CN105012291A - Application of ERK inhibitor PD98059 in preparation of medicine for treatment of alveolar hydatid disease - Google Patents

Abstract

The present invention discloses application of ERK inhibitor PD98059 in preparation of a medicine for treatment of alveolar hydatid diseasein, and the ERK inhibitor PD98059 is used for killing alveolar hydatid diseasein pathogen-bubble hydatid tapeworm, has significant hydatid protoscolex inhibition and killing effect, and can be used for treatment of alveolar hydatid disease.

Description

The application of ERK inhibitor PD98059 in preparation treatment alveolar hydatid medicine

Technical field

The present invention relates to the pharmaceutical applications of PD98059, belong to medicinal chemistry art.

Background technology

Echinococcosis multilocularis (echinococcosis multilocularis alveolar echinococcosis, AE) is a kind of infecting both domestic animals and human parasitic disease can bringing serious harm to health and animal husbandry development caused in people and other animal bodies by silk ribbon phase parasitized larvae in echinococcus cestode.Although the sickness rate of echinococcosis multilocularis is only 2% ~ 3% of echinococcosis granulosa, its harm far exceedes echinococcosis granulosa.In China, echinococcosis multilocularis is mainly distributed in northwest and Southwestern animal husbandry is flourishing and economically underdeveloped area, and prevalence is 0.3% ~ 4.8%, may be 10% in some areas.Effective Therapeutic Method of the Alveolar echinococcosis of generally acknowledging at present is radical-ability hepatectomy, but echinococcosis multilocularis onset is comparatively hidden, therefore when medical, oneself causes greatly infringement to liver and misses best therapic opportunity most of patients, so this sick surgical radical treatment rate is low, be only 20% ~ 26%, and Postoperative recurrent rate is relatively high.Therefore, medicine and other Therapeutic Method become important supplementing.

This area attempts adopting albendazole to treat alveolar hydatid disease, but because its dissolubility is low, gastrointestinal absorption is poor and whole body is widely distributed, therefore clinical practice is undesirable.Though the development and application of novel drugs and dosage form constantly carries out, medication effect also increases, but because it can cause numerous serious side effects, clinical practice is still not ideal enough, especially less for suppressing the drug research of alveolar hydatid invasion and m etastasis growth.

Therefore, obtain a kind ofly act on definite, clear curative effect, the medicine that effectively can prevent and treat hydatid disease,alveolar remains this area problem urgently to be resolved hurrily.

Summary of the invention

In the chronic study procedure of applicant, surprisingly find that cattle ERK inhibitor PD98059 can effectively suppress and kill the growth of protoscolex, thus can be applicable to the clinical treatment of hydatid disease,alveolar.

In the present invention, PD98059 is specificity mek inhibitor, is combined stops its phosphorylation with the inactive form of MEK1/2, and then suppresses the phosphorylation of ERK1/2 and disabling signal Signal Transduction Pathways, thus the growth of T suppression cell.Current numerous research confirms, adopts PD98059 can the increment of the multiple cancerous cell such as vitro inhibition breast carcinoma, gastric cancer, hepatocarcinoma.

First, the invention discloses the application of ERK inhibitor PD98059 in preparation treatment alveolar hydatid medicine.

Above-mentioned hydatid disease,alveolar both can be on animal body, also can be on human body, according to the clinical onset position of this disease, be commonly referred to as many rooms hepatic echinococcosis.

Therefore, the invention also discloses the application of ERK inhibitor PD98059 in preparation treatment many rooms hepatic echinococcosis medicine.

As required, can use the PD98059 of variable concentrations, in general, in order to effectively kill cause of disease, the concentration of PD98059 is not less than 6.25 μm of ol/L.

For common protoscolex stand density, preferably, the concentration of PD98059 is 12.5-50 μm of ol/L.

Preferably, the continuous use time is not less than 3 days.

Under above-mentioned concentration, to alveolar hydatid protoscolex, there is more satisfactory suppression and killing effect.

On this basis, the invention also discloses the application of PD98059 as alveolar hydatid protoscolex inhibitor.

It will be understood by those skilled in the art that under kinds of experiments environment, especially in animal isolated experiment situation, in order to prevent due to the alveolar hydatid protoscolex growth in vitro in animal body, need to add necessary inhibitor, such as 20% hypertonic saline etc.

Similar to the above, the concentration of PD98059 is not less than 6.25 μm of ol/L, and preferably, the concentration of PD98059 is 12.5-50 μm of ol/L.

Accompanying drawing explanation

Fig. 1 is the impact of PD98059 on alveolar hydatid protoscolex form, wherein, and A, B:DMEM culture medium blank group, DMSO group; The protoscolex of C:12.5 μm of ol/L PD98059 effect 6d; The protoscolex of D:12.5 μm of ol/LPD98059 effect 12d; The protoscolex of E:50 μm of ol/L PD98059 effect 6d; The protoscolex of F:50 μm of ol/L PD98059 effect 12d.

Fig. 2 is that the PD98059 of variable concentrations is on the impact of alveolar hydatid protoscolex vigor.

Fig. 3 is PD98059 on the inner Ultrastructural impact of alveolar hydatid protoscolex, wherein, and A, B:DMEM culture medium blank group, DMSO group; C:25 μm of ol/LPD98059 effect 1d protoscolex; D:50 μm of ol/LPD98059 effect 1d protoscolex; E:25 μm of ol/LPD98059 effect 3d protoscolex; F:50 μm of ol/LPD98059 effect 3d protoscolex.

Detailed description of the invention

In order to the effect of PD98059 is described, applicant carried out experiment in vitro and illustrate that PD98059 is to the killing effect of alveolar hydatid protoscolex.

Experiment 1:PD98059 interaction in vitro is in the effect of alveolar hydatid protoscolex

The inoculation alveolar hydatid protoscolex meriones unguiculatus of 4-6 month, after anesthesia, its disconnected neck is put to death, aseptically dissect, take out complete alveolar hydatid piece of tissue, be placed in the sterilized dressing-changing bowl of appropriate PBS (PH=7.2), change drug bowl is moved in super-clean bench, reject the host tissue on piece of tissue surface, alveolar hydatid piece of tissue 3-5 time is cleaned with PBS, then grind through homogenizer, screen filters, collect filtrate in sterile centrifugation tube, leave standstill 5 minutes, protoscolex is washed repeatedly to clarification with PBS after abandoning supernatant, make alveolar hydatid protoscolex suspension with normal saline and be inoculated in hamster to prepare kind of a Mus through lumbar injection, dissect after 4 months and obtain protoscolex, protoscolex 3 extremely clarifications are cleaned with PBS (PH=7.2), dye exclusion experiment is done with 0.1% eosin stain, more than 85% refuses dye.Perusal protoscolex is tenderly white husky sample single-size, and the active good protoscolex rate of settling is fast, and the active protoscolex rate of settling that is bad or death is relatively slow.

Observing protoscolex polypide under inverted microscope is interior swaged, is dispersed in densely covered, and polypide structure is full, ovalize, and clear in structure is complete, and calcium granule is limpid and obvious.Then protoscolex is divided (penicillin 100U/mL, streptomycin 100U/mL) in the DMEM culture medium be filled to containing 10% hyclone, put 37 DEG C, the interior cultivation of 5%CO2 incubator.Experiment grouping: DMEM culture medium blank group, DMSO group, 6.25 μm of ol/L PD98059 groups, 12.5 μm of ol/L PD98059 groups, 25 μm of ol/L PD98059 groups, 50 μm of ol/LPD98059 groups.Get six orifice plates, 5mL system is established in every hole, by above grouping, prepares corresponding culture fluid.

Alveolar hydatid protoscolex PBS (pH=7.2) after cultivating 5 days is in vitro cleaned 3 times, often organizes in culture fluid and add about 2000 protoscolexs, put 37 DEG C, 5%CO ₂cultivate in incubator, under inverted microscope, observe the impact of PD98059 on alveolar hydatid protoscolex form and vigor.

Data statistical approach: after dosing, 24h starts, often organizes and evenly extracts 200 μ L culture fluid (average containing procephalon saving 90-120) every day, with 0.1% eosin stains 15min, observe the vigor of protoscolex under inverted microscope.Cephalomere of living refuses dye, not painted, and has activeness; The protoscolex that dead or vigor weakens is red stained with color, with structural deterioration.Calculate and often organize average mortality every day.Change liquid once (when often organizing system and concentration dosing of same first time) every 4d, Continuous Observation, until the protoscolex of maximum effect concentration is all dead.Experiment in triplicate.The change of protoscolex internal structure after experiment 2:PD98059 effect

While survey vigor, get corresponding protoscolex at random and do Electronic Speculum, observe the Ultrastructural change of protoscolex.PBS washs 3 times, each 5min; Fixing 24h before putting 4% glutaraldehyde.Centrifugal with little centrifuge tube, remove supernatant, use Agarose embedding cephalomere, take out gel and continue to put into 4% glutaraldehyde internal fixtion 24h.The protoscolex PBS (PH7.4) fixed is rinsed 3 times, and after in each 15min, 2%OsO4, fixing 1h, rinses twice again with PBS.Ethanol is serial dehydration step by step, 30% → 50% → 60% each 10min, and 70% spends the night, and puts 80% → 90% each 10min, 100%30min next day again.Acetone dewaters step by step afterwards, epoxy resin (Epon812) embeds, and repaiies type after fixing, and LKB2088V type ultramicrotome is cut into slices, acetic acid uranium and plumbi nitras double staining, be placed in the inner Ultrastructural change of Hitachi H-600 type transmission electron microscope observation protoscolex.

Experimental result:

With reference to figure 1, be the metamorphosis of PD98059 In vitro culture alveolar hydatid protoscolex 6d and 14d, observe under inverted microscope, protoscolex after PD98059 effect is many in the type that turns up, the little hook arrangement disorder on protoscolex rostellum, partial exfoliation, sucker projection, distortion, hypoergy or death.After drug effect, along with the increase of PD98059 concentration and the prolongation of time, the metamorphosis of protoscolex is obvious.The protoscolex rostellum of blank group and DMSO group protrudes or is recessed into, and volume is comparatively large, movable good.

With reference to figure 2, for PD98059 is to the lethal effect of external alveolar hydatid protoscolex.The alveolar hydatid protoscolex vitality test of variable concentrations PD98059 (6.25 μm of ol/L, 12.5 μm of ol/L, 25 μm of ol/L, 50 μm of ol/L) In vitro culture 12d the results are shown in Figure 2, and display variable concentrations PD98059 all has inhibitory action to external alveolar hydatid protoscolex.Wherein 50 μm of ol/L PD98059 effect 14d protoscolex mortality rates reach 100%.25 μm of ol/L effect 14d protoscolex mortality rates are 18.5%.As can be seen from Dynamic Curve figure, along with the increase of drug level and the prolongation of administration time, the suppression ratio of protoscolex increases, and this effect has dose dependent and time dependence.Each experimental group protoscolex mortality rate and matched group comparing difference all have statistical significance (P < 0.05).

, observe under transmission electron microscope, the normal alveolar hydatid protoscolex syncytium band structure cultivated is finer and close meanwhile, inside have differ in size, vesicle that shape differs, arrange more regular, there is the microtriche of more marshalling in outside; Containing dissimilar cell in protoscolex, the core of parenchyma is large and obviously, kernel is placed in the middle, and in core, chromatin is finer and smoother, and heterochromatin is less.Alveolar hydatid protoscolex syncytium band after PD98059 effect is thinning, loosely organized, and vesicle is expanded, and cilium reduces, and irregular arrangement; Part parenchyma substrate concentrates, and perinuclear space is slightly broadening, and kernel disappears; Syncytium band and intracavity occur that cavity and fat drip (Fig. 3).

Comprehensive above-mentioned experimental result can be seen, variable concentrations PD98059 all has inhibitory action to external alveolar hydatid protoscolex, concentration is that the PD98059 of 50 μm of ol/L is the most obvious to alveolar hydatid protoscolex lethal effect, along with the prolongation of time, inhibitory action significantly increases (P < 0.01).Have on inhibiting basis at confirmation PD98059 to protoscolex, explore the PD98059 of variable concentrations to the Morphology Effects of alveolar hydatid protoscolex.Result of study shows, and after PD98059 effect, can cause the form generation change in various degree of external alveolar hydatid protoscolex.Alveolar hydatid protoscolex after variable concentrations PD98059 effect, observes under transmission electron microscope, and defect in various degree appears in microtriche, and cell in stratum germinativum by cavity and fat drip substitute.It is unfavorable for forming brood capsule, the growth of protoscolex is suppressed and dead.Generally speaking, PD98059 has lethal effect to external alveolar hydatid protoscolex.

Claims (7)

1. The application of 1.ERK inhibitor PD98059 in preparation treatment alveolar hydatid medicine.
2. 2. application according to claim 1, is characterized in that the concentration of PD98059 is not less than 6.25 μm of ol/L.
3. 3. application according to claim 2, is characterized in that the concentration of PD98059 is 12.5-50 μm of ol/L.
4. The application of 4.ERK inhibitor PD98059 in preparation treatment many rooms hepatic echinococcosis medicine.
5. 5.PD98059 as the application of alveolar hydatid protoscolex inhibitor.
6. 6. application according to claim 5, is characterized in that the concentration of PD98059 is not less than 6.25 μm of ol/L.
7. 7. application according to claim 6, is characterized in that the concentration of PD98059 is 12.5-50 μm of ol/L.