WO2024253473A1 - Procédé de synthèse à écoulement continu unique pour synthétiser une banque de lipides fonctionnels utilisée dans un agent thérapeutique à base d'acide nucléique - Google Patents

Procédé de synthèse à écoulement continu unique pour synthétiser une banque de lipides fonctionnels utilisée dans un agent thérapeutique à base d'acide nucléique Download PDF

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WO2024253473A1
WO2024253473A1 PCT/KR2024/007830 KR2024007830W WO2024253473A1 WO 2024253473 A1 WO2024253473 A1 WO 2024253473A1 KR 2024007830 W KR2024007830 W KR 2024007830W WO 2024253473 A1 WO2024253473 A1 WO 2024253473A1
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cationic lipid
reactor
propane
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methyl sulfate
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김동표
카왈리산켓 아쇽라오
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Definitions

  • the present invention relates to a single continuous flow synthesis method for synthesizing a functional lipid library used in nucleic acid therapeutics, and more particularly, to a single continuous flow synthesis method for synthesizing an ionizable cationic lipid library including an ionizable cationic lipid such as 1,2-dioleoyl-3-dimethylammonium-propane and a cationic lipid library including a cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane, and a method for preparing lipid nanoparticles or lipid nanoliposomes using the method.
  • LNPs Clinically effective lipid nanoparticles
  • cationic lipids play a key role in liposomal transfection of DNA, RNA, and other negatively charged molecules.
  • cationic lipids typically consist of an ionizable or cationic head group attached to a saturated or unsaturated hydrophobic tail via a linker group.
  • Cationic lipids are positively charged at physiological pH, whereas ionizable cationic lipids are neutral at physiological pH but positively charged at pH below their pKa ( ⁇ 7) due to protonation of the head group-free amines responsible for encapsulation and endosomal release of RNA.
  • cationic lipids in which a head group is linked to a hydrophobic tail via an ester bond exhibit more clinical efficacy due to their biodegradability in the human body.
  • 1,2-Dioleoyl-3-dimethylammonium-propane is an ionizable cationic lipid with low cytotoxicity and high transfection efficiency. It is composed of two hydrophobic oleoyl tails and a hydrophilic head group containing a dimethylammonium moiety and a propane group. In contrast, 1,2-dioleoyl-3-trimethylammonium-propane (3) (DOTAP), a derivative of DODAP (2), contains a cationic trimethylammonium hydrophilic head group ( Figure 1). DODAP(2) and its derivatives are widely used to encapsulate bioactive molecules including mRNA, siRNA and plasmid DNA for disease treatment.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DODAP(2) The synthesis of DODAP(2) involves the synthesis of oleoyl chloride from oleic acid followed by esterification with 3-(dimethylamino)propane-1,2-diol.
  • the synthesis and purification in batch reactors are challenging due to the toxicity of essential chlorinating agents such as oxalyl chloride and the toxic HCl and CO gas byproducts generated during the formation of oleoyl chloride.
  • an alternative synthetic method using the coupling reaction of oleic acid and 3-(dimethylamino)propane-1,2-diol is time-consuming, difficult to purify, and relatively expensive.
  • Patent Documents 1 and 2 report two different approaches to prepare cationic lipid series that utilize highly toxic and carcinogenic methylating agents (chloromethane, iodomethane, etc.), which limit their application in large-scale production ( Figure 2 a and b).
  • Patent Document 3 and Non-Patent Document 2 utilized non-toxic trimethylamine to introduce a cationic head group for DOTAP bromide synthesis (Fig. 2c).
  • Fig. 2c the high cost of using bromopropanediol as a starting material has limited its industrial application.
  • An attempt to use glycerol, a cheap starting material, together with the trimethylamine reagent of Patent Document 4 requires a multi-step process that is time-consuming and labor-intensive and affects the quality of the lipid (Fig. 2d).
  • the shortest route involves the Steglich esterification reaction of oleic acid with 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride starting from 3-chloropropane-1,2-diol (Fig. 2e), but it takes 16 h for complete completion, and it is highly desirable to utilize flow chemistry to shorten the reaction time and productivity.
  • Patent Document 3 DE4013632A1
  • Patent Document 4 WO2005049549
  • Non-patent Document 2 Interactions of Cationic Lipid Vesicles with Negatively Charged Phospholipid Vesicles and Biological Membranes, Leonidas Stamatatos, Rania Leventis, Martin J. Zuckermann, John R. Silvius, Biochemistry 1988, 27 , 3917-3925.
  • Non-patent Document 3 Overcoming solid handling issues in continuous flow substitution reactions through ionic liquid formation, Saeed K. Kashani, Ryan J. Sullivan, Mads Andersen, Stephen G. Newman, Green Chem., 2018 , 20 , 1748-1753.
  • the present invention aims to provide a method for producing lipid nanoparticles or lipid nanoliposomes using a single continuous flow synthesis method for the aforementioned functional lipids.
  • a step of separating HCl and CO gases may be additionally included after step (a).
  • the synthetic method can be performed in a microfluidic device for a single continuous flow process
  • the microfluidic device can include: a first micromixer in which a first channel and a second channel are fluidly connected; a first reactor in which the first micromixer is fluidly connected; a first back pressure regulator in which the first reactor is fluidly connected; a T-shaped liquid-gas separator in which the first back pressure regulator is fluidly connected to separate a solution and a gas; a second micromixer in which a third channel is fluidly connected to the T-shaped liquid-gas separator; a second reactor in which the second micromixer is fluidly connected; and a second back pressure regulator in which the second reactor is fluidly connected.
  • the T-shaped liquid-gas separator may include a first supply channel fluidly connected to the first back pressure regulator, a first discharge channel through which separated gas is discharged, and a second supply channel fluidly connected to the second micro-mixer so that the separated solution is supplied to the second micro-mixer.
  • oxalyl chloride can be injected into the second path
  • HCl and CO gas can be separated through the T-shaped liquid-gas separator
  • 3-(dimethylamino)propane-1,2-diol and a basic compound can be injected into the third path
  • an ionizable cationic lipid having a structure of the chemical formula 1 can be generated in the second reactor.
  • the residence time in the first reactor may be 1 to 2 minutes
  • the residence time in the second reactor may be 2 to 10 minutes
  • the reaction temperature in the second reactor may be 20 to 60°C.
  • the basic compound may be any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine.
  • the present invention provides a method for synthesizing 6-bromohexyl 2-hexyldecanoate, an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminum, which is an ionizable cationic lipid, comprising the following steps (a) and (b):
  • the present invention includes a step of mixing and reacting an ionizable cationic lipid synthesized by the above-described synthetic method with dimethyl sulfate to produce cationic lipid methyl sulfate having a structure of chemical formula 2,
  • the synthesis method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first flow path and a second flow path are fluidly connected; a first reactor fluidly connected with the first micromixer; and a first back pressure regulator fluidly connected with the first reactor.
  • an ionizable cationic lipid having a structure of chemical formula 1 can be injected into the first flow path, and dimethyl sulfate can be injected into the second flow path.
  • cationic lipid methyl sulfate having the structure of the chemical formula 2 can be produced.
  • the ionizable cationic lipid having the structure of the chemical formula 1 and the dimethyl sulfate can be injected at a flow rate of 0.4 to 2.0 ml/min, the reaction temperature in the first reactor can be 40 to 70°C, and the residence time in the first reactor can be 3 to 7 minutes.
  • the cationic lipid methyl sulfate having the structure of the chemical formula 2 may be any one of the following (1) to (7):
  • the synthesis method may additionally include solvent evaporation and recrystallization steps.
  • the recrystallization can be performed in an acetone solvent.
  • the present invention provides a method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride.
  • DOTAP cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane
  • the synthesis method can be performed in a microfluidic device for a single continuous flow process, and the microfluidic device can include: a first micromixer in which a first flow path and a second flow path are fluidly connected; a first reactor fluidly connected with the first micromixer; and a first back pressure regulator fluidly connected with the first reactor.
  • the reaction temperature in the first reactor may be 60 to 80°C, and the residence time in the first reactor may be 3 to 7 minutes.
  • the synthesis method may additionally include solvent evaporation and recrystallization steps.
  • the recrystallization can be performed in isopropanol and acetone solvents.
  • the present invention provides a method for preparing lipid nanoparticles comprising the following steps (a) and (b):
  • the present invention provides a method for preparing lipid nano liposomes comprising the following steps (a) and (b):
  • the method for synthesizing a functional lipid used in a nucleic acid therapeutic agent according to the present invention is implemented by a continuous flow synthesis method in a microfluidic reactor, which has significantly higher productivity and provides an ionizable cationic lipid or cationic lipid of superior quality compared to a synthesis method in a conventional batch reactor.
  • DODAP and DOTAP synthesized by the method of the present invention can be utilized as lipid nanoparticles and lipid nanoliposomes that encapsulate physiologically active molecules including mRNA, siRNA, and plasmid DNA for disease treatment.
  • Figure 1 shows the structures of common cationic lipids (1), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) (2) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (3), which are used in lipid nanoparticles or liposomes.
  • DODAP 1,2-dioleoyl-3-dimethylammonium-propane
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • Figure 2 shows a conventionally known method for synthesizing DOTAP (3), and (B) shows a continuous flow method for synthesizing DOTAP using two types of counter ions of the present invention.
  • Figure 3 shows the experimental setup for continuous flow synthesis of oleoyl chloride in a capillary reactor including in-line FTIR.
  • Figure 4 is a photograph of the experimental setup of the in-line FTIR flow cell.
  • Figure 5 shows the experimental results of in-line FTIR monitoring.
  • Figure 6(a) is a photograph showing a custom T-junction used as an effective liquid-gas separator
  • b) is a photograph showing that plugging occurs in the R2 reactor due to high concentration of solid pyridinium hydrochloride salt (8 eq. of base is required) if HCl gas is not removed from the reactor
  • c) shows the results of monitoring the conversion of oleoyl chloride to DODAP(2) using in-line FTIR monitoring.
  • Figure 7 shows the single-flow synthesis of the ionizable cationic lipid DODAP(2) and a library of ionizable cationic lipids in a capillary reactor.
  • Figure 8 shows the experimental setup for the single-flow synthesis of ionizable cationic lipids.
  • Figure 9 shows the experimental setup for continuous flow synthesis of DODAP.
  • Figure 10 shows the experimental setup for continuous flow synthesis of cationic lipids DOTAP methyl sulfate (4), DLinTAP methyl sulfate (17) and other analogues (18-22) in a capillary reactor.
  • Figure 11 shows the experimental setup for continuous flow synthesis of DOTAP methyl sulfate.
  • Figure 12 shows the experimental setup for continuous flow synthesis of a cationic lipid library.
  • Figure 13 shows the continuous flow synthesis of DOTAP chloride in a capillary reactor.
  • Figure 14 shows the experimental setup for continuous flow synthesis of DOTAP chloride using a size-up method.
  • Figure 15 shows the preparation and characterization of nanoparticles generated from synthesized DODAP and DOTAP, where A of Figure 15 shows a series of processes for nanoparticle production, dialysis and analysis by DLS, and B to E show the normalized size distributions of the prepared DODAP LNPs (B), DODAP liposomes (C), DOTAP-chloride liposomes (D) and DOTAP methyl sulfate liposomes (E) before and after purification by dialysis process, respectively.
  • Figure 11 shows the production and analysis of lipid-based nanoparticles, where A is a schematic diagram of the overall procedure and B shows the numerical size and PDI of nanoparticles produced before and after dialysis.
  • Figure 16 shows the numerical sizes and PDI values of the generated DODAP LNPs (A), DODAP liposomes (B), DOTAP-chloride liposomes (C), and DOTAP methyl sulfate liposomes (D).
  • the inventors of the present invention sought to solve the above-mentioned problems by developing a novel synthetic route capable of synthesizing a high-quality functional lipid library with high productivity using a single continuous flow process.
  • a facile single-flow synthesis method for a library of ionizable lipids developed in a simple two-step manner was developed, achieving scalable productivity of up to 10 g/hr through a scale-up approach.
  • acyl chloride synthesis for continuous flow esterification was successfully explored, and clogging was prevented by effectively removing toxic HCl and CO gases using a T-junction liquid-gas separator. Rapid optimization of the continuous flow process was achieved using in-line FTIR monitoring.
  • the flow protocol of the present invention enables the synthesis of a wide range of lipids, including both established commercial types and novel ionizable lipids, in a much shorter time (approximately 4.5 min) compared to conventional batch protocols.
  • a library of biodegradable ionizable lipids was designed with ester or disulfide functionalities in the long chain group. These lipids were synthesized in a facile capillary reactor, and were obtained in 88-92% yields. In addition, rapid alkylation was achieved using dimethyl sulfate while maintaining the flow reactor for 5 min in the present invention to convert the ionizable lipids into cationic lipids. In particular, DOTAP methyl sulfate was produced with a productivity of 7 g/h. Furthermore, DOTAP chloride was effectively synthesized with a yield of 88% via the Steglich esterification reaction using EDC.HCl, resulting in a productivity of 10.2 g/h.
  • DODAP was converted into uniformly sized 64 mm LNPs with a PDI of 0.07 and 72 mm liposomes with a PDI of 0.05 using a self-developed micromixer.
  • DOTAP formed 55 mm liposomes with a PDI of 0.08. This novel fluid-assisted platform for lipid libraries holds great potential to advance gene therapy and serve as next-generation nanomedicine.
  • the present invention in its first aspect, provides a method for synthesizing an ionizable cationic lipid comprising the steps of:
  • the unsubstituted C10 to C20 saturated aliphatic hydrocarbon radical may be a linear or branched saturated aliphatic hydrocarbon radical containing 10 to 20 carbon atoms.
  • it may be a linear or branched decenyl group, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group or an eicosenyl group, but is not limited thereto.
  • all reactants of the above-mentioned synthetic method can be used in a state dissolved in an organic solvent that does not adversely affect the reactions of steps (a) and (b).
  • organic solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane can be used, but are not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform steps (a) and (b).
  • oxalyl chloride in step (a) was selected as a chlorinating agent for synthesizing oleoyl chloride in the presence of a dimethylformamide (DMF) catalyst.
  • DMF dimethylformamide
  • CH 2 Cl 2 was considered as a general solvent for flow synthesis considering the solubility of the formed base.HCl complex.
  • the 3-(dimethylamino)propane-1,2-diol may be replaced with another type of diol compound containing a dimethylamino group.
  • the synthesis method can be performed in a microfluidic device (1) for a single continuous flow process, wherein the microfluidic device (1) comprises: a first micro-mixer (100) in which a first flow path (110) and a second flow path (120) are fluidly connected; a first reactor (200) fluidly connected with the first micro-mixer (100); a first back pressure regulator (300) fluidly connected with the first reactor (200); a T-shaped liquid-gas separator (400) fluidly connected with the first back pressure regulator (300) to separate a solution and a gas; a second micro-mixer (500) in which the T-shaped liquid-gas separator (400) and a third flow path (510) are fluidly connected; a second reactor (600) fluidly connected with the second micro-mixer (500); and may include a second back pressure regulator (700) fluidly connected to the second reactor (600).
  • a first micro-mixer (100) in which a first flow path (110) and a second flow path (120) are fluidly
  • the T-shaped liquid-gas separator (400) may include a first supply path (410) fluidly connected to the first back pressure regulator (300), a first discharge path (420) through which separated gas is discharged, and a second supply path (430) fluidly connected to the second micro-mixer (500) so that the separated solution is supplied to the second micro-mixer (500).
  • the first flow path (110) and the second flow path (120) of the microfluidic device (1) are flow paths into which the reactants of step (a) are injected, respectively, and can be fluidly connected to the first micro-mixer (100).
  • reactants flowing in from the first path (110) and the second path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).
  • These byproduct gases can be separated and removed through the T-shaped liquid-gas separator (400).
  • the second micro-mixer (500) may be fluidly connected to the third channel (510).
  • 3-(dimethylamino)propane-1,2-diol and a basic compound may be injected into the third channel (510).
  • reactants flowing in from the second supply path (430) and the third path (510) are mixed to produce a mixture, and the produced mixture can be injected into the second reactor (600) that is fluidly connected to the second micro-mixer (500).
  • step (b) occurs in the second reactor (600), and an ionizable cationic lipid having the structure of the chemical formula 1 is produced as a result of the reaction.
  • the ionizable cationic lipid having the structure of the chemical formula 1 as the final product can be discharged after passing through the second back pressure regulator (700) fluidly connected to the second reactor (600).
  • a first spectrometer (800) may be additionally fluidly connected between the first reactor (200) and the first back pressure regulator (300), and a second spectrometer (900) may be additionally fluidly connected between the second reactor (600) and the second back pressure regulator (700).
  • the residence time in the first reactor (200) may be 1 to 2 minutes.
  • reaction temperature in the first reactor (200) may be 20 to 30°C.
  • the residence time in the second reactor (600) may be 2 to 10 minutes.
  • the reaction temperature in the second reactor (600) may be 20 to 60°C.
  • step (b) It is preferable to perform the reaction of step (b) within the range of the residence time (i.e., reaction time) and reaction temperature in the second reactor (600), and if it is outside the range, the yield of the ionizable cationic lipid having the structure of the chemical formula 1 may decrease.
  • the residence time in the second reactor can be achieved by injecting a solution containing 3-(dimethylamino)propane-1,2-diol and a basic compound into the third flow path (510) at a flow rate of 0.4 to 2.0 ml/min into the second micro-mixer.
  • the basic compound of step (b) may be any one selected from the group consisting of pyridine, a mixture of pyridine and DMAP, DMAP, diisopropylethylamine, and triethylamine.
  • the most preferred basic compound may be triethylamine.
  • triethylamine When triethylamine is used as the basic compound, an ionizable cationic lipid having the structure of the chemical formula 1 can be obtained in a maximum yield of 92% through a reaction at 25°C for 3 minutes in the second reactor (600).
  • the ionizable cationic lipid having the structure of the chemical formula 1 may be any one of the following (1) to (11):
  • step (a) to synthesize 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), an ionizable cationic lipid, step (a) was performed as follows.
  • the reaction was optimized at room temperature (25 °C) with various residence times by varying the flow rates of the reagents.
  • the flow apparatus was connected to an FTIR spectrometer using a microfluidic cell (Fig. 4).
  • IR spectroscopy monitored the formation of acid chloride with a characteristic CO stretching band at 1780 cm -1 and the disappearance of the CO stretching at 1708 cm -1 of the acid functional group of oleic acid (Fig. 5).
  • a 100 psi back pressure regulator (BPR) was used at the FTIR flow cell outlet to prevent the outgassing of by-products such as HCl and CO gases. After some optimization, it was confirmed that complete consumption of oleic acid occurred at a reaction temperature of 25 °C and a residence time of 1.5 min for smooth formation of oleoyl chloride. Further increase in the residence time and temperature in reactor R1 did not result in further increase in the intensity of the CO stretching peak of oleoyl chloride.
  • HCl and CO gases that are not dissolved in the solution are successfully separated using a liquid-gas separator (T-junction). If HCl is not separated, an excessive amount of base is required in step (b), and the concentration of insoluble hydrochloric acid salt in the base is high, which may cause clogging in R2.
  • DODAP 1,2-dioleoyl-3-dimethylammonium-propane
  • the reaction in R2 was optimized at various residence times by changing the length of the PTFE tube reactor.
  • the ester bond formation during the DODAP(2) synthesis was monitored using inline FTIR spectroscopy, and a characteristic CO stretching band was formed at 1735 cm -1 for the ester group ( FIG. 7 ).
  • the productivity of the ionizable cationic lipid library was improved by using the first reactor (200) and the second reactor (600) whose inner diameters were increased to 1.6 mm.
  • the tube diameter of the first reactor of the microfluidic device may be 0.8 to 2.0 mm
  • the tube length may be 200 to 300 cm
  • the tube diameter of the second reactor may be 0.8 to 2.0 mm
  • the tube length may be 500 to 900 cm.
  • a second aspect of the present invention relates to a method for synthesizing 6-bromohexyl 2-hexyldecanoate, which is an intermediate of 6-((2-hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-1-aminium, which is an ionizable cationic lipid, comprising the following steps (a) and (b).
  • the method for synthesizing 6-bromohexyl 2-hexyldecanoate of the present invention can be performed under the same conditions and in the same microfluidic device as the method for synthesizing an ionizable cationic lipid described above, except that 2-hydroxydecanoic acid is used instead of oleic acid and 6-bromohexanol is used instead of trimethylamine.
  • the residence time in the first reactor can be 1 to 3 minutes, and the reaction temperature can be 20 to 30°C, the residence time in the second reactor can be 2 to 7 minutes, and the reaction temperature in the second reactor can be 20 to 60°C.
  • a third aspect of the present invention comprises a step of mixing and reacting an ionizable cationic lipid synthesized by the synthetic method described above with dimethyl sulfate to produce a cationic lipid methyl sulfate having a structure of chemical formula 2.
  • the method for synthesizing the cationic lipid methyl sulfate can be performed in a microfluidic device (2) for a single continuous flow process, and the microfluidic device (2) can include: a first micro-mixer (100) in which a first channel (110) and a second channel (120) are fluidly connected; a first reactor (200) fluidly connected with the first micro-mixer (100); and a first back pressure regulator (300) fluidly connected with the first reactor (200).
  • all reactants of the above cationic lipid methyl sulfate synthesis method can be used in a state dissolved in an organic solvent that does not adversely affect the reaction.
  • organic solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), and dichloromethane can be used, but are not limited thereto, and a person skilled in the art can select an appropriate organic solvent and perform the above reaction step.
  • the first flow path (110) and the second flow path (120) of the microfluidic device (2) are flow paths into which reactants are injected, respectively, and can be fluidically connected to the first micro-mixer (100).
  • an ionizable cationic lipid having the structure of the chemical formula 1 can be injected into the first flow path (110), and dimethyl sulfate can be injected into the second flow path (120).
  • reactants introduced from the first flow path (110) and the second flow path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).
  • the ionizable cationic lipid and dimethyl sulfate react to produce cationic lipid methyl sulfate having the structure of the chemical formula 2 as a reaction product.
  • the cationic lipid methyl sulfate having the structure of the chemical formula 2 as the final product can be discharged after passing through the first back pressure regulator (300) fluidly connected to the first reactor (200).
  • a first spectrometer (800) may additionally be fluidly connected between the first reactor (200) and the first back pressure regulator (300) of the above microfluidic device.
  • the residence time in the first reactor (200) may be 3 to 7 minutes.
  • the time required for the reactants introduced into the first reactor (200), that is, the ionizable cationic lipid having the structure of the chemical formula 1 and dimethyl sulfate, to react to produce cationic lipid methyl sulfate having the structure of the chemical formula 2 may be 3 to 7 minutes.
  • reaction temperature in the first reactor (200) may be 40 to 70°C.
  • reaction time i.e., reaction time
  • reaction temperature in the above first reactor (200)
  • yield of the cationic lipid methyl sulfate having the structure of the chemical formula 2 may decrease.
  • the residence time in the first reactor (200) can be achieved by injecting a solution containing an ionizable cationic lipid having the structure of the chemical formula 1 and a solution containing dimethyl sulfate into the first micro-mixer at a flow rate of 0.4 to 2.0 ml/min, respectively, into the first flow path.
  • the cationic lipid methyl sulfate having the structure of the chemical formula 2 may be any one selected from the group consisting of the following (1) to (7):
  • the alkylation of DODAP(2) synthesized using dimethyl sulfate in flow chemistry was selected to synthesize cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) methyl sulfate.
  • DOTAP cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane
  • complete alkylation of DODAP (2) was achieved by using 10 equivalents of dimethyl sulfate at 60 °C and a residence time of 5 min in the R1 reactor.
  • the accelerated reaction kinetics due to the inherent superiority in mixing efficiency are highly advantageous compared to the batch process which takes 48 h to complete the alkylation.
  • another ionizable lipid or a fatty acid having an ester or disulfide functional group introduced into a long chain of an ionizable lipid is (9Z,9'Z,12Z,12'Z)-3-(dimethylamino)propane-1,2-diylbis(octadeca-9,12-dienoate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-dihexyl bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-bis((S)-3,7-dimethyloct-6-en-1-yl) bis(decanedioate), O'1,O1-(3-(dimethylamino)propane-1,2-diyl) 10-Bis((E)-3,7-dimethylocta-2
  • the productivity of the cationic lipid methyl sulfate library was improved by using the first reactor (200) with the inner diameter increased to 1.6 mm.
  • a fourth aspect of the present invention relates to a method for synthesizing cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride, comprising the step of mixing and reacting a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP with (3-dimethylamino-propyl)-ethyl-carbodiimide chloride to cause a Steglich esterification reaction to produce cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) chloride.
  • DOTAP cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane
  • DOTAP methyl sulfate (4) can be converted to DOTAP chloride (5) by treatment with Amberlite® IRA-400(Cl ) ion exchange resin, which is labor- and cost-intensive.
  • Direct synthesis of DOTAP chloride (5) without using methyl chloride as an alkylating agent can be accomplished in a batch reactor by esterifying oleoyl chloride with commercially available and readily synthesizable 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) dissolved in CH 2 Cl 2 in the presence of DMAP as a base.
  • diols (23) are insoluble in common organic solvents except dimethyl sulfoxide (DMSO), making flow chemistry difficult to perform, and furthermore, DMSO was not a suitable solvent for esterification with acyl chlorides.
  • DMSO dimethyl sulfoxide
  • DOTAP chloride (5) was obtained in 78% yield via N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl)-mediated Steglic esterification of oleic acid and diol (23) in the presence of DMAP in DMSO in 16 h. Therefore, it is worth exploring the Steglic esterification reaction in a continuous flow reactor for the direct synthesis of DOTAP chloride (5).
  • the experimental setup including a PTFE tube reactor with an inner diameter of 1.0 mm is shown in Figure 14.
  • Diol (23) was initially prepared in batch by refluxing an ethanolic solution of trimethylamine dissolved in 3-chloropropane-1,2-diol and methanol solvent.
  • all reactants of the method for synthesizing the DOTAP chloride (5) can be used in a state dissolved in an organic solvent that does not adversely affect the reaction.
  • an organic solvent that does not adversely affect the reaction.
  • DMSO dimethyl sulfoxide
  • a person skilled in the art can select an appropriate organic solvent and perform the above reaction step.
  • the first flow path (110) and the second flow path (120) of the microfluidic device (2) are flow paths into which reactants are injected, respectively, and can be fluidly connected to the first micro-mixer (100).
  • a mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride, and DMAP can be injected into the first flow path (110), and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride can be injected into the second flow path (120).
  • reactants introduced from the first flow path (110) and the second flow path (120) are mixed to generate a mixture, and the generated mixture can be injected into the first reactor (200) that is fluidly connected to the first micro-mixer (100).
  • the final product, DOTAP chloride (5) can be discharged after passing through the first back pressure regulator (300) fluidly connected to the first reactor (200).
  • the residence time in the first reactor (200) may be 3 to 7 minutes.
  • the time required for the reactants introduced into the first reactor (200), that is, the mixture of oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP, and (3-dimethylamino-propyl)-ethyl-carbodiimide chloride, to react to produce DOTAP chloride (5) may be 3 to 7 minutes.
  • reaction temperature in the first reactor (200) may be 60 to 80°C.
  • reaction time i.e., reaction time
  • reaction temperature in the above first reactor (200)
  • yield of DOTAP chloride (5) may decrease.
  • the residence time in the first reactor (200) can be achieved by injecting a solution containing oleic acid, 2,3-dihydroxy-N,N,N-trimethylpropane-1-aminium chloride and DMAP into the first flow path and a solution containing (3-dimethylamino-propyl)-ethyl-carbodiimide chloride into the first micro-mixer at a flow rate of 0.4 to 1.5 ml/min.
  • the method for synthesizing the DOTAP chloride (5) may additionally include solvent evaporation and recrystallization steps. At this time, the recrystallization may be performed in isopropanol and acetone solvents, but may be performed without limitation in a solvent suitable for recrystallization of the DOTAP chloride (5).
  • the reaction in R1 was optimized at various temperatures and residence times by varying the flow rate of the reagents using a syringe pump. Initially, the yield of DOTAP chloride (5) was low due to incomplete de-esterification of the diol (23) with a residence time of 15 min at room temperature. However, when the temperature was raised to 70 °C to further promote the de-esterification, DOTAP chloride (5) was obtained in 88% yield after purification with only 5.0 min of residence time in R1, which was much shorter than the 16 h of the batch process.
  • the productivity of DOTAP chloride (5) was improved by using a first reactor (200) with an inner diameter increased to 1.6 mm.
  • a first reactor (200) with an inner diameter increased to 1.6 mm.
  • a fifth aspect of the present invention relates to a method for producing lipid nanoparticles, comprising the following steps (a) and (b):
  • step (a) In the method for manufacturing lipid nanoparticles of the present invention, the description of step (a) is the same as described above, so its description is omitted.
  • the ionizable cationic lipid used in step (b) may include all types of ionizable cationic lipids synthesized by the above-described method.
  • the helper lipids used in the step (b) are DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPI (1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPI (1,2-distearoyl-sn-glycero-3-phosphoinositol), and It may be at least one selected from the group consisting of DLPC (1,2-dirinoley
  • the polyethylene glycol (PEG) lipid may be at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, but is not limited thereto.
  • the PEG lipid includes a PEG moiety having a size of 100 Da to 20 kDa, and more preferably, it may be at least one selected from the group consisting of DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], and Ceramide PEG2000 (N-palmitoyl-sphingosine-1- ⁇ succinyl[methoxy(polyethylene glycol)2000] ⁇ ), but is not limited thereto.
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
  • DSPE-PEG2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)
  • the molar ratio of the ionizable cationic lipid, helper lipid, cholesterol and polyethylene glycol lipid may be, but is not limited to, 40-60 : 5-15 : 30-45 : 1-3.
  • the buffer used in step (b) may be, but is not limited to, sodium acetate buffer, sodium citrate buffer, phosphate buffered saline (PBS), or citrate buffer.
  • a sixth aspect of the present invention relates to a method for preparing lipid nano liposomes comprising the following steps (a) and (b):
  • step (a) In the method for producing the lipid nano liposome of the present invention, the description of step (a) is the same as described above, so its description is omitted.
  • the ionizable cationic lipid or cationic lipid used in step (b) may include all types of ionizable cationic lipid or cationic lipid synthesized by the above-described method.
  • the molar ratio of the ionizable cationic lipid, cationic lipid or DOTAP:cholesterol may be 25 to 75:25 to 75, but is not limited thereto.
  • the buffer used in step (b) may be, but is not limited to, sodium acetate buffer, Tris buffer, phosphate buffered saline (PBS), or citrate buffer.
  • DODAP and DOTAP chloride are biomimetic molecules that take the form of liposomes or lipid nanoparticles, enabling efficient delivery of genetic and various therapeutics by enhanced endosomal escape.
  • the uniform size of the nanoparticles encapsulating the therapeutics is very important in terms of delivery by interaction with cells.
  • the on-demand manufacturing of nanoparticle carriers is difficult because complex variables such as mixer type, feed rate, and composition are involved in the nanoprecipitation process.
  • empty LNPs were synthesized using four types of components similar to those reported, i.e., ionizable lipid, helper lipid, cholesterol, and PEG-lipid.
  • ionizable lipid i.e., ionizable lipid
  • helper lipid i.e., helper lipid
  • cholesterol i.e., glycerol
  • PEG-lipid lipid-lipid-lipid-lipid-lipid
  • DCM Dichloromethane
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofuran
  • ACN acetonitrile
  • MeOH methanol
  • DMF dimethylformamide
  • EtOH absolute ethanol
  • Oleic acid (99%), linoleic acid (99%), palmitic acid (99%), stearic acid (99%), myristic acid (99%), citronellol, geraniol, nerol, sebic acid, 6-bromohexan-1-ol, 2-hexyldecanoic acid, ( ⁇ )-3-chloro-1,2-propanediol, 3-(dimethylamino)-1,2-propanediol, oxalyl chloride, and triethylamine were purchased from Sigma-Aldrich.
  • 1,2-Distearoyl-sn-glycero-3-phosphocholine DSPC
  • cholesterol Chol
  • 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 DMG-PEG 2000 were purchased from Avanti Polar Lipids, Inc.
  • Inline FT-IR spectra were recorded on a routine FT-IR spectrometer (Jasco FT/IR-4600) equipped with a sealed flow cell accessory (Specac®) based on ZnSe windows (pathlength 0.1 mm). The masses of lipids were confirmed using a BRUKER MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer.
  • DODAP(2) was considered as a parent molecule for the synthesis of a single-flow ionizable lipid library. Therefore, it was first decided to synthesize oleoyl chloride in a continuous flow system to achieve high-quality synthetic DODAP(2). The purity of commercially available oleoyl chloride is only 85%.
  • the experimental setup for the optimization of oleoyl chloride synthesis in flow chemistry is shown in Fig. 3.
  • the reaction was optimized in a flow system consisting of a PTFE tube with an inner diameter of 1.0 mm.
  • IR spectroscopy monitored the formation of acid chloride with a characteristic C-O stretching band at 1780 cm -1 and the disappearance of the C-O stretching band of the acid functional group of oleic acid at 1708 cm -1 . After some optimization, complete consumption of oleic acid was observed within a residence time of 1.5 min in reactor R1 at a flow rate of 0.6 ml/min of each reagent to form oleoyl chloride (Fig. 5).
  • An FTIR sample cell was connected to reactor R2 for in-line FTIR monitoring.
  • the residence time in R2 was varied by changing the length of the reactor R2 (254 cm, 570 cm, 760 cm, 1020 cm, 2040 cm, etc.). After reaching steady state, the resulting solution was monitored using inline FTIR monitoring. IR spectroscopy monitored the formation of DODAP (2) with the characteristic CO stretching band of ester at 1735 cm -1 and the disappearance of the CO stretching of the acid chloride functional group of oleoyl chloride at 1780 cm -1 .
  • triethylamine was found to be a suitable base, affording the highest DODAP(2) yield of 92% of the isolated yield with a residence time of 3 min at R2 at 25 °C. In batch process, complete conversion took about 8 h.
  • DODAP(2) The successful synthesis of DODAP(2) was extended to develop various routes for the synthesis of a library of ionizable cationic lipids by modifying the long chain acid (Fig. 8).
  • Lipid library development is an important aspect of pharmaceutical research aimed at identifying lipids that can effectively encapsulate therapeutic agents and deliver them to target sites while minimizing toxicity. Accordingly, we attempted to synthesize a library of ionizable cationic lipids by co-incorporating commercially available fatty acids such as linoleic acid, myristic acid, stearic acid, and palmitic acid instead of oleic acid.
  • the experimental setup for continuous flow synthesis of ionizable cationic lipids is shown in Fig. 8.
  • monoterpenoids which have many health benefits such as anti-inflammatory, antioxidant, anticancer, antidiabetic, and antifungal activities, are used in ionizable lipids such as lipids 11 (derived from ⁇ -citronellol), 12 (derived from geraniol), and 13 (derived from nerol).
  • the synthetic method of compound 10 is as follows. First, 10-(hexyloxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 1.
  • the synthetic method of compound 12 is as follows. First, (E)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 3.
  • the synthetic method of compound 13 is as follows. First, (Z)-10-((3,7-dimethylocta-2,6-dien-1-yl)oxy)-10-oxodecanoic acid was prepared in a batch reactor as shown in the following reaction scheme 4.
  • the synthetic method of compound 14 is as follows. First, 3-(octadecyldisulfanyl)propanoic acid was prepared in a batch reactor as shown in the following reaction scheme 5.
  • the synthetic method of compound 15 is as follows. First, 3-(dodecyldisulfanyl)propanoic acid was prepared in a batch reactor as shown in the following reaction scheme 6.
  • a separate experiment was performed using a PTFE tubing reactor with a larger inner diameter of 1.6 mm.
  • the experimental setup for continuous flow synthesis of DODAP using a 1.6 mm PTFE tube reactor is shown in Fig. 9.
  • the resulting solution was passed into the reactor R1 for the synthesis of oleoyl chloride accompanied by HCl and CO gaseous byproducts.
  • a 250 psi back pressure regulator was connected to the R1 outlet.
  • the experimental setup for the flow synthesis of DOTAP methyl sulfate (4) starting from DODAP (2) is shown in Fig. 10.
  • the 100 psi back pressure regulator was connected to the R1 outlet.
  • the reaction in R1 was monitored using thin layer chromatography at various retention times by varying the flow rate of the reactants using an HPLC pump. After some optimization, complete conversion of DODAP (2) to DOTAP methyl sulfate (4) was achieved under the following conditions: flow rate of 0.40 ml/min of each reactant, 60°C, and residence time of 5.0 min in R1. After reaching steady state, the resulting solution was collected from the end of reactor R1 for 5 min. The organic solution was evaporated on a rotary evaporator to give DOTAP methyl sulfate (4), which was purified using crystallization from acetone to give a clear crystalline compound in 90% yield (0.27 g).
  • the experimental setup for the flow synthesis of DOTAP methyl sulfate (4) starting from DODAP (2) is shown in Fig. 11.
  • the resulting solution was then passed into the reactor R1 for the synthesis of DOTAP methyl sulfate.
  • the 250 psi back pressure regulator was connected to the R1 outlet. After reaching steady state, the resulting solution was collected at the end of the reactor R1 for 5 min. The organic solution was evaporated on a rotary evaporator to obtain DOTAP methyl sulfate (4), which was purified using crystallization from acetone to obtain a transparent crystalline compound in 86% yield (0.67 g).
  • Spectroscopic identification results of the synthesized final products confirmed that a cationic lipid library having structural formulas of compounds 17 to 22 was successfully synthesized.
  • the experimental setup for the flow synthesis of DOTAP chloride (5) is shown in Fig. 13.
  • a 40 psi back pressure regulator was connected to the R1 outlet.
  • the reaction in R1 was monitored using thin layer chromatography at various retention times by varying the flow rate of the reactants using a syringe pump. After some optimization, complete conversion of 2,3-dihydroxy-N,N,N-trimethylpropan-1-aminium chloride (23) to DOTAP chloride (5) was observed under the following conditions: 70 °C, residence time of 5 min in R1. After reaching steady state, the resulting solution was collected at the end of reactor R1 for 5 min. The solution was slowly quenched with 1 N HCl (10.0 mL) and the resulting aqueous layer was washed three times with dichloromethane (50.0 mL ⁇ 3).
  • DOTAP chloride (5) was purified using flash silica gel column chromatography containing 30% MeOH in CH 2 Cl 2 solvent system to give DOTAP chloride (5) (0.45 g) in 88% yield.
  • the experimental setup for the flow synthesis of DOTAP chloride (5) using the size-increasing method is shown in Fig. 14.
  • a custom-made micro-mixer was fabricated using 3D printing.
  • the mixer consists of hemispherical baffles to enable rapid mixing of the injected fluids.
  • each inlet and outlet were modified with a 5 mm thread (1/4-28 flat bottom for 1/16") to enable connection with XP-235 (IDEX Health & Science) fittings.
  • the virtual model created through CAD was fabricated using a DLP-based 3D printer (Pico 2 HD, Asiga) with commercial resin (PlasCLEAR), and each layer was laminated through photocuring and the resin was removed using isopropanol. Finally, the cleaned device was post-cured in a UV chamber for complete polymerization.
  • Lipid-based nanoparticles were prepared by rapid mixing of lipid solutions and buffer solutions. Each lipid solution was fixed at a total concentration of 10 mg/mL.
  • DODAP/DSPC/Chol/DMG-PEG 2000 was dissolved in ethanol at a molar ratio of 50/10/38.5/1.5.
  • 3 M sodium acetate solution was diluted with deionized water to achieve a concentration of 25 mM.
  • the pH of the sodium acetate solution was adjusted to 5.5 using 100 mM acetic acid solution.
  • DODAP (DOTAP) and cholesterol were dissolved at the same total concentration and molar ratio of 50:50.
  • lipid solution and buffer were passed through a 0.22 ⁇ m syringe filter.
  • the solutions were mixed via a micromixer using a syringe pump.
  • the LNP and liposome samples were dialyzed overnight against 1,000-fold volume of buffer (10 mM PBS, pH 7.4 for DODAP; 10 mM Tris buffer, pH 7.2 for DOTAP) using a dialysis bag (MWCO 12,000 Da, Sigma-Aldrich) to remove residual ethanol.
  • DLS analysis was performed using a Zetasizer Nano ZS instrument (Malvern Instruments, Ltd.).
  • DODAP LNPs and liposomes had an average size of 64 nm and 72 nm and a polydispersity index (PDI) of 0.07 and 0.05, respectively (Figs. 15B, C and 16A, B).
  • PDI polydispersity index
  • For DOTAP liposomes with different counterions a size of about 55 nm and a PDI of 0.08 were produced in both cases (Figs. 15D, E and 16C, D).
  • the production of highly uniform nanoparticles, especially the same properties of cationic liposomes with different counterions, is partly due to the high purity of each major lipid component.
  • DODAP LNPs and liposomes were reduced in size to 40 nm and 45 nm with PDI of 0.12 and 0.10, respectively, whereas DOTAP liposomes reached a size of about 39 nm with PDI of 0.11 (Figs. 15B to E and 16A to D). In all cases, a decrease in the average particle size and a slight broadening of the particle distribution were observed. It is worth noting that at this point, membrane fusion occurred due to coexistence with ethanol, whereas removal of ethanol enhanced the stability.
  • the flow-synthesized DODAP and DOTAP lipids exhibited a size of about 50 nm and a PDI of about 0.10, demonstrating their potential for effective drug delivery after self-assembly even after purification.
  • nanoparticle carriers of less than 100 nm allow for prolonged blood circulation and effective endosomal escape, while the narrow size distribution prevents accumulation in unwanted organs, facilitating optimal therapeutic delivery.
  • Microfluidic device 100 First micro mixer
  • T-type liquid-gas separator 410 1st supply path
  • Second reactor 700 Second pressure regulator

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Abstract

La présente invention concerne un procédé de synthèse à écoulement continu unique pour synthétiser une banque de lipides fonctionnels utilisée dans un agent thérapeutique à base d'acide nucléique. Plus particulièrement, l'invention concerne : une banque de lipides cationiques ionisables comprenant des lipides cationiques ionisables tels que le 1,2-dioléoyl-3-diméthylammonium-propane ; un procédé de synthèse à écoulement continu unique pour une banque de lipides cationiques comprenant des lipides cationiques tels que le 1,2-dioléoyl-3-triméthylammonium-propane ; et un procédé de préparation de nanoparticules lipidiques ou de nanoliposomes lipidiques utilisant ledit procédé.
PCT/KR2024/007830 2023-06-07 2024-06-07 Procédé de synthèse à écoulement continu unique pour synthétiser une banque de lipides fonctionnels utilisée dans un agent thérapeutique à base d'acide nucléique Pending WO2024253473A1 (fr)

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Citations (3)

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WO2023086514A1 (fr) * 2021-11-11 2023-05-19 Arcturus Therapeutics, Inc. Lipides cationiques ionisables pour l'acheminement d'arn

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US5869715A (en) 1995-09-27 1999-02-09 The Reagents Of The University Of California Polyfunctional cationic cytofectins
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CN108129338A (zh) * 2018-02-01 2018-06-08 爱斯特（成都）生物制药股份有限公司 一种阳性脂质体dotap的制备方法
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