WO2015188838A1

WO2015188838A1 - Self-assembling structures

Info

Publication number: WO2015188838A1
Application number: PCT/DK2015/050162
Authority: WO
Inventors: Sami HIETALA; Stefania G. BALDURSDOTTIR; Lene JØRGENSEN; Claus Greve MADSEN; Lærke ARNFAST; Korbinian LÖBMANN
Original assignee: University Of Copenhagen
Priority date: 2014-06-13
Filing date: 2015-06-12
Publication date: 2015-12-17

Abstract

The invention relates to a self-assembling multi-layered structure that is capable of self-assembling in an aqueous environment. The structure is capable of entrapping a compound, whereby the toxicity and/or aggregation capacity of the compound can be decreased, and/or its solubility and thermal stability can be increased. The structure can further be used for delivering a compound to an organism.

Description

Self-assembling structures Field of invention

The present invention relates to a multi-layered structure comprising one or more bilayers, said one or more bilayers comprising a plurality of modified polymer moieties, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, wherein each polymer moiety is modified by end-group substitution with a hydrophobic moiety. The structure is capable of self-assembling in an aqueous environment. Also disclosed herein is a method for manufacturing such a self- assembling structure. Also disclosed is the use of the multi-layered structure of the invention to entrap a compound. The multi-layered structure of the invention provides numerous advantages, and can be used for increasing solubility and/or thermal stability of a compound as well as reducing toxicity and/or denaturation or ability to aggregate a compound. The present structure can be used for preventing denaturation of compounds upon heat treatment. Also disclosed herein is a method for targeted or untargeted delivery of a compound to an organism using a multi-layered structure of the invention.

Background of invention

Virtually all bodily processes are regulated by proteins. Due to their complex structure, proteins interact almost exclusively with their intended targets. As therapeutics, proteins therefore show high potency with limited adverse effects. However, the proteins must maintain their delicate spatial structure in order to remain active. This means that many processes traditionally employed for pharmaceutical manufacturing, such as heat sterilization, are not viable with proteins. Instead costly and time- consuming processes, such as sterile-filtering, must be employed. This has led to an extremely high cost for therapeutic proteins, with even the oldest therapeutic protein (insulin) costing over two million euros per kilo in April 2014 [1]. Extensive research has gone into finding ways of making proteins more stable, thereby decreasing production cost while making a wider range of proteins feasible drug candidates. A gap has emerged between the number of proteins currently identified as drug candidates, and the amount of protein-based pharmaceuticals currently marketed or in development. As of 2012, an estimated 64 % of all human proteins, almost 13000 proteins, had been ascribed a gene and a function [2]. With the Human Proteome Project, started in 201 1 , this number is expected to rapidly increase. Meanwhile, only around 200 proteins were approved for clinical use in the European Union and USA as of 2010 [3], despite most universities having whole departments dedicated to protein research, and the first therapeutic protein being marketed in 1923. Nanomedical technologies aiming at enhancing drug delivery by enhancing e.g. drug solubility are currently being heavily investigated. This has so far been pursued with the use of nanosystems such as encapsulating vesicles, e.g. micelles, liposomes or nanoparticles. Micelles are structures formed by a surfactant and can be charged or neutral. In an aqueous environment, the hydrophilic heads of surfactant molecules face the solvent, while the hydrophobic tails are grouped within the central region of the micelle. In a non-aqueous environment, the micelle can be an inverse micelle, with the hydrophilic tails facing the solvent and the hydrophilic heads grouped in the central region. Micelles can thus be used to separate poorly soluble lipophilic compounds in a solution. Liposomes are charged artificially-prepared spherical vesicles composed of a lipid bilayer. In an aqueous solution, the hydrophobic tails are grouped within the core of the bilayer, while the hydrophilic heads are arranged on the inner and outer surfaces of the liposome.

WO 2009/091 103 describes methods for producing micelles of biopolymers such as drugs, proteins, DNA, RNA, saccharides and insoluble biomolecules. The micelles self- assemble in an aqueous solution. Such complexes are prepared by mixing a hydrophilic biopolymer with e.g. PEG in aqueous solution after removing salts, lyophilising the conjugate and dissolving it in an organic solvent; reacting the resulting nano-conjugate with a coupling agent to activate the biopolymer; and adding insoluble biomolecules dissolved in another organic solvent so that the activated biopolymer reacts with the insoluble biomolecules. WO 2009/091 103 requires the use of organic solvents and also necessitates lyophilisation and dialysis steps.

WO 2009/061515 describes self-assembling micelle-like nanoparticles (MNP). The MNPS are generated by conjugating a cationic polymer to a phospholipid; the conjugate is mixed with a nucleic acid, resulting in the formation of an MNP due to electrostatic interaction between the polymer and the nucleic acid. The lipid monolayer is supplemented by addition of unconjugated phospholipids such as cholesterol and the MNP is stabilised by addition of PEG or PEG derivatives, such as PEG coupled to lipids. The MNPs are charged on their surface and consist of a monolayer. The MNPs are not suitable for increasing thermal stability of proteins.

US5885613 discloses fusogenic liposomes, i.e. liposomes that can release any encapsulated or associated drug or therapeutic agent in the vicinity of the target cell, or fuse with the target cell plasma membrane and introduce the drug or therapeutic agent into the cell cytoplasm. The liposome comprises a bilayer stabilising component reversibly associated with a lipid capable of adopting a non-lamellar phase which is further capable of assuming a bilayer structure in the presence of the bilayer stabilising component. The fusogenic liposomes are not suitable for increasing thermal stability of proteins.

There is a need for methods allowing stabilisation of therapeutic compounds such as proteins.

Summary of invention

The invention is as described in the claims.

The present disclosure provides a multi-layered structure which can be used to entrap a compound such as a small molecule drug, a peptide or a protein, whereby several unexpected advantageous effects are achieved: the aggregation capacity and/or the toxicity of the entrapped compound are reduced compared to the aggregation capacity and/or toxicity of the compound in a non-entrapped state; the solubility and/or stability, including thermal stability, of the entrapped compound are reduced compared to the solubility and/or stability, including thermal stability, of the compound in a non- entrapped state. The entrapped compound is protected from denaturation, in particular when exposed to high temperatures. The entrapped compound can be released from the multi-layered structure as described below. In one aspect, the present invention relates to a multi-layered structure comprising one or more bilayers, said one or more bilayers comprising a plurality of modified polymer moieties, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, wherein each polymer moiety is modified by end-group substitution with a hydrophobic moiety. In another aspect, the present invention relates to the use of the multi-layered structure of the invention to entrap a compound.

In another aspect, the present invention relates to a method for manufacturing a self- assembling structure, comprising the steps of:

i) providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety;

ii) dissolving said modified polymer in an aqueous solution, thereby obtaining a self- assembling structure.

Also provided herein is a pharmaceutical composition comprising a multi-layered structure described herein or a self-assembling structure obtainable by the present methods.

In yet another aspect the invention relates to a method of delivery of a compound to an organism, said method comprising the steps of:

i) providing a compound entrapped in a multi-layered structure of the invention;

ii) administering said multi-layered structure to an individual.

Also provided herein is a controlled-release pharmaceutical dosage form of a poorly soluble or insoluble drug comprising a multi-layered structure comprising and/or entrapping said drug, one or more controlled-release materials and one or more pharmaceutically acceptable excipients.

The present disclosure also relates to a method for the preparation of a controlled- release pharmaceutical dosage form of a poorly soluble or insoluble drug comprising a multi-layered structure comprising and/or entrapping said drug, one or more controlled- release materials and one or more pharmaceutically acceptable excipients.

The present disclosure also relates to a method for increasing the solubility and/or the thermal stability and/or the bioavailability of a compound, said method comprising the steps of: i) providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety;

ii) dissolving said modified polymer in an aqueous solution, thereby obtaining a self-assembling structure;

iii) contacting said self-assembling structure with said compound, thereby obtaining a self-assembling structure comprising and/or entrapping said compound;

thereby increasing the solubility and/or the thermal stability and/or the bioavailability of said compound.

Also disclosed herein is a method for reducing aggregation and/or denaturation of a compound upon heat treatment, said method comprising:

i) providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety ;

iv) subjecting the self-assembling structure comprising and/or entrapping said compound to heat treatment;

whereby aggregation and/or denaturation of the compound is reduced.

The structure of the invention comprises one or more bilayers comprising a plurality of polymer moieties, each of said polymer moieties having two hydrophobic heads and a hydrophilic tail. The structure of the invention is thus different from liposomes in that the hydrophobic heads are grouped in the internal region of the bilayer, while the hydrophilic bridge is on the surface.

Description of Drawings

Fig. 1 shows possible configurations of some embodiments of the present disclosure, dubbed PEGosomes. Fig. 1A shows a multi-layered structure according to the invention, comprising one bilayer. The polymer is shown in grey, the hydrophobic moieties in black. P1 : polymer moiety 1 , modified by hydrophobic moieties H1 and H1 '. P2: polymer moiety 2, modified by hydrophobic moieties H2 and H2'. R: internal region. I: inner surface of the multi-layered structure; O: outer surface of the multi-layered structure. Fig. 1 B shows a multi-layered structure according to the invention, comprising one bilayer. The polymer is shown in black circles, the hydrophobic moieties in black. P1 : polymer moiety 1 , modified by hydrophobic moieties H1 and HV. P2: polymer moiety 2, modified by hydrophobic moieties H2 and H2'. R: internal region. I: inner surface of the multi-layered structure; O: outer surface of the multi-layered structure. The structures shown in Figs. 1A and 1 B are suggestive representations supplied for clarity, and should not be mistaken for an exact illustration of the

PEGosome construct.

Fig. 2 shows the chemical structure of the designed polymers used for the PEGosome constructs. H indicates a hydrophobic moiety. R indicates the place for substitution with cholesterol, linoleic acid, oleic acid or stearic acid. Also represented are examples for R: cholesterol (I), linoleic acid (II), oleic acid (III), stearic acid (IV).

Fig. 3 shows the synthesis of cholesterol-substituted poly(ethylene glycol). DCM:

Dichloromethane; TEA: Triethylamine; RT: Room temperature; N₂: N₂ purge. The reaction also results in the production of 2 HCI (not shown).

Fig. 4 shows the synthesis of fatty acid-substituted poly(ethylene glycol). p-TSA: p- toluenesulfonic acid; N₂: N₂ purge. The reaction also results in the production of 2 H₂0 (not shown).

Fig. 5 shows dynamic Light Scattering (DLS) data of 20000 Da poly(ethylene oxide) (PEG20K) alone or modified with cholesterol (cholesterol-PEG20K), with and without lysozyme or bovine serum albumin (BSA) in 10 mM phosphate buffer. The bars indicate the intensity of scattered light at a given construct size; the grey curve indicates cumulative counts as percentage, and the white dotted line indicates 50 % cumulative counts (half of the graphs intensity lying on either side of the line). Each measurement was repeated in triplicate.

Fig. 6 shows Dynamic Light Scattering (DLS) data of 20000 Da poly(ethylene oxide) modified with cholesterol (cholesterol-PEG20K), unfiltered and filtered in 10 mM phosphate buffer (no protein present). The bars indicate intensity of scattered light at a given construct size; the grey curve indicates cumulative counts as percentage, and the white dotted line indicates 50 % cumulative counts (half of the graphs intensity lying on either side of the line). Each measurement was repeated in triplicate. A: unfiltered; B: immediately following after filtering through a 0.22 μηι sterile filter; C: 12 hours after filtering.

Fig. 7 shows Dynamic Light Scattering (DLS) data of 4000 Da poly(ethylene oxide) (PE04K) modified with different fatty acids, with and without lysozyme (Lys) or bovine serum albumin (BSA) in ultrapure water. The bars indicate intensity of scattered light at a given construct size, the grey curve indicates cumulative counts as percentage, and the white dotted line indicates the 50 % cumulative counts (meaning that half of the graphs intensity lies on either side of the line). Each sample was dissolved in filtered ultrapure water, filtered again upon transfer to a cuvette and measured. Each sampling was repeated 3 times, and each repeat measured 5 times (totalling 15 measurements for each graph, all of which are displayed).

Fig. 8 shows Dynamic Light Scattering (DLS) data of 10000 Da poly(ethylene oxide) (PEO10K) modified with different fatty acids, with and without lysozyme (Lys) or bovine serum albumin (BSA) in ultrapure water. The bars indicate intensity of scattered light at a given construct size; the grey curve indicates cumulative counts as percentage, and the white dotted line indicates 50 % cumulative counts (half of the graphs intensity lying on either side of the line). Each sample was dissolved in filtered ultrapure water, filtered again upon transfer to a cuvette and measured. Each sampling was repeated 3 times, and each repeat measured 5 times (totalling 15 measurements for each graph, all of which are displayed).

Fig. 9 shows Dynamic Light Scattering (DLS) data of 20000 Da poly(ethylene oxide) (PEO20K) modified with different fatty acids, with and without lysozyme (Lys) or bovine serum albumin (BSA) in ultrapure water. The bars indicate intensity of scattered light at a given construct size; the grey curve indicates cumulative counts as percentage, and the white dotted line indicates 50 % cumulative counts (half of the graphs intensity lying to either side of the line). Each sample was dissolved in filtered ultrapure water, filtered again upon transfer to a cuvette and measured. Each sampling was repeated 3 times, and each repeat measured 5 times (totalling 15 measurements for each graph, all of which are displayed).

Fig. 10 shows box and whisker plot of delay time (τ) distribution in samples, as measured by DLS. All data points are shown. Whiskers extend from minimum to maximum while the box indicates 25 and 75 percentiles. Line in box indicates the median, while the + indicates the mean. Larger constructs are slower and have higher delay times. Note that the maximum and minimum ranges are parameters of the measurement method, which means that the median is a better indicator of construct size distribution (compare figures 7, 8 and 9).

Fig. 1 1 shows photographs of different samples. Fig. 11 A: Cryo-Transmission Electron Microscopy images of cholesterol-PEG20K with bovine serum albumin (BSA). CG: carbon grid. P: PEGosome (cholesterol-PEG20K). I: ice. Fig. 11 B: samples of lysozyme and bovine serum albumin (BSA) with and without Iinoleic acid-PEG20K after heating. Equivalent amounts (w/w) of protein and PEGosome were mixed, and diluted to about 1 % (w/w) with ultrapure water. Samples were then covered and heated to 60°C for 24 hours, 80°C for 24 hours and finally to 118 °C for 12 hours (photographs are taken after all three heating steps). Lost solvent was replaced as needed. A:

lysozyme + Cholesterol-PEG20K; B: lysozyme; C: BSA + cholesterol-PEG20K; D: BSA. Fig. 1 1C: samples of bovine serum albumin (BSA) with and without PEG20K, cholesterol-PEG20K (chol-PEG20K) and stearic acid-PEG20K (SA-PEG20K) after autoclaving. Samples were prepared in equivalent amounts and diluted to 1 % w/w with ultrapure water. Autoclaving was conducted at 121 °C for 15 minutes. Lower panel

(arrow): bottom-up view. Fig. 1 1 D: emulsification of cholestrol-PEG20K. Samples of 1 % PEG20K and vitamin E, cholesterol-PEG20K and vitamin E, and vitamin E alone were prepared with 10 mM phosphate buffer (panel A). The samples were mixed by end-to-end mixing for 48 hours and resulting emulsions were imaged by light microscopy at 40x magnification (panel B).

Fig. 12 shows cytotoxicity data. Fig. 12A: cytotoxicity of Iinoleic acid constructs on Caco-2 cells. Caco-2 cells were incubated for 24 hours with either Hank's Balanced Salt Solution (HBSS), or solutions of HBSS and either polyethylene glycol p-(1 , 1 ,3,3- tetramethylbutyl)-phenyl ether (Triton X-100; a detergent), or different concentrations of Iinoleic acid (LA) constructs (concentrations are given as that of the fatty acid). Fig. 12B shows cytotoxicity of oleic acid constructs on Caco-2 cells. Caco-2 cells were incubated for 24 hours with either Hank's Balanced Salt Solution (HBSS), or solutions of HBSS and either polyethylene glycol p-(1 , 1 ,3,3-tetramethylbutyl)-phenyl ether (Triton X-100; a detergent), or different concentrations of oleic acid (OA) constructs

(concentrations are given as that of the fatty acid). Fig. 12C shows cytotoxicity of stearic acid constructs on Caco-2 cells. Caco-2 cells were incubated for 24 hours with either Hank's Balanced Salt Solution (HBSS), or solutions of HBSS and either polyethylene glycol p-(1 , 1 ,3,3-tetramethylbutyl)-phenyl ether (Triton X-100; a detergent), or different concentrations of stearic acid (SA) constructs (concentrations are given as that of the fatty acid). Fig. 13 shows differential scanning Calorimetry of PEGosome with and without bovine serum albumin (BSA). Equimolar samples equivalent to a 6 % w/w BSA solution were prepared and measured at a heating rate of 30 °C/min. Linoleic acid-PEG20K+BSA subtracted (corrected) by linoleic acid-PEG20K is presented for comparison.

Fig. 14 shows Fourier transformed infrared spectroscopy (FTIR) measurements of autoclaved bovine serum albumin with cholesterol-PEG20K (chol-PEG20K), stearic acid-PEG20K (SA-PEG20K) and stearic acid-PEG20K with air interface removed (SA- PEG20K in Eppendorf tube). Native BSA (dashed line) displayed for reference (BSA). Presented spectra were obtained by solvent subtraction, second derivative Savitzky- Golay smoothening, baseline correction and normalization. All samples were measured in triplicates.

Fig. 15 shows solubility increase studies by UV absorbance. The UV absorbance of PEG20K conjugated with cholesterol (chol-PEG20K) and PEG20K was measured either by itself or mixed with the model drugs Probenecid or Furosemide. Samples were prepared by mixing equal amounts of PEGosome and model drug in, and diluting to about 10 mg/mL in phosphate buffer. Samples were mixed by end-to-end mixing for at least 48 hours, filtered and measured against the sample's respective reference. Each sample was repeated in triplicate.

Fig. 16 shows solubility increase studies by HPLC. Solubility of probucol, indomethacin and amiodarone in 0.01 M phosphate buffer, with and without added PEGosome (1 % w/w). Note the broken y-axis. Numbers above columns denote column means. Results below detection limit are denoted '< V. Bars indicate data-range (n = 3).

Detailed description of the invention

The present disclosure provides a multi-layered structure which can be used to entrap a compound such as a small molecule drug, a peptide or a protein, whereby several unexpected advantageous effects are achieved: the aggregation capacity and/or the toxicity of the entrapped compound are reduced compared to the aggregation capacity and/or toxicity of the compound in a non-entrapped state; the solubility and/or stability, including thermal stability, of the entrapped compound are reduced compared to the solubility and/or stability, including thermal stability, of the compound in a non- entrapped state. The entrapped compound is protected from denaturation, in particular when exposed to high temperatures. The entrapped compound can be released from the multi-layered structure as described below. In one aspect, the present invention relates to a multi-layered structure comprising one or more bilayers, said one or more bilayers comprising a plurality of modified polymer moieties, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, wherein each polymer moiety is modified by end-group substitution with a hydrophobic moiety.

In another aspect, the present invention relates to the use of the multi-layered structure of the invention to entrap a compound. In another aspect, the present invention relates to a method for manufacturing a self- assembling structure, comprising the steps of:

ii) administering said multi-layered structure to an individual.

The present disclosure also relates to a method for increasing the solubility and/or the thermal stability and/or the bioavailability of a compound, said method comprising the steps of:

whereby aggregation and/or denaturation of the compound is reduced.

In one aspect, the present invention relates to a multi-layered structure comprising one or more bilayers, said one or more bilayers comprising a plurality of modified polymer moieties, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, wherein each polymer moiety is modified by end-group substitution with a hydrophobic moiety.

In another aspect, the present invention relates to the use of the multi-layered structure of the invention to entrap a compound.

ii) dissolving said modified polymer in an aqueous solution, thereby obtaining a self- assembling structure. In yet another aspect the invention relates to a method of delivery of a compound to an organism, said method comprising the steps of:

ii) administering said multi-layered structure to an individual.

The present inventors have shown that the multi-layered structures of the invention present numerous unexpected advantages.

The multi-layered structure of the invention is flexible and deformable. Thus in some embodiments the structure can pass a filter having a pore size smaller than the size of the structure when it is in a "resting conformation", i.e. when it is not deformed (see example 2).

The multi-layered structure of the invention spontaneously adopts a uniform size distribution. As a consequence, in some embodiments the methods of the invention do not comprise a step of size homogenisation, i.e. a size homogenisation step is optional.

The multi-layered structure of the invention can be manufactured without organic solvent. Thus the methods of the invention do not require a step of purification to remove the organic solvent, whereby in some embodiments the toxicity and chaotropicity of the structure of the invention are reduced.

The multi-layered structure of the invention is thermodynamically favourable in aqueous solutions. Thus in some embodiments the lifetime and/or storage time the compound entrapped within a multi-layered structure of the invention are increased. Moreover, storage may be carried out under less stringent conditions than with other methods. Entrapment of compounds within the multi-layered structure of the invention results in reduced toxicity and/or aggregation capacity of the compound, and/or increased solubility and thermal stability of the compound. The present structure can be used for preventing denaturation of compounds such as proteins upon heat treatment. Definitions

Bioavailability: The term bioavailability refers to the rate and the extent to which the active ingredient or active moiety of a compound becomes available at the desired target site. Compounds have low bioavailability when their bioavailability is so low that it restricts the action of the active ingredient or active moiety at the desired target site. The bioavailability of a compound such as a drug depends of the efficacy and toxicology of the compound, and depends on factors such as, but not limited to, the effective concentration of the drug and its toxicology. The skilled person knows how to determine whether a compound has low bioavailability.

Branched: The term "branched" is herein used to refer to a polymer, wherein a substituent on a monomer subunit, e.g., a hydrogen atom, is replaced by another covalently bonded chain of that polymer or by a chain of another type (graft polymer). Chaotropic: The term chaotropic refers to a molecule having the ability to disrupt the hydrogen bonding network between water molecules in water solutions. This has an impact on the stability of the native state of other molecules in the solution, mainly macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect. A chaotropic agent may cause protein denaturation.

Deformable: As used herein, the term "deformable" is to be construed as having the ability to deform or to change shape. A multi-layered structure of the invention may change shape and recover its initial or thermodynamically favourable shape when not submitted to physical constraints, such as being forced through the pores of a filter. Flexible: The term "flexible" is used herein to refer to a polymer or a multi-layered structure which can assume different three-dimensional configurations or

conformations.

Linear: The term "linear" is used herein to refer to a polymer which has a linear chain, as opposed to a branched chain. Linear polymers are essentially unbranched.

Micelle: A micelle is an aggregate of surfactant molecules, forming a colloid when dispersed in a liquid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding aqueous solvent, sequestering the hydrophobic single-tail regions in the micelle centre.

Polyethylene glycol (PEG): is also termed polyethylene oxide (PEO) or polyoxyethylene (POE) and refers to a polymer of ethylene oxide. PEG is available with a wide variety of molecular weights, ranging from 300 g/mol to 10,000,000 g/mol. The molecular weight is directly dependent on chain length. PEGs with different chain lengths may have distinct physical properties, such as different crystallinities or glass transition temperatures, but their chemical properties, i.e. their behaviour in chemical reactions, are nearly identical.

PEGosome: The term "PEGosome" refers to a PEG molecule modified according to the invention by end-substitution with a hydrophobic moiety.

Self-assembly: In the present context, self-assembly refers to the spontaneous and reversible organisation of molecular entities by non-covalent interactions. Self- assembly describes a process in which a system of pre-existing components, under specific conditions, adopts a more organised structure through interactions between the components themselves. Self-assembling structures described herein may form multi-layered structures.

Stability: As understood herein, the term stability refers to the chemical or

thermodynamic stability of a system, which occurs when the system is at a low energy state. A system is typically thermodynamically stable when it is in chemical equilibrium with its environment. The equilibrium may be dynamic.

Solubility: The term solubility refers herein to the ability of a compound to dissolve in a solvent to form a solution. Particularly relevant for the present disclosure is the definition of the terms 'poorly soluble or insoluble' according to the Biopharmaceutics Classification System (BCS) as provided and defined by the US Food and Drug Administration (FDA) for drugs for oral administration. Four different classes of drugs are defined. Class I - High Permeability, High Solubility (neither permeability not solubility limits the oral bioavailability of the drug compound).

Class II - High Permeability, Low Solubility (solubility limits the oral bioavailability of the drug compound).

Class III - Low Permeability, High Solubility (permeability limits the oral bioavailability of the drug compound).

Class IV - Low Permeability, Low Solubility (both permeability and solubility limit the oral bioavailability of the drug compound).

According to this classification, compounds have low solubility if their highest dose strength is not soluble in 250 mL of an aqueous medium or less over the pH range of 1 to 6.8.

Structure: The term "structure" is to be construed herein as referring to a physical structure, as opposed to a chemical structure. The term may for example refer to a macromolecular construct which has a structured physical organisation.

Thermal stability: A compound is herein referred to as "thermally stable" if it does not have a propensity to degradation, denaturation and/or inactivation upon temperature increase.

Thermodynamically favourable: A thermodynamically favourable product is a product which is energetically most stable of all the possible products of a given reaction and/or under given conditions.

Water soluble: In the present context, a water soluble polymer is a polymer which is soluble or dispersible in an aqueous environment, such as, but not limited to, water or a solution in which the solvent is water.

Polymer and hydrophobic moieties

Thus, the structure of the invention comprises one or more bilayers formed of a polymer having two hydrophobic heads and a hydrophilic bridge. The structure of the invention is thus different from liposomes in that the hydrophobic heads are grouped in the internal region of the bilayer, while the hydrophilic bridge is on the surface.

In some embodiments, the uncharged, water soluble, flexible polymer is linear. In other embodiments, the polymer is branched. Examples of suitable uncharged, water soluble, flexible polymers include, but are not limited to, polyethylene glycol (PEG), in which case the multi-layered structure will hereinafter be referred to as PEGosome. The polymer moiety may be a derivative of an uncharged, water soluble, flexible polymer, such as, but not limited to, an acylated (such as methylated), aminated or amidated polymer. Such polymers are known to the skilled person. In a preferred embodiment, the polymer moiety is a PEG moiety. Examples of suitable PEG derivatives include, but are not limited to: PEG substituted with one or more hydroxyl groups; alkyl modified PEG substituted with one or more alkyl groups; amine modified PEG substituted with one or more amine groups; thiol modified PEG substituted with one or more thiol groups; carboxyl modified PEG substituted with one or more carboxyl groups; acryl modified PEG substituted with one or more acryl groups; and mixtures thereof. An example of the alkyl modified PEG is dimethyl PEG. Other derivatives known to the skilled person are also envisaged. The molecular weight of the PEG moiety is directly correlated with the length of its chain. The molecular weight of the PEG moiety may in some embodiments be comprised between 400 and 100000 Da, such as between 600 and 90000 Da, such as between 1000 and 80000 Da, such as between 2000 and 70000 Da, such as between 4000 and 60000 Da, such as between 4000 Da and 50000 Da, such as between 4000 Da and 40000 Da, such as between 4000 Da and 30000 Da, such as between 4000 Da and 20000 Da, such as 4000 Da, such as 10000 Da, such as 20000 Da. In some embodiments, the molecular weight of the PEG moiety is 20000 Da or more, such as 50000 Da or more, such as 100000 Da or more, such as 250000 Da or more, such as 500000 Da or more, such as 750000 Da or more, such as 1000000 Da or more. In some embodiments, the molecular weight of the PEG moiety is lower than 1000 Da, such as lower than 900 Da, such as lower than 800 Da, such as lower than 700 Da, such as lower than 600 Da, such as lower than 500 Da. In some embodiments, the PEG moiety or the PEG derivative comprises less than 10 ethylene glycol units, such as less than 9 ethylene glycol units, such as less than 8 ethylene glycol units, such as less than 7 ethylene glycol units. In a preferred embodiment, the PEG moiety or the PEG derivative comprises 8 ethylene glycol units.

The polymer moiety is modified by end substitution with a hydrophobic moiety. The hydrophobic moiety may be selected from the group comprising cholesterol, fatty acid moieties, phospholipids, triglycerides, sterols and other natural or synthetic lipid molecules and derivatives thereof. In some embodiments, the hydrophobic moiety is a saturated fatty acid or an unsaturated fatty acid. In some embodiments, the fatty acid moiety is selected from the group comprising a stearic acid moiety, an oleic acid moiety or a linoleic acid moiety, an elaidic acid moiety, an arachidic acid moiety, a palmitic acid moiety, an arachidonic acid moiety or a linolenic acid moiety, as well as isoforms and oxidative products thereof.

In some embodiments, the polymer moiety is modified by end substitution with two hydrophobic moieties, where each end of the polymer is modified by one hydrophobic moiety. The two hydrophobic moieties may be identical or different.

Thus in some embodiments, the polymer moiety is PEG-4000 and the hydrophobic moiety is cholesterol or a fatty acid. In some embodiments, the polymer moiety is PEG- 4000 and the hydrophobic moiety is a stearic acid moiety. In some embodiments, the polymer moiety is PEG-4000 and the hydrophobic moiety is an oleic acid moiety. In some embodiments, the polymer moiety is PEG-4000 and the hydrophobic moiety is a linoleic acid moiety. In other embodiments, the polymer moiety is PEG-10000 and the hydrophobic moiety is cholesterol or a fatty acid. In some embodiments, the polymer moiety is PEG-10000 and the hydrophobic moiety is a stearic acid moiety. In some embodiments, the polymer moiety is PEG-10000 and the hydrophobic moiety is an oleic acid moiety. In some embodiments, the polymer moiety is PEG-10000 and the hydrophobic moiety is a linoleic acid moiety. In other embodiments, the polymer moiety is PEG-20000 and the hydrophobic moiety is cholesterol or a fatty acid. In some embodiments, the polymer moiety is PEG-20000 and the hydrophobic moiety is a stearic acid moiety. In some embodiments, the polymer moiety is PEG-20000 and the hydrophobic moiety is an oleic acid moiety. In some embodiments, the polymer moiety is PEG-20000 and the hydrophobic moiety is a linoleic acid moiety. The hydrophobic moiety may substitute one or both ends of the polymer moiety. Preferably, the hydrophobic moiety substitutes both ends. The polymer moiety may be substituted in both ends with two different hydrophobic moieties.

Method of modifying a polymer

Methods for manufacturing a modified polymer of the invention are known to the skilled person. For example, such methods may comprise esterification reactions suitable for attaching the hydrophobic moieties to an end of the polymer moiety. In some embodiments, the polymer is modified following hydroxyl-end activation. Direct esterification, e.g. with acid chlorides, or activation of the polymer, e.g. by amidation or action with p-toluenesulfonic acid, may also be performed. Methods of activation of the polymer prior to hydrophobic modification depend on the nature of the polymer. The appropriate method will be obvious to the skilled person. Non-limiting examples can be found in example 1 below. The composition of modified polymer may optionally be precipitated and dried in order to obtain an essentially anhydrous composition of modified polymer.

By way of example only, in order to manufacture a cholesterol-PEGosome, wherein the hydrophobic moiety is cholesterol and the polymer moiety is PEG, the hydroxyl end groups of PEG may be esterified (see example 1). The reaction product may optionally be precipitated and dried in order to obtain a dry composition of PEGosome. In order to manufacture a fatty acid-PEGosome, wherein the hydrophobic moiety is a fatty acid and the polymer moiety is PEG, the PEG may be activated by addition of p- toluenesulfonic acid. The resulting composition may optionally be precipitated and dried.

Multi-layered structure

Modified polymers as described above have the capability of self-assembling into a multi-layered structure of the invention when in an aqueous environment. Such a multi- layered structure is shown by way of example in figure 1. As described above, the polymer is uncharged, water soluble and flexible. Thus it follows that the inner and outer surfaces of the multi-layered structure (I and O on figure 1 , respectively), where the hydrophilic chain of the polymer is located, are uncharged. The multi-layered structure is preferably capable of self-assembling. In some embodiments, each polymer moiety is modified by end-group substitution with two hydrophobic moieties, each end being substituted with one hydrophobic moiety. Some examples of such a structure comprising one bilayer is shown in figure 1A and figure 1 B. A first polymer moiety P1 is modified by hydrophobic moieties H1 and H1 '. A second polymer moiety P2 is modified by hydrophobic moieties H2 and H2'. Each modified polymer forms a hairpin structure, wherein the hydrophobic moieties form the free ends of the hairpin. Two modified polymer moieties can assemble as shown, with the hydrophobic moieties interacting, and the chains of the modified polymers facing outwards. A plurality of modified polymers can thus assemble in one (as shown) or more bilayers. The region where the hydrophobic moieties interact will hereinafter be referred to as the internal region (R in figure 1). The interaction between hydrophobic moieties may be intramolecular (H1 interacting with H1') and/or intermolecular (H 1 interacting with H2). The two hydrophobic moieties substituting a same polymer moiety may be identical (Η 1=Η1 ') or different (Η1≠Η1 '). The hydrophobic moieties substituting different polymer moieties may be identical (H1=H2) or different (H1≠H2).

The diameter of the multi-layered structure of the invention is dependent on the length of the chain of the polymer. In some embodiments, the structure has a diameter in the range of 10 to 10000 nm, such as between 10 and 7500 nm, such as between 20 and 5000 nm, such as between 30 and 4000 nm, such as between 40 and 3000 nm, such as between 50 and 2000 nm, such as between 50 and 1000, such as between 40 and 500 nm, such as between 60 and 500 nm, such as between 60 and 400 nm, such as between 60 and 300 nm, such as between 60 and 200 nm, such as between 60 and 150 nm, such as between 60 and 100 nm. Thus in preferred embodiments, the structure has a diameter comprised between 60 and 100 nm. In some embodiments, the structure forms a nanostructure having a diameter between 0.1 nm and 1000 nm, such as between 1 nm and 900 nm, such as between 10 nm and 800 nm, such as between 25 nm and 700 nm, such as between 50 nm and 600 nm, such as between 75 nm and 500 nm, such as between 100 nm and 400 nm, such as between 150 nm and 300 nm, such as between 170 nm and 250 nm, such as 200 nm.

The multi-layered structure is thermodynamically favourable; in other words, the structure is stable in an aqueous environment. This can be determined by methods of structural analysis as known in the art, e.g. cryogenic transmission electron microscopy (cryo-TEM) or dynamic light scattering (DLS). Such structural analyses are preferably performed once the system has reached equilibrium.

In particular embodiments, the multi-layered structure disclosed herein is not a micelle.

Multi-layered structure comprising an entrapped compound

In some embodiments, the multi-layered structure may comprise a compound within said internal region (R in figure 1 ; compound not shown). Thus the compound is entrapped within the multi-layered structure.

In order to entrap a compound within a multi-layered structure of the invention, a composition of modified polymer as described above is contacted with a composition of said compound. Contacting of the modified polymer with the compound may preferably be performed in an aqueous environment.

Entrapping a compound within a multi-layered structure

Thus it is one aspect of the invention to use a multi-layered structure of the invention to entrap a compound. In some embodiments, the compound is entrapped upon contact with the modified polymer described above. In other embodiments, the compound is entrapped upon contact with the modified polymer described above in an aqueous environment.

Thus also disclosed herein is a method for entrapping a compound, said method comprising the steps of:

i) providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety; dissolving said modified polymer in an aqueous solution, thereby obtaining self-assembling structure;

contacting said self-assembling structure with said compound, thereby obtaining a self-assembling structure comprising and/or entrapping said compound.

It will be understood that the compound can be contacted directly with the modified polymer in step ii), so that both the modified polymer and the compound are dissolved in an aqueous solution, whereby a self-assembling structure comprising and/or entrapping said compound is obtained in step ii). Thus in some embodiments, a self- assembling structure comprising and/or entrapping said compound can be obtained by the steps of:

i) providing a modified polymer, said polymer being an uncharged, water

soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety ;

ii) dissolving said modified polymer and said compound in an aqueous

solution, thereby obtaining a self-assembling structure comprising and/or entrapping said compound. Without being bound by theory, the inventors propose that the multi-layered structure's size has an upper limit depending on the chain length of the polymer moiety. Once this upper size limit is reached, the structure may not be thermodynamically favourable any longer. Likewise, it is possible that the size of the compound to be entrapped influences the stability of the structure. As a consequence, some polymers may be better suited for entrapping large compounds and other polymers for entrapping smaller compounds. The skilled person will know how to determine which polymers are best suited depending on the compound to entrap. In some embodiments modified polymers, wherein the polymer moiety has a long chain, are preferably used for entrapping large compounds.

The compound to be entrapped and the multi-layered structure may be brought into contact in different relative amounts. Thus in some embodiments, the compound and the structure are in a molar ratio between 1 :100 and 100: 1 , such as between 1 :75 and 75: 1 , such as between 1 :50 and 50:1 , such as between 1 :40 and 40: 1 , such as between 1 :30 and 30: 1 , such as between 1 :25 and 25: 1 , such as between 1 :20 and 20:1, such as between 1:10 and 10:1, such as between 1:5 and 5:1, such as between 1:4 and 4:1, such as between 1:3 and 3:1, such as between 1:2 and 2:1, such as 1:1. The optimal molar ratio may depend upon the nature of the compound to be entrapped, of the polymer moiety, of the hydrophobic moieties, and on the strength of the interactions between the structure and the compound.

Likewise, the compound to be entrapped and the multi-layered structure may be contacted in a weight ratio between 1:100 and 100:1, such as between 1:75 and 75:1, such as between 1:50 and 50:1, such as between 1:40 and 40:1, such as between 1:30 and 30:1, such as between 1:25 and 25:1, such as between 1:20 and 20:1, such as between 1:10 and 10:1, such as between 1:5 and 5:1, such as between 1:4 and 4:1, such as between 1:3 and 3:1, such as between 1:2 and 2:1, such as 1:1.

The compound to be entrapped may be any compound of interest. In some

embodiments, the compound is lipophilic. The compound may be selected from the group comprising proteins and peptides. In other embodiments, the compound is a toxic compound, and may e.g. have a lethal or damaging effect on cell viability. In other embodiments, the compound is poorly soluble. In yet other embodiments, the compound is prone to aggregation. In yet other embodiments, the compound has poor thermal stability.

The inventors have shown that entrapment of compounds with the multi-layered structures of the invention presents numerous unexpected advantages. The multi-layered structure of the invention is flexible and deformable. Thus in some embodiments the structure can pass a filter having a pore size smaller than the size of the structure when it is in a "resting conformation", i.e. when it is not deformed (see example 2). The multi-layered structure of the invention spontaneously adopts a uniform size distribution. As a consequence, in some embodiments the methods of the invention do not necessarily comprise a step of size homogenisation.

The multi-layered structure of the invention can be manufactured without the use of organic solvent. Thus the methods of the invention do not require a step of purification to remove the solvent, whereby in some embodiments the toxicity and chaotropicity of the structure of the invention are reduced.

The multi-layered structure of the invention is thermodynamically favourable. Thus the lifetime and/or storage time the compound entrapped within a multi-layered structure of the invention are increased. Moreover, storage of the entrapped compound may be carried out under non-stringent conditions.

Thus the invention relates to the use of a multi-layered structure of the invention to entrap a compound and thereby reduce its aggregation capacity and/or denaturation capacity. The invention also relates to increasing the thermal stability of a compound. In particular, aggregation upon heat treatment such as sterilisation or autoclaving is reduced. Conversely, thermal stability of the compound is increased. In some embodiments, aggregation of a compound upon heat treatment, e.g. autoclaving, is at least partially prevented. In other embodiments, denaturation of a compound upon heat treatment is at least partially prevented. In some embodiments, aggregation of a compound upon heat treatment may be completely prevented. The absence of liquid- air interface during the heat treatment facilitates reduction of aggregation (see example 4). In some embodiments, no aggregation is visible upon autoclaving of the compound entrapped within the multi-layered structure. Without being bound by theory, the inventors hypothesise that steric hindrance by the modified polymer is one of the mechanisms which results in decreased possibilities for aggregation of the compound. The use of a multi-layered structure of the invention to entrap a compound and thereby increase its thermal stability is particularly interesting for compounds which are not stable upon heat treatment under their free form. Thus in some embodiments the use of a multi-layered structure of the invention is for entrapping a protein or a peptide. In some embodiments the protein or peptide may be subjected to a heat treatment such as autoclaving or another heat sterilisation method, and retain at least part of its structure. In other embodiments, the protein or peptide suffers no structure loss.

The invention also relates to the use of a multi-layered structure of the invention to entrap a compound and thereby increase its solubility. The compound to be entrapped may be an insoluble compound. The inventors surprisingly found that solubility of such compounds may be increased when they are entrapped in a multi-layered structure of the invention. Releasing the entrapped compound

The complex consisting of a multi-layered structure of the invention and an entrapped compound is reversible. In other words, the compound can be released from the multi- layered structure. This can be achieved by contacting the multi-layered structure comprising the entrapped compound in its internal region with another compound.

In some embodiments, the other compound has the ability to bind strongly to the entrapped compound. Although the interaction between the entrapped compound and the multi-layered structure is sufficient to provide a thermodynamically favourable and thus a stable complex, the interaction is hypothesised to be a hydrophobic interaction in some embodiments and is as such relatively weak. Therefore, contacting the complex with another compound capable of strongly binding the entrapped compound because of its high affinity results in disruption of the multi-layered structure and release of the entrapped compound. The multi-layered structure may self-assemble after having released the compound, but is then empty, i.e. the compound is no longer entrapped.

In embodiments where the entrapped compound is a protein or a peptide, the other compound may be a receptor with a strong affinity for the protein or peptide.

Alternatively, the other compound may be an enzyme of which the protein or peptide is a substrate, or an inhibitor of which the protein or peptide is a target, or an antibody which recognises and binds the protein or peptide with strong affinity. In some embodiments, releasing the compound may be performed via disruption of the multi-layered structure itself. This can be achieved by contacting the multi-layered structure comprising the entrapped compound in its internal region with another compound which can bind to the modified. In preferred embodiments, the other compound can bind to the hydrophobic moieties. The other compound having a high affinity towards the hydrophobic moieties, its binding prevents the formation of the multi-layered structure, which is based on weaker hydrophobic interactions.

The skilled person will know which other compound to use depending on the nature of the entrapped compound to be released. Method for manufacturing a multi-layered structure

It is an object of the invention to provide a method for manufacturing a multi-layered structure as described above. Such a method comprises the steps of:

In some embodiments, the modified polymer is provided in a dry form, such as a precipitate. The precipitate may be obtained by methods known in the art. The method of manufacturing the multi-layered structure may optionally comprise a step of drying said structure. The precipitate may optionally be dried prior to dissolution in an aqueous solution.

In some embodiments, the method may comprise a further step of contacting the modified polymer dissolved in said aqueous solution with a compound to be entrapped, thereby obtaining a multi-layered structure comprising an entrapped compound as detailed above. In other embodiments, the compound to be entrapped is dissolved in the aqueous solution prior to dissolution of the modified polymer, whereby a multi- layered structure comprising an entrapped compound is obtained. In other

embodiments, the modified polymer and the compound to be entrapped are contacted with the aqueous solution simultaneously, whereby a multi-layered structure comprising the entrapped compound is obtained.

Method of delivery

In another aspect the invention relates to a method of delivery of a compound to an organism, said method comprising the steps of:

ii) administering said multi-layered structure to an individual.

As described above, releasing a compound entrapped in a multi-layered structure of the invention may occur by contacting the structure with another compound capable of binding either to the entrapped compound or to the modified polymer of the structure. Thus the multi-layered structure of the invention may be used for delivering a compound to an organism, where the other compound is found in said organism. The delivery may be targeted or untargeted. In some embodiments, the other compound is only present in specific parts of the organism and said other compound will only cause release of the entrapped compound in said specific parts. This may be advantageous for delivery of compounds such as drugs, where side-effects may be prevented or diminished using targeted delivery. For example, anti-cancer drugs may thus be delivered only to cancerous cells. In other embodiments, the other compound is present in all parts of the body, and said other compound will cause release of the entrapped compound in all parts of the organism.

The organism may be any organism, such as an animal organism, a plant organism, a microorganism. In some embodiments, the organism is an animal organism, such as a mammalian organism, such as a human organism.

In some embodiments, the entrapped compound is a therapeutically active compound.

Pharmaceutical composition

The present disclosure also relates to a pharmaceutical composition comprising a multi-layered structure as described herein above. It also relates to a pharmaceutical composition comprising a self-assembling structure obtainable by the methods described herein.

The pharmaceutical compositions disclosed herein may further comprise at least one therapeutically active agent, such as at least two therapeutically active agents, such as at least three therapeutically active agents, such as at least four therapeutically active agents, such as at least five therapeutically active agents.

The at least one therapeutically active agent may be comprised within the internal region of the multi-layered structure (I on figure 1). The at least one therapeutically active agent may be entrapped within the multi-layered structure. The therapeutically active agent may comprise an immunosuppressant, an alpha- adrenergic antagonist, a steroid, a prostaglandin EP2 agonist, a muscarinic, a prostaglandin, an alpha agonist, an antibiotic, an anti-infective agent, an oncology agent, a psychotropic agent, an anti-inflammatory, a beta blocker, or a combination thereof. In particular embodiments, the therapeutically active agent is an

immunosuppressant, an alpha-adrenergic antagonist, a steroid, a prostaglandin EP2 agonist, a muscarinic, a prostaglandin, an alpha agonist, an antibiotic, an anti-infective agent, an oncology agent, a psychotropic agent, an anti-inflammatory, a beta blocker, or a combination thereof. Combinations of the above listed agents may comprise two or more elements.

It follows that in some embodiments, the method of delivery described herein above comprises the steps of:

ii) administering said multi-layered structure to an individual,

where the compound is at least one compound such as a therapeutically active agent entrapped in a multi-layered structure as described herein, where the multi-layered structure is comprised in a pharmaceutical composition, and where administering of the multi-layered structure comprises administering of said pharmaceutical composition.

The methods of delivery described herein may comprise a step of oral administration of a multi-layered structure disclosed herein or of a pharmaceutical composition comprising a multi-layered structure.

Controlled-release pharmaceutical dosage

In one aspect, the present disclosure relates to a controlled-release pharmaceutical dosage of a poorly soluble or insoluble drug comprising a multi-layered structure comprising and/or entrapping said drug, one or more controlled-release materials and one or more of pharmaceutically acceptable excipients.

The term 'poorly soluble or insoluble' is as defined above. Drugs for oral administration can be classified according to the Biopharmaceutics Classification System (BCS) as provided and defined by the US Food and Drug Administration (FDA). Four different classes of drugs are defined depending on their bioavailability, as detailed above.

In some embodiments, the poorly soluble or insoluble drug belongs to Class II (high permeability, low solubility) or to Class IV (low permeability, low solubility) as defined by the BCS. Thus in some embodiments, the poorly soluble or insoluble drug has a solubility such that the highest dose strength of the drug is not soluble in 250 ml_ of an aqueous medium or less over the pH range of 1 to 6.8. The solubility of such drugs can be increased by contacting such drugs with the multi-layered of self-assembling structures described herein, whereby the drugs may be entrapped within said structures. Without being bound by theory, the inventors believe that this is the mechanism resulting in increased solubility of Class II and Class IV drugs.

The dosage may comprise one or more controlled-release materials. Such materials are known in the art. The term "controlled-release", as used herein, includes matrix- type controlled-release pharmaceutical dosage forms, reservoir-type controlled-release pharmaceutical dosage forms, or combinations of both. The matrix-type dosage forms are those in which the drug is distributed uniformly in one or more of controlled-release materials and reservoir-type compositions utilize polymeric coating over a core comprising the drug. A combination of the reservoir and matrix types includes controlled-release coatings on controlled-release matrices.

The controlled-release materials as used in the dosage form may comprise hydrophilic polymers, hydrophobic polymers, water-swellable polymers, hydrophobic material, and mixtures thereof. The controlled-release material may comprise from about 2% to about 95% by weight of the composition.

Examples of hydrophilic polymers include, but are not limited to, cellulose derivatives, alginates, polyvinyl alcohol, povidone, carbomer, xanthan gum, guar gum, locust bean gum, potassium pectate, potassium pectinate, polyvinylpyrrolidone, polysaccharide, polyalkylene oxides, polyalkyleneglycol, starch and derivatives, and mixtures and combinations thereof.

Examples of hydrophobic polymers include, but are not limited to, ethyl cellulose, hydroxyethylcellulose, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate, poly (alkyl) methacrylate, and copolymers of acrylic or methacrylic acid esters, polyvinyl acetate, and mixtures and combinations thereof. Examples of water-swellable polymers include, but are not limited to, polyethylene oxide; poly(hydroxy alkyl methacrylate); poly(vinyl) alcohol; a mixture of methyl cellulose, cross-linked agar and carboxymethyl cellulose; Carbopol® carbomer;

Cyanamer® polyacrylamides; cross-linked water swellable indene-maleic anhydride polymers; Goodrich® polyacrylic acid; starch graft copolymers; Aqua Keep's® acrylate polymer polysaccharides; Amberlite® ion exchange resins; Explotab® sodium starch glycolate; and Ac-Di-Sol® croscarmellose sodium.

Examples of hydrophobic materials include, but are not limited to, waxes, fatty acids, fatty alcohols, fatty acid esters, vegetable oil and mineral oil.

In addition, the dosage form may further include pharmaceutically acceptable excipients. For example, one or more pharmaceutically acceptable excipients such as binders, fillers/diluents, disintegrants, anti-adherents, lubricants/glidants, plasticizers, coloring agents, flavoring agents and stabilising agents such as sugars.

Suitable examples of binders include, but are not limited to, acacia, sodium alginate, starch, gelatin, saccharides (including glucose, sucrose, dextrose and lactose), molasses, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husk, carboxymethylcellulose, methylcellulose, veegum, larch arabolactan, polyethylene glycols, ethylcellulose, water, alcohols, waxes, polyvinylpyrrolidone such as PVP K90, or mixtures and combinations thereof.

Suitable examples of fillers/diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose or sucrose or other disaccharides, cellulose, cellulose derivatives, kaolin, mannitol, dry starch, glucose or other monosaccharides, dextrin or other polysaccharides, sorbitol, inositol, or mixtures and combinations thereof.

Suitable examples of disintegrants include, but are not limited to, starches, clays, cellulose derivatives including crosscarmellose, gums, algins, various combinations of hydrogencarbonates with weak acids (e.g., sodium hydrogencarbonate/tartaric acid or citric acid) crosprovidone, sodium starch glycolate, agar, cation exchange resins, citrus pulp, veegum HV, natural sponge, bentonite, or mixtures and combinations thereof.

Suitable examples of lubricants/glidants include, but are not limited to, talc, magnesium stearate, calcium stearate, steeric acid, hydrogenated vegetable oils, sodium benzoate, sodium chloride, leucine, carbowax 4000, magnesium lauryl sulfate, colloidal silicon dioxide, and mixtures and combinations thereof,

Suitable examples of plasticizers include, but are not limited to, phosphate esters; phthalate esters; mineral oils; fatty acids and esters; fatty alcohols, vegetable oils and hydrogenated vegetable oils including acetylated hydrogenated cottonseed glyceride and acetylated hydrogenated soybean oil glycerides; acetyl tributyl citrate; acetyl triethyl citrate; Castor oil; diacetylated monoglycerides; dipropylene glycol salicylate glycerin; glyceryl cocoate; mono- and di-acetylated monoglycerides; phthalyl glycolate; diocyl phthalate; sorbitol, sorbitol glyceryl tricitrate; sucrose octaacetate; a-tocopheryl; polyethylene glycol succinate; phosphate esters; phthalate esters; amides; mineral oils; fatty acids and esters; fatty alcohols including cetostearyl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol and myristyl alcohol; methyl abietate; acetyl tributyl citrate; acetyl triethyl citrate; diisooctyl adipate; amyl oleate; butyl ricinoleate; benzyl benzoate; butyl and glycol esters of fatty acids; butyl diglycol carbonate; butyl oleate; butyl stearate; di(beta-methoxyethyl) adipate; dibutyl sebacate; dibutyl tartrate; diisobutyl adipate; dihexyl adipate; triethylene glycol; di(beta-ethyl butyrate); polyethylene glycol;

diethylene glycol monolaurate; monomeric polyethylene ester; hydrogenated methyl ester of rosin; methoxyethyl oleate; butoxyethyl stearate; butyl phthalyl butyl glycolate; glycerol tributyrate; and triethylene glycol.

Suitable examples of coloring agents include, but are not limited to, water-soluble FD&C dyes and mixtures thereof with corresponding lakes and direct compression sugars such as Di-Pac from Amstar. In addition, colored dye migration inhibitors such as tragacanth, acacia or attapulgite talc may be added. Specific examples include calcium carbonate, chromium-cobalt-aluminium oxide, ferric ferrocyanide, ferric oxide, iron ammonium citrate, iron (III) oxide hydrated, iron oxides, magnesium carbonate, and titanium dioxide. In addition, the controlled-release pharmaceutical dosage form can optionally have one or more coatings, which are functional or non- functional.

Suitable examples of polymers useful for coating include, but are not limited to, cellulose acetate, ethyl cellulose, polyamide, polyethylene, polyethylene tereppthalate, polypropylenem polyurethane, polyvinyl acetate, polyvinyl chloride,

polyhydroxybutyrate, polyhydroxyvalerate, polylactic acid or polyglycolic acid and copolymers thereof, copolymers such as ethylene vinyl acetate (EVA), styrene- butadienestyrene (SBS) and styrene-isoprene-styrene (SIS).

The controlled-release dosage forms according to the present disclosure may take the form of tablets, which may be produced by compressing the final mix of granules and/or powders into tablets. Alternatively, controlled-release dosage forms according to the present invention may take the form of pellets which may be coated with one or more coaters, optionally followed by the controlled-release coating. The coating of the pellets may be carried out in any conventional coating system. The final pellets may be filled into capsules, such as hard or soft gelatin capsules, or compressed into tablets. The controlled-release dosage forms may also be formulated as capsules, caplets, pills, granules or mini-tablets.

The present disclosure also relates to a method for the preparation of a controlled- released pharmaceutical dosage form of a poorly soluble or insoluble drug comprising a multi-layered structure comprising and/or entrapping said drug, one or more controlled-release materials and one or more of pharmaceutically acceptable excipients.

Method for increasing the solubility, thermal stability and/or bioavailability of a compound

The present disclosure relates in another aspect to a method for increasing the solubility and/or the thermal stability and/or the bioavailability of a compound, said method comprising the steps of:

i) providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety ; ii) dissolving said modified polymer in an aqueous solution, thereby obtaining a self-assembling structure;

iii) contacting said self-assembling structure with said compound, thereby

obtaining a self-assembling structure comprising and/or entrapping said compound;

It will be understood that the compound can be contacted directly with the modified polymer in step ii), so that both the modified polymer and the compound are dissolved in an aqueous solution, whereby a self-assembling structure comprising and/or entrapping said compound is obtained in step ii). Thus in some embodiments, the method for increasing the solubility and/or the thermal stability and/or the bioavailability of a compound comprises the steps of:

iii) providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety ;

iv) dissolving said modified polymer and said compound in an aqueous

solution, thereby obtaining a self-assembling structure comprising and/or entrapping said compound;

In some embodiments, the method is a method for increasing the solubility of a compound. In other embodiments, the method is a method for increasing the thermal stability of the compound. In yet other embodiments, the method is a method for increasing the bioavailability of the compound. In some embodiments, the solubility and the thermal stability of the compound are increased. In some embodiments, the solubility and the bioavailability of the compound are increased. In some embodiments, the thermal stability and the bioavailability of the compound are increased. In some embodiments, the solubility, the thermal stability and the bioavailability of the compound are increased.

The inventors have found that contacting a self-assembling structure as described herein with a compound can lead to an increase in the solubility, the thermal stability and/or the bioavailability of the compound as compared to its solubility, thermal stability and/or bioavailability when said compound has not been contacted with a self- assembling structure. In some embodiments, the increase is a significant increase. In some embodiments, at least one of the solubility, thermal stability and bioavailability of the compound when contacted with a self-assembling structure is significantly increased as compared to the solubility, thermal stability and bioavailability of the compound when not contacted with a self-assembling structure. In some embodiments, the increase is at least 1.2-fold, such as at least 1.5-fold, such as at least 1.75-fold, such as at least 2-fold, such as at least 3-fold, such as at least 4-fold, such as at least 5-fold, such as at least 10-fold, such as at least 25-fold, such as at least 50-fold, such as at least 100-fold, such as at least 250-fold, such as at least 500-fold, such as at least 750-fold, such as at least 1000-fold.

Drugs for oral administration can be classified according to the Biopharmaceutics Classification System (BCS) provided by the US Food and Drug Administration (FDA). Four different classes of drugs are defined depending on their bioavailability, as detailed above.

In some embodiments, the compound is a poorly soluble or insoluble drug belonging to Class II (high permeability, low solubility) or to Class IV (low permeability, low solubility) as defined and provided by the BCS. Thus in some embodiments, the compound when not contacted with a self-assembling structure has a solubility such that the highest dose strength of the drug is not soluble in 250 ml_ of an aqueous medium or less over the pH range of 1 to 6.8, while the compound after contacting with a self-assembling structure has a solubility such that the highest dose strength of the drug is soluble in 250 ml_ of an aqueous medium or less over the pH range of 1 to 6.8.

Compounds have a low thermal stability when they are liable to degradation, inactivation and/or denaturation upon temperature increase. Such compounds typically show a rate of degradation, inactivation and/or denaturation that correlates with the temperature.

Accordingly, in some embodiments, the compound having low thermal stability is a compound, the degradation and/or inactivation and/or denaturation rate of which correlates with temperature. In some embodiments, the compound having low thermal stability is a compound which, when the temperature is raised to a pre-defined target temperature, gets degraded and/or inactivated and/or degraded prior to reaching said target temperature. The inventors have found that the methods disclosed herein may allow heat sterilisation such as autoclaving of compounds which under normal conditions would be denatured, inactivated and/or degraded upon such treatment; the term "normal conditions" refers herein to conditions where the compound is not or has not been contacted with a self-assembling structure as described herein. In some embodiments, the target temperature is 138°C or less, such as 130°C or less, such as 125°C or less, such as such as 121 °C or less, such as 120°C or less, such as 110°C or less, such as 100°C or less, such as 90°C or less, such as 80°C or less, such as 70°C or less, such as 60°C or less, such as 55°C or less, such as 54°C or less, such as 53°C or less, such as 52°C or less, such as 51 °C or less, such as 50°C or less. In some embodiments, the present methods can be used to increase the thermal stability of compounds which are under normal conditions degraded, inactivated and/or denatured at a temperature of 138°C or less, such as 130°C or less, such as 125°C or less, such as such as 121 °C or less, such as 120°C or less, such as 110°C or less, such as 100°C or less, such as 90°C or less, such as 80°C or less, such as 70°C or less, such as 60°C or less, such as 55°C or less, such as 54°C or less, such as 53°C or less, such as 52°C or less, such as 51 °C or less, such as 50°C or less. The present methods thus can be used to increase the thermal stability of compounds which under normal conditions have low thermal stability at such temperatures. In particular embodiments, the compound is a therapeutically active compound which has low thermal stability. In some embodiments, the compound is a compound having low bioavailability. The term bioavailability refers to the rate and the extent to which the active ingredient or active moiety of a compound becomes available at the desired target site. Compounds have low bioavailability when their bioavailability is so low that it restricts the action of the active ingredient or active moiety at the desired target site. The bioavailability of a compound such as a drug depends of the efficacy and toxicology of the compound, and depends on factors such as, but not limited to, such as the effective concentration of the drug and its toxicology. The skilled person knows how to determine whether a compound has low bioavailability. Thus the present methods can be used to increase bioavailability of a compound by contacting said compound with a self-assembling structure as described herein. In some embodiments, the self-assembling structure is a multi-layered structure as described herein. In some embodiments, the self-assembling structure is obtainable by the methods described herein above. Accordingly, in some embodiments the compound and the self-assembling structure may be brought into contact in different relative amounts. Thus in some embodiments, the compound and the structure are in a molar ratio between 1 : 100 and 100: 1 , such as between 1 :75 and 75: 1 , such as between 1:50 and 50:1, such as between 1:40 and 40:1, such as between 1:30 and 30:1, such as between 1:25 and 25:1, such as between 1:20 and 20:1, such as between 1:10 and 10:1, such as between 1:5 and 5:1, such as between 1:4 and 4:1, such as between 1:3 and 3:1, such as between 1:2 and 2:1, such as 1:1. The optimal molar ratio may depend upon the nature of the compound to be entrapped, of the polymer moiety, of the hydrophobic moieties, and on the strength of the interactions between the structure and the compound.

Method for reducing aggregation and/or denaturation

It is another aspect of the present disclosure to provide a method for reducing aggregation and/or denaturation of a compound upon heat treatment, said method comprising:

iii) contacting said self-assembling structure with said compound, thereby obtaining a self-assembling structure comprising and/or entrapping said compound; subjecting the self-assembling structure comprising and/or entrapping said compound to heat treatment;

whereby aggregation and/or denaturation of the compound is reduced.

ii) dissolving said modified polymer and said compound in an aqueous

iii) subjecting the self-assembling structure comprising and/or entrapping said compound to heat treatment

whereby aggregation and/or denaturation of the compound is reduced.

In some embodiments, the aggregation and/or denaturation of the compound is decreased compared to the aggregation and/or denaturation of the compound when not comprised nor entrapped within the self-assembling structure.

The method may optionally comprise a cooling step after subjecting the self- assembling structure comprising and/or entrapping said compound to heat treatment.

In some embodiments, the heat treatment of step iv) is performed in the absence of a gas-liquid interface, such as an air-liquid interface. In some embodiments, the self-assembling structure is a multi-layered structure as described herein. In particular embodiments, the self-assembling structure is obtainable by the methods described herein. In some embodiments, the self- assembling structure is a multi-layered structure as described herein. In some embodiments, the self-assembling structure is obtainable by the methods described herein above. Accordingly, in some embodiments the compound and the self- assembling structure may be brought into contact in different relative amounts. Thus in some embodiments, the compound and the structure are in a molar ratio between 1 :100 and 100: 1 , such as between 1 :75 and 75: 1 , such as between 1 :50 and 50: 1 , such as between 1 :40 and 40: 1 , such as between 1 :30 and 30: 1 , such as between 1 :25 and 25: 1 , such as between 1 :20 and 20:1 , such as between 1 :10 and 10: 1 , such as between 1 :5 and 5:1 , such as between 1 :4 and 4: 1 , such as between 1 :3 and 3: 1 , such as between 1 :2 and 2: 1 , such as 1 : 1. The optimal molar ratio may depend upon the nature of the compound to be entrapped, of the polymer moiety, of the hydrophobic moieties, and on the strength of the interactions between the structure and the compound.

Examples

Example 1 - Synthesis Cholesterol-substitution

The synthesis, involving esterification of the hydroxyl end groups of the bifunctional polyethylene glycol polymers, was performed as shown in figure 3. Polyethylene glycol (1 equivalent) was weighed into a round bottomed flask and dissolved in

dichloromethane (q.s.). The solution was saturated with N₂ for half an hour at room temperature, under magnetic stirring. Cholesteryl chloroformate (10 equivalents) was weighed into a round-bottomed flask and dissolved in dichloromethane (q.s.). The solution was bubbled with N₂ for half an hour at room temperature, under magnetic stirring. Keeping both solutions cooled on an ice-bath, the polyethylene glycol was added drop-wise to the cholesteryl chloroformate mixture. The species were allowed to react for 48-72 hours at room temperature under N₂-purge and magnetic stirring. The reaction mixture was precipitated in ice-cooled diethyl ether by drop-wise addition of the solution. The precipitate was filtered under suction, re-dissolved in dichloromethane and precipitated once more in ice-cold diethyl ether and filtered again. The semi-dry precipitate was allowed to dry overnight in a fume hood. Modifications of 5 polyethylene glycol commodities of different chain-lengths were obtained. The substitution was confirmed by H-NMR (results not shown). Fatty-acid substitution

The synthesis was performed as shown in figure 4. Poly(ethylene glycol) (1 equivalent) and p-toluenesulfonic acid (1.5 weight % of the fatty acid) was transferred to a reaction flask, and dissolved in refluxing xylene (q.s.) under magnetic stirring and nitrogen atmosphere. The fatty acid (2 equivalents) were then added and allowed to react for at least 4 hours at 130°C. The reaction solution was then filtered, and the precipitated products were dissolved in chloroform (q.s.), and re-precipitated in excess of cold hexane (q.s.). The precipitate was then filtered, and dried for three days. The substitution was confirmed by H-NMR, and found to be 70-100 % (results not shown).

Example 2 - Dynamic Light Scattering studies of cholesterol constructs

Dynamic Light Scattering (DLS) measurements were carried out at 25.0 ± 0.1 °C on a Zetasizer Nano ZS instrument (Malvern Instruments, UK) at a wavelength of 632.8 nm and a 173° detection angle. Samples were prepared by mixing PEO20K or cholesterol- PEO20K and lysozyme or BSA before dissolving in 10 mM phosphate buffer (pH adjusted to 7.4). The resulting solutions were clear and without noticeable aggregates. Each measurement was performed in triplicate.

Cholestrol-PEG20K filtered and unfiltered

Unfiltered cholesterol-PEG20K showed two construct clusters, corresponding to unstructured (3-20 nm) and structured PEGosomes (60-1000 nm). These remained after the solution was filtered through a 0.22 μηι sterile filter. However, 12 hours after the initial measurement, the smaller construct cluster was no longer present, and the bigger cluster had become narrower. The results presented in figure 6 show that the cholesterol-PEG20K PEGosomes are actively forming into a uniform construct, even after being disrupted by the filter.

PEG20K, cholesterol-PEG20K, lysozyme and BSA measurements

PEG20K solutions contained two major construct clusters at about 9 nm and 500 nm corresponding to fully dissolved and partly undissolved polymer (figure 5). Cholestrol- PEG20K solutions on the other hand showed one major construct cluster at 100-1000 nm. The variation in construct size may stem from variations in the chain length of the PEG20K reagent used, as this is a combination of multiple polymer chain lengths, averaging 20000 Da. This size variation may not be apparent from the DLS results of PEG20K by itself as a structure large enough to be measured should only be present when PEG20K is conjugated with cholesterol. Solutions of BSA showed a single construct cluster around 10 nm. This corresponds well with the reported size of BSA being about 7 nm in diameter. Solution of lysozyme showed two construct size clusters corresponding to native (2-6 nm) and aggregated/undissolved (100-5000 nm) lysozyme. Some of the larger constructs may also be contaminates such as dust. When mixed (in a 1 :1 ratio), solutions of lysozyme or BSA and PEG20K contained the same construct size clusters as each protein by itself. This indicates that no interaction is taking place. In solutions of lysozyme and cholesterol-PEG20K (in a 1 : 1 ratio) the cluster for free lysozyme was absent. This may be caused by an interaction of cholesterol-PEG20K with lysozyme, or by the cholesterol-PEG20K being so large that they overshadow the signal from free lysozyme. Solutions of BSA with cholesterol- PEG20K (in a 1 :1 ratio) showed a construct cluster similar to that of cholesterol- PEG20K alone (100-1000 nm) and that of the lysozyme/cholesterol-PEG20K solution. The cluster for free BSA (3-18 nm) was also present but reduced in size. This may indicate that while all of the lysozyme is incorporated into the 100-1000 nm sized constructs of the cholesterol-PEG20K, the larger BSA protein is not. This may be indicative of an upper loading capacity for the modified polymer-protein network construct obtained. In addition, cryo-TEM imaging of the constructs with BSA did not show signs of interactions (Fig. 1 1 A).

The results show that cholesterol-PEG20K forms a structure whereas PEG20K does not. This structure is present both with and without protein.

Example 3 - Dynamic Light Scattering studies of fatty acid constructs

A sample of the fatty acid constructs was dissolved in filtered ultrapure water either by itself or with lysozyme or BSA, filtered again upon transfer to a cuvette and measured at 25°C. The filters used had a pore size of 0.45 μηι. Each sampling was repeated 3 times, and each repeat measured 5 times (totaling 15 measurements for each graph). Samples were measured at 25°C with a 488 nm laser at an angle of 90°.

Measurements were conducted with a custom build DLS, equipped with a Coherent Sapphire laser and a Brookhaven Instruments detector.

Fatty acid constructs with PEG4K

The apparent average size of the constructs was about 10 nm for all fatty acid/protein combinations (figure 7). Constructs of stearic acid contained some larger constructs (> 100 nm), in amounts of about 10-20 % with and without lysozyme. In the presences of BSA, the amount of larger constructs fell to 2-3 %, despite BSA being a larger protein than lysozyme. Oleic acid-PE04K with BSA also contained slight amounts of larger construct sizes. As larger constructs scatter light with higher intensity, DLS

overemphasizes larger constructs, making them appear more abundant than they really are. As constructs larger than about 100 nm were observed in relatively low amounts, the true average construct size of PEG4K constructs and protein

combinations may be expected to be well below 100 nm.

The results indicate that PEG4K creates constructs of about 10 nm in size,

independent of fatty acid and protein present, expect for constructs of stearic acid, in which BSA lowers the amount of larger constructs.

Fatty acid constructs with PEG 10K

Constructs of linoleic acid averaged construct sizes of about 40-50 nm in the absences and presence of lysozyme (figure 8). Larger constructs (>100 nm) were observed for both of these samples in abundances of about 25 %. However, when mixed with BSA, the average construct size fell to about 10 nm, with the amount of larger constructs falling to about 3 %. Constructs of oleic acid with and without lysozyme or BSA contained constructs averaged about 10 nm regardless of the presences of protein. Stearic acid constructs averaged about 500 nm without and with lysozyme, the content of larger constructs being about 100 % and 99 % respectively. In the presence of BSA, the average construct size dropped to about 300 nm, with the amount of larger constructs falling to about 60 %.

The results indicate an interaction of linoleic acid-PEO10K and stearic acid-PEO10K with BSA, which does not occur with lysozyme. While the average construct size in samples of oleic acid-PEO10K was not affected by either protein, the presence of BSA significantly lowered the amount of constructs over 100 nm, indicating that an interaction may be taking place.

Fatty acid constructs with PEO20K

The average construct size of constructs of linoleic acid was about 170 nm with the amount of larger constructs (>100 nm) being about 65 % (figure 9). In the presence of lysozyme, the average construct size decreased to about 130 nm, the amount of larger constructs falling to 55 %. The decrease in average construct size is therefore likely to be caused by the presences of smaller constructs, rather than a decrease in construct size. In the presence of BSA, the average construct size fell to about 10 nm, with the amount of larger constructs falling to about 15 %. For oleic acid constructs, the average construct size was about 100 nm, with the amount of larger constructs being about 50 %. In the presence of lysozyme the average construct size increased to 180 nm, the amount of larger constructs being about 60 %. In the presence of BSA, the average construct size fell to about 10 nm, the amount of larger constructs being about 3 %. For stearic acid constructs, the average construct size was about 300 nm in the absence and presence of lysozyme, the amount of larger constructs being about 97 % in both cases. In the presence of BSA the average construct size fell to about 10 nm, and the amount of larger constructs to about 12 %.

The results indicate an interaction of linoleic, oleic and stearic acid-PEO20K with BSA, causing a structural loss of the constructs and a lowering of the average construct size. The same type of interaction was not observed with lysozyme. This may be explained by the fact that BSA is known to bind fatty acids, while lysozyme does not. While some construct size decrease was observed in the presence of lysozyme for linoleic and stearic acid constructs, the effect was significantly more pronounced in the presence of BSA.

The sizes of the PEGosomes were in some cases (e.g. stearic-acid-PEG20K) bigger than the pore size of the filter should allow. This can be explained by the PEGosomes being flexible enough to deformed going through the filter and then reforming in the cuvettete prior to measurements.

However, in order to evaluate the relative sizes of PEGosomes created with different PEG'S, proteins and fatty acids, the raw DLS data (construct delay times, figure 10, see below) must be evaluated as well.

Size comparison by delay time

Dynamic light scattering essentially measures the time it takes for a construct to move a certain distances due to Brownian motion (the delay time). The time is measured by monitoring the scattered light that arises when light of a certain wavelength is shined through the sample. Larger constructs will move more slowly than smaller constructs and the measured time can therefore be used to calculate the size of the construct. However, larger constructs will also scatter light with a higher intensity, which may lead to an overestimation of their amount in samples containing both larger and smaller constructs. In these cases, the delay time is a superior method of evaluating relative construct sizes. Other parameters are also measured and used for the calculation, and the above description is oversimplified for the sake of discussion. The median delay time of linoleic acid constructs by itself and in the presences of lysozyme increased with PEG chain length, representing larger constructs in samples with longer chain length (figure 10). However, samples containing BSA had the approximate same median delay time, indicating that these all had the same construct size regardless of PEG chain length. In addition, BSA containing samples had a median delay time comparable to constructs of PEG4K, indicating a lower size limit for the PEGosomes. While the same tendency was observed for oleic acid constructs of PEG20K, the delay times did not greatly differ for constructs of PEG4K and PEG10K. While the presence of BSA did lower the median delay time for oleic acid constructs, this was as pronounced for PEG4K and PEG10K constructs as for PEG20K. For stearic acid constructs, the median delay time increased with PEG chain length for PEG4K and PEG20K constructs, while PEG10K constructs had the longest delay times observed. The presence of BSA decreased the median delay time, but most markedly for PEG20K. The nature of the fatty acid used for the constructs did not seem to affect the delay times in a similar manner as the PEG chain length.

The results show that the PEGosome size is dependent on the PEG chain length. The results also show that the presence of BSA decreases PEGosome size, and that a lower limit for PEGosome size may exist which is comparable to constructs of PEG4K. Cryo-Transmission Electron Microscopy images of cholesterol-PEG20K with bovine serum albumin (BSA)

Samples for cryo- Transmission Electron Microscopy (cryo-TEM) imaging of cholesterol-PEG20K with bovine serum albumin (BSA) were obtained by adsorbing a small amount of sample (1 % w/w in equal amounts) to a small pored carbon grid, and freezing in liquid ethane. The grid with sample was then imaged with a FEI Tecnai G2 20 TWIN Transmission Electron Microscope (figure 11 A).

The images showed the PEGosomes as circular/spherical noisome-like structures, with no apparent BSA association as the protein could not be located on the images. Example 4 - Toxicity studies

Toxicity of fatty acid based constructs

In a 96 well plate, 20000 Caco-2 cells were seeded and allowed to attach overnight. The medium was then removed and the wells washed twice with fresh Hank's Balanced Salt Solution (HBSS). The fatty acid-PEG structures were dissolved at a concentration of 1 mg/mL and then filtered through a 0.2 μηι poly(tetrafluoroethene) (PTFE) filter and diluted to the testing concentrations. Then, 100 μΙ_ of the fatty acids- PEG structures were added to each well and incubated for 24 h. After that, the samples were removed, the wells were washed once with fresh HBSS and 50 μΙ of HBSS mixed with 50 μΙ_ of Celltiter GLO reagent for Luminescence viability assessment. The plates were then read by Luminescence. The testing concentrations were chosen to be comparable with studies on fatty acid toxicity [4].

Linoleic acid based PEGosomes

Linoleic acid based PEGosomes did not decrease the viability of Caco-2 cells in concentrations of 15-100 μg/mL (figure 12A). At 250 μg/mL, a slight decrease in viability was seen for construct of PEG4K (85 % viability) and PEG10K (90 % viability). For constructs of PEG20K at concentrations of 250 μg/mL no decrease in viability was observed. Other studies have shown linoleic acid to be cytotoxic at the concentrations used [4], and the results indicate that conjugation with PEG considerably decreases the cytotoxicity of linoleic acid. Linoleic acid has been found to induce both necrosis and apoptosis in cells. It is believed that linoleic acid triggers necrosis by disrupting the cell membrane through oxidation, and apoptosis by damaging the cell's DNA. Conjugation to PEG may interfere with both these mechanisms, as PEG would prevent linoleic acid from crossing the cell membrane (as PEG cannot cross the membrane due to being hydrophilic).

Oleic acid based PEGosomes

Oleic acid based PEGosomes did not decrease the viability of Caco-2 cells at any concentration (figure 12B). A slight increase (10-20 %) in viability was observed for oleic acid constructs with PEG10K and PEG20K at concentrations of 50 and 100 μg/mL. This may be explained by the fatty acids being used as energy source for the cells. Other studies have shown oleic acid to be cytotoxic at the concentrations used [4], and the results indicate that conjugation with PEG decreases the cytotoxicity of oleic acid. Oleic acid has been found to induce both necrosis and apoptosis in cells. It is believed that oleic acid triggers necrosis by disrupting the cell membrane through oxidation, and apoptosis by damaging the cell's DNA. Conjugation to PEG may interfere with both these mechanisms, as PEG would prevent the fatty acid from crossing the cell membrane (as PEG cannot cross the membrane due to being hydrophilic). Stearic acid based PEGosomes

Stearic acid based PEGosomes did not decrease the viability of Caco-2 cells at any concentration (figure 12C). However, constructs of PEG4K and PEG10K increased cell viability (20-50 %) at all concentrations. This may be explained by the fatty acids being used as a source of energy for the cells.

Conclusion

The results show that polymer linking of fatty acids and thus their internalisation within the PEGosomes removes the cytotoxic effects observed for the same fatty acids alone. Some constructs, especially those of stearic acid, surprisingly increased the viability of the Caco-2 cells.

Example 5 - Stability studies

Thermal stability of protein

Equivalent amounts (w/w) of protein and linoleic acid-PEG20K were mixed, and diluted to about 1 % (w/w) with ultrapure water. Samples were then covered and heated to 60°C for 24 hours, 80° for 24 hours and finally to 1 18°C for 12 hours. Lost solvent was replaced as needed.

Following heating the solvent volume was doubled in order to better assess changes in the solutions. While the solutions of protein showed substantial amounts of

aggregation, solutions containing linoleic acid-PEG20K did not (figure 11 B; pictures are taken after the three heating steps). After about a day of cooling, the solutions of lysozyme with linoleic acid-PEG20K formed some aggregation; however not as substantial as the protein by itself. This indicates that while both BSA and lysozyme were stabilized by the PEGosome, the effect was stronger for BSA. This may be explained by there being more lysozyme molecules in the solutions of this protein, than those of BSA, as lysozyme has a lower molecular mass than BSA (almost 1/3 of BSA), and an upper concentration limit thereby being exceed. Also, as BSA has intrinsic fatty acid binding capabilities, the effect of the PEGosome may be enhanced. The results show that both lysozyme and BSA are stabilized against heat induced aggregation by the PEGosome, and indicate that there is an optimum concentration ratio for stabilization/protein binding. Differential Scanning Calorimetry studies

Samples of linoleic acid-modified PEG20K (linoleic acid-PEG20K) and bovine serum albumin (BSA) both by itself or in equimolar mixtures, were prepared and diluted to a concentration of 0.9 mM (equivalent to 6 % w/w BSA) in ultrapure water. The samples were then measured in hermetically sealed aluminium pans at a heating rate of 30°C/min, on a TA Instruments Discovery DSC.

Differential Scanning Calorimetry (DSC) works by measuring the energy needed to heat a sample to a certain temperature, relative to a reference without sample (empty sample holder). Any energy differences between the sample and the reference must be caused by the sample changing as it is heated. In the case of proteins, energy is typically expended to change protein structure, before finally collapsing and

aggregating. An endothermic slope was observed for all samples from 0-20 °C (figure 13). This slope may correspond to changes in water as it goes from near solid to liquid. The difference in the magnitude of the slope may be due to different chaotropic effects of the sample molecules. A sudden increase in energy was observed for samples of BSA at about 75 °C, corresponding to complete collapse of the protein, and subsequent aggregation. This correlates well with the denaturation temperature for BSA, which is reported as being a 2 step process, with the first step occurring at 50-52 °C, and the second step occurring at 80-82 °C [7].. Earlier events may have been observed had the heating rate been decreased. When mixed with linoleic acid-PEG20K, the denaturation peak is not observed. The endothermic slopes go lower however, which may indicate that structural changes are taking place even if the protein is has not been completely denatured. Linoleic acid-PEG20K displays an even lower endothermic slope, indicating that the lowering of the slope is caused by changes in the PEGosome, and not by BSA. Indeed, when corrected for the energy lost by changes in the PEGosome the slope almost disappears, indicating that little or no changes are taking place from 0-80 °C.

The results show that the PEGosome protects BSA from heat induced denaturation, and that the PEGosome is actively changing conformation as it is heated. Note that as the mixtures in this study were equimolar, the ratio of PEGosome was substantially higher than in the examples which employed equivalent masses (w/w). Autoclaving of bovine serum albumin (BSA) with and without PEG20K, cholesterol- PEG20K (chol-PEG20K) and stearic acid-PEG20K (SA-PEG20K)

Samples of Bovine serum albumin (BSA) with and without PEG20K, cholesterol- PEG20K and stearic acid-PEG20K were prepared in equivalent amounts and diluted to 1 % w/w with ultrapure water. The samples were then autoclaved by heating to 121 °C over a period of 40 minutes, holding for 15 minutes at a pressure of about 100 kPa, release of pressure, and cooling to room temperature. Samples were held above 50°C for at least 4 hours following autoclaving. While aggregation was observed in all samples containing protein, samples contain either PEG20K or PEGosome was heavily decreased (figure 11 C). While BSA by itself formed a dense, uniform aggregated mass, the presence of PEG20K decreased the aggregation, which was heaviest near solid-liquid interfaces and in the middle of the liquid. In the presence of cholesterol-PEG20K, the aggregation was limited to the solid- liquid interfaces, and less pronounced than observed BSA/PEG20K solutions. For solutions containing stearic acid-PEG20K PEGosomes, aggregation was almost eliminated, and only present as a thin flake on top of the liquid, indicating an even stronger stabilizing effect than observed for cholesterol-PEG20K/BSA solutions. As discussed for the heating study (figure 11 B), the observed aggregation may be caused by an excess of protein relative to PEGosome. The results show that aggregation is heaviest in interfacial regions, indicating that altering other parameters than the PEGosome-protein ratio may also help to optimize the method. In order to confirm this, the experiment was repeated for stearic acid-PEG20K/BSA mixtures in fully filled Eppendorf tubes, thereby removing the liquid-air interface. This did indeed remove all visible aggregation (results not shown).

The results show that it may be possible to remove all visible protein aggregation at temperatures allowing for heat sterilization. While optimization is needed, this may only be a matter of finding the correct protein-PEGosome ratio, and optimal container use during autoclaving.

Fourier transform infrared spectroscopy (FTIR) measurements of autoclaved bovine serum albumin with PEGosome

Fourier transform infrared spectroscopy (FTIR) measurements of autoclaved bovine serum albumin (BSA) with either cholesterol-PEG20K, stearic acid-PEG20K (figure 1 1 C) or with stearic acid-PEG20K in a fully filled Eppendorf tube were performed on a Bomem Arid ZoneTM of the MB series (Bomem, Canada) with a resolution of 4 cm^"1. Samples of 15 μΙ_ were measured in a CaF₂ cell at a path length of 6 μηι. For each spectrum, 256 scan were recorded, and each samples was measured in triplicates. For data analysis, the GRAMS/AI 7.00 (Thermo Galactic, USA) software was used to subtract the buffer spectrum in the region 1800-2600 cm^"1 and the water vapor spectrum in the amide I and II region, 1500-1700 cm^"1. The second derivative spectra were then obtained by Savitzky-Golay smoothening, and the spectra were cut to fit the amide I region, 1595-1705 cm^"1, after which the baseline was corrected. The spectra were normalized to an area under the curve of 1.

While almost all structural similarity to native protein was lost for BSA autoclaved with either cholesterol-PEG20K or stearic acid-PEG20K in glass vials (figure 14), some structure was preserved for samples with stearic acid-PEG20K in Eppendorf tubes. Most notability, the strong band at about 1650 cm^"1 is present, if diminished, in both native and autoclaved BSA. This band corresponds to a-helix, and as BSA is almost entirely composed of a-helixes, this indicates that some BSA has maintained its structure during autoclaving. However, as FTIR compiles structural information of all protein species present in the sample, it is not possible to assess whether all the protein in the sample has lost a moderate amount of structure, or if the sample is composed of native and denatured protein.

The results show that some BSA structure was maintained during autoclaving.

Combined with the results from DSC (figure 13), in which heat denaturation was prevented in equimolar solutions of BSA and PEGosome, the results also indicate that simply increasing the amount of PEGosome in the mixture could allow for heat sterilization without any loss of protein structure.

Example 6 - solubility enhancement studies

Solubility enhancement of poorly soluble drugs

The UV absorbance of PEG20K modified with cholesterol (chol-PEG20K) and PEG20K was measured either by itself or mixed with the model drugs Probenecid or

Furosemide. Samples were prepared by mixing equal amounts of PEGosome and model drug in, and diluting to about 10 mg/mL in phosphate buffer. Samples were mixed by rotation for at least 48 hours, filtered and measured against the sample's respective reference. Each sample was repeated in triplicate.

Probenecid is a poorly soluble (27.1 mg/L in water) lipophilic (logP = 3.21) compound used for the treatment of gout [5]. Furosemide is also a poorly soluble compound (118 mg/L in water), while being less lipophilic (logP = 2.71) than Probenecid [6].

Furosemide belongs to Class IV. Furosemide is used for the treatment of congestive heart failure and oedema. Solutions of Probenecid absorbed UV light at wavelengths from 200-300 nm, with a maximum absorbance of 0.4 at 200 nm. When mixed with chol-PEG20K, the UV absorbance of Probenecid increased at wavelengths from 200 nm to about 340 nm (figure 15). Most markedly, the absorbance at wavelength 209 nm increased from 0.25 to 0.85, indicating a solubility increase of at least threefold. When mixed with PEG20K, the UV absorbance became negative, indicating over subtraction of the reference, and no solubility increase of Probenecid. Conversely, neither chol-PEG20K nor PEG20K showed any remarkable effect on the UV absorbance of Furosemide, indicating that no increase in solubility took place. This difference in solubility increase may be explained by Furosemide being less lipophilic than Probenecid and therefore not having as high affinity for the PEGosome as Probenecid.

The results indicate that the PEGosome can be used for increasing the solubility of poorly soluble, lipophilic drugs.

Emulsifying studies

Samples of 1 % PEG20K and vitamin E, cholesterol-PEG20K and vitamin E, and vitamin E by alone were prepared with 10 mM phosphate buffer. The samples were mixed by rotation for 48 hours and resulting emulsions were imaged by light microscopy at 40x magnification.

Vitamin E did not mix with the phosphate buffer and phase separation was apparent (Fig. 11 D, panel A). This is expected due to the lipophilic nature of vitamin E (logP = 10). PEG20K did not cause any apparent changes to the mixture as compared to vitamin E by itself. Mixtures of cholesterol-PEG20K and vitamin E produced milky white solutions, indicating that cholesterol-PEG20K emulsified vitamin E. This was confirmed by light microscopy, by which small droplets were seen suspended in the buffer (Fig. 11 D, panel B). This is characteristic of an oil-in-water emulsion, and confirms that the PEGosome does indeed emulsify vitamin E. After three months, the emulsion was stable. Currently, the emulsion has been stable for six months, with experiments still ongoing.

Example 7 - solubility of therapeutic molecules

Example 6 presents UV absorption data showing that addition of PEGosome to solutions of poorly soluble drugs increases their solubility. In this example, we studied additional drug molecules, and measured the concentration of these with and without PEGosome by a HPLC method.

Method

The therapeutically active (drug) compounds amiodarone (class II compound), clotrimazole (Class II compound), fenofibrate (Class II compound), furosemide (class IV compound), indomethacin (class II compound) and probucol (Class II) were investigated. Samples were prepared by transferring 15 mg of the drug compound and 15 mg of the PEGosome (in solid form) to 35 x 12 mm culture vials. 1.5 ml phosphate buffer (0.01 M, pH 7.2) was added to the vials, and these were closed with an aluminium cap, covered with tinfoil and attached to an end-to-end mixer. Samples were mixed for 24 hours at 30 RPM. Controls were treated identically except that no PEGosome was added. All samples and controls were prepared in triplicate.

After the mixing, the samples and controls were filtered with 0.22 μηι Q-Max RR Syringe Filters into 1.5 ml HPLC vials and measured.

The HPLC was equipped with a C18 (10 cm x 2.1 , 5 μηι) column kept at 30°C, an autosampler and a Chrom UV/VIS detector. The flow rate was set to 1 mL/min.

Different mobile phases, injection volumes and detection wavelengths were used for each drug compound. The detection limit of the method was about 1 μΜ/mL.

Results

Fenofibrate showed a significant but unremarkable increase in solubility (controls were under the detection limit, while samples contained about 0.9 μΜ/mL fenofibrate; results not shown). The results obtained for probucol, indomethacin and amiodarone are shown in Figure 16.

Our studies indicated that drug solubility of probucol, indomethacin and amiodarone increases exponentially with PEGosome concentration (results not shown). Absence of solubility increase with the PEGosome concentration used (1 % w/w) for some compounds does therefore not mean that an increase in solubility will not occur with higher PEGosome concentrations. This example shows that the PEGosome increases the aqueous solubility of a broad range of therapeutically relevant molecules

References

1. Sigma-Aldrich. Insulin human. 2014 [cited 2014 24-04-2014]; Available from: http://www.sigmaaldrich.com/catalog/product/fluka/1342106.

2. Farrah, T., et al., The state of the human proteome in 2012 as viewed through PeptideAtlas. J Proteome Res, 2013. 12(1): p. 162-71.

3. Dimitrov, D.S., Therapeutic proteins. Methods Mol Biol, 2012. 899: p. 1- 26.

4. Andrade, L.N., et al., Toxicity of fatty acids on murine and human melanoma cell lines. Toxicol In Vitro, 2005. 19(4): p. 553-60.

5. Law V, K.C., Djoumbou Y, Jewison T, Guo AC, Liu Y, Maciejewski A, Arndt D, Wilson M, Neveu V, Tang A, Gabriel G, Ly C, Adamjee S, Dame ZT, Han B, Zhou Y, Wishart DS, Drugbank: Probenecid, in DrugBank 4.0: shedding new light on drug metabolism. 2014: Nucleic Acids Res.

6. Law V, K.C., Djoumbou Y, Jewison T, Guo AC, Liu Y, Maciejewski A, Arndt D, Wlson M, Neveu V, Tang A, Gabriel G, Ly C, Adamjee S, Dame ZT, Han B, Zhou Y, Wishart DS, Drugbank: Furosemide, in DrugBank 4.0: shedding new light on drug metabolism. 2014: Nucleic Acids Res.

7. Lu, R., Li, W.-W., Katzir.A., Raichlin.R., Yu H.-Q., Mizaikoff B. Probing the secondary structure of bovine serum albumin during heat-induced denaturation using mid-infrared fiberoptic sensors. Analyst, 2015,140, 765-770

Claims

1. A multi-layered structure comprising one or more bilayers,

said one or more bilayers comprising a plurality of modified polymer moieties, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof,

wherein each polymer moiety is modified by end-group substitution with a hydrophobic moiety. 2. The structure of claim 1 , wherein said polymer is linear.

3. The structure of any of the preceding claims, wherein said polymer is

polyethylene glycol (PEG). 4. The structure of any of the preceding claims, wherein the polymer is

methylated, aminated or amidated.

5. The structure of any one of the preceding claims, wherein each polymer moiety is modified by end-group substitution with two hydrophobic moieties, each end being substituted with one hydrophobic moiety.

6. The structure of claim 5, wherein the two hydrophobic moieties are

identical. 7. The structure of any one of the preceding claims, wherein the hydrophobic moiety is selected from the group consisting of cholesterol, fatty acid moieties, phospholipids, triglycerides, sterols and natural or synthetic lipid molecules. 8. The structure of claim 7, wherein the hydrophobic moiety is selected from the group consisting of saturated fatty acids and unsaturated fatty acids.

9. The structure of claim 8, wherein the hydrophobic moiety is a stearic acid moiety, an oleic acid moiety or a linoleic acid moiety, an elaidic acid moiety, an arachidic acid moiety, a palmitic acid moiety, an arachidonic acid moiety or a linolenic acid moiety, as well as isoforms and oxidative products thereof.

10. The structure of any one of the preceding claims, wherein the structure is capable of self-assembling in an aqueous environment.

1 1. The structure of any one of the preceding claims, wherein the inner and outer surfaces of the structure are uncharged. 12. The structure of any one of the preceding claims, wherein the hydrophobic moieties interact with each other within an internal region of the one or more bilayers.

13. The structure of any one of the preceding claims, wherein the molecular weight of the PEG moiety is comprised between 400 and 100000 Da, such as between 600 and 90000 Da, such as between 1000 and 80000 Da, such as between 2000 and 70000 Da, such as between 4000 and 60000 Da, such as between 4000 Da and 50000 Da, such as between 4000 Da and 40000 Da, such as between 4000 Da and 30000 Da, such as between 4000 Da and 20000 Da, such as 4000 Da, such as 10000 Da, such as

20000 Da.

14. The structure of any one of the preceding claims, wherein the molecular weight of the PEG moiety is lower than 1000 Da, such as lower than 900 Da, such as lower than 800 Da, such as lower than 700 Da, such as lower than 600 Da, such as lower than 500 Da.

15. The structure of any one of the preceding claims, wherein the PEG

comprises less than 10 ethylene glycol units, such as less than 9 ethylene glycol units, such as less than 8 ethylene glycol units, such as less than 7 ethylene glycol units.

16. The structure of any one of the preceding claims, wherein said structure has a diameter in the range of 10 to 10000 nm, such as between 10 and 7500 nm, such as between 20 and 5000 nm, such as between 30 and 4000 nm, such as between 40 and 3000 nm, such as between 50 and 2000 nm,

53 such as between 50 and 1000, such as between 40 and 500 nm, such as between 60 and 500 nm, such as between 60 and 400 nm, such as between 60 and 300 nm, such as between 60 and 200 nm, such as between 60 and 150 nm, such as between 60 and 100 nm.

17. The structure of any one of the preceding claims, wherein said structure forms a nanostructure having a diameter between 0.1 and 1000 nm.

18. The structure of any one of the preceding claims, wherein the diameter of the structure is determined by the molecular weight of the polymer moiety.

19. The structure of any one of the preceding claims, wherein said structure is thermodynamically favourable in an aqueous environment.

20. The structure according to any one of the preceding claims, further

comprising a compound within said internal region.

21. The structure of claim 17, wherein said compound interacts with the

hydrophobic moieties.

The structure of any one of the preceding claims, wherein the construct i not a micelle.

23. Use of the multi-layered structure of any one of claims 1 to 22 to entrap a compound.

24. The use of claim 23, wherein said compound is contacted with said

structure and is thereby entrapped.

25. The use of claim 24, wherein said compound and said structure are

contacted in an aqueous environment.

26. The use of any one of claims 23 to 25, wherein the compound and the structure are in a molar ratio comprised between 1 :100 and 100: 1 , such as between 1 :75 and 75: 1 , such as between 1 :50 and 50: 1 , such as between 1 :40 and 40:1 , such as between 1 :30 and 30: 1 , such as between 1 :25 and 25: 1 , such as between 1 :20 and 20: 1 , such as between 1:10 and 10:1, such as between 1:5 and 5:1, such as between 1:4 and 4:1, such as between 1:3 and 3:1, such as between 1:2 and 2:1, such as 1:1.

27. The use of any one of claims 23 to 26, wherein the compound and the structure are in a weight ratio comprised between 1:100 and 100:1, such as between 1:75 and 75:1, such as between 1:50 and 50:1, such as between 1:40 and 40:1, such as between 1:30 and 30:1, such as between 1:25 and 25: 1 , such as between 1 :20 and 20: 1 , such as between 1:10 and 10:1, such as between 1:5 and 5:1, such as between 1:4 and 4:1, such as between 1:3 and 3:1, such as between 1:2 and 2:1, such as 1:1.

28. The use of any one of claims 23 to 27 for reducing the aggregation capacity of said compound.

29. The use of any one of claims 23 to 28 for increasing the solubility of said compound.

30. The use of any one of claims 23 to 29 for increasing the stability of said compound.

31. The use of any one of claims 23 to 30 for decreasing the toxicity of said compound.

32. The use of any one of claims 23 to 31 , wherein said compound is lipophilic.

33. The use of any one of claims 23 to 32, wherein said compound is selected from the group comprising peptides and proteins.

34. A method for manufacturing a self-assembling structure, comprising the steps of:

providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety ; i) dissolving said modified polymer in an aqueous solution, thereby obtaining a self-assembling structure.

35. The method of claim 30, wherein the self-assembling structure is a multi- layered structure according to any one of claims 1 to 22.

36. The method of any one of claims 34 to 35, further comprising contacting the self-assembling structure with a compound, thereby entrapping said compound within said structure.

37. The method of any one of claims 34 to 36, wherein the compound is a therapeutically active agent.

38. A pharmaceutical composition comprising a multi-layered structure

according to any one of claims 1 to 22 or a self-assembling structure obtainable by the method of any one of claims 34 to 37.

39. The pharmaceutical composition of claim 38, further comprising at least one therapeutically active agent.

40. The pharmaceutical composition of any one of claims 38 to 39, wherein the at least one therapeutically active agent is comprised within the internal region of the multi-layered structure.

41. The pharmaceutical composition of any one of claims 38 to 40, wherein the at least one therapeutically active agent is entrapped within the multi- layered structure.

42. The pharmaceutical composition of any one of claims 38 to 41 , wherein the therapeutically active agent comprises an immunosuppressant, an alpha- adrenergic antagonist, a steroid, a prostaglandin EP2 agonist, a

muscarinic, a prostaglandin, an alpha agonist, an antibiotic, an anti- infective agent, an oncology agent, a psychotropic agent, an antiinflammatory, a beta blocker, or a combination thereof.

43. A method of delivery of a compound to an organism, said method

comprising the steps of: i) providing a compound entrapped in a multi-layered structure according to any one of claims 1 to 18 or a composition according to any one of claims 38 to 41 ;

ii) administering said multi-layered structure or said composition to an individual in need thereof.

The method of claim 43, wherein the delivery is a targeted delivery.

45. A controlled-release pharmaceutical dosage form of a poorly soluble or insoluble drug comprising a multi-layered structure comprising and/or entrapping said drug, one or more controlled-release materials and one or more pharmaceutically acceptable excipients.

46. The controlled-release pharmaceutical dosage form of claim 45, wherein the dosage form is in the form of hard or soft gelatin capsules, tablets, capsules, caplets, pills, granules or mini-tablets.

47. A method for the preparation of a controlled-release pharmaceutical

dosage form of a poorly soluble or insoluble drug comprising a multi- layered structure comprising and/or entrapping said drug, one or more controlled-release materials and one or more pharmaceutically acceptable excipients.

48. A method for increasing the solubility and/or the thermal stability and/or the bioavailability of a compound, said method comprising the steps of:

providing a modified polymer, said polymer being an uncharged, water soluble, flexible polymer or a derivative thereof, said polymer being modified by end-group substitution with a hydrophobic moiety ;

dissolving said modified polymer in an aqueous solution, thereby obtaining a self-assembling structure;

contacting said self-assembling structure with said compound, thereby obtaining a self-assembling structure comprising and/or entrapping said compound;

49. The method of claim 48, where the compound is a poorly soluble compound.

50. The method of any one of claims 48 to 49, where the compound is

classified according to the Biopharmaceutics Classification System as belonging to Class II or Class IV.

51. The method of any one of claims 48 to 50, where the compound at the highest dose strength is insoluble in 250 ml_ or less of an aqueous medium over the pH range of 1 to 6.8.

52. The method of any one of claims 48 to 51 , where the compound has a low thermal stability.

53. The method of claim 52, wherein the compound having low thermal stability is a compound, the degradation and/or inactivation and/or denaturation rate of which correlates with temperature.

54. The method of any one of claims 52 to 53, wherein the compound having low thermal stability is a compound which, when the temperature is raised to a pre-defined target temperature, gets degraded and/or inactivated and/or degraded prior to reaching said target temperature.

55. The method of claim 54, wherein the target temperature is 138°C or less, such as 130°C or less, such as 125°C or less, such as such as 121 °C or less, such as 120°C or less, such as 110°C or less, such as 100°C or less, such as 90°C or less, such as 80°C or less, such as 70°C or less, such as 60°C or less, such as 55°C or less, such as 54°C or less, such as 53°C or less, such as 52°C or less, such as 51 °C or less, such as 50°C or less.

The method of any one of claims 48 to 55, where the compound has a low bioavailability.

57. The method of any one of claims 48 to 56, where the solubility and/or the thermal stability and/or the bioavailability of the compound comprised and/or entrapped within the self-assembling structure is increased compared to the solubility and/or thermal stability and/or the bioavailability bo of the compound when not comprised nor entrapped within the self- assembling structure.

58. The method of any one of claims 48 to 57, wherein the self-assembling structure is a multi-layered structure according to any one of claims 1 to 22.

59. The method of any one of claim 48 to 58, wherein the self-assembling

structure is obtainable by the method of any one of claims 34 to 37. 60. A method for reducing aggregation and/or denaturation of a compound upon heat treatment, said method comprising:

iii) contacting said self-assembling structure with said compound, thereby

whereby aggregation and/or denaturation of the compound is reduced.

The method of claim 60, where the aggregation and/or denaturation of the compound is decreased compared to the aggregation and/or denaturation of the compound when not comprised nor entrapped within the self- assembling structure.

The method of any one of claims 60 to 61 , where the heat treatment of step iv) is performed in the absence of a gas-liquid interface, such as an air- liquid interface.

63. The method of any one of claims 60 to 62, where the self-assembling

structure is a multi-layered structure according to any one of claims 1 to 33.

64. The method of any one of claim 60 to 63, wherein the self-assembling

structure is obtainable by the method of any one of claims 34 to 44.