CN85104452A - Make the method for water-soluble iron dextran - Google Patents

Abstract

Produce water miscible, has iron level and is 27 to 33 weight percentage and the average mol quality dextran component and be the method for 2000 to 4000 ferridextran, it is characterized in that, this aqueous solution in an aqueous solution, in α (1 → 6)-dextrorotation-Transglucosylase of per 1000 units, contain dextrorotation-grape glucose more than 200 millimoles, 265 to 310K and the pH value be 4.5 to 8 o'clock, add a kind of aqueous sucrose solution, its quantity is that the molecular ratio of sucrose and glucose is 2.0 to 5.0, after sugar consumption, isolate glucose, the fructose of emitting and undesirable oligose, the dextran of Jing Huaing transforms with new sedimentary ferric oxyhydroxide like this, and further purifies sometimes.

Description

Make the method for water-soluble iron dextran

The ferridextran preparation is used for vein and injection muscle in the animal medical treatment, the anemia of treatment iron deficiency.Said preparation is made by transforming with the colloidal ferric hydroxide as the complex compound of dextran.

Especially for the preparation of intravenous administration, very high requirement has been proposed for the stability of solution and the bioavailability of iron.Have the ferridextran of high Fe content, that is to say,, have 20% or every gram dry-matter of more iron, can not satisfy this requirement if when having used the dextran of too high molecular-weight average when making.Owing between different dextran molecules,, formed colloid and throw out then again by the formation of iron atom at the intramolecule complex compound.Therefore typically use the synthetic ferruginous dextran of the low-molecular-weight dextran of 4000-5000.The dextran of this molecular weight ranges is that the acidification hydrolization effect of the high molecular dextran by nature obtains, and as byproduct, for example when producing medical molecular-weight average and be the dextran of 4000-7500, produces.

But the molecular weight distribution of this by-product dextran is very wide, and generally reaches relevant glucose and approximately be no more than 50000 molecular weight.Therefore the general common practice is, under alkaline condition, dextran transforms with tervalent molysite and obtains thick ferridextran, and is settlement separate or pass through the film separation method by the many solvents of change expense, and the part of unwelcome higher molecular weight is removed.

If the waste material fraction that medical dextran is produced contains too much glucose and different-Fructus Hordei Germinatus-low its molecular weight of saccharides approximately is no more than 1000, this saccharides is before changing into ferridextran, equally also must remove,, can further decompose the deleterious product of generation because under conversion condition.

Because institute is spoken of, to wish to produce a kind of water-soluble ferridextran with high Fe content, this means that also the used glucose of people must be that molecular-weight average is 2000 to 4000, the dextran of narrow molecular weight distribution.

This task, obtained solution by method of the present invention, this method is characterized in that, in a kind of aqueous solution, per 1000 α of unit (1 → 6)-dextrorotation-Transglucosylases of this solution contain the dextrorotation-glucose more than 200 millimoles, 265 to 310K and the PH-value be 4.5 to 8 o'clock, add following quantity-aqueous sucrose solution, the mole ratio of sucrose in water ratio glucose is 2.0 to 5.0, after sugar consumption, separate fructose and unwelcome low saccharides that glucose is emitted, to have an average mol quality be 2000 to 4000 to the glucose of Jing Huaing like this, transform with the ferric hydroxide that newly is settled out, purify sometimes and further.

Success has made that a kind of water miscible to have iron level be that 27 to 33 weight percentage and the average mol quality dextran component are 2000 to 4000 ferridextran by this way.

Reaction mixture mainly is to remain on 290 to 300K and be in 5 to 6.5 in PH-value scope.These two indexs are influential to the structure that generates product.

According to the classification of " the enzyme council ", the dextrorotation-glucopyranosyl that makes sucrose is transferred to enzyme on the suitable acceptor, be called α (1 → 6)-dextrorotation-Transglucosylase.A kind of so extracellular enzyme is Sucrose:glucan alpha1 (E, C, 2,4,1,5), and this enzyme is the bacterial classification by certain Lactobacilleae, for example leuconostoc mesenteroides, particularly bacterial classification B-512, and leuconostoc dextranicum, suis and lactobacillus constitute.Produce natural dextran, at first use sucrose to make acceptor, and agent is worked as the chain skirt in chain polymerization, in the middle of this, shift dextrorotation-glucopyranosyl group by sucrose to the chain that increases by successive, formation has the polyose dextran of millions of molar masss, and simultaneously to the sucrose molecules of each conversion, produces a part fructose.

If added other list in this reaction, two or three-carbohydrate is as acceptor, will impair dextran and among a small circle in the generation oligose.When using glucose as acceptor, generate two of about 78% natural glucose and about 13%-and oligose be no more than IM-12.(Robyt and Eklund, carbohydrate research, 121, (1983) 279-286 page or leaf).The oligosaccharides of Sheng Chenging typically, along with the increase of the polymerization degree, its quantity then reduces gradually.

Such control of success is shifted the glucosyl group group to glucose by sucrose under the reaction conditions of invention, makes it not generate natural dextran, but constitute with high yield have 15 to 25 glucose anhydrides-unitary different-Fructus Hordei Germinatus-oligosaccharides.Beyond expectationly in the middle of this be, the different-Fructus Hordei Germinatus-oligosaccharides of generation, along with the increase of the polymerization degree, its quantity no longer is to reduce gradually, but relevant with the sucrose quantity that transforms, and mainly is to generate a kind of certain polymerization degree.

In the method for invention, for obtain desirable different-high yield of Fructus Hordei Germinatus-oligosaccharides, can adopt a kind of such speed successive to add the aqueous solution of sucrose, the enzyme of existing quantity can make the sucrose quantity of inflow transform immediately, and avoid the sucrose enrichment in reaction mixture, this enrichment can cause not controlling the high molecular dextran of generation.In any case the sucrose component of the sugar one butt material of reaction mixture in the equilibrium state that transforms continuously, should not surpass 25%.

Dextran-the sucrase that need not purify also can use the mixture of being made up of the bacterium of enzyme and production enzyme.

Reaction structure can be described below:

Glucose+n sucrose (Sucrose:glucan alpha1)/()

Different-Fructus Hordei Germinatus-(n+1)-carbohydrate+n fructose

Here n is the sucrose mole number of putting into, and its dextrorotation one glucopyranosyl group is used to constitute low molecular dextran, and emits respective number fructose simultaneously.

This reaction can be controlled like this according to invention, makes different-Fructus Hordei Germinatus-oligose or the polysaccharide of producing each desired molecular weight.Under the condition of temperature of being spoken of and hydrogen ion concentration, the molecular weight that in this building-up reactions, reaches, and with certain enzymic activity, the molal quantity of the relevant acceptor in existing solution and total sucrose and molar ratio of acceptor that adds have relation.

U(=unit of unit of enzyme activity) be the quantity of α (1 → 6)-dextrorotation-glucanotransferase, this quantity is at PH5, and 2 and 298K, per minute transforms the molar sucrose of 1 μ.

If more than the sucrose that the existing enzymic activity of adding can transform, control bulk of molecule so and just can not.

If with enzymic activity 1000 units as the basis, so the sucrose addition total be that per 1000 units of 1000 millimoles and glucose are 100 to 500 millimoles, 200 to 500 millimoles particularly can obtain the desirable mixture that a molecular-weight average is approximately 2000 to 4000 oligose that has.

Therefore also may, in several exploratory experiments, use conversion glucose molecule quantity in indicated scope, pre-determining α (1 → 6)-dextrorotation-glucosyl transferase activity (for example 1000 units) and a constant sucrose quantity (for example 1000 millimoles), an amount of interpolation sucrose, it is directly transformed by enzyme, dextrorotation-Glucopyranose the group of control sucrose makes it synthesize desirable the have different-Fructus Hordei Germinatus-oligose of narrow molecular weight distributions or the fraction of polyose with high yield as the addition reaction on the glucose of acceptor like this.

Can take out total essential amount of glucose, or keep remaining reaction conditions, particularly concentration ratio, the same an amount of replacement glucose of successive, as it as acceptor used up.Therefore, making building-up reactions also is possible fully continuously.

A unexpected advantage of the inventive method is, the sugar of reaction mixture-butt substances content can be very high, and it can be 30 to 50%, particularly 40 to 50%.

Though the enzymatic building-up reactions of invention is aseptic carrying out, as the common practice of for example synthesis of natural dextran, for fear of the female growth of unwelcome enzyme, can add anti-nuclear fission material (Mytosehemmer to reaction mixture, wheat head department hold back agent), as sulfuric acid, quantity is no more than 1000 milligrams/kilogram, particularly 400 to 600 milligrams/kilogram.

Mixture by reaction, by separating the glucose do not transform, the fructose of emitting and be less than the different-Fructus Hordei Germinatus-oligose of 6 anhydroglucoses-unit is produced the dextran of low average molecular weight 2000 to 4000, can be according to being known method originally, for example by carrying out with ethanol precipitation.

By with a chromatographic separation byproduct that is full of highly acid cationite acid, proved very suitable.

In order to produce iron-dextran, outstanding turbid with the water of different-Fructus Hordei Germinatus-oligosaccharide mixture heats until 373K with the new sedimentary ferric oxyhydroxide of washing then, is dissolved in the solvent up to ferric oxyhydroxide.Advise this conversion, carry out having citric acid or alkaline lemon salt to exist under the situation, as known by Deutsches Reichs-Patent specification sheets 1768912.

In order to remove negatively charged ion or positively charged ion, these ions also are contained in the solution of ferridextran after transforming, and this will stand cationite and anionite is handled.

Example 1

In the aqueous solution of 43 gram iron trichlorides-hexahydrated 250 milliliters, by splashing into 28 gram soda solutions, be dissolved in the 600ml water, ferric oxyhydroxide then is precipitated out.This trivalent ironic hydroxide filtering separation is come out, and use distilled water wash, the trivalent ironic hydroxide is poured in the liter stirred flask, and the oligosaccharide mixture of obtaining by chromatogram with 15 grams, the average mol material is 3000 gram/mol and 0.5 gram citric acid mixing.

After adding 9 milliliters 20% NaOH-solution, this mixture is stirred under the condition of 368-373K, up to the complete pass into solution of ferric oxyhydroxide.

Dark red solution with the ferridextran that generates at room temperature cools off, and utilizes the thorough desalination of ion-exchanger.

After sterile filtration, making solution concentration to a content by evaporation is 10% iron/milliliter, and the aseptic ampoule of packing into.With the dry-matter is benchmark, and iron level is 29.5%.

The ferridextran solution of obtaining according to the nineteen sixty-five code of Britain veterinarian, is checked relevant toxicity.

Test for this reason, used weight in average be 20 grams the NMI-mouse (raise: Winkelmann, Winkler is graceful, Paderborn, Bai Debao).

The dosage of test usefulness is 0.25 milliliter/animal, and after inject, substances causes that immediately slight motion lacks of proper care, but this imbalance is in 1 hour, completely dissolve gradually again.To for some time of observing after remaining, do not find symptom again, do not occur dead yet.

Example 2

At the enzyme of 16 liters, in the aqueous solution of Sucrose:glucan alpha1, it has an activity is 5400 units/liter, has dissolved 7.3 kilograms of crystalline glucose at 298K.The PH-value of solution is 5.4.Continuously 5.4 sucrose solution in this solution with the PH-value of 2.6 kilograms/hour 40% of pump inputs.After 48 hours, stop to add sucrose, and make afterwards 2 hours of enzyme, ℃ lose activity by reacting by heating mixture to 70.

A sample by reaction mixture, by gel chromatography single saccharides component and two saccharides component are separated, and according to Suo Maokeyi-phosphoric acid salt-method (carbohydrate chemical process, volume 1(1962), the 384-386 page or leaf), measure the mean value Mn of oligose fraction molecular weight.It is Mn=2540, and this and an average polymerization degree are that 15.7 glucose anhydride units adapt.The fructose component of the butt material of sugar is 45.0%, and the glucose component is 3.6%.This saccharides solution of 45 liters is added in the chromatogram tripping device, and it is highly acid that this device contains 400 liters, carries the cation exchange resin agent with sodium ion, and by adding 43 liters/hour distilled water, goes out each saccharides by elution in the pillar.

After singlings 60 liters, following in the 22 later liters, go out different-Fructus Hordei Germinatus-oligosaccharide mixture by the separator column elution, it is 3000 gram/mol that this mixture has an average mol quality.

Trial report

The piglet that one tire produces is divided into two comparative group, and per 4 piggys are one group.A group was injected 2 milliliters of ferridextrans at the 3rd day that gives birth to according to british patent specification 1200902 every pigs, 2 milliliters of ferridextrans of the present invention of every pig injection of another group.The variable color that cell tissue in the injection site occurs, after one day, all those have all disappeared with the piggy of ferridextran injection of the present invention, oppose that mutually as seen this variable color is also known with the piggy of commercial general ferridextran injection.To these piggys, the variable color of cell tissue, second talent after having an injection disappears.

This result proves, the iron of ferridextran of the present invention utilizes than the tissue that the iron of commercial general ferridextran is configured blood faster.

Claims (17)

1, produce water miscible, has iron level and is 27 to 33 weight percentage and the average mol quality dextran component and be the method for 2000 to 4000 iron dextran, it is characterized in that, in an aqueous solution, this solution, in α (1 → 6)-dextrorotation-glucosyl transferase of per 1000 units, contain dextrorotation-glucose more than 200 millimoles, 265 to 310K and the pH-value be 4.5 to 8 o'clock, adding-aqueous sucrose solution, its quantity is that the molecular ratio of sucrose and grape glucose is 2.0 to 5.0, after sugar consumption, isolate grape glucose, the fructose of emitting and undesirable oligose, the dextran of Jing Huaing transforms with new sedimentary ferric oxyhydroxide like this, and further purifies sometimes.

According to the method for claim 1, it is characterized in that 2, the aqueous solution of this glucose in the enzyme of per 1000 units, contains the glucose of 400 to 600 millimoles.

According to the method for one of claim 1 or 2, it is characterized in that 3, reaction is carried out 290 to 300K the time.

According to the method for one of claim 1 to 3, it is characterized in that 4, the pH-value of reaction mixture is 5 to 6.5.

According to the method for one of claim 1 to 4, it is characterized in that 5, the molecular ratio of sucrose in water ratio glucose is 3.0 to 4.0.

6, according to the method for one of claim 1 to 5, it is characterized in that, add sucrose solution continuously.

7, according to the method for one of claim 1 to 6, it is characterized in that, add sucrose solution, sucrose is directly transformed by α (1 → 6)-dextrorotation-Transglucosylase with a kind of like this speed.

According to the method for one of claim 1 to 7, it is characterized in that 8, the sugared butt substances content of reaction mixture is 30% to 50%, particularly 40% to 50%.

9, according to the method for one of claim 1 to 8, it is characterized in that, used the midgut bacterium by the bacterium leuconos toc, particularly the Sucrose:glucan alpha1 of bacterial classification B-512 discharge is as (1 → 6)-dextrorotation-Transglucosylase.

10, according to the method for one of claim 1 to 8, it is characterized in that, used the Sucrose:glucan alpha1 of discharging, as α (1 → 6)-dextrorotation-Transglucosylase by the dextran bacterium of bacterium leuconos toc.

11, according to the method for one of claim 1 to 10, it is characterized in that, like this an amount of continuous replacement dextrorotation-glucose, as it as acceptor consume.

12, according to the method for one of claim 1 to 11, it is characterized in that,, in the mixture of reaction, add anti-nuclear fission material (Mytose-hemmer, wheat head department hold back agent) for fear of undesirable yeast growth.

13, according to the method for claim 12, it is characterized in that, added sulfuric acid, its quantity is lower than 1000 milligrams/kilogram, is mainly 400 to 600 milligrams/kilogram.

14, according to the method for one of claim 1 to 13, it is characterized in that,,, isolate the average mol quality and be 2000 to 4000 dextran by precipitation or chromatogram step by step by the mixture of monose, disaccharide and oligose.

15, according to the method for one of claim 1 to 14, it is characterized in that, will be not less than 373K, be dissolved in the solution up to ferric oxyhydroxide by the aqueous suspension heating that different one Mai Ya-oligosaccharide mixture and sedimentary ferric oxyhydroxide constitute.

16, according to the method for claim 15, it is characterized in that existing under the situation at citric acid or alkaline Citrate trianion, carry out conversion reaction.

17, according to one of method of claim 15 or 16, it is characterized in that the solution of the ferridextran that obtains after the conversion is handled with anionite and cationite.