Stereoisomer phosphatidyl oligoglycerol

Abstract

The present invention relates to stereoisomer phosphatidyl oligoglycerols having a predetermined arrangement of at least three stereocenters, and in particular to a phosphatidyl oligoglycerol that is a single stereoisomer. Furthermore, the present invention relates to a stereospecific method for providing stereoisomer phosphatidyl oligoglycerols having a predetermined arrangement of at least three stereocenters, and in particular to a phosphatidyl oligoglycerol that is a single stereoisomer, as well as liposomes containing novel stereoisomer phosphatidyl oligoglycerols, and in particular to liposomes containing novel stereoisomer phosphatidyl oligoglycerols that are a single stereoisomer.

Description

TECHNICAL FIELD

[0001] The present invention relates to stereoisomeric phosphatidyl oligoglycerols having a predefined configuration at at least three stereogenic centers, and in particular to phosphatidyl oligoglycerols that are a single stereoisomer. Furthermore, the present invention relates to stereospecific methods for providing stereoisomeric phosphatidyl oligoglycerols having a predefined configuration at at least three stereogenic centers, and in particular for providing phosphatidyl oligoglycerols that are a single stereoisomer, as well as liposomes comprising the novel stereoisomeric phosphatidyl oligoglycerols, and in particular liposomes comprising the novel stereoisomeric phosphatidyl oligoglycerols that are a single stereoisomer.

BACKGROUND ART

[0002] 1,2-Dipalmitoyl-sn-glycero-3-phospho-oligoglycerol (DPPG x ), such as 1,2-diacyl-sn-glycero-3-phospho-oligoglycerol, is a phosphatidyl oligoglycerol (PG x ), which is a naturally occurring glycerophospholipid. Phosphatidyl oligoglycerols are composed of a diacylglycerol group, a phosphate group, and an oligoglycerol group comprising two or more glycerol moieties.

[0003] In particular, phosphatidyl oligoglycerols have been developed as a constituent component of liposomes for increasing the circulation half-life of liposomes (WO 97 / 30058 A1). The head group of naturally occurring phosphatidyl monoglycerols such as 1,2-dipalmitoyl-sn-glycero-3-phospho-glycerol (DPPG) was modified by an additional glycerol unit linked via an ether bond to increase the hydrophilicity of the head group. DPPG xDPPG can be used to replace PEGylated phospholipids in cholesterol-containing liposomes (WO 97 / 30058 A1) and cholesterol-free liposomes (Hossann et al. 2021), extending the cyclic half-life of these nanocarriers. x A key advantage of phosphatidyl oligoglycerols such as 1,2-diacyl-sn-glycero-3-phospho-diglycerol (DPPG2), 1,2-dipalmitoyl-sn-glycero-3-phospho-triglycerol (DPPG3), and 1,2-dipalmitoyl-sn-glycero-3-phospho-tetraglycerol (DPPG4) form lamellar structures instead of the micelle structures seen in PEGylated lipids (Lokerse et al. 2021). Therefore, DPPG2 can be incorporated into thermosensitive liposome (TSL) formulations up to 70 mol% (Lindner et al. 2004). The effects of DPPG2 and PEGylated lipids as components of liposomes on protein corona, interactions with blood cells, complement activation in human plasma / blood, and hypersensitivity reactions in rats have recently been investigated (Lokerse et al. 2021). In recent years, the use of PEGylated liposomes in human pharmaceuticals has been questioned for several reasons (Hoang et al. 2020), and therefore DPPG x Substitutes like these are attracting attention as novel excipients for liposomal drug delivery systems.

[0004] Phosphatidyl oligoglycerol, especially DPPG x(x≧2) can be produced by organic synthesis without the use of enzymes or polymerization reactions (Hossann et al. 2021), thereby preventing the generation of mixed oligoglycerols in lipid batches. The synthesis route to 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-oligoglycerol is described in WO 97 / 30058 A1. In this route, the stereocenter of the oligoglycerol unit could not be controlled.

[0005] WO 2014 / 202680 A1 provides a synthetic pathway that controls two stereocenters, producing 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1'-diglycerol. However, the third and further stereocenters in the oligoglycerol unit remain racemic due to the lack of stereocontrol in the synthesis of the oligoglycerol constituent blocks.

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

[0007] WO 2022 / 008471 A1 describes means for obtaining a thermosensitive drug delivery system based on DPPG2-TSL with appropriate pharmaceutical qualities. A challenge in the preclinical development of thermosensitive liposomal formulations for use in human patients is to achieve a stable and long-lasting formulation without compromising the instability required at temperatures >39°C for thermally induced local drug delivery to solid tumors in patients. The optimal formulation must withstand industrial-scale manufacturing processes and be long-lasting without altering quality-critical specifications that could affect patient therapeutic efficacy and safety.

[0008] WO 2023 / 021024 A1 describes an administration schedule for DPPG2-TSL-DOX in combination with local hyperthermia (HT) for human patients. SUMMARY OF THE INVENTION

[0009] The object of the present invention is to provide novel phosphatidyl oligoglycerols having a predetermined configuration of all stereocenters in the head group of oligoglycerol, and processes for producing such stereocontrolled forms of phosphatidyl oligoglycerols. Such stereoisomeric phosphatidyl oligoglycerols make it possible in particular to provide thermosensitive drug delivery systems that address the requirements and needs identified above.

[0010] The present invention relates to formula (I)

[0011] [Chemical Formula]

[0012] (wherein the three stereocenters *1, *2 and *3 have a predetermined configuration; and *p denotes a further stereocenter; and R 1 and R 2 each independently are a hydrocarbon group having 12 to 24 C 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 at each occurrence) stereoisomeric phosphatidyl oligoglycerols.

[0013] The inventors of the present application have succeeded in providing a stereospecific synthesis of phosphatidyl oligoglycerols that allows control of the three stereocenters *1, *2 and *3. In the case of diglycerols (n=0), such as diacylglycerophosphodiglycerols, in particular dipalmitoylglycerophosphodiglycerol, the compounds are single stereoisomers.

[0014] The phosphatidyl oligoglycerol of the present invention comprises an oligoglycerol group having at least two glycerol moieties. These glycerol moieties include stereocenters*2 and *3. The stereospecific method of the present invention provided herein enables the synthesis of a stereoisomer phosphatidyl oligoglycerol of formula (I) in which three stereocenters*1, *2 and *3 have a predetermined configuration. The predetermined configuration means that stereocenter*1 has either an (R) or (S) configuration, stereocenter*2 has either an (R) or (S) configuration, and stereocenter*3 has either an (R) or (S) configuration. Thus, the predetermined configuration means that each center is determined to be either pure (R) or pure (S). In particular, the predetermined configuration means that each stereocenter does not exist in a racemic configuration.

[0015] The phospholipid of the present invention may further contain glycerol and / or glycol groups bound thereto. At m=1, an additional glycerol moiety is present, and at m=0, an ethylene glycol moiety is included. *p indicates an additional stereocenter in the additional glycerol moiety. Preferably, each of the additional stereocenters *p has a predetermined configuration, meaning that each of the additional stereocenters *p has either an (R) or (S) configuration.

[0016] Therefore, the most preferred is phosphatidyl oligoglycerol, which is a single stereoisomer of formula (I). In such a most preferred phosphatidyl oligoglycerol, which is a single stereoisomer of formula (I), all stereocenters*1,*2,*3 and *p in each occurrence have a predetermined configuration, where stereocenter*1 has either the (R) or (S) configuration, stereocenter*2 has either the (R) or (S) configuration, stereocenter*3 has either the (R) or (S) configuration, and each stereocenter*p in each occurrence has either the (R) or (S) configuration.

[0017] According to the present invention, n is a number from 0 to 20. Preferably, n is a number from 0 to 10, and more preferably from 0 to 5. In particular, n is 0, 1 or 2, and m=1, in which case the stereoisomer phosphatidyl oligoglycerol of formula (I) is diglycerol, triglycerol or tetraglycerol, respectively. Even more preferably, n is 0 or 1, and most preferably, n is 0. The stereoisomer phosphatidyl oligoglycerol of formula (I) with n=0 is phosphatidyl diglycerol. The stereoisomer phosphatidyl oligoglycerol of formula (I) with n=1 and m=1 is phosphatidyl triglycerol.

[0018] In the stereoisomer phosphatidyl oligoglycerol of formula (I), residue R 1 and R 2 Each of these is a hydrocarbon group having 12 to 24 C atoms independently. Preferably R 1 and R 2 Each of these is a hydrocarbon group having 13 to 19 carbon atoms, particularly 14 to 18 carbon atoms. Preferably R 1 and R 2 Each of these is independently a linear or branched hydrocarbon group, preferably a linear hydrocarbon group. More preferably R 1 and R 2 Each of these is independently a linear or branched alkyl group, and is particularly a linear alkyl group. 1 and R 2 R may be a saturated, monounsaturated, or polyunsaturated alkyl group, preferably a saturated alkyl group. More preferably R 1 and R 2 Each of these is independently a linear saturated alkyl group. More preferably R 1 and R 2 Each of these is independently a linear saturated C12-C24 alkyl group, particularly a linear saturated C13-C19 alkyl group. Most preferably R 1 and R 2 It is a linear saturated C15 alkyl group.

[0019] According to the present invention, the three stereocenters*1,*2, and*3, and preferably stereocenter*p, also have a predetermined stereoconfiguration. The stereoconfiguration of each of the three stereocenters can be predetermined to either the S configuration or the R configuration. Most preferably, stereocenter*1 is in the R configuration, stereocenter*2 is in the S configuration, and stereocenter*3 is in the R configuration. The stereospecific method provided herein enables the formation of a compound having a desired predetermined configuration at all stereocenters. In the stereoselective as well as stereospecific method of the present invention, the product obtained starting from a single stereoisomer starting material is phosphatidyl oligoglycerol, which is a single stereoisomer of formula (I).

[0020] In a more general sense, the predetermined configuration means that, in the preparation, each stereocenter, for example, stereocenter*1, or stereocenter*2, or stereocenter*3, is present in a predetermined configuration, i.e., either the (R) configuration or the (S) configuration, at a rate of at least 95%, preferably at least 99%, and most preferably at least 99.9%.

[0021] The stereoisomer phosphatidyl oligoglycerol of formula (I) is shown as anion and can pair with any cation as a counterion. The preferred counterion is Na. + That is the case.

[0022] A desired arrangement can be predetermined for each of the three solid centers *1, *2, and *3, but the most preferred is given by equation (II)

[0023] [ka]

[0024] (In the formula, the center of solid *1 is in the R configuration, the center of solid *2 is in the S configuration, and the center of solid *3 is in the R configuration, and *p indicates a further center of solid; and R 1 and R 2(Each of these is a hydrocarbon group having 12 to 24 C atoms independently; and n is a number from 0 to 20, especially from 0 to 2.) It is a stereoisomerized phosphatidyl oligoglycerol having [specific properties].

[0025] The stereoisomer phosphatidyl oligoglycerol having formula (II) is also referred to herein as 1,2-diacyl-sn-glycero-3-phospho-sn-1'-glycero-sn-3”-glycerol.

[0026] The stereospecific method provided herein enables the formation of a 1,2-diacyl-sn-glycero-3-phospho-sn-1'-glycero-sn-3”-glycerol compound having a desired predetermined configuration at all stereocenters, where stereocenter*1 is in the R configuration, stereocenter*2 is in the S configuration, and stereocenter*3 is in the R configuration. The method of the present invention is not only stereoselective but also stereospecific, and the product obtained starting from a single stereoisomer starting material is phosphatidyl oligoglycerol, which is a single stereoisomer of formula (II).

[0027] More generally, in stereoisomer phosphatidyl oligoglycerol having formula (II), at least 95%, preferably at least 99%, and most preferably at least 99.9%, the stereocenter*1 is in the R configuration, the stereocenter*2 is in the S configuration, and the stereocenter*3 is in the R configuration.

[0028] The most preferred of the present invention is formula (III)

[0029] [ka]

[0030] It is phosphatidyl oligoglycerol, a single stereoisomer of phosphatidyl oligoglycerol. This compound is referred to herein as stereoisomer DPPG2 or DPPG2 ***It is also called 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1'-glycero-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. The counterion is Na. + If so, the IUPAC name is sodium (2R)-2,3-bis(palmitoyloxy)propyl(2S)-3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl phosphate or sodium (2R)-2,3-bis(hexadecanoyloxy)propyl(2S)-3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl phosphate.

[0031] Each of the stereoisomerized phosphatidyl oligoglycerols of the present invention, represented by formula (I), formula (II), or formula (III), exhibits remarkable advantages. In particular, the stereoisomerized phosphatidyl oligoglycerols of formulas (I), (II), and (III) show advantages compared to phosphatidyl-rac-oligoglycerol.

[0032] In particular, stereoisomerized phosphatidyl oligoglycerols and TSL formulations containing such stereoisomerized phosphatidyl oligoglycerols and cytotoxic agents exhibit differences in temperature-dependent drug release in vitro, and in vivo in terms of pharmacokinetics (PK), organ distribution, and therapeutic efficacy. Furthermore, they tended to exhibit slower metabolic rates in vitro.

[0033] Liposomes containing stereoisomerized phosphatidyl oligoglycerols exhibit altered plasma clearance in vivo. Furthermore, stereoisomerized phosphatidyl oligoglycerols show different biological interactions with the complement system and / or plasma proteins compared to racemic molecules. Additionally, liposomes containing stereoisomerized phosphatidyl oligoglycerols show different uptake in organs, particularly the spleen.

[0034] Furthermore, we found that the stereoisomerized phosphatidyl oligoglycerols of the present invention exhibit differences in liposome packing and liposome structure. In particular, liposomes containing the stereoisomerized phosphatidyl oligoglycerols of the present invention showed a steeper phase transition in the temperature-dependent release profile, which indicates the effect of the lipid excipient on packing.

[0035] Even more surprisingly, according to the present invention, drug-filled DPPG2***-TSL in which all glycerol moieties are stereospecific exhibits faster drug clearance from the bloodstream compared to drug-filled DPPG2-TSL in which all glycerol moieties are not stereospecific. This effect was particularly pronounced when doxorubicin was used as the drug. Furthermore, the use of DPPG2***-TSL-DOX liposomes increased drug accumulation in tumor tissue, particularly doxorubicin accumulation, but decreased it in the spleen.

[0036] We have found that the stereoisomerized phosphatidyl oligoglycerol of the present invention can increase the temperature-dependent drug release of thermosensitive liposomes. It has also been observed that liposomes containing the stereoisomerized phosphatidyl oligoglycerol of the present invention feature a narrower phase transition in the temperature-dependent release profile. Therefore, the stereoisomerized phosphatidyl oligoglycerol of the present invention can be used to increase the temperature-dependent drug release of thermosensitive liposomes and / or to narrow the phase transition in the temperature-dependent release profile.

[0037] In particular, the provision of stereoisomers of formula (I), and especially formulas (II) and (III), allows for their use in liposomal compositions for human application, even when considering the strict standards set by pharmaceutical authorities, by handling absolute stereochemistry. The chirality of lipid excipients plays an important role in stereoselective metabolism by enzymes (e.g., phospholipases) and prevents potential accumulation of lipid excipients in patients.

[0038] The present invention further includes stereoisomer phosphatidyl oligoglycerols described herein, particularly formula (I)

[0039] [ka]

[0040] (In the formula, the three stereocenters *1, *2 and *3 have a predetermined stereoconfiguration; and *p indicates a further stereocenter; and R 1 and R 2 (where each is a hydrocarbon group having 12 to 24 C atoms independently; 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 in each occurrence.) The present invention relates to a stereospecific method for preparing stereoisomer phosphatidyl oligoglycerols of formula (IV) or formula (IV'), wherein the synthetic pathway is of formula (IV) or formula (IV').

[0041] [ka]

[0042] (In the formula, the two stereocenters *2' and *3' have a predetermined configuration, and PG, PG', and PG'' are independent protecting groups.) The compound is characterized by containing a central intermediate. In particular, PG and PG'' are benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropan-2-yl (MOP), silyl, or paramethoxybenzyl (PMB), and preferably PG and PG'' are benzyl (Bn). In particular, PG' is CH2 (methylene), CHCH3 (ethylidene), CHPh (benzylidene), CHPhp-Ome (paramethoxybenzylidene), C(CH3)2 (isopropylidene), or SitBu2 (di-tert-butylsilylene), Si(iPr)2-O-Si(iPr)2 (tetraisopropyldisiloxanylidene), and preferably PG' is C(CH3)2.

[0043] According to the present invention, a synthetic pathway comprising a central intermediate of formula (IV) or (IV') having two stereocenters *2' and *3' in predetermined configurations enables the stereospecific preparation of phosphatidyl oligoglycerols that can confer predetermined configurations to three stereocenters *1, *2, and *3. The predetermined configurations specifically mean that stereocenter *1 has either an (R) or (S) configuration, stereocenter *2 has either an (R) or (S) configuration, and stereocenter *3 has either an (R) or (S) configuration.

[0044] The central intermediate of formula (IV) or (IV') can be prepared from glycidol and 1,2-isopropylidene-glycerol. The arrangement of the stereocenters *2' and *3' in the central intermediate of formula (IV) or (IV') is derived from the arrangement of the enantiomerally pure starting materials, glycidol and 1,2-isopropylidene-glycerol, used (see below).

[0045] To obtain stereoisomerized sphatidyl diglycerol, the central intermediate of formula (IV) or (IV') is reacted with the compound of formula (V) (particularly preferably compound 12a). To obtain higher oligoglycerols (particularly triglycerol or tetraglycerol), the intermediate of formula (IV) or (IV') is repeatedly reacted with glycidol to form a constituent block having an increased number of glycerol units. In particular, compound 10a is provided as a constituent block for preparing stereoisomerized phosphatidyl triglycerol, and constituent block 11a is provided for preparing stereoisomerized phosphatidyl tetraglycerol.

[0046] The synthesis overview is provided in the following scheme.

[0047] [ka]

[0048] [ka]

[0049] [ka]

[0050] [ka]

[0051] Formula (II)

[0052] [ka]

[0053] (In the formula, the solid center*1 is in the R configuration, the solid center*2 is in the S configuration, and the solid center*3 is in the R configuration.) To obtain the stereoisomer phosphatidyl oligoglycerol, the synthetic route in particular is based on formula (IVa) or (IVa').

[0054] [ka]

[0055] (In the formula, the stereocenter *2' is in the R configuration, and the stereocenter *3' is in the S configuration in (IVa) and the R configuration in (IVa'), and PG, PG', and PG'' are protecting groups independently.) It includes the central intermediate.

[0056] Preferably, the central intermediate of formula (IVa) or (IVa') is prepared from glycidol and 1,2-isopropylidene-glycerol, particularly from (R)-(+)-glycidol and (S)-(+)-1,2-isopropylidene-glycerol (sn-1,2-IPG), resulting in the central intermediate of formula (IVa) or (IVa'), respectively.

[0057] A more preferable option is the central intermediate 4.1a.

[0058] [ka]

[0059] Furthermore, the method of the present invention uses formula (IV) or (IV'), particularly formula (IVa) or (IVa'), as the central intermediate of formula (V).

[0060] [ka]

[0061] (In the formula, R 1 and R 2 (Each of these is a hydrocarbon group that independently has 12 to 24 carbon atoms.) This includes reacting with the compound R. 1 and R2 The above definition is preferred. R' represents a leaving group LG. In particular, R' is 2-cyanoethyl (CNE). R'' is a C1-C12 hydrocarbon group, and in particular R'' is methyl (Me), ethyl (Et), isopropyl (iPr), or benzyl (Bn), and preferably R'' is isopropyl.

[0062] Preferably, the compound of formula (V) is 1,2-diacyl-sn-glycerol and the following structure

[0063] [ka]

[0064] (In the formula, R' and R'' are defined independently as described above.) It is prepared from phosphorylation reagents. In particular, R' is 2-cyanoethyl (CNE). In particular, R'' is methyl (Me), ethyl (Et), isopropyl (iPr), and benzyl (Bn), and preferably R'' is isopropyl.

[0065] The most preferred is the central intermediate 12a

[0066] [ka]

[0067] This is the result of the reaction between 1,2-diacyl-sn-glycerol and 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite (PAMCl).

[0068] The present invention's method for preparing stereoisomerized phosphatidyl oligoglycerols comprises, in particular, at least one or more of the process steps outlined herein.

[0069] In particular, the present invention relates to formula (III)

[0070] [ka]

[0071] This invention relates to the synthesis of stereoisomerized DPPG2 having the following properties.

[0072] The central intermediate of formula (IVa) has two chiral centers. Both configurations are derived from the enantiomerically pure starting materials (R)-(+)-glycidol and (S)-(+)-1,2-isopropylidene-glycerol (sn-1,2-IPG) and are converted without isomerization to the final product 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1'-glycero-sn-3”-glycerol (also referred to herein as stereoisomerized DPPG2). The central intermediate of formula (IVa) has a (2R),(6S) configuration and is used for the stereospecific synthesis of stereoisomerized DPPG2. This single stereoisomer can be obtained in four steps by the synthesis shown below.

[0073] [ka]

[0074] PG, PG 1 And PG' are protecting groups independently. This pathway allows control of two stereocenters and generates the central intermediate of formula (IVa) as a single stereoisomer. (R)-(+)-glycidol and sn-1,2-IPG are used as commercially available starting materials. First, the OH group of (R)-(+)-glycidol is protected with, for example, 4-methoxybenzyl (PMB) chloride to form compound 1a. 1a reacts with sn-1,2-IPG under basic conditions to form compound 2a. Subsequently, the newly formed hydroxyl group is protected with, for example, benzyl ether to obtain compound 3a. Finally, the central intermediate of formula (IVa) is obtained by cleavage of the PMB ether using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0075] Furthermore, using phosphoramidite chemistry, DPPG2 is prepared as a single stereoisomer from the central intermediate of formula (IVa) and 1,2-dipalmitoyl-sn-glycerol (sn-1,2-DPG) in synthesis.

[0076] [ka]

[0077] Compound 13a can be produced in a three-step one-pot synthesis. Preferably, sn-1,2-DPG is first reacted with 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (PAMCl) to obtain compound 12a. The central intermediate of formula (IVa) is added to obtain a substituted phosphite triester, which is then oxidized, for example, with H2O2. The resulting fully protected DPPG2 derivative 13a is then treated to cleave the protecting group. This can be done according to known procedures. The phosphate protecting group can be cleaved with, for example, 1,8-diazabicyclo[5.4.0]undeca-7-ene (DBU) or TEA; the ketal group can be cleaved with acids, such as trifluoroacetic acid (TFA), oxalic acid, and formic acid; and the benzyl ether can be cleaved by catalytic hydrogenation using Pd. The deprotected product is purified by chromatography and crystallized from hot ethanol to obtain DPPG2(22a) as a single stereoisomer.

[0078] The stereoisomerized phosphatidyl oligoglycerols of the present invention are particularly suitable as components of liposomes. Accordingly, the present invention further relates to liposomes comprising at least one stereoisomerized phosphatidyl oligoglycerol as defined herein.

[0079] Liposomes are spherical vesicles formed by a membrane bilayer composed of lipid excipients, and are widely used and recognized as drug delivery nanocarriers. The stereoisomerized phosphatidyl oligoglycerol of the present invention forms a lamellar structure and is therefore particularly suitable as a component of liposomes.

[0080] The stereoisomerized phosphatidyl oligoglycerols of the present invention have been found to increase the circulating half-life of liposomes. Furthermore, the oligoglycerol moiety, which is a highly hydrated group, can alter both electrostatic and hydrophobic interactions with blood components when present on the liposome surface. As a result of protein binding to liposomes, a so-called protein corona is formed, and the composition of this protein corona can be controlled by the content and type of the stereoisomerized phosphatidyl oligoglycerols (e.g., stereoisomerized DPPG2) of the present invention contained in the liposomes. In addition, liposomes containing at least one stereoisomerized phosphatidyl oligoglycerol of the present invention exhibit a strong negative surface charge and confer high dispersion stability.

[0081] In particular, the stereoisomerized phosphatidyl oligoglycerol of the present invention can be incorporated into irritation-sensitive, preferably thermosensitive, liposomes. The use of stereoisomerized phosphatidyl oligoglycerol makes it possible to prepare thermosensitive liposomes with extended blood circulation time. These liposomes are stable in the bloodstream under physiological conditions (37-38°C) and release little to no pre-encapsulated active ingredients. The majority of the liposomes remain in blood circulation for 2 hours or longer, thus being utilized for thermally controlled release of the active ingredient. Due to the rapid release kinetics of these liposomes, which release the active ingredient by heat induction in seconds, preferably <30 seconds, more preferably <20 seconds, the pre-encapsulated active ingredient can be released immediately by targeted heating of the liposomes to a temperature of >39°C, preferably 40-42°C.

[0082] The liposomes of the present invention, particularly thermosensitive liposomes, preferably comprise (i) at least one stereoisomerized phosphatidyl oligoglycerol as described herein, and (ii) at least one additional neutral or zwitterionic phospholipid, preferably phosphatidylcholine. Preferably, the phosphatidylcholine has a main transition temperature of 0°C to 80°C, particularly 37°C to 45°C.

[0083] (i) thermosensitive liposomes containing at least one stereoisomerized phosphatidyl oligoglycerol and (ii) at least one phosphatidylcholine have a long half-life in serum. Furthermore, such nanocarrier systems, in particular the contents of liposomes, are rapidly released under the action of a stimulus, for example, when a certain temperature is exceeded, especially in less than 30 seconds.

[0084] The amount of stereoisomerized 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 up to 70 mol%, more preferably up to 50 mol%, and even more preferably up to 35 mol%, based on the lipid content of the liposome.

[0085] A further preferred component of the liposomes of the present invention, particularly thermosensitive liposomes, is at least one phosphatidylcholine. The at least one phosphatidylcholine is present in an amount preferably 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 preferably up to 99 mol%, more preferably up to 90 mol%, particularly preferably up to 80 mol%, and most preferably up to 75 mol%, based on the lipid content of the liposome.

[0086] Particularly preferred are liposomes containing 25-35 mol% stereoisomerized phosphatidyl oligoglycerol (especially stereoisomerized DPPG2) and 65-75 mol% phosphatidylcholine, based on the lipid content of the liposomes.

[0087] The liposomes of the present invention, particularly thermosensitive liposomes, preferably contain at least one of formula (VII)

[0088] [ka]

[0089] (In the formula, R 3 and R 4 (Each of these independently represents a hydrocarbon functional group having 12 to 24 carbon atoms.) It contains phosphatidylcholine.

[0090] R 3 and R 4 Preferably, each is independently saturated, monovalent, or polyunsaturated, preferably linear alkyl groups, particularly saturated linear alkyl groups. 3 and R 4 More preferably, each is independently a C13-C19, particularly C15-C17 hydrocarbon group. Preferably R 3 and R 4 Each of these is independently a linear saturated C13-C23, particularly C13-C21 alkyl group. Most preferably R 3 and R 4 These are independently linear saturated C15 or C17 alkyl groups.

[0091] The liposomes of the present invention, particularly the thermosensitive liposomes, preferably contain phosphatidylcholine of the natural structure of formula (VII).

[0092] Suitable phosphatidylcholines include, for example, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-lauroyl-sn-glycero-3-phosphocholine, 1-behenoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1-stearoyl-2-lauroyl-sn- Glycero-3-phosphocholine, 1,3-dimyristoyl-sn-glycero-2-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine n, 1,3-palmitoyl-sn-glycero-2-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-stearoyl-3-myristoyl-sn-glycero-2-phosphocholine, 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2- These are stearoyl-sn-glycero-3-phosphocholine, 1,3-distearoyl-sn-glycero-2-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diaraquinoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, and 1,2-dilignoceroyl-sn-glycero-3-phosphocholine. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) are particularly preferred.

[0093] The thermosensitive liposome contains at least one phosphatidylcholine having a main transition temperature particularly preferably in the range of 35°C to 43°C, and more preferably in the range of 39°C to 41°C. Such thermosensitive liposomes containing phosphatidylcholine have a release temperature that allows the liposome to be stable in normal circulation in healthy humans (37°C) and allows the release of contents at a temperature of 39°C to 43°C by the action of heat, particularly local heat. Thermosensitive liposomes having a release temperature of 40°C to 43°C are particularly preferred.

[0094] The thermosensitive liposomes of the present invention particularly preferably comprise at least one phosphatidylcholine selected from 1,3-dipalmitoyl-sn-glycero-2-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,3-distearoyl-sn-glycero-2-phosphocholine, and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0095] Generally, various further excipients can be included in liposome formulations, such as phosphatidylethanolamine (PE), phosphatidylglycerol, and phosphatidylserine (PS); as well as cholesterol (Chol), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1-acyl-sn-glycero-3-phosphocholine (lysoPC), hexadecyl-phosphocholine (HePC), or polyoxyethylene (20) stearyl ether (Brij78).

[0096] Liposomes may further contain cholesterol. However, the most preferred liposomes of the present invention do not contain cholesterol. Since cholesterol broadens the transition temperature from solid to liquid disordered phase, thus resulting in a wide thermal transition range, the liposomes of the present invention are preferably cholesterol-free. At high cholesterol concentrations, the phase transition temperature is suppressed, and non-thermosensitive liposomes are formed. In particular, the liposomes of the present invention, specifically the thermosensitive liposomes, contain cholesterol in amounts of <0.1 mol%, more preferably <0.01 mol%. Particularly preferably, the liposomes contain no cholesterol at all and are completely cholesterol-free.

[0097] In one embodiment, thermosensitive liposomes having a main phase transition temperature of approximately 40°C to 43°C are formed from a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, optionally 1,2-distearoyl-sn-glycero-phosphocholine, and stereoisomer DPPG2 and / or stereoisomer DPPG3. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine functions as the basic matrix for setting the phase transition temperature to approximately 42°C, 1,2-distearoyl-sn-glycero-3-phosphocholine slightly increases the phase transition temperature, and stereoisomer DPPG2 and / or stereoisomer DPPG3 functions to establish dispersion stability, serum stability, and stability in bloodstream, and to accelerate the release rate of the encapsulated compound.

[0098] In the present invention, particularly preferred is a stereoisomerized DPPG2 or stereoisomerized DPPG3 in a concentration of 20-40 mol%, preferably 25-35 mol%, based on the lipid content of the liposomes. The liposomes also contain a total of 65-75 mol% of phosphatidylcholine selected from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and / or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0099] The most preferred liposomes in this invention are those containing 25-35 mol% stereoisomerized DPPG2 and 65-75 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), based on the lipid content of the liposomes.

[0100] Furthermore, the most preferred liposomes in the present invention are those containing 20-40 mol%, preferably 25-35 mol%, of stereoisomerized DPPG2, 40-60 mol%, preferably 45-60 mol%, of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 15-25 mol%, of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), based on the lipid content of the liposomes.

[0101] The liposomes containing stereoisomerized phosphatidyl oligoglycerols of the present invention exhibit high stability in systemic circulation and extremely low nonspecific release of the active ingredient. As a result, these liposomes are well-suited for highly specific methods of local treatment of tumors, particularly soft tissue sarcomas and bladder cancer. After intravenous administration of the liposomes of the present invention, the release of the pre-encapsulated active ingredient at a desired site, such as soft tissue, can be induced by targeted heating of that site.

[0102] The active ingredient encapsulated in a thermosensitive liposome can be released by targeted heating of either the liposome or the target tissue, i.e., the body part where the tumor is located. This site can be heated using a wide range of methods. For example, in addition to simple flushing with warm water, heating can also be performed using electromagnetic waves, ultrasound, light, or laser technology.

[0103] Thermotherapy can be administered in the form of local, regional, or superficial thermotherapy, preferably regional deep thermotherapy. In particular, thermotherapy can be administered in the form of ultrasound-guided thermotherapy, microwave frequency-guided thermotherapy, or hyperthermic perfusion, where ultrasound or microwave frequency-guided thermotherapy may optionally be administered externally. Generally, the choice of thermotherapy depends on the type of tumor being treated and the specific condition of the patient being treated. The various options for administering thermotherapy are summarized by Paulides et al. (2020).

[0104] In thermotherapy, different types of energy can be used to apply heat, including light, laser light, microwaves, radio waves, and ultrasound.

[0105] Regional or locoregional hyperthermia can involve heating a moderate-sized body area, such as the chest or pelvis, including the cancerous area and surrounding healthy tissue. The rest of the body is kept as close to normal temperature as possible. Regional hyperthermia can be administered, for example, as regional deep hyperthermia or regional perfusion hyperthermia.

[0106] In preferred embodiments, the thermotherapy according to the present invention is administered in the form of regional deep or locoregional deep thermotherapy.

[0107] Preferably, the thermal therapy according to the present invention is performed in the form of electromagnetic wave-based thermotherapy techniques, such as radio wave or microwave frequency-induced thermotherapy, ultrasound-induced thermotherapy, or thermoperfusion therapy.

[0108] In the use of liposomes for targeted therapy of local tumors, the physical properties of the liposomes are extremely important. Thermosensitive liposomes filled with active ingredients should be stable at 36-37°C. For example, a local temperature increase to 42°C in the tumor area causes the active ingredient to be rapidly released, particularly in <60 seconds, preferably <20 seconds. The lipid components of the liposomes of this invention pass through a phase transition temperature in the region of about 40-45°C. Below 40°C, the phospholipids are arranged lamellae and in the solid gel phase; above that temperature, they are in the liquid disordered phase. In the transition region within a narrow temperature range (e.g., between 40 and 43°C), the active ingredient is spontaneously and rapidly released.

[0109] The main transition temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is approximately 41°C. In particular, by using the stereoisomer phosphatidyl oligoglycerol of the present invention in combination with 1,2-distearoyl-sn-glycero-3-phosphocholine, we were able to provide thermosensitive liposomes with an extended circulation time in the blood.

[0110] Thermosensitive liposomes are particularly suitable for local or topical therapies of cancer. The stereoisomerized phosphatidyl oligoglycerols provided herein enable clinical use in cancer therapy because the phospholipids are guaranteed to have a clearly defined structure with respect to their structure, fatty acid composition, and arrangement. In preferred embodiments, the cancer to be treated is selected from soft tissue sarcoma, 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), fibroplastic round cell sarcoma (DSRCT), desmoid, Kaposi's sarcoma, or osteosarcoma, preferably Ewing's sarcoma, osteosarcoma, or chondrosarcoma. In further embodiments, the cancer is a bladder tumor.

[0111] The liposomes of the present invention can be used to treat tumors, particularly solid and / or localized tumors. Superficial tumors and metastases, in particular, can be treated with the liposomes of the present invention because they can be heated in a simple manner. However, other tumors, such as tumors in hollow organs, can also be treated. In this case, the heating required to release the liposome contents can be achieved, for example, by flushing with warm water. The most desirable treatment is for soft tissue sarcoma (STS).

[0112] The liposomes of the present invention preferably further comprise an active ingredient, particularly a cell proliferation inhibitor. The most preferred liposomes of the present invention comprise doxorubicin (DOX), a cytotoxic anthracycline. Further suitable cytostatic anthracyclines currently used in cancer treatment include epirubicin, idarubicin, daunorubicin, and mitoxantrone. In further embodiments of the present invention, other cell proliferation inhibitors such as mitomycin C, gemcitabine, trabectedin, or platinum derivatives such as cisplatin, carboplatin, or oxaliplatin may be included in the liposomes.

[0113] In a further preferred embodiment, the thermosensitive liposomes of the present invention include anthracyclines such as doxorubicin, daunorubicin, idarubicin, epirubicin, aracrubicin, amrubicin, pirarubicin, barurubicin, and zolbicin; anthracendions such as mitoxantrone and picantrone; antitumor antibiotics such as mitomycin and bleomycin; vinca alkaloids such as vinblastine, vincristine, and vinorelbine; alkylating agents such as cyclophosphamide and mechloretamine hydrochloride; camptothecins such as topotecan, irinotecan (CPT-11), lulutotecan, 9-aminocamptothecin, 9-nitrocampotecin, and 10-hydroxycampotecin; 5-fluorouracil, gemcitabine (2,2'-difluoro-2-deoxycytidine, dFdC), floxlidine The active ingredient is selected from the group consisting of: purines and pyrimidine derivatives such as furin (FUDR), cytarabine (cytosine arabinoside), and 6-azauracil (6-AU); oxazaphosphorines such as cyclophosphamide, ifosfamide, and trophosfamide; taxanes such as paclitaxel and docetaxel; podophyllotoxin derivatives such as etopside and teniposide; platinum compounds such as cisplatin, carboplatin, oxaliplatin, and nedaplatin; methotrexate; tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib, adavocertib, and lapatinib; and cytarabines such as cytosine arabinoside.

[0114] By using the liposomes of the present invention, particularly thermosensitive liposomes, the active ingredient encapsulated within the liposome can be released by stimulation, especially local heating of the liposome. Therefore, liposomes, especially thermosensitive liposomes, are particularly suitable for local or topical therapy of tumors, especially soft tissue sarcomas. Since the release of the active ingredient encapsulated in the liposome can be induced in a targeted manner, the active ingredient, especially a cell proliferation inhibitor, can be released directly at the desired site. These findings can also be applied to the therapy of other solid and topical tumors.

[0115] The liposomes described herein are particularly suitable for the local release of active ingredients by thermotherapy. Liposomes that are thermosensitive and contain at least one phosphatidylcholine and at least one stereoisomerized phosphatidyl oligoglycerol having a phase transition temperature of about 40-43°C are preferred.

[0116] Therefore, the basic requirements for clinical application, particularly for local treatment of tumor diseases such as soft tissue sarcomas, are met. The effectiveness of these therapies has already been confirmed in animal studies.

[0117] Upon passing the phase transition temperature, the membrane changes its phase state, becoming permeable, and the liposome contents are released accordingly. This effect can be utilized in the treatment of soft tissue sarcoma according to the present invention. By raising the temperature within the tumor to the temperature necessary for the release of liposome contents, and releasing the liposome contents specifically and almost exclusively within the tumor, the active ingredient can be effectively used for tumor treatment.

[0118] Accordingly, the present invention further relates to thermosensitive liposomes as described herein, combined with thermotherapy and / or ultrasound. In this case, heating can be applied by a number of methods, such as simple flushing with warm water, heating by means of electromagnetic waves, ultrasound, light, or lasers. Preferred are ultrasound-induced thermotherapy, microwave frequency-induced thermotherapy, or thermoperfusion therapy.

[0119] The liposomes of the present invention, particularly thermosensitive liposomes (including liposomes containing the active ingredient), can be prepared by a wide range of different techniques, such as lipid membrane hydration, ethanol injection, or extrusion. The liposomes of the present invention are preferably prepared by ethanol injection. Furthermore, preferred buffer exchange is performed by tangential flow filtration (TFF).

[0120] Accordingly, the present invention also includes liposomes disclosed herein, particularly thermosensitive liposomes, for use in the treatment of tumors, especially soft tissue sarcomas.

[0121] The cancer is preferably a soft tissue sarcoma (STS). STS is a group of cancers that occur in tissues that connect, support, and surround other body structures, such as muscles, fat, blood vessels, nerves, tendons, and the lining of joints. Preferably, STS may 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; fibroplastic round cell sarcoma (DSRCT), desmoid, Kaposi's sarcoma, or osteosarcoma, especially Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

[0122] The present invention further comprises liposomes, particularly thermosensitive liposomes, for use in 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, desmoid tumors, glioblastoma, and other tumor diseases, preferably having localized extent.

[0123] Such liposomes preferably contain an active ingredient suitable for treating the tumor, and the active ingredient is subsequently released in response to stimulation within or near the tumor.

[0124] References Brooks WH, Guida WC, Daniel KG. The significance of chirality in drug design and development. Curr Top Med Chem 2011;11:760-70. DOI: 10.2174 / 156802611795165098

[0125] Hoang Thi TT, Pilkington EH, Nguyen DH, Lee JS, Park KD, Truong NP. The Importance of Poly(ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation. Polymers 2020;12:298. DOI: 10.3390 / polym12020298

[0126] Hossann M, Wang T, Wiggenhorn M, Schmidt R, Zengerle A, Winter G, et al. Size of thermosensitive liposomes influences content release. J Control Release 2010;147:436-43. DOI: 10.1016 / j.jconrel.2010.08.013

[0127] Hossann M, Hirschberger J, Schmidt R, Baumgartner C, Zimmermann K, Baer S, et al. A heat‐activated drug delivery platform based on phosphatidyl‐(oligo)‐glycerol nanocarrier for effective cancer treatment. Adv NanoBiomed Res 2021;1:2000089. DOI: 10.1002 / anbr.202000089

[0128] Kneidl B, Peller M, Winter G, Lindner LH, Hossann M. Thermosensitive liposomal drug delivery systems: state of the art review. Int J Nanomedicine 2014;9:4387-98. DOI: 10.2147 / IJN.S49297

[0129] Lehnert R, Eibl H, Mueller K. FT-IR and NMR studies on the conformational and structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyloligoglycerols. J Phys Chem 2003;107:75-85. DOI: 10.1021 / jp026509t

[0130] Limmer S, Hahn J, Schmidt R, Wachholz K, Zengerle A, Lechner K, et al. Gemcitabine treatment of rat soft tissue sarcoma with phosphatidyldiglycerol-based thermosensitive liposomes. Pharm Res 2014;31:2276-86. DOI: 10.1007 / s11095-014-1322-6

[0131] Lindner LH, Eichhorn ME, Eibl H, Teichert N, Schmitt-Sody M, Issels RD, et al. Novel temperature-sensitive liposomes with prolonged circulation time. Clin Cancer Res 2004;10:2168-78. DOI: 10.1158 / 1078-0432.CCR-03-0035

[0132] Lokerse WM, Lazarian A, Kleinhempel A, Petrini M, Schwarz P, Hossann M, et al. Mechanistic investigation of thermosensitive liposome immunogenicity and understanding the drivers for circulation half-life: A polyethylene glycol versus 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol study. J Control Release 2021;333:1-15. DOI: 10.1016 / j.jconrel.2021.03.014

[0133] Nutt DJ, Feetam CL. What one hand giveth the other taketh away: some unpredicted effects of enantiomers in psychopharmacology. J Psychopharmacology 2010;24:1137-41. DOI: 10.1177 / 0269881110374782

[0134] Paulides MM, Dobsicek Trefna H, Curto S, Rodrigues DB. Recent technological advancements in radiofrequency- and microwave-mediated hyperthermia for enhancing drug delivery. Adv Drug Deliv Rev. 2020;163-164:3-18. DOI: 10.1016 / j.addr.2020.03.004

[0135] Schagon O. Liposomen als potentielle Arzneistofftraeger: Variation der biopharmazeutischen Eigenschaften durch 1,2-Dipalmitoyl-sn-glycero-oligo-glycerine. Shaker Verlag, Aachen; 1997. ISBN: 978-3-8265-2479-0

[0136] van Valenberg FJP, Brummelhuis ISG, Lindner LH, Kuhnle F, Wedmann B, Schweizer P, et al. DPPG2-based thermosensitive liposomes with encapsulated doxorubicin combined with hyperthermia lead to higher doxorubicin concentrations in the bladder compared to conventional application in pigs: a rationale for the treatment of muscle-invasive bladder cancer. Int J Nanomedicine 2021;16:75-88. DOI: 10.2147 / IJN.S280034.

[0137] Willerding L, Limmer S, Hossann M, Zengerle A, Wachholz K, Hagen TL ten, et al. Method of hyperthermia and tumor size influence effectiveness of doxorubicin release from thermosensitive liposomes in experimental tumors. J Control Release 2016;222:47-55. DOI: 10.1016 / j.jconrel.2015.12.004

[0138] The present invention will be further illustrated with reference to the enclosed drawings and embodiments. [Brief explanation of the drawing]

[0139] [Figure 1] Figure 1: Evaluation of the optical purity of the central intermediate of formula (IVa) by chiral HPLC. Figure 1A shows the complete racemic intermediate, Figure 1B shows a mixture of epimers 2(S),6(R,S) (98.9%), Figure 1C shows the desired stereoisomer 2(S),6(R) (99.5%), and Figure 1D shows the stereoisomer 2(R),6(S) (100.0%). [Figure 2] Figure 2: Cleavage of DPPG2 by phospholipase PLA2. Figure 2A shows cleavage of glycerophospholipids, and Figure 2B shows formation of lysoglycerophospholipids. Values ​​with units (%) are normalized to the glycerophospholipid content (%m / m) at time t=0 min (without phospholipase). This accounts for differences in purity and other substances such as residual solvent or residual substances. [Figure 3] Figure 3: Comparison of biophysical properties of different DPPG2-TSL-DOX batches. Batches were prepared by one of the following methods: 1) ethanol injection and use of DPPG2 (Formula III), 2) ethanol injection and use of DPPG2 (WO97 / 30058), 3) lipid membrane hydration at 100 nm extrusion using DPPG2 (WO97 / 30058), and 4) lipid membrane hydration at 200 nm extrusion using DPPG2 (WO97 / 30058). [Figure 4]Figure 4: DPPG2-TSL-DOX in a rat sarcoma model. (A) PK profiles of DOX and (B) DPPG2 after IV bolus injection of different DOX formulations (2 mg / kg DOX, applied as a fixed volume of 1 mL). Application of 60 minutes of normothermic (NT) treatment with a water bath set at 37°C (blue area). N=6 for each formulation. (C) PK profile of DOX after application of DPPG2-TSL-DOX, prepared using DPPG2 of formula III by the preparation method for ethanol injection, as an IV bolus injection of 2 mg / kg DOX. Rats were treated with a water bath at either 37°C (without HT) or 41.5°C (with HT) for 60 minutes in one hind limb. N=6 for each formulation. (D) DOX accumulation in tumors and (E) organs 60 minutes after IV bolus injection of different DOX formulations (2 mg / kg DOX, applied as a fixed volume of 1 mL). All animals received ramp HT treatment for one tumor, while the tumor on the opposite side (the other hind limb) was not treated. N=6 for each formulation. Student's t-test for statistical analysis (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). (F) Therapeutic effects of different DOX formulations (2 mg / kg DOX) combined with 60-minute ramp HT (41°C) on tumors in a BN175 rat sarcoma model. Kaplan-Meier plot (survival, tumor volume increased 5-fold). NT: Animals did not receive HT treatment. N=6 for each formulation. [Figure 5] Figure 5: Effect of DPPG2 structure on in vitro release of carboxyfluorescein (CF) from TSL in DPPC / DSPC / DPPG2 50:20:30 (mol / mol) concentrations in physiological saline at 37°C and 42°C within 1 hour. [Examples]

[0140] Examples 1.1 Prior art 1.1.1 Synthesis of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-diglycerol (Comparative Example) The synthesis route for the production of DPPG2 is described in patent application WO 97 / 30058 A1, and is phosphatidyl oligoglycerol (PG). x It was described as part of the synthesis of glycerophospholipids of the molecular class. DPPG2 was synthesized in a multi-step organic synthesis in which the stereocenter of the glycerol unit could not be controlled.

[0141] 1.1.2 Synthesis of 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1'-diglycerol (Comparative Example) WO 2014 / 202680 A1 describes a synthetic pathway enabling the synthesis of 1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1'-glycero-3'-rac-glycerol. The objective was to obtain DPPG2 with an sn-1' configuration, similar to naturally occurring PG glycerophospholipids. This configuration is related to phospholipase activity and prevents potential lipid accumulation after patient administration. The synthetic pathway can only control two stereocenters within the molecule.

[0142] [ka]

[0143] Comparison of the synthetic routes for DPPG2 disclosed in (A) WO9730058 (1,2-dipalmitoyl-sn-glycero-3-phospho-rac-diglycerol) and (B) WO 2014 / 202680 A1 (1,2-dipalmitoyl-sn-glycero-3-phospho-sn-1'-diglycerol). In both routes, it is not possible to control all three stereocenters due to the epoxide ring cleavage step.

[0144] 1.2 Synthesis of (2R)-2,3-bis(palmitoyloxy)propyl(2S)3-{[(2R)-2,3-dihydroxypropyl]oxy}-2-hydroxypropyl) phosphate 1.2.1 Preparation of a Preferred Example of the Central Intermediate of Equation 4.1a Intermediate 4.1a (a preferred version of central intermediate (IVa)) has two chiral centers in a (2R),(6S) configuration. The configurations of both chiral centers are derived from the enantiomerically pure starting materials (R)-(+)-glycidol and (S)-(+)-1,2-isopropylidene-glycerol (sn-1,2-IPG), which are converted to the final product DPPG2 without isomerization. The single stereoisomer 4.1a is obtained in four steps by the following synthesis:

[0145] [ka]

[0146] Other possible stereoisomers can be obtained from the following combinations of enantiomerically pure starting materials: 4.1b: (2S), (6R) configuration: (S)-(-)-glycidol and (R)-(-)-1,2-isopropylidene-glycerol (sn-2,3-IPG) 4.1c:(2R),(6R) configuration:(R)-(+)-glycidol and (R)-(-)-1,2-isopropylidene-glycerol(sn-2,3-IPG) 4.1d: (2S), (6S) configuration: (S)-(-)-glycidol and (S)-(+)-1,2-isopropylidene-glycerol (sn-1,2-IPG)

[0147] Step 1: 3.8 kg of (R)-(+)-glycidol is dissolved in 22.8 L of NMP, then 8.02 kg of PMBCl is added, followed by 2.26 kg of NaH at 0-5°C. After 4 hours at RT, the reaction mixture is poured into ice water and extracted with siRNA. The organic phase is washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The resulting residue is purified by silica gel chromatography (n-heptane / ethyl acetate) to obtain 8.0 kg of 1.1a as a colorless oil. This is then subjected to HPLC, TLC, and 1 The characteristics are evaluated by 1H-NMR.

[0148] 1.1a1 1H NMR spectrum (400 MHz, CDCl3): δ ppm 7.29 (d, J = 10.8 Hz, 2H), 6.89 (d, J = 10.8 Hz, 2H), 4.53 (q, J= 11.6 Hz, 2H), 3.79 (s, 3H), 3.74 (dd, J = 3.2, 11.6 Hz, 1H), 3.41 (dd, J = 6.0, 11.6 Hz, 1H), 3.17 (m, 1H), 2.79 (dd, J = 4.0, 9.2 Hz, 1H), 2.61 (dd, J = 2.4, 4.8 Hz, 1H).

[0149] Steps 2+3: 8.0 kg of 1.1a was dissolved in 64 L of DMF, then 8.16 kg of sn-1,2-IPG was added, followed by the addition of 6.94 kg of tBuOK at 0-5°C. The resulting reaction mixture was stirred at RT for 12 hours to obtain 2.1a. Characterization was performed by TLC and HPLC. Subsequently, without workup or further purification, an additional 6.91 kg of tBuOK and 12.6 kg of benzyl bromide were added at 0-5°C, and the resulting reaction mixture was stirred at RT for 4 hours. The reaction mixture was then poured into ice water and extracted with dimethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. After purification by silica gel chromatography (n-heptane / ethyl acetate), 7.0 kg of pure compound 3.1a was obtained as a yellow oil. 3.1a was characterized by TLC, HPLC, and 1 The characteristics are evaluated by 1H-NMR.

[0150] 3.1a 1 1H NMR spectrum (400 MHz, CDCl3): δ ppm 7.39 - 7.26 (m, 8H), 6.92 (d, J= 8.4 Hz, 2H), 4.71 (s, 2H), 4.50 (s, 2H), 4.27 (d, J = 6.0 Hz, 1H), 4.10 - 4.02 (m, 1H), 3.85 (s, 3H), 3.83 - 3.47 (m, 9H), 1.44 (s, 3H), 1.39 (s, 3H).

[0151] Step 4: Dissolve 6.9 kg of 3.1a in 41.8 L of DCM / H2O 10:1 (vol / vol) and cool the mixture to 0°C. Add 4.89 kg of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at RT for 4 hours. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 solution and water. Dry the organic phase over Na2SO4 and remove the solvent under reduced pressure. After workup, purify crude 4.1a by silica gel chromatography (n-heptane / ethyl acetate) and purify 2.6 kg of pure 4.1a obtained as brown oil, and perform HPLC, TLC and SFC. 1 The properties are characterized by 1H NMR.

[0152] 4.1a 1 1H NMR (400MHz, CD3OD) δ ppm 7.41 - 7.23 (m, 5H), 4.67 (s, 2H), 4.29 - 4.19 (m, 1H), 4.03 (dd, J = 6.4, 8.4 Hz, 1H), 3.76 - 3.69 (m, 1H), 3.68 - 3.56 (m, 5H), 3.55 - 3.46 (m, 2H), 1.37 (s, 3H), 1.32 (s, 3H).

[0153] 1.2.2 Evaluation of the optical purity of the central intermediate The optical purity of the central intermediate (particularly 4.1a) of formula (IVa) was confirmed by chiral HPLC (hexane / isopropanol containing isopropylamine) using a Chiralpak IA-3 column (3 μm, 0.46 cm ID × 25 cm length) from Daicel Chiral Technologies. Samples of all possible stereoisomers and mixtures thereof were prepared to correctly assign the corresponding peaks (see Figure 1).

[0154] 1.2.3 Preparation of DPPG2 from the central intermediate of formula (IVa) and 1,2-dipalmitoyl-sn-glycerol (sn-1,2-DPG)

[0155] [ka]

[0156] This synthesis allows control over all three stereocenters of DPPG2. The starting materials for introducing the stereocenters are 1,2-dipalmitoyl-sn-glycerol (sn-1,2-DPG) and 4.1a. The three stereocenters of DPPG2 are denoted by (S) and (R) to indicate absolute stereochemistry.

[0157] Step 5 : To a suspension of 500 g of sn-1,2-DPG in 1 L of MeTHF and 1.25 L of DCM, 230 mL of DIPEA is added, and the mixture is then cooled to -15°C. 206 mL of 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (PAMCl) is dissolved in DCM, and the solution is cooled to -20°C before being added to the reaction mixture. The resulting mixture is stirred at -15°C for 1 hour, followed by stirring at 0°C for 1.5 hours and then at 20°C for 1 hour. 114 g of 4,5-dicyanoimidazole (DCI) and 274 g of 4.1a are then dissolved in 1.5 L of MeTHF, and the solution is slowly added to the reaction mixture at 20°C. After stirring for 1 hour, an additional 52 g of DCI is added as a solution dissolved in 500 ml of DCM, and the entire mixture is then stirred overnight at 20°C. Next, 91 ml of 35% w / w aqueous H2O2 solution is added over 1 hour at 0°C, and then the mixture is stirred for another 1 hour at 20°C. The reaction mixture is diluted with 5% by weight aqueous Na2S2O5 solution and extracted with MeTHF. The organic phase is dried over Na2SO4 and the solvent is removed under reduced pressure. The crude intermediate 13.1a is purified by flash silica chromatography (DCM / acetone) to obtain 664 g of 13.1a as a white glassy solid.

[0158] 13.1a 1 1H NMR spectrum (400 MHz, CDCl3): δ ppm 7.91 - 7.94 (m,7 H), 7.66 - 7.74 (m, 7 H), 5.31 - 5.40 (m, 15 H), 5.19 - 5.28 (m, 1 H), 2.23 - 2.38 (m, 4 H).

[0159] Step 6: To a solution of 664 g of 13.1a dissolved in MeTHF, 165 g of DBU was added, and the reaction mixture was stirred in RT for 2 hours. Then 72 g of Pd / C was added, and the resulting reaction suspension was stirred under an H2 atmosphere (1.2 bar) for 1 hour. The reaction mixture was filtered through Celite, washed with 1 M aqueous citric acid solution, and the organic phase was separated. The organic phase was washed with 5 wt% aqueous NaHCO3 solution, dried over Na2SO4, and concentrated under reduced pressure. 725 g of crude 16.1a was obtained.

[0160] 16.1a 1 1H NMR spectrum (400 MHz, CDCl3): δ ppm 7.83 - 8.01 (m, 6 H), 7.63 - 7.79 (m, 6 H), 5.28 - 5.38 (m, 12 H), 5.16 - 5.26 (m, 1 H), 2.19 - 2.33 (m, 4 H).

[0161] Step 7 : 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 distilled twice with 2-propanol, then dissolved in MeTHF, and the pH was adjusted to 7 with an 8 wt% NaHCO3 solution. The organic phase was separated, dried over Na2SO4, and concentrated under reduced pressure to obtain 840 g of crude 19.1a.

[0162] 19.1a 1 1H NMR spectrum (400 MHz, CDCl3) δ ppm 7.87 - 7.98 (m, 5 H), 7.65 - 7.76 (m, 5 H), 5.28 - 5.40 (m, 11 H), 5.16 - 5.27 (m, 1 H), 4.51 - 4.69 (m, 2 H), 2.14 - 2.32 (m, 4 H), 0.81 - 0.96 (m, 7 H).

[0163] Step 8: 615 g of 19.1a is dissolved in 2.8 L of MeTHF, then washed with 3.1 L of 8 wt% NaHSO4 solution, and subsequently the pH is adjusted to 4 with 60 g of NaOAc in 3.1 L of water. The organic phase is separated and concentrated to 9.3 L. A suspension of 20% Pd(OH)2 (50% wet, 31.7 g) on ​​charcoal in 1 L of MeOH is added, and the resulting suspension is stirred under an H2 atmosphere (3.5 bar) for 18 hours. The pH is adjusted to 7 by adding 35 g of NaOAc, then the mixture is filtered through Decalite and concentrated under reduced pressure. The concentrate is diluted with DCM, filtered again through Decalite, then the organic phase is separated and concentrated under reduced pressure. The crude concentrate containing 8 is purified by flash silica chromatography (DCM / MeOH), Pd scavenging, and subsequent crystallization from ethanol to obtain 322 g of DPPG2 as a white powder.

[0164] DPPG2 1 1H NMR spectrum (400 MHz, CDCl3): δ ppm 7.88 - 7.98 (m, 5 H), 7.66 - 7.74 (m, 5 H), 5.34 (s, 10 H), 5.19 - 5.28 (m, 1 H), 2.21 - 2.37 (m, 4 H).

[0165] IPC is performed by HPLC / CAD measurement at each synthesis step. Chromatographic purification of intermediate 13.1a is performed as needed to control and adjust the quality of this intermediate.

[0166] 1.3 Synthesis of (R)-2,3-bis(palmitoyloxy)propyl((S)-3-((R)-3-((R)-2,3-dihydroxypropoxy)-2-hydroxypropoxy)-2-hydroxypropyl) phosphate Intermediate 10.1a has three chiral centers in the (2R), (6S), and (10S) configurations. All three chiral center configurations originate from the enantiomerically pure starting materials (S)-(+)-1,2-isopropylidene-glycerol (sn-1,2-IPG), (S)-(-)-glycidol, and (R)-(+)-glycidol, and are converted to the final product DPPG3 without isomerization. The (10S) configuration is determined by the use of sn-1,2-IPG. The use of the enantiomer (R)-(-)-1,2-o-isopropylidene glycol leads to (10R). A single stereoisomer is obtained in 7 steps in the following synthesis:

[0167] [ka]

[0168] Step 1: (S)-(-)-glycidol is dissolved in NMP, then PMBCl is added, followed by the addition of NaH at 0-5°C. The resulting reaction mixture is stirred at RT until the reaction is complete. The mixture is then poured into ice water and extracted with ethyl acetate. The organic phase is washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The isolated residue is purified by silica gel chromatography (n-heptane / ethyl acetate) to obtain 1.1b. Using the same procedure, obtain 1.1a from (R)-(+)-glycidol.

[0169] Steps 2+3: 1.1b is dissolved in DMF, then sn-1,2-IPG is added, followed by tBuOK at 0-5°C. The resulting reaction mixture is stirred at RT until the reaction is complete to obtain 2.1b. Then, without workup or further purification, additional tBuOK and benzyl bromide are added at 0-5°C, and the resulting reaction mixture is stirred at RT until the reaction is complete. The reaction mixture is then poured into ice water and extracted with ethyl acetate. The organic phase is washed with brine, dried over Na2SO4, and concentrated under reduced pressure. After purification by silica gel chromatography (n-heptane / ethyl acetate), pure compound 3.1b is obtained.

[0170] Step 4: Dissolve 3.1b in DCM / H2O 10:1 (vol / vol) and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at RT until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 solution and water. Dry the organic phase over Na2SO4 and remove the solvent under reduced pressure. Purify crude 4.1b by silica gel chromatography (n-heptane / ethyl acetate).

[0171] Steps 5+6: 1.1a is dissolved in DMF, then 4.1b is added, followed by the addition of tBuOK at 0-5°C. The resulting reaction mixture is stirred at RT until the reaction is complete to obtain 8.1a. Then, without workup or further purification, additional tBuOK and benzyl bromide are added at 0-5°C, and the resulting reaction mixture is stirred at RT until the reaction is complete. The reaction mixture is then poured into ice water and extracted with ethyl acetate. The organic phase is washed with brine, dried over Na2SO4, and concentrated under reduced pressure. After purification by silica gel chromatography (n-heptane / ethyl acetate), pure compound 9.1a is obtained.

[0172] Step 7: Dissolve 9.1a in DCM / H2O 10:1 (vol / vol) and cool the mixture to 0°C. Add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the mixture at RT until the reaction is complete. Filter the reaction mixture and wash the filtrate with aqueous NaHCO3 solution and water. Dry the organic phase over Na2SO4 and remove the solvent under reduced pressure. Purify crude 10.1a by silica gel chromatography (n-heptane / ethyl acetate).

[0173] Step 8 : DIPEA is added to a suspension of sn-1,2-DPG in MeTHF and DCM, and the mixture is then cooled to -15°C. 2-Cyanoethyl-N,N-diisopropyl chlorophosphoramidite (PAMCl) is dissolved in DCM, the solution is cooled to -20°C, and then added to the reaction mixture. The resulting mixture is stirred at -15°C for 1 hour, followed by stirring at 0°C for 1.5 hours and then at 20°C for 1 hour. 4,5-Dicyanoimidazole (DCI) and 10.1a are then dissolved in MeTHF, and the solution is slowly added to the reaction mixture at 20°C. If necessary, after stirring for 1 hour, an additional amount of DCI is added as a DCM solution, and the entire mixture is then stirred overnight at 20°C. A 35% w / w aqueous H2O2 solution is then added over 1 hour at 0°C, followed by stirring at 20°C for another 1 hour. The reaction mixture is diluted with a 5 wt% aqueous Na2S2O5 solution and extracted with MeTHF. The organic phase is dried with Na2SO4, and the solvent is removed under reduced pressure. Crude intermediate 14.1a is purified by flash silica chromatography.

[0174] Step 9: To a solution of 14.1a dissolved in MeTHF, DBU is added, and the reaction mixture is stirred in RT for 2 hours. Then Pd / C is added, and the resulting reaction suspension is stirred under an H2 atmosphere (1.2 bar) for 1 hour. The reaction mixture is filtered through Celite, washed with 1 M aqueous citric acid solution, and the organic phase is separated. The organic phase is washed with 5 wt% aqueous NaHCO3 solution, dried over Na2SO4, and concentrated under reduced pressure to obtain 17.1a.

[0175] Step 10 : Crude 17.1a is suspended in 2-propanol, then TFA is added, and the resulting reaction mixture is stirred at 45°C for 1 hour. The TFA is removed under reduced pressure, the crude product is distilled twice with 2-propanol, then dissolved in MeTHF, and the pH is adjusted to 7 with an 8 wt% NaHCO3 solution. The organic phase is separated, dried over Na2SO4, and concentrated under reduced pressure to obtain crude 20.1a.

[0176] Step 11: Dissolve 20.1a in MeTHF, then wash with 8 wt% NaHSO4 solution, and subsequently adjust the pH to 4 with NaOAc in water. Separate and concentrate the organic phase. Add a suspension of 20% Pd(OH)2 (50% wet) on charcoal in MeOH, and stir the resulting suspension under an H2 atmosphere (3.5 bar) until the reaction is complete. Adjust the pH to 7 with the addition of NaOAc, then filter the mixture through Decalite and concentrate under reduced pressure. Dilute the concentrate with DCM, filter again through Decalite, then separate the organic phase and concentrate under reduced pressure. Purify crude DPPG3 by flash silica chromatography.

[0177] 1.4 Stereospecific cleavage of DPPG2 by phospholipase A2 Phospholipases are enzymes that selectively cleave bonds in glycerophospholipids based on their stereochemistry. They are involved in the in vivo metabolism of glycerophospholipids. Phospholipase A2 hydrolyzes 1,2-acyl-sn-glycero-3-phospholipids into their 1-acyl-sn-glycero-3-phospholipids and corresponding fatty acids. For the experiment, a commercially available enzyme (extracted from pig pancreas) was used. Lipid samples (2 μmol) were dissolved in 1.5 wt% deoxycholate and then incubated with 0.5 U of enzyme in Tris buffer (pH 8.5) with added CaCl2 at 37°C in a thermoshaker. Samples were taken at different time points, and cleavage was analyzed by HPLC. A Waters XBridge Phenyl column (3.5 μm, 2.1 × 150 mm) was used, and analytes were detected by a charged aerosol detector (CAD). Analytes were separated using gradient elution with 100 mM NH4Ac (pH 6.0) and MeOH in water, respectively, as eluents A and B. The column oven was set to 35°C.

[0178] Three batches of DPPG2 with different stereocenter configurations in the diglycerol head group were tested, and DPPC was used as the control glycerophospholipid (Figure 2). DPPC was significantly slower cleaved than DPPG2. The DPPG2 batches tended to cleave slowly, depending on the stereocenter configuration in the diglycerol head group. After 10 minutes, 14.8%, 20.4%, and 24.3% of WO 9730058, WO 2014 / 202680 A1, and DPPG2 according to Formula III, respectively, were still detectable in the samples.

[0179] 1.5 Manufacturing of DPPG2-TSL-DOX Thermosensitive liposomes (TSLs) and their compositions, also known as thermosensitive (TSL) formulations, can be prepared by different preparation methods (e.g., lipid membrane hydration and extrusion, ethanol injection, or other methods). The active ingredients are filled by either passive or active processes. Active remote filling can be achieved, for example, by using an ammonium gradient, an acid gradient, or an EDTA salt gradient. A method for preparing TSLs containing encapsulated DOX (DPPG2-TSL-DOX) is described in WO 2022 / 008471 A1.

[0180] 1.5.1 Lipid membrane hydration method using DPPG2 according to WO 97 / 30058 DPPG2 according to WO 97 / 30058 was used for batch preparation. Fifteen batches were prepared using a 100 nm filter film during extrusion. Twenty-five batches were prepared using a 200 nm filter film during extrusion. These batches were used for comparison purposes (Hossann et al. 2021).

[0181] All solutions were prepared with deionized and purified water from ultrapure water systems (Milli Q Advantage, Millipore) and filtered through a 0.2 μm filter before subsequent use. Using a round-bottom flask, phospholipids in the desired molar ratio (DPPC / DSPC / DPPG 250:20:30) were dissolved in chloroform / methanol 9:1 (vol / vol). The solvent was evaporated under vacuum in a rotary vaporizer until a thin, uniform lipid film was formed. To remove any residual organic solvent, the lipid film was dried at 10 mbar / 70°C for at least 1 hour. The film was hydrated with 300 mM citric acid (pH 4) under shaking at 60°C for 30 minutes. The resulting lipid concentration was 50 mM. (LIPEX Thermobarrel Extruder) TMMonolayer vesicles were obtained by extruding 10 times through two polycarbonate filters of the desired pore size (e.g., 100 nm, 200 nm) at a maximum N2 pressure of 20 bar at 60°C using Northern Lipids Inc. (Burnaby, BC, Canada). The dispersion was then cooled to 2–8°C and the buffer was replaced with phosphate-buffered saline (PBS) buffer (pH 7.4) by applying a PD10 column (GE Healthcare). The desired active DOX packing conditions were obtained by mixing the liposome dispersion with PBS (pH 7.4) and stock DOX solution (5.7 mg / ml DOX HCl). The dispersion was heated in an Eppendorf Thermomixer comfort with a 50 ml thermoblock under shaking (e.g., 37°C, up to 60 minutes). Traces of unsealed DOX were removed by centrifugation at 75,000 × g (Beckman Coulter Avanti J-26 XP with JA-25.50 rotor). The supernatant was discarded, and the pellet was carefully resuspended in storage buffer (e.g., 10% (w / v) trehalose, 10.5 mM Na / K phosphate (pH 7.4)) if storage at -20°C was assumed. If the batch was to be used immediately, the buffer was changed with PBS (pH 7.4).

[0182] 1.5.2 Ethanol injection method using stereoisomerized DPPG2 The stereoisomer DPPG2 of formula III was used to prepare eight independent batches, as follows.

[0183] DPPG2-TSL-DOX was prepared and characterized as described in WO 2022 / 008471 A1. All application solutions were prepared with deionized and purified water obtained from an ultrapure water system and filtered through a 0.2 μm filter before subsequent use. The pH of 300 mM ammonium phosphate buffer and physiological PBS buffer was adjusted to 7.4, respectively, with phosphate as needed. A phospholipid excipient in a molar ratio of DPPC / DSPC / stereoisomerized DPPG250:20:30 was dissolved in ethanol at 60°C. The ethanol-lipid solution and ammonium phosphate buffer were pumped independently through two heating coils (60°C) and the flows were combined at 60°C using a T-fitting. The 60°C thermal dispersion was then pumped into a 60°C thermal extrusion chamber equipped with multiple extrusion membranes of appropriate pore sizes to generate liposomes with vesicle sizes in the range of 100–150 nm. After extrusion, the flow was passed through a 5°C cooling coil and pumped into an ice-cooled recovery container. A gradient for active DOX loading was created by exchanging the extraliposomal buffer to PBS (pH 7.4) using tangential flow filtration (TFF). The desired DOX:lipid molar ratio of 0.08 was obtained by mixing the liposome dispersion with PBS buffer (pH 7.4) and aqueous DOX stock solution (5.7 mg / ml DOX HCl). The dispersion was heated at 37°C for 30 minutes with stirring in a round-bottom flask equipped with a heating mantle. After cooling the dispersion to 2-8°C, any traces of unfilled DOX were removed, and the extraliposomal buffer was replaced with a storage buffer by TFF. Finally, the dispersion was subjected to sterile filtration (0.2 μm).

[0184] 1.5.3 Ethanol injection method using DPPG2 according to WO 97 / 30058 Liposome batches were prepared as controls by using DPPC, DSPC, and DPPG2 (according to WO 97 / 30058) in a molar ratio of 50:20:30. These liposomes were packed with DOX using 250 mM ammonium sulfate (pH 5.4) (4 batches) or 300 mM ammonium phosphate (pH 7.4) (4 batches) as the packing buffer, as described in Example 1.5.2. The batches were immediately characterized and no buffer change to storage buffer was performed as they were not intended for storage at -20°C.

[0185] 1.6 In vitro characterization of TSL formulations The TSL formulations prepared in Examples 1.5.1, 1.5.2, and 1.5.3 were characterized as follows: Hydrodynamic diameter (z-mean), size intensity distribution plot, and zeta potential were measured 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 with a fluorescence detector or charged aerosol 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). Furthermore, pH, osmotic molar concentration, intracellular toxins, and sterility were measured according to the corresponding European Pharmacopoeia (EP) methods.

[0186] Each batch was compared to assess the effects of stereochemistry and / or the manufacturing method on their respective biophysical properties. The results are shown in Figure 3.

[0187] In batches prepared by ethanol injection (DPPG2 of formula III), ethanol injection (DPPG2 according to WO 97 / 30058), lipid membrane hydration (100 nm extrusion), and lipid membrane hydration (200 nm extrusion), the vesicle sizes (expressed as z-mean) 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. In batches prepared by ethanol injection (DPPG2 of Formula III), lipid membrane hydration (100 nm extrusion), and lipid membrane 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.

[0188] Overall, the ethanol injection preparation method produced TSL formulations with smaller variability (lower standard deviation) in vesicle size, size distribution, and zeta potential compared to the lipid membrane hydration preparation method. This demonstrates the superior robustness and controllability of the ethanol injection preparation method.

[0189] All prepared batches showed temperature-dependent DOX release profiles for TSL formulations (Figure 3). While there were no significant differences in TDR among batches prepared by the lipid membrane hydration method, vesicle sizes (expressed as zeta-mean) differed. Therefore, the DOX release kinetics of DPPG2-based TSL were not affected by vesicle size in the 117 nm to 161 nm range (investigated temperature range: 37 to 41 °C) (Hossann et al. 2010). When comparing batches from different preparation methods, it is important to note that the excipients used for DOX loading do not affect TDR for data interpretation (WO 2022 / 008471 A1).

[0190] In batches prepared by ethanol injection (DPPG2 of formula III), ethanol injection (DPPG2 by WO 97 / 30058), lipid membrane hydration (100 nm extrusion), and lipid membrane hydration (200 nm extrusion), the in vitro DOX release at 37°C for 5 minutes in FCS was 6.8±3.6%, 1.1±1.2%, 3.5±3.5%, and 6.5±6.2%, respectively. There were minor differences between batches in stability at body temperature, but all showed acceptable low DOX release <10%. The in vitro DOX release at 40°C for 5 minutes in FCS was 75.4±9.9%, 65.7±8.5%, 46.7±26.2%, and 43.6±23.3%, respectively. All batches showed comparable thermally induced DOX emissions at temperatures >40°C, with over 75% emissions during a 5-minute incubation (data not shown).

[0191] Surprisingly, batches prepared by ethanol injection using DPPG2 of formula III showed higher in vitro DOX release compared to batches prepared by ethanol injection using DPPG2 according to WO 97 / 30058, at 37°C, 38°C, 39°C, and 40°C for 5 minutes in FCS. This indicates that DOX crosses the membrane bilayer faster in batches prepared with DPPG2 of formula III than in batches prepared with DPPG2 according to WO 97 / 30058. This is caused by more uniform packing of lipid excipients into the bilayer, resulting in TSLs being released into their T m When heated to higher temperatures, a phase transition from a narrower solid gel to a liquid, disordered phase may occur. Faster drug release under hyperthermia is a desirable effect for the in vivo application of such TSLs because the encapsulated drug becomes more bioavailable faster within the heated area of ​​the body, potentially leading to a greater accumulation of DOX in tumor tissue.

[0192] 1.7 Protein Coronavirus in DPPG2-TSL Preparations The molar content of DPPG2 affects the protein corona composition of DPPC / DSPC / DPPG280-x / 20 / x (mol / mol) (x=5, 10, 20, 30) (Lokerse et al. 2021). This example investigates the potential stereoselective effects on protein binding to DPPG2-based TSL.

[0193] Preparation and characterization of liposome batches: Batches of DPPC / DSPC / DPPG2 50:20:30 (mol / mol) TSLs were prepared using either DPPG2 (according to WO 97 / 30058), DPPG2 (according to WO 2014 / 202680), or DPPG2 (of Formula III), respectively, and filled with either physiological saline or 100 mM carboxyfluorescein (CF) (pH 7.2). Furthermore, non-thermal liposomes (NTSLs) mimicking the lipid composition of the approved liposome formulation Caelyx (HSPC / cholesterol / DSPE-PEG2000 55:40:5 (mol / mol)) were prepared as control formulations without DPPG2 and filled with physiological saline. The batches were prepared by lipid membrane 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 shown as average values ​​from measurements of two batches prepared independently for each formulation. Measurements were performed before protein corona formation. Vesicle size (z-mean) and PDI were similar across all formulations and are shown in Table 1.

[0194] DPPG2-based TSLs exhibited a negative zeta potential due to anionic DPPG2, while the control formulation showed a neutral zeta potential. CF-filled TSL batches showed a typical encapsulation efficiency of approximately 5–6.5% for passive filling methods.

[0195] Measurements in physiological saline show the effect of the DPPG2 material used on heat-induced CF release (Figure 5). In vitro CF release within 1 hour at 42°C was higher with TSL using DPPG2 (Formula III) compared to TSL using either DPPG2 (according to WO 97 / 30058) or DPPG2 (according to WO 2014 / 202680). This result is consistent with the in vitro DOX release results described in Example 1.6 and indicates that this effect is independent of the TSL preparation method and the presence of serum components, respectively.

[0196] Protein corona formation and characterization: Human plasma, collected from healthy human donors using dextrose citrate phosphate, was from Biowest SAS (0.2 μm filtration), aliquot, and stored at -80°C. Before use, thawed plasma was centrifuged (2 min / 1000 × g) to remove any coagulations that could block the size exclusion column (SEC). A TSL batch (25 mM total lipid content) was mixed with human plasma in a 1:9 ratio. The mixture was incubated in a thermoshaker (0 rpm) at 37°C for 30 minutes and then packed into CL-4B SECs preconditioned with saline. The fraction containing TSL was transferred to a 1000 kDa Float-A-Lyzer dialysis chamber and dialyzed overnight in physiological saline at 4°C. Subsequently, to wash the pellet with physiological saline, the dialyzed TSL samples were centrifuged and concentrated at 4°C using a 10 kDa Amicon centrifuge filter in one centrifugation step (4500 × g for 1 hour) and a 1000 kDa VivaSpin centrifuge concentrator in four centrifugation steps (8600 × g for 1 × 1 hour, and 8600 × g for 3 × 30 minutes). The final TSL pellet was resuspended in 100 μl of physiological saline, and the total protein (BCA protein assay) and lipid content were quantified.

[0197] Liquid chromatography-mass spectrometry (LC-MS) analysis: For protein digestion, 3 μg of each sample was used, and the fractions were injected into LC-MS / MS. For absolute quantification, six heavy peptides of the Hi3 E. coli standard (Waters) were added to the samples. The samples were processed with a Preomics iST kit to obtain peptides from the protein corona. The peptides were measured by LC-MS / MS in DDA mode. The raw data were searched for human proteins (total human proteome) and E. coli using MaxQuant.

[0198] The isolated samples had protein concentrations of approximately 250 μg / ml and 400 μg / ml in DPPG2-TSL packed with saline or CF, 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. Table 1 shows the top 10 most abundant proteins for each formulation. DPPG2-based TSLs primarily bind to different proteins from the apolipoprotein class. Apolipoproteins CI, C-II, and AI were the major components of their protein corona, regardless of the DPPG2 used. Interestingly, the molar ratio of apolipoprotein CI to apolipoprotein C-II depended on the structure of the DPPG2 used. These ratios were 2.5, 1.8, and 3.0 for DPPG2-based TSLs containing DPPG2 (according to WO 97 / 30058), DPPG2 (according to WO 2014 / 202680), or DPPG2 (of formula III), respectively. Furthermore, DPPG2-TSL containing DPPG2 (of formula III) showed little binding to proteins from the keratin class, while DPPG2-TSL containing DPPG2 (according to WO 97 / 30058) and DPPG2 (according to WO 2014 / 202680) showed binding to different types of keratin proteins. These data suggest that the stereochemistry of DPPG2 influences the affinity of protein binding. In contrast to DPPG2-TSL, the most abundant proteins in the control formulation belonged to the immunoglobulin class.

[0199] [Table 1]

[0200] JPEG2026534184000025.jpg209133

[0201] JPEG2026534184000026.jpg74132

[0202] 1.8 In vivo testing of DPPG2-TSL-DOX in a rat model DPPG2-TSL-DOX batches prepared by either ethanol injection or lipid membrane hydration (comparative example) were investigated in a rat BN175 sarcoma model. The former batch contained DPPG2 of formula III, while the latter batch contained DPPG2 according to WO 97 / 30059. The batches were characterized as described in Example 1.6. DPPG2-TSL-DOX (DPPG2 of formula III) prepared by ethanol injection showed a vesicle size (z-mean) of 118 nm, a PDI of 0.08, a zeta potential of -28.3 mV, and a DOX:lipid ratio of 0.06. DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) prepared by the lipid membrane hydration method exhibited a vesicle size (z-mean) of 178 nm, a PDI of 0.13, a zeta potential of -29.4 mV, and a DOX:lipid ratio of 0.10.

[0203] In the PK study, 18 healthy male Brown Norway rats (approximately 300g) under anesthesia received either non-liposomal DOX (n=6), DPPG2-TSL-DOX (DPPG2 of formula III) (n=6), or DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) (n=6) as an iv bolus injection at a dose of 2 mg / kg of DOX. Animal body temperature was controlled using a rectal temperature probe. During anesthesia, animal body temperature was maintained in a water bath (37°C). Blood samples were collected at predetermined time points. DOX and DPPG2 content were measured in plasma by HPLC and LC / MS, respectively, using fluorescence detectors.

[0204] The DOX plasma clearances of both DPPG2-TSL-DOX batches were similar (Figure A), showing the expected long-circulating properties. The plasma half-life of DOX (t 1 / 2,α ) was 141±5 minutes and 180±14 minutes for DPPG2-TSL-DOX (DPPG2 of Formula III) and DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059), respectively. The percentage of the initial dose in plasma at 240 minutes (%ID) was also similar, being 6.3% and 17.2% respectively, which indicates that the clearance of DOX was equivalent for both formulations. t 1 / 2,α The slight difference in can be explained by the known effect of vesicle size on the circulation half-life of drugs encapsulated in DPPG2-TSL (Limmer et al. 2014). In contrast, non-liposomal DOX was cleared within minutes, and DOX plasma levels were undetectable at time points >15 minutes.

[0205] The difference in the DOX:lipid molar ratio in both DPPG2-TSL-DOX formulations resulted in a difference in the maximum observed concentration (C max ) for DPPG2 (Figure B). The dose of DPPG2 was higher for DPPG2-TSL-DOX (DPPG2 of Formula III). This is because the DOX:lipid ratio in this formulation was smaller, and all rats were administered the same amount of DOX (2 mg / kg). Both liposomal formulations showed similar DPPG2 clearance with equivalent plasma concentrations after 240 minutes.

[0206] High blood concentrations of DPPG2 (nanocarrier) and DOX (drug) in the first hour after intravenous (i.v.) administration are an important prerequisite for TSL formulations (Kneidl et al. 2014). This allows a large portion of the administered TSL to be activated by local or regional HT in the tumor, subsequently generating high local DOX levels. Only DOX released in the tumor exerts a therapeutic effect (see below).

[0207] In an independent series of PK experiments, 12 anesthetized healthy male Brown Norway rats (approximately 300 g) received DPPG2-TSL-DOX (DPPG2 of Formula III) as an i.v. bolus injection at a DOX dosage of 2 mg / kg. 6 rats each received local HT treatment (41.5°C, water bath) on one hind limb, while the other 6 rats received local NT treatment (37°C, water bath) on one hind limb. The body temperature of these animals was controlled with a rectal temperature probe. Blood samples were collected at defined time points. DOX content was measured in plasma by HPLC equipped with a fluorescence detector. A 1-hour local HT treatment was shown to be sufficient to almost completely deplete circulating DPPG2-TSL-DOX via heat-induced DOX release. The PK parameters decreased significantly, indicating remarkable heat-induced DOX release from nanocarriers in the heated (41.5°C) hind limb of rats. For example, the t of rats that received NT and HT 1 / 2,α decreased from 141±5 minutes to 29±3 minutes, respectively. The area under the concentration curve (AUC) for the first 60 minutes decreased from 1410 to 412 μg*h / ml, respectively. Induced DOX release in heated body regions has been previously demonstrated for DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) (Hossann et al. 2021).

[0208] Next, we investigated the accumulation of DOX in tumor tissue (Figure D) and selected organs (Figure E). Eighteen anesthetized male brown Norwegian rats (approximately 300 g) with tumors received either non-liposomal DOX (n=6), DPPG2-TSL-DOX (DPPG2 of formula III) (n=6), or DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) (n=6) as an iv bolus injection at a dose of 2 mg / kg of DOX. All animals each had two BN175 tumors (soft tissue sarcomas) subcutaneously transplanted into each hind limb. One tumor was treated with local HT therapy for 60 minutes with a light source at 41.5°C, while the second tumor remained untreated (room temperature, NT). A light source was used instead of a water bath to apply HT therapy. This is because it allows for more intensive (localized) heating of the tumor tissue (Willerding et al. 2016). The animals were sacrificed after 60 minutes, the organs were perfused, and the tumors and organs were collected. DOX content was measured in plasma by HPLC with a fluorescence detector.

[0209] Tumor DOX concentrations were strongly dependent on the application conditions (HT vs. NT) and formulation (non-liposomal vs. TSL). HT application increased DOX tumor accumulation by 1.6-fold, 16.2-fold, and 12.7-fold (compared to untreated tumors) 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), respectively. The enrichment factors were comparable to published data (Willerding et al. 2016, Hossann et al. 2021). Heat-treated tumor DOX concentrations were 3.1±0.5 ng / mg, 32.3±23.0 ng / ml, and 41.7±19.0 ng / ml, respectively. The DOX concentrations in untreated tumors were 1.9±0.3 ng / mg, 2.0±0.3 ng / ml, and 3.3±0.3 ng / ml, respectively. Interestingly, the DOX content in both tumors of rats treated with DPPG2-TSL-DOX (DPPG2 of formula III) was higher than that in the corresponding tumors of rats treated with DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059). This is consistent with in vitro DOX release data (Example 1.6) indicating that DOX becomes more rapidly bioavailable with DPPG2-TSL-DOX (DPPG2 of formula III).

[0210] The organ distribution of DOX was similar for both DPPG2-TSL-DOX formulations, except in the spleen. The DOX content in the spleen was significantly lower for DPPG2-TSL-DOX (DPPG2 of formula III) at 13.7±1.0 ng / mg compared to 20.6±2.4 ng / mg for DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059). It has been previously shown that the preparation method does not affect organ distribution (including spleen content) (WO 2022 / 008471 A1), suggesting that the use of DPPG2 of formula III may reduce the rate of liposome uptake into the spleen.

[0211] The literature shows examples where slight changes in molecular structure can produce unexpectedly strong effects in vitro or in vivo. (DPPC / DPPG) x In liposomes with a cholesterol ratio of 40:10:50 (mol / mol), substitution of DPPG2 with DPPG4 increased uptake into the rat spleen (Schagon 1997).

[0212] As a result of the application of non-liposomal DOX, significantly higher DOX accumulation was observed in the lungs and heart compared to both DPPG2-TSL-DOX formulations (Figure 4E). This may reduce the risk of DOX-related cardiotoxicity after the application of DPPG2-TSL-DOX.

[0213] Finally, the therapeutic effects of both DPPG2-TSL-DOX formulations were investigated in vivo and compared with clinically relevant selected DOX formulations (e.g., non-liposomal DOX, Caelyx). Thirty-six anesthetized male brown Norwegian rats with tumors were treated with HT-enhanced DPPG2-TSL-DOX (DPPG2 of formula III) (n=6), HT-enhanced DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059) (n=6), HT-enhanced non-liposomal DOX (n=6), HT-enhanced Caelyx (n=6), HT-enhanced saline (n=6), and HT-less DPPG2-TSL-DOX (DPPG2 of formula III) (n=6), respectively. All animals had one BN175 tumor (soft tissue sarcoma) sc-grafted in one hind limb. Local HT treatment was performed with a light source. The target temperature was 41°C. The animals were preheated at this temperature for 30 minutes, and the temperature was maintained at 41°C for 60 minutes after IV bolus injection. Animals treated with DOX received 2 mg / kg of DOX as a single IV bolus injection after a single tumor diameter >5 mm. After completion of treatment, the animals were returned to cages and tumor growth was monitored every two days until endpoints were reached (e.g., tumor diameter >3 cm, ulceration, bleeding, skin toxicity).

[0214] Figure 4F shows the Kaplan-Meier plots (survival, 5-fold increase in tumor volume) for all experimental groups. DPPG2-TSL-DOX (DPPG2 of formula III) combined with HT achieved the longest survival, followed by DPPG2-TSL-DOX (DPPG2 by WO 97 / 30059) combined with HT treatment. This may be due to faster DOX release at 40°C and reduced splenic uptake compared to DPPG2-TSL-DOX (DPPG2 by WO 97 / 30059). In the absence of HT, the therapeutic effect of DPPG2-TSL-DOX (DPPG2 of formula III) was significantly reduced, comparable to 0.9% saline, Caelyx, and non-liposomal DOX, respectively. Only rats treated with the DPPG2-TSL-DOX formulation in combination with HT showed a transient reduction in tumor size after treatment. All other rats showed a steady increase in tumor volume after treatment. These results indicate that high local DOX concentrations in tumor tissue are necessary for effective tumor treatment.

[0215] In conclusion, DPPG2-TSL-DOX (DPPG2 of formula III) showed slightly faster DOX clearance from the bloodstream compared to DPPG2-TSL-DOX (DPPG2 according to WO 97 / 30059). Surprisingly, DOX accumulation in tumor tissue increased, and DOX content in the spleen decreased, demonstrating the superior therapeutic effect of this formulation.

[0216] This application also discloses the following items: Item 1. Formula (I)

[0217] [ka]

[0218] (In the formula, the three stereocenters *1, *2 and *3 have a predetermined stereoconfiguration; and *p indicates a further stereocenter; and R 1 and R 2Each is a hydrocarbon group having 12 to 24 C atoms independently; n is a number from 0 to 20; and (m is either 0 or 1 independently in each occurrence.) Stereoisomer phosphatidyl oligoglycerol.

[0219] Item 2. A stereoisomer phosphatidyl oligoglycerol according to Item 1, having formula (I) where n is a number from 0 to 10, preferably a number from 0 to 5, more preferably n is 0, 1 or 2, and even more preferably n is 0 or 1.

[0220] Item 3. A stereoisomer phosphatidyl oligoglycerol according to either item 1 or 2, having formula (I) where n is 0.

[0221] Item 4. A stereoisomer phosphatidyl oligoglycerol described in any one of items 1-3 of formula (I), where m is 1 in each occurrence.

[0222] Item 5.R 1 and R 2 However, each of the stereoisomer phosphatidyl oligoglycerols described in any one of items 1 to 4 has a hydrocarbon group of formula (I) in which each independently has 13 to 19 carbon atoms, particularly 14 to 18 carbon atoms.

[0223] Item 6.R 1 and R 2 However, each is independently a linear or branched hydrocarbon group, preferably a linear hydrocarbon group, and / or R 1 and R 2 However, each of the stereoisomer phosphatidyl oligoglycerols described in any one of items 1 to 5 has a linear or branched alkyl group, particularly a linear alkyl group, represented by formula (I).

[0224] Item 7.R 1 and R 2is a saturated or monounsaturated or polyunsaturated alkyl group, preferably a saturated alkyl group, the stereoisomeric phosphatidyl oligoglycerol according to any one of items 1 to 6 having formula (I).

[0225] Item 8. R 1 and R 2 are each independently a linear saturated alkyl group; preferably R 1 and R 2 are each independently a linear saturated C12 to C24 alkyl group; more preferably R 1 and R 2 are a linear saturated C13 to C19 alkyl group; and most preferably R 1 and R 2 is a linear saturated C15 alkyl group, the stereoisomeric phosphatidyl oligoglycerol according to any one of items 1 to 7 having formula (I).

[0226] Item 9. The stereoisomeric phosphatidyl oligoglycerol according to any one of items 1 to 8 having formula (I), wherein stereocenter *1 is in the R configuration, stereocenter *2 is in the S configuration, and stereocenter *3 is in the R configuration.

[0227] Item 10. The stereoisomeric phosphatidyl oligoglycerol according to any one of items 1 to 9 having formula (I), wherein the stereoisomeric phosphatidyl oligoglycerol is a single stereoisomer.

[0228] Item 11. The stereoisomeric phosphatidyl oligoglycerol according to any one of items 1 to 10 having formula (I), wherein stereocenter *p has a predetermined configuration.

[0229] Item 12. Formula (II)

[0230] [Chemical Formula]

[0231] (In the formula, the solid center *1 is in the R configuration, the solid center *2 is in the S configuration, and the solid center *3 is in the R configuration, and *p indicates a further solid center.) A stereoisomer phosphatidyl oligoglycerol described in any one of items 1 to 11.

[0232] Item 13. Stereoisomer phosphatidyl oligoglycerols described in any one of items 1-12, in particular, where n=0 or n=0.

[0233] Item 14.R 1 and R 2 However, it is a linear saturated alkyl group having 13 to 19 C atoms independently, and especially R 1 and R 2 However, a stereoisomer phosphatidyl oligoglycerol as described in any one of items 1 to 13, which is a linear saturated C15 alkyl group.

[0234] Item 15. Formula (III)

[0235] [ka]

[0236] A stereoisomer phosphatidyl oligoglycerol described in any one of items 1 to 14.

[0237] Item 16. Formula (III')

[0238] [ka]

[0239] (In the formula, the center of the solid *p has a predetermined configuration.) A stereoisomer phosphatidyl oligoglycerol described in any one of items 1 to 14.

[0240] Item 17. The synthesis pathway is equation (IV) or equation (IV')

[0241] [ka]

[0242] (In the formula, the two stereocenters *2' and *3' have a predetermined arrangement, and PG, PG', and PG'' independently represent protecting groups.) A method for preparing a stereoisomerized phosphatidyl oligoglycerol according to any one of items 1 to 16, characterized by containing a central intermediate.

[0243] Item 18. PG is independently benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropan-2-yl (MOP), silyl, or paramethoxybenzyl (PMB) in each occurrence, preferably PG is benzyl (Bn); PG is independently benzyl (Bn), tetrahydropyranyl (THP), ethoxyethyl (EE), 2-methoxypropan-2-yl (MOP), silyl, or paramethoxybenzyl (PMB) in each occurrence, preferably PG is A method for preparing a stereoisomerized phosphatidyl oligoglycerol according to item 17, wherein the PG' is benzyl (Bn); and PG' is CH2 (methylene), CHCH3 (ethylidene), CHPh (benzylidene), CHPhp-Ome (paramethoxybenzylidene), C(CH3)2 (isopropylidene), or SitBu2 (di-tert-butylsilylene), Si(iPr)2-O-Si(iPr)2 (tetraisopropyldisiloxanylidene), and preferably PG' is C(CH3)2.

[0244] Item 19. The synthesis pathway is equation (IVa) or equation (IVa')

[0245] [ka]

[0246] (In the formula, the stereocenter *2' is in the R configuration, and the stereocenter *3' is in the S configuration in (IVa) and the R configuration in (IVa'), and PG, PG', and PG'' independently represent protecting groups.) A method for preparing stereoisomerized phosphatidyl oligoglycerol according to item 17 or 18, characterized by comprising a central intermediate.

[0247] Item 20. A method for preparing a stereoisomer phosphatidyl oligoglycerol according to any one of items 17 to 19, characterized in that the central intermediate of formula (IV) is prepared from glycidol and 1,2-isopropylidene glycol, particularly from (R)-(+)-glycidol and (S)-(+)-1,2-isopropylidene glycol (sn-1,2-IPG).

[0248] Item 21. The central intermediate of formula (IV) or formula (IVa) is formula (V)

[0249] [ka]

[0250] (In the formula, R 1 and R 2 (Each of these is a hydrocarbon group having 12 to 24 carbon atoms, R' is a leaving group, and R'' is a hydrocarbon group having 1 to 12 carbon atoms.) A method for preparing a stereoisomer phosphatidyl oligoglycerol as described in any one of items 17 to 20, comprising reacting it with a compound.

[0251] Item 22. A method for preparing a stereoisomer phosphatidyl oligoglycerol according to any one of items 17 to 21, wherein R' is 2-cyanoethyl (CNE) and R'' is a C1-C12 hydrocarbon group, particularly R'' being methyl (Me), ethyl (Et), isopropyl (iPr) or benzyl (Bn), preferably R'' being isopropyl.

[0252] Item 23. A liposome comprising the stereoisomeric phosphatidyl oligoglycerol according to any one of Items 1 to 16.

[0253] Item 24. The liposome comprising the stereoisomeric phosphatidyl oligoglycerol according to Item 23, wherein the stereoisomeric phosphatidyl oligoglycerol is a single stereoisomer of formula (I).

[0254] Item 25. The stereoisomeric phosphatidyl oligoglycerol according to Item 23 or 24, which is a single stereoisomer of formula (III)

[0255]

Chemical Formula

[0256] , the liposome according to Item 23 or 24.

[0257] Item 26. The liposome according to any one of Items 23 to 25, further comprising at least one phosphatidylcholine of formula (VII)

[0258]

Chemical Formula

[0259] (wherein R 3 and R 4 are each independently a hydrocarbon group having 12 to 24 C atoms) .

[0260] Item 27. Based on the lipid content of the liposome, 20 to 40 mol% of the stereoisomeric phosphatidyl oligoglycerol according to any one of claims 1 to 16, particularly the phosphatidyl oligoglycerol of formula (III) which is a single stereoisomer; 40 to 60 mol% of 1,2-dipalmitoylphosphatidylcholine; and 15 to 25 mol% of 1,2-distearoylphosphatidylcholine A liposome described in any one of items 23-26, including the following:

[0261] Item 28. Based on the lipid content of liposomes 25-35 mol% of stereoisomer DPPG2 having formula (III); and 65-75 mol% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) A liposome described in any one of items 23-26, including the following:

[0262] Item 29. A thermosensitive liposome as described in any one of items 23-28.

[0263] Item 30. Liposomes as described in any of items 23-29, further comprising an active agent, particularly doxorubicin.

[0264] Item 31. Liposomes described in any one of items 23-30 for use in the treatment of tumors.

[0265] Item 32. The liposome described in Item 31, where the tumor is a soft tissue sarcoma.

[0266] Item 33. Use of stereoisomerized phosphatidyl oligoglycerols described in any one of items 1-16 to increase temperature-dependent drug release of thermosensitive liposomes and / or to narrow the phase transition in the temperature-dependent release profile.

Claims

1. Equation (I) 【Chemistry 1】 (In the formula, the three stereocenters *1, *2 and *3 have a predetermined stereoconfiguration; and *p indicates a further stereocenter; and R １ and R ２ Each is a hydrocarbon group having 12 to 24 C atoms independently; n is a number from 0 to 20; and (m is either 0 or 1 independently in each occurrence.) Stereoisomer phosphatidyl oligoglycerol.

2. Formula (II) 【Chemistry 2】 (In the formula, the solid center *1 is in the R configuration, the solid center *2 is in the S configuration, the solid center *3 is in the R configuration, and *p indicates a further solid center.) A stereoisomer phosphatidyl oligoglycerol according to claim 1, having the following characteristics:

3. The stereoisomer phosphatidyl oligoglycerol according to claim 1 or claim 2, in particular n = 0 or 1, and n = 0.

4. R １ and R ２ However, it is a linear saturated alkyl group having 13 to 19 C atoms independently, and especially R １ and R ２ However, the stereoisomer phosphatidyl oligoglycerol according to any one of claims 1 to 3, wherein the stereoisomer is a linear saturated C15 alkyl group.

5. Formula (III) 【Transformation 3】 A stereoisomer phosphatidyl oligoglycerol according to any one of claims 1 to 4.

6. The synthesis pathway is either formula (IV) or formula (IV'). 【Chemistry 4】 (In the formula, the two stereocenters *2' and *3' have a predetermined arrangement, and PG, PG', and PG'' independently represent a protecting group.) A method for preparing stereoisomerized phosphatidyl oligoglycerol according to any one of claims 1 to 5, characterized by comprising a central intermediate.

7. The synthesis pathway is either formula (IVa) or formula (IVa'). 【Transformation 5】 (In the formula, the stereocenter *2' is in the R configuration, and the stereocenter *3' is in the S configuration in (IVa) and the R configuration in (IVa'), and PG, PG' and PG'' independently represent protecting groups.) A method for preparing stereoisomerized phosphatidyl oligoglycerol according to claim 6, characterized by comprising a central intermediate.

8. A method for preparing a stereoisomer phosphatidyl oligoglycerol according to claim 6 or 7, characterized in that the central intermediate of formula (IV) is prepared from glycidol and 1,2-isopropylidene-glycerol, particularly from (R)-(+)-glycidol and (S)-(+)-1,2-isopropylidene-glycol (sn-1,2-IPG).

9. The central intermediate of formula (IV) or formula (IVa) is formula (V) 【Transformation 6】 (In the formula, R １ and R ２ (Each of these is a hydrocarbon group having 12 to 24 carbon atoms independently, R' is a leaving group, and R'' is a hydrocarbon group having 1 to 12 carbon atoms.) A method for preparing a stereoisomer phosphatidyl oligoglycerol according to any one of claims 6 to 8, comprising reacting it with a compound.

10. A liposome comprising stereoisomerized phosphatidyl oligoglycerol as described in any one of claims 1 to 5.

11. At least one equation (VII) 【Transformation 7】 (in the formula, R ３ and R ４ are each independently a hydrocarbon group having 12 to 24 C atoms) The liposome according to claim 10, further comprising phosphatidylcholine.

12. 20 to 40 mol% of the stereoisomer phosphatidyl oligoglycerol according to any one of claims 1 to 5, particularly the phosphatidyl oligoglycerol of formula (III); 40-60 mol% of 1,2-dipalmitoylphosphatidylcholine; and 15-25 mol% 1,2-distearoylphosphatidylcholine A liposome according to claim 10 or 11, comprising:

13. A liposome according to any one of claims 10 to 12, further comprising an activator, particularly doxorubicin.

14. Liposomes according to claims 10 to 13 for use in the treatment of tumors.

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