CN105012317A - Application of ursodesoxycholic acid as active component in preparing medicine for treating echinococcus granulosas - Google Patents

Abstract

The invention discloses an application of ursodesoxycholic acid as an active component in preparing a medicine for treating echinococcus granulosas. When ursodesoxycholic acid is adopted to kill pathogen of echinococcus granulosas, namely, echinococcus granulosas, protoscoleces of echinococcus granulosas can be remarkably inhibited and killed, so that treatment on echinococcus granulosas can be achieved.

Description

Ursodesoxycholic acid is as the application of active component in preparation treatment Echinococcus Granulosus Cysts medicine

Technical field

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

Background technology

Echinococcosis granulosa (echinococcosis) is also known as cystic echinococcosis (cystic echinococcosis, CE), by a kind of serious harm people ' s health of middle silk ribbon phase of Echinococcus granulosus (Echinococcus granulosus, Eg) caused by larva and the parasitic zoonoses of animal husbandry development.This disease is global distribution, at China's Major Epidemic in Xinjiang, Qinghai, the Inner Mongol, the animal husbandry prosperity such as Tibet provinces and regions.Cystic echinococcosis major effect liver, also can develop in lung.At present, the treatment mainly operating method of this disease is directed to, i.e. external capsule Complete excision in Bile fistula adventitia.Although the complete enucleation of external capsule can reduce the relapse rate of echinococcosis greatly in Bile fistula adventitia adopted in recent years, farthest reduce operation to hepatic parenchymal damage, but when hydatidoma volume is comparatively large or when pressing close to important organ or pipeline (hepatic portal or bile duct), then need to select the complete enucleation of external capsule in open adventitia.This kind of art formula may have leaking outside of cephalomere and remain, and prevents Echinococcus hydatid cyst recurrence in art with the postoperative medicine auxiliary treatment that needs.Therefore all in all, in art, dealing with improperly of the excessive and cephalomere of cephalomere is the defect being difficult to overcome that operative treatment cystic echinococcosis exists.

For the problems referred to above, this area attempts adopting high-concentration acidic wastewater (HCl) and alkaline solution (KOH) in vitro, and clinical conventional local chemotherapeutic drug has 20% hypertonic saline, but has certain destruction to surrounding normal hepatic tissue; This area also once studies have found that bile can suppress the growth of hydatidoma, but bile component is complicated, effect target is many, therapeutic effect is changeable, such as the delay in liver and blood of bile, bile acid can produce multiple toxic action, comprise cytotoxicity and acute and chronic toxic action, cause hepatic injury and whole body multisystem, multiple organ injury, finally cause liver cirrhosis, liver failure and death; Also once studies have found that bile acid formed damage etc. to pancreatic acinar cell.

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

Summary of the invention

Applicant surprisingly finds that in chronic study procedure ursodesoxycholic acid (UDCA) can effectively suppress and kill the growth of protoscolex, thus for the clinical treatment of echinococcosis granulosa.

In the present invention, ursodesoxycholic acid (UDCA), its chemical name is: 3 α, 7 beta-dihydroxy-5 β-cholestane-24-acid, it is a kind of hydrophilic cholic acid, be present in normal person's bile, account for 3% of human body total bile acid, can relax sphincter of common bile duct, promote liver glycogen accumulation, protecting liver and detoxication, was applied to the treatment of primary biliary cirrhosis and primary sclerosing cholangitis from 1989, be the sole drug of FDA approval treatment primary biliary cirrhosis.

Ursodesoxycholic acid, as a kind of medicine of dissolving cholesterol calculus, is widely used in the treatment of hepatic diseases because having stronger subduing the hyperfunction of the liver effect.Nearest domestic scholars also finds that ursodesoxycholic acid has the curative effect for the treatment of fatty liver, glutamate pyruvate transaminase (ALT) can be reduced, millet straw turns by enzyme (AST), r-glutamyl transpeptidase (r-GT), reduce blood fat, obvious alleviation fatty liver symptom, improve the Radiologic imaging of fatty liver, and untoward reaction is slight.

First, the invention discloses ursodesoxycholic acid as the application of active component in preparation treatment Echinococcus Granulosus Cysts medicine.

Above-mentioned medicine, both can be simple active raw materials, can also be the various preparations that ursodesoxycholic acid is made as active component and other adjuvants, such as common tablet, capsule, drop pill, transfusion, freeze-dried powder etc., for the selection of pharmaceutical dosage form and corresponding adjuvant thereof, those skilled in the art can adjust as required.

Above-mentioned echinococcosis granulosa 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 Cystic hydatidosis.

Therefore, the invention also discloses ursodesoxycholic acid as the application of active component in preparation treatment Cystic hydatidosis medicine.

As required, can use the ursodesoxycholic acid of variable concentrations, in general, in order to effectively kill cause of disease, the concentration of ursodesoxycholic acid is not less than 1000 μm of ol/L.

For common protoscolex stand density, preferably, the concentration of ursodesoxycholic acid is 2000-4000 μm of ol/L.

Preferably, the continuous use time is not less than 6 days.

Under above-mentioned concentration, to Echinococcus Granulosus Cysts protoscolex, there is more satisfactory suppression and killing effect.

On this basis, the invention also discloses the application of ursodesoxycholic acid as Echinococcus Granulosus Cysts protoscolex inhibitor.

It will be appreciated 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 growth in vitro of the Echinococcus Granulosus Cysts protoscolex in animal body and cephalomere excessive, need to add necessary inhibitor, such as 20% hypertonic saline etc.

Similar to the above, the concentration of ursodesoxycholic acid is not less than 1000 μm of ol/L, and preferably, the concentration of ursodesoxycholic acid is 2000-4000 μm of ol/L.

Accompanying drawing explanation

Fig. 1 is the impact of ursodesoxycholic acid on Echinococcus Granulosus Cysts protoscolex form, wherein, and A: blank group protoscolex; B:DMSO group protoscolex; The protoscolex of C:2000 μm of ol/L UDCA effect 6d; The protoscolex of D:4000 μm of ol/L UDCA effect 6d; The protoscolex of E:2000 μm of ol/L UDCA effect 12d; The protoscolex of F:4000 μm of ol/L UDCA effect 12d.

Fig. 2 is that the ursodesoxycholic acid of variable concentrations is on the impact of Echinococcus Granulosus Cysts protoscolex vigor.

Fig. 3 is the impact of ursodesoxycholic acid on Echinococcus Granulosus Cysts protoscolex Surface Ultrastructure, wherein, and A: contrast

Group protoscolex; B, C:4000 μm of ol/L ursodesoxycholic acid effect 6d protoscolex.

Detailed description of the invention

In order to the effect of ursodesoxycholic acid (UDCA) is described, applicant carried out experiment in vitro and illustrate that ursodesoxycholic acid is to the killing effect of Echinococcus Granulosus Cysts protoscolex, applicant carried out following experiment.

Experiment 1:UDCA interaction in vitro is in the effect of Echinococcus Granulosus Cysts protoscolex

Get the sheep liver of fresh natural infection Echinococcus Granulosus Cysts, clean Hepar Caprae seu ovis surface, with 75% alcohol disinfecting surface, extract the capsule liquid containing protoscolex under aseptic condition, move in aseptic bottle.Clean protoscolex 3 extremely clarifications with PBS (PH=7.2), do dye exclusion experiment with 0.1% eosin stain, more than 98% 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 RPMI1640 culture medium be filled to containing 10% calf serum, put 37 DEG C, the interior cultivation of 5%CO2 incubator.

Experiment grouping: RMPI-1640 culture medium blank group, 1000 μm of ol/L UDCA groups, 2000 μm of ol/LUDCA groups, 3000 μm of ol/L UDCA groups, 4000 μm of ol/L UDCA groups, DMSO groups.Get six orifice plates, 5mL system is established in every hole, by above grouping, prepares corresponding culture fluid.

Echinococcus Granulosus Cysts 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 UDCA on Echinococcus Granulosus Cysts 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 surface texture after experiment 2:UDCA 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; Put 4% glutaraldehyde and fix 24h.Protoscolex is put into the ethanol serial dehydration step by step that concentration is 50%, 70%, 80%, 90%, 100%, dewatering time is respectively 5min, 10min, 30min, 1h to spending the night, critical point drying, vacuum metal spraying LOOA, observes protoscolex top layer Change of Ultrastructure under LEO1430VP scanning electron microscope.

Experimental result:

With reference to figure 1, be the metamorphosis of UDCA In vitro culture Echinococcus Granulosus Cysts protoscolex 6d and 12d, observe under inverted microscope, protoscolex after UDCA 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.Along with the increase of UDCA concentration, 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 UDCA is to the lethal effect of external Echinococcus Granulosus Cysts protoscolex.The Echinococcus Granulosus Cysts protoscolex vitality test of variable concentrations UDCA (1000 μm of ol/L, 2000 μm of ol/L, 3000 μm of ol/L, 4000 μm of ol/L) In vitro culture 12d the results are shown in Figure 2, and display variable concentrations UDCA all has inhibitory action to external Echinococcus Granulosus Cysts protoscolex.Wherein 4000 μm of ol/L UDCA effect 12d protoscolex mortality rates reach 100%.3000 μm of ol/L effect 12d protoscolex mortality rates are 74.6%.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).

Meanwhile, applicant observes the protoscolex Change of Ultrastructure after UDCA effect under a scanning electron microscope, and observe display blank group protoscolex smooth surface, many in interior swaged, cephalomere is spherical in shape or oval; After 4000 μm of ol/L UDCA effect Echinococcus Granulosus Cysts protoscolex 6d, observe discovery under a scanning electron microscope, protoscolex is many in the type that turns up, rostellum destruction, the distortion of head hook defect, sucker, there is many lacunas in body surface, cortex is in sample infringement (Fig. 3) of damaging by worms.

To sum up can see, variable concentrations UDCA all has inhibitory action to external Echinococcus Granulosus Cysts protoscolex, concentration is that the UDCA of 4000 μm of ol/L is the most obvious to Echinococcus Granulosus Cysts protoscolex lethal effect, along with the prolongation of time, inhibitory action significantly increases (P < 0.05).Have on inhibiting basis at confirmation UDCA to protoscolex, explore the UDCA of variable concentrations to the Morphology Effects of Echinococcus Granulosus Cysts protoscolex.Result of study shows, and after UDCA effect, can cause the form generation change in various degree of external Echinococcus Granulosus Cysts protoscolex.Interior swaged and the type protoscolex that turns up are the different existence forms of polypide.When condition is suitable, protoscolex rostellum indent, can protect a hook, sucker, microtriche to be without prejudice; After adding UDCA, the protoscolex of the type that turns up increases.The microtriche of the Echinococcus Granulosus Cysts protoscolex stratum germinativum of normal cultivation physically well develops, quantity is many, adhere to and extend in horny layer, stratum germinativum and cuticular contact area can be increased, be conducive to the absorption of nutrient substance, without when blood supply when echinococcus still can comparatively fast grow and constantly form brood capsule.

Generally speaking, UDCA has lethal effect to external Echinococcus Granulosus Cysts protoscolex.

Claims (7)

1. ursodesoxycholic acid is as the application of active component in preparation treatment Echinococcus Granulosus Cysts medicine.

2. application according to claim 1, is characterized in that the concentration of ursodesoxycholic acid is not less than 1000 μm of ol/L.

3. application according to claim 2, is characterized in that the concentration of ursodesoxycholic acid is 2000-4000 μm of ol/L.

4. ursodesoxycholic acid is as the application of active component in preparation treatment Cystic hydatidosis medicine.

5. ursodesoxycholic acid is as the application of Echinococcus Granulosus Cysts protoscolex inhibitor.

6. application according to claim 5, is characterized in that the concentration of ursodesoxycholic acid is not less than 1000 μm of ol/L.

7. application according to claim 6, is characterized in that the concentration of ursodesoxycholic acid is 2000-4000 μm of ol/L.