CN110368394B - Application of avilamycin in inhibition drug of clostridium perfringens of pigs - Google Patents

Abstract

The invention discloses an application of avilamycin in a medicament for inhibiting clostridium perfringens of pigs, wherein a wild critical value is established by measuring the MIC of 120 clinically separated pathogenic strains; collecting intestinal contents, measuring the distribution of the avilamycin in the intestinal contents by using a high performance liquid chromatography, and then obtaining main pharmacokinetic parameters of the avilamycin, wherein a pharmacokinetic critical value is formulated by Monte Carlo simulation; clinical cut-off values were obtained from clinical trials of different MIC strains. And obtaining a drug resistance judgment fold point value through comprehensive analysis of a wild type critical value, a pharmacodynamic critical value and a clinical critical value. The invention comprehensively analyzes the antibacterial action characteristics, the pharmacokinetic change and the clinical application effectiveness of the avilamycin in the pig body for the first time, not only predicts and monitors the antibiotic drug resistance, but also can keep good treatment effect, and has deeper and wider application value.

Description

Application of avilamycin in inhibition drug of clostridium perfringens of pigs

Technical Field

The invention belongs to the technical field of veterinary pharmacology, and particularly relates to application of avilamycin in a swine clostridium perfringens inhibition drug.

Background

Clostridium perfringens (Clostridium perfringens) is widely distributed in nature as a pathogenic bacterium of pig digestive tract diseases, and seriously harms the development of animal husbandry in various countries. It has the ability to produce spores under harsh environmental conditions, and is widely present in the digestive tract of most animals, which is also one of its most potential threats. A certain amount of clostridium perfringens exists in the intestinal tract of healthy pigs, but does not cause the animal to suffer from diseases, and only when the clostridium perfringens accumulates to a certain extent, a large amount of exotoxin is generated to cause the diseases. The genotypes which are currently responsible for the major pathogenicity of swine clostridium perfringens disease are clostridium perfringens type a and type C, with the major clinical manifestations including piglet diarrhea and hemorrhagic necrotic enteritis. The death rate of the disease is extremely high, which causes serious economic loss in animal husbandry. In addition, clostridium perfringens is at risk of transmission to humans through the food chain.

Bacterial resistance is becoming increasingly problematic due to the improper use of antibiotics. Clinical treatment of clostridium perfringens disease has also become very tricky due to the development of resistance. The drug resistance of clostridium perfringens of different animal sources to tetracycline and minocycline is 33-73% and 17-40% respectively. A drug sensitivity experiment is carried out on 258 strains of clostridium perfringens of pigs by Rood et al, and 200 strains of the clostridium perfringens are resistant to tetracycline, and 58 strains of the clostridium perfringens are resistant to multiple drugs of tetracycline, clindamycin and lincomycin.

Avilamycin belongs to oligosaccharide antibiotics, and known structures of Avilamycin (Avilamycin) are 16 types from A to N, wherein the Avilamycin A has the main antibacterial effect, and the rest of Avilamycin components have small effects on clostridium perfringens and low content, so the effects can be approximately ignored, and therefore, the Avilamycin A is mainly used as a research object in the invention. The Li Lai company research report states that the MIC of a metabolite flambic acid of the avilamycin to clostridium perfringens is up to 128 mu g/mL and is more than forty times of the avilamycin. Therefore, there is no need to consider the problem of the antibacterial activity of the metabolite. The research reports that the avilamycin is used for controlling necrotic enteritis of pigs and chickens caused by clostridium perfringens and assisting in controlling diarrhea of weaned piglets caused by escherichia coli, and is not easy to generate cross resistance with other antibiotics. However, the breakpoint value of avilamycin is not included in the CLSI of the veterinary version, and a specific administration technique is not clearly given, so that there is a phenomenon of abuse of the drug in clinical application.

Disclosure of Invention

The purpose of the invention is as follows:

(1) Providing an acquisition scheme of the wild-type critical value of the avilamycin by the clostridium perfringens of pigs;

(2) Providing an acquisition scheme of the swine clostridium perfringens on the pharmacodynamic critical value of the avilamycin;

(3) Providing an acquisition scheme of the swine clostridium perfringens on the clinical critical value of the avilamycin;

(4) Comprehensively analyzing and establishing drug resistance judgment sensitivity break values of the clostridium perfringens of the pigs to the avilamycin by using the three critical values;

(5) And updating the administration dosage of the avilamycin for treating the swine clostridium perfringens by using the acquired drug resistance judgment sensitivity break point value.

The purpose of the invention is realized by the following technical scheme:

the clostridium perfringens of pigs is purchased from British microbial strain collection center, and the preservation number is as follows: NCTC3180.

The operation process of the software related to the present invention is a conventional technical means of those skilled in the art, and is not the essential point of the invention, and is not described herein again.

The invention provides an application of avilamycin in a swine clostridium perfringens inhibition drug, and the usage amount optimization method comprises the following steps:

(1) Obtaining a wild critical value of the swine clostridium perfringens on the avilamycin;

(2) Acquiring a pharmacodynamic critical value of the swine clostridium perfringens to the avilamycin;

(3) Obtaining a clinical critical value of the swine clostridium perfringens to the avilamycin;

(4) Establishing a drug resistance judgment sensitivity break point value of the clostridium perfringens of the pig on the avilamycin by utilizing the three critical values;

(5) The drug-resistance judgment sensitivity breakpoint value is used for optimizing the drug administration dosage of the avilamycin for treating the swine clostridium perfringens, the specific formula is shown in formula (1),

in the formula (1), dose represents the administration dosage, and CL represents the clearance rate of avilamycin in the pig digestive tract; (AUC) _24h /MIC) _ex Refers to the PK/PD parameter break value corresponding to different curative effects; MIC is the minimum inhibitory concentration of the drug on bacteria; f is a biological advantageThe usage degree; fu is the free drug proportion of the drug in the target tissue; BP is the drug resistance judgment sensitivity break point value.

Further, the drug resistance judgment sensitivity break point value is obtained by adopting a break point value judgment decision tree Susceptibility breakdown tree according to a wild type critical value, a pharmacodynamic critical value and a clinical critical value.

Further, the wild type cut-off value is obtained by adopting Ecoffinder software simulation based on the MIC of avilamycin to 120 clinical swine clostridium perfringens. And (3) determining the wild critical value by adopting an agar dilution method to determine the MIC of the separated strain, and executing a program and a method according to the CLSI critical value to obtain the wild critical value of the swine clostridium perfringens to the avilamycin.

Further, the pharmacodynamic cutoff values were established based on pharmacokinetic data of avilamycin in porcine digestive tracts, and were simulated by winnonlin software and Crystal Ball software.

Further, the obtaining of pharmacodynamic cutoff values comprises: (1) in vitro antibacterial characteristics: the action characteristics of the medicament on bacteria are judged by measuring the MIC, MBC and time sterilization curve of the avilamycin on the clostridium perfringens of pigs, and proper pharmacokinetic parameters are selected.

(2) Porcine intrahalfbody PK/PD model study: after the avilamycin is orally taken, the intestinal contents are collected, the collection method is a conventional technical means, which is not described herein, and the distribution of the avilamycin in the intestinal contents is measured by using a high performance liquid phase. Collecting plasma and ileum contents at different time points, and detecting the content of the medicine in one part by using an HPLC method; the other was used for in vivo drug susceptibility testing after aseptic processing.

(3) And (4) according to Monte Carlo simulation pharmacokinetic parameters, obtaining a pharmacodynamic target and a pharmacodynamic critical value through statistical analysis.

Further, clinical cut-off values were obtained: establishing a disease model by adopting an artificial infection method, establishing a healthy control group, a negative control group and an experimental dose group (set on the basis of a treatment dose obtained based on a PK/PD model), observing clinical symptoms, weight, feed intake and the like, calculating indexes such as cure rate, effective rate, death rate, daily gain and the like, scoring symptoms of animals of each group, counting viable bacteria colonies of clostridium perfringens, simulating a clinical critical value by using software, and comprehensively analyzing to obtain a final breaking point value.

The clinical critical value is established according to clinical effectiveness experiments of the avilamycin to the clostridium perfringens of pigs, is obtained by CART software, "Window" and nonlinear regression simulation, and is calculated by using PK/PD parameter break point values to give a medicament dosage formula (2):

in equation (2): dose is expressed as Dose, and CL refers to clearance of avilamycin in pig digestive tract; (AUC) _24h /MIC) _ex Refers to the PK/PD parameter break value corresponding to different curative effects; MIC is the minimum inhibitory concentration of the drug on bacteria; f is bioavailability; fu is the free drug ratio of the drug in the target tissue.

The invention has the beneficial effects that:

and determining the MIC of the isolated strain through a drug sensitivity experiment of the avilamycin to swine clostridium perfringens, and acquiring a wild critical value of the swine clostridium perfringens to the avilamycin according to a relevant standard. Through in vitro and in vivo pharmacodynamic studies, the antibacterial action characteristics of the avilamycin on clostridium perfringens are inspected, appropriate pharmacokinetic parameters are selected through establishing an in vivo PK/PD model of porcine ileum contents, the pharmacokinetic parameters are simulated according to Monte Carlo, and pharmacodynamic critical values are obtained through statistical analysis. Clinical critical values are obtained through clinical tests, and bases are provided for clinical reasonable medication of the avilamycin.

The invention comprehensively analyzes the antibacterial action characteristics, the pharmacokinetic change and the clinical application effectiveness of the avilamycin in the pig body for the first time, not only predicts and monitors the antibiotic drug resistance, but also can keep good treatment effect, and has deeper and wider application value.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the minimum inhibitory concentration of avilamycin against 120 strains of Clostridium perfringens;

FIG. 2 is a non-linear regression fit of avilamycin to Clostridium perfringens;

FIG. 3 growth curves of Clostridium perfringens in FT broth and ileal fluid;

FIG. 4 is a graph of in vitro and in vivo bactericidal profiles of avilamycin against Clostridium perfringens;

FIG. 5 is a graph of mean time course concentration of avilamycin in the contents;

FIG. 6 is PTA at different MICs for Avanamycin and Clostridium perfringens;

FIG. 7 is a graph of a non-linear regression simulation of clinical data;

FIG. 8 is the results of CART regression tree analysis clinical treatment;

FIG. 9 is a technical roadmap for the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

The technical scheme of the invention is shown in figure 9.

Window is a general method for calculating a break point value published in journals in the field, and the operation process of software related to the invention is a conventional technical means of technicians in the field, is not an inventive point, and is not described herein.

Example 1 acquisition of wild type cut-off values

Obtaining the wild critical value of the swine clostridium perfringens to the avilamycin

(1) Determination of MIC (minimum inhibitory concentration) value by agar dilution method

The specific method participates in CLSI M11-A7 anaerobic bacteria drug susceptibility test method.

(2) MIC data analysis and wild type cut-off establishment

The data analysis software chosen for this trial was ecofmder. The cut-off value determined should comprise a minimum of 95% of the wild-type strain.

Software automatically simulates the upper limit of distribution of wild strains in different confidence intervals of 95%,97.5%,99.0%,99.5% and 99.9%, and the upper limit of the MIC distribution of the wild clostridium perfringens in the confidence interval of 95% is taken as a final wild critical value, namely 0.25 mu g/mL. This result can also be obtained by non-linear regression analysis simulation with SPSS software. The minimum inhibitory concentration of avilamycin to 120 clostridium perfringens is shown in table 1, and fig. 1 shows the minimum inhibitory concentration of avilamycin to 120 clostridium perfringens.

TABLE 1 minimum inhibitory concentration of avilamycin against 120 strains of Clostridium perfringens

The results of curve fitting by ecofmder software are shown in fig. 2, with the thick solid line representing the measured MIC cumulative distribution curve and the thin solid line representing the fitting results.

Example 2 acquisition of pharmacodynamic cutoff values

Obtaining the pharmacodynamic critical value of the swine clostridium perfringens to the avilamycin

(1) Clostridium perfringens growth curve plotting

Preparation of sterile ileal contents: weighing the blank ileum content in a 10mL centrifugal tube, centrifuging at 12000r/min for 10min, sucking the supernatant, filtering the supernatant in a super clean bench with a 0.22 μm sterile filter membrane for sterilization, collecting the filtrate in a sterilized fine bottle, and storing in a refrigerator at-20 ℃. Inoculating 10 μ L of the extract to nutrient agar culture medium for overnight culture before each use, and storing if no bacteria grow; if bacteria grow, the bacteria are filtered again for sterilization.

Preparing seed bacteria liquid: on a reinforced Brookfield agar plate, 2 colonies are selected and inoculated in reinforced FT broth, and are statically cultured for 6 h-8 h at 37 ℃ in an anaerobic incubator. Subjecting the cultured bacterial solution to turbidimetry with standard turbidimetric tube, diluting with blank broth to obtain bacterial solution (10) with turbidity of 0, mcLeod standard ⁸ CFU/mL), and diluting 100 times to obtain seed bacterial liquid for later use.

Measurement of growth curves: 2 sterile vials were taken, one with 1.9mL FT broth and one with 1.9mL sterile ileum contents. And sucking 100 mu L of prepared seed bacterial liquid, uniformly whirling, counting colonies with 0 point, and then placing the colonies into an anaerobic incubator for standing culture at 37 ℃. Inoculating bacteria for 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 12h and 24h, respectively sucking 20 mu L of the bacteria, diluting by times, and obtaining the number of viable bacteria colonies at each time point by adopting a pouring counting method. The growth curve was plotted on the ordinate, which is the logarithmic value of the number of viable bacteria units contained in 1mL of the bacterial suspension, and on the abscissa, which is the sampling time point. The growth curves of clostridium perfringens in FT broth and ileal fluid are shown in fig. 3.

(2) Broth dilution method for determination of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration)

Determination of Minimum Inhibitory Concentration (MIC): the MIC values of avilamycin against selected clostridium perfringens were determined with reference to the broth dilution procedure of CLSI M11-A7.

Determination of Minimum Bactericidal Concentration (MBC): the measurements were carried out according to the method recommended in the CLSI file under M26-AE number.

Determination of MIC, MBC in vivo: the in vivo MIC and MBC assay method is similar to that of the in vitro MIC and MBC assay except that the broth is replaced with sterile ileal contents and the results are interpreted as required for the acquired antimicrobial susceptibility test. The susceptibility test of avilamycin to clostridium perfringens from pigs is shown in table 2.

TABLE 2 drug susceptibility testing of avilamycin to Clostridium perfringens in swine

(3) In vitro and in vivo sterilization curve drawing

In vitro sterilization curve: according to the results of the MIC measurement, 10mL of FT broth containing concentrations of 32 XMIC, 16 XMIC, 8 XMIC, 4 XMIC, 2 XMIC, 1/2 XMIC avamycin was prepared, and a drug-free blank was set up. The broths containing different concentrations of avilamycin were placed in sterile bacterial flasks and 100. Mu.L of a 0.5M turbidity standard (10) was added to each ⁸ CFU/mL), the same treatment is carried out on the clostridium perfringens bacterial liquid of a control group, and the clostridium perfringens bacterial liquid is placed in an anaerobic incubator at 37 ℃ for static culture after being evenly mixed by vortex. And taking a blank control bottle for bacterial colony counting to obtain the initial bacterial concentration. And sucking 100 mu L of culture solution after 0h, 1h, 2h, 4h, 6h, 8h, 12h and 24h respectively, performing bacterial counting after serial dilution, and setting 3 parallels for averaging during counting. After the number of the surviving colonies with different concentrations at each time point is obtained, a sterilization curve is drawn by taking the logarithmic value as the vertical axis and the time point as the horizontal axis.

Semi-in vivo bactericidal curve: ileum contents collected at different time points after the gavage administration of the pharmacokinetic experiment are treated by high-speed centrifugation and sterile filtration membrane, 1.9mL of the ileum contents is taken at each time point and added into a sterile bacterial bottle, then 100 mu L of log phase bacterial liquid is inoculated, and blank ileum contents are taken as growth control. Mixing, standing at 37 deg.C in anaerobic incubator, respectively sucking 100 μ L culture solution for 0h, 1h, 2h, 4h, 6h, 8h, 12h, and 24h, diluting in series, counting bacteria, and setting three parallel averages. And (4) after the number of the surviving colonies with different concentrations at each time point is obtained, taking the logarithm value as a vertical axis, and drawing a semi-body sterilization curve by taking the time point as a horizontal axis to obtain semi-body sterilization data.

The in vitro and in vivo sterilization curves are shown in figure 4, and the sterilization curves show that the avilamycin has stronger sterilization effect on clostridium perfringens, and the sterilization speed and degree are obviously enhanced along with the increase of the concentration of the medicament. The avilamycin exhibits a concentration dependence on the bactericidal power of clostridium perfringens.

(4) Pharmacokinetics study of avilamycin in pigs

The test pigs enter the test site to adapt to the environment 7 days in advance, and are fasted and forbidden 24 hours before the operation. An ileal fistula placement procedure was performed. Ileum and caecum were distinguished using ileocecal valve as a marker. The position of the ileum about 10cm from the cecum opening was selected as the fistula-mounting site. And 7d, removing stitches after the operation, and performing subsequent tests after the pigs to be tested recover stable diet and defecate. The experimental method uses a T-shaped fistula installation operation for the first time to collect intestinal contents, and uses high performance liquid chromatography to determine the distribution of the avilamycin in the intestinal contents.

Since avilamycin is not distributed in plasma, this test only measures changes in drug concentration in the intestinal tract of the target tissue. The administration mode of the pig is single-time intragastric administration, and the administration dosage is 4mg/kg body weight. When the stomach is filled, the pig is bound on the back, the mouth of the pig is opened by the mouth gag, and the avilamycin raw material which is weighed according to the weight of the pig is prepared into suspension with 0.5 percent of sodium carboxymethylcellulose to be filled into the stomach for administration. The intestinal contents of the animals are collected at 0h, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h, 10h, 12h, 24h, 36h and 48h after administration. The obtained ileum content sample is marked and then placed in a refrigerator at the temperature of-70 ℃ for storage and detection.

Accurately weighing 1g of ileum content, adding 3mL of acetone into a 50mL plastic centrifuge tube, vortexing for 5min, centrifuging for 10min at 10000r/min, and taking supernatant to be put into a 100mL centrifuge tube. Adding 2mL of acetone into the residue, repeatedly extracting twice, combining 2 times of supernate, adding 10mL of 10% NaCl solution, removing fat by 3mL of saturated n-hexane, and removing the n-hexane layer. 15mL of dichloromethane and 10mL of dichloromethane are respectively added, the mixture is kept stand for layering, and then a dichloromethane layer is taken out to be arranged in a centrifuge tube and dried by nitrogen at 50 ℃. The residual material was re-dissolved in chloroform 3 mL. The mobile phase was acetonitrile and ammonium acetate. The drug concentrations in the ileal contents (n = 6) at different time points after a single gavage administration in pigs are shown in table 3. The mean time-course concentration of avilamycin in the contents is shown in figure 5.

Table 3 drug concentration in ileal contents at different time points after a single gavage administration in pigs (n = 6)

Note: "ND" means not detected

(5) Data analysis and establishment of pharmacodynamic thresholds

Pharmacokinetic model fitting: pharmacokinetic data for ileum contents of piglets after intragastric administration of avilamycin at a dose of 4mg/kg were processed using Winnonlin 5.2 (Pharsight, USA) software, and a non-compartmental model was selected to calculate major pharmacokinetic parameters such as peak concentration (C) _max ) Time to peak (T) _max ) Half life, half life

Clearance (CL), area under the curve (AUC) at time of drug (Beauchene 1995), etc. The data are described by means of mean values plus or minus standard deviations, excel for Windows is adopted for calculating all the mean values, and SPSS software is used for carrying out statistical analysis on pharmacokinetic parameters of avilamycin in pigs.

Semi-in vivo PK/PD model fitting: substituting the sterilization data of the avilamycin to the interior of the half body of the clostridium perfringens into the inhibitory Sigmoid E _max Model, processing in vivo PK/PD parameter values (AUC) _24h /MIC) _ex And obtaining the corresponding equation parameter values through fitting according to the relation of the antibacterial effect (bacterial log reduction). Inhibitory Sigmoid E _max The model equation is:

e represents the difference of the bacteria concentration of the ileum content sample after being cultured for 24h and the initial inoculation concentration logarithmic value at each time point; e _max Shows the bacterial concentration of blank ileum content samples after inoculation and culture for 24hDifference from initial inoculated colony log value; e ₀ Represents the maximum change value of the bacteria concentration logarithm value after the initial inoculation bacteria culture for 24h in the ileum content sample; c represents the in vivo PK/PD parameter value; EC (EC) ₅₀ Represents the intra-half-body PK/PD parameter value when half of the maximum bactericidal effect is achieved in the ileal content sample; n is the Hill coefficient and describes the slope of the half-in-vivo PK/PD parameter values after effect E is linearized. E _max And E ₀ Can be directly obtained by a sterilization curve test in a half body, EC ₅₀ And N are obtained by fitting with Winnonlin software.

After the Hill equation is fitted, calculating PK/PD parameter break values corresponding to different treatment effects by using the equation: when E =0, it indicates that the bacterial concentration does not change significantly before and after the culture, and the bacterial growth can be inhibited; when E = -3, the culture medium shows that 99.9% of bacteria can be killed by proper culture time, and the therapeutic effect is achieved; when E = -4, it means that 99.99% of bacteria are killed at an appropriate time of culture, and pathogenic bacteria can be eradicated to prevent recurrence.

Monte Carlo simulation of AUC of 10000 pigs Using Crystal ball according to the calculation results _24h Simulating pharmacodynamic targets, and respectively calculating the standard-reaching rate of different MICs to obtain the maximum MIC with the standard-reaching rate higher than 90%, which is the pharmacodynamic critical value.

According to AUC ₂₄ Data 428.62 h. Mu.g/mL and standard deviation 14.23, monte Carlo simulations using Crystalball 7 software, generated AUC for 10000 pigs ₂₄ And (6) data. PK/PD parameter value (AUC) when E = -3 is taken _24h /MIC) _ex 36.15h as pharmacodynamic target, calculated according to simulation results (AUC) _24h /MIC) _ex The achievement rate at different MIC values is shown in figure 6. It can be seen that the achievement rate is 100% when the MIC is 8. Mu.g/mL, and 0% when the MIC is 16. Mu.g/mL. According to the regulations, the maximum MIC with the standard reaching rate of more than 90% is a pharmacodynamic break point. Thus, a pharmacodynamic cutoff value of 8. Mu.g/mL was obtained. The integrated pharmacokinetic parameters in the intestine after administration of 4mg/kg b.w. avilamycin to pigs are shown in table 4. PTA for avilamycin versus clostridium perfringens at different MICs are shown in fig. 6.

TABLE 4 Integrated pharmacokinetic parameters in the intestinal tract after administration of 4mg/kg b.w. avilamycin to pigs

Note: c _max To reach peak concentration, T _max In order to achieve the peak time,

to eliminate half-life, AUC _24h Area under the curve for drug hours from 0h to 24h, AUMC _24h Area under first moment of drug time curve, MRT, within 24h _24h Mean retention time within 24h, CL/F is clearance corrected by bioavailability.

Table 5 avilamycin in half of porcine ileum content (AUC) _24h /MIC) _ex And log reduction of bacteria

Note: a represents the drug concentration at different time points, b represents the drug concentration in the 24h half-body (AUC) incubated with bacteria at different drug concentrations _24h /MIC) _ex (ii) a c represents the log of the bacterial count at different drug concentrations when incubated with the bacteria for 0 h; d represents the log of the bacterial count for 24h incubation with different drug concentrations; e represents the log change value of the bacteria after incubation of the drug with the bacteria at the corresponding time point (d-c).

TABLE 6 in vivo pharmacodynamic parameters of avilamycin against Clostridium perfringens

Note: e is the difference of the colony log values after the intestinal contents are inoculated and cultured for 24 hours at each time point; e _max Difference value between the colony logarithm value after inoculation and culture of intestinal contents for 24h and the colony logarithm value of initial inoculation; e ₀ Maximum difference between the log values of the initial inoculated colonies and the log values before and after 24h of inoculation culture of the intestinal content sample; c is a half-body internal PK/PD parameter value; EC (EC) ₅₀ The in vivo PK/PD parameter value for the time at which 50% of maximal bactericidal effect was produced in the intestinal content sample; and N is a Hill coefficient, describes the slope of the half-in-vivo PK-PD parameter value and the effect E after linearization, and determines the steepness of the S-shaped curve relation.

Example 3 acquisition of clinical Critical values

Obtaining clinical critical value of swine clostridium perfringens to avilamycin

(1) Therapeutic trials of avilamycin against different MIC Clostridium perfringens porcine bacteria

Determination of binding rate: taking a dialysis bag pretreated before an experiment, cutting the dialysis bag into small sections with the length of about 8cm, fastening one end of the dialysis bag by using a cotton rope, removing redundant water outside the bag, putting 1g of blank intestinal contents into the dialysis bag, fastening the opening of the bag to enable the dialysis bag to be suspended in 20mL of dialyzates containing 0.2 mu g/mL, 5 mu g/mL and 20 mu g/mL of medicinal contents respectively, adjusting the position of the dialysis bag to enable the liquid level inside and outside the dialysis bag to be kept at the same horizontal plane, avoiding the dialysis bag from being tightly attached to the wall of the tube as much as possible, sealing the opening of the tube, and placing the dialysis bag in a refrigerator at 4 ℃ for dialysis until the balance is achieved. Each concentration was set to 3 replicates. After dialysis, respectively taking internal and external samples of the dialysis bag, measuring the concentration of the drug in the samples, and calculating the binding rate according to the concentration, wherein the binding rate = (Dt-Df)/Dt x 100%, and the binding rate is as follows: dt is the concentration of the drug in the dialysis bag, and Df is the concentration of the drug in the external dialysate.

Calculating the dosage formula (2) by using the PK/PD parameter breakpoint value:

in equation (2): CL refers to the clearance rate of avilamycin in pig digestive tract; (AUC) _24h /MIC) _ex Referring to PK/PD parameter break-point values corresponding to different curative effects; MIC is the minimum inhibitory concentration of the drug on bacteria; f is bioavailability; fu is the free drug ratio of the drug in the target tissueFor example. The binding rate of avilamycin in porcine ileum contents is shown in table 7, and the administration technique and the herd feeding technique are shown in table 8.

Table 7 binding rates of avilamycin in porcine ileum contents

TABLE 8 dosing technique and group-fed dosing technique

A mechanism PK/PD model based on bacterial growth kinetics is used, a population wild type critical value of 0.25 mu g/mL is selected as a reference, and the growth condition of clostridium perfringens under different administration doses and administration intervals is predicted through MlxPlare software.

66 test piglets (about 15 kg) were divided into 11 groups, one group was blank control group, five groups were negative control groups of different MIC strains, five groups were experimental groups of different MIC strains, and each group had six piglets. The blank group was not administered with no infection, the negative control group was not administered with infection, the experimental group was administered after infection, and the dose and interval of administration were performed according to the therapeutic dose obtained for PK/PD.

Preparing an infectious bacterium solution: inoculating clostridium perfringens in a liquid thioglycollate culture medium, performing anaerobic amplification culture at 37 ℃ for 8-12 h, centrifuging at 8000r/min for 10min, and concentrating the bacterial liquid to 10 ⁹ CFU/mL～ 10 ¹⁰ CFU/mL, this is the bacterial suspension for infection.

The infection method comprises the following steps: by adopting a method of intragastric infection, 50mL of infection suspension is injected into pigs before feeding every morning and afternoon, fasting and water deprivation are carried out 3h after inoculation, which is beneficial to the colonization of bacteria in intestinal tracts, and then water is fed normally for 3d.

Scoring the symptoms according to a symptom scoring principle, wherein 0 score indicates that the mental state is normal, the stool is in a strip shape or a granular shape, and the diet is normal; 1 point indicates slight lassitude, softer than ordinary feces, and anorexia; 2, the food is obviously sleepy, thin and thick excrement, no separation of excrement and water, obvious loss of appetite and emaciation; 3, no response to external stimulation, separation of liquid excrement from liquid excrement, no eating and obvious emaciation. When each symptom reaches 2 minutes, indicating that the infection is successful, starting to administer the medicine, continuously observing for 14 days, and counting the cure rate. The general clinical symptoms and the special symptoms of each group of animals after the test is started, before the test is administered, after the test is administered and after the test is stopped, and the occurrence, development, regression and disappearance of the symptoms are observed and recorded in detail, the death number and death time of each group of animals after the test is started, the symptom improvement and symptom disappearance of the animals after the test is administered are observed and recorded, and the death rate, effective rate and cure rate of each group of animals during the test are calculated. When the infection is carried out to the third day, the negative control group and the experimental group have the states of mental depression, loose stool and the like. The PCR electrophoresis result shows that the bacteria separated from the collected fecal sample detects a fragment with the length of 402bp and is an A-type strain. The cure rates for avilamycin against clostridium perfringens at different MICs are given in table 9. The results of the colony counts of live clostridium perfringens bacteria are shown in table 10.

TABLE 9 curative ratio of Avermectin to Clostridium perfringens at different MICs

TABLE 10 Clostridium perfringens viable bacterial colony counts

Note: the same column of different letters indicated significant differences (P < 0.05) as analyzed by the Duncan's multiple range test method.

(2) Statistical data analysis and clinical threshold acquisition

And carrying out data statistics on clinical treatment results and bacteriological prognosis results. The clinical treatment results are success or failure, when all symptoms score 0, the treatment is successful, otherwise the treatment fails. The bacteriological prognosis is mainly from the perspective of bacteria, and the bacteria are separated in the sick pig body, because the clostridium perfringens is a common flora in the digestive tract of a normal animal, the bacteria number is judged to be reduced below a normal value, namely the cure is successful. A cure is indicated when both clinical and bacteriology indicate successful treatment. And (4) counting the cure rate of each bacterium in the experimental group, and finishing the relationship between MIC and cure rate.

Substituting POC and MIC calculation parameters Maxdiff and CAR with Window to obtain the selection range of clinical critical values formed by Maxdiff and CAR. And then POC = 1/(1 + e) according to a formula proposed by EUCAST ^-a+bf(MIC) ) Fitting the obtained data by using nonlinear regression in SPSS software, taking MIC as an independent variable and POC as a dependent variable to obtain a corresponding model expression, and then calculating the MIC value corresponding to the cure rate of 90%. And finally, analyzing the obtained data by using a CART regression tree, and segmenting the MIC to obtain the cure rate of different MIC intervals.

And finally, obtaining a final drug resistance judgment standard according to a breakpoint branch tree (Susceptibility breakdown trees). The results of the Maxdiff and CAR simulation are shown in Table 11.

TABLE 11 Maxdiff and CAR simulation results

The non-linear regression simulation of clinical data is shown in FIG. 7, and the regression analysis model expression is y =83.771-3.542x-4.24x ² -0.054x ³ Calculating the independent variable Log corresponding to the cure rate of 90 percent ₂ MIC is-2.17, MIC is 0.22 μ g/mL, and the recommended clinical critical value is 0.06 μ g/mL-0.22 μ g/mL.

And finally, substituting the data of the clinical test into Salford Predictive Modeler software, and obtaining the cure rate of 100% when the MIC is less than or equal to 0.16 mu g/mL and 86.7% when the MIC is more than 0.16 mu g/mL from a regression tree. When POC is equal to 90%, MIC should be greater than 0.16. Mu.g/mL, and therefore the resulting clinical cutoff should be between 0.16. Mu.g/mL and 2. Mu.g/mL.

Results of the CART regression tree analysis clinical treatment are shown in figure 8.

Firstly, the visual observation can obtain that the MIC interval corresponding to the cure rate of 90 percent is between 0.06 mu g/mL and 0.25 mu g/mL (1) the selection WindoW of the clinical critical value recommended by the 'Window W' method is between 0.06 mu g/mL and 2 mu g/mL; (2) Nonlinear regression analysis shows that when the cure rate is 90%, the MIC is 0.22 mu g/mL, and the recommended clinical critical value is 0.06 mu g/mL-0.22 mu g/mL; (3) The clinical threshold range obtained by CART regression tree analysis is 0.16-2 mug/mL. From the MIC profiles determined in this experiment, the closest MIC of 0.22. Mu.g/mL and 0.16. Mu.g/mL, i.e., 0.125. Mu.g/mL, was selected as the clinical cutoff.

In combination with the above experiments, it is known that the wild type cutoff value is 0.25. Mu.g/mL, the pharmacodynamic cutoff value is 8. Mu.g/mL, and the clinical cutoff value is 0.125. Mu.g/mL. The three critical values are substituted into a flow chart formulated by a break point published by CLSI, which conforms to the CO _PD >CO _WT >CO _CL The final standard value of drug resistance judgment of the clostridium perfringens of the pig to the avilamycin is 0.25 mug/mL of the wild critical value.

Example 4 optimization of dosing technical formula dosing technique for the treatment of clostridium perfringens in swine using a breakpoint value optimization for avilamycin

The traditional PK/PD recommended dose formula is:

the dosage can be changed by calculating the dosage by using the break point value

Wherein BP represents the breaking point value of drug resistance judgment sensitivity, and CL represents the clearance rate of the avilamycin in the pig digestive tract; (AUC) _24h /MIC) _ex Refers to the PK/PD parameter break value corresponding to different curative effects; MIC is the minimum inhibitory concentration of the drug on bacteria; f is bioavailability; fu is the free drug ratio of the drug in the target tissue.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (3)

1. The method for establishing the drug resistance judgment standard of the clostridium perfringens of pigs to the avilamycin is characterized by comprising the following steps of: the method is for non-diagnostic and non-therapeutic purposes, comprising the steps of:

(1) Acquiring a wild type critical value of the swine clostridium perfringens for the avilamycin, wherein the wild type critical value is obtained by adopting Ecofrider software simulation on the basis of the MIC of the avilamycin for 120 clinical swine clostridium perfringens, the MIC of an isolated strain is determined by adopting an agar dilution method, and the wild type critical value of the swine clostridium perfringens for the avilamycin is acquired by executing a program and a method according to the CLSI critical value;

(2) Acquiring a pharmacodynamic critical value of clostridium perfringens of pigs on avilamycin, wherein the pharmacodynamic critical value is established according to pharmacokinetic data of avilamycin in pig digestive tracts and is obtained by simulating winnonlin software and Crystal Ball software, and the method specifically comprises the following steps: (1) the in vitro antibacterial characteristic: determining the action characteristics of the medicament on bacteria by measuring MIC, MBC and time sterilization curves of the avilamycin on the clostridium perfringens of pigs, and selecting proper pharmacokinetic parameters;

(2) porcine intrahalfbody PK/PD model study: after oral administration of the avilamycin, collecting intestinal contents, determining the distribution of the avilamycin in the intestinal contents by using a high performance liquid phase, collecting plasma and ileum contents at different time points, and detecting the content of a medicine by using an HPLC method in one part; the other part is used for drug sensitivity test in half body after being processed aseptically;

(3) according to Monte Carlo simulation pharmacokinetic parameters, a pharmacodynamic target and a pharmacodynamic critical value are obtained through statistical analysis;

(3) Obtaining a clinical critical value of the swine clostridium perfringens to the avilamycin, wherein the clinical critical value is established according to a clinical effectiveness experiment of the avilamycin to the swine clostridium perfringens: establishing a disease model by adopting an artificial infection method, establishing a healthy control group, a negative control group and an experimental dose group, wherein the experimental dose group is set on the basis of a treatment dose obtained based on a PK/PD model, observing clinical symptoms, body weight and food consumption, calculating cure rate, effective rate, mortality and daily gain indexes, scoring symptoms of animals, counting viable bacteria colonies of clostridium perfringens, simulating clinical critical values by using software, comprehensively analyzing to obtain a final breaking point value, simulating by CART software, windoW and nonlinear regression, and calculating a drug delivery dose formula (2) by using a PK/PD parameter breaking point value:

（2），

in equation (2): dose represents the administration Dose, and CL refers to the clearance rate of avilamycin in pig digestive tract; (AUC 24 h/MIC) ex refers to PK/PD parameter break-point values corresponding to different therapeutic effects; MIC is the minimum inhibitory concentration of the drug on bacteria; f is bioavailability; fu is the free drug proportion of the drug in the target tissue;

(4) Acquiring a drug resistance judgment sensitivity break point value of the clostridium perfringens of the pig on the avilamycin by using a Susceptibility breakthrough tree according to a wild critical value, a pharmacodynamic critical value and a clinical critical value;

(5) The drug-resistance judgment sensitivity breakpoint value is used for optimizing the drug administration dosage of the avilamycin for treating the swine clostridium perfringens, the specific formula is shown in formula (1),

（1），

in the formula (1), dose represents the administration dosage, and CL refers to the clearance rate of the avilamycin in the pig digestive tract; (AUC 24 h/MIC) ex refers to PK/PD parameter break-point values corresponding to different therapeutic effects; MIC is the minimum inhibitory concentration of the drug on bacteria; f is bioavailability; fu is the free drug proportion of the drug in the target tissue; BP is the drug resistance determination sensitivity break-point value.

2. The method of claim 1, wherein: the determination of the wild type cut-off value comprises:

(1) Determining MIC value by agar dilution method;

(2) Analyzing MIC data;

(3) Establishing a wild type cut-off value.

3. The method of claim 1, wherein: the acquisition of the pharmacodynamic cut-off value comprises the following steps:

(1) Drawing a clostridium perfringens growth curve;

(2) Broth dilution method for determining minimum inhibitory concentration and minimum bactericidal concentration;

(3) Drawing in-vitro and half-body-outside sterilization curves;

(4) And analyzing the data and establishing a pharmacodynamic critical value.

Images