JP2007106704A - Visualization agent of vitreous body - Google Patents
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Abstract
Description
本発明は、硝子体可視化剤に関する。 The present invention relates to a vitreous body visualization agent.
硝子体切除術は、多くの眼疾患を治療する上で不可欠な手術手技である。当該手術は、透明な硝子体を見極めながら切除する（図１ａ）ため、熟練を要する上、いかに硝子体を残さず切除するかが問題となる局面が多く（図１ａ）、難易度の高い手術であった。 Vitreous resection is an indispensable surgical technique for treating many eye diseases. Since this surgery involves excision while identifying the transparent vitreous body (FIG. 1a), it requires skill, and there are many situations in which excision without leaving the vitreous body is problematic (FIG. 1a), which is a highly difficult operation. Met.
２００１年頃より、硝子体切除術時にトリアムシノロンアセトニド（ケナコルト、登録商標）を硝子体腔内に投与して硝子体を可視化する手技が普及し始めた。この手技は、トリアムシノロンアセトニドを投与することにより、硝子体に当該薬剤の白い粒子を付着させ、透明な硝子体の辺縁を白く浮かび上がらせ、硝子体と眼房水及び眼内灌流液とを明確に区別する方法であり、これにより硝子体手術が従来よりも容易かつ安全になった（図１ｂ）。 From around 2001, a technique for visualizing the vitreous body by administering triamcinolone acetonide (Kenacort, registered trademark) into the vitreous cavity at the time of vitrectomy began to spread. In this procedure, by administering triamcinolone acetonide, the white particles of the drug adhere to the vitreous body, the edges of the transparent vitreous body appear white, and the vitreous body and aqueous humor and intraocular perfusate are removed. This is a distinct method, which makes vitrectomy easier and safer than before (FIG. 1b).
しかし、トリアムシノロンアセトニドは、ステロイド製剤であり、副作用も多く、特に眼圧上昇と易感染性が問題になっている。眼圧上昇の例としては、トリアムシノロンアセトニドを硝子体内に注射した場合の合併症として、黄斑変性において３０％に高眼圧が出現したという報告（非特許文献１）や、網膜静脈閉塞に伴う黄斑浮腫においては７８％に高眼圧が出現し、そのうち１１％で手術が必要だったという報告（非特許文献２）などがある。易感染性の例としては、硝子体への注射での眼内炎の発生頻度は０．３８％であったという報告（非特許文献３）がある。硝子体切除術時にトリアムシノロンアセトニドを使用した場合の眼内炎の発生頻度は低いといえるが（非特許文献４）、硝子体内への注射では眼内炎の頻度は高く、眼内にステロイドが存在する時点で、感染症には十分に注意する必要があった。 However, triamcinolone acetonide is a steroid preparation and has many side effects, and in particular, increased intraocular pressure and easy infection are problems. Examples of elevated intraocular pressure include reports of high intraocular pressure appearing in 30% of macular degeneration as a complication when triamcinolone acetonide is injected intravitreally (Non-Patent Document 1), and accompanying retinal vein occlusion In macular edema, high intraocular pressure appeared in 78%, of which 11% reported that surgery was necessary (Non-patent Document 2). As an easily infectious example, there is a report (Non-patent Document 3) that the incidence of endophthalmitis upon injection into the vitreous was 0.38%. Although it can be said that the incidence of endophthalmitis when triamcinolone acetonide is used during vitrectomy is low (Non-patent Document 4), intraocular inflammation is frequent and steroids are present in the eye. At present, it was necessary to pay close attention to infections.
本発明の目的は、眼疾患治療時に用いる、安全で副作用の少ない硝子体可視化剤及び可視化方法を提供することである。 An object of the present invention is to provide a vitreous body visualization agent and a visualization method that are safe and have few side effects for use in the treatment of eye diseases.
本発明者らは、前記課題の解決について研究を重ねた結果、炭酸脱水素酵素阻害剤が、硝子体可視化剤として利用できることを見出し、かつ副作用も低減できることを確認して本発明を完成させるに至った。
すなわち、本発明は以下のとおりのものである。
（１）炭酸脱水素酵素阻害作用を有する化合物を含有する硝子体可視化剤。
（２）液剤である（１）記載の硝子体可視化剤。
（３）散剤又は顆粒剤である（１）記載の硝子体可視化剤。
（４）炭酸脱水素酵素阻害作用を有する化合物がブリンゾラミド、アセタゾラミド及びその塩である（１）〜（３）いずれか１つに記載の硝子体可視化剤。
（５）糖尿病網膜症、網膜剥離、増殖硝子体網膜症、硝子体出血、黄斑円孔、加齢黄斑変性、黄斑上膜、黄斑浮腫、網膜静脈閉塞症、未熟児網膜症、外傷性眼疾患、眼内炎、眼内異物、前部硝子体切除術、白内障手術、緑内障手術、眼内レンズ挿入術、又は眼内レンズ逢着術の手術時に用いられる（１）〜（４）いずれか１つに記載の硝子体可視化剤。
（６）（３）記載の硝子体可視化剤、及び水性媒体を別々の容器に含んでなる、硝子体可視化用キット。
（７）（１）〜（５）いずれか１つに記載の硝子体可視化剤又は（６）に記載のキットを用いることを特徴とする硝子体を可視化する方法。
（８）硝子体可視化剤を製造するための炭酸脱水素酵素阻害作用を有する化合物の使用。（９）炭酸脱水素酵素阻害作用を有する化合物を含有する、眼疾患の改善のための硝子体内注射用組成物。
（１０）眼疾患が眼圧上昇又は黄斑浮腫である（９）記載の注射用組成物。
（１１）（１）〜（５）いずれか１つに記載の硝子体可視化剤、及び当該硝子体可視化剤を硝子体を可視化するために使用することができること、又は使用すべきであることを記載した当該硝子体可視化剤に関する記載物を含む商業用パッケージ。
（１２）（９）又は（１０）記載の組成物、及び当該組成物を眼疾患の改善に使用することができること、又は使用すべきであることを記載した当該組成物に関する記載物を含む商業用パッケージ。
As a result of repeated studies on the solution of the above problems, the present inventors have found that a carbonic acid dehydrogenase inhibitor can be used as a vitreous body visualization agent and confirmed that side effects can also be reduced, thereby completing the present invention. It came.
That is, the present invention is as follows.
(1) A vitreous body visualization agent containing a compound having a carbonic acid dehydrogenase inhibitory action.
(2) The vitreous body visualization agent according to (1), which is a liquid agent.
(3) The vitreous body visualization agent according to (1), which is a powder or granule.
(4) The vitreous body visualization agent according to any one of (1) to (3), wherein the compound having a carbonic acid dehydrogenase inhibitory action is brinzolamide, acetazolamide and a salt thereof.
(5) Diabetic retinopathy, retinal detachment, proliferative vitreoretinopathy, vitreous hemorrhage, macular hole, age-related macular degeneration, macular degeneration, macular edema, retinal vein occlusion, retinopathy of prematurity, traumatic eye disease , Endophthalmitis, intraocular foreign body, anterior vitrectomy, cataract surgery, glaucoma surgery, intraocular lens insertion, or intraocular lens ablation surgery (1)-(4) any one Vitreous body visualization agent according to 1.
(6) A vitreous body visualization kit comprising the vitreous body visualization agent according to (3) and an aqueous medium in separate containers.
(7) A method for visualizing a vitreous body comprising using the vitreous body visualization agent according to any one of (1) to (5) or the kit according to (6).
(8) Use of a compound having a carbonic acid dehydrogenase inhibitory action for producing a vitreous body visualization agent. (9) A composition for intravitreal injection for improving eye diseases, comprising a compound having a carbonic acid dehydrogenase inhibitory action.
(10) The injectable composition according to (9), wherein the eye disease is increased intraocular pressure or macular edema.
(11) The vitreous body visualization agent according to any one of (1) to (5) and that the vitreous body visualization agent can or should be used to visualize the vitreous body. A commercial package containing a description of the described vitreous visualization agent.
(12) Commercial including the composition according to (9) or (10), and a description relating to the composition that states that the composition can be used for improvement of eye diseases or should be used For package.
本発明の硝子体可視化剤は、硝子体処置時において、硝子体の視認性に優れ、処置後の眼圧上昇を有意に抑制する。高眼圧症や易感染症などの副作用もない。
本発明のキットは、硝子体処置時に、硝子体可視化剤を液剤に用時調製するために供されるものであり、可視化剤の長期安定性と利便性とを兼ね備えるものである。
本発明の硝子体を可視化する方法は、硝子体処置時において、硝子体を容易に可視化するとともに、処置後の眼圧上昇を有意に抑制できる。高眼圧症や易感染症などの副作用も伴わない。
本発明の注射用組成物は、即効性の眼疾患の改善作用を有する。
The vitreous body visualization agent of the present invention has excellent visibility of the vitreous body during treatment of the vitreous body and significantly suppresses an increase in intraocular pressure after the treatment. There are no side effects such as ocular hypertension or infection.
The kit of the present invention is used for preparing a vitreous body visualization agent as a liquid agent during treatment of the vitreous body, and combines the long-term stability and convenience of the visualization agent.
The method for visualizing a vitreous body of the present invention can easily visualize the vitreous body and significantly suppress an increase in intraocular pressure after the treatment during the vitreous treatment. There are no side effects such as ocular hypertension or susceptibility.
The injectable composition of the present invention has an immediate effect of improving eye diseases.
本発明において硝子体可視化とは、硝子体と眼房水及び眼内灌流液との境界を明確にさせ、硝子体を視認可能にすることを意味する。硝子体可視化剤とは、上記の目的のために用いるものであり、手術時に注射等で硝子体腔内に注入し、手術の確実性を高めるものである。 In the present invention, the vitreous body visualization means that the boundary between the vitreous body, aqueous humor and intraocular perfusate is clarified so that the vitreous body is visible. The vitreous body visualization agent is used for the above-mentioned purpose, and is injected into the vitreous cavity by injection or the like at the time of surgery to enhance the certainty of the surgery.
炭酸脱水素酵素とは、 What is carbonic acid dehydrogenase?
を可逆的に触媒する酵素である。炭酸脱水素酵素阻害作用を有する化合物とは、炭酸脱水素酵素を阻害して、利尿作用又は眼圧低下作用を示す化合物である。眼圧下降の作用機序は毛様体における炭酸脱水素酵素抑制に起因する房水分泌抑制である。 It is an enzyme that catalyzes reversibly. The compound having a carbonic acid dehydrogenase inhibitory action is a compound that inhibits carbonic acid dehydrogenase and exhibits a diuretic action or an intraocular pressure reducing action. The mechanism of intraocular pressure reduction is suppression of aqueous humor secretion caused by carbonic acid dehydrogenase suppression in the ciliary body.
本発明の炭酸脱水素酵素阻害作用を有する化合物としては、例えば、アセタゾラミド、ブリンゾラミド、ドルゾラミドが挙げられる。好ましくは、アセタゾラミド又はブリンゾラミである。前記化合物は、市販品、又は自体公知の方法により製造したものを使用することが可能である。 Examples of the compound having a carbonic acid dehydrogenase inhibitory action of the present invention include acetazolamide, brinzolamide, and dorzolamide. Preferably, it is acetazolamide or brinzolami. As the compound, a commercially available product or a product produced by a method known per se can be used.
本発明において使用するアセタゾラミド、ブリンゾラミド等は、塩の形態で使用することもできる。塩の形態には金属塩、アンモニウム塩、無機酸との塩、有機酸との塩、無機塩基との塩、有機塩基との塩、塩基性又は酸性アミノ酸との塩等を挙げることができるが、難水溶性の塩を選択することが好ましい。 Acetazolamide, brinzolamide and the like used in the present invention can also be used in the form of a salt. Examples of the salt form include metal salts, ammonium salts, salts with inorganic acids, salts with organic acids, salts with inorganic bases, salts with organic bases, salts with basic or acidic amino acids, and the like. It is preferable to select a slightly water-soluble salt.
前記化合物は、水に対して溶解性が低いものが望ましい。通常、水に溶かした時に懸濁状態になるものが望ましい。可視化剤として機能を果たすためには、水に対する溶解度が、１ｍｇ／ｍＬ以下のものが望ましい。 The compound preferably has low solubility in water. Usually, it is desirable that it becomes suspended when dissolved in water. In order to function as a visualization agent, it is desirable that the solubility in water is 1 mg / mL or less.
本発明の硝子体可視化剤中の前記化合物の含有量は、０．００１〜１００重量％であり、好ましくは、０．０１〜１重量％である。
本発明の硝子体可視化剤中の前記化合物は、少なくとも１種を含んでいればよく、２種以上を混合して含んでいてもよい。
Content of the said compound in the vitreous body visualization agent of this invention is 0.001 to 100 weight%, Preferably, it is 0.01 to 1 weight%.
The said compound in the vitreous body visualization agent of this invention should just contain at least 1 sort (s), and may contain it in mixture of 2 or more types.
本発明の硝子体可視化剤は、液剤の形態でもよい。液剤は、前記化合物に薬学的に許容されうる添加剤を加えて、自体公知の方法で製造することができる。 The vitreous body visualization agent of the present invention may be in the form of a liquid agent. The liquid preparation can be produced by a method known per se by adding a pharmaceutically acceptable additive to the compound.
また本発明の硝子体可視化剤は、散剤又は顆粒剤の形態であってもよい。前記化合物をそのまま、又は、本発明の目的の効果を損なわない範囲内で、前記化合物に薬学的に許容されうる添加剤を加えて、自体公知の方法で粉末又は微粒状とすることにより、散剤又は顆粒剤とすることができる。これらの散剤又は顆粒剤は、硝子体投与の便宜の観点から、使用時に、滅菌精製水、生理食塩水、眼内灌流液等の水性媒体で液剤に調製して硝子体に投与することが望ましい。 The vitreous body visualization agent of the present invention may be in the form of powder or granules. By adding a pharmaceutically acceptable additive to the compound as it is or within a range not impairing the object effect of the present invention, the powder is made into powder or fine particles by a method known per se. Or it can be set as a granule. From the viewpoint of convenience of vitreous administration, it is desirable that these powders or granules are prepared in liquid form with an aqueous medium such as sterile purified water, physiological saline, intraocular perfusate and administered to the vitreous body. .
本発明は、前記散剤又は顆粒剤、及び前記水性媒体を別々の容器に含む硝子体可視化用キットを提供する。 The present invention provides a vitreous body visualization kit comprising the powder or granule and the aqueous medium in separate containers.
本発明の硝子体可視化剤の投与形態は、注射投与、とりわけ注射による局所投与が望ましい。 As the dosage form of the vitreous body visualization agent of the present invention, injection administration, particularly local administration by injection is desirable.
本発明の硝子体可視化剤の投与量は、投与する患者の症状、特に眼の状態、年齢、投与方法等によって異なるが、硝子体可視化剤を硝子体腔内に投与して、硝子体が視認出来得る量であればよい。炭酸脱水素酵素阻害を有する化合物は、硝子体手術時に、通常０．０１〜１ｍｇを投与すると硝子体が視認できる。好ましくは、０．０４〜０．４ｍｇの投与である。これを硝子体腔内（体積は約４ｍｌ）に均等に拡散した場合の濃度に換算すると、通常は０．００２５〜０．２５ｍｇ／ｍｌであり、好ましくは０．０１〜０．１ｍｇ／ｍｌに該当する。例えば、アセタゾラミド１０ｍｇ／ｍｌを１回あたり１眼に１００μｌを硝子体腔内に投与することにより、硝子体腔内のアセタゾラミドの濃度は０．２５ｍｇ／ｍｌになり、硝子体を視認可能にすることができる。 The dose of the vitreous body visualization agent of the present invention varies depending on the symptom of the patient to be administered, particularly the state of the eye, age, administration method, etc. Any amount can be obtained. A compound having carbonic acid dehydrogenase inhibition can visually recognize a vitreous body by administering 0.01 to 1 mg usually at the time of vitreous surgery. Preferably, the dose is 0.04 to 0.4 mg. When this is converted into the concentration when uniformly diffused into the vitreous cavity (volume is about 4 ml), it is usually 0.0025 to 0.25 mg / ml, preferably 0.01 to 0.1 mg / ml To do. For example, when 100 μl of acetazolamide 10 mg / ml per dose is administered into the vitreous cavity, the concentration of acetazolamide in the vitreous cavity becomes 0.25 mg / ml, and the vitreous body can be made visible. .
本発明の硝子体可視化剤の使用対象は、ヒト、非ヒト霊長類、イヌ、ネコ、ウサギ、ラット、又はマウスなど、眼疾患に罹患し得る任意の動物とする。 The target for use of the vitreous body visualization agent of the present invention is any animal that can suffer from an eye disease, such as a human, non-human primate, dog, cat, rabbit, rat, or mouse.
本発明における硝子体手術とは、硝子体自体に障害があり該硝子体を切除する手術及び眼疾患を治療する上で硝子体を切除しなければならない手術を意味する。 The vitreous surgery in the present invention means an operation in which the vitreous body itself is damaged, and an operation in which the vitreous body has to be removed in order to treat an eye disease.
本発明の硝子体可視化剤を適応できる眼疾患としては、網膜はく離、眼内炎、硝子体出血、硝子体混濁、糖尿病網膜症、増殖性硝子体網膜症、増殖糖尿病網膜症、黄斑部疾患（黄斑円孔、黄斑前膜、黄斑浮腫、加齢黄斑変性）、網膜中心静脈閉塞症、網膜静脈分枝閉塞症などが挙げられる。好ましくは、網膜はく離、黄斑部疾患又は糖尿病網膜症である。 Examples of eye diseases to which the vitreous body visualization agent of the present invention can be applied include retinal detachment, endophthalmitis, vitreous hemorrhage, vitreous opacification, diabetic retinopathy, proliferative vitreoretinopathy, proliferative diabetic retinopathy, macular disease (macular disease) A hole, anterior macular membrane, macular edema, age-related macular degeneration), central retinal vein occlusion, branch retinal vein occlusion, and the like. Preferably, retinal detachment, macular disease, or diabetic retinopathy.
本発明の硝子体可視化剤を硝子体腔内に投与した場合、硝子体を切除しても、手術後眼内に残留した硝子体可視化剤が炭酸脱水素酵素阻害作用を有するため、頻度の高い術後合併症である高眼圧症を抑制することができる。さらに、本発明の硝子体可視化剤にはトリアムシノロンアセトニドに見られる易感染性の副作用はなく、トリアムシノロンアセトニド使用による術後感染症の潜在的な危険性を回避できる。 When the vitreous body visualization agent of the present invention is administered into the vitreous cavity, even if the vitreous body is excised, the vitreous body visualization agent remaining in the eye after surgery has a carbonic acid dehydrogenase inhibitory action, so that a high frequency operation is performed. Ocular hypertension, a post-complication, can be suppressed. Furthermore, the vitreous visualization agent of the present invention has no infectious side effects seen with triamcinolone acetonide, and avoids the potential risk of postoperative infections due to the use of triamcinolone acetonide.
本発明の硝子体内注射用組成物は、前記炭酸脱水素酵素阻害作用を有する化合物を含有する。眼疾患、特に眼圧上昇又は黄斑浮腫に対して、硝子体内に局所注射することにより、即効性を有し、経口投与により認められる副作用を回避することができる。 The composition for intravitreal injection of the present invention contains the compound having the carbonic acid dehydrogenase inhibitory action. For eye diseases, particularly increased intraocular pressure or macular edema, local injection into the vitreous has immediate effect and avoids side effects observed by oral administration.
以下に、本発明を実施例によってさらに詳細に説明するが、本発明はこれらの例によって何ら限定されるものではない。 Examples The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
（実験１）
屠殺されたブタの眼球から硝子体を摘出し標本ビンに入れ、ブリンゾラミド（エイゾプト（登録商標）点眼液）１０ｍｇ／ｍｌを０．１ｍｌ、アセタゾラミド（シグマ社）１０ｍｇ／ｍｌを０．１ｍｌ投与した。薬剤投与前後の硝子体の視認性を目視で比較した。
(Experiment 1)
Vitreous bodies were extracted from the eyes of slaughtered pigs and placed in specimen bottles, and 0.1 ml of 10 mg / ml of brinzolamide (Eizopt (registered trademark) ophthalmic solution) and 0.1 mg of acetazolamide (Sigma) were administered. Visibility of the vitreous before and after drug administration was compared visually.
結果を、図２に示す。ブタ硝子体は、ブリンゾラミドやアセタゾラミドを投与することにより、白色の粒子が硝子体表面に付着し、可視化されることが明らかになった。 The results are shown in FIG. It was clarified that porcine vitreous body was visualized by the administration of brinzolamide or acetazolamide, with white particles adhering to the vitreous surface.
（実験２）
培養網膜色素上皮細胞ＡＰＲＥ−１９を、様々な濃度のブリンゾラミド、アセタゾラミド、トリアムシノロンアセトニド（ケナコルト、登録商標）を含む培地で培養し、ＷＳＴ−１ ａｓｓａｙにより５０％細胞生存濃度を求めた。
ＡＰＲＥ−１９細胞はＡｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎから購入した。培地は１０％ Ｆｅｔａｌ Ｂｏｖｉｎｅ ＳｅｒｕｍとＰｅｎｉｃｉｌｌｉｎ（５０ｕｎｉｔｓ／ｍｌ）、 Ｓｔｒｅｐｔｏｍｙｃｉｎ（５０μｇ／ｍｌ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ／Ｈａｍ’ｓ Ｆ１２ Ｍｅｄｉｕｍを使用し、３７℃、５％ＣＯ２で培養した。９６ｗｅｌｌ ｐｌａｔｅに１００，０００ｃｅｌｌｓ／ｗｅｌｌとなるように細胞をまき、インキュベータで培養した。３日後培地を吸引し、Ｈａｎｋｓ’ Ｂａｌａｎｃｅｄ Ｓａｌｔ Ｓｏｌｕｔｉｏｎ（ＨＢＳＳ）で前記細胞を洗浄し、様々な濃度のアセタゾラミド、ブリンゾラミド、トリアムシノロンアセトニドを含む培地で培養した。３０分後、薬剤含有培地を吸引し、ＨＢＳＳで洗浄し、通常の培地で培養した。３時間後、ＷＳＴ−１試薬（Ｐｒｅｍｉｘ ＷＳＴ−１、登録商標）を１０μｌ加え培養した。１時間後、マイクロプレートリーダーでホルマザン色素の吸光度（ＯＤ４５０ｎｍ）を測定した。薬剤を含まない培地で培養した細胞における吸光度を１００％として、細胞生存率を算出した。
(Experiment 2)
Cultured retinal pigment epithelial cells APRE-19 were cultured in media containing various concentrations of brinzolamide, acetazolamide, and triamcinolone acetonide (Kenacort, registered trademark), and 50% cell viability was determined by WST-1 assay.
APRE-19 cells were purchased from American Type Culture Collection. The medium was Dulbecco's Modified Eagle Medium / Ham's F12 Medium containing 10% Fetal Bovine Serum, Penicillin (50 units / ml) and Streptomycin (50 μg / ml) at 37 ° C., 2 %. Cells were seeded on a 96-well plate at 100,000 cells / well and cultured in an incubator. Three days later, the medium was aspirated, the cells were washed with Hanks' Balanced Salt Solution (HBSS), and cultured in a medium containing various concentrations of acetazolamide, brinzolamide, and triamcinolone acetonide. After 30 minutes, the drug-containing medium was aspirated, washed with HBSS, and cultured in a normal medium. After 3 hours, 10 μl of WST-1 reagent (Premix WST-1, registered trademark) was added and cultured. After 1 hour, the absorbance (OD450 nm) of the formazan dye was measured with a microplate reader. The cell viability was calculated with the absorbance in cells cultured in a medium containing no drug as 100%.
結果を、図３及び４に示す。
ブリンゾラミドのＡＰＲＥ−１９細胞における細胞生存率は、原液である１％（１０ｍｇ／ｍｌ）製剤では１％であったが、２倍希釈（５ｍｇ／ｍｌ）では１０％に上昇し、４倍希釈（２．５ｍｇ／ｍｌ）では９９％となった。アセタゾラミドでは、１００ｍｇ／ｍ
ｌで３０％、１０ｍｇ／ｍｌで５４％、５ｍｇ／ｍｌで７７％、２．５ｍｇ／ｍｌで８７％、１．２５ｍｇ／ｍｌで９２％となだらかに変化した（図３）。トリアムシノロンアセトニドでは原液である４０ｍｇ／ｍｌ製剤では７％であったが、２倍希釈（２０ｍｇ／ｍｌ）では１０４％となった（図４）。
ブリンゾラミド、アセタゾラミド及びトリアムシノロンアセトニドの今回の実験条件でのＡＰＲＥ−１９細胞における５０％細胞生存濃度は、それぞれ約３ｍｇ／ｍｌ、１０ｍｇ／ｍｌ、３０ｍｇ／ｍｌであった。
The results are shown in FIGS.
The cell viability of brinzolamide in APRE-19 cells was 1% in the stock solution of 1% (10 mg / ml), but increased to 10% at 2 times dilution (5 mg / ml) and diluted 4 times ( 2.5 mg / ml) was 99%. For acetazolamide, 100 mg / m
It gradually changed to 30% at 10 mg, 54% at 10 mg / ml, 77% at 5 mg / ml, 87% at 2.5 mg / ml, and 92% at 1.25 mg / ml (FIG. 3). Triamcinolone acetonide was 7% in the 40 mg / ml formulation as the stock solution, but it was 104% in the 2-fold dilution (20 mg / ml) (FIG. 4).
The 50% cell viability concentrations in APRE-19 cells under the current experimental conditions for brinzolamide, acetazolamide and triamcinolone acetonide were about 3 mg / ml, 10 mg / ml and 30 mg / ml, respectively.
（実験３）
各薬剤につき、雄性日本白色ウサギ（体重２．５−３．０ｋｇ）３羽３眼を使用した。ウサギを固定具に固定し、ペントバルビタール（ネンブタール、登録商標）静脈注射で全身麻酔後、片眼に対して２７Ｇ鋭針を前房に刺入し前房水を１００μｌ除去し、硝子体内に２７Ｇ鋭針にて薬剤１００μｌを注射した。ブリンゾラミド（エイゾプト、登録商標）、アセタゾラミド（シグマ社）及びトリアムシノロンアセトニド（ケナコルト、登録商標）の濃度は、実験２から算出した５０％細胞生存濃度、それぞれ３ｍｇ／ｍｌ、１０ｍｇ／ｍｌ、３０ｍｇ／ｍｌを用いた。コントロールとしては、生理食塩水を注射した。
処置前、処置後６時間、１日、３日、７日後に眼圧測定、眼底検査、網膜電位（ＥＲＧ）の測定を行った。まず塩酸オキシブプロカイン（ベノキシール、登録商標）にて点眼麻酔後、開瞼器で開瞼し、電子眼圧計（トノペンＸＬ、登録商標）にて眼圧を測定した。眼圧測定は各時点で３回行い、平均値を測定値とした。次に散瞳剤であるトロピカミド・塩酸フェニレフリン（ミドリンＰ、登録商標）を点眼し、ペントバルビタールで全身麻酔し、２０分間暗順応させ、ポータブルＥＲＧ（ＬＥ−２０００、登録商標）でＥＲＧを測定した。フラッシュＥＲＧを４回加算平均し、ａ波、ｂ波の潜時と振幅を測定した。その後、倒像鏡にて眼底検査を行い、眼底写真を撮影した。全検査終了後、ペントバルビタール過剰投与でウサギを屠殺し、眼球摘出を行い、組織標本を作製した。
眼圧、ＥＲＧの値はｐａｉｒｅｄ ｔ−ｔｅｓｔにて検定し、投与前の値と比較した。
(Experiment 3)
For each drug, 3 male Japanese white rabbits (body weight 2.5-3.0 kg) 3 eyes were used. A rabbit is fixed on a fixture, and after general anesthesia by intravenous injection of pentobarbital (Nembutal, registered trademark), a 27G sharp needle is inserted into the anterior chamber for one eye, and 100 μl of anterior aqueous humor is removed. 100 μl of drug was injected with a sharp needle. The concentrations of brinzolamide (Eizopt®), acetazolamide (Sigma) and triamcinolone acetonide (Kenacort®) were 50% cell viability calculated from Experiment 2, 3 mg / ml, 10 mg / ml, and 30 mg / ml, respectively. Was used. As a control, physiological saline was injected.
Before treatment, 6 hours after treatment, 1 day, 3 days, and 7 days later, intraocular pressure measurement, fundus examination, and retinal potential (ERG) measurement were performed. First, after ophthalmic anesthesia with oxybuprocaine hydrochloride (Benoxeal, registered trademark), the eyelid was opened with an eyelid opener, and the intraocular pressure was measured with an electronic tonometer (Tonopen XL, registered trademark). The intraocular pressure measurement was performed three times at each time point, and the average value was taken as the measured value. Next, myopic tropicamide / phenylephrine hydrochloride (Midrin P, registered trademark) was instilled, general anesthetized with pentobarbital, dark-adapted for 20 minutes, and ERG was measured with portable ERG (LE-2000, registered trademark). . The flash ERG was averaged four times and the latency and amplitude of the a and b waves were measured. Thereafter, the fundus was examined with an inversion mirror and a fundus photograph was taken. After completion of all examinations, the rabbits were sacrificed by pentobarbital overdose, and the eyeballs were removed to prepare tissue specimens.
The intraocular pressure and ERG values were tested by paired t-test and compared with the values before administration.
結果を、図５〜７に示す。
ＥＲＧは各波の潜時と振幅を計測することで網膜障害を鋭敏に判定できる。ウサギＥＲＧａ波はヒトと同様に視細胞に発生母体を有し、視細胞活動を反映する。ｂ波はグリア細胞の活動を主に反映するとされ、さらに双極細胞の電気活動関与も考えられている。今回の実験ではすべての薬剤においてＥＲＧａ波、ｂ波の潜時、振幅に変化はなく、網膜機能障害を認めなかった（図５、６）。
眼圧はトリアムシノロンアセトニドでは投与前後で変化はなかった。ブリンゾラミドとアセタゾラミドでは投与後６時間で投与前より有意に眼圧下降を認め、さらに術後１日以降の眼圧が安定していることを認めた。その他の測定時点では有意差はなかった（図７）。
眼底検査では、すべての薬剤において投与後に網膜、視神経、脈絡膜に異常はなく、眼内の炎症所見等の異常所見はみられなかった。眼組織を光学顕微鏡にて観察したところ、毛様体や網膜に細胞の変性や壊死、炎症細胞の遊走などの異常所見はなかった。
これらの結果から今回の条件での炭酸脱水素酵素阻害剤の眼組織における安全性が確認できた。さらに硝子体可視化剤として使用した炭酸脱水素酵素阻害剤が術後高眼圧を抑制する可能性が示された。
The results are shown in FIGS.
ERG can determine retinal damage sensitively by measuring the latency and amplitude of each wave. Rabbit ERGa waves, like humans, have a developmental matrix in photoreceptor cells and reflect photoreceptor activity. The b wave is considered to reflect mainly the activity of glial cells, and the electric activity of bipolar cells is also considered to be involved. In this experiment, ERGa wave and b wave latencies and amplitudes did not change in all drugs, and no retinal dysfunction was observed (FIGS. 5 and 6).
Intraocular pressure did not change before and after administration of triamcinolone acetonide. In the case of brinzolamide and acetazolamide, the intraocular pressure was significantly decreased 6 hours after the administration, and the intraocular pressure after 1 day after the operation was further stabilized. There was no significant difference at other time points (FIG. 7).
In the fundus examination, all drugs had no abnormalities in the retina, optic nerve and choroid after administration, and there were no abnormal findings such as inflammatory findings in the eye. When the ocular tissue was observed with an optical microscope, there were no abnormal findings such as cell degeneration or necrosis or migration of inflammatory cells in the ciliary body or retina.
From these results, the safety of the carbonic acid dehydrogenase inhibitor in the eye tissue under the present conditions was confirmed. Furthermore, it was shown that a carbonic acid dehydrogenase inhibitor used as a vitreous body visualizing agent may suppress postoperative high intraocular pressure.
Claims (11)
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002501017A (en) * | 1998-01-23 | 2002-01-15 | メルク・シャープ・エンド・ドーム・ベー・ベー | Method for increasing oxygen tension in the optic nerve and retina |
| JP2003313119A (en) * | 2002-02-22 | 2003-11-06 | Santen Pharmaceut Co Ltd | Drug delivery system for subconjunctival administration of fine particle |
| WO2004014352A2 (en) * | 2002-08-07 | 2004-02-19 | Pharmacia Corporation | Methods for treating carbonic anhydrase mediated disorders |
| WO2004066980A2 (en) * | 2003-01-24 | 2004-08-12 | Control Delivery Systems, Inc. | Sustained release device and method for ocular delivery of carbonic anhydrase inhibitors |
| WO2005072701A1 (en) * | 2004-01-20 | 2005-08-11 | Allergan, Inc. | Compositions for localized therapy of the eye, comprising preferably triamcinolone acetonide and hyaluronic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002501017A (en) * | 1998-01-23 | 2002-01-15 | メルク・シャープ・エンド・ドーム・ベー・ベー | Method for increasing oxygen tension in the optic nerve and retina |
| JP2003313119A (en) * | 2002-02-22 | 2003-11-06 | Santen Pharmaceut Co Ltd | Drug delivery system for subconjunctival administration of fine particle |
| WO2004014352A2 (en) * | 2002-08-07 | 2004-02-19 | Pharmacia Corporation | Methods for treating carbonic anhydrase mediated disorders |
| WO2004066980A2 (en) * | 2003-01-24 | 2004-08-12 | Control Delivery Systems, Inc. | Sustained release device and method for ocular delivery of carbonic anhydrase inhibitors |
| WO2005072701A1 (en) * | 2004-01-20 | 2005-08-11 | Allergan, Inc. | Compositions for localized therapy of the eye, comprising preferably triamcinolone acetonide and hyaluronic acid |
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