CN114366803A

CN114366803A - Medicine for treating bacterial endophthalmitis and preparation method thereof

Info

Publication number: CN114366803A
Application number: CN202210077823.9A
Authority: CN
Inventors: 吴宏; 王湛云; 王烁
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2022-01-24
Filing date: 2022-01-24
Publication date: 2022-04-19

Abstract

The invention discloses a medicament for treating bacterial endophthalmitis and a preparation method thereof, wherein the medicament comprises antibacterial peptide, and the content of the antibacterial peptide in liquid medicine is 2-512 mug/ml. The antibacterial peptide is any one of ik-12, pp-12, rk-12, ip-12 and wp-12, and the sequences of the five antibacterial peptides are respectively as follows: ilrwwpwrrwrrk, pwirwrwrwrkwwp, rwiprwrwrpwwk, irlrwrwrwrkwwp and wpirlrwrwrkwwp. The preparation method comprises the following steps: firstly, preparing instruments and reagents; secondly, solid phase polypeptide synthesis; step three, experiment of each antibacterial peptide; has the advantages that: the antibacterial peptides IK-12, IP-12 and WP-12 have low minimum inhibitory concentration to methicillin-resistant staphylococcus aureus and low toxicity to erythrocytes, and the antibacterial peptides IK-12, IP-12 and WP-12 injected into the early vitreous cavity can effectively treat bacterial endophthalmitis of rabbits.

Description

Medicine for treating bacterial endophthalmitis and preparation method thereof

Technical Field

The invention relates to a medicament and a preparation method thereof, in particular to a medicament for treating bacterial endophthalmitis and a preparation method thereof.

Background

Endophthalmitis, which is currently referred to as vitreous and/or aqueous humor infection caused by bacteria or fungi, can attack the retina, choroid, sclera, and cornea, causing an inflammatory response, is a destructive ocular infection that can lead to inevitable visual loss within hours or days after symptoms appear. Although the disease has low morbidity, once the disease occurs, serious consequences can be caused, such as vision loss, blindness and even eyeball extirpation, and the burden is increased on the life of a patient. Although it can be treated by local and systemic application of antibiotics, in the past decades, antibiotic resistance has increased dramatically worldwide due to improper use of antibiotics and few new antibiotics, which has become one of the most serious global public health threats in the 21 st century, and humans have stepped into the post-antibiotic age. The World Health Organization (WHO) emphasizes that 1000 million people will die by 2050 due to antibiotic resistance if the use of antibiotics is not regulated.

The antibacterial peptide is host defense molecules commonly existing in a natural immune system in an organism, is a short peptide chain consisting of 5-100 amino acid residues generally, has the molecular weight of 1-5 kDa, is used as a new weapon in antibacterial fight, has wide antibacterial spectrum, can kill bacteria and fungi, and also has the functions of resisting virus, parasites and biofilm; the sterilization is rapid, and can be completed within dozens of minutes; in addition, the compound has the advantages of anti-tumor, immunoregulation, difficult drug resistance generation and small harm to human bodies, and is expected to become a new medicine for treating endophthalmitis.

Studies have found that the eye and related tissues, such as the lacrimal apparatus, express a range of human defensins which help prevent infection and may be involved in wound healing responses and tissue remodeling, and that these endogenous defensins may also be involved in ocular inflammation and immune responses, which provide support for the use of antimicrobial peptides to treat ocular diseases.

Disclosure of Invention

The main object of the present invention is to solve the problem of antibiotic resistance that occurs during the treatment of endophthalmitis;

it is another object of the present invention to provide a novel drug for treating endophthalmitis;

the invention provides a medicine for treating bacterial endophthalmitis and a preparation method thereof, aiming at solving the problems and achieving the aim.

The medicine for treating bacterial endophthalmitis provided by the invention comprises antibacterial peptide, wherein the content of the antibacterial peptide in the liquid medicine is 2-512 mu g/ml.

The antibacterial peptide is any one of ik-12, pp-12, rk-12, ip-12 and wp-12, and the sequences of the five antibacterial peptides are respectively as follows: ilrwwpwrrwrrk, pwirwrwrwrkwwp, rwiprwrwrpwwk, irlrwrwrwrkwwp and wpirlrwrwrkwwp.

The preparation method of the medicine for treating bacterial endophthalmitis provided by the invention comprises the following steps:

first step, preparing the instruments and reagents: a polypeptide solid phase synthesis tube; analytical HPLC; an amino acid; a resin; DMF; piperidine; ninhydrin; phenol; an HBTU; HoBt; DCM; methanol; EDT; TIS; water; acetonitrile; TFA;

step two, solid-phase polypeptide synthesis, wherein the synthesis conditions are as follows: the method comprises the following specific steps at room temperature:

step 1, swelling resin: putting the resin into a reaction tube, adding DCM with the DCM content of 15mL/g for 30 min;

step 2, deprotection: removing DCM, adding 20% piperidine DMF solution with DMF content of 15mL/g for 5min, and adding 20% piperidine DMF solution with DMF content of 15mL/g for 15 min;

step 3, detection: pumping out the piperidine solution, taking a plurality of resins, washing the resins with ethanol for three times, adding ninhydrin and phenol solution, respectively one drop, heating the resins at 105-110 ℃ for 5min, and turning dark blue to be a positive reaction;

step 4, washing: adding DMF with the DMF content of 10mL/g, and washing for six times;

step 5, condensation: and (3) dissolving protected amino acid and HOBt with three times of excess by using DMF (dimethyl formamide), adding into a reaction tube, immediately adding DIC with three times of excess, and reacting for 90 min.

Step 6, washing: adding DMF with the DMF content of 10mL/g, and washing for three times;

step 7, repeating the operations from the step 2 to the step 6, and sequentially connecting amino acids;

step 8, washing for the last time, adding DMF (dimethyl formamide) with the DMF content of 10mL/g, and washing for three times; adding DCM with the DCM content of 10mL/g, washing for three times, adding methanol with the methanol content of 10mL/g, and washing for three times;

step 9, cracking: preparing a lysis solution, wherein the lysis solution comprises 86% of TFA, 4% of thioanisole, 3% of water, 5% of EDT and 2% of phenol by weight, and performing lysis for 120 min;

step 10, suction filtration and washing: separating the lysate from the resin by using a sand core funnel, stirring the lysate into ethyl ether with eight times of the volume of the lysate, performing suction filtration separation by using a Buchner funnel, washing polypeptide solids in the funnel by using the ethyl ether for three times after pumping, volatilizing at normal temperature, drying and packaging;

step 11, HPLC purification: performing analytical HPLC purification, namely performing C18 reversed-phase column, taking water and acetonitrile as a mobile phase, adding 0.1% TFA into the mobile phase, and performing purification from 10% to 90% in a gradient manner;

step three, the experimental steps of each antibacterial peptide are as follows:

step 1, IK-12, the sequence is ilrwwpwpwrrk, and the molecular weight is as follows: 1779.173, respectively;

Fmoc-linker Am resin

︱1

Lys 2

︱1

Arg 2

︱1

Arg 2

firstly, carrying out 1 operation on Fmoc amino acid resin, wherein the 1 operation is 20% of Piperidine reaction for 20 minutes, and detecting that the color is blue by using a detection reagent;

performing operation 2, wherein the operation 2 comprises the steps of adding Fmoc-Lys or Boc-OH into HBTU + DIEA for reaction for 1 hour, detecting the color to be colorless by using a detection reagent, then performing operation 1, reacting 20% of Piperidine for 20 minutes, and detecting the color to be blue by using the detection reagent;

2, HBTU + DIEA is added with Fmoc-Arg or pbf-OH for reaction for 1 hour, a detection reagent is used for detecting that the color is colorless, then the operation 1 is carried out, and the operation is repeated until the Fmoc of the last amino acid Ile is removed, the resin is dried and cut to obtain crude peptide, the crude peptide is sent to HPLC for purification, and the fine peptide is obtained after freeze-drying;

step 2, PP-12, the sequence is pwirrwrkwwp, the molecular weight is as follows: 1779.173, respectively;

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Trp 2

firstly, carrying out operation 1 on the resin, wherein the operation 1 is 20% of Piperidine reaction for 20 minutes, and detecting that the color is blue by using a detection reagent;

2, carrying out HBTU + DIEA and Fmoc-Pro-OH reaction for 1 hour, detecting the color to be colorless by using a detection reagent, then carrying out 1 operation, carrying out 20% Piperidine reaction for 20 minutes, and detecting the color to be blue by using the detection reagent;

2, performing HBTU + DIEA reaction for 1 hour by adding Fmoc-Trp or Boc-OH, detecting the color to be colorless by using a detection reagent, performing 1 operation, repeating the steps until the Fmoc is removed from the last amino acid Ile, then drying and cutting the resin to obtain crude peptide, purifying the crude peptide by HPLC, and performing freeze-drying to obtain a fine product;

step 3, RK-12, with the sequence of rwiprlwrwwk, molecular weight: 1779.173, respectively;

Rink Amide MBHA resin

︱1

Lys 2

︱1

Trp 2

︱1

Trp 2

carrying out the operation 2, adding Fmoc-Trp or Boc-OH into HBTU + DIEA to react for 1 hour, detecting the color to be colorless by using a detection reagent, carrying out the operation 1, repeating the operation until the Fmoc is removed from the last amino acid Ile, then carrying out suction drying and cutting on the resin to obtain crude peptide, purifying the crude peptide by HPLC, and carrying out freeze-drying to obtain a fine product;

and 4, IP-12, wherein the sequence is irlrwrwrkwwpwp, and the molecular weight is as follows: 1779.173, respectively;

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Pro 2

and 5, WP-12, the sequence is wpirlrwrwrkwwp, and the molecular weight is as follows: 1779.173

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Lys 2

and 2, carrying out HBTU + DIEA reaction by adding Fmoc-Trp or Boc-OH for 1 hour, detecting the color by using a detection reagent to be colorless, carrying out 1 operation, repeating the operation until the Fmoc is removed from the last amino acid Ile, then carrying out suction drying and cutting on the resin to obtain crude peptide, purifying the crude peptide by HPLC, and freeze-drying to obtain a fine product.

The mechanism of the invention is as follows:

the medicine for treating bacterial endophthalmitis and the preparation method thereof provided by the invention are designed into any one of antibacterial peptides of IK-12, PP-12, RK-12, IP-12 and WP-12, and the sequences of the five antibacterial peptides are respectively as follows: ilrwwpwrrwrrk, pwirwrwrwrkwwp, rwiprwrwrpwwk, irlrwrwrwrkwwp and wpirlrwrwrkwwp.

The sequences are rich in: isoleucine (I), leucine (L), arginine (R), tryptophan (W), proline (P), and lysine (K).

Wherein hydrophobic isoleucine (I), tryptophan (W), proline (P) and cationic arginine (R), lysine (K) residues interact and perturb microbial cell walls and membranes; isoleucine (I), leucine (L), and tryptophan (W) have a tendency to form strong β -sheets; arginine (R) high charge density improves the antibacterial effect.

The invention has the beneficial effects that:

the medicine for treating bacterial endophthalmitis and the preparation method thereof have the advantages that the minimum inhibitory concentration of the antibacterial peptides IK-12, IP-12 and WP-12 to methicillin-resistant staphylococcus aureus is low, the toxicity to red blood cells is low, and the antibacterial peptides IK-12, IP-12 and WP-12 injected in an early stage of a vitreous cavity can effectively treat bacterial endophthalmitis of rabbits.

Drawings

FIG. 1 is a schematic diagram showing the therapeutic effect of the intravitreal injection of PBS, ampicillin, vancomycin, IK-12, IP-12 and WP-12 for treating bacterial endophthalmitis of rabbits for 72 hours.

FIG. 2 is a graph showing the clinical scores of the groups according to the present invention, wherein the clinical scores of endophthalmitis in the vancomycin group, the IK-12 group, the IP-12 group and the WP-12 group were lower than those in the PBS group and the ampicillin group at 24h, 48h and 72h after injection of the drugs, and the difference was statistically significant (. p <0.05VS PBS).

FIG. 3 is a graph showing the counting results of the dilution coating plate method of each group according to the present invention, wherein the counting results of the vancomycin group, the IK-12 group, the IP-12 group and the WP-12 group are lower than those of the PBS group and the ampicillin group, and the difference is statistically significant (. p <0.05VS PBS).

FIG. 4 is a graph showing HE staining results of various retinal groups according to the present invention, in which PBS group is shown at magnification x 80; ampicillin group, magnification times 80; vancomycin group, magnification x 200; IK-12 group, magnification x 200; IP-12 group, magnification times 200; WP-12 group, magnification times 200.

FIG. 5 is a graph showing GMS staining results for various groups of retinas according to the invention, in PBS, magnification x 80; ampicillin group, magnification times 80; vancomycin group, magnification x 200; IK-12 group, magnification x 200; IP-12 group, magnification times 200; WP-12 group, magnification times 200.

Detailed Description

Please refer to fig. 1 to 5:

Fmoc-linker Am resin

︱1

Lys 2

︱1

Arg 2

︱1

Arg 2

carrying out the operation 2, adding Fmoc-Arg or pbf-OH into HBTU + DIEA for reaction for 1 hour, detecting the color by using a detection reagent to be colorless, carrying out the operation 1, repeating the operation until the Fmoc of the last amino Fmoc-Ile-OH is removed, draining and cutting the resin to obtain crude peptide, purifying the crude peptide by HPLC, and freeze-drying to obtain a fine product;

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Trp 2

Rink Amide MBHA resin

︱1

Lys 2

︱1

Trp 2

︱1

Trp 2

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Pro 2

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Lys 2

The specific verification test is as follows:

firstly, the method comprises the following steps: minimum inhibitory concentration step:

taking out the standard strain methicillin-resistant staphylococcus aureus (ATCC43300) from a refrigerator at-80 ℃, taking out the bacterial liquid after melting, marking a 4-zone line on a brain-heart culture dish, and putting the brain-heart culture dish into a constant temperature incubator for overnight culture at 37 ℃. Picking up single colony of recovered methicillin-resistant staphylococcus aureus,culturing in 5ml MHB culture medium overnight for 12 hr, inoculating the culture bacterial liquid into 5ml MHB culture medium, continuously inoculating for 3 times, culturing for 4 hr, measuring the bacterial liquid with turbidity of 0.35, 0.38, 0.41, 044, 0.47, 0.50, 0.53, 0.56 with McLeeb turbidimeter, diluting by 10 times, coating, and counting to determine bacteria 1 × 10⁸Turbidity in Mach relative to CFU/ml. According to bacteria 1X 10⁸The turbidity in McLeod corresponding to CFU/ml was found to correspond to 1X 10⁸Diluting the bacterial suspension with CFU/ml, and mixing the diluted bacterial suspension with 1X 10⁸Diluting the bacterial liquid at CFU/ml 1000 times to obtain 1 × 10⁵CFU/ml bacterial fluid. In a 96 well plate, 100. mu.l of 1X 10⁵Mixing the bacterial solution of CFU/mL with 100 mul antibiotic or antibacterial peptide (2 times serial dilution, concentration ranges are respectively 10-2560 mug/mL ampicillin solution, 2-512 mug/mL antibacterial peptide solution and 0.25-64 mug/mL vancomycin solution), and the final drug concentration ranges are respectively: ampicillin concentration: 5-1280 μ g/ml, antimicrobial peptide concentration: 1-256 μ g/ml, vancomycin concentration: 0.125-32 mu g/ml, incubating at 37 ℃ for 16-20h by using other medicines except for vancomycin at 37 ℃ and measuring the OD value of a 96-well plate by using an enzyme-labeling instrument. In a 96 well plate, 100. mu.l of 1X 10⁸Mixing the bacterial liquid of CFU/ml with 100 mul antibiotic or antibacterial peptide (2 times serial dilution, concentration ranges are respectively ampicillin concentration: 10-2560 mug/ml, antibacterial peptide concentration: 2-5120 mug/ml and vancomycin concentration: 0.25-64 mug/ml), and the final drug concentration ranges are respectively: ampicillin concentration: 5-1280 μ g/ml, antimicrobial peptide concentration: 1-256 μ g/ml, vancomycin concentration: 0.125-32 mu g/ml, measuring the OD value of the 96-well plate by using an enzyme-labeling instrument, incubating the 96-well plate for 16-20h at 37 ℃ by using other medicines except for 24h of vancomycin incubation, and measuring the OD value of the 96-well plate by using the enzyme-labeling instrument. The experiment was performed using a blank control group in wells to which only 200. mu.l of medium was added, and a negative control group in wells to which only 100. mu.l of medium and 100. mu.l of bacteria were added. 3 replicate wells were set for each concentration gradient.

All drug sensitive experiments are repeated three times, and the results show that the fluctuation range of the minimum inhibitory concentration does not exceed 1 drug concentration gradient (table 1.1), and table 1.2 shows the minimum inhibitory concentration of the quality control strain methicillin-resistant staphylococcus aureus (ATCC29213), wherein the minimum inhibitory concentration is within the standard range of the minimum inhibitory concentration of the quality control strain published in the American national committee for standardization in Clinical Laboratories (CLSI) M100-S19 (table 1.3).

TABLE 1.1 minimum inhibitory concentration (μ g/ml) of methicillin-resistant Staphylococcus aureus (ATCC43300)

TABLE 1.2 minimum inhibitory concentration (. mu.g/ml) of methicillin-resistant Staphylococcus aureus (ATCC29213)

TABLE 1.3 minimum inhibitory concentration Range (μ g/ml) for methicillin-resistant Staphylococcus aureus (ATCC29213) in CLSI

II, secondly: hemolytic activity step:

on a 96-well plate, 100. mu.l of antimicrobial peptide or antibiotic were serially diluted 2-fold with 100. mu.l of PBS at concentrations ranging from 15.625 to 2000. mu.g/ml, with 3 replicates per concentration setting. Wells containing 100. mu.l of 0.1% Triton X-100 were used as positive controls, and wells containing 100. mu.l PBS were used as negative controls. Positive and negative controls were set for each 96-well plate.

Injecting 0.2-0.3ml/kg of domperidone into hind legs of New Zealand white rabbits, after anesthesia takes effect, collecting 2ml of blood from ear marginal veins after local disinfection, and diluting fresh blood with PBS 25 times to obtain 50ml of 4% erythrocyte suspension. Mu.l of 4% rabbit red blood cell suspension and 100. mu.l of antibiotic or antimicrobial peptide (2-fold serial dilution, concentration range 15.625-2000. mu.g/ml) were mixed in a 96-well plate to a final drug concentration of 7.8125-1000. mu.g/ml. Each well of the positive and negative controls was filled with 100. mu.l of 4% rabbit red blood cell suspension. After putting the 96-well plate into a constant temperature box at 37 ℃ and incubating for 1h, centrifuging at 1000rpm for 5min, taking 100 mu l of centrifuged supernatant, adding the supernatant into a new 96-well plate, and measuring the OD value at 570mm by using a microplate reader.

The hemolytic activity detection experiment was repeated three times, and the hemolytic fractions of the antimicrobial peptides and antibiotics at different concentrations were calculated according to the following calculation formula.

Hemolysis fraction ═ OD 570%_{(drug group)}-OD570_{(negative control group)})/(OD570_{(Positive control group)}-OD570_{(negative control group)})]×100％。

And (3) analysis: even if the concentration of vancomycin and ampicillin reaches 1000 mug/ml, the hemolysis fraction does not exceed 2 percent, and good biocompatibility is shown. Among 5 antibacterial peptides, the hemolysis fraction of PP-12 is higher, and when the concentration reaches 1000 mu g/ml, the hemolysis fraction exceeds 30 percent; although the hemolysis fraction of the IK-12 is over 30 percent when the concentration is 1000 mu g/ml, the hemolysis fraction of the IK-12 is reduced to about 6 percent when the concentration is 500 mu g/ml, and the method is safer; the hemolysis fractions of RK-12 and WP-12 are low and do not exceed 10% when the concentration reaches 1000. mu.g/ml. When the concentration is 250 mug/ml, the hemolytic fraction of all antibacterial peptides does not exceed 4%.

TABLE 2.1 hemolytic fractions (%) -of antibacterial peptides and antibiotics

Hemolytic fractions (%) of antibacterial peptide and antibiotic

Thirdly, the method comprises the following steps: curative effect analysis of antibacterial peptide on rabbit bacterial endophthalmitis model

The experiment has passed ethical review of experimental animals by the military veterinary institute.

After the compound tropicamide eye drops are applied to the right eye of a rabbit and pupils are dilated, 0.2-0.3ml/kg of domestris is injected into the rabbit through muscles for anesthesia. Before operation, the conjunctival sac is washed by sterile normal saline and the eyes are disinfected. Oxybuprocaine eye drops were applied to the right eye once for 5 minutes four times in a row. The eyelid retractor opens the eyelid. Injecting 1X 10 insulin injection needle with sterile disposable insulin injection needle at 2.0mm posterior to temporal limbus of right eye, parallel to pars plana and limbus perpendicular to scleral wall, and inserting into vitreous body cavity with visible needle point in the pupillary region³And (3) 50 mu l of CFU/ml of methicillin-resistant staphylococcus aureus liquid. The levofloxacin eye ointment is smeared on the postoperative conjunctival sac once. All rabbits were used as experimental groups for the right eye and as placebo group for the left eye.

After 24h of bacteria injection, 36 rabbits were randomly divided into 6 groups, group A was PBS group, 100. mu.l of PBS was intravitreally injected; group B is ampicillin group, 1mg/0.1ml ampicillin solution is injected into vitreous body; group C is vancomycin group, and 1mg/0.1ml vancomycin solution is injected into the vitreous body; group D was IK-12, and 1mg/0.1ml IK-12 solution was intravitreally injected; group E is IP-12 group, and intravitreal injection of 1mg/0.1ml IP-12 is performed; group F was WP-12, and 1mg/0.1ml WP-12 solution was injected intravitreally. The inflammation of the rabbit eyes was evaluated according to table 3.1 with the time of bacteria injection as DAY0, the time of drug injection as DAY 1, and the time of drug injection as DAY 1, after 0h, 24h, 48h, and 72h, and are respectively designated as DAY 1, DAY 2, DAY 3, and DAY 4.

TABLE 3.1 clinical scoring Table for endophthalmitis

At 72h, overnarcotizing, killing the rabbits, picking the right eyes of the rabbits under aseptic conditions, putting one right eye of each rabbit into a 4% paraformaldehyde solution, immediately performing HE staining and Giemsa staining, and scoring according to the table 3.2; grinding the rest eyeball in 4 deg.C physiological saline, performing 10 times serial gradient dilution, coating on brain-heart agar plate, and counting after 18-24 hr.

TABLE 3.2 endophthalmitis pathological inflammation scoring table

24h (DAY 1) infected by methicillin-resistant staphylococcus aureus, the right eyes of 36 rabbits all have endophthalmitis, corneal focal edema, conjunctival edema and mild hyperemia, iris mild hyperemia, vitreous opacity, visible part of fundus oculi and visible red light reflection.

Injecting 24h (DAY 2) of medicine such as PBS group corneal diffuse edema, conjunctival edema, obvious hyperemia and slight exudation, iris hyperemia, vitreous moderate turbidity, invisible fundus details, and invisible red reflection; ampicillin group has cornea diffuse edema, conjunctiva obvious hyperemia, iris hyperemia, vitreous body moderate turbidity, invisible fundus details and invisible red reflection; the vancomycin group cornea is normal, the conjunctiva is slightly hyperemic, the iris is hyperemic, the vitreous body is slightly turbid, part of the eyeground is visible, and the red light reflection can be seen; IK-12, IP-12 and WP-12 groups showed similar manifestations, localized corneal edema, conjunctival edema, mild hyperemia, conjunctival hyperemia, vitreous moderate turbidity, invisible fundus details, and a faint red reflex.

48h (DAY 3) injection of PBS group with opaque cornea, conjunctival edema, obvious hyperemia, serious exudation, iris hyperemia, vitreous opacity, and disappearance of red light reflection of fundus oculi; ampicillin group has opaque cornea, conjunctival edema, obvious hyperemia, slight exudation, iris hyperemia, vitreous body weight turbidity and disappearance of fundus red light reflection; the vancomycin group cornea is normal, the conjunctiva is slightly edematous, the iris is slightly hyperemic, the vitreous body is slightly turbid, the visible part of the eyeground is detailed, and the red light reflects (+); IK-12, IP-12 and WP-12 groups showed similar appearance, with normal cornea, conjunctival edema, mild hyperemia, mild exudation, mild hyperemia of iris, moderate vitreous opacity, invisible details of fundus, and a red reflex.

Injecting medicine 72h (DAY 4) with PBS group cornea opaque, conjunctiva congestion and exudation, pupil adhesion, irregular shape, vitreous body weight turbidity, fundus red light reflex (-); ampicillin group is opaque in cornea, conjunctiva is obviously hyperemic, pupil adhesion is caused, the shape is irregular, the weight of glass is turbid, and fundus oculi red light is reflected (-); vancomycin group normal cornea, conjunctiva normal, iris mild hyperemia, vitreous opacity, visible fundus detail, red light reflex (+); IK-12, IP-12 and WP-12 groups showed similar appearance, with normal cornea, conjunctival edema, mild hyperemia, mild exudation, mild hyperemia of iris, moderate vitreous opacity, invisible details of fundus, and a red reflex.

Clinical scores for rabbit ocular endophthalmitis at various times are plotted in Table 3.3 and statistically analyzed.

Bacteria were injected for 24h (DAY 1) with no statistical difference between the different groups (p ═ 0.993).

The drug was injected for 24h (DAY 2), and there was no statistical difference before the group of IK-12, IP-12, and WP-12 (p >0.05), the group of IK-12, IP-12, and WP-12 was statistically different from the PBS group and the vancomycin group (p <0.05), and the group of IK-12, IP-12, and WP-12 was statistically different from the ampicillin group (p 0.36, p 0.154, and p 0.47).

No statistical difference (p >0.05) was observed before the group of IK-12, IP-12 and WP-12 after injection of the drug for 48h (DAY 3), and the groups of IK-12, IP-12 and WP-12 were statistically different from those of PBS, ampicillin and vancomycin (p < 0.05). There was no statistical difference between the PBS group and the ampicillin group (p ═ 0.233).

The drugs were injected for 72h (DAY 4), and there were no statistical differences before the groups IK-12, IP-12 and WP-12 (p >0.05), and IK-12, IP-12 and WP-12 were statistically different from the groups PBS, ampicillin and vancomycin (p < 0.05). There was no statistical difference between the PBS group and the ampicillin group (p ═ 0.252).

TABLE 3.3 clinical scoring results for endophthalmitis in each group

LOG for bacterial liquid concentration of each rabbit eye dilution coating plate method counting result₁₀(CFU/ml) and statistically analyzed. No statistical difference was observed between PBS and ampicillin groups (p 0.58), and between IK-12, IP-12 and WP-12 groups (p)>0.05), the IK-12, IP-12 and WP-12 groups were statistically different (p) from the PBS and ampicillin groups<0.05), there was a statistical difference (p) between the IK-12, IP-12 and WP-12 groups and the vancomycin group<0.05)。

From the results of the dilution-spread plate counting for each rabbit eye, the sterilization rates of the different drugs were calculated using the following calculation formula.

Percent sterilization rate [ [ (average bacterial liquid concentration)_{(PBS group)}-drug group_{(drug group)}) Average bacteria liquid concentration_{(PBS group)}]×100％。

The different groups were analyzed for differences in bactericidal rate, with no statistical differences between the IK-12, IP-12, WP-12 and vancomycin groups (p >0.05), and the IK-12, IP-12, WP-12 and vancomycin groups were statistically different from the PBS and ampicillin groups (p < 0.05).

TABLE 3.4 results of different groups of dilution coating plate method counts and sterilization rates

HE staining results showed that the PBS group vitreous body was filled with inflammatory cells, retinal necrosis, without any intact retinal layer, the ampicillin group vitreous body was filled with inflammatory cells, retinal partial necrosis, visible retinal layer, vancomycin group: there was no vitreous chamber infiltration, retinal tissue was essentially normal, IK-12 group of vitreous humor disseminated inflammatory cells, retinal tissue was approximately normal, IP-12 group of vitreous humor disseminated inflammatory cells, retinal tissue was approximately normal, WP-12 group of vitreous chamber disseminated inflammatory cells, retinal tissue was approximately normal.

The GMS staining results showed that the PBS group vitreous body was filled with inflammatory cells, the retina was necrotic without any intact retinal layer, the ampicillin group vitreous body was filled with inflammatory cells, the retina was partially necrotic, the visible part of the retinal layer, the vancomycin group: there was no vitreous chamber infiltration, retinal tissue was essentially normal, IK-12 group of vitreous humor disseminated inflammatory cells, retinal tissue was approximately normal, IP-12 group of vitreous humor disseminated inflammatory cells, retinal tissue was approximately normal, WP-12 group of vitreous chamber disseminated inflammatory cells, retinal tissue was approximately normal. The pathological scores of each group were plotted in table 3.5 and statistically analyzed. Compared with the PBS group and the ampicillin group, the pathological scoring results of the vancomycin, the IK-12 group, the IP-12 group and the WP-12 group are lower, the difference has statistical significance, the differences among the antibacterial peptides IK-12 group, the IP-12 group and the WP-12 group have no statistical significance, and the pathological scoring results of the antibacterial peptides IK-12 group, the IP-12 group and the WP-12 group and the vancomycin group have statistical difference.

TABLE 3.5 pathological Scoring results for each group

Sequence listing

<110> Jilin university

<120> a medicine for treating bacterial endophthalmitis and preparation method thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys

1 5 10

<210> 2

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Pro Trp Ile Arg Arg Leu Trp Arg Lys Trp Trp Pro

1 5 10

<210> 3

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Arg Trp Ile Pro Arg Leu Trp Arg Pro Trp Trp Lys

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ile Arg Leu Arg Trp Arg Trp Lys Trp Pro Trp Pro

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Trp Pro Ile Arg Leu Arg Trp Arg Trp Lys Trp Pro

1 5 10

Claims

1. A medicament for the treatment of bacterial endophthalmitis characterized by: the medicine contains antibacterial peptide, wherein the content of the antibacterial peptide in the liquid medicine is 2-512 mug/ml.

2. A medicament for the treatment of bacterial endophthalmitis according to claim 1, wherein: the antibacterial peptide is any one of ik-12, pp-12, rk-12, ip-12 and wp-12, and the sequences of the five antibacterial peptides are respectively as follows: ilrwwpwrrwrrk, pwirwrwrwrkwwp, rwiprwrwrpwwk, irlrwrwrwrkwwp and wpirlrwrwrkwwp.

3. A preparation method of a medicine for treating bacterial endophthalmitis is characterized in that: the method comprises the following steps:

step 5, condensation: protecting amino acid, dissolving with DMF, adding into reaction tube, adding DIC, and reacting for 90 min;

Fmoc-linker Am resin

︱1

Lys 2

︱1

Arg 2

︱1

Arg 2

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Trp 2

Rink Amide MBHA resin

︱1

Lys 2

︱1

Trp 2

︱1

Trp 2

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Pro 2

Rink Amide MBHA resin

︱1

Pro 2

︱1

Trp 2

︱1

Lys 2