeial structure of the precursor emul sions.

[0093] The PLGA nanoparticle charge was varied by using either DMAS or PVA as a stabiliser during the nanoparticle synthesis, in order to investigate the influence of emulsion stabilit on dry particle nanosfrueture. DMAB produced nanoparticles with a highly cationie charge, whereas PVA produced nanoparticles with aft anionic charge due to the hydrolysis of poiy(laefic acid) groups on the particle surfaces [ 30], Lecithin induced negative charge onto the emulsion droplets, causing oppositely charged PLGA- 1 nanoparticles- to electrostatically coat the lipid droplet interface. By contrast _. , negatively charged PLGA-2 nanoparticles required high nanopartiele concentrations to show droplet nanoparticle adsorption d¾e to the electrostatic repulsion interactions ^' between the two interfaces. Due to the high concenti'ation of PLGA nanoparticles (80 wt% relative to oil concenti'ation), the zeta potential of the stabilised emulsions and tlie dry PLH inieropartieles was dependent on the charge of the nanoparticle. Extrapolation of the data gives aa isoelectric point between pH 9 and 10 for PLH- 1 microparticles and pH 3 and 4 for PLH-2 micropartieles (Figure 2).

[0094] Spray dried stabilised emulsions of PLGA nanoparticles formed a cohesive and sticky solid material due to the aggregation of individual microparticles. T his was independent of nanoparticle charge at 80 wt% PLGA relative to lipid, as it was demonstrated for both PLGA-1 and PLGA-2 nanoparticles _^ Consequently,, it was hypothesised that this was not due to emulsion stability but rathe the high temperatures and shea forces of spra drying inducin cohesion between individual PLGA nanoparticles. Unlike other robust materials commonly used in spray drying, PLGA has a low glass transition temperature which introduces difficulties in. maintaining the integrity of PLGA nanoparticles during this process. Carbohydrates, such as mannitol, act as bulking, agents and provide protection to PLGA nanoparticles- from shear forces and high temperatures durin spra drying [ 31, 32 j. As a result, mannitol was administered during the preparation method to investigate the effect of a carbohydrate eryoprotectant on the nanoparticle integrit and micropartiele aggregation during the water removal step. The zeta potential, and particle size, increased for PLGA iipid-mannitot hybrid microparticies (Table 2) designated PLMH-1 and PLMH-2; yielding a pKa between 10 and 1 1 for PLMH-1 microparticies and 6 and 7 for PLMH-2 mic oparticies. Both positively and negatively charged nanoparticle-stabilised emulsions formed free flowing powders when spray dried with an atpeous solution of mann toi .

[00951 Table !

The ¾s Potentials reported are in the pH range of 7.0-7.5. j 0096 ) SEM and CLSM images clearly showed that porous aggregates are formed for PLH

micropartieles in the slze range of 20-100 pm, consisting of individual micropartiel.es in the size range of 2-5.5 pxa (Figure 3). Ήιε difference in morpholog and particle aggregation between individual PLH and PLCH -microparticies was attributed to the addition of mannitol during the water removal process. Examination, of g£M images of PLH mieropartieles demonstrates a smooth spherical morphology, with the absence of indi vidual PLGA nanopaiticies on the particle surface indicating that the nanoparticle integrity was not main tained during spray drying. I n comparison, the inclusion of mann itol appears to increase the ability for the self-assembly of PLGA nanoparticks from the continuous phase to the droplet interface during spray dry ing, whilst maintaining the: integrity of individual nanopartieles and forming a three dimensional porous structure whereby lipid droplets are encapsulated within a PLGA matrix. This is further highligh ted i Fi gure 4, whereby the reeonstitution of PLCH micropartieies into heterogeneous dispersions of PLGA nanopartieles and lipid droplets is demonstrated by the reductio of particle size over time from 5.63 ± 1.2 μηι to 1.76 ± 0.3 pm and 5. 5 * 1.0 μιη to 0.98 i 0.2 μιη for PLMH-1 mietOparticies and PLMH-2 micropartieles, respectively. Minimal change in particle size was observed for PLH microparticies (4.85 ± 0.9 p.m to 4.55 ± 0.75 um and 4.23 ± 0.7 um to 2.75 ± 0.51 pm for PLH-1 microparticies and PLH-2 mieroparticles, respectively) confirming that the PLG

nanoparticks were disrupted due to the high temperatures and shear stress of ^" the water removal process, causing the nanopartieles to morph into a mierocapsifle-iike structure whereby lipid is encapsulated within a solid PLG outer shell (Figure 1). 10097 ] It was found that eationic PLGA ..nanoparticles exerted a greater level of stability on negatively charged lipid droplets due to strong electrostatic attraction, increasing the stability of the PLH

microparticle three-dimensional structure in the dry composition. Thereby, the difference in PLMH-1 and ^' PLMH-2 teconstitution can be attributed to the difference in electrostatic interactions between PLGA nanoparticles and. emulsion drop lets. Both PLH-2 and PLMH-2 experienced a greater rate and extent of particle redistribution than PLH-1 and PLMH-1 , respectively, most likely due to the electrostatic repulsion between the PLGA-2 nanoparticles and lipid droplets. It is also hypothesised that the floecuiation between eationic mieropartieles is greater than anionic micioparticles due to the interaction between posi tively charge mieropartieles with small patches of negatively charged raiero article

interface. This floecuiation further reduced the redispersion of PLH- i mieropartieles to primary- nanoparticle and lipid droplets. j 0098 ) in viteo lipid digestion studies

[0099] Influence of PLG nanoparticles o digestion of lipid droplets - The effect of PL A

•nanoparticles- on li id digestion was examined by assessing pancreatic lipase action in a stabilised emulsion system, in particular, different concentrations of PLGA- 1 and PLG A-2 nanoparticles were added to a suhmicron MCT emulsion stabilised by lecithin to investigate this stabilising effect on lipid digestibility (Figure 5). It was found that the initial rate of lipolysis was significantl reduced when emulsion droplets were stabilised with PLGA-I nanepariicies, compared to PLGA-2 nanoparticles. This coincided with the enhanced ability for eationic .nanoparticles (at pH 7,5} t stabilise and coagulate with anionic emulsion droplets (at pH 7.5) due to the electrostatic attraction between both interfaces. This is demonstrated in Figure 5 A inset, as the average particle size of the PLG A- .! stabilised emulsio increased 6-fold over a 60-minute period from 0.5 ± 0.2 pm to 2.9 ± 0.6 urn due -to heterocoagulatiott between the nanoparticles- and droplets, leading to physiea l shieldi ng of the emulsio droplets and a reduced abil ity for lipase to adsorb to the lipid interface; PLG -2 nanoparticles, however, catty a small negative charge at neutral pH and, consequently, electrostatic repulsive interactions between nanoparticles and emulsion droplets: are believed to generate an energy barrier at: close approach, restricting the numbe of nanoparacies that can weakly adsorb to the droplet: interface through hydrophobic interactions, leading to less particle aggregation (0.4 μιη to 1.82 pm over 60 minutes; Figure 5.B inset) than, for the positively charged PLG A nanoparticles, A similar initial rate of digestion to that of a conventional submicron emulsion wa observed for PLG -2 nanoparticle-stabilised emulsions due to reduced physical shielding of the lipid by the nanoparticles, thereby allowing the enzyme to gain access to the- lipid interface.

However, as the lipid digestion proceeded for both PLGA-1 and PLGA-2 nanoparticles, emulsion droplets decreased in size, increasing die shielding effect of the PLGA nanoparticles and restricting the acces of lipase to the oil-water interface, leading id reduced extents of lipolysis over a 60 minute period 22J . * both instances, as the conceotrati ^' oa of nanoparticles increased from 10: t% to 50 wt%, the rate and extent of digestion decreased due to increased physical shielding and sterie hindrance. However., the degree of inhibition of lipase was significantly greater in PLGA- 1 nanopartides than in PLGA-2 nanoparticles due to the difference in tmno artkle charge. Thus, the overall extent of Iipolysis for 1 wt% and.50 wt% PLGA-. I stabilised emulsions was 64.0 sfe 3.6% and 55.8 ± 2.9%, respectively, compared to 78.9 ± 5.6% and 66,9 ± 5.1% for 1 wt% and 50 wt% PLGA-2 stabilised emulsions, respectively.

[00100] Digestion kinetics: for spray dried, hybrid micfoparfieics - Figure depicts the percent of lipid hydrolysis as a function of time for the four de veloped dr hybrid microparticles (ie, PLH-i, PLH-2, PLMH-i and PLMH-2 rnteroparttcles) compared to a simmicron MCT emulsion stabilised with lecithin. Under simulated fasted intestinal conditions, lipid digestion for the suhmtcron emulsion occurred at a rapid initial rate due to a greater exposure of lipid substrate to lipase during the early phase of digestion before prompt inhibition at approxi mately 60% digestion, in comparison, Iipolysis of all four hybrid microparticles occurred at a slowe initial rate due to the sterie hindrance of PLGA on enzyme adsorption hu t the overall extent of digestion was greater than for the submieron emulsion (100% for PLMH- i and PLMH-2 mieropartieles versus 94.5■± 3.3% for submieron emulsion);. Both the submiero emulsion and emulsion droplet within the dry rmcroparticies were stabilised with lecithin. At low to moderate concentrations of lecithi n, relative to lipid concentration, lecithin appears to enhance lipase action by removing the surface active digestion products from the emulsified interface. However, at high concentrations, or a the emulsio droplet sizes decreases, lecithin seems to interfere with lipase adsorption by competing with the enzyme for sites on the oil-water interface |3 |. In addition to this, at insufficient bile salt concentrations, digestion products (ie. free fatty acids and motioglyeerides) adsorb to the oil-water interface due to their amphiphilie structure, which further restricts lipase adsorption and inhibits Iipolysis. The rapid decrease in emulsion droplet size and rapid rel ease of digestion products from the submieron emulsion leads to an inhibition of enzymatic ^' degradation due to a saturation of the oil- water interface. In comparison, a mechanism must exist for PLH and PLCH microparticles whereby lecithin and the released digestion products are removed from the pores, leaving a bare lipid interface for lipase adsorption and thereby facilitating complete digestion, it is hypothesised that the electrostatic interaction between the PLGA naiiopaiticles and negatively charged digestion products reduces the number of amphiphilie components that adsorb to the oil-water interface, and increases the level of vesicular and mi cellar structure formation [25].

[00101] Lipid hydrolysis of all dry hybrid micropariicies demonstrated sustained digestion eompared to PLGA nanoparticie-stabilised emulsions, indicating that there is a greater level of physical shielding o lipase by PLGA in. the dry phase compared to the wet phase. The overall extent of Iipolysis for PLH-I micropariicies was 1.8 ± 3.7% over a 60-mInute digestion period, which was consistent with the sateymatic degradation of lipid for a. 10 wt% PLGA-1 stabilised emulsion. Hence, the rate of lipid hydrolysis was significantly slower for PLH mieropartieles, but the overall extent of digestion was equal to a PLGA-stabilised emulsion. Slow initial digestion kinetics suggests slow adsorption of the enzyme ^■ from the aqueous continuous phase before becoming cataiytically active at the interface [22 ], however, equivalent extent of digestion suggests: that the microstructure facilitates, sustained digestion and a reduced interference effect of digestion products and PLGA nanopartieles on lipase adsorption.

[0O102 j The hydrolysis of lipid encapsulated within PLH and PLCH mieropartieles showed pseudo-first order kinetics (Figure 6 inset). In particular, the first order rate constants, k, were determined, for each lipid system b fitting a curve to the /«(_¾.¾-—H!¾) verses time- (Figure 6 inset) and arc gi ven in Table 3, along with the extent of hydrolysis after hours digestion,. Him*.

[00103] Tablc 3

[00104 j Previous kinetic analysis demonstrated the inability to fit first-order kinetics t the digestion of a submicfon .emulsion as a result of the shar decrease its reaction rate after 5 minutes of digestion, due to the role of amp ophilic compounds in inhibiting lipase adsorption as the emulsion droplet decreases in size [25 ]. Consequently, the first order rate constant of 0.305 mitt ^! is significantly greater than those for the hybrid mieropartieles, but only describes the initial digestion kinetics. Along with this, digestion, of lipid within siliea-lipid hybrid (SLH) mieropartieles prepared accordmg to Tan at al [22] by spray drying silica nanoparticle-stabiiised emulsion droplets, demonstrated tri-phasie pseudo-first order kinetics and thus required three first order rate constants to precisely describe the data. Digestion kinetics were enhanced in SLH mieropartieles compared to a submicron emulsion due to the increased surface area o lipid and the hydrophific silica matrix facilitating the interracial activation of lipase. The difference in digestion kinetics between the hybrid systems o f silica and PLGA indicates that the change in surface chemistry and larger particle size of PLGA nanopartieles compared to silica alters the interaction between lipase and the lipid interface. While not to be bound b theory,, it is considered that lipase adsorbs to the mostly hydrophobic PLGA surface in its inactive., "closed-lid" orientation, reducing the number of enzyme molecules, available for catalysis and, thereby, reducing the lipid digestion rate. Further, the increased particle size that constitutes the solid matrix increases the steric hindrance of enzyme molecules to the lipid interface,

[00105] The digestion kinetics were enhanced when negatively charged PLGA-2 nanoparticies were used to prepare hybrid mieroparticks, both in the presence and absence of mamii ol, when compared with positi vely charged PLGA-1 nanoparticies. The pseudo-first order rate constant increased from O.Q _. 17 and 0,037 mm ^'1 f r positively charged PLH-1 and PLMH- microparticles, respectively, to 0,024 and 0.049 mm ^"! for negatively charged PLB-2 and PL H-2 microparticies, respectivel (Table 3). This was consisten t wi th previous studies that have demonstrated the effect of surface charge on. digestion of conventional emulsion droplets; whereby, positively charged lipid droplets- coagulate due to electrostatic interactions between the positively charged droplet interface and negatively ciiarged digestion products that adsorb to the interface, reducing the accessible surface area tor lipase adsorption. [33, 34). In. addition to this, the rate and extent: of lipol sis increased when mannitof. was used in the composition.

[00106] The influence of surface charge and.manni.tol on. the digestibility of lipid, within the formulated hybrid microparticles is attributed to the effect that each component has on particle redispersibility. Thus, micropartiele redispersibilit was enhanced when PLGA nanoparticies were prepared with an. anionic stabiliser and mannitol was administered as a ciyepixsteciani (Figure 4). Attractive electrostatic interaction between positively charged PLGA nattoparticles and negatively charged lipid droplets appear to increase the stability of the micro-particle structure causing a reduction in redispersion properties, Marmito! was found to act as a cryoprotectant during spray drying by main taining the integrit of PLGA nanoparticies, thereby increasing the redispersion of mieroparticles to- heterogeneous coagulations of PLGA nanoparticies and lipid droplets. The rate constant and extent of lipid digestion was linearly dependent on the micropartiele redispersion/disintegmtlon (Figure 7), which is consistent: with previous findings where the rate and extent was found to be linearly dependent, to the surface area of lipid when adsorbed i porous silica [25 ). That is, the lipid digestion is controlled by the ability for lipase to access the lipid interface [35, 36] and thereby, an increase in particle disintegration increases the accessible surface area of lipid, which in turn increases lipase adsorption (Figure 8). The formation of ^' heterogeneous dispersions of PLGA nanoparticies and emulsion droplets for PLCH microparticles increases the interfacial surface are of lipid, whereas the inability for PLM microparticles to redisperse due to the microeapsule-like structure (ie. where the micropartiele consists of lipid droplets encapsulated within a sol i d outer shel l of nanoparticies) restri cts t he number of l i pase molecules that can bind to lip i d interface, producing digestion kinetics similar to a coarse emulsion. 100107] Whilst previously prepared PLGA-tipid hybrid microparticics with lipid shell-polymer core architecture have demonstrated .promising drug deliver}' applications, the novel three-dimensional porous structure of ^' microparticles synthesised in thi example address stability issues for the lipid

component of hybrid mictopartscles. That is:, by controlling lipase ^' action through surface charge and eryoproteetaat concentration, if is considered that this novel structure can facilitate controlled drug release and adsorption from the lipid component, in comparison to the common burst release mechanism of lipid shell-polymer core microparticles. It is proposed that microparticles according to the present invention presented will act as novel ding carriers with a wide range of applications, such as combinational therapy, oral delivery of poorly water soluble drugs and targeted delivery.

Example 2 Polymer-lipid hybrid (PLH) and poiyroer-lipid-cryo protectant hybrid (PLCH) micropartkle-based compositions for einnarizine

Materials a ^r Methods jOO!OSJ The poorly water soluble, anti-emetic dreg, cinnariztne (CIN) (Sigma-Aklrich Co, LLC, St Louts, MO, United States of America), was loaded into both the lipid droplets and polymer nanoparticies of PLH and PLCH micropartic les prepared according to the method described in Example 1 , but wherein einnarizine was dissolved in the lipid preparation (ie Migiyoi® ¹ 8.12 or a preparation of long chain length triglycerides (LCT), soybean oil) at a concentration of 5% and in the PLGA/ethyi acetate solution (with PV A or DMAB) at a concentration of 5%. The PLG used in these experiments were either of low- molecular weight (ie. MW = 7000-17000 Da.) or high molecular weight (fa MW = 30000-60060). Where included, the cryoprotectant used was mannitoi. Various dissolution studies were conducted according to standard protocols to assess the release of the cinnariztne from the PLH and PLCH mieroparticie:

compositions. A snbmicron MCT emulsion, s tabilised by lecithin, with a droplet size of approximatel 1 0 nm, a PLGA naaopartiele preparation and pure CIN were used for comparison.

Memlts and Bisa sion

[00 09] CIN release (ie. dissolution) into 0.1% sodium !anryl sulfate (SLS) solution was followed over 120 minutes, it was clearly apparent that CIN release from a PLCH mieroparticie composition (including high molecular weight PLGA) was enhanced compared to the emulsion alone, PLGA-1 nanoparticies and pure CIN (see Figure 9A). Similar enhancement of C N release was observed ton the PLCH mieroparticie composition when assessed in simulated gastric conditions and simulated intestinal conditions uring a two-step dissolution (ie. 0-60 mins in. the simulated gastric conditions and -60-120 minutes in the simulated intestinal conditions) (see Figure 9B). [001 10] in. further: studies of release into 0.1% SLS, it was found that the rate and extent of O dissolution was greater for negatively charged mieropartiele compo itions (ie including high molecular weight PLGA- 1 nanopartieles) compared to positively charged mieropartiele compositions (including high. molecula weight PLGA-2 nanoparticles) (see ^■■ Figure ^' 10), which may be due to enhanced redispersion of anionte niieropai icles compared to catlonic m.icropa:rlieies> in: addition, it. was found that the presence of mannitol within the PLCH micropartieles facilitated more rapid release kinetics due to increased mieropartiele redispersihi!ity (see Figure 10).

[0011 1] In addition, it was observed that the release kinetics of CIN were enhanced when low molecular weight (M W = 7000-57000) PLGA was used to formulate PLCH nricroparticles compared to hig molecular weight PLGA (Figure 1 1..), demonstrating the ease in hich drug release can be controlled in PLH/PLCH inieropartieles. CIN release kinetics are also greatly affected by substituting MCT lipid with long chain length triglycerides (LCT) (see Figure 12). in this case, LCT caused a. decrease in Ci release from PLCH mieropartiele composition.

Example 3 Polymer-lipid hybrid (PLH) mierepartkle-based composition for

ciiinarizine

[0 1 12] The pharmacokinetic behaviour of a PLH micropartieie-based composition for cinnarizine (CIN) was investigated against comparative silica-containing compositions.

Materials and Methods

[001 13] CoHipositioH re aratian

[00114] A silica-lipid hybrid (SLH) composition comprising CIN was prepared in accordance with previously described methods [ 18-20], 160 rng of the dried composition was pie-weighed int the 10 mL centrifuge tube, and then dispersed with 2 niL saline by vigorously vorte ing the mixture. For a 300 g rat, 1.5 mL of this mix was dosed vi oral gavage to rats for a dosage of 1 mg/kg. The CZ content was estimated to be about L5wt¾.

[00.115] A polymer-lipid hybrid (PLH) mieropartiele composition comprising CIN was prepared according to the method described in Example 1 , but wherein cinnarizine was dissolved in the lipid preparation (ie Migiyol® 812). The PLGA was of low molecular weight (MW = 7000-1.7000). The PLH composition was dosed into rats in a similar manner to that of the SLH composition, with 370 mg of dried composition weighed to allow for the same C content. A milky solution was formed upon hydration with 2 mL of saline. [001 16] A sitica-stabfiised cisbosomes (ioewtral) composition .comprising ON was. prepared according to Bhatt ef. &L [44]. This composition was dosed, into rats without ftrther preparation at a lower CZ content. (~-0.S wt%). However, all PK concentrations were dose-normalised following .Liquid Chromatography Mass Spectroscopy (LCMS) analysis,

[001 17] Surgical procedures

2:50-300 g male Sprague Dawley rats were used in this study. All forinulations were conducted in quadruplicates (n=4). Surgical procedures were conducted as described by ^' Nguyen et al. [42 j. Briefly, rats were anaesthetised via inhalation of isoflurane (5% v/v for induction, 2.5% v v for maintenance) and weighed. The incision sites above the breast bone and the scruff were shaved, and analgesic (0.1 mL of Bupivacaine 0,5%) was subcutaneously injected. The right carotid artery was i olated and eaanulated with a 0.96 mm x 0.54 mm polyethylene tubing, with the cannula flushed with 2 EJ heparin saiine solution. The rats were then attached to a hamess swivei system and placed in individual metabolism cages. Rats were also fasted and fed in the same manner as described by Nguyen ei l [42]. To allow for accurate determination of the dose amount, the syringe and gavage were weighed before and after dosing rats. Blood sampling from the rats was performed at 0 h, 1 2 h, 3 h, 4 , 6 h, 8 h, 1 h and 24 h. A volume of 200 was drawn each time, then immediately dispensed into 1.5 mL centrifuge tubes prc- filied with 10 Ιϋ of heparin. To allow cannula patency, 2 1U of heparin saline solution w ^? as flushed through the cannula after each blood sampling. The blood samples were then centrifuged for 5 minutes at. 7378 x.g. Plasma was collected as 2 aliquot of 50 fiL and f ozen at -20 °C until analysis. j 001 18 | Plasma extraction for C1N content

CIN plasma extraction was performed according to procedures described, by Sahbaz et al [43].. However, briefly, 50 uL blank plasma was spiked with cinnarizine and haiofantrme (internal standards), with CIN standard concentrations of 5, 10, 25, 50, 100 and 250 ng/mL. Saturated ammonium sulphate was used to precipitate plasma protein, and then acetonitrile was added, Aliquots of 25 μΐ, were collected from the supernatant, which was then, transferred into vials for LCMS analysis.

[001 19] LCMS analysis f r O

Processed plasma analysis was conducted on. a single-quadrupole LCMS ^' (Model 201 ; Shimadzu Corporation, Kyoto. Japan), using methods and settings described by Sahbaz el al, [43 j.

Results andD ^' iscussion

10 120] The results of this study arc provided in Table 4 and i Figure 13 , The results indicate that the oral bioavailability of CIN was enhanced when encapsulated in PLH rnieropartleles compared to two other hybrid mieropartieulate systems, given by greater AUC and C„ values. The bioavailability of PLH i -2,5 times that of the SLH composition and ~5 time that of the silica- stabilised cubosome composition. Thus, the results highlight die- promising release, characteristics: and soinbiiisation capacity of PLH PLCH mieropartiel es., whereby they may be used to increase the absorption of poorly water- soluble drugs. While- not wishing to be- bound by theory, this may be achieved by multiple-delivery raeehamsies; that is. Lipid digestion and increased dru solu !isation in mixed micelles, slow release of drug from the PLGA na»o rt es into the GIT,, and the pote-ntlai for direct absorptio of dfug-containing PLO-A nanopaitielcs (ie following microcapsule breakdown, which maybe particularl suited for the administration of an active substance to the Jang). The PLH/PLCH formulations may also be expected to extend the absorption half-life of a acti ve substance, due to the release behaviour and drug deli very ^' mechanisms.

[00121] Table 4 Pharmacokinetic parameters following oral administratiori of

incorporated in PLH, SLH and neutral silica-stabilised cabosnme compositions... Doses were normalised to 1 rng kg ON administered to rats. Each data is expressed as the mean ± SEM, n=4.

Composition AUQ _M last (ng/ral,h) Mean T _111SX (h) Mean C _mai (ng/ml)

SLH 108.6 ± 26.4 0.6 ± (U 20.8 ÷ 1.7

PLH 245.0 ± 77.5 4.0 ± 2.0 39.0 ± 7.3

Silica-stabilised 54.3 ± 13.7 3.5 J. 1.4 14.7 ± 5.2

100122] Throughout the specification and the claims that follow, unless the context-requires otherwise, the words -'comprise'- and "include" and variations suc as "comprising" and ''including" will he understood to -haply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers,

[00123] The reference to any prior art fa this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prio art forms rt of the common general knowledge.

[00124] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described.. Neither is the present invention, restricted in its preferred embodiment with regard to the particular demerits and/or features described or depicted herein. It will be appreciated that the invention is not limited to th embodiment or embodiments disclosed, but is capabie of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

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