CN102603865A

CN102603865A - Dipeptide derivative and application of the dipeptide derivative

Info

Publication number: CN102603865A
Application number: CN2011100255168A
Authority: CN
Inventors: 罗楹
Original assignee: Individual
Current assignee: Beijing Kangdini Pharmaceutical Co ltd
Priority date: 2011-01-24
Filing date: 2011-01-24
Publication date: 2012-07-25
Anticipated expiration: 2031-01-24
Also published as: CN102603865B

Abstract

The invention provides a dipeptide derivative and an application of the dipeptide derivative. Particularly, the invention provides an N-substituted 2(1H) dipeptide derivative or a pharmaceutically acceptable salt of the N-substituted 2(1H) dipeptide derivative and a pharmaceutical composition containing the compound. The compound is capable of effectively treating various hepatic failures such as acute hepatic failure and chronic-on-acute hepatic failure.

Description

Dipeptidase derivant and application thereof

Technical field

The present invention relates to pharmaceutical field, relate more specifically to a kind of dipeptidase derivant of treating liver failure, and the application aspect the medicine of preparation treatment liver failure.

Background technology

Liver failure is meant the serious liver damage that is caused by multiple factor; Cause that liver itself is synthetic, function generation serious hindrance such as detoxifcation, drainage and bio-transformation or lose compensatoryly, one group of syndrome with performances such as zymoplasm mechanism impediment, jaundice, hepatogenic encephalopathy, dehydrations appears clinically.The case fatality rate of liver failure is high.

2006 China " liver failure practice guidelines " with following four types of being divided into of liver failure:

(a) acute hepatic failure refers to Acute onset, and occurring the above hepatogenic encephalopathy of II degree in 2 weeks is the liver failure of characteristic;

(b) subacute liver failure refers to that onset is more anxious, and the liver failure of the clinical manifestation of liver failure appears in 15 days～26 weeks;

(c) slow extra urgaent dispatch (subacute) liver failure refers on the basis of chronic hepatopathy, acute liver function occurs and loses compensatory liver failure;

(d) chronic liver failure refers on the basis of liver cirrhosis, chronic liver function occurs and loses compensatory liver failure.

The pathogenesis of liver failure is very complicated, and hepatitis virus, medicine, intracellular toxin and shock etc. all can cause liver failure, and multiple factor can interact and influence.

It is generally acknowledged that at present all types of hepatitis viruss are to cause one of paathogenic factor that liver failure takes place.In addition, intracellular toxin and liver injury are closely related, and liver is a metabolism organ maximum in the body, are to remove intracellular toxin and toxicide main organs, also are the primary organs that attacked by intracellular toxin.Cytokine and liver injury are also closely related, and cytokine is the main molecules of participating in liver failure, hepatic necrosis generating process, are again to constitute the important molecule that suppresses the liver cell regeneration extracellular environment.In addition, apoptosis also is considered to relevant with liver failure with factors such as multiple organ dysfunction syndromes.

Think that at present the liver failure of American-European countries is mainly infected by medicine or alcohol or HCV and causes, and China is very many because of HBV the infected, the liver failure that is caused by hepatitis B has accounted for suitable vast scale, but fatty liver, alcohol, and factor such as HCV also have ascendant trend.Yet as far as concrete liver failure case, its morbidity cause possibly be one or more above-mentioned reasons.

At present, mainly be the internal medicine complex therapy for the treat-ment of liver failure, comprise (a) infrastructural support treatment, for example, correct water, electrolyte disturbance through a large amount of FFPs that use, means such as prevention digestive tract hemorrhage are carried out.(b) stimulated hepatic cell regeneration; (c) antiviral therapy; (d) immune modulating treatment for example uses Thymosin alpha 1; (e) control of complication.

Yet above-mentioned treatment means still can not be satisfactory, in addition, still lacks gratifying to the effective medicine of liver failure at present in the world.Therefore, the mortality ratio of liver failure is very high clinically, and especially the mortality ratio of acute hepatic failure and slow extra urgaent dispatch liver failure is up to about 30% or higher.

Therefore, this area presses for the novel drugs that exploitation was effectively treated or prevented liver failure or alleviates its symptom.

Summary of the invention

The object of the invention just provides a kind of compound and application thereof of effective treatment liver failure.

In first aspect of the present invention, a kind of formula I compound or its pharmacy acceptable salt that can be used for treating liver failure is provided:

In second aspect of the present invention, a kind of pharmaceutical composition that is used to treat or prevent liver failure is provided, it contains formula I compound or its pharmacy acceptable salt described in the first aspect present invention of pharmaceutically acceptable carrier and safe and effective amount.

In another preference, it contains formula I compound or its pharmacy acceptable salt that accounts for gross weight 0.01-99%.

In another preference, the formulation of said pharmaceutical composition is tablet, capsule, injection (injection), pill.

In the third aspect of the invention, the formula I compound described in the first aspect present invention or the purposes of its pharmacy acceptable salt are provided, it is used to prepare the medicine of treatment or prevention liver failure.

In another preference, described liver failure comprises: acute hepatic failure, subacute liver failure and slow extra urgaent dispatch liver failure, chronic liver failure.

In another preference, the formulation of described medicine is injection or oral prepns.

In another preference, described medicine also is used to improve liver failure patient's survival rate; Or be used to improve liver failure patient's liver function index.In another preference, the described liver function index that improves comprises: reduce SGPT (ALT), reduce SGOT (AST) and/or reduce total bilirubin (TBil).

In fourth aspect of the present invention, the method for a kind of treatment or prevention liver failure is provided, comprise step: the object of treating for needs is used formula I compound or its pharmacy acceptable salt described in the first aspect present invention of safe and effective amount.

In another preference, described using comprises: intravenous injection, subcutaneous injection, abdominal injection or injection into liver.

In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described each technical characterictic can mutual combination in (like embodiment) hereinafter, thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.

Description of drawings

When Fig. 1 has shown after modeling 2 hours and has given compound F 17-hydroxy-corticosterone 573, observations under the light microscopic of each experimental group.

When Fig. 2 has shown after modeling 2 hours and has given compound F 17-hydroxy-corticosterone 573, the photo of the TUNEL method half-quantitative detection hepatocellular apoptosis of each experimental group.

Fig. 3 has shown in modeling simultaneously (0 hour) when giving compound F 17-hydroxy-corticosterone 573, observations under the light microscopic of each experimental group.

Fig. 4 has shown at modeling simultaneously (0 hour) when giving compound F 17-hydroxy-corticosterone 573, the photo of the TUNEL method half-quantitative detection hepatocellular apoptosis of each experimental group.

Embodiment

The inventor is surprised to find that through extensive and deep research one type of dipeptidase derivant can significantly suppress or reverse liver failure, for liver failure significant curative effect is arranged, and pair cell does not have tangible toxicity.Accomplished the present invention on this basis.

As used herein, term " The compounds of this invention ", " formula I compound ", " dipeptidase derivant of the present invention " interchangeable use all refer to have compound in structural formula I or its pharmacy acceptable salt,

A kind of preferred compound is the compound of salt-independent shape, i.e. 3-(2-benzyloxycarbonyl amino-3-methyl-amide-based small)-5-fluoro-4-oxo-valeric acid, and this compound is called F573, and molecular weight is 382.38.This compound can the synthetic or purchase acquisition by ordinary method.

The compounds of this invention has molecular weight little (～400), water-soluble high characteristics.The experimentation on animals result shows that F573 has good curing liver failure effect, and in the treatment liver failure, can obviously alleviate hepatocellular necrosis, promotes liver cell to recover normal function, is particularly useful for the treatment of slow extra urgaent dispatch liver failure.Evidence, this compound is safer in cell tissue and animal, and the anxious poison experiment of mouse stomach shows half lethal agent (LD ₅₀) amount is for i.g.3000 milligram/kg body weight.

The compounds of this invention can be used for various liver failure treatments and or prevention.Representational example comprises (but being not limited to): acute hepatic failure, subacute liver failure, slow extra urgaent dispatch (subacute) liver failure and add subacute liver failure slowly are particularly useful for slow extra urgaent dispatch liver failure and add subacute liver failure slowly.

The compounds of this invention also comprises by pharmaceutically or acceptable acid of physiology or alkali deutero-salt form.These salt include, but is not limited to the salt with following mineral acid formation: example hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the salt that forms with organic acid, organic acid then refers to acetate, oxalic acid, Succinic Acid, tartrate, methylsulfonic acid and toxilic acid.Other salt comprise the salt that forms with basic metal or earth alkali metal (like sodium, potassium, calcium or magnesium), with the form (when with this form administration, can change into active part in vivo) of ester, carbamate or other conventional " prodrugs ".

The present invention also comprises pharmaceutical composition and treat-ment and prevention method, and it comprises the formula I compound to the administration medicine effective quantity.

When The compounds of this invention is used for such use; Can with one or more pharmaceutically acceptable carrier or mixed with excipients; Like solvent, thinner etc.; And can use following form oral administration: tablet, pill, capsule, dispersible powder, particle or suspension-s (containing 0.05-5% suspension agent according to appointment), syrup (containing 10-50% sugar according to appointment) and elixir (containing the 20-50% ethanol of having an appointment); Or with the administration of external application mode: ointment, gel, pastille adhesive plaster etc., perhaps carry out the parenteral administration with sterile injectable solution or form of suspension (containing the 0.05-5% suspension agent of having an appointment in the medium waiting to ooze).For example, these pharmaceutical prepns can contain and the about 0.01-99% of carrier blended, more preferably are about the activeconstituents of 0.1%-90% (weight).

The effective dose of used activeconstituents can change with the severity of the pattern of used compound, administration and disease to be treated.Yet, when compound of the present invention every day gives with the dosage of about 0.25-1000mg/kg the weight of animals, can obtain gratifying effect usually, preferably give with the dosage that separates for 2-4 time every day, or with the slowly-releasing form administration.As far as most of large mammal, the total dose of every day is about 1-100mg/kg, preferably is about 2-80mg/kg.Be applicable to dosage form for oral administration, comprise active compound with the intimately mixed about 0.25-500mg of solid-state or liquid pharmaceutically acceptable carrier.This dosage of adjustable is replied so that optimal treatment to be provided.For example, by an urgent demand of treatment situation, but give the dosage that several times separate every day, or dosage is reduced pari passu.

But these active compound administered through oral and intravenously, intramuscular or administration such as subcutaneous.Solid-state carrier comprises: starch, lactose, Lin Suanergai, Microcrystalline Cellulose, sucrose and white bole; And liquid carrier comprises: sterilized water, polyoxyethylene glycol, non-ionics and edible oil (like Semen Maydis oil, peanut oil and til), as long as be fit to the characteristic of activeconstituents and required specific administration mode.Normally used adjuvant also can advantageously be comprised in pharmaceutical compositions, for example seasonings, pigment, sanitas and inhibitor such as vitamin E, vitamins C, BHT and BHA.

See that from the position that is easy to prepare preferred pharmaceutical composition is a solid-state composition, especially the capsule of filling of tablet and solid or liquid filling with administration.The oral administration of compound is preferred.

But these active compounds are parenteral or intraperitoneal administration also.The solution or the suspension-s that also can in the water that suitably is mixed with tensio-active agent (like hydroxypropylcellulose, Vinylpyrrolidone polymer), prepare these active compounds (as free alkali or pharmacy acceptable salt).Also can in glycerine, liquid, polyoxyethylene glycol and the mixture in oil thereof, prepare dispersion liquid.Under routine storage and working conditions, contain sanitas in these preparations to prevent microorganism growth.

The medicament forms that is adapted to inject comprises: aseptic aqueous solution or dispersion liquid and aseptic powder (being used for preparing aseptic injectable solution or dispersion liquid) temporarily.In all situations, these forms must be aseptic and must be that fluid is discharged fluid to be easy to syringe.Under manufacturing and condition of storage must be stable, and must be able to prevent the pollution effect of mikrobe (like bacterium and fungi).Carrier can be solvent or dispersion medium, wherein contains just like water, alcohol (like glycerine, Ucar 35 and liquid polyethylene glycol), their suitable mixture and vegetables oil.

In addition, The compounds of this invention also can with medicine (like Interferon, rabbit, lamivudine, the Adefovir etc.) coupling of other treatment liver failure.

Major advantage of the present invention is:

(a) to the effect highly significant of liver failure;

(b), can significantly reduce the mortality ratio and raising survival rate of liver failure to liver failure effect highly significant; With

(c) can suppress relevant apoptosis and tissue necrosis with liver failure.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment

1. material

1.1 medicine

Compound F 17-hydroxy-corticosterone 573 is a white powder, and lot number MC08261-27-2, chemical name are 3-(2-benzyloxycarbonyl amino-3-methyl-amide-based small)-5-fluoro-4-oxo-valeric acid, are provided by Shanghai Genomics.Face the time spent with the ultra 30min of UW after, with the F573 of the saline water of 0.5% Xylo-Mucine (CMC) preparation high dosage (H) 1mg/ml (5.0mg/kg), middle dosage (M) 0.5mg/ml (2.5mg/kg), 3 concentration of low dosage (L) 0.25mg/ml (1.25mg/kg).

Positive drug N-acetylcystein (N-acetyl-L-cysteine:NAC) is available from Japanese Kyowa ltd.Be formulated as 31mg/ml (155mg/kg) with the saline water dilution.

Modeling medicine (1) D-Gal (D-GalN) is available from Canadian BBI company.With saline water preparation 100mg/ml (500mg/kg).

Modeling medicine (2) intracellular toxin (LPS) available from U.S. Sigma company, is prepared 4 μ g/ml (20 μ g/kg) with saline water.

1.2 test kit and reagent

SGPT (ALT), SGOT (AST) and total bilirubin (TBil) (vannadate method) test kit are Zhongsheng Beikong Biological Science & Technology Co., Ltd.'s product, and lot number is respectively 100321,100291,100351.

Flow cytometer is used reagent

Propidium iodide (PI), RNase A and Triton X-100 are U.S. Sigma Company products, and lot number is respectively 059k3526,039k13562 and 57H0650.

TUNEL apoptosis test regent box is German Roche Applied Science Company products, lot number 11684817910.

1.3 key instrument

BECKMAN GS-15R refrigerated centrifuge; Hitachi's 7080 type automatic clinical chemistry analyzers; BD FACSCalibur flow cytometer; OLYMPUS 2BX41 microscope; Electronic balance Mettler-240.

1.4 animal and animal divide into groups

Rat, the Wistar kind, the SPF level, male, body weight 150～170g provides available from Nat'l Pharmaceutical & Biological Products Control Institute's Experimental Animal Center.Rat was stablized in the laboratory 1 day, and rat fasting 16h before the experiment freely drinks water.Rat is divided at random 10 every group.

2. universal method

2.1 put to death animal

10 hours femoral artery sacrificed by exsanguination rats after the modeling, it is to be checked from serum and part hepatic tissue to get blood system.

2.2 detect index and method

2.2.1 rat body weight

Before the administration and behind the modeling 10h, every weighing rat body weight.

2.2.2 liver weight and liver coefficient

Behind the sacrificed by exsanguination rat, cut open and round liver and weigh, and to calculate the heavy per-cent with body weight of liver be the liver coefficient: liver weight (g)/body weight (g) * 100%=liver coefficient (%).

2.2.3 serum biochemistry index

After giving modeling medicine 10h; Each treated animal femoral artery is got the about 1.5ml of blood, and blood leaves standstill more than the 1h centrifugal in 4 ℃, and 3000 change; Centrifugal 15min; Separation of serum 100 μ l on Hitachi's 7020 type automatic clinical chemistry analyzers, press the test kit specification sheets and survey SAIT (ALT), SGOT (AST) and total bilirubin (TBil) content.

2.2.4 pathological examination

Get right lobe of liver, 1.5 * 1.5 * 0.5 big or small liver is got at every same position, and 10% neutral formalin is fixed, paraffin embedding, section (make a slice more for every and give over to TUNEL usefulness), and pathological study is carried out in conventional H E dyeing under the light microscopic.Change with sxemiquantitative pathology index (PI) evaluation hepatic tissue pathology; Be every group and measure 20 high power fields; Count the ratio (under the situation of not knowing to divide into groups, passing a judgement) of pathology liver cell number in each visual field, obtain every group of mean and SD and carry out statistical analysis by exper ienced pathology expert.

2.2.5 flow cytometer PI quantitative analysis hepatocyte apoptosis rate

Get left lobe of liver 0.1-0.2g with 70% alcohol fixation, hepatic tissue blocking is gently rubbed with the hands on 120 purpose stainless (steel) wires, simultaneously with the downward cells washed of saline water; Rub with the hands clean until tissue; The collecting cell suspension filters with 300 order copper mesh and removes cell mass, centrifugal collecting cell; Be made into single cell suspension, make every part of contained cell count be no less than 1 * 10 ⁶Individual.Get above-mentioned cell suspension 0.2ml; Add propidium iodide (PI) 1ml, the 30min that in 4 ℃ of refrigerators, dyes detects at BD FACSCalibur flow cytometer; Collect 20,000 cells behind the last machine, use Modifit LT 3.0 analysis of cells apoptosis rate and collection of illustrative plates thereof then.

2.2.6TUNEL method semi-quantitative analysis hepatocyte apoptosis index

Be deoxyribonucleotide terminal enzyme (DNA) mediation the breach end-labelling (terminal-deoxynucleotidyl transferase mediated nick end labeling, TUNEL), get the conventional dewaxing dehydration of hepatic pathology section of paraffin embedding, section after; Operate by TUNEL test kit specification sheets: drip Proteinase K (20 μ g/ml are dissolved among the PBS), incubated at room 30 minutes, twice of PBS flushing; The TUNEL reaction mixture of Dropwise 50 μ l was hatched 60 minutes for 37 ℃ in wet box, the PBS flushing; Add 50 μ l transforming agent POD, in constant temperature incubator moisture releasing box, hatched 30 minutes for 37 ℃, twice of PBS flushing; Add 50 μ lDAB substrate solutions, incubated at room 10 minutes, twice of PBS flushing; Hematorylin is redyed, mounting; The per-cent of apoptotic cell in 500 cells is counted in 5 visuals field of every sections observation under opticmicroscope, calculate apoptotic index (apoptosis index, AI)=positive cell number/total cell count * 100%.

2.2.7 statistical procedures: measurement data is expression with

, relatively adopts the t check between two groups.

Embodiment 1

Give F573 after the modeling

1. method

The Wistar rat is divided into 6 groups at random: normal control group, model group, positive drug N-acetylcystein (NAC 155mg/kg) group and F573 high dosage (H 5.0mg/kg), middle dosage (M 2.5mg/kg), low dosage (L 1.25mg/kg) group; Every group 10; Except that blank control group, each organizes abdominal injection (ip) modeling medicine D-Gal500mg/kg and LPS 20 μ g/kg, and ip corresponding N AC, F573 are respectively organized in modeling after 2 hours; Blank control group gives the respective volume solvent, that is:

1. blank control group: ip solvent (saline water of 0.5%CMC), by body weight ip 2ml/200g

2. model group: ip D-Gal 500mg/kg and LPS 20 μ g/kg and ip solvent 1ml/200g

3. NAC organizes: ip D-Gal 500mg/kg and LPS 20 μ g/kg+NAC 155mg/kg

4. F573H organizes: ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 5.0mg/kg

5. F573M organizes: ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 2.5mg/kg

6. F573L organizes: ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 1.25mg/kg

Rat femoral is got blood system from serum behind the modeling 10h, measures SGPT (ALT), SGOT (AST) and total bilirubin (TBil), behind the sacrificed by exsanguination rat; Get liver and weigh, calculate the liver coefficient, and the big or small liver of 1.5 * 1.5 * 0.5 (cm) is got at every same position; 10% neutral formalin is fixed, paraffin embedding, section, conventional H E dyeing; Do pathological study, change with sxemiquantitative pathology index (PI) evaluation hepatic tissue pathology; Use the variation of TUNEL method and flow cytometry analysis hepatocellular apoptosis.

2 results

2.1 rat body weight

Each organizes rat administration front and back changes of weight little (P＞0.05), sees table 1, table 2-b, shows this experiment modeling medicine and supplies reagent that rat body weight is not had influence.

Body weight (g) before and after the administration of table 1 rat

2.2 liver weight and liver coefficient

Respectively organize rat liver weight and liver coefficient after the administration and than difference statistical significance (P＜0.01) is arranged with the normal control group; And each administration group difference be more or less the same not statistically significant (P＞0.05) see the table 2-a; 2-b; Show that modeling medicine Gal N/LPS is bigger to the rat liver influence, causes certain inflammation to hepatomegaly, but rat body weight is not had influence; And F573 does not all have influence to rat liver weight and body weight.

Rat liver weight and liver coefficient after the table 2-a administration

Annotate: ^##Compare P＜0.01 with the normal control group

Rat body weight, liver weight and liver index variation after the table 2-b administration (

, n=10)

Annotate: ^##Compare P＜0.01 with the blank group

2.3 serum biochemistry index

After the D-GalN/LPS modeling, rat blood serum ALT, AST and T Bil level sharply raise significantly, and model group shows the model establishment apparently higher than normal control group (P＜0.01) (model group ALT, AST and T Bil raise 49,17 and 22 times respectively); F573H, M, each dose groups rat blood serum ALT of L, AST and T Bil level very obviously reduce (P＜0.01) than model group, and wherein the ALT of F573H, M and L group reduces by 42%, 56% and 49% respectively; AST reduces by 58%, 54% and 62% respectively; T Bil reduces by 77%, 84% and 70% respectively, and is close with positive drug NAC group, sees table 3-a, 3-b.

The result shows, but F573 highly significant ground reduces ALT, AST and T Bil, obviously improves liver function, has therapeutic action.

Respectively organize rat blood serum ALT, AST and T Bil after the table 3-a administration

Annotate: ^##Compare P＜0.01 with the blank group; * and model group be P＜0.01 relatively

Respectively organize the comparison

of rat blood serum ALT, AST and T Bil level after the table 3-b administration

2.4 liver histopathology is observed

2.4.1 visual inspection

Normal control group liver form is normal, and tunicle is smooth, complete, color and luster is ruddy, quality is soft.The obvious swelling of model group liver shows slightly pale, the visible diffuse hemorrhage point in surface and be dispersed in ecchymosis, and the quality softness is tough partially.All the other are respectively organized, and liver surface is dispersed in point, sheet blutpunkte area is little than model group.

2.4.2 light microscopic is observed down

Rats in normal control group liver lobule clear in structure, hepatic cords are that the center is radial arrangement with the central vein, and cell infiltration is not seen in the portal area, and sinus hepaticus does not have expansion and extravasated blood; And the model group liver cell is large stretch of downright bad, or is the distribution of being dispersed in property, the visible downright bad structureless liver cell of dissolving and the liver cell (possibly be non-viable apoptotic cell) of coagulation necrosis spherical in shape and the apoptotic body that is dispersed in distribution in the necrosis region; Visible on every side normal liver cell is residual, and original liver lobule structure disappears; Apoptotic cell is rounded, karyon pyknosis engrain, and chromatin is lumps, and kytoplasm concentrates; F573H, M, each dosed administration group hepatic necrosis of L, apoptosis alleviate than model group is obvious; Sxemiquantitative pathology index (PI: measure 20 high power fields for every group, count the ratio of pathology liver cell number in each visual field) than the obvious minimizing of model group (P＜0.01), reduces 92%, 96% and 85% respectively; Positive drug NAC group also obviously reduces; The PI that is F573H, M, L dose groups and NAC group is higher than control group and is starkly lower than model group, sees table 4, table 7, Fig. 1.

The result shows: F573 can very obviously alleviate liver injury, obviously improves liver function, has therapeutic action.

Table 4F573 is to the influence of rat liver pathology indices P I

2.4.3 flow cytometer detects apoptosis rate in the liver tissues of rats

Compare with the normal control group, model group hepatocyte apoptosis rate obviously raises (P＜0.01) (raising 5 times than normal control group hepatocellular apoptosis rate), and model is set up; Compare with model group, each administration group hepatocellular apoptosis rate of F573H, M and L obviously reduces, and reduces by 63% (P＜0.01), 44% (P＜0.01) and 37% (P＜0.05) respectively than model group, presents dose-effect relationship (table 3, table 7); Positive drug NAC group also has identical performance, reduces by 52%.Experimental result shows that F573 can obviously suppress hepatocellular apoptosis.

Table 5F573 convection type cell art detects the influence

of hepatocyte apoptosis rate %

Annotate: ## and blank group be P＜0.01 relatively; *, * and model group be P＜0.01 relatively, P＜0.05.

2.4.4TUNEL method half-quantitative detection hepatocellular apoptosis

Compare with the normal control group, model group hepatocyte apoptosis Index A I (per-cent of apoptotic cell in 500 cells of every section 5 visuals field counting) (P＜0.01) (raising 108 times) model that sharply raises is set up; Compare with model group, each administration group hepatocellular apoptosis Index A I highly significant ground of the H of F573, M and L reduces, and reduces by 67%, 55% and 42% respectively, all has highly significant property meaning (P＜0.01), and presents dose-effect relationship; Positive drug NAC organizes also has identical performance reduction by 55% (table 6,7, Fig. 2).

The result shows once more: F573 can obviously suppress hepatocellular apoptosis.

Table 6F573 is to the influence of TUNEL method half-quantitative detection hepatocellular apoptosis Index A I

Table 7F573 is to rat liver pathology and apoptotic influence

Annotate ^##Compare P＜0.01 with the blank group; *, * and model group be P＜0.01 relatively, P＜0.05

3. discuss and conclusion

Liver cell bulk apoptosis plays a leading role in FLF with downright bad, and the course of disease is dangerous, and patient's death in about 10 days is commonly called as acute severe hepatitis.LPS and D-GaLN combined utilization synergy cause that the rat acute liver injury model is acknowledged as, pathology similar with human diseases certain evolution is arranged, and rate of formation is high, and mortality ratio is low, good reproducibility, and modeling is simple and easy to do, so select this model for use.

Biochemical indicators such as Serum ALT, AST and TBil have begun to raise behind trial test demonstration ip D-GalN/LPS (the 500mg/kg/20 μ g/kg) 2h, and apoptosis occurs in the early stage of damage, so select to begin to give F573 behind the 2h.Being main with apoptosis before the acute liver damage 10h that causes because of bibliographical information D-GalN/LPS, is main with necrosis behind the 10h, thus detect each index when selecting 10h, to investigate the therapeutic action of F573.

F573 is a kind of dipeptidase derivant, and it is the suppressor factor of caspase.

Experimental result shows that rat the performance of typical FLF occurred after D-GalN/LPS handles, and Serum ALT, AST and T Bil level sharply raise significantly, and apoptosis is remarkable, and model is set up.

F573H group 5mg/kg, M group 2.5mg/kg, the L group 1.25mg/kg rat FLF that liver injury causes to rat acute all have the obvious suppression effect; Can obviously improve rational apoptosis of HCD and necrosis due to D-Gal and the LPS; Highly significant reduces ALT, AST and T Bil, and (H of F573, M and L group reduce by 42%, 56% and 49% respectively than model group ALT; AST reduces by 58%, 54% and 62% respectively; T Bil reduces by 77%, 84% and 70% respectively), very obviously improve liver function; The histopathology demonstration can very obviously improve rational apoptosis of HCD and the necrosis due to the D-GalN/LPS; Alleviate liver injury; Sxemiquantitative pathology index reduces (reduce by 92%, 96% and 85% respectively than model group, all have statistical significance P＜0.01) significantly.

TUNEL method, two kinds of tests of fluidic cell method combine to have verified that F573 can suppress apoptosis in testing in vivo effectively; With the apoptosis rate highly significant reduction by 63%, 44% and 37% respectively of model group comparison type, the apoptotic index of TUNEL highly significant respectively reduces by 67%, 55% and 42% (all having statistical significance P＜0.01).

Conclusion: F573 can obviously improve liver function; Alleviate liver injury; Significantly suppress hepatic necrosis and apoptosis, the liver injury of acute severe is had therapeutic action, be expected to become the medicine of leading acute hepatic failure (FHF) disease (comprising slow extra urgaent dispatch liver failure) of cell a large amount of downright bad and apoptosis.

Embodiment 2

Modeling gives F573 simultaneously

1. method

Repeat embodiment 1; Difference is: 70 Wistar rats are divided into 7 groups at random: normal control group, model group, positive drug N-acetylcystein (NAC 155mg/kg) group and F573 high dosage (H:5mg/kg), middle dosage (M:2.5mg/kg), low dosage (L:1.25mg/kg) group and very low dose (LL:0.625mg/kg) group; Every group 10; Except that blank control group, each organizes abdominal injection (ip) modeling medicine D-Gal 500mg/kg and LPS20 μ g/kg, and ip corresponding N AC, F573 are respectively organized in (0 hour) in the time of modeling; Blank control group gives the respective volume solvent, that is:

2. model group: ip D-Gal 500mg/kg and LPS 20 μ g/kg+ip solvent 1ml/200g

3. NAC organizes: ip D-Gal 500mg/kg and LPS 20 μ g/kg+NAC 155mg/kg

4. F573H organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 5.0mg/kg

5. F573M organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 2.5mg/kg

6. F573L organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F5731.2 5mg/kg

7. F573LL organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 0.625mg/kg

Rat femoral is got blood system from serum behind the modeling 10h, measures SGPT (ALT), SGOT (AST) and total bilirubin (TBil).Behind the sacrificed by exsanguination rat, get liver and weigh, calculate the liver coefficient; And every same position gets 1.5 * 1.5 * 0.5cm size liver, and 10% neutral formalin is fixed, paraffin embedding, section, and pathological study is done in conventional H E dyeing, with the variation of sxemiquantitative pathology index (PI) evaluation hepatic tissue pathology; And the variation of using TUNEL method and flow cytometry analysis hepatocellular apoptosis.

2. result

2.1 rat body weight

Each organizes rat administration front and back changes of weight little (P＞0.05), sees table 8, table 9-b, shows this experiment modeling medicine and supplies reagent that rat body weight is not had influence.

Body weight (g) before and after the administration of table 8 rat

2.2 liver weight and liver coefficient

Respectively organize rat liver weight and liver coefficient after the administration and than difference statistical significance (P＜0.01) is arranged with the normal control group; And each administration group difference be more or less the same not statistically significant (P＞0.05) see the table 9-a; 2-b; Show that modeling medicine Gal N/LPS is bigger to the rat liver influence, causes certain inflammation to hepatomegaly, but rat body weight is not had influence; And F573 does not all have influence to rat liver weight and body weight.

Rat liver weight and liver coefficient after the table 9-a administration

Annotate: ^##Compare P＜0.01 with the normal control group

Rat body weight, liver weight and liver index variation

after the table 9-b administration

Annotate: ^##Compare P＜0.01 with the blank group

2.3 serum biochemistry index

After the D-GalN/LPS modeling, rat blood serum ALT, AST and T Bil level sharply raise significantly, and model group shows the model establishment apparently higher than normal control group (P＜0.01) (model group ALT, AST and T Bil raise 45,18 and 5 times respectively); Each dose groups rat blood serum ALT of F573H, M, L and LL, AST and T Bil level obviously reduce (P＜0.01) than model group, and wherein the ALT of F573H, M, L and LL group reduces by 86%, 65%, 67% and 28% respectively, presents dose-effect relationship; AST reduces by 82%, 61%, 62% and 7% respectively, presents dose-effect relationship; T Bil reduces by 96%, 52%, 30% respectively, and the LL group does not have reduction, presents dose-effect relationship; Positive drug NAC group also has ALT, AST and the T Bil effect of reduction.

Test shows that F573 can reduce ALT, AST and T Bil significantly, obviously improves liver function, has the prevention provide protection.See table 10-a, 10-b.

Respectively organize rat blood serum ALT, AST and T Bil after the table 10-a administration

Respectively organize the comparison

of rat blood serum ALT, AST and T Bil level after the table 10-b administration

2.4 liver histopathology is observed

2.4.1 visual inspection

2.4.2 light microscopic is observed down

Pathological study is carried out in conventional H E dyeing under the light microscopic, rats in normal control group liver lobule clear in structure, and hepatic cords is that the center is radial arrangement with the central vein, and cell infiltration is not seen in the portal area, and sinus hepaticus does not have expansion and extravasated blood; The big sheet of model group liver cell is downright bad or be the distribution of being dispersed in property; Cellularstructure is smudgy, the apoptotic body that the liver cell that visible liver cell liquefactive necrosis is accompanied by more spherical coagulation necrosis in the hepatic tissue is dispersed in distribution (possibly be non-viable apoptotic cell) and is dispersed in distribution; Original liver lobule structure disappears; Apoptotic cell is rounded, karyon pyknosis engrain, and chromatin is lumps, and kytoplasm concentrates; Visible on every side normal liver cell.Except that F573 very low dose group, each dose groups of other F573 and positive drug NAC group hepatic necrosis, apoptosis and obviously alleviate than model group.Wherein LL organizes: pathology and model group are suitable, and the liver cell of visible more spherical coagulation necrosis is dispersed in distribution in the hepatic tissue, and scope is bigger.L group: visible more special mess shape liver cell is liquefactive necrosis in the hepatic tissue, simultaneously in the hepatic tissue also the liver cell of visible more spherical coagulation necrosis be dispersed in distribution; Apoptotic cell quantity is starkly lower than model group, and the liver cell of visible point-like and little focal necrosis has monocyte infiltration on every side and necroses, and the hepatocellular degeneration necrosis alleviates than model group to some extent, sinus hepaticus extravasated blood, every visible spot, kitchen range shape hepatic necrosis district; The M group: lesion degree further alleviates than low dose group; Spherical coagulation necrosis and liquefactive necrosis take place in the visible part liver cell in the hepatic tissue; Spotty necrosis takes place in the minority liver cell; Be dispersed in distribution, the liver cell of also visible special mess property hepatic necrosis and single necrosis is with cell infiltration and necrose; The H group: the middle dose groups of lesion degree further alleviates; Visible special mess shape liver cell liquefactive necrosis is with the spherical coagulation necrosis of minority liver cell in the hepatic tissue; Be the distribution of being dispersed in property, the nucleus of visible a large amount of pyknosis in the necrosis region, the liver cell of single necrosis is common.The NAC group: pathology is attached most importance to than the M group of F573, and visible more special mess shape hepatic necrosis is the distribution of being dispersed in property in the hepatic tissue, with the liver cell of more spherical coagulation necrosis, possibly be the liver cell of apoptosis in late period.F573H, M group pathology index (sxemiquantitative pathology indices P I: measure 20 high power fields for every group; Count in each visual field the pathology liver cell and count proportion) highly significant is lower than model group (P＜0.01), and also be lower than positive drug NAC group (P＜0.05), the PI of F573H, M, L group reduces 83%, 69%, 17% respectively than model group; LL organizes no change; Present dose-effect relationship, see table 11, table 14, Fig. 3.

Table 11F573 is to the influence of rat liver pathology indices P I

Annotate: ^##Compare P＜0.01 with the blank group; *, * and model group be P＜0.01 relatively, P＜0.05

2.4.3 flow cytometer detects apoptosis rate in the liver tissues of rats

Compare with the normal control group, model group hepatocyte apoptosis rate obviously raises (P＜0.01) (raising nearly 3 times than normal control group hepatocellular apoptosis rate), and model is set up; Compare with model group, the hepatocellular apoptosis rate of each administration group streaming of F573H, M, L and LL obviously reduces, and reduces by 54% (P＜0.01), 54% (P＜0.01), 25% (P＜0.05) and 9% respectively than model group, presents dose-effect relationship; Positive drug NAC group also has identical performance to reduce by 64% (P＜0.01) (table 12, table 14).

The result shows: F573 can obviously suppress hepatocellular apoptosis.

Table 12F573 convection type cell art detects the influence

of hepatocyte apoptosis rate %

5.4.4TUNEL method half-quantitative detection hepatocellular apoptosis

Compare with the normal control group, model group hepatocyte apoptosis Index A I (per-cent of apoptotic cell in 500 cells of every section 5 visuals field counting) (P＜0.01) (the raising 52 times) that obviously raise, model is set up; Compare with model group, each administration group hepatocellular apoptosis index of F573H, M, L and LL obviously reduces, and reduces by 57% (P＜0.01), 35% (P＜0.01), 27% (P＜0.05) and 22% respectively, presents dose-effect relationship; Positive drug NAC group also has identical performance, and AI reduces by 38% (P＜0.05) and (shows 13-14, Fig. 4).The result shows that once more F573 can obviously suppress hepatocellular apoptosis.

Table 13F573 is to the influence of TUNEL method half-quantitative detection hepatocellular apoptosis Index A I

Table 14F573 is to rat liver pathology and apoptotic influence

Rat the performance of typical FLF due to the acute liver damage occurred after D-GalN/LPS handles, Serum ALT, AST and T Bil level sharply raise significantly, and apoptosis is remarkable, and model is set up; Each dose groups of H, M, L and LL of caspase suppressor factor F573 does not have influence to rat body weight and liver coefficient; But can obviously reduce ALT, AST and T Bil level, wherein F573H, M, L and LL group reduce by 86%, 65%, 67% and 28% respectively than the ALT of model group; AST reduces by 82%, 61%, 62% and 7% respectively; T Bil reduces by 96%, 52%, 30% (the LL group does not have reduction) respectively, presents dose-effect relationship, and liver injury has obvious improvement effect to rat acute to show F573, obviously improves liver function; Histopathology shows except that the LL group; Other each group of F573 can significantly suppress rational apoptosis of HCD and the necrosis due to the D-GalN/LPS; Alleviate liver injury, F573H, M group pathology index (PI) highly significant are lower than model group (P＜0.01), and also are lower than positive drug NAC group (P＜0.05); The PI of F573H, M, L group reduces 83%, 69%, 17% respectively than model group, has dose-effect relationship; TUNEL method, two kinds of tests of fluidic cell method combine to have verified that F573 can suppress the hepatic necrosis apoptosis in testing in vivo effectively; Wherein the fluidic cell method is measured each administration group hepatocellular apoptosis rate of F573H, M, L and LL and is obviously reduced; Reduce by 54% (P＜0.01), 54% (P＜0.01), 25% (P＜0.05) and 9% respectively than model group; The apoptotic index of TUNEL reduces by 57% (P＜0.01), 35% (P＜0.01), 27% (P＜0.05) and 22% respectively; All have dose-effect relationship, demonstration F573 can significantly suppress rational apoptosis of HCD and the necrosis due to D-Gal and the LPS.

Conclusion: F573 obviously improves liver function, can significantly suppress hepatic necrosis and apoptosis, and the liver injury of acute severe is had prevention and provide protection, is expected to become the medicine of leading acute hepatic failure (FHF) disease of cell a large amount of downright bad and apoptosis.

Embodiment 3

Give F573 before the modeling

1. method

Repeat embodiment 1, difference is: respectively organized ip corresponding N AC, F573 in 4 hours before the modeling, blank control group gives the respective volume solvent, that is:

2. model group: ip D-Gal 500mg/kg and LPS 20 μ g/kg+ip solvent 1ml/200g

3. NAC organizes: ip D-Gal 500mg/kg and LPS 20 μ g/kg+NAC 155mg/kg

4. F573H organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 5.0mg/kg

5. F573M organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 2.5mg/kg

6. F573L organizes ip D-Gal 500mg/kg and LPS 20 μ g/kg+F573 1.25mg/kg

Give F573H group 5mg/kg, M group 2.5mg/kg, the L group 1.25mg/kg rat FLF that liver injury causes to rat acute in advance and all have the obvious suppression effect; Can obviously improve rational apoptosis of HCD and necrosis due to D-Gal and the LPS; Highly significant reduces ALT, AST and T Bil, very obviously improves liver function; The histopathology demonstration can very obviously improve rational apoptosis of HCD and the necrosis due to the D-GalN/LPS, alleviates liver injury, and sxemiquantitative pathology index reduces (reduce 80-92% respectively than model group, all have statistical significance P＜0.01) significantly.

TUNEL method, two kinds of tests of fluidic cell method combine to have verified that F573 can suppress apoptosis in testing in vivo effectively; Reduce 35-66% with the apoptosis rate difference highly significant of model group comparison type, the apoptotic index of TUNEL highly significant respectively reduces 40-70% (all having statistical significance P＜0.01).

Conclusion: giving F573 in advance can obviously improve liver function, alleviates liver injury, significantly suppresses hepatic necrosis and apoptosis, and the liver injury of acute severe is had prophylactic effect.

Embodiment 4

F573 is to the provide protection of people's normal liver cell L02

Whether this experimental observation F573 has provide protection to the growth by the apoptosis-induced people's normal liver cell L02 of inducer of apoptosis TNF α.

Experimental technique is following:

1) mtt assay detects F573 to apoptosis-induced cytoprotection: to the L02 cell, a collection of trial-product F573 establishes 4 dose groups, is respectively 0,0.5,5,50 μ M.Another batch trial-product F573 establishes 10 dose groups, and since three times of dilutions of 10 μ M, last point is 0 μ M.The L02 cell that conventional serum is cultivated adds medicine F573 and cultivates and add TNF α again behind the 2hr and dactinomycin stimulates 24hr altogether.Mtt assay detects the propagation situation of cell, and calculates EC ₅₀

2) the Western-blot method detects F573 apoptosis-induced product active-PAP is sheared segmental restraining effect: F573 establishes 4 dose groups to L02 cell trial-product, is respectively 0,0.5,5,50 μ M.L02 cell adding TNF α that conventional serum is cultivated and dactinomycin add medicine F573 cultured continuously 6hr after stimulating 18-24hr altogether.Collect albumen, the Western-blot method detects apoptosis product active-PAP expression in the born of the same parents.

3) Caspase-Glo 3&7 test kit (Promega) detects the restraining effect of different time points adding F573 to apoptosis enzyme Caspase 3 in the born of the same parents: the blank well of L02 cell trial-product F573 being established 10 points and not dosing; Since three times of dilutions of 10 μ M; The L02 cell that conventional serum is cultivated adds medicine F573 preincubate 2hr, preincubate 2hr flush away F573 and add TNF α simultaneously and dactinomycin continues to cultivate 6hr altogether then.The expression level that adds Caspase-Glo 3&7 test kit substrate detection Caspase 3.

4) experimental result:

MTT result shows: F573 can obviously be alleviated the apoptosis that inducer of apoptosis TNF α and dactinomycin cause, and reduces provide protection with the drug level gradient and also decrease.And the EC50 of F573 protection normal liver cell L02 is 1.14 μ M.

Western-blot result shows: F573 can obviously reduce the interior apoptosis product active-PARP of born of the same parents and shear segmental generation.

Caspase-Glo 3&7 reagent detected result shows: F573 can suppress the activity of Caspase 3 in the born of the same parents, is nM-μ M level to the IC50 of Caspase 3, and the change that does not add mode with F573 changes.

Above-mentioned in vitro study result of experiment shows: F573 can obviously protect the L02 cell of vitro culture.

Embodiment 5

F573 is to the raising effect of survival rate

120 of Kunming (km) mouse (spf level); Male and female half and half; Body weight 20-22g is divided into 6 groups by 20 every group: model group, model+group of solvents, model+positive drug N-acetylcystein (NAC 200mg/kg) group and F573 high dosage (H 5.0mg/kg), middle dosage (M 2.5mg/kg), low dosage (L 1.25mg/kg) group.

Each organizes abdominal injection (ip) modeling medicine D-Gal 600mg/kg and LPS 20 μ g/kg, (0 hour) ip corresponding N AC or subcutaneous injection F573 in the time of modeling, and blank control group gives the respective volume solvent.Observe 24h, survival is as shown in Tble 15.

Table 15F573 is to the raising effect of survival rate

Can find out that from table 15 F573 can significantly improve the survival rate of liver failure animal pattern, and increase rate is relevant with F573 dosage.This prompting, F573 can be used for improving the survival rate of liver failure patient (especially acute hepatic failure and slow extra urgaent dispatch liver failure patient).

Embodiment 6

Preparation contains the pharmaceutical composition of F573

(a) injection liquid

F573 30mg

Non-water-soluble matchmaker (injection) adds to 2ml

By above-mentioned prescription, take by weighing F573 and an amount of non-water-soluble matchmaker, main ingredient is sterilized into sterilized powder, after packing respectively.During injection at present with join at present.

(b) capsule

F573 50-200mg

Vinylpyrrolidone polymer 2-10mg

Starch 50-100mg

Lactose 2-10mg

By 1000 capsular consumptions, mix after taking by weighing above each auxiliary material porphyrize sieving respectively, add F573 with the equivalent method of progressively increasing then, fully grind, it is uniformly dispersed, after 80 mesh sieves, record into capsule then.

All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims

1. formula I compound or its pharmacy acceptable salt that can be used for treating liver failure:

2. a pharmaceutical composition that is used to treat or prevent liver failure is characterized in that, it contains the described formula I compound of claim 1 or its pharmacy acceptable salt of pharmaceutically acceptable carrier and safe and effective amount.

3. pharmaceutical composition as claimed in claim 2 is characterized in that, it contains formula I compound or its pharmacy acceptable salt that accounts for gross weight 0.01-99%.

4. pharmaceutical composition as claimed in claim 2 is characterized in that, the formulation of said pharmaceutical composition is tablet, capsule, injection (injection), pill.

5. the purposes of the described formula I compound of claim 1 or its pharmacy acceptable salt is characterized in that, is used to prepare the medicine of treatment or prevention liver failure.

6. purposes as claimed in claim 5 is characterized in that, described liver failure comprises: acute hepatic failure, subacute liver failure and slow extra urgaent dispatch liver failure, chronic liver failure.

7. purposes as claimed in claim 5 is characterized in that, the formulation of described medicine is injection or oral prepns.

8. purposes as claimed in claim 5 is characterized in that described medicine also is used to improve survival rate; Or be used to reduce SGPT (ALT), reduce SGOT (AST) and/or reduce total bilirubin (TBil).

9. the method for treating or preventing liver failure is characterized in that, comprises step: the object of treating for needs is used the described formula I compound of claim 1 or its pharmacy acceptable salt of safe and effective amount.

10. method as claimed in claim 8 is characterized in that, described using comprises: intravenous injection, subcutaneous injection, abdominal injection or injection into liver.