JP2006291028A - Low-molecular heparin or salt thereof, and manufacturing method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a low-molecular heparin or a salt thereof which is obtained efficiently in a simple method unnecessary for a large scale facilities and which is curtailed extremely in the contents of impurities; a pharmaceutical composition containing this; and a method for manufacturing a low-molecular heparin or a salt thereof. <P>SOLUTION: This low-molecular heparin or a salt thereof can be obtained by the steps of depolymerizing a heparin and/or a salt thereof, carrying out the treatment by using a reducing agent, and removing and/or decomposing the above reducing agent, then adjusting a pH of the above solution to 4 or lower. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to low molecular weight heparin or a salt thereof, and a method for producing the same.

  Heparin is a kind of mucopolysaccharide having a broad molecular weight distribution and an average relative molecular weight of 10,000 to 30,000, and is a living tissue of animals (for example, intestinal mucosa, lung, spleen, liver, muscle, tendon) and cells (for example, Are widely present in mast cells). Since heparin has a strong blood anticoagulant effect, it can be used for the treatment and prevention of various thromboembolisms such as myocardial infarction and cerebral embolism, as well as insertion of a vascular catheter or injection needle during hemodialysis, blood transfusion, blood tests, etc. Sometimes used to prevent blood clotting.

  In particular, when an extracorporeal circulation device such as a hemodialysis device or artificial cardiopulmonary device is used, the membrane may be clogged due to coagulation of perfused blood on the dialysis membrane or filtration membrane. In order to prevent this, it is necessary to control blood coagulation ability.

  On the other hand, low molecular weight heparin adjusted to a molecular weight within a certain range has a feature that it has little action to promote bleeding while maintaining blood anticoagulant activity and antithrombotic action. For this reason, low molecular weight heparin is suitably used when treating and preventing various thromboembolisms such as myocardial infarction and cerebral embolism, as well as when using an extracorporeal circulation device such as a hemodialyzer or a cardiopulmonary device.

An example of a method for efficiently producing high-purity low-molecular-weight heparin is a purification method using a dialysis membrane (see Patent Document 1). However, in order to industrially produce low molecular weight heparin or a salt thereof by this method, a large-scale industrial facility is required, so that it is difficult to carry out easily in terms of cost and facilities.
Japanese Patent No. 2628507

  An object of the present invention is to obtain a low-molecular-weight heparin or a salt thereof, which can be efficiently obtained by a simple method without requiring a large-scale facility, and the content of impurities is extremely reduced, a pharmaceutical composition containing the same, And a method for producing a low molecular weight heparin or a salt thereof.

  In order to achieve the above object, the present inventors have conducted extensive research on a method for reducing the molecular weight of heparin or a salt thereof. As a result, the molecular weight of heparin and / or the salt thereof is reduced, and the treatment is performed using a reducing agent. After removing and / or decomposing the reducing agent, it is found that the content of impurities can be effectively reduced without reducing the yield by adjusting the pH of the solution to 4 or less, and the present invention is completed. It came to.

  The low molecular weight heparin or a salt thereof of the present invention is prepared by reducing the molecular weight of heparin and / or a salt thereof, treating with a reducing agent, removing and / or decomposing the reducing agent, and then reducing the pH of the solution to 4 or less. It is obtained by adjusting.

  Here, in the low molecular weight heparin or the salt thereof of the present invention, the molecular weight reduction can be performed by a reaction between the heparin and / or a salt thereof and nitrite.

  Here, in the low molecular weight heparin of the present invention or a salt thereof, the content of the nitrite may be 5 ppm or less.

  Here, in the low molecular weight heparin of the present invention or a salt thereof, the pH of the solution is adjusted to 4 or less, and then the pH of the solution is adjusted to 5.5 to 8, and then a solvent is added to form a precipitate. And collecting the precipitate. In this case, the solvent may be at least one selected from ethanol, methanol, and acetone.

  Here, in the low molecular weight heparin or the salt thereof of the present invention, when the pH of the solution is adjusted to 4 or less, it can be obtained by adjusting the pH to 2.5 or less.

Here, in the low molecular weight heparin of the present invention or a salt thereof, a singlet peak is not substantially detected in the range of 3.65-3.75 ppm in the ¹ H-NMR spectrum, and in the ¹³ C-NMR spectrum. No peaks can be detected substantially in the range of 61.5-62.5 ppm.

  The pharmaceutical composition of the present invention contains the low molecular weight heparin of the present invention or a salt thereof as an active ingredient.

The method for producing the low molecular weight heparin or a salt thereof of the present invention includes:
(1) a step of reducing the molecular weight of heparin and / or a salt thereof and then reacting with a reducing agent; and (2) a step of adjusting the pH of the solution to 4 or less after removing and / or decomposing the reducing agent. including.

  Here, in the method for producing low molecular weight heparin or a salt thereof of the present invention, after the step (2), (3) the pH of the solution is adjusted to 5.5 to 8, and then a solvent is added to the precipitate. And a step of recovering the precipitate.

  Here, in the method for producing a low molecular weight heparin or a salt thereof according to the present invention, the step of adjusting the pH of the solution in the step (2) to 4 or less and the step (3) as one set, the set Can be repeated several times.

  Here, in the method for producing a low molecular weight heparin or a salt thereof according to the present invention, the molecular weight reduction can be performed by a reaction between the heparin and / or a salt thereof and nitrite.

  In this case, the content of the nitrite may be 5 ppm or less.

  Here, in the method for producing a low molecular weight heparin or a salt thereof according to the present invention, in the step (2), when the pH of the solution is adjusted to 4 or less, the pH can be adjusted to 2.5 or less.

  According to the low molecular weight heparin or a salt thereof of the present invention, it can be efficiently obtained by a simple method without requiring a large-scale facility, and the content of impurities is extremely reduced.

  According to the method for producing low molecular weight heparin or a salt thereof of the present invention, since it can be simply and efficiently purified, a large-scale facility is not required, a long purification process is not required, and the yield is reduced. Low molecular weight heparin or a salt thereof can be obtained economically without reducing the rate.

  According to the pharmaceutical composition of the present invention, by containing the low molecular weight heparin of the present invention or a salt thereof as an active ingredient, an anticoagulant action can be exhibited and a correlation with bleeding is suggested. The effect of extending the activated partial thromboplastin time (APTT) is weak. That is, according to the pharmaceutical composition of the present invention, blood coagulation action and antithrombotic action can be achieved without promoting bleeding. More specifically, the pharmaceutical composition of the present invention can be used for coagulation of perfused blood during extracorporeal blood circulation and prevention of generalized intravascular blood coagulation.

  The present invention will be described below. In the present invention, “%” means “mass%” and “part” means “part by mass”.

1. Low molecular weight heparin and its salt The low molecular weight heparin or its salt (hereinafter also referred to as "low molecular weight heparin") of the present invention is obtained by reducing the molecular weight of heparin and / or its salt and treating it with a reducing agent. After removing and / or decomposing the reducing agent, the pH of the solution is adjusted to 4 or less.

  In the present invention, “heparin” means a disaccharide structural unit in which glucuronic acid and N-acetylglucosamine are α1 → 4 linked and a disaccharide structural unit in which iduronic acid and N-acetylglucosamine are α1 → 4 linked. It is a polysaccharide containing. In heparin, at least one of the hydroxyl group at the C6 position and the amino group at the C2 position of N-acetylglucosamine may be sulfated, and the hydroxyl group at the C2 position of glucuronic acid or iduronic acid is sulfated. May be.

  In the present invention, the “heparin salt” is not particularly limited, but is preferably a pharmaceutically acceptable salt, for example, sodium salt, potassium salt, lithium salt, calcium salt, barium salt, zinc salt, magnesium Examples thereof include salts and ammonium salts.

1.1. Raw Material Heparin and its salt (hereinafter also referred to as “raw material heparin and its salt” or “raw material heparin etc.”) that can be used as a raw material for the low molecular weight heparin of the present invention have an average relative molecular weight of 10,000 to 30,000. It is preferable to use those having an average relative molecular weight of 10,000 to 20,000.

  As the raw material heparin and its salt used in the present invention, either the crude extract or the purified product may be used. Heparin is widely distributed in animal organs. For example, as the raw material heparin and its salt used in the present invention, those derived from porcine intestinal mucosa can be used.

1.2. Low molecular weight heparin or a salt thereof In the present invention, “low molecular weight heparin or a salt thereof” refers to a substance having a molecular weight reduced as compared with a raw material heparin and a salt thereof.

  The low molecular weight heparin or a salt thereof of the present invention has an average relative molecular weight of usually 2,000 to 10,000, preferably 3,000 to 8,000, more preferably 4,000 to 6,000. .

  Further, the low molecular weight heparin or a salt thereof of the present invention is a mixture of those having different molecular weights, and 90% thereof is preferably distributed between the molecular weights of 2,000 and 9,000.

  The low molecular weight heparin salt of the present invention is not particularly limited, but is preferably a pharmaceutically acceptable salt of the low molecular weight heparin of the present invention. The pharmaceutically acceptable salt is not particularly limited as long as it is a salt that can be used as a pharmaceutical product. For example, sodium salts and calcium salts are common as heparin preparations as represented by dalteparin sodium. .

The presence or absence of impurities in the low molecular weight heparin or a salt thereof of the present invention can be confirmed by a known analysis method (for example, ¹ H-NMR, ¹³ C-NMR).

In the low molecular weight heparin or a salt thereof of the present invention, a singlet peak is not substantially detected in the range of 3.65 to 3.75 ppm in the ¹ H-NMR spectrum, and 61.5- in the ¹³ C-NMR spectrum. No substantial peak is detected in the 62.5 ppm range. Thereby, the content of impurities in the low molecular weight heparin or a salt thereof of the present invention is extremely reduced. The ¹ H-NMR and ¹³ C-NMR measurements are performed at 30 ° C. in heavy water.

  The low molecular weight heparin or a salt thereof of the present invention preferably has a nitrite content of 5 ppm or less. The content of nitrite can be measured by absorbance measurement to which a grease / lomen nitrite reagent is added, and can be measured, for example, by the method described in Examples described later.

  For example, inhalation or ingestion of nitrites such as sodium nitrite or potassium nitrite may cause symptoms such as cyanosis (lips, nails, skin, etc.), confusion, convulsions, dizziness, headache, nausea, and loss of consciousness. For this reason, when using nitrite in the manufacturing process of the low molecular weight heparin or its salt of this invention, it is necessary to fully remove nitrite from the low molecular weight heparin or its salt of this invention. According to the low molecular weight heparin or a salt thereof of the present invention, impurities such as nitrite are extremely reduced because it can be obtained by the production method described later. For this reason, the above symptom is not caused by the remaining nitrite.

2. 2. Method for producing low molecular weight heparin or a salt thereof 2.1. Process (1)
The method for producing low molecular weight heparin and the like of the present invention includes (1) a step of reacting with a reducing agent after reducing the molecular weight of raw material heparin and the like.

  In the present invention, the molecular weight reduction of raw material heparin or the like can be performed by reaction of raw material heparin or the like with nitrite. More specifically, for example, there is a method in which a reaction liquid obtained by adding raw material heparin and the like and nitrite (sodium nitrite, potassium nitrite and the like) to the solvent is stirred for a predetermined time. In this case, the solvent that can be used is not particularly limited as long as it can dissolve the raw material heparin and the like and nitrite, but water, alcohol (for example, methanol, ethanol, n-propanol, 2-propanol, n- Butanol, sec-butanol), or aqueous solvents such as a mixed solvent of water and alcohol.

  In the reaction with nitrite, the hexosaminide bond is cleaved, so that the molecular weight reduction (depolymerization reaction) proceeds. Thereby, low molecular weight compounds, such as raw material heparin, are obtained.

  The degree of lowering the molecular weight of the raw material heparin can be controlled by appropriately adjusting the reaction time, the reaction temperature, the type of solvent used, the concentration of the raw material heparin, etc. in the reaction solution.

  In the present invention, a low molecular weight product such as raw material heparin is treated with a reducing agent. By this treatment, the terminal group of the low molecular weight product can be reduced. Thereby, the low molecular weight heparin of this invention can be obtained.

Here, the reducing agent is not particularly limited. For example, the reaction in an aqueous solvent is possible, and the carboxyl group or acetyl group in the low molecular weight product can be retained without reduction. And sodium borohydride (NaBH ₄ ) or sodium cyanoborohydride (NaBH ₃ CN) is preferred.

  For example, when raw material heparin or the like is reduced in molecular weight using nitrite, it is presumed that at least a part of terminal groups of the resulting reduced molecular weight is an -ONO group. By treating the low molecular weight product with a reducing agent, the -ONO group can be converted to an -OH group.

2.2. Step (2)
The method for producing low molecular weight heparin and the like of the present invention includes a step of (2) removing and / or decomposing the reducing agent and then setting the pH of the solution to 4 or less after the step (1).

  The removal and / or decomposition of the reducing agent is preferably performed by a method suitable for the reducing agent used. That is, the reducing agent may be removed from the reaction solution, the reducing agent may be decomposed in the reaction solution, or both the reducing agent may be decomposed and removed. Further, a reducing agent may be removed by adding a solvent that is hardly soluble in a low molecular weight product such as raw material heparin to the reaction solution, precipitating the low molecular weight product, and collecting the precipitate. Here, as the hardly soluble solvent, a solvent described later can be used.

  For example, when using sodium borohydride as the reducing agent, after the reaction between the reducing agent and a low molecular weight product such as raw material heparin, for example, hydrochloric acid is added to the reaction solution to decompose excess sodium borohydride. Furthermore, for example, an aqueous sodium hydroxide solution is added to the reaction solution for neutralization, and then a solvent is added to precipitate a precipitate (low molecular weight heparin of the present invention), which can be recovered. According to this method, the low molecular weight heparin of the present invention and the reducing agent can be efficiently separated. Here, the solvent used for generating the precipitate is not particularly limited as long as it can generate the precipitate, but preferably has low solubility in the low molecular weight heparin of the present invention, for example, Ethanol, methanol, isopropyl alcohol, diethyl ether, acetone, ethyl acetate and the like, or a mixture of two or more thereof, and can contain water as appropriate. The solvent is preferably at least one selected from ethanol, methanol, and acetone, and more preferably water-containing ethanol (90-98 (V / V)% ethanol aqueous solution).

  Next, the obtained precipitate is dissolved in a solvent to prepare a solution. Here, the solvent used for preparing the solution is not particularly limited as long as the precipitate can be dissolved. For example, a solvent (for example, water) used when reducing the molecular weight of the above-described raw material heparin or the like, an aqueous sodium chloride solution, or the like can be exemplified.

  Next, after removing and / or decomposing the reducing agent, the pH of the solution is adjusted to 4 or less. Here, as what is used in order to make pH of the said solution into 4 or less, an acid and acidic cation exchange resin are mentioned, for example.

  Although it does not specifically limit as an acid, The thing which can be used in manufacture of a pharmaceutical is preferable. Examples of the acid include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as acetic acid, glacial acetic acid, fumaric acid, and malic acid.

  Although it does not specifically limit as acidic cation exchange resin, For example, strong acidic cation exchange resin (Diaion SK1B, Dowex 50W), weak acidic cation exchange resin (Duolite C-464), etc. are mentioned, Strong acid A cationic cation exchange resin is preferred.

  In the step (2), when the pH of the solution is adjusted to 4 or less, the pH of the solution is preferably adjusted to 4 or less, preferably 2.5 or less. Here, when the pH of the solution is adjusted to be higher than 4, the obtained low molecular weight heparin and the impurities are not sufficiently dissociated, and the removal rate of the impurities is lowered even if a purification process using a column or the like is performed thereafter.

  In the step (2), the lower limit value of the pH when the pH of the solution is adjusted to 4 or less is not particularly limited, but in particular, it is used as a medicine by adjusting the pH to 2.5 or less. Thus, low-molecular-weight heparin with sufficient purity can be obtained. Even when the pH is adjusted to 2.5 or less, the effect is almost the same as that when the pH is adjusted to 2.5.

2.3. Process (3)
In the method for producing low molecular weight heparin and the like of the present invention, after the step (2), (3) the pH of the solution is adjusted to 5.5 to 8, and then a solvent is added to form a precipitate. The method may further include a step of recovering the product. Here, the collected precipitate is a low molecular weight heparin salt.

  In the step (3), the pH of the solution is more preferably adjusted to 6.5 ± 0.8, and further preferably adjusted to 6.5 ± 0.5.

  In the step (3), a base can be used in adjusting the pH of the solution. As a base, what is necessary is just a base accept | permitted in manufacture of a pharmaceutical, For example, sodium hydroxide, potassium hydroxide, sodium carbonate etc. are mentioned.

  In the present invention, the efficiency is improved by adjusting the pH of the solution to 4 or less in the step (2) and then adjusting the pH of the solution to 5.5 to 8 in the step (3). Impurities can be removed well. The mechanism of action at that time is presumed to be as follows.

  In the step (2), by adjusting the pH of the solution to 4 or less, a reagent (for example, nitrite and other impurities (for example, ethylene glycol, EDTA, acetic acid, (Ammonium methyl sulfate, sodium methyl sulfate, etc.) are dissociated from low molecular weight heparin, and in the step (3), the pH of the solution is adjusted to 5.5-8, and then the low molecular weight is reduced with a heparin hardly soluble solvent (ethanol, etc.). By precipitating the heparin salt, it is considered that the impurities and the low molecular weight heparin salt can be separated, whereby a low molecular weight heparin having high purity or a salt thereof can be obtained.

  More preferably, the set is repeated a plurality of times, with the step of adjusting the pH of the solution in step (2) to 4 or less and the step (3) as one set. According to this method, the content of impurities can be further reduced. In this method, the pH of the solution obtained by dissolving the precipitate collected in the step (3) in a solvent is adjusted to 4 or less. The solvent used in this case may be any solvent that can dissolve low molecular weight heparin. For example, the solvent used in dissolving the precipitate in the step (2) can be used.

  The low molecular weight heparin salt of the present invention can be obtained by performing the above-mentioned steps, performing other purification (for example, column purification) as necessary, and then performing a drying treatment such as vacuum drying or freeze drying. .

2.4. An example of the manufacturing method of the present invention An example of the manufacturing method of the present invention is shown below, but the manufacturing method of the present invention is not limited to this.

  (A) A sodium heparin aqueous solution is acidified with hydrochloric acid, and an aqueous sodium nitrite solution is added to carry out a low molecular weight reaction (depolymerization reaction). During the reaction, the reaction solution is kept acidic with hydrochloric acid.

  (B) Next, an aqueous sodium hydroxide solution is added to the reaction solution to neutralize it, and the reaction is stopped.

  (C) Next, the reaction solution is heated, and a reducing agent (sodium borohydride) is added to carry out a reduction reaction (corresponding to step (1) above).

  (D) Next, hydrochloric acid is added to the reaction solution to decompose excess sodium borohydride, followed by neutralization with an aqueous sodium hydroxide solution.

  (E) Next, a solvent (ethanol) is added to the reaction solution until a precipitate is deposited, and the mixture is allowed to stand, and then the precipitate is collected by centrifugation.

  (F) Next, this precipitate was dissolved in an aqueous sodium chloride solution to prepare a solution.

  (G) Next, the pH of this solution is adjusted to 4 or less (2.2 ± 0.3) with hydrochloric acid (corresponding to the above step (2)).

  (H) The pH of this solution is then adjusted to 5.5-8 with sodium hydroxide.

  (I) Next, a solvent (ethanol) is added to the solution until the precipitate is precipitated, and the mixture is allowed to stand, and then the precipitate is recovered by centrifugation (corresponding to the step (3) above).

  (K) Next, this precipitate is dissolved in distilled water and subjected to column purification.

  (L) Next, membrane filtration is performed, and ethanol is added to the obtained eluate until a precipitate is deposited, which is allowed to stand, and then the precipitate is collected by centrifugation.

  (M) Low molecular weight sodium heparin is obtained by vacuum drying the collected precipitate.

3. Pharmaceutical Composition The pharmaceutical composition of the present invention is a pharmaceutical preparation comprising the low molecular weight heparin of the present invention or a salt thereof as an active ingredient. As one aspect | mode of the pharmaceutical composition of this invention, a dalteparin sodium injection solution is mentioned, for example. This dalteparin sodium injection solution is a pharmaceutical preparation for intravenous injection formulated with heparin sodium, which has been reduced in molecular weight using sodium nitrite, as the main ingredient. Dalteparin sodium injection solution can be used for preventing coagulation of perfused blood during blood extracorporeal circulation during hemodialysis and for treating generalized intravascular blood coagulation.

4). Examples Next, the present invention will be described in more detail based on examples, comparative examples, test examples, and application examples. The present invention is not limited to these.

4.1. Measurement Method About the low molecular weight heparin obtained in this example, ¹ H-NMR and ¹³ C-NMR were measured. The measurement conditions of ¹ H-NMR and ¹³ C-NMR in this example are shown below. Moreover, it was measured whether or not nitrite remained in the low molecular weight heparin obtained in this example and the comparative example.

4.1.1. Preparation of sample solution About 50 mg of each sample (low molecular weight heparin or a salt thereof) was weighed and dissolved in 0.75 mL of heavy water (D ₂ O), and about 3 mg of sodium trimethylpropanesulfonate (DSS) was added as an internal standard reagent. .

4.1.2. Measurement conditions for ¹ H-NMR Apparatus: VXR-400S (manufactured by Varian)
Observation frequency: 399.9 MHz
Temperature: 30 ° C
Standard: DSS (0 ppm)
Under the above conditions, a ¹ H-NMR spectrum of each sample was obtained. ^{In the 1} H-NMR spectrum, it was confirmed whether a singlet peak (impurity signal) was observed in the range of 3.65-3.75 ppm.

4.1.3. ¹³ C-NMR measurement conditions Apparatus: VXR-400S (manufactured by Varian)
Observation frequency: 100.5 MHz
Temperature: 30 ° C
Standard: DSS (0 ppm)
Under the above conditions, a ¹³ C-NMR spectrum of each sample was obtained. Especially in the ¹³ C-NMR spectrum, it was confirmed whether or not a peak (impurity signal) was observed particularly in the range of 61.5-62.5 ppm.

4.1.4. Method for Measuring Nitrite Content In this example, the content of nitrite was measured by the following method. In addition, according to this measuring method, content can be measured not only about sodium nitrite but also about other nitrites (potassium nitrite).

  (A) A sample (low molecular weight heparin or a salt thereof) was diluted 100 times with distilled water to prepare a sample solution of 1 (W / V)%.

  (B) A nitrite aqueous solution was prepared with respect to the sample so that the nitrites would be 2, 4, 6, 8, and 10 ppm, respectively, and this was used as a standard solution.

  (C) A grease / lomen nitrous acid reagent was added to each of the sample solution and the standard solution, and the mixture was allowed to stand for 10 minutes. Then, the absorbance was measured at a wavelength of 525 nm using distilled water as a control.

  (D) The content of nitrite contained in the sample was measured from a calibration curve obtained from the absorbance measurement result of the standard solution.

4.2. Example 1
(A) 15.0 g of pharmacopoeia sodium heparin was dissolved in 250 mL of a solvent (ion distilled water) to prepare a reaction solution, and then the pH of this reaction solution was adjusted to 2.5 with a 4 mol / L hydrochloric acid aqueous solution.

  (B) Next, 10 mL of 5% sodium nitrite was added to the reaction solution, and a molecular weight reduction reaction (depolymerization reaction) was performed at room temperature for 1 hour. During the reaction, the pH of the reaction solution was maintained at pH 2.5 using 4 mol / L hydrochloric acid aqueous solution.

  (C) Subsequently, the pH of the reaction solution was adjusted to 7.0 using a 4 mol / L sodium hydroxide aqueous solution to stop the reaction.

  (D) Next, the reaction solution was heated to 40 ° C., 1.0 g of a reducing agent (sodium borohydride) was added, and the reduction reaction was performed for 2 hours.

  (E) Next, the pH of the reaction solution was adjusted to 4.0 using 4 mol / L hydrochloric acid aqueous solution, and excessive sodium borohydride was decomposed.

  (F) Next, the pH of the reaction solution was adjusted to 7.0 using a 4 mol / L sodium hydroxide aqueous solution.

  (G) Next, a solvent (ethanol) was added to the reaction solution until a precipitate was deposited, and the mixture was allowed to stand, and then the precipitate was collected by centrifugation.

  (H) Next, this precipitate was dissolved in 150 mL of 5% aqueous sodium chloride solution to prepare a solution.

  (I) Subsequently, the pH of this solution was adjusted to 4 or less (2.2 ± 0.3) using a 4 mol / L hydrochloric acid aqueous solution.

  (J) Next, the pH of this solution was adjusted to 7.0 using a 4 mol / L aqueous sodium hydroxide solution.

  (K) Next, a solvent (ethanol) was added to the solution until a precipitate was deposited, and the mixture was allowed to stand, and then the precipitate was collected by centrifugation.

  (L) Next, this precipitate was dissolved in 200 mL of distilled water and subjected to column purification.

  (M) Next, membrane filtration was performed, ethanol was added to the eluate until a precipitate was deposited, the mixture was allowed to stand, and the precipitate was collected by centrifugation.

  (N) The collected precipitate was vacuum-dried to obtain white and powdery low-molecular-weight heparin sodium (dalteparin sodium).

4.3. Example 2
Low molecular weight heparin sodium (dalteparin sodium) was obtained in the same manner as in Example 1, except that the pH of the solution was adjusted to 3.8 ± 0.3 in step (i).

4.4. Example 3
In Example 1, in the steps (g), (k), and (m), the same method as in Example 1 was used except that a solvent mixture of methanol-acetone (1: 1) was used instead of ethanol. Thus, low molecular weight heparin sodium (dalteparin sodium) was obtained.

4.5. Example 4
In Example 1, except that the column purification of the step (l) was performed after the step (g), and then the processes of the steps (h) to (k) were performed, the same method as in Example 1, Low molecular weight heparin sodium (dalteparin sodium) was obtained.

4.6. Comparative Example 1
In the same manner as in Example 1 except that steps (h) to (k) were deleted (ie, step (l) was performed after step (g)), low molecular weight heparin was obtained. Sodium (dalteparin sodium) was obtained.

4.7. Comparative Example 2
Low molecular weight heparin sodium (dalteparin sodium) was obtained in the same manner as in Example 1 except that the pH was adjusted to 5.0 ± 0.3 in Example (i).

4.8. Test Example The purity of the low molecular weight heparin sodium obtained in Example 1 and Comparative Example 1 was evaluated by detecting the content of sodium nitrite and impurity spectra by ¹ H-NMR and ¹³ C-NMR measurements.

  About the content of sodium nitrite, the light absorbency was measured and quantified by the said method.

Moreover, about the measurement of < ¹ > H-NMR and < ¹³ > C-NMR, the < ¹ > H-NMR spectrum and the < ¹³ > C-NMR spectrum were calculated | required by the said method. More specifically, whether a singlet peak is substantially detected in the range of 3.65-3.75 ppm in the ¹ H-NMR spectrum, and 61.5-62 in the ¹³ C-NMR spectrum. It was confirmed whether or not a peak was substantially detected in the range of 0.5 ppm.

From Table 1, in Examples 1-4, content of sodium nitrite is within 5 ppm. From ¹ H-NMR and ¹³ C-NMR measurement results, 3.65-3.75 ppm in ¹ H-NMR spectrum. No single line peak was substantially detected in the range, and no peak was substantially detected in the range of 61.5-62.5 ppm in the ¹³ C-NMR spectrum. From the above results, it was confirmed that the content of impurities in the low molecular weight heparin sodium (dalteparin sodium) of Examples 1 to 4 was reduced to a sufficient level as a pharmaceutical product.

  On the other hand, the low molecular weight heparin sodium obtained in Comparative Example 1 is unsuitable as a pharmaceutical product, and the low molecular weight heparin sodium obtained in Comparative Example 2 requires a further purification step to become a pharmaceutical product. Was suggested. From the above results, the low molecular weight heparin sodium obtained in Comparative Example 1 is a step corresponding to the step (i) of Example 1 (after removing and / or decomposing the reducing agent, the pH of the solution is 4 or less). It is presumed that the impurities were not sufficiently removed because the adjustment step) was not performed. Moreover, it is estimated that the low molecular weight heparin sodium obtained in Comparative Example 2 was not able to sufficiently remove impurities because the pH of the solution was adjusted to about 4 or more in the step corresponding to step (i) of Example 1. The

4.9. Application Examples The low molecular weight heparin (dalteparin sodium) powder obtained in Examples 1 to 4 was mixed with a buffering agent so that the final pH was 6 to 7.5, and dissolved in isotonic distilled water for injection. Then, it was prepared semi-aseptically to be 1000 low molecular heparin international units per mL.

  Next, this solution was filtered through a 0.1 μm-diameter membrane filter, washed and sterilized in an aseptic environment using an automatic vial filling and sealing machine (manufactured by Tachibana Seisakusho), and 5 mL in a pyrogen-free vial. Filled, sealed (plugged) and formulated.

  The obtained preparation (blood coagulation inhibitor) has an average relative molecular weight of about 5,000, 90% of which is 1000 low molecular weight heparin international units per mL of dalteparin sodium distributed in the range of 2,000 to 9,000. contains. Thereby, the preparation obtained in this application example had a quality suitable for the preparation for intravenous injection.

It is a ¹ H-NMR spectrum of the low molecular weight heparin Na obtained in Example 1 (no single-line peak is observed in the range of 3.65-3.75 ppm). It is a ¹³ C-NMR spectrum of the low molecular weight heparin Na obtained in Example 1 (no peak is observed in the range of 61.5-62.5 ppm). It is a ¹ H-NMR spectrum of the low molecular weight heparin Na obtained in Comparative Example 1 (single-line peaks (indicated by “A”) are observed in the range of 3.65-3.75 ppm). It is a ¹³ C-NMR spectrum of the low molecular weight heparin Na obtained in Comparative Example 1 (a peak (indicated by “A”) is observed in the range of 61.5-62.5 ppm).

Claims (14)

  Low molecular weight heparin obtained by reducing the molecular weight of heparin and / or a salt thereof, treating with a reducing agent, removing and / or decomposing the reducing agent, and then adjusting the pH of the solution to 4 or less. Its salt. In claim 1,
The low molecular weight reduction is performed by a reaction of the heparin and / or a salt thereof and nitrite, and the low molecular weight heparin or a salt thereof. In claim 2,
Low molecular weight heparin or a salt thereof, wherein the nitrite content is 5 ppm or less. In any one of Claims 1 thru | or 3,
A low molecular weight obtained by adjusting the pH of the solution to 4 or less and then adjusting the pH of the solution to 5.5 to 8, then adding a solvent to form a precipitate, and collecting the precipitate Heparin or its salt. In any of claims 1 to 4,
Low molecular weight heparin or a salt thereof, wherein the solvent is at least one selected from ethanol, methanol, and acetone. In any of claims 1 to 5,
Low molecular weight heparin or a salt thereof obtained by adjusting the pH to 2.5 or lower when the pH of the solution is adjusted to 4 or lower. In any one of Claims 1 thru | or 6.
^A singlet peak is substantially not detected in the range of 3.65-3.75 ppm in the ¹ H-NMR spectrum, and a peak is substantially detected in the range of 61.5-62.5 ppm in the ¹³ C-NMR spectrum. Low molecular weight heparin or its salt not detected.   A pharmaceutical composition comprising the low molecular weight heparin according to any one of claims 1 to 7 or a salt thereof as an active ingredient. (1) a step of reducing the molecular weight of heparin and / or a salt thereof and then reacting with a reducing agent; and (2) a step of adjusting the pH of the solution to 4 or less after removing and / or decomposing the reducing agent. A method for producing a low molecular weight heparin or a salt thereof. In claim 9,
After the step (2), the method further comprises a step of (3) adjusting the pH of the solution to 5.5 to 8, then adding a solvent to form a precipitate, and collecting the precipitate. Or the manufacturing method of the salt. In claim 9 or 10,
A method for producing low molecular weight heparin or a salt thereof, wherein the step of adjusting the pH of the solution in the step (2) to 4 or less and the step (3) are repeated as a set, and the set is repeated a plurality of times. In any of claims 9 to 11,
The method for producing a low molecular weight heparin or a salt thereof, wherein the molecular weight reduction is performed by a reaction of the heparin and / or a salt thereof and a nitrite. In claim 12,
The manufacturing method of the low molecular weight heparin or its salt whose content of the said nitrite is 5 ppm or less. In any of claims 9 to 13,
In the step (2), when the pH of the solution is adjusted to 4 or less, the pH is adjusted to 2.5 or less, and the method for producing low molecular weight heparin or a salt thereof.

Images