Stereoisomeric phosphatidyl oligoglycerols

Abstract

The present invention relates to stereoisomeric phosphatidyl oligoglycerols having a predetermined configuration in at least three stereocenters, in particular to phosphatidyl oligoglycerols as a single stereoisomer. The present invention also relates to a stereospecific method for providing a stereospecific phosphatidyl oligoglycerol having a predetermined configuration in at least three stereocenters, in particular for providing a phosphatidyl oligoglycerol as a single stereoisomer, and to a liposome comprising the novel stereoisomeric phosphatidyl oligoglycerol, in particular, liposomes comprising novel stereoisomeric phosphatidyl oligoglycerols as a single stereoisomer.

Description

[0001] This invention relates to stereoisomers of phosphatidyl oligoglycerides having a predetermined configuration at at least three stereocenters, and particularly to phosphatidyl oligoglycerides as a single stereoisomer. The invention also relates to a stereospecific method for providing stereoisomers of phosphatidyl oligoglycerides having a predetermined configuration at at least three stereocenters (particularly for providing phosphatidyl oligoglycerides as a single stereoisomer), and to liposomes comprising said novel stereoisomeric phosphatidyl oligoglycerides, particularly to liposomes comprising novel stereoisomeric phosphatidyl oligoglycerides as a single stereoisomer.

[0002] As a 1,2-diacyl- sn -Glyceryl-3-phosphate-oligoglycerides (such as 1,2-dipalmitoyl-... sn 3-glycerol-3-phosphate-oligoglycerides (DPPG) x )) of phosphatidyl oligoglycerides (PG) x Phosphatidyl oligoglycerides are non-naturally occurring glycerol phospholipids. Phosphatidyl oligoglycerides consist of a diacylglycerol group, a phosphate group, and an oligoglycerol group containing two or more glycerol moieties.

[0003] Phosphatidyl oligoglycerides have been specifically developed as components of liposomes to prolong their circulating half-life (WO 97 / 30058 A1). Naturally occurring phosphatidyl monoglycerides, such as 1,2-dipalmitoyl- sn The head group of glycerol-3-phosphate-glycerol (DPPG) is modified with additional glycerol units linked via ether bonds to enhance the hydrophilicity of the head group. x This technology can be used to replace polyethylene glycol-modified phospholipids in cholesterol-containing liposomes (WO 97 / 30058 A1) and cholesterol-free liposomes (Hossann et al. 2021) to extend the cycling half-life of these nanocarriers. Phosphatidyl oligoglycerides (such as DPPG) x A significant advantage over PEGylated lipids is the significantly smaller head group (74 Da per glycerol unit, compared to approximately 2000 Da for PEG). As a result, 1,2-diacyl- sn -Glyceryl-3-phosphate-oligoglycerides, such as 1,2-dipalmitoyl- sn -glycerol-3-phosphate-diglycerol (DPPG2), such as 1,2-dipalmitoyl- sn -glycerol-3-phosphate-triglycerol (DPPG3), such as 1,2-dipalmitoyl- snDPPG2 (3-glycerol-3-phosphate-tetraglycerol) forms a layered structure (Lehnert et al. 2003) rather than the micellar structure exhibited by PEGylated liposomes. Therefore, DPPG2 can be incorporated into thermosensitive liposome (TSL) formulations at concentrations up to 70 mol% (Lindner et al. 2004). The effects of DPPG2 and PEGylated liposomes as components of liposomes on protein corona, hemocyte-cell interactions, complement activation in human plasma / blood, and hypersensitivity in rats have been recently investigated (Lokerse et al. 2021). In recent years, the use of PEGylated liposomes in human pharmaceutical products has been questioned for several reasons (Hoang et al. 2020), therefore, DPPG2... x Such alternatives have attracted attention as novel excipients for liposome drug delivery systems.

[0004] Phosphatidyl oligoglycerides, especially DPPG x (x≥2) can be prepared by organic synthesis without the use of enzymes or polymerization reactions (Hossann et al. 2021), thus preventing the mixing of oligoglycerides in lipid batches. 1,2-Dipalmitoyl- sn -glycerol-3-phosphate- racemic - Synthetic pathway of oligoglycerol. This pathway lacks control over the stereocenter in the oligoglycerol unit.

[0005] WO 2014 / 202680 A1 provides a synthetic route with control over two stereocenters to obtain 1,2-dipalmitoyl-sn-glycerol-3-phosphate-sn-1'-diglycerol. However, due to the lack of stereocontrol in the synthesis of the oligoglycerol structural unit, the third and subsequent stereocenters in the oligoglycerol unit remain racemic.

[0006] DPPG x In particular, DPPG2 has attracted attention in the clinical development of thermosensitive liposomes (TSLs) of the encapsulated drug doxorubicin (DOX) for the treatment of solid tumors, particularly soft tissue sarcoma (STS) and bladder cancer (DPPG2-TSL-DOX) (Hossann et al. 2021, van Valenberg et al. 2021). DPPG2-TSL-DOX is currently undergoing a phase 1 clinical trial in patients (NCT05858710).

[0007] WO 2022 / 008471 A1 describes measures to obtain a DPPG2-TSL-based thermosensitive drug delivery system with appropriate pharmaceutical quality. A particular challenge in the preclinical development of thermosensitive liposome formulations for human patients is obtaining stable and long-term storable formulations without compromising the instability required at temperatures >39°C for heat-induced local drug delivery to patient solid tumors. Optimal formulations must withstand industrial-scale manufacturing processes and require long-term storability without changes in quality-critical specifications that could affect efficacy and patient safety.

[0008] WO 2023 / 021024 A1 describes the administration regimen of DPPG2-TSL-DOX in combination with local hyperthermia (HT) for use in human patients.

[0009] The object of this invention is to provide novel phosphatidyl oligoglycerides with a predetermined configuration having all stereocenters in the oligoglyceride head group, and to provide a method for producing such phosphatidyl oligoglycerides in a stereocontrolled manner. Such stereoisomeric phosphatidyl oligoglycerides should in particular be able to provide thermosensitive drug delivery systems that meet the requirements and needs identified above.

[0010] This invention relates to stereoisomers of phosphatidyl oligoglycerides of formula (I).

[0011] (I)

[0012] Among the three solid-states 1 2 3. It has a predetermined configuration; p represents another solid center; and

[0013] Where R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms; n is a number from 0 to 20, in particular, n is a number from 0 to 2; and m is independently 0 or 1 each time it appears.

[0014] The inventors of this application have successfully provided a stereospecific synthesis of phosphatidyl oligoglycerides, which allows control over three stereochemical phases. 1 2 3. In the case of diglycerides (n=0) (such as diacylglycerol diglyceride phosphate, especially dipalmitoylglycerol diglyceride phosphate), the compound is a single stereoisomer.

[0015] The phosphatidyl oligoglycerol of the present invention comprises an oligoglycerol group having at least two glycerol moieties. These glycerol moieties include stereochemically oriented oligoglycerols. 2 3. The stereospecific method of the present invention provided herein allows for the synthesis of stereoisomers of phosphatidyl oligoglycerides of formula (I), wherein the three stereoisomers are... 1 2 3. It has a predetermined configuration. A predetermined configuration means that the solid... 1 has an (R) or (S) configuration, in a solid state 2 has an (R) or (S) configuration, in a solid state 3 has an (R) or (S) configuration. Therefore, a predetermined configuration means that the corresponding center is determined to be pure (R) or pure (S). In particular, a predetermined configuration means that the corresponding solid center does not exist in a racemic configuration.

[0016] The phospholipids of the present invention may further comprise glycerol and / or ethylene glycol groups attached thereto. For m = 1, an additional glycerol portion is present; for m = 0, an ethylene glycol portion is included. p represents the center of another stereochemistry within the other glycerol fractions. Preferably, in each additional stereochemistry fraction... p has a predetermined configuration, which means that in each additional solid... p has an (R) or (S) configuration.

[0017] Therefore, the most preferred is phosphatidyl oligoglycerol as a single stereoisomer of formula (I). In this most preferred single stereoisomer of formula (I), all stereoisomers... 1 2 3 p has a predetermined configuration each time it appears, which means that in the solid... 1 has an (R) or (S) configuration, in a solid state 2 has an (R) or (S) configuration, in a solid state 3 has (R) or (S) configuration, and in each solid... p has either an (R) or (S) configuration each time it appears.

[0018] According to the present invention, n is a number from 0 to 20. Preferably, n is a number from 0 to 10, more preferably a number from 0 to 5. In particular, n is 0, 1, or 2, and m = 1, such that the stereoisomers of formula (I) are diglycerides, triglycerides, or tetraglycerides. Even more preferably, n is 0 or 1, and most preferably n is 0. Wherein the stereoisomers of formula (I) with n = 0 are phosphatidyl diglycerides. Wherein the stereoisomers of formula (I) with n = 1 and m = 1 are phosphatidyl triglycerides.

[0019] In the stereoisomer of phosphatidyl oligoglycerol of formula (I), residue R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms. Preferably, R 1 and R 2 Each is independently a hydrocarbon group having 13 to 19 carbon atoms, particularly 14 to 18 carbon atoms. Preferably, R 1 and R 2 Each is independently a straight-chain or branched hydrocarbon group, preferably a straight-chain hydrocarbon group. More preferably, R 1 and R 2 Each is independently a straight-chain or branched alkyl group, especially a straight-chain alkyl group. R 1 and R 2 It can be a saturated, monounsaturated, or polyunsaturated alkyl group, preferably a saturated alkyl group. More preferably, R 1 and R 2 Each is independently a straight-chain saturated alkyl group. Even more preferably, R 1 and R 2 Each is independently a straight-chain saturated C12 to C24 alkyl group, particularly a straight-chain saturated C13 to C19 alkyl group. Most preferably, R... 1 and R 2 It is a straight-chain saturated C15 alkyl group.

[0020] According to the present invention, in three solids 1 2 3 and preferred options also include three-dimensional... p has a predetermined configuration. The configuration of each of the three solid centers can be predetermined as either an S-configuration or an R-configuration. The most preferred solid center... 1 is an R-configuration, in solid form 2 is an S-configuration, in three dimensions 3 represents the R-configuration. The stereospecific method provided herein allows for the formation of compounds with the desired predetermined configuration at all stereocenters. Utilizing the stereospecific, rather than stereoselective, method of the present invention, starting from a starting material as a single stereoisomer, the product obtained is a phosphatidyl oligoglycerol of formula (I), which is a single stereoisomer.

[0021] In a more general sense, predetermined configuration specifically refers to the corresponding stereocenter (e.g., stereocenter) in a formulation. 1 or three-dimensional 2 or three-dimensional 3) It exists in a predetermined configuration, that is, in a (R) configuration or (S) configuration of at least 95%, preferably at least 99%, most preferably at least 99.9%.

[0022] The stereoisomers of phosphatidyl oligoglycerides of formula (I) are described as anions and can pair with any cation (as a counterion). A preferred counterion is Na. + .

[0023] Although it can be found in three solids 1 2 Each of the three is predetermined to have a desired configuration, but the most preferred is a phosphatidyl oligoglyceride with a stereoisomer of formula (II).

[0024] (II)

[0025] In the three-dimensional 1 is an R-configuration, in solid form 2 is an S-configuration, in three dimensions 3 is an R-configuration, and p represents another solid center; and R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms; and where n is a number from 0 to 20, especially from 0 to 2.

[0026] Stereoisomer phosphatidyl oligoglycerides of formula (II) are also referred to herein as 1,2-diacyl- sn -glycerol-3-phosphate- sn -1′-glycerol- sn -3″-glycerol.

[0027] The stereospecific method presented in this paper allows for the formation of 1,2-diacyl- sn -glycerol-3-phosphate- sn -1′-glycerol- sn -3″-glycerol compounds having the desired predetermined configuration at all stereocenters, wherein the stereocenters 1 is an R-configuration, in solid form 2 is an S-configuration, in three dimensions 3 is the R-configuration. Utilizing the stereospecificity, rather than stereoselectivity, of the present invention, starting from a starting material that is a single stereoisomer, the product obtained is a phosphatidyl oligoglycerol of formula (II) as a single stereoisomer.

[0028] In a more general manner, in the stereoisomer of phosphatidyl oligoglycerol having formula (II), at least 95%, preferably at least 99%, most preferably at least 99.9% of the stereoisomer is contained in the phosphatidyl oligoglycerol. 1 is an R-configuration, with a solid proportion of at least 95%, preferably at least 99%, and most preferably at least 99.9%. 2 is an S-configuration, with a solid proportion of at least 95%, preferably at least 99%, and most preferably at least 99.9%. 3 is the R-configuration.

[0029] According to the present invention, the most preferred form is phosphatidyl oligoglycerol, which is a single stereoisomer of formula (III).

[0030] (III)

[0031] This compound is also referred to herein as stereoisomer DPPG2 or DPPG2. Or 1,2-dipalmitoyl- sn -glycerol-3-phosphate- sn -1′-glycerol- sn -3″-glycerol. The IUPAC name is (2R)-2,3-bis(palmitoyloxy)propyl(2S)-3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl phosphate or (2R)-2,3-bis(hexadecanoyloxy)propyl(2S)-3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl phosphate. + In the case of counterions, the IUPAC name is (2R)-2,3-bis(palmitoyloxy)propyl(2S)-3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl phosphate sodium or (2R)-2,3-bis(hexadecanoyloxy)propyl(2S)-3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl phosphate sodium.

[0032] The stereoisomers of phosphatidyl oligoglycerides of formula (I), (II), or (III) of the present invention exhibit surprisingly advantageous aspects. In particular, those with phosphatidyl- racemic Compared to oligoglycerols, the stereoisomers of phosphatidyl oligoglycerols of formulas (I), (II) and (III) show advantages.

[0033] Specifically, stereoisomeric phosphatidyl oligoglycerides exhibited differences in temperature-dependent drug release in vitro, and in vivo, differences in pharmacokinetics, organ distribution, and therapeutic efficacy of TSL formulations containing such stereoisomeric phosphatidyl oligoglycerides and cytotoxic drugs. Furthermore, there was a trend towards slower in vitro metabolic rates.

[0034] Liposomes containing stereoisomerized phosphatidyl oligoglycerides exhibit altered plasma clearance in vivo. Furthermore, compared to racemic molecules, stereoisomerized phosphatidyl oligoglycerides demonstrate different biological interactions with the complement system and / or plasma proteins. Additionally, liposomes containing stereoisomerized phosphatidyl oligoglycerides show varying uptake in organs, particularly the spleen.

[0035] Furthermore, the stereoisomers of phosphatidyl oligoglycerides of the present invention were found to exhibit differences in liposome packaging and structural liposomes. In particular, for liposomes containing the stereoisomers of phosphatidyl oligoglycerides of the present invention, a sharper phase transition was observed in the temperature-dependent release profile, indicating an influence on the packaging of the lipid excipient.

[0036] Furthermore, according to the present invention, it has been surprisingly found that, compared to DPPG2-TSL, where not all glycerol fractions are stereospecific, DPPG2-TSL, where all glycerol fractions are stereospecific, is a more effective drug delivery system. -TSL showed faster drug clearance from the bloodstream. This effect was particularly evident when using doxorubicin as the drug. Furthermore, the use of DPPG2... -TSL-DOX liposomes increased drug accumulation in tumor tissue, particularly doxorubicin, but reduced drug accumulation in the spleen.

[0037] It has been found that the stereoisomeric phosphatidyl oligoglycerol of the present invention can increase the temperature-dependent drug release from thermosensitive liposomes. Liposomes containing the stereoisomeric phosphatidyl oligoglycerol of the present invention were also observed to exhibit a narrower phase transition in their temperature-dependent release profiles. Therefore, the stereoisomeric phosphatidyl oligoglycerol of the present invention can be used to increase temperature-dependent drug release and / or narrow the phase transition in the temperature-dependent release profile of thermosensitive liposomes.

[0038] In particular, by addressing absolute stereochemistry, the provision of stereoisomers of phosphatidyl oligoglycerides of formula (I), especially formulas (II) and (III), allows them to be used in liposome compositions for human application, even when considering the stringent standards set by pharmaceutical regulatory agencies. The chirality of the lipid excipient plays a crucial role in the stereoselective metabolism by enzymes (e.g., phospholipases) and prevents the potential accumulation of the lipid excipient in the patient's body.

[0039] This invention also relates to a stereospecific method for preparing the stereoisomers of phosphatidyl oligoglycerides described herein, particularly the stereoisomers of phosphatidyl oligoglycerides of formula (I).

[0040] (I)

[0041] Among the three solid-states 1 2 3. It has a predetermined configuration; p represents another solid center; and

[0042] Where R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms; n is a number from 0 to 20, in particular, n is a number from 0 to 2; and m is independently 0 or 1 each time it appears;

[0043] Its characteristic is that the synthetic route includes a key intermediate of formula (IV) or formula (IV′).

[0044]

[0045] Among the two solid-states 2′ 3′ has a predetermined configuration, wherein PG, PG′, and PG″ are independently protecting groups. PG and PG″ are particularly benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropyl-2-yl (MOP), silyl, or p-methoxybenzyl (PMB), preferably benzyl (Bn). PG′ is especially CH2 (methylene), CHCH3 (ethylene), CHPh (benzylene), CHPh p -Ome (p-methoxybenzyl), C(CH3)2 (isopropylidene), or Si t Bu2 (di-tert-butylmethylenesilyl), Si(iPr)2-O-Si(iPr)2 (tetraisopropyldisiloxanediol), preferably PG′ is C(CH3)2.

[0046] According to the present invention, a key intermediate comprising formula (IV) or (IV′) is discovered (wherein two stereotypes are present). 2′ The 3′ synthetic route (with a predetermined configuration) allows for the stereospecific preparation of phosphatidyl oligoglycerides, wherein the three stereo groups are... 1 2 3 can be assigned a predetermined configuration. A predetermined configuration specifically refers to a solid within a given space. 1 has an (R) or (S) configuration, in a solid state 2 has an (R) or (S) configuration, in a solid state 3 has (R) or (S) configuration.

[0047] The key intermediate of formula (IV) or (IV′) can be prepared from glycidyl and 1,2-isopropylglycerol. The stereochemical intermediate in formula (IV) or (IV′) is... 2′ The 3′ configuration is derived from the configuration of the enantiomeric pure starting materials glycidol and 1,2-isopropylglycerol (described below).

[0048] To obtain stereoisomeric phosphatidyl diglyceride, a key intermediate of formula (IV) or (IV′) is reacted with a compound of formula (V), particularly preferably with compound 12a. To obtain oligoglycerides with higher degrees of polymerization, especially tri- or tetra-glycerides, an intermediate of formula (IV) or (IV′) is repeatedly reacted with glycidyl to form a structural unit having an increased number of glycerol units. In particular, compound 10a is provided as a structural unit for the preparation of stereoisomeric phosphatidyl triglyceride, and structural unit 11a is provided for the preparation of stereoisomeric phosphatidyl tetraglyceride.

[0049] An overview of the synthesis is provided in the following scheme.

[0050]

[0051]

[0052]

[0053] Obtain stereoisomers of phosphatidyl oligoglycerides according to formula (II)

[0054] (II)

[0055] In the three-dimensional 1 is an R-configuration, in solid form 2 is an S-configuration, and in three dimensions 3 is the R-configuration; in particular, this synthetic pathway includes a key intermediate of formula (IVa) or (IVa′).

[0056] (IVa) and (IVa′)

[0057] Among the solids in (IVa) 2′ is an R-configuration, in the solid. 3′ is in the S-configuration in (IVa) and in the R-configuration in (IVa′), wherein PG, PG′ and PG″ are independently protecting groups.

[0058] Preferably, it is composed of glycidyl glycerol and 1,2-isopropylglycerin, particularly (R)-(+)-glycidyl glycerol and (S)-(+)-1,2-isopropylglycerin. sn The preparation of key intermediates of formula (IVa) or (IVa′) by -1,2-IPG yields key intermediates of formula (IVa) or (IVa′), respectively.

[0059] The preferred option is the key intermediate 4.1a.

[0060]

[0061] Furthermore, the method of the present invention specifically includes reacting an intermediate of formula (IV) or (IV′), particularly a key intermediate of formula (IVa) or (IVa′), with a compound of formula (V).

[0062] (V)

[0063] Where R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms. R 1 and R 2 Preferably, it is as defined above. R′ represents the leaving group LG. R′ is particularly 2-cyanoethyl (CNE). R″ is a C1-C12 hydrocarbon group, particularly methyl (Me), ethyl (Et), isopropyl (iPr) or benzyl (Bn), preferably isopropyl.

[0064] Preferably, the compound of formula (V) consists of 1,2-diacyl- sn -Preparation of glycerol and phosphorylation reagents with the following structures:

[0065]

[0066] R′ and R″ are independently defined as above. R′ is in particular 2-cyanoethyl (CNE). R″ is in particular methyl (Me), ethyl (Et), isopropyl (iPr) and benzyl (Bn), preferably isopropyl.

[0067] The preferred option is intermediate 12a.

[0068]

[0069] Composed of 1,2-diacyl- sn It is produced by the reaction of ethylene glycol and 2-cyanoethyl N,N-diisopropylchlorophosphamide (PAMCl).

[0070] The method for preparing stereoisomeric phosphatidyl oligoglycerides of the present invention specifically includes at least one or more process steps outlined herein.

[0071] In particular, the present invention relates to a synthetic method for preparing stereoisomers of DPPG2 having formula (III).

[0072] (III)

[0073] The key intermediate of formula (IVa) has two chiral centers. Both configurations are derived from enantiomeric pure starting materials, (R)-(+)-glycidyl and (S)-(+)-1,2-isopropylene-glycerol. sn -1,2-IPG), and in the final product 1,2-dipalmitoyl- sn -glycerol-3-phosphate- sn -1'-glycerol- sn The isomerization-free transfer of -3"-glycerol (also referred to herein as the stereoisomer DPPG2) is achieved. The key intermediate of formula (IVa) has a (2R), (6S)-configuration for the stereospecific synthesis of the stereoisomer DPPG2. This single stereoisomer can be obtained in four steps using the synthetic method shown below.

[0074]

[0075] PG, PG 1 PG′ and PG′ are independent protecting groups.

[0076] This pathway allows control of two stereocenters, producing the key intermediate of formula (IVa) (as a single stereoisomer (R)-(+)-glycidyl), and sn -1,2-IPG is used as a commercially available starting material. First, the OH- group in (R)-(+)-glycidyl chloride is protected, for example, with 4-methoxybenzyl (PMB) chloride, leading to the formation of 1a. 1a reacts with... sn The -1,2-IPG reaction yields compound 2a. Subsequently, the newly formed hydroxyl group is protected (e.g., as a benzyl ether) to give compound 3a. Finally, the PMB ether is cleaved using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to give the key intermediate of formula (IVa).

[0077] In further synthesis, phosphoramidide chemistry was used from the key intermediate of formula (IVa) and 1,2-dipalmitoyl- sn -glycerin( sn DPPG2 was prepared as a single stereoisomer using 1,2-DPG.

[0078]

[0079] Compound 13a can be synthesized in a three-step one-pot process. Therefore, it is preferred that, firstly... sn -1,2-DPG and 2-cyanoethyl- N,N - The reaction of diisopropylphosphorous chloride (PAMCl) yields compound 12a. The addition of the key intermediate of formula (IVa) yields a substituted phosphite triester, which is subsequently oxidized, for example, with H2O2. The fully protected DPPG2 derivative 13a thus generated is treated to remove the protecting group. This can be done according to a known procedure. The phosphate protecting group can be removed, for example, with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or TEA; the ketal group can be removed with acids such as TFA, oxalic acid, and formic acid; and the benzyl ether can be removed by Pd-catalyzed hydrogenation. The deprotected product is purified by chromatography and crystallized from hot ethanol to obtain DPPG2 (22a) as a single stereoisomer.

[0080] The stereoisomeric phosphatidyl oligoglycerides of the present invention are particularly suitable as components of liposomes. Therefore, the present invention also relates to liposomes comprising at least one stereoisomeric phosphatidyl oligoglyceride as defined herein.

[0081] Liposomes are spherical vesicles formed from a membrane bilayer composed of lipid excipients, and are widely used and accepted as drug delivery nanocarriers. The stereoisomeric phosphatidyl oligoglycerides of this invention form a layered structure, making them particularly suitable as a component of liposomes.

[0082] The stereoisomers of phosphatidyl oligoglycerides of the present invention have been found to prolong the circulating half-life of liposomes. Furthermore, the oligoglyceride moiety, as a highly hydrated group, can alter electrostatic and hydrophobic interactions with blood components when located on the liposome surface. The binding of proteins to liposomes results in a so-called protein crown, the composition of which can be controlled by the content and type of stereoisomers of phosphatidyl oligoglycerides of the present invention (such as stereoisomer DPPG2) contained in the liposomes. Moreover, liposomes containing at least one stereoisomer of phosphatidyl oligoglyceride of the present invention exhibit a strong negative surface charge, which imparts high dispersion stability.

[0083] The stereoisomeric phosphatidyl oligoglycerides of the present invention can be particularly contained in irritation-sensitive, preferably thermosensitive, liposomes. By using stereoisomeric phosphatidyl oligoglycerides, it is possible to prepare thermosensitive liposomes with prolonged blood circulation time. These liposomes are stable in the bloodstream under physiological conditions (37-38°C) and release little or no of the active ingredient previously encapsulated in the liposomes. Most liposomes remain in the bloodstream for 2 hours or longer and are therefore suitable for the thermally controlled release of the active ingredient. Due to the rapid release kinetics of the thermally induced release of the active ingredient within seconds (preferably <30 seconds, more preferably <20 seconds) by these liposomes, the previously encapsulated active ingredient can be released immediately by targeted heating of the liposomes to a temperature >39°C (preferably 40 to 42°C).

[0084] The liposomes according to the invention, particularly the thermosensitive liposomes, preferably comprise (i) at least one stereoisomer of phosphatidyl oligoglyceride as described herein and (ii) at least one additional neutral or zwitterionic phospholipid, preferably phosphatidylcholine. Preferably, the principal phase transition temperature of the phosphatidylcholine is from 0°C to 80°C, particularly from 37°C to 45°C.

[0085] Thermosensitive liposomes comprise (i) at least one stereoisomer of phosphatidyl oligoglycerol and (ii) at least one phosphatidylcholine having a long half-life in serum. Furthermore, upon application of stimulation, such as when a specific temperature is exceeded, the contents of such nanocarrier systems (particularly liposomes) are rapidly released, particularly within less than 30 seconds.

[0086] Based on the lipid content of the liposomes, the amount of stereoisomeric phosphatidyl oligoglycerol is preferably at least 1 mol%, more preferably at least 10 mol%, even more preferably at least 15 mol%, most preferably at least 25 mol% and at most 70 mol%, more preferably at most 50 mol%, even more preferably at most 35 mol%.

[0087] Another preferred component of the liposomes of the present invention, particularly the thermosensitive liposomes, is at least one phosphatidylcholine. Based on the lipid content of the liposomes, the at least one phosphatidylcholine is preferably present in an amount of at least 1 mol%, more preferably at least 10 mol%, even more preferably at least 30 mol%, even more preferably at least 50 mol%, particularly at least 60 mol%, most preferably at least 65 mol%, and at most 99 mol%, more preferably at most 90 mol%, particularly preferably at most 80 mol%, and most preferably at most 75 mol%.

[0088] Particularly preferred is that, based on the lipid content of the liposomes, it contains 25 to 35 mol% of stereoisomeric phosphatidyl oligoglycerides (especially stereoisomeric DPPG2) and 65 to 75 mol% of phosphatidylcholine.

[0089] The liposomes according to the invention, particularly the thermosensitive liposomes, preferably contain at least one phosphatidylcholine of formula (VII).

[0090] (VII)

[0091] Where R 3 and R 4 Each of them independently represents a hydrocarbon functional group with 12 to 24 carbon atoms.

[0092] R 3 and R 4 Preferably, they are saturated, monounsaturated, or polyunsaturated independently of each other, and straight-chain alkyl groups are preferred, especially saturated straight-chain alkyl groups. R 3 and R 4 It is also preferably that each is independently a C13 to C19, particularly a C15 to C17 hydrocarbon group. Preferably, R 3 and R 4 Each is independently a straight-chain saturated C13- to C23-alkyl group, particularly C13- to C21-alkyl. Most preferably, R... 3 and R 4 It is independently a straight-chain saturated C15- or C17-alkyl group.

[0093] Liposomes, particularly the thermosensitive liposomes according to the invention, preferably contain phosphatidylcholine of formula (VII) in its natural configuration.

[0094] Suitable phosphatidylcholine is, for example, 1-palmitoyl-2-oleoyl- sn -glycerol-3-phosphocholine, 1-stearoyl-2-oleoyl- sn -glycerol-3-phosphocholine, 1-palmitoyl-2-lauroyl- sn -glycerol-3-phosphocholine, 1-behenyl-2-oleoyl- sn -glycerol-3-phosphocholine, 1-stearoyl-2-lauroyl- sn -glycerol-3-phosphocholine, 1,3-dimyristic- sn -glycerol-2-phosphocholine, 1,2-dimyristoyl- sn -glycerol-3-phosphocholine, 1-palmitoyl-2-myristoyl- sn -glycerol-3-phosphocholine, 1-stearoyl-2-myristoyl- sn -glycerol-3-phosphocholine, 1-myristoyl-2-palmitoyl- sn -Glyceryl-3-phosphatecholine, 1,3-palmitoyl- sn -Glyceryl-2-phosphocholine, 1,2-dipalmitoyl- sn-glycerol-3-phosphocholine, 1-myristoyl-2-stearoyl- sn -glycerol-3-phosphocholine, 1-stearoyl-3-myristoyl- sn -Glyceryl-2-phosphocholine, 1-stearoyl-2-palmitoyl- sn -glycerol-3-phosphocholine, 1-palmitoyl-2-stearoyl- sn -glycerol-3-phosphocholine, 1,3-distearate- sn -glycerol-2-phosphocholine, 1,2-distearate- sn -glycerol-3-phosphocholine, 1,2-diarachido- sn -glycerol-3-phosphocholine, 1,2-disorbonyl- sn -glycerol-3-phosphocholine and 1,2-dicarboxyl- sn -Glyceryl-3-phosphate choline. 1,2-Dipalmitoyl- sn -glycerol-3-phosphocholine (DPPC) and 1,2-distearate- sn 3-glycerol-3-phosphocholine (DSPC) is particularly preferred.

[0095] Thermosensitive liposomes particularly preferably contain at least one phosphatidylcholine with a main phase transition temperature in the range of 35°C to 43°C, more preferably in the range of 39°C to 41°C. Thermosensitive liposomes containing such phosphatidylcholine have a release temperature that allows the liposomes to be stable in normal circulation in a healthy human body (at 37°C) and to release their contents due to the effects of heat (especially localized heat at temperatures above 39°C to 43°C). Thermosensitive liposomes with a release temperature of 40°C to 43°C are particularly preferred.

[0096] According to the present invention, the thermosensitive liposomes particularly preferably contain at least one component selected from 1,3-dipalmitoyl- sn -Glyceryl-2-phosphocholine, 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate choline (DPPC), 1,3-distearate- sn -glycerol-2-phosphocholine and 1,2-distearate- sn -glycerin- 3-phosphoric acid Phosphatidylcholine (DSPC) is a component of choline.

[0097] Typically, liposome formulations may contain various other excipients, such as phosphatidylethanolamine (PE), phosphatidylglycerol, and phosphatidylserine (PS); as well as cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearate- sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1-acyl- sn -Glyceryl-3-phosphate choline (Lyso-PC), hexadecyl-phosphate choline (HePC) or polyoxyethylene (20) stearyl ether (Brij78).

[0098] Furthermore, liposomes may contain cholesterol. However, the most preferred liposomes of the present invention are cholesterol-free. According to the present invention, liposomes are preferably cholesterol-free because cholesterol causes a broadening of the solid-to-liquid disorder phase transition temperature, thereby widening the thermal transition range. At high cholesterol concentrations, the phase transition temperature is suppressed, resulting in non-thermally sensitive liposomes. Specifically, the liposomes according to the present invention, particularly the thermosensitive liposomes, contain <0.1 mol%, more preferably <0.01 mol%, of cholesterol. Particularly preferred liposomes are cholesterol-free, but completely cholesterol-free.

[0099] In one embodiment, thermosensitive liposomes with a primary phase transition temperature of approximately 40°C to 43°C are composed of 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate choline, optionally 1,2-distearate- sn It is formed from a mixture of glycerol-phosphocholine and stereoisomers DPPG2 and / or DPPG3. 1,2-Dipalmitoyl- sn glycerol-3-phosphate choline is used as the base matrix to set the phase transition temperature at approximately 42°C, 1,2-distearate- sn -Glyceryl-3-phosphate choline causes a slight increase in phase transition temperature. Stereoisomers DPPG2 and / or DPPG3 are used to establish dispersion stability, serum stability, blood flow stability, and promote the release rate of the encapsulated compound.

[0100] A particularly preferred liposome according to the invention comprises, based on the lipid content of the liposome, 20 to 40 mol%, preferably 25 to 35 mol%, of stereoisomer DPPG2 or stereoisomer DPPG3, and a total of 65 to 75 mol% of a compound selected from 1,2-dipalmitoyl- sn -glycerol-3-phosphocholine (DPPC) and / or 1,2-distearate- sn 3-glycerol-3-phosphocholine (DSPC) liposomes of phosphatidylcholine.

[0101] The most preferred liposome according to the present invention comprises, based on the lipid content of the liposome, 25 to 35 mol% of stereoisomerized DPPG2 and 65 to 75 mol% of 1,2-dipalmitoyl- sn Liposomes of glycerol-3-phosphate choline (DPPC).

[0102] Furthermore, the most preferred liposome according to the present invention comprises, based on the lipid content of the liposome, 20 to 40 mol%, preferably 25 to 35 mol%, of stereoisomerized DPPG2 and 40 to 60 mol%, preferably 45 to 55 mol%, of 1,2-dipalmitoyl- sn -Glyceryl-3-phosphate choline (DPPC) and 15 to 25 mol% of 1,2-distearate- sn Liposomes of glycerol-3-phosphate choline (DSPC).

[0103] Due to the high stability of the liposomes containing stereoisomeric phosphatidyl oligoglycerides according to the invention in systemic circulation and with only very low nonspecific release of the active ingredient, these liposomes are suitable for the local treatment of tumors (particularly soft tissue sarcomas and bladder cancer) in a highly specific manner. Following intravenous administration of the liposomes according to the invention, the release of the previously encapsulated active ingredient at the desired site (e.g., in soft tissue) can be induced by targeted heating of the desired site.

[0104] The active ingredients encapsulated in thermosensitive liposomes can be released by targeting and heating either the liposomes or the target tissue (i.e., the body part where the tumor is located). The site can be heated using various methods. For example, in addition to simple rinsing with warm water, heating can be achieved using electromagnetic waves, ultrasound, light, or laser technology.

[0105] Hyperthermia can be applied in the form of localized, regional, or superficial hyperthermia, with deep regional hyperthermia being preferred. Hyperthermia can be specifically applied in the form of ultrasound-induced hyperthermia, microwave frequency-induced hyperthermia, or hyperthermic perfusion, with ultrasound or microwave frequency-induced hyperthermia optionally administered externally. Typically, the choice of hyperthermia regimen will depend on the type of tumor to be treated and the specific circumstances of the patient. Paulides et al. (Paulides et al., 2020) outlined various options for applying hyperthermia.

[0106] For thermotherapy, different types of energy can be used to heat the body, including light, laser, microwave, radio frequency, and ultrasound.

[0107] In regional or localized hyperthermia, moderately large body parts, such as the chest cavity or pelvis, including cancerous areas and surrounding healthy tissue, can be heated. The rest of the body is kept as close to normal temperature as possible. Regional hyperthermia can be performed, for example, in the form of regional deep hyperthermia or regional perfusion hyperthermia.

[0108] In a preferred embodiment, the thermotherapy according to the invention is applied in the form of regional deep thermotherapy or localized regional deep thermotherapy.

[0109] Preferably, the thermotherapy according to the invention is applied in the form of electromagnetic-based thermotherapy techniques (e.g., radio wave or microwave frequency induced thermotherapy, ultrasound induced thermotherapy, or thermotherapy perfusion).

[0110] For liposomes to be used for targeted therapy of local tumors, their physical properties are crucial. Thermosensitive liposomes carrying the active ingredient should be stable at 36 to 37°C. By locally (e.g., in the tumor region) raising the temperature to 42°C, the active ingredient is rapidly released (particularly within <60 seconds, preferably <20 seconds). The lipid components of the liposomes of the present invention undergo a phase transition temperature in the range of approximately 40 to 45°C. Below 40°C, the phospholipids are arranged in layers and are in a solid gel phase; above this temperature, they are in a liquid disordered phase. Within a narrow temperature range (e.g., between 40°C and 43°C) of phase transition, the active ingredient is spontaneously and rapidly released.

[0111] 1,2-Dipalmitoyl- sn The principal phase transition temperature of glycerol-3-phosphocholine is approximately 41°C. This invention utilizes stereoisomers of phosphatidyl oligoglycerides (particularly those with 1,2-distearate-...). sn (a combination of glycerol-3-phosphate choline) could potentially provide thermosensitive liposomes with prolonged circulation time in the blood.

[0112] Thermosensitive liposomes are particularly suitable for local or regional treatment of cancer. The stereoisomeric phosphatidyl oligoglycerides provided herein can be used in the clinical application of cancer treatment because they ensure that the phospholipids have a well-defined structure, defined in terms of structure, fatty acid composition, and configuration. In a preferred embodiment, the cancer to be treated is selected from soft tissue sarcomas, preferably undifferentiated pleomorphic sarcoma (UPS), liposarcoma (well-differentiated liposarcoma, dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma), leiomyosarcoma, synovial sarcoma, angiosarcoma, epithelioid sarcoma, malignant peripheral nerve sheath tumor (MPNST), rhabdomyosarcoma (alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), solitary fibroma, myxofibrosarcoma, fibrosarcoma, uterine sarcoma (uterine leiomyosarcoma, endometrial stromal sarcoma), desmoplastic small round cell sarcoma (DSRCT), desmoidoma, Kaposi's sarcoma, or osteosarcoma, preferably Ewing sarcoma, osteosarcoma, or chondrosarcoma. In another implementation, the cancer is a bladder tumor.

[0113] Using the liposomes according to the invention, tumors, particularly solid and / or localized tumors, can be treated. Superficial tumors and metastatic tumors, in particular, can be treated with the liposomes according to the invention because they can be heated in a simple manner. However, other tumors, such as those in hollow organs, can also be treated. In this case, the heating required to release the contents of the liposomes can be achieved, for example, by rinsing with warm water.

[0114] The preferred treatment is for soft tissue sarcoma (STS).

[0115] The liposomes according to the invention preferably further comprise an active ingredient, particularly a cell inhibitor. Most preferably, the liposomes of the invention comprise doxorubicin (DOX), a cytotoxic anthracycline. Other suitable anthracyclines currently used for cancer treatment that inhibit cell growth include epirubicin, idarubicin, daunorubicin, and mitoxantrone. In further embodiments of the invention, other cell inhibitors such as mitomycin C, gemcitabine, trabectedin, or platinum derivatives such as cisplatin, carboplatin, or oxaliplatin may be included in the liposomes.

[0116] In another preferred embodiment, the thermosensitive liposome according to the invention comprises an active pharmaceutical ingredient selected from the group consisting of: anthracyclines, such as doxorubicin, daunorubicin, idarubicin, epirubicin, arubicin, amararubicin, pirarubicin, pentorubicin, and zorararubicin; anthraquinones, such as mitoxantrone and pisacocele; antitumor antibiotics, such as mitomycin and bleomycin; vinca alkaloids, such as vincristine, vinblastine, and vinorelbine; alkylating agents, such as cyclophosphamide and nitrogen mustard hydrochloride; camptothecins, such as topotecan, irinotecan (CPT-11), letopecan, 9-aminocamptothecin, 9-nitrocamptothecin, and 10-hydroxycamptothecin; purines And pyrimidine derivatives, such as 5-fluorouracil, gemcitabine (2,2′-difluoro-2-deoxycytidine, dFdC), fluorouridine (FUDR), cytarabine (cytosine arabinoside), 6-azauracil (6-AU); oxazolidinyl phosphates, such as cyclophosphamide, ifosfamide, and trefophosphamide; taxanes, such as paclitaxel and docetaxel; podophyllotoxin derivatives, such as etoposide and teniposide; platinum-based compounds, such as cisplatin, carboplatin, oxaliplatin, and nedaplatin; methotrexate; tyrosine kinase inhibitors, such as imatinib, gefitinib, erlotinib, sunitinib, adavoritetinib, and lapatinib; and cytarabine derivatives, such as cytosine arabinoside.

[0117] By using liposomes according to the invention, particularly thermosensitive liposomes, the active ingredient encapsulated in the liposomes can be released by applying stimulation, particularly local heating of the liposomes. Liposomes, especially thermosensitive liposomes, are therefore particularly suitable for the local or regional treatment of tumors (especially soft tissue sarcomas). The release of the active ingredient encapsulated in the liposomes can be induced in a targeted manner, so that the active ingredient, particularly cell growth inhibitors, can be released directly at the desired site. These experiences can also be applied to the treatment of other solid tumors and localized tumors.

[0118] The liposomes described herein are particularly suitable for the local release of active pharmaceutical ingredients via thermotherapy. Preferably, they are thermosensitive liposomes comprising at least one phosphatidylcholine and at least one stereoisomeric phosphatidyl oligoglyceride with a phase transition temperature of approximately 40 to 43°C.

[0119] Therefore, these treatments meet the basic requirements for clinical application (especially for the local treatment of tumor diseases such as soft tissue sarcoma). The effectiveness of these therapies has been demonstrated in animal studies.

[0120] If the phase transition temperature is exceeded, the membrane changes phase and becomes permeable, and the liposome contents are released accordingly. This effect can be used to treat soft tissue sarcomas according to the present invention. By raising the temperature within the tumor to the temperature required for the release of the liposome contents, the liposome contents are then specifically and almost exclusively released within the tumor, thereby enabling the active ingredient to be used efficiently for tumor treatment.

[0121] Therefore, the present invention also relates to the combination of thermosensitive liposomes with thermotherapy and / or ultrasound as described herein. In this case, heating can be achieved by a variety of methods, such as simply rinsing with warm water, or heating by electromagnetic waves, ultrasound, light, or laser. Preferred methods include ultrasound-induced thermotherapy, microwave frequency-induced thermotherapy, or hyperthermic perfusion.

[0122] Liposomes according to the present invention, particularly thermosensitive liposomes, including those containing active pharmaceutical ingredients, can be prepared by a variety of different techniques (such as lipid membrane hydration, ethanol injection, or extrusion). The liposomes of the present invention are preferably prepared by ethanol injection. A further preferred method is tangential flow filtration (TFF) for buffer exchange.

[0123] Therefore, the present invention also includes liposomes, particularly the thermosensitive liposomes disclosed herein, for the treatment of tumors, particularly soft tissue sarcomas.

[0124] The preferred cancer is soft tissue sarcoma (STS). STS is a class of cancers originating from tissues that connect, support, and surround other body structures, including muscle, fat, blood vessels, nerves, tendons, and joint linings. Preferably, STS can be undifferentiated pleomorphic sarcoma (UPS), liposarcoma (especially differentiated liposarcoma, dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma), leiomyosarcoma, synovial sarcoma, angiosarcoma, epithelioid sarcoma, malignant peripheral nerve sheath tumor (MPNST), rhabdomyosarcoma (especially alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), solitary fibroma, myxofibrosarcoma, fibrosarcoma, uterine sarcoma (especially uterine leiomyosarcoma, endometrial stromal sarcoma); desmoplastic small round cell sarcoma (DSRCT), desmoidoma, Kaposi's sarcoma, or osteosarcoma (especially Ewing sarcoma, osteosarcoma, or chondrosarcoma).

[0125] The present invention also includes liposomes, particularly thermosensitive liposomes, for the treatment of other tumors such as osteosarcoma, bladder cancer (muscle-invasive bladder cancer [MIBC] and non-muscle-invasive bladder cancer [NMIBC]), ovarian cancer, gastric cancer, breast cancer (especially triple-negative breast cancer [TNBC]), hepatocellular carcinoma, uterine cancer, thyroid cancer, head and neck tumors, prostate cancer, chordoma, desmoidoma, glioblastoma, and other tumor diseases, preferably those with local spread.

[0126] These liposomes preferably contain an active pharmaceutical ingredient suitable for treating the tumor, and then release the active pharmaceutical ingredient in response to stimulation within or near the tumor.

[0127] References

[0128]

[0129]

[0130] The present invention will be further described with reference to the accompanying drawings and embodiments.

[0131] Figure 1 The optical purity of key intermediates of formula (IVa) was evaluated by manual HPLC. Figure 1 A shows a completely racemic intermediate. Figure 1 B shows a mixture of epimers 2(S), 6(R,S) (98.9%). Figure 1 C displays the required stereoisomers 2(S), 6(R) (99.5%). Figure 1 D shows stereoisomers 2(R), 6(S) (100.0%).

[0132] Figure 2 DPPG2 was removed using phospholipase PLA2. Figure 2 A shows the breakdown of glycerophospholipids. Figure 2 B shows the formation of lysophosphatidylcholine. Values ​​expressed in units (%) are normalized to the content of phosphatidylcholine (% m / m) at time point t=0 min (without phospholipase). This method takes into account differences in purity and other substances (such as residual solvents or residues).

[0133] Figure 3Comparison of biophysical properties of different DPPG2-TSL-DOX batches. The batches were prepared using the following methods: 1) DPPG2 (Formula III) prepared via ethanol injection; 2) DPPG2 (WO97 / 30058) prepared via ethanol injection; 3) DPPG2 (WO97 / 30058) prepared via lipid film hydration combined with 100nm extrusion; 4) DPPG2 (WO97 / 30058) prepared via lipid film hydration combined with 200nm extrusion.

[0134] Figure 4: DPPG2-TSL-DOX in a rat sarcoma model. Pharmacokinetics of (A) DOX and (B) DPPG2 after intravenous bolus administration of different DOX formulations (2 mg / kg DOX, administered in a fixed volume of 1 mL). Treatment at room temperature (NT) for 60 minutes was performed by setting a water bath at 37°C (blue area). N=6 for each formulation. (C) PK curve of DOX after intravenous bolus administration of DPPG2-TSL-DOX, which was prepared from DPPG2 according to Formula III using the ethanol injection method. One hind leg of the rat was treated in a water bath at 37°C (without HT) or 41.5°C (with HT) for 60 min, respectively. N=6 for each formulation. Accumulation of DOX in (D) tumor and (E) organ 60 minutes after intravenous bolus administration of different DOX formulations (2 mg / kg DOX, administered in a fixed volume of 1 mL). All animals received light-induced HT treatment on one side of the tumor, while the contralateral tumor (the other hind leg) was not treated. N=6 for each preparation. Statistical analysis was performed using Student's t-test. p<0.05; p<0.01; p<0.001; p<0.0001. (F) The therapeutic effects of different DOX formulations (2 mg / kg DOX) combined with 60-minute light HT (41°C) exposure to tumors in a BN175 rat sarcoma model. Kaplan-Meier plot (survival rate, 5-fold increase in tumor volume). NT: Animals not treated with HT. N=6 for each formulation.

[0135] Figure 5 Effect of DPPG2 structure on the in vitro release of carboxyfluorescein (CF) from DPPC / DSPC / DPPG2 (molar ratio 50:20:30) thermosensitive liposomes (TSL) in physiological saline, at 37°C and 42°C for 1 hour. Detailed Implementation

[0136] 1.1 Existing Technology

[0137] 1.1.1 1,2-Dipalmitoyl- sn Synthesis of glycerol-3-phosphate-racemic diglycerol (Comparative Example)

[0138] The synthetic route for preparing DPPG2 is described in patent application WO 97 / 30058 A1, which is a phosphatidyl oligoglyceride (PG) synthesis route. x DPPG2 is part of the synthesis of glycerophospholipids, a molecular class of molecules. It is synthesized in a multi-step organic synthesis process where there is a lack of control over the stereocenter in the glycerol unit.

[0139] 1.1.2 1,2-Dipalmitoyl- sn -glycerol-3-phosphate- sn Synthesis of -1′-diglycerol (Comparative Example)

[0140] WO 2014 / 202680 A1 describes a standard that allows for the synthesis of 1,2-dipalmitoyl- sn -glycerol-3-phosphate- sn The synthetic pathway of -1′-glycerol-3′-racemic-glycerol. The aim is to obtain... sn DPPG2 has a -1′ configuration (as in naturally occurring PG glycerophospholipids). This configuration is associated with phospholipase activity and will prevent potential lipid accumulation after administration to patients. The synthetic pathway allows control over only two stereocenters in the molecule.

[0141]

[0142] (A) WO9730058 (1,2-dipalmitoyl-sn-glycerol-3-phosphate-racemic-digrazol) and (B) DPPG disclosed in WO2014 / 202680 A1 (1,2-dipalmitoyl-sn-glycerol-3-phosphate-sn-1′-diglycerol) 2 of Comparison of synthetic routes. Due to the ring-opening step of epoxides, neither route can control all three stereocenters.

[0143] 1.2 Synthesis of (2R)-2,3-bis(palmitoyloxy)propyl(2S)3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl)phosphate

[0144] 1.2.1 Preparation of a preferred example of the key intermediate in Equation 4.1a

[0145] Intermediate 4.1a is the preferred form of the key intermediate (IVa), possessing two chiral centers with (2R), (6S)-configurations. Both chiral centers are derived from enantiomeric pure starting materials (R)-(+)-glycidyl and (S)-(+)-1,2-isopropylene-glycerol. sn -1,2-IPG), and is transferred without isomerization in the final product DPPG2. The single stereoisomer 4.1a is obtained through the following four-step synthesis:

[0146]

[0147] All other possible stereoisomers were obtained by combining the following enantiomeric starting materials:

[0148] 4.1b: (2S), (6R)-configuration: (S)-(-)-glycidyl and (R)-(-)-1,2-isopropylene-glycerol (sn-2,3-IPG)

[0149] 4.1c: (2R), (6R)-configuration: (R)-(+)-glycidyl and (R)-(-)-1,2-isopropylglyceryl ( sn -2,3-IPG)

[0150] 4.1d: (2S), (6S)-configuration: (S)-(-)-glycidyl and (S)-(+)-1,2-isopropylidene-glycerol ( sn -1,2-IPG)

[0151] Step 1:

[0152] 3.8 kg of (R)-(+)-glycidyl was dissolved in 22.8 L of NMP, followed by the addition of 8.02 kg of PMBCl at 0–5 °C, and then 2.26 kg of NaH. After 4 hours at room temperature, the reaction mixture was poured into ice water and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The separated residue was purified by silica gel chromatography (n-heptane / ethyl acetate) to give 8.0 kg of 1.1a as a colorless oil. It was further analyzed by HPLC, TLC, and... 1 Characterized by H-NMR.

[0153] 1.1a 1 H NMR (400 MHz, CDCl3):

[0154]

[0155] Steps 2+3:

[0156] Dissolve 8.0 kg of 1.1a in 64 L of DMF, then add 8.16 kg of... sn-1,2-IPG was added, followed by 6.94 kg tBuOK at 0–5 °C. The resulting reaction mixture was stirred at room temperature for 12 hours to give 2.1a. Characterized by TLC and HPLC. Subsequently, without post-treatment or further purification, another 6.91 kg tBuOK and 12.6 kg benzyl bromide were added at 0–5 °C, and the resulting reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then poured into ice water and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Purification by silica gel chromatography (n-heptane / ethyl acetate) yielded 7.0 kg of pure compound 3.1a as a yellow oil. 3.1a was characterized by TLC, HPLC, and... 1 Characterized by H-NMR.

[0157] 3.1a 1 H NMR (400 MHz, CDCl3):

[0158]

[0159] Step 4:

[0160] Dissolve 6.9 kg of 3.1a in 41.8 L of DCM / H2O 10:1 (by volume). / Add 4.89 kg of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to a container and cool the mixture to 0°C. Stir the mixture at room temperature for 4 hours. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 and water. Dry the organic phase with Na2SO4. ， The solvent was removed under reduced pressure. After post-treatment, crude 4.1a was purified by silica gel chromatography (n-heptane / ethyl acetate) to give 2.6 kg of pure 4.1a as a brown oil. 4.1a was further purified by HPLC, TLC, and SFC. 1 Characterized by HNMR.

[0161] 4.1a 1 H NMR (400 MHz, CD3OD)

[0162]

[0163] 1.2.2 Evaluation of the optical purity of key intermediates

[0164] The optical purity of the key intermediates of formula (IVa), particularly 4.1a, was confirmed by chiral HPLC (hexane / isopropanol and isopropylamine) using a Chiralpak IA-3 column (3 µm, 0.46 cm ID x 25 cm length) from Daicel Chiral Technologies. Samples of all possible stereoisomers and mixtures thereof were prepared to ensure proper assignment of the corresponding peaks (see [link to sample citation]). Figure 1 ).

[0165] 1.2.3 From the key intermediate of formula (IVa) and 1,2-dipalmitoyl- sn -glycerin( sn Preparation of DPPG2 by -1,2-DPG)

[0166] Synthesis of DPPG2 as a single stereoisomer:

[0167]

[0168] This synthesis allows control over all three stereocenters in DPPG2. The starting material for introducing the stereocenters is 1,2-dipalmitoyl. -sn- glycerin( sn -1,2-DPG) and 4.1a. The three stereocenters in DPPG2 are denoted by (S) and (R) to show absolute stereochemistry.

[0169] Step 5:

[0170] Add 230 mL of DIPEA to 500 g sn -1,2-DPG was suspended in 1 L MeTHF and 1.25 L DCM, and the mixture was then cooled to -15 °C. 206 mL of 2-cyanoethyl- N,N1,5-Diisopropylphosphonamide chloroform (PAMCl) was dissolved in DCM, and the solution was cooled to -20°C and then added to the reaction mixture. The resulting mixture was stirred at -15°C for 1 hour, then at 0°C for 1.5 hours, and finally at 20°C for 1 hour. Then, 114 g of 4,5-dicyanimidazole (DCI) and 274 g of 4.1a were dissolved in 1.5 L of MeTHF, and the solution was slowly added to the reaction mixture at 20°C. After stirring for 1 hour, 52 g of DCI was added as a solution in 500 ml of DCM, and the entire mixture was stirred overnight at 20°C. Then, 91 ml of 35% w / w H2O2 aqueous solution was added over 1 hour at 0°C, and the mixture was stirred again at 20°C for 1 hour. The reaction mixture was diluted with 5 wt% Na2S2O5 aqueous solution and extracted with MeTHF. The organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure. The crude intermediate 13.1a was purified by rapid silica gel chromatography (DCM / acetone) to give 664 g of 13.1a in the form of a white glassy solid.

[0171] 13.1a 1 H NMR (400 MHz, CDCl3):

[0172]

[0173] Step 6:

[0174] 165 g of DBU was added to a solution of 664 g of 13.1a dissolved in MeTHF, and the reaction mixture was stirred at room temperature for 2 hours. Then, 72 g of Pd / C was added, and the resulting reaction suspension was stirred for 1 hour under a H2 atmosphere (1.2 bar). The reaction mixture was filtered through diatomaceous earth, washed with 1M citric acid aqueous solution, and the organic phase was separated. The organic phase was washed with 5 wt% NaHCO3 aqueous solution, dried over Na2SO4, and concentrated under reduced pressure to obtain 725 g of crude 16.1a.

[0175] 16.1a 1 H NMR (400 MHz, CDCl3):

[0176]

[0177] Step 7:

[0178] 707 g of crude 16.1a was suspended in 1.7 L of 2-propanol, then 688 g of TFA was added, and the resulting reaction mixture was stirred at 45 °C for 1 hour. The TFA was removed under reduced pressure, and the crude product was co-distilled twice with 2-propanol. The product was then dissolved in MeTHF, and the pH was adjusted to 7 with 8 wt% NaHCO3 solution. The organic phase was separated, dried over Na2SO4, and concentrated under reduced pressure to give 840 g of crude 19.1a.

[0179] 19.1a 1 H NMR (400 MHz, CDCl3)

[0180]

[0181] Step 8:

[0182] 615 g of 19.1a was dissolved in 2.8 L of MeTHF, then washed with 3.1 L of 8 wt% NaHSO4 solution, followed by pH adjustment to 4 with 60 g of NaOAc in 3.1 L of water. The organic phase was separated and concentrated to 9.3 L. A suspension of 20% Pd(OH)2 / charcoal (50% moisture content; 31.7 g) in 1 L of MeOH was added, and the resulting suspension was stirred for 18 hours under H2 atmosphere (3.5 bar). 35 g of NaOAc was added to adjust the pH to 7, and the mixture was then filtered through a Decalite filter and concentrated under reduced pressure. The concentrate was diluted with DCM, filtered through a Decalite filter again, and the organic phase was separated and concentrated under reduced pressure. The crude concentrate containing 8 was purified by rapid silica gel chromatography (DCM / MeOH), treatment with a Pd scavenger, and subsequent crystallization from ethanol to give 322 g of DPPG2 as a white powder.

[0183] DPPG2 1 H NMR (400 MHz, CDCl3):

[0184]

[0185] In each synthetic step, IPC was performed by HPLC / CAD measurements. If necessary, intermediate 13.1a was purified by chromatography to control and regulate its quality.

[0186] 1.3 Synthesis of (R)-2,3-bis(palmitoyloxy)propyl((S)-3-((R)-3-((R)-2,3-dihydroxypropoxy)-2-hydroxypropoxy)-2-hydroxypropyl)phosphate

[0187] Intermediate 10.1a has three chiral centers with (2R), (6S), and (10S)- configurations. The configurations of all three chiral centers originate from the enantiomeric pure starting material (S)-(+)-1,2-isopropylene-glycerol. sn -1,2-IPG), (S)-(-)-glycidylglycerol and (R)-(+)-glycidylglycerol, and transferred without isomerization in the final product DPPG3. This was achieved through the use of... sn -1,2-IPG defines the (10S) configuration. Using the enantiomer (R)-(-)-1,2-o-isopropylglycerol results in (10R). A single stereoisomer is obtained in 7 steps via the following synthetic method:

[0188]

[0189] Step 1:

[0190] (S)-(-)-glycidyl was dissolved in NMP, then PMBCl was added, followed by NaH at 0-5°C. The resulting reaction mixture was stirred at room temperature until the reaction was complete. The mixture was then poured into ice water and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (n-heptane / ethyl acetate) to give 1.1b.

[0191] 1.1a was obtained from (R)-(+)-glycidyl ether using the same procedure.

[0192] Steps 2+3:

[0193] Dissolve 1.1b in DMF, then add sn -1,2-IPG was added, followed by the addition of tBuOK at 0–5 °C, and the resulting reaction mixture was stirred at room temperature until the reaction was complete, yielding 2.1b. Subsequently, without post-treatment or further purification, additional tBuOK and benzyl bromide were added at 0–5 °C, and the resulting reaction mixture was stirred at room temperature until the reaction was complete. The reaction mixture was then poured into ice water and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Purification by silica gel chromatography (n-heptane / ethyl acetate) yielded pure compound 3.1b.

[0194] Step 4:

[0195] Dissolve 3.1b in DCM / H₂O at a ratio of 10:1 (volume / volume) and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at room temperature until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO₃ and water. Dry the organic phase with Na₂SO₄. ， The solvent was removed under reduced pressure. The crude product 4.1b was purified by silica gel chromatography (n-heptane / ethyl acetate).

[0196] Steps 5 and 6:

[0197] 1.1a was dissolved in DMF, then 4.1b was added, followed by the addition of tBuOK at 0–5 °C. The resulting reaction mixture was stirred at room temperature until the reaction was complete, yielding 8.1a. Subsequently, without post-treatment or further purification, additional tBuOK and benzyl bromide were added at 0–5 °C, and the resulting reaction mixture was stirred at room temperature until the reaction was complete. The reaction mixture was then poured into ice water and extracted with EtOAc. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Purification by silica gel chromatography (n-heptane / ethyl acetate) yielded pure compound 9.1a.

[0198] Step 7:

[0199] Dissolve 9.1a in DCM / H₂O at a ratio of 10:1 (volume / volume) and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at room temperature until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO₃ and water. Dry the organic phase with Na₂SO₄. ， The solvent was removed under reduced pressure. The crude product 10.1a was purified by silica gel chromatography (n-heptane / ethyl acetate).

[0200] Step 8:

[0201] DIPEA was added to a suspension of sn-1,2-DPG in MeTHF and DCM, and the mixture was then cooled to -15°C. 2-Cyanoethyl- N,N1,4-Diisopropylphosphonamide chloroform (PAMCl) was dissolved in DCM, the solution was cooled to -20°C, and then added to the reaction mixture. The resulting mixture was stirred at -15°C for 1 hour, then at 0°C for 1.5 hours, and finally at 20°C for 1 hour. 4,5-Dicyanimidazole (DCI) and 10.1a were then dissolved in MeTHF, and the solution was slowly added to the reaction mixture at 20°C. If necessary, additional DCI was added to the solution in DCM after 1 hour of stirring, and the entire mixture was stirred overnight at 20°C. Then, 35% w / w H2O2 aqueous solution was added over 1 hour at 0°C, followed by stirring at 20°C for another 1 hour. The reaction mixture was diluted with 5 wt% Na2S2O5 aqueous solution and extracted with MeTHF. The organic phase was dried over Na2SO4, and the solvent was removed under reduced pressure. The crude intermediate 14.1a was purified by rapid silica gel chromatography.

[0202] Step 9:

[0203] DBU was added to a solution of 14.1a dissolved in MeTHF, and the reaction mixture was stirred at room temperature for 2 hours. Pd / C was then added, and the resulting reaction suspension was stirred for 1 hour under a H2 atmosphere (1.2 bar). The reaction mixture was filtered through diatomaceous earth and washed with 1M citric acid aqueous solution to separate the organic phase. The organic phase was washed with 5 wt% NaHCO3 aqueous solution, dried over Na2SO4, and concentrated under reduced pressure to obtain 17.1a.

[0204] Step 10:

[0205] The crude product 17.1a was suspended in 2-propanol, then TFA was added, and the resulting reaction mixture was stirred at 45°C for 1 hour. TFA was removed under reduced pressure, and the crude product was co-distilled twice with 2-propanol. The product was then dissolved in MeTHF, and the pH was adjusted to 7 with 8 wt% NaHCO3 solution. The organic phase was separated, dried over Na2SO4, and concentrated under reduced pressure to obtain crude product 20.1a.

[0206] Step 11:

[0207] 20.1a was dissolved in MeTHF and washed with 8 wt% NaHSO4 solution, followed by pH adjustment to 4 with NaOAc aqueous solution. The organic phase was separated and concentrated. A suspension of 20% Pd(OH)2 / carbon (50% water content) in methanol was added, and the suspension was stirred under H2 atmosphere (3.5 bar) until the reaction was complete. NaOAc was added to adjust the pH to 7, and the mixture was then filtered through a Decalette filter and concentrated under reduced pressure. The concentrate was diluted with DCM, filtered through a Decalette filter again, and the organic phase was separated and concentrated under reduced pressure. Crude DPPG3 was purified by rapid silica gel chromatography.

[0208] 1.4 Stereospecific cleavage of DPPG2 with phospholipase A2

[0209] Phospholipases are enzymes that selectively cleave bonds in glycerophospholipids based on their stereochemistry. They play a role in the metabolism of glycerophospholipids in vivo. Phospholipase A2 cleaves 1,2-acyl- sn -Glyceryl-3-phospholipids hydrolyze to their 1-acyl- sn -Glyceryl-3-phospholipids and their corresponding fatty acids. For experiments, commercially available enzymes (extracted from porcine pancreas) were used. Lipid samples (2 µmol) were dissolved in 1.5 wt% deoxycholate and then incubated at 37°C in a heated shaker in pH 8.5 Tris buffer supplemented with CaCl2 and 0.5 U of enzyme. Samples were taken at different time points and analyzed by HPLC. A Waters XBridge Phenyl column (3.5 μm, 2.1 μm) was used. (150 minutes), analytes were detected using an electrosol detector (CAD). Gradient elution was performed using a 100 mM NH4Ac aqueous solution (pH 6.0) and a methanol solution as eluent A and eluent B, respectively, to separate the analytes. The column oven was set to 35°C.

[0210] Three batches of DPPG2 were tested (their stereoconfigurations in the diglyceride head group differed), and DPPC was used as a control glycerophospholipid. Figure 2 DPPC cleaved significantly slower than DPPG2. DPPG2 batches showed a trend toward slower cleavage, depending on the configuration of the stereocenter in the diglyceride head. After 10 minutes, 14.8%, 20.4%, and 24.3% of DPPG2 according to WO 9730058, WO 2014 / 202680 A1, and Formula III, respectively, were still detectable in the samples.

[0211] 1.5 Preparation of DPPG2-TSL-DOX

[0212] Thermosensitive liposomes (TSLs) and compositions thereof, also known as thermosensitive (TSL) formulations, can be prepared by various methods, such as lipid film hydration and extrusion, ethanol injection, or other methods. The active pharmaceutical ingredient is either passively loaded or loaded via an active process. Active remote loading can be achieved using, for example, ammonium gradients, acid gradients, or EDTA salt gradients. A method for preparing TSLs with encapsulated DOX (DPPG2-TSL-DOX) is described in WO2022 / 008471 A1.

[0213] 1.5.1 Lipid film hydration method using DPPG2 according to WO 97 / 30058

[0214] DPPG2 was used for batch preparation according to WO97 / 30058. Fifteen batches were prepared using a 100 nm filter membrane during extrusion. Twenty-five batches were prepared using a 200 nm filter membrane during extrusion. These batches were used for comparative purposes (Hossann et al. 2021).

[0215] All solutions were prepared using deionized and purified water from an ultrapure water system (Milli Q Advantage, Millipore) and then filtered through 0.2 μm before use. The desired molar ratio (DPPC / DSPC / DPPG 250:20:30) of phospholipids was dissolved in chloroform / methanol 9:1 (v / v) using a round-bottom flask. The solvent was evaporated under vacuum in a rotary evaporator until a thin and homogeneous lipid membrane was formed. The lipid membrane was dried at 10 mbar / 70°C for at least 1 hour to remove residual trace organic solvents. The membrane was then hydrated at 60°C with 300 mM citric acid at pH 4 under shaking for 30 minutes. The resulting lipid concentration was 50 mM. The membrane was then processed using a hot-tube extruder (LIPEX). TMMonolayer vesicles were obtained by extrusion 10 times through two polycarbonate membranes of desired pore sizes (e.g., 100 nm and 200 nm, Whatman, GE Healthcare Europe GmbH, Freiburg, Germany) at 60°C and a maximum N2 pressure of 20 bar. The dispersion was then cooled to 2–8°C, and the buffer was replaced with physiological phosphate-buffered saline (PBS) buffer at pH 7.4 using a PD10 column (GE Healthcare). The desired active DOX loading conditions were obtained by mixing the liposome dispersion with PBS at pH 7.4 and DOX stock solution (5.7 mg / ml DOX HCl). The dispersion was then heated by shaking in an Eppendorf Thermomixer comfort model equipped with a 50 ml heating module (e.g., 37°C for up to 60 minutes). Trace amounts of unencapsulated DOX were removed by centrifugation at 75,000 xg (Beckman Coulter Avanti J-26XP with a JA-25.50 rotor). The supernatant was discarded, and if storage at -20°C was required, the precipitate was carefully resuspended in storage buffer (e.g., 10% (w / v) trehalose, 10.5 mM sodium phosphate / potassium phosphate, pH 7.4). If the batch was to be used immediately, the buffer was replaced with PBS at pH 7.4.

[0216] 1.5.2 Ethanol Injection Method Using Stereoisomer DPPG2

[0217] The stereoisomer DPPG2 according to Formula III was used in the preparation of the following eight independent batches.

[0218] As described in WO2022 / 008471 A1, DPPG2-TSL-DOX has been prepared and characterized. All solutions used were prepared from deionized and purified water from an ultrapure water system, and then filtered through 0.2 µm before use. If necessary, the pH of 300 mM ammonium phosphate buffer and physiological PBS buffer was adjusted to 7.4 with phosphoric acid. A phospholipid excipient with a molar ratio of DPPC / DSPC / stereoisomeric DPPG2 of 50:20:30 was dissolved in ethanol at 60°C. The ethanol lipid solution and ammonium phosphate buffer were pumped independently through two heated coils (60°C) and the streams were combined at 60°C using a T-connector. The 60°C hot dispersion was then pumped through a 60°C hot extrusion chamber containing multiple extrusion membranes with suitable pore sizes to produce liposomes with vesicle sizes in the range of 100 to 150 nm. After extrusion, the stream was pumped through a 5°C cold coil into an ice bath collection container. An active loading gradient of DOX was created by replacing the liposome in vitro buffer with pH 7.4 PBS via tangential flow filtration (TFF). The desired DOX:liposome molar ratio of 0.08 was obtained by mixing the liposome dispersion with pH 7.4 PBS buffer and DOX stock solution (5.7 mg / ml DOXHCl). The dispersion was heated at 37°C for 30 min with stirring in a round-bottom flask equipped with a heating jacket. After cooling the dispersion to 2–8°C, trace amounts of unencapsulated DOX were removed, and the liposome in vitro buffer was replaced with storage buffer via TFF. Finally, the dispersion was sterilely filtered (0.2 µm).

[0219] 1.5.3 Use the ethanol injection method for DPPG2 according to WO 97 / 30058

[0220] Liposome batches were prepared as controls using DPPC, DSPC, and DPPG2 in a molar ratio of 50:20:30 (according to WO 97 / 30058). These liposomes were loaded with DOX using either 250 mM ammonium sulfate at pH 5.4 (4 batches) or 300 mM ammonium phosphate at pH 7.4 (4 batches) as loading buffers, as described in Example 1.5.2. No buffer-to-storage buffer replacement was performed because the batches were characterized immediately and were not intended for storage at -20°C.

[0221] 1.6 In vitro characterization of TSL formulation

[0222] The TSL formulations produced in Examples 1.5.1, 1.5.2, and 1.5.3 were characterized as follows: Hydrodynamic diameter (z-mean), particle size distribution, and zeta potential were determined by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). The purity and concentration of DOX and phospholipid excipients were quantified by HPLC equipped with a fluorescence detector or an electrosol detector (CAD), respectively. In vitro temperature-dependent DOX release (TDR) was analyzed in fetal bovine serum (FCS). Residual solvent was measured by gas chromatography (headspace). In addition, pH, osmolarity, bacterial endotoxin, and sterility were measured according to the corresponding European Pharmacopoeia (EP) methods.

[0223] The biophysical properties of all batches were compared to assess the effects of stereochemistry and / or manufacturing methods, respectively. Results are as follows: Figure 3 As shown.

[0224] For batches prepared using ethanol injection (DPPG2 according to Formula III), ethanol injection (DPPG2 according to WO 97 / 30058), lipid film hydration (100 nm extrusion), and lipid film hydration (200 nm extrusion) methods, the vesicle sizes (expressed as z-means) were 118 ± 8 nm, 119 ± 2 nm, 120 ± 11 nm, and 135 ± 14 nm, respectively. The PDIs were 0.10 ± 0.03, 0.13 ± 0.04, 0.13 ± 0.07, and 0.13 ± 0.05, respectively. For batches prepared by ethanol injection (DPPG2 according to Formula III), lipid film hydration (100 nm extrusion), and lipid film hydration (200 nm extrusion), the zeta potentials were -28.3 ± 1.2 mV, -22.7 ± 7.0 mV, and -26.3 ± 3.5 mV, respectively.

[0225] In summary, compared with the lipid film hydration method, the TSL formulation prepared by ethanol injection exhibits less variation in vesicle size, particle size distribution, and zeta potential (low standard deviation). This indicates that the ethanol injection preparation process possesses excellent robustness and controllability.

[0226] All prepared batches showed temperature-dependent DOX release profiles for the TSL formulation. Figure 3The batches of TDR produced by lipid film hydration showed no significant differences, but their vesicle sizes differed (expressed as z-mean). Therefore, the DOX release kinetics of DPPG2-based TSLs were unaffected by vesicle sizes in the range of 117 nm to 161 nm (studied temperature range: 37 to 41 °C) (Hossann et al. 2010). When comparing batches from different preparation methods, it must be noted that the excipients used to load DOX had no effect on TDR (WO 2022 / 008471 A1).

[0227] For batches prepared using ethanol injection (DPPG2 according to Formula III), ethanol injection (DPPG2 according to WO 97 / 30058), lipid film hydration (100 nm extrusion), and lipid film hydration (200 nm extrusion) methods, the in vitro DOX release within 5 minutes at 37°C in FCS was 6.8 ± 3.6%, 1.1 ± 1.2%, 3.5 ± 3.5%, and 6.5 ± 6.2%, respectively. The batch-to-batch stability at body temperature was relatively small, but all showed acceptable low DOX release (<10%). In FCS, the in vitro DOX release within 5 minutes at 40°C was 75.4 ± 9.9%, 65.7 ± 8.5%, 46.7 ± 26.2%, and 43.6 ± 23.3%, respectively. All batches showed considerable heat-induced DOX release at temperatures >40°C, with over 75% released during a 5-minute incubation period (data not shown).

[0228] Surprisingly, compared to batches prepared with ethanol injection and batches prepared with DPPG2 according to Formula III, the latter showed higher in vitro DOX release in the FCS over a 5-minute period at 37°C, 38°C, 39°C, and 40°C. This indicates that the batches prepared with DPPG2 according to Formula III exhibited faster DOX translocation across the membrane bilayer compared to batches prepared with DPPG2 according to WO 97 / 30058. This is likely due to a more uniform distribution of the lipid excipients in the bilayer, resulting in faster DOX translocation when TSLs are heated above their TL. m The phase transition from the solid gel phase to the liquid disordered phase is narrower. Faster drug release under thermotherapy is a beneficial effect for the in vivo application of this type of TSL, as the encapsulated drug is more readily bioavailable in heated body regions, and more DOX can accumulate in tumor tissue.

[0229] 1.7 Protein crown of DPPG2-TSL formulation

[0230] The molar content of DPPG2 affects the composition of the protein crown of DPPC / DSPC / DPPG2 80-x / 20 / x (mol / mol) (x = 5, 10, 20, 30) (Lokerse et al. 2021). In this example, the potential stereoselective effect of protein binding to DPPG2-based TSL was investigated.

[0231] Batch preparation and characterization of liposomes:

[0232] Batches of DPPC / DSPC / DPPG2 50:20:30 (mol / mol) TSLs were prepared using DPPG2 (according to WO 97 / 30058), DPPG2 (according to WO2014 / 202680), or DPPG2 (according to Formula III) and loaded with physiological saline or 100 mM carboxyfluorescein (CF) at pH 7.2. Additionally, non-thermally sensitive liposomes (NTSLs) mimicking the lipid composition of the approved liposomal formulation Caelyx (HSPC / cholesterol / DSPE-PEG2000 55:40:5 (mol / mol)) were prepared as a DPPG2-free control and loaded with physiological saline. All batches were prepared by lipid film and hydration methods and characterized by vesicle size (z-mean), PDI, and heat-induced CF release, as described elsewhere (Hossann et al. 2010). The following results are presented as the average of measurements from two independently prepared batches for each formulation. Measurements were performed prior to protein crown formation. The vesicle size (z-mean) and PDI of all formulations were similar, as shown in Table 1.

[0233] While DPPG2-based TSL exhibits a negative zeta potential due to the anionic DPPG2, the control formulation shows a neutral zeta potential. CF-loaded TSL batches showed approximately 5 to 6.5% of the typical encapsulation efficiency of passive loading methods.

[0234] Measurements in physiological saline demonstrated the effect of the DPPG2 material used on thermally induced CF release. Figure 5 Compared with TSL using DPPG2 (according to WO 97 / 30058) or DPPG2 (according to WO 2014 / 202680), TSL using DPPG2 (according to formula III) showed higher in vitro CF release over 1 hour at 42°C. This result is consistent with the in vitro DOX release results described in Example 1.6 and demonstrates that this effect is independent of the TSL preparation method and the presence of serum components.

[0235] Formation and characterization of protein crowns:

[0236] Human plasma collected from healthy donors using citrate phosphate glucose was obtained from a Biowest SAS (0.2 μm filtered), aliquoted, and stored at -80°C before use. Thawed plasma was centrifuged (2 min / 1000 xg) to remove aggregates that could clog the size exclusion column (SEC). TSL fractions (25 mM total lipid content) were mixed with human plasma at a 1:9 ratio. The mixture was incubated on a heated shaker (0 rpm) at 37°C for 30 min, then loaded onto a saline-pretreated CL-4B SEC. The TSL-containing fraction was transferred to a 1000 kDa Float-A-Lyzer dialysis chamber in physiological saline and dialyzed overnight at 4°C. Subsequently, the dialyzed TSL samples were concentrated by centrifugation at 4°C using a single centrifugation step (4500 x g for 1 hour) with a 10 kDa Amicon centrifuge filter and four centrifugation steps (1 x 1 hour 8600 x g, 3 x 30 min 8600 x g) with a 1000 kDa VivaSpin centrifuge concentrator. The precipitate was washed with physiological saline. The final TSL precipitate was resuspended in 100 µl of physiological saline, and the total protein (BCA protein assay) and lipid content were quantified.

[0237] Liquid chromatography-mass spectrometry (LC-MS) analysis:

[0238] 3 μg of each sample was used for protein digestion, and a small portion of it was injected into LC-MS / MS. Hi3 was then used. E. coli Six standard (Waters) peptides were incorporated into the samples for absolute quantification. Samples were processed using the Preomics iST kit to obtain peptides from the protein crown. Peptides were measured by LC-MS / MS in DDA mode. The raw data were searched using MaxQuant for human proteins (whole human proteome) + E. coli.

[0239] For DPPG2-TSL loaded with saline or CF, the isolated samples had protein concentrations of approximately 250 µg / ml and 400 µg / ml, respectively. Liposome concentrations in the samples were approximately 3 mM and 4 mM, respectively. The protein concentration in the control formulation was approximately 575 µg / ml. The top 10 most abundant proteins for each formulation are shown in Table 1. DPPG2-based TSLs bind to most of the different proteins in the apolipoprotein class. Apolipoproteins CI, C-II, and AI are the major components of their protein crown, independent of the DPPG2 used. Interestingly, the ratio of apolipoprotein CI to apolipoprotein C-II depends on the structure of the DPPG2 used. The ratios of DPPG2-based TSLs to DPPG2 (according to WO97 / 30058), DPPG2 (according to WO2014 / 202680), or DPPG2 (according to Formula III) were 2.5, 1.8, and 3.0, respectively. Furthermore, DPPG2-TSL with DPPG2 (according to Formula III) showed almost no binding to keratin-like proteins, while DPPG2-TSL with DPPG2 (according to WO 97 / 30058) and DPPG2 (according to WO 2014 / 202680) showed binding to different keratin types. These data indicate the influence of stereochemistry in DPPG2 on protein binding affinity. In contrast to DPPG2-TSL, the most abundant protein in the control formulation belonged to the immunoglobulin class.

[0240] Table 1: Protein crown composition of selected formulations. List of the top 10 most abundant proteins for each formulation.

[0241]

[0242]

[0243]

[0244]

[0245] 1.8 In vivo detection of DPPG2-TSL-DOX in a rat model

[0246] In a rat BN175 sarcoma model, batches of DPPG2-TSL-DOX produced by either ethanol injection or lipid film hydration (comparative example) were investigated. The former batch contained DPPG2 according to Formula III, while the latter batch contained DPPG2 according to WO97 / 30059. The batches were characterized as described in Example 1.6. DPPG2-TSL-DOX prepared by ethanol injection (DPPG2 according to Formula III) exhibited a vesicle size of 118 nm (z-mean), a PDI of 0.08, a zeta potential of -28.3 mV, and a DOX:lipid ratio of 0.06. DPPG2-TSL-DOX prepared by lipid film hydration (DPPG2 according to WO97 / 30059) exhibited a vesicle size of 178 nm (z-mean), a PDI of 0.13, a zeta potential of -29.4 mV, and a DOX:lipid ratio of 0.10.

[0247] For the PK study, 18 anesthetized healthy male brown Norwegian rats (~300 g) were administered non-liposomal DOX (n = 6), DPPG2-TSL-DOX (DPPG2 according to Formula III) (n = 6), or DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) (n = 6) via intravenous bolus injection. Animal body temperature was controlled using a rectal temperature probe. During anesthesia, animal body temperature was maintained by a water bath (37°C). Blood samples were collected at specified time points. Plasma DOX and DPPG2 levels were determined by HPLC and LC / MS equipped with fluorescence detectors, respectively.

[0248] The two DPPG2-TSL-DOX batches showed similar DOX plasma clearance rates (Figure A) and exhibited the expected long circulation characteristics. For DPPG2-TSL-DOX (DPPG2 according to Formula III) and DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059), the plasma half-life (t) of DOX was... 1 / 2,α The initial plasma concentrations (ID%) were 141 ± 5 min and 180 ± 14 min, respectively. After 240 min, the plasma concentrations were also similar, at 6.3% and 17.2%, respectively, indicating that the two formulations had comparable clearance rates of DOX. 1 / 2,α The slight difference can be explained by the known effect of vesicle size on the circulating half-life of drugs encapsulated in DPPG2-TSL (Limmer et al. 2014). In contrast, non-liposomal DOX was cleared within minutes, and no DOX plasma levels were detected at time points >15 min.

[0249] The difference in the DOX:lipid molar ratio in these two DPPG2-TSL-DOX formulations resulted in different maximum observed concentrations of DPPG2 (Figure B) (C). max For DPPG2-TSL-DOX (DPPG2 according to formulation III), a higher DPPG2 dosage was administered. This is because the DOX:lipid ratio is lower in this formulation, and all rats were given the same amount of DOX (2 mg / kg). After 240 min, both liposomal formulations showed similar DPPG2 clearance and comparable plasma concentrations.

[0250] High blood levels of DPPG2 (nanocarrier) and DOX (drug) within the first hour after intravenous (iv) administration are an important prerequisite for TSL formulations (Kneidl et al. 2014). This allows for the activation of as much of the administered TSL as possible via local or regional HT in the tumor, subsequently resulting in high local DOX levels. Only DOX released within the tumor will be therapeutically effective (see below).

[0251] In a series of independent pharmacokinetic (PK) experiments, 12 anesthetized healthy male brown Norwegian rats (~300g) received DPPG2-TSL-DOX (DPPG2 according to Formula III) via intravenous bolus injection at a dose of 2 mg / kg DOX. Six rats received local HT treatment (41.5°C, water bath) on one hind leg, while the other six rats received local NT treatment (37°C, water bath) on one hind leg. The body temperature of these animals was controlled by a rectal temperature probe. Blood samples were collected at specified time points. The DOX content in plasma was determined by HPLC with a fluorescence detector. The results showed that 1 hour of local HT treatment was sufficient to almost completely deplete circulating DPPG2-TSL-DOX through heat-induced DOX release. The PK parameters decreased significantly, indicating significant heat-induced DOX release from the nanocarrier in the heated (41.5°C) rat hind legs. For example, for rats receiving NT and HT, t 1 / 2,α The concentration decreased from 141 ± 5 min to 29 ± 3 min, respectively. The area under the curve (AUC) of the concentration in the first 60 min decreased from 1410 µg to 29 ± 3 min, respectively. h / ml reduced to 412 µg h / ml. Prior to this (Hossann et al., 2021), it had been demonstrated that DPPG2-TSL-DOX (DPPG2 according to WO97 / 30059) induces DOX release at heated body sites.

[0252] The accumulation of DOX in tumor tissue (Figure D) and selected organs (Figure E) was investigated next. Eighteen anesthetized male brown Norwegian rats (~300 g) bearing tumors were administered non-liposomal DOX (n=6), DPPG2-TSL-DOX (DPPG2 according to Formula III) (n=6), or DPPG2-TSL-DOX (DPPG2 according to WO97 / 30059) (n=6) via intravenous bolus injection. Two BN175 tumors (soft tissue sarcomas) were subcutaneously implanted in each hind leg of all animals. One tumor was subjected to local heat treatment (HT) at a light source of 41.5 °C for 60 min, while the second tumor remained untreated (room temperature, NT). A light source was used instead of a water bath for HT treatment because it allowed for more concentrated (local) heating of the tumor tissue (Willerding et al. 2016). After 60 min, the animals were sacrificed, organs were perfused, and tumors and organs were collected. The DOX content in plasma was determined by HPLC with a fluorescence detector.

[0253] Tumor DOX concentrations were strongly dependent on administration conditions (HT vs. NT) and formulation (non-liposomal vs. TSL). In rats treated with non-liposomal DOX, DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059), and DPPG2-TSL-DOX (DPPG2 according to Formula III), HT application increased DOX tumor accumulation (compared to untreated tumors) by 1.6-fold, 16.2-fold, and 12.7-fold, respectively. Enrichment factors were comparable to published data (Willerding et al. 2016, Hossann et al. 2021). In heated tumors, DOX concentrations were 3.1 ± 0.5 ng / mg, 32.3 ± 23.0 ng / ml, and 41.7 ± 19.0 ng / ml, respectively. In untreated tumors, DOX concentrations were 1.9 ± 0.3 ng / mg, 2.0 ± 0.3 ng / ml, and 3.3 ± 0.3 ng / ml, respectively. Interestingly, the two tumors in rats treated with DPPG2-TSL-DOX (DPPG2 according to Formula III) showed higher DOX levels compared to the corresponding tumors in rats treated with DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059). This is in good agreement with in vitro DOX release data (Example 1.6), which indicates that DPPG2-TSL-DOX (DPPG2 according to Formula III) enables faster bioavailability of DOX.

[0254] Except for the spleen, the organ distribution of DOX in both DPPG2-TSL-DOX formulations was comparable. The spleen DOX content of DPPG2-TSL-DOX (DPPG2 according to Formula III) was significantly lower at 13.7 ± 1.0 ng / mg, compared to 20.6 ± 2.4 ng / mg for DPPG2-TSL-DOX (DPPG2 according to Formula III). It has previously been shown that the preparation method has no effect on organ distribution (including spleen content) (WO 2022 / 008471 A1), suggesting that the use of DPPG2 according to Formula III may reduce the rate of liposome uptake into the spleen.

[0255] The literature provides examples of how even minute changes in molecular structure can produce unexpectedly powerful effects, either in vitro or in vivo. In DPPC / DPPG... X The replacement of DPPG2 with DPPG4 in liposomes with cholesterol 40:10:50 (mol / mol) increased uptake into the spleen of rats (Schagon 1997).

[0256] Compared to these two DPPG2-TSL-DOX formulations, the use of non-liposomal DOX resulted in significantly higher DOX accumulation in the lungs and heart (Figure 4E). This may reduce the risk of DOX-related cardiotoxicity following DPPG2-TSL-DOX administration.

[0257] Finally, the efficacy of these two DPPG2-TSL-DOX formulations was investigated in vivo and compared with selected clinically relevant DOX formulations (e.g., non-liposomal DOX, Caelyx). Thirty-six anesthetized tumor-bearing male brown Norwegian rats were treated with DPPG2-TSL-DOX (DPPG2 according to Formula III) and HT (n = 6), DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) and HT (n = 6), non-liposomal DOX and HT (n = 6), Caelyx and HT (n = 6), saline and HT (n = 6), and DPPG2-TSL-DOX (DPPG2 according to Formula III) without HT (n = 6). A BN175 tumor (soft tissue sarcoma) was subcutaneously implanted in one hind leg of all animals. Local HT treatment was performed using a light source. The target temperature was 41°C. Animals were preheated to this temperature for 30 min, and then maintained at 41°C for 60 min after intravenous bolus injection. Once a tumor diameter >5 mm was observed, animals treated with the DOX formulation received a single intravenous bolus injection of 2 mg / kg DOX. After treatment, animals were returned to their cages, and tumor growth was monitored every 2 days until endpoints were reached (e.g., tumor diameter >3 cm, ulceration, hemorrhage, skin toxicity).

[0258] Figure 4F depicts the Kaplan-Meier plots (survival rate, 5-fold increase in tumor volume) for all experimental groups. The combination of DPPG2-TSL-DOX (DPPG2 according to Formula III) and HT achieved the longest survival, followed by the combination of DPPG2-TSL-DOX (DPPG2 according to WO97 / 30059) and HT treatment. This is likely due to the faster release of DOX at 40°C and reduced splenic uptake compared to DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059). In the absence of HT, the efficacy of DPPG2-TSL-DOX (DPPG2 according to Formula III) was significantly reduced, comparable to 0.9% saline, Caelyx, and non-liposomal DOX, respectively. Only rats showing transient tumor size reduction after treatment were treated with the DPPG2-TSL-DOX formulation in combination with HT. All other rats showed a stable increase in tumor volume after treatment. These results suggest that high local DOX concentrations in tumor tissue are necessary for effective tumor therapy.

[0259] In summary, compared with DPPG2-TSL-DOX (DPPG2 according to Formula III), DPPG2-TSL-DOX showed slightly faster clearance of DOX from the bloodstream. Surprisingly, DOX accumulation in tumor tissue was increased, DOX content in the spleen was reduced, and this formulation showed superior therapeutic efficacy.

[0260] This application also discloses the following:

[0261] Item 1. A stereoisomer of phosphatidyl oligoglycerol of formula (I)

[0262] (I)

[0263] Among the three solid-states 1 2 3 has a predetermined configuration p represents another solid center; and

[0264] Where R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms; n is a number from 0 to 20; and

[0265] m is 0 or 1 independently each time it appears.

[0266] Item 2. The stereoisomer of phosphatidyl oligoglycerol according to Item 1, having formula (I), wherein n is a number from 0 to 10, preferably a number from 0 to 5, more preferably a number from 0 to 5, and even more preferably a number from 0 to 1.

[0267] Item 3. A stereoisomer of phosphatidyl oligoglycerol according to either item 1 or 2, having formula (I), where n is 0.

[0268] Item 4. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-3, having formula (I), where m is 1 each time it appears.

[0269] Item 5. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-4, having formula (I), wherein R 1 and R 2 Each is independently a hydrocarbon group having 13 to 19 carbon atoms, especially 14 to 18 carbon atoms.

[0270] Item 6. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-5, having formula (I), wherein R 1 and R 2Each is independently a straight-chain or branched hydrocarbon group, preferably a straight-chain hydrocarbon group, and / or wherein R 1 and R 2 Each is independently a straight-chain or branched alkyl group, especially a straight-chain alkyl group.

[0271] Item 7. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-6, having formula (I), wherein R 1 and R 2 It is a saturated, monounsaturated, or polyunsaturated alkyl group, preferably a saturated alkyl group.

[0272] Item 8. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-7, having formula (I), wherein R 1 and R 2 Each is independently a straight-chain saturated alkyl group; preferably, R 1 and R 2 Each is independently a straight-chain saturated C12 to C24 alkyl group; more preferably, R 1 and R 2 It is a straight-chain saturated C13 to C19 alkyl group; most preferably, R 1 and R 2 It is a straight-chain saturated C15 alkyl group.

[0273] Item 9. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-8, having formula (I), wherein the stereoisomer is... 1 is an R-configuration, in solid form 2 is an S-configuration, and in three dimensions... 3 is the R-configuration.

[0274] Item 10. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-9, having formula (I), wherein the stereoisomer of phosphatidyl oligoglycerol is a single stereoisomer.

[0275] Item 11. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-10, having formula (I), wherein the stereoisomer... p has a predetermined configuration.

[0276] Item 12. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-11, having formula (II)

[0277] (II)

[0278] In the three-dimensional 1 is an R-configuration, in solid form 2 is an S-configuration, in three dimensions 3 is an R-configuration, and p represents the center of another solid.

[0279] Item 13. Phosphatidyl oligoglycerides with stereoisomers according to any one of items 1-12,

[0280] Where n = 0 or 1, especially where n = 0.

[0281] Item 14. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-13, wherein R 1 and R 2 Independently, it is a straight-chain saturated alkyl group having 13 to 19 carbon atoms, and particularly, wherein R 1 and R 2 It is a straight-chain saturated C15 alkyl group.

[0282] Item 15. A phosphatidyl oligoglycerol having a stereoisomer of formula (III) according to any one of items 1-14

[0283] (III)

[0284] Item 16. A stereoisomer of phosphatidyl oligoglycerol according to any one of items 1-14, having formula (III′)

[0285] (III′)

[0286] In the three-dimensional p has a predetermined configuration.

[0287] Item 17. A method for preparing stereoisomeric phosphatidyl oligoglycerides according to any one of items 1-16, characterized in that the synthetic route includes a key intermediate of formula (IV) or formula (IV′).

[0288]

[0289] In the two solids mentioned 2′ 3′ has a predetermined configuration, and PG, PG′ and PG″ independently represent protecting groups.

[0290] Item 18. The method for preparing stereoisomeric phosphatidyl oligoglycerides according to Item 17, wherein PG, each time it appears, is independently benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropyl-2-yl (MOP), silyl or p-methoxybenzyl (PMB), preferably PG is benzyl (Bn); wherein PG″, each time it appears, is independently benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropyl-2-yl (MOP), silyl or p-methoxybenzyl (PMB), preferably PG″ is benzyl (Bn); and wherein PG′ is CH2 (methylene), CHCH3 (ethylene), CHPh (benzylene), CHPhp-Ome (p-methoxybenzylene), C(CH3)2 (isopropylene) or Si t Bu2 (di-tert-butylsilyl alkyl), Si(iPr)2-O-Si(iPr)2 (tetraisopropyldisiloxane alkyl alkyl), preferably PG′ is C(CH3)2.

[0291] Item 19. A method for preparing stereoisomeric phosphatidyl oligoglycerides according to Item 17 or 18, characterized in that the synthetic route includes a key intermediate of formula (IVa) or formula (IVa′).

[0292]

[0293] In the three-dimensional 2′ is an R-configuration, in the solid. 3′ is S-configuration for (IVa) and R-configuration for (IVa′), where PG, PG′ and PG″ independently represent protecting groups.

[0294] Item 20. A method for preparing stereoisomeric phosphatidyl oligoglycerol according to any one of items 17-19, characterized in that it is prepared from glycidyl and 1,2-isopropylglycerol, particularly from (R)-(+)-glycidyl and (S)-(+)-1,2-isopropylglycerol ( sn Intermediate for the preparation of (IV) 1,2-IPG.

[0295] Item 21. A method for preparing stereoisomeric phosphatidyl oligoglycerides according to any one of items 17-20, comprising reacting a key intermediate of formula (IV) or (IVa) with a compound of formula (V).

[0296] (V)

[0297] Where R 1 and R 2Each is independently a hydrocarbon group with 12-24 carbon atoms, R′ is a leaving group, and R″ is a hydrocarbon group with 1-12 carbon atoms.

[0298] Item 22. A method for preparing stereoisomeric phosphatidyl oligoglycerides according to any one of items 17-21, wherein R′ is 2-cyanoethyl (CNE), R″ is a C1-C12 hydrocarbon group, particularly R″ is methyl (Me), ethyl (Et), isopropyl (iPr) or benzyl (Bn), preferably R″ is isopropyl.

[0299] Item 23. A liposome comprising a stereoisomer of phosphatidyl oligoglycerol from any one of items 1-16.

[0300] Item 24. The liposome containing stereoisomeric phosphatidyl oligoglycerol according to Item 23, wherein the stereoisomeric phosphatidyl oligoglycerol is a single stereoisomer of formula (I).

[0301] Item 25. Liposomes according to Item 23 or 24, wherein the stereoisomer of phosphatidyl oligoglycerol is a single stereoisomer of formula (III).

[0302] .

[0303] Item 26. The liposome according to any one of items 23-25 ​​further comprises at least one phosphatidylcholine of formula (VII).

[0304] (VII)

[0305] Where R 3 and R 4 Each is an independent hydrocarbon group with 12 to 24 carbon atoms.

[0306] Item 27. Liposomes according to any one of items 23-26, comprising: lipid content based on the liposomes,

[0307] 20 mol% to 40 mol% of stereoisomeric phosphatidyl oligoglycerol according to any one of claims 1 to 16, especially phosphatidyl oligoglycerol of formula (III) as a single stereoisomer;

[0308] 40 mol% to 60 mol% of 1,2-dipalmitoylphosphatidylcholine; and

[0309] 15 mol% to 25 mol% of 1,2-distearate phosphatidylcholine.

[0310] Item 28. A liposome according to any one of items 23-26, comprising: a lipid content based on the liposome,

[0311] 25 mol% to 35 mol% of the stereoisomer DPPG2 of formula (III); and

[0312] 65 mol% to 75 mol% of 1,2-dipalmitoyl- sn 3-glycerol-3-phosphate choline (DPPC).

[0313] Item 29. The liposomes according to any one of items 23-28 are thermosensitive liposomes.

[0314] Item 30. Liposomes according to any one of items 23-29, which also contain an active agent, particularly doxorubicin.

[0315] Item 31. Liposomes according to any one of items 23-30, used for the treatment of tumors.

[0316] Item 32. According to Item 31, the liposome is a soft tissue sarcoma.

[0317] Item 33. Use of stereoisomers of phosphatidyl oligoglycerides according to any one of items 1-16 for increasing temperature-dependent drug release from thermosensitive liposomes and / or narrowing phase transitions in temperature-dependent release profiles of thermosensitive liposomes.

Claims

1. A stereoisomer of phosphatidyl oligoglycerol of formula (I) (I) Among the three solid-states 1 2 3. Has a predetermined configuration; p represents another solid center; and Where R 1 and R 2 Each is independently a hydrocarbon group having 12 to 24 carbon atoms; n is a number from 0 to 20; and m is 0 or 1 independently each time it appears.

2. The stereoisomeric phosphatidyl oligoglycerol according to claim 1, having formula (II) (II) In three-dimensional 1 is an R-configuration, in solid form 2 is an S-configuration and a three-dimensional configuration. 3 is an R-configuration, and p represents the center of another solid.

3. The stereoisomeric phosphatidyl oligoglycerol according to claim 1 or 2, Where n = 0 or 1, especially where n = 0.

4. The stereoisomeric phosphatidyl oligoglycerol according to any one of claims 1 to 3, wherein R 1 and R 2 Independently, it is a straight-chain saturated alkyl group having 13 to 19 carbon atoms, and particularly, wherein R 1 and R 2 It is a straight-chain saturated C15 alkyl group.

5. The stereoisomeric phosphatidyl oligoglycerol according to any one of claims 1-4, having formula (III) (III)。 6. A method for preparing stereoisomeric phosphatidyl oligoglycerides according to any one of claims 1-5, characterized in that, The synthetic pathway includes a key intermediate of formula (IV) or formula (IV′). In the two solids mentioned 2′ 3′ has a predetermined configuration, and PG, PG′ and PG″ independently represent protecting groups.

7. The method for preparing stereoisomeric phosphatidyl oligoglycerides according to claim 6, characterized in that, The synthetic pathway includes a key intermediate of formula (IVa) or formula (IVa′). In three-dimensional 2′ is an R-configuration, and in the solid... 3′ is S-configuration for (IVa) and R-configuration for (IVa′), wherein PG, PG′ and PG″ independently represent protecting groups.

8. The method for preparing stereoisomeric phosphatidyl oligoglycerides according to claim 6 or 7, characterized in that, The intermediate of formula (IV) is prepared from glycidyl glycerol and 1,2-isopropylglycerol, particularly from (R)-(+)-glycidyl glycerol and (S)-(+)-1,2-isopropylglycerol (sn-1,2-IPG).

9. A method for preparing stereoisomeric phosphatidyl oligoglycerides according to any one of claims 6-8, the method comprising reacting a key intermediate of formula (IV) or (IVa) with a compound of formula (V). (V) Where R 1 and R 2 Each is independently a hydrocarbon group with 12-24 carbon atoms, R′ is a leaving group, and R″ is a hydrocarbon group with 1-12 carbon atoms.

10. A liposome comprising stereoisomeric phosphatidyl oligoglycerol according to any one of claims 1-5.

11. The liposomes according to claim 10, wherein the liposomes further comprise at least one phosphatidylcholine of formula (VII). (VII) Where R 3 and R 4 Each is an independent hydrocarbon group with 12 to 24 carbon atoms.

12. The liposome of claim 10 or 11, comprising: 20 mol% to 40 mol% of stereoisomeric phosphatidyl oligoglycerol according to any one of claims 1 to 5, especially stereoisomeric phosphatidyl oligoglycerol of formula (III); 40 mol% to 60 mol% of 1,2-dipalmitoylphosphatidylcholine; and 15 mol% to 25 mol% of 1,2-distearate phosphatidylcholine.

13. The liposomes according to any one of claims 10-12, further comprising an active agent, particularly doxorubicin.

14. The liposomes according to claims 10-13, used for the treatment of tumors.

Images