CN115869262A

CN115869262A - Novel PEG lipid compound, preparation method, composition and application thereof

Info

Publication number: CN115869262A
Application number: CN202111133235.4A
Authority: CN
Inventors: 黄才古; 孙辉; 曾维霖
Original assignee: Guangzhou Anovent Pharmaceutical Co Ltd
Current assignee: Guangzhou Anovent Pharmaceutical Co Ltd
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2023-03-31
Also published as: WO2023045372A1

Abstract

The invention relates to a novel PEG lipid compound, a preparation method, a composition and an application thereof, and discloses a novel PEG lipid compound shown as a formula A, or an isomer thereof, or a pharmaceutically acceptable salt and a prodrug thereof. Also discloses a preparation method, a composition and application of the PEG lipid compound. The compound is used as functional PEG lipid, is suitable for a pharmaceutical composition for nucleic acid delivery, and can realize better stability and intracellular delivery efficiency of the drug, wherein the formula A is as follows.

Description

Novel PEG lipid compound, preparation method, composition and application thereof

The technical field is as follows:

the invention relates to the technical field of medicines, in particular to a preparation method, a composition and an application of a novel PEG lipid compound, and the functional PEG lipid is a pharmaceutical composition suitable for nucleic acid delivery.

The background art comprises the following steps:

since Watson and Crick discovered DNA double helix structure in 1953, nucleic acid drugs for disease treatment were successfully marketed over 1998 for about half a century. The nucleic acid medicine is DNA or RNA with disease treating function, and the medicine can block the expression of corresponding functional gene and the transmission of molecular information and act on pathogenic gene or mRNA effectively and specifically. Compared with the traditional micromolecular medicines and antibody medicines, the nucleic acid medicine can realize the expression of radically regulating and controlling pathogenic genes, can achieve the sequence specificity on the level of a single base, and has the characteristics of treating both symptoms and root causes. The nucleic acid medicine has the advantages of high treatment efficiency, low medicine toxicity, strong specificity, wide application field and the like.

Therapeutic nucleic acids include: messenger RNA (mRNA), antisense oligonucleotides, small interfering nucleic acids (siRNA), micro nucleic acids (miRNA), and aptamers (aptamers). mRNA is transcribed by using a single strand of DNA as a template, is single-stranded nucleotide which carries genetic information and guides protein synthesis, can correct gene expression defects or abnormalities by exogenously introducing mRNA, and has the advantages of low cost, short development cycle, fast protein expression and the like. The delivery barrier of RNA drugs is specifically as follows: 1. the Chinese medicinal composition has poor medicinal properties, high hydrophilicity, high density of negative charges and poor stability, and is easy to degrade by plasma RNA enzyme; 2. the ability of cell membranes to block free RNA from entering intracellular compartments where relevant translation mechanisms exist is limited. Chemical modification and nano-carrier technology are key technologies for clinical transformation of RNA drugs, so that the RNA drugs are protected and the biomembrane barrier is effectively overcome.

Lipid nanoparticles formed from cationic lipids, other lipid components (e.g., neutral lipids, steroids, pegylated lipids), and nucleic acids have been used to prevent degradation of RNA in plasma and facilitate intracellular delivery of nucleic acids. PEG lipids, cationic lipids, and lipid nanoparticles for delivery of nucleic acids can have a large room for improvement and development in terms of physicochemical properties (pKa, stability), in vivo nucleic acid protection and efficient delivery, safety, and the like. The development of new functionalized lipid compounds and optimized formulations of lipid nanoparticles that can protect nucleic acids from degradation and clearance in serum, that are suitable for systemic delivery, and that provide intracellular delivery of nucleic acids, while these lipid-nucleic acid particles should be well-tolerated and provide sufficient therapeutic index to treat with an effective dose of nucleic acids that does not result in unacceptable toxicity and/or risk to the patient.

Disclosure of Invention

The invention provides a novel PEG lipid compound shown as a formula A, or an isomer, a pharmaceutically acceptable salt and a prodrug thereof, and also discloses a preparation method, a composition and an application of the PEG lipid compound. The functional PEG lipid provided by the invention can realize better physicochemical property-stability and intracellular delivery efficiency of nucleic acid.

The invention provides a pegylated lipid shown as a formula A, or an isomer thereof, or a pharmaceutically acceptable salt and a prodrug thereof;

wherein:

R ₁ and R ₂ Each independently a linear or branched, saturated or unsaturated hydrocarbyl chain containing from 10 to 30 carbon atoms, wherein said hydrocarbyl chains are optionally linked by one or more ester linkages; w has an average value of 30 to 60.

The pegylated lipids of the present invention, wherein R ₁ And R ₁ Each independently a linear, saturated or unsaturated hydrocarbyl chain containing from 12 to 22 carbon atoms.

The average value of w of the pegylated lipids of the present invention is 40 to 50.

The compound of the invention is characterized in that: wherein R is ₁ And R ₂ Or both have one of the following structures:

the preparation method of the polyethylene glycol lipid compound A comprises the following steps:

(1) In an organic solvent, carrying out condensation ring-opening reaction on a compound H, ammonia water and benzaldehyde to prepare an intermediate G, and carrying out deprotection reaction under the catalysis of hydrochloric acid to prepare an intermediate F;

(2) In an organic solvent, under the action of an acid-binding agent, carrying out condensation reaction on the intermediate F and acetic anhydride to obtain an intermediate E;

(3) In an organic solvent, under the action of an acid-binding agent, carrying out substitution and alcoholysis reaction on the intermediate D with the general formula and the intermediate E to obtain an intermediate C with the general formula;

(4) In an organic solvent, under the action of a condensing agent, carrying out condensation reaction on the general formula intermediate C and the general formula compound B to prepare a polyethylene glycol lipid compound with a general formula A;

compositions comprising the PEG lipid compounds and therapeutic agents are disclosed. The composition further comprises one or more components selected from the group consisting of cationic lipids, neutral lipids, and steroids.

The neutral lipid in the composition comprises one or more components of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM, preferably DSPC. The molar ratio of cationic lipid to neutral lipid in the composition is from about 2 to 8.

The steroid in the composition of the present invention comprises one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, preferably cholesterol. The molar ratio of cationic lipid and steroid in the composition is from about 1 to 5.

The molar ratio of cationic lipid and said pegylated lipid in said composition of the invention is from about 100.

The therapeutic agent in the composition of the invention is a nucleic acid selected from the group consisting of antisense nucleic acids, small interfering nucleic acids (siRNA), micro nucleic acids (miRNA), and messenger nucleic acids (mRNA).

The invention discloses the use of said composition in the manufacture of a medicament or vaccine for the treatment of infectious, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative, cardiovascular and renal vascular and metabolic diseases. The composition, the route of administration to the patient comprising: intravenous, intramuscular, subcutaneous, intradermal, intranasal or inhalation administration.

The cationic lipid in the composition of the invention has the following structure (formula I):

wherein:

L ₁ selected from the following structures: -C-, -O (C = O) -, - (C = O) O-, -C (= O) -, -O-, -S (O) _X -、-S-S-、-C(＝O)S-、-SC(＝O)-、-N-C(＝O)-、-C(＝O)-N-，L ₂ Selected from the following structures: -C-, - (C = O) O-, -C (= O) -, -S (O) _X -、-C(＝O)S-、-C(＝O)-N-；

R ₁ And R ₂ Each independently is C ₆ -C ₂₄ Alkyl or C ₆ -C ₂₄ Alkenyl, said hydrocarbyl chain optionally being linked by one or more ester or ether linkages; r is ₃ And R ₄ Each independently is C ₁ -C ₁₂ Alkyl or C ₁ -C ₁₂ Alkenyl, or R ₃ And R ₄ Combine with each other to form a 4-to 10-membered heterocyclic ring, the heteroatoms comprising one or more heteroatoms of N, O, S, the heterocyclic ring being optionally substituted with 1-6 heteroatoms;

x is C, N, O, S, -S-S-; m is C ₁ -C ₁₂ Alkyl or C ₁ -C ₁₂ An alkenyl group; x is 0, 1 or 2.

The cationic lipid compounds in the compositions described herein include all cationic lipid compounds disclosed in the following patent application nos.: CN2021109304214, CN2021109693918, CN2021109698790, CN2021109700095, CN202110970112X, CN2021109787347, CN2021110306263, CN2021110307694, CN2021110439195, CN2021110310644, CN2021110311986, CN2021111108325, CN2021111057272.

The above-mentioned conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention without departing from the common general knowledge in the field.

The positive benefit of the invention is that: the novel PEG lipid compound can realize better physicochemical property-stability and intracellular delivery efficiency of nucleic acid, is easy to metabolize in vivo and has hydrophilic and low-toxicity metabolites.

The following examples are provided for the purpose of illustration and are not intended to be limiting.

The following examples, unless otherwise indicated, all solvents and reagents used were commercially available and used as received.

The procedures described below can be used to synthesize the compounds contained in Table 1

The following abbreviations are used herein:

DCM dichloromethane

THF: tetrahydrofuran (THF)

DMF: n, N-dimethylformamide

Ac2O acetic anhydride

HATU N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) urea hexafluorophosphate

DMAP 4-dimethylaminopyridine

DIEA: n, N-diisopropylethylamine

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Example 1

Representative synthetic route to PEG lipids

Preparation of the Compound PEGL-3-3 (reference: ORG. PROCESS. RES. DEV.2003,7 (4), 533-546,)

To benzaldehyde (59.54g, 0.561mol, 1.03eq) in ethanol (150 mL) at 18 ℃ was added aqueous ammonia (28.8 wt%,49.9g,0.861mol, 1.58eq) and rinsed with ethanol (6 mL). Epichlorohydrin (50.5g, 0.546 mol) was added with stirring, and after the addition, the mixture was rinsed with ethanol (22 mL). Heating the reaction solution to 40 ℃ for 1 hour, controlling the temperature at 35-40 ℃, keeping the temperature, stirring for 6 hours, cooling to 20-25 ℃, and stirring for reaction for 13.5 hours. The raw epichlorohydrin was detected by GC to be about 1.4% residual. The reaction mixture was concentrated under reduced pressure to 133mL, and 115mL of toluene was added. An aqueous solution (77 mL) of concentrated hydrochloric acid (37 wt%,80.6g,0.827mol, 1.52eq) was slowly added over a period of about 5 minutes while maintaining the temperature at 36-41 ℃. Stirring the obtained two-phase mixed solution at 35-45 ℃ for 3 hours under the condition of heat preservation, separating liquid, washing an upper water layer with water (28 mL), combining water phases, adding 28mL of ethanol, concentrating under reduced pressure to 95mL, repeating the steaming for 7 times, and distilling under reduced pressure to 95mL each time. After the last steaming, 95mL of ethanol is added. The resulting suspension was heated to reflux, cooled to-25 ℃ and incubated for crystallization for 18 hours. The solid was collected by filtration and washed with 28mL of cold ethanol at-25 ℃. Nitrogen flow drying at room temperature gave 61.4g of white solid in 77.0% yield.

LC-MS(ESI ⁺ ):mass calculated for C ₃ H ₈ ClNO,109.03,m/z found 110.03,[M+H] ⁺

HNMR(CD ₃ OD,400MHz):δ4.85(s,4H)，4.2-4.0(m,1H),3.66-3.57(m,2H),3.22(dd,J＝17.2,3.6Hz,1H)，2.94(dd,J＝12.4,16.8Hz,1H)

Synthesis of Compound PEGL-3-4 (reference: ORG. PROCESS. RES. DEV.2003,7 (4), 533-546,)

To a suspension of compound PEGL-3-3 (20.0 g, 0.137mol) in DCM (50 mL) was added acetic anhydride (32.2 g,0.315mol, 2.3eq). The resulting suspension was warmed to 38 deg.C, pyridine (14.0 g,0.173mol, 1.26eq) was added, controlling the temperature at 36-38 deg.C and, after addition was complete, rinsed with DCM (32 mL). The obtained solution is kept at the temperature of 37-40 ℃ and stirred for reaction for 5 hours, and then the temperature is reduced to 20-25 ℃ and stirred for reaction for 14 hours. GC showed about 0.9% of amino monoacylated intermediate remaining. After the reaction, water (24 mL) was added, the reaction solution was cooled to 0-5 ℃ and potassium carbonate solution (47 wt%,80g,0.27mol, 1.98eq) was slowly added, and the temperature was controlled at 5-7 ℃ for about 7 minutes. Water (65 mL) and DCM (25 mL) were added, the mixture was stirred, the aqueous phase was extracted with DCM (12mL. Times.2), the organic phases were combined, washed with saturated sodium chloride, and the aqueous phase was back-extracted with DCM. The organic phases were combined and concentrated to 540mL under reduced pressure. Toluene was added to 40mL portions and the portions were steamed 2 times to 54mL portions. And after the steaming is finished, cooling to 28 ℃, separating out solid, adding 90mL of isooctane, cooling to 3 ℃, preserving heat, crystallizing for 1 hour, filtering, leaching 28mL of filter cake isooctane, and drying by nitrogen flow to obtain 22g of white solid with the yield of 82.9%.

LC-MS(ESI ⁺ ):mass calculated for C ₇ H ₁₂ ClNO ₃ ,193.05,m/z found 194.05,[M+H] ⁺

HNMR(CDCl ₃ ,400MHz):δ6.20(bs,4H),5.09(p,J＝4.8Hz，1H),3.70(dd,J＝12.0,4.8Hz,1H),3.64-3.47(m,3H),2.12(s,3H),2.00(s,3H)

Preparation of Compound PEGL-3-5

A mixed solution of polyethylene glycol monomethyl ether (68.2g, PEG MW about 2000, n = about 45,34.1mmol, 1.2eq) in THF (55 mL) and DMF (10 mL) is ice-cooled at 0-10 deg.C, naH (60% mass fraction, 1.4g,34.1mmol, 1.2eq) is added, stirring is carried out at constant temperature for 0.5hr, a THF solution (10 mL) of PEGL-3-4 (5.5g, 28.4mmol, 1.0eq) is slowly added, and the mixture is naturally warmed to room temperature after the addition and stirred for 18 hours. TLC showed that PEGL-3-4 was essentially complete, the reaction was poured slowly into saturated ammonium chloride solution. THF was added and extracted 3 times. The organic phases were combined and washed with saturated sodium chloride. The organic phase was stirred with silica gel and purified by column chromatography (EtOAc: PE = 5)

The semi-solid obtained above was dissolved in methanol (350 mL), naOH (0.8g, 22.2mmol, 1.2eq) was added, and the reaction was allowed to proceed for 0.5 hour at 50-55 ℃ with incubation. After TLC detection, water (110 mL) is added into the reaction solution, the volume is reduced to 250mL, solid is separated out, the mixture is filtered after being pulped for 2 hours, the filter cake is washed by water and is dried in vacuum to obtain 30.6g, and the yield is 80%.

HNMR(CDCl ₃ ,400MHz):δ3.78-3.80(m,3H),3.52-3.56(m,180-200H),3.78-3.80(m,1H),3.38(s,3H)，2.80-2.82(m,2H)

Preparation of Compound PEGL-3

HATU (0.66g, 1.74mmol, 2.2eq) and DIEA (0.44g, 3.48mmol, 4.4eq) were added to 15mL DCM (15 mL) of tetradecanoic acid (0.5g, 1.74mmol, 2.2eq) at 0-10 deg.C, and after the reaction was kept at constant temperature and stirred for 0.5 hour, PEGL-3-5 (1.65g, 0.79mmol, 1.0eq) was added, and after the addition was completed, the reaction was naturally stirred at constant temperature for 18 hours. TLC detects the reaction is complete, DCM and saturated sodium chloride are added, liquid separation is carried out by stirring, and the organic phase is washed with sodium chloride for 2 times. Mixing organic phase silica gel, purifying by column chromatography to obtain white solid 1.28g, yield 65%

HNMR(DMSO-d6,400MHz):δ8.01(bs,1H),5.49(m,1H),3.72-3.78(m,1H),3.62-3.65(m,1H),3.51-3.56(m,180-200H),3.46-3.48(m,1H),3.38(s,3H),3.33-3.36(m,1H),2.35-2.38(m,2H),2.10-2.15(m,2H),1.52-1.60(m,4H),1.27-1.33(m,44H),0.88(t,J＝6.6Hz,6H)

By employing the above-described techniques and related procedures, which are well known to those skilled in the art, and using the corresponding fatty acids, according to the preparation method of example 1, the following compounds PEGL-1 through PEGL-11 can be prepared:

TABLE 1 novel PEG lipid Compounds

(n＝45)

Example 2

PEGL-1 (Compound 1, table 1), white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),5.47(m,1H),3.71-3.76(m,1H),3.62-3.64(m,1H),3.52-3.57(m,180-200H),3.46-3.49(m,1H),3.35(s,3H),3.32-3.36(m,1H),2.35-2.39(m,2H),2.13-2.16(m,2H),1.52-1.61(m,4H),1.26-1.35(m,36H),0.88(t,J＝6.6Hz,6H)

Example 3

PEGL-2 (Compound 2, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),5.48(m,1H),3.72-3.78(m,1H),3.62-3.65(m,1H),3.50-3.56(m,180-200H),3.44-3.48(m,1H),3.38(s,3H),3.32-3.36(m,1H),2.35-2.39(m,2H),2.12-2.15(m,2H),1.53-1.60(m,4H),1.25-1.33(m,40H),0.88(t,J＝6.6Hz,6H)

Example 4

PEGL-4 (Compound 4, table 1), white solid

HNMR((DMSO-d6,400MHz):δ8.02(bs,1H),5.48(m,1H),3.70-3.78(m,1H),3.62-3.66(m,1H),3.51-3.56(m,180-200H),3.46-3.50(m,1H),3.38(s,3H),3.33-3.36(m,1H),2.35-2.38(m,2H),2.13-2.16(m,2H),1.52-1.60(m,4H),1.23-1.33(m,48H),0.87(t,J＝6.6Hz,6H)

Example 5

PEGL-5 (Compound 5, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.02(bs,1H),5.49(m,1H),3.71-3.78(m,1H),3.62-3.65(m,1H),3.52-3.56(m,180-200H),3.46-3.48(m,1H),3.38(s,3H),3.34-3.36(m,1H),2.35-2.37(m,2H),2.13-2.15(m,2H),1.53-1.62(m,4H),1.27-1.33(m,52H),0.88(t,J＝6.6Hz,6H)

Example 6

PEGL-6 (Compound 6, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),5.47(m,1H),3.71-3.79(m,1H),3.62-3.65(m,1H),3.51-3.56(m,180-200H),3.46-3.48(m,1H),3.38(s,3H),3.34-3.36(m,1H),2.35-2.37(m,2H),2.13-2.15(m,2H),1.53-1.61(m,4H),1.26-1.34(m,56H),0.88(t,J＝6.6Hz,6H)

Example 7

PEGL-7 (Compound 7, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.02(bs,1H),5.49(m,1H),3.73-3.78(m,1H),3.62-3.65(m,1H),3.52-3.58(m,180-200H),3.46-3.48(m,1H),3.38(s,3H),3.34-3.36(m,1H),2.35-2.37(m,2H),2.13-2.15(m,2H),1.53-1.60(m,4H),1.26-1.33(m,60H),0.88(t,J＝6.6Hz,6H)

Example 8

PEGL-8 (Compound 8, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),5.48(m,5H),5.35-5.37(m,4H),3.72-3.78(m,1H),3.62-3.66(m,1H),3.52-3.57(m,180-200H),3.46-3.48(m,1H),3.38(s,3H),3.34-3.36(m,1H),2.80(t,J＝8.8Hz,4H),2.35-2.37(m,2H),2.13-2.16(m,10H),1.55-1.60(m,4H),1.26-1.39(m,28H),0.89(t,J＝6.6Hz,6H)

Example 9

PEGL-9 (Compound 9, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),3.71-3.76(m,1H),3.62-3.66(m,1H),3.52-3.57(m,180-200H),3.46-3.49(m,1H),3.38(s,3H),3.33-3.36(m,1H),2.27-2.14(m,2H),1.49-1.60(m,8H),1.27-1.33(m,24H),0.88(t,J＝6.6Hz,6H)

Example 10

PEGL-10 (Compound 10, table 1), white solid

HNMR((DMSO-d6,400MHz):δ8.02(bs,1H),3.71-3.78(m,1H),3.62-3.65(m,1H),3.52-3.56(m,180-200H),3.46-3.48(m,1H),3.38(s,3H),3.34-3.36(m,1H),2.27-2.13(m,2H),1.49-1.60(m,8H),1.26-1.33(m,32H),0.88(t,J＝6.6Hz,6H)

Example 11

PEGL-11 (Compound 11, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),3.71-3.77(m,1H),3.62-3.64(m,1H),3.52-3.54(m,180-200H),3.46-3.49(m,1H),3.35(s,3H),3.34-3.36(m,1H),2.27-2.15(m,2H),1.49-1.60(m,8H),1.26-1.33(m,36H),0.88(t,J＝6.6Hz,6H)

Example 12

Preparation of mono-9-heptadecyl adipate

EDCI (2.0g, 10.3mmol, 1.2eq) and a catalytic amount of DMAP (0.2g, 1.7mmol, 0.2eq) were added to a solution of adipic acid (1.5g, 10.3mmol, 1.2eq) in DCM (20 mL) at 0-10 ℃ and the reaction was stirred for 0.5 hour. 9-heptadecanol (2.2g, 8.6mmol, 1.0eq) was added to the reaction system, and after the addition, the reaction was stirred at natural temperature for 18 hours. After TLC detection, DCM and 2N sodium hydroxide are added into the reaction system, liquid separation is carried out by stirring, and the water tank is washed for 2 times by 2N sodium hydroxide. Mixing organic phase silica gel, and purifying by vermilion chromatography to obtain 2.7g of adipic acid mono-9-heptadecyl ester as light yellow oily substance with yield of 84%.

LC-MS(ESI ^- ):mass calculated for C ₂₃ H ₄₄ O ₄ 384.13,m/z found:383.03,[M-H] ^-

HNMR(CDCl ₃ ,400MHz):δ11.87(bs,1H),4.47-4.49(m,1H),2.32(t,J＝6.4Hz,2H),1.64-1.66(m,2H),1.26-1.29(m,24H),0.88(t,J＝6.6Hz,6H)

Example 13

The following compounds can be prepared according to the preparation method of example 1 by using the above-mentioned techniques and related procedures well known to those skilled in the art and using mono-9-heptadecyl adipate.

PEGL-12 (Compound 12, table 1), a white solid

HNMR((DMSO-d6,400MHz):δ8.01(bs,1H),4.46-4.49(m,1H),3.73-3.78(m,1H),3.62-3.65(m,1H),3.52-3.58(m,180-200H),3.45-3.48(m,1H),3.38(s,3H),3.34-3.37(m,1H),2.32-2.37(m,6H),2.27-2.11(m,2H),1.52-1.64(m,8H),1.46-1.49(m,8H),1.25-1.33(m,48H),0.88(t,J＝6.6Hz,12H)

Example 14

Luciferase mRNA in vivo evaluation using lipid nanoparticle composition

The Cationic Lipid (CLPP), DSPC, cholesterol and PEG-lipid (shown in table 1) were dissolved in ethanol at a molar ratio of 50. Lipid Nanoparticles (LNPs) were prepared at a weight ratio of total lipid to mRNA of about 10. Briefly, mRNA was diluted to 0.15mg/mL in 10mL to 50mL citrate buffer (pH = 4). The lipid ethanol solution and the mRNA aqueous solution are mixed using a syringe pump at a ratio of about 1 to 1 (volume/volume) in the range of 10mL/min or more for the total flow rate. The ethanol was then removed and the external buffer was replaced by PBS by dialysis. Finally, the lipid nanoparticles were filtered through a sterile filter with a pore size of 0.2 μm. The particle size of the lipid nanoparticles as determined by quasielastic light scattering using a Malvern Zetasizer Nano ZS was approximately 65-105nm in diameter, and in some cases approximately 75-100nm in diameter.

The study was carried out on 6-8 week old female C57BL/6 mice, 8-10 week old CD-1 mice, according to the guidelines set by the national institute of science and technology. Different doses of mRNA lipid nanoparticles were administered systemically by tail vein injection and animals were euthanized at specific time points (e.g., 5 hours) post-dose. Liver and spleen were collected in pre-weighed tubes, weighed, immediately snap frozen in liquid nitrogen, and stored at-80 ℃ until used for analysis.

For the liver, approximately 50mg was cut for analysis in 2mL FastPrep tubes (MP Biomedicals, solon OH). 1/4 "ceramic spheres (MP Biomedicals) were added to each tube, and 500. Mu.L of Glo lysis buffer-GLB (Promega, madison Wis.) equilibrated to room temperature was added to the liver tissue. Liver tissue was homogenized at 2x6.0 m/s for 15 seconds using a FastPrep24 instrument (MP Biomedicals). The homogenate was incubated at room temperature for 5 minutes, then diluted 1. Specifically, 50. Mu.L of the diluted tissue homogenate was reacted with 50. Mu.L of SteadyGlo substrate, shaken for 10 seconds, followed by incubation for 5 minutes, and then quantified using a SpectraMAX _ L chemiluminescence-type microplate reader (Meigu Motors, inc.). The amount of the protein determined was determined by using BCA protein quantification kit (shanghai chromophil medical science and technology ltd). The Relative Luminescence Units (RLU) were then normalized to the total μ g of protein assayed. To convert RLU to μ g luciferase, a standard curve was generated with QuantiLum recombinant luciferase (Promega).

Fluciferase protein will be expressed by Fluuc mRNA from Trilink Biotechnologies (L-6107), which was originally isolated from fireflies (Photinus pyralis). Fluc is commonly used in mammalian cell cultures to measure gene expression and cell viability. Which emits bioluminescence in the presence of the substrate luciferin. This capped and polyadenylated mRNA was completely replaced by 5-methylcytidine and pseudouridine.

Example 15

Comparative Activity of PEG lipids

Table 2 shows the comparative activity of different PEG lipids. LNP was formulated using the following molar ratios: 50% Cationic Lipid (CLPP)/10% Distearoylphosphatidylcholine (DSPC)/40% cholesterol/10% PEG-lipid. Activity was determined by measuring luciferase expression in the liver over 4 hours after tail vein injection administration as described in example 14. The activity was compared at doses of 0.3, 1.0mg/kg and expressed as ng luciferase/g liver measured 4 hours after administration as described in example 14.

TABLE 2 LNP mRNA activity prepared from different PEG lipids (0.3 &1.0mg/kg)

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(n＝45)

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent disclosure. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Claims

1. A PEGylated lipid represented by formula A, or its isomer, or its pharmaceutically acceptable salt, prodrug;

wherein:

R ₁ and R ₂ Each independently a linear or branched, saturated or unsaturated hydrocarbyl chain containing from 10 to 30 carbon atoms, wherein said hydrocarbyl chains are optionally linked by one or more ester linkages;

w has an average value of 30 to 60.

2. The pegylated lipid of claim 1, wherein R ₁ And R ₁ Each independently a linear, saturated or unsaturated hydrocarbyl chain containing from 12 to 22 carbon atoms.

3. The pegylated lipid of claim 1, wherein w has an average value of from 40 to 50.

4. The compound of claim 1, wherein: wherein R1 and R2 or both have one of the following structures:

5. a process for the preparation of a pegylated lipid compound A, as defined in any one of claims 1 to 4, comprising the steps of:

6. a composition comprising a compound of any one of claims 1-5 and a therapeutic agent.

7. The composition of claim 6, further comprising one or more components selected from the group consisting of cationic lipids, neutral lipids, and steroids.

8. The composition of claim 7, wherein the neutral lipids comprise one or more components of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM, preferably DSPC.

9. The composition of any one of claims 6-8, wherein the molar ratio of the cationic lipid and the neutral lipid is from about 2 to 1.

10. The composition according to any one of claims 6-9, wherein the steroid comprises one or more of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, preferably cholesterol.

11. The composition of claim 10, wherein the molar ratio of cationic lipid to steroid is from about 1.

12. The composition of any one of claims 6-11, wherein the molar ratio of cationic lipid and pegylated lipid is from about 100 to about 20.

13. The composition of any one of claims 6-12, wherein the therapeutic agent is a nucleic acid.

14. The composition of any one of claims 13, wherein the nucleic acid is selected from the group consisting of an antisense nucleic acid, a small interfering nucleic acid (siRNA), a micro nucleic acid (miRNA), and a messenger nucleic acid (mRNA).

15. Use of a composition according to any one of claims 6 to 14 in the manufacture of a medicament or vaccine for the treatment of infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular and renal vascular diseases and metabolic diseases.

16. The composition of any one of claims 6-15, administered to a patient by a route of administration comprising: intravenous, intramuscular, subcutaneous, intradermal, intranasal or inhalation administration.

17. The composition of any one of claims 6-16, wherein the cationic lipid has the following structure (formula I):

wherein:

R ₁ And R ₂ Each independently is C ₆ -C ₂₄ Alkyl or C ₆ -C ₂₄ Alkenyl, said hydrocarbyl chain optionally being linked by one or more ester or ether linkages;

R ₃ and R ₄ Each independently is C ₁ -C ₁₂ Alkyl or C ₁ -C ₁₂ Alkenyl, or R ₃ And R ₄ Combine with each other to form a 4-to 10-membered heterocyclic ring, the heteroatoms including one or more of N, O, S, the heterocyclic ring being optionally substituted with 1-6 heteroatoms;

x is C, N, O, S, -S-S-;

m is C ₁ -C ₁₂ Alkyl or C ₁ -C ₁₂ An alkenyl group;

x is 0, 1 or 2.