1281404 A7 經濟部智慧財產局員工消費合作社印製 Β7 五、發明說明（）） 水溶液的組成物之成分。添加齊的實施例係爲水溶性的蛋 白質，例如血淸白蛋白，如人類血淸白蛋白（在此以後稱 爲H S A )，其選擇性地以重組形式存在，酵素以及磷酸 甘油酯，例如磷脂醯乙醇胺、磷脂醯膽鹼、磷脂醯絲氨酸 、磷脂醯肌醇。 較佳地，本發明的組成物具有的水含量低於10% 、 較佳爲低於5% 、更佳爲低於1% (重量/重量）。 較佳地，本發明的組成物具有的MA S含量低於1 % 、較佳爲低於0 · 1 %、更佳爲低於0 · 0 5 % (重量/重量 )° 較佳地，本發明的組成物具有的添加劑含量高於9 9 %、更佳爲高於9 9.9% (重量/重量）。 本發明的較佳組成物係爲那些可被一種不含或僅含低 濃度的有機溶劑的水性介質處理而得到一含有MA S的溶 液者。較佳地，這些水性介質含有低於1%、較佳低於〇 · 5%、更佳低於0.1%的有機溶劑（重量/重量）。 稍早之前，一些欲製備符合此需求的嘗試已失敗。 在此，MA S—詞係表示調節卵細胞減數分裂之化合 物。更特定而言，MA S s係爲下述實施例1所述測試中 具有明顯高於對照組的胚泡破裂百分率（在此以後稱爲G V B )之化合物。較佳的MA S係爲那些具有G V B百分 率至少50% 、較佳至少80%者。較佳的MAS之實施 例係爲4,4-二甲基-5α-膽固-8，H，24-三烯-3β-醇（在此以後 稱爲 FF—MAS) ; 4,4-二甲基-5α-膽固-8，14,24-三烯- 4 ----^„------------訂---------^ (請先閱讀背面之注意事項再填窝本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 經濟部智慧財產局員工消費合作社印製 1281404 A/ ______B7 五、發明說明（3 ) 3β-醇半琥珀酸酯；5α_膽固-8，14 _二烯-3β-醇；5α-膽固-8，14 -二烯-3β-醇半琥珀酸酯；（20S)-膽固-5-烯-3β，20-二醇 ;3 β-經基-4,4- 一甲基·5α-膽固-8，14 -—嫌- 24-酸-Ν-(甲硫 胺酸)醯胺；以及膽固-5-烯-16β-醇。MA Ss的其他實施例 係在WO 96/0 0 235、96/27658、97/ 00884、98/28323、98/54965 以及 98/55498中提及，更明確地在其申請專利範圍第 1項中。 一種製備本發明的組成物之方法係將於有機溶劑例如 乙醇中的MA S溶液與一添加劑的水溶液混合，之後等到 該溶劑蒸發爲止。可使用連續氣流於該產品之上、真空或 任何其他可行方法來移除該溶劑而加速蒸發作用。市面販 售的產品可以一種具有一或多個凹陷（depressions)或洞 (hollows)的運送系統，在此以後，凹陷或洞係相互地以凹 洞稱呼。這些凹洞的至少一個係含有一種根據本發明的組 成物。一種放置其中的固體MA S之方便方法係首先將一 種含有MA S以及添加劑之溶液放置於凹洞中且之後使該 ι 溶液蒸發，以此種方法，蒸發的殘留物（即根據本發明的 組成物）係直接至於該裝置（傳送系統）中的凹洞內。 由於本發明的組成物係用於處理卵細胞，故本發明的 組成物不含有負面影響卵細胞的成分係爲重要的。 使用本發明的組成物之一種方法係微將該組成物溶解 於一水性介質例如水中，若需要時，加入可對卵細胞成熟 具有有利影響之其它成分。 5 .. -------^--------- (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） A7 1281404 _B7 ___ 五、發明說明（4 ) 使用該組成物的其他方法係爲將其溶解於一種一般用 於活體外成熟的介質中。 本發明係進一部由下列實施例來說明，但是這些實施 例係不應被闡述爲限至保護的範圍。先前敘述以及下列實 施例所揭示的特性可以其任何組合方式爲瞭解呈現其不同 形式之本發明的基礎。 實施例1 用於測定一化合物是否爲MA S之方法 卵細胞係得自重量爲13至16公克之未成熟的雌性老 鼠（C57BL/6J X DBA/2J Fl，Bomholtgaard，Denmark) ，其維持在受控制的溫度（2 0 - 2 2°C)、光線（光罩 於06.00— 18.0 0)以及相對溼度（5 0 - 7 0% ) 下。該等老鼠係接受0.2毫升之含有2 0 I U F § Η的 促性腺激素之腹膜內注射，且在4 8小時之後，該等動物 係以頸部錯位(cervical dislocation)的方式被殺死。將卵巢 切開，且在立體顯微鏡下藉由使用一對2 7標準規格 (gauge)針頭人爲操作使濾泡破裂而將卵細胞分離於Η X-介 質中（參見如下所述）。表現完整胚泡（在此以後稱爲G V )之球狀卵細胞係分爲小丘(cumulus)包圍的卵細胞（在 此以後稱爲C E〇）以及裸露卵細胞（在此以後稱爲N〇 )，且使其等置於α-最小基礎培養基（不具核糖核苷之心 Μ Ε Μ，Gibco BRL，Cat· No· 22561 )，該培養基係補充 3 毫克/毫升的牛血淸白蛋白（B S A，Sigma Cat. No. A-7030 )、5毫克/毫升的人類血淸白蛋白（HS A，State 6 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ^—.------------^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 1281404 B7 五、發明說明（女）1281404 A7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Β7 V. Invention description ()) The composition of the composition of the aqueous solution. The added embodiment is a water-soluble protein, such as blood albumin, such as human blood albumin (hereinafter referred to as HSA), which is selectively present in recombinant form, enzymes and phosphoglycerides such as phospholipids.醯Ethanolamine, phospholipid choline, phospholipid 醯 serine, phospholipid 醯 inositol. Preferably, the composition of the present invention has a water content of less than 10%, preferably less than 5%, more preferably less than 1% (weight/weight). Preferably, the composition of the present invention has a MA S content of less than 1%, preferably less than 0.1%, more preferably less than 0.5% (weight/weight). Preferably, The composition of the invention has an additive content of more than 99%, more preferably more than 9.9% (weight/weight). Preferred compositions of the invention are those which are treated with an aqueous medium which contains no or only a low concentration of organic solvent to provide a solution containing MAS. Preferably, these aqueous media contain less than 1%, preferably less than 5%, more preferably less than 0.1%, of organic solvent (weight/weight). Earlier attempts to prepare for this requirement have failed. Here, the MA S-word system refers to a compound that regulates egg cell meiosis. More specifically, the MA S s is a compound having a blastocyst rupture percentage (hereinafter referred to as G V B ) which is significantly higher than that of the control group in the test described in the following Example 1. Preferred MA S systems are those having a G V B percentage of at least 50%, preferably at least 80%. A preferred embodiment of MAS is 4,4-dimethyl-5α-cholester-8,H,24-triene-3β-ol (hereinafter referred to as FF-MAS); 4,4-di Methyl-5α-cholester-8,14,24-triene- 4 ----^„------------ order---------^ (please first Read the notes on the back and fill in the nest. This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm). Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1281404 A/ ______B7 V. Invention description (3 3β-alcohol hemisuccinate; 5α_cholester-8,14-diene-3β-ol; 5α-cholester-8,14-diene-3β-alcoholic semisuccinate; (20S)-biliary固-5-ene-3β,20-diol; 3 β-radio-4,4-methyl-5α-cholester-8,14------------methionine Guanamine; and cholesterol-5-ene-16β-alcohol. Other examples of MA Ss are in WO 96/0 0 235, 96/27658, 97/00884, 98/28323, 98/54965, and 98/55498. Mention is made more specifically in the scope of claim 1 of the patent application. A method of preparing the composition of the present invention is to mix an MA S solution in an organic solvent such as ethanol with an aqueous solution of an additive, and then wait until The agent may evaporate using a continuous stream of gas over the product, vacuum or any other feasible means to accelerate the evaporation. Commercially available products may have one or more depressions or holes (hollows) The transport system, after which the depressions or caves are referred to each other by a recess. At least one of the recesses contains a composition according to the invention. A convenient method of placing the solid MA S therein is first A solution containing MA S and an additive is placed in a cavity and then the solution is evaporated, in such a way that the evaporated residue (ie the composition according to the invention) is directly concave into the device (transport system) Since the composition of the present invention is for treating egg cells, it is important that the composition of the present invention does not contain a component which adversely affects egg cells. One method of using the composition of the present invention is to microly dissolve the composition. An aqueous medium such as water, if necessary, is added to other ingredients which have a beneficial effect on egg cell maturation. 5 .. -------^------ --- (Please read the note on the back and fill out this page.) This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 1281404 _B7 ___ V. Description of invention (4) Use of this composition The other method is to dissolve it in a medium that is generally used for in vitro maturation. The invention is further illustrated by the following examples, which are not to be construed as limited. The foregoing description, as well as the features disclosed in the following examples, may be used in any combination to understand the basis of the invention in its various forms. Example 1 Method for determining whether a compound is MA S The egg cell line was obtained from an immature female mouse (C57BL/6J X DBA/2J Fl, Bomholtgaard, Denmark) weighing 13 to 16 grams, which was maintained under control. Temperature (2 0 - 2 2 ° C), light (photomask at 06.00 - 18.0 0) and relative humidity (50 - 70%). The mice received an intraperitoneal injection of 0.2 ml of gonadotropin containing 20 I U F § ,, and after 48 hours, the animals were killed in a cervical dislocation. The ovaries were dissected and the egg cells were separated into ΗX-media by a human microscope using a pair of 27 gauge gauge needles (see below). A globular egg cell line exhibiting a complete blastocyst (hereinafter referred to as GV) is divided into an egg cell surrounded by cumulus (hereinafter referred to as CE〇) and a naked egg cell (hereinafter referred to as N〇), and The cells were placed in α-minimum basal medium (not ribonucleosides, Gibco BRL, Cat. No. 22561) supplemented with 3 mg/ml bovine serum albumin (BSA, Sigma Cat). No. A-7030 ), 5 mg/ml human blood albumin (HS A, State 6 This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) ^—.---- --------^--------- (Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed Economy Ministry Intellectual Property Bureau Staff Consumer Cooperatives System 1281404 B7 V. Description of invention (female)
Serum Institute，Denmark)、〇·2 3mM 的丙酮酸鹽（ Sigma，Cat· No S-8636)、2 mM的榖醯胺（Flow Cat· No. 16-801)、1 0 0 I U/毫升的盤尼西林以及100微克/ 毫升的鏈黴素（Flow Cat· No· 16-700)。此培養基係補充 3 m Μ的次黃嘌呤（Sigma Cat. No· H-9377)且稱爲Hx-培 養基。 該等卵細胞係於Η X -培養基中沖洗三次，且相同尺 寸的卵細胞係分成C Ε〇以及Ν〇組。將C Ε〇以及Ν〇 培養於四個凹槽的多重培養皿（Nunclon，Denmark)中， 其中每一個凹槽包括0 ·4毫升的Η X -培養基以及濃度爲 1 〇μΜ之欲測試的化合物。一個對照組凹槽（即培養於未 添加測試化合物的相同培養基中3 5至4 5個卵細胞）係 通常與三個測試凹槽（即添加測試化合物的每一個凹槽係 有3 5至4 5個卵細胞）同時培養。 卵細胞係在3 7°C於空氣中一 5% C〇2的濕化大氣下 培養2 4小時。在培養時期結束之前，分別使用立體顯微 鏡（Wildt，Leica MZ 12)來計數具有GV、GVB及極體 (在此以後稱爲P B )的卵細胞之數目。GVB之百分率 係定義爲於該凹槽中每總數目的卵細胞中進行G V B之卵 細胞百分率，其係如下計算： % GVB = ( (GVB數目+ PB數目）/卵細胞的 總數目）X 1 0 0 實施例2 用於測定一化合物是否可作爲本發明組成物中的添加 7 紙張尺度適用中國國家標準(CNS)A4規格（210 x 297公釐） ^ -------------訂--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1281404 A/ ___ B7 五、發明說明（心） 劑之方法 一種用於F F -MA S組成物之添加劑的特徵係在於 增進於乙醇/水（1 ·· 2 · 5 v/v)中F F — M A S 的溶解度。 確保在於Μ Ε Μα培養基中組成物重組之後的一淸澈 溶液。 確認當測試得自未成熟的雌性老鼠時G V Β百分率係 爲至少50% 、較佳爲80% 。 製備一種飽和之F F -MA S的乙醇溶液，將其與添 加劑水溶液以1 : 2.5的比例混合。藉由目視檢查，可以 於該溶液中控制過剩的F F -MA S。使該溶液在室溫下 旋轉2 4小時，使該溶液通過0 · 2 2微米的濾紙而過濾， 以Η P L C測定F F —MA S的含量且計算其溶解度。將 3 5 0微升轉移至四個凹槽的培養皿中且使其於室溫下蒸 發至乾燥。加入 5 0 0 微升的MEMα培養基（GibCObal) 。若是在半小時內得到一淸澈的溶液時，該組成物係測試 於得自未成熟的雌性老鼠的卵細胞上。參閱實施例1，所 得到的％ GVB係爲至少5 0%、較佳爲8 0%。 實施例3 含有人類血淸白蛋白（H S A)之組成物 在此實施例中，3個產品被製備。參照下表，用於產 品1、2及3之F F - M A S的貯備溶液係分別含有5 Q 、500及3330微克/毫升。對於每一產品而言，η 8 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） Ί—·—. —-------"訂—----- (請先閱讀背面之注意事項再填寫本頁)Serum Institute, Denmark), 2·3 mM pyruvate (Sigma, Cat· No S-8636), 2 mM guanamine (Flow Cat· No. 16-801), 100 IU/ml of penicillin And 100 μg/ml of streptomycin (Flow Cat· No. 16-700). This medium was supplemented with 3 m 次 of hypoxanthine (Sigma Cat. No. H-9377) and was referred to as Hx-culture medium. The egg cell lines were washed three times in ΗX-medium, and the same size egg cell line was divided into C Ε〇 and Ν〇 groups. C Ε〇 and Ν〇 were cultured in four wells in a multi-well dish (Nunclon, Denmark), each of which included 0. 4 ml of ΗX-medium and a compound of 1 〇μΜ at the concentration of 1 〇μΜ . One control groove (ie, 35 to 45 egg cells cultured in the same medium to which no test compound was added) is usually associated with three test grooves (ie, each groove of the test compound is added with 3 to 45) Egg cells) were cultured at the same time. The egg cell line was cultured for 24 hours at 37 ° C in a humidified atmosphere of 5% C 〇 2 in air. Before the end of the culture period, stereoscopic microscopes (Wildt, Leica MZ 12) were used to count the number of egg cells having GV, GVB and polar bodies (hereinafter referred to as P B ). The percentage of GVB is defined as the percentage of eggs per unit of GVB in the total number of target eggs in the groove, which is calculated as follows: % GVB = ((number of GVB + number of PBs) / total number of egg cells) X 1 0 0 Example 2 For the determination of whether a compound can be used as an additive in the composition of the present invention. 7 Paper scale applicable to China National Standard (CNS) A4 specification (210 x 297 mm) ^ ------------- --------- (Please read the note on the back and then fill out this page) Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed 1281404 A/ ___ B7 V. Invention Description (Heart) Method of One Agent The additive of the FF-MA S composition is characterized by an increase in the solubility of FF-MAS in ethanol/water (1·· 2 · 5 v/v). Make sure that there is a clear solution after the composition of the Μ Μ Μα medium is reconstituted. It was confirmed that the percentage of G V 当 when tested from immature female mice was at least 50%, preferably 80%. A saturated solution of F F -MA S in ethanol was prepared and mixed with an aqueous solution of the additive at a ratio of 1:2.5. Excess F F -MA S can be controlled in the solution by visual inspection. The solution was spun at room temperature for 24 hours, the solution was filtered through a 0.22 μm filter paper, and the F F -MA S content was measured by L P L C and its solubility was calculated. Transfer 350 liters to a four-well petri dish and evaporate to dryness at room temperature. Add 500 μl of MEMα medium (GibCObal). If a clear solution is obtained within half an hour, the composition is tested on egg cells obtained from immature female mice. Referring to Example 1, the % GVB obtained is at least 50%, preferably 80%. Example 3 Composition containing human blood albumin (H S A) In this example, three products were prepared. Referring to the table below, the stock solutions for F F - M A S for products 1, 2 and 3 contained 5 Q, 500 and 3330 μg/ml, respectively. For each product, η 8 paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) Ί—·—. —-------" order------ (Please read the notes on the back and fill out this page)