This application claims the benefit of priority of European applications 22193021 .7 submitted 30 August 2022 and 22198195.4 submitted 27 September 2022, the content of both of which is incorporated herein by reference.

Field

The present invention relates to compositions for parenteral sustained release drug delivery of hydrophilic active pharmaceutical ingredients (API). Particular embodiments of the compositions comprise phosphatidylcholine (PC) phospholipids and 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) in a multiphase spherulite structure, in which concentric lipid bilayers alternate with continuous aqueous compartments, the aqueous compartments comprising the hydrophilic drug substance.

In certain aspects and embodiments of the invention presented herein, the compositions for sustained release parenteral drug delivery comprise inositol phosphate derivatives, particularly 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1 ,2,3,5-tetrakis(phosphate) ((OEG2)2-IP4). These compositions may be used for the treatment or prevention of conditions associated with the formation of crystals or other precipitates, particularly precipitates comprising calcium phosphate.

Background

Most parenteral sustained release formulations, such as liposomes, lipid nanoparticles and microspheres have been developed for the delivery of hydrophobic and/or lowly-dosed and relatively high molecular weight (MW) drugs (/.e., nucleic acids and peptides). On the other hand, only a few systems have been reported for small hydrophilic compounds that are administered at relatively high doses. Among them, one can cite multivesicular liposomes (MVLs) that consist of numerous discontinuous internal aqueous compartments separated by several nonconcentric lipid bilayers, which afford the delivery of large quantities of drugs in a small volume of injection. MVLs have been commercialized under the trademark DepoFoam®, and have been shown to release drugs over several days to weeks after parenteral extravascular administration. The DepoFoam® technology is found in DepoCyt® (cytarabine, FDA-approved in 2007, production discontinued in 2017) and DepoDur® (morphine, FDA- approved in 2004). The relatively limited success of DepoFoam® is partially explained by the laborious preparation method consisting of a multi-step double emulsification process, together with the impossibility of sterilizing the final product which increases the complexity of the production. In this work, a lipid-based spherulitic vesicular system capable of entrapping large amounts of low MW ionized molecules (/.e., in the milligram range) and spontaneously triggering the drug release over time was engineered with a simple and green process. Spherulites are nano- to micro-sized vesicles that were first reported by D. Roux and collaborators in 1993 (Diat, O. et al., J. Phys. II 1993. https://doi.Org/10.1051/jp2:1993106.). They are considered a special class of liposomes characterized by a closely packed and well- organized onion-like structure, where concentric lipid bilayers alternate with continuous aqueous compartments. Spherulites possess the advantages of conventional liposomes (e.g., safety) while overcoming drawbacks related to the use of organic solvents or heat, and allowing a higher loading efficiency (LE). So far, several drug-delivery applications employing the spherulite technology have been described, covering a wide range of molecules (low MW organic molecules, proteins, DNA) and functions (oral delivery, drug detoxification, immune stimulation and imaging). Spherulites are usually made of PC-based lipids, a phospholipid having a cylindrical shape that assembles in stable bilayers (critical packing parameter (CPP) value of ~ 1 ). However, due to the multiple lipid lamellae that form conventional spherulites, the vesicles might be too stable in-vivo to release the non-membrane permeable API cargo, and would too heavily rely on the degradation of the phospholipid, a process that might be too slow to achieve therapeutic drug levels. Hence, herein, the inventors engineered spherulites that would become kinetically unstable upon injection and subsequent dilution. These vesicles contain 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and an injectable nonionic cone-shaped surfactant, e.g., polysorbate 20 (P20). Differently from PC-based lipids, DOPE forms hexagonal phases, having an inverted-cone shape (negative curvature, CPP value > 1 ). However, in the presence of cone-shaped surfactants (positive curvature, CPP value < 1 ), DOPE can form bilayers that might undergo lamellar to hexagonal phase transition over time, triggering the API release. As a model API, (OEG2)2-IP4 was selected, owing to its high hydrophilicity, ionized structure, low MW (748.92 g/mol) and relatively high dosage. (OEG2)2- IP4 is a novel inositol phosphate-based inhibitor of soft-tissue calcification, currently under clinical investigation for the treatment of aortic valve stenosis, in patients suffering from chronic kidney disease. This work shows that spherulites prepared with DOPE/P20 can i) be formulated with a green and simple preparation process, ii) entrap a high amount of very hydrophilic and low MW compound and iii) be destabilized upon dilution (e.g. subcutaneous (s.c.) injection), leading to the complete release of (OEG2)2-IP4 over an extended time.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to facilitate sustained release drug delivery of hydrophilic active pharmaceutical ingredients.

This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification. Summary of the Invention

A first aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutical drug. The composition forms spherulitic vesicles of a lipid phase in an aqueous phase. The pharmaceutical drug is comprised in an aqueous phase. This aqueous phase is comprised in vesicles constituted by a plurality of concentric lipid layers of a lipid phase. The concentric lipid layers are separated by layers of said aqueous phase.

The lipid phase comprises a cone-shaped first component consisting of a surfactant characterized by a CPP value of <1 (i.e., an intrinsic curvature of >0), and an inverse-cone- shaped second component consisting of a bis-acyl surfactant characterized by a CPP value of >1 (i.e. an intrinsic curvature of <0).

In particular embodiments, the pharmaceutical drug is an inositol phosphate derivative. For clarity, an isositol phosphate derivative is any molecule comprising an inositol moiety and at least one phosphate covalently attached to an inositol oxygen.

Another aspect of the invention relates to a lipid precursor composition suitable for preparing the pharmaceutical composition as disclosed as the first aspect of the invention. This lipid precursor composition comprises between 35% and 50 (w/w) of the first component consisting of a (cone-shaped) surfactant characterized by a CPP value of <1 , and between 50 and 65% (w/w) of the inverse-cone-shaped second component consisting of a bis-acyl surfactant characterized by a CPP value of >1 .

Yet another aspect of the invention relates to a biphasic precursor composition from which the pharmaceutical composition as disclosed as the first aspect of the invention may be prepared by application of shear stress. This biphasic precursor composition comprises the lipid precursor composition and an aqueous solution of a pharmaceutical drug.

The invention further encompasses methods for the preparation of the compositions described here, as well as the use of these compositions in the treatment or prevention of conditions associated with calcium crystallization.

Terms and definitions

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

"And/or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et aL, Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

The term spherulitic vesicle in the context of the present specification relates to a particle type formed by an outer lipid bilayer in an aqueous phase, the outer lipid bilayer comprising a plurality of concentric lipid bilayers inside the outer bilayer, each bilayer separated from another by an aqueous phase in an onion-like fashion. The term “cone-shaped” as used herein in relation to a surfactant or membrane I lipid phase constituent relates to a molecule which when assayed in pure form, displays a positive curvature (a critical packing parameter CPP<1 ). The term “inverse cone-shaped” as used herein in relation to a surfactant or membrane I lipid phase constituent relates to a molecule which when assayed in pure form, displays a negative curvature (a critical packing parameter CPP>1 ).

Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

As used herein, the term pharmaceutically acceptable earner includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treating or treatment of any disease or disorder (e.g. pathology associated crystallization) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.

Detailed Description of the Invention

A first aspect of the invention relates to a pharmaceutical composition comprising a pharmaceutical drug in the form of spherulitic vesicles. The pharmaceutical drug is comprised in a continuous aqueous phase, said aqueous phase being comprised in vesicles constituted by a plurality of concentric lipid layers of a lipid phase, said concentric lipid layers being separated by layers of said aqueous phase.

The lipid phase comprises two components characterized by distinct intrinsic curvature when assayed for this parameter in pure form: a cone-shaped first component consisting of a surfactant characterized by a CPP value of <1 (i.e. an intrinsic curvature of >0), and an inverse-cone-shaped second component consisting of a bis-acyl surfactant characterized by a CPP value of >1 (i.e., an intrinsic curvature of <0).

A method to measure intrinsic curvature is disclosed in Hamai et al. Effect of Average Phospholipid Curvature on Supported Bilayer Formation on Glass by Vesicle Fusion. Biophys. J. 2006, Volume 90, Issue 4, 15 February 2006, Pages 1241-1248, fully incorporated by reference herein. Unless specified otherwise, the value is determined for the individual pure compound by the cited method.

Another description for a method of measuring intrinsic curvature is disclosed in “Emulsions” (Tharwat Thadros); Chapter ?: Formation, Stability, Industrial Applications, Berlin, Boston: De Gruyter, 2016, pp. 73-94. https://doi.org/10.1515/9783110452242-008.

The cone-shaped first component characterized by a CPP value of <1 may be a single-acyl surfactant such as the polysorbate (“Tween”) surfactants used in the examples, but may also comprise more than a single acyl group. Kolliphor HS15 is formed by a mixture of a single- and a bis-acyl surfactant.

In another set of embodiments, surfactants useful for practicing the invention include polyethylene glycol aliphatic esters and polyethylene glycol aliphatic ethers.

In certain embodiments, nonionic surfactants (characterized by a CPP < 1 ), are polyethylene glycol aliphatic ethers commonly known in the art as Brij 35 and 58, CAS 9002-92-0 and 9004- 95-9 respectively. Alternatively or additionally, polyethylene glycol aliphatic esters can be employed, particularly PEG stearates, CAS 9004-99-3, as well as sorbitans (i.e., Span 60, CAS 1338-41-6. These non-ionic surfactants can be used together with an inverse cone shaped component, exemplified herein by DOPE for the spherulite formation. These components are added to the mixture at the same weight ratio ranges as the surfactants described in the examples (i.e., P20 and HS15), by using the same preparation process.

Negative curvature I CPP>1 inverse-cone shaped surfactants include the group comprised of monogalactosyldiacylglycerol (MGDG), monoglucosyldiacylglycerol (MGIcDG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylserine (PS), and phosphatidic acid (PA).

In certain embodiments, the vesicles constituting the pharmaceutical composition are characterized by a mean hydrodynamic diameter in the range of 10 nm to 10 pm, particularly by a mean hydrodynamic diameter in the range of 100 nm to 5 pm. In certain particular embodiments, the vesicles constituting the pharmaceutical composition are characterized by a mean hydrodynamic diameter in the range of 150 to 3000 nm. The hydrodynamic diameter is measured as specified in the example (see “Characterization of the vesicles”, using a Malvern ZetaSizer Advance Pro, at a scattering angle of 179° at 25 °C).

In certain embodiments, the vesicles constituting the pharmaceutical composition are characterized by a zeta-potential value in the range of -40 and +40 mV.

In certain embodiments, the vesicles constituting the pharmaceutical composition are characterized a concentric lipid bilayer structure comprising 2 to 100 bilayers per particle. In certain particular embodiments, the vesicles constituting the pharmaceutical composition are characterized a concentric lipid bilayer structure comprising 5 to 50 bilayers per particle.

The distribution of numbers of bilayers per particle depends on the composition of the lipid phase. The vesicles are visualized as Maltese crosses under polarized light. The pharmaceutical composition is made of a certain number of bilayers ranging from 2 to 100 that form an onion like structure.

In certain embodiments, the molecules constituting the first component and second component are present in the lipid phase at a ratio of between 20:80 and 70:30 (w/w), respectively. In certain particular embodiments, the ratio of first component molecules and second component molecules is 59:41 and 54:46 (w%), respectively.

Another factor influencing lamellae formation is the water content. In certain embodiments, when the water content per mass (w%) is taken into account, the ratios of two exemplary formulations designated 16 and 24 in the examples, are 39:28:33 (DOPE/P2O/H2O) and 36: 15.5:15.5:33 (DOPE/P20/HS15/H ₂ O).

In certain embodiments, the cone-shaped first component comprises a conjugate comprising polyethoxylated sorbitan and a C10 to C12 fatty acid.

In certain particular embodiments, the cone-shaped first component comprises polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate; CAS No 9005-64-5).

In certain particular embodiments, the cone-shaped first component comprises polysorbate 60 (polyoxyethylene (20) sorbitan monostearate; CAS No 9005-67-8).

In certain particular embodiments, the cone-shaped first component comprises polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate; CAS No 9005-66-7).

In certain particular embodiments, the cone-shaped first component comprises polysorbate 80 (polyoxyethylene (20) sorbitan monooleate; CAS No 9005-65-6).

In more particular embodiments, the conjugate is polysorbate 20. In certain embodiments, said cone-shaped first component comprises a polyethoxylated 12- hydroxystearic acid.

In certain particular embodiments, the first component comprises, or essentially consists of, Kolliphor® HS15, also known as Solutol HS15 CAS No 70142-34-6, which consists of polyglycol mono- and di-esters of 12-hydroxystearic acid (making up the lipophilic part of the compound) and of about 30% of free polyethylene glycol (= hydrophilic part).

In certain embodiments, the inverse-cone shaped second component comprises 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine; DOPE; CAS No. 4004-05-1 ).

In certain embodiments, the cone-shaped first component comprises a conjugate comprising polyethoxylated sorbitan and a C14-C20 fatty acid. In particular embodiments the conjugate is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate; CAS No 9005-65-6).

In certain embodiments, the lipid phase, in addition to the first and second component comprises a fatty acid component comprising C12-C20 fatty acids.

In certain embodiments, the lipid phase in addition to the first and second component and optionally, to the fatty acid component, comprises a compound selected from the group consisting of cholesterol and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy(polyethylene glycol) (DSPE- PEG).

In certain embodiments, the cholesterol content relative to the mass of the entire lipid phase (w%), ranges from 1 to 20%. In particular embodiments, the cholesterol content relative to the mass of the entire lipid phase (w%), ranges from 5 to 15%, more particularly from 7.5 to 10%, even more particularly the cholesterol content of the lipid phase is 7.7%.

When defining the weight (mass) ratios of the compositions described herein, this specification accounts for the weight of the water where necessary.

The weight of water is considered for the preparation process of the vesicles (it is important for the lamellae formation). However, after dilution (step 3 of Fig. 1 B) it is not relevant anymore and only the weight of lipid and surfactant can be considered.

In certain embodiments, the composition comprises the following weight percentages of components: the cone-shaped first component is present at 5 to 50% (w%); the inverse-cone-shaped second component is present at between 30 and 70% (w%); cholesterol is present at between 0 and 20 w%.

In certain particular embodiments thereof, the composition comprises the following weight percentages of components: the cone-shaped first component is present at 25 to 35% (w%); the inverse-cone-shaped second component is present at between 33 and 43% (w%), particularly from 36 to 40%.

The cited mass ratios add up to 100% when the aqueous phase is considered. In other words, the aqueous phase containing the API is not cited here explicitly.

In certain particular embodiments, the cone-shaped first component is present at 5 to 50% (w%); the inverse-cone-shaped second component is present at between 30 and 70% (w%); cholesterol is present at between 0 and 20 (w%). the cone-shaped first component is polysorbate 20 or a mixture of polysorbate 20 and Solutol HS15; the inverse-cone-shaped second component is DOPE.

In certain particular embodiments, the cone-shaped first component is present at 25 to 35% (w%); the inverse-cone-shaped second component is present at between 36 to 40%. the cone-shaped first component is polysorbate 20 or a mixture of polysorbate 20 and Solutol HS15; the inverse-cone-shaped second component is DOPE.

In certain embodiments, the composition comprises 38 to 41 % (w/w) DOPE and 27 to 29% polysorbate 20, and the remainder is made up by the aqueous phase comprising the drug substance.

In certain particular embodiments, the composition comprises 35 to 38% (w/w) DOPE, 14 to 16% Solutol HS15, and 14 to 16% polysorbate 20 (P20), and the remainder is made up by the aqueous phase comprising the drug substance.

Inositol derivatives as API

The pharmaceutical composition according to any one of the preceding claims, wherein said pharmaceutical drug is an inositol phosphate derivative.

Inositol phosphate derivatives useful for inhibiting pathological crystallization processes are shown, inter alia, in PCT/EP2012/004088 (also published as US9358243B2), PCT/EP2016/080657 (also published as US10624909B2), and PCT/EP2019/074986 (US2021347793A1 ).

Other inositol phosphate derivatives of medical interest include those disclosed in PCT/EP2016/080545 (US10487097B2, ETH Zurich), WO2020157362A1 (Sanifit Therapeutics); WO2021094331 A1 (Sanifit Therapeutics). Any patent document mentioned herein is deemed to be incorporated herein by reference in its entirety.

Mono-inositol derivatives

In certain embodiments, the inositol phosphate derivative used as the pharmaceutical drug in the composition is characterized by a general formula (I) wherein one or two or three X are R ¹ and each of the remaining X is an anionic moiety independently selected from the group consisting of OPOs ² ', OPSO? ² ', and OSC '; and each R ¹ independently of any other R ¹ is a polyethylene glycol or a polyglycerol.

In certain particular embodiments, two X of formula (I) are R ¹ .

In certain specific embodiments, the inositol phosphate derivative is characterized by a general formula selected from the group of (Ila), (lib), (He) and (lid): with each X being an anionic moiety independently selected from the group consisting of OPO ₃ ² ’, OPSO? ² ', and OSO ₃ ‘; and R ¹ being a polyethylene glycol or a polyglycerol.

Particular embodiments thereof relate to compounds in which each X is phosphate.

In certain other embodiments, one X of formula (I) is R ¹ . In certain specific embodiments, the inositol phosphate derivative is characterized by a general formula selected from the group of (Illa), (I I lb), (I He) and (Hid): with each X being an anionic moiety independently selected from the group consisting of OPO ₃ ² ’, OPSO? ² ', and OSO ₃ ‘; and R ¹ being a polyethylene glycol or a polyglycerol.

Particular embodiments thereof relate to compounds in which each X is phosphate.

In certain other embodiments, three X of formula (I) are R ¹ .

In certain specific embodiments, the inositol phosphate derivative is characterized by a general formula selected from the group of (IVa) and (IVb): with each X being an anionic moiety independently selected from the group consisting of

OPO3 , OPSO2 , and OSOs'; and R ¹ being a polyethylene glycol or a polyglycerol.

Particular embodiments thereof relate to compounds in which each X is phosphate.

Particular embodiments of any of the compounds specified above, relate to compounds wherein each R ¹ is a poly(ethylene glycol).

Particular embodiments of any of the compounds specified above, relate to compounds wherein each R ¹ is a poly(ethyleneglycol) described by the formula -O-((CH2)2-O) _n -R ^T , wherein n is an integer selected from the range between 2 and 50, particularly wherein n is an integer between 2 and 12, more particularly wherein n is selected from 2, 3, 4, 5, 6, 7 , 8 and 9; and wherein

R ¹ is selected from the group consisting of CH3 and H.

In certain very specific embodiments, the inositol phosphate derivative is described by formula (II e),

Oligo-inositol derivatives

In certain embodiments, the inositol phosphate derivative used as the pharmaceutical drug in the composition is an oligo-inositol compound comprising, particularly consisting of, two or more cyclohexanolpentakisester moieties described by a general formula (V) linked by a common central linker L wherein X is selected from the group consisting OPOs ² ', OPSO? ² ', and OSOs',

L is a common central linker to which n individual moieties characterized by the formula in brackets are attached, and n is an integer selected from 2, 3 and 4, wherein the common central linker L has a molecular weight <1000 g/mol, and wherein L comprises or essentially consists of a linear or branched poly(ethylene glycol) or polyglycerol.

Particular embodiments relate to compounds wherein each anionic moiety X is a phosphate.

In certain embodiments, any one of the cyclohexanolpentakisester moieties described by the general formula (V) of the oligo-inositol derivative is independently selected from a moiety described by general formulae (Va) or (Vb),

In certain embodiments, the pharmaceutical drug is present in the aqueous phase at a concentration from 1 mg/mL to 1000 mg/mL. In particular embodiments thereof, the drug is an inositol derivative as specified herein.

In certain particular embodiments, the pharmaceutical drug is present in the aqueous phase at a concentration of from 50 mg/mL to 800 mg/mL. In more particular embodiments, the drug is present at a concentration of from 200 mg/mL to 600 mg/mL.

In certain embodiments, the composition comprises between 30 and 50% (w%) DOPE, between 20 and 40% (w%) P20, and the remainder being the aqueous phase of between 25 and 40% (w%), the aqueous phase comprising the pharmaceutical drug at between 200mg/mL to 600mg/mL. In particular embodiments thereof, the drug is an inositol derivative as specified herein, particularly He.

In certain embodiments, the composition comprises between 30 and 50% (w%) DOPE, between 10 and 30% (w%) P20, between 10 and 30% Solutol HS15, and the remainder being the aqueous phase of between 25 and 40% (w%), the aqueous phase comprising the pharmaceutical drug at between 200 mg/mL to 600 mg/mL. In particular embodiments thereof, the drug is an inositol derivative as specified herein, particularly He.

In certain particular embodiments, the composition comprises between 38 and 41 % (w%) DOPE, between 27 and 29% (w%) P20, and the remainder being the aqueous phase of between 32 and 34% (w%), the aqueous phase comprising the pharmaceutical drug at between 400 mg/mL to 600 mg/mL. In particular embodiments thereof, the drug is an inositol derivative as specified herein, particularly He.

In certain particular embodiments, the composition comprises between 35 and 38% (w%) DOPE, between 14 and 17% (w%) P20, between 14 and 17% Solutol HS15, and the remainder being the aqueous phase of between 32 and 37% (w%), the aqueous phase comprising the pharmaceutical drug at between 400 mg/mL to 500 mg/mL. In particular embodiments thereof, the drug is an inositol derivative as specified herein, particularly He.

Another aspect of the invention relates to a lipid precursor composition suitable for preparing the pharmaceutical composition according to the above aspect of the invention in any one of its embodiments. This lipid precursor composition comprises: between 35 and 50% (w/w) of a cone-shaped first component consisting of a surfactant characterized by a CPP value of <1 ; and between 50 and 65% (w/w) of an inverse-cone-shaped second component consisting of a bis-acyl surfactant characterized by a CPP value of >1 .

In particular embodiments thereof, the precursor composition’s first component is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and I or solutol HS15.

In particular embodiments, the second component is DOPE.

The precursor composition may optionally comprise cholesterol.

In addition, the precursor composition may optionally comprise precursor composition may optionally comprise DSPE- PEG, particularly at a concentration between 1 % and 10% (w/w).

The mass ratios given for this lipid precursor composition naturally do not account for the water later added to the precursor to obtain the vesicles.

In certain embodiments, the precursor composition comprises cholesterol and DSPE-PEG at a combined mass ratio of between 1 and 20%. In certain embodiments, the precursor composition comprises up to 10% (w%) DSPE-PEG and cholesterol, the combination of the masses of DSPE-PEG and cholesterol making up no more than 20% of the precursor composition. Another aspect of the invention relates to a biphasic precursor composition suitable for preparing the pharmaceutical composition according to the first aspect of the invention in any one of its embodiments, said biphasic precursor composition comprising: the lipid precursor composition as disclosed as the preceding aspect of the invention, and an aqueous solution of a pharmaceutical drug. From this biphasic precursor composition, the pharmaceutical composition according to the invention can be prepared by application of shear stress.

Another aspect of the invention relates to a method of preparation of a pharmaceutical composition according to the first aspect of the invention in any one of its embodiments. This method comprises the steps: providing a lipid precursor solution according to the first aspect of the invention in any of its embodiments; combining the lipid precursor solution with an aqueous solution of a pharmaceutical drug, yielding a lamellar phase precursor composition; applying a shear stress to said lamellar phase precursor composition; diluting the resultant composition with a physiologically acceptable buffer under agitation (shaking).

In certain embodiments, lipids are mixed up to 48 h at the range temperatures of 20 and 50 °C, more particularly for 24 h at 37 °C. The hydration time is up to 48 h at the range temperatures of 20 and 50 °C, more particularly for 24 h at 37 °C.

The method is of particular utility for formulating any of the inositol derivatives disclosed in the present specification.

In certain embodiments, the pharmaceutical drug is present at a concentration of between 200 mg/mL and 600 mg/mL in the aqueous phase. In certain particular embodiments, the pharmaceutical drug is present at a concentration of between 400 mg/mL and 500 mg/mL.

The invention encompasses a pharmaceutical composition obtained by the method according to the previously discussed aspect relating to a method of preparation of a pharmaceutical composition.

The pharmaceutical composition according to the invention may be administered for use in treatment or prevention of a condition associated with pathological calcium crystallization.

In certain embodiments, the condition for treatment or prevention of which the composition is administered, is selected from vascular calcification, coronary artery disease, vascular stiffening, valvular calcification, nephrocalcinosis, calcinosis cutis, kidney stones, chondrocalcinosis, osteoporosis, peripheral arterial disease, critical limb ischemia, calciphylaxis, general arterial calcification of infancy, aortic stenosis, atherosclerosis, pseudogout, primary hyperoxaluria and pseudoxanthoma elasticum). Another aspect encompassed by the invention relates to a method of treating a condition associated with pathological calcium crystallization in a patient, said method comprising administering to the patient a pharmaceutical composition as specified according to the first aspect of the invention in any one of its embodiments.

Likewise, the composition as specified according to the first aspect of the invention in any one of its embodiments may be used in the manufacture of a medicament for treatment of a condition associated with pathological calcium crystallization.

Administration/Dosage Forms and Salts

The skilled person is aware that any specifically mentioned drug compound mentioned herein may be present as a pharmaceutically acceptable salt of said drug. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, etc. Procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

Method of Manufacture and Method of Treatment according to the invention

The invention further encompasses, as an additional aspect, the use of a pharmaceutical composition as specified in detail above, for use in a method of manufacture of a medicament for the treatment or prevention of a condition pathology-associated crystallization of calcium salts, particularly of calcium phosphate and/or oxalate salts.

Similarly, the invention encompasses methods of treatment of a patient having been diagnosed with a disease associated with a condition pathology-associated crystallization of calcium salts. This method entails administering to the patient an effective amount of pharmaceutical composition as specified in detail above.

Wherever alternatives for single separable features such as, for example, first composition component or medical indication are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention further encompasses the following items:

Item 1. A pharmaceutical composition comprising a pharmaceutical drug, said pharmaceutical drug being comprised in a continuous aqueous phase, said aqueous phase being comprised in vesicles constituted by a plurality of concentric lipid layers of a lipid phase, said concentric lipid layers being separated by layers of said aqueous phase; said lipid phase comprising: a. a first component consisting of a surfactant characterized by a CPP value of <1 , and b. a second component consisting of a surfactant characterized by a CPP value of >1.

Item 2. The pharmaceutical composition according to item 1 , wherein the vesicles constituting the pharmaceutical composition are characterized by a. a mean hydrodynamic diameter in the range of 10 nm to 10 pm, particularly in the range of 150 to 3000 nm, b. a zeta-potential value in the range of -40 and +40 mV; c. a concentric lipid bilayer structure comprising 2 to 100 bilayers per particle, particularly 5 to 50 bilayers per particle.

Item 3. The pharmaceutical composition according to item 1 or 2, wherein the first component and second component are present in the lipid phase at a ratio of between 20:80 and 70:30 (w/w), respectively, particularly wherein the ratio of first component molecules and second component molecules is 59:41 and 54:46 (w%), respectively.

Item 4. The pharmaceutical composition according to any one of the preceding items, wherein said first component comprises a conjugate comprising polyethoxylated sorbitan and a C10 to C12 fatty acid, particularly wherein the conjugate is selected from polysorbate 20, polysorbate 60, polysorbate 40, polysorbate 80, more particularly wherein the conjugate is polysorbate 20.

Item 5. The pharmaceutical composition according to any one of the preceding items, wherein said first component comprises a polyethoxylated 12-hydroxystearic acid.

Item 6. The pharmaceutical composition according to any one of the preceding items, wherein said second component comprises 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine.

Item 7. The pharmaceutical composition according to any one of the preceding items, wherein the composition comprises the following weight percentages of components: a. the first component is present at 5 to 50% (w%); b. the second component is present at between 30 and 70% (w%); c. cholesterol is present at between 0 and 20 (w%).

Item 8. The pharmaceutical composition according to item 7, wherein a. the first component is present at 25 to 35% (w%); b. the second component is present at between 33 and 43% (w%), particularly from 36 to 40%.

Item 9. The pharmaceutical composition according to item 7 or 8, wherein a. the first component is polysorbate 20 or a mixture of polysorbate 20 and Solutol HS15; b. the second component is DOPE.

Item 10. The pharmaceutical composition according to item 9, wherein the composition comprises 38 to 41% (w/w) DOPE and 27 to 29% polysorbate 20.

Item 11 . The pharmaceutical composition according to item 9, wherein the composition comprises 35 to 38% (w/w) DOPE, 14 to 16% Solutol HS15, and 14 to 16% polysorbate 20 (P20).

Item 12. The pharmaceutical composition according to any one of the preceding items, wherein said pharmaceutical drug is an inositol phosphate derivative.

Item 13. The pharmaceutical composition according to any one of the preceding items, wherein said pharmaceutical drug is characterized by a general formula (I) wherein

- one or two or three X are R ¹ and each of the remaining X is an anionic moiety independently selected from the group consisting of OPOs ² ', OPSO? ² ', and OSOs'; and

- each R ¹ independently of any other R ¹ is a polyethylene glycol or a polyglycerol.

Item 14. The pharmaceutical composition according to item 13, wherein two X are R ¹ .

Item 15. The pharmaceutical composition according to item 14, wherein said pharmaceutical drug is characterized by a general formula selected from the group of (Ila), (lib), (He) and (lid): with each X being an anionic moiety independently selected from the group consisting of OPOs ² ', OPSO? ² ', and OSC '; and R ¹ being a polyethylene glycol or a polyglycerol.

Item 16. The pharmaceutical composition according to item 13, wherein one X is R ¹ .

Item 17. The pharmaceutical composition according to item 16, wherein said pharmaceutical drug is characterized by a general formula selected from the group of (Illa), (lllb), (lllc) and (Hid):

with each X being an anionic moiety independently selected from the group consisting of OPO3 ² ; OPSO? ² ', and OSOs'; and R ¹ being a polyethylene glycol or a polyglycerol.

Item 18. The pharmaceutical composition according to item 13, wherein three X are R ¹ .

Item 19. The pharmaceutical composition according to item Item 18, wherein said pharmaceutical drug is characterized by a general formula selected from the group of (IVa) and (IVb): with each X being an anionic moiety independently selected from the group consisting of OPC ² ', OPSO? ² ', and OSOs'; and R ¹ being a polyethylene glycol or a polyglycerol.

Item 20. The pharmaceutical composition according to any one of the preceding items Item 13 to Item 19, wherein each R ¹ is a poly(ethylene glycol). Item 21 . The pharmaceutical composition according to any one of the preceding items, wherein said pharmaceutical drug is described by formula (II e),

Item 22. The pharmaceutical composition according to any one of the preceding items Item 13 to Item 20, wherein each R ¹ is a poly(ethyleneglycol) described by the formula -O-((CH2)2-O) _n - ^T , wherein a. n is an integer selected from the range between 2 and 50, particularly wherein n is an integer between 2 and 12, more particularly wherein n is selected from 2, 3, 4, 5, 6, 7, 8 and 9; and b. wherein R ¹ is selected from the group consisting of CH3 and H.

Item 23. The pharmaceutical composition according to any one of the preceding items, wherein said pharmaceutical drug is a compound comprising, particularly consisting of, two or more cyclohexanolpentakisester moieties described by a general formula (I) linked by a common central linker L wherein X is selected from the group consisting OPOs ² ', OPSO? ² ', and OSC ', L is a common central linker to which n individual moieties characterized by the formula in brackets are attached, and n is an integer selected from 2, 3 and 4, wherein the common central linker L has a molecular weight <1000 g/mol, and wherein L comprises or essentially consists of a linear or branched poly(ethylene glycol) or polyglycerol.

Item 24. The pharmaceutical composition according to any one of the preceding items Item 13 to 23, wherein each anionic moiety is a phosphate. Item 25. The pharmaceutical composition according to any one of the preceding items, wherein said pharmaceutical drug is present in the aqueous phase at a concentration from 1 mg/mL to 1000 mg/mL, particularly at a concentration of from 50 mg/mL to 800 mg/mL, more particularly at a concentration of from 200 mg/mL to 600 mg/mL.

Item 26. The pharmaceutical composition according to any one of the preceding items, wherein the composition comprises between 30 and 50% (w%) DOPE, between 20 and 40% (w%) P20, and the remainder being the aqueous phase of between 25 and 40% (w%), the aqueous phase comprising the pharmaceutical drug at between 200 mg/mL to 600 mg/mL.

Item 27. The pharmaceutical composition according to any one of the preceding items, wherein the composition comprises between 30 and 50% (w%) DOPE, between 10 and 30% (w%) P20, between 10 and 30% Solutol HS15, and the remainder being the aqueous phase of between 25 and 40% (w%), the aqueous phase comprising the pharmaceutical drug at between 200 mg/mL to 600 mg/mL.

Item 28. The pharmaceutical composition according to any one of the preceding items, wherein the composition comprises between 38 and 41 % (w%) DOPE, between 27 and 29% (w%) P20, and the remainder being the aqueous phase of between 32 and 34% (w%), the aqueous phase comprising the pharmaceutical drug at between 400 mg/mL to 600 mg/mL.

Item 29. The pharmaceutical composition according to any one of the preceding items, wherein the composition comprises between 35 and 38% (w%) DOPE, between 14 and 17% (w%) P20, between 14 and 17% Solutol HS15, and the remainder being the aqueous phase of between 32 and 37% (w%), the aqueous phase comprising the pharmaceutical drug at between 400 mg/mL to 500 mg/mL.

Item 30. A lipid precursor composition suitable for preparing the pharmaceutical composition according to any one of items 1 to Item 29, said lipid precursor composition comprising: a. between 35 and 50% (w/w) of a first component consisting of a surfactant characterized by a CPP value of <1 ; particularly wherein the first component is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and I or solutol HS15; and b. between 50 and 65% (w/w) of a second component consisting of a surfactant characterized by a CPP value of >1 particularly wherein the second component is DOPE and optionally, a compound selected from the group consisting of cholesterol and DSPE-PEG.

Item 31 . A biphasic precursor composition suitable for preparing the pharmaceutical composition according to any one of items 1 to Item 29, said biphasic precursor composition comprising: the lipid precursor composition according to item 0, and an aqueous solution of a pharmaceutical drug.

Item 32. A method of preparation of a pharmaceutical composition according to any one of items 1 to Item 29, said method comprising the steps: a. providing a lipid precursor solution according to item 0; b. combining the lipid precursor solution with an aqueous solution of a pharmaceutical drug, yielding a lamellar phase precursor composition; c. applying a shear stress to said lamellar phase precursor composition; d. diluting the resultant composition with a physiologically acceptable buffer under agitation.

Item 33. The method according to item Item 32, wherein the pharmaceutical drug is a compound as specified in any one of items 12 to Item 24.

Item 34. The method according to item Item 32 or Item 33, wherein the pharmaceutical drug is present at a concentration of between 200 mg/mL and 600 mg/mL in the aqueous phase; particularly, wherein the pharmaceutical drug is present at a concentration of between 400 mg/mL and 500 mg/mL.

Item 35. A pharmaceutical composition obtained by the method according to items 32 to 34.

Item 36. The pharmaceutical composition according to any one of items 12 to 29, for use in treatment of a condition associated with pathological calcium crystallization.

Item 37. The pharmaceutical composition for use according to item 36, wherein said condition is selected from vascular calcification, coronary artery disease, vascular stiffening, valvular calcification, nephrocalcinosis, calcinosis cutis, kidney stones, chondrocalcinosis, osteoporosis, peripheral arterial disease, critical limb ischemia, calciphylaxis, general arterial calcification of infancy, aortic stenosis, atherosclerosis, pseudogout, primary hyperoxaluria and pseudoxanthoma elasticum).

Item 38. The pharmaceutical composition for use according to item 36 or 37, wherein the pharmaceutical drug is lie. Item 39. A method of treating a condition associated with pathological calcium crystallization in a patient, said method comprising administering to the patient a pharmaceutical composition as specified in any one of items 12 to 29.

Item 40. Use of a composition as specified in any one of items 12 to 29 in the manufacture of a medicament for treatment of a condition associated with pathological calcium crystallization.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows (A) Schematic representation of the lipid lamellae composition of the main spherulite formulations. The main components of LE80 are PC (80-85, w/w) as well as palmitic and oleic acid (28-34 and 26-30, w/w respectively). HS15 mainly consists of polyglycol mono- and di-esters of 12-hydroxystearic acid. CPP, T _m and HLB values of phospholipids, nonionic surfactants and CH are reported. The CPP value of HS15 was not available (N.A.). (B) Steps of the optimized formation process of spherulites. The API ((OEG2)2lP4(formula lie)) is added as an aqueous solution in step 1 .

Fig. 2 shows physicochemical characterization of drug-free PC vs PE spherulites. (A) and (B) show the dh (nm) and ^-potential (mV), respectively, of F8, F10, F15, F16 and F24 (Table 1 ) measured with Zeta-Sizer, at the concentration of 10 mg/mL. Data are shown as means ± s.d. (n = 3) of three independent batches. Images refer to the LE80- (F8, top panel) vs DOPC- (F15, middle panel) vs DOPE-based (F16, bottom panel) spherulite formulations (Table 1), dispersed in 5% dextrose. Most representative polarized light (C-E), low and high magnification cryo-SEM (F-K) and cryo-TEM (L-N) micrographs.

Fig. 3 shows physicochemical characterization of drug-loaded PC vs PE spherulites. (A) dh (nm) and (B) ^-potential (mV) values measured with the Zeta-Sizer, at the vesicle concentration of 10 mg/mL of F8d, F10, F15, F16 and F24 (Table 3). Mean ± s.d. (n = 3). Images refer to the main (OEG2)2lP4-loaded spherulite formulations recorded immediately after step 3 (Figure 1B) in 5% dextrose, at pH 7.2. Representative polarized light (C-E), low and high magnification cryo- SEM (F-K) and cryo-TEM (L-N) micrographs of F8d, F15 and F16 (Table 3). Fig. 4 shows morphology of PC- vs PE-based (OEG2)2lP4-loaded spherulites (F15, F16 and F24, Table 3) over time, shown by cryo-TEM images at 37 °C. (A-C) display representative micrographs of F15 immediately after resuspension (t = 1 h), after 6 h and 5 days, respectively. (D) shows F16 at t = 1 h, while (E) and (F) show F16 at t = 6 h. Two images were chosen for the sample at 6 h, to illustrate the heterogeneity of the formulation. (G) displays F16 at 5 days. (H) and (I) show F24 at t = 1 h. Only a few spherulitic structures were detected and the formulation already displayed high heterogeneity. (J-M) show F24 at t = 6 h and 5 days. All samples were dispersed in 5% dextrose, at pH 7.2.

Fig. 5 shows (A) comparison of the (OEG2)2lP4 LE achieved with spherulites vs MLVs and LUVs prepared by REV. Statistical significance was calculated using ordinary one-way ANOVA followed by T ukey’s multiple comparisons test and is depicted as ** p < 0.01 , *** p < 0.001 , **** p < 0.0001. Mean ± s.d. (n = 3). (B) and (C) display cryo-TEM images of F8d before and after extrusion (MLVs and LUVs, respectively), as well as (D) and (E) show cryo-TEM micrographs of F16 before (MLVs) and after (LUVs) extrusion, respectively.

Fig. 6 shows in-vitro (OEG2)2lP4 release (A) from spherulites (F8d, F10, F15, F16, F24) from 0 to 96 h (A) and from 6 to 96 h (B, magnification). The diffusion of OEG2)2lP4 from the donor to receptor cells is also represented. Studies were conducted at 37 °C in PBS (pH 7.2). Mean ± s.d. (n = 3).

Fig. 7 shows PK studies of the lead (OEG2)2lP4-loaded spherulite formulations (F15, F16, F24) compared to the (OEG2)2lP4 solution (control), in healthy rats following the s.c. injection of 10 mg/kg (OEG2)2lP4. All the samples were dispersed in 5% dextrose, at pH 7.2. (A) Illustration of the formulations tested in vivo. (B) Area under the plasma concentration vs. time curve (AUC) values. (C) Plasma concentration profile in a non-logarithmic scale between 0 and 10 h and (D) in a semi-logarithmic scale between 0 and 72 h. Mean ± s.d. (n = 3). Statistical significance was calculated using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test and is depicted as * p < 0.05, **** p < 0.0001.

Fig. 8 shows chemical structures of (A) DOPC, (B) P80, (C) DOPE, (D) P20 and (E) HS15.

Fig. 9 shows LC-CAD method for the detection and quantification of (OEG2)2lP4. (A) Mobile phase gradient. (B) (OEG2)2lP4 calibration curve in the concentration range of 30-500 pg/mL. (C) Limit of quantification (LOQ) of (OEG2)2lP4 was set to 30 pg/mL, whereas the lowest limit of detection (LLOD) was set to 25 pg/mL. Fig. 10 shows representative polarized light micrographs of (OEG2)2lP4-loaded spherulites (F15, F16 and F24, upper (A-C) middle (D-F) and bottom (G-M) panels, respectively, from Table 3), at t = 1 h, 6 h and 5 days. Two images have been selected for F24 after t = 6 h and 5 days, to show the heterogeneity of the formulation. In the images F, H-M the contrast was increased in order to show the presence of the vesicles. However, these structures lost the ability to polarize the light as spherulites. All samples were dispersed in 5% (w/v) dextrose, pH 7.2

Fig. 11 shows morphological change of (OEG2)2lP4-loaded PE spherulites (F16, Table 3) over time. Representative cryo-TEM micrographs of F16 immediately after resuspension (t = 1 h, A and B) at t = 6 h (C and D) and t = 5 days (E and F). All samples are dispersed in 5% dextrose pH 7.2.

Fig. 12 shows size distribution, PDI and LE of liposomes prepared by ethanol injection (A-C), thin-film hydration (D-F) and REV (G-l). Liposomes were prepared with the same starting materials as formulation F8d (Table 2). (A-l) show the dh (nm), size distribution and PDI, respectively, of unloaded LUVs and MLVs and (OEG2)2lP4-loaded LUVs. (J) displays the LE of LUVs prepared by the three different approaches. Statistical significance was calculated using ordinary oneway AN OVA followed by Tukey’s multiple comparisons test and is depicted as * p < 0.05, ** p < 0.01 , *** p < 0.005, **** p < 0.001 . Mean ± s.d. (n = 3).

Fig. 13 shows workflow of the REV approach for producing MLVs and LUVs.

Fig. 14 shows (A) dh (nm), (B) size distribution and (C) PDI of unloaded MLVs and

(OEG2)2lP4/MLVs prepared by REV. Liposomes were prepared with the same starting materials as spherulite formulations F8d, F10, F15, F16 and F24 (Table 2). Mean ± s.d. (n=3) for (A) and (C), whilst (B) shows one representative profile.

Fig. 15 shows (A) dh (nm), (B) size distribution and (C) PDI of unloaded LUVs and (OEG2)2lP4/LUVs prepared by REV. Liposomes were prepared with the same starting materials as spherulite formulations F8d, F10, F15, F16 and F24 (Table 2). Mean ± s.d. (n=3) for (A) and (C), whilst (B) shows one representative profile.

Fig. 16 shows (A) dh (nm) and (B) ^-potential (mV) of F8d, F10, F15, F16 and F24 (Table 2), over one week at 37 °C, in PBS at pH 7.2. Mean ± s.d. (n=3).

Fig. 17 shows MTS assay on HDFa of (A) spherulites and (B) (OEG2)2lP4/spherulites (F8d, F10, F15, F16 and F24) at concentrations ranging from 12.5 to 200 pg/mL after 4 and 24 h incubation. Mean ± s.d. (n = 3). Fig. 18 shows PK data of F8d and F10 compared to the (OEG2)2lP4 solution (control, same data points as those shown in Figure 6), in healthy rats following the s.c. injection of 10 mg/kg (OEG2)2lP4. All the samples were dispersed in 5% dextrose, at pH 7.2. (A) Area under the plasma concentration i/s. time curve (AUC) values. (B) (OEG2)2lP4 plasma concentration profile in a non-logarithmic scale between 0 and 10 h and (C) in a semi-logarithmic scale between 0 and 72 h. (B) Mean ± s.d. (n = 3 for the control and n = 2 for F8d and F10). AUC for control is the same as that shown in Figure 6

Fig. 19 shows the increase of the LE of the formulation shown in example 2A.

Examples

Example 1: Development and screening of the spherulite formulations

A library of spherulite formulations was prepared with PC phospholipids (Lipoid E80 (LE80) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)), DOPE, nonionic surfactants (/.e., polysorbate 80 (P80), P20 and Kolliphor HS15 (HS15)), cholesterol (CH) and water (Table 1). PC phospholipids and DOPE were selected due to their distinct packing arrangements (Figure 1 A and Figure 8). DOPC has two hydrophobic acyl chains that take up a similar width as the polar head group, thus assembling into bilayers (e.g., F8, F10 and F15, Table 1). On the other hand, DOPE has an inverted-cone shape, hence forming hexagonal phases (e.g., F16 and F24, Table 1). Nevertheless, with a suitable surfactant (e.g., cone-shaped P20), DOPE can pack in bilayers. Hence, the inventors hypothesized that DOPE-based bilayers should be destabilized once the surfactant diffuses out of the spherulite structure (lamellar to hexagonal phase transition) and, subsequently allow the release of the encapsulated drug. Among PC phospholipids, LE80 and DOPC were chosen due to their different transition temperatures (T _m , + 26.0 and - 16.5 °C, respectively), thus forming bilayers with different fluidity. The nonionic surfactants P80, P20 and HS15 (Figure 8B, D and E, respectively) were chosen due to their well-established safety profile. Further, P80 and P20 possess slightly different hydrophilic- lipophilic balance (HLB) values (15 and 16.7, respectively), which should result in the formation of lamellar phases with variable stability (Figure 1A). On the other hand, HS15 has a different structure compared to polysorbates, which should affect the bilayer packing and subsequent release profile. Table 1. Composition of the engineered spherulite formulations. Formulations in bold were selected for the API loading.

*Milli-Q water or dextrose solution at 5% (w/v).

The previously reported preparation process of spherulites (Crauste-Manciet, S. et al., https://doi.org/10.4155/tde.14.81 ) consists of three consecutive steps that can include the use of organic solvents: (1) formation of a lamellar phase with phospholipids, surfactants and water; (2) application of shearing stress to the lamellar phase and; (3) dispersion of the sheared lamellar phase in an aqueous medium to suspend the vesicles. In our optimized procedure (Figure 1B), all components were mixed (phospholipids, surfactants and eventually the API and CH), left to rest for 24 h at 37 °C and hydrated with water for a further 24 h at 37 °C (step 1), without organic solvents. Samples were then transferred onto the static sample stage of a cone-plate rheometer and sheared (y = 10 s' ¹ ) for 10 min (step 2) at 37 °C. The vesicles were finally diluted in an isotonic medium (dextrose 5%, w/v) by gentle shaking (step 3).

Table 2 shows the mean hydrodynamic diameter (dh, nm) and ^-potential (mV) of the screened spherulite formulations. Overall, Figure 2A shows that PC spherulites (F8, F10 and F15, Table 1) exhibit larger dh (nm) than spherulites prepared with DOPE (F16 and F24, Table 1). Aside from F15, all formulations had a negative ^-potential (mV) value in 5% dextrose (Figure 2B).

Figure 2C-E display the typical Maltese crosses of PC- and DOPE-based spherulites that are visible under polarized light microscopy. Cryo-scanning electron microscopy (cryo-SEM, Figure 2F-K) and cryo-transmission electron microscopy (cryo-TEM, Figure 2L-N) analyses confirmed the onion-like structures of the vesicles. It should be noted that cryo-SEM showed bigger particles than cryo-TEM as the latter only detect particles with sizes smaller than 1 pm.

Among all, F8, F10, F14-16, F21 and F24 exhibited the best size distribution and appropriate morphology and were therefore selected for the loading with (OEG2)2lP4.

Table 2. Mean hydrodynamic diameter (dh, nm) and ^-potential (mV) of the spherulite formulations. Formulations in bold were selected for the API loading. Mean ± s.d. (n=3).

Example 2: Loading of spherulites with (OEG ₂ ) ₂ IP4

To encapsulate (OEG2)2lP4, the latter was added as an aqueous solution in step 1 (Figure 1B). The loaded spherulites were separated from the unloaded API by dialysis (using a Float- A-Lyzer device) against 5% dextrose, then dissolved with MeOH/water (99/1 , v/v) and the LE of (OEG2)?IP4 was measured by liquid chromatography-charged aerosol detection (LC-CAD) (Figure 9). The LE was first assessed with F8 (F8a-8d . Table 3) at different API concentrations, and the one providing the highest LE (469 mg/mL) was used in subsequent formulations. LEs ranged from ~ 20 to 53%, as shown in Table 3. Table 3. Preparation parameters and LE% of the (OEG2)2lP4-loaded spherulites. Data are shown as the mean ± s.d. of three independent batches.

8a 35.0 286 25.5 + 1.6

8b 35.0 429 27.9 + 3.6

8c 35.0 571 22.7 + 2.2

8d 32.7 469 30.9 + 5.9

10 32.7 469 37.3 + 6.7

14 32.7 469 33.0 + 6.6

15 32.7 469 53.3 + 5.4

16 32.7 469 27.9 + 4.6 27 32.7 469 19.6 + 2.1

24 32.7 469 29.3 + 3.4

*Concentration of (OEG2)?IP4 in the aqueous phase added to the I ipid/su rfactant mixtures. F8a- 8c possess the same lipid/surfactant composition, but different (OEG2)?IP4 amounts (F8, Table 1 ). ExF8d corresponds to the optimized formulation. Formulations in bold were selected for the next physicochemical characterization studies and in vitro experiments.

Overall, F8d, F10, F15, F16 and F24 showed high (OEG2)2lP4 loadings, and were, therefore, selected for further characterization and in vitro release kinetics. F15 displayed the highest LE (53.3 ± 5.4%, Table 3). This might be due to the positive ^-potential value (Figure 2B) that allows the establishment of electrostatic interactions with the negatively charged (OEG2)2lP4. Figures 3A and 3B show the dh and ^-potential of the selected formulations, respectively. The inclusion of the API, did not seem to influence the dh, with an exception of F15 and F16 which showed a larger vesicle size (550 i/s 819 nm and 385 vs 564). Furthermore, all the spherulite formulations displayed negative ^-potential values after the (OEG2)2lP4 loading. Polarized light images (Figures 3C-E) revealed the typical Maltese crosses of (OEG2)2lP4-loaded spherulites, whereas cryo-EM micrographs confirmed the retention of the onion-like structure of the vesicles after the drug entrapment (Figures 3F-N).

The morphological change of the (OEG2)2lP4-loaded DOPE-based spherulites (F16 and F24, Table 3) was investigated over time by cryo-TEM (Figure 4) and polarized light microscopy (Figure 10) and compared to that of the (OEG2)2lP4-loaded DOPC-based vesicles (F15, Table 2). As expected, F15 showed the presence of intact spherulites (blue arrow) at all the time points (Figure 3A-C) with no significant change in the vesicle structure over time. This is due to the property of DOPC to form stable bilayers. By contrast, F16 showed a lamellar disruption of some vesicles (black arrow, Figure 4D), 1 h after step 3 (Figure 1B). After 6 h (purification step), spherulites (blue arrow) started to disappear (Figures 4E and F and Figure 11). The spherulite structure collapsed (red arrow) and sponge-like nanoparticles were observed (yellow arrow). After 5 days, spherulites completely lost their structural organization and perforated smaller vesicles appeared (Figure 4G). This reflects the transformation pathway of the vesicular bilayer membranes into sponge-like structures. Such a phenomenon might be due to the intrinsically unstable nature of DOPE lamellae that are destabilized once the surfactant diffuses out of the membrane, forming inverted hexagonal lipid phases. F24 displayed only a few spherulitic vesicles 1 h after step 3 (Figures 4H and I). Sponge-like structures were also detected. After 6 h and 5 days, perforated smaller vesicles and spongelike structures totally replaced spherulites (Figures 4J-M). These results were confirmed by the polarized light micrographs (Figure 10). Maltese crosses of F16 and F24 started to disappear after 6 h and they were not detected anymore after 5 days. By contrast, F15 displayed stable Maltese crosses at all the time points.

Spherulites have been claimed to possess higher LE than liposomes prepared by conventional processes. This can be explained by the small volume of the aqueous phase needed in the hydration step (step 1), and its near-complete conversion into the spherulite structure. This is in contrast to the other passive loading procedures used to incorporate hydrophilic drugs into liposomes, where usually high volumes of aqueous phase are required for lipid-film rehydration, and only a small fraction of the drug gets entrapped into the liposomal core. Remote loading procedure such as those based on transmembrane pH-gradients, while extremely efficient to load ionizable low MW drugs into liposomes cannot be applied to (OEG2)2lP4 due to its non-permeability across phospholipid membranes. The LE achieved with spherulites was then compared to that of liposomes prepared with the same starting materials as formulation F8d (Table 4 and Table 3) using three different procedures: ethanol injection, thin-film hydration and reverse-phase evaporation (REV). A fraction of the multilamellar vesicles (MLVs) produced by thin-film hydration and REV methods was also subjected to an extrusion step in order to generate the large unilamellar vesicles (LUVs). The dh, size distribution, polydispersity index (PDI) and LE of liposomes were characterized by DLS and LC-CAD by following the same protocol as (OEG2)2lP4-loaded spherulites (Figure 12). MLVs produced by ethanol injection had the smallest size and PDI, without needing the extrusion step (Figure 12A-C), but also the lowest LE (Figure 12J). Liposomes formed by thin-film hydration displayed larger size and PDI before extrusion (Figure 12D-F) than those prepared by REV. Liposomes formulated by REV showed the highest LE (Figure 12J), and were therefore compared to the spherulite formulations. In fact, REV (Figure 13) is a process that is generally yielding higher LE due to the high fraction of the aqueous phase that gets entrapped within the vesicles. The dh of both unloaded and (OEG2)2lP4-loaded MLVs prepared by the REV ranged from 200 to 400 nm, while PDI values were higher than 0.4 (F8d, F10, F15, F16 and F24), reflecting large size distributions (Figure 14). After extrusion, the dh decreased to 150-180 nm as well as PDI reached values lower than 0.2 in all the formulations (Figure 15). Cryo-TEM micrographs of F8d prepared by REV revealed the presence of MLVs and LUVs after the extrusion step (Figures 5B and C, respectively). F16 lead LUVs by REV, even prior to the extrusion step (Figure 5D). More importantly, as shown in Figure 5A (with the exception of F8d) all spherulites were associated with significantly higher LE than liposomes (both LUVs and MLVs), reaching values up to 12.5-fold higher for the DOPE vesicles (F16).

Example 2A: Addition of PEG-DSPE to DQPE/P20-based spherulites

DOPE (38 mg), P20 (27 mg) and 1 , 2-Distearoyl-sn-glycero-3-phosphoethanolamine- Poly(ethylene glycol) (PEGylated-DSPE; DSPE- PEG ) (PEG MW: 2 kDa, amount range of 0.3-2 mg) were precisely weighed, mixed with a spatula and left to rest at 37° C for 24 h. Next, 15 mg of INS-3001 (formula He) in 32 pL of Milli-Q water were added to the lipid mixture for 24 h at 37°C. Samples were transferred onto the static sample stage of a cone-plate rotational rheometer. INS-3001 -loaded spherulites were obtained by applying a shear stress for 10 min at 37° C, using a shear rate of y = 10 s-1 with a HAAKE RheoStress 600 device (Thermo Electron GmbH, Karlsruhe, Germany) mounted with a C 35/2 Ti cone and equipped with a HAAKE GH-D1 apparatus and Universal Temperature Controller (Thermo Electron GmbH). INS-3001 /spherulites were finally re-suspended with 0.8 mL of 5% (w%) dextrose solution and purified with the Float-A-Lyzer (MWCO 100 kDa, 1 mL) against 20 mL of D(+) glucose solution for 5 h. The loading efficiency (LE) was checked by UPLC-CAD as previously described.

The results are shown in Fig. 19.

Example 3: In vitro drug release of (OEG?)?IP4/spherulites

In vitro release kinetics experiments were carried out in side-by-side diffusion cells to investigate the ability of spherulite formulations to release (OEG2)2lP4 in a sustained fashion (Figure 6). This kind of in vitro set-up, while useful to assess any burst release, cannot, reliably mimic in vivo conditions (lack of enzymatic degradation, migration of vesicle from the injection site to the bloodstream, etc.). It should, therefore, be interpreted with caution. It was decided not to separate the free (OEG2)2lP4 from the loaded one, to keep the preparation process as simple as possible and to avoid the kinetic destabilization of the DOPE formulations that can occur during the purification step. As expected, the release observed during the first 4 h corresponded to the diffusion of the unloaded compound in the acceptor chamber. From 6 to 96 h, the release rate from PC spherulites was 0.21 h' ¹ which was slower than that from DOPE vesicles (0.33 and 0.32 h' ¹ for F16 and F24, respectively). Only F16 and F24 fully released its content within 4 days (Figure 6). The physicochemical stability of (OEG2)2lP4/spherulites was also assessed over one week under the same experimental conditions (Figure 16). The mean vesicle size of F10 significantly decreased compared to F8d, which might be due to the lack of CH that usually stabilizes bilayers. More interestingly, the ^-potential net value of F16 and F24 increased significantly over time, approaching the values of the unloaded vesicles (Figure 2B and Figure 16) and suggesting the progressive release of the (OEG2)2lP4.

Example 4: Pharmacokinetics of (OEG2 lP4/spherulites in rats

The impact of the encapsulation of (OEG2)2lP4 on its pharmacokinetics (PK) was investigated in healthy Sprague Dawley rats (Figure 7). Before injecting the formulation, it was verified in cell culture that the vesicles did not induce any cytotoxicity (Figure 17). The rats received 10 mg/kg (OEG2)2lP4 via the s.c. route. No treatment-dependent clinical signs were observed at the injection site during the observation time (up to 5 days). The PK profiles of the spherulite formulations were compared to that of the solution of (OEG2)2lP4 (Figure 7). The spherulites were diluted (Figure 1 B, step 3) just prior to their injection to avoid the premature destabilization of the vesicles, and the free (OEG2)2lP4 was therefore not separated from the API-loaded vesicles. This is also a mean to provide a bolus dose of (OEG2)2lP4. In a preliminary screening, F8d and F10 (formulations based on Lipoid E80) were injected in only 2 rats (Figure 18). These formulations that are expected to be more stable than F15 due to the presence of lipid with a high phase T _m (+ 26 vs. - 16.5 °C, respectively) provided lower plasma levels than the control solution. Indeed, the calculated areas under the plasma concentration vs. time curve (AUCs) were 44 and 55% (F8d and F10, respectively) lower than that of the free (OEG2)2lP4, which likely corresponded to the fraction of the unloaded compound (Table 2). These data suggest that the release of the encapsulated (OEG2)2lP4 from F8d and F10 vesicles in vivo was extremely slow leading to undetectable concentrations. Subsequently, F16 and F24 were selected for their ability to completely release (OEG2)2lP4 in vitro within a few days, whereas F15 was also chosen for its higher LE and the presence of lipid (DOPC) with low phase T _m (Figure 7A).

Compared to the control solution, F15 did not increase the (OEG2)2lP4 AUC (Figures 7B and C) nor did it lead to sustained plasma concentration of the API. In fact, the compound could be detected only up to 8 h after injection (Figure 7D). By contrast, the two DOPE-based formulations provided 3.1- and 2.6-fold higher AUCs (F16 and F24, respectively) than the control (OEG2)2lP4 solution, with F16 displaying the highest systemic exposure (Figures 7B and C). The higher AUCs obtained with formulations F16 and F24 compared to F15 might result from the reduced drug metabolism at the injection site and gradual destabilization of spherulites. In addition, the decrease in size of F16 and F24 vs. F15 over time (Figure 4) might allow the migration to the systemic circulation via the lymphatic system and impact the AUC. This has been observed for instance upon the s.c. and intraperitoneal administration of liposomes. Furthermore, compared to the (OEG2)2lP4 solution, the animals treated with F16 exhibited measureable drug concentrations in the blood up to 3 days after injection (Figure 6D). The better performance of F16 over F24 could be related to its higher propensity to be destabilized over time, as shown by the cryo-TEM images (Figure 4). Further experiments should be performed to improve the loading as well as the destabilization kinetics of DOPE spherulites and to characterize the drug release mechanism from the vesicles in vivo.

Example 5: Conclusion

This research established a subcutaneous lipid technology for the sustained release of small hydrophilic compounds that are dosed in the milligram range (/.e., (OEG2)2lP4). The system is based on spherulites made of DOPE and a cone-shaped surfactant, such as P20. Despite its high hydrophilicity, this compound was passively entrapped into spherulites with high yields for a vesicular system. Spherulites were spontaneously destabilized upon dilution, displaying a vesicle-to-sponge transition by cryo-TEM images. Such a phenomenon might be due to the diffusion of the surfactant out of the bilayer, triggering the DOPE lamellar-hexagonal phase transition, thereby promoting the sustained release of the encapsulated compound. After s.c. injection in rats, it was shown that DOPE spherulites increased the systemic exposure of (OEG ₂ ) ₂ IP4 to 3.1 -fold.

Example 6: Material and Methods

Materials

Trehalose (D-(+)-trehalose dihydrate; 99%) was purchased from Aber GmbH (Karlsruhe, Germany). Dextrose (D-(+)-glucose anhydrous) and polysorbate 80 (P80) were purchased from AppliChem GmbH (Darmstadt, Germany). Kolliphor HS15 (HS15) was purchased from BASF ChemTrade GmbH (Burgbernheim, Germany). 1 ,2-Dioleoyl-sn-glycero-3- phosphatidylcholine (DOPC) and 1 ,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) were purchased from Cayman Chemical (Ann Arbor, USA). (OEG ₂ ) ₂ IP4 was kindly provided by Inositec AG (Zurich, Switzerland). Lipoid E 80 (LE80, egg phospholipids with 80% phosphocholine) was purchased from Lipoid AG (Ludwigshafen, Germany). Nanopure Milli-Q water was deionized and purified with a Synergy UV water purification system by Merck Millipore (Darmstadt, Germany). Chloroform (> 99.8% stab, amylene), diethyl ether (99.5%, stab, butylhydroxytoluene for analysis), ethanol (EtOH, absolute 99.8% for analysis), cholesterol (CH, Sigma Grade 99%) and polysorbate 20 (P20) were all purchased from Sigma- Aldrich (St. Louis, MO, USA). Acetonitrile (AcCN, Optima LC/MS grade), formic acid (FA, Optima LC/MS grade), methanol (MeOH, Optima HPLC-MS) and phosphate-buffered saline (PBS, 1X, pH 7.2) were from Thermo Fisher Scientific (Waltham, MA, USA).

Cell culture conditions

Primary adult human dermal fibroblasts (HDFa) were kindly provided by Dr. Hans-Dietmar Beer (Department of Dermatology, University Hospital Zurich, Zurich, Switzerland) and tested for mycoplasma contamination before the experiments. Cells were cultured in high glucose DMEM (GlutaMAX™, supplement pyruvate, Gibco, Bleiswijik, The Netherlands) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) at 37 °C in a humidified atmosphere with 5% CO ₂ . Cells were grown to 70-80% semi-confluence, washed with 1X PBS pH 7.4 (Gibco) and detached with 0.25% trypsin-EDTA (Gibco) for 5 min at 37 °C. HDFa were treated with (OEG ₂ ) ₂ IP4-free or loaded spherulites as well as (OEG ₂ ) ₂ IP4 alone, at several time points and concentrations. All experiments contained untreated cells (either PBS or 5% glucose) processed in parallel as negative controls. Animals

In healthy animals, PK studies were performed by the contract research organization Pharmacelsus GmbH (Saarbrucken, Germany). In this study, (OEG2)2l 4 was administered as a saline solution (control) or loaded into spherulite formulations. Healthy male Sprague Dawley (SD) rats (~ 250 g at delivery and purchased from Janvier Labs, Le Genest-Saint-lsle, France) were housed in a temperature-controlled room (20-24 °C) and maintained in a 12 h light/12 h dark cycle on Tapvei GLP Aspen Bedding (WH-4K. Altromin, Germany). Food (1324 TPF maintenance diet for rats and mice, Altromin, Germany) and tap water were available ad libitum. All experimental procedures were approved by and conducted in accordance with the regulations of the local Animal Welfare authorities (Germany, approval number GB 4 2.4.2.2 18/2021 , Landesamt fur Gesundheit und Verbraucherschutz, Abteilung Lebensmittel- und Veterinarwesen, Saarbrucken).

Preparation of spherulites

Different ratios of phospholipids, surfactants and eventually CH were weighed and mixed with a spatula into an Eppendorf Tube® (Table 1 ) and left to rest at 37 °C for 24 h. The total amount of materials was 65 mg. Next, the mixtures were hydrated with 35 or 32 pL of Milli-Q water and left for a further 24 h at 37 °C. Samples were then transferred onto the static sample stage of a cone-plate rotational rheometer (HAAKE RheoStress 600 device, Thermo Electron GmbH, Karlsruhe, Germany). Spherulites were obtained by applying a shear stress for 10 min at 37 °C, using a shear rate (y) of 10 s' ¹ . The rheometer was equipped with a C 35/2 Ti cone and a HAAKE GH, D1 and Universal Temperature Controller (Thermo Electron GmbH). Afterward, the spherulites were diluted in 1 or 0.8 mL of Milli-Q water or 5% dextrose solution (w/v) by gentle shaking. Three independent batches of all formulations were prepared.

Preparation of (OEG2)2lP4-loaded spherulites

Different ratios of phospholipids, surfactants and eventually CH were weighed and mixed with a spatula into an Eppendorf Tube® (Table 4) and left to rest at 37 °C for 24 h. The total amount of materials was 65 mg. Next, the mixtures were hydrated with 35 or 32 pL of Milli-Q water where 10, 15 or 20 mg of (OEG2)2lP4 were previously dissolved and left for a further 24 h at 37 °C. Samples were then transferred onto the static sample stage of the cone-plate rheometer. (OEG2)2lP4 -loaded spherulites were obtained by applying shear stress as described above and dispersed with 0.8 mL of 5% dextrose. The free (OEG2)2lP4 was purified from the loaded spherulites by using the Float-A-Lyzer device (MWCO 100 kDa, 1 mL). The suspension (0.8 mL) was added to the Float-A-Lyzer and dialyzed against 20 mL of 5% dextrose, under magnetic stirring at 25 °C for 5 h. The medium was replaced every hour with the fresh dextrose solution. Then, 10 pL of the purified (OEG2)2lP4/spherulites suspension were added to 1 mL of MeOH/H2O (99/1 , v/v) solution, and injected into the LC-CAD apparatus to quantify the amount of loaded (OEG2)2lP4. Three independent batches of all formulations were prepared.

Table 4. Chemical composition of liposomes used in comparison experiments.

Preparation of (OEG2)2lP4 -loaded liposomes

LUVs and MLVs (Table 1 ) were prepared by following three different approaches, i.e. ethanol injection, thin-film formation and REV.

Ethanol injection

Phospholipids and surfactants (Table 2) were dissolved in 0.4 mL EtOH and subsequently added drop by drop into a glass vial, under magnetic stirring, containing 0.6 mL 5% dextrose (unloaded liposomes) or 3 mg of (OEG2)2lP4 dissolved in 5% dextrose ((OEG2)2lP4 -loaded liposomes). The volume ratio of organic to aqueous solvent was kept constant at 40:60 (v/v).

Thin-film formation

Liposomes were prepared as described elsewhere ⁴⁵ . Phospholipids and surfactants were dissolved in chloroform (Table 2) and the latter was removed by rotary evaporation (Buchi Labortechnik AG) at 45 °C. Subsequently, the lipid film was dried in vacuo. The addition of either 5% dextrose (unloaded liposomes) or 3 mg (OEG2)2lP4 dissolved in 5% dextrose ((OEG2)2lP4-loaded liposomes) was followed by vortexing (Heidolph Instruments GmbH & Co., Germany). To obtain LUVs with a homogeneous size distribution, the aqueous suspension containing MLVs was extruded under N2 atmosphere with an extruder connected to a water bath set at 30 °C. Two stacked polycarbonate (PCTE) membrane filters with a pore size of 0.2 pm (Sterilitech, Auburn, AL, USA) were used and the extrusion was repeated 9 times.

REV

Phospholipids and surfactants (Table 2) were dissolved in chloroform in round-bottom flasks and the solvent was removed with a rotary evaporator (Buchi Labortechnik AG, Flawil, Switzerland) at 45 °C under reduced pressure. Phospholipid films were further dried in a vacuum for 1 h. Next, the films were re-dissolved in 3 mL of diethyl ether before adding either 5% dextrose (unloaded liposomes) or 3 mg (OEG2)2lP4 dissolved in dextrose 5% ((OEG2)2lP4- loaded liposomes). The ratio of organic to aqueous solvents was always maintained at 3:1 (v/v). The obtained two-phase system was ultra-sonicated (Sonorex sonication bath, Bandelin, Berlin, Germany) at a temperature below 10 °C until the mixture became a homogeneous, turbid dispersion (5-20 min). Subsequently, the organic solvent was removed by rotary evaporation at 25 °C under reduced pressure, leading to the aqueous suspension containing MLVs. LUVs were produced by extrusion as described above. Three independent batches of all formulations were prepared.

Characterization of the vesicles

The dh (nm), size distribution and ^-potential (mV) of the vesicles were measured with a Malvern ZetaSizer Advance Pro (Instrumat AG, Renens, Switzerland), at a scattering angle of 179° at 25 °C. Polarized light microscopy images of spherulites were taken with the Leica Fluorescent microscope (CTR6000, Leica Camera AG, Wetzlar, Germany). Five pL of 50 mg/mL spherulite suspension in 5% dextrose was added onto the microscope slide, covered with a coverslip (Thermo Scientific, Gerhard Menzel B.V. & Co. KG, Braunschweig, Germany) and images were recorded at 40x magnification. The shape and structure of spherulites were visualized with cryo-TEM and cryo-SEM. For cryo-TEM images, lacey carbon-coated 300- mesh copper grids (EMS, Hatfield, PA) were glow discharged for 30 s (Emitech K100X, Quorum Technologies Ltd., Laughton, U.K.). An aliquot (3.7 pL) of sample in water (50 mg/mL) was applied onto the grids in a Vitrobot Mark II (Thermo Fisher Scientific), and the sample excess was removed by controlled blotting at 95% humidity. A mixture of liquid ethane/propane was used for sample vitrification. The grids were then transferred on a Gatan cryo-holder (AMETEK, Pleasanton, CA, USA) into a Tecnai F20 cryo-TEM (Thermo Fisher Scientific) and kept at -180 °C during observation. Micrographs were recorded under low dose conditions (<20 e-/A2) using a Falcon II 4K camera (Thermo Fisher Scientific) at the acceleration voltage of 200 kV in bright field mode. For cryo-SEM images, 5 pL samples were added into 6-mm aluminum dishes, frozen in a high-pressure freezer HPM 100 (Bal-Tec/Leica, Austria) and successively stored in liquid nitrogen. Vitrified specimens were mounted on the cryo-holder and transferred into a precooled (-130 °C) freeze-fracturing system BAF 060 (Bal-Tec/Leica, Austria) at 1.10' ⁴ Pa. The coating was made with 3 nm tungsten at an elevation angle of 45° followed by 3 nm at 90°. The transfer to the precooled cryo-SEM was done under a high vacuum (< 5 x 10“ ⁴ Pa) with an air-lock shuttle. Cryo-SEM micrographs were recorded with a field emission SEM Leo Gemini 1530 (Carl Zeiss, Germany) equipped with a cold stage to maintain the specimen temperature at -120 °C (VCT Cryostage, Bal-Tec/Leica). Inlens-SE- and Everhart-Thornley SE-signals were used for image acquisition at the acceleration voltage of 2 kV. Quantification of (OEG2)2lP4

(OEG2)?IP4 was quantified with a Dionex Ultimate™ 3000 Ultra-High-Performance liquid chromatography (UHPLC) system (ThermoFisher Scientific) equipped with an auto-sampler and coupled to a charged aerosol detector (CAD, Corona Veo, ThermoFisher Scientific). Data were recorded and processed with Chromeleon 7 software. HILIC stationary phase (Luna Omega 3 pm, Sugar 100 A, 150 x 2.1 mm, Phenomenex, Germany) was used and the column temperature was set at 25 °C. The mobile phase consisted of the gradient made of 0.1 % (v/v) formic acid (FA) in Milli-Q water (solvent A) and 0.1 % (v/v) FA in acetonitrile (AcCN) (solvent B), at the flow rate of 0.8 mL/min (Figure 9A). The (OEG2)2lP4 calibration curve was established at the concentration range of 30-500 pg/mL (Figure 9B), by injecting 4 pL of samples in MeOH/H2O (99/1 v/v). D (+) trehalose in MeOH/H2O (250 pg/mL) was used as internal standard (i.s.). The sensitivity of the method was measured in terms of the lower limit of detection (LLOD) and limit of quantification (LOQ) (Figure 9C). The LE, % was calculated with equation 1 : 100 Eq. 1

In vitro release studies

The release of (OEG2)2lP4 from spherulites was monitored for 4 days with the side-bi-side (SBS) diffusion cell (PermeGear, Hellertown, PA, USA). SBS cells made of two half-cells with an equal chamber volume of 3.4 mL and an orifice diameter of 9 mm were separated by a 0.2- pm PCTE membrane filter (Sterilitech). The unpurified (OEG2)2lP4/spherulite suspension (0.8 mL) was added to the donor chamber and both chambers were filled up to full volume (3.4 mL) with PBS (pH 7.4). The chambers were stirred with a magnetic stirrer and connected to a water heating jacket that ensured circulation of water at 37 °C. At predefined time points, 3 mL were sampled from the acceptor chamber, and fresh PBS was added to replace the removed volume. Samples were first solubilized with MeOH/H2O (99/1 , v/v) by using appropriate dilution factors (df) and, successively, the released (OEG2)2lP4 was assayed with LC-CAD, as described above. All release experiments were conducted in triplicate.

Cell viability assay

MTS Test: 200 pL HDFa (2,500 cells per well in complete DMEM) was seeded in a 96 well plate (sterile and flat bottom polystyrene microplates, TPP, Switzerland) and incubated for 24 h at 37 °C. Cell monolayers were then added by 2 pL of free or (OEG2)2lP4-loaded spherulites as well as free (OEG2)2lP4 in 5% dextrose, at specific final spherulite concentrations (ranging from 12.5 to 200 pg/mL, df: 1 :2) and (OEG2)2lP4 concentrations (ranging from 6.3 to 50 pg/mL, df: 1 :2), and incubated for 4 and 24 h. As a negative control, cells received either 2 pL 5% glucose or PBS. Then, the medium was removed, cells were gently washed with PBS and 100 pL complete DMEM was added. Twenty pL MTS solution (Promega, Switzerland) was added and HDFa were incubated for 1 h at 37 °C. Absorbance was recorded at 490 nm, using a Tecan microplate reader (Infinite M200, Austria). Results were processed using i-control 2.0 Software. Three independent batches of all formulations were tested.

PK studies in rats

Surgery. Rats were catheterized 3 days before the experiments. For this purpose, the animals were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg), and the right jugular vein was cannulated with silicone tubing. The catheter was tunneled subcutaneously, exteriorized in the neck region and connected to a vascular access harness (Instech Solomon, USA). For analgesia, rats were treated twice with carprofen (Rimadyl, 1 mL/kg, 5 mg/kg s.c.) directly after induction of anesthesia for catheter surgery and the following day. Catheters were rinsed daily with 50 pL Li-heparin solution (500 lE/mL).

Pharmacokinetics. The body weight of the animals was determined before s.c. application of the formulations. All samples were freshly prepared on the day of the in-life phase and rats were administered s.c. 10 mg/kg of (OEG2)2lP4. The compound was given as saline solution or formulated in spherulites (n = 3). Blood was collected at 0.5, 2, 4, 8, 24 and 48 h (saline solution) or 0.5, 2, 4, 8, 24, 72 and 120 h (spherulites) post-injection. Aliquots of 100 pL Li- Heparin blood were obtained from the vascular access harness. Samples were stored on ice and subsequently centrifuged at 3000 x g for 10 min at 4 °C. Plasma was prepared within 45 min after sampling and was kept at -20 °C until being assayed. AUCs were calculated using the linear-log trapezoidal method (linear up. log down), from 0 to 72 h.

Sample preparation. Working solutions for calibration and quality control (QC) samples were prepared as follows. A stock solution of (OEG2)2lP4 (1 mg/mL in 20% MeOH) was diluted with 10% MeOH to a final concentration of 500 pg/mL (starting solution). Further working solutions were prepared by dilution of the starting solution in 10% MeOH and were used for the preparation of calibration standards and QCs by spiking 25 pL blank plasma with 2.5 pL of the corresponding working solution. A volume of 25 pL of each sample was spiked with 2.5 pL 10% MeOH. The mixtures were vortexed and 5 pL 10 % MeOH containing the i.s. ((OEG2)2lP4- D6, 46 pg/mL) was added to each sample, whilst 5 pL MeOH 10% was added to all blanks. After vortexing, 25 pL of 10% trichloroacetic acid were added to all samples, which were subsequently vigorously shaken and centrifuged for 5 min at 6,000 x g and 20 °C. Finally, samples were injected into LC-mass spectrometry (LC-MS) for the (OEG2)2lP4 detection and quantification.

LC-MS analysis. Concentration data were extrapolated with Chromeleon™ 7.2 SR5 MUf software (Thermo Fisher Scientific Inc., Germany) and quantified using the ISTD method. The system was calibrated using a quadratic regression as a mathematical model with a 1/X ² or a 1/Y ² weighting for improved accuracy. LC-MS system consisted of a Vanquish quaternary pump with a Vanquish column compartment and a Vanquish Split sampler (Thermo Fisher Scientific, USA). MS was performed on a Q-Exactive mass spectrometer (Orbitrap™ technology with accurate mass) equipped with an H-ESI (heated electrospray interface, Thermo Fisher Scientific, USA). Analyses were performed with a Poroshell 120 EC-C18 stationary phase (2.7 pm, 150 x 2.1 mm, Agilent Technologies, Germany) equipped with a C6- Phenyl (4 x 2.0 mm) pre-column. The mobile phase consisted of a gradient of AcCN as organic phase (A) and 0.01 % ammonium hydroxide in water as aqueous phase (B), at the flow rate of 0.3 mL/min. An optimized MS tune file was used and the [M+H] ⁺ ion of the diisooctyl phthalate (m/z 391.28429) was taken as a lock mass for internal mass calibration. For the detection of (OEG2)2lP4, the MS was operated in the PRM negative mode. Further settings were as follows: max. injection time 200 ms, sheath gas 40, auxiliary gas 10, sweep gas 2, spray voltage 4 kV, capillary temperature 350 °C and heater 350 °C. The lower limit of quantification (LLOQ) was set to 10 ng/mL. The selectivity expressed as signal-to-noise ratio was > 5.

Statistical analysis

Data analysis was carried out using Prism (GraphPad, version 9.3.1 ). Assuming a normal distribution of analyzed datasets, statistical tests include one-way analysis of variance (ANOVA) followed up with Tukey’s multiple comparisons test. The p-value threshold for all analyses was set as 0.05 and values above this threshold are indicated as not significant (ns).

Spontaneous lipid curvature

The lipid geometry can be expressed by the critical packing parameter (CPP). CPP is related to the hydrocarbon chain volume (v) and length (I) and the interfacial area occupied by the polar head group (a) of lipids. Specifically, CPP is calculated by the following equation:

The volume difference between head group and tail blocks can be thought of as a measure of the lipid spontaneous curvature. The spontaneous curvature of a monolayer is dictated by a packing of the lipids in the membrane, i.e., the interaction of a lipid with its surrounding molecules. Thus, the spontaneous curvature of a monolayer may slightly differ in a bilayer membrane and a monolayer at an oil-water interface because the oil chains may differently interdigitate into the hydrophobic part of the monolayer than in the case of a bilayer. Lipids with large head groups and small tails (cone-like shaped, positive curvature, CPP < 1 ) will prefer to self-assemble into convex structures, such as micelles, whereas lipids with comparable volumes of their head group and tail blocks (cylindrical lipids, CPP ~ 1 ) will form planar bilayers or large vesicles, and lipids with small head groups and large tails (inverted cone-like, negative curvature, CPP > 1 ) will yield inverted aggregates, such as the inverted hexagonal phase.

Different approaches (i.e., X-ray diffraction and osmotic stress) can be used to measure the spontaneous curvature of lipids and their membranes. These methods and techniques are disclosed in the following works:

J. Sodt, R. W. Pasto, Bending Free Energy from Simulation: Correspondence of Planar and Inverse Hexagonal Lipid Phases, Biophys. J., 104, 2202, 2013

LA. Barragan Vidal, M. Muller, Generalization of the swelling method to measure the intrinsic curvature of lipids, J. Chem. Phys. 147, 224902, 2017

R. P. Rand, N. L. Fuller, S. M. Gruner and V. A. Parsegian, Membrane Curvature, Lipid Segregation, and Structural Transitions for Phospholipids under Dual-Solvent Stress, Biochemistry, 29, 76, 1990.

C. Hamai, T. Yang, S. Kataoka, P.S. Cremer, S. M. Musser, Effect of Average Phospholipid Curvature on Supported Bilayer Formation on Glass by Vesicle Fusion. Biophys. J. 90, 1241 , 2006.

Cited prior art documents:

Diat, O.; Roux, D. Preparation of Monodisperse Multilayer Vesicles of Controlled Size and High Encapsulation Ratio. J. Phys. II 1993. https://doi.Org/10.1051/ip2:1993106.

Crauste-Manciet, S.; Khawand, K.; Mignet, N. Spherulites: Onion-like Vesicles as Nanomedicines. Therapeutic Delivery. 2015. https://doi.org/10.4155/tde.14.81.

PCT/EP2012/004088 (US9358243B2)

PCT/EP2016/080657 (US10624909B2)

PCT/EP2019/074986 (US2021347793A1 )

PCT/EP2016/080545 (US10487097B2)

WO2020157362A1 (Sanifit Therapeutics)

WO2021094331 A1 (Sanifit Therapeutics)