Cocaine esterase-loaded w / o / w nanoemulsion, preparation method and application thereof

Abstract

The application discloses a cocaine esterase-loaded W / O / W nanoemulsion and a preparation method and application thereof, wherein the nanoemulsion is composed of cocaine esterase, a phosphate buffer, soybean phospholipid, vitamin E polyethylene glycol succinate, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and medium-chain triglyceride; the preparation method comprises the following steps: preparing an inner water phase; preparing an oil phase; slowly injecting the inner water phase into the oil phase under shearing to obtain a W / O primary emulsion; slowly adding the primary emulsion into an outer water phase under shearing, and obtaining a secondary emulsion through high-speed shearing; and performing high-pressure homogenization on the secondary emulsion to obtain a final W / O / W nanoemulsion. The cocaine esterase is combined with the nanoemulsion for the first time, the nanoemulsion prepared through the method has the effects of improving the temperature stability of the cocaine esterase and prolonging the in-vivo half-life, and can be applied to the rescue or prevention of cocaine acute poisoning.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and specifically relates to a cocaine esterase-loaded W / O / W nanoemulsion and a preparation method and application thereof. BACKGROUND

[0002] As a powerful central nervous stimulant, cocaine has strong hallucinogenicity and addiction. Taking a large dose of cocaine can cause heart failure and severely inhibit the respiratory center of the brain, leading to death. According to statistics, more than 20,000 people died of cocaine overdose in the United States in 2020-2021, and the number of deaths due to cocaine overdose increased by 22% compared with the previous year (Spencer, M. R., Hedegaard, H., & Warner, M. (2022). Drug Overdose Deaths in the United States, 1999-2021. NCHS data brief, (474), 1-8.). A. M, & Garnett, M. F. (2023). Co-involvement of Opioids in Drug Overdose Deaths Involving Cocaine and Psychostimulants, 2011-2021. NCHS data brief, (474), 1-8.). Cocaine acute poisoning has become a global problem that endangers human health. The current clinical treatment method for cocaine acute poisoning mainly relies on symptomatic treatment, such as the use of benzodiazepines, short-acting barbiturates, etc., which can only relieve symptoms and there is no specific drug to directly eliminate cocaine in the body.

[0003] Effective enzyme therapy is considered the most promising strategy for treating cocaine overdose. Bacterial cocaine esterase (CocE) shows high catalytic activity to cocaine and can catalyze the hydrolysis of cocaine to produce non-toxic metabolite benzoic acid. Its quadruple mutant (T172R / G173Q / L196C / I301C, hereinafter referred to as E196-301, K M = 1.5 μM, k cat = 3450 min -1 ) can quickly eliminate cocaine in the body and is a highly potential cocaine antidote drug. However, as an exogenous protein drug, cocaine esterase has defects such as temperature sensitivity and short half-life in the body, which limits its application in clinical practice. Therefore, preparing a preparation for loading and delivering unstable protein drugs, improving the thermal stability and in vivo half-life of cocaine esterase, and thus improving the drug efficacy, is of great significance for the application of cocaine esterase in the rescue or prevention of cocaine acute poisoning.

[0004] Lipid nanoparticles are drug delivery carriers composed of materials such as phospholipids, with particle sizes ranging from tens to hundreds of nanometers, and mainly include the following dosage forms: liposomes, solid lipid nanoparticles, nanostructured lipid carriers, microemulsions, and nanoemulsions. In the research of loading enzymes through lipid nanoparticles, liposomes and nanoparticles are usually used. This is because liposomes are composed of phospholipid bilayers, which are similar in structure to cell membranes and have high biocompatibility. Their closed structure can effectively protect enzymes from degradation by body fluids (such as proteases and extreme pH). Polymer nanoparticles (such as PLGA and chitosan) can immobilize enzymes through physical embedding or chemical bonding, providing a mechanical barrier to extend the circulation time and achieving targeted delivery through surface modification. However, nanoemulsions often require high-energy homogenization, ultrasonication, or high-temperature and high-pressure treatment during preparation, which may cause enzyme denaturation and inactivation due to excessive temperature, so they are generally not used for enzyme loading. This leads to certain limitations in the administration of enzyme substances.

[0005] Therefore, it is of great significance to develop a water-in-oil-in-water nanoemulsion based on cocaine esterase E196-301 for the rescue or prevention of acute cocaine poisoning in the delivery of unstable protein drugs for the treatment of diseases. SUMMARY

[0006] The present application aims to solve the problems of the prior art and provide a W / O / W type nanoemulsion containing cocaine esterase and a preparation method thereof. For the first time, cocaine esterase is combined with nanoemulsion to improve the thermal stability of cocaine esterase through the special structure of water-in-oil-in-water nanoemulsion.

[0007] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a preparation method of a W / O / W type nanoemulsion loaded with cocaine esterase, which greatly reduces the impact of high temperature on thermally unstable enzymes. The preparation method comprises the following steps:

[0009] Step (1), preparing the inner water phase: dilute cocaine esterase with phosphate buffer solution as the inner water phase;

[0010] Step (2), preparing the oil phase: add soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 to medium-chain triglyceride, and melt at high temperature to obtain the oil phase;

[0011] Step (3), preparing the primary emulsion: after cooling the oil phase under high-speed shearing, slowly inject the inner water phase into the oil phase, and continue to shear at high speed to obtain the W / O type primary emulsion;

[0012] Step (4), preparation of secondary emulsion: the W / O primary emulsion is slowly added into the external water phase under high shear, and the secondary emulsion is obtained by continuing high-speed shearing under ice bath;

[0013] Step (5), preparation of final emulsion: the secondary emulsion is subjected to high-pressure homogenization at 4℃ to obtain the final W / O / W nanoemulsion;

[0014] Step (6), ultrafiltration concentration: the nanoemulsion is concentrated by centrifugation at 4000r / min through an ultrafiltration tube, and the enzyme-containing nanoemulsion is separated from the unencapsulated enzyme.

[0015] Preferably, the amount of cocaine esterase (E196-301) in step (1) is 0.5-1 mg / mL based on the total volume of the W / O / W nanoemulsion.

[0016] Preferably, the mass ratio of soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 to cocaine esterase in step (2) is (10-30):1, and more preferably the mass ratio is 20:1. The method of the application prolongs the in vivo half-life of the enzyme by surface modification of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 in the nanoemulsion.

[0017] Preferably, the mass ratio of soybean phospholipid to vitamin E polyethylene glycol succinate in step (2) is (4-5):1, and more preferably 4.4:1; the mass ratio of soybean phospholipid to distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is 40:1.

[0018] Preferably, the temperature of the oil phase melting in step (2) is 60-80℃.

[0019] Preferably, the shearing speed in steps (3) and (4) is 8000-12000r / min, and the shearing time is 10-15min.

[0020] Preferably, the cooling temperature in step (3) is 20-30℃, and more preferably the temperature is 25℃.

[0021] Preferably, the high-pressure homogenization pressure in step (5) is 1200-1500bar, and the homogenization frequency is 3-6 times.

[0022] Preferably, the molecular weight cut-off of the ultrafiltration tube in step (6) is 100kDa, and the centrifugation time is 2-4h.

[0023] Preferably, in the W / O / W nanoemulsion, the volume ratio of the oil phase to the internal water phase is (10-12):1, and more preferably 10:1; the volume ratio of the external water phase to the oil phase is (10-12):1, and more preferably 10:1.

[0024] Preferably, the purity of the soybean phospholipid is not less than 95%.

[0025] Preferably, the pH value of the phosphate buffer is 7.2-7.6.

[0026] In the second aspect, the application provides a cocaine esterase W / O / W nanoemulsion for cocaine poisoning rescue or prevention, which is prepared by the above method.

[0027] Specifically, the nanoemulsion is composed of an inner water phase, an oil phase and an outer water phase, the inner water phase includes cocaine esterase (E196-301) and phosphate buffer, the oil phase includes soybean phospholipid, vitamin E polyethylene glycol succinate (TPGs), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) and medium-chain triglyceride (MCT), and the outer water phase is phosphate buffer.

[0028] In the third aspect, the application provides application of the above cocaine esterase-loaded W / O / W nanoemulsion in preparation of a preparation for cocaine acute poisoning rescue or prevention.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] The application selects soybean phospholipid as the main material and encapsulates the enzyme in the nanoemulsion. The E196-301 is encapsulated in the nanoemulsion (CocE NEs) composed of soybean phospholipid, vitamin E polyethylene glycol succinate (TPGs), medium-chain triglyceride (MCT), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000). The application optimizes the preparation method of the nanoemulsion, controls the temperature of the preparation of the colostrum at room temperature or around room temperature for the first time, overcomes the technical prejudice that the colostrum can only be prepared at high temperature in the prior art, and makes the residual enzyme activity of the prepared enzyme-containing nanoemulsion higher than 90%. The nanoemulsion is in the form of water-in-oil-in-water emulsion, the E196-301 is encapsulated in the innermost water, the oil phase plays a role in isolating the external environment, the stability of the enzyme is enhanced, and the surface of the nano-particle is modified with MPEG2000, so that the nanoemulsion has the ability to prolong the in-vivo half-life. The application combines the cocaine esterase with the nanoemulsion for the first time through a unique preparation method, prolongs the in-vivo half-life while improving the thermal stability, addresses the deficiencies of the existing cocaine esterase in storage and treatment, prepares a more stable long in-vivo half-life detoxification enzyme preparation by using pharmaceutical means, provides a basis for further application of the cocaine esterase in the clinic, and provides a new idea for the research and development of long-acting enzymes.

[0031] In summary, the application has the following beneficial effects:

[0032] (1) The nanoemulsion prepared by the present application can improve the stability of E196-301 at 37℃ and 40℃, and can maintain more than 95% activity at 37℃ within 100h and more than 90% activity at 40℃ within 720min.

[0033] (2) The nanoemulsion prepared by the present application can improve the in vivo half-life of E196-301, and the improvement effect is 3-4 times when the dose is 3mg / kg.

[0034] (3) The nanoemulsion prepared by the present application can effectively degrade the subsequent injection of cocaine within 60min after intravenous injection (1mg / kg), greatly reducing the content of cocaine in the blood, and weakening the influence of cocaine on the excitation state of the mouse brain. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a process flow chart for the preparation method of the cocaine esterase-containing nanoemulsion of embodiments 1-7 of the present application.

[0036] Figure 2 It is a transmission electron microscope image of the cocaine esterase-containing nanoemulsion in embodiment 1 of the present application.

[0037] Figure 3 It is a graph showing the changes in particle size (A) and PDI (B) of the cocaine esterase nanoemulsion (CocE NEs) in embodiment 1 of the present application placed at 4℃ for 28 days in different media.

[0038] Figure 4 It is a graph showing the changes in enzyme activity of the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in embodiment 1 of the present application over time at different temperatures (A, B, C) and a graph showing the thermal drift results of E196-301 in three states (D, E, F).

[0039] Figure 5 It is a graph showing the changes in the concentration of the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in embodiment 1 of the present application over time after injection at a dose of 1mg / kg (A) and 3mg / kg (B).

[0040] Figure 6 It is a graph showing the changes in the concentration of cocaine (A) and benzoic acid (B) in the body when cocaine is re-injected 1h after the injection of the cocaine esterase nanoemulsion (CocE NEs) and E196-301 in embodiment 1 of the present application.

[0041] Figure 7The whole process of the mouse moving distance is shown in the figure. (A) The total moving distance of the mouse; (B) The time course of the moving distance of the mouse, with the injection of cocaine as 0 min. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be further described in detail below with reference to the drawings and specific embodiments, but the protection scope of the present application is not limited by this.

[0043] The present application provides a preparation method of the W / O / W type nanoemulsion loaded with cocaine esterase, comprising the following steps:

[0044] Step (1), preparing the inner water phase: diluting the cocaine esterase with a phosphate buffer solution as the inner water phase; wherein the pH value of the phosphate buffer solution is 7.2-7.6;

[0045] Step (2), preparing the oil phase: adding soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 into medium-chain triglyceride, and melting at 60-80℃ to obtain the oil phase; wherein the mass ratio of soybean phospholipid to vitamin E polyethylene glycol succinate is (4-5):1, the mass ratio of soybean phospholipid to distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 is 40:1, and the mass ratio of the total mass of soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 to cocaine esterase is (10-30):1; the purity of the soybean phospholipid is not less than 95%;

[0046] Step (3), preparing the primary emulsion: the oil phase is cooled to 20-30℃ under high-speed shearing at 8000-12000 r / min for 10-15 min, the inner water phase is slowly injected into the oil phase through a 1 mL syringe, and the high-speed shearing is continued to obtain the W / O type primary emulsion; the volume ratio of the oil phase to the inner water phase is 10:1;

[0047] Step (4), preparing the secondary emulsion: the W / O type primary emulsion is slowly moved into the outer water phase through a pipette under high-speed shearing, and the high-speed shearing is continued under ice bath to obtain the secondary emulsion; the volume ratio of the outer water phase to the oil phase is 10:1;

[0048] Step (5), preparing the final emulsion: the secondary emulsion is subjected to high-pressure homogenization at 4℃ and 1200-1500 bar for 3-6 times to obtain the final W / O / W type nanoemulsion;

[0049] Step (6), ultrafiltration concentration: the nanoemulsion is concentrated by centrifugation at 4000 r / min for 2-4 h through an ultrafiltration tube with a molecular weight cut-off of 100 kDa, and the enzyme-containing nanoemulsion is separated from the unencapsulated enzyme.

[0050] The application is further described below.

[0051] Example 1:

[0052] (1) Preparation of the internal water phase: 50 mg of E196-301 was diluted with 0.5 mL of a phosphate buffer as the internal water phase;

[0053] (2) Preparation of the oil phase: 0.8 g of soybean phospholipid, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglyceride, and the oil phase was obtained by melting at 80°C;

[0054] (3) Preparation of the primary emulsion: the oil phase was cooled to 25°C under high-speed shearing at 8000 r / min, and the internal water phase was slowly injected into the oil phase using a syringe, and high-speed shearing was continued at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0055] (4) Preparation of the secondary emulsion: the primary emulsion was slowly added to 50 mL of an external water phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0056] (5) Preparation of the final emulsion: the secondary emulsion was subjected to high-pressure homogenization at 4°C, 1200 bar, and repeated homogenization 5 times to obtain a final W / O / W type nanoemulsion.

[0057] (6) The enzyme-containing nanoemulsion was concentrated by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0058] Example 2:

[0059] (1) Preparation of the internal water phase: 50 mg of E196-301 was diluted with 0.5 mL of a phosphate buffer as the internal water phase;

[0060] (2) Preparation of the oil phase: 0.4 g of soybean phospholipid, 0.09 g of vitamin E polyethylene glycol succinate, and 0.01 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglyceride, and the oil phase was obtained by melting at 80°C;

[0061] (3) Preparation of the primary emulsion: the oil phase was cooled to 25°C under high-speed shearing at 8000 r / min, and the internal water phase was slowly injected into the oil phase using a syringe, and high-speed shearing was continued at 10000 r / min for 10 min to obtain a W / O type primary emulsion;

[0062] (4) Preparation of secondary emulsion: slowly add the primary emulsion into 50 mL of the outer aqueous phase phosphate buffer under high shear at 12000 r / min, continue to shear under ice bath for 10 min to obtain the secondary emulsion;

[0063] (5) Preparation of final emulsion: high pressure homogenization of the secondary emulsion at 4℃, 1200 bar, repeated homogenization for 5 times to obtain the final W / O / W nanoemulsion.

[0064] (6) Concentration of the enzyme-containing nanoemulsion by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0065] Example 3:

[0066] (1) Preparation of the inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0067] (2) Preparation of the oil phase: 1.2 g of soybean phospholipid, 0.27 g of vitamin E polyethylene glycol succinate, and 0.03 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglyceride, and melted at 80℃ to obtain the oil phase;

[0068] (3) Preparation of the primary emulsion: the oil phase was cooled to 25℃ under high shear at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, and high shear was continued at 10000 r / min for 10 min to obtain the W / O primary emulsion;

[0069] (4) Preparation of the secondary emulsion: slowly add the primary emulsion into 50 mL of the outer aqueous phase phosphate buffer under high shear at 12000 r / min, continue to shear under ice bath for 10 min to obtain the secondary emulsion;

[0070] (5) Preparation of the final emulsion: high pressure homogenization of the secondary emulsion at 4℃, 1200 bar, repeated homogenization for 5 times to obtain the final W / O / W nanoemulsion.

[0071] (6) Concentration of the enzyme-containing nanoemulsion by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0072] Example 4:

[0073] (1) Preparation of the inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0074] (2) Preparation of the oil phase: 0.8 g of soybean phospholipid, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglyceride, and melted at 80℃ to obtain the oil phase;

[0075] (3) Preparation of the primary emulsion: the oil phase was cooled to 4°C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, and high-speed shearing at 10000 r / min was continued for 10 min to obtain a W / O type primary emulsion;

[0076] (4) Preparation of the secondary emulsion: the primary emulsion was slowly added to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0077] (5) Preparation of the final emulsion: the secondary emulsion was subjected to high-pressure homogenization at 4°C, 1200 bar, and repeated homogenization for 5 times to obtain a final W / O / W type nanoemulsion.

[0078] (6) The enzyme-containing nanoemulsion was concentrated by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0079] Example 5:

[0080] (1) Preparation of the inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0081] (2) Preparation of the oil phase: 0.8 g of soybean phospholipid, 0.18 g of vitamin E polyethylene glycol succinate, and 0.02 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added to 5 g of medium-chain triglyceride, and melted at 80°C to obtain an oil phase;

[0082] (3) Preparation of the primary emulsion: the oil phase was cooled to 40°C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, and high-speed shearing at 10000 r / min was continued for 10 min to obtain a W / O type primary emulsion;

[0083] (4) Preparation of the secondary emulsion: the primary emulsion was slowly added to 50 mL of the outer aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0084] (5) Preparation of the final emulsion: the secondary emulsion was subjected to high-pressure homogenization at 4°C, 1200 bar, and repeated homogenization for 5 times to obtain a final W / O / W type nanoemulsion.

[0085] (6) The enzyme-containing nanoemulsion was concentrated by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0086] Example 6:

[0087] (1) Preparation of the inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as the inner aqueous phase;

[0088] (2) Preparation of oil phase: 0.8 g of soybean phospholipid, 0.18 g of vitamin E polyethylene glycol succinate, 0.02 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added into 5 g of medium chain triglyceride, and melted at 80°C to obtain an oil phase;

[0089] (3) Preparation of primary emulsion: the oil phase was cooled to 60°C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, and high-speed shearing at 10000 r / min was continued for 10 min to obtain a W / O type primary emulsion;

[0090] (4) Preparation of secondary emulsion: the primary emulsion was slowly added into 50 mL of external aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0091] (5) Preparation of final emulsion: the secondary emulsion was subjected to high-pressure homogenization at 4°C, 1200 bar, and repeated homogenization for 5 times to obtain a final W / O / W type nanoemulsion.

[0092] (6) The enzyme-containing nanoemulsion was concentrated by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0093] Example 7:

[0094] (1) Preparation of inner aqueous phase: 50 mg of E196-301 was diluted with 0.5 mL of phosphate buffer as an inner aqueous phase;

[0095] (2) Preparation of oil phase: 0.8 g of soybean phospholipid, 0.18 g of vitamin E polyethylene glycol succinate, 0.02 g of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 were added into 5 g of medium chain triglyceride, and melted at 80°C to obtain an oil phase;

[0096] (3) Preparation of primary emulsion: the oil phase was cooled to 60°C under high-speed shearing at 8000 r / min, and the inner aqueous phase was slowly injected into the oil phase using a syringe, and high-speed shearing at 10000 r / min was continued for 10 min to obtain a W / O type primary emulsion;

[0097] (4) Preparation of secondary emulsion: the primary emulsion was slowly added into 50 mL of external aqueous phase phosphate buffer under high-speed shearing at 12000 r / min, and high-speed shearing was continued for 10 min under ice bath to obtain a secondary emulsion;

[0098] (5) Preparation of final emulsion: the secondary emulsion was subjected to high-pressure homogenization at 4°C, 1200 bar, and repeated homogenization for 5 times to obtain a final W / O / W type nanoemulsion.

[0099] (6) The enzyme-containing nanoemulsion was concentrated by centrifugation at 4000 r / min for 2 h through a 100 kDa ultrafiltration tube.

[0100] The nanoemulsions prepared in Examples 1-7 were subjected to performance tests as follows:

[0101] I. Parameter characterization of cocaine esterase nanoemulsion

[0102] The particle size, PDI, encapsulation efficiency, and relative enzyme activity of the cocaine esterase nanoemulsion prepared in the above examples were detected. The remaining enzyme activity was detected by detecting the amount of benzoic acid generated after the nanoemulsion reacted with cocaine. Relative enzyme activity = (benzoic acid concentration / reaction time / enzyme concentration) 纳米乳液 / (benzoic acid concentration / reaction time / enzyme concentration) E196-301 × 100%. The unencapsulated enzyme concentration was detected by a BCA kit, and the encapsulation efficiency = (total enzyme concentration - unencapsulated enzyme concentration) / total enzyme concentration × 100%. The results are shown in Table 1. As can be seen from Table 1, when the mass ratio of total mass of soybean phospholipid, vitamin E polyethylene glycol succinate, and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 to E196-301 is 20:1, and the cooling temperature is 25°C (Example 1), the particle size, PDI (Polymer dispersity index) of the nanoemulsion is optimal, and the encapsulation efficiency and enzyme activity are higher.

[0103] Table 1: Parameter characterization results of cocaine esterase nanoemulsion (mean ± SD)

[0104]

[0105] II. Morphology observation of cocaine esterase nanoemulsion

[0106] Figure 2 The transmission electron micrograph of the cocaine esterase nanoemulsion of Example 1 of the present application is shown in the figure. In order to more directly observe the morphology of the nanoemulsion, it was observed by transmission electron microscopy. As shown in the figure, the nanoemulsion morphology presents uniform spherical shape, and the light-colored oil phase is wrapped around the transparent water phase in the large spherical particles, which indicates that a water-in-oil-in-water nano system is formed. The diameter of the large particles is between 60-100 nm, and the particles are relatively uniform.

[0107] III. Storage stability of cocaine esterase nanoemulsion

[0108] Figure 3Figure 4 shows the particle size and PDI of cocaine esterase nanemulsion (CocE NEs) in Example 1 in different media at 4°C for 28 days. The storage stability of cocaine esterase nanemulsion was evaluated by measuring the particle size and PDI of the nanemulsion in PBS, 10% FBS / PBS, 10% FBS / DMEM at 4°C for 28 days. As shown in the figure, the particle size (A) and PDI (B) of cocaine esterase nanemulsion in the above three media at 4°C for 28 days did not change significantly, indicating that cocaine esterase nanemulsion in Example 1 has excellent storage stability under this condition. Figure 3 Figure 3

[0109] IV. Temperature stability of cocaine esterase nanemulsion

[0110] Figure 4 Figure 5 shows the change in enzyme activity of cocaine esterase nanemulsion (CocE NEs) in Example 1 and E196-301 over time at different temperatures (A, B, C) and the thermal drift results of E196-301 in three states (D, E, F). In order to reflect the improvement of temperature tolerance of E196-301 by nanemulsion, the stability of E196-301 and nanemulsion at 37°C, 40°C, 45°C was compared. The method was as follows: 500 μL of E196-301 (0.2 mg / ml, in 0.1M PB / in 0.1M PB+0.5 mg / ml BSA) and cocaine esterase nanemulsion (0.2 mg / ml, in 0.1M PB) were placed in glass tubes and placed in a 37°C oven / 40°C metal bath / 45°C metal bath, and 50 μL was sampled every hour to dilute and detect enzyme activity. In addition, the melting temperature (Tm) of E196-301 (PB / PB with BSA) and CocE NEs was detected by thermal drift experiment.

[0111] Figure 4 ​​Figures (A), (B), and (C) show that E196-301 is relatively sensitive to temperature changes, and its stability at 37℃ and 40℃ is better when diluted with 0.1M PB containing 0.5 mg / ml BSA than when diluted with 0.1M PB alone. When diluted with 0.1M PB, the remaining enzyme activity after 100 hours at 37℃ is approximately 70%, and after 720 minutes at 40℃, the remaining enzyme activity is only about 40%. However, when diluted with 0.1M PB containing 0.5 mg / ml BSA, the remaining enzyme activity after 100 hours at 37℃ is about 80%, and after 720 minutes at 40℃, the remaining enzyme activity is about 50%. When the temperature is increased to 45℃, the remaining enzyme activity of both is below 10% after 120 minutes. However, the cocaine esterase nanoemulsion maintained over 95% activity within 100 hours at 37°C, over 90% activity within 720 minutes at 40°C, and over 30% activity within 120 minutes at 45°C. The cocaine esterase nanoemulsion improved the temperature stability of E196-301, enabling it to maintain over 90% activity for an extended period between 37°C and 40°C. Figure 4 Results (D), (E), and (F) show that the Tm value of E196-301 diluted with PB was 44℃, while the Tm value of E196-301 diluted with PB containing BSA increased to 50℃. Most importantly, CocE NEs provided good thermal protection for the enzyme, increasing its Tm value to 73℃.

[0112] V. In vivo half-life of cocaine esterase nanoemulsion

[0113] Figure 5 This is a time-varying process of the concentration of cocaine esterase nanoemulsion (CocE NEs) and E196-301 in vivo after injection in Example 1 of this invention (values ​​are expressed as mean ± SEM). To verify the effect of the nanoemulsion of this invention in increasing the in vivo half-life of E196-301, cocaine esterase nanoemulsion and E196-301 were injected intravenously into rats at doses of 1 mg / kg and 3 mg / kg, respectively. Blood samples were collected at 2, 5, 10, 15, 30, 45, 60, 90, and 120 min after injection. The blood samples were diluted and reacted with cocaine to determine the reaction rate. The enzyme concentration at each time point was then calculated by back-calculating the reaction rate. The results are as follows: Figure 5 As shown in (A), at a dose of 1 mg / kg, the in vivo half-life of E196-301 was 6.076 ± 1.2 min, while that of cocaine esterase nanoemulsion was 13.56 ± 4.8 min; Figure 5Figure 6 shows the in vivo half-life of E196-301 and cocaine esterase nanoemulsion (CocE NEs) in rats. (A) shows the concentration of cocaine in blood samples taken at 5, 10, 15, 30, 45, 60 min after intravenous injection of cocaine (5 mg / kg) in rats pretreated with cocaine esterase nanoemulsion (1 mg / kg) or E196-301 (1 mg / kg) 1 h before. (B) shows the concentration of benzoic acid in blood samples taken at 5, 10, 15, 30, 45, 60 min after intravenous injection of cocaine (5 mg / kg) in rats pretreated with cocaine esterase nanoemulsion (1 mg / kg) or E196-301 (1 mg / kg) 1 h before. The results show that cocaine esterase nanoemulsion can effectively prolong the in vivo half-life of E196-301 and increase the in vivo retention time.

[0114] Six, Effect of cocaine esterase nanoemulsion on elimination of cocaine in vivo

[0115] Figure 6 Figure 6 shows the in vivo half-life of E196-301 and cocaine esterase nanoemulsion (CocE NEs) in rats. (A) shows the concentration of cocaine in blood samples taken at 5, 10, 15, 30, 45, 60 min after intravenous injection of cocaine (5 mg / kg) in rats pretreated with cocaine esterase nanoemulsion (1 mg / kg) or E196-301 (1 mg / kg) 1 h before. (B) shows the concentration of benzoic acid in blood samples taken at 5, 10, 15, 30, 45, 60 min after intravenous injection of cocaine (5 mg / kg) in rats pretreated with cocaine esterase nanoemulsion (1 mg / kg) or E196-301 (1 mg / kg) 1 h before. The results show that cocaine esterase nanoemulsion can effectively prolong the in vivo half-life of E196-301 and increase the in vivo retention time. Figure 6 Figure 6(A) shows that the concentration of cocaine in the cocaine esterase nanoemulsion group was significantly lower than that in the E196-301 group. Figure 6 Figure 6(B) shows that the concentration of benzoic acid in the cocaine esterase nanoemulsion group was significantly higher than that in the E196-301 group. The results show that after 1 h of injection at a dose of 1 mg / kg, the content of cocaine esterase nanoemulsion in the blood is still sufficient to metabolize 5 mg / kg of cocaine, and can convert cocaine to benzoic acid in a short time, which is a significant improvement compared to E196-301.

[0116] The pharmacokinetic parameters (mean ± SD) of cocaine in this example are shown in Table 2.

[0117] Table 2: Comparison of pharmacokinetic parameters of cocaine esterase nanoemulsion and E196-301 in rats (Mean ± SD)

[0118]

[0119]

[0120] (wherein, λz is the terminal elimination rate constant, t 1 / 2z is the half-life, C max is the maximum concentration, AUC 0-t is the area under the curve of the drug-time curve within a certain time, AUC 0-∞The area under the drug-time curve for infinite time is AUC. ％Extrap MRT is the percentage of the extrapolated area to the total AUC. 0-∞ (where Vz is the average residence time, CL is the apparent volume of distribution, and CL is the clearance rate.)

[0121] VII. Effects of Cocaine Esterase Nanoemulsion on Open Field Activity in ICR Mice After Cocaine Injection

[0122] Figure 7 The graph shows the distance traveled by mice during the entire process, 45 minutes after a tail vein injection of CocE NEs and E196-301 at a dose of 3 mg / kg, followed by an intraperitoneal injection of 25 mg / kg of cocaine (values ​​are expressed as mean ± SEM). Cocaine rapidly reaches the brain after entering the body, stimulating the mice to produce an excited state. Recording the distance traveled by the mice in the open field provides an indicator of the degree of excitement. To verify that CocE NEs can rapidly metabolize cocaine into the body over a prolonged period after injection, thereby reducing or completely eliminating the effects of cocaine on the body, this invention set up four groups: Control group (PBS injected intraperitoneally 45 min after tail vein injection), Cocaine group (cocaine injected intraperitoneally 45 min after tail vein injection), E196-301 group (cocaine injected intraperitoneally 45 min after tail vein injection), and CocENEs group (cocaine injected intraperitoneally 45 min after tail vein injection of CocE NEs). The dosage of E196-301 and CocE NEs was 3 mg / kg, and the dosage of cocaine was 25 mg / kg. The distance traveled by the mice throughout the process was recorded and compared. The results showed ( Figure 7 A& Figure 7 In the study (B), E196-301 had essentially lost its effect of rapidly degrading cocaine by 45 minutes, and the level of excitement in mice was comparable to that of the cocaine control group. In contrast, CocE NEs rapidly cleared subsequently entering cocaine from the body within 45 minutes after injection, preventing the excitatory effects of cocaine. These results indicate that CocE NEs can significantly increase the duration of action of cocaine esterase in vivo, preventing the effects of cocaine on mouse behavior for a longer period (45 minutes). (Compared to the Cocaine group, P<0.0001 in the CocE NEs group, ****; compared to the E196-301 group, P<0.001 in the CocE NEs group, ***).

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for preparing a W / O / W nanoemulsion loaded with cocaine esterase, characterized in that, The preparation method includes the following steps: Step (1) Preparation of the inner aqueous phase: Dilute cocaine esterase with phosphate buffer to prepare the inner aqueous phase; Step (2), Preparation of oil phase: Soybean phospholipids, vitamin E polyethylene glycol succinate, and distearate phosphatidylethanolamine-polyethylene glycol 2000 are added to medium-chain triglycerides and melted to obtain oil phase; Step (3) Preparation of primary emulsion: The oil phase is cooled to 20℃-30℃ under high-speed shearing, and the inner aqueous phase is slowly injected into the oil phase. High-speed shearing continues to obtain W / O type primary emulsion; Step (4) Preparation of secondary emulsion: Using phosphate buffer as the external aqueous phase, the W / O type primary emulsion is added to the external aqueous phase under high-speed shearing, and the secondary emulsion is obtained by continuing high-speed shearing under ice bath. Step (5) Preparation of final emulsion: The secondary emulsion is repeatedly subjected to high-pressure homogenization to obtain the final W / O / W nanoemulsion, which is then concentrated by centrifugation through an ultrafiltration tube.

2. The preparation method according to claim 1, characterized in that, In step (1), the amount of cocaine esterase used is 0.5-1 mg / mL based on the total volume of the W / O / W nanoemulsion.

3. The preparation method according to claim 1, characterized in that, In step (2), the total mass of soybean phospholipids, vitamin E polyethylene glycol succinate, distearate phosphatidylethanolamine-polyethylene glycol 2000 and the mass ratio of cocaine esterase are (10-30):

1.

4. The preparation method according to claim 3, characterized in that, In step (2), the mass ratio of soybean lecithin to vitamin E polyethylene glycol succinate is (4-5):1; the mass ratio of soybean lecithin to distearate phosphatidylethanolamine-polyethylene glycol 2000 is 40:

1.

5. The preparation method according to claim 1, characterized in that, In step (2), the temperature at which the oil phase melts is 60-80℃.

6. The preparation method according to claim 1, characterized in that, The shearing speed in steps (3) and (4) is 8000-12000 r / min, and the shearing time is 10-15 min.

7. The preparation method according to claim 1, characterized in that, In step (5), the pressure of high-pressure homogenization is 1200-1500 bar, and the number of homogenization cycles is 3-6.

8. The preparation method according to claim 1, characterized in that, In the W / O / W type nanoemulsion, the volume ratio of the oil phase to the inner aqueous phase is (10-12):1, and the volume ratio of the outer aqueous phase to the oil phase is (10-12):

1.

9. A W / O / W nanoemulsion loaded with cocaine esterase, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. The use of the W / O / W nanoemulsion loaded with cocaine esterase as described in claim 9 in the preparation of antidotes or preventative agents for acute cocaine poisoning.

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