CN114507178A - Fentanyl protein conjugate and application thereof - Google Patents

Fentanyl protein conjugate and application thereof Download PDF

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CN114507178A
CN114507178A CN202210146811.7A CN202210146811A CN114507178A CN 114507178 A CN114507178 A CN 114507178A CN 202210146811 A CN202210146811 A CN 202210146811A CN 114507178 A CN114507178 A CN 114507178A
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狄斌
江程
刘婕
徐莉莉
陈小意
崔芷涵
王丽
褚凤鑫
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Abstract

The invention discloses a fentanyl protein conjugate and application thereof. The protein conjugate disclosed by the invention is proved by experiments to be capable of generating an antibody against fentanyl in a mouse body and combining with fentanyl to form an antigen-antibody complex, so that fentanyl at the periphery in the body cannot pass through a blood brain barrier, and the risks of using obstacle and excessive toxicity of fentanyl are resisted.

Description

Fentanyl protein conjugate and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a fentanyl protein conjugate and application thereof.
Background
While drug use disorders (OUDs) are a global health problem, the prevalence of opioid use disorders and the incidence of opioid-related overdoses has increased at a dramatic rate in north america and throughout the world over the past decade. In the united states, at least 250 million people have OUDs and a number of opioid-related fatal overdose events occur. Fentanyl is a potent synthetic opioid and is used legally as a prescription analgesic. Fentanyl, however, exhibits a significant abuse liability because it produces a pleasant sensation by activating the μ -opioid receptors (MOR) in the brain, and excessive MOR activation can lead to respiratory depression, which can be fatal. Thus, when fentanyl is taken from an unregulated source, it poses a significant risk of over-use. In addition, the ease of synthesis of fentanyl makes illegal production and manufacture of tailor-made drug analogs possible. The pharmacology of these analogs has not been properly characterized, which makes them particularly dangerous, especially when certain modifications, even methyl additions, can increase efficacy. Approved approaches to combat drug overdose include the opioid receptor agonists methadone, the partial agonists buprenorphine, the antagonists naltrexone and naloxone, among others. These drugs are safe and effective, but clinical results remain less than ideal due to side effects, regulatory disorders, and the potential for abuse and distraction, and therefore there is an urgent need for safe, long-lasting, and effective treatments to combat the toxicity and excess caused by fentanyl.
In recent years, an immunotherapy has been used to combat the harmful and addictive effects of fentanyl and new psychoactive substances. The basis of this strategy involves active vaccination with protein-drug conjugates to generate in vivo immune antagonists, effectively minimizing the concentration of the drug of interest at the site of action. Fentanyl-specific antibodies reduced hot plate antinociception and fentanyl-induced respiratory depression, and these studies involved passive immunization with polyclonal antibodies or intradermal active vaccination with Freund's complete adjuvant or intraperitoneal administration with other adjuvants, which may not be feasible in humans. Michael d. et al investigated the efficacy of intramuscular fentanyl vaccine injection in mice and rats. The fentanyl-based hapten was conjugated to a natural Keyhole Limpet Hemocyanin (KLH) carrier protein or GMP grade subunit KLH (sKLH) to develop protein conjugates, yielding F-KLH and F-sKLH, respectively, by tetraglycine linker using carbodiimide chemistry. Both mice and rats immunized with F-KLH had lower fentanyl-induced antinociception than the control group. The fentanyl concentration in the brains of rats immunized with F-KLH was lower after intravenous fentanyl compared to the control group. In another group of rats, F-sKLH reduced fentanyl-induced hot plate antinociception, respiratory depression, and bradycardia under a series of cumulative subcutaneous fentanyl doses.
The use of fentanyl analogs is rapidly changing with the advent of new analogs and their supply. However, such vaccines are still in the development stage of new drugs, and the dosage of the vaccines and the concentration of the produced antibodies are still to be improved. Therefore, the study of a novel fentanyl vaccine is a viable strategy to combat the barrier and toxicity to fentanyl use.
Disclosure of Invention
The invention aims to provide a fentanyl protein conjugate taking 4-phenylamino-1-phenylethylpiperidine as a parent nucleus and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fentanyl hapten is shown in a formula I-a:
Figure BDA0003509311090000021
in the formula, R1Is C1-C20 alkyl and amido;
R2is C1-C20 alkyl, amide, olefinic bond, optionally containing 1-3 heteroatoms selected from N, O or S, and C1-6Alkyl radical, C3-8-A 3-8 membered saturated or unsaturated ring of heterocycloalkyl;
preferably, R1Selected from C1-C4 alkyl, R2Is C1-C4 alkyl.
A fentanyl antigen has a structural formula shown in formula I:
Figure BDA0003509311090000022
in the formula: r1Selected from alkyl or amido of C1-C20;
R2selected from C1-C20 alkyl, amide, olefinic bond, optionally containing 1-3 heteroatoms selected from N, O or S, and C1-6Alkyl radical, C3-8-3-to 8-membered saturated or unsaturated ring of heterocycloalkyl group, preferably C1-C20 alkyl group or amide group;
the Protein is hemocyanin (KLH) or cholera toxin subunit B (CTB).
Further, the fentanyl antigen is selected from one of the following structural formulas:
Figure BDA0003509311090000031
the preparation method of the fentanyl antigen is shown as the following formula:
Figure BDA0003509311090000041
wherein: r1Is C1-C20 alkyl and amido; r2Is C1-C20 alkyl and amido; the Protein is hemocyanin (KLH) or cholera toxin subunit B (CTB).
Specifically, the method comprises the following steps: the method comprises the following steps:
step 1, Compound 1 with CH3OCOR2COCl reaction to obtain a compound 2;
step 2, hydrolyzing the compound 2 to obtain a compound 3
And 3, condensing the compound 3 with an amino compound to respectively obtain a compound 4 and a compound 5, and respectively removing protecting groups from the compound 4 and the compound 5 to obtain a compound I-a.
And 4, coupling the compound I-a with protein to obtain the fentanyl protein conjugate shown in the formula I.
As a preferred technical scheme of the application, the step 1 is to add the organic solvent into the compound 1 and then add CH at the temperature of 0 DEG C3OCOR2COCl, and moving to room temperature for reaction to obtain the compound 2. Wherein the organic solvent is anhydrous dichloromethane.
As a preferred technical scheme of the application, the compound 2 is subjected to hydrolysis and demethylation to obtain the compound 2 shown in the formula, wherein the used alkali is 2M lithium hydroxide or sodium hydroxide aqueous solution.
As a preferred technical scheme of the application, the compound I-a is activated by N-acyl succinimide at 4 ℃, added with triethylamine, coupled with protein and dialyzed for 24 hours at 4 ℃ to obtain the series compounds in the formula I. Wherein the protein is KLH or CTB.
The fentanyl hapten or the fentanyl antigen is applied to preparation of drugs for treating abuse of opioid drugs and opioid drug use disorder.
The invention achieves the following beneficial effects:
(1) the fentanyl hapten with the parent nucleus of 4-phenylamino-1-phenylethylpiperidine provided by the invention is novel in structure and simple in preparation method;
(2) according to the invention, cholera mycin B subunit protein is introduced, and fentanyl hapten is connected with the protein, so that the cholera mycin B subunit protein can better act on T cells to generate more antibodies.
(3) The present invention employs active vaccination with protein-drug conjugates to generate in vivo immune antagonists, thereby effectively minimizing the concentration of the target drug at the site of action. Finally, fentanyl vaccines can reduce the addictive and overdose potential of fentanyl.
Drawings
FIG. 1 is a graph showing the titer of antibodies produced by the vaccine in an ELISA assay.
FIG. 2 shows the characterization of the I-5(BSA) protein-MALDI-TOF.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
In the following examples, BSA was used as a protein for the detection of antibody titers.
Example 1
Preparation I-1 a
1. Synthesis of the Compound methyl 2 (6-oxo-6- ((1-phenethylpiperidin-4-yl) (phenyl) amino) hexanoate)
4-phenylamino-1-phenylethylpiperidine (1.0g, 3.56mmol) was dissolved in anhydrous dichloromethane (5mL), the reaction was brought to 0 deg.C, pyridine (950.43 μ L, 31.60mmol) was added and stirred, then methyl fatty acid chloride (695.7 μ L,4.63mmol) was slowly added dropwise and stirred for 10 mIN, and then brought to room temperature for reaction for 2 h. After the reaction was complete, it was quenched with saturated NaHCO3 solution, extracted with ethyl acetate, and the aqueous layer was extracted 3 times with EtOAc. The organic phases were combined, washed with saturated NaCl solution and dried over anhydrous Na2SO 4. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 4:1) gave 980mg of a pale yellow solid in 86% yield. 1H NMR (300MHz, Chloroform-d) δ 7.52-7.34 (m,3H),7.28(dt, J ═ 6.9,1.4Hz,2H),7.20 (tt, J ═ 7.9,1.5Hz,3H), 7.15-7.02 (m,2H),4.71(tt, J ═ 12.2,4.0Hz,1H),3.66(s,3H), 3.11-2.98 (m,2H), 2.83-2.71 (m,2H), 2.63-2.48 (m,2H), 2.29-2.15 (m,4H),1.96(t, J ═ 7.0Hz, 2H), 1.90-1.77 (m,2H), 1.68-1.43 (m,6H).
2. Synthesis of the Compound 3 (6-oxo-6- ((1-phenethylpiperidin-4-yl (phenyl) amino) hexanoic acid)
After compound 2(200mg, 0.489mmol) was dissolved in 1mL of methanol and all dissolved, an aqueous solution of lithium hydroxide (2mL) was added and stirred at room temperature for 2 hours, the pH was adjusted to 3 to 4 with 1N HCl solution, and 190mg of a white solid was filtered off, yielding 80%. 1H NMR (300MHz, Chloroform-d) δ 7.47(d, J ═ 6.7Hz,3H),7.31(q, J ═ 5.7,5.3Hz,3H),7.26 to 7.21(m,2H), 7.18 to 7.03(m,2H),4.86 to 4.72(m,1H),3.61(d, J ═ 11.9Hz,2H),3.25 to 3.10(m,4H),2.91 (t, J ═ 12.3Hz,2H),2.25(t, J ═ 6.9Hz,2H),2.21 to 1.93(m,6H),1.55(qt, J ═ 10.1,5.3Hz,4H).
3. Synthesis of Compound I-1 a
The method comprises the following steps: 3- (6-oxo-6- ((1-phenethylpiperidin-4-yl) (phenyl) amino) hexa-amino) propionic acid tert-butyl ester (Compound 5)
Compound 3(100mg,0.244mol) was dissolved in DCM (1mL), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) (70.39mg,0.367mmol), 1-Hydroxybenzotriazole (HOBT) (49.61mg,0.367mmol), N, N-diisopropylethylamine (63.59. mu.L, 0.367mmol) were added, after stirring for 30mIN, β -alanine tert-butyl ester hydrochloride (66.70mg,0.292mmol) was added, after completion of the reaction, water was added, ethyl acetate was extracted (3 times), the organic phases were combined, washed with saturated brine, and dried over anhydrous Na2SO 4. Purification by silica gel column chromatography (1: 1 petroleum ether/ethyl acetate) gave 69mg of a white solid in 59% yield. 1H NMR (300MHz, Chloroform-d) δ 7.46-7.31 (m,3H), 7.31-7.21 (m,2H), 7.21-7.10 (m,3H),7.06(dd, J ═ 7.4,2.0Hz,2H),6.28(t, J ═ 6.0Hz,1H), 4.67(tt, J ═ 12.1,3.9Hz,1H),3.44(q, J ═ 6.1Hz,2H), 3.07-2.96 (m,2H), 2.79-2.60 (m,2H), 2.60-2.48 (m,2H),2.42(t, J ═ 6.1Hz,2H), 2.24-2.01 (m,4H), 1.98-1.73 (m,4H), 1.62-1H (m,4H), 1.44(s).
Step two: 3- (6-oxo-6- ((1-phenethylpiperidin-4-yl) (phenyl) amino) hexaamido) propanoic acid (Compound I-1 a)
Compound i-1-5 (100mg,0.186mmol) was dissolved in 1:1 solution (500. mu.L) was mixed and reacted for 2 hours, and the solvent was spin-dried to obtain 97mg of a pale yellow oily liquid with a yield of 90%. 1H NMR (300MHz, DMSO-d6) δ 7.87 (t, J ═ 5.6Hz,1H),7.53(q, J ═ 7.8,7.0Hz,3H),7.34(d, J ═ 6.9Hz,2H), 7.30-7.23 (m,5H), 4.81-4.68 (m,1H),4.05(q, J ═ 7.1Hz,2H),3.20(p, J ═ 5.5,4.6Hz,6H),2.98(dd, J ═ 11.2,5.7Hz, 2H),2.36(t, J ═ 6.9Hz,2H),1.95(t, J ═ 6.8Hz,3H),1.85(t, J ═ 6.9Hz,2H), 1.70H (t, 1.70H), 1.51H (m,5H), 1.48H).
Compound I-1 a (20mg, 0.037mmol) was dissolved in DMF-H2O (10%) (500. mu.L) was added to 1-ethyl- (3-dimethylaminopropyl) carbonyldiimide hydrochloride (EDC) (42.97mg,0.224mmol), N-hydroxysuccinimide (NHS) (8.59mg,0.0746mmol) and triethylamine (31.59. mu.L, 0.224mmol), reacted at room temperature for 6 hours, the solvent was added to the solution, BSA-PBS solution (6mg/mL, 300. mu.L) was added thereto, stirred at 4 ℃ for 12 hours, dialyzed for 24 hours, and the reaction solution was filteredThe reaction solution was lyophilized to obtain a white powder.
The KLH coupling vaccine is consistent with the preparation method, and finally I-1 is obtained.
Examples 2 to 5
Compounds I-3 a, I-4 a, I-6 a, I-8 a were prepared by the preparation method of reference example 1.
Figure BDA0003509311090000071
Figure BDA0003509311090000081
Example 6
Preparation of Compound I-2 a:
the method comprises the following steps: 4- (6-oxo-6- ((1-phenethylpiperidin-4-yl) (phenyl) amino) hexa-amido) butyric acid methyl ester (Compound 4)
Compound 3(200mg,0.489mmol) was dissolved in DCM (1mL), 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC) (140.77mg,0.734mmol), 1-Hydroxybenzotriazole (HOBT) (99.23mg,0.734mmol), N, N-diisopropylethylamine (170.55. mu.L, 0.734mmol) were added, after stirring for 30min, methyl 4-aminobutyrate hydrochloride (90.24mg,0.586mmol) was added, after completion of the reaction, water was added, ethyl acetate was extracted (3 times), the organic phases were combined, washed with saturated brine, and dried over anhydrous Na2SO 4. Purification by silica gel column chromatography (petroleum ether/ethyl acetate 1:1) gave 160mg of a white solid in 70% yield. 1H NMR (300MHz, Chloroform-d) δ 7.48-7.38 (m,3H), 7.37-7.15 (m,6H),7.11(dt, J ═ 7.4,1.7Hz,2H),6.13(s,1H),4.71(tt, J ═ 12.2,3.9Hz,1H),3.71 (s,3H),3.32(q, J ═ 6.6Hz,2H),3.05(d, J ═ 11.3Hz,2H),2.77(dd, J ═ 10.7,5.8Hz,2H),2.58(dd, J ═ 10.7,5.9Hz,2H),2.41(t, J ═ 7.3Hz,2H),2.18(dt, J ═ 13.4,8, 5.7, 5.9Hz,2H), 1.41 (t, J ═ 7.3Hz,2H),2.18(dt, J ═ 13.4,8, 1.8, 1.7, 1.78H), 1.1.78 (1H, 1.78H).
Step two: 4- (6-oxo-6- ((1-phenethylpiperidin-4-yl) (phenyl) amino) hexa-amino) butanoic acid (I-2 a)
The compound of step one (200mg, 0.405mmol) was dissolved in 1mL of methanol, after all dissolved, an aqueous solution of lithium hydroxide (2mL) was added and stirred at room temperature for 2h, the pH was adjusted to 3-4 with 1N HCl solution, and 170mg of a white solid was filtered off, giving a yield of 84%. 1H NMR (300MHz, DMSO-d6) δ 12.08(s,1H),7.81(t, J ═ 5.6Hz,1H),7.49(q, J ═ 7.7,7.1 Hz,3H), 7.38-7.19 (m,7H),4.72(t, J ═ 12.4Hz,1H),3.52(d, J ═ 11.9Hz,2H), 3.23-3.06 (m,4H), 2.99(p, J ═ 6.0,5.2Hz,4H),2.19(t, J ═ 7.4Hz,2H), 2.00-1.87 (m,4H),1.83(t, J ═ 6.9Hz,2H), 1.73-1.51 (m,4H),1.36(d, J ═ 9, 3.5, 11.5, 3H).
Compound I-2 a (20mg, 0.041mmol) was dissolved in DMF-H2To O (10%) (500. mu.L) was added 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (46.60mg,0.246mmol), N-hydroxysuccinimide (NHS) (9.32mg,0.082mmol) and triethylamine (33.78. mu.L, 0.246mmol), and after 6 hours of reaction at room temperature, the solvent was dried by spinning, BSA-PBS protein solution (6mg/mL, 300. mu.L, stirred at 4 ℃ for 12 hours, dialyzed for 24 hours, and the reaction mixture was lyophilized to obtain a white powder.
The KLH-coupled hapten is prepared by the same method as the preparation method, and finally I-2 is obtained.
Examples 7 to 8
Compounds I-5 a and I-7 a were prepared by the preparation method of reference example 6.
Figure BDA0003509311090000101
Test examples
ELISA test antibody titer and competitive IC50Value of
On 96-well plates, each well was coated with 1.5. mu.g/mL of Fent-BSA coated protein, overnight at 4 ℃ and washed 3 times with PBST solution. After blocking with skim milk for 1 hour at room temperature, 5 × PBS solution was washed, starting from 1:1000, inoculated mouse serum 1% BSA solution was serially diluted 1:1 and 12 gradients were added to 96-well plates. After 1.5 hours incubation at room temperature, plates were washed 5 times, then donkey anti-mouse IgG Horse Radish Peroxidase (HRP) diluted 1:600 in 1% BSA was added and incubated for 1.5 hours at room temperature. 5 XWash and addition of 3,3',5,5' -Tetramethylbenzidine (TMB) substrate (Thermo Pierce) were carried out,then 2M H was added 5 minutes after TMB addition2SO4. The plates were incubated for 10 min and then the absorbance OD450 read at 450 nm. Values with a P/N value greater than 2 were determined as antibody titer values.
Competitive ELISA was also performed in a similar manner, but with the addition of one step: will IC50The diluted sera were incubated with 60uM to 0.1nM dilutions of free fentanyl (11 3-fold dilutions) in Fent-BSA coated plates for 2 hours. In GraphPad PRISM, the absorbance values were normalized to the highest absorbance value for each sample, and the response-variable slope equation was normalized to determine the midpoint titer and standard error. The non-vaccinated mice did not contain any detectable anti-fentanyl titers.
The experimental results are as follows:
table one: competitive IC50 value for compound on fentanyl
Figure BDA0003509311090000111
As can be seen from the results in the Table, the antibodies from the Fent-KLH (I) immunized mice, in which serially diluted free fentanyl competed with the hapten for the immobilized drug, had a high affinity for fentanyl, resulting in a binding curve with a low nanomolar IC50The value is obtained.
As can be seen from the results in the figure, different protein conjugates have significant antibody titer values at 21 days and 35 days of inoculation, and the titer values rise along with the time, which indicates that the protein conjugates have certain immune curative effect.
The vaccine I-5 has a higher affinity for fentanyl as shown by competitive ELISA tests, and also has a significant antibody titer value in vivo.

Claims (6)

1. A fentanyl hapten, which is characterized in that: the structural formula is shown as formula I-a:
Figure FDA0003509311080000011
in the formula, R1Is C1-C20 alkyl and amido;
R2is C1-C20 alkyl, amide, olefinic bond, optionally containing 1-3 heteroatoms selected from N, O or S, and C1-6Alkyl radical, C3-8-3-8 membered saturated or unsaturated ring of heterocycloalkyl.
2. The fentanyl-based hapten according to claim 1, wherein: r1Selected from C1-C4 alkyl, R2Is selected from alkyl of C1-C4.
3. A fentanyl-like antigen characterized by: the structural formula is shown as formula I:
Figure FDA0003509311080000012
in the formula: r1Selected from alkyl or amido of C1-C20;
R2selected from C1-C20 alkyl, amide, olefinic bond, optionally containing 1-3 heteroatoms selected from N, O or S, and C1-6Alkyl radical, C3-8-3-to 8-membered saturated or unsaturated ring of heterocycloalkyl group, preferably C1-C20 alkyl group or amide group;
the Protein is hemocyanin or cholera toxin subunit B.
4. The fentanyl-based antigen of claim 1, wherein: r1Selected from C1-C4 alkyl, R2Is selected from alkyl of C1-C4.
5. The fentanyl-based antigen of claim 3, wherein: the fentanyl antigen is selected from one of the following structural formulas:
Figure FDA0003509311080000021
6. use of the fentanyl hapten as defined in claim 1, the fentanyl antigen as defined in claim 3 or 5, for the manufacture of a medicament for the treatment of opioid abuse and opioid use disorders.
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Citations (4)

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US20140093525A1 (en) * 2012-09-17 2014-04-03 Minneapolis Medical Research Foundation Compositions and methods of treating opioid addiction
WO2014124317A1 (en) * 2013-02-08 2014-08-14 Regents Of The University Of Minnesota Analgesic conjugates
WO2020018596A1 (en) * 2018-07-16 2020-01-23 The Scripps Research Institute Opioid haptens, conjugates, vaccines, and methods of generating antibodies
CN113292634A (en) * 2021-06-23 2021-08-24 中国人民解放军军事科学院军事医学研究院 Mu/delta opioid receptor dual agonist and medical application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140093525A1 (en) * 2012-09-17 2014-04-03 Minneapolis Medical Research Foundation Compositions and methods of treating opioid addiction
WO2014124317A1 (en) * 2013-02-08 2014-08-14 Regents Of The University Of Minnesota Analgesic conjugates
WO2020018596A1 (en) * 2018-07-16 2020-01-23 The Scripps Research Institute Opioid haptens, conjugates, vaccines, and methods of generating antibodies
CN113292634A (en) * 2021-06-23 2021-08-24 中国人民解放军军事科学院军事医学研究院 Mu/delta opioid receptor dual agonist and medical application thereof

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