JP2016216518A - Extract and formulation containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an extract from inflamed rabbit skin inoculated with vaccinia virus and a formulation etc. containing the extract as an active ingredient, which have greater stability in quality.SOLUTION: By using the amount of N-acetyl neuraminic acid contained in the extract from inflamed rabbit skin inoculated with vaccinia virus and the formulation containing the extract as an index, greater stability in quality of the extract and the formulation can be ensured for each production lot. The extract from inflamed rabbit skin inoculated with vaccinia virus and the formulation containing the extract, which are produced in this manner so as to have greater stability in quality, are extremely useful with effectiveness and safety more rigorously secured.SELECTED DRAWING: None

Description

  The present invention relates to a vaccinia virus-inoculated rabbit inflammation skin extract whose quality is more stable by confirming by test or inspection that it contains a predetermined amount of N-acetylneuraminic acid, a preparation containing the extract as an active ingredient, etc. About.

  A pharmaceutical product can only be approved for production and sales once its quality has been ensured. In Japan, this is stipulated in Article 14 of the Pharmaceutical Affairs Law. In other countries, such handling is basically the same due to the nature of pharmaceuticals. Thus, ensuring quality in pharmaceutical products is important because quality ensures the effectiveness and safety of pharmaceutical products. In other words, a drug whose quality is not ensured is not guaranteed to be effective and safe, and is not qualified as a drug.

  In Japan, the raw materials used in the production of pharmaceuticals, which are the active ingredients of pharmaceuticals, are called “Drugs”. As with pharmaceuticals, the quality of the drug substance is also required to be ensured. This is because the quality of the drug depends on the quality of the drug substance. According to Japanese pharmaceutical regulations, the drug substance is exclusively used for the manufacture of other drugs, and by definition, drug substances are included in drugs. However, in the present application, for convenience, the drug and the drug substance may be referred to separately, and the drug in this case means a drug excluding the drug substance.

  In general, pharmaceuticals and drug substances are manufactured by a predetermined manufacturing method so that products of a predetermined quality are manufactured. Therefore, manufacturing management is also important in order to protect the quality of drugs and drug substances. Therefore, in Japan, “Ministerial Ordinance on Standards for Manufacturing Management and Quality Control of Drugs and Quasi-drugs” is established. The ministerial ordinance is also called GMP (short for Good Manufacturing Practice) in Japan. GMP defines “Manufacturing Management” and “Quality Control” for pharmaceuticals. In “Manufacturing Management”, the concept of protecting the quality by managing the procurement of raw materials and the manufacturing process from the beginning to the end is adopted. “Quality control” is centered on confirming whether or not an actually manufactured drug or drug substance has a predetermined quality by a test or inspection performed after the manufacture. If the results of such tests and inspections do not conform to predetermined standards, the drug or drug substance cannot be shipped, sold, or used. In this way, the quality of drugs and drug substances is controlled through manufacturing control and quality control.

  Vaccinia virus inoculated rabbit inflamed skin extract (hereinafter sometimes referred to as “this extract”) is an extract containing non-protein active substances extracted and isolated from inflamed rabbit tissue inoculated with vaccinia virus. It is a thing. The extract is liquid in the extracted state, but can also be made solid by drying.

  A preparation containing this extract as an active ingredient (hereinafter sometimes referred to as “the present preparation”) is very useful as a pharmaceutical product, as described later. In this case, since this extract is an active ingredient of this formulation, this extract is the drug substance of this formulation. The specific product manufactured and sold by the applicant in Japan as this preparation is “Vaccinia virus inoculated rabbit inflammation skin extract-containing preparation” (trade name: Neurotropin / NEUROTROPIN (registered trademark), hereinafter referred to as “NTP preparation”) ) NTP preparations include injections (hereinafter referred to as “NTP injections”) and tablets (hereinafter referred to as “NTP tablets”), both of which are ethical drugs. Vaccinia virus inoculated rabbit inflammation skin extract (hereinafter referred to as “NTP extract”), which is an active ingredient of NTP preparation, is the drug substance of NTP preparation. NTP extract is included in this extract, and NTP preparations (NTP injections and NTP tablets) are included in this preparation.

  Indications for NTP injections are: “Low back pain, neck-shoulder arm syndrome, symptomatic neuralgia, pruritus associated with skin diseases (eczema, dermatitis, hives), allergic rhinitis, cold feeling / abnormalities of SMON aftereffects “Perception / Pain”. The indications for NTP tablets are “postherpetic neuralgia, low back pain, cervical-arm syndrome, peri-shoulderitis, osteoarthritis”. The NTP formulation was created by the applicant and developed as a pharmaceutical product. NTP preparations have been evaluated for their excellent effectiveness and safety, have been sold for many years, and have established a firm position in the Japanese pharmaceutical market. Currently, NTP products are exported to China and sold under the trade name “NEUROTROPIN”. The indications for NTP products in China are the same as in Japan.

  Thus, this preparation is very useful as a pharmaceutical product, and this extract is also very useful as a drug substance for this preparation. However, as described above, this extract was extracted and isolated from the inflamed skin tissue of rabbits inoculated with vaccinia virus. Therefore, the present extract contains a very large number of substances (components), and the present preparation produced using the extract also contains a very large number of substances (components). Therefore, how to control the quality of the extract and the preparation to be stable is a very important matter.

  Many pharmaceuticals are preparations containing one kind or at most two to three kinds of substances (components) as active ingredients, and these substances are usually chemically synthesized compounds. Therefore, if the content of the compound in the preparation is measured and a predetermined amount is contained, the quality in terms of the content of the active ingredient in the preparation is ensured. However, this extract is an extract from the inflamed skin tissue of rabbits inoculated with vaccinia virus and contains an extremely wide variety of substances. Of course, the present preparation containing the present extract as an active ingredient also contains a very wide variety of substances. As described above, since the present extract and the present preparation do not contain one or several kinds of specific substances as active ingredients, quality control should be performed as in the case of normal pharmaceuticals in which the active ingredients are specified as substances. I can't. Therefore, this extract and this preparation produced by the applicant, that is, quantitative determination of the active ingredient of the NTP extract and NTP preparation is performed by a method of measuring its biological activity (titer). .

The method is a biological test method for obtaining an analgesic factor using a SART stress (repeated cold load) animal, which is a chronic stress animal whose pain threshold is lower than that of a normal animal (“Nichi Pharmacology”, Vol. 72). No. 5, pp. 573-584, 1976). By the method described in this document, an analgesic coefficient is obtained by performing an analgesic test by the Randall-Selitto method using a SART stress (repeated cold load) animal that is a chronic stress animal whose pain threshold is lower than that of a normal animal. Ask for. In this method, pressure is applied to the tail of the mouse, and the analgesic effect is measured using the pressure weight until the mouse shows an escape reaction as an index. The analgesic coefficient is a value obtained by dividing the pressurized weight measured after administering a drug by the value before drug administration. For the NTP extract and NTP preparation, when the analgesic coefficient exceeds a certain value determined by the applicant, the analgesic effect is positive, the percentage of animals determined to be positive is obtained, and the analgesic effective rate (%) To do. From this value, the ED ₅₀ value is determined from the results of measurement of standard products diluted to various concentrations. “Neurotropin unit (NU)”, the unit of biological activity (titer) used by applicants for NTP preparations, refers to 1 mg of this extract with an ED ₅₀ value of 100 mg / kg (mouse body weight) The activity is defined as 1 neurotropin unit. And for each NTP formulations by measuring the ED ₅₀ values, which quantify the analgesic activity (active ingredient amount) as compared to the standard. Hereinafter, in the present application, the indication of “unit” is used as a measure of the amount of active ingredient (titer) of the present extract and the present preparation, but substantially, it is used in the NTP extract and NTP preparation. It has the same meaning as “neurotropin unit”.

By the way, this extract and this preparation manufactured by the applicant, that is, NTP extract and NTP preparation should be subjected to the following multiple confirmation tests in addition to the above analgesic activity quantification and should be adapted to these. Is stipulated. In other words, for the extract and the preparation produced by the applicant, the following lots of confirmation tests are conducted instead of ensuring that the production lot is appropriate only by the biological activity (titer). In order to meet these requirements, the extract and preparation should be used and shipped.
・ Amino acid confirmation test by liquid chromatographic method ・ Ultraviolet absorption substance confirmation test by ultraviolet visible absorbance measurement method ・ Phosphorus confirmation test by color reaction method ・ Nucleic acid base confirmation test by liquid chromatographic method ・ In vitro test method Confirmation test of kallikrein-like substance production inhibitory action

  Even if such a test is conducted, the amino acid, ultraviolet light absorbing substance, phosphorus, nucleobase, and the like, which are the subject of the test, are not the core active ingredients of the extract or the preparation. Of these tests, the confirmation tests for amino acids, UV-absorbing substances, phosphorus, and nucleobases are qualitative tests, as long as the test target amino acid is present, and how much is contained. It doesn't matter whether or not. Nonetheless, the applicants approved the manufacture of pharmaceuticals from the authorities as a measure to reduce the variation in quality among production lots and to ensure homogeneity in both the extract and the preparation with unknown active ingredients. It is the one that was determined in the get.

  Generally speaking, for those who produce extracts from organisms such as animals and plants, and preparations containing such extracts as active ingredients, the fewer standards that the preparations must conform to, This is advantageous because it does not require time, labor, and expense for the manufacturing process, and the possibility of non-conformity of the manufactured product is reduced. However, from the viewpoint of ensuring the quality of the extracts and preparations as described above, it is desirable that the standards to be adapted are more strictly defined. From this point of view, the present invention has been made as a result of intensive studies on the present extract and the preparation produced by the applicant as appropriate as a new standard to be met. That is, when the amount of N-acetylneuraminic acid contained in the extract and the preparation according to the present invention is measured and the content is within a certain range, the extract and the preparation are appropriate. In other words, it is handled as manufactured, that is, it is allowed to use or ship. Thereby, the dispersion | variation for every manufacturing lot of this extract and this preparation is further reduced, and quality becomes a more stable thing. This also ensures the effectiveness and safety of the extract and the preparation more strictly. If there is this extract or this preparation whose N-acetylneuraminic acid content deviates from a certain fixed range, this extract and It is possible to control the quality of this preparation to be more stable. That is, it is possible to set a voluntary or official standard for the amount of N-acetylneuraminic acid contained in the extract or the preparation. In addition, the applicant does not set a standard for the amount of N-acetylneuraminic acid contained in NTP extracts and NTP preparations produced so far, and measures the content, and the content Is confirmed to be within a certain range, and the NTP extract or NTP preparation is not used or shipped.

  Patent documents 1 to 3 exist as documents disclosing the present extract or the present preparation. These include a description of the content of amino acids and nucleobases in the extract or preparation. Similarly, regarding the present extract or the present preparation, Patent Document 4 describes the content of silicon. However, these Patent Documents 1 to 4 do not describe how much the extract or the preparation contains a specific substance called N-acetylneuraminic acid. Furthermore, in these documents, the content of N-acetylneuraminic acid in the extract or the preparation is used as an index for managing the quality of the extract or the preparation more stably. Neither listed nor suggested.

Chinese Patent Publication CN1205233A International Publication WO2004 / 060381 Chinese Patent Publication CN1613305A JP-A-7-97336

  As described above, in the present extract and the present preparation, no single substance has been identified as an active ingredient. Therefore, the quality of the extract and the preparation is ensured by a titer test by a biological test for obtaining an analgesic coefficient using a SART stress mouse, a plurality of confirmation tests, and the like. Nevertheless, it is inevitable that the various components contained in the present extract and the present preparation have considerable variations among production lots. However, as long as the preparation is used as a medicine for treating diseases, it is desirable that its quality be as constant as possible. Ensuring that the quality of the extract and the preparation is more stable leads to a more constant effectiveness and safety of the extract and the preparation, which is very significant. .

  The present invention ensures the quality of the extract and the preparation with the amount of N-acetylneuraminic acid contained in the extract and the preparation as an index. That is, the present invention provides the present extract, the present preparation, and the like that are appropriately produced by confirming that N-acetylneuraminic acid is contained in a prescribed amount.

  The present extract and the present preparation according to the present invention contain a predetermined amount of N-acetylneuraminic acid. Thereby, this extract and this formulation which concern on this invention can be handled as what was manufactured appropriately. The present invention contributes to ensuring that the quality of the extract and the preparation is more stably secured, and that the effectiveness and safety of the extract and the preparation are more constant.

  N-acetylneuraminic acid is a type of sialic acid. Sialic acid is a generic name for acyl derivatives of neuraminic acid, and there are many types of sialic acid in nature. Among them, N-acetylneuraminic acid is the most common, followed by N-glycolylneuraminic acid. Sialic acid is widely distributed in the body as a constituent of glycoproteins, glycolipids, glycopeptides, etc., and is particularly important on the surface of cell membranes of animals or bacteria and is involved in the specific recognition mechanism of cells. It is responsible for biological functions. In addition, sialic acid is regarded as medically and pharmacologically important as a substance involved in cancer, inflammation, immunity, viral infection, cell differentiation, hormone receptor, etc., and there are various sialic acid-containing biological components, sialic acid and its derivatives. Has been studied.

  With regard to its sialic acid, in particular N-acetylneuraminic acid, the applicant has surprisingly found that the substance is analgesic, in particular non-inflammatory pain diseases such as neuropathic pain disease. It has been found to have an effect on diseases), and a patent has been obtained based on such knowledge (Japanese Patent No. 4005115, Chinese Patent Notice CN101600438B). Since the characteristics of the NTP preparation manufactured and sold by the applicant is also a therapeutic effect for neuropathic pain, N-acetylneuramin as an index for managing the quality of the extract and the preparation as more stable Acid is a reasonable substance (component).

Next, the method for producing the present extract and the present preparation will be described.
This extract was obtained by inoculating rabbit skin with vaccinia virus and collecting the inflamed skin tissue, crushing it, adding extraction solvent, treating it, removing the tissue fragment, and deproteinizing it. It can be obtained by adsorbing to an adsorbent in acidic conditions and then eluting the active ingredient in basic conditions.

  Here, the vaccinia virus may be of any strain. Examples include Lister strains, Daren strains, Ikeda strains, EM-63 strains, New York City Board of Health strains, and the like.

  Moreover, any rabbit may be used as long as it belongs to the order of rabbit eyes. Examples include rabbits, rabbits (rabbits made from rabbits), rabbits (Japanese rabbits), rabbits, snow rabbits, and the like. Of these, chira rabbits are suitable for use. In Japan, there is a so-called rabbit that has been bred from the past and used widely as domestic animals or experimental animals. There are many varieties (breeds) of the rabbit, but varieties such as Japanese white varieties and New Zealand white varieties (New Zealand white) can be suitably used.

As a basic extraction process of the present extract, for example, the following processes are used.
(A) The skin tissue of rabbits inoculated with vaccinia virus intradermally is collected, the germed tissue is crushed, and an extraction solvent such as water, phenol water, physiological saline, or phenol-added glycerin water is added to the number. After performing the extraction process for one day, a crude extract (filtrate or supernatant) from which tissue pieces have been removed is obtained by filtration or centrifugation.
(B) The crude extract obtained in (A) is adjusted to an acidic pH, heated, filtered or centrifuged to remove protein. Next, the deproteinized solution is adjusted to a basic pH, heated, and further filtered or centrifuged to obtain a deproteinized filtrate or supernatant.
(C) The filtrate or supernatant obtained in (B) is adjusted to an acidic pH and adsorbed on an adsorbent such as activated carbon or kaolin.
(D) Add an extraction solvent such as water to the adsorbent obtained in (C), adjust to basic pH, and elute the adsorbed component to remove the inflammatory skin extract (this extract) inoculated with vaccinia virus. obtain.
The above is the basic process, and these processes are described in more detail as follows.

About (A) Inflamed skin tissue obtained by inoculating the skin of rabbits with vaccinia virus intradermally is collected. The collected skin tissue is washed and disinfected with a phenol solution. This inflamed skin tissue is crushed and 1 to 5 times the amount of extraction solvent is added. Here, crushing means crushing finely into a mince using a mincing machine or the like. In addition, as extraction solvents, distilled water, physiological saline, weakly acidic to weakly basic buffer solutions, and the like can be used. Sterilizers and preservatives such as phenol, stabilizers such as glycerin, sodium chloride, potassium chloride Further, salts such as magnesium chloride may be appropriately added. At this time, extraction can be facilitated by disrupting the cell tissue by treatment with freeze-thaw, ultrasound, cell membrane lytic enzyme, surfactant or the like. The resulting suspension is left for 5 to 12 days. Meanwhile, the mixture may be heated to 30 to 45 ° C. with or without stirring as appropriate. The obtained liquid is subjected to solid-liquid separation (filtration, centrifugation, etc.) to remove the tissue piece to obtain a crude extract (filtrate or supernatant).

About (B) A protein removal process is performed about the crude extract obtained by (A). Deproteinization can be carried out by known methods commonly used, such as heat treatment, treatment with a protein denaturant (for example, an organic solvent such as acid, base, urea, guanidine, acetone, etc.), isoelectric precipitation, salting out. Etc. can be applied. Subsequently, the insoluble protein that has been precipitated by a usual method for removing insoluble matters, for example, filtration using filter paper (cellulose, nitrocellulose, etc.), glass filter, celite, zeit filtration plate, etc., ultrafiltration, centrifugation, etc. A removed filtrate or supernatant is obtained.

About (C) The filtrate or supernatant obtained in (B) is adjusted to acidic, preferably pH 3.5 to 5.5, and the adsorption operation to the adsorbent is performed. Examples of usable adsorbents include activated carbon, kaolin, and the like. Add the adsorbent to the extract and stir, or pass the extract through an adsorbent-filled column to add the active ingredient to the adsorbent. Can be adsorbed. When an adsorbent is added to the extract, the adsorbent can be obtained by adsorbing the active ingredient by removing the solution by filtration or centrifugation.

About (D) In order to elute (desorb) the active ingredient from the adsorbent obtained in (C), an elution solvent is added to the adsorbent, and it is adjusted to basic, preferably pH 9 to 12, at room temperature or appropriately. Elution is carried out by heating or stirring, and the adsorbent is removed by a usual method such as filtration or centrifugation. As an elution solvent to be used, a basic solvent, for example, water adjusted to a basic pH, methanol, ethanol, isopropanol or the like, or an appropriate mixed solution thereof can be used, preferably water adjusted to pH 9 to 12. Can be used. The amount of the elution solvent can be appropriately set. In order to use the eluate thus obtained as a drug substance, the pH is appropriately adjusted to near neutral, etc. to finally obtain a vaccinia virus-inoculated rabbit inflammation skin extract (this extract). Can do.

  Since this extract is liquid at the time of completion, it can be made to have a desired concentration by appropriately concentrating and diluting. When manufacturing a formulation from this extract, it is preferable to heat-sterilize. In order to obtain an injection, for example, sodium chloride or the like can be added to prepare a solution that is isotonic with physiological saline. Moreover, oral solid preparations, such as a tablet, can also be manufactured by performing operation, such as concentration and drying suitable for this extract in a liquid state. Specific methods for producing such an oral solid preparation from this extract are described in the specifications of Japanese Patent Nos. 3818657 and 4883798. This preparation is an injection or a solid preparation for oral use thus obtained.

  The content of N-acetylneuraminic acid in the present extract or the present preparation can be measured by a commonly used quantitative method. Specifically, for example, a measurement method using a liquid chromatograph mass spectrometer (LC-MS) or a capillary electrophoresis mass spectrometer (CE-MS), DMB (1,2-diamino-4,5-methylenedioxybenzene) Fluorescent labeling method, enzymatic method, etc. can be used. In any method, the N-acetylneuraminic acid in the present extract or the present preparation can be quantified using a calibration curve prepared with a calibration N-acetylneuraminic acid sample.

  The content of N-acetylneuraminic acid contained in the present extract and the present preparation produced by the applicant as described above was measured. As a result, the extract and the preparation contained 4,000 ng or more of N-acetylneuraminic acid per unit, although there were variations. Furthermore, the extract and the preparation contained 4000-18000 ng N-acetylneuraminic acid per unit. Therefore, when the quality of the extract and the preparation was controlled using the content of N-acetylneuraminic acid in the extract and the preparation as an index, it was determined that such content could be provided as a reference. . The applicant confirmed that the content of N-acetylneuraminic acid may be less than 4000 ng per unit when there is a variation in the production method of the extract and the preparation.

  Here, “per unit” means, as understood from the above description, per content of the active ingredient in the present extract or the present preparation. The present extract (NTP extract) produced by the applicant contains 1.2 units / mL of active ingredient. The present preparation for injection (NTP injection) produced using this also contains 1.2 units / mL of the active ingredient. NTP injections are available in 3 mL and 1 mL volumes. Therefore, 3.6 mL of the active ingredient is contained in the 3 mL preparation, and 1.2 units of the active ingredient is contained in the 1 mL preparation. On the other hand, the present oral preparation (NTP tablet) manufactured by the applicant contains 4 units of active ingredient per tablet.

  By the way, the present extract can be concentrated or diluted. In addition, the present preparation can also be prepared containing various units. In such a case, the amount of the active ingredient contained in the extract or the amount of the preparation (per mL, per mg, per tube, per tablet, etc.) can also vary. Therefore, basically, it is meaningful to define the content of N-acetylneuraminic acid in relation to the amount of the active ingredient of the present extract or the present preparation. This is because it leads to a relationship with the effectiveness and safety of the extract and the preparation. Therefore, the applicant has defined the content of N-acetylneuraminic acid in the present extract or the present preparation per content of active ingredient ("per unit"). On the other hand, since the applicant currently manufactures and sells NTP preparations, it is possible to specify specific injections per mL and per tube, and per tablet, depending on the dose. Such provisions were also made because of their significance.

  By the way, NTP injections are generic drugs or similar drugs manufactured by companies other than the applicant (hereinafter referred to as “other companies”) in Japan and China (hereinafter referred to as “other company injections”). ")". Similarly to the NTP preparation, these other injections are also subjected to quantification of the inflammatory skin extract of rabbit skin inoculated with vaccinia virus (this extract), which is an active ingredient, using SART stress mice as an index of analgesic effect. In terms of labeling, the active ingredient content of these competitors' injections is “Rose Morgen Injection / ROSEMORGEN Inj.” (Registered trademark), “Nabutopin Injection / NABUTOPIN Inj.” (Registered) Trademark) and “NOLPORT Inj.” (Registered trademark), there are those that are simply indicated as “unit”, while other companies' injections in China, “ANALGECINE” (registered trademark) ) And “Analgecine unit” or “AGC unit” are also displayed. However, in any of these preparations, the active ingredient content is 1.2 units or 1.2 Analgecine (1.2AGC) units per mL as in the case of the NTP injection, and 3.6 units or 3.6 Analgecine ( 3.6AGC) unit. After all, "Neurotropin units" used in NTP preparations and "Units" and "Analgecine units" used by other companies differ only in the display method, and the same scale is used to regulate the content of active ingredients. It has become. Therefore, in the present application, “unit” is used as a display indicating the content of the present extract, which is an active ingredient, including not only NTP preparations but also injections of other companies. In this way, a preparation comprising a plurality of vaccinia virus-inoculated rabbit inflammation skin extracts as an active ingredient displays the amount of the active ingredient in “units”, and the “unit” indication for the preparation is clear to those skilled in the art.

  As is clear from the above, in the present application, “the present preparation” is a concept including NTP preparations (NTP injections and NTP tablets) and injections from other companies. In addition, when another company manufactures and sells tablets (hereinafter referred to as “other company tablets”) as generic products or similar products of NTP tablets, it is a concept that includes other company tablets.

  Hereinafter, specific examples of the production of the present extract and the present preparation, and the measurement results of the content of N-acetylneuraminic acid in the present extract and the present preparation are shown as examples. It is not limited at all.

Example 1 (Production of the present extract)
The skin of healthy mature rabbits was inoculated intradermally with vaccinia virus, and the cut skin was cut out and collected. The collected skin is washed and disinfected with a phenol solution, then the excess phenol solution is removed, crushed, the phenol solution is added and mixed, left to stand for 3 to 7 days, and then stirred for 3 to 4 days. Warmed to 35-40 ° C. Thereafter, the extract obtained by solid-liquid separation was adjusted to pH 4.5 to 5.2 with hydrochloric acid, heat-treated at 90 to 100 ° C. for 30 minutes, and then filtered to deproteinize. Further, the filtrate was adjusted to pH 9.0 to 9.5 with sodium hydroxide, heated at 90 to 100 ° C. for 15 minutes, and then separated into solid and liquid.

  The obtained deproteinized solution was adjusted to pH 4.0 to 4.3 with hydrochloric acid, activated carbon in an amount of 2% of the deproteinized solution mass was added and stirred for 2 hours, followed by solid-liquid separation. Water was added to the collected activated carbon, adjusted to pH 9.5 to 10 with sodium hydroxide, stirred at 60 ° C. for 90 to 100 minutes, and then centrifuged to obtain a supernatant. Water was added again to the activated carbon precipitated by centrifugation, and then adjusted to pH 10.5 to 11 with sodium hydroxide, stirred at 60 ° C. for 90 to 100 minutes, and then centrifuged to obtain a supernatant. Both supernatants were combined and neutralized with hydrochloric acid to obtain the present extract.

Example 2 (Method for measuring N-acetylneuraminic acid content)
The N-acetylneuraminic acid content of this extract and this preparation was measured with a high performance liquid chromatograph mass spectrometer (LC-MS) as follows.
The present extract prepared according to Example 1 (1.2 units / mL) was diluted 10-fold with water and injected into LC-MS.
This preparation (NTP injection) produced using this extract produced according to Example 1 was similarly diluted 10-fold with water and injected into LC-MS.
This preparation (NTP tablet) produced using this extract produced according to Example 1 was washed 3 times with 3 mL of methanol / chloroform (1: 1) 3 times to remove the film coat layer and dried. Thereafter, 12 mL of water was added to suspend the mixture (1 unit / mL). After centrifugation, the supernatant was diluted 10-fold with water and injected into LC-MS.
For N-acetylneuraminic acid, a standard solution of an aqueous solution was prepared to prepare a calibration curve.

For LC-MS, an Agilent 1100 series was used for the HPLC section, and API3000 (Applied Biosystems / MDS Sciex) was used for the mass spectrometer. The analysis conditions are as follows.
Column: Inertsil ODS-3 (φ2.1 × 150 mm)
Column temperature: 25 ° C., flow rate: 200 μL / min Mobile phase: methanol / 0.05% formic acid methanol% / min: 0 / 0-0 / 1-44 / 8-100 / 8.1-100 / 11
Injection volume: 5 μL, sample chamber set temperature: 4 ° C
Detection: Positive ion detection MRM
Table 1 shows the measurement conditions for LC-MS. The meaning of each parameter in Table 1 is as follows.
・ DP: Voltage applied to orifice plate ・ FP: Voltage applied to focus ring ・ CE: Collagen energy ・ CXP: Voltage applied to Q2 outlet ・ NEB: Nebulizer gas pressure ・ CUR: Curtain gas pressure ・ IS: Ion spray -CAD: pressure of the collage gas -TEM: turbo gas temperature

Example 3 (Measurement result of N-acetylneuraminic acid content of this extract)
Table 2 shows the results of measuring the content of N-acetylneuraminic acid in this extract by the method described in Example 2 above. The active ingredient content in this extract is 1.2 units / mL. The content of N-acetylneuraminic acid in the extract was expressed both per unit of the extract (“/ unit”) and per mL of the extract (“/ mL”). In addition, the symbols A to C in the lot number represent differences in the manufacturing location (facility) of the applicant. In addition, the measured values were aligned with 3 significant figures (hereinafter the same for all measured values).

Example 4 (Measurement result 1 of N-acetylneuraminic acid content of this preparation)
Table 3 shows the results of measuring the N-acetylneuraminic acid content in the NTP injection (containing 1.2 units per mL) among the present preparations by the method described in Example 2 above. The results are shown as the content per unit (“/ unit”) of the active ingredient of this preparation, 1 mL of this preparation (“/ mL”), and 1 ampule of 3 mL (“/ tube”). It was.

Example 5 (Measurement result 2 of N-acetylneuraminic acid content of this preparation)
Table 4 shows the results obtained by measuring the N-acetylneuraminic acid content in NTP tablets (containing 4 units per tablet) by the method described in Example 2 above. The results are shown in terms of each content of active ingredient per unit (“/ unit”) and per tablet (“/ tablet”).

Comparative Example 1 (Measurement result of N-acetylneuraminic acid content of other injections)
Next, Table 5 shows the results of measuring the content of N-acetylneuraminic acid in other companies' injections in the same manner as in NTP injections. “Rose Morgen Injection” has been sold at the time of filing of the present application and does not exist in the market. The following measurement results are the results obtained and measured by the applicant in the market in the past. .

  From the above results, it can be seen that even in the same vaccinia virus-inoculated rabbit inflammatory skin extract preparation, the content of N-acetylneuraminic acid varies greatly depending on the manufactured company (Tables 3 to 5). In addition, it can be seen that the content of N-acetylneuraminic acid may be considerably different even if it is a preparation of the same company including the applicant (Tables 3 to 5). Since vaccinia virus-inoculated rabbit inflammation skin extract, which is the drug substance for vaccinia virus-inoculated rabbit inflammation skin extract preparation, is not available from other companies, only those manufactured by the applicant (NTP extract) are measured. It was. And also about this, it turns out that there is a certain amount of variation in the content of N-acetylneuraminic acid (Table 2).

  However, the extract and the preparation produced by the applicant contained 4000 ng / unit or more of N-acetylneuraminic acid (Tables 2 to 4). Further, the extract and the preparation produced by the applicant contained N-acetylneuraminic acid in the range of 4000 to 22500 ng / unit. On the other hand, none of the preparations produced by other companies had such N-acetylneuraminic acid content (Table 5). Although it is not clear what causes the difference in the content of N-acetylneuraminic acid in the preparations manufactured by each company, it is strongly presumed that it is caused by the difference in the manufacturing method of the preparations in each company. In any case, since N-acetylneuraminic acid has an analgesic action, particularly an effect on non-inflammatory pain diseases such as neuropathic pain, it is as described above. It is understood that the present preparation (NTP injection) produced by the applicant is more characteristic than the other company's injection, which is a characteristic per se and a preferable characteristic.

  As described above, the presence of 4000 ng / unit or more of N-acetylneuraminic acid in the extract and the preparation is a feature of the extract and the preparation. Similarly, in the present extract and the present preparation, a method for measuring the content of N-acetylneuraminic acid in the present extract and the present preparation (injection, tablet) described in Example 2 (hereinafter referred to as “10-fold dilution method”). ) That N-acetylneuraminic acid is contained in the range of 4000 to 15000 ng / unit is a feature of the present extract and the present preparation. This is a measurement method (hereinafter referred to as “separate dilution method”) where the 10-fold dilution method with water is set to 10-fold, for example, with another dilution rate such as 50-fold dilution or 250-fold dilution with water. When the content of N-acetylneuraminic acid in the extract and the preparation is measured, the fact that N-acetylneuraminic acid is contained in the range of 4000 to 22500 ng / unit is a feature of the extract and the preparation. It will be. The reason why the upper limit is 1.5 times that of the 10-fold dilution method will be described later.

  In addition, among these preparations, for injections containing 1.2 units per mL (hereinafter referred to as “present injections”), it is indicated that N-acetylneuraminic acid is contained in an amount of 4800 ng / mL or more. It is a characteristic. Similarly, when measured by a 10-fold dilution method, the fact that N-acetylneuraminic acid is contained in the range of 4800 to 18000 ng / mL is a feature of this injection. When the content of N-acetylneuraminic acid in this injection is measured by another dilution method, the fact that N-acetylneuraminic acid is contained in the range of 4800 to 27000 ng / mL It will be. Furthermore, when the injection is in a 3 mL ampule (hereinafter referred to as “the 3 mL injection”), N-acetylneuraminic acid is 14400 ng / tube or more per tube The inclusion is a feature of the injection containing 3 mL. Similarly, when measured by a 10-fold dilution method, the fact that N-acetylneuraminic acid is contained in the range of 14400 to 54000 ng / tube is a feature of this 3 mL injection. When the content of N-acetylneuraminic acid in the injection containing 3 mL of this is measured by another dilution method, N-acetylneuraminic acid is contained in the range of 14400 to 81000 ng / tube. It is a characteristic of the agent. The reason why the upper limit is 1.5 times that of the 10-fold dilution method will also be described later.

  In addition, among the preparations, tablets containing 4 units per tablet (hereinafter referred to as “present tablets”) contain N-acetylneuraminic acid in an amount of 16000 ng / tablet or more. It will be. Similarly, when measured by a 10-fold dilution method, N-acetylneuraminic acid is contained in the range of 16000 to 60000 ng / tablet, which is a feature of this tablet. When the content of N-acetylneuraminic acid in this tablet is measured by another dilution method, it is a feature of this tablet that N-acetylneuraminic acid is contained in the range of 16000 to 90000 ng / tablet. It is. The reason why the upper limit is 1.5 times that of the 10-fold dilution method will also be described later.

  For this reason, the amount of N-acetylneuraminic acid contained in the extract and the preparation is measured, and whether the extract and the preparation are properly manufactured is controlled. Is possible. That is, the amount of N-acetylneuraminic acid contained in the present extract and the present preparation is measured, and if it is 4000 ng / unit or more, it can be appropriately produced. In addition, 4000 to 15000 ng / unit if the amount of N-acetylneuraminic acid contained in the extract and the preparation is measured by a 10-fold dilution method, 4000 to if it is measured by another dilution method It is possible to check whether the range is 22500 ng / unit and to make the extract and the preparation appropriately manufactured.

  In the case of this injection, measure the amount of N-acetylneuraminic acid contained in this injection, and if it is 4800 ng / mL or more, the said injection shall be appropriately manufactured. Is possible. In addition, if the amount of N-acetylneuraminic acid contained in the injection is measured and measured by a 10-fold dilution method, 4800 to 18000 ng / mL, if measured by another dilution method It is possible to confirm that the dose is in the range of 4800 to 27000 ng / mL and to make the present injection appropriately produced. Similarly, in the case of the injection containing 3 mL, the amount of N-acetylneuraminic acid contained is measured and it is 14400 ng / tube or more, or when it is measured by a 10-fold dilution method. 14400-54000ng / tube, if measured by another dilution method, it can be confirmed that it is in the range of 14400-81000ng / tube, and it is possible that the injection containing 3 mL of this is appropriately manufactured. is there.

  In the case of this tablet, the amount of N-acetylneuraminic acid contained in this tablet is measured, and if it is 16000 ng / tablet or more, it can be said that this tablet has been produced appropriately. is there. In addition, if the amount of N-acetylneuraminic acid contained in the tablet is measured by a 10-fold dilution method, 16000 to 60000 ng / tablet, and if measured by another dilution method, 16000 to 90000 ng / tablet. It is also possible to confirm that the range is within the range and to make the tablet appropriately manufactured.

  In addition, when the numerical value which corresponds to the upper limit when the content of the N-acetylneuraminic acid is shown in the range of the lower limit to the upper limit is measured by another dilution method, the measurement results obtained in Examples 3 to 5 ( The fact that it is considerably larger than Tables 2 to 4) (about 1.5 times) is due to the following reasons and grounds.

  That is, in the said Example, although content of N-acetylneuraminic acid is measured by LC-MS, N-acetylneuraminic acid is easy to receive the influence (ion suppression) of a contaminating ion. The present inventors basically measure a sample such as the present extract by diluting 10 times, but when diluted 50 times or 250 times, a value higher than the measurement value at the time of 10 times dilution is obtained. It was sometimes measured. This phenomenon was thought to be because the influence of the concentration of contaminating ions decreased when the dilution was high. As a result of comparing and verifying the quantitative values at each dilution ratio with a plurality of samples by the present inventors, when diluted 50 times or 250 times, a value of about 1.5 times at the maximum when diluted 10 times is measured. This was confirmed (Table 6). Based on this fact, in the present application, the numerical value corresponding to the upper limit of the content of N-acetylneuraminic acid was set as described above.

From the above, the following can be derived as the present invention. However, these are examples, and the present invention is not limited to these. In each of the inventions shown below, the phrase “measure the amount of N-acetylneuraminic acid” is usually described as N-acetylneuraminic acid for each production lot of the extract or the preparation. Is used to measure the amount of
(1) It is a rabbit inflammation skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4000 ng or more per unit of the extract A vaccinia virus inoculated rabbit inflamed skin extract that was properly manufactured by confirming.
(2) Rabbit inflammation skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4000-22500 ng per unit of the extract Rabbit inflammation skin extract inoculated with vaccinia virus, which was properly manufactured by confirming that.
(3) A rabbit inflammatory skin extract inoculated with vaccinia virus, which is a liquid, and the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4800 ng or more per 1 mL of the extract Vaccinia virus inoculated rabbit inflamed skin extract that is a liquid that has been properly manufactured by confirming that it is present.
(4) A rabbit inflammatory skin extract inoculated with vaccinia virus which is a liquid, the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4800-27000 ng per 1 mL of the extract A vaccinia virus inoculated rabbit inflamed skin extract that is a liquid that has been properly manufactured by confirming that
(5) Vaccinia virus inoculated rabbit A preparation containing an inflammatory skin extract, the amount of N-acetylneuraminic acid contained in the preparation was measured, and the content was determined to be vaccinia virus inoculated rabbit in the preparation A preparation containing an inflammatory skin extract inoculated with vaccinia virus, which was appropriately manufactured by confirming that it was 4000 ng or more per unit of the inflammatory skin extract.
(6) Vaccinia virus inoculated rabbit Inflammatory skin extract containing an extract of inflammatory skin, the amount of N-acetylneuraminic acid contained in the preparation was measured, and the content was determined to be vaccinia virus inoculated rabbit in the formulation Formulation containing inflammatory skin extract inoculated with vaccinia virus inoculated appropriately by confirming that it is 4000-22500 ng per unit of inflammatory skin extract.
(7) A vaccinia virus-inoculated rabbit containing a inflammatory skin extract, the amount of N-acetylneuraminic acid contained in the preparation being measured, and the content of the vaccinia virus-inoculated rabbit inoculated with vaccinia virus Formulation containing vaccinia virus inoculated rabbit inflammatory skin extract shipped as properly manufactured by confirming that it is 4000 ng or more per unit of inflammatory skin extract.
(8) A vaccinia virus-inoculated rabbit containing a inflammatory skin extract, the amount of N-acetylneuraminic acid contained in the preparation being measured, and the content of the vaccinia virus-inoculated rabbit inoculated with vaccinia virus Formulation containing vaccinia virus inoculated rabbit inflammatory skin extract shipped as properly manufactured by confirming 4000-22500 ng per unit of inflammatory skin extract.
(9) Vaccinia virus inoculated rabbit A preparation containing an inflammatory skin extract, the amount of N-acetylneuraminic acid contained in the preparation was measured, and the content was determined to be vaccinia virus inoculated rabbit in the preparation A preparation containing inflammatory skin extract inoculated with vaccinia virus that has been shipped to meet the standard of 4000 ng or more per unit of inflammatory skin extract.
(10) A preparation containing an inflammatory skin extract of a vaccinia virus-inoculated rabbit, the amount of N-acetylneuraminic acid contained in the preparation being measured, Formulation containing a vaccinia virus inoculated rabbit inflammatory skin extract shipped to meet the criteria of 4000-22500 ng per unit of inflammatory skin extract.
(11) An injection containing an inflammatory skin extract of rabbit vaccinia virus inoculated rabbit, the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 ng per 1 mL of the injection The injection containing the inflammatory skin extract inoculated with the vaccinia virus inoculated appropriately by confirming the above.
(12) An injection containing a inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content being 4800 per mL of the injection Injection containing vaccinia virus inoculated rabbit inflamed skin extract that was made properly by confirming that it was ~ 27000ng.
(13) An injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 ng per 1 mL of the injection An injection containing the inflammatory skin extract inoculated with vaccinia virus, which has been properly manufactured by confirming the above.
(14) An injection containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 per mL of the injection Injection containing vaccinia virus inoculated rabbit inflammation skin extract shipped as properly manufactured by confirming ~ 27000ng.
(15) An injection containing an inflammatory skin extract of rabbit vaccinia virus inoculated rabbit, the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 ng per 1 mL of the injection An injection containing an inflammatory skin extract of a rabbit inoculated with vaccinia virus that has been shipped satisfying the above criteria.
(16) An injection containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content being 4800 per mL of the injection Injection containing vaccinia virus inoculated rabbit inflammation skin extract shipped to meet the criteria of ~ 27000ng.
(17) A 3 mL injection containing a inflammatory skin inoculated rabbit inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content of the injection being the injection 3 mL injection containing vaccinia virus inoculated rabbit inflamed skin extract, which has been properly produced by confirming that it is at least 14400 ng per tube.
(18) A 3 mL-containing injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is determined by the injection 3 mL injection containing vaccinia virus inoculated rabbit inflammatory skin extract made appropriate by confirming 14400-81000 ng per tube.
(19) A 3 mL injection containing a inflammatory skin extract of rabbits inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content of the injection being the injection 3 mL injection containing vaccinia virus inoculated rabbit inflammation skin extract shipped as properly manufactured by confirming that it is at least 14400 ng per tube.
(20) A 3 mL injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is determined by the injection 3 mL injection containing vaccinia virus inoculated rabbit inflammation skin extract shipped as properly manufactured by confirming 14400-81000 ng per tube.
(21) A 3 mL injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is determined by the injection 3mL injection containing vaccinia virus inoculated rabbit inflammation skin extract shipped to meet the standard of 14400ng or more per tube.
(22) A 3 mL injection containing a vaccinia virus inoculated rabbit inflammation skin extract, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content of the injection being the injection 3 mL injection containing vaccinia virus inoculated rabbit inflammation skin extract shipped to meet the standard of 14400-81000 ng per tube.
(23) A tablet containing a skin inflammation extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 ng or more per tablet. A tablet containing an extract of inflammatory skin of rabbits inoculated with vaccinia virus, which has been properly manufactured by confirming that it is present.
(24) A tablet containing a skin inflammation extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 to 90000 ng per tablet A tablet containing an extract of inflammatory skin of rabbits inoculated with vaccinia virus, which was properly manufactured by confirming that
(25) A tablet containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 ng or more per tablet. Tablets containing vaccinia virus inoculated rabbit inflammation skin extract shipped as properly manufactured by confirming that there is.
(26) A tablet containing an extract of inflammatory skin of rabbits inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the tablet was measured, and the content was 16000-90000 ng per tablet A tablet containing vaccinia virus inoculated rabbit inflammation skin extract shipped as properly manufactured by confirming that.
(27) A tablet containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 ng or more per tablet. Tablets containing vaccinia virus inoculated rabbit inflammation skin extract shipped to meet certain criteria.
(28) A tablet containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 to 90000 ng per tablet Tablets containing vaccinia virus inoculated rabbit inflammation skin extract shipped to meet the criteria of
(29) The preparation according to any one of (5) to (10), which is an analgesic.
(30) The injection according to any one of (11) to (22), which is an analgesic.
(31) The tablet according to any one of (23) to (28), which is an analgesic.

(32) A rabbit inflammatory skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the extract being measured, and the content being 4000 ng or more per unit of the extract A rabbit inflammatory skin extract inoculated with vaccinia virus, characterized in that
(33) A rabbit inflammatory skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the extract being measured, and the content being 4000 to 22500 ng per unit of the extract A inflammatory skin extract inoculated with vaccinia virus, characterized in that it has been confirmed.
(34) A inflammatory skin extract inoculated with vaccinia virus inoculated as a liquid, wherein the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4800 ng or more per mL of the extract A rabbit inflammatory skin extract inoculated with vaccinia virus, which is a liquid characterized by being found.
(35) A inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus which is a liquid, the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4800-27000 ng per 1 mL of the extract A inflammatory skin extract inoculated with vaccinia virus, which is a liquid characterized by
(36) A preparation containing an inflammatory skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the preparation being measured, and the content of the extract being inoculated with vaccinia virus in the preparation A preparation containing inflammatory skin extract inoculated with vaccinia virus, characterized in that the amount is 4000 ng or more per unit of inflammatory skin extract.
(37) A vaccinia virus-inoculated rabbit containing a inflammatory skin extract, the amount of N-acetylneuraminic acid contained in the preparation being measured, and the content of the vaccinia virus-inoculated rabbit inoculated with vaccinia virus Formulation containing inflammatory skin extract inoculated with vaccinia virus, characterized in that it is confirmed to be 4000-22500 ng per unit of inflammatory skin extract.
(38) An injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 ng per 1 mL of the injection An injection containing an inflammatory skin extract of rabbits inoculated with vaccinia virus, characterized in that the above is confirmed.
(39) An injection containing an inflammatory skin extract of rabbit vaccinia virus inoculated rabbit, the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 per mL of the injection An injection containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, which was confirmed to be ˜27000 ng.
(40) A 3 mL injection containing a vaccinia virus inoculated rabbit inflammation skin extract, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content of the injection being the injection 3 mL injection containing vaccinia virus inoculated rabbit inflamed skin extract characterized by being confirmed to be 14400 ng or more per tube.
(41) An injection containing 3 mL of an inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is determined by the injection 3. 3 mL injection containing vaccinia virus inoculated rabbit inflammation skin extract characterized by 14400-81000ng per tube.
(42) A tablet containing a skin inflammation extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 ng or more per tablet. A tablet containing an inflammatory skin extract of rabbit inoculated with vaccinia virus, characterized in that it is confirmed to be present.
(43) A tablet containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000 to 90000 ng per tablet A tablet containing an inflammatory skin extract of rabbits inoculated with vaccinia virus, characterized in that
(44) The preparation according to any one of (36) to (37), which is an analgesic.
(45) The injection according to any one of (38) to (41), which is an analgesic.
(46) The tablet according to any one of (42) to (43), which is an analgesic.

(47) A preparation containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the preparation being measured before shipment, and the content of vaccinia in the preparation being determined A preparation containing, as an active ingredient, a vaccinia virus-inoculated rabbit inflammation skin extract, which is confirmed to be 4000 ng or more per unit of virus-inoculated rabbit inflammation skin extract.
(48) A preparation containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the preparation being measured before shipment, and the content of vaccinia virus in the preparation A preparation containing, as an active ingredient, a vaccinia virus-inoculated rabbit inflammation skin extract, characterized in that the amount is 4000-22500 ng per unit of virus-inoculated rabbit inflammation skin extract.
(49) An injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured before shipment, and the content is determined by the injection. An injection containing, as an active ingredient, a vaccinia virus-inoculated rabbit inflammation skin extract characterized in that it is confirmed to be 4800 ng or more per mL.
(50) An injection containing a inflammatory skin extract inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured before shipment, and the content is determined by the injection. An injection containing an inflammatory skin extract inoculated with vaccinia virus, which has been confirmed to be 4800 to 27000 ng per mL.
(51) A 3 mL injection containing a inflammatory skin extract inoculated with vaccinia virus, and measuring the amount of N-acetylneuraminic acid contained in the injection before shipment; An injection containing 3 mL containing a inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, characterized in that the amount was 14400 ng or more per tube.
(52) A 3 mL injection containing a inflammatory skin extract inoculated with vaccinia virus, and measuring the amount of N-acetylneuraminic acid contained in the injection before shipment; 3. 3 mL injection containing vaccinia virus-inoculated rabbit inflammation skin extract characterized by being confirmed to be 14400-81000 ng per tube.
(53) A tablet containing a inflammatory skin extract of rabbits inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the tablet measured before shipment, and the content per tablet A tablet containing a inflammatory skin extract inoculated with vaccinia virus, which has been confirmed to be 16000 ng or more, as an active ingredient.
(54) A tablet containing a inflammatory skin extract of rabbits inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the tablet measured before shipment, and the content per tablet A tablet containing an inflammatory skin extract of rabbit inoculated with vaccinia virus, characterized in that it was confirmed to be 16000-90000 ng.
(55) The preparation according to any one of (47) to (48), which is an analgesic.
(56) The injection according to any one of (49) to (52), which is an analgesic.
(57) The tablet according to any one of (53) to (54), which is an analgesic.

(58) The amount of N-acetylneuraminic acid contained in the inflammatory skin extract inoculated with vaccinia virus is measured, and when the content is 4000 ng or more per unit of the extract, the extract can be produced. A method for examining the extract, which is appropriately performed.
(59) Measurement of the amount of N-acetylneuraminic acid contained in a inflammatory skin extract inoculated with vaccinia virus, and production of the extract when the content is 4000 to 22500 ng per unit of the extract A method for inspecting the extract, wherein
(60) The amount of N-acetylneuraminic acid contained in the preparation containing vaccinia virus inoculated rabbit inflammation skin extract was measured, and the content was 1 unit of vaccinia virus inoculated rabbit inflammation skin extract in the preparation The method for inspecting the preparation, wherein the preparation is appropriately manufactured when the weight is 4000 ng or more.
(61) The amount of N-acetylneuraminic acid contained in the preparation containing the inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus was measured, and the content was 1 unit of the inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus in the preparation The method for inspecting the preparation, in which the preparation is appropriately produced when the amount is 4000 to 22500 ng.
(62) In the injection containing the inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 ng or more per 1 mL of the injection. A method for inspecting the injection, wherein the injection is appropriately manufactured in some cases.
(63) In an injection containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800-27000 ng per 1 mL of the injection In such a case, the method for inspecting an injection, wherein the injection is appropriately manufactured.
(64) In an injection containing 3 mL of vaccinia virus-inoculated rabbit inflammation skin extract, the amount of N-acetylneuraminic acid contained in the injection is measured, and the content of the injection is one tube of the injection. The method for testing an injection, wherein the injection containing 3 mL is appropriately manufactured when the amount is 14400 ng or more per unit.
(65) In a 3 mL injection containing a inflammatory skin extract of rabbit inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection was measured, and the content was 1 tube of the injection The method for inspecting an injection, wherein the production of the injection containing 3 mL is appropriately performed when the amount is 14400 to 81000 ng per unit.
(66) When the amount of N-acetylneuraminic acid contained in a tablet containing a vaccinia virus inoculated rabbit inflammation skin extract is measured and the content is 16000 ng or more per tablet And a method for inspecting the tablet.
(67) In a tablet containing a inflammatory skin extract of rabbit inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the tablet is measured, and the content is 16000-90000 ng per tablet A method for inspecting the tablet, wherein the tablet is appropriately manufactured.

(68) Use of the extract having a content of N-acetylneuraminic acid of 4000 ng or more per unit of the extract in the preparation of a preparation containing a inflammatory skin extract inoculated with vaccinia virus.
(69) Use of the extract having a N-acetylneuraminic acid content of 4000 to 22,500 ng per unit of the extract in the manufacture of a preparation containing a inflammatory skin extract inoculated with vaccinia virus.
(70) Use of the extract having a content of N-acetylneuraminic acid of 4800 ng or more per mL of the extract in the preparation of a preparation containing an inflammatory skin extract inoculated with vaccinia virus.
(71) Use of the extract having an N-acetylneuraminic acid content of 4800 to 27000 ng per mL of the extract in the manufacture of a preparation containing an inflammatory skin extract inoculated with vaccinia virus.

(72) The amount of N-acetylneuraminic acid contained in the preparation containing a vaccinia virus-inoculated rabbit inflammation skin extract was measured, and the content was determined to be vaccinia virus-inoculated rabbit inflammation skin in the preparation A method for shipping the preparation, wherein when it is confirmed that the amount is 4000 ng or more per unit of the extract, it is determined that the preparation can be shipped as properly manufactured.
(73) The amount of N-acetylneuraminic acid contained in the preparation containing a vaccinia virus-inoculated rabbit inflammation skin extract was measured, and the content was determined to be vaccinia virus-inoculated rabbit inflammation skin in the preparation A method for shipping the preparation, wherein it is determined that the preparation can be shipped as properly manufactured when it is confirmed that the amount is 4000 to 22500 ng per unit of the extract.
(74) The amount of N-acetylneuraminic acid contained in the injection containing the inflammatory skin extract of rabbit inoculated with vaccinia virus was measured, and the content was 4800 ng or more per mL of the injection A method for shipping the injection, wherein it is determined that the injection can be shipped as properly manufactured when it is confirmed that the injection is properly manufactured.
(75) The amount of N-acetylneuraminic acid contained in the injection containing the inflammatory skin extract of rabbit inoculated with vaccinia virus was measured, and the content was 4800-27000 ng per 1 mL of the injection When it is confirmed that the injection is shipped, it is determined that the injection can be shipped as properly manufactured.
(76) The amount of N-acetylneuraminic acid contained in the injection containing 3 mL of the vaccinia virus-inoculated rabbit inflammation skin extract was measured, and the content was one tube of the injection. A method of shipping the injection containing 3 mL, wherein it is determined that the injection containing 3 mL can be shipped as properly manufactured when it is confirmed that the amount is 14400 ng or more per unit.
(77) The amount of N-acetylneuraminic acid contained in the injection containing 3 mL of the vaccinia virus-inoculated rabbit inflammation skin extract was measured, and the content was 1 tube of the injection A method for shipping the injection containing 3 mL, wherein it is determined that the injection containing 3 mL can be shipped as properly manufactured when it is confirmed that the amount is 14400 to 81000 ng per unit.
(78) The amount of N-acetylneuraminic acid contained in the tablet containing the inflammatory skin extract inoculated with vaccinia virus is measured, and the content is 16000 ng or more per tablet. If the tablet is confirmed, it is determined that the tablet can be shipped as being properly manufactured.
(79) The amount of N-acetylneuraminic acid contained in the tablet containing a inflammatory skin extract inoculated with vaccinia virus is measured, and the content is 16000 to 90000 ng per tablet When it is confirmed that the tablet is shipped, it is determined that the tablet can be shipped as properly manufactured.
(80) A preparation containing the inflammatory skin extract of rabbit inoculated with vaccinia virus, shipped by the shipping method according to any one of (72) to (73).
(81) An injection containing a inflammatory skin extract inoculated with vaccinia virus that has been shipped by the shipping method according to any one of (74) to (77).
(82) A tablet containing the inflammatory skin extract inoculated with vaccinia virus, which was shipped by the shipping method according to any one of (78) to (79).

  As described above, the present invention measures the content of N-acetylneuraminic acid and confirms that it contains a predetermined amount, and the inflammatory skin extract inoculated with vaccinia virus inoculated appropriately was produced. Alternatively, a preparation containing the extract is provided. Further, the present invention confirms that the extract or preparation is appropriately produced by confirming that a predetermined amount of N-acetylneuraminic acid is contained in each production lot of the extract or preparation. An inspection method is provided. Since such extracts and preparations are produced using biological tissues, it is possible to more strictly ensure the constancy of quality for each production lot and are very useful.

Claims (15)

  A inflammatory skin extract inoculated with vaccinia virus, which measures the amount of N-acetylneuraminic acid contained in the extract and confirms that the content is 4000 ng or more per unit of the extract Rabbit inflammatory skin extract inoculated with vaccinia virus, which has been produced appropriately.   Rabbit inflammatory skin extract inoculated with vaccinia virus, measuring the amount of N-acetylneuraminic acid contained in the extract, and confirming that the content is 4000-22500 ng per unit of the extract Rabbit inflammation skin extract inoculated with vaccinia virus, which was properly produced by   A vaccinia virus-inoculated rabbit inflammation skin extract that is liquid, the amount of N-acetylneuraminic acid contained in the extract is measured, and the content is 4800 ng or more per mL of the extract Vaccinia virus inoculated rabbit inflamed skin extract that has been properly produced by confirmation.   A vaccinia virus inoculated rabbit inflammation skin extract that is liquid, the amount of N-acetylneuraminic acid contained in the extract is measured, and the content thereof is 4800-27000 ng per 1 mL of the extract A vaccinia virus inoculated rabbit inflamed skin extract that was properly manufactured by confirming.   A preparation containing a inflammatory skin extract inoculated with vaccinia virus inoculated rabbit, wherein the amount of N-acetylneuraminic acid contained in the preparation was measured, and the content was extracted from the inflamed rabbit skin inoculated with vaccinia virus in the preparation A preparation containing a vaccinia virus-inoculated rabbit inflammation skin extract that has been properly manufactured by confirming that it is 4000 ng or more per unit.   A preparation containing a inflammatory skin extract inoculated with vaccinia virus inoculated rabbit, wherein the amount of N-acetylneuraminic acid contained in the preparation was measured, and the content was extracted from the inflamed rabbit skin inoculated with vaccinia virus in the preparation A preparation containing a inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, which has been properly produced by confirming that it is 4000 to 22500 ng per unit.   An injection containing an inflammatory skin extract of rabbits inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800 ng or more per mL of the injection An injectable preparation containing an extract of inflammatory skin of rabbits inoculated with vaccinia virus, which was appropriately manufactured by confirming the above.   An injection containing an inflammatory skin extract of rabbit inflammation inoculated with vaccinia virus, wherein the amount of N-acetylneuraminic acid contained in the injection is measured, and the content is 4800-27000 ng per 1 mL of the injection An injection containing an extract of inflammatory skin of rabbits inoculated with vaccinia virus, which has been appropriately manufactured by confirming that it exists.   An injection containing 3 mL of an inflammatory skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content of the injection per tube of the injection 3 mL injection containing vaccinia virus inoculated rabbit inflamed skin extract that has been properly manufactured by confirming that it is 14400 ng or more.   An injection containing 3 mL of an inflammatory skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the injection being measured, and the content of the injection per tube of the injection 3mL injection containing vaccinia virus inoculated rabbit inflammation skin extract, which was made properly by confirming 14400-81000ng.   A tablet containing a inflammatory skin extract inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the tablet was measured, and the content was 16000 ng or more per tablet. A tablet containing a inflammatory skin extract of a rabbit inoculated with vaccinia virus that has been properly manufactured by confirmation.   A tablet containing an inflammatory skin extract of rabbit inoculated with vaccinia virus, the amount of N-acetylneuraminic acid contained in the tablet being measured, and the content being 16000-90000 ng per tablet A tablet containing a vaccinia virus inoculated rabbit inflamed skin extract, which has been properly manufactured by confirming the above.   The preparation according to claim 5 or 6, which is an analgesic.   The injection according to any one of claims 7 to 10, which is an analgesic.   The tablet according to claim 11 or 12, which is an analgesic.