CN102614502A

CN102614502A - Application of ulinastatin to preparing anti-HIV (Human Immunodeficiency Virus)-1 virus medicament

Info

Publication number: CN102614502A
Application number: CN2012101231589A
Authority: CN
Inventors: 徐宗昆
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-04-25
Filing date: 2012-04-25
Publication date: 2012-08-01

Abstract

The invention discloses application of ulinastatin to preparing an anti-HIV (Human Immunodeficiency Virus)-1 virus medicament, wherein the concentration of the ulinastatin is 200-400 mug/ml. The medicament can be immediately used at a window phase after HIV infection, and can also be used at different stages after occurrence of AIDS (Acquired Immune Deficiency Syndrome). According to the medicament, the concentration of the ulinastatin is 400 mug/ml, the suppression rate of 100 percent for the HIV-1 virus can be achieved, and no obvious cytotoxicity effect exists in cytotoxicity test.

Description

Ulinastatin is as the application of preparation anti-HIV-1 virus drugs

Technical field

The present invention relates to the application of ulinastatin as preparation anti-HIV-1 virus drugs.

Background technology

AIDS has just spread in the whole world since 1970 with surprising rapidity, obtains huge progress aspect the AIDS preventing and treating in recent years, but still lacks the active drug that radical cure HIV infects.The therapeutic goal of present stage is: to greatest extent with persistent reduction virus load; Obtain immunity function restructuring and keep immunologic function; Improve the quality of living; Reduce the relevant M & M of HIV.Therapeutic Method comprises general treatment, antiviral therapy, recovery and improves the treatment of immunologic function and the treatment of opportunistic infection and malignant tumor.Wherein antiviral is the key of treatment.Appearance along with the hiv protease inhibitor; Efficient anti-retroviral conjoint therapy (highly active anti-retroviral therapy has just appearred; HAART) application; Improved the curative effect of anti-HIV greatly, significantly improved AIDS patient's quality of life and prognosis, the treatment of AIDS is a a progressive step.

Current main antiviral drugs: granted come into the market have 16 kinds, mainly divide three major types: ucleosides reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitor (PI).

Wherein protease inhibitor (protease inhibitor, PI): HIV-1 protease is a symmetric dimer, under the catalysis of this enzyme, HIV has two hatching egg Rhizoma Cynanchi Stauntonii bodies to be cracked into maturation protein, so this enzyme is extremely important to duplicating of virus.Protease inhibitor can through the CKIs enzyme promptly block HIV duplicate with maturation process in necessary protein synthesis, thereby suppress duplicating of HIV.This type of preparation comprises:

A. Saquinavir (saquinavir): the hiv protease inhibitor of efficient high selectivity.These article act on the later stage of HIV breeding, combine with the activation point of hiv protease, make it to lose the function of combination and hydrolytic cleavage polypeptide.These article suppress hiv protease and other anti HIV-1 virus medicine such as azidothymidine AZT, and the effect target enzyme system that suppresses hiv reverse transcriptase is different, and no crossing drug resistant virus produces.600mg, 2 times/d or 3 times/d.Side effect can have symptoms of digestive tract such as nausea,vomiting,diarrhea, also visible headache, transaminase's rising etc.

B. indinavir (indinavir): (hiv protease inhibitor and other antiretroviral drugs are united use.800mg, 3 times/d.That side effect has is nauseating, vomiting, headache, weak, dizzy, erythra etc., and can cause unconjugated bilirubin rising, thrombocytopenia, renal calculus, blood sugar increasing and fat redistribution etc.

C. viracept see nelfinaivr (nelfinavir): 750mg, 3 times/d.Side effect has diarrhoea, hyperglycemia etc.

D. oral effective inhibitor of ritonavir (ritonavir): HIV-1 and HIV-2 aspartic protease; Block this enzymatic and make the required polyprotein of ripe HIV granule in the generation morphology; Make the HIV granule thereby remain on immature state; Thereby slow down HIV spreading in cell, to prevent the generation that a new round infects and to postpone advancing of disease.These article are generally all effective with zidovudine and the drug-fast HIV strain of Saquinavir to the zidovudine sensitivity.300～600mg, 2 times/d.Side effect has nausea,vomiting,diarrhea, weak, paraesthesia, hepatic disfunction, triglyceride rising, uric acid increase, blood sugar increasing etc.

Above-mentioned three types of medicine majorities all have untoward reaction, and are single with very difficult effective duplicating of virus of suppressing, and are prone to the generation drug resistance.Therefore, stress drug combination.The scheme of drug combination is a lot, at present manyly adds two kinds of NRTI preparations with a kind of protease inhibitor, or two kinds of albumen enzyme inhibitors add one or two kind of NRTI preparation, and common scheme for combining has: Saquinavir ZDV 3TC; Or viracept see nelfinaivr D4T ddi; Or indinavir ZDV ddi; Or ritonavir ZDV ddc; Or Saquinavir ritonavir D4T 3TC.Use these scheme for combining treatments, can suppress a plurality of links of HIV reproduction process simultaneously, can efficiently suppress HIV and duplicate, reduce drug resistance to greatest extent, improve patients ' life quality and survival rate, significantly reduce the danger of mother and baby's vertical transmission etc.

HAART scheme for combining on opportunity about antiviral therapy is used the curative effect that is obtained, and arouses the confidence that people defeat AIDS again.Many doctors were thought getting over early treatment, effect is good more.But prolongation along with the time of application; Can suppress virus load in the blood to the level that does not detect although it is found that therapeutic alliance; But remove the virus of concealing in lymphocyte, macrophage and the nervous tissue and be not easy, treat decades possibly even take medicine all the life.Yet; Period in a medicine, except digestive tract reaction and bone marrow depression etc. the common adverse effect, these drug-induced other serious toxic and side effects; Patient's compliance, compliance are poor; The Drug resistance of HIV is increasing in addition, and the propagation of Drug resistance strain is more and more serious or the like, forces the doctor to consider treatment opportunity carefully.

Advocate at present: 1. be developed to AIDS person,, all should have treated regardless of the value height of CD4 and virus load; 2. asymptomatic person, < 200 * 106/L should treat though virus load can not surveyed also CD4; 3. cd4 cell>200 * 106/L, but < 350 * 106/L, virus load are not high, and the patient is asymptomatic, and the treatment of this moment still has arguement; 4. asymptomatic person's CD4>350 * 106/L, virus load>55000 copy numbers, advocate treatment at present mostly; 5. asymptomatic person's CD4>350 * 106/L, < 55000 copy numbers advocate to continue monitoring CD4 amount to virus load mostly at present; 6. be interrupted treatment.

Summary of the invention

Goal of the invention: the objective of the invention is to deficiency to prior art; A kind of morbidity that both can the window phase after infected by HIV prevents AIDS is provided; Can therapeutical effect be arranged to the patient who suffers from AIDS again, prevent recurrence, and not have the medicine of the anti-HIV-1 virus of obvious toxic and side effects.

Technical scheme: ulinastatin of the present invention is as the application of preparation anti-HIV-1 virus drugs, and the concentration of ulinastatin is 200～400 μ g/ml.

Preferably, the concentration of said ulinastatin is 400 μ g/ml, under this concentration, can reach 100% suppression ratio to HIV-1 virus.

Preferably, the concentration of said ulinastatin is 200 μ g/ml, under this concentration, can reach 99% suppression ratio to HIV-1 virus, and simultaneously, the toxic action of pair cell is littler, and Financial cost is lower.

Also comprise pharmaceutically acceptable auxiliaries in the said medicine.

Said medicine can be lyophilized injectable powder, and said pharmaceutically acceptable auxiliaries is any one or a few in mannitol, lactose, gelatin hydrolysate, sodium chloride or the glucose sugar.

Said medicine also can be injection, and said pharmaceutically acceptable auxiliaries is any one or a few in water for injection, mannitol, sodium chloride or the glucose.

Beneficial effect: 1, medicine of the present invention can use by the window phase after infected by HIV immediately, also can be used for the different phase that AIDS has taken place, and to compare toxic and side effects little with the medicine that has gone on the market; 2, medicine of the present invention when the concentration of ulinastatin is 400 μ g/ml, can reach 100% suppression ratio to HIV-1 virus, and in cytotoxicity test, not have the significant cytotoxicity effect; 3, for many years, in the clinical observation of using active constituents of medicine ulinastatin treatment acute pancreatitis of the present invention, not finding obvious toxic and side effects, is comparatively safe therefore.

The specific embodiment

Be elaborated in the face of technical scheme of the present invention down, but protection scope of the present invention is not limited to said embodiment.

Embodiment:Ulinastatin of the present invention is as the application of preparation anti-HIV-1 virus drugs, and the concentration of ulinastatin is 200～400 μ g/ml, and concentration can reach 100% suppression ratio when being 400 μ g/ml.

Utilize the anti-HIV-1 virus drugs of ulinastatin preparation, can use immediately by the window phase after infected by HIV, also can be used for the different phase that AIDS has taken place.The window phase dosage is ulinastatin 100,000 U, with 5% glucose injection 500ml dissolving 1-2 time/day, intravenous drip.When get into AIDS sick period then with various RTI Combined application.Medicine is usually with the intravenous route administration, and main dosage form is lyophilized injection or injection liquor.

When medicine was lyophilized injectable powder, pharmaceutically acceptable auxiliaries was any one or a few in mannitol, lactose, gelatin hydrolysate, sodium chloride or the glucose sugar, can be dissolved in use in go out water bacterium or various other injection sterile medium.

When medicine was injection, pharmaceutically acceptable auxiliaries was any one or a few in water for injection, mannitol, sodium chloride or the glucose.

The pharmacodynamic study test of AIDS virus resisting of the present invention is provided below:

1, material and medicine

1.1 medicine: after the ulinastatin water fully dissolves, after the bag filter dialysis, cultivate base with 1640 and be diluted to variable concentrations (the ulinastatin medicine is produced by the Dalian northern biological factory);

1.2 reagent: the P24 test kit is available from U.S. Organon Teknika company, and MTT (Thiazoll blue) is available from Sigma company;

1.3 cell: MT4 cell line is adopted to cultivate and is obtained, and culture medium is that RPMI-1640 adds 10% hyclone, 1% glutamine, 1% mycillin;

1.4 virus: the German strain of HIV-1 virus, Virology Inst., China Academy of Preventive Medicine Sciences preserves goes down to posterity.

2, experimental technique

2.1 inhibition test to the HIV-1 actute infection

Be diluted to 10,000 TCID 50/ml after HIV-1 melted, get the 5000000 MT4 cells (1800 rev/mins centrifugal 5 minutes) that viral liquid after the 1ml dilution adds centrifugal back and abandons supernatant, put in 37 ℃ of incubators effect 2 hours.It is centrifugal to take out the back, abandons supernatant, washes once with 1640, with culture medium to the 50 ten thousand cells/ml of 20% hyclone, with the ulinastatin adding culture plate of the infection cell for preparing and the variable concentrations of correspondence.Put 37 ℃, 5% CO ₂In the incubator.Change liquid after 48 hours, continued to hatch 48 hours.Get basic supernatant and measure P24 antigen with the U.S. P24 of Organon Teknika company antigen test box.According to calculating virus concentration in each hole, calculate the suppression ratio of medicine to virus replication.

2.2 MTT cytotoxicity test

The MT4 cell is assigned to 500,000/ml with the culture medium that contains 20% hyclone, adds cell that 100 μ l prepare and the ulinastatin of 100 μ l and go into 96 well culture plates, 37 ℃, 5% CO ₂Cultivated 6 days in the incubator; Take out every hole, back and absorb 150 μ l supernatants; Add MTT (0.25%) the 25 μ l that newly joins, 37 ℃ act on 4 hours down, in every hole, add 100 μ lDMSO and fully dissolve formed crystallization; With BIO-RAD company 550 type ELIASA readings (570nm), calculate cell survival rate.The base computational methods are: cell inhibitory rate=(cell control well OD value-dosing cell hole OD value) ÷ cell control well OD value * 100%.

3, result

3.1 anti-HIV-1 experimental result

Ulinastatin concentration (μ g/ml)	The OD value	Virus quantity in the supernatant (pg)	Suppression ratio
				1000	0.832	57	52%
800	0.305	22	84%
				500	0.021	3	97%
400	0.011	0	100%
				200	0.015	1	99%
100	0.399	25	78.3%
				50	0.996	64	44.4%
Virus control	1.798	115.3	?
				The cell contrast	0.013	?	?

3.2 cytotoxicity result

Ulinastatin concentration (μ g/ml)	The OD value	Pair cell toxicity rate
			1000	0.503	47.7%
800	0.592	38.5%
			500	0.685	28.8%
400	0.707	26%
			200	0.725	24%
100	0.89	7.5%
			50	0.87	9.5%
The cell contrast	0.962	?

Find by above result, ulinastatin to HIV-1 virus actute infection inhibition test in, in the culture medium to 20% hyclone of 500,000 cells/ml of containing HIV-1; Ulinastatin pair cell toxicity rate is linear; But ulinastatin is not linear to the suppression ratio of HIV-1 virus, add ulinastatin 0.4mg/ml and can reach 100% suppression ratio, and along with the further increase of ulinastatin concentration; Suppression ratio to HIV-1 virus reduces, and pair cell toxicity rate continues to increase.Therefore, the ulinastatin of 0.2～0.4mg/ml concentration is best to the inhibition effect of HIV-1 virus, the interval that pair cell toxicity is the most moderate.

As stated, although represented and explained the present invention that with reference to specific preferred embodiment it shall not be construed as the restriction to the present invention self.Under the spirit and scope of the present invention prerequisite that does not break away from the accompanying claims definition, can make various variations in form with on the details to it.

Claims

1. ulinastatin is as the application of preparation anti-HIV-1 virus drugs, and the concentration of ulinastatin is 200～400 μ g/ml.

2. application according to claim 1 is characterized in that: the concentration of said ulinastatin is 400 μ g/ml.

3. application according to claim 1 is characterized in that: the concentration of said ulinastatin is 200 μ g/ml.

4. application according to claim 1 is characterized in that: also comprise pharmaceutically acceptable auxiliaries in the said medicine.

5. application according to claim 4 is characterized in that: said medicine is a lyophilized injectable powder, and said pharmaceutically acceptable auxiliaries is any one or a few in mannitol, lactose, gelatin hydrolysate, sodium chloride or the glucose sugar.

6. application according to claim 4 is characterized in that: said medicine is an injection, and said pharmaceutically acceptable auxiliaries is any one or a few in water for injection, mannitol, sodium chloride or the glucose.