CN108743616B

CN108743616B - Method for removing superparamagnetic iron oxide endotoxin

Info

Publication number: CN108743616B
Application number: CN201810536323.0A
Authority: CN
Inventors: 袁建栋; 张强; 李荣山
Original assignee: Borui Pharmaceutical Suzhou Co Ltd; Borui Biomedical Suzhou Co ltd
Current assignee: Borui Pharmaceutical Suzhou Co Ltd; Borui Biomedical Suzhou Co ltd
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2020-12-25
Anticipated expiration: 2038-05-30
Also published as: CN108743616A

Abstract

The invention provides a method for simply, conveniently and effectively removing endotoxin in a superparamagnetic iron oxide bulk drug. The method removes endotoxin by using a mixed solution of strong alkali and sodium hypochlorite with a certain concentration.

Description

Method for removing superparamagnetic iron oxide endotoxin

Technical Field

The invention belongs to the field of medicinal chemistry, relates to preparation of a crude drug of a venous iron preparation, and particularly relates to a method for removing endotoxin in a superparamagnetic iron oxide crude drug.

Background

Superparamagnetic iron oxide (Ferumoxyytol) is a nanoparticle formed by polydextrose sorbitol carboxymethylether coated superparamagnetic 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.

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.

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.

Endotoxin is a component of the cell wall of gram-negative bacteria called lipopolysaccharide. Lipopolysaccharides are toxic to the host. Common methods for removing endotoxin include high-concentration acid-base removal, ultrafiltration membrane and charged microporous membrane methods, asbestos and activated carbon adsorption, chemical degradation, ion exchange chromatography, and affinity chromatography.

However, in the case of drugs, endotoxin control is mainly carried out by environmental control or physical adsorption using activated carbon because of their physiological activity.

The superparamagnetic iron oxide bulk drug contains a large amount of free sugar, the pH is neutral, the superparamagnetic iron oxide bulk drug is very suitable for the growth of microorganisms, endotoxin is extremely difficult to control, the requirements on production environment are strict, and the production cost is high. The endotoxin condition is controlled to be harsh through the environment, and the failure rate is high; if activated carbon is selected to remove endotoxin, the activated carbon is worried about whether the activated carbon can be completely removed, and the activated carbon is not recommended to remove the endotoxin in the injection at home and abroad at present. At present, no method for effectively and simply removing endotoxin in a superparamagnetic iron oxide bulk drug is reported in the prior art.

Disclosure of Invention

Because endotoxin in the bulk drug of superparamagnetic iron oxide is difficult to remove by an environmental control method or an activated carbon physical adsorption method, technicians in the invention need to explore other methods.

The technical personnel of the invention find that when strong base such as sodium hydroxide, potassium hydroxide, lithium hydroxide and the like is used simply to remove endotoxin in the superparamagnetic iron oxide raw material drug, and the concentration of the strong base is more than 5%, although the endotoxin content can be reduced to 6.25-12.5Eu/ml, the quality standard is met, the particle size of the raw material drug is more than 51nm, and the superparamagnetic iron oxide nanoparticles are damaged and do not meet the quality standard. The concentration of the strong base is reduced to 3 percent, and the endotoxin and the particle size do not meet the quality standard. When the concentration of the strong base is 1%, the nanoparticles are not damaged, but the endotoxin does not meet the quality standard. The endotoxin is removed by using the strong base alone, so that the endotoxin content and the particle size of the nanoparticles are difficult to meet the quality standard. And (3) removing endotoxin in the superparamagnetic iron oxide bulk drug by using an oxidant sodium hypochlorite, wherein when a sodium hypochlorite solution with the concentration of 10% is used, the content of the endotoxin still does not meet the quality standard, and meanwhile, the nanoparticles are damaged.

The technical personnel of the invention unexpectedly discover that strong base and sodium hypochlorite are added into the concentrated solution of the raw material medicine simultaneously, so that the concentration of sodium hydroxide in the concentrated solution of the raw material medicine is 1 percent, and when the concentration of the sodium hypochlorite is 0.5 to 2.0 percent, the endotoxin level can be reduced to 3.0 to 6.25Eu/ml, and meanwhile, the raw material medicine nano-particles are not damaged and meet the quality standard.

The reaction time for removing endotoxin in the present invention is more than 0.5 hour, preferably 2 hours. The reaction temperature for removing endotoxin is 50-100 ℃, preferably 70-90 ℃ and most preferably 80 ℃.

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

Dissolving 100g of dextran 10(PSC) in 200ml of water, adding 2g of 50% sodium hydroxide solution, adding 1.6g of sodium borohydride, reacting for 4 hours at room temperature, adding 80.0g of 50% sodium hydroxide and 27.8g of bromoacetic acid at the temperature of not higher than 25 ℃, reacting for 16 hours at room temperature, adjusting the pH of the system to 6.2 by using 6M hydrochloric acid, adding 5000ml of ethanol to form white precipitate, removing supernatant, dissolving the residue in 240ml of water, adding 800mg of sodium chloride and adding 120ml of ethanol to form white precipitate, repeating the purification for 2 times, dissolving the residue in 120ml of water, adding 1L of ethanol to precipitate white solid, filtering, and drying for 24 hours at 50 ℃ to obtain the PSC.

PSC (40g) is dissolved in 850ml of water, ferric trichloride-hexahydrate (29.9g) and ferrous chloride-tetrahydrate (14.9g) are dissolved in 373ml of water, the mixture is filtered by a 0.2-micron filter membrane, the temperature is reduced to 10 ℃ in a reaction bottle, nitrogen is introduced for protection, 114ml and 28% ammonia water are added in the reaction bottle while stirring, the mixture is heated to 78 ℃ after the dropwise addition, the temperature is kept for 60min at 78 ℃, air is introduced for oxidation at 78 ℃, 1.5L of water is added for dilution after the oxidation is finished, the reaction solution is filtered by the 0.2-micron filter membrane, and the filtrate is subjected to ultrafiltration purification and concentration by a 10-ten-thousand molecular-weight ultrafiltration membrane to obtain the superparamagnetic iron oxide bulk drug.

Example 2

The crude drug obtained in example 1 was subjected to endotoxin measurement according to the following method:

bacterial endotoxin test (Chinese pharmacopoeia 2015 year edition general rule 1143)

The operation process is as follows:

(1) instrument and apparatus

Vortex mixer, precision pipettor, electric thermostat.

(2) Test device

A non-heat source sampling spoon, a non-heat source empty ampoule and a disposable non-heat source suction head.

(3) Reagent

0.125EU/ml limulus reagent, bacterial endotoxin working standard, and bacterial endotoxin test water

(4) Procedure of the test

Bacterial endotoxin detection was performed according to the general rule 1143 of Chinese pharmacopoeia 2015 edition, the method was as follows:

according to the interference test result, the sensitivity of the limulus reagent selected in the test is 0.125EU/ml, and the maximum effective dilution multiple of the test article is 100 times.

(4-1) reaction item settings

Name of item	Examination of test articles	Positive control for test article	Positive control	Negative control
					Solution numbering	A	B	C	D
Contents of solution	S₁₀₀	S₁₀₀E_0.25	E_0.25	W
					Number of parallel tubes	2	2	2	2

(4-2) preparation of respective reaction solutions

Solution C: and dissolving the bacterial endotoxin working standard product with water for bacterial endotoxin detection, uniformly mixing for 15 minutes on a vortex mixer, and then gradually diluting to prepare the bacterial endotoxin standard solution 2. Each dilution step should be mixed on a vortex mixer for 30 s.

Solution A: absorbing a proper amount of superparamagnetic iron oxide, adding BET water, and gradually diluting the test solution by 100 times. Each dilution step should be mixed on a vortex mixer for 30 s. Reference may be made to the following dilution steps:

solution B: 0.5ml of the S50 and E0.5 solutions are respectively sucked into a non-heat source empty ampoule bottle and mixed by swirling for 30S to obtain a test article positive solution S50E0.5. The schematic diagram is as follows:

(4-3) sample application:

taking 8 redissolved 0.1 ml/branch limulus reagent ampoules, wherein 0.1ml of solution A with the dilution concentration of S100 is added into 2 of the limulus reagent ampoules to serve as test tube bodies; adding 0.1ml of endotoxin working standard solution from 2 to 2 as a positive control tube; adding 0.1ml of water for bacterial endotoxin detection into 2 branches as negative control tubes; 0.1ml of S100E0.25-concentrated solution B was added to 2 of the test tubes as a positive control tube. After the sample adding is finished, sealing with a sealing film, gently mixing uniformly to avoid generating bubbles, putting the test tube rack and the test tube rack into a water bath with the temperature of 37 +/-1 ℃ or a suitable thermostat, keeping the test tube rack in a horizontal state, preserving the heat for 60 +/-2 minutes, and observing the result. The process of heat preservation and taking the test tube should avoid false negative results caused by vibration.

(4-4) judgment:

gently taking out the test tube from the thermostat, slowly turning over the test tube for 180 degrees, and recording as (+); negative results were obtained when no gel formed or when the formed gel was not firm, deformed and slipped off the vessel wall, and the record was (-) thereby.

If the parallel canals of the negative control solution D are all negative, the parallel canals of the positive control solution B of the test article are all positive, and the parallel canals of the positive control solution C are all positive, the test is effective. If both parallel channels of solution A are negative, the test article is judged to be in accordance with the specification. If both parallel channels of solution A are positive, the test article is judged not to be in accordance with the specification. If one of the two parallel channels of solution A is positive and the other is negative, a retest is performed. And (3) during retesting, making 4 parallel tubes for the solution A, judging that the test article is in accordance with the specification if all the parallel tubes are negative, and otherwise, judging that the test article is not in accordance with the specification.

The quality standard of the superparamagnetic iron oxide endotoxin is less than 12.5 Eu/ml.

Through determination, the endotoxin level of the bulk drug obtained in the example 1 is 50-100 Eu/ml.

The particle size is one of the key quality attributes of the nano-iron preparation, and whether the nano-particles are damaged or not can be observed through the change of the particle size.

The particle size of the drug substance obtained in example 1 was measured by the following method:

according to the particle size and particle size distribution determination method (the third method of 0982 on the basis of the 2015 pharmacopoeia of China pharmacopoeia, the Ministry of edition) (Malvern Mastersizer Nano ZS90 or a laser particle size analyzer with equivalent performance), taking a proper amount of the product, adding water to dissolve the product, preparing a solution containing 0.3mg of iron in each 1ml of the solution, carrying out ultrasonic treatment for 10-15 seconds, and carrying out examination according to the method, wherein the light intensity average particle size is 19-51 nm.

The particle size of the drug substance obtained in example 1 was determined to be 31.15 nm.

Example 3

Adding sodium hydroxide into the concentrated solution of the raw material medicine obtained in the example 1 until the overall concentration of the sodium hydroxide in the concentrated solution is 10%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling to room temperature, performing ultrafiltration to remove added strong base, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 6.25-12.5Eu/ml and the particle size to be 60.39 nm.

Example 4

Adding sodium hydroxide into the concentrated solution of the raw material medicine obtained in the example 1 until the concentration of the whole sodium hydroxide in the concentrated solution is 5%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling to room temperature, performing ultrafiltration to remove added strong base, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 6.25-12.5Eu/ml and the particle size to be 58.47 nm.

Example 5

Adding sodium hydroxide into the concentrated solution of the raw material medicine obtained in the example 1 until the overall concentration of the sodium hydroxide in the concentrated solution is 3%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling to room temperature, performing ultrafiltration to remove added strong base, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 12.5-25Eu/ml and the particle size to be 58.02 nm.

Example 6

Adding sodium hydroxide into the concentrated solution of the raw material medicine obtained in the example 1 until the overall concentration of the sodium hydroxide in the concentrated solution is 1%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling to room temperature, performing ultrafiltration to remove added strong base, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 12.5-25Eu/ml and the particle size to be 31.59 nm.

Example 7

Adding sodium hypochlorite to the concentrated solution of the raw material medicine obtained in the example 1 until the concentration of the whole sodium hypochlorite in the concentrated solution is 0.5%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling to room temperature, performing ultrafiltration to remove the added sodium hypochlorite, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 50-100Eu/ml and the particle size to be 32.01 nm.

Example 8

Adding sodium hypochlorite to the concentrated solution of the raw material medicine obtained in example 1 until the concentration of the whole sodium hypochlorite in the concentrated solution is 5%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling the solution to room temperature, removing the added sodium hypochlorite through ultrafiltration, adjusting the concentration, and then measuring endotoxin and particle size according to the method of example 2, wherein the endotoxin level is 25-50Eu/ml, and the particle size is 32.23 nm.

Example 9

Adding sodium hypochlorite into the concentrated solution of the raw material medicine obtained in the example 1 until the concentration of the whole sodium hypochlorite in the concentrated solution is 10%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling the solution to room temperature, removing the added sodium hypochlorite through ultrafiltration, adjusting the concentration, and then measuring endotoxin and particle size according to the method of the example 2, wherein the endotoxin level is 25-50Eu/ml, and the particle size is 60.64 nm.

Example 10

Adding sodium hydroxide and sodium hypochlorite into the concentrated solution of the raw material medicine obtained in the example 1 respectively until the overall concentration of the sodium hydroxide in the concentrated solution is 1% and the overall concentration of the sodium hypochlorite in the concentrated solution is 0.5%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling the solution to room temperature, performing ultrafiltration to remove the added sodium hydroxide and the added sodium hypochlorite, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 3.0-6.25Eu/ml and the particle size to be 31.80 nm.

Example 11

Adding sodium hydroxide and sodium hypochlorite into the concentrated solution of the raw material medicine obtained in the example 1 respectively until the overall concentration of the sodium hydroxide in the concentrated solution is 1% and the overall concentration of the sodium hypochlorite in the concentrated solution is 1.5%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling the solution to room temperature, performing ultrafiltration to remove the added sodium hydroxide and the added sodium hypochlorite, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 3.0-6.25Eu/ml and the particle size to be 39.27 nm.

Example 12

Adding sodium hydroxide and sodium hypochlorite into the concentrated solution of the raw material medicine obtained in the example 1 respectively until the overall concentration of the sodium hydroxide in the concentrated solution is 1% and the overall concentration of the sodium hypochlorite in the concentrated solution is 2.0%, heating the concentrated solution to 80 ℃ for reaction for 2 hours, naturally cooling the solution to room temperature, performing ultrafiltration to remove the added sodium hydroxide and the added sodium hypochlorite, adjusting the concentration, measuring endotoxin and particle size according to the method of the example 2, and measuring the endotoxin level to be 3.0-6.25Eu/ml and the particle size to be 35.78 nm.

Claims

1. A method for removing endotoxin from superparamagnetic iron oxide comprises adding strong alkali solution and sodium hypochlorite solution into superparamagnetic iron oxide raw material for reaction; the concentration of the strong alkali solution is 1 percent, and the concentration of the sodium hypochlorite is 0.5 to 2.0 percent; the strong base is sodium hydroxide, potassium hydroxide or lithium hydroxide; the reaction temperature is 50-100 ℃.

2. The process of claim 1, wherein the reaction temperature is from 70 ℃ to 90 ℃.

3. The process of claim 2, wherein the reaction temperature is 80 ℃.