Application of ultrasonic microvesicle and amphotericin B in preparation of medicine for treating cryptococcus meningitis

Abstract

The invention discloses an application of ultrasonic microvesicle combined amphotericin B in preparation of a medicine for treating cryptococcus meningitis. The invention provides a treatment strategy combining ultrasound microvesicles with amphotericin B, and aims to improve the prognosis of cryptococcus meningitis. Experiments show that after ultrasound is combined with microbubble intervention, the intracerebral concentration of amphotericin B of a CM mouse is increased to 1.7 times of the original concentration, the load capacity of cryptococcus is reduced by 97%, the survival rate in 60 days is increased to 100% from 50%, and the curative effect is superior to that of single-drug treatment. Effectiveness is verified in an animal model, the antifungal drug is adaptive to existing antifungal drugs (amphotericin B deoxycholate and liposome amphotericin B), new drugs do not need to be developed, and the research and development cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of ultrasound microbubbles combined with amphotericin B in the preparation of a drug for treating cryptococcal meningitis. Background Art

[0002] Cryptococcal meningitis (CM) is a severe central nervous system infectious disease. The mortality rate of untreated patients is nearly 100%, and about 30% of the treated survivors have permanent neurological deficits (such as vision loss, hearing impairment, or cognitive decline). In areas with scarce medical resources, problems such as delayed diagnosis, shortage of antifungal drugs, and lack of intracranial pressure monitoring equipment often lead to an actual mortality rate exceeding 60%. In addition, the ability of Cryptococcus to form biofilms results in a high fungal load in cerebrospinal fluid. The increasing resistance rate of azole drugs and the spread of amphotericin B resistance genes have further plunged the treatment into a vicious cycle of "insufficient drug concentration - prolonged treatment course - cumulative toxicity - treatment interruption". Currently, the treatment of CM relies on amphotericin B, but despite the treatment regimen using amphotericin B, the mortality rate is still 20 - 60%. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of ultrasound microbubbles combined with amphotericin B in the preparation of a drug for treating cryptococcal meningitis.

[0004] Preferably, the amphotericin B is amphotericin B deoxycholate or liposomal amphotericin B or a pharmaceutical composition containing them.

[0005] Preferably, the drug may contain pharmaceutically acceptable excipients.

[0006] The present invention proposes a treatment strategy of ultrasound microbubbles combined with amphotericin B, aiming to improve the prognosis of cryptococcal meningitis. The present invention has found through experiments that after the intervention of ultrasound combined with microbubbles, the intracerebral concentration of amphotericin B in CM mice is increased to 1.7 times the original level, the cryptococcal burden is decreased by 97%, and the 60 - day survival rate is increased from 50% to 100%. The curative effect is better than that of single - drug treatment. The present invention has verified its effectiveness in an animal model, is compatible with existing antifungal drugs (amphotericin B deoxycholate, liposomal amphotericin B), and does not require the development of new drugs, reducing the R & D cost. Brief Description of the Drawings

[0007] Figure 1Ultrasound combined with microbubbles can increase the concentration of amphotericin B in the brains of CM mice; after 7 days of treatment with L-AMB, the concentration of amphotericin B in the brain was 43.4425 ng / g, while after 7 days of treatment with Sonovue+Ul+L-AMB, the concentration of amphotericin B in the brain was 72.8867 ng / g; after 7 days of treatment with AMB-D, the concentration of amphotericin B in the brain was 37.4428 ng / g, while after 7 days of treatment with Sonovue+Ul+AMB-D, the concentration of amphotericin B in the brain was 57.4325 ng / g.

[0008] Figure 2 Ultrasound combined with microbubble delivery of amphotericin B can reduce the cryptococcal burden in the brains of CM mice; after 7 days of treatment, the cryptococcal burden in the brains of mice in the CM group was 2039215 CFU / g, the cryptococcal burden in the brains of mice in the CM+AMB-D group was 711806 CFU / g, the cryptococcal burden in the brains of mice in the CM+L-AMB group was 526150 CFU / g, the cryptococcal burden in the brains of mice in the CM+Sonovue+Ul group was 1509402 CFU / g, the cryptococcal burden in the brains of mice in the CM+Sonovue+Ul+AMB-D group was 145122 CFU / g, and the cryptococcal burden in the brains of mice in the CM+Sonovue+Ul+L-AMB group was 39394 CFU / g.

[0009] Figure 3 Ultrasound combined with microbubble delivery of amphotericin B can improve the 60-day survival rate of CM mice. The 60-day survival rate of mice in the CM group was 0%, the 60-day survival rate of mice in the CM+AMB-D group was 16.7%, the 60-day survival rate of mice in the CM+L-AMB group was 50%, the 60-day survival rate of mice in the CM+Sonovue+Ul group was 0%, the 60-day survival rate of mice in the CM+Sonovue+Ul+AMB-D group was 100%, and the 60-day survival rate of mice in the CM+Sonovue+Ul+L-AMB group was 100%.

[0010] Note: Group 1 is CM, Group 2 is CM + AMB-D, Group 3 is CM + L-AMB, Group 4 is CM + Sonovue + Ul, Group 5 is CM + Sonovue + Ul + AMB-D, Group 6 is CM + Sonovue + Ul + L-AMB. CM - cryptococcal meningitis, CM + AMB-D - cryptococcal meningitis treated with amphotericin B deoxycholate, CM + L-AMB - cryptococcal meningitis treated with liposomal amphotericin B, CM + Sonovue + Ul - cryptococcal meningitis treated with ultrasound microbubbles, CM + Sonovue + Ul + AMB-D - cryptococcal meningitis treated with ultrasound microbubbles combined with amphotericin B deoxycholate, CM + Sonovue + Ul + L-AMB - cryptococcal meningitis treated with ultrasound microbubbles combined with liposomal amphotericin B. Detailed implementation manners

[0011] The following examples are further descriptions of the present invention rather than limitations thereof.

[0012] Example 1:

[0013] (1) Establishment of CM mouse model

[0014] Take out the cryopreserved Cryptococcus neoformans and place it at room temperature for 24 hours for resuscitation. Under sterile conditions, transfer the resuscitated Cryptococcus neoformans to SDA solid medium with an inoculation loop and incubate it in an incubator at 37°C for 3 days. Prepare the bacterial suspension 2 hours before inoculation. The process is as follows: Inject 1 ml of 0.9% normal saline into a sterile test tube, pick an appropriate amount of colonies with a sterile inoculation loop and put them into the test tube, gently shake to dissolve the colonies in the saline to form a bacterial suspension. Use a micropipette to draw a drop of the bacterial suspension and place it at the corresponding position on the counting plate, cover it with a cover glass, count under a microscope, and adjust it with normal saline according to the number of spores to adjust the concentration of the Cryptococcus neoformans suspension to 1.0×10 7 CFU / ml. Enter the biosafety level 2 laboratory, wear protective clothing and double-layer latex gloves. First, intraperitoneally inject 3% pentobarbital sodium (2.5 mL / kg) into the mice to anesthetize them, then fix the mice with a stereotaxic apparatus, locate the lateral ventricles of the mice (0.7 mm behind the bregma, ±1 mm beside the sagittal line, 2 mm below the skull surface), and then inject 5 μL (containing 5×10 4 CFU) of the Cryptococcus neoformans suspension at a rate of 0.5 ml / min for 10 min. After injection, leave the needle for 2 min and then pull out the micropipette. After inoculating the Cryptococcus neoformans suspension for 24 hours, culturing Cryptococcus neoformans from the mouse brain tissue homogenate is used as the standard for successful modeling. The results of previous experiments show that this method can successfully establish the model.

[0015] (2) Intervention method

[0016] Ultrasound combined with microbubble group: First, 200 μL of microbubbles (SonoVue, 59 mg / mL, dissolved in 5 ml of 0.9% sodium chloride solution) were intravenously injected into CM mice. Then, CM mice were anesthetized with isoflurane. After anesthesia, ultrasonic coupling agent was applied to the scalp of CM mice, and then a low-frequency ultrasonic therapy instrument (Intelect 2776 mobile low-frequency ultrasonic therapy instrument) was used to stimulate for 1 minute (stimulation frequency 1.0 MHz, intensity 1 w / cm 2 , duty cycle 50%). After that, amphotericin B (0.02 mg of amphotericin B deoxycholate or 0.1 mg of liposomal amphotericin B, both dissolved in sterile injection water and then diluted with 5% glucose solution) or 200 μl of 0.9% sodium chloride solution was intravenously injected into CM mice.

[0017] Control group: CM mice were anesthetized with isoflurane, and then amphotericin B (0.02 mg of amphotericin B deoxycholate or 0.1 mg of liposomal amphotericin B, both dissolved in sterile injection water and then diluted with 5% glucose solution) or 200 μl of 0.9% sodium chloride solution was intravenously injected into CM mice.

[0018] (3) Evaluation of amphotericin B concentration

[0019] Take 50 mg of amphotericin B standard, dissolve it in 1 ml of DMSO, and then transfer it to a 100-ml volumetric flask. Dilute it to the mark with acetonitrile to prepare a standard stock solution with a mass concentration of 500 μg / ml. Take 1 ml of the standard stock solution and dilute it with acetonitrile to prepare standard working solutions with concentrations of 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2 μg / ml, 3 μg / ml, 4 μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml. Take 300 μl of blank plasma, add the above 10 concentration gradients of standard working solutions, and prepare dilutions with concentrations of 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml. Vortex for 30 seconds, let stand for 5 minutes, then centrifuge (10800 rpm, 5 minutes, 4 °C), and take the supernatant. Take 300 μl of blank brain tissue homogenate, add the above 10 concentration gradients of standard working solutions, and prepare dilutions with concentrations of 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml. Vortex for 30 seconds, let stand for 5 minutes, then centrifuge (10800 rpm, 5 minutes, 4 °C), and take the supernatant. According to the pharmacokinetic data and literature, collect the brain tissue and plasma of mice 4 hours after the last administration on the 4th day of intervention. Measure the weight of the brain tissue, add it to 1 ml of physiological saline, homogenize it with a multi-functional tissue grinder (60 HZ, 60 s), then centrifuge with a centrifuge (3000 rpm, 10 minutes), take 300 μl of the upper brain tissue homogenate, add 300 μl of acetonitrile, vortex for 30 seconds, let stand for 5 minutes, then centrifuge (10800 rpm, 5 minutes, 4 °C), and take the supernatant. Take 700 μl of eyeball blood, centrifuge with a centrifuge (3000 rpm, 10 minutes), take 300 μl of the upper plasma, add 300 μl of acetonitrile, vortex for 30 seconds, let stand for 5 minutes, then centrifuge (10800 rpm, 5 minutes, 4 °C), and take the supernatant. Use a 1-ml syringe to extract the supernatant of brain tissue and plasma supernatant, pass through an organic filter head, and inject it into a sample vial. Prepare the mobile phase (mobile phase A: 0.01% potassium dihydrogen phosphate aqueous solution, mobile phase B: acetonitrile), connect the vacuum pump, prepare a microporous filter membrane (aqueous system), take 0.01% potassium dihydrogen phosphate aqueous solution into the solvent filter, start the vacuum pump for filtration, and transfer it to a 500-ml volumetric flask after filtration. Connect the vacuum pump, prepare a microporous filter membrane (aqueous system), take acetonitrile into the solvent filter, start the vacuum pump for filtration, and transfer it to a 500-ml volumetric flask after filtration.Install an Agilent TC-C18 column (4.6 mm × 150 mm, 5 μm) into an Agilent Series 1200 high-performance liquid chromatograph. Open the computer software, connect the tubing, connect the detection system, and expel any bubbles from each line. Design a sampling method (select UV detector, mobile phase A: methanol; mobile phase B: 70:30 EDTA-2Na, isocratic elution, detection wavelength: 383 nm; flow rate: 1 mL / min; injection volume: 10 μL; column temperature: 25°C, sampling time: 15 min). Before sampling, run a baseline for 20 min, then begin sampling and data acquisition. After all samples have been run, rinse with 90% water:10% acetonitrile, gradually transitioning to 100% acetonitrile for storage.

[0020] (4) Assessment of cryptococcal load in brain tissue

[0021] The brain tissue was placed in a 2 ml EP tube containing 1 ml of normal saline. The tissue homogenate was thoroughly mixed using a multifunctional tissue grinder (60 Hz, 60 s) and then diluted at a 1:10 v / v ratio to obtain tissue homogenates of different dilution gradients. 20 μL of tissue homogenates of different dilution gradients were taken using a pipette and evenly spread on 2 plates of SDA culture medium. The homogenate tissue was evenly spread on the surface of the culture medium using a spreading rod. The culture plates were then placed in a 37 °C constant temperature incubator for culture. The number of colonies in the culture plates 48 h after inoculation was recorded and the average value was calculated.

[0022] 1. Significantly improved drug delivery efficiency:

[0023] like Figure 1 、 2 As shown in Figure 3, after ultrasound combined with microbubble intervention, the concentration of amphotericin B in the brain of CM mice increased to 1.7 times the original, the cryptococcal load decreased by 97%, and the 60-day survival rate increased from 50% to 100%, with better efficacy than single-drug treatment.

[0024] 2. Strong potential for clinical transformation:

[0025] Its effectiveness has been verified in animal models and it is compatible with existing antifungal drugs (amphotericin B deoxycholate, liposomal amphotericin B), eliminating the need for new drug development and reducing R&D costs.

Claims

1. Use of ultrasound microbubbles combined with amphotericin B in the preparation of a drug for treating cryptococcal meningitis.

2. The application according to claim 1, wherein The amphotericin B described above is amphotericin B deoxycholate or liposomal amphotericin B or a pharmaceutical composition containing them.

3. The application according to claim 1, characterized in that, The drug described above may contain pharmaceutically acceptable excipients.

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