Gemcitabine amphiphile prodrugs

Abstract

To provide amphiphilic gemcitabine prodrugs or amphiphilic prodrugs of other biologically active molecules with the capacity to make liquid crystalline nanostructured nanoparticles, and uses thereof to treat animals, including humans.SOLUTION: The invention provides compounds such as 2'-deoxy-2',2'-difluoro-N4-(((cis-9, octadecenyl)oxy)carbonyl) cytidine (Gem-oleyl).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to improved prodrugs and compositions thereof. In particular, the present invention relates to amphiphilic gemcitabine prodrugs, or prodrugs of other biologically active molecules, that have the ability to form liquid crystalline nanostructured nanoparticles and are used to treat animals, including humans. [Background technology]

[0002] Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside-based chemotherapy agent for pancreatic, colorectal, lung, breast, ovarian and bladder cancer. It has very limited bioavailability in vivo, poor pharmacokinetic properties, and is susceptible to deamination and inactivation in body fluids and the bloodstream.

[0003] Current chemotherapeutics, including gemcitabine, are generally nonselective and cause severe side effects due to high clearance rates, poor pharmacokinetic profiles, inactivation in the circulation, lack of tumor selectivity, and intrinsic or acquired resistance by tumors.

[0004] Efficient delivery of chemotherapeutic agents to tumor sites requires site-specific targeting. It is well known that nanoparticles and polymers passively accumulate in tumor and inflamed tissues due to unique vascular disorders and properties, as well as the lack of lymphatic restitution system. -The so-called enhanced penetration and retention effect (EPR) (MatsumuraY et al, 1986) Applications of nanotechnology in medicine (nanomedicine) include, but are not limited to, the use of nanoscale particles as drug delivery carriers for anticancer drugs. Nanomedicines improve the therapeutic index, safety and efficacy of cancer treatment by improving pharmacokinetics and pharmacodynamics and providing efficient delivery of active agents to the site of action.

[0005] The use of nanoparticles as drug delivery carriers for anticancer drugs can effectively target the delivery of drugs to cancer tissues and cells.

[0006] Encapsulation of anticancer drugs in nanoparticles is being investigated as a second generation chemotherapy. Nanoparticles based on phospholipids or polymeric materials have been demonstrated to provide high payloads of anticancer drugs and protect the drugs from premature inactivation in some circumstances, resulting in increased efficacy and reduced systemic toxicity. Several products are on the market, such as Doxil®, the first FDA approved nano drug (Barenholz Y).

[0007] Another approach involves chemically modifying anticancer drugs to form prodrugs that can form nanoscale particles (i.e., third generation chemotherapeutic agents). This has been demonstrated with active 5-fluorouracil (5-FU), which is covalently attached to hydrophobic molecules to provide a 5-FU prodrug that can form nanoscale particles (US8603999). Chemically modified prodrug derivatives of active drugs reduce systemic toxicity, improve pharmacokinetics and pharmacodynamics, and have excellent stability in body fluids. In vivo, the prodrug undergoes a biotransformation reaction, preferably at the desired site of action (such as a tumor), for example, by hydrolysis, oxidation, reduction, or enzymatic cleavage.

[0008] Gemcitabine has been incorporated into nanoparticles by conjugating the active drug to squalene (US20090130214A1), however this system requires the use of organic solvents to form the nanoparticles, making it undesirable for end use in aqueous biological fluids.

[0009] There remains a need to generate better methods of drug delivery.

[0010] The reference to prior art in this specification is not, and should not be construed as, an acknowledgment or form or suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction, or that this prior art is reasonably possible and would be expected to be identified, understood and deemed relevant by those skilled in the art. Summary of the Invention

[0011] The present invention aims to provide prodrugs capable of forming self-assembled structures. The present invention also provides pharmaceutical compositions thereof. These higher order phases modify the release profile of the drug.

[0012] In one embodiment, the present invention provides prodrugs of general formula I:

[0013] [ka]

[0014] where A is a biologically active agent; Y is a cleavable bond between the biologically active agent and X; and X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), and a substituent according to formula (d):

[0015] [ka]

[0016] where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and the like; S is (polyethylene glycol)n, n=1-10; L1 is a linker group covalently attached to one SR group at one attachment site and to A at a second attachment site via a biolabile bond Y; L2 is a linker group covalently attached to two SR groups (which may be the same or different) at two independent attachment sites and to A at the third attachment site via a biolabile bond Y; and L3 is a linker group covalently attached via a biolabile bond Y to three SR groups (which may be the same or different) at three independent attachment sites and to A at the fourth attachment site.

[0017] In an embodiment of this aspect of the invention, there is provided a prodrug of general formula (I):

[0018] [ka]

[0019] where A is a therapeutically active agent; Y is a cleavable bond between the therapeutically active agent and X; and X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), and a substituent according to formula (d):

[0020] [ka]

[0021] where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and the like; S is (polyethylene glycol)n, n=1-10.

[0022] L1 is a linker group covalently attached via a biolabile bond Y to A to the SR group at one binding site and to a therapeutically active agent A at the second binding site; L2 is a linker group that is covalently attached to two SR groups (which may be the same or different) at two independent attachment sites and is attached to the therapeutically active agent A at a third attachment site via a biolabile bond Y to A; and L3 is a linker group that is covalently attached to three SR groups (which may be the same or different) at three independent attachment sites and is attached to the therapeutically active agent A at the fourth attachment site via a biolabile bond Y to A.

[0023] In one embodiment, A is a therapeutically active nucleoside agent, preferably a nucleoside agent containing a cytosine base, more preferably gemcitabine.

[0024] In one embodiment, Y is a selectively cleavable bond independently selected from the group consisting of amide, carbamate, sulfonamide, imide, imine, and carbonate. Preferably, Y is independently selected from the group consisting of amide and carbamate. In a particularly preferred embodiment, Y is a carbamate bond.

[0025] In a preferred embodiment, X is a substituent according to formula (a) of the prodrug of general formula (I).

[0026] In a preferred embodiment, general formula (I) represents a compound according to formula (II):

[0027] [ka]

[0028] where X is a substituent according to formula (e) selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), a substituent according to formula (d), and a substituent according to formula (b):

[0029] [ka]

[0030] where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and the like; S is (polyethylene glycol)n, n=1-10; L1 is a linker group covalently attached to the SR group at one binding site and to gemcitabine via a carbamate bond to gemcitabine at the second binding site; L2 is a linker group that is covalently attached to two SR groups (which may be the same or different) at two independent attachment sites and that is attached to gemcitabine via a carbamate bond to gemcitabine at the third attachment site; and L3 is a linker group that is covalently attached to three SR groups (which may be the same or different) at three independent binding sites and is attached to gemcitabine at the fourth binding site via a carbamate bond to gemcitabine.

[0031] In a preferred embodiment, X is a substituent according to formula (a) or (e) of the prodrug of formula (II). R is generally hydrophobic. Optionally, R has a linear chain length corresponding to 10 to 30 carbon atoms. In one embodiment, R is alpha-tocopherol. In another embodiment, R is an isoprenoid group. In another embodiment, R is a hydroxylated alkyl or hydroxylated alkenyl group. Preferred embodiments of R are alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl (isoprenoid), branched alkynyl, substituted alkyl, substituted alkenyl and substituted alkynyl groups and their analogs, such as alpha-tocopherol, hydroxylated alkyl or alkenyl groups. In a preferred embodiment, each R has a chain length corresponding to 10 to 24 carbon atoms, more preferably corresponding to 12 to 24 carbon atoms, more preferably corresponding to 14 to 20 carbon atoms. When X is a substituent according to formula (c) or formula (d), each R may be independently selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl and substituted alkynyl groups and the like. Alternatively, the R groups may be identical. In general, R is intended to impart self-assembly properties to A.

[0032] In a preferred embodiment, R is a myristyl, myristoyl, myristoylamide, palmityl, palmitoyl, palmitoylamide, stearyl, stearoyl, stearoylamide, oleyl, oleoyl, oleoylamide, linoleyl, linoleoyl, linoleoylamide, linolenyl, linolenoyl, linolenoyl, linolenoyl arachidonoyl, arachidonoylamide, phytanyl, phytanoyl, phytanoylamide, hexahydrofarnesyl, hexahydrofarnesoyl, and hexahydrofarnesoylamide chain. Most preferably, R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, phytanyl, and phytanoyl chains.

[0033] In one embodiment, S is polyethylene glycol (PEG).1-10 , α-carboxypolyethylene glycol (HOOC-PEG) 1-10 , and α-aminopolyethylene glycol (H2N-PEG) 1-10 Preferably, S is selected from the group consisting of (PEG). 1-10 and more preferably S is (PEG) 1-6 In a particularly preferred embodiment, S is (PEG). 3-6 It is.

[0034] In one embodiment, L1 is a selectively cleavable linker group selected from the group consisting of succinic anhydride, maleic anhydride, glycolic acid, chloroacetic acid, allyl isocyanoacetate, hydroxypropanesulfonic acid, glycine and alanine. In another embodiment, L2 is a selectively cleavable linker group selected from the group consisting of diethanolamine, propane-1,2,3-tricarboxylic acid, cysteine, glutamic acid, glutamine, aspartic acid, serine, tyrosine, lysine, arginine, histidine and threonine. In another embodiment, L3 is a selectively cleavable linker group selected from the group consisting of tris(hydroxymethyl)aminoethane (tris) and citric acid.

[0035] Compounds according to formula (II) can be made as depicted in Scheme 1 below.

[0036] In a particularly preferred embodiment, the general formula (II) represents a compound selected from the group consisting of:

[0037] [ka]

[0038] Here, Z is O or NH.

[0039] More preferably, general formula (II) represents a compound selected from the group consisting of:

[0040] [ka]

[0041] Preferably, the prodrug of general formula (I) or (II) can form a self-assembled structure having a lamellar phase, a cubic phase, a hexagonal phase, a sponge, an emulsion, or a lyotropic phase exhibiting a crystalline lamellar morphology. More preferably, the phase is a lamellar phase, a cubic phase, a hexagonal phase, or a sponge phase. Even more preferably, the phase is an inverse phase. In a particularly preferred embodiment, the phase is an inverse phase to a lamellar phase.

[0042] In a second aspect of the present invention, there is provided a self-assembled structure of the prodrug of general formula (I) or (II) of the above aspect. In one embodiment, the self-assembled structure of the prodrug of general formula (I) or (II) further comprises a component selected from the group consisting of phospholipids, cholesterol lipids, glycerol lipids, prodrug amphiphiles, and combinations thereof. Preferably, the self-assembled structure of the prodrug of general formula (II) where x is a substituent according to formula (e) further comprises a component selected from the group consisting of phospholipids, cholesterol lipids, glycerol lipids, prodrug amphiphiles, and combinations thereof.

[0043] Preferably, the self-assembled structure is a lyotropic phase exhibiting a lamellar, cubic, hexagonal, sponge, emulsion, or crystalline lamellar morphology. More preferably, the phase is a lamellar, cubic, hexagonal, or sponge (L3) phase. Even more preferably, the phase is a reverse phase. In general, reverse phases are advantageous as drug delivery carriers due to their thermodynamic stability in excess water, larger surface area, and controlled channel dimensions. The latter property is particularly important for the release of actives embedded within the self-assembled matrix. Thus, there is provided a prodrug that can self-assemble into a lamellar, reverse cubic, reverse sponge (L3), reverse hexagonal, or reverse micellar phase (L2), preferably a lamellar, reverse cubic, or L3 phase. In another embodiment, the self-assembled prodrug has a crystalline structure.

[0044] The self-assembled structure of the prodrug according to the invention may be a bulk phase or may be a colloidal particle derived therefrom. Particularly preferred colloidal particles may be selected from the following group: liposomes, cubosomes, hexosomes, "sponge-like" particles or reverse micelles. Under certain conditions, the self-assembled structure may have more than one phase.

[0045] In a particularly preferred embodiment, the self-assembled structure is of a gemcitabine prodrug of formula (II). Preferably, the self-assembled structure is a lamellar phase, a cubic phase, an L2 or an L3 phase. Such particles can be suitably stabilized for pharmaceutical use by surfactant stabilizers such as polyethylene glycol lipids, polysorbates or poloxamers.

[0046] In another aspect of the present invention, there is provided a method for modulating the release of a biologically active agent, such as a drug or prodrug, comprising covalently linking a biologically active agent A to at least one tail moiety R via a spacer S and a cleavable covalent bond Y to form an amphiphile capable of self-assembly under physiological conditions, and wherein the amphiphile is cleavable in vivo to release the biologically active agent.

[0047] In one embodiment of this aspect, a method of modulating the bioavailability of a biologically active agent A is provided, comprising covalently linking a biologically active agent A to at least one tail moiety R via a cleavable covalent bond Y to form an amphiphile, where the covalent bond is cleavable in vivo to release the biologically active agent from the self-assembled structure; and administering the amphiphile to a patient such that the amphiphile self-assembles into the self-assembled structure.

[0048] In another aspect of the present invention, a pharmaceutical composition for the treatment of a medical condition is provided, comprising a self-assembled structure of formula (I) or (II) as an active ingredient. In some embodiments, the pharmaceutical composition for the treatment of a medical condition consists essentially of an active ingredient that is a self-assembled structure of formula (I) or (II), optionally in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles of the self-assembled structure. In some embodiments, the self-assembled structure exhibits a lamellar, cubic, hexagonal, micellar cubic, micellar or sponge phase. Preferably, in some embodiments, the active ingredient is a self-assembled structure of formula (II) in a liposome, cubosome, hexosome, reverse micelle or less ordered sponge-like nanoparticle or solid lipid nanoparticle or combination thereof.

[0049] In some embodiments, the disease state is a tumor state and the pharmaceutical composition comprises, as an active ingredient, solid lipid particles or the self-assembled structure of formula (II).

[0050] The self-assembled structure / active ingredient is preferably present in the pharmaceutical composition in a therapeutically active amount.

[0051] In another aspect of the present invention, a method for the treatment of a disease condition is provided, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition for the treatment of a disease condition comprising a self-assembled structure of formula (I) or (II) as an active ingredient, optionally in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles in a self-assembled colloidal particle. In some embodiments, the self-assembled structure displays liposomes, cubosomes, hexosomes, micelles cubosomes, micelles, or sponge-like nanoparticles. Preferably, in some embodiments, the active ingredient is a self-assembled structure of formula (II), optionally in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles in a self-assembled structure such as a liposome, cubosome, hexosome, reverse micelle, or sponge-like nanoparticle of lower order, or solid lipid nanoparticle, or combinations thereof.

[0052] In some embodiments, the disease state is due to the presence of a tumor, in which case the pharmaceutical composition preferably comprises an active ingredient in a self-assembled structure of formula (II), optionally in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles, including liposomes, reverse cubosomes, hexosomes, micelles, disordered sponge nanoparticles or solid lipid nanoparticles or combinations thereof.

[0053] In another aspect of the invention, the medicament provides a self-assembled structure according to the invention of formula (II), optionally in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles, to provide a self-assembled structure for the manufacture of a medicament for the treatment of a disease condition. The medicament comprises a self-assembled bulk or colloidal particle structure as in the previous aspect of the invention.

[0054] In some embodiments, the disease state is due to the presence of a tumor, in which case the pharmaceutical composition preferably comprises the self-assembled structure of formula (II) as an active ingredient, optionally in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles, including liposomes, reverse cubosomes, hexosomes, micelles, sponge-like nanoparticles, or solid lipid nanoparticles, or combinations thereof.

[0055] In another aspect of the present invention, a method for delivering a biologically active agent by utilizing enhanced permeation and retention effects by forming a self-assembled structure containing the active agent is provided, the method comprising covalently linking the biologically active agent to at least one hydrophobic tail. The component R forms an amphiphile capable of self-assembling into the self-assembled structure under physiological conditions via a spacer S and a cleavable bond Y, the amphiphile being cleavable in vivo to release the biologically active agent. Preferably, the biologically active agent is gemcitabine.

[0056] One embodiment of this aspect provides a method of delivering a biologically active agent, the method comprising covalently linking the biologically active agent to at least one tail moiety to form an amphiphile, where the link is cleavable in vivo to release the biologically active agent from the self-assembled structure; administering the amphiphile to a patient, such that the amphiphile self-assembles into the self-assembled structure.

[0057] Preferably, the amphiphiles self-assemble to form self-assembled structures of lyotropic mesophases displaying lamellar, cubic, hexagonal, sponge and / or emulsified morphologies and similar nanoparticles, solid lipid nanoparticles or combinations thereof.

[0058] A further aspect of the invention relates to a process for preparing bulk phases; lamellar, cubic, hexagonal, sponge and crystalline mesophases are prepared by the process of this aspect.

[0059] A further aspect of the invention relates to a method for preparing colloidal particles from the bulk phase according to the invention. There is further provided colloidal particles according to the invention prepared by the method of this aspect.

[0060] Any embodiment herein is intended to apply mutatis mutandis to any other embodiment unless otherwise stated.

[0061] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

[0062] Further aspects of the invention and further embodiments of the aspects described in the previous paragraphs will become apparent from the following description, given by way of example with reference to the accompanying drawings, in which:

[0063] It will be understood that the term "comprises" (or grammatical variations thereof) as used herein is equivalent to the term "includes" and should not be interpreted as excluding the presence of other elements or features. [Brief description of the drawings]

[0064] [Figure 1] Schematic of the different phases that can occur upon hydration of different amphiphiles. The abbreviations for the different mesophases are micellar (L1), micellar cubic (I1), normal hexagonal (H1), bicontinuous cubic (V1), lamellar (La), reverse bicontinuous cubic (V2), reverse hexagonal (H2), reverse micellar cubic (I2), and reverse micellar (L2), with the subscripts 1 and 2 referring to the "normal" and "reverse" phases, respectively. [Diagram 2] DSC of representative gemcitabine amphiphiles: (a) without spacer and (b) with PEG spacer. [Diagram 3] 1D SAXS diffraction patterns of neat and lyotropic mesophase behavior of representative gemcitabine amphiphiles without spacers: (a) gem-oleyl, (b) gem-linoleyl, (c) gem-phytanyl; --bulk_25 °C, --hydration excess water_25 °C, ···hydration excess water_37 °C. [Figure 4-1] 1D SAXS diffraction patterns of neat and lyotropic mesophase behavior of representative gemcitabine amphiphiles with PEG spacers: (a) gem-PEG6-oleoyl, (b) gem-PEG6-linolenoyl, (c) gem-PEG3-linoleoyl, (d) gem-PEG6-linoleoyl, (e) gem-PEG4-linoleoyl, (f) gem-PEG2-linoleoyl; --Bulk_25 °C, --Hydrated excess water_25 °C, ···Hydrated excess water_37 °C. [Figure 4-2] 1D SAXS diffraction patterns of neat and lyotropic mesophase behavior of representative gemcitabine amphiphiles with PEG spacers: (a) gem-PEG6-oleoyl, (b) gem-PEG6-linolenoyl, (c) gem-PEG3-linoleoyl, (d) gem-PEG6-linoleoyl, (e) gem-PEG4-linoleoyl, (f) gem-PEG2-linoleoyl; --Bulk_25 °C, --Hydrated excess water_25 °C, ···Hydrated excess water_37 °C. [Diagram 5] Cryo-TEM images of gemcitabine amphiphilic nanoparticles in the presence or absence of phospholipid / cholesterol: (a) gem-phytanyl / DMPC / cholesterol (18.38 / 70.52 / 8.10 w %), (b) gem-PEG3-oleoyl / DMPC / cholesterol (31.04 / 62.07 / 6.89 w %), (c) gem-PEG3-linoleoyl, and (d) gem-PEG6-linoleoyl. [Figure 6-1]Size distribution of gemcitabine amphiphilic nanoparticles in the presence and absence of phospholipids / cholesterol as determined by dynamic light scattering (DLS): (a) gem-phytanyl / DMPC / cholesterol (18.38 / 70.52 / 8.10 w %), (b) gem-PEG3-oleoyl / DMPC / cholesterol (31.04 / 62.07 / 6.89 w %), (c) gem-PEG3-linoleoyl, and (d) gem-PEG6-linoleoyl. [Figure 6-2] Size distribution of gemcitabine amphiphilic nanoparticles in the presence and absence of phospholipids / cholesterol as determined by dynamic light scattering (DLS): (a) gem-phytanyl / DMPC / cholesterol (18.38 / 70.52 / 8.10 w %), (b) gem-PEG3-oleoyl / DMPC / cholesterol (31.04 / 62.07 / 6.89 w %), (c) gem-PEG3-linoleoyl, and (d) gem-PEG6-linoleoyl. [Figure 7] Biotransformation reaction of gemcitabine amphiphilic nanoparticles containing phospholipids / cholesterol and 50 uM gem-phytanyl (■), gem-PEG3-oleoyl (diamond), or gem-PEG3-oleoylamide (▲) using CES enzyme (33.7 units / mL) at 37 °C. [Figure 8] The tumor inhibition of gemcitabine amphiphilic nanoparticles (gem-phytanyl / DMPC / cholesterol: 18.38 / 70.52 / 8.10 w%) was compared with gemcitabine drug and PBS control groups. [Figure 9] Average ex vivo tumor weight of tumors treated with gemcitabine amphiphilic nanoparticles (gem-phytanyl / DMPC / cholesterol: 18.38 / 70.52 / 8.10 w%), gemcitabine drug, and PBS control. [Figure 10]Biodistribution of fluorescent gemcitabine amphiphilic nanoparticles (Gem-phytanyl / DMPC / cholesterol / DiD fluorescent lipid: 18.38 / 70.52 / 7.10 / 1w%) in NOD / SCID mice: (a) for periods of 4, 24, 48, 72 and 96 h. (b) Distribution in different organs excised after culling the mice. Excitation and emission wavelengths were 650 and 700 nm. [Figure 11] Tumor inhibition of gemcitabine-amphiphilic nanoparticles (Gem-Oleyl / DOPC / PEG4000-Oleoyl 26 / 60.8 / 13.2 w%) in PDXCXF260 xenograft model in nude mice versus Avastin. [Figure 12] Tumor inhibition of gemcitabine amphiphilic nanoparticles Gem-PEG3-oleoyl / phospholipid / cholesterol 6 / 9 / 1w%+PEG4K-oleoyl (15% of total lipid weight). The nanoparticle dispersion contained 15mg / Kg of active gemcitabine compared to 15mg / Kg of gemcitabine drug, and PBS, and was tested in Mia paca2 pancreatic cancer xenograft model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] It should be noted that various terms used in the specification, examples, and claims have meanings that would be understood by one of ordinary skill in the art, however, in order to clarify the meaning intended in this document, certain terms are defined below.

[0066] The term "prodrug" as used throughout this specification refers to a biologically active compound that includes a structural modification thereof such that, in vivo, the prodrug undergoes one or more reactions or steps to produce the biologically active compound, for example, by hydrolysis, oxidation, reduction or enzymatic cleavage. This includes active substances that require a common metabolic step to generate the active biomolecule. Thus, the term is understood to also include "preprodrugs."

[0067] The term gemcitabine prodrug as used throughout this specification refers to a compound of general formula (II) that can be converted in vivo, for example by enzymatic and / or chemical reaction, to gemcitabine.

[0068] [ka]

[0069] where X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), a substituent according to formula (d), and a substituent according to formula (e).

[0070] [ka]

[0071] where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and analogs thereof; S is (polyethylene glycol)n, where n=1-10; L1 is a linker group that is covalently attached to the SR group at one attachment site and is linked to gemcitabine at a second attachment site via a carbamate bond to gemcitabine; L2 is a linker group that is covalently attached to two SR groups (which may be the same or different) at two independent binding sites and is attached to gemcitabine at the third binding site via a carbamate bond to gemcitabine; and L3 is a linker group that is covalently attached to three SR groups (which may be the same or different) at three independent binding sites and is attached to gemcitabine at the fourth binding site via a carbamate bond to gemcitabine.

[0072] Here, X is a molecule capable of imparting self-assembly properties to the compound.

[0073] The term "self-assembled structure" as used throughout this specification is meant to refer to an assembly of amphiphiles that has some degree of internal organizational order. Self-assembled structures can be formed by contacting amphiphiles with a solvent. Self-assembled structures are meant to refer to either bulk lyotropic phases, colloidal particles derived therefrom (so-called "colloidosomes"), or solid lipid particles.

[0074] The term "bulk phase" as used throughout this specification includes micellar cubic (I1); normal hexagonal (H1); bicontinuous cubic (V1); lamellar (L α ); reverse bicontinuous cubic (V2); reverse hexagonal (H2); lyotropic phases, including but not limited to reverse micellar (L2) and sponge (L3) phases.

[0075] The term "colloidal particles" as used throughout the specification should be understood to refer to "colloidosomes" and solid lipid particles. The term "colloidosomes" as used throughout the specification should be understood to refer to colloidal particles that have the same internal nanostructure as the bulk lyotropic phase. The term solid lipid particles as used throughout the specification should be understood to mean colloidal particles of the prodrugs of the present invention, which include a core of neat prodrug and are usually stabilized by a surface layer of surfactant. The core of the neat prodrug may be in crystalline, microcrystalline, liquid crystalline, or amorphous form. The term "particles" will be understood to refer to particles that may be nanoparticles or microparticles based on their average size. Often such particles are referred to as "solid lipid nanoparticles", but may actually be in the size range of microparticles. This form of self-assembled structure does not swell when contacted with excess solvent.

[0076] The term "lamellar phase" as used throughout this specification refers to a phase in which opposing monolayers of the hydrophilic parts of amphiphilic molecules are separated by polar solvent domains, and back-to-back layers of the hydrophobic parts of amphiphilic molecules are in intimate contact to form a hydrophobic layer. A planar lamellar phase is called a "lamellar phase". α It is called "phase".

[0077] The term "cubic phase" as used throughout this specification refers to two major classes of phases: micellar cubic and bicontinuous cubic. A "micellar cubic phase" refers to a phase consisting of spherical micelles arranged in a cubic array. A "normal micellar cubic phase" or "L I The term "reverse micellar cubic phase" is composed of spherical reverse micelles arranged in a cubic array.

[0078] "Bicontinuous cubic phases" refers to a family of closely related phases consisting of a single curved lipid bilayer that forms a complex network that separates the polar solvent space into two successive but nonintersecting volumes. Bicontinuous cubic phases have a long-range order based on a cubic unit cell. The average curvature of a bicontinuous cubic phase is zero; that is, at every point on the surface of an amphiphilic bilayer, the surface is as convex as it is concave. Bicontinuous cubic phases are usually ("v I "v phase") or inverse ("v II In bicontinuous cubic phases, several types of long-range orientation order have been observed. The orientation orders of these phases correspond to the space groups Ia3d, Pn3m, and Im3m. When colloidosomes have an internal structure of a bulk cubic phase, they are sometimes called "cubosomes".

[0079] The term "hexagonal phase" as used throughout this specification should be understood to mean an amphiphilic phase consisting of long rod-shaped micelles packed in a hexagonal arrangement. A "normal hexagonal phase" is a hexagonal phase consisting of long rod-shaped normal micelles, and a "reverse hexagonal phase" is a hexagonal phase consisting of long rod-shaped reverse micelles. A normal hexagonal phase is also known as a "H I phase" and the inverted hexagonal phase is called "H II When a colloidosome has an internal structure of a bulk hexagonal phase, the colloidosome may be referred to as a "hexosome."

[0080] The term "sponge phase" or "L3 phase" as used throughout this specification refers to phases that resemble bicontinuous cubic phases in that they have an amphiphilic bilayer that separates the polar solvent space into two unconnected volumes, but they do not possess long-range order. These phases are therefore analogous to "molten cubic phases."

[0081] As used throughout this specification, the term "lattice constants" refers to the set of lattice constants that define the unit cell of a crystalline solid or liquid crystal, and may include values ​​such as the length of the unit cell.

[0082] The term "isoprenoid" as used throughout this specification means an alkyl chain consisting of isoprene (2-methyl-1,3-butadiene) monomers or subunits. As used herein, the use of the term "isoprenoid" is intended to encompass unsaturated, partially saturated or fully saturated isoprene analogs and derivatives.

[0083] The term "pharmaceutical composition" as used throughout this specification means a composition comprising a therapeutically effective amount of at least one prodrug according to the present invention and at least one pharma- ceutically acceptable carrier, excipient, diluent, additive or carrier selected based on the intended mode of administration and consistent with conventional pharmaceutical practice.

[0084] The terms "biologically active agent", "therapeutically active agent", "pharmacologically active agent", "active agent" and "active ingredient" as used throughout this specification refer to a substance intended, but not limited to, for the diagnosis, cure, mitigation, treatment, prevention and / or modification of a condition in a biological system. References to "biologically active agents" are broader than references to "therapeutically active agents". The terms "drug" and therapeutic agent are used interchangeably throughout this specification.

[0085] The biologically active agent may be a drug. In this case, AYX represents a prodrug. In another embodiment, the biologically active agent may be a prodrug. Thus, the biologically active agent may be a drug that can be metabolized to a biologically active agent. In this case, AYX represents a pre-prodrug. The biologically active agent is preferably a therapeutically active agent.

[0086] As used herein, a "therapeutically effective amount" refers to an amount or dosage of an agent, such as a gemcitabine prodrug or a composition thereof, that results in one or more desired effects, particularly the inhibition or cessation of tumor growth. A therapeutically effective amount of a substance will vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the ability of the substance to elicit a desired response in the subject.

[0087] In one aspect, the present invention provides prodrugs of general formula I:

[0088] [ka]

[0089] where A is a biologically active agent; Y is a cleavable bond between the therapeutically active agent and X; and X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), and a substituent according to formula (d).

[0090] [ka]

[0091] where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and analogs thereof; S is (polyethylene glycol)n, n=1-10; L1 is a linker group covalently attached to one SR group at one attachment site and to A at the second attachment site via a biolabile bond Y to A; L2 is a linker group covalently attached to two SR groups (which may be the same or different) at two independent attachment sites and covalently attached to A at the third attachment site via a biolabile bond Y to A; and L3 is a linker group that is covalently attached to three SR groups (which may be the same or different) at three independent attachment sites and to A at the fourth attachment site via a biolabile bond Y to A.

[0092] In a preferred embodiment, X is a substituent according to formula (a) of the prodrug of general formula (I).

[0093] R is generally hydrophobic. Optionally, R has a linear chain length equivalent to 10 to 30 carbon atoms. In one embodiment, R is α-tocopherol. In another embodiment, R is an isoprenoid group. In another embodiment, R is a hydroxylated alkyl or hydroxylated alkenyl group. Preferred embodiments of R are alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, alkynyl (isoprenoid), substituted alkyl, substituted alkenyl and substituted alkynyl groups and analogs thereof, such as alpha-tocopherol, hydroxylated alkyl or alkenyl groups. In a preferred embodiment, R has a chain length equivalent to 10 to 24 carbon atoms, more preferably 14 to 20 carbon atoms. When X is a substituent according to formula (c) or formula (d), each R may be independently selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl and substituted. Alkynyl groups and analogs thereof. Alternatively, both R groups can be the same. In general, R is intended to impart self-assembly properties to A.

[0094] In a preferred embodiment, R is selected from the group consisting of myristyl, myristoyl, myristoylamide, palmityl, palmitoyl, palmitoylamide, stearyl, stearoyl, stearoylamide, oleyl, oleoyl, oleoylamide, linoleyl, linoleoyl, linoleoylamide, linolenyl, linolenoyl, linoyl, arachidonoylamide, phytanyl, phytanoyl, phytanoylamide, hexahydrofarnesyl, hexahydrofarnesoyl, and hexahydrofarnesoylamide chains. Most preferably, R is selected from the group consisting of oleyl, oleoyl, linoleyl, linoleoyl, phytanyl, phthanoyl, hexahydrofarnesyl, and hexahydrofarnesoyl chains.

[0095] As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon radical having 10 to 30 carbon atoms, or any range therebetween. An alkyl group is optionally substituted with a substituent, allowing multiple degrees of substitution. Examples of "alkyl" as used herein include, but are not limited to, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, icosane, henicosane, docosane, tricosane, tetracosane, pentacosane, hexacosane, heptacosane, octacosane, nonacosane, triacontane, and the like.

[0096] As used herein, "C 10 -C 30 The term "alkyl" refers to an alkyl group, as defined above, containing at least 10 and at most 30 carbon atoms, or any range between 12-24. (For example, an alkyl group containing 12-24 carbon atoms is also referred to as C 10 -C 30 in the range.

[0097] As used herein, the term "alkenyl" refers to an alkyl group that contains a double bond, which can be optionally substituted with substituents, with multiple degrees of substitution being possible.

[0098] As used herein, the term "alkynyl" refers to an alkyl group that contains a triple bond, which can be optionally substituted with substituents, with multiple degrees of substitution being possible.

[0099] As used throughout this specification, terms such as "optionally substituted" or "may be substituted" indicate that a group may or may not be substituted or fused with one or more non-hydrogen substituents (to form a polycyclic ring system). Chemically feasible substituents suitable for a particular functional group will be apparent to one of ordinary skill in the art. Examples of suitable substituents include, but are not limited to, oxygen or sulfur substituted analogs.

[0100] In some embodiments according to the invention, L1, L2 and L3 are linker groups. "Linker" refers to a group that acts as a multifunctional domain between the biologically active agent A and the group SR. The linker is at least bifunctional in the case of L1, at least trifunctional in the case of L2 and at least tetrafunctional in the case of L3, and contains at least one functional group ("attachment site") for anchoring the group SR at one site in the molecule and a selectively cleavable functional group to another attachment site for anchoring the drug A via a Y bond. Preferably, the linker group is a covalent bond.

[0101] L1, L2, and L3 may be at least one functional group linked to at least one selectively cleavable functional group. Preferably, L1, L2, and L3 include a moiety that links at least one functional group and at least one selectively cleavable functional group. The moiety may be selected, for example, from the group consisting of heteroatoms, alkyls, alkenyls, alkynes, which may be cyclic, and / or contain additional heteroatoms and functional substituents (e.g., carbonyls, carboxylic acids, amides, hydroxyls, ethers, amines, etc.), or any combination thereof.

[0102] L1 comprises a selectively cleavable functional group selected from the group consisting of succinic anhydride, maleic anhydride, glycolic acid, allylsulfonamide, allyl isocyanate, hydroxypropanesulfonic acid, chloroacetic acid, glycine and alanine. L2 is a selectively cleavable linker group selected from the group consisting of diethanolamine, propane-1,2,3-tricarboxylic acid, cysteine, glutamic acid, glutamine, aspartic acid, serine, tyrosine, arginine, lysine, arginine, histidine and threonine. L3 is a selectively cleavable linker group selected from the group consisting of tris(hydroxymethyl)aminoethane (tris) and citric acid. The examples provided for L1, L2 and L3 are not intended to be an exhaustive list and the present invention contemplates other embodiments of L1, L2 and L3.

[0103] The bond Y between A and L may be cleaved in vivo during metabolism of the prodrug AYLSR. Examples of bond Y include selectively cleavable bonds, including but not limited to amides, carbamates, esters, imides, sulfonamides, and imines. In a preferred embodiment, the covalent bond is labile so that it can be cleaved when necessary to release the active drug, but is stable enough to resist premature activation. Preferably, Y is a carbamate or amide bond. In a particularly preferred embodiment, Y is a carbamate bond.

[0104] The bond (Y) between the drug (A) linker (L) or spacer (S) and the hydrophobic tail group (R) may include a carbamate or amide bond between the drug and the spacer. Preferably, there is a carbamate, amide, ester or thioester bond between the spacer and the hydrophobic tail. Other bifunctional groups, such as chloroacetate, hydroxyl or thiol groups, and combinations thereof, can also be added as needed and are known in the art.

[0105] In some embodiments according to the invention, S is a spacer. "Spacer" refers to a bifunctional group that links to the therapeutically active domain A directly or via a linker L at one end and a hydrophobic group R at the other end. The spacer supports improved solvation of the head groups in polar solvents and provides better self-assembly properties. The spacer is usually bifunctional, containing one functional group that anchors to the R group at one end of the molecule and one functional group that is attached to the drug A or via the linker (L) using a selective, cleavable Y bond. In one embodiment, S is a polyethylene glycol (PEG) 1-10 , α-carboxypolyethylene glycol (HOOC-PEG) 1-10 , or α-aminopolyethylene glycol (H2N-PEG) 1-10 Preferably, S is selected from the group consisting of (PEG). 1-10 and more preferably, S is (PEG). 1-6 In a particularly preferred embodiment, S is (PEG). 3-6 A person skilled in the art will understand which compounds and methods are suitable for linking A to X.

[0106] Preferably, A is a hydrophilic biologically active agent. For example, A is a biologically active agent with a logP value of less than 0. In another embodiment, A is a drug that can be metabolized to a biologically active agent, and the biologically active agent is hydrophilic with a logP value of less than 0. In one embodiment, A is itself a prodrug that is converted to the biologically active agent by one or more reaction steps, for example, by hydrophilic, oxidative, reductive, or enzymatic cleavage. When A is itself a prodrug, general formula (I) can be considered to describe a compound called a preprodrug.

[0107] The present invention contemplates that the therapeutically active agent A itself may be a prodrug rather than a drug or active substance. It will be recognized by those skilled in the art that in the compound according to formula (II) above, A is a prodrug that undergoes modification in vivo to release the therapeutically active agent gemcitabine triphosphate. That is, A is a precursor of the biologically active agent that is formed in vivo after cleavage of the prodrug, for example by an enzyme.

[0108] Compound A may or may not require a further chemical modification step before it is rendered into a therapeutically active form. That is, compound A may itself be a prodrug, in which case AYLSR may be described as a pre-prodrug. In embodiments in which the biologically active agent is itself a prodrug, at least a further chemical modification step may be required before the amphiphilic prodrug is converted into a biologically active form.

[0109] In one embodiment, A is a therapeutically active nucleoside agent, preferably a nucleoside agent that contains a cytosine base, more preferably gemcitabine.

[0110] Preferably, the amphiphile is an enzyme substrate for an enzyme that promotes the formation of a biologically active form of the biologically active agent present in the amphiphile. Preferably, the amphiphile is pre-determined to be one that can be acted upon by an enzyme present in the patient. More preferably, the enzyme acts on a cleavable linker.

[0111] In one preferred embodiment, general formula (I) represents a compound according to formula (II):

[0112] [ka]

[0113] wherein X is as defined above in formula (II) and is a molecule capable of imparting self-assembly properties to the compound. In a preferred embodiment, X is a substituent according to formula (a) or (e) of the prodrug of formula (II). Preferably, R is an alkyl, alkenyl, alkynyl or isoprenoid with a chain length between 12-24 carbon atoms. Preferably, Y is an amide or carbamate, more preferably a carbamate. Preferably, S is a PEGyl group with n=1-10, more preferably n=1-6, more preferably n=3-6. n It is.

[0114] In a particularly preferred embodiment, general formula (II) represents a compound selected from the group consisting of:

[0115] [ka]

[0116] Here, Z is O or NH.

[0117] More preferably, general formula (II) represents a compound selected from the group consisting of:

[0118] [ka]

[0119] Particularly preferred embodiments of the compounds of the invention are 2'-deoxy-2',2'-difluoro-N4-(3,7,11,15-tetramethylhexdecanyl-1-oxycarbonyl)cytidine (gem-phytanyl), 2'-deoxy-2',2'-difluoro-N4-(cis-9-octadecenyl-1-oxycarbonyl)cytidine (gem-oleyl), 2'-deoxy-2',2'-difluoro-N4-(cis-9,cis-12-octadecadien-1-oxycarbonyl)cytidine (gem-linoleyl), 2'-deoxy-2',2'-difluoro-N4-(cis-6,cis-9,cis-12-octadecatrien-1-oxycarbonyl)cytidine (gem-linolenoyl), 2'-deoxy-2',2'-difluoro-N 4 -(triethyleneglycoxyl-cis-9-octadecenyl-1-oxycarbonyl)cytidine (Gem-PEG-3-oleoyl), 2'-deoxy-2',2'-difluoro-N 4 -(hexaethyleneglycoxyl-cis-9-octadecenyl-1-oxycarbonyl)cytidine(Gem-PEG-6-oleoyl),2'-deoxy-2',2'-difluoro-N 4 -(hexaethyleneglycoxyl-cis-9,cis-12-octadecenyl-1-oxycarbonyl)cytidine (Gem-PEG-6-linoleoyl), 2'-deoxy-2',2'-difluoro-N 4 -(tetraethyleneglycoxyl-cis-9,cis-12-octadecenyl-1-oxycarbonyl)cytidine(Gem-PEG-4-linoleoyl)or2'-deoxy-2',2'-difluoro-N 4-(diethyleneglycoxyl-cis-9,cis-12-octadecenyl-1-oxycarbonyl)cytidine (gem-PEG-2-linoleoyl), or a pharma- ceutically acceptable form thereof, including solvates, hydrates, and salts thereof.

[0120] Preferably, the prodrug of general formula (I) or (II) can form a self-assembled structure having a lyotropic phase exhibiting a lamellar, cubic, hexagonal, sponge, emulsion, or crystalline lamellar morphology. More preferably, the phase is a lamellar, cubic, hexagonal, or sponge phase. Even more preferably, the phase is an inverse phase.

[0121] In some embodiments, the prodrugs of formula (I) or (II) can be used in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles or hydrophobic drugs in self-assembled structures to form nanoparticles capable of delivering combinations of chemotherapeutic agents, or combinations of chemotherapeutic and diagnostic agents.

[0122] In some embodiments, the prodrugs of formula (I) or (II) may be used in combination with a targeting ligand, including a targeting molecule, peptide, antibody, protein, or aptamer, to more efficiently deliver the targeted active agent.

[0123] In a particularly preferred embodiment, the self-assembled structure is a combination of the compound of formula (II) gemcitabine, optionally with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles in the self-assembled structure. Such structures can benefit from surfactant stabilizers such as poloxamers, pegylated lipids, and polysorbates to be appropriately stabilized for pharmaceutical use.

[0124] A preferred embodiment of the present invention is a self-assembled structure comprising the compound according to formula (II) above.Preferably, the self-assembled structure is in the form of lamellar, cubic, hexagonal and sponge phase.When the self-assembled structure is in the form of nanoparticles, the average particle size is preferably between 10-500nm, more preferably 10-200nm.

[0125] The reaction conditions for the synthesis of the compounds according to the invention can be easily determined by those skilled in the art with a minimal amount of experimentation and are also illustrated in the accompanying examples. In a particularly preferred embodiment, the compounds are prepared according to Scheme 1.

[0126] [ka]

[0127] Scheme 1 When X is defined as described herein, Lg is a leaving group, preferably a halide or pseudohalide, most preferably chloride, and Pg is a protecting group, preferably a trimethylsilyl group. The selection of protecting groups is readily determined by one of skill in the art with minimal experimentation and is also illustrated in the accompanying examples.

[0128] The reaction conditions for the synthesis of compounds according to the present invention are readily determined by one of ordinary skill in the art with a minimal amount of experimentation, and are also illustrated in the accompanying Examples.

[0129] Starting materials and reagents used to synthesize compounds according to the present invention are available from commercial suppliers, such as, for example, Sigma-Aldrich Chemical Company (St. Louis, Missouri) and Merck (Australia), unless otherwise specified or prepared by methods known to those skilled in the art.

[0130] Another preferred embodiment according to the present invention is a self-assembled structure comprising one according to formula (I), where A is another therapeutic nucleoside, such as cytarabine and different forms of gemcitabine, such as 5-fluorocytosine.

[0131] [ka]

[0132] wherein X is defined as in formula II, and X is a molecule capable of imparting self-assembly properties to the compound.

[0133] The self-assembled structures of the present invention represent desirable prodrug delivery systems due to their modified release properties compared to prodrugs that do not undergo self-assembly into lamellar, inverse cubic, inverse hexagonal and inverse sponge and micellar phases. Without wishing to be bound by theory or mode of action, it is believed that the self-assembled structures of the present invention have modified release properties, firstly due to the difference in hydrolysis effect on the self-assembled amphiphilic molecules and the complexity of access to single molecules in self-assembled systems compared to that of isolated single molecules in non-assembled systems. Secondly, in the case of the preferred compounds, it is believed that the hydrophobic tail R and spacer S of the preferred prodrugs result in compounds with less substrate activity for the initial enzyme required to convert the prodrug and its analogues to gemcitabine or cyatravine or 5-flurocytidine. Thus, resulting in an improved release profile of the compounds according to the present invention. Finally, it is believed that hydrolysis of the compounds of the present invention releases aliphatic chain moieties that may themselves form self-assembled structures and then change the local environment of the enzymatic reaction, thus affecting the enzymatic behavior.

[0134] Similarly, the self-assembled structures according to the present invention are believed to be more desirable than prodrug self-assembled structures that exhibit micellar morphology. The prodrug lamellar, cubic, hexagonal, sponge phase or reverse micelle nanoparticles according to the present invention have a much larger amphiphile-solvent interface area than any of the previously disclosed prodrug self-assembled structures. Moreover, unlike conventional micelles, the reverse phases according to the present invention are stable in excess aqueous solvent.

[0135] In one embodiment, the self-assembled structure of the present invention comprises at least one solvent domain and at least one amphiphilic domain, the amphiphilic domain comprising at least one compound according to formula (II) or (III), where X is defined as any group capable of imparting self-assembly properties to the prodrug.

[0136] The solvent domains of the present invention comprise at least one polar solvent, including those commonly used in amphiphilic self-assembly, such as water, glycerol, propylene glycol, butylene glycol, N-methylformamide, hydrazine, propylene carbonate, methanol, ethanol, selected ionic liquids such as ammonium nitrate, and mixtures thereof.

[0137] The solvent may also contain other components including, for example, the solvent, salts, pH buffers, sugars such as glucose and sucrose, and stabilizing agents such as polysorbate 80 and PEG-PPO-PEG copolymers, more specifically poloxamer 127, poloxamer 108, PEG lipid chains of various PEG lengths or lipid chains, such as PEG 4000-oleoyl, PEG 4000-linoleoyl, PEG 2000-oleoyl, PEG 2000-linoleoyl, PEG 10000-oleoyl, or PEG 10000-linoleoyl.

[0138] In some embodiments, the prodrugs of formula ((I) or (II) or (III)) will be used in combination with other phospholipids, cholesterol, glycerol lipids, or other prodrug amphiphiles in self-assembled structures selected from liposomes, cubosomes, hexosomes, reverse micelles, and sponge-like nanoparticles.

[0139] Pharmaceutically active agents that can be incorporated into amphiphilic drug delivery carriers are known to those skilled in the art.See, for example, WO2005 / 0210046 (DBL Australia Pty Ltd) and WO9830206.Examples of pharmaceutical or biologically active agents that can be incorporated into carriers include, but are not limited to: global proteins and glycoproteins, highly reactive lipids such as prostaglandins, large biologically active drug molecules such as proteins, polysaccharides, DNA and RNA, and smaller drug molecules such as cyclosporine, paclitaxel, indomethacin, fenofibrate, progesterone, amphotericin B (AMB) and irinotecan.

[0140] The self-assembled structure of the present invention may also contain at least one other component intended to stabilize the self-assembled structure. Examples of stabilizing agents are triblock copolymers of different building blocks PEG-PPO-PEG, more specifically poloxamer 407, poloxamer 127, poloxamer 108, as well as polysorbate 80, PEG-lipid chains of various PEG lengths, such as PEG 4000-oleoyl, PEG 4000-linoleoyl, PEG 2000-oleoyl, PEG 2000-linoleoyl, PEG 2000-linoleoyl, PEG 2000-linoleoyl, etc. Those skilled in the art will recognize that the formation of the desired lyotropic liquid crystal phase of the present invention requires a critical balance between the specific hydrophilic and hydrophobic domains. Thus, those skilled in the art will recognize that the selection of X in relation to A will determine whether the prodrug of the present invention will form either a lyotropic phase and / or solid lipid particles in accordance with the present invention.

[0141] In general, the interactions between surfactant head groups, tails and volume are very important in determining the lyotropic phase behavior. The relationship between molecular shape and phase behavior can be described by the critical packing parameter (CPP). CPP is given by CPP=v / a0l c where v is the molecular volume, a0 is the cross-sectional area of ​​the surfactant head group, and l c where corresponds to the length of the hydrophobic tail. Since the development of this equation, CPP has been widely used to predict mesophase behavior based on molecular curvature. For molecules with small head groups and bulky hydrophobicity, the CPP value will be greater than 1, thereby inducing an average negative interfacial curvature and potentially the formation of inverse mesophases.

[0142] The cleavable tails according to the invention are selected based on the formation of more than one CPP when considered in the context of the head groups according to the invention. Figure 1 illustrates this interaction between the head and tail groups. Phases to the left of the lamellar phase have a critical packing density less than 1 and often occur at low concentrations of amphiphile. Phases to the right of the lamellar phase have more than one CPP and usually occur at high concentrations of amphiphile. The CPP is not constant for an amphiphilic molecule and depends on external factors such as temperature, pressure, concentration and pH of the amphiphilic molecule, as well as some additional solvents and additives. However, this parameter can be used as a simple guess of the phases that may occur upon hydration of the amphiphile at room temperature or physiological temperature and physiological pH and pressure.

[0143] The self-assembly behavior of amphiphiles in solvents occurs due to preferential interactions between the solvent and either the hydrophilic or hydrophobic portions of the amphiphilic molecules. When an amphiphile is exposed to a polar solvent, the hydrophilic portions of the amphiphile tend to interact preferentially with the polar solvent, resulting in the formation of hydrophilic domains ("solvent domains"). The hydrophobic portions of the amphiphile tend to be excluded from this domain, resulting in the formation of hydrophobic domains ("hydrophobic domains").

[0144] Lyotropic liquid crystals are formed by adding a solvent to a suitable solid or liquid amphiphile. They may be classified by the curvature of the interface between the hydrophilic and hydrophobic domains. The curvature between these domains depends on several factors, including the concentration of the amphiphile and the molecular structure. When the interface displays a net curvature towards the hydrophobic domains, the phase is called "normal". When the interface displays a net curvature towards the hydrophilic domains, the phase is called "inverted". When the net curvature of the system approaches zero, the resulting phase may have a lamellar type structure consisting of planar amphiphilic bilayers separated by solvent domains. Alternatively, if every point on the surface is convex in one dimension and concave in another, the net curvature may approach zero, and such a phase is called a "bicontinuous cubic phase". Examples of specific phases that can be formed by self-assembled structures include, but are not limited to, micelles (normal and reverse), hexagonal (normal and reverse), lamellar, cubic phases (normal, reverse and bicontinuous), and other mesophases such as reverse micellar cubic phases, ribbons, meshes, or non-cubic "sponge" bicontinuous phases.

[0145] The bulk phases described above can be dispersed to form colloidal particles that retain the internal structure of the non-dispersed bulk phase. These colloidal particles have also been investigated for application as drug delivery carriers. U.S. Patent 5,531,925 discloses colloidal particles that include an interior of an amphiphilic phase surrounded by a surface phase that is anchored in a bilayer or monolayer of the internal phase. The internal phase of the particle can be selected from lamellar, inverted cubic, hexagonal or L3 ("sponge") phases, or mixtures thereof.

[0146] When these particles have an internal structure of an inverse bicontinuous cubic phase, the particles are colloquially called cubosomes. Similarly, when the particles have an internal structure of an inverse hexagonal phase, they are called hexosomes. When the particles have an internal structure of a lamellar phase, they are called liposomes. Colloidal particles may also be formed from "sponge" phases.

[0147] An alternative drug delivery carrier is solid lipid particles. Solid lipid particles consist of a crystalline amphiphilic core stabilized by a surfactant surface layer, such as polysorbate 80, poloxamer, and PEGylated lipids. Solid lipid particles have been used as carriers for hydrophobic drugs. For example, camptothecin, an anticancer drug, adsorbed to solid lipids and dispersed as SLNs showed increased drug levels in brain tissue (Yang 1999, J. control release, 59(3):299-307). However, the drug loading of conventional SLNs is limited by the solubility of the drug in the lipid melt and the structure of the lipid matrix.

[0148] To increase drug loading, the "pharmacosome" approach has been adopted. This method involves generating prodrugs that can be assembled into micelles or liposomes. Gin-Etoile identifies several lipid-nucleoside analogues that can form regular lamellar vesicles (2005, 2006) or higher-order nanostructures (WO2010063080A1, US8603999B2). However, micelles have substantial drawbacks as a suitable phase for drug delivery. Micellar systems can collapse under dilution and below the critical micelle concentration (CMC).

[0149] Bulk Phase In one embodiment, the self-assembled structures of the present invention include at least one bulk phase.

[0150] The bulk phase of the present invention comprises at least one phase selected from the group consisting of: normal hexagonal, normal micellar cubic, normal bicontinuous cubic phase, lamellar, reverse bicontinuous cubic phase, L2 "reverse micelle", L3 "sponge" and reverse hexagonal. Preferably, the bulk phase comprises at least one phase selected from the group consisting of reverse hexagonal phase, reverse cubic phase, L2 "reverse micelle", L3 "sponge" phase and lamellar phase. Most preferably, the bulk phase comprises lamellar phase, reverse hexagonal phase and reverse cubic phase.

[0151] In a preferred embodiment, the bulk phase according to the present invention can be readily produced in a temperature range of about room temperature to about 50° C., and can be stable within this temperature range for at least several months.

[0152] A preferred embodiment according to the present invention is a bulk lyotropic reverse phase. The thermodynamic stability of the lyotropic phase according to the present invention against dilution in excess aqueous solvent means that the bulk phase maintains its primary conformation, although the lattice parameters may change upon swelling of the amphiphile in water. Most preferably, the lyotropic phase according to the present invention is a lamellar phase, a reverse bicontinuous cubic phase, or a sponge mesophase.

[0153] It will be appreciated by those skilled in the art that the observed lyotropic phase is temperature dependent. The bulk phase according to the present invention is stable between room temperature and physiological temperatures, preferably at temperatures from about 35° C. to about 40° C., and most preferably at temperatures from about 35° C. to about 37° C.

[0154] The process for preparing the bulk phase according to the present invention is known to those skilled in the art. In one embodiment, the bulk phase according to the present invention can be prepared by dissolving each amphiphile in a suitable buffer solution to a suitable concentration. Examples of suitable buffer solutions include, but are not limited to, physiologically acceptable buffer solutions, such as phosphoric acid, phosphate buffered saline (PBS), tris(hydroxymethyl)aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris-sucrose, Tris-glycine, and glycine buffer solutions.

[0155] In another embodiment, the preferred phase according to the present invention is prepared by mechanically mixing the molten lipids between room temperature and 50° C. until a visually homogenous sample is obtained. Optionally, the addition of a co-solvent, such as ethanol, for example in the range of 10-20% by weight, can aid the homogenization process.

[0156] Colloidal particles: colloidosomes or nanoparticles A further aspect of the invention relates to a self-assembled structure of the invention comprising one or more particles that retain the internal structure of a bulk phase. Such particles are called "colloidosomes" or "nanoparticles."

[0157] In one embodiment, the self-assembled structures of the present invention comprise colloidosomes or are selected from the following group: liposomes, cubosomes, hexosomes and "sponge-like" particles. In a preferred embodiment, the colloidal particles are selected from the following group: liposomes, cubosomes, hexosomes and sponge-like particles. Most preferably, the colloidal particles are liposomes and cubosomes and sponge-like particles.

[0158] In a particularly preferred embodiment according to the invention, the colloidosomes are derived from a lamellar phase and an inverse phase. The thermodynamic stability of the lyotropic phase according to the invention means that the bulk phase can be gradually diluted in an excess of aqueous solvent and dispersed in the colloidosomes while maintaining the same liquid crystalline structure as the bulk phase.

[0159] Colloidosomes according to the present invention can be prepared according to processes known to those skilled in the art. For example, colloidosomes / nanoparticles can be prepared by hydrating a lipid thin film in water or saline (e.g., phosphate buffered saline). In addition, sugars such as glucose, dextrose, etc. may be added to the medium. Reverse phase colloidosomes, such as reverse cubosomes and hexosomes, can be hydrated in water to form a gel-like bulk phase and dispersed into particles using shear forces such as sonication or high pressure homogenization in the presence of stabilizers.

[0160] It will be appreciated by those skilled in the art that in order to prepare stable colloidosomes, it is necessary to add a stabilizing or fragmenting agent. Suitable fragmenting agents are known to those skilled in the art and include, for example, poloxamers or polysorbates or PEGylated lipids. Poloxamers are the most widely used stabilizers for reverse phase colloidosomes and are block copolymers of polyethylene glycol (PEG) and polypropylene oxide (PPO). In a preferred embodiment according to the invention, the stabilizer is a triblock copolymer of different building blocks PEG-PPO-PEG. In a particularly preferred embodiment according to the invention, the stabilizer is poloxamer 407, poloxamer 108, and PEGylated lipid (PEG2k-10K-oleoyl).

[0161] In one embodiment, colloidal particles are prepared by dispersing a bulk phase. The bulk phase of the present invention can be dispersed by dropping an ethanol solution of the bulk phase into water containing a stabilizing reagent. Alternatively, the bulk phase can be dispersed by adding water containing at least one stabilizing reagent to the bulk phase. The size of these particles can be controlled by vortexing, sonication, filtration, extrusion and homogenization, which are techniques well known to those skilled in the art.

[0162] In a preferred embodiment, the colloidosome or nanoparticle dispersion according to the present invention is prepared by dispersing the bulk phase of the prodrug-amphiphile mixture in water containing a stabilizing reagent using vortexing and preferably with shear forces such as a probe-type ultrasonic homogenizer or ultrasonic bath. The colloidosome prepared according to this embodiment may optionally undergo one or more additional processing steps. Such processing methods are known to those skilled in the art and include high pressure homogenization and stepwise extrusion through membranes. The membranes used for stepwise extrusion may have pore sizes including, for example, 0.8, 0.4, 0.2, 0.1 and 0.05 μm. In one embodiment, the processing step is a size selection process.

[0163] In a preferred embodiment, the course colloidosome or nanoparticle preparation is further processed by passing it through a series of polycarbonate (PC) membranes. The size range of the membranes will be selected by the skilled artisan according to the desired particle size of the final product. Equipment that can be used for this processing step is known to those skilled in the art, but may include, for example, an extruder.

[0164] It will be recognized by those skilled in the art that the size of the nanoparticles of the present invention depends on the intended use.For example, for intravenous administration, the preferred colloidosome size range is generally between about 30 nm and about 400 nm.More preferably, the size range is between about 30 nm and about 200 nm for intravenous application.

[0165] For delivery of nanoparticles to specific organs such as the liver and passive targeting to tumors, particle sizes between about 30nm and about 400nm are contemplated. More preferably, particle sizes are about 30nm to less than about 200nm. Without wishing to be bound by theory, it is believed that particles with a size of 30-200nm passively target cancer cells due to enhanced penetration and retention time in the leaky and chaotic neovasculature of solid tumors. See, for example, Brannon-Peppas L.et al (Brannon-Peppas 2012 and Matsumura et al) 7,1.

[0166] Colloidal particles: solid lipid particles A preferred embodiment of the present invention is directed to providing solid-lipid particles comprising at least one gemcitabine prodrug. The solid lipid particles according to the invention can be prepared by methods known to those skilled in the art. See, for example, Mehnert and Mader. (Mehnert 2001). 8 .

[0167] The suitable method used to prepare solid lipid particles according to the present invention can be selected according to the physicochemical properties of the prodrug of the present invention. Some of the typical methods for preparing solid lipid particles, such as the method requiring melting lipids in a lipid liquid, have a melting point higher than 100° C. In one embodiment, the solid lipid particles of the present invention are prepared according to a mechanical method. According to this embodiment, one or more stabilizers are added to the neat amphiphile. Examples of stabilizers include, but are not limited to, triblock polymers (e.g., poloxamer 407, poloxamer 108, and PEGylated lipids). The amount of stabilizer added to the neat amphiphile can be between about 5-30% (w / w), preferably between about 10-30% (w / w), and most preferably between about 15-30% (w / w). To prepare the initial bulk phase, typically 20-30% by weight of water is added to the amphiphile, typically at room temperature (about 22-about 25° C.). The amphiphile-water mixture is then sheared using methods known to those skilled in the art. The mixture can then be further processed to produce particles of the desired size and polydispersity. Methods of further processing are known to those skilled in the art and can include, for example, high pressure homogenization, sonication, and extrusion through different membranes with known pore sizes.

[0168] Pharmaceutical Compositions A further aspect of the present invention relates to a pharmaceutical composition of the present invention. In one embodiment, the pharmaceutical composition according to the present invention comprises at least one compound according to formula (I). In another embodiment, the pharmaceutical composition comprises at least one self-assembled structure according to the present invention. In a further embodiment, the composition comprises at least one solid-lipid particle of the present invention.

[0169] In one embodiment, a pharmaceutical composition according to the present invention may be a lyophilized, spray freeze dried, lyophilized, or spray dried powder.

[0170] Pharmaceutical compositions according to the invention may contain pharma- ceutically acceptable carriers, excipients, diluents, additives and vehicles that are selected based on the intended mode of administration and consistent with conventional pharmaceutical practice. Suitable pharmaceutical carriers, excipients, diluents, additives and vehicles are known to those skilled in the art and described, for example, in publications such as Remington: The Science and Practice of Pharmacy.

[0171] The pharmaceutical compositions according to the invention may further comprise adjuvants, including but not limited to preservatives, wetting agents or antibacterial agents. Other adjuvants include but are not limited to cryoprotectants, spray drying aids, buffers, tonicity adjusting agents, and pH adjusting materials.

[0172] The amount of active ingredient which can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain from about 1 mg to about 5,000 mg of active ingredient, preferably 20 to 1000 mg, and most preferably 10 to 750 mg.

[0173] References to the mass of the active ingredient are not to the mass of the self-assembled structure or solid lipid particles thereof; It will be understood that this refers to the mass of the gemcitabine prodrug.

[0174] treatment method Another aspect of the present invention relates to the use of the self-assembled structures, solid lipid particles or pharmaceutical compositions thereof according to the present invention for tumor growth inhibition.In a preferred embodiment, the pharmaceutical compositions according to the present invention are used to inhibit the growth of solid tumors and metastatic tumors.In a particularly preferred embodiment, the pharmaceutical compositions according to the present invention are used to inhibit the growth of solid tumors or metastatic tumors associated with pancreatic cancer, colon cancer, colorectal cancer or breast cancer.

[0175] In one embodiment, there is provided a method of treating or preventing cancer in an individual comprising administering to a person in need thereof a self-assembled structure according to the present invention.

[0176] "Treatment" generally refers to both therapeutic treatment and prophylactic or preventative measures.

[0177] The purpose or outcome of treatment is to reduce the number of cancer cells; to reduce the size of the primary tumor; to inhibit (i.e., slow to some extent and preferably stop) invasion of cancer cells into peripheral organs, to inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, to inhibit to some extent tumor growth, and / or to alleviate to some extent one or more symptoms associated with the disorder.

[0178] The efficacy of treatment can be measured by assessing survival, time to disease progression, response rate (RR), duration of response, and / or quality of life.

[0179] In one embodiment, the method is particularly useful for slowing the progression of the disease.

[0180] In one embodiment, the method is particularly useful for extending human survival, including overall survival as well as progression-free survival.

[0181] In one embodiment, the method is particularly useful for providing a complete response to treatment, whereby all signs of the cancer have disappeared in response to the treatment, which does not necessarily mean that the cancer has been cured.

[0182] In one embodiment, the method is particularly useful for providing a partial response to treatment in which the size of one or more tumors or lesions, or the extent of cancer in the body, is reduced in response to the treatment.

[0183] In another embodiment, a method is provided for providing a prodrug of an active agent to an individual in need thereof in vivo, preferably the active agent is gemcitabine, and the prodrug undergoes a bioconversion reaction, for example, by hydrolysis, oxidation, reduction or enzymes. Cleavage to the active agent, preferably at the desired site of action (such as a tumor). The prodrug may exhibit one of a number of features compared to the active agent: reduced systemic toxicity, improved pharmacokinetics and / or pharmacodynamics, and improved stability in body fluids. The prodrug may allow for the delivery of a higher payload of the active agent and protect the active agent from premature inactivation, resulting in increased efficacy and reduced systemic toxicity. Preferably, the prodrug is provided in the form of a self-assembled structure. More preferably, the self-assembled structure includes an additional component selected from the group consisting of phospholipids, cholesterol, glycerol lipids, prodrug amphiphiles, and combinations thereof. Most preferably, the self-assembled structure includes an additional component selected from the group consisting of phospholipids, cholesterol, and combinations thereof.

[0184] It will be appreciated that the intended administration form of the self-assembled structure is either as its bulk phase, as a colloidal particle derived therefrom, or as a solid lipid particle.

[0185] The dosing regimen for the self-assembled structures, solid lipid particles or pharmaceutical compositions thereof according to the present invention will vary depending on known factors such as the pharmacodynamic properties of the compounds, the self-assembled structures, colloidal particles and their present compositions, their method and route of administration; and will also vary depending on the patient's age, sex, health, medical condition, weight, nature and extent of symptoms, type of concurrent treatment, frequency of treatment, renal, hepatic, cardiovascular and otherwise general health of the patient in need of such treatment, which can be readily determined by standard clinical techniques.

[0186] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention. EXAMPLES

[0187] The following examples are intended to illustrate the invention but not to limit it in any way.

[0188] Materials: All solvents were analytical grade and purchased from Merck Australia. All reagents were obtained from Sigma-Aldrich. Gemcitabine was purchased from Xingcheng Chempharm Co., LTD, China. Fatty acids and alcohols were purchased from NucheckPrep INC. (MN, USA). N-BOC-amino(PEG)3_alcohol linker was purchased from Leo Biochem (China). DiD' (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate) was purchased from Molecular Probes (Life Technologies).

[0189] instrumentation: Nuclear magnetic resonance (NMR): 1 H NMR spectra (200 MHz) were recorded on a Bruker AC400 spectrometer in deuterated solvents using tetramethylsilane ((CH3)4Si, TMS) as an internal standard unless otherwise stated. Solute concentrations were approximately 15 mg / ml in standard 5 mm NMR tubes. Spectra were analyzed using mNova software. Chemical shift values ​​(δ) were expressed in ppm and coupling constants were expressed as J values ​​in Hertz.

[0190] High performance liquid chromatography (HPLC): Analytical HPLC is available from Phenomenex Gemini C18 column (5μM, 4.6X150mm) and Altech 3300 evaporative light scattering (ELS) and Shimadzu UV-Vis (λ=260nm) detectors. The mobile phase for the analysis of the prodrug amphiphiles consisted of (A) 50:50 water / acetonitrile and (B) 60:40 tetrahydrofuran / acetonitrile.

[0191] Flash column chromatography: Purification of the prodrug amphiphiles was performed on a Reveleris® iES flash chromatography system (Grace Division Discover Sciences, Deerfield, IL, USA) using Reveleris® C1812 or 40 g and 80 g columns. The mobile phase for the analysis of the prodrug amphiphiles consisted of (Buffer A) water / ethanol (90 / 10), (Buffer B) ethanol.

[0192] Electrospray ionization mass spectrometry (ESI-MS): Electrospray ionization mass spectrometry (ESI-MS) was performed using an ESI and APCI interface. Samples were injected using a syringe pump. Methanol was used as the mobile phase unless otherwise stated.

[0193] Samples were typically delivered dissolved in DCM and the solvent was removed using a rotary evaporator under reduced pressure with a water bath temperature below 50°C.

[0194] Physicochemical properties Differential scanning calorimetry (DSC): Differential scanning calorimetry (DSC) measurements were performed on a Mettler Toledo DSC822 system equipped with a Mettler TSO 801RO sample robot (Mettler Toledo, Switzerland). Samples were prepared by weighing 4–8 mg of sample into 40 μL aluminum crucibles and sealing them. Samples were cooled to -130 °C and then heated to 300 °C at a rate of 2.5 °C / min. DSC thermograms were recorded using the STARe software package (Mettler Toledo, Switzerland). Indium was used for instrument calibration.

[0195] Polarized Optical Microscopy (POM): Samples used for polarized optical microscopy (POM) were prepared by placing a small amount of prodrug amphiphile on a microscope slide and covering with a coverslip. Water was placed on the edge of the coverslip, allowing the water to interact with 50 °C, and heated at 3 °C / min between room temperature and 50 °C. The amphiphile was observed in the presence and absence of cross-polarized lenses under an Olympus GX51 inverted optical microscope (Olympus Australia All specimens were observed at the National Agricultural Research Center (NARC), Stockholm, Sweden (Pty Ltd), Melbourne, Australia. Images were taken with a Nikon DS-Ri-1 camera. All images were taken under a microscope at 100x magnification.

[0196] Dynamic Light Scattering (DLS): Particle size distribution of the dispersions was analyzed using a Malvern Zetasizer (Nano ZS, Worcestershire, UK) equipped with a He-Ne laser (4 mW, 633 nm). Disposable 40 μL cuvettes with a scattering angle of 90° were used for all measurements. Samples were equilibrated for 1 min at 25 °C. Viscosity and refractive index values ​​were set to 0.8872 cP and 1.330, respectively, for all dispersions. Size distribution was recorded in terms of intensity and number.

[0197] Small-angle X-ray scattering (SAXS): SAXS analysis of the bulk and lyotropic phases of the amphiphiles was performed on a Bruker NanoSTAR laboratory SAXS instrument (Brucker AXS, Karlsruhe, Germany).

[0198] The equilibrated lyotropic phase was placed in a removable button sample cell. A multi-capillary holder was used for the analysis of the dispersions. The button cell and the capillary holder were interchangeably fitted into a block setup that was temperature controlled with a Peltier heater-cooler system. The 2D scattering images were radially averaged against conventional 1D scattering plots to obtain q values ​​(Å). -1 ) where q is the length of the scattering vector calculated using the equation q = (4π / λ) sin(θ / 2), where λ is the wavelength and θ is the scattering angle.

[0199] The mesophase formed was determined by indexing the peaks using the reflection law as described by De Campo et al., Langmuir, 2004. 20(13), 5254-5261. The small angle X-ray scattering patterns of emulsions and liposomes show large, broad peaks, whereas the liquid crystalline phases of highly ordered nanostructured particles such as cubosomes and hexosomes show distinct, sharp peaks. The relative position ("spacing") of the peaks allows elucidating the symmetry of the structure. The interplanar distance (d) between two reflecting planes can be calculated using the formula d=(2π) / q, where q is the absolute position of the peak. This allows the calculation of the lattice constant (a), which is the size of the unit cell.

[0200] Example 1: Synthesis of 2'-deoxy-2',2'-difluoro-N4-(((cis-9,octadecenyl)oxy)carbonyl)cytidine (gem-oleyl) Gemcitabine base (2.00 g, 7.60 mmol) was dissolved in DMF (10 mL) and placed in a round bottom flask. Anhydrous pyridine (3.06 mL, 38 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (4.82 mL, 38 mmol) was added dropwise to the solution and the reaction was stirred for 1 h. The product showed a cation m / Z of 408. Oleyl chloroformate (2.56 g, 7.6 mmol, molar ratio with gemcitabine 1:1) was dissolved in DMF (2 mL) and added dropwise to the solution. The reaction was left stirring at room temperature for 2 h. The reaction was quenched and the protecting silyl ether was removed by adding deionized water (3 mL) at the end of the reaction followed by stirring for 3 h. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water and then separated in an extraction funnel. The organic phase was collected, dried over Na2SO4, and then evaporated under reduced pressure to obtain the crude product. The crude product was purified in preparative HPLC using 40 g of Reveleris® C18. The pure fractions were collected and evaporated to dryness to obtain 2.30 g of a white wax (yield: 54.3%). 1 H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H J = 7.6 Hz, 6-CH); 7.09 (d, 1H J = 7.6 Hz, 5-CH); 6.3 (b, 1H, 1'-CH), 6.16 (t, 1H, 5'-OH); 5.35 - 5.28 (m, 3H, CH=CH and 3'-OH); 4.18 (dt, 1H. 4'-CH); 4.09 (t, 2H, J = 6.65 Hz, CO2-CH2); 3.8 - 3.67 (dd, 2H, 5'-CH2); 1.97 (q, 4H, -CH2-); 1.58 (m, 2H, -OH); 1.36 - 1.15 (m, 22H -CH2-); 0.84 (t, 3H, -CH3). ESI - MS (MeOH) +ve mode m / z: 558.10 [M + H]+, 1115.13 [2M +H]+.

[0201] Synthesis of intermediate compounds Example 1.1 Oleyl chloroformate The method used for the synthesis of oleyl chloroformate was based on a modified version of the method reported by Ding and Fritz. Oleyl alcohol (5 g, 18.62 mmol) was dissolved in DCM (30 mL), combined with a 1 / 3 molar ratio of triphosgene (1.84 g, 6.02 mmol) and stirred on an ice bath. Pyridine (1.5 mL, 18.62 mmol) was added dropwise to the solution and stirred for 1 h in the pre-cooled solution and for an additional 2 h at room temperature. The reaction mixture was then quickly extracted with ice-cold water and dried over Na2SO4. The solution was evaporated to an oil and used in further reactions without further purification (4.81 g, 78% yield).

[0202] Example 2: Synthesis of 2'-deoxy-2',2'-difluoro-N4-(((cis-9,12,octadecadienyl)oxy)carbonyl)cytidine (gem-linoleyl) Gemcitabine base (2.00 g, 7.60 mmol) was dissolved in DMF (10 mL) and placed in a round bottom flask. Anhydrous pyridine (3.06 mL, 38 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (4.82 mL, 38 mmol) was added dropwise to the solution and the reaction was stirred for 1 h. The product showed a cation m / Z of 408. Linoleyl chloroformate (2.5 g, 7.60 mmol, dissolved in DMF (2 mL) was added dropwise to the solution. The reaction was left stirring at room temperature for 2 h. The reaction was quenched and the protected silyl ether was removed by adding deionized water (6 mL) at the end of the reaction and stirring for 3 h. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water and then separated in an extraction funnel. The organic phase was collected and dried over Na2SO4 and then evaporated under reduced pressure to give the crude product. The crude product was purified using 40 g of Reveleris® C18. Mobile phase (A) 90:10 H2O / ethanol and (B) 100% ethanol. The pure fractions were collected and evaporated to dryness to give 2.6 g of a pale yellow wax (yield: 63.5%). 1H NMR (400 MHz, DMSO-d6): δ 10.82 (s, 1H, -NHCO); 8.21 (d, 1H, 6-CH); 7.10 (d, 1H, 5-CH); 6.16 (t, 1H, 5'-OH); 5.37 - 5.26 (m, 5H, CH=CH, 3'OH); 4.18 (q, 1H, 4'-CH); 4.10 (t, 2H, NHCO2-CH2); 3.89 -3.78 (dd, 2H, 5'-CH2); 2.73 (t, 2H, -CH2-) 2.00 (q, 4H, -CH2-); 1.59 (m, 2H, -CH2-); 1.36 - 1.20 (m, 16H, -CH2-); 0.84 (t, 3H, -CH3). ESI - MS (MeOH) -ve mode m / z: 554.27 [M - H]-, 590.07[M - H + Cl]-, 1109.13 [2M - 2H]-.

[0203] Synthesis of intermediate compounds Example 2.1: Linoleyl chloroformate The method used for the synthesis of linoleoyl chloroformate was based on a modified version of the method reported by Ding and Fritz. Linoleyl alcohol (3.5 g, 13.14 mmol) was combined with triphosgene (1.29 g, 4.35 mmol) in a 1 / 3 molar ratio in DCM (35 mL) on an ice bath. Pyridine (1.06 mL, 13.14 mmol) was added dropwise to the solution and the reaction mixture was stirred on ice for 1 h and then at room temperature for an additional 2 h. At the end of the reaction, the mixture was quickly extracted using ice-cold water and the organic layers were combined and dried over Na2SO4. The mixture was then evaporated under reduced pressure to give a yellow oil, which was used for further reactions without further purification (3.50 g, 81% yield).

[0204] Example 3: Synthesis of 2'-deoxy-2',2'-difluoro-N4-(((3,7,11,15-tetramethyl-hexadecyl)oxy)carbonyl)cytidine (gem-phytanyl) Gemcitabine base (2.00 g, 7.6 mmol) was dissolved in DMF (10 mL) and placed in a round bottom flask. Anhydrous pyridine (3.06 mL, 38 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (4.82 mL, 38 mmol) was added dropwise to the solution and the reaction was stirred for 1 h. The product showed a cation m / Z of 408. Phytanyl chloroformate (2.74 g, 7.6 mmol) was dissolved in DMF (2 mL) and added dropwise to the solution. The reaction was left stirring at room temperature for 2 h. The reaction was quenched and the protecting silyl ether was removed by adding deionized water (6 mL) at the end of the reaction followed by stirring for 3 h. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water and then separated in an extraction funnel. The organic phase was collected and dried over Na2SO4 before being evaporated under reduced pressure to give the crude product. The crude product was purified on a preparative HPLC using a Revelis® C18 12 g column. Mobile phase (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 3.22 g of a white crystalline wax (yield: 72.1%). 1 H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H J = 7.6 Hz, 6-CH); 7.10 (d, 1H J = 7.6 Hz, 5-CH); 6.3 (b, 1H, 1'-CH), 6.15 (t, 1H J = 7.5 Hz, 5'-OH); 5.25 (dd,1H, 3'-OH), 4.25 - 4.07 (m, 3H, 4'-CH, NHCO2-CH2); 3.89- 3.66 (dd, 1H 5'-CH2); 1.69 - 0.95 (m, 24H -CH, CH2); 0.90 - 0.77 (m, 15H, -CH3). ESI - MS (MeOH) -ve mode m / z: 586.10 [M - H]-, 621.95 [M - H + Cl]-, 1172.99 [2M - 2H]-.

[0205] Synthesis of intermediate compounds Example 3.1: 3,7,11,15-tetramethyl-hexadecyl chloroformate Phytanoyl (4.8 g, 16.35 mmol) and triphosgene (1.62 g, 5.46 mmol) were dissolved in 30 ml of dichloromethane, stirred and cooled on an ice bath. Anhydrous pyridine (1.3 g, 16.35 mmol) was added dropwise over 1 h. The reaction mixture was stirred at room temperature for an additional 1 h and then quickly extracted with additional methylene chloride and ice water. The organic layer was collected, dried over Na2SO4 and evaporated to dryness to give 5.22 g (yield: 88.7%) of phytanyl chloroformate as a yellow liquid. 1 H NMR in CDCl3: δ0.83, 0.85, 0.86, 0.88, (4s, each 3H, CH3), 0.91 (d, 3H, J=6.0Hz, CH3), 0.95-1.40 (m, 20H, CH2), 1.39-1.68 (m, 4H, CH), 1.68-1.88 (m, 1H, OH), 4.27-4.47 (m, 2H, CH2OH).

[0206] Example 4: Synthesis of 2'-deoxy-2',2'-difluoro-N4-((((cis-9,octadecenoyl)oxy-triethyleneglycol)oxy)carbonyl)cytidine (gem-PEG-3-oleoyl) Gemcitabine base (1.26 g, 4.8 mmol) was dissolved in 10 mL DMF. Anhydrous pyridine (2.0 mL, 24 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (2.61 mL, 24 mmol) was added dropwise to the solution and the reaction mixture was stirred for 1 h. The product showed a cation m / Z of 408. A solution of PEG 3-oleate (4.74 mmol) in chloroformic acid was dissolved in DMF (2 mL) and added dropwise to the reaction mixture. The reaction was left stirring at room temperature for 2 h. At the end of the reaction, deionized water (6 mL) was added followed by stirring for 1 h to remove the protected silyl ether. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water before being separated in an extraction funnel. The organic phase was collected and dried over Na2SO4 before being evaporated under reduced pressure to obtain the crude product. The crude product was purified on a preparative HPLC using 40 g of Reveleris® C18. Mobile phase (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 2.42 g of a white crystalline wax (yield: 72.1%). 1 H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H, 6-CH); 7.09 (d, 1H, 5-CH); 6.3 (b, 1H, 1'-CH), 6.16 (t, 1H, 5'-OH); 5.35 - 5.28 (m, 3H, CH=CH and 3'-OH); 4.25 (m, 2H, CH2-OCO), 4.18 (dt, 1H. 4'-CH); 4.09 (t, 2H,-CH2-OCO); 3.89 - 3.67 (dd, 1H, 5'-CH2); 3.67-3.45 (m, 8H, CH2-O,), 2.33 (t, 2H,CH2), 1.97 (dt, 4H, -CH2-); 1.48 (m, 2H, -CH2); 1.36 - 1.15 (m, 22H -CH2-); 0.84 (t, 3H, -CH3). ESI-MS (MeOH) +ve mode m / z: 704[M + H].

[0207] Synthesis of intermediate compounds Example 4.1: PEG3-Oleoyl Oleic acid (5 g, 17.7 mmol) was dissolved in 50 mL of DCM and cooled in an ice bath. Oxalyl chloride (5 g, 39.39 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred in the ice bath for an additional 10 min and then allowed to warm to room temperature and stirred for an additional 2 h. Excess oxalyl chloride was removed and the residue was redissolved in DCM and evaporated to dryness to remove oxalyl chloride and give an oily residue of oleoyl chloride. Oleoyl chloride (2.8 g, 8.89 mmol) was dissolved in 20 mL of DCM and pre-cooled on ice. Triethylene glycol (4.84 mL, 35.56 mmol) and TEA (1.28 mL, 8.89 mmol) were added to the reaction mixture. TEA was added as needed to adjust the pH to 9. The reaction mixture was stirred overnight. Products tested by ESI / MS show that PEG-3-oleate predominates with additional bis-PEG-oleate. The reaction mixture was evaporated under reduced pressure and the resulting residue was redissolved in DCM and extracted with water. The DCM phase was evaporated to dryness, redissolved in ethanol and purified by preparative HPLC using a Revelis® C18, 40 g column. Mobile phases (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 3.0 g of oil (yield: 81.5%). ESI-MS(MeOH)+ve mode / z:415 [M+H].

[0208] Example 4.2: PEG-3-oleoyl chloroformate PEG-3-oleoyl (2 g, 4.76 mmol) and triphosgene (0.470 g, 1.58 mmol) were dissolved in 20 mL of dichloromethane, stirred, and cooled on an ice bath. Anhydrous pyridine (0.3 mL, 7.24 mmol) was added dropwise to the reaction mixture, and the reaction mixture was stirred in the ice bath for 30 min, followed by stirring at room temperature for 2 h. The reaction mixture was quickly extracted with additional methylene chloride and ice water. The organic layers were pooled, dried over Na2SO4, evaporated to dryness, and used in the next step reaction with protected gemcitabine.

[0209] Example 5: Synthesis of 2'-deoxy-2',2'-difluoro-N4-((((cis-9,cis-12-octadecadienoyl)-oxy-triethyleneglycol)oxy)carbonyl)cytidine (gem-PEG-3-linoleoyl) Gemcitabine base (3.48 g, 13.2 mmol) was dissolved in 30 mL of DMF. Anhydrous pyridine (5.4 mL, 66 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (8.32 mL, 66 mmol) was added dropwise to the solution and the reaction mixture was stirred for 1 h. The product showed a cation m / Z of 408. A solution of PEG-3-linoleic acid in chloroformate (6.27 g, 13.2 mmol) was dissolved in DMF (6 mL) and added dropwise to the reaction mixture. The reaction was left stirring at room temperature for 2 h. At the end of the reaction, the protective silyl ether was removed by adding deionized water (12 mL) followed by stirring for 1 h. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water before being separated in an extraction funnel. The organic phase was collected and dried over Na2SO4 before being evaporated under reduced pressure to give the crude product. The crude product was purified on a preparative HPLC using a Reveleris® C18 40 g column. Mobile phase (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 6.27 g of a white crystalline wax (yield: 76%). 1 H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H, 6-CH); 7.09 (d, 1H, 5-CH); 6.3 (d, 1H, 1'-CH), 6.16 (t, 1H, 5'-OH); 5.55 (b, 3'OH) 5.35 - 5.28 (m, 4H, CH=CH); 4.25 (m, 2H, CH2-OCO), 4.18 (m, 4'-CH); 4.09 (t, 2H, CO2-CH2); 3.89 (dt, 1H. 4'-CH); 3.79 -3.67 (dd, 1H, 5'-CH2); 8H, CH2-O), 2.7 (m, 1H, CH), 2.33 (t, 4H,CH2), 1.97 (m, 2H, -CH2-); 1.48 (m, 2H, -CH2); 1.36 - 1.15 (m, 18H -CH2-); 0.84 (t, 3H, -CH3).ESI - MS (MeOH) +ve mode m / z: [M + H]: 702.20, [M + Na]: 724.33.

[0210] Synthesis of intermediate compounds Example 5.1: PEG-3-linoleoil Linoleic acid (5.61 g, 20 mmol) was dissolved in 50 mL of DCM and cooled in an ice bath. Oxalyl chloride (5 g, 40 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred in the ice bath for an additional 10 min, then allowed to warm to room temperature and stirred for an additional 2 h. Excess oxalyl chloride was removed and the residue was redissolved in DCM and evaporated to dryness to remove oxalyl chloride, leaving an oily residue of linoleoyl chloride.

[0211] The linoleoyl chloride obtained above was redissolved in 50 mL of DCM and pre-cooled in an ice bath. Triethylene glycol (12 g, 80 mmol) and TEA were added to the reaction mixture and the pH was adjusted to 9. The reaction mixture was stirred overnight. The products tested by ESI / MS showed that PEG3-lyoleoyl was predominant with additional bis-PEG3-linoleoyl. The reaction mixture was evaporated under reduced pressure and the resulting residue was redissolved in DCM and extracted with water. The DCM phase was evaporated to dryness, redissolved in ethanol and purified by preparative HPLC using a Revelis® C18, 40 g column. Mobile phases (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 6.18 g of oil (yield: 81.5%).

[0212] ESI-MS(MeOH)+ve mode m / z: 413 [M+H].

[0213] Example 5.2: PEG-3-linoleoyl chloroformate PEG-3-linoleoyl (6.18 g, 15 mmol) and triphosgene (1.48 g, 5 mmol) were dissolved in 100 mL of dichloromethane, stirred, and cooled on an ice bath. Anhydrous pyridine (1.209 mL, 15 mmol) was added dropwise to the reaction mixture, and the reaction mixture was stirred in the ice bath for 30 min, followed by stirring at room temperature for 2 h. The reaction mixture was quickly extracted with additional methylene chloride and ice water. The organic layers were pooled, dried over Na2SO4, evaporated to dryness, and used in the next step reaction with protected gemcitabine.

[0214] Example 6: Synthesis of 2'-deoxy-2',2'-difluoro-N4-((((cis-9,octadecenoyl)-oxy-hexaethyleneglycol)oxy)carbonyl)cytidine (gem-PEG6-oleoyl) Gemcitabine base (1 g, 3.80 mmol) was dissolved in 10 mL of DMF. Anhydrous pyridine (1.52 mL, 19 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (2.15 mL, 19.0 mmol) was added dropwise to the solution and the reaction mixture was stirred for 1 h. The product showed a cation m / Z of 408. A solution of PEG 6-oleate in chloroformic acid (2.1 g, 3.8 mmol) was dissolved in DMF (6 mL) and added dropwise to the reaction mixture. The reaction was left stirring at room temperature for 2 h. At the end of the reaction, the protecting silyl ether was removed by adding deionized water (12 mL) followed by stirring for 1 h. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water before being separated in an extraction funnel. The organic phase was collected and dried over Na2SO4 before being evaporated under reduced pressure to give the crude product. The crude product was purified on a preparative HPLC using a Revelis® C18 40 g column. Mobile phase (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 0.73 g of a yellow wax (yield: 23%). 1H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H, 6-CH); 7.09 (d, 1H, 5-CH); 6.3 (d, 1H, 1'-CH), 6.16 (t, 1H, 5'-OH); 5.35 - 5.28 (m, 2H, CH=CH, 3'-OH); 4.25 (m, 2H, CH2-OCO), 4.18 (m, 4'-CH); 4.09 (t, 2H, CO2-CH2); 3.79 -3.67 (dd, 1H, 5'-CH2); 3.67-3.45 (m, 20H, CH2-O), 2.33 (t, 2H,CH2), 1.97 (m, 4H, -CH2-); 1.48 (m, 2H, -CH2); 1.36 - 1.15 (m, 22H -CH2-); 0.84 (t, 3H, -CH3). ESI - MS (MeOH) +ve mode m / z: [M + H]: 836.20, [M + Na]: 858.33.

[0215] Synthesis of intermediate compounds Example 6.1: PEG-6-oleoyl Oleic acid (2.5 g, 8.85 mmol) was dissolved in 25 mL of DCM and cooled in an ice bath. Oxalyl chloride (2.5 g, 17.7 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred in the ice bath for an additional 10 min, then allowed to warm to room temperature and stirred for an additional 2 h. Excess oxalyl chloride was removed and the residue was redissolved in DCM and evaporated to dryness to remove oxalyl chloride and give an oily residue of oleoyl chloride.

[0216] The oleoyl chloride obtained above was redissolved in 50 mL of DCM and pre-cooled in an ice bath. Hexaethylene glycol (4.99 g, 17.7 mmol) and TEA were added to the reaction mixture and the pH was adjusted to 9. The reaction mixture was stirred overnight. The product tested by ESI / MS shows that PEG6-oleoyl predominates with additional bis-PEG6-oleoyl. The reaction mixture was evaporated under reduced pressure and the resulting residue was redissolved in DCM and extracted with water. The DCM phase was evaporated to dryness, redissolved in ethanol and purified by preparative HPLC using a Revelis® C18 40 g column. Mobile phases (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 2.9 g of oil (yield: 60%). ESI-MS (MeOH)+ve mode m / z: 547 [M+H].

[0217] Example 6.2: PEG-6-oleoyl chloroformate PEG-6-oleoyl (2.9 g, 5.32 mmol) and triphosgene (0.53 g, 1.77 mmol) were dissolved in 30 mL of dichloromethane, stirred, and cooled on an ice bath. Anhydrous pyridine (0.43 mL, 5.32 mmol) was added dropwise to the reaction mixture, and the reaction mixture was stirred in the ice bath for 30 min, followed by stirring at room temperature for 2 h. The reaction mixture was quickly extracted with additional methylene chloride and ice water. The organic layers were pooled, dried over Na2SO4, evaporated to dryness, and used in the next step reaction with protected gemcitabine.

[0218] Example 7: Synthesis of 2'-deoxy-2',2'-difluoro-N4-((((cis-9,12,15 octadecatrienoyl)-oxy-hexaethyleneglycol)oxy)carbonyl)cytidine (gem-PEG-6-linolenoyl) Gemcitabine base (0.42 g, 1.6 mmol) was dissolved in 5 mL DMF. Anhydrous pyridine (0.64 mL, 8 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (0.91 mL, 8 mmol) was added dropwise to the solution and the reaction mixture was stirred for 1 h. The product showed a cation m / Z of 408. A solution of PEG-6-oleic acid in chloroformate (0.97 g, 1.6 mmol) was dissolved in DMF (6 mL) and added dropwise to the reaction mixture. The reaction was left stirring at room temperature for 2 h. At the end of the reaction, the protecting silyl ether was removed by adding deionized water (12 mL) followed by stirring for 1 h. The reaction mixture was evaporated under reduced pressure and the resulting residue was taken up in diethyl ether and a small amount of water before being separated in an extraction funnel. The organic phase was collected and dried over Na2SO4 before being evaporated under reduced pressure to give the crude product. The crude product was purified on a preparative HPLC using a Reveleris® C18 40 g column. Mobile phase (A) 90:10 H2O / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 0.58 g of a yellow wax (yield: 50%). EI - 1H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H, 6-CH); 7.09 (d, 1H, 5-CH); 6.3 (d, 1H, 1'-CH), 6.16 (t, 1H, 5'-OH); 5.35 - 5.28 (m, 7H, CH=CH, 3'-OH); 4.25 (m, 2H, CH2-OCO), 4.18 (m, 4'-CH); 4.09 (t, 2H, CO2-CH2); 3.79 -3.67 (dd, 1H, 5'-CH2); 3.67-3.45 (m, 20H, CH2-O), 2.7 (m, 1H, CH), 2.33 (t, 2H,CH2), 1.97 (m, 4H, -CH2-); 1.48 (m, 2H, -CH2); 1.36 - 1.15 (m, 22H -CH2-); 0.84 (t, 3H, -CH3), ESI-MS (MeOH) +ve mode m / z: [M + H]: 832.20, [M + Na]: 854.33.

[0219] Synthesis of intermediate compounds Example 7.1: PEG 6- Linolenoyl Linolenic acid (1 g, 3.5 mmol) was dissolved in 10 mL of DCM and cooled in an ice bath. Oxalyl chloride (0.9 g, 7 mmol) was added dropwise to the reaction mixture. The reaction mixture was stirred in the ice bath for an additional 10 min, then allowed to warm to room temperature and stirred for an additional 2 h. Excess oxalyl chloride was removed and the residue was redissolved in DCM and evaporated to dryness to remove oxalyl chloride and give an oily residue of linolenoyl chloride.

[0220] The linolenoyl chloride obtained above was redissolved in 20 mL of DCM and pre-cooled in an ice bath. Hexaethylene glycol (0.56 g, 7 mmol) and TEA were added to the reaction mixture and the pH was adjusted to 9. The reaction mixture was stirred overnight. The product tested by ESI / MS shows that PEG6-linolenoyl is predominant with additional bis-PEG6-oleoyl. The reaction mixture was evaporated under reduced pressure and the resulting residue was redissolved in DCM and extracted with water. The DCM phase was evaporated to dryness, redissolved in ethanol and purified by preparative HPLC using a Revelis® C18 40 g column. Mobile phase (A) 90:10 HO / ethanol and (B) 100% ethanol. Pure fractions were collected and evaporated to dryness to give 0.95 g of oil (yield: 50%). 1 H NMR (400 MHz, DMSO-d6): δ 10.83 (s, 1H, -NHCO); 8.21 (d, 1H, 6-CH); 7.09 (d, 1H, 5-CH); 6.3 (d, 1H, 1'-CH), 6.16 (t, 1H, 5'-OH); 5.5 (b, 3'-OH), 5.35 - 5.28 (m, 6H, CH=CH); 4.25 (m, 2H, CH2-OCO), 4.18 (m, 4'-CH); 4.09 (t, 2H, 1.36 - 1.15 (m, 8H -CH2-); 0.84 (t, 3H, -CH3). ESI - MS (MeOH) +ve mode m / z: 545.13 [M + H].

[0221] Example 7.2: PEG-6-linolenoyl chloroformate PEG 6-linolenoyl (0.59 g, 1.75 mmol) and triphosgene (0.166 g, 0.58 mmol) were dissolved in 10 mL of dichloromethane, stirred and cooled on an ice bath. Anhydrous pyridine (0.15 mL, 1.75 mmol) was added dropwise to the reaction mixture and the reaction mixture was stirred in the ice bath for 30 min followed by stirring at room temperature for 2 h. The reaction mixture was quickly extracted with additional methylene chloride and ice water. The organic layers were pooled, dried over Na2SO4, evaporated to dryness and used in the next step reaction with protected gemcitabine.

[0222] Example 8: Synthesis of 2'-deoxy-2',2'-difluoro-N4-((((cis-9,octadecenoyl)amino-triethyleneglycol)oxy)carbonyl)cytidine (gem-PEG-3-oleoylamide) Gemcitabine base (1.052 g, 4 mmol) was dissolved in DMF (3 mL) and added to a round bottom flask and sealed under nitrogen. Anhydrous pyridine (1.62 mL, 20 mmol) was added to the solution and stirred on an ice bath using a magnetic stirrer. Chlorotrimethylsilane (2.172 mL, 20 mmol) was added dropwise to the solution and the reaction was stirred for 1 h. The product showed a cation m / Z of 408. N-Boc(PEG3)-chloroformate (4 mmol), the product obtained from the reaction described in 8.1, was added to the gemcitabine solution. Anhydrous pyridine (1.62 mL) was added and the reaction mixture was stirred at room temperature for 1 h. The product showed a cation m / Z of 682.7. The pyridine salt was removed by filtration and the filtrate was evaporated to dryness. The residue was redissolved in DCM (5 mL) and cooled to 4 °C. Trifluoroacetic acid (TFA, 5 mL) was added to the DCM solution and stirred at 4 °C for 30 min and at room temperature for 1 h. The product showed a cation (m / Z was 438.93). TFA and DCM were then evaporated under reduced pressure. The residue was redissolved in water and purified by preparative HPLC using 40 g of Reveleris® C18. Water and water / ethanol (50 / 50) were used as solvents A and B, with a linear gradient program from 100% solvent A to 100% solvent B. Appropriate fractions were pooled and evaporated to dryness. To this product (0.876 g, 2 mmol) was added oleoyl succinimide obtained from Example 8.2. The pH of the reaction was adjusted to 7.5-8 by adding triethylamine (0.7 mL). The title compound was formed after 30 min of reaction as confirmed by MS, showing the correct MS+ve mode m / Z: 703.14. The solvent was evaporated to dryness and the residue was redissolved in ethanol / water and purified by preparative HPLC using 40 g of Reveleris® C18. Water / ethanol (90 / 10) and ethanol were used as solvents A and B, with a linear gradient program from 100% solvent A to 100% solvent B. Appropriate fractions were pooled to give 450 mg of pure compound. Total yield 36%. ESI - MS (MeOH) +ve mode m / z: 703.14, 725.27, 1427.10.

[0223] Synthesis of intermediate compounds Example 8.1 N-Boc-PEG-3-chloroformate N-Boc-amino(PEG)3-OH (0.997 g, 4 mmol) was dissolved in 4 mL of DCM and added to a round bottom flask and sealed under nitrogen. Triphosgene (0.395 g, 1.33 mmol) dissolved in 3 mL of DCM was added to the reaction mixture at 4° C., followed by dropwise addition of pyridine (0.33 mL). The mixture was stirred at 4° C. for 30 min and at room temperature for 2 h. The product solution was further added to the protected gemcitabine solution (Example 8) without further purification.

[0224] Example 8.2 Oleoyl succinimide Oleic acid (0.564 g, 2 mmol) was dissolved in 2 mL of DCM and mixed with N-hydroxysuccinimide (HOSU, 0.276 g, 2.4 mmol). The mixture was sealed under nitrogen and stirred at 4° C. N,N'-dicyclohexylcarbodiimide (DCC, 0.453 g, 2.2 mmol) dissolved in 4 mL of DCM was added dropwise to the reaction mixture and stirred at 4° C. for 30 min and at room temperature for 3 h. The white DCU precipitate was filtered and the filtrate was used in the next step reaction without purification as described in Example 8.

[0225] Example 9: DSC of Gemcitabine-amphiphiles DSC was used to measure the temperatures associated with the phase transitions of various gemcitabine prodrug amphiphiles (Figure 2A). The DSC thermogram of gem-Ole shows endothermic transitions at 59.97 °C and 76.31 °C. This may be due to changes in the crystal structure. The endothermic peak at 98 °C corresponds to the melting point of the prodrug, which was confirmed by visual observation under an optical microscope. The large endothermic peak at 167.72 °C may be due to degradation of the sample, as this peak was not observed in subsequent heating and cooling samples (data not shown). A glass transition was observed at 4.81 °C in the DSC of gem-linoleyl. An exothermic peak occurred at higher temperatures starting at approximately 74 °C. This may be due to oxidation of the unsaturated linoleoyl chains. A second endothermic peak at ~137.82 °C was not observed in subsequent heating and cooling cycles, which may be due to degradation of the sample. Two glass transitions were observed in the DSC of gem-phytanyl at 2.92° C. and 79.31° C. A large endothermic peak was observed at 152.64° C. This indicates degradation of the sample.

[0226] DSC of gem amphiphiles using PEG spacers is shown in Figure 2B. Gem amphiphiles with a PEG3 spacer with either an oleoyl or linoleoyl chain showed large endothermic peaks at 85.6 and 79.1 °C. Adding the PEG length to 6 decreased the melting point of the samples to 59.7, while the corresponding melting point of linoleoyl decreased to 34.26 with a much smaller enthalpy of melting. The linolenoyl counterpart was an oil sample and did not show a melting point.

[0227] Demonstration of self-organizing behavior Example 10: Analysis of the mesophase behavior of gem amphiphiles using polarized optical microscopy (POM) and small angle X-ray scattering (SAXS) The water penetration scan and SAXS results are summarized in Tables 1 and 2 and show that a variety of self-assembled lyotropic liquid crystalline phases were formed for different gem-amphiphiles, ranging from lamellar crystalline structures to lamellar liquid crystals, cubic mesophases and sponge mesophases.

[0228] Textures obtained from water penetration scans along an established concentration gradient from pure amphiphile to pure water provide rapid insight into the lyotropic phase behavior of surfactant / water systems. The dissolved neat amphiphile was placed on a microscope slide and then water was introduced around the edge. Gem-phytanyl has the ability to first form a 3D inverse bicontinuous cubic phase (Pn3m space group) and then transform to a 2D hexagonal phase at elevated temperatures or prolonged equilibration. Similarly, the prodrug Gem-Ole was able to form a conventional lamellar (Lα) phase at room temperature and a 3D cubic (Pn3m) phase at elevated temperatures above 33 °C.

[0229] [Table 1]

[0230] [Table 2]

[0231] Example 11: Preparation of colloidal particles or dispersions Preferred prodrugs according to the present invention can be dispersed in aqueous solutions to form colloidal particles with very fine internal nanostructures in the size range of 100-1000 nm by using the following procedure.

[0232] Exemplary nanoparticle dispersions are prepared from gem-amphiphiles in the presence or absence of phospholipids (optionally containing cholesterol), where the phospholipids are selected from dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), and combinations thereof. An exemplary method is as follows: Gem-amphiphiles were hydrated with warm PBS containing 15-30% PEG-4K-oleoyl and sonicated in a sonicator bath using a probe sonicator resulting in lipid emulsions. Gem-amphiphile nanoparticles were processed through a polycarbonate membrane (3x200nm, 3x100nm) using an extruder (Avestin, LipoFast LF-50) to ensure homogenous sizing. Gemcitabine amphiphile nanoparticles containing phospholipids were prepared by mixing (i) DOPC and gem-oleoyl, (ii) DMPC / cholesterol and gem-phytanyl, or (iii) DMPC / cholesterol and gem-PEG3-oleoyl in ethanol followed by vigorous mixing. Gentle evaporation under reduced pressure produces a thin film. The thin film was then hydrated with warm PBS containing 10-15% PEG-4K-oleoyl solution and sonicated in a sonicator bath using a probe sonicator resulting in lipid emulsions. The nanoparticles were then processed through a polycarbonate membrane (3x200nm, 3x100nm) using an extruder (Avestin, LipoFast LF-50) to ensure uniform sizing.

[0233] Dispersions used for in vitro or in vivo studies were sterile filtered using a 220 μM filter. The final concentration of the nanoparticle solutions was 10 mg / mL. The particle size distribution and morphology of the above suspensions were determined using the methods described above with a Zetasizer and cryo-TEM characterization.

[0234] Solid lipid particles: Solid lipid particles of gem amphiphiles were prepared (manufactured) using a mechanical method according to the method described above.

[0235] Example 12: Morphology and size distribution 12.1 Cryo-TEM: Cryo-TEM images of gem-amphiphile nanoparticles, optionally containing phospholipids and cholesterol, were obtained using a laboratory-built vitrification system that allowed humidity to be kept close to 90% during sample plunging and vitrification. 4-5 μl of sample solution was applied to a 300 mesh copper TEM grid coated with lacey carbon film (ProSciTech, Thuringowa Qld4817Australia), allowed to settle for 30 s, the grid was manually blotted for 10-15 s, and the resulting thin film was vitrified by plunging into liquid ethane. The grid was stored in liquid nitrogen before being transferred to a Gatan626-DH cryo-holder. Imaging was performed using a FEITecnai 12TEM operating at 120 kV equipped with a MegaView III CCD camera and Analysis imaging software (Olympus Soft Imaging Solutions). Samples were kept at a temperature of -180 °C and standard low-dose procedures were used to minimize radiation damage. Representative cryo-TEM images of DMPC / Gem-phytanyl / cholesterol stabilized with PEG4K-oleoyl and DMPC / Gem-PEG3-oleoyl / cholesterol nanoparticles stabilized with PEG4K-oleoyl are shown in Figures 5(a) and 5(b), respectively. Nanoparticles made from Gem-PEG3-linoleoyl and Gem-PEG6-linoleoyl are shown in Figures 5(c) and 5(d), respectively.

[0236] 12.2 Characterization of dispersions: Particle size distribution: Determination of particle size distribution of nanoparticle dispersions (colloidal domes) was performed using a Zetasizer (nano zs, Malvern, England) equipped with a He-Ne laser (4mw, 633nm) and an avalanche photodiode detector. Dynamic light scattering (DLS) analysis was performed on the dispersions in a disposable sizing cuvette at 25 °C with a scattering angle θ = 90 °. Each measurement was repeated at least three times. In the data calculations, viscosity and RI values ​​of 0.8872 cp and 1.330, respectively, were used. Size distribution was recorded by intensity.

[0237] Representative nanoparticle dispersions prepared from various gem-amphiphiles are shown in Figure 6 .

[0238] Example 13: In vivo results The cytotoxicity of gemcitabine nanoparticle dispersions made from gemcitabine amphiphiles in the presence or absence of DMPC-cholesterol was evaluated in comparison to gemcitabine alone in several pancreatic cell lines including CF Pac-1, BxPC3, Panc-1 and MIA PaCa-2, Mcf7 and CACO2 cell lines. Cell lines were exposed to media with various concentrations of treatment (100-0.001 μM) and incubated for 72 h. Cell viability was determined using the MTS assay and IC 50 Triplicates were performed to derive values.

[0239] Treatment resulted in a dose-dependent decrease in cell viability in all cell lines. Experimental results showed that gemcitabine alone had IC50 values ​​ranging from 6nM to >100 in the PANC1 cell line. The IC50 of gemcitabine for the CFPAC-1 cell line at 72 hours ranges from 6.1-33nM. All gemcitabine amphiphilic nanoparticles were less toxic to cells. Without wishing to be bound by theory, we hypothesize that the reason for this phenomenon may be due to the longer time frame required for gemcitabine nanoparticles to convert to the active and cytotoxic free gemcitabine, resulting from further phosphorylation reactions to mono-, di- and triphosphates. On the other hand, free gemcitabine enters via transport proteins, which is different from the mechanism of nanoparticle uptake by endocytosis. Gemcitabine prodrugs also need to be bioconverted to gemcitabine to exert their cytotoxic effects, in contrast to gemcitabine already in its active state.

[0240] [Table 3]

[0241] Example 14: In-situ enzyme reaction 4.1: Preparation of nanoparticles for reaction kinetics studies Nanoparticle dispersions of a mixture of dimyristoylphosphatidylcholine and cholesterol were made at 50 μM concentration of (i) gemcitabine-phytanyl, (ii) gemcitabine-PEG3-oleoyl or (iii) gemcitabine-PEG3-oleoylamide gemcitabine prodrugs. A carboxylesterase solution from porcine liver obtained from Sigma-Aldrich was added to the nanoparticle dispersions to set the enzyme concentration in each nanoparticle dispersion to 33.7 units / mL.

[0242] 14.2: Enzymatic biotransformation reactions The time when the enzyme solution was added was designated as time zero for the hydrolysis reaction. The mixture was maintained at 37°C throughout the experiment. The concentrations of the hydrolysis products were monitored by LC / MS and LC / MS for the detection of the prodrug converted to active gemcitabine. 2 The reaction time of such a sample was determined as the time at which the sample was injected into the LC column. The bioconversion reaction to gemcitabine was monitored by MS. 2 The hydrolysis progress curves showing the decrease in the concentration of the original substrate were plotted versus time, whereas the concentration of the original substrate was determined by the method. Standard curves of both the prodrug and the active bioconverted gemcitabine were evaluated using standards.

[0243] A Thermo Finnigan (Finnigan LCQ series, Thermo Scientific, USA) LC / MS equipped with electrospray ionization (ESI) was used to evaluate the enzymatic reaction using both MS and MS2 modes. The depletion of the prodrug was detected by the negative ion form. The conversion of the prodrug to gemcitabine was detected by MS2 at 112.1. A 10 μl sample taken from the reaction solution was directly injected onto a Phenomenex C8 150 mmX2 mm5 uLuna column (Phenomenex, Australia). A 100% methanol solution was used as the mobile phase at a flow rate of 0.5 ml / min. The sample after column separation was linked to the ESI source. The capillary temperature and ion spray voltage were set at 350 °C and 4.60, respectively. The sample temperature was well maintained at 37 °C during the entire measurement by incubating the sample in a temperature-controlled autosampler. The data were acquired and processed with Xcalibur Quan chromatography software.

[0244] The retention times of the synthesized prodrugs were 5.26, 4.80, and 4.75 min, and the m / z were 585.97, 702.07, and 701.05, corresponding to gem-phytanyl, gem-PEG3-oleoyl, and gem-PEG3-oleoylamide, respectively.

[0245] The amount of reduced prodrug was calculated as the percentage of the area under the gemcitabine-prodrug peak compared to the area before addition of the enzyme. The amount of gemcitabine produced was calculated as the percentage of the area under the gemcitabine-prodrug peak compared to the area before addition of the enzyme. 2 was calculated from the area under the peak at 3.4 min associated with

[0246] The enzymatic hydrolysis reaction at comparable enzyme concentrations (33.7 units / mL) was plotted as the ratio of prodrug to original prodrug concentrations at specific incubation times (Figure 7a), and Figure 7b shows the amount of gemcitabine produced at the compared time intervals.

[0247] Gem-PEG3-oleoyl, which has an ester bond between the hydrophobic chain and PEG3, was rapidly reduced to Gem-PEG3 and oleic acid, whereas gemcitabine was bioconverted very slowly. Gem-PEG3-oleoylamide was reduced at a slower rate of approximately 6 h (Figure 7a), but produced gemcitabine persistently. Gem-phytanyl was the slowest prodrug to reduce completely, but produced gemcitabine at similar levels as Gem-PEG3-oleoylamide.

[0248] Example 15: In vivo tumor inhibition The antitumor efficacy of Gem-phytanyl-DMPC-cholesterol nanoparticles was evaluated over 34 days in comparison to free gemcitabine in a CFPAC-1 cell line-derived xenograft animal model. All treatments were administered by IV injection twice weekly for 4 weeks. The amount of gemcitabine administered in the Gem nanoparticles was 4.45 mg / kg, which is an order of magnitude smaller than the usual dose used in the clinic. The dose was equimolar to the gemcitabine control group, allowing for a direct comparison of antitumor efficacy between groups. The amount of total lipid injected was 1 mg / dose. Gemcitabine is administered intravenously at a dose of 1000 mg / m2 to treat pancreatic cancer in humans. The dose administered here is equivalent to 13.35 mg / m2 of gemcitabine injected into humans, which is 1.33% of the clinical dose of gemcitabine in humans. In other studies, the amount of gemcitabine injected into mice in in vivo studies ranged from 7-100 mg / kg.

[0249] Thirty mice were used in this experiment. However, one mouse in the saline control group failed to grow a tumor, so data analysis was performed on 29 mice. Thus, there were nine mice in the control group, ten mice in the gemcitabine group, and ten mice in the gem amphiphilic phospholipid / cholesterol nanoparticle group. Raw tumor volumes were graphed over time and displayed in Figure 8. While mice treated with native gemcitabine and gem amphiphilic phospholipid / cholesterol nanoparticles showed a decrease in tumor size over time, tumor size growth over time. The tumor volume of mice treated with native gemcitabine was 54% of the control tumor size, while the tumor volume of mice treated with gem amphiphilic phospholipid / cholesterol nanoparticles was 11% of the control tumor size, respectively. Thus, the general trend indicates that the size of tumors in the control group increased. Gemcitabine-treated tumors increased in size but at a slower rate compared to the control PBS group, and gemcitabine-treated tumors did not increase in size substantially.

[0250] Considering the relatively low doses used in the current experiments, a higher dose of gemcitabine would be ideal, but the limiting factor was the combination of the volume injected into the mice and the gem nanoparticle concentration. The gem nanoparticle stock solution was 5mg / mL. Due to injection volume limitations, a stock solution of 30mg / mL or higher is necessary, but dispersions above 10mg / mL were not stable and the gem nanoparticles precipitated out of solution.

[0251] At the end of the study, tumors were harvested. A general trend by visual inspection suggested control group > gemcitabine > gem nanoparticles, based on the mean tumor size. Tumors were weighed and determined to have the largest mean weight in control mice, followed by mice treated with gemcitabine, and finally tumors treated with gem nanoparticles (Figure 9). Thus, a general trend shows an increase in tumor weight in the following order: control group > gemcitabine group > gemcitabine nanoparticles (gem-phytanyl / DMPC / cholesterol: 18.38 / 70.52 / 8.10 w%). Statistical significance was observed between tumors in the control group and gemcitabine nanoparticles. Pairing indicates statistical significance (p<0.05).

[0252] Example 16: Biodistribution of Gem Nanoparticles To elucidate the in vivo biodistribution, mice were injected with fluorescently labeled Gem-amphiphilic phospholipid / cholesterol nanoparticles (fluorescent lipids of Gem-phytanyl / DMPC / cholesterol / DiD: 18.38 / 70.52 / 7.10 / 1w%). Mice were imaged at times 4, 24, 48, 72, and 96 h using a Carestream fluorescent imager (Figure 10). The results show the accumulation of fluorescent Gem nanoparticles in the tumor. A small amount of autofluorescence was observed in the ears and legs. The intensity / area of ​​fluorescence was quantified using ImageJ and these values ​​were graphed (maximum intensity was achieved after 72 h, suggesting that circulation continued for 72 h and then eventually accumulated in the tumor. After this period, the amount of fluorescence gradually decreased. The ex vivo distribution showed that the biological fate of Gem nanoparticles was mainly in the tumor and liver, and Gem nanoparticles were able to efficiently enter the tumor (passive targeting).

[0253] Example 17: In vivo tumor inhibition in a colorectal cancer xenograft model In another independent test, Gem-oleyl-DOPC nanoparticles were tested in a colorectal cancer xenograft model, as shown in Figure 11. The four groups in this set of experiments were prodrug nanoparticles, the active drug at 5mg / Kg / dose; control nanoparticles without the prodrug and bevacizumab (Avastin). The NPs were used at a low dose, about 1 / 10th the dose of gemcitabine normally used in mouse tumor models; and Avastin at a high dose of 25mg / Kg / dose, which is almost 10 times the usual dose for mouse experiments. The results show that very low levels of gem nanoparticles, 1 / 10th the clinical dose, efficiently inhibited tumor growth to levels not significantly lower than those of the high dose of 25mg / Kg Avastin. The general trend shows that the tumors in the control group increased in size, whereas the gemcitabine nanoparticle group inhibited tumor growth at a fraction of the clinical dose of gemcitabine (4mg / Kg).

[0254] Example 18: In vivo tumor inhibition in the Miapaca2 pancreatic cancer xenograft model In another test, Gem-PEG3-oleoyl / DMPC / cholesterol nanoparticles were tested in the highly resistant Mia paca2 pancreatic cancer xenograft model shown in Figure 12. The three groups in this series of experiments were: Gem nanoparticles at 15mg / Kg / dose of active gemcitabine, Gemcitabine drug solution at 15mg / Kg / dose and a control PBS group (Control PBS group).

[0255] Results show that the prodrug nanoparticle group inhibited tumor growth more efficiently than gemcitabine and significantly better than PBS. Although tumor growth in this highly resistant pancreatic cancer xenograft was not completely inhibited, the growth rate was slower with the prodrug nanoparticles compared to similar doses of gemcitabine.

[0256] References 1) Matsumura Y, Maeda H. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs. Cancer Research. 1986;46(12 Part 1):6387-92 2) Barenholz Y. Doxil(R)--the first FDA-approved nano-drug: lessons learned. Journal of controlled release: official journal of the Controlled Release 3) Drummond C.J, Sagnella S.M., Moghaddam M.J, Gong, X, Amphiphile prodrugs, US8603999B2. 4) Couvreur P., Stella B., Cattel L., Rocco F., Renoir J.M. Rosillo V, Gemcitabine derivatives nanoparticles, US20090130214A1. 5) Yangab S.C., Lub, L.F., Caic Y., Ahub,J.B. Liangc B.W., Yanga Z., Body distribution in mice intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain. Journal of controlled release, 1999, 3, 299-307. 6) Jin Y., Tong L, Ai P., Li M. and Hou X. Self-assembled drug delivery systems 1. properties and in vitro / in vivo behaviour of acyclovir self-assembled nanoparticles (SAN), International Journal of Pharmaceutics, 2006, 309: 199-207 7) Brannon-Peppas L. Blanchtte J.O., Nanopartiles and targeted systems for cancer theapy, Adv drug delivery reviews, 2012, 64, 206-212 8) Mehnert W., Maeder K., Solid lipid nanoparticles: production, characterisation and applications, Adv drug selivery rev. 2001, 472-3):165-96 9) Jin, et al. 2005. Colloid. Surf. B Biointerfaces 42, 45-51。

Claims

1. Prodrugs of general formula (II): 【Chemistry 1】 where X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), a substituent according to formula (d), and a substituent according to formula (e): 【Chemistry 2】 where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and analogs thereof; S is PEG n, n=1-10; L １ is a linker group covalently attached to the group S-R at one attachment site and to gemcitabine at a second attachment site via a carbamate bond to gemcitabine; L ２ is a linker group that is covalently attached to two S-R groups (which may be the same or different) at two independent attachment sites and is attached to gemcitabine at the third attachment site via a carbamate bond to gemcitabine; and L ３ is a linker group that is covalently attached to three S-R groups (which may be the same or different) at three independent binding sites and is attached to gemcitabine at the fourth binding site via a carbamate bond to gemcitabine.

2. Prodrugs of General Formula I: 【Chemistry 3】 where A is a biologically active agent; Y is a cleavable bond between the biologically active agent and X; and X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), and a substituent according to formula (d): 【Chemistry 4】 where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and analogs thereof; S is (polyethylene glycol)n, where n=1-10; L １ is a linker group covalently attached to one S-R group at one attachment site and to A at a second attachment site via a biolabile bond Y to A; L ２ is a linker group covalently attached via a biolabile bond Y to A to two S-R groups (which may be the same or different) at two independent attachment sites and to A at a third attachment site; and L ３ is a linker group that is covalently attached to three S-R groups (which may be the same or different) at three independent attachment sites and to A at a fourth attachment site via a biolabile bond Y to A.

3. 3. The prodrug of claim 2, wherein Y is selected from the group consisting of amides, carbamates, imides, sulfonamides, and amines.

4. The prodrug of claim 3, wherein Y is a carbamate.

5. 5. The prodrug of claim 1, wherein R is selected from the group consisting of myristyl, myristoyl, myristoylamide, palmityl, palmitoyl, palmitoylamide, stearyl, stearoyl, stearoylamide, oleyl, oleoyl, oleoylamide, linoleyl, linoleoyl, linoleoylamide, linolenyl, linolenoyl, linolenoylamide, arachidonyl, arachidonoyl, arachidonoylamide, phytanyl, phytanoyl, phytanoylamide, hexahydrofarnesyl, hexahydrofarnesoyl, and hexahydrofarnesoylamide.

6. 6. The prodrug of claim 5, wherein R is selected from the group consisting of oleyl, linoleyl, linolenoyl and phytanyl.

7. The prodrug of any one of claims 1 to 6, wherein X is a substituent according to formula (a).

8. S is peg ３－６ The prodrug of claim 7,

9. 9. The prodrug of claim 7 or 8, wherein R is selected from the group consisting of oleoyl, oleoylamide, linoleoyl, linoleoylamide, linolenoyl, linolenoylamide, phytanoyl, and phytanoylamide.

10. 7. The prodrug of any one of claims 1, 5 or 6, wherein X is a substituent according to formula (e).

11. 11. The prodrug of claim 10, wherein R is selected from the group consisting of oleyl, linoleyl, linolenoyl and phytanyl.

12. 12. A self-assembled structure formed from the prodrug of any one of claims 1 to 11, said structure exhibiting a mesophase under physiological conditions selected from the group consisting of a lamellar phase, a bicontinuous cubic phase, a hexagonal phase and a sponge phase.

13. 13. The self-assembled structure of claim 12, wherein the mesophases exhibited are a lamellar phase and a reverse phase.

14. A self-assembled structure formed from the prodrug of any one of claims 1 to 11, said structure being a solid lipid particle.

15. The self-assembled structure of any one of claims 12 to 14, further comprising a component selected from the group consisting of phospholipids, cholesterol, glycerol lipids, prodrug amphiphiles and combinations thereof.

16. 1. A method of modulating the release of a biologically active compound or agent that can be metabolized to a biologically active agent A, comprising covalently linking A to at least one tail moiety X to form an amphiphile capable of self-assembling into a self-assembled structure under physiological conditions, said amphiphile being cleavable in vivo to release A in a biologically active form, wherein: A is gemcitabine; X is selected from the group consisting of a substituent according to formula (a), a substituent according to formula (b), a substituent according to formula (c), a substituent according to formula (d), and a substituent according to formula (e): 【Chemistry 5】 where R is selected from the group consisting of alkyl, alkenyl, alkynyl, branched alkyl, branched alkenyl, branched alkynyl, substituted alkyl, substituted alkenyl, and substituted alkynyl groups and analogs thereof; S is PEG n, n=1-10; L １ is a linker group covalently attached to the group S-R at one attachment site and to a therapeutically active agent A at a second attachment site, exhibiting a biolabile bond to A; L ２ is a linker group that is covalently attached to two S-R groups (which may be the same or different) at two independent attachment sites and to a therapeutically active agent A at a third attachment site via a biolabile bond to A; and L ３ is a linker group that is covalently attached to three S-R groups (which may be the same or different) at three independent attachment sites and is attached to a therapeutically active agent A at the fourth attachment site by a biolabile bond to A.

17. 17. The method of claim 16, wherein the amphiphile is an enzyme substrate for an enzyme that promotes the formation of a biologically active form of the compound.

18. A method for treating or preventing cancer in a human, comprising administering to a human in need thereof a self-assembled structure according to any one of claims 12 to 15.

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