CN108675356A - The endotoxic minimizing technology of superparamagnetic iron oxide - Google Patents

Abstract

The invention provides a simple and effective method for removing endotoxin in superparamagnetic iron oxide raw materials. This method removes endotoxins by using a mixed solution of a certain concentration of strong alkali and sodium hypochlorite.

Description

Superparamagnetic Iron Oxide Endotoxin Removal Method

technical field

The invention belongs to the field of medicinal chemistry and relates to the preparation of raw materials of intravenous iron preparations, in particular to a method for removing endotoxin in superparamagnetic iron oxide raw materials.

Background technique

Superparamagnetic iron oxide (Ferumoxytol) is a nanoparticle formed by wrapping superparamagnetic iron oxide with polydextrose sorbitol carboxymethyl ether. The injection of the drug was approved by the FDA in 2009 for the treatment of adult patients with chronic kidney disease (CKD) of iron deficiency anemia.

Iron-deficiency anemia (IDA) is anemia in which iron stores in the body cannot meet the needs of normal erythropoiesis. It is caused by insufficient iron intake, decreased absorption, increased requirement, impaired iron utilization, or excessive iron loss. Morphology showed microcytic hypochromic anemia. Iron deficiency anemia is not a disease, but a symptom of the disease, and the symptoms are related to the degree of anemia and the priority of onset.

Iron deficiency and anemia are common complications of many serious diseases, including chronic kidney disease, chronic heart failure, anemia caused by cancer chemotherapy, inflammatory bowel disease, heavy menstrual bleeding and postpartum hemorrhage. Patients with chronic kidney disease, women of childbearing age, pregnant women, and developing children are high-risk groups for iron deficiency anemia.

Iron deficiency anemia will seriously reduce the quality of life of patients, increase the risk of hospitalization and even death, and also increase the medical burden of patients. Data show that the medical expenses of patients with chronic diseases combined with iron deficiency anemia will increase by 30-40%. Adopting an effective treatment plan is therefore an important part of patient blood management.

The treatment of IDA mainly includes oral iron, parenteral preparations (ie intravenous iron), blood transfusion therapy, dietary adjustment and other treatments. Oral iron is the first choice for the treatment of IDA. For patients who cannot tolerate oral iron, do not respond adequately to oral iron, and patients with intestinal absorption disorders, intravenous iron can be used. Mainstream intravenous iron preparations in the U.S. market include: ① iron dextran injection (iron dextran); ② iron sucrose injection (iron sucrose); ③ carboxymaltose iron injection (ferric carboxymaltose); gluconate complex); ⑤ superparamagnetic iron oxide injection (ferumoxytol). Mainstream intravenous iron preparations in the Chinese market include dextran injection and iron sucrose injection.

Ferumoxytol is a colloidal iron-carbohydrate complex. The molecule is centered on iron oxide and covered with polydextrose-sorbitol-carboxymethylcellulose sodium shell, which can prevent the biologically active ferrous iron and plasma components before the drug reaches the macrophages of the liver, spleen and bone marrow touch. Iron ions are released from the complex in macrophages and either enter intracellular iron storage pools (eg, ferritin) or are transported via plasma transferrin to erythroid progenitor cells for synthesis of hemoglobin.

Studies have shown 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, and the easier it is to be cleared by the body. The clinical application interval and single dosage can only smaller. Iron preparations with higher molecular weight and larger iron oxide core particles mean safer and more convenient. The molecular weight of superparamagnetic iron oxide reaches 750kD, which has high safety.

In a clinical phase III study of "iron deficiency anemia in CKD patients", compared with oral iron, two injections of Ferumoxytol can significantly increase the amount of hemoglobin, and it is well tolerated. Compared with oral iron and iron sucrose, superparamagnetic iron oxide is more effective, because its iron loading is higher than iron sucrose, and it is easy to use, only needs to be injected twice, which improves patient compliance and reduces medical operations , and reduced some costs.

Endotoxins are a component of the cell walls of Gram-negative bacteria called lipopolysaccharides. Lipopolysaccharides are toxic to the host. Common endotoxin removal methods include high-concentration acid-base removal method, ultrafiltration membrane and charged microporous membrane method, asbestos and activated carbon adsorption method, chemical degradation method, ion exchange chromatography, and affinity chromatography.

However, for pharmaceuticals, due to their physiological activity, the control of endotoxins is mainly through environmental control methods or physical adsorption with activated carbon.

The superparamagnetic iron oxide raw material contains a large amount of free sugar, and the pH is neutral, which is very suitable for the growth of microorganisms, and the endotoxin is extremely difficult to control, which has strict requirements on the production environment and high production costs. Controlling endotoxins through the environment is harsh and has a high failure rate; if you choose to use activated carbon to remove endotoxins, you are worried about whether activated carbon can completely remove endotoxins, and it is not recommended to use activated carbons to remove endotoxins in injections at home and abroad. At present, the prior art has not reported an effective and simple method for removing endotoxin from superparamagnetic iron oxide raw materials.

Contents of the invention

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

The invention provides a simple and effective method for removing endotoxin in superparamagnetic iron oxide raw materials. This method removes endotoxins by using a mixed solution of a certain concentration of strong alkali and sodium hypochlorite.

The technicians of the present invention have found that simply using a strong base, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, etc., to remove endotoxins in superparamagnetic iron oxide raw materials, when the strong base concentration is greater than 5%, although the endotoxin content can be reduced Reduced to 6.25-12.5Eu/ml, which meets the quality standard, but the particle size of the raw material drug is greater than 51nm, showing that the superparamagnetic iron oxide nanoparticles are destroyed and does not meet the quality standard. The strong base concentration was reduced to 3%, and neither the endotoxin nor the particle size met the quality standards. When the strong alkali concentration is 1%, although the nanoparticles are not destroyed, the endotoxin does not meet the quality standard. Using a strong base alone to remove endotoxins is difficult to make both the endotoxin content and the particle size of nanoparticles meet the quality standards. The oxidant sodium hypochlorite is used to remove the endotoxin in the superparamagnetic iron oxide raw material drug. When using a sodium hypochlorite solution with a concentration as high as 10%, the endotoxin content still does not meet the quality standard, and at the same time, the nanoparticles are destroyed.

The technicians of the present invention unexpectedly found that strong alkali and sodium hypochlorite were added to the concentrated liquid of raw material medicine at the same time, so that the concentration of sodium hydroxide in the concentrated liquid of raw material medicine was 1%, and when the concentration of sodium hypochlorite was 1%, the endotoxin level could be reduced to Below 3.0Eu/ml, at the same time, the raw material drug nanoparticles are not damaged, which meets the quality standard.

The reaction time of the present invention to remove endotoxin is greater than 0.5 hours, preferably 2 hours. The reaction temperature for removing endotoxin in the present invention is 50°C to 100°C, preferably 70°C to 90°C, most preferably 80°C.

specific embodiment

The present invention will be described in detail below. However, the invention may be embodied in many different forms, and it should not be limited to the embodiments described herein, which are provided so that this disclosure will be complete and comprehensive. All the reagents and raw materials used are commercially available except those provided for the preparation methods. 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

Dissolve 100g of dextran 10 (PSC) in 200ml of water, add 2g of 50% sodium hydroxide solution, add 1.6g of sodium borohydride, react at room temperature for 4 hours, add 80.0g of 50% sodium hydroxide at no higher than 25°C, Bromoacetic acid 27.8g, react at room temperature for 16 hours, adjust the pH of the system to 6.2 with 6M hydrochloric acid, add 5000ml of ethanol to form a white precipitate, remove the supernatant, dissolve the residue in 240ml of water, add 800mg of sodium chloride, and add 120ml of ethanol to form White precipitate, repeat the above purification twice, dissolve the residue in 120ml of water, add 1L of ethanol, a white solid precipitates, filter, and dry at 50°C for 24 hours to obtain PSC.

PSC (40g) was dissolved in 850ml of water, ferric chloride hexahydrate (29.9g) and ferrous chloride tetrahydrate (14.9g) were dissolved in 373ml of water, filtered with a 0.2um filter, mixed, and In the reaction bottle, cool down to 10°C, protect it with nitrogen, add 114ml of 28% ammonia water under stirring, after the dropwise addition, heat to 78°C, keep it warm for 60min at 78°C, and then heat it at 78°C Under certain conditions, air was introduced to oxidize. After the oxidation was completed, 1.5 L of water was added to dilute, and the reaction solution was filtered through a 0.2um filter membrane.

Example 2

The crude drug that embodiment 1 obtains carries out the mensuration of endotoxin according to the following method:

Bacterial endotoxin test method (General Rule 1143 of Chinese Pharmacopoeia 2015 Edition)

Operation process:

(1) Instruments and equipment

Vortex mixer, precision pipette, electric thermostat.

(2) Test equipment

Non-pyrogenic sampling spoon, non-pyrogenic empty ampoule, disposable non-pyrogenic tip.

(3) Test drug

>>＝0.125EU/ml Limulus Reagent, Bacterial Endotoxin Working Standard, Bacterial Endotoxin Test Water

(4) Test process

According to the "Chinese Pharmacopoeia" 2015 edition of the Chinese Pharmacopoeia General Rule 1143, the bacterial endotoxin detection method is as follows:

According to the results of the interference test, the sensitivity of the LAL reagent was selected in this test as 0.125 EU/ml, and the maximum effective dilution factor of the test product was 100 times.

(4-1) Reaction item setting

project name Test article inspection Test article positive control positive control negative control Solution number A B C D. Solution content S ₁₀₀ S ₁₀₀ E _0.25 E _0.25 W Number of parallel tubes 2 2 2 2

(4-2) Preparation of each reaction solution

Solution C: Bacterial endotoxin working standard is dissolved in bacterial endotoxin test water, mixed on a vortex mixer for 15 minutes, and then gradually diluted to prepare 2 "bacterial endotoxin standard solution. Each dilution step should be mixed on a vortex mixer for 30s.

Solution A: Take an appropriate amount of superparamagnetic iron oxide, then add BET water, and gradually dilute the test solution by 100 times. Each dilution step should be mixed on a vortex mixer for 30s. Refer to the following dilution steps:

Solution B: pipette 0.5ml of S50 and E0.5 solutions into the empty ampoule without pyrogen, vortex and mix for 30s to obtain the positive solution of the test product S50E0.5. The schematic diagram is as follows:

(4-3) Sample addition:

Take 8 reconstituted 0.1ml/branches of the original ampoules of LAL reagent, 2 of which are added with 0.1ml of solution A with a dilution concentration of S100 as the test tube; 2 of them are added with 0.1ml of 2>> endotoxin working standard solution As a positive control tube; 2 tubes were added with 0.1ml of water for bacterial endotoxin inspection as a negative control tube; 2 tubes were added with 0.1ml of solution B with a concentration of S100E0.25 as a positive control tube for the test product. After adding the sample, seal it with a parafilm, mix gently to avoid air bubbles, put it together with the test tube rack in a 37°C±1°C water bath or a suitable thermostat, keep the test tube rack in a horizontal state, keep warm for 60±2 minutes, and observe the results . The process of heat preservation and taking of test tubes should avoid false negative results caused by vibration.

(4-4) Judgment:

Gently take the test tube out of the thermostat and slowly turn it over 180°. If a gel is formed in the tube, and the gel is not deformed and does not slip off the tube wall, it is positive and recorded as (+); If the gel is not solid, deformed and slipped from the tube wall, it is negative and recorded as (-).

If the parallel tubes of the negative control solution D are all negative, the parallel tubes of the positive control solution B of the test product are all positive, and the parallel tubes of the positive control solution C are all positive, the test is valid. If the two parallel tubes of solution A are negative, it is judged that the test product complies with the regulations. If the two parallel tubes of solution A are positive, it is determined that the test product does not meet the requirements. If one of the two parallel tubes of solution A is positive and the other is negative, a retest is required. During the retest, four parallel tubes should be prepared for solution A. If all the parallel tubes are negative, it is determined that the test product meets the requirements, otherwise it is determined that the test product does not meet the requirements.

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

It has been determined that the endotoxin level of the bulk drug obtained in Example 1 is 50-100 Eu/ml.

Particle size is one of the key quality attributes of nano-iron preparations, and the change of particle size can be used to observe whether the nanoparticles are damaged.

The crude drug that embodiment 1 obtains is carried out the mensuration of particle diameter according to the following method:

According to the particle size and particle size distribution determination method (Chinese Pharmacopoeia 2015 edition general rule 0982 third method) (MalvernMastersizerNano ZS90 or laser particle size analyzer with equivalent performance), take an appropriate amount of this product, add water to dissolve and make iron containing about 0.3mg per 1ml Solution, ultrasonic for 10-15 seconds, check according to law, the average particle size of light intensity should be 19-51nm.

After measurement, the particle diameter of the crude drug obtained in Example 1 is 31.15nm.

Example 3

Add sodium hydroxide to the bulk drug concentrate obtained in Example 1 until the overall concentration of sodium hydroxide in the concentrate is 10%, heat it to 80°C for 2 hours, cool down to room temperature naturally, and remove the added strong base by ultrafiltration , after adjusting the concentration, the endotoxin and particle size were measured according to the method of Example 2. The measured endotoxin level was 6.25-12.5Eu/ml, and the particle size was 60.39nm.

Example 4

Add sodium hydroxide to the bulk drug concentrate obtained in Example 1 until the overall concentration of sodium hydroxide in the concentrate is 5%, heat it to 80°C for 2 hours, cool down to room temperature naturally, and remove the added strong base by ultrafiltration , after adjusting the concentration, the endotoxin and particle size were measured according to the method of Example 2. The measured endotoxin level was 6.25-12.5Eu/ml, and the particle size was 58.47nm.

Example 5

Add sodium hydroxide to the bulk drug concentrate obtained in Example 1 until the overall concentration of sodium hydroxide in the concentrate is 3%, heat it to 80°C for 2 hours, cool down to room temperature naturally, and remove the added strong base by ultrafiltration , after adjusting the concentration, the endotoxin and particle size were measured according to the method of Example 2. The measured endotoxin level was 12.5-25Eu/ml, and the particle size was 58.02nm.

Example 6

Add sodium hydroxide to the bulk drug concentrate obtained in Example 1 until the overall concentration of sodium hydroxide in the concentrate is 1%, heat it to 80°C for 2 hours, cool down to room temperature naturally, and remove the added strong base by ultrafiltration , after adjusting the concentration, the endotoxin and particle size were measured according to the method of Example 2. The measured endotoxin level was 12.5-25Eu/ml, and the particle size was 31.59nm.

Example 7

Add sodium hypochlorite to the bulk drug concentrate obtained in Example 1 until the overall sodium hypochlorite concentration in the concentrate is 0.5%, heat it to 80°C for 2 hours, then drop to room temperature naturally, remove the added sodium hypochlorite by ultrafiltration, after adjusting the concentration, The endotoxin and particle size were measured according to the method of Example 2. The endotoxin level was determined to be 50-100Eu/ml, and the particle size was 32.01nm.

Example 8

Add sodium hypochlorite to the concentrated solution of bulk drug obtained in Example 1 until the overall concentration of sodium hypochlorite in the concentrated solution is 5%, it is heated to 80 ° C for 2 hours, and it is naturally lowered to room temperature, and the sodium hypochlorite added is removed by ultrafiltration. After adjusting the concentration, The endotoxin and particle size were measured according to the method of Example 2, and the endotoxin level was determined to be 25-50Eu/ml, and the particle size was 32.23nm.

Example 9

Add sodium hypochlorite to the concentrated solution of bulk drug obtained in Example 1 until the overall concentration of sodium hypochlorite in the concentrated solution is 10%, it is heated to 80 ° C for 2 hours, and it is naturally lowered to room temperature, and the sodium hypochlorite added is removed by ultrafiltration. After adjusting the concentration, The endotoxin and particle size were measured according to the method of Example 2, and the endotoxin level was determined to be 25-50Eu/ml, and the particle size was 60.64nm.

Example 10

Add sodium hydroxide and sodium hypochlorite respectively in the crude drug concentrated solution that embodiment 1 obtains, be 1% to integral sodium hydroxide concentration in the concentrated solution, sodium hypochlorite is 1%, it is heated to 80 ℃ of reaction 2 hours, naturally drops to At room temperature, the added sodium hydroxide and sodium hypochlorite were removed by ultrafiltration. After adjusting the concentration, the endotoxin and particle size were measured according to the method in Example 2. The endotoxin level was determined to be lower than 3.0Eu/ml, and the particle size was 32.63nm.

Claims (5)

1. a kind of endotoxic minimizing technology of superparamagnetic iron oxide, this method is that highly basic is added in superparamagnetic iron oxide bulk pharmaceutical chemicals Solution and liquor natrii hypochloritis, are reacted,

Wherein, a concentration of the 1% of strong base solution, a concentration of the 1% of sodium hypochlorite.

2. method as claimed in claim 1, the highly basic is sodium hydroxide, potassium hydroxide or lithium hydroxide.

3. method as claimed in claim 1, reaction temperature is 50 DEG C~100 DEG C.

4. method as claimed in claim 3, reaction temperature is 70 DEG C~90 DEG C.

5. method as claimed in claim 4, reaction temperature is 80 DEG C.