CN116036006B - Phage suppository and application thereof in treating bacterial prostatitis - Google Patents
Phage suppository and application thereof in treating bacterial prostatitis Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to a phage suppository, which comprises the following raw materials in percentage by mass of 0.5-1.5: 3-4: 4-8: 1-3 of gelatin, glycerol, phage solution and sterilized water, the preparation method comprises: weighing gelatin, placing the gelatin in sterilized water, and heating the gelatin at 85-95 ℃ after the gelatin absorbs water and swells; adding glycerol into the system, uniformly mixing the glycerol and the system, transferring the glycerol to 40-50 ℃ for heating after the glycerol is homogeneous; adding phage solution, mixing, pouring into suppository mould, and refrigerating. The invention also relates to the application of the phage suppository or the preparation method thereof in preparing a preparation for treating bacterial prostatitis and/or in evaluating pharmacokinetics and pharmacodynamics parameters of the phage suppository. The invention obtains the phage suppository with water-soluble matrix, has the advantages of good uniformity, quick drug release, high drug loading and the like, and can be used for evaluating pharmacokinetics and pharmacodynamics of phage rectal administration.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a phage suppository, a preparation method thereof and application thereof in treating bacterial prostatitis.
Background
Bacterial prostatitis is often caused by lower urinary tract infection, and the effects of antibiotic treatment are often limited due to poor tissue permeability and other reasons. With the advent and popularity of resistant bacteria, the failure rate of antibiotic therapy is increasing. In recent years, infections caused by gram-positive bacteria have been on the rise, with enterococci accounting for about 5% -10% of bacterial prostatitis.
A suppository is a dosage form for delivering drugs by the vaginal and rectal routes. When being locally administrated, the suppository can treat intestinal diseases and has the effects of lubrication, bacteriostasis and the like. When the medicine is administrated in a whole body, the medicine is absorbed by intestinal mucosa, enters the rectal circulation through the vein under the rectum, avoids the liver from being affected and has stimulation effect on the gastrointestinal tract. Compared with the conventional treatment means, the phage suppository can exert certain clinical treatment advantages. The rectal administration not only can achieve the aim of slow release and improve the bioavailability of phage, but also can improve the compliance of patients and relieve the pain of the patients. However, the suppository has little research on the pharmacokinetic properties and the like, and needs further research. Compared with antibiotic treatment, the phage suppository has the advantages of large administration dosage, strong penetrating power and the like, and is a scheme hopeful to replace antibiotic treatment.
However, the current phage therapy field lacks pharmacokinetic and pharmacodynamic related studies, and the formulation of clinical treatment protocols is dependent only on phage in vitro related study data. The pharmacokinetics and pharmacodynamics parameters of the phage suppository have important reference significance for evaluating the therapeutic effect of phage and formulating a reasonable dosing scheme.
Currently, there is no phage suppository available for treatment on acute bacterial prostatitis rat models, and there is no associated pharmacokinetic and pharmacodynamic assessment.
Disclosure of Invention
In order to overcome at least one problem in the prior art, the invention provides a phage suppository for treating acute bacterial prostatitis of rats and a preparation method thereof, and pharmacokinetics and pharmacodynamics parameters of the phage suppository can be obtained so as to better evaluate the treatment effect of phage.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a phage suppository comprising the following raw materials: gelatin, glycerol, phage solution and sterile water.
Further, the mass ratio of the raw materials is as follows: gelatin: glycerol: phage solution: sterilized water=0.5 to 1.5: 3-4: 4-8: 1 to 3; still further, gelatin: glycerol: phage solution: sterilized water=0.8 to 1.2:3.3 to 3.7:5 to 7:1.5 to 2.5; most preferably, gelatin: glycerol: phage solution: sterilized water = 1:3.5:6:2.
further, the sterilized water may be replaced with a PBS solution or physiological saline.
Further, the phage suppository is a suppository of enterococcus faecalis phage; still further, the enterococcus faecalis phage is enterococcus faecalis phage EFap02. Among them, phage EFap02 is from the institute of phage and drug resistance, reference: liu, J.et al bacteriophage-Resistant Mutant of Enterococcus faecalis Is Impaired in Biofilm formation. Front Microbiol 13,913023 (2022), genBank accession No. OL505084.
Further, the phage suppository has the following specification: each containing 10 5 ～10 9 PFU phage (e.g., 10 per each) 5 PFU phage, 10 per each 7 PFU phage or 10 per each 9 PFU phage); more specifically, each content is 10 9 The average weight of the suppositories for PFU was 0.443.+ -. 0.01g.
In a second aspect, the present invention provides a method for preparing a phage suppository according to any one of the first aspects of the present invention, comprising the steps of:
s1, weighing a predetermined amount of gelatin, placing the gelatin in a predetermined amount of sterilized water, and heating the gelatin at 85-95 ℃ after the gelatin absorbs water and swells to obtain a first system;
s2, adding a predetermined amount of glycerol into the first system, uniformly mixing the glycerol and the first system, transferring the glycerol to 40-50 ℃ for heating after forming a homogeneous phase, and obtaining a second system;
s3, adding a predetermined amount of phage solution into the second system, uniformly mixing, pouring the mixture into a suppository mould while the mixture is hot, and obtaining the phage suppository, and refrigerating and preserving the phage suppository.
Further, the heating temperature in step S1 was 90 ℃, the heating temperature in step S2 was 45 ℃, and the refrigerated storage temperature in step S3 was 4 ℃. Specifically, the heating manners in the steps S1 and S2 are both water bath heating, and other suitable heating manners may also be adopted.
In one embodiment, the phage suppository is prepared by a method comprising: weighing 1 part of gelatin, placing the gelatin in 2 parts of sterilized water, placing the gelatin in a water bath at 90 ℃ after the gelatin absorbs water and swells, then adding 3.5 parts of glycerol into the system, and uniformly mixing the glycerol and the system; after the 2 matrixes are homogeneous, transferring the matrixes into a water bath at 45 ℃, adding 6 parts of phage solution into the matrixes, gently mixing the matrixes to prevent bubbles, pouring the mixed matrixes into a suppository mold when the mixed matrixes are hot, and refrigerating and preserving the mixed matrixes at 4 ℃.
Further, the phage solution preparation step includes: after the phage is subjected to gradient dilution, absorbing a solution containing the phage, mixing the solution with bacterial liquid of enterococcus faecalis in the logarithmic phase, and adding upper agar at 50-60 ℃; after a single phage spot is picked, placing the phage spot in normal saline to release phage; repeating the above operations, and continuously purifying phage; infecting enterococcus faecalis with the purified solution containing phage according to the ratio of MOI=0.0001-0.01, and carrying out phage amplification; dialyzing the amplified phage-containing solution in physiological saline; the dialyzed phage-containing solution is filtered by a polyethersulfone membrane, the filtered phage-containing solution is serially diluted to obtain the phage solution, the titer is measured by a double-layer plate method, and the phage solution is refrigerated and stored for standby.
In one embodiment, phage is diluted in a gradient (specifically 10-fold diluted with physiological saline, 100. Mu.L of phage-containing solution to be diluted is taken and 900. Mu.L of physiological solution is added)Mixing with salt water, and diluting to 10 6 ) After that, 50. Mu.L of phage-containing solution was pipetted and mixed with 300. Mu.L of log phase bacterial liquid, and then 55℃upper agar was added. After picking individual phage plaques, phage were released by placing in 200. Mu.L of physiological saline. The above procedure was then repeated, and phage were purified 3 times in succession. Bacteria were infected at a ratio of moi=0.001 and phage amplification was performed. The amplified phage-containing solution was dialyzed in physiological saline for 24 hours. After passing the dialyzed phage-containing solution through a 0.22 μm Polyethersulfone (PES) membrane filter, the phage-containing solution was serially diluted, and the titer was measured by a two-layer plate method, and then stored at 4 ℃ for later use. The phage may be enterococcus faecalis EFap02 (GenBank accession No. OL505084), and the bacterial liquid or bacteria may be enterococcus faecalis b2137904 (NCBI: SRR 22764493), both from Shanghai phage and drug resistance institute.
A third aspect of the present invention provides the use of a phage suppository according to any one of the first aspects of the invention, or a method of preparation according to any one of the second aspects of the invention, selected from at least one of the following uses: the use in the preparation of a formulation for the treatment of bacterial prostatitis, the use in the evaluation of phage suppository pharmacokinetics and pharmacodynamics parameters.
Further, the bacterial prostatitis is acute bacterial prostatitis of rats. Further, the acute bacterial prostatitis in rats is acute bacterial prostatitis caused by enterococcus faecalis.
In one embodiment, the phage suppository is at 10 9 The phage rapidly detected a relatively high titer in the prostate 15 minutes after rectal administration of CFU/mouse, approximately 5.61±0.03log10 PFU/prostate, reached a peak after 2 hours of administration, and the phage titer was approximately 6.41±0.53log10 PFU/prostate; the elimination rate constant of phage after rectal administration was 0.769h-1 (R 2 =0.965), t1/2 is about 0.901h; the retention time of phage in the prostate is 12-48 hours (specifically, 10 5 CFU/mouse dose, retention time 12 hours; 10 7 CFU/mouse dose, retention time 24 hours；10 9 CFU/mouse dose, retention time of 48 hours, etc.).
In one embodiment, the rats have a significantly reduced inflammatory response in the ventral and dorsal prostates following phage suppository treatment.
Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:
the invention obtains the phage suppository with water-soluble matrix by optimizing the raw material proportion and the preparation steps, has the advantages of good uniformity, quick drug release, high drug loading and the like, and can be used for evaluating the pharmacokinetics and pharmacodynamics of phage rectal administration, thereby being used for evaluating the therapeutic effect of phage and further formulating a reasonable administration scheme.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:
FIG. 1 is a schematic flow diagram of a phage suppository according to an embodiment of the present invention;
FIG. 2 is a schematic diagram showing quality inspection results of phage suppository according to an embodiment of the present invention; wherein, part A is the determination of the EOP contained in the phage suppository, part B is the in vitro sterilization effect of the phage suppository, in vitro experiments are carried out in LB liquid medium, the experiments at each time point are repeated 3 times, and error bars represent standard deviations;
FIG. 3 is a graphical representation of the results of pharmacokinetic profile following rectal administration of a phage suppository according to an embodiment of the present invention; wherein, part A is: after rectal administration, 3 doses (10 9 PFU/mouse, 10 7 PFU/mouse, 10 5 PFU/mouse) and intravenous (10) 9 PFU/mouse), phage titer time profile in prostate; the part B and the part C are as follows: high dose group (10) 9 PFU/mouse) phage titer time curves in liver, spleen, lung, kidney, bladder and seminal vesicles, error bars represent standard deviation (n=5, n=4 after rectal administration except liver and spleen), p.r.: transrectal; i.v. the process comprises: intravenous injection;
FIG. 4 is a graphical representation of the results of histological features of ventral and dorsal lobes of the prostate treated with phage suppositories in an embodiment of the invention; wherein, part A and part B are: prostate ventral and dorsal lobes treated with phage; the C part and the D part are as follows: prostate ventral and dorsal lobes (magnification x 40) treated without phage, red arrows directed to neutrophils, black arrows directed to lymphocytes, blue arrows directed to plasma cells.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.
In the following examples: enterococcus faecalis b2137904 (NCBI: SRR 22764493), phage EFap02 (GenBank accession No. OL505084) and enterococcus faecalis b2137904 can be used as the bacterial liquid or bacteria; gelatin and glycerol are purchased from the biological engineering (Shanghai) stock company; the water bath is purchased from Shanghai Ke Du medical science and technology Co., ltd; 1.25% of Afodine was purchased from Nanjing Aibei Biotechnology Co.
In the following examples, the preparation flow of phage suppository is shown in FIG. 1, comprising the steps of: weighing 0.5-1.5 parts of gelatin, placing the gelatin in 1-3 parts of sterilized water, placing the gelatin in a water bath at 85-95 ℃ after the gelatin absorbs water and swells, and then adding 3-4 parts of glycerin into the system and uniformly mixing the glycerin; after the 2 matrixes form homogeneous phase, transferring the homogeneous phase into a water bath with the temperature of 40-50 ℃, and adding 4-8 parts of phage solution into the matrixes; gently mixing the system to prevent bubbles; mixing, pouring into a suppository mold, and refrigerating at 4deg.C. The sterilized water used as described above may be replaced with a PBS solution or physiological saline.
EXAMPLE 1 preparation of phage suppositories
The embodiment is a preferred phage suppository preparation method, which specifically comprises: weighing 2g of gelatin, placing in 4g of sterilized water, placing in 90 ℃ water bath, melting, and stirring gently; adding 7g of glycerol into melted gelatin, and uniformly mixing; after evenly mixing the matrix, transferring the matrix into a water bath at 45 ℃, adding 12g of phage solution, and lightly mixing the phage solution to avoid generating bubbles; mixing, pouring into a suppository mold, and refrigerating at 4deg.C. The specifications of the prepared phage suppository are respectively as follows: each content of 10 5 PFU phage, 10 per count 7 PFU phage, 10 per count 9 PFU phage.
In the above steps, the phage solution preparation step includes: gradient diluting phage (specifically phage φEFAP 02) with physiological saline 10 times, by adding 100 μl of phage-containing solution to be diluted into 900 μl of physiological saline, mixing with vortex oscillator, and continuously diluting to 10 6 ) Then, 50. Mu.L of phage-containing solution is sucked and mixed with 300. Mu.L of bacterial liquid of enterococcus faecalis in logarithmic phase, and then upper agar at 55 ℃ is added; single phage selectionAfter body spots, the phage were released by placing in 200. Mu.L of physiological saline; then repeating the above operation, and continuously purifying phage for 3 times; enterococcus faecalis was infected at a ratio of moi=0.001 and phage amplification was performed; dialyzing the amplified phage-containing solution in physiological saline for 24 hours; after passing the dialyzed phage-containing solution through a 0.22 μm Polyethersulfone (PES) membrane filter, the phage-containing solution was serially diluted to obtain the phage solution, and the titer was measured by a two-layer plate method (about 2 to 5X 10 9 PFU/mL) and then stored at 4℃for later use.
EXAMPLE 2 preparation of phage suppositories
The present embodiment is another preferred phage suppository preparation method, which specifically comprises: weighing 1.6g of gelatin, placing in 3g of sterilized water, placing in 85 ℃ water bath, melting, and stirring gently; adding 8g of glycerol into melted gelatin, and uniformly mixing; after evenly mixing the matrix, transferring the matrix into a water bath with the temperature of 50 ℃, adding 14g of phage solution, and lightly mixing the phage solution to avoid generating bubbles; mixing, pouring into a suppository mold, and refrigerating at 4deg.C. In the above steps, the phage solution was prepared in the same manner as in example 1.
EXAMPLE 3 preparation of phage suppositories
The present embodiment is a preparation method of a phage suppository, which specifically comprises: weighing 3g of gelatin, placing in 4g of sterilized water, placing in 95 ℃ water bath, melting, and stirring gently; adding 6g of glycerol into melted gelatin, and uniformly mixing; after evenly mixing the matrix, transferring the matrix into a water bath at 40 ℃, adding 8g of phage solution, and lightly mixing to avoid generating bubbles; mixing, pouring into a suppository mold, and refrigerating at 4deg.C. In the above steps, the phage solution was prepared in the same manner as in example 1.
Example 4 quality control of phage suppositories
In this example, the ratio of gelatin, glycerol, sterile water and phage solution was 1:3.5:2:6. after gelatin has fully absorbed the heated sterilized water and swelled, glycerol is added. After the substrate melted to form a homogeneous solution, the water bath temperature was adjusted to 45℃and phage solution was added to the substrate. All ingredients were mixed with gentle agitation, then poured into bullet-type molds to obtain phage suppositories, and stored at 4 ℃.
The 10 suppositories for each dose were weighed to calculate the weight change. 3 suppositories were randomly selected for quality control, and the 3 suppositories were placed in a 37 ℃ water bath and the melting time of the suppositories was recorded. After complete thawing of the suppositories, serial gradient dilutions were performed with pre-warmed 37 ℃ physiological saline to determine the plating Efficiency (EOP) and to compare the sharpness and titer of phage plaques formed by phage suppositories, phage solutions and matrices over a period of time. Respectively adding phage suppository, empty suppository and phage solution into liquid LB culture medium, measuring OD600 value of bacterial liquid, and comparing sterilizing effect of suppository and phage solution in liquid environment.
The experimental results show that after weighing: the content is 10 9 PFU suppositories, each having an average weight of 0.443+ -0.01 g weight coefficient of variation (WV) divided into 4.61%, showed good suppository weight uniformity. The suppositories were rapidly thawed at 37℃5 minutes after the water bath, as shown in part A of FIG. 2, and phage plaque assays showed that the titer, transparency and plaque formation time of the thawed phage suppositories were the same as those of the assays performed with phage solutions, whereas the phage suppositories had no antibacterial activity as a matrix. Also, as shown in part B of fig. 2, in the liquid medium, the activity of the phage suppository is the same as that of the phage solution, and the matrix of the phage suppository has no antibacterial activity.
EXAMPLE 5 construction of acute bacterial prostatitis rat model
In order to evaluate the pharmacokinetic and pharmacodynamic parameters of the phage suppositories prepared in the above examples, the present example was used to construct a model of acute bacterial prostatitis rats using SPF-grade male Sprague-Dawley (SD) rats, 220+ -10 g in weight, purchased from Shanghai Ji Hui animals Inc.
Rats were raised for environmental adaptation one week before the start of the experiment. The temperature was set at 22.+ -. 1 ℃ and the relative humidity was set at 50.+ -. 1% and the light/dark cycle was 12/12 hours. All animal studies, including the mouse euthanasia procedure, were in compliance with the Shanghai public health center ethics committee and were conducted according to AAALAC and IACUC guidelines (2021-a 065-02).
Individual enterococcus faecalis colonies were picked and transferred to medium, shake-cultured at 37℃and 250rpm until OD600 was 0.5. After centrifugation, the solution was resuspended in PBS to ensure that about 200. Mu.L of bacterial solution contained 10 8 Bacteria of CFU. A PE-10 hose with the length of 2cm is cut and connected with a 27G islet injection needle head, and the PE-10 hose needs to be soaked in alcohol in advance and sterilized by ultraviolet rays for 1h. Before administration, the food and water of the rats were removed. Prior to anesthesia, the bladder was gently pressed to empty the bladder of urine. Then, 1.25% avermectin solution was injected intraperitoneally at a dose of 10mL/kg to anesthetize the rat, and after the rat lost plantar reflex, the bacterial solution was injected. The rat's penis is gripped with blunt forceps to expose the urethral meatus, and the catheter tip with lubricant is slowly inserted into the rat's urethral meatus. A needle containing 200 μl of bacterial solution was attached to the catheter. Then, the genitals of the rat are gently lifted to maintain a vertical angle with the operating table to facilitate intubation. The catheterization procedure is performed slowly to avoid local bleeding in the rat, and the catheter should conform to the natural curvature of the urethra. If an obstruction is encountered during insertion, the catheter is withdrawn in time and slowly reinserted. After insertion of the catheter, the syringe is slowly advanced to inject the bacterial fluid. Sections of the prostate, bladder and seminal vesicles were obtained 24 hours, 72 hours and 4 weeks after bacterial injection. Tissues were divided into two parts, one for bacterial enumeration after tissue homogenization treatment and the other for hematoxylin-eosin (HE) staining to analyze histological and pathological features after infection.
EXAMPLE 6 evaluation of phage suppositories for pharmacokinetic parameters
This example used 3 doses of phage suppository (10) prepared in example 1 9 PFU/mouse, 10 7 PFU/mouse, 10 5 PFU/mouse) to evaluate pharmacokinetic parameters for treating acute bacterial prostatitis, using the following steps:
the rat model constructed in example 5 was anesthetized with 1.25% aftidine solution, and after the leg-shrinking movement disappeared, the scrotum was gently pressed to the anus with the finger along the discharge direction to discharge the residual feces. Applying a lubricant containing lidocaine gently around the anus of the rat; the suppositories were injected into the rectum of rats and the anus was clamped with hemostats for about 1 hour, preventing the suppositories from flowing out. During anesthesia, the rats remained in a lateral position with the buttocks slightly raised. Rats were sacrificed 15min, 30min, 1h, 2h, 4h, 6h, 8h, 24h, 48h post-dose. Femoral artery blood collection was performed on rats and organs such as lung, liver, spleen, kidney, prostate, bladder and seminal vesicles were collected. After homogenizing the collected organs, the homogenate was centrifuged at 4℃and the supernatant was collected. The supernatant was passed through a 0.22 μm membrane filter to further determine phage titer.
The experimental results show that: as shown in parts A-C of FIG. 3, phage titers in the prostate and other organs gradually increased after 2h of administration, i.e., the suppositories released the phage rapidly after administration, and relatively high titers could be detected rapidly in the prostate 15 minutes after rectal administration (5.61.+ -. 0.03log10 PFU/prostate, initial dose 10 9 CFU/rate), phage titer reached peak (6.41±0.53log10 PFU/prostate) 2 hours after phage embolus rectal administration, followed by onset of phage titer decrease; phage titers in the prostate were higher after intravenous administration of phage of the same titer than after rectal administration.
After rectal administration, phages can reach urinary organs such as the kidney and bladder, but the peak titer of phages in other organs is lower than in the prostate, and the titers of phages in major organs such as the liver, kidney and lung vary greatly at the same time points. The elimination rate constant of phage after rectal administration was calculated to be 0.769h-1 (r2=0.965) when the dose was 10 9 At PFU/mouse, the corresponding T1/2 is 0.901h. The retention time of phage in the target organ is different after rectal administration of different doses. High dose group (10) 9 Phage could still be detected 24 hours after PFU/mouse administration, whereas the low dose group (10 5 PFU/mouse) was rapidly attenuated and was undetectable after 12 hours.
Example 7 histological Change after phage suppository treatment
In this example, histological studies of the ventral and dorsal prostates were performed in experimental rats with or without phage suppository therapy, and histological changes of the ventral and dorsal prostates were observed by HE staining sections, and the results are shown in parts A to D of FIG. 4, which show that phage suppositories were received (at a dose of 10 9 PFU/plug) treatment, the inflammatory response was significantly reduced, whereas neutrophil infiltration and the characteristics of acute suppurative inflammation were still observed in the prostate of rats not receiving phage treatment.
From the above examples, it can be seen that the phage suppository prepared by the invention can be used for treating acute bacterial prostatitis in rats, and can be used for evaluating pharmacokinetic and pharmacodynamic parameters of phage suppository, so as to better evaluate the therapeutic effect of phage, and provide reference significance for formulating a reasonable administration scheme.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, and it will be appreciated by those skilled in the art that equivalent substitutions and obvious variations may be made using the description and illustrations of the invention, and are intended to be included within the scope of the invention.
Claims (9)
1. Use of a phage suppository in the preparation of a formulation for the prevention and/or treatment of bacterial prostatitis, characterized in that the bacterial prostatitis is acute bacterial prostatitis caused by enterococcus faecalis, the phage suppository is a suppository of enterococcus faecalis phage, comprising the following raw materials: gelatin, glycerol, phage solution and sterile water; wherein, the mass ratio of the raw materials is as follows: gelatin: glycerol: phage solution: sterilized water=0.5 to 1.5: 3-4: 4-8: 1-3, wherein the enterococcus faecalis phage is phage EFap02.
2. The use according to claim 1, wherein the mass ratio of the raw materials is: gelatin: glycerol: phage solution: sterilized water=0.8 to 1.2:3.3 to 3.7: 5-7: 1.5-2.5.
3. The use according to claim 1, wherein the sterile water is replaced by a PBS solution or physiological saline.
4. The use according to claim 1, wherein said phage suppository is of the specification: each containing 10 5 ~10 9 PFU phage.
5. The use according to any one of claims 1 to 4, wherein the preparation method of the phage suppository comprises the following steps:
s1, weighing a predetermined amount of gelatin, placing the gelatin in a predetermined amount of sterilized water, and heating the gelatin at 85-95 ℃ after the gelatin absorbs water and swells to obtain a first system;
s2, adding a predetermined amount of glycerol into the first system, uniformly mixing the glycerol and the first system, transferring the glycerol to 40-50 ℃ for heating after forming a homogeneous phase, and obtaining a second system;
s3, adding a predetermined amount of phage solution into the second system, uniformly mixing, pouring the mixture into a suppository mould while the mixture is hot, and obtaining the phage suppository, and refrigerating and preserving the phage suppository.
6. The use according to claim 5, wherein the heating temperature in step S1 is 90 ℃, the heating temperature in step S2 is 45 ℃, and the refrigerated storage temperature in step S3 is 4 ℃.
7. The use according to claim 5, wherein the phage solution preparation step comprises: after the phage is subjected to gradient dilution, absorbing a solution containing the phage, mixing the solution with bacterial liquid of enterococcus faecalis in the logarithmic phase, and adding upper agar at 50-60 ℃; after a single phage spot is picked, placing the phage spot in normal saline to release phage; repeating the above operations, and continuously purifying phage; infecting enterococcus faecalis with the purified solution containing phage according to the ratio of MOI=0.0001-0.01, and carrying out phage amplification; dialyzing the amplified phage-containing solution in physiological saline; the dialyzed phage-containing solution is filtered by a polyethersulfone membrane, the filtered phage-containing solution is serially diluted to obtain the phage solution, the titer is measured by a double-layer plate method, and the phage solution is refrigerated and stored for standby.
8. The use according to any one of claims 1 to 4, further comprising the use of said phage suppository for assessing pharmacokinetic and pharmacodynamic parameters of phage suppositories.
9. The use according to claim 1, wherein the bacterial prostatitis is acute bacterial prostatitis in rats.
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| Bacteriophage-Resistant Mutant of Enterococcus faecalis Is Impaired in Biofilm Formation;Jiazhen Liu等;Bacteriophage-Resistant Mutant of Enterococcus faecalis Is Impaired in Biofilm Formation;第13卷;摘要、第2页左栏第2段 * |
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