JP4125512B2 - Powder formulation and method for producing the same - Google Patents

Description

【００２８】
上記方法にて、日本薬局方乳糖（吉田製薬）及びグルカゴン乳糖百倍散凍結乾燥品について約 10 g ずつ粉砕化を行った。10 g というある程度の量を処理する場合にはそれぞれの収率は表 1 に示すとおりほぼ同等であり、目視による性状確認においても両者ほぼ同等であった。懸念されていたグルカゴンの凝集性は目で見える範囲では少なくとも問題は解決されており、殆ど乳糖と同じ性状を示す粉体であることを確認した。さらに両者の粒度分布についてレーザー回折/散乱式粒径分布測定装置 (日機装社製) にて評価を行った。図１及び２に示すように、分布の幅は多少異なるものの、ピークトップは約 3 μm 程度であり、またほぼ100％の粉末が粒径10μm以下であった。
【００２９】
【表１】

【００３０】
〔実施例３〕 グルカゴン粉末の微量製造
実施例１と同様の方法にて、グルカゴン乳糖百倍散凍結乾燥品およびグルカゴンエリスリトール百倍散について２ g の粉砕化を行った。この際の収率を表２に示す。この結果が示すとおり、少量の製造になった場合には吸着、吸湿をはじめとする収率を低下させる多くの要因がより明確な形で表面に出てくるため，エリスリトール利用混合物の方が製造収率が高くなった。懸念されていたグルカゴンの凝集性は目で見える範囲では少なくとも問題は解決されており、殆ど実施例１において作製した賦形剤微粉末と同じ性状を示す粉体であることを確認した。
【００３１】
【表２】

【００３２】
〔実施例４〕 酢酸ブセレリン粉末の作製
酢酸ブセレリン（伊藤ハム株式会社製）、酢酸ブセレリン-乳糖混合物（570 倍散）、酢酸ブセレリン-マンニトール混合物（570 倍散）、酢酸ブセレリン-エリスリトール混合物（570 倍散）について実施例１と同様にして微粉化を行った。
【００３３】
収率はそれぞれ次の通りであった。
酢酸ブセレリンのみ 潮解のため未確認
酢酸ブセレリン-乳糖混合物 27.0%
酢酸ブセレリン-マンニトール混合物 41.7%
酢酸ブセレリン-エリスリトール混合物 71.7%
この結果から明らかに、ペプチドだけの粉砕は非常に難しいことが示され、乳糖もしくはエリスリトール等の賦形剤添加の効果が明らかとなった。乳糖混合物は非常に付着性が強く、バグフィルターからの回収はやや難しかったが、その反対にマンニトール、エリスリトール混合物は非常に付着性も低く、回収率の飛躍的な向上が確認された。
【００３４】
〔実施例５〕 グルカゴン高濃度含有粉末の作製
実施例１記載の方法と同様にジェットミルを用いてグルカゴン-エリスリトール混合物（3, 5, 10, 20 倍散）について次の条件にて微粉化を行った。すなわち、グルカゴンとエリスリトールを下記表の様に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから微粉化されたグルカゴン-エリスリトール混合物を得た。回収率はいずれも30〜40％前後であった。グルカゴンの粉体中含量はほぼ理論値であることを HPLCによる定量法にて確認した。本製剤処方では、このような高濃度ペプチド製剤の作製も可能であることが示された。
【００３５】
【表３】

【００３６】
〔実施例６〕 血管作動性腸管ペプチド（VIP）誘導体粉末の作製
実施例１記載の方法と同様にジェットミルを用いてVIP-エリスリトール混合物（400 倍散）について次の条件にて微粉化を行った。すなわち、特開平 8-333276 に記載の VIP 誘導体 His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Met-Ala-Val-Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Arg-Arg-NH₂（配列番号１）5 mg をエリスリトール 2 g と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから約 70% の回収率で微粉化された VIP 誘導体-エリスリトール混合物を得た。VIP 誘導体の粉体中含量はほぼ理論値であることを HPLCによる定量法にて確認した。
【００３７】
〔実施例７〕 インスリン粉末の作製
実施例１記載の方法と同様にジェットミルを用いてインスリン-エリスリトール混合物（20 倍散）について次の条件にて微粉化を行った。すなわち、ブタインスリン（伊藤ハム株式会社製） 100 mg をエリスリトール 1.9 g と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから約 60% の回収率で微粉化されたインスリン-エリスリトール混合物を得た。インスリンの粉体中含量はほぼ理論値であることを HPLC による定量法にて確認した。
【００３８】
〔実施例８〕 サケカルシトニン粉末の作製
実施例１記載の方法と同様にジェットミルを用いてサケカルシトニン-乳糖混合物（200 倍散）およびサケカルシトニン-エリスリトール混合物（200 倍散）について次の条件にて微粉化を行った。すなわち、サケカルシトニン（伊藤ハム株式会社製）15 mg を乳糖またはエリスリトール約 3.0 g と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから約 70%（サケカルシトニン-乳糖混合物）および約 80%（サケカルシトニン-エリスリトール混合物）の回収率で微粉化されたサケカルシトニン混合粉末を得た。サケカルシトニンの粉体中含量はほぼ理論値であることを HPLC による定量法にて確認した。
【００３９】
〔実施例９〕 エルカトニン粉末の作製
実施例１記載の方法と同様にジェットミルを用いてエルカトニン-乳糖混合物（200 倍散）およびエルカトニン-エリスリトール混合物（200 倍散）について次の条件にて微粉化を行った。すなわち、エルカトニン（伊藤ハム株式会社）15 mg を乳糖またはエリスリトール約 3.0 g と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから約 20%（エルカトニン-乳糖混合物）および約 70%（エルカトニン-エリスリトール混合物）の回収率で微粉化されたエルカトニン混合粉末を得た。エルカトニンの粉体中含量はほぼ理論値であることを HPLC による定量法にて確認した。
【００４０】
〔実施例１０〕 ヒト成長ホルモン（GH）粉末の作製
実施例１記載の方法と同様にジェットミルを用いてヒト成長ホルモン-エリスリトール混合物（50 倍散）について次の条件にて微粉化を行った。すなわち、ヒト成長ホルモン混合物（Jiangxi Chinese Import & Export Co. Ltd） 30 mg（ヒト成長ホルモン 10 mg, ヒトアルブミン 10 mg, グリシン 10 mg）をエリスリトール約 470 mg と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから約 50% の回収率で微粉化されたヒト成長ホルモン-エリスリトール混合物を得た。ヒト成長ホルモンの粉体中含量はほぼ理論値であることをHPLCによる定量法にて確認し、なおかつその安定性についても同時に確認した。これにより、非常に不安定で扱い難いとされるヒト成長ホルモンのような高分子化合物においても本製剤処方は適用可能であることが示された。
【００４１】
〔実施例１１〕 シクロスポリン粉末の作製
実施例１記載の方法と同様にジェットミルを用いてシクロスポリン-エリスリトール混合物（10 倍散）について次の条件にて微粉化を行った。すなわち、シクロスポリンA（和光純薬株式会社製） 100 mg をエリスリトール約 900 mg と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施し、粉体捕集用バグフィルターから約 60% の回収率で微粉化されたシクロスポリン-エリスリトール混合物を得た。シクロスポリンの粉体中含量はほぼ理論値であることを HPLC による定量法にて確認した。
【００４２】
〔実施例１２〕 クエン酸含有グルカゴン粉末の作製
実施例１記載の方法と同様にジェットミルを用いてグルカゴン-クエン酸混合物およびグルカゴン-クエン酸-エリスリトール混合物について次の条件にて微粉化を行った。すなわち、グルカゴン（伊藤ハム株式会社製） 80 mg をクエン酸 80 mg と混和するか、あるいはグルカゴン 80 mg をクエン酸 80 mg とエリスリトール約 640 mg と適度に混和し、ジェットミルを用いて実施例１にて示した条件下で微細粒子加工を施した。グルカゴン-クエン酸混合物の粉砕物は速やかに潮解を起こし、回収が極めて難しかったが、グルカゴン-クエン酸-エリスリトール混合物は吸湿による潮解を認めず非常に取り扱いやすく、粉体捕集用バグフィルターからの回収率は約 40% であった。このことから，潮解性を示す添加物混入においても本製剤設計は有用であることが示された。グルカゴンの粉体中含量はほぼ理論値であることを HPLC による定量法にて確認した。
【００４３】
〔実施例１３〕 グルカゴンのリポソーム封入物作製
グルカゴン（伊藤ハム株式会社製）を 0.1% トリフルオロ酢酸水溶液にて 10 mg/mL に調製し、これを L-α-ジパルミトイルホスファチジルコリン、コレステロール、L-α-ジパルミトイルホスファチジルグリセロールから構成される乾燥中空リポソーム（粒径：100_300 nm, コートソーム EL シリーズ，日本油脂株式会社）に添加した。数分間振盪後、凍結乾燥（バーチス社製凍結乾燥機，ユニトップ HL，フリーズモビル 25 EL）した。凍結乾燥前のサンプルから 約 200 μL採取し、遠心式少量限外ろ過ユニット ULTRAFREE-0.5 (Mw 10000 以下カット) を用いて遠心（10 分間，12,000 x g）した。ろ液中のグルカゴン含量を定量し、これをリポソームに封入されなかった量として算出した。定量 (HPLC) 条件は下記の通りである。
(HPLC 条件)
使用カラム： TSK gel ODS-120 T (TOSO)
検出器： RF_535 (Shimadzu)
励起波長: 280 nm
蛍光波長： 346 nm
Pump: LC-10AD (Shimadzu)
Mobile Phase: 35% CH₃CN (0.1% TFA acidified)
移動相流速： 1.0 mL/min
HPLC による定量の結果、グルカゴンのリポソームへの封入率は 98％であり、極めて高い封入効率であることが示された。
【００４４】
〔実施例１４〕 リポソーム封入グルカゴン粉末（1% グルカゴン粉体）の作製
実施例１３記載の方法によって得られたグルカゴン-リポソーム封入物約200 mg に、エリスリトール 約 800 mg を適度に混和し、実施例１と同様にしてジェットミルにて微粉化を行った。粉体捕集用バグフィルターから約 50% の回収率で微粉化されたグルカゴン-リポソーム封入物-エリスリトール混合物を得た。得られた粉体は極めて流動性が高く、製剤特性上良好な粒子であった。この粉体中グルカゴン含量はほぼ理論値であることを HPLC による定量法にて確認した。これによって、リポソーム製剤にも本製剤処方が利用可能であり、製造中に薬物が潮解することなく生産可能であることが示された。
【００４５】
〔実施例１５〕 粉末と担体の混合
実施例２にて得られた乳糖およびグルカゴン乳糖百倍散のそれぞれの粉末を速やかに表４記載の条件で担体（Pharmatose 325 M；DMV社製；平均粒径 50±10μm）と混合し、50 mL ディスポチューブにて乾燥・保管した。なお、その際の（目視での）性状を併せて表４に記載する。乳糖粉末とグルカゴン乳糖百倍散粉末を担体に添加した際には、その流動性を含む多くの性状は両者同じであり、これはグルカゴンの望ましくない性質である自己凝集性を抑制できたものと判断することができた。なお、担体との混合をしていないグルカゴン乳糖百倍散微粉化物について着目した際、少なくとも 24 時間経過時には顕著な凝集塊の形成を認めた。
この結果から明らかな如く、本発明の粉末製剤は、自己凝集が抑制され、投与にとって非常に好適なものであった。
【００４６】
【表４】

【００４７】
〔実施例１６〕担体とグルカゴン百倍散粉砕物の混合物粒度分布について
SK レーザーマイクロンサイザー LMS-30 (セイシン企業) にて、担体（Pharmatose 325 M）のみ、および担体とグルカゴン百倍散粉砕物を混和したものの双方についてそれぞれ粒度分布を検討した（図３及び４）。その結果、2.0 kg/cm² の圧力にて粉体を分散させたところ、図４に示すように、明瞭に担体と薬物の分離が示されており、しかも分離したグルカゴン百倍散は自己凝集による粒度増大を示さなかった。
この結果から明らかな如く、本発明の粉末製剤は、自己凝集が見られない上、投与の際には容易に薬剤が担体と分離し、薬剤を含有する微細粒子のみが目的部位まで到達可能となる。
【００４８】
〔実施例１７〕グルカゴン含量の均一性評価
グルカゴンの自己凝集性を抑制し、なおかつ流動性を向上させることにより、担体混合後の薬物含量均一性が極めて向上するであろうと推測される。そこで、実施例１６で得られた粉末からランダムに薬物を採取し、HPLC による定量分析を行うことにより、その均一性を評価した。以下方法を示す。
【００４９】
なお、製剤含有グルカゴンの高速液体クロマトグラフィー（HPLC) 分析条件は以下の通りである。
（HPLC分析条件）
使用カラム： TOSO ODS-120 T（トーソー）
検出器： Jusco 870-UV
UV 検出波長： 220 nm
移動相流速： 1.0 mL/min
移動相： 35 % CH₃CN aq (0.1 % TFA acidified)
ポンプ： Jusco 880-PU
Inject 量： 50 μL
カラムオーブン温度 : 40 ℃
（方法）
対象サンプル
グルカゴン粉体（DPG080700）Pharmatose 混合物
（混合比、グルカゴン乳糖百倍散：担体 ＝ 1：2）
【００５０】
本検体（容量：25 mL）を 50 mL ディスポチューブにてほぼ均等に三等分し、それぞれのセクションからランダムに 3 検体（サンプル１〜３）を約 10 mg 量り取る。0.1 N HCl にて10 mg/mL に調製し、RP-HPLC にて分析を行い、各検体間のグルカゴン相当ピーク面積の値に関して比較検討する。
各セクション間でのグルカゴン相当ピークについて、図５に示す。
図５から明らかな如く、ランダムに採取したサンプルにおいてほぼ同量の薬剤が検出でき、本発明の粉末製剤は、流動性が改善され、ほぼ均一に担体と混和していることが示されている。
【００５１】
〔実施例１８〕 カスケードインパクターによるグルカゴンおよびエルカトニン製剤評価
粉体の空気力学的粒径に関して調査を行うため、人工気道および肺モデルであるカスケードインパクターにて検討を行った。本体は８段のステージと最終フィルターを重ねたものであり、これに流速計と吸引ポンプを組み合わせたものである。基本的な方法はUSP 2000 "Physical Tests and Determinations/Aerosols" 中 "Multistage Cascade Impactor Apparatus"記載の手法を適用した。具体的な方法は次の通りである。

（結果）
HPLCにおける各定量値を下記に示す。
【００５２】
【表５】

【００５３】
【表６】

【００５４】
本モデルによればステージ３以降に捕集される薬物は気管支および肺へと移行するが、両製剤についてはステージ３以降に約３〜４割の主剤が存在していることを考慮に入れれば、体内に入る薬物のうち約３〜４割に望ましい薬効を期待できる。
【００５５】
〔実施例１９〕 担体-薬物微粒子混合比による製剤特性への影響
実施例１に記載した製造方法と同様に日局乳糖（吉田製薬）を微粉化し、得られた粉末を非帯電袋を用いて下記の条件で担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。
【００５６】
【表７】

【００５７】
この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。RF 値について表８に示す。この RF 値は各ステージに捕集された粉体重量を秤量し、ステージ 2〜5 における捕集総量を製剤中含有微粉化物総量で割った数字である。
【００５８】
【表８】

【００５９】
この結果から、賦形剤と粒子の混合比率が大きく製剤特性に関与していることが示され、担体に対する薬物の含量は少ない方が好ましいことが明らかとなった。
【００６０】
〔実施例２０〕 リポソーム封入グルカゴン製剤の含量均一性評価
実施例１４にて作製したグルカゴン-リポソーム封入物の粉末を非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。実施例１７にて示した方法に従い、3 つのセクションから一定量秤量し、その中に含まれるグルカゴン含量について HPLC にて定量を行った。HPLC 条件を下記に示す。
（HPLC 条件）
使用カラム： TOSO ODS-120 T（トーソー）
検出器： RF-535 (Shimadzu)
励起波長： 280 nm
蛍光波長： 346 nm
移動相流速： 1.0 mL/min
移動相： 35% CH₃CN aq (0.1% TFA acidified)
ポンプ： LC-10 AD (Shimadzu)
カラムオーブン温度 : 40 ℃
3 つのセクションから抜き出した検体について上記条件における定量を行ったところ、それぞれ 2.32 μg/mg-製剤、2.43 μg/mg-製剤、2.41 μg/mg-製剤であり、平均値は 2.39 μμg/mg-製剤 であった。リポソームのように会合・凝集しやすい分子を用いた本ケースのような場合においても、発明製剤処方は概ね含量均一な製剤を供給可能であることを確認した。
【００６１】
〔実施例２１〕 サケカルシトニン製剤の含量均一性評価
実施例８にて作製したサケカルシトニン粉末を非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。実施例１７にて示した方法に従い、3 つのセクションから一定量秤量し、その中に含まれるサケカルシトニン含量について HPLC にて定量を行った。HPLC 条件を下記に示す。
（HPLC 条件）
使用カラム： TOSO ODS-120 T（トーソー）
検出器： RF-535 (Shimadzu)
励起波長： 280 nm
蛍光波長： 320 nm
移動相流速： 1.0 mL/min
移動相： 38% CH₃CN aq (0.1% TFA acidified)
ポンプ： Jusco 880-PU
カラムオーブン温度 : 40 ℃
3 つのセクションから抜き出した検体について上記条件における定量を行ったところ、それぞれ 3.95 μg/mg-製剤、4.29 μg/mg-製剤、4.05 μg/mg-製剤であり、平均値は 4.09 μg/mg-製剤 であった。含量が極めて低い本ケースのような場合においても、発明製剤処方は概ね含量均一な製剤を供給可能であることを確認した。
【００６２】
〔実施例２２〕 カスケードインパクターによるグルカゴン製剤評価
実施例３の方法で作成したグルカゴン粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。RF 値とカプセル残存について表９に示す。この RF 値は各ステージに捕集された製剤中グルカゴンを HPLC にて定量し、ステージ 2〜 5 におけるグルカゴン総量をカプセル充填製剤中グルカゴン総量で割った数字である。なお、HPLC における分析条件を下記に示す。
（対象製剤）
グルカゴン-乳糖百倍散-乳糖担体混合物
グルカゴン-エリスリトール百倍散-乳糖担体混合物
（HPLC分析条件）
使用カラム： ODS-120 T （トーソー）
検出器： RF-535 （島津）
ポンプ： LC-10 AD （島津）
励起波長： 280 nm
蛍光検出波長： 346 nm
移動相流速： 1.0 mL/min
移動相： 35 % CH₃CN aq (0.1 % TFA acidified)
カラムオーブン温度： 室温
この検討によって乳糖使用製剤とエリスリトール使用製剤のカプセルからの放出特性およびその後の分散性、そして担体との解離の容易さは明瞭に示された。
【００６３】
【表９】

【００６４】
〔実施例２３〕 カスケードインパクターによるグルカゴン高濃度含有製剤評価
実施例３の方法で作成したグルカゴン-エリスリトール混合粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。 RF 値について表１０に示す。この RF 値は各ステージに捕集された製剤中グルカゴンを HPLC にて定量し、ステージ 2〜 5 におけるグルカゴン総量をカプセル充填製剤中グルカゴン総量で割った数字である。なお、HPLC における分析条件は実施例２２と同様である。
【００６５】
【表１０】

【００６６】
この結果から、賦形剤添加の効果が明瞭に示されており、5倍散というペプチド製剤としては極めて高濃度な条件においても良好な製剤特性を保持していることを確認した。先の実施例２２においても認められたように賦形剤非存在下では、臨床使用に耐えうる製剤供給が極めて難しいことから、本発明製剤処方の有用性が強く示唆される。
【００６７】
〔実施例２４〕 カスケードインパクターによるクエン酸含有グルカゴン製剤評価
実施例１２の方法で作成したグルカゴン-クエン酸-エリスリトール混合粉末（グルカゴン 10 倍散）について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。RF 値について表１１に示す。この RF 値は各ステージに捕集された製剤中グルカゴンを HPLC にて定量し、ステージ 2〜 5 におけるグルカゴン総量をカプセル充填製剤中グルカゴン総量で割った数字である。なお、HPLC における分析条件は実施例１７と同様である。
【００６８】
【表１１】

【００６９】
ペプチド及びタンパク質だけでなく、添加剤にも製剤学上取り扱いにくい化合物は多々知られている。今回使用したクエン酸もその一つであり、エリスリトール非存在下においては速やかな潮解を示しており、臨床使用においては非常に大きな問題点を提示している。しかしながら、本発明による製剤処方によれば、クエン酸存在下で、かつペプチド性薬剤の濃度が非常に高い本実施例のようなケースでも非常に良好な製剤特性を示すことが明らかとなった。
【００７０】
〔実施例２５〕 カスケードインパクターによるリポソーム封入グルカゴン製剤評価
実施例１４の方法で作成したリポソーム封入グルカゴン粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。各ステージに捕集された粉体重量を測定した結果、気管支または肺に相当するステージ 2〜5 には約 30% の微細粒子が捕集されていることを確認した。脂質のみで構成されるリポソーム封入薬剤は脂質の特性上凝集塊を形成しやすいが、本実施例にて示されるように、臨床使用に耐えうる肺到達率を獲得させることができる。
【００７１】
〔実施例２６〕 カスケードインパクターによる VIP 誘導体製剤評価
実施例６の方法で作成した VIP 誘導体-エリスリトール混合粉末（VIP 誘導体 400 倍散）について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。各ステージに捕集された粉体重量を測定した結果、気管支または肺に相当するステージ 2〜5 には約 40% の微細粒子が捕集されていることを確認した。
【００７２】
〔実施例２７〕 カスケードインパクターによるインスリン製剤評価
実施例７の方法で作成したインスリン-エリスリトール混合粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。なお、HPLC における分析条件は下記の通りである。
（HPLC分析条件）
使用カラム： ODS-120 T （トーソー）
検出器： SPD-10 A （島津）
ポンプ： LC-10 AD （島津）
検出波長： 220 nm
移動相流速： 1.0 mL/min
移動相： 34% CH₃CN aq (0.1 % TFA acidified)
カラムオーブン温度 : 室温
本検討の結果、インスリン製剤の RF 値は 22% であることが示され、本インスリン吸入製剤は充分臨床応用可能であることを確認した。
【００７３】
〔実施例２８〕 カスケードインパクターによるサケカルシトニン製剤評価
実施例８の方法で作成したサケカルシトニン−乳糖およびサケカルシトニン−エリスリトール混合粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV社製）と混和し DPI 製剤とした。この製剤についてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。分析後算出された RF 値について表１２に示す。この RF 値は各ステージに捕集された製剤中カルシトニンを HPLC にて定量し、ステージ 2〜 5 におけるカルシトニン総量をカプセル充填製剤中カルシトニン総量で割った数字である。なお、HPLC における分析条件を下記に示す。
【００７４】
【表１２】

【００７５】
この結果でも乳糖使用製剤とエリスリトール使用製剤のカプセルからの放出特性およびその後の分散性、そして担体との解離の容易さは明瞭に示された。
【００７６】
〔実施例２９〕 カスケードインパクターによるエルカトニン製剤評価
実施例９の方法で作成したエルカトニン-エリスリトール混合粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。各ステージに捕集された粉体重量を測定した結果、気管支または肺に相当するステージ 2〜5 には約 40% の微細粒子が捕集されていることを確認した。
【００７７】
〔実施例３０〕 カスケードインパクターによる成長ホルモン (GH) 製剤評価
実施例１０の方法で作成した成長ホルモン (GH)-エリスリトール混合粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。各ステージに捕集された粉体重量を測定した結果、気管支または肺に相当するステージ 2〜5 には約 47％ の微細粒子が捕集されていることを確認した。
【００７８】
〔実施例３１〕 カスケードインパクターによるシクロスポリン製剤評価
実施例１１の方法で作成したシクロスポリン-エリスリトール混合粉末について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。この製剤を対象としてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。各ステージに捕集された粉体重量を測定した結果、気管支または肺に相当するステージ 2〜5 には約 40% の微細粒子が捕集されていることを確認した。
【００７９】
〔実施例３２〕 カスケードインパクターによる賦形剤粉砕物評価
実施例１の方法で作成した各種賦形剤粉末（乳糖，マンニトール，エリスリトール）について非帯電袋を用いて担体（Pharmatose 325M, DMV 社製）と混和し DPI 用製剤とした。
【００８０】
本DPI 用製剤を対象に人工気道および肺モデルであるカスケードインパクターにて USP 記載の方法に従い分析を行い、各種サンプルの分散性および組織到達度を検討した。検討方法は実施例１８に記載している内容と同様である。

賦形剤微粉化物-担体混合物のカスケードインパクターによる分析結果を図６〜８に示す（棒グラフ部位は各ステージにおける捕集量の装置総捕集量に対するパーセンテージを表し、折れ線グラフ部位は累積捕集 % を示す）。
【００８１】
図６はエリスリトール混合物、図７はマンニトール混合物、図８は乳糖混合物を示すが、この結果から明らかにエリスリトールとマンニトール粉砕物は担体である乳糖との付着性が低く、その結果同担体からの解離が容易であることを見出した。特筆すべきはエリスリトールの低吸湿性であり、これによって経時的な重量変化が顕著に抑えられることも、製剤特性の向上に大きく貢献していると考えられる。
【００８２】
〔実施例３３〕 賦形剤の異なる酢酸ブセレリン製剤の特性
実施例４にて作成した酢酸ブセレリン含有吸入製剤についてカスケードインパクターにて分析を行い、それぞれの特性について比較検討した。基本的分析方法は実施例１８と同様であるが、本検討においてはデバイスを使用した。分析に際しての変更点を下記に示す。

【００８３】
カスケードインパクターでの分析の結果、表１３に示すように エリスリトールを賦形剤として適用した製剤は極めてカプセルからの放出能が高く、かつ高い RF 値を示すことが明らかとなった。また、マンニトールは流動性が悪く、カプセルに残存する製剤の量が比較的多いため、 RF 値も必然的に低くなることが推測される。
【００８４】
【表１３】

【００８５】
〔実施例３４〕 賦形剤の異なるグルカゴン製剤の特性
実施例３の方法で作成したグルカゴン製剤についてカスケードインパクターによる分析を検討した。基本的な分析条件は実施例１８と同様である。 RF 値とカプセル残存について表１４に示す。この RF 値は各ステージに捕集された製剤中グルカゴンを HPLC にて定量し、ステージ 2〜 5 におけるグルカゴン総量をカプセル充填製剤中グルカゴン総量で割った数字である。なお、HPLC における分析条件を下記に示す。
（対象製剤）
グルカゴン-乳糖百倍散-乳糖担体混合物（[微粉化物]/[325M] = 0.4）
グルカゴン-エリスリトール百倍散-乳糖担体混合物（[微粉化物]/[325M] = 0.2）
（HPLC分析条件）
使用カラム： TOSO ODS-120 T （トーソー）
検出器： RF-535 （島津）
ポンプ： LC-10 AD （島津）
励起波長： 280 nm
蛍光検出波長： 346 nm
移動相流速： 1.0 mL/min
移動相： 35 % CH₃CN aq (0.1 % TFA acidified)
カラムオーブン温度 : 室温
この結果でも乳糖使用製剤とエリスリトール使用製剤のカプセルからの放出特性およびその後の分散性、そして担体との解離の容易さは明瞭に示された。
【００８６】
【表１４】

【００８７】
〔実施例３５〕グルカゴン製剤類のラット肺からの吸収率
実施例３にて作製したグルカゴン-DPI 製剤および実施例１４にて作製したリポソーム封入グルカゴン-DPI を下記の通り実験動物に投与し、経時的に血糖値をモニタリングした。体重 300 〜 400 g の雄性 SD ラットをネンブタール麻酔下、気道内投与デバイスにて各種グルカゴン製剤を肺内に投与した。投与後、経時的に頸静脈より約 1 mL の採血を行い、4.8 mg の EDTA (2 Na) を添加後、遠心分離処理を行い、血漿サンプルを得た。血漿は直ちに −40℃ に保存した。血漿中グルコース濃度は酵素法にて算出した。リポソームに封入していないグルカゴン-DPI の経肺投与における血糖値変化を図９(a) に示す。リポソーム封入グルカゴン-DPI の投与結果を図９(b) に示す。両者共に投与前よりも血糖値が有意に上昇しており、更に特筆すべきは図９(c) にて示されるリポソーム封入グルカゴン-DPI のより強力な効果である。
【００８８】
【発明の効果】
本発明の粉末製剤は、生体内において非常に有用であるペプチド・タンパク類が特定条件下にて自己凝集を起こすことを阻害し、含量均一な製剤として提供できるのみならず、かつ当該医薬化合物の性質も損なうことなく臨床に提供され、医療経済学的な見地からもその有用性がある。
【００８９】
【配列表】

【００９０】
【配列表フリーテキスト】
配列番号１：VIP誘導体
【図面の簡単な説明】
【図１】担体との混合前の乳糖のみの微細粒子の粒子径分布を示す。
【図２】担体との混合前のグルカゴン及び乳糖を含有する微細粒子の粒子径分布を示す。
【図３】担体（Pharmatose325M）のみの粒子径分布を示す。
【図４】微細粒子と担体（Pharmatose325M）との混合後の複合体の粒子径分布を示す。
【図５】 HPLCにおける各サンプルのグルカゴンピーク面積を示す。
【図６】エリスリトール微粉化物-乳糖担体混合物のカスケードインパクターによる分析結果を示す。
【図７】マンニトール微粉化物-乳糖担体混合物のカスケードインパクターによる分析結果を示す。
【図８】乳糖微粉化物-乳糖担体混合物のカスケードインパクターによる分析結果を示す。
【図９】グルカゴン-DPIの経肺投与における血糖値変化を示す。
（ａ）リポソームに封入していないグルカゴン-DPIの投与前に対する投与後（１５分）の血糖値上昇率
（ｂ）リポソーム封入グルカゴン-DPIの投与前に対する投与後（１５分）の血糖値上昇率
（ｃ）リポソームに封入していないグルカゴン-DPIとリポソーム封入グルカゴン-DPIにおける血糖値変化の比較[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a carrier-based powder formulation, and in particular, it is possible to easily make a peptide or protein that is difficult to formulate into a good inhalation formulation, and is easy to handle in pharmacology and improves dispersibility. The present invention relates to a powder formulation characterized in that the content can be kept uniformly.
[0002]
[Prior art]
Inhalation therapy is used as a medication for the respiratory tract, not only for treatment of airway diseases, but also for diagnosis of diseases, administration of systemic drugs of the respiratory tract, prevention of diseases, and trans-respiratory immunity desensitization therapy. However, in any case, the method for determining the indication of this therapy has not been sufficiently studied, and the development of a corresponding inhalant is desired. When applying as a targeting therapy, it is necessary to consider the method for selecting an inhalant not only based on its effectiveness on diseases, but also on the relationship between the method of generating and reaching the drug particles, and the basic physical properties of the drug. Currently, aerosol inhalation therapy is widely used as this form of drug. Actually, bronchodilators, mucosal solubilizers, antibiotics, antiallergic agents, steroids, vaccines, physiological saline, etc. are used, and in these clinical applications, the site of action of inhalants, The mechanism of action, composition, and usage are considered important factors.
[0003]
In recent years, powder inhalers (DPI) have been attracting attention in the treatment of bronchial asthma and chronic obstructive pulmonary disease. Other inhalants include the aforementioned aerosols (quantitative nebulizer, Metered Dose Inhaler, MDI) and nebulizers, but these drugs have a large surface area that allows the target lung to be comparable to the small intestine. And has many excellent properties such as no first-pass effect after being absorbed. Common inhalation features include 1) rapid onset of efficacy, 2) progressive reduction of side effects, 3) administration of small doses, and 4) avoidance of first-pass effects.
[0004]
As mentioned above, although MDI is widely used and is still being researched vigorously, environmental problems have been pointed out because chlorofluorocarbon must be used as a propellant. In order to solve this problem, chlorofluorocarbon alternatives have been developed, but rather, chlorofluorocarbons that do not require chlorofluorocarbon and have excellent portability are being actively developed. Nebulizers also have the problem that the drug must be stored in the device in solution, so it is considered unsuitable for unstable drugs and the drug can exist in a stable form DPI is one of the formulations that are useful for long-term stability.
[0005]
DPI is classified into capsule type, blister type, reservoir type, etc. according to the packaging form. At the time of DPI administration, in order to take out the medicines packed in these, an inhalation hole is opened in a capsule or the like by device operation, and the drug is released by inhalation operation and is administered to bronchi or lungs.
[0006]
In DPI, there is a close relationship between the particle size of drug particles inhaled by patients and deposition in the respiratory tract [Pharmacia (1997) Vol. 33, No. 6, 98-102]. There is also an aerodynamic correlation of whether it deposits inside the lung. It is generally known that the optimal size of drug particles that can reach the bronchial and alveolar sites is particles with an aerodynamic diameter of about 1-6 μm [Int. J. Pharm. (1994) 101, 1-13].
[0007]
Particle sizes of several μm reach the alveoli, are efficiently absorbed from the lung mucosa and migrate into the blood, so this particle size is especially important when systemic effects are expected. However, the finer the particles, the worse the fluidity of the powder, and there is a concern about the accompanying decrease in filling accuracy and handling during production. Furthermore, it is assumed that it adheres to the inside of the device or capsule at the time of administration. In order to overcome these problems in handling DPI preparations, a method of mixing these fine powders with coarse particles such as lactose as a carrier and a refined drug is well known. This is because the micronized drug is adsorbed on the surface of lactose by intermolecular interaction to weaken the cohesive force of the micronized drug, the particle size becomes larger as a whole, and the fluidity of the preparation is improved. Other methods include drug granulation and surface modification. For example, International Publication No. WO99 / 27911 “Soft Pellet Drug and Method for Producing the Same” suggests improvement in fluidity by drug granulation.
[0008]
For a dosage form using a carrier unlike a soft pellet, a uniform drug distribution is a prerequisite for its pharmaceutical chemistry. However, there are many drugs that have been confirmed to be self-associate regardless of whether they are natural products or synthetic products, and uniform mixing with a carrier is often difficult.
[0009]
In addition, peptides and proteins generally have extremely high hygroscopicity, and many compounds exhibit deliquescence when left for a long period of time, depending on the compound. It is easy to predict that the effect of moisture absorption on the formulation characteristics is very large. This can have a very large effect on the production efficiency during the production of the preparation. Therefore, one of the most important factors is how to effectively pulverize peptides and proteins and maintain them as a dry powder.
[0010]
As described above, there are many factors unsuitable for DPI in peptides and proteins, but there are many useful drugs in these peptides and proteins, and a very small dose is sufficient. A pharmacological effect is often obtained. Therefore, in the medical economics, there is a demand for formulation design that makes these usefully pulmonary agents and inhalants.
[0011]
[Problems to be solved by the invention]
An object of the present invention is to develop a drug product design in which a self-aggregating drug can be uniformly mixed with a carrier, and to develop a drug form in which a very small amount of drug can be uniformly mixed with a carrier. It is to provide a new dosage form for many drugs that are unstable but indeed useful, while maintaining dryness and without pain. It is also an important issue to maintain good production efficiency during the production of the preparation by devising the prescription form.
[0012]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have mixed appropriate excipients and drugs in a solution or in an aerodynamic pulverizer, and the object is achieved by a pulverizer such as a jet mill. After producing a drug with a particle size, the obtained drug was mixed with a carrier, thereby succeeding in obtaining a very uniform preparation and completing the present invention. In addition, at this time, the solubility was dramatically improved by using lactose or erythritol as an excipient. This formulation can be used as a powder nasal preparation in addition to a powder pulmonary preparation. That is, the present invention provides the following (1) to (14).
[0013]
(1) A powder formulation obtained by mixing fine particles containing a powdery drug and an excipient with an average particle size of 20 μm or less with a carrier having an aerodynamically acceptable particle size.
(2) The powder formulation according to (1) above, wherein the drug has a self-aggregation ability due to intermolecular interaction.
(3) The powder formulation according to (1) above, wherein the drug has hygroscopicity and / or deliquescence.
(4) The powder formulation according to any one of (1) to (3) above, wherein the drug is a peptide or protein.
(5) The powder formulation according to any one of (1) to (4) above, wherein the drug is encapsulated in a lipid membrane.
(6) The powder formulation according to any one of (1) to (5) above, wherein the excipient is easily soluble in water.
(7) The powder formulation according to (6) above, wherein the excipient is erythritol.
(8) The powder formulation according to any one of (1) to (7) above, wherein the ratio of the powdery drug and the excipient is in the range of 1: 5000 to 10: 1.
(9) The powder formulation according to any one of (1) to (8) above, wherein the ratio of the fine particles to the carrier is in the range of 1: 100 to 10: 1.
(10) The powder formulation according to any one of (1) to (9) above, wherein the carrier has a particle size in the range of 10 to 200 μm.
(11) A dry product obtained by dissolving or suspending a powdered drug in a solution in which an excipient is dissolved and making a fine powder formulation by spray drying as it is, or freeze-drying the dissolved solution or suspension. And then mixing with a carrier having an aerodynamically acceptable particle size.
(12) A method for producing a powder preparation, wherein an excipient and a powdered drug are simultaneously pulverized and mixed by an aerodynamic pulverizer and mixed with a carrier having an aerodynamically acceptable particle size.
(13) The method for producing a powder preparation according to (11) or (12) above, wherein the drug has a self-aggregation ability due to intermolecular interaction.
(14) The method for producing a powder preparation according to any one of (11) to (13) above, wherein the carrier has an average particle size of 10 μm or more.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[1] drugs
The drug used in the present invention is not particularly limited, and includes drugs used as medicines or used in the future. Drugs include those that can be obtained in powder form and those that can be processed into powder form by processing such as grinding. The shape and particle size of the powdered drug that can be used in the present invention are not particularly limited, but the present invention is particularly effective when the drug has self-aggregation ability, hygroscopicity, and deliquescence due to intermolecular interaction. Can be used. Examples of drugs having self-aggregation ability by intermolecular interaction include glucagon [J. Biol. Chem. (1984) 259, 7031-7], FGF [Biochem. J. (1999) 341, 613-20], growth hormone. [Biochemistry (1993) 32, 1555-62], endothelin [Pept. Res. (1992) 5, 97-101], calcitonin [Biochem. Biophys. Res. Commun. (1998) 245, 344-8], insulin [ Int. J. Pharm. (1999) 191, 51-64] glucagon-like peptide-1 [Pharmaceutical Research (1998) 15, 254-62] and the like. Further, in the present invention, it is particularly preferable that the drug has a very small dose, so that uniform mixing for the preparation is difficult. For example, without limitation, a series of angiotensin converting enzyme inhibitors, LH-RH, ACTH, Caerulein, CCK, salmon calcitonin, EGF, G-CSF, GM-CSF, oxytocin, neurotensin, ghrelin (Ghrelin), PTH, PTHrP, somatostatin, VIP, PACAP, vasopressin, enkephalin, endorphin, dynorphin, substance P, β-NGF, TGF-β, erythropoietin, interferon α, β, or γ, interleukin, tumor necrosis factor , GHRF, c-GRP, ANP, CRF, secretin, cyclosporine and other immunosuppressants, a series of serine proteases such as α-MSH, thrombin and plasmin, or derivatives containing agonists and antagonists thereof, etc. . In addition, the preparation of the present invention can be used as a method for directly administering lung diseases such as lung cancer and pneumothorax.
[0015]
Although peptides and proteins are very expensive, they have been shown to aggregate under certain conditions, including high concentration conditions.For example, calcitonin described above is self-reacting at concentrations of 50 mg / mL and higher. Aggregation has been observed. Hydrogen bonds, hydrophobic interactions and many other interactions are observed in the group of peptide compounds and protein compounds, and it has been pointed out that physicochemical modification such as aggregation or sedimentation can occur during production. It is possible to prevent them in advance.
[0016]
[2] excipients
Usually, excipients are added for the purpose of increasing the amount of solid preparations such as powders and tablets, diluting, filling, and prosthesis, and have a great influence on the release characteristics of the active ingredient. Cost. In the present invention, the excipient is effective for increasing the solubility of the drug and / or reducing the self-aggregation ability. Therefore, as the excipient, those readily soluble in water are preferable, but those that absorb moisture significantly are not preferable in terms of the properties of this preparation. In the present invention, generally used starches, lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, etc. are used as excipients, but they are biologically inert. Those that are present and expected to have a certain degree of metabolism may be used. Others, erythritol, mannitol, sorbitol, trehalose, sucrose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carmellose sodium, pullulan, dextrin, gum arabic, agar, gelatin, tragacanth, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, stearic acid Fatty acids such as these or salts thereof, waxes and the like may be used. Particularly preferred excipients in the present invention are lactose and erythritol.
[0017]
In the preparation of the present invention, the ratio of the powdery drug and the excipient is preferably in the range of 1: 5000 to 10: 1 by weight. If the amount of the drug is larger than this range, the content uniformity may be hindered. If the amount of the excipient is large, the pharmacological activity may be lost in certain drugs.
[0018]
On the other hand, in recent years, as a technique for improving the absorption rate in DPI, a preparation technique in which an enhancer is added and a preparation formula in which a drug is encapsulated in a lipid layer have come to be well known. Known enhancers include organic acids such as citric acid and capric acid, enzyme inhibitors such as bacitracin, and NO generators (Drug Delivery System (2001) 16, 297; Drug Delivery System (2001 ) 16, 299). As a liposome substrate, a polymer such as chitosan is well known. These are only intended to improve the membrane permeability of the drug, and do not necessarily improve the target tissue (pulmonary / bronchial) reach of the preparation. Even if the absorption rate is dramatically improved, it cannot be said that clinical application is possible unless the drug reaches the target tissue. Therefore, application of the present formulation to a formulation design that has been devised for some purpose makes it possible to obtain even better formulation characteristics.
[0019]
[3] carriers
In the present invention, the carrier is used for preventing aggregation of the drug until administration of the powder preparation, and at the time of administration, for efficient separation during the inhalation operation using the inhaler to increase absorption efficiency. When using a carrier for DPI formulation design, it is desirable that the drug is reliably released from the capsule or device and that the drug is separated from the carrier surface with a high probability. When using a carrier, the fluidity of the preparation, prevention of drug aggregation, the possibility of dose increase / decrease, etc. are important. Therefore, not only toxicity and physicochemical stability, but also ease of handling and workability are questions as a selection criterion for carriers, and in order to clear this problem, its stability has been established in the past, Lactose, which is neutral and has low reactivity and is slightly sweet, is useful in many respects and has been confirmed to be useful as a carrier for DPI [Int. J. Pharm. (1998) 172, 179-188]. Examples of carriers that can be used in the present invention include lactose, glucose, fructose, mannitol, sucrose, maltose and dextran sugars, and general excipients such as calcium sulfate, calcium carbonate, talc, and titanium oxide. There is no particular limitation.
[0020]
The powder formulation of the present invention is to be administered using an inhaler, and therefore the carrier has an aerodynamically acceptable particle size. Specifically, the particle size of the carrier is in the range of 10 to 200 μm.
It is known that the particle size should be increased when it is desired to act on the formulation design only as a carrier, but it is also a well-known fact that the carrier can remain in the throat or mouth if the particle size is increased at the same time. is there. Therefore, if the carrier itself is biologically inert but it is desirable to prevent it from reaching the lungs, there is no problem if the particle size is at least 10 μm. In addition, when seeking the best conditions, it is desirable to select the material in consideration of compatibility with the excipient mixed with the main agent, but the same as the excipient unless a major problem is observed. It is preferable to select a material carrier.
[0021]
The ratio of the fine particles containing the powdered drug and the excipient to the carrier is preferably in the range of 1: 100 to 10: 1. If the amount of fine particles is larger than this range, the content uniformity may be hindered, and if the amount of the carrier is increased, the pharmacological activity may be lost in certain drugs.
[0022]
[4] Mixing and grinding process of drug and excipient
The production of the powder preparation of the present invention first includes a mixing and pulverizing step of a powdery drug and an excipient. The pulverization is performed, for example, by dissolving or suspending the powdered drug in a solution in which the excipient is dissolved, and pulverizing a dried product obtained by freeze-drying the solution or suspension. Alternatively, the excipient and the powdered drug can be simultaneously pulverized and mixed by an aerodynamic pulverizer. The manufacturing method of the powder formulation of this invention is not specifically limited, The method normally used by those skilled in the art can be used suitably. Which method is used can be appropriately determined depending on the types of drugs and excipients, the final particle size, and the like. In many cases, the latter treatment method should preferably be selected in this step, since the crystalline state of the compound and the formulation characteristics such as adhesion and dispersibility often correlate. However, when erythritol is used, a good pulverized mixture can be obtained regardless of which step is selected due to the extremely high crystal orientation of the present compound.
[0023]
In the present invention, general dry pulverization can be used for pulverization of the drug and excipient, but it is particularly preferable to use an aerodynamic pulverizer. Specifically, as a general dry pulverizer, an apparatus for efficiently pulverizing a small amount such as a mortar or a ball mill is frequently used for a laboratory. As the ball mill, a rolling ball mill, a centrifugal ball mill, a vibration ball mill, and a planetary ball mill are known, and these can be pulverized on the principle of grinding, rotation, vibration and impact. For industrial use, there are many devices for efficiently pulverizing a large amount of raw materials such as a medium stirring mill, a high-speed rotary milling / impact mill, and a jet mill. High-speed rotary grinding mills include disk mills and roller mills. High-speed rotary impact mills include cutter mills (knife mills), hammer mills (atomizers), pin mills, screen mills, etc. in addition to rotational impacts, and grinding is also performed by shearing force. There is something to do. Many jet mills are mainly pulverized by impact, but the most orthodox particle / particle collision type, particle / collision plate collision type, and nozzle suction type (blowing) type are available.
By this pulverization step, the powdered drug is uniformly mixed with the excipient and pulverized so that the average particle diameter becomes fine particles of 20 μm or less. By setting it as this particle size, it can isolate | separate from a support | carrier after administration and can reach | attain target sites, such as a bronchi and an alveoli.
[0024]
[5] Mixing process of carrier and fine particles
The fine particles obtained in the above mixing / pulverization step are then mixed with a carrier to form a stable complex until administration.
For mixing the carrier and the fine particles, a generally known mixer can be used. There are mainly batch type and continuous type. There are two types of batch type: rotary type and fixed type. Rotary type includes horizontal cylindrical mixer, V type mixer, double cone type mixer, and cubic type mixer. Fixed type is screw type (vertical and horizontal) mixer, swivel screw type mixer, ribbon There are type (vertical, horizontal) mixers. The continuous type is also divided into a rotary type and a fixed type. The rotary type is a horizontal cylindrical mixer, a horizontal cone type mixer, and the fixed type is a screw type (vertical, horizontal) mixer, ribbon type (vertical, Horizontal) mixers and rotary disk mixers are known. In addition to this, by using a mixing method using an aerodynamic pulverizer such as a medium stirring mill, a high-speed rotary milling / impact mill, a jet mill, or a bag made of nylon or similar properties, and stirring. It is possible to make a uniform mixed preparation.
[0025]
[6] inhalers
The powder formulation of the present invention obtained in the above step can be administered to a subject by transmucosal administration such as pulmonary administration or nasal administration. Specifically, if the route of administration is pulmonary administration, it can be administered using any inhaler used in the art.
As an inhaler, inhalation transpulmonary devices such as spin heller, eherer, FlowCaps, jet hella, disc heller, rotor heller, inspear ease, inhalation eight, and quantitative nebulizers can be used. It is not something.
[0026]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
[Example 1] Preparation of excipient powder
Among the compounds that can be used as excipients in the present invention, lactose, mannitol, and erythritol were micronized under the following conditions.
(Crushing conditions)
Equipment used: A-O-Jet Mill (Seishin company)
Raw material supply method: Auto feeder
Supply air pressure: 6.0 kg / cm²G
Grinding air pressure: 6.5 kg / cm²G
Dust collection method: Outlet bug (polyethylene)
The yields were as follows.
Lactose 32.0%
Mannitol 50.5%
Erythritol 75.7%
From this result, it was revealed that sugar alcohol is superior in yield to lactose.
[0027]

[0028]
About 10 g of Japanese Pharmacopoeia Lactose (Yoshida Pharmaceutical) and Glucagon Lactose Hypotrophic Lyophilized Product were pulverized by the above method. When a certain amount of 10 g was processed, the yields were almost the same as shown in Table 1, and both were almost the same in visual property confirmation. It was confirmed that the glucagon aggregation, which had been a concern, had been solved at least in the visible range, and was a powder having almost the same properties as lactose. Furthermore, the particle size distribution of both was evaluated with a laser diffraction / scattering particle size distribution measuring device (manufactured by Nikkiso Co., Ltd.). As shown in FIGS. 1 and 2, although the width of the distribution is slightly different, the peak top is about 3 μm, and almost 100% of the powder has a particle size of 10 μm or less.
[0029]
[Table 1]

[0030]
[Example 3] Microproduction of glucagon powder
In the same manner as in Example 1, 2 g of pulverized glucagon lactose 100-fold freeze-dried product and glucagon erythritol 100-fold were pulverized. The yield at this time is shown in Table 2. As this result shows, when a small amount is produced, many factors that lower the yield, including adsorption and moisture absorption, appear on the surface in a clearer form, so the erythritol-based mixture is produced more. The yield was high. The problem of glucagon aggregation, which was a concern, has been solved at least within the visible range, and it was confirmed that the powder had almost the same properties as the excipient fine powder produced in Example 1.
[0031]
[Table 2]

[0032]
[Example 4] Preparation of buserelin acetate powder
Buserelin acetate (manufactured by Itoham Co., Ltd.), buserelin acetate-lactose mixture (570 times powder), buserelin acetate-mannitol mixture (570 times powder), and buserelin acetate-erythritol mixture (570 times powder) are the same as in Example 1. Micronization was performed.
[0033]
The yields were as follows.
Only buserelin acetate not confirmed due to deliquescence
Buserelin acetate-lactose mixture 27.0%
Buserelin acetate-mannitol mixture 41.7%
Buserelin acetate-erythritol mixture 71.7%
This result clearly shows that it is very difficult to grind only the peptide, and the effect of adding excipients such as lactose or erythritol became clear. The lactose mixture was very sticky and it was somewhat difficult to recover from the bag filter. On the other hand, the mannitol and erythritol mixture was very low in adhesion, confirming a dramatic improvement in the recovery rate.
[0034]
[Example 5] Preparation of powder containing high concentration of glucagon
In the same manner as described in Example 1, the glucagon-erythritol mixture (3, 5, 10, 20 folds) was pulverized using the jet mill under the following conditions. That is, glucagon and erythritol are mixed as shown in the table below, and fine particles are processed using a jet mill under the conditions shown in Example 1, and then glucagon-erythritol is pulverized from a bag filter for collecting powder. A mixture was obtained. The recovery rate was about 30 to 40% in all cases. It was confirmed by HPLC that the content of glucagon in the powder was almost theoretical. It was shown that such a high-concentration peptide preparation can be prepared by this preparation formulation.
[0035]
[Table 3]

[0036]
[Example 6] Preparation of vasoactive intestinal peptide (VIP) derivative powder
In the same manner as described in Example 1, the VIP-erythritol mixture (400 times dispersion) was pulverized using the jet mill under the following conditions. That is, the VIP derivative His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Arg-Gln-Met-Ala-Val- described in JP-A-8-333276 Arg-Arg-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-Gly-Arg-Arg-NH₂(SEQ ID NO: 1) 5 mg is mixed with 2 g of erythritol in an appropriate amount, processed with fine particles using a jet mill under the conditions shown in Example 1, and about 70% from the bag filter for collecting powder. A finely pulverized VIP derivative-erythritol mixture was obtained. It was confirmed by HPLC quantitative method that the content of VIP derivative in powder was almost theoretical value.
[0037]
[Example 7] Preparation of insulin powder
The insulin-erythritol mixture (20-fold powder) was micronized under the following conditions using a jet mill in the same manner as described in Example 1. That is, 100 mg of porcine insulin (manufactured by Itoham Co., Ltd.) was mixed with 1.9 g of erythritol in an appropriate amount, and fine particle processing was performed under the conditions shown in Example 1 using a jet mill to produce a powder collecting bug. A finely divided insulin-erythritol mixture was obtained from the filter with a recovery rate of about 60%. It was confirmed by HPLC that the content of insulin in the powder was almost the theoretical value.
[0038]
[Example 8] Preparation of salmon calcitonin powder
In the same manner as described in Example 1, the salmon calcitonin-lactose mixture (200 times trituration) and the salmon calcitonin-erythritol mixture (200 times trituration) were pulverized under the following conditions using a jet mill. That is, 15 mg of salmon calcitonin (manufactured by Itoham Co., Ltd.) is mixed with lactose or about 3.0 g of erythritol, and fine particle processing is performed under the conditions shown in Example 1 using a jet mill to collect powder. The salmon calcitonin mixed powder which was micronized with the recovery rate of about 70% (salmon calcitonin-lactose mixture) and about 80% (salmon calcitonin-erythritol mixture) from the collecting bag filter was obtained. It was confirmed by HPLC that the salmon calcitonin content in powder was almost theoretical.
[0039]
[Example 9] Preparation of elcatonin powder
In the same manner as described in Example 1, the elcatonin-lactose mixture (200 folds) and the elcatonin-erythritol mixture (200 folds) were pulverized using the jet mill under the following conditions. That is, elcatonin (ITO HAM Co., Ltd.) 15 mg was mixed with lactose or erythritol approximately 3.0 g in a moderate amount, and fine particle processing was performed under the conditions shown in Example 1 using a jet mill to collect powder. Elcatonin mixed powder micronized with a recovery rate of about 20% (elcatonin-lactose mixture) and about 70% (elcatonin-erythritol mixture) from the bag filter was obtained. It was confirmed by HPLC that the content of elcatonin in the powder was almost theoretical.
[0040]
[Example 10] Preparation of human growth hormone (GH) powder
In the same manner as described in Example 1, a human growth hormone-erythritol mixture (50-fold powder) was pulverized under the following conditions using a jet mill. That is, human growth hormone mixture (Jiangxi Chinese Import & Export Co. Ltd) 30 mg (human growth hormone 10 mg, human albumin 10 mg, glycine 10 mg) is mixed with erythritol approx. 470 mg, using a jet mill. Fine particle processing was performed under the conditions shown in Example 1 to obtain a human growth hormone-erythritol mixture that was micronized with a recovery rate of about 50% from a powder collecting bag filter. It was confirmed by HPLC that the content of human growth hormone in powder was almost the theoretical value, and the stability was also confirmed at the same time. As a result, it was shown that this pharmaceutical formulation can be applied even to a high molecular compound such as human growth hormone which is considered to be very unstable and difficult to handle.
[0041]
[Example 11] Preparation of cyclosporin powder
The cyclosporin-erythritol mixture (10-fold dispersion) was pulverized under the following conditions using a jet mill in the same manner as described in Example 1. That is, 100 mg of cyclosporin A (manufactured by Wako Pure Chemical Industries, Ltd.) is appropriately mixed with about 900 mg of erythritol, and fine particle processing is performed under the conditions shown in Example 1 using a jet mill to collect powder. A finely divided cyclosporin-erythritol mixture was obtained from the bag filter for use with a recovery rate of about 60%. It was confirmed by HPLC that the content of cyclosporine in the powder was almost theoretical.
[0042]
[Example 12] Production of citric acid-containing glucagon powder
The glucagon-citric acid mixture and the glucagon-citrate-erythritol mixture were micronized under the following conditions using a jet mill in the same manner as described in Example 1. That is, 80 mg of glucagon (manufactured by Itoham Co., Ltd.) is mixed with 80 mg of citric acid, or 80 mg of glucagon is mixed with 80 mg of citric acid and about 640 mg of erythritol, and Example 1 using a jet mill. Fine particle processing was performed under the conditions indicated by. The pulverized glucagon-citric acid mixture rapidly deliquesced and was extremely difficult to recover. The recovery rate was about 40%. From this, it was shown that this formulation design is useful even in the presence of additives that exhibit deliquescence. It was confirmed by HPLC that the content of glucagon in the powder was almost the theoretical value.
[0043]
[Example 13] Preparation of encapsulated liposome of glucagon
Glucagon (manufactured by Ito Ham Co., Ltd.) was prepared to 10 mg / mL with 0.1% trifluoroacetic acid aqueous solution, and this was composed of L-α-dipalmitoylphosphatidylcholine, cholesterol, and L-α-dipalmitoylphosphatidylglycerol. It added to the hollow liposome (particle size: 100_300 nm, coatsome EL series, NOF Corporation). After shaking for several minutes, it was freeze-dried (freeze dryer made by Vertis, UNITOP HL, Freezemobile 25 EL). About 200 μL was collected from the sample before lyophilization, and centrifuged (10 minutes, 12,000 x g) using a centrifugal ultrafiltration unit ULTRAFREE-0.5 (cut below 10,000 Mw). The glucagon content in the filtrate was quantified and calculated as the amount not encapsulated in the liposomes. The quantitative (HPLC) conditions are as follows.
(HPLC conditions)
Column used: TSK gel ODS-120 T (TOSO)
Detector: RF_535 (Shimadzu)
Excitation wavelength: 280 nm
Fluorescence wavelength: 346 nm
Pump: LC-10AD (Shimadzu)
Mobile Phase: 35% CH_ThreeCN (0.1% TFA acidified)
Mobile phase flow rate: 1.0 mL / min
As a result of quantification by HPLC, the encapsulation rate of glucagon in liposomes was 98%, indicating that the encapsulation efficiency was extremely high.
[0044]
[Example 14] Preparation of liposome-encapsulated glucagon powder (1% glucagon powder)
About 200 mg of erythritol was appropriately mixed with about 200 mg of glucagon-liposome inclusions obtained by the method described in Example 13, and pulverized by a jet mill in the same manner as in Example 1. A finely divided glucagon-liposome inclusion-erythritol mixture was obtained from the bag filter for powder collection at a recovery rate of about 50%. The obtained powder had extremely high fluidity and was a particle having good formulation characteristics. It was confirmed by HPLC that the glucagon content in the powder was almost the theoretical value. Accordingly, it was shown that the present pharmaceutical formulation can be used for liposome preparations, and that the drug can be produced without deliquescent during production.
[0045]
[Example 15] Mixing of powder and carrier
Each powder of lactose and glucagon lactose obtained in Example 2 was quickly mixed with a carrier (Pharmatose 325 M; manufactured by DMV; average particle size 50 ± 10 μm) under the conditions shown in Table 4 and 50 mL. Dried and stored in a disposable tube. The properties (visually) at that time are also shown in Table 4. When lactose powder and glucagon lactose powdered powder were added to the carrier, many of the properties including the fluidity were the same, and it was determined that the self-aggregation property, which is an undesirable property of glucagon, could be suppressed. We were able to. When attention was paid to the glucagon lactose hundred powdered powder that had not been mixed with the carrier, formation of significant aggregates was observed at least after 24 hours.
As is apparent from this result, the powder preparation of the present invention was highly suitable for administration because self-aggregation was suppressed.
[0046]
[Table 4]

[0047]
[Example 16] Mixture particle size distribution of carrier and pulverized glucagon powder
With SK Laser Micronizer LMS-30 (Seishin company), the particle size distribution was examined for both the carrier (Pharmatose 325 M) alone and the mixture of the carrier and the glucagon 100-fold powder (FIGS. 3 and 4). As a result, 2.0 kg / cm² As shown in FIG. 4, when the powder was dispersed at a pressure of 1, the separation of the carrier and the drug was clearly shown, and the separated glucagon hundred times did not show an increase in particle size due to self-aggregation.
As is apparent from the results, the powder formulation of the present invention does not show self-aggregation, and the drug is easily separated from the carrier upon administration, and only fine particles containing the drug can reach the target site. Become.
[0048]
[Example 17] Uniformity evaluation of glucagon content
By suppressing the self-aggregation property of glucagon and improving the fluidity, it is presumed that the drug content uniformity after mixing the carrier will be greatly improved. Thus, the drug was randomly collected from the powder obtained in Example 16, and the uniformity was evaluated by quantitative analysis by HPLC. The method is shown below.
[0049]
The conditions for high-performance liquid chromatography (HPLC) analysis of glucagon containing the preparation are as follows.
(HPLC analysis conditions)
Column used: TOSO ODS-120 T
Detector: Jusco 870-UV
UV detection wavelength: 220 nm
Mobile phase flow rate: 1.0 mL / min
Mobile phase: 35% CH_ThreeCN aq (0.1% TFA acidified)
Pump: Jusco 880-PU
Inject volume: 50 μL
Column oven temperature: 40 ° C
(Method)
Target sample
Glucagon powder (DPG080700) Pharmatose mixture
(Mixing ratio, glucagon lactose 100 times powder: carrier = 1: 2)
[0050]
This sample (volume: 25 mL) is divided into three equal parts in a 50 mL disposable tube, and about 10 mg of 3 samples (samples 1 to 3) are randomly weighed from each section. Prepare 10 mg / mL with 0.1 N HCl, analyze with RP-HPLC, and compare the glucagon-equivalent peak area values between each sample.
FIG. 5 shows the glucagon-equivalent peaks between the sections.
As is apparent from FIG. 5, almost the same amount of drug can be detected in a sample collected at random, and it is shown that the powder formulation of the present invention has improved fluidity and is almost uniformly mixed with the carrier. .
[0051]
[Example 18] Evaluation of glucagon and elcatonin preparations by cascade impactor
In order to investigate the aerodynamic particle size of the powder, a cascade impactor, an artificial airway and a lung model, was examined. The main body is an eight-stage stage and a final filter, which are combined with a current meter and a suction pump. As a basic method, the method described in “Multistage Cascade Impactor Apparatus” in USP 2000 “Physical Tests and Determinations / Aerosols” was applied. The specific method is as follows.

(result)
Each quantitative value in HPLC is shown below.
[0052]
[Table 5]

[0053]
[Table 6]

[0054]
According to this model, drugs collected after stage 3 migrate to the bronchi and lungs, but if both preparations are taken into account that about 30 to 40% of the main drug is present after stage 3 In addition, about 30 to 40% of the drugs that enter the body can be expected to have desirable effects.
[0055]
[Example 19] Effect of carrier-drug fine particle mixing ratio on formulation characteristics
In the same manner as in the production method described in Example 1, Japanese lactose (Yoshida Pharmaceutical) is micronized, and the obtained powder is mixed with a carrier (Pharmatose 325M, manufactured by DMV) under the following conditions using an uncharged bag. Preparation for use.
[0056]
[Table 7]

[0057]
Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. Table 8 shows the RF values. This RF value is a number obtained by weighing the weight of powder collected at each stage and dividing the total amount collected at stages 2 to 5 by the total amount of fine powder contained in the preparation.
[0058]
[Table 8]

[0059]
From this result, it was shown that the mixing ratio of the excipient and the particles is large and is related to the formulation characteristics, and it is clear that the smaller content of the drug with respect to the carrier is preferable.
[0060]
[Example 20] Content uniformity evaluation of liposome-encapsulated glucagon preparation
The glucagon-liposome-encapsulated powder prepared in Example 14 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. According to the method shown in Example 17, a fixed amount was weighed from three sections, and the glucagon content contained therein was quantified by HPLC. The HPLC conditions are shown below.
(HPLC conditions)
Column used: TOSO ODS-120 T
Detector: RF-535 (Shimadzu)
Excitation wavelength: 280 nm
Fluorescence wavelength: 346 nm
Mobile phase flow rate: 1.0 mL / min
Mobile phase: 35% CH_ThreeCN aq (0.1% TFA acidified)
Pump: LC-10 AD (Shimadzu)
Column oven temperature: 40 ° C
Samples extracted from the three sections were quantified under the above conditions.The results were 2.32 μg / mg-formulation, 2.43 μg / mg-formulation, and 2.41 μg / mg-formulation, respectively, with an average value of 2.39 μμg / mg-formulation. Met. Even in the case of this case using molecules that are likely to associate and aggregate, such as liposomes, it was confirmed that the pharmaceutical preparation of the invention can supply a preparation with a substantially uniform content.
[0061]
[Example 21] Content uniformity evaluation of salmon calcitonin preparation
The salmon calcitonin powder prepared in Example 8 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. According to the method shown in Example 17, a fixed amount was weighed from three sections, and the salmon calcitonin content contained therein was quantified by HPLC. The HPLC conditions are shown below.
(HPLC conditions)
Column used: TOSO ODS-120 T
Detector: RF-535 (Shimadzu)
Excitation wavelength: 280 nm
Fluorescence wavelength: 320 nm
Mobile phase flow rate: 1.0 mL / min
Mobile phase: 38% CH_ThreeCN aq (0.1% TFA acidified)
Pump: Jusco 880-PU
Column oven temperature: 40 ° C
Samples extracted from the three sections were quantified under the above conditions.These were 3.95 μg / mg-formulation, 4.29 μg / mg-formulation, and 4.05 μg / mg-formulation, respectively, with an average value of 4.09 μg / mg-formulation. Met. Even in the case of this case where the content is extremely low, it was confirmed that the pharmaceutical preparation of the invention can supply a preparation with a substantially uniform content.
[0062]
[Example 22] Evaluation of glucagon preparation by cascade impactor
The glucagon powder prepared by the method of Example 3 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. Table 9 shows the RF values and capsule remaining. This RF value is obtained by quantifying glucagon in the preparation collected at each stage by HPLC and dividing the total amount of glucagon in stages 2 to 5 by the total amount of glucagon in the capsule-filled preparation. The analytical conditions for HPLC are shown below.
(Target product)
Glucagon-Lactose Hypotrophic-Lactose Carrier Mixture
Glucagon-Erythritol Hypotrophic-Lactose Carrier Mixture
(HPLC analysis conditions)
Column used: ODS-120 T (Tosoh)
Detector: RF-535 (Shimadzu)
Pump: LC-10 AD (Shimadzu)
Excitation wavelength: 280 nm
Fluorescence detection wavelength: 346 nm
Mobile phase flow rate: 1.0 mL / min
Mobile phase: 35% CH_ThreeCN aq (0.1% TFA acidified)
Column oven temperature: room temperature
This study clearly showed the release characteristics and subsequent dispersibility of the lactose and erythritol formulations from the capsules and the ease of dissociation from the carrier.
[0063]
[Table 9]

[0064]
[Example 23] Evaluation of preparation containing high concentration of glucagon by cascade impactor
The glucagon-erythritol mixed powder prepared by the method of Example 3 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. Table 10 shows the RF values. This RF value is obtained by quantifying glucagon in the preparation collected at each stage by HPLC and dividing the total amount of glucagon in stages 2 to 5 by the total amount of glucagon in the capsule-filled preparation. The analytical conditions in HPLC are the same as in Example 22.
[0065]
[Table 10]

[0066]
From this result, the effect of excipient addition was clearly shown, and it was confirmed that the peptide formulation of 5 times san retained good formulation characteristics even under extremely high concentration conditions. As recognized in Example 22 above, in the absence of excipients, it is extremely difficult to supply a preparation that can withstand clinical use, which strongly suggests the usefulness of the preparation of the present invention.
[0067]
[Example 24] Evaluation of citric acid-containing glucagon preparation by cascade impactor
The glucagon-citric acid-erythritol mixed powder (glucagon 10-fold powder) prepared by the method of Example 12 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Table 11 shows the RF values. This RF value is obtained by quantifying glucagon in the preparation collected at each stage by HPLC and dividing the total amount of glucagon in stages 2 to 5 by the total amount of glucagon in the capsule-filled preparation. The analytical conditions in HPLC are the same as in Example 17.
[0068]
[Table 11]

[0069]
There are many known compounds that are difficult to handle not only for peptides and proteins but also for additives. Citric acid used this time is one of them, showing rapid deliquescence in the absence of erythritol, and presenting a very big problem in clinical use. However, according to the pharmaceutical formulation according to the present invention, it has been clarified that very good pharmaceutical properties are exhibited even in the case of this example in the presence of citric acid and the concentration of the peptide drug is very high.
[0070]
[Example 25] Evaluation of liposome-encapsulated glucagon preparation by cascade impactor
The liposome-encapsulated glucagon powder prepared by the method of Example 14 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. As a result of measuring the weight of the powder collected in each stage, it was confirmed that about 30% of fine particles were collected in stages 2 to 5 corresponding to bronchi or lung. A liposome-encapsulated drug composed only of lipids tends to form aggregates due to the characteristics of lipids, but as shown in this example, can achieve a lung reach that can withstand clinical use.
[0071]
[Example 26] VIP derivative preparation evaluation by cascade impactor
The VIP derivative-erythritol mixed powder (VIP derivative 400 times dispersion) prepared by the method of Example 6 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. As a result of measuring the weight of the powder collected in each stage, it was confirmed that about 40% of fine particles were collected in stages 2 to 5 corresponding to bronchi or lung.
[0072]
[Example 27] Insulin preparation evaluation by cascade impactor
The insulin-erythritol mixed powder prepared by the method of Example 7 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. The analytical conditions in HPLC are as follows.
(HPLC analysis conditions)
Column used: ODS-120 T (Tosoh)
Detector: SPD-10 A (Shimadzu)
Pump: LC-10 AD (Shimadzu)
Detection wavelength: 220 nm
Mobile phase flow rate: 1.0 mL / min
Mobile phase: 34% CH_ThreeCN aq (0.1% TFA acidified)
Column oven temperature: room temperature
As a result of this study, it was shown that the RF value of the insulin preparation was 22%, and it was confirmed that this insulin inhalation preparation was sufficiently clinically applicable.
[0073]
[Example 28] Evaluation of salmon calcitonin preparation by cascade impactor
The salmon calcitonin-lactose and salmon calcitonin-erythritol mixed powder prepared by the method of Example 8 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to obtain a DPI preparation. This formulation was analyzed by a cascade impactor. Basic analysis conditions are the same as in Example 18. Table 12 shows the RF values calculated after the analysis. This RF value is obtained by quantifying the calcitonin in the preparation collected at each stage by HPLC and dividing the total amount of calcitonin in stages 2 to 5 by the total amount of calcitonin in the capsule-filled preparation. The analytical conditions for HPLC are shown below.
[0074]
[Table 12]

[0075]
This result clearly showed the release characteristics and the subsequent dispersibility of the preparations using lactose and erythritol, and the ease of dissociation from the carrier.
[0076]
[Example 29] Evaluation of elcatonin preparation by cascade impactor
The elcatonin-erythritol mixed powder prepared by the method of Example 9 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. As a result of measuring the weight of the powder collected in each stage, it was confirmed that about 40% of fine particles were collected in stages 2 to 5 corresponding to bronchi or lung.
[0077]
[Example 30] Growth hormone (GH) formulation evaluation by cascade impactor
The growth hormone (GH) -erythritol mixed powder prepared by the method of Example 10 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. As a result of measuring the weight of the powder collected in each stage, it was confirmed that about 47% of fine particles were collected in stages 2 to 5 corresponding to bronchi or lung.
[0078]
[Example 31] Evaluation of cyclosporine preparation by cascade impactor
The cyclosporin-erythritol mixed powder prepared by the method of Example 11 was mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation. Analysis using a cascade impactor was examined for this preparation. Basic analysis conditions are the same as in Example 18. As a result of measuring the weight of the powder collected in each stage, it was confirmed that about 40% of fine particles were collected in stages 2 to 5 corresponding to bronchi or lung.
[0079]
[Example 32] Evaluation of pulverized excipients by cascade impactor
Various excipient powders (lactose, mannitol, erythritol) prepared by the method of Example 1 were mixed with a carrier (Pharmatose 325M, manufactured by DMV) using an uncharged bag to prepare a DPI preparation.
[0080]
This DPI preparation was analyzed using the artificial airway and a cascade impactor, a lung model, according to the method described in USP, and the dispersibility and tissue reachability of various samples were examined. The examination method is the same as that described in Example 18.

The analysis results of the excipient micronized product-carrier mixture by the cascade impactor are shown in FIGS. 6 to 8 (the bar graph portion represents the percentage of the total amount collected in each stage, and the line graph portion represents the cumulative collection) Indicates%).
[0081]
FIG. 6 shows an erythritol mixture, FIG. 7 shows a mannitol mixture, and FIG. 8 shows a lactose mixture. From these results, erythritol and mannitol pulverized product clearly have low adhesion to the carrier lactose, resulting in dissociation from the carrier. Found it easy. Of particular note is the low hygroscopicity of erythritol, and the fact that the change in weight over time can be remarkably suppressed is considered to contribute greatly to the improvement of the formulation characteristics.
[0082]
[Example 33] Properties of buserelin acetate preparations with different excipients
The inhaled preparation containing buserelin acetate prepared in Example 4 was analyzed with a cascade impactor, and the characteristics of each were compared and examined. The basic analysis method was the same as in Example 18, but a device was used in this study. The changes in the analysis are shown below.

[0083]
As a result of the analysis by the cascade impactor, it was revealed that the formulation to which erythritol was applied as an excipient as shown in Table 13 had extremely high ability to release from the capsule and showed a high RF value. In addition, since mannitol has poor fluidity and the amount of the formulation remaining in the capsule is relatively large, it is assumed that the RF value is inevitably low.
[0084]
[Table 13]

[0085]
[Example 34] Properties of glucagon preparations with different excipients
The analysis by the cascade impactor was examined for the glucagon preparation prepared by the method of Example 3. Basic analysis conditions are the same as in Example 18. Table 14 shows the RF value and capsule remaining. This RF value is obtained by quantifying glucagon in the preparation collected at each stage by HPLC and dividing the total amount of glucagon in stages 2 to 5 by the total amount of glucagon in the capsule-filled preparation. The analytical conditions for HPLC are shown below.
(Target product)
Glucagon-Lactose Hypotrophic-Lactose Carrier Mixture ([Micronized] / [325M] = 0.4)
Glucagon-Erythritol 100-fold-Lactose carrier mixture ([micronized product] / [325M] = 0.2)
(HPLC analysis conditions)
Column used: TOSO ODS-120 T
Detector: RF-535 (Shimadzu)
Pump: LC-10 AD (Shimadzu)
Excitation wavelength: 280 nm
Fluorescence detection wavelength: 346 nm
Mobile phase flow rate: 1.0 mL / min
Mobile phase: 35% CH_ThreeCN aq (0.1% TFA acidified)
Column oven temperature: room temperature
This result clearly showed the release characteristics and the subsequent dispersibility of the preparations using lactose and erythritol, and the ease of dissociation from the carrier.
[0086]
[Table 14]

[0087]
[Example 35] Absorption rate of glucagon preparations from rat lungs
The glucagon-DPI preparation prepared in Example 3 and the liposome-encapsulated glucagon-DPI prepared in Example 14 were administered to experimental animals as described below, and blood glucose levels were monitored over time. Male SD rats weighing 300-400 g were administrated intrapulmonary with various glucagon preparations under Nembutal anesthesia using an intratracheal administration device. After administration, approximately 1 mL of blood was collected from the jugular vein over time, and 4.8 mg of EDTA (2 Na) was added, followed by centrifugation to obtain a plasma sample. Plasma was immediately stored at -40 ° C. Plasma glucose concentration was calculated by the enzymatic method. FIG. 9 (a) shows changes in blood glucose level by transpulmonary administration of glucagon-DPI not encapsulated in liposomes. The result of administration of liposome-encapsulated glucagon-DPI is shown in FIG. 9 (b). In both cases, the blood glucose level was significantly higher than before administration, and it should be noted that the liposome-encapsulated glucagon-DPI shown in FIG. 9 (c) is more potent.
[0088]
【The invention's effect】
The powder formulation of the present invention not only prevents the peptide / proteins that are very useful in vivo from causing self-aggregation under specific conditions, but also provides a uniform content formulation, It is provided clinically without any loss of properties, and is useful from a medical economic point of view.
[0089]
[Sequence Listing]

[0090]
[Sequence Listing Free Text]
SEQ ID NO: 1 VIP derivative
[Brief description of the drawings]
FIG. 1 shows the particle size distribution of fine particles of only lactose before mixing with a carrier.
FIG. 2 shows a particle size distribution of fine particles containing glucagon and lactose before mixing with a carrier.
FIG. 3 shows the particle size distribution of only the carrier (Pharmatose 325M).
FIG. 4 shows the particle size distribution of a composite after mixing fine particles and a carrier (Pharmatose 325M).
FIG. 5 shows the glucagon peak area of each sample in HPLC.
FIG. 6 shows the analysis results of a erythritol micronized product-lactose carrier mixture by a cascade impactor.
FIG. 7 shows the analysis results of a mannitol micronized product-lactose carrier mixture by a cascade impactor.
FIG. 8 shows the analysis results of a lactose micronized product-lactose carrier mixture by a cascade impactor.
FIG. 9 shows changes in blood glucose level after pulmonary administration of glucagon-DPI.
(A) Rate of increase in blood glucose level after administration (15 minutes) compared to before administration of glucagon-DPI not encapsulated in liposomes
(B) Increase rate of blood glucose level after administration (15 minutes) compared to before administration of liposome-encapsulated glucagon-DPI
(C) Comparison of blood glucose level change between glucagon-DPI not encapsulated in liposomes and glucagon-DPI encapsulated in liposomes

Claims (10)

Powdered medicament powder formulation average particle diameter containing erythritol as an excipient is obtained the following fine particles 20μm in admixture with lactose carrier having a particle size of 10~200μ m.   The powder formulation according to claim 1, wherein the drug has a self-aggregation ability by intermolecular interaction.   The powder preparation according to claim 1, wherein the drug has hygroscopicity and / or deliquescence.   The powder formulation according to any one of claims 1 to 3, wherein the drug is a peptide or protein.   The powder formulation of any one of Claim 1 to 4 which is a form with which the chemical | medical agent was enclosed with the lipid membrane. The powder formulation according to any one of claims 1 to 5 , wherein the ratio of the powdered drug to erythritol is in the range of 1: 5000 to 10: 1. The powder formulation according to any one of claims 1 to 6 , wherein the ratio of the fine particles to the lactose carrier is in the range of 1: 100 to 10: 1. Dissolve or suspend the powdered drug in a solution in which erythritol is dissolved as an excipient and make a fine powder formulation by spray-drying as it is, or freeze-dry the resulting solution or suspension after grinding, characterized by mixing with lactose carrier having a particle size of 10～200Myu m, method of producing a powder formulation. Erythritol and powdered medicament pulverized and mixed simultaneously by aerodynamic grinder characterized by mixing with lactose carrier having a particle size of 10～200Myu m, method of producing a powder formulation. The method for producing a powder preparation according to claim 8 or 9 , wherein the drug has a self-aggregation ability by an intermolecular interaction.

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