CN109701009A - Vaccine preparation and its application - Google Patents
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- CN109701009A CN109701009A CN201910005563.2A CN201910005563A CN109701009A CN 109701009 A CN109701009 A CN 109701009A CN 201910005563 A CN201910005563 A CN 201910005563A CN 109701009 A CN109701009 A CN 109701009A
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Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to vaccines arts, a kind of vaccine preparation and its application are disclosed.Vaccine preparation provided by the invention is by the way that dependence producing drug haptens and adjuvant to be combined to obtain in a manner of being covalently attached or mix self assembly, structure is simple, immune performance is good (IgG antibody for causing high titre), and thermal stability is good, is easy to storage and transport.
Description
Technical Field
The invention relates to the field of vaccines, in particular to a vaccine preparation and application thereof.
Background
The abuse of the addictive drugs seriously affects the physical health and causes huge loss and waste of social wealth. At present, psychological treatment and drug combination are usually adopted for treatment, however, the effect of the treatment method is not satisfactory, and the search for more effective methods is still a challenge in modern medicine. Vaccines may be a unique, potentially attractive treatment to stimulate the immune system and produce corresponding specific antibodies.
It is often necessary to link the addictive substances to a carrier protein because their molecular weight is too small to stimulate the immune system to produce antibodies. Semi-synthetic vaccines of hapten-protein complexes (hapten-proteins) are currently the most common method for the production of IgG antibodies. Covalent attachment of haptens to proteins containing T cell epitopes allows the haptens to be recognised by the immune system, resulting in the production of high affinity IgG antibodies. The antibody generated by the vaccine can be combined with antigen to form an antibody-antigen complex, the complex has large molecular weight and cannot pass through a blood brain barrier, and the dependence of the brain on drugs is reduced, so that the aim of controlling drug addiction is fulfilled. Wherein the protein carrier comprises Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), chicken Ovalbumin (OVA), diphtheria CRM197 protein, etc. Semi-synthetic vaccines have some disadvantages, such as uncertain conjugation site, unstable conjugation rate, and the protein itself causing some immune response. In addition, the hapten-protein complex has poor thermal stability, which imposes additional burden on storage and transportation.
The study of fully synthetic vaccine molecules has received much attention in recent years. The classical total synthetic vaccine molecule is composed of hapten, helper T cell epitope (Th epitope) peptide and Toll-like receptor (TLR) agonist through covalent coupling. It has been reported that the immunological performance of a vaccine molecule can be improved by constructing several different Th epitopes in the same vaccine molecule. In addition, the Major Histocompatibility Complex (MHC) has polymorphism, and different individuals require different Th epitope peptides and MHC binding, so that a universal vaccine molecule requires introduction of multiple Th epitope peptides. As the components increase, the difficulty of organic synthesis also increases, which poses a great challenge to industrial production.
Disclosure of Invention
The invention aims to overcome the problems that in the prior art, an addictive drug vaccine generates IgG antibody through a semi-synthetic vaccine of a hapten-protein complex, so that coupling sites are uncertain, coupling rate is unstable, protein can cause certain immune reaction, and the heat stability of the hapten-protein complex is poor.
In order to achieve the above object, the present invention provides, in one aspect, a vaccine formulation comprising:
component (a): a hapten, the hapten being an addictive drug;
a component (b): a linker unit covalently linked at one end to the component (a) to form components a-b;
a component (c): an adjuvant selected from at least one of the compounds represented by formula (1) to formula (4);
wherein,
the component (c) is covalently linked with the other end of the component (b) in the components a-b to form the effective component shown as a-b-c; or
The preparation also comprises a component (d): a carrier which is covalently linked to the other end of the component (b) to form a component a-b-d, and the component a-b-d and the component (c) are mixed and self-assembled into an effective component represented by a-b-d/c;
each X in formulas (1) to (4) is independently selected from-CH3、-NH2-OH or-SH, each Y is independently selected from-CH2-, -NH-, -O-or-S-;
r is selected from
One of (1);
and the linking unit is selected from structural units represented by formula (5) to formula (16):
in formula (5) -formula (16), m1-m5、n1-n7、p1、p2And q is1Each independently is any one integer of 1-60, X1-X7、Y1-Y9Each independently selected from-CH2-, -NH-, -O-or-S-;
and the carrier is selected from the group consisting of those represented by the formulae (17) to (20),
in the formulae (17) to (20), each m is independently any one integer of 0 to 30, each n is independently any one integer of 0 to 6, and each X is8Each independently selected from-CH2-, -NH-, -O-or-S-.
In a second aspect, the invention provides the use of the vaccine preparation in the preparation of a medicament for the treatment of addiction.
Through the technical scheme, the vaccine preparation provided by the invention can simplify the molecular structure of the vaccine and ensure the generation of IgG antibodies with high titer and high affinity; the vaccine has good molecular heat stability, and is easy to store and transport.
Drawings
FIG. 1 is a graph showing the results of evaluation of anti-nicotine titer produced by vaccine preparations A-E, F1-F3, wherein a) is the result of evaluation of IgG antibody titer, b) is the result of evaluation of IgM antibody titer, c) is the result of evaluation of IgG1 antibody titer, d) is the result of evaluation of IgG3 antibody titer, e) is the result of evaluation of IgG2a antibody titer, and F) is the result of evaluation of IgG2b antibody titer;
FIG. 2 is a result of evaluation of anti-nicotine titer produced by the vaccine preparations obtained in examples 7 to 11, wherein a) is a result of evaluation of IgG antibody titer, b) is a result of evaluation of IgM antibody titer, c) is a result of evaluation of IgG1 antibody, IgG3 antibody, IgG2a antibody, IgG2b antibody titer produced by the vaccine preparations after 42 d;
FIG. 3 is a graph showing the results of the affinity assay for the antibodies generated by vaccine formulation A-E, F1-F3;
FIG. 4 shows the results of the affinity determination of the antibodies produced by the vaccine preparations obtained in examples 7 to 11;
FIG. 5 is the in vivo cytokine assay of vaccine formulation A-E, F1-F3, wherein the left panel is the interferon- γ (24h) assay and the right panel is the interleukin-4 (2h) assay;
FIG. 6 is the results of the hotplate test of vaccine formulation A-E, F1-F3;
FIG. 7 is the results of the hot plate test of the vaccine formulations obtained in examples 7-11;
FIG. 8 is a test result of the body temperature of mice after injection of vaccine formulation A-E, F1-F3;
FIG. 9 is a test result of the body temperature of mice after injection of the vaccine preparations obtained in examples 7 to 11;
FIG. 10 shows the results of body weight change measurements of mice injected with vaccine formulations A-E, F1-F3;
FIG. 11 is the results of measurement of body weight change of mice after injection of the vaccine preparations obtained in examples 7 to 11.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect the present invention provides a vaccine formulation comprising:
component (a): a hapten, the hapten being an addictive drug;
a component (b): a linker unit covalently linked at one end to the component (a) to form components a-b;
a component (c): an adjuvant selected from at least one of the compounds represented by formula (1) to formula (4);
wherein,
the component (c) is covalently linked with the other end of the component (b) in the components a-b to form the effective component shown as a-b-c; or
The preparation also comprises a component (d): a carrier which is covalently linked to the other end of the component (b) to form a component a-b-d, and the component a-b-d and the component (c) are mixed and self-assembled into an effective component represented by a-b-d/c;
each X in formulas (1) to (4) is independently selected from-CH3、-NH2-OH or-SH, each Y is independently selected from-CH2-, -NH-, -O-or-S-;
r is selected from
One of (1);
and the linking unit is selected from structural units represented by formula (5) to formula (16):
in formula (5) -formula (16), m1-m5、n1-n7、p1、p2And q is1Each independently is any one integer of 1-60, X1-X7、Y1-Y9Each independently selected from-CH2-, -NH-, -O-or-S-;
and the carrier is selected from the group consisting of those represented by the formulae (17) to (20),
in the formulae (17) to (20), each m is independently any one integer of 0 to 30, each n is independently any one integer of 0 to 6, and each X is8Each independently selected from-CH2-, -NH-, -O-or-S-.
In the invention, the effective components comprise two composition modes:
1) the component (c) is covalently linked with the other end of the component (b) in the components a-b to form the effective component shown as a-b-c;
2) the component (d) is covalently linked to the other end of the component (b) in the components a-b to form the components a-b-d, and the components a-b-d and the component (c) are mixed and self-assembled into an effective component represented by a-b-d/c. In the present invention, the manner of the hybrid self-assembly may be various conventional manners, and may include, for example: stirring, vortexing, shaking or inverting repeatedly, etc.
The inventor finds in research that the compound shown in the formula (1) to the formula (4) is introduced into a vaccine preparation as an adjuvant through the two composition modes, so that the structure of the vaccine can be simplified, high-titer and high-affinity IgG antibodies can be guaranteed to be generated, a protein complex used in a semi-synthetic vaccine can be omitted, the stability of the vaccine can be improved, and the vaccine is easy to store and transport.
In the present invention, preferably, the addictive drug is selected from at least one of nicotine, methamphetamine, heroin, cocaine, fentanyl, opiates, ketamine and caffeine.
Further preferably, the structural formula of nicotine is selected from At least one of (1).
Preferably, the structural formula of the methamphetamine is selected from At least one of (1).
Preferably, said heroin has a formula selected from At least one of (1).
Preferably, the cocaine has the structural formula
Preferably, the fentanyl has the structural formula
Preferably, the structural formula of the opium is selected from At least one of (1).
Preferably, the structural formula of the ketamine is
Preferably, the caffeine has a formula of
According to a preferred embodiment of the present invention, the effective components represented by a-b-c have the following structures:
according to another preferred embodiment of the invention, in the active ingredient represented by a-b-d/c, the component a-b-d has the following structure:
and the component (c) is at least one selected from the group consisting of compounds represented by the formulae (21) to (25),
according to the invention, in the effective component represented by a-b-d/c, the content molar ratio of the component a-b-d to the component (c) is preferably 1: (0.1-10);
more preferably, the content molar ratio of the components a-b-d to the component (c) is 1: (0.2-0.8).
In the present invention, preferably, the vaccine formulation further comprises: a component (e): a lipid carrier, wherein the lipid carrier is one or more of cholesterol, 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine and 1, 2-dioleoyl lecithin. Preferably, the lipid carrier is a mixture of cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine in a molar ratio of (0.1-10): 1.
According to a preferred embodiment of the invention, the formulation comprises: the cholesterol-containing oral liquid comprises the effective components shown as a-b-c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, wherein the effective component shown as a-b-c is cholesterol, 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, 1, (1-10) and (1-10) in molar ratio.
According to another preferred embodiment of the invention, the formulation comprises: the effective component shown in a-b-d/c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, and the effective component shown in a-b-d/c is cholesterol, 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, 1, (1-10) and (1-10) in molar ratio.
The present invention provides a preferred embodiment, the vaccine formulation comprising:
the composition comprises an active ingredient represented by a-b-c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, wherein the active ingredient represented by a-b-c has the following structure:
the effective components shown in a-b-c are cholesterol 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine 1 (1-10) and (1-10) in molar ratio.
The present invention provides another preferred embodiment, the vaccine formulation comprising:
a-b-d/c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine;
wherein the components a-b-d are of the following structures:
the component (c) is at least one selected from the group consisting of compounds represented by the formulae (21) to (25),
and the content molar ratio of the components a-b-d to the component (c) is 1: (0.2-0.8);
the effective component shown as a-b-d/c is cholesterol 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine 1 (1-10) to (1-10) in molar ratio.
The present invention provides a particularly preferred embodiment, the vaccine formulation comprising:
a-b-d/c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine;
wherein the components a-b-d are of the following structures:
the component (c) is selected from compounds shown in a formula (21), a formula (25) or a formula (24),
and the content molar ratio of the components a-b-d to the component (c) is 1: (0.2-0.8);
the effective component shown as a-b-d/c is cholesterol 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine 1 (1-10) to (1-10) in molar ratio. The inventor finds in research that under the embodiment, the vaccine preparation has better immunity performance, higher IgG antibody titer and better affinity.
In a second aspect, the invention provides the use of a vaccine formulation according to the invention in the manufacture of a medicament for the treatment of addiction.
The present invention will be described in detail below by way of examples. In the following examples, the test methods used were all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The particle size and Zeta potential of the vaccine formulation were characterized by a Malvern Zetasizer ZEN 3600analyzer (Malvern Instruments ltd, Malvern, u.k.).
Example 1
The method is used for illustrating the preparation process of the components a-b-d in the effective components shown as a-b-d/c.
1) Preparation of vaccine antigen
Preparation of compound 7: dissolving compound 6(0.26mmol) in dry DMF, placing in an ice bath, stirring for 10min, protecting with argon, adding NaH (2.6mmol) into the reaction solution, reacting for 1h, dropwise adding p-methylsulfonic acid azide dissolved in dry DMF into the reaction system, stirring at room temperature overnight, detecting by TLC plate that the raw material 6 disappears, removing the solvent under reduced pressure, purifying the product by column chromatography, and eluting with an eluent in proportion of CH2Cl2Solvent was dried on methanol 10:1 to give product 7 as a pale yellow oil (90% yield).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3)δ8.57–8.17(m,2H),7.65(dt,J＝7.9,2.0Hz,1H),7.17(dd,J＝7.9,4.8Hz,1H),3.60–3.22(m,10H),3.18–3.08(m,1H),2.81(d,J＝8.2Hz,1H),2.33–2.20(m,2H),2.05(d,J＝5.3Hz,4H),1.60(dddd,J＝13.1,8.1,5.4,1.9Hz,1H)；13CNMR(400MHz,CDCl3)δ149.64,148.35,137.67,134.93,123.24,72.90,72.04,70.37,70.26,69.86,55.82,50.50,47.82,40.21,26.82；ESI-MS calcd.for C15H23N5O2 +[M]+305.19,found305.18.
preparation of compound 4: dissolving the compound 7(0.10mmol) with 5mL of MeOH, replacing the air in the bottle with argon, adding Pd/C (10 percent, 10mg), introducing hydrogen, stirring for 2-3h, monitoring by TLC that the reaction of the raw material 7 is finished, filtering the Pd/C by using kieselguhr, continuously washing with methanol for 2-3 times, combining organic phases, and performing vacuum spin-drying on the solvent to obtain the compound 8, wherein the next step is directly performed without purification. Dissolving Compound 8 in CH2Cl2MeOH (4mL,2:1, v/v), diselenyl adipate additionReacting in the reaction solution until TLC monitors that the raw materials are completely reacted, concentrating the reaction mixed solution in vacuum, and purifying by column chromatography, wherein the eluent ratio is CH2Cl210/MeOH: 1 to obtain compound 4 (70% yield in two steps).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3)δ8.60(d,J＝2.3Hz,1H),8.43(dd,J＝4.9,1.7Hz,1H),7.87(s,1H),7.45(dt,J＝7.5,2.3Hz,3H),7.33(dd,J＝5.3,2.1Hz,3H),7.27(dd,J＝7.9,4.7 Hz,1H),3.6-3.35(m,10H),3.33–3.21(m,1H),3.15–3.05(m,1H),2.70(pd,J＝6.2,5.6, 2.4Hz,2H),2.46–2.28(m,2H),2.28–2.10(m,6H),1.78–1.63(m,J＝3.6Hz,4H),1.56(s, 1H)；13C NMR(400MHz,CDCl3)δ200.16,172.73,150.72,148.11,137.94,135.75,134.83,129.29,128.84,126.36,123.77,73.76,72.88,70.41,70.25,70.21,55.84,47.37,47.19,40.30,39.36,35.90,26.64,24.89,24.87；ESI-MS calcd.For C27H38N3O4Se+[M+H]+548.20,found 548.16.
2) preparation of S2 β
Preparation of hemiselenoate compound 2: N-Boc-glycine (2.5mmol) and diphenyl diselenide were dissolved in 10mL of DMMF, and 2.5mL of PBu were added under argon3In the reaction solution, the reaction is monitored by TLC until the raw material disappears completely, the solvent is pumped off, the reaction mixture is purified by column chromatography, and the eluent ratio is PE/CH2Cl21:1, the pool was concentrated to give compound 2 as a pale yellow solid (85% yield).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3)δ7.49(d,J＝6.7Hz,2H),7.37(d,J＝6.1Hz,3H),5.45(s,1H),4.04(d,J＝6.0Hz,2H),1.53(s,9H)；13C NMR(100MHz,CDCl3)δ200.96,155.37,135.77,129.12,128.75,125.16,80.57,53.17,28.43,28.29；ESI-MS calcd.forC13H17NNaO3Se+[M+Na]+:338.03,found:337.95.
preparation of compound 3: compound 1(0.04mmol) was dissolved in wet THF/MeOH (10mL, 1: 9) and PMe was added under an argon atmosphere3(0.4mmol) and stirred at room temperature until the TLC plate monitors the disappearance of starting material. The solvent was dried by water-spinning, the residue was pumped to an oil pump and the reaction flask was placed in an environment at 30 ℃ for a further 24h to remove Me3PO, compound S1 β, was directly reacted in the next step without purification S1 β was dissolved in 3mL CH2Cl2To this solution, 15. mu.L of DIPEA was added, and the solution was dissolved in CH2Cl2Stirring at room temperature for 2 hr, removing solvent under reduced pressure, and purifying the mixture by column chromatography with eluent at CH ratio2Cl2Concentrate the pool to give 3 as a white solid (96% yield over two steps).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3)δ7.37–7.23(m,20H),7.04–6.98(m,1H),6.06(d,J＝8.7Hz,1H),5.59(s,1H),4.78(d,J＝11.2Hz,1H),4.72–4.64(m,4H),4.60(d,J＝11.8Hz,1H),4.55(d,J＝3.4Hz,2H),4.29(d,J＝7.7Hz,1H),3.88(d,J＝3.5Hz,1H),3.85–3.74(m,5H),3.70(dd,J＝17.0,5.5Hz,2H),3.64–3.56(m,2H),3.43(ddd,J＝16.0,9.1,3.6Hz,2H),3.25(ddd,J＝13.1,8.6,3.9Hz,1H),2.59(s,1H),1.81(q,J＝7.0Hz,2H),1.71–1.61(m,2H),1.42(s,9H),1.23(d,J＝16.1Hz,70H),0.90–0.84(t,6H).13C NMR(100MHz,CDCl3)δ172.90,170.16,155.71,138.22,138.16,137.42,128.33,128.29,128.23,128.18,127.92,127.83,127.70,127.60,127.52,104.36,81.10,80.33,79.61,78.78,75.13,72.90,72.40,72.00, 69.03,67.15,50.59,44.12,40.22,36.69,32.04,30.60,29.83,29.49,29.15,28.47,27.78,25.96, 25.71,22.84,14.32.ESI-MS calcd.for C57H111N3NaO11 +[M+Na]+:1397.00,found:1397.30.
of compound S2 βPreparation: dissolve Compound 3(0.039mmol) in CH2Cl2MeOH (4mL,2:1) with addition of Pd (OH)2C (25mg, 20% Pd on carbon), reaction in H2Stirring in the atmosphere until the starting material disappeared. The hydrogen in the reaction solution was replaced by argon, and then the reaction solution was passed through MeOH and CH2Cl2Pre-washed diatomaceous earth was filtered to remove Pd (OH)2Collecting the filtrate, concentrating, and purifying the mixture by column chromatography with eluent at CH ratio2Cl2Concentrate the pool to give S2 β as a white solid (82% yield).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3/CD3OD 1:1)δ4.24(s,1H),4.19(d,J＝7.2Hz,1H),4.07(d,J＝5.6Hz,1H),3.77–3.66(m,4H),3.63–3.42(m,7H),2.21(t,J＝7.6Hz,2H),1.62(m, 2H),1.46(s,9H),1.27(s,70H),0.89(t,J＝6.6Hz,6H).ESI-MS calcd.For[M+H]+:1014.82,found:1015.06.
3) covalent attachment of vaccine antigen Ends to S2 β
Dissolving the compound S2 β in CH2Cl2(4mL) and cooling to 0 ℃, adding anhydrous trifluoroacetic acid (0.013mmol), stirring the reaction solution at 0 ℃ for 15min, then moving to room temperature and stirring for 30min, removing the solvent under reduced pressure to obtain a crude product S3 β, dissolving S3 β in CH2Cl2MeOH (4mL,1:1) then CH was added2Cl2Dissolve Compound 4 (1mL) and add 50. mu.L Et3N, stirring at room temperature until the raw material S3 β is reacted completely, concentrating under reduced pressure to remove the solvent, separating the mixture by Sephadex LH-20, and eluting with CH solvent2Cl2The purified fractions were collected and lyophilized to give Nic- β GalCer as a white solid (53% yield over two steps).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3/CD3OD 1:1)δ8.55(dd,J＝14.4,3.5Hz,1H),7.95(d,J＝8.0Hz,1H),7.47(dd,J＝7.9,4.7Hz,1H),4.24(s,1H),4.21(d,J＝6.9Hz,1H),4.11(dd,J＝10.3,4.6Hz,1H),3.86(d,J＝6.3Hz,1H),3.77(d,J＝2.9Hz,1H),3.64(q,J＝7.1Hz,4H),3.60–3.45(m,11H),3.38(s,6H),2.44–2.11(m,10H),1.65(s,9H),1.27(s,70H),0.89(t,J＝6.7Hz,6H).13C NMR(100MHz,CDCl3)δ174.31,174.03,173.95,170.33,148.94,148.35,135.94,123.80,103.29,73.70,72.65,72.41,71.70,71.47,70.63,69.75,69.39,68.97,68.48, 56.75,55.16,49.78,48.60,48.38,48.17,47.96,47.74,47.53,47.32,47.11,46.08,42.06,39.23, 38.76,38.58,35.75,35.01,34.73,31.39,31.28,29.14,29.05,29.00,28.92,28.81,28.70,28.68, 25.48,25.29,24.55,24.38,21.98,16.90,13.07.ESI-MS calcd.For[M+H]+:1304.01,found: 1304.30.
example 2
To illustrate the preparation of the active ingredients shown in a-b-c.
Preparation of compound S5 α the procedure for the preparation of compound S5 α starting from 5 was similar to that of preparation 3 from 1, and the two-step reaction gave S5 α as a white solid (82% yield in two steps).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3)7.42–7.13(m,20H),6.71(d,J＝6.0Hz,1H),5.76(d,J＝8.7Hz,1H),5.54(t,J＝6.2Hz,1H),4.73(d,J＝3.8Hz,1H),4.67(dt,J＝22.3,11.8Hz,4H),4.56–4.48(m,2H),4.43(dd,J＝28.6,11.5Hz,2H),4.21(s,1H),3.87(s,1H),3.79–3.65(m,6H),3.59(q,J＝8.7,7.6Hz,3H),3.44(s,1H),3.20(dd,J＝12.1,6.4Hz,1H),2.80(s,1H),1.98–1.78(m,4H),1.65–1.49(m,3H),1.42(t,J＝8.8Hz,3H),1.36(s,9H),1.18(m,J＝7.4Hz,64H),0.82(t,J＝6.9Hz,6H).13C NMR(100MHz,CDCl3)δ173.91,171.15,156.51,138.38,138.30,137.87,137.39,128.38,128.32,128.27,128.13,128.06,127.80,127.76,127.67,127.58,127.52,97.79,79.98,79.67,77.59,75.75,74.24,73.80,71.77,71.25,68.67, 66.81,66.55,49.75,43.74,39.77,36.54,31.83,29.65,29.61,29.40,29.28,28.14,25.84,22.60, 14.01.ESI-MS calcd.for C85H135N3NaO11 +[M+Na]+:1398.00,found:1398.21.
preparation of Compound S6 α preparation of S6 α starting from S5 α (0.018mmol) was carried out in analogy to the preparation of S2 β from 3 the mixture was purified by column chromatography using CH as eluent2Cl2Concentrate the pool to give S6 α as a white solid (82% yield).
1H NMR(400MHz,CDCl3/CD3OD 1:1)δ7.51(s,1H),7.43(d,J＝8.6Hz,1H),6.37 (s,1H),4.77(d,J＝3.7Hz,1H),4.09(s,1H),3.78–3.60(m,7H),3.56–3.42(m,4H),3.27(q, J＝1.8Hz,1H),2.12(t,J＝7.7Hz,2H),1.60–1.43(m,4H),1.37(s,9H),1.18(s,68H),0.79(d,J＝7.0Hz,6H).13C NMR(100MHz,CDCl3/CD3OD 1:1)δ174.34,171.22,156.48,98.97,79.70,73.78,71.57,69.56,69.19,68.53,68.38,66.46,50.10,48.95,48.74,48.53,48.31,48.10, 47.89,47.68,43.29,39.48,36.07,31.81,31.55,29.47,29.36,29.31,29.13,29.04,28.99,27.78, 25.57,22.29,13.58.ESI-MS calcd.for C57H111N3NaO11 +[M+Na]+:1036.81,found:1037.00.
Preparation of Nic- α GalCer preparation of Nic- α GalCer starting from S6 α (0.015mmol) the procedure for the preparation of Nic- α GalCer from S2 β was similar to the preparation of Nic- β GalCer. the mixture was separated by Sephadex LH-20, eluting with CH as solvent2Cl2The purified fractions were collected and lyophilized to give Nic- α GalCer as a white solid (61% yield over two steps).
The nuclear magnetic data are:
1H NMR(400MHz,CDCl3/CD3OD 1:1)δ8.58–8.40(m,2H),7.92(d,J＝7.9Hz,1H),7.41(dd,J＝7.9,4.9Hz,1H),4.76(d,J＝3.5Hz,1H),4.09(d,J＝5.2Hz,1H),3.77–3.60(m,7H),3.56–3.35(m,13H),3.25(dd,J＝12.6,3.8Hz,4H),2.39(s,2H),2.21(d,J＝6.7Hz,3H),2.12(dd,J＝9.3,5.8Hz,5H),1.55(d,J＝7.0Hz,9H),1.17(s,70H),0.79(t,J＝6.6Hz,6H).13C NMR(100MHz,CDCl3/CD3OD 1:1)δ174.25,174.04,173.90,170.10,149.08,148.88,136.04,123.91,98.95,73.76,71.44,71.25,70.53,69.75,69.41,69.34,69.15,68.91, 68.30,68.21,66.21,56.71,55.02,49.91,48.34,48.12,47.91,47.70,47.49,47.27,47.06,41.96, 39.33,38.53,38.28,35.72,34.99,34.71,31.63,31.24,29.14,29.05,29.02,28.96,28.79,28.71, 28.67,28.64,25.31,25.22,25.06,24.55,24.34,21.95,16.86,13.04.ESI-MS calcd.for C73H134N6NaO13 +[M+Na]+:1325.99,found:1326.19.
examples 3 to 6
Dissolving 1, 2-distearoyl-Sn-propanetriyl phosphorylcholine (DSPC), Cholesterol (Cholesterol), vaccine molecules and adjuvant in CH at a molar ratio of 5:4:1:0.52Cl2The vaccine preparations A, F1, F2 and F3 are prepared according to the compositions in the table 1, wherein, the structural formula of α GalCer is shown in the specification
The particle size and potential characterization results for each vaccine formulation are shown in table 2.
Comparative examples 1 to 4
Dissolving 1, 2-distearoyl-Sn-propanetriyl phosphorylcholine (DSPC), Cholesterol (Cholesterol), vaccine molecules and adjuvant in CH at a molar ratio of 5:4:1:0.52Cl2In MeOH (v/v, 1:1), sonicated for 20 minutes, and lyophilized. Vaccine formulations B, C, D and E were prepared according to the composition in Table 1, in which the group C vaccine was first immunised with complete Freund's adjuvantThe second and third emulsification with incomplete Freund's adjuvant, and the particle size and potential characterization results of the vaccine formulations are shown in Table 2.
TABLE 1
Note: molar ratio of*Is the molar ratio of nicotine antigen, adjuvant, DSPC and cholesterol.
TABLE 2
Examples 7 to 11
Dissolving 1, 2-distearoyl-Sn-propanetriyl phosphorylcholine (DSPC), Cholesterol (Cholesterol), vaccine molecules and adjuvant in CH at a molar ratio of 5:4:1:0.52Cl2In MeOH (v/v, 1:1), sonicated for 20 minutes, and lyophilized. Vaccine formulations were prepared according to the compositions in table 3. Wherein, the adjuvants are respectively:
TABLE 3
| Nicotine antigens | Adjuvant | DSPC | Cholesterol | Molar ratio of* | |
| Example 7 | Nic-βGalcer(3.04μg) | α-C-Galcer(1μg) | 9.23μg | 3.61μg | 10:5:50:40 |
| Example 8 | Nic-βGalcer(3.13μg) | C34(1μg) | 9.48μg | 3.71μg | 10:5:50:40 |
| Example 9 | Nic-βGalcer(3.05μg) | α-Galcer(1μg) | 9.23μg | 3.61μg | 10:5:50:40 |
| Example 10 | Nic-βGalcer(3.38μg) | C20:2(1μg) | 10.27μg | 4.02μg | 10:5:50:40 |
| Example 11 | Nic-βGalcer(4.17μg) | C10:0(1μg) | 12.63μg | 4.95μg | 10:5:50:40 |
Note: molar ratio of*Is the molar ratio of nicotine antigen, adjuvant, DSPC and cholesterol.
Test example
1) Preparation of coating antigen
BSA (10mg) was dissolved in PBS (pH 7.5,2mL) buffer, Compound 4 was dissolved in DMF (100L) and slowly added to the protein solution, gently shaken in a shaker, reacted at room temperature for 24h, extracted with a small amount of EtOAc, the aqueous phase was collected and transferred to a 30kDa ultrafiltration tube for separation and concentration, and the tube was rinsed with deionized water and the concentrate was lyophilized to 20 ℃ for storage, and the resulting protein was confirmed by MALDI-TOF mass spectrometry to have 18 nicotine antigenic molecules attached to the protein.
2) Animal immunization
BALB/c female mice were purchased from the animal laboratories center of Wuhan university and the university of Huazhong agriculture for 6-8 weeks and were housed in the people hospital of Wuhan university, all of which were operated according to animal ethics guidelines. 5 mice are distributed to each group of vaccine, blood is taken from the mice before immunization as a blank control, the vaccine preparations obtained in the examples and the comparative examples are subjected to immunization injection on the 1 st day, the 15 th day and the 29 th day, blood is taken at the 2h and the 24h after the immunization on the first day for detecting the content of the cell factors, and blood is taken at the 14 th day, the 28 th day and the 42 th day for detecting the antibody titer. Blood is obtained by cutting tail, standing for 1h, centrifuging at 4 deg.C to obtain serum, and storing at-80 deg.C. Animal behavioral tests, i.e., anti-labor and body temperature tests, were performed on day 56.
3) Indirect non-competitive ELISA method for determining antibody content in blood
mu.L of Nic-BSA solution (1. mu.g/mL, pH 9.0-9.6 carbonate buffer) was coated in a 96-well plate and then incubated overnight at 4 ℃. PBST (PBS containing 0.05 vol% Tween-20) plate 3 times, then with 1.5% (w/v) BSA blocking, 37 degrees 1h incubation. The plates were washed 3 times with PBST and incubated for 1h at 37 ℃ with 0.1% BSA diluted serum. PBST plates were washed 3 times, adding 1: 5000 dilution of horseradish peroxidase-labeled goat anti-mouse secondary antibodies, IgG, IgM, IgG1, IgG2a, IgG2b and IgG3, and incubation at 37 ℃ for 1 h. Washing the PBST for 3 times, adding TMB developing solution, incubating for 15min, and adding 2mol/L H2SO4The solution was stopped and the absorbance was measured by a microplate reader at a wavelength of 450 nm. The results of the vaccine formulations at 42d for the anti-nicotine titer evaluation are shown in figure 1.
Wherein, the test adopts Nic-BSA coated plate, and the serum is obtained after 3 rd immunization. Each bar represents the mean of the titers of 5 mice, and each value is repeated independently twice, with the mean standard error being identified by the error bars. Significant differences relative to group D: p<0.05；**P<0.01；***P<0.001；****P<0.0001. The vaccines were compared with each other:##P<0.01；#P<0.05; ns, significant differences were not significant. Data analysis was performed using GraphPad Prism software (GraphPad software, San Diego, CA) by one-way analysis of variance and Turkey's posthoc testing.
From FIG. 1, it can be seen that α GalCer is used as an additional adjuvant, compared with Pam3CSK4Group B and Freund's adjuvant group C and combination α GalCer and Pam3CSK4Group E elicited higher titers of IgG and IgM antibodies, while group A produced a comparable amount of IgG as seen from the IgG subtype resultsIgG1, IgG3, and IgG2a antibodies, where IgG1 is the Th2 immune response and IgG3 and IgG2a are the Th1 immune response, indicate that group A elicits a Th1/Th2 mixed response.
The results of evaluation of anti-nicotine titer produced by the vaccine formulations obtained in examples 7 to 11 are shown in FIG. 2. From the results of fig. 2, it can be seen that the vaccine prepared in the self-assembly manner can rapidly produce IgG and IgM antibodies at 14d, and the antibody titer can be maintained at the same level for a long period of time, wherein the IgG antibody titer produced by the vaccine with a-C-GalCer as adjuvant is equivalent to that of the vaccine with aggacer as adjuvant. From the subtype results, the antibody subtypes produced were mainly IgG1 and IgG 3.
The results show that the vaccine preparation provided by the invention can generate high-titer antibodies.
4) Indirect competitive ELISA for determining antibody affinity
Antibody affinity was measured by a competition ELISA, which was essentially similar to the non-competition ELISA. The ELISA plate was coated with Nic-BSA solution, incubated overnight at 4 ℃ and then washed 3 times with PBST. Blocking was performed by adding 1.5% BSA in PBS (w/v) and incubation at 37 ℃ for 1 h. The serum was diluted to a multiple of 1.0 absorbance (step see previous non-competitive ELISA), the standard nicotine tartrate was diluted from 100mmol to 2nmol in 0.1% BSA PBS solution, and equal volumes of serum dilution (65L) and nicotine standard (65L) were added to the unbound plates and incubated at 37 ℃ for 1 h. The blocked plates were washed 3 times with PBST, and then the combined nicotine standards and serum were added to the conjugate plates and incubated for 0.5h at 37 ℃. PBST washing plate 3 times, adding sheep anti mouse two IgG, incubation, color, reading, the same steps as non-competitive ELISA. The binding rate is determined by B/B0Wherein B is a serum plate hole containing nicotine standard, B0Serum plate wells without nicotine standards. The test results are shown in FIGS. 3 and 4 for half inhibitory concentrations.
Wherein, antibody affinity is obtained by competitive ELISA, serum is obtained after 3 rd immunization, each column represents the titer average of 5 mice, each value is independently repeated twice, and error bars indicate the average standard error.
It can be seen from FIG. 3 that group A immunization with the addition of α GalCer produced IgG antibodies with higher recognition of nicotine than Pam3CSK4Group B and combination α GalCer and Pam of3CSK4Group E of (1). Although the groups F2 and F3, to which an adjuvant was covalently attached, produced low antibody titers, their nicotine recognition ability was comparable to that of group a.
From the results in fig. 4, it can be seen that the IgG antibodies produced by different adjuvants in a self-assembly manner have better affinity with nicotine, wherein the IC50 of the IgG antibodies produced by the vaccine adjuvanted with C34 is the smallest, indicating that the generated antibody has the highest affinity.
The results show that the vaccine preparation provided by the invention has better affinity.
5) In vivo cytokine assay
The level of cytokines in serum was determined by ELISA kit (interferon- γ and interleukin-4, Biolegend) and the experimental procedure followed the cytokine protocol provided with the kit. The 96-well microplate was coated with the capture antibody and incubated overnight at 4 ℃. And then, oscillating and sealing the enzyme label plate for 1h by using 1% BSA PBS at room temperature, adding the diluted serum after washing into the plate, oscillating for 2h at room temperature, then adding a detection antibody and a secondary antibody, developing the color for 20-30min by TMB, stopping developing the color by using 2mol/L sulfuric acid, and reading at 450 nm. The results of the experiment are shown in FIG. 5.
Wherein, sera taken 2h and 24h after the first immunization were used for interleukin-4 and interferon-gamma determination, respectively, each bar represents the mean value of the content of 5 mice, and each value was repeated twice independently, and the error bar indicates the mean standard error. Significant differences relative to group D: p<0.05；**P<0.01；***P<0.001；****P<0.0001. The vaccines were compared with each other:##P<0.01；#P<0.05; ns, significant differences were not significant. Data analysis was performed using GraphPad Prism Software (GraphPad Software, San Diego, Calif.) by one-way analysis of variance and Turkey's posthoc testing.
It can be seen from FIG. 5 that group A with the addition of adjuvant α GalCer both secreted interferon-gamma of the Th1 type and interleukin-4 of the Th2 type, indicating that the mixed response of Th1 and Th2 was consistent with the antibody production results, although binding α GalCer to Pam3CSK4Group E of (2) secretes interferon-gamma and interleukin-4, but elicits antibody titers and semi-inhibitory concentrations IC50Lower than group a. Vaccines with covalently linked adjuvants can secrete higher levels of interferon- γ and interleukin-4, with a low dose of the F1 group secreting predominantly interferon- γ, biased towards the t h1 response. The results show that the vaccine preparation provided by the invention has better immunological activity.
6) Determination of the resistance to injury
And performing an anti-injury test 2 weeks after the third immunization, specifically, placing the mouse on a hot plate at 55 ℃ to record the time of paw lifting or violent reaction as an initial value, then subcutaneously injecting 0.5mg/kg nicotine to the mouse, placing the mouse on the hot plate at 55 ℃ after 15min to record the reaction time as a test value, wherein 35s is set as a maximum value for preventing the mouse from being scalded due to long-time non-reaction on the hot plate. Antinociceptive effects are expressed by% MPE, where% MPE is (test value-initial value)/(maximum value-initial value). The results are shown in FIGS. 6 and 7.
Where each bar represents the mean of 5 mice, the error bars identify the mean standard error. Significant differences relative to the non-immunized group: p < 0.05; p < 0.01; p < 0.001. Data analysis was performed using GraphPad Prism Software (GraphPad Software, San Diego, Calif.) by one-way analysis of variance and Turkey's posthoc testing.
From the hot plate test results in fig. 6, it can be seen that the group a anti-nociceptive ability of the adjuvant α GalCer was lower than that of other adjuvant vaccines when stimulated with a certain amount of nicotine, indicating that the vaccine with adjuvant α GalCer produced antibodies with better ability to bind nicotine than other adjuvant vaccines.
From the results of FIG. 7, the results are shown in the following formulas a-C-GalCer, C34 and C20: the self-assembly vaccine with the adjuvant 2 has the best anti-injury effect within 5min and 15min, and the mice immunized by the vaccine in the mixed self-assembly mode are all recovered to the normal state within 30 min.
7) Determination of body temperature changes
Mouse body temperature was measured 5min, 15min, 30min after receiving nicotine injection. The results are shown in FIGS. 8 and 9.
Where each bar represents the mean of 5 mice, the error bars identify the mean standard error. Significant differences relative to the non-immunized group: p < 0.05; p < 0.01; p < 0.001. Data analysis was performed using GraphPad Prism Software (GraphPad Software, San Diego, Calif.) by one-way analysis of variance and Turkey's posthoc testing.
As can be seen from FIG. 8, the group A with the adjuvant α GalCer has the least change of body temperature with time after receiving nicotine compared with other vaccines with the adjuvant, and the body temperature recovery is fast.
From the results in fig. 9, it can be seen that the antibody generated by the vaccine mixed with the self-assembly mode can neutralize nicotine in the injected mice, and the body temperature is fast recovered compared with that of the vaccine not immunized, indicating that the vaccine mixed with the self-assembly mode has better immune activity.
8) Determination of body weight Change
Mice were observed for changes in body weight after receiving the vaccine. As shown in fig. 10 and 11.
As can be seen from fig. 10, the change in body weight over time using the vaccine formulation provided by the present invention was substantially the same as that of normal non-immunized mice, indicating that the vaccine formulation provided by the present invention is relatively safe.
From the results in fig. 11, it can be seen that the vaccine mixed with the self-assembly method has a small effect on the body weight of the mice, indicating that the self-assembly method is relatively safe.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (11)
1. A vaccine formulation, comprising:
component (a): a hapten, the hapten being an addictive drug;
a component (b): a linker unit covalently linked at one end to the component (a) to form components a-b;
a component (c): an adjuvant selected from at least one of the compounds represented by formula (1) to formula (4);
wherein,
the component (c) is covalently linked with the other end of the component (b) in the components a-b to form the effective component shown as a-b-c; or
The preparation also comprises a component (d): a carrier which is covalently linked to the other end of the component (b) to form a component a-b-d, and the component a-b-d and the component (c) are mixed and self-assembled into an effective component represented by a-b-d/c;
each X in formulas (1) to (4) is independently selected from-CH3、-NH2-OH or-SH, each Y is independently selected from-CH2-, -NH-, -O-or-S-;
r is selected from
One of (1);
and the linking unit is selected from structural units represented by formula (5) to formula (16):
in formula (5) -formula (16), m1-m5、n1-n7、p1、p2And q is1Each independently is any one integer of 1-60, X1-X7、Y1-Y9Each independently selected from-CH2-, -NH-, -O-or-S-;
and the carrier is selected from the group consisting of those represented by the formulae (17) to (20),
in the formulae (17) to (20), each m is independently any one integer of 0 to 30, each n is independently any one integer of 0 to 6, and each X is8Each independently selected from-CH2-, -NH-, -O-or-S-.
2. The vaccine formulation of claim 1, wherein the addictive drug is selected from at least one of nicotine, methamphetamine, heroin, cocaine, fentanyl, opiates, ketamine, and caffeine.
3. The vaccine formulation of claim 2, wherein the structural formula of nicotine is selected from the group consisting ofAt least one of;
the structural formula of the methamphetamine is selected from At least one of;
the structural formula of the heroin is selected from At least one of;
the structural formula of the cocaine is shown in the specification
The fentanyl has a structural formula
The structural formula of the opium is selected from At least one of;
the structural formula of the ketamine is shown in the specification
The structural formula of the caffeine is shown in the specification
4. The vaccine formulation according to any one of claims 1 to 3, wherein the effective ingredient represented by a-b-c has the following structure:
5. the vaccine preparation according to any one of claims 1 to 3, wherein the effective component a-b-d/c has the following structure:
and the component (c) is at least one selected from the group consisting of compounds represented by the formulae (21) to (25),
6. the vaccine formulation of any one of claims 1-3 and 5,
in the effective components shown by a-b-d/c, the content molar ratio of the components a-b-d to the component (c) is 1: (0.1-10);
preferably, the content molar ratio of the components a-b-d to the component (c) is 1: (0.2-0.8).
7. The vaccine formulation of any one of claims 1-6, further comprising:
a component (e): a lipid carrier, wherein the lipid carrier is one or more of cholesterol, 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine and 1, 2-dioleoyl lecithin.
8. The vaccine formulation of claim 7, wherein the lipid carrier is a mixture of cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine in a molar ratio of (0.1-10): 1.
9. The vaccine formulation of claim 7, wherein the formulation comprises: the cholesterol-containing oral liquid comprises the effective components shown as a-b-c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, wherein the effective component shown as a-b-c is cholesterol, 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, 1, (1-10) and (1-10) in molar ratio.
10. The vaccine formulation of claim 7, wherein the formulation comprises: the effective component shown in a-b-d/c, cholesterol and 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, and the effective component shown in a-b-d/c is cholesterol, 1, 2-distearoyl-Sn-propanetriyl-3-phosphorylcholine, 1, (1-10) and (1-10) in molar ratio.
11. Use of a vaccine formulation according to any one of claims 1 to 10 in the manufacture of a medicament for the treatment of addiction to related drugs.
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