CN108403716B

CN108403716B - Preparation method of superparamagnetic iron oxide

Info

Publication number: CN108403716B
Application number: CN201810536325.XA
Authority: CN
Inventors: 袁建栋; 张强; 李荣山
Original assignee: Brightgene Bio Medical Technology Co Ltd
Current assignee: Brightgene Bio Medical Technology Co Ltd
Priority date: 2018-06-25
Filing date: 2018-06-25
Publication date: 2021-11-05
Anticipated expiration: 2038-06-25
Also published as: CN108403716A

Abstract

The invention discloses a preparation method of superparamagnetic iron oxide, which comprises the following steps: (1) reducing dextran 10 and carboxymethylating to obtain polydextrose sorbitol carboxymethyl ether reaction liquid; (2) regulating the pH of the polydextrose sorbitol carboxymethyl ether reaction liquid to be neutral; (3) mixing polydextrose sorbitol carboxymethyl ether with a ferric chloride solution and a ferrous chloride solution, injecting air under an alkaline condition, and performing an oxidation reaction to obtain superparamagnetic ferric oxide; (4) ultra-filtration of superparamagnetic iron oxide. Compared with the prior art, the method omits the steps of purifying and solidifying the polydextrose sorbitol carboxymethyl ether reaction liquid, shortens the production steps, saves the production time and greatly reduces the production cost under the condition of keeping the quality index of the superparamagnetic iron oxide unchanged.

Description

Preparation method of superparamagnetic iron oxide

Technical Field

The invention belongs to the field of medicinal chemistry, relates to preparation of bulk drugs of a vein iron preparation, and particularly relates to a method for preparing superparamagnetic iron oxide by taking dextran 10 as a starting material.

Background

Superparamagnetic iron oxide (Ferumoxytol) is a nanoparticle formed by poly glucose sorbitol carboxymethyl ether coated iron oxide, and an injection of the drug is approved by FDA to be marketed in 2009 and is used for treating iron deficiency anemia in adult patients with Chronic Kidney Disease (CKD).

Iron Deficiency Anemia (IDA) is an anemia that occurs when the storage of iron in the body fails to meet the needs of normal erythropoiesis. This is due to insufficient iron intake, decreased absorption, increased demand, impaired iron utilization or excessive loss. The morphology is manifested as microcytic hypopigmented anemia. Iron deficiency anemia is not a disease, but is a symptom of a disease, which is associated with the degree of anemia and the urgency of onset.

Iron deficiency and anemia are common complications of many serious diseases, including chronic kidney disease, chronic heart failure, anemia from tumor chemotherapy, inflammatory bowel disease, massive menstrual bleeding, and postpartum hemorrhage. Patients with chronic kidney disease, women of childbearing age, pregnant women and children in development stage are high risk groups of iron deficiency anemia.

Iron deficiency anemia can severely reduce the quality of life of patients, increase the risk of hospitalization and even death, and also increase the medical burden of patients. The data show that the medical costs for patients with chronic iron deficiency anemia can be increased by 30-40%. The use of effective treatment regimens is therefore an important aspect of patient blood management.

IDA is mainly treated by oral iron preparation, parenteral preparation (i.e. intravenous iron preparation), transfusion therapy, and diet adjustment and other treatments. The treatment of IDA is preferably oral iron, and intravenous iron can be used for patients who cannot tolerate oral iron and do not respond well to oral iron, and patients suffering from intestinal absorption diseases. The mainstream intravenous iron preparations in the American market are as follows: dextran iron injection (iron dextran); ② iron sucrose injection (iron sucrose); ③ ferric carboxymaltose injection (ferric carboxymaltase); sodium ferric gluconate complex; superparamagnetic iron oxide injection (ferumoxytol). The main intravenous iron preparations in Chinese market include dextran injection and ferric saccharate injection.

Ferumoxoytol is a colloidal iron-carbohydrate complex. The molecule is centered on ferric oxide and coated with a polydextrose-sorbitol-sodium carboxymethylcellulose shell to prevent bioactive ferrous iron from contacting plasma components before the drug reaches macrophages in the liver, spleen, and bone marrow. Iron ions are released from the complex within macrophages and either enter the intracellular iron storage pool (e.g., ferritin) or are transported by plasma transferrin to erythroid progenitor cells for hemoglobin synthesis. The size of the whole colloidal particle is 17-31 nanometers in diameter.

A clinical phase III study of 'iron deficiency anemia of CKD patients' shows that compared with an oral iron preparation, the two-time injection of Ferumoxyytol can significantly improve the amount of hemoglobin and has good tolerance.

Compared with oral iron preparation and iron sucrose, the superparamagnetic iron oxide has better effect, because the iron loading amount of the superparamagnetic iron oxide is higher than that of iron sucrose, and the superparamagnetic iron oxide is convenient to use, only needs to be injected twice, improves the compliance of patients, reduces medical operation, and reduces part of cost.

Research shows that the smaller the molecular weight and the iron oxide core, the more unstable the iron preparation, the faster the active iron is released, the more adverse reactions caused by the active iron are, and the more easily the active iron is eliminated by the organism, and the clinical application interval time and the single dosage can only be smaller. While iron preparations with higher molecular weight and larger iron oxide core particles mean greater safety and convenience. The molecular weight of the superparamagnetic iron oxide reaches 750kD, and the superparamagnetic iron oxide has higher safety.

The polydextrose sorbitol carboxymethyl ether is a key starting material of superparamagnetic iron oxide, and is prepared from dextran 10 through several steps of sodium borohydride reduction, carboxymethylation, purification, solidification and the like.

The conventional polydextrose sorbitol carboxymethyl ether purification and solidification mainly adopts the following two modes:

refining polydextrose sorbitol carboxymethyl ether with ethanol for 3 times, removing impurities in reaction liquid, solidifying with a large amount of ethanol, and finally drying under reduced pressure. The method needs a large amount of ethanol, has long drying time and high production cost, and the ethanol cost in the purification and refining process accounts for about 30 percent of the total cost, thereby being not beneficial to industrialization.

② the polydextrose sorbitol carboxymethyl ether is ultrafiltered by 3000Da molecular weight ultrafiltration membrane, purified by membrane separation means, and dried into solid by spray drying mode, the cost of said method is lower than that of first method, but it needs to introduce membrane purification equipment and spray drying equipment, and its operation is complex, and is not favourable for industrialization.

The inventor finds that the purification steps of the superparamagnetic iron oxide starting material polydextrose sorbitol carboxymethyl ether are complicated, and the industrialization cost is increased.

Disclosure of Invention

In order to reduce the production cost and shorten the production period, the invention takes the polydextrose sorbitol carboxymethyl ether reaction liquid as a material to prepare the superparamagnetic iron oxide, and omits complicated steps such as purification, solidification and the like.

The preparation method of the superparamagnetic iron oxide comprises the following steps:

(1) reducing dextran 10 and carboxymethylating to obtain polydextrose sorbitol carboxymethyl ether reaction liquid;

(2) regulating the pH of the polydextrose sorbitol carboxymethyl ether reaction liquid to be neutral;

(3) preparing a superparamagnetic iron oxide bulk drug;

(4) purifying the superparamagnetic iron oxide material.

Specifically, in the step (1), dextran 10 and sodium borohydride are subjected to reduction reaction and then subjected to carboxymethylation with bromoacetic acid to obtain a polydextrose sorbitol carboxylmethyl ether reaction solution.

And (2) regulating the pH of the polydextrose sorbitol carboxymethyl ether reaction liquid to be neutral by using hydrochloric acid, wherein the pH can be 6.2-7.0.

And (3) mixing the polydextrose sorbitol and carboxymethyl ether reaction liquid obtained in the step (2) with a ferric trichloride solution, filtering to remove part of insoluble impurities, mixing with a ferrous chloride solution, injecting air under an alkaline condition, performing an oxidation reaction, and finally performing ultrafiltration filtration.

Compared with the prior art, the method omits the steps of purifying and curing the polydextrose sorbitol carboxymethyl ether reaction liquid, namely the step of preparing the superparamagnetic iron oxide does not comprise the step of purifying and curing the polydextrose sorbitol carboxymethyl ether reaction liquid.

The omitted purification and solidification steps are as follows: adding ethanol into the polydextrose sorbitol carboxymethyl ether reaction liquid with the pH value adjusted to be neutral, stirring, standing, dissolving the sticky substance in water, adding sodium chloride, adding ethanol again, stirring, standing, dissolving the sticky substance in water again, adding ethanol for the third time, stirring, crystallizing, filtering, and drying under reduced pressure to obtain a polydextrose sorbitol carboxymethyl ether pure product.

A large amount of solvent is used in the whole operation of purification and solidification, and if 384L of ethanol is needed for producing 12kg of polydextrose sorbitol methyl ether, the material drying time is 48 hours. In the whole process of purifying and curing the polydextrose sorbitol carboxymethyl ether, the material loss is 20 percent, the operation is complicated, the production time is long, and the ethanol cost in the purification and refining process accounts for about 30 percent of the total cost.

By the method, the production steps are shortened, the production time is saved and the production cost is greatly reduced under the condition of keeping the quality index of the superparamagnetic iron oxide unchanged.

Superparamagnetic iron oxide is a high molecular nano-drug, and mainly judges whether products are consistent from the aspects of molecular weight and particle size. In addition, because the purified polydextrose sorbitol carboxymethyl ether is mainly used for removing a large amount of inorganic salts (mainly sodium chloride), chloride ions in the concentrated solution of the polydextrose sorbitol carboxymethyl ether are monitored; the inventor also monitors the content of ammonium ions by using a large amount of ammonia water in the reaction liquid. To ensure consistent impurity removal levels. Wherein, the content of chlorine is less than 50ppm and the content of ammonia ion is less than 1000ppm in the quality standard.

Drawings

FIG. 1: example 1 liquid phase diagram

FIG. 2: example 2 liquid phase diagram

The superparamagnetic iron oxide products obtained by the methods illustrated in example 1 and example 2 are of consistent quality.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention will be described in detail below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are provided for the purpose of making the disclosure more complete and complete. The reagents and starting materials used were all commercially available except for the preparation provided. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.

Example 1 preparation of superparamagnetic iron oxides according to the prior art

Preparation of Polydextrose Sorbitol Carboxymethylether (PSC):

15kg of dextran 10 is stirred and dissolved in 30L of purified water; respectively adding 312g of 50% sodium hydroxide solution and 246g of sodium borohydride, and stirring and reacting for 4 hours at room temperature after the addition is finished; respectively adding 12kg of 50% sodium hydroxide and 3.9kg of bromoacetic acid, reacting at room temperature for 10 hours, and adjusting the pH value of the system to 6.2 by using 6N hydrochloric acid; adding 72L of ethanol into the reaction solution after the pH is adjusted under stirring, stirring for 10min, and standing for 10 min; the supernatant was decanted off; dissolving the viscous substance in 24L water, adding 120g sodium chloride, stirring for dissolving, adding 24L ethanol, stirring for 10min, standing for 10min, and pouring off supernatant (washing for 3 times); dissolving the viscous substance in 24L of water, slowly adding 240L of absolute ethyl alcohol, stirring for crystallization for 1 hour, filtering, and drying the sample in a reduced pressure drying oven at 60 ℃ for 48 hours to obtain 12kg of the pure product of the polyglucose sorbitol carboxymethyl ether.

Preparation of superparamagnetic iron oxide:

dissolving 12kg of PSC in 132kg of purified water; dissolving 5.15kg of ferric chloride hexahydrate in 51.5kg of purified water; pouring the prepared ferric trichloride solution into the PSC solution while stirring, filtering the PSC solution into a 300L reaction kettle through a filter element, introducing nitrogen to exhaust air, and cooling the PSC solution to 1-6 ℃ in an ice bath; dissolving 2.58kg of ferrous chloride and tetrasodium in 9kg of purified water; adding into the above mixed solution; under the condition of stirring, dropwise adding an ammonia water solution, heating to 78-83 ℃, and carrying out heat preservation reaction for 1 hour; replacing nitrogen with an air injection method, and heating the mixture at 78-83 ℃ for reaction for 4h for full oxidation. Cooling, transferring to an ultrafiltration device equipped with a filter element with molecular weight cut-off of 10 ten thousand daltons, adding purified water to one time volume, and performing ultrafiltration on the suspension for 8 times in total; the concentrated solution is used for testing iron content, molecular weight, particle size, chloride ions and ammonia ions.

Example 2 preparation of superparamagnetic iron oxides by the process of the invention

Preparing a polydextrose sorbitol carboxymethyl ether reaction solution:

15kg of dextran 10 is stirred and dissolved in 30L of purified water; respectively adding 312g of 50% sodium hydroxide solution and 246g of sodium borohydride, and stirring and reacting for 4 hours at room temperature after the addition is finished; respectively adding 12kg of 50% sodium hydroxide and 3.9kg of bromoacetic acid, reacting at room temperature for 10 hours, and adjusting the pH value of the system to 6.2 by using 6N hydrochloric acid;

preparation of superparamagnetic iron oxide:

adding purified water into the solution to 165kg, dissolving 6.4kg of ferric trichloride hexahydrate in 64kg of purified water, pouring the prepared ferric trichloride solution into a PSC solution under stirring, filtering the solution into a 300L reaction kettle through a filter element, introducing nitrogen to discharge air, and cooling the solution to 1-6 ℃ in an ice bath; dissolving 3.2kg of ferrous chloride and tetrasodium in 9kg of purified water; adding into the above mixed solution; under the condition of stirring, dropwise adding an ammonia water solution, heating to 78-83 ℃, and carrying out heat preservation reaction for 1 hour; replacing nitrogen with an air injection method, and heating the mixture at 78-83 ℃ for reaction for 4h for full oxidation. Cooling, transferring to an ultrafiltration device equipped with a filter element with molecular weight cut-off of 10 ten thousand daltons, adding purified water to one time volume, and performing ultrafiltration on the suspension for 8 times in total; the concentrated solution is used for testing iron content, molecular weight, particle size, chloride ions and ammonia ions.

Example 3 quality index of superparamagnetic iron oxides prepared in example 1 and example 2 was examined

1. The particle size detection method comprises the following steps:

the checking method comprises the following steps: determination of particle size and particle size distribution (third method of 0982 general rules of the Chinese pharmacopoeia 2015 edition)

The method is numbered:

the operation process is as follows:

(1) instrument and reagent

The instrument comprises the following steps: nano ZS90 of Malvern laser scattering particle size instrument

Reagent: ultrapure water

(2) Parameters of the instrument

Dispersing agent: water (W)

Refractive index of the dispersant: 1.330

Viscosity (cP): 0.8872

Fe2O3 refractive index: 3.01

Fe2O3 absorption: 0.010

Measurement mode: automatic

(3) Preparation of Standard solutions

(3-1)60nm Standard substance solution

Adding 0.22 mu m filtered water into the test tube, adding 10-12 drops of 60nm standard substance and 1-4 drops of 4mM sodium chloride, uniformly mixing, and carrying out ultrasonic treatment for 10-15 seconds to suspend.

(4) Test solution

Taking about 40 mu l of the product, placing the product in a test tube, adding 4ml of 0.22 mu m filtered water, mixing, and carrying out ultrasonic treatment for 10-15 seconds to obtain a test solution.

(5) Procedure of the test

(5-1) Instrument calibration

And (3) injecting a 60nm standard substance solution into the sample cell, placing the sample cell into an instrument detection window, setting instrument parameters, and testing the particle size of the standard substance, wherein the result is required to be +/-15% of the standard particle size.

(5-2) measurement of test article

And injecting a sample solution into the sample cell, placing the sample cell into a detection window of the instrument, setting instrument parameters, and testing the particle size of the sample.

2. The molecular weight detection method comprises the following steps:

the checking method comprises the following steps: molecular exclusion chromatography (Chinese pharmacopoeia 2015 year edition general rule 0514)

The method is numbered:

the operation process is as follows:

(1) instrument, reagent and reference substance

The instrument comprises the following steps: high performance liquid chromatograph, differential refraction detector and electronic analytical balance

Reagent: ultrapure water, sodium sulfate (analytical grade or higher), sodium azide (analytical grade or higher) controls: amylopectin reference substance (P-5, P-10, P-20, P-50, P-100, P-200, P-400, P-800) (2) chromatographic conditions

A chromatographic column: two chromatographic columns of Waters ultrahydrogel 250(7.8 mm. times.30 cm) and ultrahydrogel 1000(7.8 mm. times.30 cm) are connected in series, and the exclusion limit is large before.

Protection of the column: waters Ultrahydrogel (6X 40mm)

A detector: differential refractive detector

Mobile phase: 0.71% sodium sulfate solution (containing 0.02% sodium azide)

Column temperature: 35 deg.C

Temperature of the differential detection cell: 35 deg.C

Flow rate: 0.5ml/min

Sample introduction amount: 20 μ l

Diluting liquid: same mobile phase

Operating time: 60 minutes

(3) Solution preparation

(3-1) flow phase formulation

Weighing 7.1g of sodium sulfate and 0.2g of sodium azide, adding 1000ml of water for dissolving, and uniformly mixing to obtain the sodium azide-sodium sulfate-sodium azide.

(3-2) blank solution: i.e. the mobile phase.

(3-3) control solution

Taking 10mg of amylopectin reference substances (P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-800) respectively, placing in a 1ml measuring flask, adding appropriate amount of mobile phase, standing overnight at 4 deg.C for dissolving, fixing volume to scale with mobile phase, and shaking slowly to obtain reference solution.

(3-4) test article solution

Weighing 1ml of the product, placing in a 10ml measuring flask, adding 8ml of mobile phase, standing overnight at 4 deg.C for dissolving, fixing volume to scale with mobile phase, and slowly shaking to obtain sample solution.

(4) Sample introduction procedure

Operating the instrument under the selected analysis method, taking blank solution, reference solution and sample solution to be tested after the chromatographic system is stable, and recording the chromatogram.

(5) Computing

Standard curve: and (3) preparing a linear regression equation of a cubic standard curve by using the logarithmic value of the molecular weight (Mp) of the standard substance and the corresponding retention time (tR), wherein the correlation coefficient is not lower than 0.99. The weight average molecular weight and molecular weight distribution of the test article were calculated by GPC software.

3. Quality comparison:

	molecular weight	Particle size	Chlorine content	Ammonium ion
					Example 1	19W	35.2nm	20ppm	423ppm
Example 2	19W	35.7nm	25ppm	451ppm

The experimental data show that: the molecular weight and the particle size of the superparamagnetic iron oxide sample prepared in the purification and solidification step are reduced to be consistent with those of the superparamagnetic iron oxide sample prepared after the PSC is purified and solidified, and because 10 ten thousand molecular weight ultrafiltration membranes are used for purification in the superparamagnetic iron oxide purification process, impurities in the PSC reaction solution can be removed at the stage.

Example 4 comparison of yields between example 1 and example 2

	Iron content	Weight of ultrafiltration concentrate	Effective iron
				Example one	45.6mg/g	28.95kg	1.32kg
Example two	50mg/g	32kg	1.60kg

It can be seen from the above table that the same dextran 10 dosage, the second example, gives 20% more superparamagnetic iron oxide concentrate than the first example.

Claims

1. A preparation method of superparamagnetic iron oxide comprises the following steps:

(1) stirring dextran 10 and dissolving in purified water; respectively adding 50% sodium hydroxide solution and sodium borohydride, and stirring at room temperature to react after the addition; respectively adding 50% sodium hydroxide and bromoacetic acid, and reacting at room temperature;

(2) after the reaction in the step (1) is finished, adjusting the pH of the reaction solution to 6.2 by using hydrochloric acid;

(3) adding purified water into the reaction liquid obtained in the step (2), pouring a ferric chloride solution into the solution while stirring, filtering the solution into a reaction kettle through a filter element, introducing nitrogen to discharge air, and cooling the solution to 1-6 ℃ in an ice bath; adding a ferrous chloride solution into the mixed solution; under the condition of stirring, dropwise adding an ammonia water solution, heating to 78-83 ℃, and carrying out heat preservation reaction for 1 hour; replacing nitrogen by using an air injection method, heating and reacting the mixture at 78-83 ℃ for 4 hours, fully oxidizing, cooling, transferring the mixture into an ultrafiltration device provided with a filter element with a molecular weight cut-off of 10 ten thousand daltons, adding purified water to one time of volume, and performing ultrafiltration on the suspension.