JPS6116248B2 - - Google Patents
Info
- Publication number
- JPS6116248B2 JPS6116248B2 JP55097096A JP9709680A JPS6116248B2 JP S6116248 B2 JPS6116248 B2 JP S6116248B2 JP 55097096 A JP55097096 A JP 55097096A JP 9709680 A JP9709680 A JP 9709680A JP S6116248 B2 JPS6116248 B2 JP S6116248B2
- Authority
- JP
- Japan
- Prior art keywords
- cytochrome
- solid
- activity
- dextran
- aqueous solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 102100030497 Cytochrome c Human genes 0.000 claims description 64
- 108010075031 Cytochromes c Proteins 0.000 claims description 64
- 239000007787 solid Substances 0.000 claims description 31
- 238000002360 preparation method Methods 0.000 claims description 26
- 229920002307 Dextran Polymers 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 238000004108 freeze drying Methods 0.000 claims description 5
- 238000001694 spray drying Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 description 33
- 239000000203 mixture Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 239000000843 powder Substances 0.000 description 7
- 239000012265 solid product Substances 0.000 description 7
- 239000003708 ampul Substances 0.000 description 6
- 210000004165 myocardium Anatomy 0.000 description 6
- 239000000523 sample Substances 0.000 description 5
- 239000003381 stabilizer Substances 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 229940031703 low substituted hydroxypropyl cellulose Drugs 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108010075027 Cytochromes a Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- 238000007922 dissolution test Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 235000010265 sodium sulphite Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical group N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 101710170467 Cytochrome c-a Proteins 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000003677 abuse test Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000004898 kneading Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical class [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Description
本発明はチトクロームcの活用を維持する固型
製剤及びその製造方法に関する。
チトクロームcは、動物、高等植物、酵母、カ
ビなどに存在しエネルギー産出に関与する酵素で
細胞の動きを活発にする等重要な働きをしてい
る。
チトクロームcはタンパク質であり、そのため
熱、空気による酸化あるいは圧力、又は有機溶媒
等との接触などにより変性失活を受け易い。この
ため、その特異的な薬理作用を安定に保持する医
薬品を製剤化する方法が種々考案されるに至つて
いる。
従来のチトクロームc製剤は注射剤として製剤
化されてきたものであり、チトクロームcを含む
水溶液に亜硫酸塩、アスコルビン酸等の還元剤の
添加あるいはグリシン、グルタミン酸ソーダ等の
アミノ酸、アルブミン等のタンパク質の添加、更
には存在する空気をチツ素ガスで置換し封入した
アンプル化剤として供給される。
このチトクロームcの注射剤はその用法が静脈
内あるいは筋肉内投与に限られ、この場合、投与
後の血中濃度の速やかな低下のためその有効濃度
の維持が困難であり、またその排泄速度が著しく
速いという欠点が確認されている。
近年、チトクロームcが腸管壁から吸収される
場合にはその有効血中濃度の持続時間が長いこと
に着目し顆粒剤、錠剤等経口剤として製剤化する
方法の研究が多々なされている。
しかし、例えば錠剤化に際し、有機溶媒との練
合、造粒、乾燥、打錠、フイルムコート及び乾燥
の各工程に於いてチトクロームcが安定であるこ
とが要求され、また得られる錠剤に於いても、空
気との接触、含有水分等によつてチトクロームc
の変性失活の虞がある。このような条件での工程
に於ける安定性は前記従来の注射剤のそれとは全
く比較にならない。
このように顆粒剤、錠剤等の経口剤として製す
るためには、その原料たるチトクロームc原末自
体安定であるのみならず、その固型製剤化するに
充分に安定なものでなければならない。
なお、チトクロームcの固型製剤化の従来例と
しては昭和46年特許出願公告第8716号があり、ゼ
ラチンを安定化剤として使用する固型製剤及びそ
の製造方法が示されているが、製造工程が長く従
つてその所要時間が長く、またその製造操作が困
難で特殊な装置を必要とするなどの欠点を有す
る。
本発明は、上記した従来方法における欠点を解
消し、チトクロームcの活性を有効に維持する固
型製剤及びこの固型製剤を得る操作容易な工程の
製造方法を提供することを目的とする。
この目的を達成するために本発明では、安定な
チトクロームc固型製剤につき、乾燥した固型状
のデキストランによりチトクロームc分子が分散
して包被されてなることを特徴とする。
上記本発明に係るチトクロームc固型製剤は次
の製造方法によつて得られる。
その第1の製造方法は、チトクロームcとデキ
ストランとの混合水溶液を凍結乾燥することによ
る。例えば、チトクロームcの10〜30(重量)%
水溶液に20〜40(重量)%高分子デキストラン水
溶液を加え、さらに崩壊性を良くするために水練
合のできる崩壊剤を少量加え混合し、凍結乾燥し
チトクロームc固型物を得る。この固型物は公知
の方法により顆粒化又は打錠化される。
上記の製造工程中チトクロームc分子には加温
されず、またその乾燥工程に於いてもデキストラ
ンにより効果的に包被保護されているため、この
チトクロームcの活性はほぼ維持される。この活
性維持効果は後掲の第2表に示されるところであ
る。
また、第2の製造方法は、チトクロームcとデ
キストランとの混合水溶液を噴霧乾燥することに
よる。例えば、5〜20%チトクロームc水溶液に
5〜20%デキストラン水溶液を加えて混合し、ノ
ズル側での熱気流温度を100℃以下に維持して噴
霧し乾燥することによりチトクロームc固型物を
得る。この固型物は公知の方法により顆粒化又は
打錠される。
この方法の場合、噴霧乾燥機のノズル側の熱気
流温度が100℃以下に維持されることによつて、
品温が40℃以下に維持されるものである。なお、
この熱気流温度は品温及び乾燥状態に関し80℃前
後が好適である。これによつてチトクロームcの
活性はほぼ維持される。この活性維持効果は後掲
の第3表に示さるところである。
このようにチトクロームc固型製剤を組成する
ことにより、虐待試験による結果からも100日以
上の長期間に亘りチトクロームc活性の90%以上
を有効に持続させることができる。
本発明に於いてはデキストランの分子量につい
ては広範囲のものについて前記した効果を認める
ことができ、またデキストランに対するチトクロ
ームcの包含量は10〜20重量%のものが好適であ
る。
次の第1表には本発明に係るチトクロームc固
型製剤の組成例に於けるチトクロームcの活性残
存割合を他の組成例のものと比較して示した。
The present invention relates to a solid preparation that maintains the utilization of cytochrome c and a method for producing the same. Cytochrome c is an enzyme that is present in animals, higher plants, yeast, molds, etc., and is involved in energy production, and plays important roles such as activating cell movement. Cytochrome c is a protein and is therefore susceptible to denaturation and inactivation due to heat, oxidation by air, pressure, or contact with organic solvents. For this reason, various methods have been devised to formulate pharmaceuticals that stably retain their specific pharmacological actions. Conventional cytochrome c preparations have been formulated as injections, and are made by adding reducing agents such as sulfites and ascorbic acid to an aqueous solution containing cytochrome c, or adding amino acids such as glycine and monosodium glutamate, and proteins such as albumin. Furthermore, it is supplied as an ampoule agent in which the existing air is replaced with nitrogen gas and sealed. The usage of this cytochrome c injection is limited to intravenous or intramuscular administration, and in this case, it is difficult to maintain an effective concentration due to the rapid drop in blood concentration after administration, and its excretion rate is low. The drawback is that it is extremely fast. In recent years, attention has been focused on the fact that when cytochrome c is absorbed through the intestinal wall, its effective blood concentration lasts for a long time, and many studies have been conducted on methods of formulating it into oral preparations such as granules and tablets. However, for example, when making tablets, cytochrome c is required to be stable in each step of kneading with an organic solvent, granulation, drying, tabletting, film coating, and drying, and in the tablets obtained. Cytochrome c is also degraded by contact with air, moisture content, etc.
There is a risk of degeneration and deactivation. The stability in the process under such conditions is completely incomparable with that of the conventional injection preparations. In order to manufacture oral preparations such as granules and tablets in this manner, the raw material cytochrome c bulk powder itself must not only be stable, but also sufficiently stable to form solid preparations. In addition, as a conventional example of solid formulation of cytochrome c, there is Patent Application Publication No. 8716 of 1972, which describes a solid formulation using gelatin as a stabilizer and its manufacturing method, but the manufacturing process It has the drawbacks that the process is long and therefore requires a long time, and the manufacturing operation is difficult and requires special equipment. An object of the present invention is to eliminate the drawbacks of the conventional methods described above and to provide a solid preparation that effectively maintains the activity of cytochrome c, and a manufacturing method with easy-to-operate steps for obtaining this solid preparation. In order to achieve this object, the present invention provides a stable solid cytochrome c preparation, which is characterized in that cytochrome c molecules are dispersed and encapsulated in dry solid dextran. The above solid cytochrome c preparation according to the present invention can be obtained by the following manufacturing method. The first manufacturing method is by freeze-drying a mixed aqueous solution of cytochrome c and dextran. For example, 10-30% (by weight) of cytochrome c
A 20 to 40% (by weight) aqueous polymer dextran solution is added to the aqueous solution, and a small amount of a disintegrant that can be mixed with water is added to improve the disintegration properties.The mixture is then freeze-dried to obtain a cytochrome c solid. This solid product is granulated or tableted by a known method. During the above manufacturing process, cytochrome c molecules are not heated, and even during the drying process, they are effectively encapsulated and protected by dextran, so the activity of cytochrome c is almost maintained. This activity maintenance effect is shown in Table 2 below. Moreover, the second manufacturing method is based on spray drying a mixed aqueous solution of cytochrome c and dextran. For example, a 5-20% dextran aqueous solution is added to a 5-20% cytochrome c aqueous solution, mixed, sprayed and dried while maintaining the hot air flow temperature at the nozzle side at 100°C or less to obtain a cytochrome c solid. . This solid product is granulated or tableted by a known method. In this method, by maintaining the hot air flow temperature at the nozzle side of the spray dryer below 100℃,
The product temperature is maintained below 40℃. In addition,
The temperature of this hot air stream is preferably around 80°C in terms of product temperature and drying state. As a result, the activity of cytochrome c is almost maintained. This activity maintenance effect is shown in Table 3 below. By composing the cytochrome c solid preparation in this way, it is possible to effectively maintain 90% or more of the cytochrome c activity for a long period of 100 days or more, as shown by the results of the abuse test. In the present invention, the above-mentioned effects can be observed with a wide range of molecular weights of dextran, and the amount of cytochrome c included in dextran is preferably 10 to 20% by weight. Table 1 below shows the residual activity percentage of cytochrome c in composition examples of the solid cytochrome c preparation according to the present invention in comparison with other composition examples.
【表】【table】
【表】
この第1表に示す結果は、馬心筋から抽出精製
したチトクロームcにつき、その水溶液を、チト
クロームcのみの形態で凍結乾燥した固型物(No.
1)、チトクロームcと低分子デキストランとの
凍結乾燥による固型物(No.2及びNo.3)、チトク
ロームcと高分子デキストランとの凍結乾燥によ
る固型物(No.4及びNo.5)、またチトクロームc
水溶液に酸性亜硫酸ナトリウムを添加して凍結乾
燥した固型物(No.6)の各検体夫々について安定
性の試験の結果を活性残存割合で示したものであ
る。なお、この場合、チトクロームcの活性残存
割合の測定は、各試料をアンプル中に充填して密
封し、45℃の恒温器中に保存した状態で所定期間
経過後のものについて行なつた。またこの測定方
法は、チトクロームa濃度測定法に準じて分光学
的方法によつた。即ち、試料溶液を弱酸性カチオ
ン交換樹脂(アンバーライトCG−50)のカラム
でチトクロームcのみを精製し、ハイドロサルフ
アイトナトリウムによりチトクロームcを還元型
とし、セフアデツクス(G−25)を用いてゲル濾
過後、一定濃度(0.5mM)の溶液にチトクロー
ムaの一定量を加えて反応させ、チトクロームc
の酸化の初速度を波長50nmの吸光度減少として
読み取り、チトクロームcの標準品の値と比較す
る方法(「チトクロームの研究」、奥貫一男教授退
官記念会編、東京大学出版会、初版)によるもの
である。
第1表に示す結果から、チトクロームcのみの
固型物(No.1)では10日後に於いてすでに20%近
い活性の低下が認められ、150日経過後では変色
して活性度が半減しその有効使用が望めないのに
対し、本発明に係るチトクロームc固型物(No.2
〜No.5)では10日後に於いても活性度の初期値を
ほぼ維持し、更に150日経過後に於いても約90%
の活性度を維持しその活性度の安定性に優れてい
ることを確認することができ、またこの効果は従
来例である酸性亜硫酸ナトリウム添加物(No.6)
以上であることも判る。
上記した本発明に係るチトクロームc組成物を
原料とし、これに低置換度ヒドロキシプロピルセ
ルロース等の結合剤兼崩壊剤を用いて通常の方法
により打錠することによつて、チトクロームc活
性の維持効果の優れた錠剤を得ることができる。
この錠剤の溶出試験の結果を第1図に示す。こ
の溶出試験は、37℃の恒温蒸留水中で崩壊試験に
準じて上記の錠剤を溶出させたものであり、第1
図に示される溶解度曲線により、この錠剤の速や
かな溶解性を確認することができる。
従来、シヨ糖、乳糖、ブドウ糖などの低分子量
の糖がチトクロームc製剤の安定剤として適用さ
れてきたが、次の第2表及び第3表には本発明に
係る固型製剤と、安定剤として各種の低分子量糖
を夫々適用して得られた固型製剤とのチトクロー
ムc活性の安定性を残存活性割合により比較して
示した。[Table] The results shown in Table 1 are based on cytochrome c extracted and purified from horse heart muscle.The aqueous solution was freeze-dried in the form of only cytochrome c (No.
1) Solid products obtained by freeze-drying cytochrome c and low-molecular-weight dextran (No. 2 and No. 3), Solid products obtained by freeze-drying cytochrome c and high-molecular-weight dextran (No. 4 and No. 5) , and also cytochrome c
The results of the stability test for each solid sample (No. 6) obtained by adding acidic sodium sulfite to an aqueous solution and freeze-drying it are shown in terms of the percentage of remaining activity. In this case, the residual activity percentage of cytochrome c was measured after a predetermined period of time had elapsed with each sample filled into an ampoule, sealed, and stored in a thermostat at 45°C. This measurement method was based on a spectroscopic method similar to the cytochrome a concentration measurement method. That is, the sample solution was purified to only cytochrome c using a column of weakly acidic cation exchange resin (Amberlite CG-50), the cytochrome c was reduced to a reduced form with sodium hydrosulfite, and the sample solution was subjected to gel filtration using Sephadex (G-25). After that, a certain amount of cytochrome a is added to a solution of a certain concentration (0.5mM) and reacted, and cytochrome c
This method is based on the method of reading the initial rate of oxidation as the decrease in absorbance at a wavelength of 50 nm and comparing it with the value of a standard cytochrome c product ("Research on Cytochrome", edited by Professor Kazuo Okunuki Retirement Memorial Association, University of Tokyo Press, first edition). be. From the results shown in Table 1, the activity of the solid product containing only cytochrome c (No. 1) was already observed to decrease by nearly 20% after 10 days, and after 150 days, the activity changed by half and the activity decreased by half. In contrast, the cytochrome c solid material (No. 2) according to the present invention cannot be expected to be used effectively.
- No. 5) maintained almost the initial value of activity even after 10 days, and even after 150 days it remained at about 90%.
It was confirmed that the activity of the acidic sodium sulfite additive (No. 6) was maintained and the stability of the activity was excellent.
It turns out that the above is also true. The effect of maintaining cytochrome c activity is achieved by using the above-mentioned cytochrome c composition according to the present invention as a raw material and tableting it using a binder/disintegrant such as low-substituted hydroxypropyl cellulose by a conventional method. You can get excellent tablets. The results of the dissolution test for this tablet are shown in FIG. In this dissolution test, the above tablet was dissolved in constant-temperature distilled water at 37°C in accordance with the disintegration test.
The solubility curve shown in the figure confirms the rapid dissolution of this tablet. Conventionally, low molecular weight sugars such as sucrose, lactose, and glucose have been applied as stabilizers for cytochrome c preparations, but Tables 2 and 3 below show solid preparations according to the present invention and stabilizers. The stability of cytochrome c activity was compared with solid preparations obtained by applying various low molecular weight sugars as a percentage of residual activity.
【表】【table】
【表】
(残存活性%)
この第2表に示す結果は、第1表の結果により
得られた知見に基づき、本発明に係る固型製剤に
ついては、馬心筋から抽出精製したチトクローム
cの20%水溶液の2mlと20%デキストラン(分子
量;200000)水溶液10mlとを4℃で混合し、これ
をアンプル中に入れ凍結乾燥して粉末とし密封し
たものを乾燥固型物の検体となし、またこの乾燥
固型物の前記粉末剤に賦形剤を混合し、この混合
剤を後記の条件で打錠して得た錠剤(錠剤特性;
重量200mg/錠、チトクロームc含量5mg/錠)
を検体とし、またシヨ糖、乳糖、ブドウ糖を夫々
安定剤とするチトクロームc固型製剤についても
上記した本発明の固型製剤と全く同一の条件下で
製した乾燥固型物及び錠剤を夫々検体とし、これ
らの検体を45℃の保温器中で保存して得られた結
果である。[Table] (Residual activity%)
The results shown in Table 2 are based on the knowledge obtained from the results in Table 1, and for the solid preparation according to the present invention, 2 ml of a 20% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 20% dextran (molecular weight: 200000) and 10 ml of an aqueous solution at 4°C, this was put into an ampoule, lyophilized, sealed as a powder, and the dried solid sample was used. and excipients, and tablets obtained by compressing this mixture under the conditions described below (tablet characteristics;
Weight 200mg/tablet, cytochrome c content 5mg/tablet)
The samples were dried solid products and tablets produced under exactly the same conditions as the above-mentioned solid preparations of the present invention for cytochrome c solid preparations containing sucrose, lactose, and glucose as stabilizers, respectively. These results were obtained by storing these specimens in a 45°C incubator.
【表】
(残存活性%)
またこの第3表に示す結果は、第1表の結果に
より得られた知見に基づき、本発明に係る固型製
剤については、馬心筋から抽出精製したチトクロ
ームcの10%水溶液50mlと5%デキストラン(分
子量176000)の水溶液1000mlを室温で混合し、ノ
ズル(入口)側80℃、出口側50℃、受器30℃に冷
却した噴霧乾燥機により噴霧乾燥して得た粉末を
アンプル中に密封したものを乾燥固型物の検体と
なし、またこの乾燥固型物の前記粉剤に賦形剤を
混合し、この混合剤を下記の条件で打錠して得た
錠剤(錠剤特性;重量200mg/錠、チトクローム
c含量5mg/錠)を検体とし、またシヨ糖、乳
糖、ブドウ糖を夫々安定剤とするチトクロームc
固型製剤についても上記した本発明の固型製剤と
全く同一条件下で製した乾燥固型物及び錠剤を
夫々検体とし、これらの検体を45℃の保温器中で
保存して得られた結果である。
なお、上記した各場合の打錠条件は、
(a) 打錠臼杵径;7.0mmφ
(b) 打錠圧力
予備圧;1200Kg/cm2
本圧 ; 800Kg/cm2
(c) 打錠速度; 300錠/分
であつた。
これら第2表及び第3表にて示す結果から、本
発明に係る固型製剤は特に打錠に際してチトクロ
ームc活性の安定性、さらに経時的な安定性にも
優れていることを確認でき、この点に於いて、低
分子量糖が適用される場合と著しい作用、効果の
差異がある。
上述したように、本発明はチトクロームcの活
性を製造段階及び保存状態で経時的に安定して維
持することができる固型製剤、及び簡単かつ迅速
な生産工程による製造方法を提供する。〔実施例
1〕
第1表の結果により得られた知見に基づき、馬
心筋から抽出精製したチトクロームcの20%水溶
液2mlと20%デキストラン(分子量;200000)水
溶液10mlとを4℃の温度条件下で混合し、アンプ
ル中に入れて凍結乾燥して粉末とし密封した。こ
のアンプルを所要数作り、50℃保温器中で保存し
たときのチトクロームcの残存活性を次の第4表
に示す。[Table] (Residual activity%)
The results shown in Table 3 are based on the knowledge obtained from the results in Table 1, and the solid preparation according to the present invention contains 50 ml of a 10% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 5% dextran. (molecular weight 176,000) was mixed at room temperature and spray-dried in a spray dryer cooled to 80°C on the nozzle (inlet) side, 50°C on the outlet side, and 30°C on the receiver, and the obtained powder was sealed in an ampoule. A sample of the dry solid was prepared by mixing an excipient with the powder of the dry solid, and tableting the mixture under the following conditions (tablet characteristics: weight 200 mg/ Tablet, cytochrome c content 5 mg/tablet) was used as the sample, and sucrose, lactose, and glucose were used as stabilizers.
Regarding solid preparations, the results obtained were obtained by using dried solid products and tablets produced under exactly the same conditions as the above solid preparations of the present invention as specimens, and storing these specimens in a heat insulator at 45°C. It is. The tableting conditions for each of the above cases are: (a) Tableting die diameter; 7.0 mmφ (b) Tableting pressure Preliminary pressure: 1200 Kg/cm 2 pressure; 800 Kg/cm 2 (c) Tableting speed; 300 It was a tablet/minute. From the results shown in Tables 2 and 3, it can be confirmed that the solid preparation according to the present invention has excellent stability of cytochrome c activity, especially during tableting, and also stability over time. In this respect, there is a significant difference in action and effect from when low molecular weight sugars are applied. As described above, the present invention provides a solid preparation capable of stably maintaining the activity of cytochrome c over time during manufacturing and storage conditions, and a manufacturing method using a simple and rapid production process. [Example 1] Based on the findings obtained from the results in Table 1, 2 ml of a 20% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 10 ml of a 20% dextran (molecular weight: 200,000) aqueous solution were mixed at a temperature of 4°C. The mixture was mixed in an ampoule, freeze-dried, and sealed as a powder. The remaining activity of cytochrome c when the required number of ampoules were made and stored in a 50°C incubator is shown in Table 4 below.
【表】
(残存活性%)
〔実施例 2〕
第1表の結果により得られた知見に基づき、馬
心筋から抽出精製したチトクロームcの25%水溶
液20mlと25%デキストラン(分子量176000)100
mlを室温下で混合し、さらに低置換度ヒドロキシ
プロピルセルロース(LH−31)5gを均一に分
散し、これを凍結乾燥し、その後低温下で粉砕又
は造粒し、次にこれに賦形剤を混合し打錠臼杵径
7.0mmφ、打錠圧力;予備圧1200Kg/cm2、本圧800
Kg/cm2、打錠速度 300錠/分の条件で打錠し錠
剤(重量200mg/錠、チトクロームc含量5mg/
錠)を得た。この錠剤を45℃の保温器中で保存し
たときのチームcの残存活性を次の第5表に示
す。[Table] (Residual activity%)
[Example 2] Based on the knowledge obtained from the results in Table 1, 20 ml of a 25% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 100 ml of 25% dextran (molecular weight 176,000)
ml at room temperature, further disperse 5 g of low-substituted hydroxypropyl cellulose (LH-31) uniformly, freeze-dry this, then crush or granulate at low temperature, and then add excipients to this. Mix and tablet with mortar diameter
7.0mmφ, tableting pressure; preliminary pressure 1200Kg/cm 2 , main pressure 800
Kg/cm 2 , tableting speed 300 tablets/min (weight 200 mg/tablet, cytochrome c content 5 mg/min)
Tablets) were obtained. The residual activity of team c when this tablet was stored in a 45°C incubator is shown in Table 5 below.
【表】
(残存活性%)
〔実施例 3〕
第1表の結果により得られた知見に基づき、馬
心筋から抽出精製したチトクロームcの10%水溶
液50mlと5%デキストラン(分子量176000)水溶
液1000mlを室温で混合し、ノズル(入口)側80
℃、出口側50℃、受器30℃に冷却した噴霧乾燥機
により噴霧乾燥して粉末を得た。なお、このと
き、品温は40℃であつた。この原末をアンプル中
に封入して50℃の保温器中で保存したときのチト
クロームcの残存活性を次の第6表に示す。[Table] (Residual activity%)
[Example 3] Based on the knowledge obtained from the results in Table 1, 50 ml of a 10% aqueous solution of cytochrome c extracted and purified from horse heart muscle and 1000 ml of a 5% dextran (molecular weight 176,000) aqueous solution were mixed at room temperature, ) side 80
A powder was obtained by spray drying in a spray dryer cooled to 50°C on the outlet side and 30°C on the receiver. At this time, the product temperature was 40°C. The residual activity of cytochrome c when this bulk powder was sealed in an ampoule and stored in a 50°C incubator is shown in Table 6 below.
【表】
(残存活性%)
[Table] (Residual activity%)
第1図……本発明に係るチトクロームc錠剤の
溶解度を示すグラフ。
図面符号の説明、A……溶解度曲線。
FIG. 1: A graph showing the solubility of cytochrome c tablets according to the present invention. Explanation of drawing symbols: A: Solubility curve.
Claims (1)
ロームc分子が分散して包被されてなることを特
徴とする安定なチトクロームc固型製剤。 2 チトクロームcとデキストランとの混合水溶
液を凍結乾燥することを特徴とする安定なチトク
ロームc固型製剤の製造方法。 3 チトクロームcとデキストランとの混合水溶
液を噴霧乾燥することを特徴とする安定なチトク
ロームc固型製剤の製造方法。[Scope of Claims] 1. A stable solid cytochrome c preparation, characterized in that cytochrome c molecules are dispersed and encapsulated in dry solid dextran. 2. A method for producing a stable cytochrome c solid preparation, which comprises freeze-drying a mixed aqueous solution of cytochrome c and dextran. 3. A method for producing a stable cytochrome c solid preparation, which comprises spray-drying a mixed aqueous solution of cytochrome c and dextran.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9709680A JPS5721315A (en) | 1980-07-15 | 1980-07-15 | Stable solid preparation of cytochrome c and its preparation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP9709680A JPS5721315A (en) | 1980-07-15 | 1980-07-15 | Stable solid preparation of cytochrome c and its preparation |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS5721315A JPS5721315A (en) | 1982-02-04 |
JPS6116248B2 true JPS6116248B2 (en) | 1986-04-28 |
Family
ID=14183092
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP9709680A Granted JPS5721315A (en) | 1980-07-15 | 1980-07-15 | Stable solid preparation of cytochrome c and its preparation |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS5721315A (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6290991B1 (en) | 1994-12-02 | 2001-09-18 | Quandrant Holdings Cambridge Limited | Solid dose delivery vehicle and methods of making same |
US20060174691A1 (en) * | 2005-02-07 | 2006-08-10 | David Chazan | Method of controlling degradation of trace gas sensors |
-
1980
- 1980-07-15 JP JP9709680A patent/JPS5721315A/en active Granted
Also Published As
Publication number | Publication date |
---|---|
JPS5721315A (en) | 1982-02-04 |
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