CN101485882A - Pharmaceutical formulation containing candida urate oxidase - Google Patents

Abstract

The invention relates to a medicinal preparation containing Candida urate oxidase, in particular to a liquid or freeze-dried composition medicinal preparation containing the Candida urate oxidase. The composition comprises the following active ingredients: the Candida urate oxidase, glycine, cysteine and a buffer system. The composition is aseptic, and can be injected into human body or animal body to take effect through hypodermic injection, intravenous injection or intramuscular injection.

Description

A kind of pharmaceutical formulation that contains candida urate oxidase

Technical field

The present invention relates to a kind of pharmaceutical formulation that contains candida urate oxidase, especially relate to the liquid or the freeze dried compositions pharmaceutical formulation that contain candida urate oxidase.

Background technology

303 aminoacid of candida urate oxidase UOX total length, molecular weight is 34.19kDa, theoretical isoelectric point, IP is 8.4.Urate oxidase (UOX) is one of key enzyme on the purine metabolism approach.The ability of different types of biological decomposition purine base is different, thereby metabolite also has nothing in common with each other.People and apes and some uricotelic animals with uric acid as the metabolic end-product of purine base.Multiple biology such as candida mycoderma and rat then can produce urate oxidase, and this enzyme further decomposes uric acid, forms allantoin.The dissolubility of the latter in water be much larger than uric acid, removes uric acid in the body thereby help mammal such as rat.In the evolution of long period of time process, human urate oxidase gene is undergone mutation, can not the normal encoding urate oxidase, and therefore when causing that easily increasing with the blood uric acid level is the gout or the urate nephropathy of feature during the uric acid level increase in the body.Clinically,, reduce the blood uric acid concentration, therefore can be used for treating metabolic arthritis disease because UOX can decompose uric acid effectively.

In order to make candida urate oxidase act on human body or animal, it must be mixed with certain pharmaceutical formulation form for clinical use, and this preparation can guarantee that urate oxidase stable and biologic activity of physicochemical properties in secular preservation process is stable.The same with numerous pharmaceutical grade proteins, candida urate oxidase is made preparation need consider many-sided factor, to guarantee Products Quality, technological feasibility, economic feasibility and rules feasibility etc.Dissolubility is one of the factor that need consider of numerous protein pharmaceutical formulation, how to select for use economy, reliable, effectively solubilizing agent or preparation technology are very important for preparing qualified preparation.

France Sanofi company has developed hyperuricemia medicine " granny rag power enzyme ", and it is a kind of recombined Aspergillus flavus bacteriuria acid oxidase lyophilized injectable powder, June calendar year 2001 in Germany and Britain's Initial Public Offering, but its product price costliness comparatively.Sanofi company has applied for patent of invention and obtaining the authorization in 1996 in China, patent name is " contain the stable liquid compositions of urate oxidase and be used to prepare the freeze-dried composition of this fluid composition ", the patent No. is 96110721.9, the pharmaceutical composition outstanding feature of describing in its patent is to contain co-emulsifier, dispersant poloxamer 188, keeps the dissolubility of medicine with this.There is the poloxamer (124,188,237,338,407) of 5 kinds of different sizes in the U.S., has only poloxamer 188 to use as the emulsifying agent of intravenous injection, and the poloxamer of other specification all is used for oral or external preparation.In the world wide, poloxamer also lacks enough clinical uses, to verify its reliability.Present poloxamer adjuvant import China not as yet, promptly China's poloxamer of still not having the injection specification at present goes on the market, and China does not approve this adjuvant as yet.Therefore, domestic protein formulation can not use this medicine as adjuvant.

In addition, the urate oxidase of different molecular has different firsts and seconds structures, and its stability of molecule and character differ bigger, and a certain preparation prescription does not have general value to the protein of different structure.Candida urate oxidase contains a plurality of cysteine, and its intramolecularly and intermolecular mispairing also merit attention in preparation research.

Summary of the invention

The object of the present invention is to provide a kind of pharmaceutical formulation that contains candida urate oxidase, adopt specific prescription to form, can keep medicine stability and activity, reduce cost.

Active component candida urate oxidase described in the present invention can obtain by gene recombinaton, and preparation method can be with reference to Chinese patent 031091504.According to the basic standard of biological product preparation process and prescription thereof and my company gone on the market the excipient experience of product and the molecular characterization of UOX, requiring the aspect to start with from outward appearance, clarity, biological activity etc. respectively, starts with and gropes the preparation process prescription in the configuration aspect of below writing out a prescription:

1. the excipient of Kao Chaing: mannitol, sucrose, glycine.

2. the activity protecting agent of Kao Chaing: alanine, arginine, glutamic acid, cysteine.

3. the cosolvent of Kao Chaing: tween, glycerol, polyoxyethylene castor oil, SDS etc.

4. the buffer system of Kao Chaing: Tris-HCL (pH7.5,8.0,8.5 and 9.0)

50mM PB buffer (pH7.0,7.5 and 8.0)

Boric acid (pH7.0,7.5,8.0,8.5).

Selecting for use of adjuvant, we have abandoned at present at the still unaccredited poloxamer 188 of China, and are apparent with the known references difference.

Above-mentioned additive different proportion is write out a prescription and is formed and lyophilizing, detect following index after the lyophilizing in order successively: outward appearance, with indexs such as water for injection dissolving back clarity, biological activity (adopting external uric acid oxidizing process, concrete activity determination method), moisture with reference to the medicine granny rag power enzyme that gone on the market.

Research by experiment; compared a large amount of different auxiliary material combinations; the present invention finally provides a kind of desirable combination that contains the pharmaceutical formulation of candida urate oxidase, and compositions comprises active component candida urate oxidase, mannitol, protective agent and buffer agent.

The screening route is as follows:

Buffer system one

PB commonly used is as buffer solution, final concentration is 50mM, and (annotate: pH8.0 is near the PB buffer capacity upper limit, and it is pH8.0 that external like product Rasburicase selects buffer system for use to select pH7.0,7.5,8.0,40mM PB), excipient is selected mannitol, sucrose, glycine respectively for use, and each plant demand is respectively 2%, 5%, 7%, 20 of each packing, are carried out orthogonal test (seeing Table) by totally 9 groups 180.

Table one 50mM PB buffer, UOX 1.8mg/ml/ props up

By result of the test as seen, buffer system pH is that 7.0,7.5 o'clock dissolving back clarity are too poor, all muddy, does not reach requirement.Mannitol during pH8.0, sucrose are that the sample of excipient be cannot see tangible muddiness after with physiological saline solution, but under umbrella canopy lamp visible opalescence, do not reach requirement equally, and each excipient final concentration is that 2% o'clock outward appearance inborn nature is relatively poor, even atrophy occurs, and profile is imperfect.The prescription that the test discovery does not meet the requirements.

Buffer system two

Select boric acid.EXPERIMENTAL DESIGN boric acid final concentration is 50mM, and pH is 7.0,7.5,8.0, and according to test one, the mannitol final concentration can select 5%, 7%, is not influencing on the excipient basis, selects low amount 5%.

Table two 50mM borate buffer, UOX 1.8mg/ml/ props up

PH value	Mannitol 5%	Clarity	Biological activity	Moisture
					7.0	Block porous solid, outward appearance is good	Qualified, meet standards of pharmacopoeia	Substantially do not detect activity	<3％

7.5	Block porous solid, outward appearance is good	Qualified, meet standards of pharmacopoeia	Substantially do not detect activity	<3％
					8.0	Block porous solid, outward appearance is good	Qualified, meet standards of pharmacopoeia	Substantially do not detect activity	<3％
8.5	Block porous solid, outward appearance is good	Qualified, meet standards of pharmacopoeia	Substantially do not detect activity	<3％

By table two as seen, boric acid is buffer, all more satisfactory after outward appearance and the dissolving, but activity is zero substantially, measuring dissolving back albumen pH value finds to have reduced to about 4.0, survey boric acid solution, mannitol solution, protein solution respectively, and the semi-finished product pH value that is mixed with, discovery semi-finished product pH value has also reduced to 4.0, and boric acid solution, mannitol solution, protein solution pH value no change, boric acid solution is mixed the back find that pH reduces to 4.0 with mannitol, as seen the two reacts and causes the pH reduction and then make enzyme deactivation.Thereby abandon using boric acid to make buffer.

Buffer system three

Select higher pH value buffer, Tris-HCL is optionally arranged, Gly-Na0H be Tris-HCL because purge process selects for use, thereby the finished product lyophilizing selects for use similar buffer comparatively suitable.Select pH7.5,8.0,8.5,9.0, final concentration 50mM, 5% mannitol, result of the test sees Table three.

Table three 50mM Tris-HCL buffer, 5% mannitol, UOX (final concentration 1.8mg/ml)

PH value	Outward appearance	Clarity	Biological activity	Moisture
					7.5	Hollow out, not full	Muddy	71％	<3％
8.0	Block porous solid raps no chip	Muddy	73％	<3％
					8.5	Atrophy, no solid shape	Indissoluble	52％	<3％
9.0	Block porous solid raps no chip	Dissolving back lamp inspection has opalescence	71％	<3％

As seen pH8.0,9.0 o'clock outward appearances are good, but clarity does not all reach requirement, and active descend more obvious.Final definite buffer system is pH9.0.

Protectant selection in the preparation prescription

For protecting activity, making dissolving back clarity reach requirement, add glycine, glycine is a hydrophobic amino acid, helps the hydrophobic proteins dissolving, improves clarity.

Table four. press table two pH9.0 prescription and add glycine

	Active U/ bottle (average)	Activity preservation rate
			Before the lyophilizing	15.86
After adding the lyophilizing of 50mM glycine	15.37	97％
			After not adding the glycine lyophilizing	11.25	71％

By table four as seen, glycine has protective effect to the UOX activity in freeze-drying process, but after adding glycine, dissolving back clarity does not still reach standard.

For protection UOX protein molecular, prevent its polymerization or oxidation in freeze-drying process, need to add another kind of protective agent, select to add SDS, Arg, L-Cys, Glu.Wherein SDS has forbidden being used for intravenous fluid, and this test is research in contrast only, and Arg is a basic amino acid, and Glu is an acidic amino acid, can be used to regulate pH value, and L-Cys has antioxidation, can prevent the disulfide bond mispairing.Result of the test sees Table five.

Table five. to 1.8mgUOX, 50mM Tris-gly, lyophilizing situation after the 5% mannitol adding protective agent

Do not improve clarity problem after the preparation dissolving by the adding of visible arginine of table five and glutamic acid, opalescence is still arranged; SDS can make active obviously decline, and this may make the loose degeneration of UOX chain relevant with SDS; Along with the cysteine addition raises, the active rising, dissolubility improves.

Be to determine the addition of cysteine, add 5,10,20 respectively, 40mM L-Cys, detect after the lyophilizing, the results are shown in Table six;

Table six. the cysteine addition is preferred

Sequence number	1	2	3	4
					L—Cys(mM)	5	10	20	40
Active (U/ml)	15.5	26.1	26.22	27.5
					Outward appearance	Well	Well	Well	Well
Clarity	Qualified	Qualified	Qualified	Qualified

Found that adding 10,20,40mM L-Cys outward appearance and clarity are all qualified, along with the L-Cys amount strengthens, active and no significant difference, thereby the final concentration of cysteine is 10mM in the selection preparation finished product.

Final every the component determining of pharmaceutical formulation prescription of the present invention is formed:

Reorganization candida urate oxidase: 1-3.6mg/ml

Buffer system: Tris-Gly 10-80mM/l

pH 7.5-10

Excipient: mannitol 1-7%

Protective agent: L-Cys 5-40mM/l

Pharmaceutical formulation of the present invention is a kind of stable liquid or freeze-dried composition.In the composition component, protective agent is selected cysteine for use, and buffer system is selected Tris-Gly for use, is used to regulate pH value.

Pharmaceutical formulation of the present invention highly preferred every the component of writing out a prescription is formed and is:

Reorganization candida urate oxidase: 1.8mg/ml

Buffer system: Tris-Gly 50mM/L

pH 9

Excipient: mannitol 5%

Protective agent: L-Cys 10mM/L

Comparative test result can be found: Tris-HCL makes buffer system; add glycine and make protective agent; clarity descends after adding the lyophilizing that cysteine can prevent that the albumen polymerization from causing; the cysteine addition selects 10mM to get final product; add and do not play more positive effect in a large number; the mannitol final concentration selects 5% for use, and this prescription biological activity is stable, and clarity, moisture reach requirement.

The present invention obtains drug solution by each component being dissolved in water for injection or lyophilized products being dissolved in water for injection, and the drug solution that contains candida urate oxidase obtains stable lyophilized formulations by lyophilizing.Said composition is aseptic, can enter in human body or the animal body by subcutaneous, vein or intramuscular injection to play a role.

Owing to adopted technique scheme, the invention provides a kind of method that is different from prior art, screen a kind of stable pharmaceutical formulation that contains candida urate oxidase, not only can reach the requirement of clinical use, and can make extensive patients benefit from it, reduce the medical expense spending.

The specific embodiment

Come the pharmaceutical formulation of candida urate oxidase of the present invention done further specifying by following examples, but be not limited only to following example.

The preparation of the candida urate oxidase solution that embodiment 1 is stable

Stock solution preparation: according to the reorganization candida urate oxidase of Chinese patent 031091504 method preparation, replace following buffer through ultrafiltration: glycine 100mM/L, Tris-HCl (pH9.0/100mM/L), L-Cys (20mM/L), being prepared into reorganization candida urate oxidase concentration is the stock solution of 3.6mg/ml;

Dosing: get this stock solution 500ml, add 25% mannitol 200ml, add water for injection and be settled to 1000ml;

Filtration sterilization: adopt 0.22 micron membrane filtration;

Packing: adopt cillin bottle to be distributed into the preparation of every 1ml;

Lyophilizing: lyophilization, make final preparation.

Claims (9)

1, a kind of pharmaceutical formulation that contains candida urate oxidase is characterized in that said preparation comprises active component candida urate oxidase, excipient, protective agent and buffering system.

2, pharmaceutical formulation according to claim 1, excipient is selected the mannitol of 1-7% for use in the said composition.

3, pharmaceutical formulation according to claim 1, protective agent is selected the L-cysteine of 5-40mM/L for use in the said composition.

4, pharmaceutical formulation according to claim 1, buffer system selection pH value is that 7.5-9, concentration are the Tris-Gly of 10-50mM/L in the said composition.

5, pharmaceutical formulation according to claim 1, wherein said active component candida urate oxidase is the gene recombinaton thing.

6, pharmaceutical formulation according to claim 1 is a kind of fluid composition.

7, pharmaceutical formulation according to claim 1 is a kind of freeze-dried composition.

8, according to each described pharmaceutical formulation in the claim 1 to 7, said composition component composition is:

Reorganization candida urate oxidase: 1-3.6mg/ml;

Tris-Gly：pH 7.5-10，10-80mM/L；

Mannitol: 1-7%;

L-cysteine: 5-40mM/L.

9, according to each described pharmaceutical formulation in the claim 1 to 8, said composition component composition is:

Reorganization candida urate oxidase: 1.8mg/ml;

Tris-Gly：pH 9， 50mM/L；

Mannitol: 5%;

L-cysteine: 10mM/L.