Patent Application
20250049931
This application claims the benefit of the European Patent Application 21383149.8 filed on 17 Dec. 2021.
The present invention relates to polysarcosine-vitamin E derivatives, and to conjugates comprising them wherein the polysarcosine-vitamin E derivatives are covalently attached to other moieties such as e.g., diagnostic agents or therapeutic agents. It also relates to compositions comprising the polysarcosine-vitamin E derivatives and/or their conjugates as well as to self-assembled particles formed from them which may encapsulate a variety of agents, and to their uses in medicinal, cosmetic or food applications.
The vitamin E family is a group of fat-soluble compounds which includes tocopherols and tocotrienols. Both tocopherols and tocotrienols include a chroman ring and a 16-carbon side chain in their structure. The difference between them lies in that while the carbon side chain of the tocopherols is saturated, the side chain of the tocotrienols is unsaturated. Each tocopherol and tocotrienol exists in four different forms: α, β, γ, δ, which differ in the number and position of methyl groups on the ring as shown below. The most common and biologically active form is α-tocopherol, which is often referred to as vitamin E.
Vitamin E is an essential food ingredient. It has been identified as an essential factor for reproduction. It has antioxidant, anti-thrombolytic and other therapeutic effects. The antioxidant properties of vitamin E derivatives are linked with the 6-hydroxy-chroman ring of their chemical structure.
Low blood levels of Vitamin E are associated with increased risk of development of degenerative diseases. This has prompted researchers to study the ability of vitamin E to prevent chronic diseases related to oxidative stress, including cardiovascular and neurodegenerative diseases, atherosclerosis, and cancer. Further, the involvement of vitamin E in cell signaling and gene expression has also been reported. Additionally, there is an emerging interest in the use of vitamin E derivatives as drug delivery systems to increase cell permeability and access to the desired targets, as well as for medical imaging as contrast agents or fluorescent probes.
Vitamin E tends to be an unstable molecule. Besides, its poor water solubility has greatly limited its application. In order to improve its shelf life, vitamin E has been supplied in the form of derivatives in which the 6-hydroxy moiety attached to the chroman ring is esterified with an acetate or a succinate moiety to render vitamin E acetate or vitamin E succinate derivatives.
α-tocopherylsuccinate was the first reported vitamin E derivative for which activity against growth inhibition of cancer cells was demonstrated. However, this derivative is highly hydrophobic and toxic. Efforts to overcome the poor water solubility of vitamin E led to the preparation of tocopheryl polyethylene glycol succinate (TPGS) also called tocophersolan, which is obtained by esterification of vitamin E succinate with poly(ethylene glycol) (PEG). TPGS has the following chemical structure:
TPGS has been approved by the US FDA as a nutritional supplement and drug delivery vehicle. It can be used as solubilizer, absorption and permeation enhancer, emulsifier as well as surface stabilizer in drug delivery. TPGS may act as anticancer agent against many cancer types. In this regard, conjugates of TPGS with e.g., doxorubicin, paclitaxel or cisplatin have been described. Additionally, TPGS-based polymers can improve the drug encapsulation efficiency, intracellular uptake, and therapeutic effect.
Despite the above, TPGS has some drawbacks associated to the inclusion of PEG in its structure. PEG is widely used in drug delivery and nanotechnology because of its stealth properties and biocompatibility, which allows particulate delivery systems and biomaterials to evade the immune system, thereby having a prolonged circulation time within the biological system. However, it has been reported that PEG may cause an unexpected immunogenic response known as the accelerated blood clearance (ABC) phenomenon, resulting in the increased clearance and reduced efficacy of PEG-conjugated substances. Furthermore, PEG conjugates show poor degradability.
Therefore, there is still the need to develop alternative vitamin E derivatives which overcome the problems of the prior art.
The inventors have developed new vitamin E derivatives based on polysarcosine (PSar), which, unlike PEG-containing derivatives such as TPGS, are biodegradable, non-immunogenic, and show excellent shelf-life. The polysarcosine-vitamin E derivatives of the invention are amphiphilic. They comprise a hydrophilic polar head portion, the PSar-based moiety, and a hydrophobic part, the vitamin E-based moiety. As hybrid compounds, they have the properties of both components. Thus, on the one hand, they show excellent solubility in water and organic solvents thanks to the PSar-based moiety, and, on the other hand, they have antioxidant properties due to the Vitamin E-based moiety.
As illustrated in the examples below, the polysarcosine-vitamin E derivatives of the invention may form self-assembled particles in solvents, such as micelles, and encapsulate molecules such as active agents or imaging contrast agents. The polysarcosine-vitamin E derivatives showed higher accumulation of the delivered agent in the epidermis layer in comparison to analogue TPGS, which demonstrates that PSar-based materials may be advantageous over TPGS for skin delivery applications, while avoiding the disadvantages associated with PEG.
Therefore, a first aspect of the invention relates to a polymer comprising a repeating sarcosine-based unit which is covalently attached directly or through a linker to a vitamin E-based moiety, or a pharmaceutically, nutraceutically, or cosmetically acceptable salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the polymer or of any of its salts; wherein the repeating sarcosine-based unit has the formula (XXIII)
wherein R5 is selected from the group consisting of methyl, —CH2F, —CHF2, and —CF3; R6 and R7 are independently selected from the group consisting of hydrogen and fluorine; and n is an integer from 5 to 500; and the vitamin E-based moiety has the formula (XXIV):
wherein R2, R3 and R4 are independently selected from the group consisting of hydrogen, fluorine, methyl, —CH2F, —CHF2, and —CF3; and each of the dashed bonds is independently a single bond or, alternatively, a double bond. In the polymer of the invention the repeating sarcosine-based unit is covalently attached directly or through a linker to the vitamin E-based moiety by any of the attachment points indicated herein with an “*”. The vitamin E-based moiety is covalently attached directly or through a linker to the repeating sarcosine-based unit by the attachment point indicated herein with a wavy line “
” that intersects a bond.
More particularly, the polymer comprising a repeating sarcosine-based unit which is covalently attached directly or through a linker to a vitamin E-based moiety is a polymer of formula (I),
wherein:
The polysarcosine-vitamin E derivatives of the invention may form covalent conjugates with other molecules. Thus, another aspect of the invention relates to a conjugate of formula (II), or a pharmaceutically, nutraceutically, or cosmetically acceptable salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the conjugate of formula (II) or of any of its salts
wherein:
As mentioned above, thanks to their amphiphilic nature, the polysarcosine-vitamin E derivatives of the invention may form self-assembled particles in solution such as for example micelles or lipid nanoparticles which may be used to encapsulate and deliver molecules of different nature. Accordingly, another aspect of the invention relates to a self-assembled particle comprising:
The polysarcosine-vitamin E derivatives of the invention as well as the conjugates of formula (II) as defined herein may be formulated in a variety of compositions such as nutraceutical, pharmaceutical, diagnostic, or cosmetic compositions. Thus, another aspect of the invention relates to a composition comprising an effective amount of:
The polysarcosine-vitamin E derivatives of the invention, their conjugates, self-assembled particles, and compositions thereof may be used in medicinal, cosmetic or food applications. Thus, another aspect of the invention relates to
Another aspect of the invention relates to
According to another aspect, the invention relates to the use of the polymer as defined herein as a carrier, as a solubilizer, as absorption and permeation enhancer, as emulsifier or as surface stabilizer.
All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply throughout the description and claims.
The term “about” or “around” as used herein refers to a range of values ±10% of a specified value. For example, the expression “about 10” or “around 10” includes ±10% of 10, i.e., from 9 to 11.
As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun.
The term (C1-Cx)alkyl refers to a saturated linear or branched hydrocarbon chain which contains from 1 to x carbon atoms and only single bonds. Examples of alkyl groups may include without limitation methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, neopentyl, n-hexyl, decyl and undecyl. The term (C2-Cx)alkenyl refers to an unsaturated branched or linear hydrocarbon chain which comprises from 2 to x carbon atoms and at least one or more double bonds. Examples of alkenyl groups may include without limitation ethenyl (i.e. vinyl), allyl, propenyl, butenyl, pentenyl and hexenyl.
The expression “biradical chain which comprises one or more moieties” as defined herein is intended to encompass the biradicals as defined herein. The biradicals may be optionally substituted as indicated along the document.
The term “—(C1-C6)alkyl-O—(C1-C6)alkyl” refers to an alkyl chain as defined above wherein one of the hydrogen atoms has been substituted by a —O—(C1-C6)alkyl moiety, i.e., al alkoxy group. Examples of this radical include, without limitation, 2-methoxyethyl, 3-methoxy propyl, 4-methoxy butyl, 2-ethoxyethyl, 3-ethoxy propyl, and 4-ethoxy butyl.
The term “—(C1-C12)alkylphenyl” refers to an alkyl chain as defined above wherein one of the hydrogen atoms has been substituted by a phenyl moiety. Examples of this radical include, without limitation, benzyl, 2-phenetyl, and 3-phenylpropyl.
The term “-phenyl(C1-C6)alkyl” refers to an phenyl radical wherein one of the hydrogen atoms has been substituted by an alkyl group as previously defined. Examples of this radical include, without limitation, (2-methyl)phenyl, (4-methyl)phenyl, and (3-ethyl)phenyl.
The terms “alkylene” or “phenylene” refer to bivalent radicals.
The term “(C6-C12)aryl” refers to an aromatic carbocyclic mono- or bicyclic ring system comprising 6 to 12 carbon ring atoms. Examples of aryl moieties include, without limitation, phenyl and naphthyl.
The term “halogen” or “halo” as used herein means fluorine, chlorine, bromine, and iodine, preferably fluorine, chlorine, and bromine, more preferably fluorine and chlorine.
The expression “substituted with one or more” means that a group can be substituted with one or more, preferably with 1, 2, 3, 4, 5 or 6 substituents, provided that this group has enough positions susceptible of being substituted.
The term “chemically feasible” as used herein refers to the connectivity of atoms that satisfies the chemical valency of each atom and is expected to be stable by a skilled person.
As used herein, a wavy line “” that intersects a bond in any chemical structure disclosed herein and the “*” represent the points of attachment to the remainder of the molecule. The wavy lines and the asterisks are used interchangeably.
A first aspect of the invention refers to a polymer comprising a repeating sarcosine-based unit of formula (XXIII) which is covalently attached directly or through a linker to a vitamin E-based moiety of formula (XXIV), or a pharmaceutically, nutraceutically, or cosmetically acceptable salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the polymer or of any of its salts.
In the polymers of the invention the repeating polysarcosine-based unit may be placed in any order. Thus, the repeating sarcosine-based unit of formula (XXIII) may be attached directly or through a linker to the vitamin E-based moiety either by the carbonyl group or by the amino group.
This different orientation of the repeating polysarcosine-based moiety does not influence the amphiphilic behaviour of the polymer described herein, and consequently the activity displayed is maintained.
There is no limitation on the type of salt of the polymers of the invention that can be used, provided that these are pharmaceutically, nutraceutically, or cosmetically acceptable when they are used for pharmaceutically, nutraceutical or cosmetic purposes, respectively. The term “pharmaceutically, nutraceutically, or cosmetically acceptable salts”, embraces non-toxic salts commonly used. The preparation of pharmaceutically, nutraceutically, or cosmetically acceptable salts of the polymers of the invention can be carried out by methods well-known in the art. Generally, such salts can be prepared by reacting the free acid or base form of a polymer of the invention with a stoichiometric amount of an appropriate base or acid, respectively, in a suitable solvent such as water, an organic solvent or a mixture of them. The polymers of the invention and their salts may differ in some physical properties, but they are equivalent for the purposes of the present invention.
Some polymers of the invention can have chiral centres that can give rise to various stereoisomers. As used herein, the term “stereoisomer” refers to all isomers of individual polymers that differ only in the orientation of their atoms in space. The term stereoisomer includes mirror image isomers (enantiomers), mixtures of mirror image isomers (racemates, racemic mixtures), geometric (cis/trans or syn/anti or E/Z) isomers, and isomers of polymers with more than one chiral center that are not mirror images of one another (diastereoisomers). The present invention relates to each of these stereoisomers and also mixtures thereof. Diastereoisomers and enantiomers can be separated by conventional techniques such as chromatography or fractional crystallization. Optical isomers may be obtained using enantiospecific synthesis. Alternatively, optical isomers can be resolved by conventional techniques to give optically pure isomers. The resolution can be carried out on any chiral synthetic intermediates or on the polymers of the invention.
In all embodiments of the invention referring to the polymers comprising a repeating sarcosine-based unit and a vitamin E-based moiety, the pharmaceutically, nutraceutically, or cosmetically acceptable salts thereof and the stereoisomers or mixtures of stereoisomers, either of any of the polymers or of any of their pharmaceutically, nutraceutically, or cosmetically acceptable salts are always contemplated even if they are not specifically mentioned.
The polymers of the invention may be in crystalline form either as free solvation polymers or as solvates (e.g., hydrates). It is intended that all these forms are within the scope of the present invention. Methods of solvation are generally known within the art. For the purposes of the invention, the solvated forms with pharmaceutically, nutraceutically, or cosmetically acceptable solvents such as water, ethanol and the like are equivalent to the unsolvated form.
Except as otherwise specified, the polymers of the present disclosure also include polymers that differ only in the presence of one or more isotope-enriched atoms. Examples of isotope-enriched atoms, without limitation, are deuterium, tritium, 13C or 14C, or a nitrogen atom enriched in 15N or a fluorine atom enriched in 18F.
The polymers of the invention are structures containing a polysarcosine-based moiety as a repeating unit. These polymers are characterized by a molecular weight (which can be average molecular weight (MW) or number average molecular weight (Mn)), a degree of polymerization, and a polydispersity index. The molecular weight can be measured by methods well-known in the art such as size exclusion chromatography (SEC) (also referred to as gel permeation chromatography (GPC)) matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) or 1H-NMR spectroscopy, for example. Some of these methods are indicated in more detail in the examples below.
As used herein, the term “repeating unit”, refers to the monomeric sarcosine-based unit and is defined by brackets depicted around it. The number on the lower right of the brackets (represented by letter “n”) refers to the degree of polymerization (DP) for each monomeric unit as a statistical number. The DP is calculated by dividing the molecular weight of the polymer given by SEC-MALS technique by the molecular weight of the monomer unit. The DP value is reported as the central value of a gaussian distribution which comprises polymers of variable DP (depending on intrinsic polydispersity). The DP value is subject to a reasonable uncertainty, due to the ring-opening polymerization mechanism, which, in the context of the present invention, may be considered within the range ±20%, preferably ±15%, more preferably ±10%, even more preferably ±5%, being particularly preferred ±2%.
The term “polydispersity index” (PDI) is used as a measure of broadness of molecular weight distribution. The larger the PDI, the broader the molecular weight. PDI of a polymer is calculated as the ratio of weight average (MW) by number average (Mn) molecular weight.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polymer of the invention comprising a repeating sarcosine-based unit and a vitamin E-based moiety has the formula (I),
wherein R1-R4, X, PAA, L, m, n and z are as defined herein.
The polymers of formula (I) include a polysarcosine-based moiety PAA (drawn in the left part of the formula) and a vitamin E-based moiety (drawn in the right part of the formula) which are covalently bonded directly or through a linker L.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular in the polymer of formula (I), the degree of polymerization of the polymer, or n, is from 5 to 400, or from 5 to 300, or from 5 to 200, or from 5 to 100, or from 5 to 50, or from 5 to 40, or from 5 to 35, or from 5 to 25, and more particularly is about 6, or 15, or 20, in particular measured by SEC or 1H-NMR spectroscopy as indicated in the examples.
According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the average molecular weight (Mw) of the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular of the polymer of formula (I), is from 700 to 50000 Da, more particularly from 1000 to 20000 Da, and even more particularly from 1000 to 5000 Da or from 1000 to 3000 Da, measured by SEC as disclosed in the examples.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the polydispersity index of the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular of the polymer of formula (I), is from 1.01 to 1.80, or from 1.01 to 1.50, or from 1.01 to 1.30, or from 1.01 to 1.20, or from 1.01 to 1.10 measured by SEC as disclosed in the examples.
In the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular in the polymer of formula (I), R5 is selected from the group consisting of methyl, —CH2F, —CHF2, and —CF3. According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, R5 is methyl.
In the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular in the polymer of formula (I), R6 and R7 are independently selected from the group consisting of hydrogen and fluorine. According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, R6 and R7 are hydrogen.
In the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular in the polymer of formula (I), R2, R3 and R4 are independently selected from the group consisting of hydrogen, fluorine, methyl, —CH2F, —CHF2, and —CF3. According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, R2, R3 and R4 are independently selected from the group consisting of hydrogen and methyl.
According to one embodiment, the polymer comprising a repeating sarcosine-based unit and a vitamin E-based moiety, and in particular in the polymer of formula (I), includes a vitamin E part which may encompass any of the 8 possible different forms of vitamin E (α, β, γ, δ tocopherols and α, β, γ, δ tocotrienols).
Thus, in one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of the invention, and in particular in the polymer of formula (I), each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond, and R2, R3 and R4 are selected from the following meanings: (i) R2, R3 and R4 are methyl, (ii) R2, R4 are methyl and R3 is hydrogen, (iii) R3 and R4 are methyl and R2 is hydrogen, and (iv) R4 is methyl and R2 and R3 are hydrogen. In a more particular embodiment, each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond and R2, R3 and R4 are methyl. Even more particularly, the vitamin E-based moiety of the polymer of the invention, and in particular in the polymer of formula (I), has the formula (XXIV′):
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (I), each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond, and R2, R3 and R4 are selected from the following meanings: (i) R2, R3 and R4 are methyl, (ii) R2, R4 are methyl and R3 is hydrogen, (iii) R3 and R4 are methyl and R2 is hydrogen, and (iv) R4 is methyl and R2 and R3 are hydrogen. In a more particular embodiment, each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond and R2, R3 and R4 are methyl.
In the polymer of formula (I), R1 is selected from the group consisting of —(C1-C12)alkyl, —(C1-C12)alkylphenyl, —(C1-C6)alkyl-O—(C1-C6)alkyl, —CO—(C1-C12)alkyl, —CO—(C1-C12)alkylphenyl, and —CO—(C1-C6)alkyl-O—(C1-C6)alkyl; wherein the alkyl groups may be optionally substituted as defined herein. More particularly, when PAA is a repeating sarcosine-based unit of formula (XXI), and z is 1, R1 is selected from the group consisting of —(C1-C12)alkyl, —(C1-C12)alkylphenyl, and —(C1-C6)alkyl-O—(C1-C6)alkyl; and when PAA is a repeating sarcosine-based unit of formula (XXII), and z is 0, R1 is selected from the group consisting of —CO—(C1-C12)alkyl, —CO—(C1-C12)alkylphenyl, and —CO—(C1-C6)alkyl-O—(C1-C6)alkyl; wherein all the alkyl groups may be optionally substituted as defined herein.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (I), particularly when PAA is a repeating sarcosine-based unit of formula (XXI), and z is 1, R1 is selected from the group consisting of —(C1-C6)alkyl, —(C1-C3)alkyl-O—(C1-C3)alkyl, and —(C1-C6)alkylphenyl; and wherein the alkyl groups may be optionally substituted as defined herein. More particularly, R1 is selected from the group consisting of isopropyl, 2-methoxyethyl, n-propyl, n-butyl, neopentyl, benzyl, and 2-(2-methoxyethoxy)ethyl, and even more particularly R1 is isopropyl.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (I), particularly when PAA is a repeating sarcosine-based unit of formula (XXII), and z is 0, R1 is selected from the group consisting of —CO—(C1-C6)alkyl, —CO—(C1-C6)alkyl-O—C1-C3)alkyl, and —CO—(C1-C6)alkylphenyl; and wherein the alkyl groups may be optionally substituted as defined herein. More particularly, R1 is acetyl.
In the polymers of the invention the repeating polysarcosine-based unit may be placed in any order. Thus, in the polymers of formula (I), the repeating sarcosine-based unit PAA is selected from formula (XXI) and formula (XXII):
wherein R5, R6, R7 and n are as defined herein, “*1” denotes the attaching point of the repeating sarcosine-based unit PAA to X or R1, and “*2” denotes the attaching point of the repeating sarcosine-based unit PAA to the linker L or the oxygen atom of the vitamin E-based moiety.
This different orientation of the repeating polysarcosine-based moiety does not influence the amphiphilic behaviour of the polymer described herein, and consequently the activity displayed is maintained.
In the polymers of formula (I), the linker L is a biradical chain which comprises one or more moieties selected from the group consisting of —CH═CH—, —C≡C—, —CH2—, —N═CH—, —CH═NH—, —NH—, —NH—NH—, —NH—N═CH—, —O—, —O(CH2)O—, —CO—, —O(CO)—, —COO—, —C(═CH2)—, —C(═NH)—, —CONH—, —NHCO—, —NH(CO)NH—, —S—, —S—S—, —SO—, —SO2—, —SO2NH2— and -phenylene-, wherein L is optionally substituted as defined herein.
As a biradical, one end of L is attached to the polysarcosine-based moiety, and the other end of L is attached to the oxygen atom of the vitamin E-based moiety. When the repeating sarcosine-based unit PAA has the formula (XXI), L is attached to the nitrogen atom of the PAA by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond, more particularly the bond being formed between a —CO—, —COO—, —(CO)NH—, or —CH2— moiety on the left side of the linker L and the nitrogen atom of the polysarcosine-based moiety. When the repeating sarcosine-based unit has the formula (XXII), and m is 1, L is attached to the carbonyl group of the PAA by a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond, more particularly the bond being formed between a —(O)— or —(N)— moiety on the left side of the linker L and the carbonyl moiety of the polysarcosine-based moiety.
Further, L is attached to the oxygen atom of the vitamin E-based moiety by a chemically feasible bond which is selected from the group consisting of an ester bond, a carbamate bond and an ether bond, more particularly the bond being formed between a —CO—, —N(CO)—, or —CH2— moiety on the right side of the linker L and the oxygen of the vitamin E derivative.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (I), L is a biradical chain which comprises from 1-50 moieties, more particularly from 2 to 30 or from 2 to 20 or from 2 to 15 moieties as defined above, provided that L can be attached to the rest of the molecule by chemically feasible bonds.
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (I), PAA is a repeating sarcosine-based unit of formula (XXI), z is 1, and m is 1, i.e., the polymer of formula (I) is a polymer of formula (Ia),
wherein R1-R7, X, L, and n are as defined herein, and L is attached to the nitrogen atom of the sarcosine-based unit by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond. More particularly, in the polymer of formula (Ia), the sarcosine-based unit is attached to X by a chemically feasible bond which is selected from the group consisting of an ester bond, and an amide bond.
In the polymers of formula (Ia), X is —O— or —NH—. In one more particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), X is —O—. In another more particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), X is —NH—.
In a more particular embodiment, the polymer of formula (Ia) is a polymer of formula (Ia′),
wherein n, R1, X, and L are as defined herein, and even more particularly the polymer of formula (Ia) is a polymer of formula (Ia″):
wherein R1, and n is as defined herein.
According to a particular embodiment, optionally in combination with any of the embodiments provided above or below, in the polymers of formula (Ia) the linker L is a biradical selected from the group consisting of —(C1-C12)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, —(C1-C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —(C1-C6)alkyl-CO—; —CO—(C1-C6)alkyl-; and a biradical of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (XII) (XIII), (XIV), (XV), (XVI), (XVII), (XVIII) or (XIX),
wherein the “*” denote the attaching points of the biradical L to the nitrogen atom of the repeating sarcosine-based unit or the oxygen atom of the vitamin E-based moiety, “*3” denotes the attaching point of the biradical L to the nitrogen atom of the repeating sarcosine-based unit, and “*4” denotes the attaching point of the biradical to the oxygen atom of the vitamin E-based moiety,
When drawn, the attachments points are indicated herein with an “*”. As long as the specific attachment is not specified, it means that the linker may be attached to the rest of the molecule interchangeably in any direction.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a —(C1-C12)alkylene-, more particularly a —(C1-C6)alkyl-, and is optionally substituted as defined herein.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is —(C1-C6)alkyl-CO— or —CO—(C1-C6)alkyl- and is optionally substituted as defined herein.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical of formula (III), more particularly a biradical of formula (III) wherein b is 0, and a+c are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and even more particularly, L is a biradical of formula (III) wherein b is 0 and a and c are 1, i.e. a biradical of formula (IIIa).
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical selected from the formulas (IV), (V), and (VI), more particularly, in the biradical of formula (IV), a is 1 and the dashed bond is a double bond, i.e., the biradical of formula (IV) has the formula (IVa), and in the biradical of formula (V), the dashed bond is more particularly a double bond, i.e., the biradical of formula (V) has the formula (Va).
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical selected from the formulas (VII), and (VIII), more particularly, in the biradical of formula (VIII), a is 1.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical of formula (IX), more particularly a biradical of formula (IX) wherein b and g are 0; and a, c, d, e, f and h are 1, i.e., a biradical of formula (IXa).
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical selected from the formulas (X) and (XI), more particularly in the biradicals of formula (X) and (XI), b and g are 0; and a, c, d, e, f and h are 1, i.e. the biradicals of formula (X), and (XI) have the formulas (Xa), and (XIa), respectively.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical selected from the formulas (XII) and (XIII), more particularly in the biradicals of formula (XII) and (XIII), b is 0; and a, c and d are 1.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical selected from the formulas (XIV), (XV), (XVI), (XVII), and (XVIII), more particularly in the biradicals of formula (XIV), (XV), (XVI), (XVII), and (XVIII), b is 0; and a, and c are 1.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical of formula (XIX), wherein p is 0, and j and k are 1. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical of formula (XIX), wherein p is 1, X is —NH—, and j and k are 1. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical selected of formula (XIX), wherein p is 1, X is —O—, and j and k are 1.
According to a more particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ia), L is a biradical of formula (III) selected from the group consisting of:
According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (I), PAA is a repeating sarcosine-based unit of formula (XXII), and z is 0, i.e., the polymer of formula (I) is a polymer of formula (Ib),
wherein R1-R7, L, m, and n are as defined herein, and when m is 1, L is attached to the carbonyl group of the PAA by a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; and when m is 0, the PAA is attached to the oxygen atom of the vitamin E-based moiety by an ester bond. More particularly, in the polymer of formula (Ib), the PAA is attached to R1 by an amine bond.
In one particular embodiment, optionally in combination with any of the embodiments provided above or below, in the polymers of formula (Ib), m is 0.
According to a particular embodiment, optionally in combination with any of the embodiments provided above or below, in the polymers of formula (Ib) m is 1, and the linker L is a biradical selected from the group consisting of —X′—(C1-C12)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, —(C1-C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —X′—(C1-C6)alkyl-CO—; and a biradical of formula (III′), (IV′), (V′), (VI′), (VII), (VIII), (IX′), (X′), (XI′), (XII′) (XIII′), (XIV′), (XV′), (XVI′), (XVII′), (XVIII′), or (XIX′),
wherein the “*” denote the attaching points of the biradical L to the carbonyl group of the repeating sarcosine-based unit or the oxygen atom of the vitamin E-based moiety, “*3” denotes the attaching point of the biradical L to the carbonyl group, and “*4” denotes the attaching point of the biradical to the oxygen atom of the vitamin E-based moiety,
When drawn, the attachments points are indicated herein with an “*”. As long as the specific attachment is not specified it means that the linker may be attached to the rest of the molecule interchangeably.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a —X′—(C1-C12)alkylene-, more particularly a X′—(C1-C6)alkyl-, and is optionally substituted as defined herein.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is —X′—(C1-C6)alkyl-CO— or —X′—(C1-C6)alkyl- and is optionally substituted as defined herein.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical of formula (III′), more particularly a biradical of formula (III′) wherein b is 0, and a+c are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and even more particularly, L is a biradical of formula (III′) wherein b is 0, a and c are 1, and X is —NH—, i.e. a biradical of formula (IIIa′).
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (IV′), (V′), and (VI′), more particularly, X′ is —NH—, and even more particularly, in the biradical of formula (IV′), a is 1 and the dashed bond is a double bond, i.e., the biradical of formula (IV′) has the formula (IVa′), and in the biradical of formula (V′), the dashed bond is more particularly a double bond, and X is —NH—, i.e., the biradical of formula (V) has the formula (Va′).
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (VII), and (VIII), more particularly, in the biradical of formula (VIII), a is 1.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical of formula (IX′), more particularly a biradical of formula (IX′) wherein b and g are 0; a, c, d, e, f and h are 1, and X is —NH—, i.e. a biradical of formula (IXa′).
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (X′) and (XI′), more particularly in the biradicals of formula (X′) and (XI′), b and g are 0; a, c, d, e, f and h are 1, and X′ is —NH—, i.e. the biradicals of formula (X′), and (XI′) have the formulas (Xa′), and (XIa′), respectively.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (XII′) and (XIII′), more particularly in the biradicals of formula (XII′) and (XIII′), b is 0; a, c and d are 1; and X is —NH—.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (XIV′), (XV′), (XVI′), (XVII′), and (XVIII′), more particularly in the biradicals of formula (XIV′), (XV′), (XVI′), (XVII′), and (XVIII′), b is 0; a, and c are 1; and X is —NH—.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (XIX′), wherein X is —NH—, and j and k are 1. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (XIX′), wherein X is —O—, and j and k are 1.
According to a more particular embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical of formula (III′) selected from the group consisting of:
For reasons of completeness, the following numbered embodiments also form part of the invention:
Embodiment 1. A polymer comprising a repeating sarcosine-based unit which is covalently attached directly or through a linker to a vitamin E-based moiety, or a pharmaceutically, nutraceutically, or cosmetically acceptable salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the polymer or of any of its salts; wherein the repeating sarcosine-based unit has the formula (XXIII)
wherein R5 is selected from the group consisting of methyl, —CH2F, —CHF2, and —CF3; R6 and R7 are independently selected from the group consisting of hydrogen and fluorine; and n is an integer from 5 to 500; and the vitamin E-based moiety has the formula (XXIV):
wherein R2, R3 and R4 are independently selected from the group consisting of hydrogen, fluorine, methyl, —CH2F, —CHF2, and —CF3; and each of the dashed bonds is independently a single bond or, alternatively, a double bond.
Embodiment 2. The polymer according to embodiment 1, wherein the repeating sarcosine-based unit is covalently attached directly or through a linker to the vitamin E-based moiety by any of the attachment points “*”, and the vitamin E-based moiety is covalently attached directly or through a linker to the repeating sarcosine-based unit by the attachment point indicated with a wavy line “” that intersects a bond.
Embodiment 3. The polymer according to any of the embodiments 1-2, which has the formula (I),
wherein:
Embodiment 4. The polymer according to any of embodiments 1 to 3, wherein R1 is selected from the group consisting of —(C1-C12)alkyl, —(C1-C12)alkylphenyl, —(C1-C6)alkyl-O—(C1-C6)alkyl, —CO—(C1-C12)alkyl, —CO—(C1-C12)alkylphenyl, and —CO—(C1-C6)alkyl-O—(C1-C6)alkyl; wherein R1 is optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3;
Embodiment 5. The polymer according to any of the embodiments 1 to 4, wherein n is from 5 to 400, or from 5 to 300, or from 5 to 200, or from 5 to 100, or from 5 to 50, or from 5 to 40, or from 5 to 35, or from 5 to 25, and more particularly n is about 6, or about 15, or about 20, in particular measured by SEC or 1H-NMR spectroscopy as indicated in the examples.
Embodiment 6. The polymer according to any of the embodiments 1 to 5, which has an average molecular weight (Mw) from 700 to 50000 Da, more particularly from 1000 to 20000 Da, and even more particularly from 1000 to 5000 Da or from 1000 to 3000 Da, measured by SEC as disclosed in the examples.
Embodiment 7. The polymer according to any of the embodiments 1 to 6, which has a polydispersity index from 1.01 to 1.80, or from 1.01 to 1.50, or from 1.01 to 1.30, or from 1.01 to 1.20, or from 1.01 to 1.10 measured by SEC as disclosed in the examples.
Embodiment 8. The polymer according to any of the embodiments 1 to 7, wherein R5 is methyl.
Embodiment 9. The polymer according to any of the embodiments 1 to 8, wherein R6 and R7 are hydrogen.
Embodiment 10. The polymer according to any of the embodiments 1 to 9, wherein R2, R3 and R4 are independently selected from the group consisting of hydrogen and methyl.
Embodiment 11. The polymer according to any of the embodiments 1 to 10, wherein each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond, and R2, R3 and R4 are selected from the following meanings: (i) R2, R3 and R4 are methyl, (ii) R2, R4 are methyl and R3 is hydrogen, (iii) R3 and R4 are methyl and R2 is hydrogen, and (iv) R4 is methyl and R2 and R3 are hydrogen.
Embodiment 12. The polymer according to embodiments 11, wherein each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond and R2, R3 and R4 are methyl, more particularly, the vitamin E-based moiety of the polymer has the formula (XXIV′).
Embodiment 13. The polymer according to any of the embodiments 1 to 10, wherein each of the dashed bonds of the alkyl chain attached to the chroman ring is a double bond, and R2, R3 and R4 are selected from the following meanings: (i) R2, R3 and R4 are methyl, (ii) R2, R4 are methyl and R3 is hydrogen, (iii) R3 and R4 are methyl and R2 is hydrogen, and (iv) R4 is methyl and R2 and R3 are hydrogen.
Embodiment 14. The polymer according to embodiment 13, wherein each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond and R2, R3 and R4 are methyl.
Embodiment 15. The polymer according to any of the embodiments 3 or 5-14, wherein R1 is selected from the group consisting of —(C1-C12)alkyl, —(C1-C12)alkylphenyl, —(C1-C6)alkyl-O—(C1-C6)alkyl, —CO—(C1-C12)alkyl, —CO—(C1-C12)alkylphenyl, and —CO—(C1-C6)alkyl-O—(C1-C6)alkyl; wherein the alkyl groups may be optionally substituted as defined herein; more particularly, when PAA is a repeating sarcosine-based unit of formula (XXI), and z is 1, R1 is selected from the group consisting of —(C1-C12)alkyl, —(C1-C12)alkylphenyl, and —(C1-C6)alkyl-O—(C1-C6)alkyl; and when PAA is a repeating sarcosine-based unit of formula (XXII), and z is 0, R1 is selected from the group consisting of —CO—(C1-C12)alkyl, —CO—(C1-C12)alkylphenyl, and —CO—(C1-C6)alkyl-O—(C1-C6)alkyl; wherein all the alkyl groups may be optionally substituted as defined herein.
Embodiment 16. The polymer according to any of the embodiments 3 or 5-15, wherein, particularly when PAA is a repeating sarcosine-based unit of formula (XXI) and z is 1, R1 is selected from the group consisting of —(C1-C6)alkyl, —(C1-C3)alkyl-O—(C1-C3)alkyl, and —(C1-C6)alkylphenyl; and wherein the alkyl groups may be optionally substituted as defined herein.
Embodiment 17. The polymer according to embodiment 16, wherein, particularly when PAA is a repeating sarcosine-based unit of formula (XXI) and z is 1, R1 is selected from the group consisting of isopropyl, 2-methoxyethyl, n-propyl, n-butyl, neopentyl, benzyl, and 2-(2-methoxyethoxy)ethyl, and more particularly R1 is isopropyl.
Embodiment 18. The polymer according to any of the embodiments 3 or 5-15, wherein, particularly when PAA is a repeating sarcosine-based unit of formula (XXII) and z is 0, R1 is selected from the group consisting of —CO—(C1-C6)alkyl, —CO—(C1-C6)alkyl-O—C1-C3)alkyl, and —CO—(C1-C6)alkylphenyl; and wherein the alkyl groups may be optionally substituted as defined herein.
Embodiment 19. The polymer according to embodiment 18, wherein, particularly when PAA is a repeating sarcosine-based unit of formula (XXII) and z is 0, R1 is acetyl.
Embodiment 20. The polymer according to any of embodiments 3 to 14, wherein R1 is H.
Embodiment 21. The polymer according to any of the embodiments 3-20, wherein the repeating sarcosine-based unit PAA has the formula (XXI).
Embodiment 22. The polymer according to embodiment 21, wherein z is 1, and m is 1, i.e., the polymer of formula (I) is a polymer of formula (Ia), wherein R1-R7, X, L, and n are as defined herein, and L is attached to the nitrogen atom of the sarcosine-based unit by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond.
Embodiment 23. The polymer according to embodiment 22, wherein the sarcosine-based unit is attached to X by a chemically feasible bond which is selected from the group consisting of an ester bond, and an amide bond.
Embodiment 24. The polymer according to any of the embodiments 21-22, wherein X is —NH—.
Embodiment 25. The polymer according to any of the embodiments 21-22, wherein X is —O—.
Embodiment 26. The polymer according to any of the embodiments 21-22, which has the formula (Ia′), more particularly the formula (Ia″).
Embodiment 27. The polymer according to any of the embodiments 21-26, wherein the linker L is a biradical selected from the group consisting of —(C1-C12)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, -(C1—C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —(C1-C6)alkyl-CO—; —CO—(C1-C6)alkyl-; and a biradical of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (XII) (XIII), (XIV), (XV), (XVI), (XVII), (XVIII) or (XIX).
Embodiment 28. The polymer according to embodiment 27, wherein the linker L is a —(C1-C12)alkylene-, more particularly a —(C1-C6)alkyl-, and is optionally substituted as defined herein.
Embodiment 29. The polymer according to embodiment 27, wherein the linker L is —(C1-C6)alkyl-CO— or —CO—(C1-C6)alkyl- and is optionally substituted as defined herein.
Embodiment 30. The polymer according to embodiment 27, wherein the linker L is a biradical of formula (III), more particularly a biradical of formula (III) wherein b is 0, and a+c are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and even more particularly, L is a biradical of formula (III) wherein b is 0 and a and c are 1, i.e. a biradical of formula (IIIa).
Embodiment 31. The polymer according to embodiment 27, wherein the linker L is a biradical selected from the formulas (IV), (V), and (VI), more particularly, in the biradical of formula (IV), a is 1 and the dashed bond is a double bond, i.e., the biradical of formula (IV) has the formula (IVa), and in the biradical of formula (V), the dashed bond is more particularly a double bond, i.e., the biradical of formula (V) has the formula (Va).
Embodiment 32. The polymer according to embodiment 27, wherein the linker L is a biradical selected from the formulas (VII), and (VIII), more particularly, in the biradical of formula (VIII), a is 1.
Embodiment 33. The polymer according to embodiment 27, wherein the linker L is a biradical of formula (IX), more particularly a biradical of formula (IX) wherein b and g are 0; and a, c, d, e, f and h are 1, i.e., a biradical of formula (IXa).
Embodiment 34. The polymer according to embodiment 27, wherein the linker L is a biradical selected from the formulas (X) and (XI), more particularly in the biradicals of formula (X) and (XI), b and g are 0; and a, c, d, e, f and h are 1, i.e. the biradicals of formula (X), and (XI) have the formulas (Xa), and (XIa), respectively.
Embodiment 35. The polymer according to embodiment 27, wherein the linker L is a biradical selected from the formulas (XII) and (XIII), more particularly in the biradicals of formula (XII) and (XIII), b is 0; and a, c and d are 1.
Embodiment 36. The polymer according to embodiment 27, wherein the linker L is a biradical selected from the formulas (XIV), (XV), (XVI), (XVII), and (XVIII), more particularly in the biradicals of formula (XIV), (XV), (XVI), (XVII), and (XVIII), b is 0; and a, and c are 1.
Embodiment 37. The polymer according to embodiment 27, wherein the linker L is a biradical of formula (XIX), wherein p is 0, and j and k are 1, or alternatively wherein p is 1, X is —NH—, and j and k are 1, or alternatively wherein p is 1, X is —O—, and j and k are 1.
Embodiment 38. The polymer according to embodiment 27, wherein the linker L is a biradical of formula (III) selected from the group consisting of:
Embodiment 39. The polymer according to any of the embodiments 21-26, wherein the linker L is a biradical selected from the formulas (XVa), and (XVIIa):
wherein the “*” denote the attaching points of the biradical L to the nitrogen atom of the repeating sarcosine-based unit or the oxygen atom of the vitamin E-based moiety, a, and c are independently an integer from 0 to 10, particularly from 0 to 6; b is an integer from 0 to 1; with the condition that when b is 0, then at least one of a or c is other than 0; more particularly in the biradicals of formula (XVa) and (XVIIa) b is 0; and a, and c are 1.
Embodiment 40. The polymer according to any of the embodiments 3-20, wherein the repeating sarcosine-based unit PAA has the formula (XXII).
Embodiment 41. The polymer according to embodiment 40, wherein z is 0, i.e., the polymer of formula (I) is a polymer of formula (Ib), wherein R1-R7, L, m, and n are as defined herein, and when m is 1, L is attached to the carbonyl group of the PAA by a chemically feasible bond which is selected from the group consisting of an amide bond and an ester bond; and when m is 0, the PAA is attached to the oxygen atom of the vitamin E-based moiety by an ester bond.
Embodiment 42. The polymer according to embodiment 41, wherein the PAA is attached to R1 by an amine bond.
Embodiment 43. The polymer according to any of the embodiments 41-42, wherein m is 1, and the linker L is a biradical selected from the group consisting of —X′—(C1-C12)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, —(C1-C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —X′—(C1-C6)alkyl-CO—; and a biradical of formula (III′), (IV′), (V′), (VI′), (VII), (VIII), (IX′), (X′), (XI′), (XII′) (XIII′), (XIV′), (XV′), (XVI′), (XVII′), (XVIII′), or (XIX′),
Embodiment 44. The polymer according to embodiment 43, wherein the linker L is a —X′—(C1-C12)alkylene-, more particularly a —X′—(C1-C6)alkyl-, and is optionally substituted as defined herein.
Embodiment 45. The polymer according to embodiment 43, wherein the linker L is —X′—(C1-C6)alkyl-CO— or —X′—(C1-C6)alkyl- and is optionally substituted as defined herein.
Embodiment 46. The polymer according to embodiment 43, wherein the linker L is a biradical of formula (III′), more particularly a biradical of formula (III′) wherein b is 0, and a+c are selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and even more particularly, L is a biradical of formula (III′) wherein b is 0, a is 1 and c is 0 or 1, and X′ is —NH—, i.e. a biradical of formula (IIIa′).
Embodiment 47. The polymer according to embodiment 43, wherein the linker L is a biradical selected from the formulas (IV′), (V′), and (VI′), more particularly, X′ is —NH—, and even more particularly, in the biradical of formula (IV′), a is 1 and the dashed bond is a double bond, i.e., the biradical of formula (IV′) has the formula (IVa′), and in the biradical of formula (V′), the dashed bond is more particularly a double bond, and X′ is —NH—, i.e., the biradical of formula (V′) has the formula (Va′).
Embodiment 48. The polymer according to embodiment 47, wherein the linker L is a biradical selected from the formulas (VII), and (VIII), more particularly, in the biradical of formula (VIII), a is 1.
Embodiment 49. The polymer according to embodiment 43, wherein the linker L is a biradical of formula (IX′), more particularly a biradical of formula (IX′) wherein b and g are 0; a, c, d, e, f and h are 1, and X′ is —NH—, i.e. a biradical of formula (IXa′).
Embodiment 50. The polymer according to embodiment 43, wherein the linker L is a biradical selected from the formulas (X′) and (XI′), more particularly in the biradicals of formula (X′) and (XI′), b and g are 0; a, c, d, e, f and h are 1, and X′ is —NH—, i.e. the biradicals of formula (X′), and (XI′) have the formulas (Xa′), and (XIa′), respectively.
Embodiment 51. The polymer according to embodiment 43, wherein the linker L is a biradical selected from the formulas (XII′) and (XIII′), more particularly in the biradicals of formula (XII′) and (XIII′), b is 0; a, c and d are 1; and X′ is —NH—.
Embodiment 52. The polymer according to embodiment 43, wherein the linker L is a biradical selected from the formulas (XIV′), (XV′), (XVI′), (XVII′), and (XVIII′), more particularly in the biradicals of formula (XIV′), (XV′), (XVI′), (XVII′), and (XVIII′), b is 0; a, and c are 1; and X is —NH—.
Embodiment 53. The polymer according to embodiment 43, wherein the linker L is a biradical selected from the formulas (XIX′), wherein X is —NH—, and j and k are 1. In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the polymer of formula (Ib), L is a biradical selected from the formulas (XIX), wherein X is —O—, and j and k are 1.
Embodiment 54. The polymer according to embodiment 43, wherein the linker L is a biradical of formula (III′) selected from the group consisting of:
Embodiment 55. The polymer according to any of the embodiments 41-42, wherein the linker L is a biradical of formula (IIIa″).
wherein “*3” denotes the attaching point of the biradical L to the carbonyl group, and “*4” denotes the attaching point of the biradical to the oxygen atom of the vitamin E-based moiety.
Embodiment 56. The polymer according to any of the embodiments 40-42, wherein m is 0.
Processes for the preparation of the polymers of formula (I) are also part of the Invention. The polymers of formula (I) may be prepared by synthetic processes which are well-known in the art.
For example, the polymers of formula (I) wherein the repeating sarcosine-based unit PAA has the formula (XXI) (i.e., polymers of formula (Ia)) may be prepared by reacting a polymer of formula (A) with a polymer of formula (B), as shown in the scheme below:
wherein RA is selected from -L1-(CO)—Y or -L2-O—H, or -L-LG, wherein -L1-(CO)— is a linker L which ends with a —CO— moiety, -L2-O— is a linker L which ends with a —O— moiety, and Y is OH or a halogen atom, LG is a leaving group, and n, X, R1, R2, R3, R4, R5, R6, R7, and L are as defined herein.
When RA is -L1-(CO)—Y and Y is OH, an amide bond is formed between compounds (A) and (B). This reaction may be carried out in the presence of an activating agent such as: 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMBF4), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) or hydroxybenzo-triazole (HOBt), preferably in the presence of a base, such as triethylamine (TEA), N-methylmorpholine (NMM), or N, N-diisopropylethylamine (DIPEA), in a suitable solvent, such as dimethylformamide, dichloromethane or chloroform, at a temperature comprised from room temperature to the temperature of the boiling point of the solvent, preferably at room temperature.
When RA is -L1-(CO)—Y and Y is a halogen atom, the reaction may be carried out in the presence of a base, such as triethylamine, in a suitable solvent, such as tetrahydrofuran, at a temperature comprised from room temperature to the reflux temperature of the solvent. The compound of formula (A) when X is a halogen atom may be prepared from the corresponding carboxylic acid by methods well-known in the art.
When RA is -L2-OH, the reaction may be carried out in the presence of a carbonyl source such as 1,1′-carbonyldiimidazole (CDI), N,N′-disuccinimidyl carbonate (DSC) or bis(trichloromethyl) carbonate (BTC), in a suitable solvent such as acetonitrile or 1,4-dioxane, at a temperature comprised from room temperature to the reflux temperature of the solvent.
When RA is -L-LG, the reaction may be carried out in the presence of a base such as triethylamine (TEA), N-methylmorpholine (NMM), or N, N-diisopropylethylamine (DIPEA), in a suitable solvent, such as acetonitrile, tetrahydrofuran, dimethylformamide, dichloromethane or chloroform, at a temperature comprised from room temperature to the temperature of the boiling point of the solvent, preferably at room temperature. Non-limiting examples of leaving groups include halogen, —OSO2R, and —OCOR, wherein R is selected from the group consisting of (C1-C4)alkyl optionally substituted with one or more halogen atoms, and (C6-C12)aryl optionally substituted with (C1-C4)alkyl, or with (C1-C4)alkyl optionally substituted with one or more halogen atoms. More particularly, LG is selected from halogen, —OSO2CH3 (-OMs), —OSO2C7H7 (-OTs), —OSO2CF3 (-OTf), —OCOCH3 (—OAc), —OCOPh (-OBz), and —OCOCF3 (-TFA).
Compounds of formula (A) wherein RA is -L1-(CO)—Y and Y is OH, i.e., compounds of formula (A1), are commercially available or may be prepared by reacting a compound of formula (C), which is a vitamin E derivative, with a compound of formula (D):
wherein R2, R3, and R4 are as defined herein, and La is a linker L which ends with two —COOH moieties. This reaction may be carried out under analogue conditions as the ones previously disclosed for the formation of ester bonds.
Compounds of formula (A) wherein RA is -L-LG, i.e. compounds of formula (A2), may be prepared by reacting a compound of formula (C), with a compound of formula (E),
wherein R′ is —COOH or an anhydride or ester derivative thereof, or a leaving group, under analogue conditions as the ones previously disclosed for the formation of ether bonds.
Compounds of formula (B) may be prepared by ring opening polymerization (ROP) of N-carboxy anhydrides (NCAs), in particular 3-alkyloxazolidine-2,5-diones (compounds of formula (F)). Amines or alcohols of formula (G) are used as initiators in order to obtain a linear poly sarcosine polymer (R1—X-PSarn). The reaction is carried out in a suitable solvent such as dimethylformamide, at a temperature comprised from 4° C. to room temperature, preferably at about 10° C.
The ratio monomer (NCA)/initiator (alcohol/amine) allow to control the degree of polymerization (DP).
Polymers of formula (I) wherein the repeating sarcosine-based unit PAA has the formula (XXII) (i,e., polymers of formula (Ib)) may be prepared by synthetic processes which are well-known in the art. For example, a compound of formula (XXII) wherein m is 1, may be prepared by ring opening polymerization (ROP) of a N-carboxy anhydride of formula (F) by using an amine or alcohol of formula (H) as initiator:
wherein -L4-X′— is a linker L which ends with a X′ moiety, wherein X is —NH— or —O—.
When m is 0, the compound of formula (XXII) may be prepared by ring opening polymerization (ROP) of a N-carboxy anhydride of formula (F) by using an amine or alcohol of formula (K) as initiator:
The obtained intermediates of formulas (J) and (J′) may be reacted with a compound of formula R1-LG, wherein LG is a leaving group, to give rise to compounds of formula (I).
Using appropriate surface functionality, the polymers of the present invention may be further decorated with other molecules that can actively target cells and aid in cellular entry, resulting in a conjugate which has improved cell-specific delivery.
Accordingly, the present invention also relates to conjugates of the polysarcosine vitamin E derivatives of the invention with other molecules which are attached to them by covalent bonds, either directly or through linkers. Thus, the invention relates to a conjugate of formula (II) or a pharmaceutically, nutraceutically, or cosmetically acceptable salt thereof, or any stereoisomer or mixtures of stereoisomers, either of the conjugate of formula (II) or of any of its salts,
wherein m, PAA, X, n, L, R2, R3, R4, and the dashed bonds are as defined herein;
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the conjugate of formula (II), PAA is a repeating sarcosine-based unit of formula (XXI), i.e. the conjugate of formula (II) is a conjugate of formula (IIa),
wherein n, X, L, L′, Z, z′, s, R2-R7, and the dashed bonds are as defined herein.
According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the conjugate of formula (II), PAA is a repeating sarcosine-based unit of formula (XXII), i.e. the conjugate of formula (II) is a conjugate of formula (IIb),
wherein n, L, L′, Z, s, m, R2-R7, and the dashed bonds are as defined herein.
In one embodiment, optionally in combination with any of the embodiments provided above or below, Z is a cell-targeting agent. As used herein, the term “cell-targeting agent” refers to any molecule, macromolecule, or biomacromolecule, such as a nutraceutically active agent, a pharmaceutically active agent, a diagnostically active agent, and a cosmetically active agent, which selectively binds to receptors that are expressed or over-expressed on specific cell types. Cell-targeting groups are well known in the art.
Examples of cell-targeting groups include, but are not limited to, galactosamine, folate, a Her-2 binding peptide, TLR agonists, β-D-Glucose, Asn-Gly-Arg peptide, angiopep2, folic acid, aptamers (A-9, A10, Anti-gp120, TTA1, sgc8, Anti MUC-1, AS1411), primaquine, zidovudine, superoxide dismutase, prednisolone, platinum, cisplatin, sulphamethoxazole, amoxicillin, etoposide, mesalzine, doxorubicin, paclitaxel, 5-amino salicylic acid, denosumab, docetaxel, calcitonin, proanthocyanidin, methotrexate, camptothecin, galactose, glycyrrhetinic acid, lactose, hyaluornic acid, octeotride, lactobionic acid, β-galactosyl moiety, arabino-galactan, chitosan, azo-based poly-phosphazene, azo group and 4-amino-benzyl-carbamate, succinate, 4,4′-dihydroxyazo benzene-3-carboxilic acid, cyclic RGD penta-peptide, Aspartic acid octapeptide, alendronate, transferrin, bisphosphonate adendronate, mono sialoganglioside GM1, gluthatione, E-selectin thioaptamer, poloxamer-407, a urokinase-type plasminogen activator receptor (uPAR) antagonist, a CXCR4 chemokine receptor antagonist, a GRP78 peptide antagonist, an RGD peptide, an RGD cyclic peptide, a luteinizing hormone-releasing hormone (LHRH) antagonist peptide, an aminopeptidase targeting peptide, a brain homing peptide, a kidney homing peptide, a heart homing peptide, a gut homing peptide, an integrin homing peptide, an angiogenic tumor endothelium homing peptide, an ovary homing peptide, a uterus homing peptide, a sperm homing peptide, a microglia homing peptide, a synovium homing peptide, a urothelium homing peptide, a prostate homing peptide, a lung homing peptide (e.g. RCPLSHSLICY), laminin receptor binding peptide (e.g. YIGSR) a skin homing peptide, a retina homing peptide, a pancreas homing peptide, a liver homing peptide, a lymph node homing peptide, an adrenal gland homing peptide, a thyroid homing peptide, a bladder homing peptide, a breast homing peptide, a neuroblastoma homing peptide, a lymphoma homing peptide, a muscle homing peptide, a wound vasculature homing peptide, an adipose tissue homing peptide, a virus binding peptide, or a fusogenic peptide.
According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the conjugate of formula (II), Z is anticancer drug. Non-limiting examples of anticancer drugs include, without limitation, cisplatin, paclitaxel, 5-fluoracil, gemcitabine, Herceptin, mitoxantrone, cantharidin, docetaxel, anthracycline drugs (doxorubicin, epirrubicin, daunorubicin, ildarubicin), irinotecan, or camptothecin.
In accordance with a particular embodiment, optionally in combination with any of the embodiments provided above or below, Z is selected from the group consisting of low molecular weight drugs, peptides, antibodies, hormones, enzymes, nucleic acids, proteins, and combinations thereof.
As used herein, the term “nucleic acid” refers to DNA or RNA. In a particular embodiment, optionally in combination with any of the embodiments provided above or below, the nucleic acid is an DNA/RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a single guide RNA (sgRNA), a donorDNA, a self-amplifying/replicating RNA, a circularRNA (oRNA), a plasmid DNA (pDNA), a closed-linear DNA (clDNA), a short hairpin RNA (shRNA), messenger RNA (mRNA), and antisense RNA (aRNA), a messenger RNA (mRNA), a CRISPR guide RNA, an antisense nucleic acid, a decoy nucleic acid, an aptamer, and a ribozyme to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as double stranded, single stranded, helical, hairpin, etc, and may contain modified or unmodified bases. When distinct nucleic acids are provided, they may be all DNA molecules or all RNA molecules or may be mixtures of DNA and RNA molecules or molecules comprising an association of DNA and RNA strands.
The nucleic acid may be a poly- or oligonucleotide, such as oligo- or poly-double stranded RNA, oligo- or poly-double stranded DNA, oligo- or poly-single stranded RNA, oligo- or poly-single stranded DNA. Each of the nucleotides contained in the nucleic acid may be a naturally occurring nucleotide or a chemically-modified non-naturally occurring nucleotide. The strand length of the nucleic acid is not particularly limited, and the nucleic acid may have a short strand ranging from 10 to 200 bases, preferably from 20 to 180 bases, preferably from 25 to 100 bases, preferably from 30 to 50 bases; or the nucleic acid may have a relatively long strand of from 200 to 20000 bases, more preferably of from 250 to about 15000 bases.
In accordance with a particular embodiment, optionally in combination with any of the embodiments provided above or below, the nucleic acid is closed-linear DNA (clDNA), i.e., molecules wherein the double stranded region is flanked and protected by two single stranded loops thereby generating dumbbell-shaped molecules.
In a more particular embodiment, optionally in combination with any of the embodiments provided above or below, the clDNA consists of a stem region comprising a double stranded DNA sequence of interest covalently closed at both ends by hairpin loops, the clDNA comprising at least two modified nucleotides.
As used herein, the term “closed linear DNA” or “clDNA” refers to a single stranded covalently closed DNA molecule that forms a “dumbbell” or “doggy-bone” shaped structure under conditions allowing nucleotide hybridization. Therefore, although the clDNA is formed by a single stranded DNA molecule, the formation of the “dumbbell” structure by the hybridization of two complementary sequences within the same molecule generates a structure consisting on a double-stranded middle segment flanked by two single-stranded loops. The skilled in the art know how to generate clDNA from open or closed double stranded DNA using routine molecular biology techniques. For instance, those skilled in the art knows that a clDNA can be generated by attaching hairpin DNA adaptors—for instance, by the action of a ligase—to both ends of an open double stranded DNA. “Hairpin DNA adaptor” refers to a single stranded DNA that forms a stem-loop structure by the hybridization of two complementary sequences, wherein the stem region formed is closed at one end by a single stranded loop and is open at the other end.
A “modified nucleotide” is any nucleotide (e.g., adenosine, guanosine, cytidine, uracil, and thymidine) that has been chemically modified-by modification of the base, the sugar, or the phosphate group- or that incorporates a non-natural moiety in its structure. Thus, the modified nucleotide may be naturally or non-naturally occurring depending on the modification
In the context of the present disclosure the expression, “diagnostically active agent”, also referred to as “labeling or imaging agent”, refers to any substance that is used as a label, or that enhances specific structures in any imaging technique. An imaging agent, hence, includes optical imaging agent, magnetic resonance imaging agent, radioisotope, and contrast agent. Imaging or labelling agents are well known in the art. Particular examples or imaging or labelling agents are gases such as sterilized air, oxygen, argon, nitrogen, fluor, perfluorocarbons, carbon dioxide, nitrogen dioxide, sulfur hexafluoride, xenon and helium; commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Examples of suitable materials for use as contrast agents in MRI include the gadolinium chelates currently available, such as diethylene triamine pentaacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium. Examples of materials useful for CAT and x-rays include iodine-based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexol, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte. Other useful materials include barium for oral use and non-soluble salts such as zinc acetate. In some molecules, an imaging agent is a dye. In some molecules, an imaging agent is a fluorescent moiety. In some molecules, a fluorescent moiety is selected from: a fluorescent protein, a fluorescent peptide, a fluorescent dye, a fluorescent material or a combination thereof. Examples of fluorescent dyes include, but are not limited to, xanthenes (e.g., rhodamines, rhodols and fluoresceins, and their derivatives); bimanes; coumarins and their derivatives (e.g., umbelliferone and aminomethyl coumarins); aromatic amines (e.g., dansyl; squarate dyes); benzofurans; fluorescent cyanines; indocarbocyanines; carbazoles; dicyanomethylene pyranes; polymethine; oxabenzanthrane; xanthene; pyrylium; carbostyl; perylene; acridone; quinacridone; rubrene; anthracene; coronene; phenanthrecene; pyrene; butadiene; stilbene; porphyrin; pthalocyanine; lanthanide metal chelate complexes; rare-earth metal chelate complexes; and derivatives of such dyes. Examples of fluorescein dyes include, but are not limited to, 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate and 6-carboxyfluorescein. Examples of rhodamine dyes include, but are not limited to, tetramethylrhodamine-6-isothiocyanate, 5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (sold under the tradename of TEXAS RED®). Examples of cyanine dyes include, but are not limited to, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, ICG. Examples of fluorescent peptides include GFP (Green Fluorescent Protein) or derivatives of GFP (e.g., EBFP, EBFP2, Azurite, mKalama1, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet). Fluorescent labels are detected by any suitable method. For example, a fluorescent label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs), photomultipliers, etc. In some molecules, the imaging agent is labeled with a positron-emitting isotope (e.g., 18F) for positron emission tomography (PET), gamma-ray isotope (e.g., 99mTc) for single photon emission computed tomography (SPECT), or a paramagnetic molecule or nanoparticle (e.g., Gd3+ chelate or coated magnetite nanoparticle) for magnetic resonance imaging (MRI). In some molecules, the imaging agent is labeled with: a gadolinium chelate, an iron oxide particle, a super paramagnetic iron oxide particle, an ultra small paramagnetic particle, a manganese chelate or gallium containing agent. Examples of gadolinium chelates include, but are not limited to diethylene triamine pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA). In some molecules, the imaging agent is a near-infrared fluorophore for near-infra red (near-IR) imaging, a luciferase (firefly, bacterial, or coelenterate) or other luminescent molecule for bioluminescence imaging, or a perfluorocarbon-filled self-assembled particle for ultrasound. In some molecules, the imaging agent is a nuclear probe. In some molecules, the imaging agent is a SPECT or PET radionuclide probe. In some molecules, the radionuclide probe is selected from: a technetium chelate, a copper chelate, a radioactive fluorine, a radioactive iodine, a indiuim chelate. Examples of Tc chelates include, but are not limited to HYNIC, DTPA, and DOTA. In some molecules, the imaging agent contains a radioactive moiety, for example a radioactive isotope such as 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 64Cu radioactive isotopes of Lu, and others.
All embodiments indicated herein for the polymers of formula (I) also apply to the conjugates of formula (II) for all the common structure they have. Also, the embodiments referred to the salts and stereoisomers indicated for the polymers of formula (I) apply to the conjugates of formula (II).
In the conjugates of formula (II), when s is 0, Z is directly attached to X or the PAA sarcosine repeating unit by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, an amine bond, a urea bond, an ether bond, and an ester bond.
More particularly, when s is 0, the sarcosine repeating unit PAA is of formula (XXI) and z′ is 0, Z is directly attached to the PAA sarcosine repeating unit by a chemically feasible bond which is selected from the group consisting of an amide bond, and an ester bond, even more particularly, the bond between Z and the PAA sarcosine repeating unit may be formed between the —(O)— or —(N)-moiety of Z and the carbonyl group of the PAA sarcosine repeating unit.
When s is 0, the sarcosine repeating unit PAA is of formula (XXI) and z′ is 1, Z is directly attached to X by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, an ester bond, and amine bond, an urea bond, and an ether bond, even more particularly, the bond between Z and X may be formed between the —(O)— or —(N)-moiety of X and a —CO—, —COO—, —CONH—, or —CH2— moiety of Z.
When s is 0, the sarcosine repeating unit PAA is of formula (XXII) and z′ is 0, Z is directly attached to the PAA sarcosine repeating unit by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond, and an amine bond, even more particularly, the bond between Z and the PAA may be formed between the —N— moiety of the PAA and a —CO—, —COO—, —CONH—, or —CH2— moiety of Z.
When s is 1 L′ is attached to Z by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, an amine bond, a urea bond, an ether bond, and an ester bond, and L′ is attached to X or the PAA sarcosine repeating unit by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, an amine bond, a urea bond, an ether bond, and an ester bond.
More particularly, when s is 1, the sarcosine repeating unit PAA is of formula (XXI) and z′ is 0, L′ is directly attached to the PAA sarcosine repeating unit by a chemically feasible bond which is selected from the group consisting of an amide bond, and an ester bond, even more particularly, the bond between L′ and the PAA sarcosine repeating unit may be formed between a —(O)— or —(N)-moiety of L′ and the carbonyl group of the PAA sarcosine repeating unit.
More particularly, when s is 1, the sarcosine repeating unit PAA is of formula (XXI) and z′ is 1, L′ is attached to X by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, an ester bond, a urea bond, an amine bond, and an ether bond, more particularly, the bond between L′ and X may be formed between the —(O)— or —(N)-moiety of X and a —CO—, —OCO—, —NHCO—, or —CH2— moiety of L′.
When s is 1, the sarcosine repeating unit PAA is of formula (XXII) and z′ is 0, L′ is directly attached to the PAA sarcosine repeating unit by a chemically feasible bond which is selected from the group consisting of an amide bond, a carbamate bond, a urea bond and an amine bond, more particularly, the bond between L′ and the PAA may be formed between the —N— moiety of the PAA and a —CO—, —COO—, —CONH—, or —CH2— moiety of L′.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, in the conjugate of formula (II), L′ is a biradical chain which comprises from 1-50 moieties, more particularly from 2 to 30 or from 2 to 20 or from 2 to 15 moieties as defined above, provided that L′ can be attached to the rest of the molecule by chemically feasible bonds.
According to a more particular embodiment, optionally in combination with any of the embodiments provided above or below, when in the conjugate of formula (II) the sarcosine repeating unit PAA is of formula (XXI), z′ is 1, and s is 1, the linker L′ is a biradical selected from the group consisting of —(C1-C12)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, —(C1-C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —(C1-C6)alkyl-CO—; —CO—(C1-C6)alkyl-; and a biradical of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (XII) (XIII), (XIV), (XV), (XVI), (XVII), (XVIII) or (XIX), as previously defined.
According to another more particular embodiment, optionally in combination with any of the embodiments provided above or below, when in the conjugate of formula (II) the sarcosine repeating unit PAA is of formula (XXII), z′ is 0, and s is 1, the linker L′ is a biradical selected from the group consisting of —(C1-C12)alkylene-optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, —(C1-C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —(C1-C6)alkyl-CO—; —CO—(C1-C6)alkyl-; and a biradical of formula (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI) (XII) (XIII), (XIV), (XV), (XVI), (XVII), (XVIII) or (XIX), as previously defined.
According to a more particular embodiment, optionally in combination with any of the embodiments provided above or below, when in the conjugate of formula (II) the sarcosine repeating unit PAA is of formula (XXI), z′ is 0, and s is 1, the linker L′ is a biradical selected from the group consisting of —X′—(C1-C12)alkylene- optionally substituted with one or more substituents selected from the group consisting of halogen, —OH, —NRaRb, —SH, —NHNH2, —COORc, —CF3, and —OCF3, wherein Ra, Rb and Rc are radicals independently selected from the group consisting of H, -phenyl, —(C1-C6)alkyl, —(C2-C6)alkenyl, —(C1-C6)alkylphenyl, and -phenyl(C1-C6)alkyl; —X′—(C1-C6)alkyl-CO—; —X′—CO—(C1-C6)alkyl-; and a biradical of formula (III′), (IV′), (V′), (VI′), (VII), (VIII), (IX′), (X′), (XI′), (XII′) (XIII′), (XIV′), (XV′), (XVI′), (XVII′), (XVIII′), or (XIX′), as previously defined.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the Z is a moiety has the formula (XX):
wherein R8, R9 and R10 are independently selected from the group consisting of hydrogen, fluorine, methyl, —CH2F, —CHF2, and —CF3; and each of the dashed bonds is independently a single bond or, alternatively, a double bond.
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, R8, R9 and R10 are independently selected from the group consisting of hydrogen and methyl.
In another embodiment, optionally in combination with any of the embodiments provided above or below, in the conjugate of formula (II), Z is a radical of formula (XX), wherein each of the dashed bonds of the alkyl chain attached to the chroman ring is a single bond, and R8, R9 and R10 are selected from the following meanings: (i) R8, R9 and R10 are methyl, (ii) R8, R10 are methyl and R9 is hydrogen, (iii) R9 and R10 are methyl and R8 is hydrogen, and (iv) R10 is methyl and R8 and R9 are hydrogen. More particularly, in the above embodiment R8, R9 and R10 are methyl.
In a more particular embodiment of the previous one, R8, R9 and R10 are methyl. More particularly, in the conjugate of formula (II), Z is a radical of formula (XXa):
and even more particularly Z is a radical of formula (XXb):
The conjugates of formula (II) may be prepared by processes well-known in the art. These processes also form part of the invention. For example, when the repeating sarcosine-based unit PAA has the formula (XXI), s is 0, and X is an amine or alcohol group, Z can be used as initiator in the ROP for the preparation of polysarcosine-based moiety as shown in the scheme below:
This reaction can be carried out under the conditions previously mentioned for the preparation of polymers (B).
The obtained polysarcosine derivative (B′) may be attached to the vitamin E derivative through a linker L′ by any of the processes disclosed for the preparation of the polymers of formula (I).
When s is 1, and X is an amine or alcohol group, Z can be used as initiator in the ROP for the preparation of polysarcosine-based moiety as shown in the scheme below:
This reaction can be carried out under the conditions previously mentioned for the preparation of polymers (B).
The obtained polysarcosine derivative (B″) may be attached to the vitamin E derivative through a linker L by any of the processes disclosed for the preparation of the polymers of formula (I).
Conjugates of formula (II) wherein the repeating sarcosine-based unit PAA has the formula (XXII) may be prepared by procedures as the ones described above for the compounds of formula (Ib).
Another aspect of the invention relates to a self-assembled particle comprising:
As used herein, the term “self-assembled particles” refers to a small, enclosed structure of any shape, typically spherical and/or tubular. It intends to encompass any of a number of structures that are known in the art to be formed from amphiphilic polymers. Non-limiting examples of self-assembled particles include micelles (also being referred to interchangeably herein as micellar worms or simply “worms”), inverted micelles, planar bilayers, crystal nanoparticles, liposomes, microbubbles or lipid nanoparticles. The self-assembled nanoparticles and microparticles can also form gels.
The self-assembled particles may be self-assembled nanoparticles or self-assembled microparticles. A “nanoparticle”, as defined herein, is any particle having a smallest end-to-end diameter of between 1 and 100 nm in size. A “microparticle”, as defined herein, is typically any particle having a smallest end-to-end between 0.1 and 100 μm in size. Typically, in a composition comprising a plurality of particles, the relevant diameter is the number average diameter.
In the case a) above, the polymer forms the external shell of the self-assembled particle, and an agent is encapsulated or loaded in the inner core. In the case b) above, the conjugate of formula (II) forms the external shell of the self-assembled particle, and an agent is optionally encapsulated, i.e., loaded, in the inner core. In the case c) above, both the polymer as defined herein and the conjugate of formula (II) form the external shell of the self-assembled particle, and an agent is optionally encapsulated or loaded in the inner core or the shell surface.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the self-assembled particle is selected from the group consisting of a micelle, inverted micelle, planar bilayer, crystal nanoparticle, liposome, and lipid nanoparticle.
For the purposes of the invention, the term “micelle” refers to the arrangement of the polysarcosine vitamin E derivatives of the invention in a solvent in which the non-polar tails (hydrophobic part) face inward and the polar head portion face outward. Typically, the micelles show a spherical or tubular shape. In particular, the micelle is a core-shell structure comprising an inner core and an external shell.
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a micelle comprising:
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the micelle is formed in water.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the micelle is formed in an organic solvent. More particularly, the organic solvent is selected from the group consisting of acetone, tetrahydrofuran, methanol, ethanol, acetonitrile and mixtures thereof.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the micelles have a hydrodynamic diameter (Dh) in water, as measured by diffusion-ordered NMR spectroscopy (DOSY) or alternatively measured by Dynamic Light Scattering (DLS), from 5 to 500 nm.
According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the micelles have a critical micelle concentration (CMC) in water at 25° C., atmospheric pressure, from 0.01 to 10 mg/mL.
According to another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the encapsulation efficiency of the polymer of formula (I) is from 0.1 to 100 wt %, more particularly from 15 to 60 wt %. The encapsulation efficiency is calculated by the following formula:
The micelles may be prepared by different methods which are well-known for a person skilled in the art, such as for example direct formulation, co-solvent formulation, thin-film formulation, formulation in supercritical CO2, and the like.
It also forms part of the invention a process for the preparation of the micelle as defined herein, which comprises either a) dissolving the polymer of the invention, the conjugate of formula (II) or combination thereof as defined herein, and optionally an agent to be encapsulated within the micelle in water, or alternatively b) dissolving the polymer of the invention, the conjugate of formula (II) or combination thereof as defined herein, and optionally an agent to be encapsulated within the micelle in an organic solvent to obtain a solution, add this solution to water and evaporate the organic solvent, or alternatively c) dissolving the polymer of the invention, the conjugate of formula (II) or combination thereof as defined herein, and optionally an agent to be encapsulated within the micelle in an organic solvent to obtain a solution, evaporate the organic solvent to obtain a film, and dissolve the obtained film with water. The present invention also relates to the micelle obtainable by the above process.
The term “obtainable” is used herein for defining the micelle by its preparation process. For the purposes of the invention, the expressions “obtainable”, “obtained” and similar equivalent expressions are used interchangeably and, in any case, the expression
“obtainable” encompasses the expression “obtained”.
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a liposome comprising:
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a lipid nanoparticle (LNP) comprising:
For the purposes of the invention, the term “liposome” refers to artificially prepared self-assembled particles composed of concentric lipidic bi-layers enclosing one or more aqueous compartments. Non-limiting examples of liposomes include monolamellar liposome, multi-lamellar liposomes, multi-vesicular-liposomes and polymer-coated liposomes.
The term “lipid nanoparticle” refers to particles typically spherical on the order of nanometers (e.g., 1-1000 nm) that includes lipids and that is stable and dispersible in aqueous media.
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a crystal nanoparticle comprising:
For the purposes of the invention, the term “crystal nanoparticle” refers to artificially prepared crystals composed of solid lipids enclosing one or more hydrophobic compartments.
The term “crystal nanoparticle” refers to particles of different shapes on the order of nanometers (e.g., 1-1000 nm) that includes lipids and that is stable and dispersible in aqueous media.
The liposomes and lipid nanoparticles disclosed herein may comprise any suitable lipid, including ionizable lipids, cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids.
Suitable ionizable lipids include, without limitation, (2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide (DOGS), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol), (1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA), 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA, also known as DLin-C2K-DMA, XTC2, and C2K), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-KC3-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminopropyl)-[1,3]-dioxolane (DLin-KC4-DMA), 1,2-dilinolenyloxy-4-(2-dimethylaminoethyl)-1,3-dioxolane (DLen-C2K-DMA), 1,2-di-y-linolenyloxy-4-(2-dimethylaminoethyl)-1,3-dioxolane (y-DLen-C2K-DMA), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 3-(dimethylamino)propanoate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA, also known as MC3) and 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (DLin-MP-DMA, also known as 1-B11).
Suitable cationic lipids include, without limitation, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDA13), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3,-ditetradecyloxy)propyll-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-(2,3,dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3β-[N—(N′,N′-dimethylamino-ethane)-carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), ethylphosphatidylcholine, (ePC), and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA).
Examples of anionic lipids include, but are not limited to, phosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-Succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine cholesterol hemisuccinate (CHEMS), lysylphos-phatidylglycerol, N-dodecanoyl phosphatidyl ethanoloamine, cardiolipin, and combinations thereof.
Suitable neutral lipids may be uncharged or zwitterionic lipids and include, without limitation steroids, phospholipids, and combinations thereof.
Examples of steroids include, without limitation, cholesterol, progesterone, cortisone, aldosterone, estradiol, testosterone, and combinations thereof. Examples of phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoyl phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyp-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidyl-ethanolamine (DSPE); lysophosphatidyl choline, lysophosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), dilauryloylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero3-phosphocholine (DEPC), and combinations thereof.
The polymers of the invention as well as their conjugates may be used as stealth lipids in lipid nanoparticles.
According to one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the invention relates to a lipid nanoparticle (LNP) or a liposome comprising the polymer, the conjugate of formula (II) or a combination of both, further comprises one or more of an ionisable lipid, a cationic lipid, a neutral lipid, and an anionic lipid. More particularly, the LNP further comprises one or more nucleic acids as previously defined.
The present invention also relates to a composition comprising an effective amount (a) the polymer of the invention as defined herein, or alternatively, (b) the conjugate of formula (II) as defined herein, or alternatively, (c) a combination of the polymer of the invention and the conjugate of formula (II), or alternatively, (d) the self-assembled particles as defined herein, together with one or more acceptable excipients or carriers.
The term “effective amount” as used herein refers to the amount of each one of the components of the composition of the present invention which provides a beneficial effect after its application.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a nutraceutical supplement comprising a nutraceutically effective amount of (a) the polymer of the invention as defined herein, or alternatively, (b) the conjugate of formula (II) as defined herein, or alternatively, (c) a combination of the polymer as defined herein and the conjugate of formula (II), or alternatively, (d) the self-assembled particles as defined herein, together with one or more nutraceutically acceptable excipients or carriers, wherein the encapsulated agent in the self-assembled particles, when present, is a nutraceutically active agent, and in the conjugate of formula (II), when present, Z is a nutraceutically active agent.
In the context of the present invention, the terms “nutraceutical supplement”, “food supplement”, “dietary supplement” or “nutritional supplement” as used herein interchangeably and refer to a preparation intended to supplement the diet and provide nutrients that may be missing or may not be consumed in sufficient quantity in a person's diet. The term “nutraceutically acceptable” excipients or carriers refers to components that may be consumed by humans without significant deleterious health consequences.
In another embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a pharmaceutical composition comprising a therapeutically effective amount of (a) the polymer as defined herein, or alternatively, (b) the conjugate of formula (II) as defined herein, or alternatively, (c) a combination of the polymer as defined herein and the conjugate of formula (II), or alternatively, (d) the self-assembled particles as defined herein, together with one or more pharmaceutically acceptable excipients or carriers, wherein the encapsulated agent in the self-assembled particles, when present, is a pharmaceutically active agent or a cell-targeting agent, and in the conjugate of formula (II), when present, Z is a pharmaceutically active agent or a cell-targeting agent.
The expression “therapeutically effective amount” as used herein, refers to the amount of a polymer that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The specific dose of the polymer of the invention to obtain a therapeutic benefit may vary depending on the particular circumstances of the individual patient including, among others, the size, weight, age and sex of the patient, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration.
For the purposes of the present invention, the term “pharmaceutically acceptable” excipients or carriers refers to components which are appropriate for use in pharmaceutical technology for the preparation of compositions for medical use. Each component should be “acceptable” in the sense of being compatible with the other ingredients of the composition. When used in contact with the tissue or organ of humans and animals should not have excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a diagnostic composition comprising a diagnostically effective amount of (a) the conjugate of formula (II) as defined herein, or alternatively, (b) the self-assembled particles as defined herein, with the condition that when the self-assembled particles comprise the polymer of the invention in the absence of a conjugate of formula (II), an encapsulated agent is present within the self-assembled particles; together with one or more diagnostically acceptable excipients or carriers, wherein the encapsulated agent in the self-assembled particles, when present, is a diagnostically active agent, and in the conjugate of formula (II), when present, Z is a diagnostically active agent.
The term “diagnostic composition” refers to a composition suitable for use in diagnostic, particularly in imaging diagnostic technology. The term “diagnostically effective amount” as used herein, refers to the effective amount of a detection polymer that, when administered, is sufficient for the diagnosis of a disease or disorder; particularly as imaging diagnostic use as contrast imaging agent. The dose of the detection polymer administered will of course be determined by the particular circumstances surrounding the case, including the polymer administered, the route of administration, the particular condition being diagnosticated, and the similar considerations. The diagnostic composition of the present invention comprises one or more diagnostically acceptable excipients or carriers. The term “diagnostically acceptable” refers to that excipients or carriers suitable for use in the diagnosing technology for preparing compositions with diagnostic use; particularly by imaging diagnostic use. The detection of these diagnostic agents in the body of the patient can be carried out by the well-known techniques used such as in imaging diagnostic with magnetic resonance imaging (MRI) and X-ray.
In one embodiment, optionally in combination with one or more features of the various embodiments described above or below, the composition is a cosmetic composition comprising a cosmetically effective amount of (a) the polymer of formula (I) as defined herein, or alternatively, (b) the conjugate of formula (II) as defined herein, or alternatively, (c) a combination of the polymer of formula (I) and the conjugate of formula (II), or alternatively, (d) the self-assembled particles as defined herein, together with one or more cosmetically acceptable excipients or carriers; wherein the encapsulated agent in the self-assembled particles, when present, is a cosmetically active agent, and in the conjugate of formula (II), when present, Z is a cosmetically active agent.
The term “cosmetically effective amount” as used herein, refers to the effective amount of a cosmetic active agent that, when administered, is intended to improve its appearance or to beautify, preserve, condition, cleanse, color or protect the skin, nails or hair without non-medical application. The term “cosmetically acceptable” or “dermatologically acceptable” excipients or carriers is used interchangeably in this document and refer to components which are appropriate for use in human skin contact without toxicity, incompatibility, instability, inappropriate allergic response, among others.
The compositions of the invention may be in solid or liquid form. Depending on the desired purpose, the skilled person will know the most appropriate formulation and excipients to be used. Examples of excipients or carriers include, without limitation, diluents, binders, glidants, disintegrants, lubricants colorants, mixtures thereof, and other components known in the state of the art. Any administration route may be used such as e.g. oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous route, etc.).
The nutraceutically active agents, pharmaceutically active agents, cell-targeting agents present in the self-assembled particles and compositions of the invention are as defined in the context of the conjugates of formula (II). Thus, all embodiments indicated for the conjugates of formula (II) regarding the active agents also apply to the self-assembled particles and compositions.
Another aspect of the invention relates to (i) the polymer of formula (I) as defined herein, or alternatively, (ii) the conjugate of formula (II) as defined herein, or alternatively,
(iii) the self-assembled particle as defined herein, or alternatively, (iv) the pharmaceutical composition as defined herein, for use in medicine, wherein the encapsulated agent in the self-assembled particles, when present, is a pharmaceutically active agent or a cell-targeting agent as defined herein, and in the conjugate of formula (II), when present, Z is a pharmaceutically active agent or a cell-targeting agent.
Non-limiting examples of diseases that may be treated and/or prevented by the derivatives of the present invention include neurodegenerative disorders, neurological diseases, cancer, infectious diseases, disorders related to aging, neuro-inflammation, demyelinating disorders, multiple sclerosis, ischemic disorders, ischemia-reperfusion damage, amyloidotic disease, cardiomyopathy, spinal cord injury, immune disorders, inflammatory disorders, rare diseases, wound healing, skin related diseases and lysosomal storage diseases.
Non-limiting examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischaemia, post-encephalitic Parkinsonisms, dystonias, Tourette syndrome, periodic limb movement pathologies, restless legs syndrome, attention deficit hyperactivity disorders, Huntington's disease, progressive supranuclear palsy, Pick's disease, fronto-temporal dementia and neuromuscular diseases.
The term “disorder” as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disease,” “syndrome,” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms.
For example, a conjugate of formula (II) wherein Z is an anticancer agent, or a self-assembled particle which comprises an anticancer agent encapsulated within the self-assembled particle, may be used for the treatment of cancer. Thus, in one embodiment, the invention relates to
Thus, this aspect of the invention relates to the use of (i) the conjugate of formula (II) as defined herein; or alternatively, (ii) the self-assembled particle as defined herein, with the condition that when the self-assembled particle comprises the polymer of formula (I) in the absence of a conjugate of formula (II), an encapsulated agent is present within the self-assembled particle; or alternatively,
(iii) the pharmaceutical composition as defined herein; for the manufacture of a medicament for the treatment and/or prevention of cancer, wherein the encapsulated agent in the self-assembled particles, when present, is an anticancer agent, and in the conjugate of formula (II), when present, Z is anticancer agent.
It may also be formulated as a method for the treatment and/or prevention of cancer, comprising administering in a subject in need thereof, including a human, an effective amount of (i) the conjugate of formula (II) as defined herein; or alternatively, (ii) the self-assembled particle as defined herein, with the condition that when the self-assembled particle comprises the polymer of formula (I) in the absence of a conjugate of formula (II), an encapsulated agent is present within the self-assembled particle; or alternatively, (iii) the pharmaceutical composition as defined herein; wherein the encapsulated agent in the self-assembled particles, when present, is an anticancer agent, and in the conjugate of formula (II), when present, Z is anticancer agent.
In the context of the present invention, the terms “treat”, “treating” and “treatment”, as used herein, refers to ameliorating symptoms associated with a disease or disorder, including preventing or delaying the onset of the disease or disorder symptoms, and/or lessening the severity or frequency of symptoms of the disease or disorder.
Another aspect of the invention relates to (i) the conjugate of formula (II) as defined herein, or alternatively, (ii) the self-assembled particle as defined herein, with the condition that when the self-assembled particle comprises the polymer of formula (I) in the absence of a conjugate of formula (II), an encapsulated agent is present within the self-assembled particle; or alternatively, (iii) the diagnostic composition as defined herein, for use in diagnostics, wherein the encapsulated agent in the self-assembled particles, when present, is a diagnostically active agent, and in the conjugate of formula (II), when present, Z is a diagnostically active agent.
This aspect of the invention relates to the use of (i) the conjugate of formula (II), or alternatively, (ii) the self-assembled particle as defined herein, with the condition that when the self-assembled particle comprises the polymer of formula (I) in the absence of a conjugate of formula (II), an encapsulated agent is present within the self-assembled particle; or alternatively, (iii) the diagnostic composition as defined herein in diagnostics, wherein the encapsulated agent in the self-assembled particles, when present, is a diagnostically active agent, and in the conjugate of formula (II), when present, Z is a diagnostically active agent. It may also be formulated as a method for the diagnostic of a disease or condition, comprising administering (i) the conjugate of formula (II), or alternatively, (ii) the self-assembled particle as defined herein, with the condition that when the self-assembled particle comprises the polymer of formula (I) in the absence of a conjugate of formula (II), an encapsulated agent is present within the self-assembled particle; or alternatively, (iii) the diagnostic composition as defined herein, in a subject in need thereof, including a human, wherein the encapsulated agent in the self-assembled particles, when present, is a diagnostically active agent, and in the conjugate of formula (II), when present, Z is a diagnostically active agent.
The detection of these imaging agents can be carried out by well-known techniques such as imaging diagnostic techniques. Examples of imaging diagnostic techniques suitable for the present disclosure include, but not limited to, are magnetic resonance imaging (MRI), X-ray, positron emission tomography (PET), single-photon emission computed tomography (SPECT), fluorescence microscopy, and in vivo fluorescence.
Another aspect of the invention relates to the use of the polymer of formula (I) as defined herein as a carrier, as a solubilizer, as absorption and permeation enhancer, as emulsifier or as surface stabilizer. When used as a carrier, the polymer of formula (I) may be in the form of self-assembled particles. Thus, it also forms part of the invention the use of the self-assembled particles which comprise the polymer of formula (I) as carriers, solubilizers, absorption and permeation enhancers, emulsifiers or as surface stabilizers.
Throughout the description and claims the word “comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
NMR spectra were recorded at 27° C. (300 K) on a 300 Ultrashield™ from Bruker (Billerica MA, USA). Data were processed with the software Topspin (Bruker GmbH, Karlsruhe, Germany). Samples were prepared at a concentration of 20-10 mg/mL approx. in the required solvent.
Pulsed-field gradient NMR spectroscopy was used to measure translational diffusion by fitting the integrals or intensities of the NMR signals to the Stejskal-Tanner equation (Fieber W. et al., Macromolecules 2007, 40(15), 5372-5378): I=I0 exp[−Dγ2 g2δ2(Δ−δ/3)] where I is the observed intensity, I0 the reference intensity (unattenuated signal intensity), D the diffusion coefficient, γ the gyromagnetic ratio of the observed nucleus, g the gradient strength, δ the length of the gradient, and Δ the diffusion time. Two-dimensional diffusion-ordered NMR spectroscopy (DOSY) was performed with a stimulated echo sequence using bipolar gradient pulses. The lengths of delays were held constant at Δ=100 ms, and 32 spectra of 64 scans each were acquired with the strength of the diffusion gradient varying between 5% and 95%. The lengths of the diffusion gradient and the stimulated echo were optimized for each sample. Typical values were δ=5 ms for the analysis of physical mixture species and cross-linked materials at 15 mg/mL. Moreover, the hydrodynamic radius (Rh) in nm through diffusion coefficient was obtained employing the Stokes-Einstein equation. Rh=(KB·T)/(6π·η·D) where KB is the Boltzman constant (1.3806488·10−23 m2·Kg/s2·K), T is the temperature (K) and η is the dynamic viscosity (1.095·10−3 Kg/m·s for D2O at 298.15 K and 0.1 MPa) (Duro-Castano A. et al., Advanced Materials 2017, 29(39), 12).
For SEC measurements in dimethylformamide (DMF) containing 0.1% (w/w) of lithium bromide as an additive, a GPC max (Malvern Instruments) autosampler was used with a flow rate of 0.7 mL/min at 60° C. as an integrated instrument, TSKgel Alpha-4000 from Tosoh. Viscoteck TDA™ 302 was used as an integrated detection system (Refractive index). The system was calibrated with polymethyl methacrylate (PMMA) (Mw=65 kDa; PDI=1.05) obtained from PSS. For this Mw determination an integrated triple detection system was used [Refractive Index, Light Scattering (two angles: 7 and 90°) and Differential Pressure Viscosimeter]. Additional SEC detector of UV-Vis was used to measure the alpha-tocopherol absorption at 230 nm.
Dynamic Light Scattering (DLS) measurements were performed using a Malvern Zetasizer NanoZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 173°. Polymer solutions were prepared under different conditions (MilliQ or KCl 1 mM at different concentrations and temperatures), solutions were sonicated for 10 min and allowed to age for the required time, filtered through a 0.45 μm cellulose membrane filter and measured. Size distribution was measured (diameter, nm) for each polymer per triplicate with n>3 measurements, automatic optimization of beam focusing and attenuation was applied for each sample.
Z-potential measurements were performed at 20° C. using a Malvern ZetasizerNanoZS instrument, equipped with a 532 nm laser using disposable folded capillary cells, provided by Malvern Instruments Ltd. (Worcestershire, UK). Polymer solutions were prepared in 1 mM KCl in MilliQ water. The solutions were filtered through a 0.45 μm cellulose membrane filter. Z-potential was measured for each sample per triplicate with n>3 measurements.
Coupling Reagent for Aqueous Conjugations: 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMMCl)
2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 50 g, 0.28 mol, 1 eq.) was dissolved in tetrahydrofuran (THF, 150 mL) at r.t. for 30 min. Then, N-methylmorpholine (NMM, 28.8 g, 0.28 mol, 1 eq.) was added slowly into reaction and a white solid was precipitated. The reaction was left 30 min and the product (DMTMMCl) was filtered under vacuum and washed 3×100 mL of THF. The white product was dried under vacuum and air current for 16 hours. Yield: 80-90%. 1H NMR (300 MHz, D2O) δ 4.70 (d, J=11.4 Hz, 2H), 4.22 (d, J=8.7 Hz, 8H), 4.06-3.87 (m, 4H), 3.63 (s, 3H).
Coupling Reagent for Organic Conjugations: 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMBF4)
DMTMMCl (25 g, 0.09 mol, 1 eq.) was dissolved in water (50 mL) at r.t. for 10 min. In parallel, sodium tetrafluoroborate (NaBF4, 9.84 g, 0.09 mol, 1 eq.) also was dissolved in water (20 mL). Then, NaBF4 solution was added drop by drop into the DMTMMCl solution and the DMTMMBF4 precipitated. The white product (DMTMMBF4) was filtered at vacuum and washed 3×20 mL of water. To remove the water, the wet product was freeze-dried. Yield: 80-90%. 1H NMR (300 MHz, D2O) δ 4.58-4.44 (m, 2H), 4.11-3.89 (m, 8H), 3.89-3.63 (m, 4H), 3.49-3.33 (m, 3H). 19F NMR (282 MHz, D2O) δ −150.52 (d, J=14.3 Hz).
1.1. Isopropyl-poly(sarcosine) Derivatives iPr-PSar6, iPr-PSar12, iPr-PSar20)
Sarcosine-N-carboxyanhydride (3-methyl-2,5-oxazolidinedione, NCA-Sar (5 g, 43.47 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (20 mL). Then, the isopropyl amine (DP6: 746.51 μL, 513.5 mg, 8.7 mmol, DP12: 311.05 μL, 213.9 mg, 4.5 mmol, DP20: 186.63 μL, 128.39 mg, 2.17 mmol) was dissolved in DMF (4 mL) and added to the reaction mixture under nitrogen atmosphere. Once the initiator was added, the round bottom flask was immediately connected to an air-lock allowing the CO2 escape. The mixture was stirred at 10° C. until the total consumption of NCA-Sar confirmed by Fourier transform infrared (FTIR) spectroscopy (disappearance of NCA signals at 1853 and 1786 cm−1). Upon completion, the reaction mixture became slightly yellow. The reaction mixture was poured into diethyl ether (1:10 to DMF:diethyl ether) to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min), washed two times with diethyl ether and dried under vacuum. For further purification, the obtained solid was redissolved in methanol at 500 mg/mL and then the solution was poured again into diethyl ether (1:10 to methanol:diethyl ether), washed two times with diethyl ether and dried under vacuum at least for 2 hours. Finally, the solid is redissolved in distilled water, freezed and lyophilized. iPrPSarn was isolated as an off-white solid. Yield: 70-90% 1H NMR (MeOD): δ=1.16 (m, 6H, CH3), 2.40 (broad s, 3H, terminal N—CH3), 2.89-3.11 (broad m, 65H, polymer N—CH3), 3.50 (broad s, 2H, terminal —CH2—) & 3.88-4.42 (broad m, 43H, —CH2—).
The obtained products were analyzed in terms of Mn distribution and DP by SEC and 1H NMR. Results are shown in table 1 below:
aAbsolute Mw was determined by SEC-RI-MALS in DMF containing LiBr at 0.1%,
b1H NMR in MeOD at 10 mg/mL.
1.2. α-tocopherol-succinate-poly(sarcosine) (iPrPSar(6)-succ-VitE, iPrPSar(12)-succ-VitE, iPrPSar(20)-succ-ViE)
Tocopherol succinate (DP6: 4.925 g, 9.28 mmol; DP12: 2.621 g, 4.94 mmol; DP20: 2.239 g, 4.22 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, dissolved in anhydrous DMF (mL) and covered to avoid the direct light contact. Then, DMTMM·BF4 coupling agent was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (mL). DMTMM·BF4 solution was added over tocopherol succinate solution under inert atmosphere and let to react at r.t. for 30 min with stirring. A few drops of N,N-diisopropylethylamine (DIPEA) were added until pH rising to 6-7. Afterwards, iPrPSar(6) (3 g, 6.19 mmol), iPrPSar(12) (3 g, 3.29 mmol) or iPrPSar(20) (4 g, 2.81 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (mL). After the 30-minutes, the iPrPSar solution was added over the reaction mixture under inert atmosphere. pH adjustment to 8-9 was performed with DIPEA addition. The mixture was stirred at r.t. for 48 hours at r.t avoiding direct light contact. The amphiphile polymer was poured into diethyl ether (1:10 DMF:diethyl ether) to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) washed two times with diethyl ether and dried under vacuum. For further purification, the obtained solid was redissolved in methanol at 500 mg/mL and then the solution was poured again into diethyl ether (1:10 to methanol:diethyl ether), washed two times with diethyl ether and dried under vacuum at least for 2 hours. Finally, the solid was dissolved in distilled water and diafiltrated in a tangential flow filtration system with the addition of some drops of HCl for total DIPEA purification. The solution was freezed and lyophilized to obtain an off-white solid. Yield: 70-90%1H NMR (MeOD): δ=0.90 (m, 12H, CH—CH3), 1.04-1.63 (broad m, 30H), 1.83 (m, 2H, —CH2—CH2—CCO), 1.98 (s, 3H, CH3—), 2.02 (s, CH3—), 2.09 (s, CH3—), 2.63 (t, 2H, —CH2—CH2—C═C), 2.86-3.15 (broad s, 71H, N—CH3), 3.92-4.55 (broad s, 46H).
The obtained product analyzed in terms of Mn distribution and DP by SEC, and 1H NMR. Results are shown in table 2 below:
aAbsolute Mn was determined by SEC-RI-MALS in DMF containing LiBr at 0.1%,
b1H NMR in MeOD at 10 mg/mL.
Sarcosine-N-carboxyanhydride (3-methyl-2,5-oxazolidinedione, NCA-Sar (0.5 g, 4.34 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMSO (3 mL). Then, the 1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-imidazo[4,5-C]quinolin-4-amine (DP20: 93.99 mg, 0.22 mmol) was dissolved in DMSO (1 mL), treated with N,N-diisopropylethylamine (75.67 μL, 56.14 mg, 0.43 mmol) and added to the reaction mixture under nitrogen atmosphere. Once the initiator was added, the round bottom flask was immediately connected to an air-lock allowing the CO2 escape. The mixture was stirred at room temperature until the total consumption of NCA-Sar confirmed by Fourier transform infrared (FTIR) spectroscopy (disappearance of NCA signals at 1853 and 1786 cm−1). Upon completion, the reaction mixture became slightly yellow. The reaction mixture was poured into tetrahydrofuran (1:10 to DMSO:tetrahydrofuran) to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min), washed two times with tetrahydrofuran and dried under vacuum. For further purification, the obtained solid was redissolved in methanol at 200 mg/mL and then the solution was poured again into diethyl ether (1:10 to methanol:diethyl ether), washed two times with diethyl ether and dried under vacuum at least for 2 hours. Finally, the solid is redissolved in distilled water, freezed and lyophilized. TLR-7,8PSar20 was isolated as an off-white solid. Yield: 70-90%. 1H NMR (MeOD): δ=0.95 (t, 3H, CH3—), 1.46 (m, 2H, —CH2—), 1.84 (m, 2H, —CH2—) 2.72 (broad t, 5H, terminal N—CH3& —CH2—), 2.82-3.13 (broad m, 71H, terminal N—CH3), 3.89 (broad s, 2H, terminal —CH2—), 3.98-4.50 (broad m, 50H, —CH2—& —CH2—), 5.93 (s, 2H, —CH2—), 7.05 (broad d, 2H, H—), 7.30 (broad m, 3H, H—), 7.60 (t, 1H, H—) 7.75 (d, 1H, H—) & 7.95 (d, 1H, H—).
The obtained product was analyzed in terms of Mn distribution and DP by SEC and 1H NMR. Results are shown in table 3 below:
aAbsolute Mw was determined by SEC-RI-MALS in DMF containing LiBr at 0.1%,
b1H NMR in MeOD at 10 mg/mL.
1.2. α-tocopherol-succinate-functionalized-poly(sarcosine) (TLR-7,8-PSar(20)-succ-tocopherol)
Tocopherol succinate (67.22 mg, 0.13 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, dissolved in anhydrous DMF (1 mL) and covered to avoid the direct light contact. Then, DMTMM·BF4 (41.55 mg, 0.13 mmol) coupling agent was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (1 mL). DMTMM·BF4 solution was added over tocopherol succinate solution under inert atmosphere and let to react at r.t. for 30 min with stirring. A few drops of N,N-diisopropylethylamine (DIPEA) were added until pH rising to 6-7. Afterwards, TLR-7,8-PSar(20) (0.15 g, 0.08 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (2 mL). After the 30-minutes, the iPrPSar solution was added over the reaction mixture under inert atmosphere. pH adjustment to 8-9 was performed with DIPEA addition. The mixture was stirred at r.t. for 48 hours at r.t avoiding direct light contact. The amphiphile polymer was poured into diethyl ether (1:10 DMF:diethyl ether) to precipitate the product. The precipitate was isolated by centrifugation (3750 rpm, 4 min) washed two times with diethyl ether and dried under vacuum. For further purification, the obtained solid was redissolved in methanol at 500 mg/mL and then the solution was poured again into diethyl ether (1:10 to methanol:diethyl ether), washed two times with diethyl ether and dried under vacuum at least for 2 hours. Finally, the solid was dissolved in distilled water and purified in a ultrafiltration unit with the addition of some drops of HCl for total DIPEA removal. The solution was freezed and lyophilized to obtain an off-white solid.
Yield: 70-90%1H NMR (MeOD): δ=0.87 (m, 12H, CH—CH3), 0.94 (t, 3H, CH3—), 1.02-1.60 (broad m, 32H), 1.80 (m, 4H, —CH2—) 1.98 (s, 3H, CH3—), 2.02 (s, 3H, CH3—), 2.09 (s, 3H, CH3—), 2.59 (broad s, 2H, —CH2—), 2.76-3.15 (broad m, 84H, terminal N—CH3), 3.80-4.57 (broad m, 57H, —CH2—& —CH2—), 5.89 (s, 2H, —CH2—), 7.03 (broad d, 2H, H—), 7.27 (broad m, 3H, H—), 7.55 (t, 1H, H—) 7.70 (d, 1H, H—) & 7.91 (d, 1H, H—).
The obtained product analyzed in terms of Mn distribution and DP by SEC, and 1H NMR. Results are shown in table 4 below:
aAbsolute Mn was determined by SEC-RI-MALS in DMF containing LiBr at 0.1%,
b1H NMR in MeOD at 10 mg/mL.
α-tocopherol (2000 mg, 4.64 mmol), Boc-Gly-OH (813 mg, 4.64 mmol) and DMAP (58.48 mg, 0.48 mmol) were added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, dissolved in anhydrous dichloromethane (16 mL). Then, DCC (958.09 mg, 4.64 mmol) was dissolved in 4 mL of anhydrous dichloromethane and added over the mixture solution. The solution turned from yellow transparent solution into white cloudy appearance. Reaction mixture was left stirring at RT for 16 h.
The resulting solution was left for 45 min at −20° C., and later it was filtered off and the dichloromethane was removed by evaporation. Afterwards, the oil obtained was purified by silica gel column chromatography with hexane:ethyl acetate (95:5). The product Boc-Gly-VitE fractions were collected, concentrated and finally dried under vacuum.
Yield: 90%
1H NMR (MeOD): δ=0.90 (m, 12H, CH—CH3), 1.04-1.63 (broad m, 24H), 1.45 (s, 9H, (CH3)3—C—), 1.83 (m, 2H, —CH2—CH2—CCO), 1.98 (s, 3H, CH3—), 2.02 (s, 3H, CH3—), 2.09 (s, 3H, CH3—), 2.63 (t, 2H, —CH2—CH2—C═C), 4.08 (s, 2H, NH—CH2—CCO).
Boc-Gly-VitE (3000 mg, 5.1 mmol), was dissolved in 15.5 mL of dichoromethane and then 5.1 mL of dichoromethane:TFA 1:1. The mixture reaction was left stirring for 30 min at 4° C., and after, 1 h at RT. Afterwards, solvents are removed by evaporation to obtain a brownish oil left for 24 h at −20° C. This oil was dissolved in MeOH at a concentration of 250 mg/mL and precipitated in 150 mL H2O. Finally, the product was lyophilized obtaining a white powder.
Yield: 56%
1H NMR (MeOD): δ=1H NMR (MeOD): δ=0.90 (m, 12H, CH—CH3), 1.04-1.63 (broad m, 24H), 1.83 (m, 2H, —CH2—CH2—CCO), 1.98 (s, 3H, CH3—), 2.02 (s, 3H, CH3—), 2.09 (s, 3H, CH3—), 2.63 (t, 2H, —CH2—CH2—C═C), 4.14 (s, 2H, NH—CH2—CCO).
VitE-Gly-NH2 (For DP x=20: 529.7 mg; x=50: 423.8 mg) was purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (For DP x=20: 3 mL; x=50: 2 mL).
Sarcosine-N-carboxyanhydride (3-methyl-2,5-oxazolidinedione, NCA-Sar (For DP x=20: 2500 mg, 21.72 mmol; x=50: 5000 mg, 43.44 mmol) was added to a round bottom flask with a stirrer bar, a stopper and purged with 3 cycles of vacuum/N2, and dissolved in anhydrous DMF (For DP x=20: 7 mL; x=50: 18 mL). Then, the corresponding initiator VitE-Gly-NH2 was added to the reaction mixture under nitrogen atmosphere at 10° C. Once the initiator was added, the round bottom flask was immediately connected to an air-lock allowing the CO2 escape. The mixture was stirred at 10° C. until the total consumption of NCA-Sar confirmed by Fourier transform infrared (FTIR) spectroscopy (disappearance of NCA signals at 1853 and 1786 cm−1). Upon completion, the reaction mixture became slightly yellow. The amphiphile polymer was poured into diethyl ether (1:10 DMF:diethyl ether) to precipitate the product and for further purification, the obtained solid was redissolved in methanol at 500 mg/mL and then the solution was poured again into diethyl ether (1:10 to methanol:diethyl ether). The solid was dried under vacuum and lyophilized, obtaining a white powder as a product.
1H NMR (MeOD): δ=0.90 (m, 12H, CH—CH3), 1.04-1.63 (broad m, 25H), 1.83 (m, 2H, —CH2—CH2—CCO), 1.98 (s, 3H, CH3—), 2.02 (s, 3H, CH3—), 2.09 (s, 3H, CH3—), 2.63 (t, 2H, —CH2—CH2—C═C), 2.86-3.15 (broad s, 81H, N—CH3), 3.92-4.55 (broad s, 57H).
Yield: 75%
1H NMR (MeOD): δ=0.90 (m, 12H, CH—CH3), 1.04-1.63 (broad m, 25H), 1.83 (m, 2H, —CH2—CH2—CCO), 1.98 (s, 3H, CH3—), 2.02 (s, CH3—), 2.09 (s, CH3—), 2.63 (t, 2H, —CH2—CH2—C═C), 2.86-3.15 (broad s, 214H, N—CH3), 3.92-4.55 (broad s, 144H).
Yield: 75%
The obtained product analyzed in terms of Mn distribution and DP by SEC, and 1H NMR. Results are shown in table 5 below:
aAbsolute Mn was determined by GPC-RI-MALS in MeOH:Guanidinium 85:15,
b1H NMR in MeOD at 10 mg/mL.
The critical micellar concentration of iPrPSar(x)-VitE polymers was determined employing the fluorescence dye Nile Red. Nile Red is a hydrophobic and solvatochromic dye with poor solubility and fluorescence in aqueous media. The excitation-mediated emission is observed when the Nile Red is associated with a hydrophobic domain, so if the concentration of polymer in water exceeds CMC and the polymeric micelles are constituted, Nile Red will emit fluorescence.
A stock solution of Nile Red at the concentration of 500 μM was prepared in acetone. Then 10 μL of the prepared stock solution was added to microcentrifuge tubes, which was allowed to evaporate the acetone in the dark. The 500-μL polymer solutions of various concentrations (range 1×10−4 to 2 mg·mL−1) were added into those microcentrifuge tubes and allowed to age overnight.
The analysis of the samples was performed in a JASCO Spectrofluorometer FP-8300 equipped with a Xe lamp. The samples were excited at 550 nm and the emission spectra between 575-740 nm were recorded. The max. emission intensity recorded was plotted against the polymer concentration logarithm. The CMC was determined as the intersection point of the two main lineal curves adjusted plotted, data is added in Table 6.
Solubilizer properties and encapsulation capability of iPrPSar-VitE polymers were tested and proven employing Carbamazepine (log P=2.67) as hydrophobic drug through solid suspension encapsulation in aqueous micelle formulation.
iPrPSar(X)-succ-VitE polymers were solubilized in ddH2O at 10 mg·mL−1. Then, solid Carbamazepine was added over aqueous polymer solution in a 15% (w/w) ratio to get a saturated solution. The micellar solution was let to stir 24 h at room temperature avoiding the direct light contact. Afterwards, the solution was centrifuged at 13300 rpm for 2 min. The supernatant was isolated and filtrated by Nylon 0.22 μm. The resultant solution was diluted 100 times in a MeOH/H2O 1/1 mixture and the carbamazepine content was determined by UV spectroscopy at 285 nm.
The polymers of the invention showed comparable or even better encapsulation efficiency with respect to TPGS.
In order to study the physico-chemical properties such as size, electrostatic charge, diffusion coefficient and morphology, empty and Dil-loaded micelles of polysarcosine-vitamin E derivatives were prepared. Dil dye fluorophore (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, Dil C18, ThermoFisher) was used as a hydrophobic active agent model at 1% in w/w. The micelles prepared from polysarcosine-vitamin E derivatives were compared to TPGS micelles. TPGS as used in these experiments was commercially available (PMC Isochem) and had the following characteristics shown in table 8 below:
aAbsolute Mw was determined by SEC-RI-MALS in DMF containing LiBr at 0.1%;
b1H NMR in MeOD at 10 mg/mL.
Micelles were formulated by 3 different approaches (direct, thin-film and co-solvent) adapted from Gaucher et al as previously described (Gaucher G. et al., Journal of Controlled Release 2005, 109(1-3), 169-188).
The block copolymer, iPrPSar(20)-succ-VitE as prepared in example 1 (TPSS) or TPGS, (10 mg/mL) was dissolved in distilled water directly with continuous stirring at r.t. until solution homogenization. The formulation was equilibrated at r.t. for 24 hours. Formulations were equilibrated at r.t. for 24 hours, followed by centrifugation at 1000 rpm for 10 minutes. Both for TPGS and TPSS, no sediment in the centrifugation step was found. The solution was freeze-dried directly obtaining a powder solid. For the preparation of fluorescently labeled micelles, the same procedure was followed with the difference that 1% w/w of Dil loading was added directly together with the block copolymer in the distilled water.
The block copolymer, iPrPSar(20)-succ-VitE as prepared in example 1 (TPSS) or TPGS, (100 mg/mL) was dissolved in MeOH and added dropwise into distilled water with continuous stirring at r.t. until the evaporation of the organic solvent. Formulations were equilibrated at r.t. for 24 hours, followed by centrifugation at 1000 rpm for 10 minutes. Both for TPGS and TPSS, no sediment in the centrifugation step was found. The solution was freeze-dried directly obtaining a powder solid. For the preparation of fluorescently labeled micelles, the same procedure was followed with the difference that 1% w/w of Dil loading was aimed by dissolving the block copolypeptides and the dye in THF/MeOH and following the same procedures described above.
The block copolymer, iPrPSar(20)-succ-VitE as prepared in example 1 (TPSS) or TPGS (100 mg/mL) was dissolved in MeOH in a round bottom flask with the adequate size. Then, MeOH was removed slowly in the rotatory evaporator forming a thin-film layer in the round bottom flask. The product was dissolved in distilled water (10 mg/mL). The solutions were equilibrated at r.t. for 24 hours, followed by centrifugation at 1000 rpm for 10 minutes. Both for TPGS and TPSS, no sediment in the centrifugation step was found. The solution was freeze-dried directly obtaining a powder solid. For the preparation of fluorescently labeled micelles, 1% w/w of Dil loading was prepared by dissolving the block copolypeptides and the dye in THF/MeOH and following the same procedures described above.
Properties of the empty and Dil-loaded micelles such as size by DLS and DOSY NMR, electrostatic charge by Z potential measurement, diffusion coefficient by DOSY NMR and morphology by TEM were analyzed. Regarding Dil C18 loading and encapsulation efficiency, since no sediments appeared in the centrifugation step, it could be assumed as indirect method that Dil C18 encapsulation was quantitative for micelles prepared by all three different routes.
The obtained micelles with and without Dil were studied by DLS in terms of size and electrostatic charge at different concentrations. Results are shown in table 9 below:
aHydrodynamic diameter by Intensity.
bHydrodynamic diameter by Number.
c Bimodal size distribution by intensity.
All micelles were studied at 10 and 1 mg/mL to determine if the concentration can induce aggregates, and this was not observed. Empty micelles were formulated using the direct method since TPGS and TPSS are completely water soluble. Micelle formulation methodologies with or without Dil seem not to affect the hydrodynamic diameters (Dh) by Intensity or Number, since all values are between 6 and 19 nm. Importantly, TPGS+DiP prepared by thin-film and TPSS+Di by the direct method resulted in bimodal Dh distributions by Intensity calculations. Regarding the Z potential, the electrostatic charge determined was neutral as expected for TPGS and TPSS at both concentrations studied. Therefore, there is not a significant difference in the micelle Dh of TPGS compared with TPSS.
Diffusion Ordered Spectroscopy by NMR (DOSY NMR) is a powerful tool to study binary systems and hybrid materials. Applying an electromagnetic field gradient the Diffusion Coefficient (DC) of molecules can be calculated (Avram, L. et al., Chemical Society Reviews 2015, 44(2), 586-602, Groves P., Polymer Chemistry 2017, 8(44), 6700-6708). Besides, employing the Stejskal-Tanner equation the profile of intensity decay can be obtained for different moieties in complex molecules. Results for TPGS and TPSS of DC and Dh in D2O are shown in table 10 below:
Dh values obtained in deuterated water (14.49 nm for TPGS and 14.54 nm for TPSS) are in good agreement with Dh determined by DLS using the intensity method being 14.1 nm for TPGS empty micelle at 10 mg/mL and 14.2 nm for TPSS in the same conditions in good agreement with previously obtained data by different techniques. Concerning face-to-face comparison between both micelles, DC and Dh values are very close between them and there are no appreciable differences in terms of DC and Dh.
In the following example the oligonucleotides used was a pDNA purchased from PlasmidFactory, with reference PF461 (pCMV-luc)), containing 6233 bp expressing luciferase. Any other oligonucleotide with the characteristics above indicated could be used to carry out the experiments below. The ionizable lipid (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or simply MC3) was purchased from Nanosoft Polymers. Shielding lipid 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) was purchased from Avanti Polar Lipids. Both polymers were used to form benchmark LNPs. Structural lipids used to form LNPs were: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, from Avanti Polar Lipids, 850365P) and Cholesterol (Chol, Sigma-Aldrich, C8667).
A microfluidic HerringboneMixer from Darwing Microfluidics was used for LNPs formulation of Table 11. The reactor presents 2 inlet-channels (one for the DNA in aqueous phase and the other for the lipid mixture in ethanol) and 1 outlet-channel. The specifications of the mixer are the following:
In addition, two programmable pumps control the fluid flow rates of the syringes (NE-1000 Programmable Single Syringe Pump, Syringe Pump, USA). The system accepts infusion rates from 0.73 μL/h (1 mL syringe) to 2100 mL/h (60 mL syringe).
This methodology provides reproducibility to the formation of LNPs as well as the possibility of scaling up the process.
Benchmark LNPs of Table 11 were formulated as follows:
After formulation, LNPs were diluted in 10 mM Phosphate buffer saline (PBS) pH 7.4 and concentrated with centrifugal concentrators (Vivaspin™ 500, Sigma) to remove ethanol. The formulation containing the polymer of the invention was prepared analogously with the difference that a polymer of formula (I) was used instead of DMG-PEG and the lipid ratio was different (shown below Table 11).
Benchmark LNPs of Table 12 were formulated with NanoScaler (KNAUER) as follows:
After formulation, LNPs were further diluted in 10 mM Phosphate buffer saline (PBS) pH 7.4 to decrease ethanol concentration and purified with centrifugal concentrators (Vivaspin™ 500, Sigma) to remove ethanol. The formulation containing the polymer of the invention was prepared analogously with the difference that a polymer of formula (I) was used instead of shielding lipid ALC-0159 and the lipid ratio was different (shown below Table 12).
Size and polydispersity (PDI) of the formulated LNPs containing mRNA or pDNA at different N/P ratios (positively-chargeable polymer amine (N=nitrogen) groups to negatively-charged nucleic acid phosphate (P)), different molar ratios and shielding polymers were performed using a Malvern ZetasizerNanoZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 173°. 50 μl of the samples were measured using a quartz glass high performance cuvette (Hellma Analytics). Size distribution was measured (diameter, nm) with n≥3 measurements.
Benchmark LNPs containing MC3 as ionizable lipid, DMG-PEG as shielding lipid and pDNA as cargo, were formulated at 50/10/38.5/1.5 molar ratios for MC3/DSPC/Cholesterol/DMG-PEG obtaining the following results:
The presence of DMG-PEG at 1.5% is enough to stabilize the LNPs obtaining a good size and PDI and reproducible formulations.
Table 11 shows the results obtained of iPrPSar(20)-succ-VitE formulated at molar ratio of 5% containing pDNA as cargo.
Results suggest that at 5% of iPrPSar(20)-succ-VitE, LNPs can be obtained showing low PDI and appropriate size.
Benchmark LNPs containing ALC-0315 as ionizable lipid, ALC-0159 as shielding lipid and mRNA as cargo, were formulated at 46.3/9.4/42.7/1.6 molar ratios for ALC-0315/DSPC/Cholesterol/ALC-0159 and the results obtained using iPrPSar(x)-succ-VitE formulated at molar ratio of 5% containing mRNA as cargo. Results are shown in Table 12 below:
Results suggest that at 5% of different iPrPSar(x)-succ-VitE, LNPs can be obtained showing low PDI and appropriate size and Encapsulation Efficiency (EE %) >90%.
To carry out the in vitro cytotoxicity studies two cell lines were used: human immortalized non-tumorigenic keratinocytes (HaCaT cells), supplied by CLS Cell Lines Service (Germany), and human fibroblasts supplied by Hospital La Fe (Valencia, Spain). High glucose Dulbecco's Modified Eagle Medium (DMEM Glutamax, Fisher, Spain) was used for keratinocyte culture and DMEM (Gibco, Spain) for fibroblast culture; both were supplemented with 2% penicillin/streptomycin and 50 mL of fetal bovine serum (FBS) in a humidified incubator (Hucoa-Erlöss S.A., Spain) at 5% CO2 and 37° C.
For the cell viability assays, 50 μL of cells were seeded in 96-well plates at a concentration of 4.000 cells/well for HaCaT cells and 2.000 cells/well for human fibroblasts. After 24 h, 50 μL of each treatment was added, reaching a final volume of 100 μL. Cells were incubated with samples or controls for 72 h before performing MTS assay. For this assay, 20 μL of phenazine methosulfate (PMS) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) (1:20) solution were added. After 3 h of incubation, absorbance values were captured at 490 nm using a Victor2Wallac™ plate reader (Perkin Elmer, Spain). The absorbance values were represented as the percentage of cell viability relative to the 100% cell viability of untreated control cells. TPGS and iPrPSar(20)-succ-VitE (TPSS) in the form of micelles prepared by the direct formulation methodology, as well as free Vitamin E were tested. The concentrations of TPGS, iPrPSar(20)-succ-tocopherol (TPSS) and the free Vitamin E were in a range from 0.1 to 2.57 μg/mL equivalents of Vitamin E.
To demonstrate the property of the of the invention as skin penetration enhancers skin permeation studies by Franz diffusion cells using human skin were performed. In this case, the penetration capacity of both amphiphilic micelles based on vitamin E derivatives were studied.
Breast skin samples were obtained from healthy women undergoing plastic surgery after written informed consent (kindly donated by Hospital la Fe, Valencia, Spain). Immediately after excision, the subcutaneous fatty tissue was carefully removed using a scalpel. The skin was cut into 4 cm2 pieces, wrapped in aluminum foil and stored at −20° C. until use. To reduce inter-individual variability and afford a better comparison of results, skin from only one donor was used in all experiments. Before starting each experiment, the skin was allowed to equilibrate at room temperature (r.t.).
Permeation studies employed the modified Franz diffusion cells (Logan Instruments Corp., EE. UU) with a diffusional area of 0.95 cm2. The skin was fixed carefully between the donor and the receptor chamber, so that stratum corneum was placed upwards. The receptor chamber was filled with 8 mL of 0.01 M phosphate-buffered saline (PBS) pH=7.4 and stirred with a magnetic stirring bar at 600 rpm with a thermostated temperature of 37° C. After an equilibration time of 30 min, 100 μL of the formulations (TPGS-Dil and TPSS-Dil as prepared by direct formulation methodology) using a fixed concentration (10 mg/mL) were introduced into the donor chamber using a standard pipette. The donor chamber was then sealed with Parafilm® and aluminum foil, and the experiments ran for 8 h.
After the permeation study, skin samples were washed twice with 0.1% Phosphate-buffered Saline with Bovine Serum Albumin (PBS-BSA) and kept in 4% paraformaldehyde (PFA) for 24 h at r.t. After that, the samples were washed with 30% sucrose in PBS solution and retained for 24 h at 4° C. Finally, skin samples were washed twice with PBS and preserved in a cryopreservation solution (40% 0.1 M Phosphate Buffer PB, 30% ethylene glycol and 30% glycerol) at 4° C. until use.
Finally, the samples were placed in the optimum cutting temperature (OCT) inclusion medium, and slides of 5 μm were generated via cryostat (version CM1850 UV, Leica, Germany) and samples were analyzed by confocal microscopy.
Images were captured with an inverted DM IRE2 microscope equipped with a λ-blue 40× oil immersion objective and handled with a TCS SP2 system, equipped with an Acoustic Optical Beam Splitter (AOBS). Dil C18(3) was excited at 549 nm, and DAPI at 405 nm. Images were captured at an 8-bit greyscale and processed with LCS software (version 2.5.1347a, Leica, Germany) containing multicolor, macro and 3D components. Control tissue that follows the same incubation time with MilliQ water was also analyzed to establish the autofluorescence. Dil intensity was quantified five times per sample using Image J software.
Confocal microscopy (not shown) and pixels quantification by ImageJ (
Activation of the complement cascade was analyzed by determining SC5b-9 (Protein S-bound terminal complement complex c5b-9) levels using an immunoassay kit MicroVue SC5b-9 Plus. Sera of three healthy individuals was used for further testing. The concentration employed 0.2 mg/mL VitE equivalents. Hepes Buffered Saline (HBS) was used as a negative control and Zymosan (0.25 mg/mL) as a positive control. All samples were incubated by mixing 1 part polymer suspension with 4 parts human serum for 1 h at 37° C. under constant agitation. Following the incubation samples were diluted 100-fold (700-fold for Zymosan) with manufacturers dilution buffer and the assay was conducted as described in the manufacturers protocol. Table 13 shows the SC5b-9 amounts and standard error mean (SEM):
iPrPSar(20)-succ-VitE shows 10× less immunogenic response than the positive control zymosan. And comparable results to negative control HBS. Therefore, iPrPSar(20)-succ-VitE has no immunogenic response in these conditions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21383149.8 | Dec 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/086403 | 12/16/2022 | WO |
