WO2014056939A1

WO2014056939A1 - Lipidic biomaterials for encapsulation and triggered release

Info

Publication number: WO2014056939A1
Application number: PCT/EP2013/070976
Authority: WO
Inventors: Ehud Moshe LANDAU; Jay Steven SIEGEL
Original assignee: Zurich Universitaet Institut fuer Medizinische Virologie
Current assignee: Zurich Universitaet Institut fuer Medizinische Virologie
Priority date: 2012-10-08
Filing date: 2013-10-08
Publication date: 2014-04-17
Anticipated expiration: 2015-04-08

Description

Lipidic Biomaterials for Encapsulation and Triggered Release

Field of the invention

The present invention relates to designed, lipid-based, biocompatible, stable, and transparent materials that can encapsulate active compounds such as drugs, and release them at will by chemical and/or photochemical triggering.

Background of the invention

Lipid materials used in drug delivery

Lipidic cubic phases (LCPs) possess the material properties described in section (c). They can be produced by mechanical mixing of the hydrophilic component (typically water or aqueous solution) with the lipophilic component (typically lipid or mixture of lipids) under conditions (composition, temperature) that have been established by independent physical experiments (phase diagram). Such mixing can be accomplished by centrifugation or by mixing in special syringes. Once formed, LCPs are stable, and can be stored and used at the temperature/ hydration range given by the respective phase diagram. Because of their unique chemical and physical nature, most notably the availability of both hydrophilic compartments and hydrophobic lipid bilayers, and the structured arrangement thereof, LCPs can incorporate various guest molecules (organic, inorganic, biological) with varied properties such as size, charge, and structure, and retain them in their active state. Diffusion in and out of hitherto known LCPs is passive, and can be rationalized in terms of effects of the lipid molecular structure (chain length, degree of unsaturation, position of double bonds, head group characteristics, etc.) on the LCP material structure (bilayer thickness and curvature, unit cell size, radius of aqueous compartment).

When more than one lipid is used, the minor lipid(s) is/are termed "additive(s)". Additives used in LCPs include lipids with various chain length, degree and posi- tion of unsaturation, and head group (cf. US 6,235,312). They include mono- glycerides, galactolipids, phospholipids, and mixtures thereof (US 5,753,259 and US 2004/0022820A1 ). Further, they include ionic anchor, a tether, and combinations thereof (cf. US 6,936,187): an„anchor" is defined as a small molecule which is not an amphiphile, for instance a charged surfactant, that has a head group that provides specific interactions in the head group region. A "tether" is defined as a molecule that is larger than an "anchor", whose head group functions in a similar way to that of an "anchor". Examples include polymers, polysaccharides, proteins and enzymes.

The major disadvantage of the additives used thus far in LCPs as compared with the additives in this invention is that they cannot be addressed to perform a desired function at a selected, chosen time. In contrast, our additives have been designed such that they can be addressed with chemical, specifically photochemical means, thereby affecting the properties of the host LCP so as to activate (switch) the release of incorporated active molecules.

US 6,936,187 describes release and entrapment due to change in pH, addition of salt, or introduction of dielectric solvents. The major disadvantage of this method is the fact that the binding between drug and "anchor" is only transient, and the counterions that are present easily displace the drug. In addition, compared with our invention, these have two disadvantages: (1 ) These affect the "tether" or "anchor" molecules, and not the amphiphiles, and (2) these are chemical means, which may not be applicable in vivo, as compared with our phototriggering. US 5,753,259 describes controlled release, which is in essence passive: there is no mention of triggering.

Brief summary of the invention

An object of the present invention is to provide novel and advantageous lipidic compounds, particularly for use as additives in lipidic cubic phase materials. Another object of the present invention is to provide novel and advantageous lipidic cubic phase materials. A further object of the present invention is to provide a novel and improved method of controlled release. These and other objects are achieved by means of the lipidic compound defined in claim 1 , by the lipidic cubic phase materials defined in claim 3 and by the method of controlled relase defined in claim 15. Advantageous embodiments are defined in the dependent claims and in the description following further below.

According to one aspect (claim 1 ), there is provided a lipidic compound, particularly for use as an additive in a lipidic cubic phase material, the lipidic compound being selected from the group consisting of the following compounds according to the Scheme found further below:

- amides: Y01 -21 , Y01 -23 (N-oleoyl-L-serine methyl ester), Y01 -17 (Dimethyl- N-oleoyl-L-glutamate), Y01 -72 (N-oleoyl-L-phenylalanine methyl ester), Y01 - 27 (Ethyl N-oleoyl-L-cysteinate);

cyclopropyl compounds: SB008 (2,3-dihydroxypropyl-8-(2-octylcyclopropyl) octanoate), No. 235 (N,N-(8-(2-octylcyclopropyl)octanoyl) bis-acetic acid),

No. 236 (N-(8-(2-octylcyclopropyl)octanoyl) glutamic acid dimethyl ester), No.

237 (N-(8-(2-octylcyclopropyl)octanoyl) sarcosine methyl ester), No. 238 (N-

(8-(2-octylcyclopropyl)octanoyl) L-serine methyl ester), No. 239 (N-(8-(2- octylcyclopropyl)octanoyl) sarcosine), No. 240 (N-(8-(2- octylcyclopropyl)octanoyl) L-serine), No. 241 (N-(8-(2- octylcyclopropyl)octanoyl) 2,3-dihydroxypropyl amine), No. 243 (N-(8-(2- octylcyclopropyl)octanoyl) glutamic acid);

- azobenzenes: SB036, SB040, SB041 , SB042, Y01 -59;

caged compounds: Y01 -89 (4,5-dimethoxy-2-nitrobenzyl oleate), Y01 -141 , SB031 (4,5-dimethoxy-2-nitrobenzyl 2-oleamido) acetate), PU251 (5-

(ethoxycarbonyl)-2-nitrobenzyl (Z)-heptadec-8-enylcarbamate), PU252 (3-

((((Z)-heptadec-8-enyl)carbamoyl)methyl)-4-nitrobenzoic acid), PU258 (5- amino-2-nitrobenzyl oleate), PU259;

esters: No. 146, No. 163 (3-amino-2-hydroxypropyl oleate);

- guanidinium compounds: Y01 -188, Y01 -190, re-SB073/Y01 -205b, re-SB074,

Y01 -195a, Y01 -199. According to an advantageous embodiment (claim 2), the lipidic compounds are selected from the group consisting of the following compounds according to the Scheme:

amides: Y01 -23 (N-oleoyl-L-serine methyl ester), Y01 -17 (Dimethyl-N- oleoyl-L-glutamate);

cyclopropyl compounds: No. 236 (N-(8-(2-octylcyclopropyl)octanoyl) glutamic acid dimethyl ester), No. 238 (N-(8-(2-octylcyclopropyl)octanoyl) L-serine methyl ester), No. 240 (N-(8-(2-octylcyclopropyl)octanoyl) L-serine), No. 243 (N-(8-(2-octylcyclopropyl)octanoyl) glutamic acid);

- caged compounds: Y01 -89 (4, 5-dimethoxy-2-nitrobenzyl oleate);

guanidinium compounds: Y01 -188, Y01 -190.

According to another aspect (claim 3), there is provided a lipidic cubic phase material, comprising a hydrophilic component and a lipophilic component, said lipo- philic component comprising a major lipophilic constituent and at least one lipidic additive, which lipidic additive comprises a hydrophilic head group and a hydrophobic tail attached thereto. In this regard, any compounds that will partition into the membrane compartment of the lipidic cubic phase will be useful. An exemplary, non-exclusive listing of compund classes is: fatty acids, alcohol derivatives from fatty acids, monoglycerides, diglycerides, lipids and their derivatives, preferably the corresponding compounds that have their acid group(s) replaced by a hydroxyl or thiol or ether or thioether group or ω-hydroxyalkenes or their ethers or homologous thiols or thioethers; monoa- cylglycerols, preferably cis monounsatu- rated monoacylglycerols, more preferably monoolein (C18: c9), monopalmitolein (C16: c9) and monovacennin (C18: c7); medium-chain length alkyl glycosides; polyalkylenglycols, poly- ethylenglycols, diacylglycerophospholipids, monoacyl- glycerophospholipids and derivatives thereof; and/or polyketides, saccharolipids, prenol lipids, sterol lipids, sphingolipids, glycerophospholipids and glycerolipids and/or derivates of lipids, in particular phosphatidylcholine (PC), in particular DOPC, phosphatidylethanolamine, in particular DOPE, phosphatidylserine, in particular DOPS, cardiolipin, lyso- phosphatidylcholine, 2-monoolein, oleamide, cholesterol, cell membrane components.

Advantageously, the hydrophobic tail comprises at least one cis double bond or cyclopropyl moiety (claim 4), which was found to be favorable with regard to LCP formation. According to a further embodiment (claim 5), the lipidic additive comprises a photochennically activatable moiety, which is particularly advantageous in applications for controlled release vehicles.

Advantageous lipidic cubic phase materials are those having a lipidic additive as defined in claims 6 to 8.

In general the major lipophilic constitutent may be any amphiphilic compound that can form a LCP with water (see, e.g. Fontell, K. Colloid & Polymer Science 1990, 268, 264-285). Advantageous embodiment regarding the major lipophilic constit- uent are defined in claims 9 to 12. Regarding claim 1 1 , it is pointed out that iso- prenoid-chained lipids are described, for example, in: Masakatsu Hato, Jun Yam- ashita and Manzo Shiono J. Phys. Chem. B, 2009, 1 13, 10196-10209.

According to another embodiment (claim 13), the hydrophilic component com- prises an aqueous solution containing a predetermined active compound. The term "active compound" shall be interpreted broadly, meaning, e.g. a drug, a prodrug or any other therapeutically or diagnostically useful compound. Accordingly, the lipidic cubic phase materials of the present invention are particularly advantageous for use in a therapeutic method (claim 14).

According to a further aspect (claim 15), there is provided a method of controlled release of a predetermined active compound, the method comprising the following steps:

forming a lipidic cubic phase material according to claim 13 by mixing said lipophilic component and said hydrophilic component comprising an aqueous solution containing said predetermined active compound; placing said lipidic cubic phase material in a region of interest;

releasing said active compound by subjecting said lipidic cubic phase material to a chemical or photochemical stimulus. In this manner, the active compound is relased in the region of interest, which will typically be a particular region in a patient's body.

According to one embodiment (claim 16), the releasing step comprises applying a photochemical stimulus generated by ultraviolet irradiation, which will lead to a rearrangement or break-up of the arrangement enclosing the active compound.

According to one embodiment (claim 17), the active compound is a DNA compound. If required, e.g. for tracking purposes, e.g. in scientific research or in diagnostics, the DNA compound may have a fluorescent or other marker.

According to another advantageous embodiment, the lipidic cubic phase material is used in the form of cubosomes, i.e. as a dispersion of small, submicron LCP particles, in which all the molecular properties of LCPs are retained. This is particularly useful, for example, for use as a vehicle for controlled delivery and/or release of drugs or other active components.

The invention concerns a novel class of designed, lipid-based, smart matrix materials which can incorporate (enclose) a broad range of guest molecules (organic, inorganic, biological) with various sizes and properties, retain them in their active state, and optionally deliver them at the required site, and at a selected, chosen time upon triggering. The guest molecules can be pharmacologically, nutritionally or cosmetically active substances. Because of the special material properties of the host matrix materials, they can be applied in a wide range of biological compartments hitherto unavailable. A significant and unique material property of this class of materials is its transparency, thereby making it ideal candidates for ocular drug delivery. These novel materials have the following set of properties: Biocompatible

Biodegradable

Stable in water or oil

Optically transparent

- Adhesive to hydrophilic as well as hydrophobic surfaces

Deformable

Loadable: can incorporate substantial amounts (mM range) of guest molecules with various polarities and charge such as hydrophobic and hydrophilic drugs, proteins, nucleic acids etc.

- Switchable: Optionally have designed, built-in switches for the control of structure-function (e.g. binding and fast release)

The combination of these properties is unique, and ensures application in a very broad spectrum of fields such as treatment of ophthalmic disorders, treatment of other medical disorders, in cosmetics, and food industry.

In the following, reference is made to monoolein (MO), which has the following molecular structure:

Moreover, reference is made to phytantriol (PHY), which has the following molecular structure:

Furthermore, reference is made to the additives listed in the enclosed Scheme. In the diffusion experiments described below, the following dyes were used:

Crocein Orange

Methylene Green

Brief description of the drawings

The above mentioned and other features and objects of this invention and the manner of achieving them will become more apparent and this invention itself will be better understood by reference to the following description of various embodiments of this invention taken in conjunction with the accompanying drawings, wherein: Figure 1: shows a monoolein (MO)/water phase diagram, depicting the exist- ence and structures of the various phases as function of composition and temperature (from: V. Cherezov, J. Clogston, M. Z. Papiz, M. Caf- frey, J. Mol. Biol. 357, 1605-1618 (2006)). Note the two bicontinuous LCP structures: The lower hydrated Ia3d, and the fully hydrated Pn3m phase, which can coexist with any amount of excess water.

Figure 2 shows photographs of LCP(s) with dyes, demonstrating their solid

consistency and transparency. The sample in the center is a pure monoolein/water cubic phase. The two colored samples are doped with dye. Figure 3\ shows the following diffusion experiments (absorbance vs. time):

(A) Diffusion of croceine orange in 60% LCP of MO (upper curve), and in a 60% LCP of MO with 1 % of the diamine compound 52 as additive (lower curve);

(B) Diffusion of methylene green in 60% LCP of MO with 4.1 % of the amino acid ester compound 22 as additive (upper curve), showing complete release; and 4.1 % of the amino acid compound 21 as additive (lower curve), showing binding;

(C) Diffusion of methylene green in 60% LCP of MO with 4.2% of the amino acid ester compound 25 (upper curve), showing complete release; and 4.2% of the amino acid compound 24 (lower curve), showing binding;

(D) Diffusion of methylene green in 60% LCP of MO with 4.2 % of the L-amino acid compound 24 additive in: Water (bottom curve, squares); 0.1 M HCI solution (crosses); buffer pH 2.0 (diamonds); and 0.1 M tetrabutyl ammonium bromide (TBAB) (circles). Binding is demonstrated in water, release in all other regimes;

(E) Binding and release of methylene green in 60% LCP of MO with 5 % of the tertiary amide lipid (compound 13) as additive. Lower curve: triggered release by chemical means (0.1 M HCI), indicated with the arrow). Upper curve: stepwise release by photochemical means.

Figure 4: shows the photochemistry of compound 48 (absorbance vs. time):

Photocleavage of compound 48 in CH₃CN (irradiation at 366 nm) as function of time.

Figure 5: shows the following diffusion experiments (absorbance vs. time) of methylene green in 80% LCP of MO with 5% of compound 48 as additive, demonstrating complete release without irradiation (upper curve), and binding upon UV irradiation at 366 nm (middle curve), plus a com- parison with 5.0% oleic acid as additive (bottom curve). UV irradiation at 366 nm.

Figure 6: shows the following diffusion experiments (absorbance vs. time):

(A) Diffusion of methylene green in 60% LCP of MO with 5.0 % of the diacid compound 13 as additive, in water (lower curve), showing complete binding, and in buffer, pH 2.0, (upper curve), showing complete release;

(B) Diffusion of methylene green in 60% LCP of MO with 4.9 % of the amino acid compound C10 as additive, in water (lower curve), showing complete binding, and in TBAB solution (upper curve), showing complete release;

(C) Diffusion of methylene green in 60% LCP of MO with 3.6 % of the acid compound 229 as additive, in water (lower curve), showing complete binding, and in TBAB solution (upper curve), showing complete release;

(D) Diffusion of methylene green in 60% LCP of MO with 6.2 % of the diacid compound 243 as additive, in water (lower curve), showing complete binding, and in TBAB solution (upper curve), showing complete release; note that compound 243 has a cyclopropyl moiety instead of the cis double bond in the middle of the hydrophobic tail;

(E) Diffusion of methylene green in 60% LCP of MO with 4.3 % of the amino acid compound 240 as additive, in water (lower curve), showing complete binding, and in 0.1 M TBAB solution (upper curve), showing complete release; note that compound 240 has a cyclopropyl moiety instead of the cis double bond in the middle of the hydrophobic tail;

(F) Diffusion of methylene green in 60% LCP of MO; lower curve: 6.2 % of the diacid compound 243 as additive, causing complete binding; upper curve: 4.7 % of the corresponding dimethyl ester compound 236 as additive, causing complete release; note that compounds 243 and 236 have a cyclopropyl moiety instead of the cis double bond in the middle of the hydrophobic tail;

(G) Diffusion of methylene green in 60% LCP of MO; lower curve: 4.3 % of the amino acid compound 240 as additive, causing complete binding; upper curve: 4.4 % of the corresponding methyl ester compound 238 as additive, causing complete release; note that compounds 240 and 238 have a cyclopropyl moiety instead of the cis double bond in the middle of the hydrophobic tail.

Figure 7: shows aborbance (at 260 nm, non-labelled DNA) vs. time courses:

binding of DNA to PEI-containing LCP (59.7% MO, 0.3% PEI, 40% H₂O) (lower trace, diamonds), and release of DNA from non-modified LCP (60% MO, 40% H₂O) (upper trace, squares).

Figure 8: shows aborbance (at 645 nm, fluorescently labelled DNA) vs. time courses: binding of DNA to LCP containing Y01 -190 (59.4% MO, 0.6% Y01 -190, 40% H₂O)(lower trace, diamonds), and release of DNA from non-modified LCP (60% MO, 40% H₂O) (upper trace, squares).

Detailed description of the invention

LCPs are ideal materials for the incorporation of various guest molecules with a wide range of properties. Diffusion in and out of hitherto known LCPs is passive, and there is no known, physiologically compatible method to trigger release from LCP. To overcome this drawback, we have synthesized a series of novel lipids that can be used as additives with designed functionalities. When mechanically mixed with the lipids that form LCPs (typically monoacylglycerols, and most commonly monoolein), they form novel LCPs. The lipids that form LCPs must have a propensity to form curved, self-assembled structures, most commonly bilayers. Such lipids have one or more cis double bonds along their hydrophobic tail, or alternatively a cyclopropyl moiety. The chain length can vary, and is typically C14 or longer. Their headgroups may vary, the only requirement being that they are hydrophilic. The additives are lipid molecules, i.e. molecules that are insoluble in water, but can be mixed with the host lipids to form a LCP. Our novel additives have one or more cis double bond(s) along the chain, or alternatively a cyclopropyl moiety, with a total chain length that is typically between C14 and C22. The linkage between the headgroup and the chain is variable, and can be an ester, thioester, amide, ether, etc. A light activatable group (the "switch") is located either at the headgroup region or along the hydrophobic chain. The switch can be any functional group that can be activated photochemically. Examples are: (a) Photolabile "caged" moieties of the substituted nitrobenzyl type; (b) azobenzene functionalities; etc.

Fabrication of LCPs is accomplished by vigorous mixing of the lipid(s) with the aqueous solution. Mixing can be by centrifugation or by any other means such as mechanical mixing, typically in special syringes. The lipid composition is based on the phase diagram(s), and is typically in the range of 60-80 % (w/w). The aque- ous composition is thus accordingly 20-40 % (w/w). The additive is in the range of 1 -5% (w/w) with respect to the total lipid content.

Triggering can be accomplished by chemical means, preferably by photochemical means. The active photochemical moiety (the "switch") undergoes well-defined structural changes upon light activation. Because the switch is covalently linked to the lipid additive, it affects the lipid's overall structure, and as a consequence alters the properties of the LCP's membrane. These structural changes thus control the binding and release properties of the host LCP. As a consequence of the availability of well-defined aqueous and lipidic compartments, and the interface between them, LCPs can be considered as universal "encapsulators" for compounds with varied size, charge, and polarity.

The range of potential applications is broad, because of the following material properties: a) LCPs have both hydrophobic and hydrophilic compartments. They can therefore be applied universally, and can be placed on and adhere to various surfaces. b) LCP are soft solid materials, therefore they can be shaped on any surface as needed. c) LCPs are biocompatible, therefore they can be applied in (bio)medical, pharmacological, cosmetics, or food contexts.

Because of their stability and biocompatibility, these novel materials that incorporate active agent can be implanted to any tissue and can also be used in transcutaneous applications. Most importantly, because of their transparency, they do not affect vision. This property makes them amenable to intraocular ap- plications: they can incorporate the active compounds (drugs) in the eye, and can deliver them at the chosen time and with a given dosage upon photo-triggering.

Syntheses of new compounds: I. Amides

Y01 -17 (compound 25), Y01 -21 , Y01 -23 (compound 22), Y01 -25, Y01 -27, Y01 - 72, FK 121 .

All amides mentioned here, except FK121 , were synthesized according to general Method A (see below). FK121 was synthesized in a 3 step synthesis as fol- lows: Synthesis of FK121 (three step synthesis)

Step 1 : Synthesis of FK1 19

FK1 19

A solution of 1 ,3-diannino-2-propanol (240 mg, 2.66 mmol; Fluka purum), dissolved in 100ml dry MeOH, was added to a solution of di-terf.-Butyl-dicarbonate (594.4 mg, 2.72 mmol) in 100 ml dry dichloromethane (DCM) within 2 min at - 10°C. The reaction mixture was stirred in an ice bath for 2 h, then allowed to warm up to RT, and stirred for further 19 h. The solvents were evaporated in vacuo, and the residue was separated by column chromatography (CC)

(CH₂CI₂:MeOH:conc. NH₃ = 10:10:1 ). Yield of FK1 19 wasl 59 mg, (31 %) as a colorless oil.

Step 2: Synthesis of FK120

159 mg (0.84 mmol) N-Boc-aminopropanol (FK1 19), 261 mg (0.86 mmol) oleoylchloride (Aldrich 85%), and 144 μΙ (0.84 mmol) N-ethyldiisopropylamine (DIPEA) were dissolved in 10 ml DCM. The reaction was carried out according to method A (see below). TLC eluent system: MeOH:DCM = 1 :2 + 1 % triethylamine (TEA); R_f = 0.49. CC: Ether:MeOH = 50:1 , yield 307 mg (80%). Step 3: Synthesis of FK121

FK121

5 ml of 4N HCI was added dropwise during 1 minute to a solution of 295 mg (0.65 mmol) of FK120 in 10 ml ethylacetate (EtOAc) at 0°C. The mixture was stirred for 24 hours at room temperature, after which it was added to water, the pH was adjusted to ca. 8 with NaOH, extracted 3 times with DCM, and one time with ether. The organic phase was washed with brine, dried over MgSO₄, filtered and the filtrate was evaporated. TLC eluent system: DCM:MeOH:NH3 = 10:10:1 ; R_f = 0.61 .

Purification: CC, eluent system DCM:MeOH:NH₃ = 10:5:1 ;

Yield: 166 mg (72%).

II. Cvclopropyl compounds

SB008, 235, 236, 237, 238, 239, 240, 241 , 243

Compounds 236, 237, 238, and 241 were synthesized according to general Method A (see below).

Compound 235 was synthesized according to general Method B (see below). Compounds 239, 240, and 243, were synthesized according to general Method D (see below). Synthesis of SB008

Cis-9,10-Methyleneoctadecanoyl chloride (297 mg, 0.89 mmol) in 0.1 ml pyridine was added to a solution of (2,2-dimethyl-1 ,3-dioxolan-4-yl)methanol (solketal) (133 mg, 126 μΙ_, 1 .01 mmol, Acros 97 %) in pyridine (0.5 ml_, Fluka puriss) at 0°C. The reaction mixture was stirred for 48 hours, poured into 10 ml DCM and 10 ml of sulfuric acid (0.25 mol/L). The aqueous phase was extracted twice with 10 ml DCM, the organic phases were washed three times with the sulfuric acid solution, water and brine. The organic phase was dried over MgSO₄ and the solvent was removed in vacuo. Flash chromatography column of the crude product (DCM:MeOH = 98:2 to 93:7) afforded 49 mg of SB008 (yield: 15 %) as a colorless oil. R_f (DCM-MeOH 95:5) = 0.16.

III. Azobenzenes

SB036, SB040, SB041 , SB042, Y01 -59

Synthesis of SB036 (two step synthesis)

Step 1 : Synthesis of SB034

SB034 was synthesized analogous to a known procedure (E.J. Harbon et al, J. Phys. Chem. B, 2004, 108, 18789-18792): A suspension of anhydrous potassium carbonate (3.46 g, 25.1 mmol), 4-phenylazophenol (0.99 g, 5 mmol, Alfa Aesar), SB033 (687 mg, 2.36 mmol) and dry acetone (15 ml) was refluxed for 24 h. The reaction mixture was brought to room temperature and filtered to remove salts, and the acetone was removed by rotary evaporation. The residue was purified by column chromatography (Et₂O:hexane = 10:90), and yielded 747 mg (76%) of SB034 as orange oil. R_f (hexane:Et₂O = 2:1 ) 0.48. Step 2: Synthesis of SB036

SB034 SB036

SB036 was obtained from SB034 as follows: A solution of KOH (0.79g, 20.4 mmol) in MeOH (6.8 ml) was added dropwise to a stirred solution of SB034 (0.7g, 1 .71 mmol) in MeOH (13 ml) at room temperature under argon. The reaction mixture was heated at 40°C for 1 h, followed by stirring overnight at room tempera- ture. The reaction mixture was poured into water, acidified with 2N HCI until pH ¾ 2, and extracted with Et₂O (3 x 30 ml_). The combined organic layers were washed with brine, dried over MgSO₄ and evaporated in vacuo. The residue was purified by recrystallization in hexane at 60°C to yield 588 mg (87%) of SB036 Synthesis of SB040

SB040 was obtained from sarcosine methylester hydrochloride (19.9 mg, 0.156 mmol), SB036 (29.7 mg, 0.076 mmol), Dicyclohexylcarbodiimide (DCC) (16.16 mg, 0.078 mmol), DMAP (10.9 mg, 0.078 mmol) in dry DCM (4 ml) following general Method D (see below). Flash (filtration) chromatography of the crude product (Et₂O:hexane = 90:10) yielded 34.6 mg (95%) of SB040 as an orange oil. R_f (hexane:Et₂O = 1 :9) 0.22.

Synthesis of SB041 (two step synthesis)

Step 1 : Synthesis of SB039

SB036 SB039 SB039 was obtained from SB036 (13.8 mg, 0.104 mmol), which was reacted with solketal (13.8 mg, 0.104 mmol, Acros 97 %), DCC (21 .46 mg, 0.104 mmol), and DMAP (12.70 mg, 0.104 mmol) in dry DCM (3 ml) following general Method D. (see below). Purification with a preparative TLC plate (Et₂O:hexane = 50:50) yielded 39 mg (86%) of SB039.

Step 2: Synthesis of SB041

SB039 SB041

740 mg (12 mmol) of boric acid was added to a solution of 30 mg (0.059 mmol) of SB039 in 3 ml of 2-methoxyethanol, and the mixture heated at 95°C for 4 h. The reaction mixture was slowly brought to room temperature, 3 ml of water and 5 ml of DCM were added, and the mixture was shaken for a few minutes. The precipi- tated boric acid was filtered off, and the solid was washed with DCM and water. The DCM phase was separated, washed with brine and dried over MgSO₄. The solvent was evaporated in vacuo and the residue was purified by column chromatography (Et₂O:hexane = 80:20) to yield 14.6 mg (57%) of SB041 as an orange waxy solid. M.p. 69.8-71 .6°. R_f (EtOAc:hexane 4:1 ) = 0.22.

Synthesis of SB042

SB040 (24.1 mg, 0.05 mmol) was reacted with LiOH (90 μΙ of a 1 mol/liter solution of LiOH in water) in 2 ml EtOH. The crude product was purified by column chromatography (EtOAc:MeOH = 90:10 with 0.5% AcOH) to yield 21 .4 mg (92%) of SB042 as an orange waxy solid. M.p. 90.2-92.3°. R_f (EtOAc:MeOH = 10:1 ) 0.22.

Synthesis of Y01-59

Compound Y01 -59 was synthesized according to general Method C (see below). V. Caged compounds

Caged compounds: SB031 , PU258, PU251 , Y01 -150, PU252, PU259, Y01 -89 (compound 48), Synthesis of SB031

SB031 was obtained from 4,5-dimethoxy-2-nitrobenzyl alcohol (180 mg, 0.85 mmol, Alfa Aesar 98%), N-oleoyl-sarcosine (300mg, 0.48 mmol), DCC (348 mg, 1 .68 mmol), DMAP (14 mg, 0.1 1 mmol) in dry DCM following general Method C (see below). Column chromatography of the crude product (EtOAc:hexane = 30:70 to 57:43) yielded 368 mg (82%) of SB031 as a slightly yellow solid. M.p. 41 .2 - 42.3°C. R_f (hexane:EtOAc = 60:40) 0.2.

Synthesis of PU258

PU258 was obtained from 5-amino-2-nitrobenzyl alcohol (60 mg, 0.35 mmol), oleic acid (100mg, 0.35 mmol, Aldrich tech 90 %), DCC (73 mg, 0.35 mmol, Fluka puriss), DMAP (43 mg, 0.35 mmol, Fluka puriss) in dry DCM (9 ml) following general Method C (see below). Preparative layer of the crude product (Et2O:pentane = 70:30) yielded 135 mg (88%) of PU258 as a light yellow solid. M.p. 57.2 - 58.4°C. R_f (Et₂O:hexane = 60:40) 0.23.

Synthesis of PU251

1 ,1 '-Carbonyldiimidazole (50 mg, 0.31 mmol, Aldrich reagent grade) was suspended in anhydrous dichloromethane (1 ml) and the reaction mixture was cooled to 0°C. Ethyl 3-(Hydroxymethyl)-4-nitrobenzoate (70 mg, 0.31 mmol) was slowly added as a solution in dichloromethane (1 ml). The mixture was stirred at room temperature for 1 h. (8Z)-8-Heptadecen-1 -amine (79 mg, 0.31 mmol) was added and the mixture was stirred at room temperature overnight. EtOAc (30 ml) was added and the mixture was washed twice with HCI 10 % (2 x 20 ml), and once with brine (1 x 20 ml). The organic layer was dried over MgSO₄, filtered and the solvent was evaporated. Preparative thin layer chromatography of the crude product (EtOAc:hexane = 40:60) yielded 123 mg (79%) of PU251 as a slightly yellow waxy solid. M.p. 62,7-64,5°. R_f (hexane:EtOAc = 3:2) 0.43.

Synthesis of Y01 -150

Analogous to the synthesis of PU251

Synthesis of PU252

PU252 was obtained from the respective ethyl ester PU251 (160 mg, 0.23 mmol), which was reacted with lithium hydroxide monohydrate (20 mg, 0.48 mmol) in EtOH/H₂O according to general Method D (see below). The crude product was purified by preparative thin layer (CH₂CI₂:MeOH 98:2 with 0.5% AcOH) to yield 59 mg (54%) of PU252. M.p. 94 - 97°C. R_f (EtOAc :hexane = 3:2 + 2% AcOH) 0.37.

Synthesis of PU259

PU259 was obtained from 5-amino-2-nitrobenzyl alcohol (30 mg, 0.18 mmol), Ν,Ν-bis-acetic acid oleamide (35 mg, 0.088 mmol), DCC (37 mg, 0.18 mmol, Fluka puriss), DMAP (22 mg, 0.18 mmol, Fluka puriss) in dry DCM (3.5 ml) following general Method C (see below). Preparative layer chromatography

(Et₂O:MeOH = 99:1 ) yielded 22 mg (37%) of PU259 as a light yellow oil. R_f (Et₂O/1 % MeOH) 0.27.

Synthesis of YO-189 (compound 48)

Compound Y01 -89 (compound 48) was synthesized according to general Method A.

VI. Esters, Thioesters

Esters, Thioesters: 146, 128

Synthesis of 146 (two step synthesis)

Step 1 : Synthesis of the tosylated monoolein 1 17

117

1 .03 g (5.40 mmol) of toluene-4-sulfochloride (Tosyl chloride, Fluka puriss) was added stepwise to 1 .49g monoolein (MO) (4.18 mmol, NuCheck 99%) dissolved in 12 ml pyridine (Fluka abs.). The mixture was stirred for 6 hours at room temperature, after which it was added to water, and extracted 3 times with ether. The organic phase was washed twice with 1 M HCI and with half saturated brine, dried over MgSO₄, filtered and the filtrate was evaporated. TLC eluent: Ether:hexane = 4;1 ; R_f = 0.33. Purification with silica column, eluent system: ether:hexane = 4:1 to yield 1 .80 g (84%) of the colorless oil 1 17.

Step 2: synthesis of 146

122 mg (1 .88 mmol) sodium azide (NaN₃, Fluka 99.5%) was added to a solution of 0.36 g (0.7 mmol) of 1 17 in 5 ml DMF (acros abs.) at room temperature. The mixture was heated to 60°C and stirred for 2.5 hours, after which it was added to water, and extracted 3 times with ether. The organic phase was washed with water and with brine, dried over MgSO₄, filtered and the filtrate was evaporated. Purification: silica column, eluent system Etherhexane = 3:2. Yield: 195 mg (73%)

Synthesis of 128

117 128 491 mg (0.96 mmol) of compound 1 17 and 142 mg (1 .24 mmol) of potassium thi- oacetate were mixed in 7.5 ml acetone (abs.) at room temperature and stirred for 16 hours under argon atmosphere. The mixture was added to half saturated brine and extracted 3 times with methyl terf-butyl ether (MTBE). The organic phase was washed with 1 M HCI, saturated NaHCO3 and brine, dried over MgSO₄, filtered and the filtrate was evaporated.

Purification: silica column, eluent system MTBE:hexane = 1 :1 . Yield: 103 mg

(26%). VII. Guanidinium compounds

75 %

PPh₃ SB 061 (X = N₃) (i) TFA, CH₂CI₂, 3h Y01 -188 {R R₂=Boc) H₂0,THF (ii) Amberlite

reflux, 24h IRA-400 (OH)

SB 062 (X = NH₂) Y01 -190

85% H₂0, 24 h (R R₂=H)

73%

The guanidinylated lipid derivative Y01 -190 was prepared from the primary azide SB061 (readily available from 2-azidoethylamine and oleoyl chloride), which was reduced to the amine derivative SB062 under neutral conditions with PPh3 in

THF/water in 85% yield. Subsequently, the guanidinylation reaction of SB062 was performed under mild conditions using N,N'-di-Boc-N"-triflylguanidine (1 .0 equiv) and Et3N (1 equiv) in CH2CI2 (0.1 M) to afford the corresponding N,N'-di-Boc- protected intermediate Y01 -188 in high yield (95%). Removal of the Boc groups using trifluroacetic acid followed by ion exchange with the Amberlite IRA-400

(OH) resin, resulting in the desired guanidine compound Y01 -190 in good yield.

General Synthetic Procedures

Method A

Solution of the corresponding acyl chloride (1 .2 mmol-range) in dichloromethane DCM (1 .0 ml_) was added dropwise within 15 min to a stirred solution of the cor- responding α-amino acid ester hydrochloride, amine, or alcohol derivative respectively (1 .3 mmol-range), and N-ethyldiisopropylamine (2-2.5 mmol) in pure DCM (3 ml_), or in a DCM/MeOH (3 ml_, 2:1 ) mixture at 0°C. The reaction mixture was stirred, brought to room temperature and stirred further until no more starting ma- terial was detected by thin layer chromatography (TLC). The reaction mixture was poured into brine and extracted three times with ethyl acetate (EtOAc). The combined organic phases were dried over MgSO₄ and concentrated under reduced pressure. The crude products were purified by column chromatography (CC). Method B

A solution of oleoyl chloride (2.6 mmol) in tetrahydrofuran (THF) (2 ml_) was added dropwise within 15 min to a stirred solution of the corresponding a-amino acid ester derivative (2.4 mmol) in 2N NaOH (5 ml_) at 0 °C. The resulting suspension was stirred at 0°C for 5 min, brought to room temperature, whereupon a clear reaction solution was obtained after a few minutes. When no more starting material was detected by TLC, the reaction mixture was diluted with 20 mL of water, cooled to 0°C, and the pH adjusted to <2 by addition of 3N HCI. The mixture was flashed with 100 ml water in a separating funnel, and extracted 3 times with EtOAc. The combined organic layers were washed with brine, dried over MgSO₄ and concentrated under reduced pressure. The crude products were purified by recrystallization or by CC.

Method C

General Procedure for Coupling Reaction using Dicyclohexylcarbodiimide (DCC): A solution of DCC (1 equivalent) and 4-Dimethylaminopyridine (DMAP) (0.068 equivalent) in dry DCM (1 .9 mL/mmol) was added dropwise to a stirred solution of 1 equivalent of the carboxylic acid derivative in dry DCM (0.82 mL/mmol) and 5 equivalents of the respective alcohol or amine in dry DCM (0.27 mL/mmol) in ice bath under argon. The mixture was stirred for 5 min at at 0°C, removed from the cooling bath and stirred at room temperature (3-6 h) until no further change was observed by TLC. The dicyclohexylurea was filtered off, and the filtrate was washed with saturated NaHCO3. The organic extracts were dried over MgSO₄ and the solvent was evaporated under reduced pressure. The residue was purified by CC on silica gel using the appropriate eluent. Method D

General Procedure for ester hydrolysis using LiOH:

A mixture of the corresponding a-amino acid ester derivative (2 mmol) in ethanol (30 ml_) and LiOH (6 mmol) in water (10 ml_) were stirred at room temperature overnight. The solvents were evaporated on a rotavap at room temperature, and the residue was dissolved in water (50 ml_) and extracted with EtOAc (50 ml_). The aqueous phase was acidified with 1 N HCI to pH 2, and extracted with EtOAc (3x40 ml_), washed with NaHCO3, brine, and dried over MgSO₄, filtered and evaporated. The residue was purified by CC. In order to test the binding and controlled release of DNA from LCP, the additive Y01 -109, i.e. the compound

was compared with an additive formed of polyethyleneimine (PEI) having a molecular weight (MW) of 1 '250.

LCP preparation

Experiment 1 : A solution containing 0.5 nmol of the 5'-TTTTTTTT-3' oligo DNA (non fluorescent) was added to the appropriate amount of MO/PEI mixture (99.5-0.5 w/w), and LCP was prepared using the standard mixing procedure. MW of PEI: V250. Experiment 2: The analogous experiment was carried out with fluorescently- labeled 16-mer oligo DNA (Cy5-16mix) and with LCP formed with MO containing Y01 -190 as additive. Binding and release profiles of oligonucleotides were established by using UV spectroscopic detection, using non-labelled (measured at 260 nm), and fluores- cently labelled oligonucleotides (cyanine dye Cy5, which exhibits absorption maximum of 645 nm in water). In each experiment, 14 g of LCP material was placed in specially designed holder and overlaid with 1 ml of mQ water, and the respec- tive DNA absorption was measured in the overlay solution.

As seen from Figure 7, the DNA compound is bound toPEI-containing LCP whereas it is quickly released from non-modified LCT. As seen from Figure 8, the DNA compound is very strongly bound to Y01 -190- containing LCP whereas it is quickly released from non-modified LCT.

Scheme: List of Additives

I. Amides

iV,iV-01eoyl-bisethanolamin Ν,Ν-diethanol oleamid

7V-oleoyl-L-alanine

A/-Oleoyl 1 - hydroxymethyl ethanolamine

7V-oleoyl-L-tyrosine

Ethyl iV-oleoyl-L-tryptophanate iV-oleoyl-L-tryptophane beschrieben

jV-oleovl-sarcosine

Methyl jV-oleoyl-L-glycinate

Methyl 4-(oleamido) prolinate

.

N-(3-amino-2-hydroxypropyl)oleamide

SB061 B062

II. Cyclopropyl compounds

2,3-dihydroxypropyl)-8-(2-octylcyclopropyl)octanoate N , N-(8-(2-octylcyclopropyl)octanoy l)-bis-acetic acid

N-(8-(2-octylcyclopropyl)octanoyl)-L-serine methylester

N-(8-(2-octylcyclopropyl)octanoyl)-sarcosine methylester

N-(8-(2-octylcyclopropyl)octanoyl) glutamicacid dimethylester N-(8-(2-octylcyclopropyl)octanoyl) 2,3-dihydroxypropyl

N-(8-(2-octylcyclopropyl)octanoyl) glutamicacid



IV. Saturated lipids rt)

N-Stearoylglycine cribed

N-Stearoyl-L-alanine N-Stearoyl-L-alanine methylester bed

-L-valine methylester

N-Stearoyl-L-valine bed

., alte Daten

N-Stearoyl-L-serine N-Stearoyl-L-serine methylester

N-Stearoyl-L-glutamic acid Dimethyl N-Stearoyl-L-glutamate

n,

N-Stearoyl-L-tyrosine N-Stearoyl-L-tyrosine methylester d compounds

,5-dimethoxy-2-nitrobenzyl oleate 3-((((Z)-heptadec-8-enyl)carbamoyl)methyl)-4-nitrobenzoic acid

re-SBD72IY01-2S3 Esters, Thioesters

Monoolein (MO) ez.

3-amino-2-hydroxypropyl oleate .

3-azido-2-hydroxypropyl oleate

-(thioactyl)-2-hydroxypropyl oleate

eschrieben

(Z)-S-2,3-dihydroxypropyl octadec-9-enethioate spez .

VII. Guanidinium compounds

NBoc

X

re-SB074

Claims

1 . A lipidic compound, particularly for use as an additive in a lipidic cubic phase material, the lipidic compound being selected from the group consisting of the following compounds according to the Scheme:

- amides: Y01 -21 , Y01 -23 (N-oleoyl-L-serine methyl ester), Y01 -17 (Di- methyl-N-oleoyl-L-glutamate), Y01 -72 (N-oleoyl-L-phenylalanine methyl ester), Y01 -27 (Ethyl N-oleoyl-L-cysteinate);

cyclopropyl compounds: SB008 (2,3-dihydroxypropyl-8-(2-octylcyclo- propyl) octanoate), No. 235 (N,N-(8-(2-octylcyclopropyl)octanoyl) bis- acetic acid), No. 236 (N-(8-(2-octylcyclopropyl)octanoyl) glutamic acid dimethyl ester), No. 237 (N-(8-(2-octylcyclopropyl)octanoyl) sarcosine methyl ester), No. 238 (N-(8-(2-octylcyclopropyl)octanoyl) L-serine methyl ester), No. 239 (N-(8-(2-octylcyclopropyl)octanoyl) sarcosine), No. 240 (N-(8-(2-octylcyclopropyl)octanoyl) L-serine), No. 241 (N-(8-(2- octylcyclopropyl)octanoyl) 2,3-dihydroxypropyl amine), No. 243 (N-(8- (2-octylcyclopropyl)octanoyl) glutamic acid);

- azobenzenes: SB036, SB040, SB041 , SB042, Y01 -59;

caged compounds: Y01 -89 (4,5-dimethoxy-2-nitrobenzyl oleate), Υ0Ί - 141 , SB031 (4,5-dimethoxy-2-nitrobenzyl 2-oleamido) acetate), PU251 (5-(ethoxycarbonyl)-2-nitrobenzyl (Z)-heptadec-8-enylcarbamate), PU252 (3-((((Z)-heptadec-8-enyl)carbamoyl)methyl)-4-nitrobenzoic acid), PU258 (5-amino-2-nitrobenzyl oleate), PU259;

esters: No. 146, No. 163 (3-amino-2-hydroxypropyl oleate);

- guanidinium compounds: Y01 -188, Y01 -190, re-SB073/Y01 -205b, re- SB074, Y01 -195a, Y01 -199.

2. A lipidic compound according to claim 1 , selected from the group consisting of the following compounds according to the Scheme:

amides: Y01 -23 (N-oleoyl-L-serine methyl ester), Y01 -17 (Dimethyl-N- oleoyl-L-glutamate); cyclopropyl compounds: No. 236 (N-(8-(2-octylcyclopropyl)octanoyl) glutamic acid dimethyl ester), No. 238 (N-(8-(2- octylcyclopropyl)octanoyl) L-serine methyl ester), No. 240 (N-(8-(2- octylcyclopropyl)octanoyl) L-serine), No. 243 (N-(8-(2- octylcyclopropyl)octanoyl) glutamic acid);

caged compounds: Y01 -89 (4,5-dimethoxy-2-nitrobenzyl oleate);

guanidinium compounds: Y01 -188, Y01 -190.

A lipidic cubic phase material, comprising a hydrophilic component and a lipophilic component, said lipophilic component comprising a major lipophilic constituent and at least one lipidic additive, which lipidic additive comprises a hydrophilic head group and a hydrophobic tail attached thereto.

The lipidic cubic phase material according to claim 3, wherein said hydrophobic tail comprises at least one cis double bond or cyclopropyl moiety.

The lipidic cubic phase material according to claim 3 or 4, wherein said lipidic additive comprises a photochemically activatable moiety.

The lipidic cubic phase material according to claim 3 or 4, wherein said lipidic additive is selected from the group consisting of the compounds listed in the Scheme.

The lipidic cubic phase material according to claim 3 or 4, wherein said lipidic additive is a compound as defined in claim 1 or 2.

The lipidic cubic phase material according to claim 3 or 4, wherein said lipidic additive is selected from the group consisting of the following compounds according to the Scheme:

amides: c10 (N-oleoyl acetic acid), Y01 -41 (N-oleoyl-L-serine), Y01 -23 (N-oleoyl-L-serine methyl ester), Y01 -37, Y01 -17 (Dimethyl-N-oleoyl-L- glutamate), No. 229 (N-oleoyl sarcosine), No. 227 (Ν,Ν-bis-acetic acid oleamide), No. 52;

cyclopropyl compounds: No. 236 (N-(8-(2-octylcyclopropyl)octanoyl) glutamic acid dimethyl ester), No. 238 (N-(8-(2-octylcyclo- propyl)octanoyl) L-serine methyl ester), No. 240 (N-(8-(2- octylcyclopropyl)octanoyl) L-serine), No. 243 (N-(8-(2-octylcyclo- propyl)octanoyl) glutamic acid);

caged compounds: Y01 -89 (4,5-dimethoxy-2-nitrobenzyl oleate);

- guanidinium compounds: Y01 -188, Y01 -190.

9. The lipidic cubic phase material according to any one of claims 3 to 8, wherein said major lipophilic constitutent is a monoacylglycerol. 10. The lipidic cubic phase material according to claim 9, wherein said monoacylglycerol is monoolein.

1 1 . The lipidic cubic phase material according to any one of claims 3 to 8, wherein said major lipophilic constitutent is an isoprenoid-chained lipid.

12. The lipidic cubic phase material according to claim 1 1 , wherein said isoprenoid-chained lipid is phytantriol.

13. The lipidic cubic phase material according to any one of claims 3 to 12, wherein said hydrophilic component comprises an aqueous solution containing a predetermined active compound.

14. The lipidic cubic phase material according to any one of claims 3 to 13, for use in a therapeutic method.

15. A method of controlled release of a predetermined active compound, com- prising the following steps:

forming a lipidic cubic phase material according to claim 13 by mixing said lipophilic component and said hydrophilic component comprising an aqueous solution containing said predetermined active compound; - placing said lipidic cubic phase material in a region of interest;

releasing said active compound by subjecting said lipidic cubic phase material to a chemical or photochemical stimulus.

16. The method of controlled release according to claim 15, wherein said re- leasing step comprises a photochemical stimulus generated by ultraviolet irradiation.

17. The method of controlled release according to claim 15 or 16, wherein said acitve compound is a DNA compound.