Trehalose-based surfactants

Abstract

Provided herein are compounds useful as surfactants and / or excipients, methods of producing such compounds, methods of using such compounds, and compositions comprising such compounds (preferably in combination with one or more additional agents or therapies).

Description

Technical Field

[0001] Provided herein are compounds that are useful as surfactants and / or excipients, methods of producing such compounds, methods of using such compounds, and compositions comprising such compounds, preferably in combination with one or more additional agents or therapies. Background Art

[0002] Excipients are a large class of molecules used in pharmaceutical formulations of chemical drugs, antibodies, and proteins. They are also involved in improving the shelf life stability of biomolecules during storage or for cryopreservation and freeze drying of biomolecules.

[0003] Excipients used in pharmaceutical formulations of antibodies and proteins can be divided into:

[0004] Bulking agents, such as the polyol / disaccharide / polysaccharide family as well as natural polysaccharides and amino acids,

[0005] Tonicity agents based on sugar materials,

[0006] Buffers, such as organic and inorganic salts,

[0007] Surfactants to reduce surface tension and increase drug solubility,

[0008] Antioxidants,

[0009] Chelating agents,

[0010] ·preservative.

[0011] Excipients are important for the biological material preservation step in the R&D industry. Biomolecules are usually stored at -80°C or freeze-dried. Maintaining materials stored at low temperatures is extremely costly in terms of electricity and nitrogen. In addition, freeze-drying is less expensive, but it causes a large loss of active materials. It is challenging to keep the material intact when the material composition (dehydration), temperature and pressure are affected. Macromolecules may face chemical instability under stress conditions (temperature changes, exposure to light, oxygen, or chemical stress and shear stress), resulting in loss of biological activity. Instability can refer to irreversible denaturation (proteins lose their tertiary structure or their secondary structure) and aggregation (proteins self-assemble in an irreversible manner).

[0012] Among surfactants, polysorbates PS20 and PS80 are widely approved for parenteral administration of antibodies. However, they are reportedly not conducive to long-term storage due to the presence of peroxide residues from poly(ethylene glycol) entities. This leads to protein oxidation and enhances protein degradation. In addition, it has been shown that polysorbates can destroy cell membrane integrity and impair mitochondrial function at concentrations of 1 v / v% to 2 v / v% on BEAES2B bronchial epithelial cells.

[0013] Therefore, better and safer excipients must be developed for the stability, long-term storage, and, if necessary, administration of macromolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Aggregation over time during storage at 4°C monitored by DLS. The figure shows the total area under the curve (AUC) of the peak corresponding to antibody aggregates after 0, 5 and 4 days of storage of 1 mg / mL antibody in different stabilizing media.

[0015] Figure 2 : Nebulization-induced aggregation assessed by DLS. The figure shows the total area under the curve (AUC) of the peak corresponding to antibody aggregates before (white bars) and after (striped bars) nebulization of the antibody at a concentration of 1 mg / mL in each stabilizing medium.

[0016] Figure 3 : Nebulization-induced aggregation assessed by the scatterometry module of Nano-DLS. The figure shows the difference in sample scattering at 70°C and 20°C before (white bars) and after (striped bars) nebulization of 1 mg / mL antibody in each stabilizing medium.

[0017] Figure 4 : Aggregation assessed by DLS after freeze drying. The figure shows the total area under the curve (AUC) of the peak corresponding to antibody aggregates in each stabilizing medium at a concentration of 1 mg / mL of antibody.

[0018] Figure 5 : Graphical representation of the proportion of HEK293 cells expressing SARS-CoV-2 spike labeled by AF488-conjugated anti-Fc IgG measured by FACS. Labeled cells should be bound to the antibody of interest in different stabilization media.

[0019] Figure 6 : Cytotoxicity of trehalose excipient and polysorbate 80 on NIH-3T3 cells after 48 h measured by colorimetric dosage after MTS staining. Summary of the invention

[0020] The present invention provides a compound of formula I

[0021]

[0022] or a salt thereof, a stereoisomer thereof, a polymorph thereof, or a mixture of stereoisomers thereof, wherein

[0023] R1 is selected from -OH; alkyl, optionally substituted by OH; or connected to R3 through oxygen;

[0024] R2 is selected from -OH; alkyl, optionally substituted with OH; or connected to R3 through oxygen;

[0025] R3 is selected from -OH; alkyl, optionally substituted by OH; or connected to R1 or R2 through oxygen;

[0026] R4 is selected from -OH; or alkyl, optionally substituted with -OH;

[0027] X1 is independently selected from -(C=O) and -(C=O)-A-(C=O), wherein A is alkyl, alkenyl, alkynyl or PEG; X2 is independently selected from H or -(C=O);

[0028] Z1 is -OH;

[0029] and,

[0030] - When X2 is C═O, then Z2 is independently selected from alkyl, alkenyl, alkynyl, PEG, succinyl, —NHalkyl, —NHalkenyl, —NHalkynyl, NH-PEG, —Salkyl, —Salkynyl, —Oalkyl, —Oalkenyl, and —Oalkynyl;

[0031] -When X2 is H, then Z2 does not exist;

[0032] And n is an integer from 1 to 20.

[0033] The present invention also provides a composition comprising a compound of formula I as described herein.

[0034] The present invention also provides a pharmaceutical composition comprising a compound of formula I as described herein.

[0035] The present invention also provides a method for preparing a compound of formula I, the method comprising

[0036] - sequentially and selectively functionalize the two primary alcohols of trehalose while leaving the other hydroxyl moieties unreacted,

[0037] - allows first binding to the fatty acid chain and then to the carboxylated side chain, where the carboxyl function is introduced in the form of a benzyl ester which is removed in the last step of the process.

[0038] The present invention also provides a method for preparing a polymer of a compound of formula I, which comprises auto-condensing an activated trehalose derivative in the presence of a coupling agent. DETAILED DESCRIPTION

[0039] As used in this disclosure, the following words and phrases are generally intended to have the meanings as set forth below, unless expressly stated otherwise or the context in which they are used indicates otherwise.

[0040] The following description sets forth exemplary methods, parameters, etc. However, it should be appreciated that such description is not intended to limit the scope of the present disclosure, but rather to provide a description of exemplary embodiments.

[0041] As used in this specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, unless otherwise indicated to some extent by the context in which they are used.

[0042] A dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -CH2 is attached through a carbon atom. Dashes before or after a chemical group are for convenience; a chemical group may be described with or without one or more dashes without losing its ordinary meaning. Unless chemically or structurally required, the order in which the chemical groups are written or named does not indicate or imply directionality.

[0043] The prefix "C u-v " indicates that the following group has u to v carbon atoms. For example, "C 1-6 "Alkyl" means an alkyl group having 1 to 6 carbon atoms.

[0044] The "about" value or parameter mentioned herein includes (and describes) embodiments for the value or parameter itself. In certain embodiments, the term "about" includes an indication of ±10%. In other embodiments, the term "about" includes an indication of ±5%. In certain other embodiments, the term "about" includes an indication of ±1%. In addition, the term "about X" includes a description of "X". In addition, unless the context clearly provides otherwise, the singular indefinite article "a" and the definite article "the" include plural referents. Therefore, for example, mentioning "the compound" includes a plurality of such compounds, and mentioning "the test" includes mentioning one or more tests known to those skilled in the art and their equivalents.

[0045] The term "substituted" means that any one or more hydrogen atoms on the designated atom or group are replaced by one or more substituents other than hydrogen, as long as the normal valence of the designated atom is not exceeded. One or more substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, guanidino, halogen, haloalkyl, heteroalkyl, heteroaryl, heterocycloalkyl, hydroxyl, hydrazine, imino, oxo, nitro, alkylsulfinyl, sulfonic acid, alkylsulfonyl, thiocyanate, thiol, thione, or a combination thereof. It is not intended herein to include polymers or similar indefinite structures obtained by further defining substituents with the attached substituents (e.g., substituted aryl with substituted alkyl, which is itself substituted by substituted aryl groups, which are further substituted by substituted heteroalkyl groups, etc.). Unless otherwise specified, the maximum number of consecutive substitutions in the compounds described herein is three. For example, a substituted aryl group that is continuously substituted by two other substituted aryl groups is limited to an aryl substituted by ((substituted aryl) substituted aryl). Similarly, the above definition is not intended to include unallowed substitution patterns (e.g., methyl substituted with 5 fluorines or heteroaryl groups with two adjacent oxygen ring atoms). Such unallowed substitution patterns are well known to the technician. When used to modify chemical groups, the term "substituted" can describe other chemical groups defined herein. For example, the term "substituted aryl" includes but is not limited to "alkyl aryl". Unless otherwise stated, when a group is described as optionally substituted, any substituent of the group is itself unsubstituted.

[0046] "Substituted" groups also include embodiments in which a monoradical substituent is attached to a single atom of the substituted group (e.g., forming a branch), and also include embodiments in which a substituent can be a diradical bridging group attached to two adjacent atoms of the substituted group, thereby forming a fused ring on the substituted group.

[0047] "Alkyl" refers to an unbranched or branched saturated hydrocarbon chain. As used herein, an alkyl group has 1 to 20 carbon atoms (i.e., C 1-20 Alkyl), 1 to 8 carbon atoms (i.e. C 1-8 Alkyl), 1 to 6 carbon atoms (i.e. C 1-6 Alkyl) or 1 to 4 carbon atoms (i.e. C 1-4Alkyl). Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a specific number of carbon atoms is named by chemical name or identified by molecular formula, all positional isomers having that number of carbon atoms may be included; thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), sec-butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and tert-butyl (i.e., -C(CH3)3); "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2).

[0048] "Alkenyl" refers to a group containing at least one carbon-carbon double bond and having 2 to 20 carbon atoms (i.e., C 2-20 alkenyl), 2 to 8 carbon atoms (i.e., C 2-8 alkenyl), 2 to 6 carbon atoms (i.e., C 2-6 alkenyl) or 2 to 4 carbon atoms (i.e. C 2-4 Examples of the alkenyl group include vinyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0049] "Alkynyl" refers to a group having from 2 to 20 carbon atoms (i.e., C 2-20 Alkynyl), 2 to 8 carbon atoms (i.e., C 2-8 Alkynyl), 2 to 6 carbon atoms (i.e., C 2-6 Alkynyl) or 2 to 4 carbon atoms (i.e. C 2-4 The term "alkynyl" also includes those groups having one triple bond and one double bond.

[0050] “Alkoxy” refers to the group “alkyl-O—” or “—O-alkyl.” Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.

[0051] "Amino" refers to the group -NR y R z , where R y and R z are independently selected from hydrogen, alkyl, haloalkyl, cycloalkyl, aryl, heterocyclyl or heteroaryl; each of which may be optionally substituted.

[0052] The term "alkylsulfinyl" refers to the group -SO-alkyl wherein alkyl is as defined above and includes optionally substituted alkyl groups as defined above.

[0053] "Cycloalkyl" refers to a saturated cycloalkyl group or a partially saturated cycloalkyl group having a single ring or multiple rings including fused ring systems, bridged ring systems, and spiro ring systems. As used herein, a cycloalkyl group has 3 to 20 ring carbon atoms (i.e., C 3-20 Cycloalkyl), 3 to 12 ring carbon atoms (i.e., C 3-12 Cycloalkyl), 3 to 10 ring carbon atoms (i.e. C 3-10 Cycloalkyl), 3 to 8 ring carbon atoms (i.e. C 3-8 Cycloalkyl) or 3 to 6 ring carbon atoms (i.e. C 3-6 Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0054] As used herein, the term "cycloalkenyl" group refers to a non-aromatic carbocyclic group having at least one double bond.

[0055] "Cyanoalkyl" refers to an alkyl group substituted with a cyano (CN) group.

[0056] "Halogen" or "halo" includes fluorine, chlorine, bromine and iodine.

[0057] The term "haloalkyl" refers to a monoradical or diradical having the indicated carbon atom of an alkyl group in which one or more hydrogen atoms have been replaced by a halogen. Examples of haloalkyl groups include -CH2F, -CHF2, -CF3, -CH2CF3, -CHFCH2F, -CF2-, -CHF-, and the like. Similarly, the term "haloalkoxy", such as -OC 1-3 Haloalkyl refers to an alkoxy group in which one or more hydrogen atoms of the alkyl group have been replaced by halogen. Examples of haloalkoxy groups include -OCH2F, -OCHF2, -OCF3, -OCH2CF3, -OCHFCH2F, etc. Those skilled in the art will appreciate that similar definitions apply to the alkenyl analogs and alkynyl analogs above (e.g., C 2-4 Halogenated alkenyl, -OC 2-4 haloalkynyl).

[0058] "Heteroalkyl" refers to an alkyl group in which one or more carbon atoms (and any associated hydrogen atoms) are each independently substituted by the same or different heteroatom groups. The term "heteroalkyl" includes an unbranched or branched saturated chain with carbon and heteroatoms. For example, 1, 2 or 3 carbon atoms can be independently substituted with the same or different heteroatom groups. Heteroatom groups include but are not limited to -NR-, -O-, -S-, -SO-, -SO2-, etc., where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl or heterocycloalkyl, each of which can be optionally substituted. Examples of heteroalkyl groups include -OCH3, -CH2OCH3, -SCH3, -CH2SCH3, -NRCH3 and -CH2NRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl or heteroaryl, each of which can be optionally substituted. As used herein, a heteroalkyl group includes 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 4 carbon atoms; and 1 to 3 heteroatoms, 1 to 2 heteroatoms, or 1 heteroatom.

[0059] "Heterocycloalkyl" refers to a saturated cycloalkyl group or an unsaturated cycloalkyl group, one or more ring heteroatoms independently selected from nitrogen, oxygen and sulfur. Heterocycloalkyl can be a single ring or multiple rings, wherein the multiple rings can be fused, bridged or spirocyclic. As used herein, heterocycloalkyl has 2 to 20 ring carbon atoms (i.e., C 2-20 Heterocycloalkyl), 2 to 12 ring carbon atoms (i.e., C 2-12 Heterocycloalkyl), 2 to 10 ring carbon atoms (i.e., C 2-10 Heterocycloalkyl), 2 to 8 ring carbon atoms (i.e., C 2-8 Heterocycloalkyl), 3 to 12 ring carbon atoms (i.e., C 3-12 Heterocycloalkyl), 3 to 8 ring carbon atoms (i.e., C 3-8 Heterocycloalkyl) or 3 to 6 ring carbon atoms (i.e. C 3-6Heterocycloalkyl);Having 1 to 5 ring heteroatoms, 1 to 4 ring heteroatoms, 1 to 3 ring heteroatoms, 1 to 2 ring heteroatoms or 1 ring heteroatom, the ring heteroatoms are independently selected from nitrogen, sulfur or oxygen. Examples of heterocycloalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, oxetanyl, dioxolanyl, azetidinyl and morpholinyl. As used herein, the term "bridged heterocycloalkyl" refers to a four-membered to ten-membered ring portion, which is connected to one or more (e.g., 1 or 2) four-membered to ten-membered ring portions having at least one heteroatom at two non-adjacent atoms of heterocycloalkyl, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur. As used herein, bridged heterocycloalkyl includes bicyclic and tricyclic systems. Also as used herein, the term "spirocyclic heterocycloalkyl" refers to a ring system in which a three- to ten-membered heterocycloalkyl has one or more additional rings, wherein the one or more additional rings are three- to ten-membered cycloalkyls or three- to ten-membered heterocycloalkyls, wherein the single atoms of the one or more additional rings are also atoms of the three- to ten-membered heterocycloalkyls. Examples of spirocyclic heterocycloalkyls include bicyclic and tricyclic systems, such as 2-oxa-7-azaspiro[3.5]nonanyl, 2-oxa-6-azaspiro[3.4]octanyl, and 6-oxa-1-azaspiro[3.3]heptanyl.

[0060] "Acyl" refers to the group -C(=O)R, where R is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroalkyl, or heteroaryl; each of which may be optionally substituted as defined herein. Examples of acyl include formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethyl-carbonyl, and benzoyl.

[0061] The term "N-alkylated" means that an alkyl group replaces one hydrogen atom of a monosubstituted amine, a disubstituted amine group, or a trisubstituted amine group. When alkylation occurs on a trisubstituted amine group, an alkyl group is produced. N-alkylation is usually associated with alkyl substitution on a ring nitrogen atom.

[0062] The term "oxo" refers to the group =0.

[0063] The term "carboxy" refers to the group -C(O)-OH.

[0064] The term "ester" or "carboxy ester" refers to the group -C(O)OR, where R is alkyl, cycloalkyl, aryl, heteroaryl or heterocyclyl, which may be optionally further substituted by, for example, alkyl, alkoxy, halogen, CF3, amino, substituted amino, cyano or -SO n R f replaced by, where R f is alkyl, aryl or heteroaryl, and n is 0, 1 or 2.

[0065] The term "substituted amino" refers to the group -NRR, wherein each R is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl, each of which may be optionally substituted, or may be a group described or exemplified herein, or wherein two R groups are linked to form a heterocyclic group described or exemplified herein (e.g., morpholino), which may also be optionally substituted.

[0066] The term "amino" refers to the group -C(O)NRR, wherein each R is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl, each of which may be optionally substituted, or may be a group described or exemplified herein, or wherein two R groups are linked to form a heterocyclic group described or exemplified herein (e.g., morpholino), which may also be optionally substituted.

[0067] The term "sulfoxide" refers to the group -SOR, where R is alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which may be optionally substituted.

[0068] As used herein, the terms "alkylcycloalkyl", "alkylaryl", "alkylheteroaryl" and "alkylheterocyclyl" are intended to refer to a cycloalkyl group, aryl group, heteroaryl group or heterocyclyl group that is bound to the remainder of the molecule via an alkyl portion, wherein the terms "alkyl", "cycloalkyl", "aryl", "heteroaryl" and "heterocyclyl" are as defined herein. Exemplary alkylaryl groups include benzyl, phenethyl and the like.

[0069] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0070] As used herein, polyethylene glycol or "PEG" refers to a polyoxyethylene glycol represented by H-(O-CH2-CH2) n -OH polymer, where n represents the number of times the O-CH2-CH2 (oxyethylene) moiety is repeated; n can vary widely because the molecular weight of PEG varies. For example, for lower molecular weight polyethylene glycol (~1500 g / mol), n can be about 33, for higher molecular weight polyethylene glycol (~10,000 g / mol), n can be as high as about 227, for example, for PEG with a molecular weight of ~20,000 g / mol, n can be about 454; for PEG with a molecular weight of ~40,000 g / mol, n can be about 908; for higher molecular weight PEG species, n is even higher.

[0071] Certain commonly used alternative chemical names may be used. For example, divalent groups such as divalent "alkyl" groups, divalent "aryl" groups, etc. may also be referred to as "alkylene" groups, "arylene" groups, respectively. In addition, unless otherwise expressly stated, where a combination of groups is referred to herein as a moiety, such as arylalkyl, the last-mentioned group includes the atoms connecting the moiety to the rest of the molecule.

[0072] If a given group (part) is described in this article as being connected to a second group, and the site of connection is unclear, then the given group can be connected at any available site of the given group or connected to any available site of the second group. For example, "alkyl-substituted phenyl", whose connection site is unclear, can connect any available site of the alkyl group to any available site of the phenyl group. In this respect, "available site" is the site of a group where the hydrogen of a group can be replaced by a substituent.

[0073] It should be understood that in all the substituent groups defined above, it is not intended herein to include polymers obtained by limiting the substituent itself to have further substituents (e.g., substituted aryl groups having substituted aryl groups substituted by substituted aryl groups, etc. as substituents). An unlimited number of substituents are also not included, whether the substituents are the same or different. In this case, the maximum number of such substituents is three. Therefore, each of the above definitions is limited, for example, substituted aryl groups are limited to -substituted aryl-(substituted aryl)-substituted aryl.

[0074] "Isomers" are different compounds that have the same molecular formula. Isomers include stereoisomers, enantiomers, and diastereomers.

[0075] "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space.

[0076] "Enantiomers" are a pair of non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. Where appropriate, the term "±" is used to refer to a racemic mixture.

[0077] "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0078] The compounds of the present disclosure may have one or more asymmetric centers and may be produced as racemic mixtures or as individual enantiomers or diastereomers. The number of stereoisomers present in any given compound of a given formula depends on the number of asymmetric centers present (there may be 2 nThe present invention discloses a single stereoisomer, wherein n is the number of asymmetric centers). The single stereoisomer can be obtained by resolving the racemic mixture or non-racemic mixture of the intermediate in a certain appropriate stage of the synthesis, or by resolving the compound by conventional methods. The single stereoisomer (including the single enantiomer and diastereomer) and the racemic mixture and non-racemic mixture of stereoisomers are included in the scope of the present disclosure, all of which are intended to be described by the structure of the present specification unless otherwise explicitly stated.

[0079] Absolute stereochemistry is specified according to the Cahn Ingold Prelog RS system. When a compound is enantiomerically pure, the stereochemistry of each chiral carbon can be indicated by R or S. Resolved compounds whose absolute configuration is unknown can be designated as (+) or (-) according to the direction (right-handed or left-handed) that they rotate plane polarized light at the wavelength of the sodium D line.

[0080] As used herein, "trehalose" refers to a non-reducing disaccharide consisting of two glucose units. Trehalose is found in many plants, microorganisms, and animals and is involved in glucose storage, signal transduction and regulation, structural and transport effects, and as a protectant and stabilizer for membranes and proteins. Trehalose is widely approved for use in biomolecule preparations for drug development. Trehalose stabilizes biomolecules through its ability to form hydrogen bonds with amino acids present on its surface.

[0081] The term "polymorph" refers to different crystal structures of a crystalline compound. Different polymorphs may result from differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism).

[0082] The term "solvate" refers to a complex formed by combining a compound of Formula (I), or any other formula disclosed herein, with a solvent.

[0083] The term "hydrate" refers to a complex formed by the combination of a compound of Formula (I) or any formula disclosed herein and water.

[0084] As used herein, the term "polymorph" refers to different crystalline forms of the same compound and other solid molecular forms including pseudopolymorphs, such as hydrates (e.g., the presence of bound water in the crystal structure as discussed above) and solvates (e.g., bound solvents other than water) of the same compound. Due to the different packing of molecules in the crystal lattice, different crystalline polymorphs have different crystal structures. This results in different crystal symmetries and / or unit cell parameters, which directly affect their physical properties such as the X-ray diffraction characteristics of crystals or powders. For example, different polymorphs will generally diffract at different angles and will give different intensity values. Therefore, X-ray powder diffraction can be used to identify different polymorphs, or solid forms containing more than one polymorph, in a reproducible and reliable manner (S. Byrn et al, Pharmaceutical Solids: A Strategic Approach to Regulatory Considerations, Pharmaceutical research, Vol. 12, No. 7, p. 945-954, 1995; JK Haleblian and W. McCrone, Pharmaceutical Applications of Polymorphism, Journal of Pharmaceutical Sciences, Vol. 58, No. 8, p. 911-929, 1969).

[0085] The term "prodrug" refers to a compound of formula (I) disclosed herein or a derivative of formula (I) that includes a chemical group that can be transformed in vivo and / or can be separated from the rest of the molecule to provide an active drug. Pharmaceutically acceptable salts of prodrugs of compounds of formula (I) or biologically active metabolites thereof are also within the scope of the present disclosure.

[0086] Any formula or structure given herein, including Formula (I) or any formula disclosed herein, is intended to represent unlabeled forms of the compounds as well as isotopically labeled forms. Isotopically labeled compounds have structures described by the formulas given herein except that one or more atoms are replaced by isotopes having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as, but not limited to, 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S.36 Cl and 125 I. Multiple isotopically labeled compounds of the present disclosure, for example, radioactive isotopes such as 3 H. 13 C and 14 C is incorporated therein, within the scope of the present disclosure. Such isotopically labeled compounds can be used for metabolic studies, reaction kinetic studies, detection techniques or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT) including drug or substrate tissue distribution determination or for the treatment of patients. Such isotopically labeled analogs of the compounds disclosed herein can also be used to treat the diseases disclosed herein because they can provide improved pharmacokinetic properties and / or pharmacodynamic properties over unlabeled forms of the same compound. Such isotopically labeled (leveled) forms or analogs of the compounds herein are within the scope of the present disclosure. Those skilled in the art can prepare and use such isotopically labeled forms according to the procedures for isotopically labeled compounds or in terms of compounds that obtain isotopically or radioactively labeled analogs of the compounds disclosed herein.

[0087] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological effectiveness and properties of a given compound and is not biologically or otherwise undesirable. Pharmaceutically acceptable base addition salts can be prepared from inorganic bases and organic bases. By way of example only, salts derived from inorganic bases include sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary amines, secondary amines, and tertiary amines, such as the following salts: alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dienylamines, trialnylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines , di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines, wherein at least two substituents on the amine are different and are selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, etc. Also included are amines in which two or three substituents together with the amino nitrogen form a heterocyclic group or a heteroaryl group. The general structure of the amine is N(R 30 )(R 31 )(R 32 ), wherein the three substituents on the nitrogen of the monosubstituted amine (R 30 , R31 and R 32 ) are hydrogen, and the three substituents on the nitrogen of the disubstituted amine (R 30 , R 31 and R 32 ) is hydrogen, and the three substituents on the nitrogen of the trisubstituted amine (R 30 , R 31 and R 32 ) in which no substituent is hydrogen. 30 , R 31 and R 32 Select from a variety of substituents such as hydrogen, optionally substituted alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocyclyl, and the like.

[0088] Specific examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucosamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0089] Pharmaceutically acceptable acid addition salts can be prepared from inorganic acids and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0090] Sugar-based surfactants with different structures are good substitutes for polysorbate surfactants. They are mainly produced by enzymatic synthesis by microorganisms. They have low toxicity, biodegradability and biocompatibility, and are produced by cheaper and renewable energy. Medicine, food, cosmetics, textiles, oils and agricultural applications show interest in the benefits of using biosurfactants. However, due to the risk of endotoxin contamination in the biosurfactants made by bacteria, their use in the pharmaceutical industry is limited. Their generation results in a glycolipid mixture with different properties, resulting in inconsistent and confusing results. Recently, sugar-based surfactants have been synthesized. Wherein trehalose lipids are composed of trehalose connected to lipids by ester bonds at different positions.

[0091] Provided herein are compounds that function as surfactants and / or excipients, methods of producing such compounds, methods of using such compounds, and compositions comprising such compounds, preferably in combination with one or more additional agents or therapies.

[0092] It is also contemplated that all aspects directed to a compound include any pharmaceutically acceptable salt, stereoisomer, polymorph, mixture of stereoisomers, solvate, or prodrug thereof.

[0093] In some aspects, the present disclosure provides compounds of formula (I)

[0094]

[0095] or a salt thereof, a stereoisomer thereof, a polymorph thereof, or a mixture of stereoisomers thereof, wherein

[0096] R1 is selected from -OH; alkyl, optionally substituted by OH; or connected to R3 through oxygen;

[0097] R2 is selected from -OH; alkyl, optionally substituted with OH; or connected to R3 through oxygen;

[0098] R3 is selected from -OH; alkyl, optionally substituted by OH; or connected to R1 or R2 through oxygen;

[0099] R4 is selected from -OH; or alkyl, optionally substituted with -OH;

[0100] X1 is independently selected from -(C=O) and -(C=O)-A-(C=O), wherein A is alkyl, alkenyl, alkynyl or PEG; X2 is independently selected from H or -(C=O);

[0101] Z1 is -OH;

[0102] and,

[0103] - When X2 is C═O, then Z2 is independently selected from alkyl, alkenyl, alkynyl, PEG, succinyl, —NHalkyl, —NHalkenyl, —NHalkynyl, NH-PEG, —Salkyl, —Salkynyl, —Oalkyl, —Oalkenyl, and —Oalkynyl;

[0104] -When X2 is H, then Z2 does not exist;

[0105] And n is an integer from 1 to 20.

[0106] In one aspect, R1 is -OH.

[0107] In one aspect, R2 is linked to R3 via oxygen.

[0108] In one aspect, R4 is OH.

[0109] In one aspect, X1 is -(C=O)-CH2-CH2-(C=O).

[0110] In one aspect, Z1 is -OH.

[0111] In one aspect, X2 is -(C=O).

[0112] In one aspect, Z2 is alkyl, alkenyl or absent. As described above, examples of alkyl include, but are not limited to, alkyl groups having 1 to 20 carbon atoms (i.e., C 1-20 Alkyl), alkyl of 1 to 8 carbon atoms (i.e. C 1-8 Alkyl), alkyl of 1 to 6 carbon atoms (i.e. C 1-6 Alkyl) or alkyl of 1 to 4 carbon atoms (i.e. C 1-4 Examples of alkenyl groups include, but are not limited to, alkenyl groups having 2 to 20 carbon atoms (i.e., C 2-20 alkenyl), alkenyl of 2 to 8 carbon atoms (i.e., C 2-8 alkenyl), alkenyl of 2 to 6 carbon atoms (i.e., C 2-6 alkenyl) or alkenyl of 2 to 4 carbon atoms (i.e. C 2-4 Examples of alkenyl groups include ethenyl, propenyl, butadienyl (including 1,2-butadienyl and 1,3-butadienyl).

[0113] In one aspect, the compound of formula (I) is selected from:

[0114]

[0115]

[0116] In some aspects, the present disclosure provides a composition comprising a compound of formula (I) of the present invention. In some preferred aspects, the compound of formula (I) is a surfactant and / or excipient and / or exhibits surfactant and / or excipient properties.

[0117] In some aspects, the present disclosure also provides a pharmaceutical composition comprising a compound of formula (I) of the present invention. The pharmaceutical composition also comprises a therapeutically effective amount of a medicament.

[0118] In some aspects, the agent is selected from a chemical compound, a peptide, a lipid, an oligonucleotide, a cellular exosome, and a combination of one or more thereof.

[0119] Examples of peptides include peptides selected from antibodies, antigen-binding fragments, and combinations thereof.

[0120] As used herein, "antibody" or "antigen binding protein or polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing a molecule that contains at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Examples of this class include, but are not limited to, a heavy or light chain complementary determining region (CDR) or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0121] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0122] The antibody can be from any animal source, including birds and mammals. Preferably, the antibody is a human antibody, a mouse antibody, a donkey antibody, a rabbit antibody, a goat antibody, a guinea pig antibody, a camel antibody, a llama antibody, a horse antibody or a chicken antibody. In another aspect, the antibody or antigen-binding protein is a humanized antibody or a humanized antigen-binding protein.

[0123] Examples of oligonucleotides include deoxyribonucleic acid (e.g., DNA, cDNA ...) or ribonucleic acid (e.g., RNA, miRNA, siRNA, piRNA, hnRNA, snRNA, sgRNA, esiRNA, shRNA, antisense oligonucleotides, lncRNA ...) polymers or combinations of deoxyribonucleic acid and ribonucleotide (e.g., DNA / RNA) polymers. The oligonucleotides of the present invention are linear or circular conformations, in single-stranded or double-stranded form. These terms should not be interpreted as limiting the length of the polymer, and may include analogs of known natural nucleotides, as well as nucleotides chemically modified at the base, sugar, and / or phosphate moieties. Typically, analogs of specific nucleotides have the same base pairing specificity; that is, analogs of A will pair with T bases.

[0124] As used herein, the term "exosomes" refers to a subset of extracellular vectors (EVs) released by prokaryotes and eukaryotes. Exosomes typically range in size from 40 nm to 160 nm in diameter (average 100 nm) and originate from endosomes. Depending on the cell of origin, exosomes can contain many components of the cell, including DNA, RNA, lipids, metabolites, cytoplasm, and cell surface proteins.

[0125] In one aspect, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers and / or diluents.

[0126] "Pharmaceutically acceptable carrier or diluent" means a carrier or diluent used to prepare a generally safe, non-toxic and desirable pharmaceutical composition, and includes carriers or diluents that are acceptable for human pharmaceutical use.

[0127] Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Physiological saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0128] The pharmaceutical composition may also include one or more pharmaceutically acceptable salts, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts, such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, gels or gel materials, flavoring agents, colorants, microspheres, polymers, suspending agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients may also be present, such as preservatives, wetting agents, suspending agents, other surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc., particularly if the dosage form is a rehydratable form. Suitable exemplary ingredients include macrocrystalline cellulose, sodium carboxymethylcellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, p-chlorophenol, gelatin, albumin, and combinations thereof. A detailed discussion of pharmaceutically acceptable excipients may be found in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), which is incorporated herein by reference.

[0129] The pharmaceutical composition of the present invention can be administered orally, buccally, parenterally, nasally, topically or rectally. Preferably, the pharmaceutical composition is administered nasally or orally, more preferably by inhalation.

[0130] Also preferably, the pharmaceutical composition of the present invention is used to treat a disease.Any disease is contemplated in the present disclosure.

[0131] In one aspect, the pharmaceutical composition of the present invention is a vaccine. Vaccines generally include attenuated organisms, inactivated organisms or dead organisms or purified products (e.g., antigens) derived therefrom. Other examples of vaccines include nucleic acid vaccines comprising antigens encoded by DNA or RNA (e.g., mRNA). The nucleic acid vaccines are generally delivered using viral vectors (e.g., adenovirus) or non-viral delivery systems (e.g., electroporation or lipid nanoparticles).

[0132] In some aspects, the present disclosure provides a method for treating and / or preventing a disease, the method comprising administering a therapeutically effective amount of a compound of formula (I) of the present invention or a pharmaceutical composition of the present invention to a subject in need thereof.

[0133] As used herein, the term "therapeutically effective amount" means an amount of a compound of formula (I) within the scope of reasonable medical judgment, which is high enough to significantly and positively change the symptoms and / or conditions to be treated, but low enough to avoid serious side effects (at a reasonable risk / benefit ratio). The therapeutically effective amount of a compound of formula (I) is selected based on a variety of factors including the type, race, age, weight, sex and medical condition of the patient, the severity of the condition to be treated, the route of administration, the patient's liver function and renal function. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of a compound of formula (I) required to prevent, control or inhibit the progression of a disease.

[0134] As used herein, the term "treatment" or "treating" refers to any administration of a composition, pharmaceutical composition, therapeutic agent, compound of formula (I), etc. disclosed herein to a subject for the purpose of:

[0135] (i) inhibiting the disease, i.e. preventing the development of clinical symptoms; and / or

[0136] (ii) Remission of the disease, i.e., regression of clinical symptoms.

[0137] As used herein, the terms "prevention" and "preventing" refer to any administration of a composition, pharmaceutical composition, therapeutic agent, compound of formula (I), etc. disclosed herein to a subject for the purpose of preventing a disease (i.e., so that clinical symptoms of the disease do not develop).

[0138] The present disclosure also provides a method for preparing a compound of formula (I). The method is described in the examples and comprises

[0139] – sequentially and selectively functionalize the two primary alcohols of trehalose while keeping the other hydroxyl moieties unreacted,

[0140] - allows first binding to the fatty acid chain and then to the carboxylated side chain, where the carboxyl function is introduced in the form of a benzyl ester which is removed in the last step of the process.

[0141] The present disclosure also provides a method for preparing a polymer of a compound of formula (I), the method comprising self-condensing an activated trehalose derivative in the presence of a coupling agent. Examples of coupling agents are selected from members of the carbodiimide family (e.g., DCC, EDCC, DIC), members of the benzotriazole family (e.g., TBTU, TATU, PyBOP), and members of the urea family (e.g., carbonyl diimidazole (CDI)).

[0142] Example

[0143] Example 1

[0144] Synthesis of succinyltrehalose fatty acids (compounds 1 to 4)

[0145]

[0146] Figure 2 : Overall synthesis of C8TreS

[0147] Synthesis of Trehalose Fatty Acid:

[0148] In a flame-dried round-bottom flask (20 mL) equipped with a magnetic stirring bar, fatty acids (1.1 equivalents) and TBTU (O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate) (1.1 equivalents) were dissolved in anhydrous pyridine (6 mL). Trehalose (D-trehalose) (1 equivalent) was then poured into the reaction mixture and continued to stir at room temperature for 72 h under an argon atmosphere. Pyridine was then removed under vacuum and the residue obtained was purified using flash column chromatography with a solvent gradient of 5% to 20% methanol in EtOAc-DCM (1:1) to obtain the desired surfactant (yield 51%).

[0149] Synthesis of Trehalose Fatty Acid Containing Succinyl Functional Group with Protecting Group:

[0150] In a flame-dried round-bottom flask equipped with a magnetic stirring bar, benzyl succinate (1.1 equivalents) and TBTU (1.1 equivalents) were dissolved in anhydrous pyridine (2 mL / 0.1 g trehalose derivative). Trehalose fatty acid (1 equivalent) was then poured into the reaction mixture and stirred at room temperature for 72 h under an argon atmosphere. Pyridine was then removed under vacuum and the resulting residue was purified by flash column chromatography with a solvent gradient of 5% to 20% methanol in EtOAc-DCM (1:1) to obtain the desired surfactant (yield 30% to 40%).

[0151] Synthesis of Succinyl Trehalose Fatty Acid:

[0152] In a 3-necked flask, fatty acid trehalose succinic acid monobenzyl ester (1 equivalent) is dissolved in MeOH (5mL), the system atmosphere is replaced with argon, and then 10% Pd / C is added. Remove argon from the system and backwash the reaction with hydrogen (3 times). Vigorous stirring at room temperature reacts 2h to 3h (monitored by TLC). After the reaction is completed, the solution is filtered through a Celite pad and the filter cake is washed with MeOH (10mL). The filtrate is concentrated under reduced pressure to obtain the desired compound (95%).

[0153] Synthesis of Trehalose Polymer (Compound 5)

[0154]

[0155] Synthesis of Trehalose Containing Succinyl Functional Groups with Protecting Groups

[0156] As described in Ballard, TE; Richards, JJ; Wolfe, AL; Melander, C. Synthesis and Antibiofilm Activity of a Second-Generation Reverse-Amide Oroidin Library: AStructure-Activity Relationship Study. Chemistry-A European Journal 2008, 14, 10745-10761, in a flame-dried round-bottom flask equipped with a magnetic stir bar, succinic acid monobenzyl ester (SAMBE) (1.1 equivalents) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (1.1 equivalents) were dissolved in anhydrous pyridine (2 mL / 0.1 g trehalose). Trehalose (1 equivalent) was then poured into the reaction mixture and stirred at room temperature for 42 h under an argon atmosphere. The pyridine was then removed under vacuum and the resulting residue was purified by flash column chromatography with a solvent gradient of 9% to 20% methanol in EtOAc-DCM (1:1) to afford the desired surfactant (yield 37%).

[0157] Synthesis of Trehalose Containing Succinic Acid Functional Group

[0158] In a 3-necked flask, trehalose succinic acid monobenzyl ester was dissolved in MeOH (1.5mL / 0.1g trehalose derivative), the system atmosphere was replaced with argon, and then 10% Pd / C (cat. about 10% mole) was added. Argon was removed from the system and the reaction was backwashed with hydrogen (3 times). The reaction was stirred vigorously at room temperature for 2hr to 3hr (monitored by TLC). After the reaction was completed, the solution was filtered through a Celite pad and the filter cake was washed with MeOH (2 times of reaction volume). The filtrate was concentrated under reduced pressure to obtain the desired compound (83%).

[0159] Synthesis of protected trehalose polymers with n=2 to 5

[0160] In a flame-dried round-bottom flask equipped with a magnetic stirring bar, trehalose succinic acid (1 equivalent) and TBTU (1 equivalent to 4 equivalents) were dissolved in anhydrous pyridine (2 mL / 0.1 g of trehalose derivative). The reaction mixture was stirred at room temperature for 42 h under an argon atmosphere. Pyridine was then removed under vacuum and the resulting residue was purified by dialysis (100 MWCO to 500 MWCO).

[0161] Synthesis of Succinyltrehalose Unsaturated Fatty Acid

[0162]

[0163] Synthesis of Succinyl Trehalose:

[0164] In a 3-necked flask, the intermediate (1 equivalent) was dissolved in MeOH (1.3 mL / 0.1 g intermediate), the solution was placed under an argon atmosphere, and then 10% Pd / C (0.1 equivalent) was added. The argon was removed from the reactor and the reaction mixture was backwashed with hydrogen (1 atm, 3 times). The reaction was stirred vigorously at room temperature for 4 hours under a hydrogen atmosphere (monitored by TLC). After the reaction was completed, the solution was filtered through a Celite pad and the filter cake was washed with MeOH. The filtrate was concentrated under reduced pressure to obtain succinyl trehalose (quantitative) as a white solid. Synthesis of succinyl trehalose unsaturated fatty acids:

[0165] In a flame-dried round-bottom flask equipped with a magnetic stirring bar, palmitoleic acid (1.2 equivalents) and TBTU (1.4 equivalents) were dissolved in anhydrous pyridine (3mL / 0.1g palmitoleic acid). The reaction mixture was stirred at room temperature for 1 hour, and TreSuc (1 equivalent) was then added. Stirring was continued at room temperature for 96 hours under an argon atmosphere. Pyridine was then removed under vacuum, and the residue obtained by reverse phase flash column chromatography (HO: MeCN 4:6→2:8) was purified. The fractions corresponding to the product were collected, concentrated in vacuo, and freeze-dried to obtain the desired compound (yield 15%) as a white solid.

[0166] Preparation process

[0167] Trehalose surfactant CnTreS solution was prepared by dissolving 5.6 mmol trehalose surfactant in 1 L of commercially available PBS buffer 1X solution. Then 6.3 mg / L antibody solution (provided in PBS (pH 6.5 to 6.7) with 100 mM L-arginine) was diluted in the trehalose surfactant solution to reach a concentration of 1 mg / mL.

[0168] Atomization process

[0169] A Briutcare mesh nebulizer (NEB-001) was used to nebulize 500 μL to 8 mL of the antibody formulation. The flow rate was set to 0.4 mL / min, and the aerosol solution was recovered into a 50 mL falcon tube.

[0170] Freeze drying process

[0171] 500 μL of 1 mg / mL antibody formulation was placed in a vial and then deep frozen in liquid nitrogen. The frozen sample was directly placed in a freeze dryer. After 24 hours, the lyophilized sample was stored in a refrigerator at 4°C for 7 days or 28 days. The sample was then added The reconstitution was performed with water, and the stability was evaluated by DLS and cell binding assays.

[0172] Stability evaluation

[0173] The stability of antibody solutions dissolved in trehalose surfactant solution was studied over 10 days at 25° C. Antibody aggregation and conformation were determined by dynamic light scattering DLS using a Nano-ZS (Malvern Panalytical) and a Nano-DSF device (NanoTemper Technology), respectively, while the ability of the Fab to interact with its functional antigen was evaluated by an ELISA assay developed for this study.

[0174] Cell binding assay

[0175] Functional stability was evaluated by studying its ability to bind antigens on the cell membrane. Wild-type HEK cells and HEK cells expressing SARS-CoV-2 spike (D614) were placed in a medium containing 10% fetal bovine serum, 100U / mL penicillin, and 100μg / mL streptomycin in RPMI matrix. Both cell lines were incubated at 4°C for 30min with 50μL of 0.5μg / mL antibody preparation. The cells were washed in PBS, and mouse anti-human FcIgG conjugated to Alexa Fluor 488 was added at a 1 / 500 dilution in PBS, followed by incubation at 4°C in the dark for 15min. The cells were washed and fluorescence-activated cells were analyzed by FACS using CANTO II (BD Bioscience, USA).

[0176] Colloid stability evaluation

[0177] Aggregation of antibodies was assessed by dynamic light scattering (DLS) operating at 25° C. Measurements were performed at different time points during storage at 25° C. and immediately after nebulization.

[0178] The average hydrodynamic diameter by intensity of the monomers is distributed at d=10 nm, while the aggregates are distributed at d>10 nm.

[0179] Cytotoxicity assay

[0180] The cytotoxicity of trehalose surfactant was evaluated on NIH-3T3 cell line. 0.0112mM to 11.2mM trehalose surfactant solution was prepared by diluting the stock solution into cell culture medium (DMEM, with 10% fetal bovine serum, supplemented with 2mM glutamine and 1% penicillin / streptomycin). The cells were incubated at 37°C for 48h with the diluted surfactant, and the solution of MTS / PMS (MTS CellTiter96 AQueous test) was added according to the supplier's instructions. The cells were then incubated at 37°C for 3h, and the absorbance was measured at 490nm using MultiSkan microplate reader (Thermo Fisher Scientific, USA). The cell viability with the change of surfactant concentration was plotted, and the cytotoxic dose median (CD50) was calculated.

[0181] Example 2

[0182] synthesis

[0183] Trehalose surfactant is produced by biocompatible, nontoxic and cheap raw materials (saturated fatty acids or unsaturated fatty acids, trehalose and succinic acid) obtained from green sources. They can be synthesized by desymmetrization strategy from unprotected trehalose. They can be easily degraded and metabolized by human body by enzymatic cleavage and hydrolytic cleavage of ester bond. In one embodiment, excipient comprises succinate functional group to enhance the interaction between biomolecule and excipient by electrostatic interaction. In another aspect, excipient comprises 2 units of trehalose to increase the hydrophilic part of surfactant, improve the solubility of biomolecule / surfactant complex in aqueous solution, and improve the stability of biomolecule by forming hydrogen bond.

[0184] About Shelf Stability

[0185] The inventors have demonstrated that trehalose surfactants can improve the solubility of biomolecules by forming drug-surfactant colloidal complexes while improving their shelf life stability. The shelf life stability of 4 days was monitored by evaluating aggregate formation by DLS ( Figure 1 ). IgG at 1 mg / mL in PBS showed aggregation after 4 days both with and without excipients.

[0186] Stability during atomization

[0187] The use of trehalose surfactant in antibody formulations reduced aggregation and conformational changes during nebulization. The stability of antibodies after nebulization was evaluated by DLS and nano-DSF to evaluate the aggregation formation and conformational changes of freshly prepared formulations after nebulization, respectively. Figure 2 As shown, nebulization in PBS alone exhibited aggregation of 9.7% of the total AUC. Nebulization appears to significantly accelerate aggregation compared to the on-shelf aggregation observed during storage at 25°C (3.5% after 10 days in PBS alone).

[0188] Aggregation was observed (approximately 5% of total volume weighted AUC) for 1 mM of C8Tre1 or C16Tre1S. Aggregates were avoided when the formulation contained a minimum concentration of 5.6 mM of C8Tre1 or C16Tre1S. The data were in good agreement with the shelf life stability studies.

[0189] The colloidal stability of the antibodies after nebulization was also evaluated by nanoDSF. The difference in scattering before and after nebulization at 70°C (highest aggregation level) and 20°C (lowest aggregation level) was calculated for each sample ( Figure 3 ).

[0190] The results show that for most samples, similar results were obtained before and after nebulization. However, 1 mM of trehalose surfactant showed aggregates that were likely generated by nebulization. The data are very consistent with DLS experiments that showed aggregation at the same concentration. C8Tre1 and C16Tre1S prevented antibody aggregation when used at a minimum concentration of 5.6 mM. The lack of changes before and after nebulization suggests that the same proportion of aggregates were present before and after heating using the nanoDESF device.

[0191] Stability during freeze drying:

[0192] 1 mg / mL antibody preparations were evaluated by adding 5.6 mM C16Tre1S and C8Tre1S trehalose surfactants as cryoprotectants. Samples were first deep frozen in liquid nitrogen and then dried for 24 hours. The lyophilized samples were stored in the refrigerator for 0, 7, or 28 days before addition. The antibody was reconstituted with water. The conformation and function of the antibody after treatment were evaluated by DLS and cell binding assays, respectively. The results were compared with antibodies formulated only in PBS and with antibodies formulated with standard cryoprotectants such as trehalose (5 wt % and 5.6 mM) and Tween 80 (5.6 mM).

[0193] DLS results ( Figure 4 ) showed that no aggregation was observed for the formulation containing C16Tre1S, and the lyophilized antibody remained stable for 28 days stored at 4°C. Similar results were obtained with two standard cryoprotectants (5.6 mM Tween 80 and 5 wt% trehalose). However, for samples containing only PBS, aggregation was observed when a low amount of trehalose (5.6 mM) was used. For samples containing C8Tre1S, aggregation was observed after freeze drying, and the amount of aggregates increased with storage time, suggesting that C8Tre1S has a cryoprotective effect but does not stabilize the antibody during storage.

[0194] The ability of the antibodies to bind to their antigen after lyophilization was then evaluated in vitro using HEK293 cells expressing SARS-CoV-2 spike ( Figure 6). After antibody treatment, cells were labeled with Alexafluor 488. When the antibody was formulated with C16TreS, high fluorescence signals were observed in all HEK293 cells expressing spike protein. Similar results were obtained with the same concentration of the standard cryoprotectant Tween 80. However, a significant reduction in antibody binding was observed for formulations containing trehalose (5 wt % and 5.6 mM), formulations containing C8TreS (5.6 mM), and formulations without surfactant. The reconstituted antibody formulations containing Tween 80 and C16TreS were then challenged with nebulization, and the antibodies were able to bind to the Spike protein at the same level as before nebulization. These results indicate that the C16Tre1S trehalose surfactant stabilizes antibodies to a degree comparable to that of the standard cryopreservative Tween 80 during freeze-drying, during storage for 28 days after lyophilization, and during nebulization.

[0195] Cytotoxicity

[0196] The cytotoxicity of all surfactants used in this study was evaluated on NIH-3T3 cells after 48 h using the MTS cell viability test. The CD50 of C8Tre1 was 5.4 mM, and the CD50 of C14Tre1, C14Tre1S or C16Tre1S was 0.30 mM, 0.84 mM and 0.24 mM, respectively. It seems that the longer the alkyl chain, the higher the cytotoxicity.

[0197] Furthermore, since the CD50 of C8TrelS is 5.4 mM, while the CD50 of C8Trel is greater than 11.2 mM, succinylation of trehalose appears to increase toxicity.Anionic surfactants are generally considered to be more toxic than nonionic surfactants.

Claims

1. A compound of formula I or a salt thereof, a stereoisomer thereof, a polymorph thereof, or a mixture of stereoisomers thereof; wherein, R1 is selected from -OH; alkyl, optionally substituted by OH; or connected to R3 through oxygen; R2 is selected from -OH; alkyl, optionally substituted with OH; or connected to R3 through oxygen; R3 is selected from -OH; alkyl, optionally substituted by OH; or connected to R1 or R2 through oxygen; R4 is selected from -OH; or alkyl, optionally substituted by -OH; X1 is independently selected from -(C=O) and -(C=O)-A-(C=O), wherein A is alkyl, alkenyl, alkynyl or PEG; X2 is independently selected from H or -(C=O); Z1 is -OH; and, -When X2 is C=O, then Z2 is independently selected from alkyl, alkenyl, alkynyl, PEG, succinyl, -NH alkyl, -NH alkenyl, -NH alkynyl, NH-PEG, -S alkyl, -S alkynyl, -O alkyl, -O alkenyl, and -O alkynyl; -When X2 is H, then Z2 does not exist; And n is an integer from 1 to 20.

2. The compound of claim 1, wherein R1 is -OH; R2 is connected to R3 through oxygen; R4 is OH; X1 is -(C=O)-CH2-CH2-(C=O); Z1 is -OH; X2 is -(C=O); and Z2 is alkyl, alkenyl or absent.

3. The compound of claim 1 or 2, wherein the compound is selected from 4. A composition comprising a compound of formula I according to any one of claims 1 to 3.

5. The composition of claim 4, wherein the compound of Formula 1 is a surfactant or an excipient.

6. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 3.

7. The pharmaceutical composition of claim 6, further comprising a therapeutically effective amount of a pharmaceutical agent.

8. The pharmaceutical composition of claim 7, wherein the agent is selected from the group consisting of chemical compounds, peptides, lipids, oligonucleotides, cellular exosomes, and a combination of one or more thereof.

9. The pharmaceutical composition of claim 8, wherein the peptide is selected from the group consisting of an antibody, an antigen binding protein, and a combination thereof.

10. The pharmaceutical composition of claim 8, wherein the oligonucleotide is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, and combinations thereof.

11. The pharmaceutical composition according to any one of claims 6 to 10, for use in treating a disease.

12. The pharmaceutical composition of any one of claims 6 to 11, wherein the pharmaceutical composition is administered orally, buccally, parenterally, nasally, topically or rectally.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is administered nasally by inhalation or orally.

14. The pharmaceutical composition of any one of claims 6 to 13, wherein the medicament is a vaccine.

15. A process for preparing a compound of formula I according to any one of claims 1 to 3, the process comprising: - sequentially and selectively functionalizing the two primary alcohols of trehalose while leaving the other hydroxyl moieties unreacted; - allows first binding to the fatty acid chain and then to the carboxylated side chain, wherein the carboxyl function is introduced in the form of a benzyl ester which is removed in the last step of the process.

16. A process for preparing a polymer of a compound of formula I according to any one of claims 1 to 3, said process comprising self-condensing an activated trehalose derivative in the presence of a coupling agent.

17. A method for treating and / or preventing a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of i) a compound of formula I according to any one of claims 1 to 3, ii) a composition according to any one of claims 4 to 5, or iii) a pharmaceutical composition according to any one of claims 6 to 15.

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