CTD 第 2 部 2.6 非臨床試験の概要文及び概要表 薬物動態試験の概要文 MSD 株式会社

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1 CTD 第 2 部 薬物動態試験の概要文 MSD 株式会社

2 2.6.4 薬物動態試験の概要文 目次 頁 表一覧... 2 図一覧... 3 略号及び用語の定義 まとめ 分析法 吸収 分布 代謝 排泄 薬物動態学的相互作用 その他の薬物動態試験 考察及び結論 薬物動態試験の概要文 1

3 2.6.4 薬物動態試験の概要文 表一覧 頁 表2.6.4: 1 バニプレビル濃度分析法の真度及び精度 薬物動態試験の概要文 2

4 2.6.4 薬物動態試験の概要文 図一覧 頁 図2.6.4: 1 バニプレビルの化学構造... 6 図2.6.4: 2 ラットにバニプレビルの2 mg/kg を単回静脈内投与又は5 mg/kg を単回経口投与 したときの血漿中濃度 時間プロファイル... 8 図2.6.4: 3 イヌにバニプレビルの2 mg/kg を単回静脈内投与又は5 mg/kg を単回経口投与 したときの血漿中濃度 時間プロファイル 図2.6.4: 4 サルにバニプレビルの2 mg/kg を単回静脈内投与又は5 mg/kg を単回経口投与し たときの血漿中濃度 時間プロファイル 図2.6.4: 5 ラットにおけるバニプレビルの血漿及び肝臓中濃度 図2.6.4: 6 イヌにおけるバニプレビルの血漿及び肝臓中濃度 図2.6.4: 7 バニプレビルの推定代謝物 図2.6.4: 8 無処置ラットにおける代表的な尿及び糞中代謝物プロファイル 図2.6.4: 9 無処置ラットに[3H]バニプレビルの114 mg/kg を単回経口投与したときの血漿 中代謝物プロファイル 投与後4時間 図2.6.4: 10 無処置イヌにおける代表的な尿 糞及び血漿中代謝物プロファイル 図2.6.4: 11 ヒト糞及び血漿における代表的な代謝物プロファイル 図2.6.4: 12 ヒト ラット イヌ又はウサギの肝 S9 もしくは マウスの肝ミクロソームに おける[14C]バニプレビルの代表的代謝物プロファイル 薬物動態試験の概要文 3

5 2.6.4 薬物動態試験の概要文 略号及び用語の定義 略語 バニプレビル ADME AUC BCRP BDC BSEP CLb CLp Cmax CYP EDTA F GLP 定義 Absorption, distribution, metabolism and excretion Area under the drug concentrationtime curve Breast cancer resistance protein Bile duct cannulation Below the lower limit of quantification Bile salt export pump Blood clearance Plasma clearance Maximum drug concentration Cytochrome P450 Ethylenediaminetetraacetic acid Absolute oral bioavailability Good laboratory practice HPLC HCV IC50 ID Ki Kinact LCMS High performance liquid chromatography Hepatitis C virus 50% inhibitory concentration Identification Inhibition constant Inactivation rate constant Liquid chromatographymass spectrometry LCMS/MS Liquid chromatographytandem mass spectrometry LIMS LLCPK LSC MDCK MDR mr MRP MS DPH Laboratory information management system Lilly laboratory cellsporcine kidney Liquid scintillation counting MadinDarby canine kidney Multiple drug resistance protein Messenger ribonucleic acid Multidrug resistanceassociated protein Mass spectrometry βnicotinamide adenine dinucleotide phosphate, reduced form Not detected Nuclear magnetic resonance Not sampled Nonstructural protein 3/4A Organic anion transporter Organic anion transporting polypeptide Organic cation transporter Polyethleneglycol Pglycoprotein Quantitative whole body autoradiography Ribonucleic acid Ritonavir Standard deviation Standard error Elimination halflife Time to the maximum drug concentration Uridine diphosphate glucuronosyltransferase Volume of distribution at steady state NMR NS NS3/4A OAT OATP OCT PEG Pgp QWBA R RTV SD SE t1/2 Tmax UGT Vdss 開発番号 MK7009 吸収 分布 代謝 排泄 薬物濃度 時間曲線下面積 乳癌耐性蛋白質 胆管カテーテル 定量下限未満 胆汁酸トランスポーター 血液クリアランス 血漿クリアランス 最高薬物濃度 チトクロム P450 エチレンジアミン四酢酸 絶対経口バイオアベイラビリティ 医薬品の安全性に関する非臨床試験の実施 基準 高速液体クロマトグラフィ C 型肝炎ウイルス 50% 阻害濃度 同定番号 阻害定数 不活性化速度定数 液体クロマトグラフィー マススペクトロ メトリー法 液体クロマトグラフィ タンデムマススペ クトロメトリー法 ラボラトリー情報管理システム ブタ腎臓近位尿細管由来の上皮細胞株 液体シンチレーション計数法 イヌ腎臓尿細管上皮細胞由来の細胞株 多剤耐性蛋白質 メッセンジャーリボ核酸 多剤耐性関連蛋白質 質量分析装置 還元型ニコチンアミドアデニンジヌクレオ チドリン酸 検出せず 核磁気共鳴 試料なし 非構造蛋白質3/4A 有機アニオントランスポーター 有機アニオン輸送ポリペプチド 有機カチオントランスポーター ポリエチレングリコール P糖蛋白質 定量的全身オートラジオグラフィー リボ核酸 リトナビル 標準偏差 標準誤差 消失半減期 最高薬物濃度到達時間 ウリジン二リン酸グルクロン酸転移酵素 定常状態分布容積 薬物動態試験の概要文 4

6 2.6.4 薬物動態試験の概要文 まとめ バニプレビル 化学名 (5R,7S,10S)10(1,1Dimethylethyl)N{(1R,2R)1[N(cyclopropanesulfonyl) carbamoyl]2ethylcyclopropyl}15,15dimethyl3,9,12trioxo2,3,5,6,7,8,9,10,11,12,14,15,16,17,18,19he xadecahydro2,23:5,8dimethano1hbenzo[n][1,10,3,6,12]dioxatriazacyclohenicosine7carboxamide [図 2.6.4: 1] は HCV NS3/4A セリンプロテアーゼの酵素活性を阻害する新規薬物であり HCV 治療 薬として開発された 本項では 薬物の非臨床安全性評価のために選択した動物種であるラット イヌ マウス及びウサギにおけるバニプレビルの吸収 分布 代謝及び排泄について述べる ア カゲザルの血漿中薬物動態及び分布 並びに チンパンジーの血漿中薬物動態も検討した 非臨 床薬物動態試験の概要一覧を[ 項]に示す 非臨床薬物動態試験の結果から ラット イヌ及びサルにおけるバニプレビルのクリアランス は中程度から高度であり 血漿中半減期は短いことが示された ラット イヌ及びサルにおける 本薬の吸収は中程度であるが バイオアベイラビリティは低かった 0% 12% ラット イヌ サル及びチンパンジーに本薬を経口投与した場合 バイオアベイラビリティは低いものの HCV 治療薬の標的臓器である肝臓への曝露は良好であった 肝臓の良好な曝露には 少なくとも一因 としてトランスポーターを介した肝取込みが寄与していると考えられる ラットに[14C]バニプレ ビルを経口投与したときの放射能の分布は主に消化管及び肝臓に限局しており すべての組織で7 日目までに定量下限未満となった 妊娠ラット及びウサギにおいて本薬の投与に由来する放射能 の胎盤通過性が認められ 授乳ラットでは乳汁移行性が確認された ラット イヌ サル チン バンジー マウス ウサギ及びヒトにおける蛋白結合率は高く 97.0% 99.5% 血液 血漿中 濃度比はラット イヌ及びヒトで であった ラット及びイヌにおけるバニプレビルの主 排泄経路は胆汁排泄であり 酸化的代謝物 約70% 75% 及び未変化体 約25% 30% として 胆汁中に排泄された 無処置ラット及びイヌに[14C]バニプレビルを投与した際 投与放射能 未 変化体及び代謝物 のほぼすべてが投与後96時間以内に糞中に回収された In vitro 及び in vivo で バニプレビル分子構造中の脂肪族 ジメチルヘキサン又はトリメチル 部分 イソインドリ ン環 ビニルシクロプロピル部分やスルホンアミド部分が酸化された数種類の酸化的代謝物が生 成した ヒト肝試料で同定した代謝物はすべて非臨床安全性試験に用いた動物種でも生成されて いた ヒト肝ミクロソームによるバニプレビルの酸化的代謝のほとんどは CYP3A を介するもの であった CYP1A2 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP2B6 CYP3A 又は UGT1A1 に対するバニプレビルの可逆的阻害作用は強力ではなかった 50%阻害濃度 IC50 19 μm ま た バニプレビルは CYP3A に対して弱い時間依存的な阻害作用を示した 阻害定数 Ki = 42 μm 不活性化速度定数 Kinact = 0.14 min1 バニプレビルはヒト肝細胞とのインキュベーションによ り 弱い CYP3A 誘導作用を示した 20 μm のバニプレビルは48時間で CYP3A4の mr を10 μm リファンピシンの12% 24%増加させた が CYP1A2又は CYP2B6は誘導しなかった バニプレ ビルは単離ヒト肝細胞に効率的に取り込まれた バニプレビルは OATP1B1 OATP1B3及び Pgp の基質であることが示されたが Pgp BCRP MRP2 MRP3及び MRP4に対して強力な阻害作 用は示さなかった IC50 8 μm が 消化管でこれらのトランスポーターを阻害する可能性は否 定できない 薬物動態試験の概要文 5

7 2.6.4 薬物動態試験の概要文 バニプレビルは MDCKIIOATP1B1細胞へのピタバスタチン取込みを阻害し IC50 = 0.30 ± 0.01 μm MKCKIIOATP1B3細胞へのスルホブロモフタレイン取込みも阻害した IC50 = 0.30 ± 0.02 μm また バニプレビルは小胞膜で BSEP を阻害した IC50 = 1.30 ± 0.2 μm バニプレビル 3 *は[ H]標識位置 #は[14C]標識位置を示す 図 2.6.4: 1 バニプレビルの化学構造 分析法 薬物動態を評価した試験におけるバニプレビルの血漿及び肝臓中濃度の測定は 沈殿除蛋白又 は液 液抽出後 ネガティブ又はポジティブイオンモードで Turbo Ion Spray を用いた LCMS/MS で実施した 定量下限は3 12 nm の範囲であった 生体試料又は HPLC 溶出液中の放射能は 直 接 LSC により測定した HPLC 溶出液はインライン型放射化学的フロー検出器で測定した [3H] バニプレビル又は[14C]バニプレビルの代謝物プロファイリングには放射能測定用検出器の付い た逆相 HPLC を使用した 代謝物構造は MS 及び NMR による分析 M7 M8 M9 M10 又は 合成標品との比較 M10 により決定した これら分析の詳細は各試験報告書を参 照のこと また 毒性試験で実施した薬物濃度評価の結果を一部 本項で記載した これらの試験では GLP に従い ラット及びイヌ血漿 並びにラット乳汁をそれぞれマトリックスとするバニプレビ ルの定量法をバリデートした 生体試料の定量範囲 真度及び精度 並びに安定性の結果を[表 2.6.4: 1]に示す 血漿は Tecan 社の Freedom 液体ハンドリング自動システムによる保持液抽出法 肝ホモジネー トは手動による液 液抽出法 乳汁は手動による希釈及び Tecan 社の Freedom 液体ハンドリング 自動システムによる液 液抽出法によって バニプレビル及び内標準物質 バニプ レビルの類縁体 を抽出した 抽出物を Chromolith SpeedROD RP18 又は Onyx Monolith C18, 薬物動態試験の概要文 6

8 2.6.4 薬物動態試験の概要文 mm HPLC カラムに注入し 以下のアイソクラティック条件を用い常温で HPLC 分析を実 施した 流量 ml/min 1.0 %A 0.1% ギ酸水溶液 35 %B アセトニトリル 65 カラムからの溶出液は ネガティブイオンモードで Turbo Ion Spray を用い 多重反応モニタリ ングによる LCMS/MS 法で測定した バニプレビル濃度は 1/x2の二次回帰式を用いて算出した これら分析法の詳細は[ 項]に掲載する資料を参照のこと 表 2.6.4: 1 動物種 ラット ラット ラット ウサギ イヌ 試料 バニプレビル濃度分析法の真度及び精度 抽出方法 血漿 自動化した保持液抽出法 血漿 自動化した保持液抽出法 母 胎児 手動による希釈及び 乳汁 自動化した保持液抽出法 血漿 自動化した保持液抽出法 母 胎児 血漿 自動化した保持液抽出法 線形範囲 ng/ml 真度 % 精度 % 約70 C で425日間 約70 C で73日間 約70 C で20日間 約70 C で309日間 約70 C で575日間 生体試料の安定性 胎児の血漿は母動物の血漿の検量線を用いて定量した 吸収 ラット ラット SpragueDawley にバニプレビルの2 mg/kg を単回静脈内投与したときの血漿中バニプ レビル濃度は時間依存的に低下した[図2.6.4: 2] バニプレビルの平均 CLp は74 ml/min/kg であり 平均 CLb は106 ml/min/kg と推定された 血液 血漿比は[ 項]に記載 したがって ラ ットにおけるバニプレビルの高いクリアランスが明らかとなった Vdss 及び t1/2の平均値はそれぞ れ1.9 L/kg 及び0.9時間であった[ 項] バニプレビルの5 mg/kg を単回経口投与したときの吸収は速やかで Tmax は投与後0.5時間 Cmax は μm であり F は0 5%であった[ 項] 無処置ラットに[14C]バニプレビルの 60 mg/kg を経口投与したとき 約33%が酸化型代謝物として糞中に回収されたことから[ 項] バニプレビルの吸収は少なくとも中程度と考えられる ラットにおける F が低値を示した原 因の一つとして 初回通過効果が挙げられる[資料 : PK003] [資料 : PK009] 薬物動態試験の概要文 7

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10 2.6.4 薬物動態試験の概要文 バニプレビルの非線形動態及び肝抽出率の検討のため ラットを用いた短時間 30分 注入に よる一連の静脈内及び門脈内投与試験が実施された[ 項] 低用量 mg/kg におけ る平均肝抽出率は0.4であった 1 5 mg/kg の用量範囲で肝抽出率は0.4から0に低下したことから この用量範囲で肝抽出が飽和することが示唆された この結果に一致して 静脈内投与したとき の血漿クリアランスは5 mg/kg における65 ml/min/kg から30 mg/kg における15 ml/min/kg に低下 した したがって バニプレビルの全身曝露の上昇が用量比を上回る非線形性を示す原因の一つ として 肝クリアランスの飽和が考えられた[資料 : PK003] イヌ イヌ ビーグル にバニプレビルの2 mg/kg を単回静脈内投与したときの平均血漿中バニプレ ビル濃度推移を[図2.6.4: 3]に示す CLp Vdss 及び t1/2の平均値はそれぞれ11 ml/min/kg 0.3 L/kg 及び1.2時間であった[ 項] 平均血液クリアランスは16 ml/min/kg と推定されたことから 血液 血漿比は[ 項]に記載 イヌにおけるバニプレビルのクリアランスは中等度であ ることが明らかとなった バニプレビルの5 mg/kg を単回経口投与したときの吸収は速やかで Tmax は投与後1.3時間 Cmax は0.5 μm であり F は12%であった[ 項] 無処置イヌに5 mg/kg を単回経口投与したとき 投与後24時間までに投与した放射能の大部分が糞中に排泄された この際 投与量の52%が酸化 型代謝物として糞中に回収されたことから[ 項] イヌにおけるバニプレビルの吸収は適 度に良好と考えられる F が低値を示した原因の一つとして 初回通過効果が大きいことが考え られる[資料 : PK003] イヌにバニプレビルの4用量 及び30 mg/kg を単回経口投与することにより 薬物 動態の用量依存性を検討した 血漿中バニプレビル濃度の AUC 及び Cmax は用量比を上回って増 加する非線形性を示したことから 初回通過効果の飽和が示唆された[ 項] [資料 : PK003] 雌雄イヌにバニプレビルの5/10 15又は45/30 mg/kg/日を1日1回25週間反復経口投与したときの 血漿中薬物動態を検討した[ 項] 雌雄イヌでバニプレビルの曝露は類似しており 性別 による影響は示されなかった 単回投与したときと同様 反復投与したときもバニプレビルの曝 露について用量比を上回る増加が認められた クロスオーバー試験ではないが 単回投与時の薬 物動態[ 項]と比較して 対応する用量で反復投与した際には全身曝露の増加が認められ たことから 反復投与によるバニプレビルの蓄積が示唆された[資料 : TT 薬物動態試験の概要文 ]

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13 2.6.4 薬物動態試験の概要文 チンパンジー 雌雄各1例のチンパンジーにバニプレビルの10 mg/kg をチョコレートミルクで調製して単回経 口投与した際の薬物動態を検討した 2匹のチンパンジーの Tmax はそれぞれ投与後2及び4時間 Cmax はそれぞれ0.7及び1.1 μm AUC はそれぞれ3.8及び6.5 μm hr であった[ 項] [資料 : PK003] ウサギ 雌ウサギ 3例 にバニプレビルの240 mg/kg を10% Tween に溶解して単回経口投与した際の薬 物動態を検討した Cmax Tmax 及び AUC はそれぞれ 1.7 ± 0.2 μm 1.3 ± 0.6時間及び7.5 ± 1.6 μm hr であった 算術平均±SD [ 項] [資料 : PK007] 分布 ラットにおける組織分布 アルビノラット SpragueDawley 及び有色ラット Long Evans を用いて QWBA によりバ ニプレビルの組織分布を検討したところ 有色ラットとアルビノラットでの結果は類似していた [ 項] アルビノラットに[14C]バニプレビルの60 mg/kg を単回経口投与したところ 放射能 の分布は主として消化管及び肝臓に限られ 全身への広範な分布は認められなかった 胃及び食 道内容物 並びに 肝臓を除いて 組織中放射能濃度 ng 当量 バニプレビル放射能/g 組織 は 投与後4時間に最高値を示した 胃及び食道内容物 並びに 肝臓における放射能濃度は 投与後 2時間 最初の試料採取時間 に最高値を示した 最も放射能濃度が高かったのは 消化管内容物 胆汁及び肝臓であり その値は胆汁で492,000 ng/g 盲腸内容物で875,000 ng/g 食道内容物で 989,000 ng/g 大腸内容物で1,100,000 ng/g 肝臓で145,000 ng/g 小腸内容物で2,600,000 ng/g 胃 内容物で1,940,000 ng/g であった 消化管及び腎臓の組織 すなわち盲腸 17,000 ng/g 腎臓 10,600 ng/g 腎皮質 12,300 ng/g 小腸 24,700 ng/g 及び胃 11,700 ng/g には低濃度の放 射能が観測された 全血中放射能濃度は投与後4時間に最高値8,910 ng/g を示した 脳組織 小脳 及び大脳 における放射能濃度は いずれの時点でも <244 ng/g であった アルビノラット SpragueDawley 及び有色ラット Long Evans のブドウ膜における放射能 濃度を比較した 投与後4時間に測定されたアルビノラット及び有色ラットのブドウ膜の放射能濃 度はそれぞれ1570及び339 ng/g であった アルビノラット及び有色ラットのブドウ膜における放 射能濃度は類似し かつ低いことから バニプレビルはメラニンに結合しないことが示された すべての組織における放射能濃度は投与後168時間までには となった[資料 : PK020] 投与後4時間における小腸及び肝臓の組織 血漿中放射能濃度比はそれぞれ1.8及び9.1であり 1 を上回った 血液 血漿中濃度比はいずれの時点でも約0.7であったことから [14C]バニプレビル 由来放射能の分布は血球選択的ではないことが示された 血液 血漿中濃度比は in vitro で得られ たデータと一致するものであった[ 項] [資料 : PK001] [資料 : PK020] アルビノラットに[3H]バニプレビルの5 mg/kg を単回経口投与した探索的試験で組織分布を検 薬物動態試験の概要文 12

14 2.6.4 薬物動態試験の概要文 討したところ [14C]バニプレビルの60 mg/kg を単回経口投与した際に得られた結果と一致してい た [3H]バニプレビルを用いた試験結果から バニプレビルの分布は消化管組織に限局しており 投与薬物に由来する物質が最も多くみられるのは肝臓であることが示された [3H]バニプレビル を用いた試験で得られた肝臓試料を LCMS を用いてプロファイリングしたところ 投与後4時間 には投与薬物に由来する物質の大部分が未変化体の形で存在することが明らかになった[資料 : PK002] [資料 : PK020] ラット イヌ サル及びチンパンジーにおける肝臓への分布 ラット SpragueDawley イヌ ビーグル 又はサル アカゲザル にバニプレビルの5 mg/kg を単回経口投与 もしくは チンパンジーに10 mg/kg を単回経口投与したときの血漿及び肝臓中 濃度データを[ 項]に要約する また ラット SpragueDawley に5 30及び300 mg/kg を 単回経口投与 又はイヌ ビーグル に2及び5 mg/kg を単回経口投与 もしくは RTV との併用 によりイヌ ビーグル に2 mg/kg を単回経口投与したときの血漿及び肝臓中濃度データを[図 2.6.4: 5]及び[図2.6.4: 6]に示す ラット イヌ又はサルにバニプレビルの5 mg/kg を単回経口投与 もしくはチンパンジーに10 mg/kg を単回経口投与したときの肝臓への曝露はいずれも良好であっ た ラット イヌ及びサルでは投与後2時間で3 35 μm チンパンジーでは投与後12時間で31 μm [ 項] ラット イヌ及びサルに5 mg/kg を単回経口投与したときの肝臓 血漿中 AUC 比は それぞれ> 及び455であった 非臨床動物種において低用量を投与した際のバニプレビル の肝臓への曝露は全身曝露 血漿 より100倍以上高かった ラットにおける肝臓中曝露の増加は 5 mg/kg と30 mg/kg の用量間では用量比を上回ったが 30 mg/kg と300 mg/kg の用量間では用量比 を下回った ラットにおいて30 mg/kg と300 mg/kg の用量間で肝臓中曝露の上昇が用量比を下回 ったことから 肝臓への取込みの飽和が示唆される また ラットでは 血漿中濃度の増加に応 じて肝臓 血漿中濃度比は減少し 肝臓 血漿中濃度比の範囲は血漿中濃度0.004 μm のときの約 4,000から血漿中濃度14 μm のときの16であった[図2.6.4: 5]ことからも 循環血中のバニプレビル 濃度の上昇とともに肝臓への取込みの飽和が示唆される イヌにおける肝臓への曝露についても 2 mg/kg と5 mg/kg の用量間で用量比を上回る増加がみられた[ 項] ラット及びイヌにおけ るバニプレビルの肝臓への分布は高いものの 終末相において血漿及び肝臓中濃度 時間プロフ ァイルは平行して減少することから 終末相の肝クリアランスは終末相血漿クリアランスで説明 できることが示唆された[図2.6.4: 5] [図2.6.4: 6] [資料 : PK003] [資料 : PK014] [資料 : PK015] 併用薬存在下で 肝臓の曝露低下と それに伴う全身曝露増加によりバニプレビルの有効性が 低下する可能性について検討するため イヌにバニプレビル 2 mg/kg と RTV 5 mg/kg を併 用経口投与したときのバニプレビルの血漿及び肝臓中曝露量を測定した 当該試験で バニプレ ビルの血漿及び肝臓中曝露量は RTV との併用投与によりそれぞれ約30倍及び約20倍増加した [ 項] 取込みトランスポーター 排出トランスポーター 代謝が複合して関与するため 当該試験デザインでは バニプレビルと RTV の相互作用機序の解明や明確な分類を行うには不十 分であった しかし 血漿及び肝臓中曝露量のいずれも同様の増加がみられたことから バニプ 薬物動態試験の概要文 13

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17 2.6.4 薬物動態試験の概要文 定した[ 項] 本薬はマウス ラット ウサギ イヌ サル チンパンジー及びヒトの血漿 蛋白に高い結合率を示した 結合率は試験したすべての動物種で同程度であり マウス ラット ウサギ イヌ サル チンパンジー及びヒトにおける結合率 算術平均 はそれぞれ99.0% 99.5% 99.0% 99.3% 98.5% 98.9%及び98.3%であった[ 項] バニプレビル濃度 ラット及びイ ヌでは1 10 μm サル及びヒトでは μm に応じた血漿蛋白結合率の変化はみられなかっ た[資料 : PK001] [資料 : PK006] [資料 : PK008] また バニプレビル 検 討濃度 1 10 μm はヒト血清アルブミン 検討濃度 40 mg/ml 及び α1酸性糖蛋白 検討濃 度 1 mg/ml のいずれにも結合することが確認された 結合率 それぞれ94.8% 95.9%及び55.2% 66.6% [資料 : PK027] また 別試験におけるバニプレビル濃度10 μm の検討で 肝機能障害患者の血漿蛋白結合率 軽 度 97.0% 中等度 97.3% 重度 97.5% は健康被験者 97.3% と同程度であった[資料 : PK017] In Vitro 全血 血漿比 抗凝固剤として EDTA を含む新鮮全血中でバニプレビルをインキュベートすることにより 本 薬の血球移行性を検討した 平衡状態における血液 血漿中濃度比には 試験したバニプレビル 濃度との関連性はみられず その平均はラット イヌ及びヒトでそれぞれ 及び0.8であっ た[ 項] これらのデータにより ラット イヌ及びヒトにおける血液クリアランスは血漿 クリアランスよりやや高いことが示された[資料 : PK001] 代謝 マウスにおける In Vivo 代謝 マウス ras トランスジェニック CB6F1 N=10 に[3H]バニプレビルの300 mg/kg を単回経口投 与し その代謝を検討した[ 項] 経口投与後1又は6時間にマウスを屠殺し 尿 糞及び末 梢血を採取して代謝物プロファイリングを行った 尿は採取量が少なく 放射能が低レベルであ ったためプロファイリングは不可能であった 血漿及び糞中では 放射能の大部分が未変化体と して認められ 少量の酸化型代謝物 M6 M7 M8 M9及び M10 も検出された[図2.6.4: 7] こ のデータにより マウスにおけるバニプレビルの消失に酸化的代謝が寄与していることが示され た[資料 : PK006] ウサギにおける In Vivo 代謝 無処置のウサギ ニュージランドホワイト N=3 に[3H]バニプレビルの240 mg/kg を単回経口 投与し その代謝を検討した[ 項] 平均血漿中曝露量 AUC は7.5 μm hr であった[ 項] 尿及び糞を投与後72時間まで採取したところ 投与放射能の平均回収率は67%であり 尿及 び糞中に投与量のそれぞれ0.5 ± 0.1%及び66.9 ± 14.1%が排泄された[ 項] 糞中に回収され た放射能は 未変化体 約75% 並びに いずれも酸化型代謝物の M6 M7 M8 M9及び M10 合計約25% であった[図2.6.4: 7] ウサギの血漿及び尿中総放射能は非常に低レベルであった 薬物動態試験の概要文 16

18 2.6.4 薬物動態試験の概要文 ため 代謝物プロファイリングは不可能であった このデータにより ウサギでバニプレビルは 適度に吸収され その消失には酸化的代謝が寄与していることが示された[資料 : PK007] ラットにおける In Vivo 代謝 BDC 処置したラット SpragueDawley に[3H]バニプレビルの2 mg/kg を静脈内投与 N=2 又は無処置ラットに[14C]バニプレビルの60 mg/kg を経口投与 N=3 することにより in vivo 代謝 を検討した[ 項] 2 mg/kg を静脈内投与後72時間までの投与放射能の回収率 算術平均 は71.7%であり 尿 胆汁及び糞中にそれぞれ15.6% 52.1%及び4.2%が回収された 尿中に回収 された放射能は酸化型代謝物 胆汁中に回収された放射能は酸化型代謝物 約67% 及び未変化 体 約33% であった ラット胆汁中には複数の酸化型代謝物 M3 M6 M7 M7a M8 M9 及び M10 が同定された[図2.6.4: 7] したがって ラットにおけるバニプレビルの主要消失経路 は代謝であることが確認された 無処置ラットに60 mg/kg を経口投与後240時間まで尿及び糞を 回収したところ 尿中には投与放射能の0.5 ± 0.5% 糞中には81.3 ± 6.7%が回収され 投与放射能 の総回収率は81.8 ± 6.3%であった[ 項] 放射能の大部分 74 ± 11% は最初の24時間に糞 中に回収された[ 項] 無処置ラットから得られた代表的な尿及び糞中代謝物プロファイル を[図2.6.4: 8]に示す 尿中に回収された放射能は低レベルであったため 代謝物プロファイリン グは不可能であった ラット糞中に回収された放射能は酸化型代謝物 約50% 及び未変化体 約 50% であった[ 項] ラット糞中に検出された酸化型代謝物は M6 M7 M8 M9及び M10であった[図2.6.4: 8] ラット糞中には1種類のグルタチオン抱合体 M13 も検出された ラ ット糞中にみられた各代謝物の割合を[ 項]に示す 全体的に見ると 経口投与された放射 能の約36%が代謝物として糞中に排泄されたことから ラットにおけるバニプレビルの吸収は中 程度あることが示された BDC 処置ラットを用いた静脈内投与試験において バニプレビルは代 謝物のほか未変化体としても胆汁中に排泄されたことから ラットに経口投与した際の実際の吸 収率は36%を超えることが予想される [3H]バニプレビルの114 mg/kg を経口投与後1 4 8及び 24時間に採取した血漿中の代謝物プロファイルを検討した 投与後4時間血漿における代表的なク ロマトグラムを[図2.6.4: 9]に示す 循環血漿中には少量の酸化型代謝物 M6 M7 M8 M9 M10及び M11 が検出された[図2.6.4: 9] [資料 : PK002] [資料 : PK009] 薬物動態試験の概要文 17

19 M1, M2 M3 M14 M6, M7a M13 M7 M11 M10 M9 M8

20 2.6.4 薬物動態試験の概要文 尿中代謝物プロファイル 60 mg/kg [14C]バニプレビル経口投与後0 24時間 cps :00 10:00 20:00 30:00 40:00 50:00 mm:ss 糞中代謝物プロファイル 60 mg/kg [14C]バニプレビル経口投与後0 24時間 cps [14C] バニプレビル M M M13 M7 M8 M :00 図 2.6.4: 8 10:00 20:00 30:00 40:00 50:00 mm:ss 無処置ラットにおける代表的な尿及び糞中代謝物プロファイル [ 項] [資料 : PK009] 薬物動態試験の概要文 19

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22 2.6.4 薬物動態試験の概要文 イリングは不可能であった 糞中に回収された放射能は多種類の酸化型代謝物 M1 M2 M3 M4 M5 M5a M6 M7 M7a M8 M9及び M10 腸内で細菌によるアミド結合の還元によ り生成すると考えられる最終加水分解産物 M14 及び未変化体であった 糞中に回収された各 代謝物の割合を[ 項]に示す 量的には 糞中放射能の約71%が酸化型代謝物 約27%が未 変化体 約2%が加水分解産物 M14であった 無処置イヌにバニプレビルを経口投与後 大量の酸 化型代謝物が糞中に回収されたことから イヌにおけるバニプレビルの吸収は良好であることが 示された また イヌにおけるバニプレビルの主要な消失の機序が酸化的代謝であることも明ら かとなった 血漿中の未変化体濃度及び総放射能濃度を測定した結果を[ 項]に示す LCMS/MS で測定した血漿中バニプレビル濃度が[14C]バニプレビル放射能当量濃度とよく一致し ていることから イヌにおける主要循環血漿中成分は未変化体である[ 項] イヌ血漿中の 総放射能は非常に低レベルであったため 投与後1時間を除き 代謝物プロファイリングは不可能 であった 投与後1時間における血漿中代謝物プロファイリング[図2.6.4: 10]において イヌ血漿 中に検出された放射能成分は未変化体のみであり 代謝物は検出されなかった[資料 : PK012] [資料 : PK021] 薬物動態試験の概要文 21

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26 2.6.4 薬物動態試験の概要文 ヒト ラット イヌ ウサギ及びマウスの In Vitro 代謝 マウス ras トランスジェニック CB6F1 ラット ウサギ イヌ アカゲザル及びヒトの in vitro 肝臓試料 S9 DPH を添加したミクロソーム又は肝細胞 と共にバニプレビルをインキュベ ートした結果 酸化型代謝物のわずかな産生が認められた ヒト肝 S9では4種の微量代謝物 M7 M8 M9及び M10 がみられた[図2.6.4: 12] ラット肝 S9では微量の M10のみが生成し イヌ肝 S9では代謝物は認められなかった[図2.6.4: 12] 雌ウサギ S9 マウス肝ミクロソーム及びマウス 肝細胞で 7種の微量酸化的代謝物 M3 M6 M7a M7 M8 M9及び M10 がみられた[図2.6.4: 12] バニプレビルをヒト肝細胞と共にインキュベートすると代謝物 M9及び M10が生成し イヌ 肝細胞と共にインキュベートすると M8が生成した バニプレビルをラット肝細胞と共にインキ ュベートしたときには代謝物はみられなかった In vitro でみられた代謝経路は in vivo でみられた ものと一致しており バニプレビルは主として酸化型代謝物を生成することが明らかになった[資 料 : PK001] [資料 : PK006] [資料 : PK008] 薬物動態試験の概要文 25

27 2.6.4 薬物動態試験の概要文 ヒト肝 S9 cps [ C] バニプレビル M M7 M M : : :00 50:00 mm:ss ラット肝 S9 cps [ C] バニプレビル M :00 図 2.6.4: 12 10:00 20:00 30: :00 mm:ss ヒト ラット イヌ又はウサギの肝 S9 もしくは マウスの肝ミクロソーム における[14C]バニプレビルの代表的代謝物プロファイル [ 項] [資料 : PK001] [資料 : PK006] [資料 : PK008] 薬物動態試験の概要文 26

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30 2.6.4 薬物動態試験の概要文 したときの総回収率は良好で 91.5% 尿及び糞中にはそれぞれ投与量の0.78及び87.3%が回収さ れた[ 項] 放射能の主要排泄経路は胆汁中排泄であった[資料 : PK002] [資料 : PK021] ウサギ 無処置の雌ウサギに240 mg/kg の[3H]バニプレビルを経口投与 N=3 し 尿及び糞を72時間採 取した 総投与量の67%が主として糞中に回収され 0.5%が尿中に回収された[ 項] [資料 : PK007] ヒト 6名の健康男性被験者に[14C]バニプレビルの575 mg を液体充填カプセル製剤で経口投与し 尿 糞及びトイレットペーパーを投与後168時間まで採取した 平均総回収率は投与量の約96%であり 各個人の回収率は94.4% 97.4%の範囲であった[ 項] 最初の96時間における平均総回収 率は約94%であり 糞 尿及びトイレットペーパー中にはそれぞれ投与量の93.3% 0.4% 0.1% が回収された[資料 : PK022] ラットの乳汁中排泄 授乳ラットの乳汁中へのバニプレビルの排泄を検討するため 妊娠6日から授乳14日まで 180 mg/kg/日のバニプレビルを経口投与し 授乳14日における母動物の血漿及び乳汁中の未変化 体濃度を測定した[ 項] 母動物の血漿及び乳汁中濃度測定には 非絶食ラット4例から授 乳14日の投与8時間後に採取した試料を用いた 母動物におけるバニプレビルの平均乳汁 血漿中 濃度比は0.147であったことから ラットにおけるバニプレビルの乳汁移行性が示された[資料 : TT 7230] 薬物動態学的相互作用 バニプレビルの CYP 基質としての評価 [3H]バニプレビル 1 10及び50 μm の酸化的代謝への CYP 分子種の関与の可能性を プール したヒト肝ミクロソームで CYP3A CYP2D6又は CYP2C に対する阻害抗体を用いて検討した 代謝物の定量は ラジオクロマトグラムの代謝物ピークを積分することにより行った その結果 バニプレビルの酸化的代謝には主として CYP3A が関与しており CYP2D6又は CYP2C の明らか な寄与は認められなかった[資料 : PK001] ヒト肝ミクロソームにおける CYP 及び UGT1A1の阻害 バニプレビルの CYP1A2 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP2B6 CYP3A 及び UGT1A1 活性に対するプレインキュベーション非依存的阻害の可能性をヒト肝ミクロソームで検討した バニプレビルは陽性対照の阻害薬と比較して CYP1A2 CYP2C8 CYP2C19又は CYP2D6に対し 薬物動態試験の概要文 29

31 2.6.4 薬物動態試験の概要文 て可逆的阻害を示さず IC50 >50 μm CYP3A IC50 = 19 μm CYP2C9 IC50 = 26 μm CYP2B6 IC50 = 51 μm 及び UGT1A1 IC50 = 19 μm に対してわずかな阻害を示したのみであった [ 項] バニプレビルは CYP3A に対して時間依存的な弱い阻害作用を示した Ki = 42 μm Kinact = 0.14 min1 [資料 : PK001] [資料 : PK010] [資料 : PK018] バニプレビルの CYP3A 2B6及び1A2の誘導作用の評価 バニプレビルの CYP3A CYP2B6及び CYP1A2の誘導作用を初代培養ヒト肝細胞で評価した ド ナー3名 培養ヒト肝細胞で バニプレビルは弱い CYP3A4の R 発現誘導作用を示したが 20 μm のバニプレビルによる平均誘導率は10 μm の濃度のリファンピシンの18% CYP3A 依 存性のテストステロン6β水酸化活性を上昇させなかった CYP3A 活性の上昇がみられなかった のは バニプレビルによる可逆性 時間依存性の CYP3A 阻害が原因であると考えられる 予想 されたように 陽性対照のリファンピシン 10 μm は CYP3A4の R 発現及び酵素活性を誘 導した それぞれ38 65倍及び5 16倍 20 μm までの濃度のバニプレビルをヒト肝細胞とイン キュベーションしたとき CYP2B6又は CYP1A2の mr 又は酵素活性の誘導はみられなかった 誘導作用を示す陽性対照のフェノバルビタール 1000 μm は CYP2B6の mr を8 24倍 酵 素活性を8 11倍に誘導し 誘導作用を示す陽性対照のオメプラゾール 50 μm は CYP1A2の mr を39 52倍 酵素活性を15 21倍に誘導した[資料 : PK001] [資料 : PK023] バニプレビルのトランスポーターの基質としての評価 バニプレビルのヒト肝細胞への時間及び温度依存的な取込みが認められた バニプレビルのヒ ト肝細胞への取込み過程は飽和性 Km = 1 μm のトランスポーター介在性輸送と非飽和性の受動 輸送から成るものであった ヒト肝細胞データは OATP1B1及び OATP1B3発現 MDCK 細胞の輸送 特性に一致していたことから バニプレビルはヒト肝取込みトランスポーターOATP1B1及び OATP1B3の基質であることが示された また バニプレビルはヒト肝排出トランスポーターであ る Pgp の基質であったが BCRP の基質ではなかった[資料 : PK001] [資料 : PK025] バニプレビルのトランスポーター阻害作用の評価 バニプレビルは MDR1発現 LLCPK1細胞で Pgp 介在性ジゴキシン輸送を阻害した IC50 = 38 ± 17 μm また バニプレビルは MDCKIIOATP1B1細胞への OATP1B1介在性ピタバスタチン取 込みを阻害し IC50 = 0.30 ± 0.01 μm MDCKIIOATP1B3細胞へのスルホブロモフタレイン取込 みを阻害した IC50 = 0.30 ± 0.02 μm また 膜小胞で バニプレビルは BCRP 介在性のメトト レキサート輸送を IC ± 2.5 μm で BSEP 介在性のタウロコール酸の輸送を IC ± 0.2 μm で MRP2介在性のエタクリン酸グルタチオン抱合体の輸送を IC50 = 42.2 ± 5.8 μm で MRP3介在 性のエストラジオール17βDグルクロン酸の輸送を IC ± 1.8 μm で MRP4介在性の葉酸の輸 送を IC ± 1.0 μm で阻害した[資料 : PK011] [資料 : PK016] [資料 : PK019] [資料 : PK024] 薬物動態試験の概要文 30

32 2.6.4 薬物動態試験の概要文 その他の薬物動態試験 その他の薬物動態試験は実施しなかった 考察及び結論 バニプレビルは非臨床動物種で中程度から高いクリアランスを有する化合物であり 血漿中半 減期は短い 非臨床動物種における本薬の吸収は中程度あるが バイオアベイラビリティは低い 0 12% バニプレビルをラット イヌ サル及びチンパンジーに経口投与したときの経口バ イオアベイラビリティは低いが HCV 治療の標的器官である肝臓への曝露は良好であることが示 された 良好な肝曝露は 少なくとも一部は肝取込み活性によるものであると考えられる ラッ トにバニプレビルを経口投与したときのバニプレビルの分布は主として消化管内容物及び肝臓に 限局しており すべての組織で放射能が7日目までに定量下限未満となった バニプレビルはラッ ト及びウサギの胎盤を通過し 授乳ラットの乳汁中にも排泄された バニプレビルは非臨床試験 における動物種及びヒトの血漿蛋白に強く結合し % 血球選択的な分布はみられな かった バニプレビルはラット及びイヌでほとんどが糞中に 主に胆汁を介して 多種類の酸化 的代謝物 約70 75% 及び未変化体 約25 30% として排泄された 非臨床安全性試験動物 種を用いて実施した試験で これらの動物種 ラット イヌ マウス及びウサギ はヒト肝画分 で同定されたすべての代謝物に曝露されることが示された バニプレビルの酸化的代謝の大部分 は CYP3A を介するものである また バニプレビルは OATP1B1 OATP1B3及び Pgp の基質で もあることが判明し CYP3A 又はこれらのトランスポーターを阻害することが知られている化合 物との薬物間相互作用を受ける可能性がある バニプレビルは CYP3A を時間依存的に弱く阻害 したことから 主に CYP3A 代謝により消失する化合物に薬物間相互作用を引き起こす可能性が ある さらに バニプレビルは OATP1B1 OATP1B3及び BSEP を阻害することから 主要消失経 路がこれらのトランスポーターに依存する化合物との薬物間相互作用を引き起こす可能性もある バニプレビルは全身レベルで その他の CYP UGT1A1 Pgp BCRP MRP2 MRP3若しくは MRP4の阻害又は CYP3A CYP1A2若しくは CYP2B6の誘導を介する薬物間相互作用は引き起こ さないと考えられるが 消化管で Pgp BCRP MRP2 MRP3及び MRP4を阻害する可能性は否 定できない 薬物動態試験の概要文 31

33 CTD 第 2 部 MSD 株式会社

34 バニプレビルカプセル剤 目次 頁略号及び用語の定義 薬物動態試験 : 一覧表 ラットにおける薬物動態 イヌにおける薬物動態 サルにおける薬物動態 チンパンジーにおける薬物動態 ウサギにおける薬物動態 組織分布 肝への分布 妊娠動物での検討 血漿蛋白への可逆的結合 血球移行性 マウスにおける in Vivo 代謝 ウサギにおける in Vivo 代謝 ラットにおける in Vivo 代謝 イヌにおける in Vivo 代謝 ヒトにおける in Vivo 代謝 推定代謝経路 In Vitro 代謝 ラット イヌ ウサギ及びヒトにおけるマスバランス ラットにおける乳汁排泄 CYP 基質としてのバニプレビルの評価 ヒト肝ミクロソーム中の CYP 及び UGT1A1の阻害 ヒト CYP(3A 2B6 及び1A2) の誘導 Pgp OATP1B1 OATP1B3 及び BCRP を介した輸送 Pgp OATP1B1 OATP1B3 BSEP BCRP MRP2 MRP3 及び MRP4を介した輸送への影響

35 略号及び用語の定義 略語 バニプレビル AMP ATP AUC BCRP BDC BSA BSEP BSP CLp CYP COV DMSO EDTA F F GD GI GLP Adenosine monophosphate Adenosine triphosphate Area under the drug concentrationtime curve Breast cancer resistance protein Bile duct cannulation Below the lower limit of quantitation Bovine serum albumin Bile salt export pump Sulfobromophthalein sodium Plasma clearance Cytochrome P450 Covance Laboratories Dimethylsulfoxide Ethylenediaminetetraacetic acid Absolute oral bioavailability Female Gestation day Gastrointestinal Good laboratory practice HBSS HEPES Hank s balanced salt solution Hydroxyethyl piperazineethanesulfonic HPLC IC50 IV Ki Kinact Km LCMS High performance liquid chromatography 50% inhibitory concentration Intravenous injection Inhibition rate constant Inactivation rate constant Michaelis constant Liquid chromatographymass spectrometry LCMS/MS Liquid chromatographytandem mass spectrometry LD LLCPK LSC M MAbs MDCK MDR mr MRL MRP βdp Lactation day Lilly laboratory cellsporcine kidney Liquid scintillation counting Male Monoclonal antibodies MadinDarby canine kidney Multiple drug resistance protein Messenger ribonucleic acid Merck research Laboratories Multidrug resistanceassociated protein Not applicable βnicotineamide adenine dinucleotide phosphate, oxidized form βnicotinamide adenine dinucleotide phosphate, reduced form Not detected / Not detectable Nuclear magnetic resonance Not sampled Not studied Not represented DPH NMR NS NS NR 定義 開発番号 MK7009 アデノシン一リン酸 アデノシン三リン酸 薬物濃度 時間曲線下面積 乳癌耐性蛋白質 胆管カテーテル 定量下限未満 ウシ血清アルブミン 胆汁酸トランスポーター スルホブロモフタレイン 血漿クリアランス チトクロム P450 コバンスラボラトリー ジメチルスルホキシド エチレンジアミン四酢酸 絶対経口バイオアベイラビリティ 雌 妊娠期間 胃腸の 医薬品の安全性に関する非臨床試験の実施 基準 ハンクス緩衝塩溶液 ヒドロキシエチル ピペラジンエタンスルホ ン酸 高速液体クロマトグラフィー 50% 阻害濃度 静脈内投与 阻害定数 不活性化速度定数 ミカエリス定数 液体クロマトグラフィー マススペクトロ メトリー法 液体クロマトグラフィー タンデムマスス ペクトロメトリー法 授乳期間 ブタ腎臓近位尿細管由来の上皮細胞株 液体シンチレーション計数法 雄 モノクローナル抗体 イヌ腎臓尿細管上皮細胞由来の細胞株 多剤耐性蛋白質 メッセンジャーリボ核酸 メルクリサーチラボラトリー 多剤耐性関連蛋白質 該当なし 酸化型ニコチンアミドアデニンジヌクレオ チドリン酸 還元型ニコチンアミドアデニンジヌクレオ チドリン酸 検出せず 核磁気共鳴 試料なし 検討しなかった 組織なし 2

36 バニプレビルカプセル剤 略号及び用語の定義 ( 続き ) 略語 定義 OAT Organic anion transporter 有機アニオントランスポーター OATP Organic anion transporting polypeptide 有機アニオン輸送ポリペプチド OCT Organic cation transporter 有機カチオントランスポーター PBS Phosphate buffered saline リン酸緩衝生理食塩水 PEG Polyethleneglycol ポリエチレングリコール Pgp Pglycoprotein P 糖蛋白質 P.O. Per os 経口投与 PK Pharmacokinetic(s) 薬物動態 QWBA Quantitative whole body autoradiography 定量的全身オートラジオグラフィー R Ribonucleic acid リボ核酸 RTV Ritonavir リトナビル SD Standard deviation 標準偏差 SEM Standard error of the mean 標準誤差 t 1/2 Elimination halflife 消失半減期 TCA Taurocholic acid タウロコール酸 T max Time to the maximum drug concentration 最高薬物濃度到達時間 UDPGA Uridine diphosphate glucuronic acid ウリジン二リン酸グルクロン酸 UGT Uridine diphosphate glucuronosyltransferase ウリジン二リン酸グルクロン酸転移酵素 V dss Volume of distribution at steady state 定常状態分布容積 3

37

38 Test Article: 薬物動態試験 一覧表 続き Type of Study Animal/Test System Method of Administration Testing Facility Location in CTD/Study Number 代謝 マウスにおける in vivo 代謝 ウサギにおける in vivo 代謝 ラットにおける in vivo 代謝 Mouse New Zealand white rabbit Rat P.O. P.O. P.O., IV MRL MRL MRL イヌにおける in vivo 代謝 Dog P.O., IV MRL ヒトにおける in vivo 代謝 Human P.O. 推定代謝経路 Mouse, Rat, Dog, Rabbit, Human P.O., IV MRL In vitro 代謝 Rat, Dog, Rabbit, Monkey, Human/liver S9, Hepatocytes In vitro MRL 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 評価 MRL = Merck Research Laboratories, = 5 [資料 : PK006] [資料 : PK007] [資料 : PK002] [資料 : PK009] [資料 : PK002] [資料 : PK012] [資料 : PK021] [資料 : PK013] [資料 : PK022] [資料 : PK002] [資料 : PK006] [資料 : PK007] [資料 : PK009] [資料 : PK012] [資料 : PK013] [資料 : PK001] [資料 : PK006] [資料 : PK008]

39 Test Article: 薬物動態試験 一覧表 続き Type of Study Animal/Test System Method of Administration Testing Facility Location in CTD/Study Number 排泄 ラット イヌ ウサギ及びヒトでの 排泄 Rat, Dog, Rabbit, Human P.O., IV MRL 乳汁排泄 薬物相互作用 CYP 基質性 CYP 及び UGT1A1阻害 Rat P.O. MRL 評価 評価 評価 評価 評価 評価 評価 評価 [資料 : PK002] [資料 : PK007] [資料 : PK009] [資料 : PK012] [資料 : PK013] [資料 : PK021] [資料 : PK022] [資料 : TT 7230] Human liver microsomes Human liver microsomes In vitro In vitro MRL MRL CYP3A CYP2B6及び CYP1A2誘導 Human hepatocytes In vitro MRL 評価 評価 評価 評価 評価 評価 [資料 : [資料 : [資料 : [資料 : [資料 : [資料 : LLCPK1 cells MDCKII cells Membrane vesicles Pgp OATP1B1 OATP1B3 BSEP LLCPK1 cells BCRP MRP2 MRP3及び MRP4を LLCMDR1 cells 介した輸送への影響 MDCKII cells Membrane vesicles MRL = Merck Research Laboratories, = In vitro MRL In vitro MRL Pgp OATP1B1 OATP1B3及び BCRP を介した輸送 6 PK001] PK001] PK010] PK018] PK001] PK023] 評価 [資料 : PK001] 評価 [資料 : PK025] 評価 [資料 : PK011] 評価 [資料 : PK016] 評価 [資料 : PK019] 評価 [資料 : PK024], Report contains a GLP Compliance Statement

40 Test Article: [資料 : PK003] ラットにおける薬物動態 単回静脈内及び経口投与後の薬物動態 ラット Species: Rat Rat Gender/Number of Animals: M/3 M/3 Feeding Condition: Fasted Fasted Vehicle/Formulation: DMSO PEG400 Method of Administration: IV P.O. Dose (mg/kg): 2 5 Sample: Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS PK Parameters (Mean SD): Total CL p (ml/min/kg) 74 ± 9 Vdss (L/kg) 1.9 ± 1.6 t1/2 (hr) 0.9 ± 0.7 AUC024hr (µm hr) 0.6 ± Cmax (µm) Tmax (hr) 0.5 F (%) 0 5 : Not applicable Additional Information: After IV dosing (2 mg/kg),the mean vaniprevir plasma clearance (CLp ) was 74 ml/min/kg. The mean values for the volume of distribution at steady state (V dss ) and the elimination halflife (t1/2) in the terminal phase were 1.9 L/kg and 0.9 hr, respectively. After oral dosing (5 mg/kg), absorption was rapid with a maximal plasma concentration (C max) of M occurring at 0.5 hr postdose. The absolute oral bioavailability (F) was 05%. F was calculated using mean IV AUC02hr and the lowest and mean P.O. AUC02hr. 7

41 Test Article: [資料 : PK003] 単回経口投与後の薬物動態 ラット Species: Gender/Number of Animals: Feeding Condition: Vehicle/Formulation: Method of Administration: Dose (mg/kg): Sample: Analyte: Assay: PK Parameters (Mean SD): AUC024hr (µm hr) Cmax (µm) T max (hr) Rat M/3 Fasted PEG400 P.O. 5 Plasma LCMS/MS Rat M/3 Fasted 10%Tween 80 P.O. 30 Plasma LCMS/MS Rat F/3 Fasted 10% Tween 80 P.O. 30 Plasma LCMS/MS Rat M/3 Fasted 10% Tween 80 P.O. 100 Plasma LCMS/MS Rat F/3 Fasted 10% Tween 80 P.O. 100 Plasma LCMS/MS ± ± ± ± ± ± ± ± ± ± ± ± 0.0 8

42 Test Article: [資料 : PK003] 単回経口投与後の薬物動態 ラット 続き Species: Rat Rat Rat Rat Gender/Number of animals: M/3 F/3 F/3 M/3 Feeding Condition: Fasted Fasted Fasted Fasted Vehicle/Formulation: 10% Tween 80 10%Tween 80 10% Tween 80 10% Tween 80 Method of Administration: P.O. P.O. P.O. P.O. Dose (mg/kg): Sample: Plasma Plasma Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS LCMS/MS LCMS/MS PK Parameters (Mean SD): AUC024hr (µm hr) 217 ± ± ± ± 379 Cmax (µm) 18.5 ± ± ± ± 23.7 T max (hr) 24 ± 0 11 ± ± ± 10 Additional Information: Following oral administration to rats at low dose ( 5 mg/kg P.O.), vaniprevir has poor oral plasma exposure and poor oral bioavailability. At higher doses (>5 mg/kg P.O.), vaniprevir demonstrates nonlinear pharmacokinetics in rats with greater than dose proportional increases in plasma exposure. No significant gender effect is observed. 9

43 Test Article: [資料 : PK003] 静脈内及び門脈内投与後の薬物動態 ラット Species: Gender/Number of Animals: Feeding Condition: Vehicle/Formulation Method of Administration: Dose (mg/kg): Sample: Analyte: Assay: PK Parameters (Mean ± SD): AUC024hr (µm hr): Hepatic Extraction Ratio: CLp (ml/min/kg): Rat M/3 Fasted 25% hydroxypropylcyclodextrin Portal vein 0.2 Plasma LCMS/MS Rat M/3 Fasted 25% hydroxypropylcyclodextrin IV 0.2 Plasma LCMS/MS Rat M/3 Fasted 25% hydroxypropylcyclodextrin Portal vein 1.0 Plasma LCMS/MS Rat M/3 Fasted 25% hydroxypropylcyclodextrin IV 1.0 Plasma LCMS/MS ± ± ± ± ± ± 6 10

44 Test Article: [資料 : PK003] 静脈内及び門脈内投与後の薬物動態 ラット 続き Species: Rat Rat Rat Gender/Number of Animals: M/3 M/3 M/2 Feeding Condition: Fasted Fasted Fasted Vehicle/Formulation 25% hydroxypropylcyclodextrin 25% hydroxypropylcyclodextrin 25% hydroxypropylcyclodextrin Method of Administration: Portal vein IV IV Dose (mg/kg): Sample: Plasma Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS LCMS/MS PK Parameters (Mean ± SD): AUC024hr (µm hr): 1.9 ± ± Hepatic Extraction Ratio: 0.0 CLp (ml/min/kg): 65 ± 6 15 : Not applicable Additional Information: At low doses (0.2 1 mg/kg) the hepatic extraction ratio was 0.4. The hepatic extraction ratio decreased from 0.4 to 0 in the dose range of 1 to 5 mg/kg indicating that hepatic extraction saturates in this dose range. Consistent with this finding, the IV plasma clearance decreased from 65 ml/min/kg at 5 mg/kg IV to 15 ml/min/kg at 30 mg/kg IV with a nonlinear increase in exposure (AUC) from 1.7 to 43 M hr, respectively. 11

45 Test Article: [資料 : TT 1004] 反復経口投与後の薬物動態 ラット Species: Rat Rat Rat Feeding Condition: Fed Fed Fed Vehicle/Formulation 10% Tween 80 10% Tween 80 10% Tween 80 Method of Administration: P.O. P.O. P.O. Dose (mg/kg): Sample: Plasma Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS LCMS/MS PK Parameters in Study Week 26 (Mean ± SEM): Gender/Number of Animals: F/15 F/15 F/15 AUC024hr (µm hr): 32.6 ± ± ± 423 Cmax (µm): 13.6 ± ± ± 29.8 Tmax (hr): Gender/Number of Animals: M/15 M/15 M/15 AUC024hr (µm hr): 78.8 ± ± 35.0 Cmax (µm): 37.3 ± ± 16.0 Tmax (hr): Gender/Number of Animals: F/M/30 F/M/30 F/15 AUC024hr (µm hr): 55.7± ± ± 423 Cmax (µm): 25.5 ± ± ± 29.8 Tmax (hr): Additional Information: The exposures are greater than dose proportional following multiple oral administrations of vaniprevir to male and female rats. 12

46 Test Article: [資料 : PK003] イヌにおける薬物動態 単回静脈内及び経口投与後の薬物動態 イヌ Species: Dog Dog Gender/Number of Animals: M/3 M/3 Feeding Condition: Fasted Fasted Vehicle/Formulation: DMSO PEG400 Method of Administration: IV P.O. Dose (mg/kg): 2 5 Sample: Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS PK Parameters (Mean ± SD): AUC024hr (µm hr): 4.1 ± ± 0.4 CLp (ml/min/kg): 11 ± 2 Vdss (L/kg): 0.3 ± 0.1 t1/2 (hr): 1.2 ± 0.0 Cmax (µm): 0.5 ± 0.2 Tmax (hr): 1.3 ± 0.6 F (%): 12 ± 2 Crossover design, : Not applicable Additional Information: Following IV administration at 2 mg/kg the means CLp, Vdss and t1/2 values were 11 ml/min/kg, 0.3 L/kg and 1.2 hr, respectively. 13

47 Test Article: [資料 : PK003] 単回経口投与後の薬物動態 イヌ Species: Dog Dog Dog Dog Gender/Number of Animals: M/3 M/2 M/F 2/2 M/1 Feeding Condition: Fasted Fasted Fasted Fasted Vehicle/Formulation: PEG400 10% Tween 10% Tween 10% Tween Method of Administration: P.O. P.O. P.O. P.O. Dose (mg/kg): Sample: Plasma Plasma Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS LCMS/MS LCMS/MS PK Parameters (Mean ± SD): AUC024hr (µm hr): 1.2 ± ± Cmax (µm): 0.5 ± ± Tmax (hr): 1.3 ± ± Additional Information: After a 5 mg/kg oral dose, vaniprevir was absorbed rapidly yielding a C max of 0.5 M at 1.3 hr postdose. The absolute oral bioavailability was 12%. Dosedependent oral pharmacokinetics was evaluated after single dose administration of vaniprevir at four dose levels (5, 10, 15 and 30 mg/kg). Plasma AUC and C max increased nonlinearly and greater than dose proportionally. The T max increased as the dose increased. 14

48 Test Article: [資料 : TT 6060] 反復経口投与後の薬物動態 イヌ Species: Dog Feeding Condition: Fed Vehicle/Formulation 10% Tween 80 Method of Administration: P.O. Dose (mg/kg): 5/10 Sample: Plasma Analyte: Assay: LCMS/MS PK Parameters in Study Week 25 (Mean ± SEM): Gender/Number of Animals: F/4 AUC024hr (µm hr): 18.9 ± 10.5 Cmax (µm): 6.24 ± 3.08 Tmax (hr): 1.0 ± 0.0 Gender/Number of Animals: M/4 AUC024hr (µm hr): 12.9 ± 3.43 Cmax (µm): 4.96 ± Tmax (hr): 1.0 ± 0.0 Gender/Number of Animals: F/M/8 AUC024hr (µm hr): 15.9 ± 5.25 Cmax (µm): 5.60 ± 1.51 Tmax (hr): 1.0 ± 0.0 Dog Fed 10% Tween 80 P.O. 15 Plasma LCMS/MS Dog Fed 10% Tween 80 P.O. 45/30 Plasma LCMS/MS F/ ± ± ± 0.3 M/ ± ± ± 0.0 F/M/ ± ± ± 0.1 F/4 816 ± ± ± 0.3 M/4 631 ± ± ± 0.3 F/M/8 723 ± ± ±

49 Test Article: [資料 : TT 6060] 反復経口投与後の薬物動態 イヌ 続き Dosage raised to 10 mg/kg/day after 6 weeks of dosing at 5 mg/kg/day. Dosed at 45 mg/kg/day from Drug Day 1 to Drug Day 37 then 30 mg/kg/day from Drug Day 38 onwards. Additional Information: After multiple oral dose administration to dogs, vaniprevir exposures were greater than dose proportional and similar in both male and female dogs. 16

50 Test Article: [資料 : PK003] サルにおける薬物動態 静脈内及び経口投与後の薬物動態 サル Species: Monkey Monkey Gender/Number of Animals: M/3 M/3 Feeding Condition: Fasted Fasted Vehicle/Formulation: DMSO PEG400 Method of Administration: IV P.O. Dose (mg/kg): 2 5 Sample: Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS PK Parameters (Mean ± SD): AUC024hr (µm hr): 2.5 ± CLp (ml/min/kg): 18 ± 2 Vdss (L/kg): 0.4 ± 0.1 t1/2 (hr): 1.3 ± 0.2 Cmax (µm): Tmax (hr): 1.3 ± 0.6 F (%): 2±1 : Not applicable Additional Information: Following IV administration at 2 mg/kg the mean CLp, Vdss and t1/2 values were 18 ml/min/kg, 0.4 L/kg and 1.3 hr, respectively. After a 5 mg/kg oral dose, vaniprevir was absorbed rapidly yielding a C max of M at 1.3 hr postdose. The absolute oral bioavailability was low at 2%. 17

51 Test Article: [資料 : PK003] 経口投与後の薬物動態 サル Species: Monkey Monkey Monkey Gender/Number of Animals: M/3 M/2 M/2 Feeding Condition: Fasted Fasted Fasted Vehicle/Formulation: PEG400 PEG400 10% Tween Method of Administration: P.O. P.O. P.O. Dose (mg/kg): Sample: Plasma Plasma Plasma Analyte: Assay: LCMS/MS LCMS/MS LCMS/MS PK Parameters (Mean ± SD): AUC024hr (µm hr): Cmax (µm): Tmax (hr): 1.3 ± Additional Information: Dosedependent oral pharmacokinetics was evaluated after single dose administration of vaniprevir at 5, 50 or 100 mg/kg. Plasma AUC and C max increased nonlinearly and greater than dose proportionally between the 50 and 100 mg/kg doses. The T max increased as the dose increased. 18

52 Test Article: [資料 : PK003] チンパンジーにおける薬物動態 Pharmacokinetics after P.O. Administration Species: Chimpanzee Gender/Number of Animals: M/1, F/1 Feeding Condition: Fasted Vehicle/Formulation: Chocolate milk Method of Administration: P.O. Dose (mg/kg): 10 Sample: Plasma Analyte: Assay: LCMS/MS PK Parameters (Mean SD): AUC0t (µm hr) 3.8, 6.5 Cmax (µm) 0.7, 1.1 T max (hr) 2, 4 Value of t is last measurable time point less than 24 hours Additional Information: The pharmacokinetics of vaniprevir was studied following a 10 mg/kg oral dose formulated in chocolate milk to one male and one female chimpanzee. The C max values were 0.7 and 1.1 M occurring at 2 and 4 hours post dose and with an AUC of 3.8 and 6.5 M hr. 19

53 Test Article: [資料 : PK007] ウサギにおける薬物動態 Pharmacokinetics after P.O. Administration Species: Rabbit Gender/Number of Animals: F/3 Feeding Condition: Fasted Vehicle/Formulation: 10% Tween Method of Administration: P.O. Dose (mg/kg): 240 Sample: Plasma Analytes: [3H] Specific Activity (μci/mg): Assay: LCMS/MS PK Parameters (Mean SD): AUC0t (µm hr) 7.5 ± 1.6 Cmax (µm) 1.7 ± 0.2 T max (hr) 1.3 ± 0.6 Value of t is last measurable time point less than 24 hours Additional Information: The oral pharmacokinetics was studied in female rabbit (n=3) dosed at 240 mg/kg. The C max value was M occurring at hours post dose and with an AUC of M hr. 20

54 Test Article: [資料 : PK020] 組織分布 Species: Gender / Number of Animals: Feeding Condition: Vehicle/Formulation: Method of Administration: Dose (mg/kg): Radionuclide: Specific Activity ( Ci/mg): Analyte: Assay: Sampling Times: Sprague Dawley (SD) Rats and Long Evans (LE) Rats M/6 (1 per time point SD) and M/4 (1 per time point LE) Fasted 10% Tween 80 P.O. 60 [14C] 6.35 Radioactivity QWBA (Quantitative Whole Body Autoradiography), LSC Group 1: 2, 4, 24, 72, 168 and 336 hr (SD), Group 2: 4, 24, 168 and 672 hr (LE) 21

55 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 1 after a single oral administration of [ 14C] vaniprevir to male nonpigmented rats (Group 1, 60 mg/kg) Matrix Adrenal gland Aorta Bile Blood Bone Bone marrow Cecum Cecum contents Cerebellum Cerebrum Choroid plexus Diaphragm Epididymis Esophagus Esophageal Contents Exorbital lacrimal gland B06855 (2 hr) B06856 (4 hr) ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) B06857 B06858 (24 hr) (72 hr) B06859 (168 hr) B06860 (336 hr)

56 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 1 (Cont.) after a single oral administration of [ 14C] vaniprevir to male nonpigmented rats (Group 1, 60 mg/kg) Matrix Eye Eye (lens) Fat (abdominal) Fat (brown) Harderian gland Intraorbital lacrimal gland Kidney Large intestinal contents Large intestine Liver Lung Lymph nodes Medulla Muscle Myocardium Nasal turbinates Olfactory lobe B06855 (2 hr) B06856 (4 hr) ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) B06857 B06858 (24 hr) (72 hr) B06859 (168 hr) B06860 (336 hr)

57 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 1 (Cont.) after a single oral administration of [ 14C] vaniprevir to male nonpigmented rats (Group 1, 60 mg/kg) Matrix Pancreas Pituitary gland Preputial gland Prostate Renal cortex Renal medulla Salivary gland Seminal vesicle Skin (nonpigmented) Small intestinal contents Small intestine Spinal cord Spleen Stomach Stomach contents Testis B06855 (2 hr) B06856 (4 hr) ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) B06857 B06858 (24 hr) (72 hr) B06859 (168 hr) B06860 (336 hr)

58 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 1 (Cont.) after a single oral administration of [ 14C] vaniprevir to male nonpigmented rats (Group 1, 60 mg/kg) ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) Matrix B06855 B06856 B06857 B06858 B06859 B06860 (2 hr) (4 hr) (24 hr) (72 hr) (168 hr) (336 hr) Thymus Thyroid Urinary bladder Urine Uveal tract ng Equivalent/g values are reported to three significant figures with a maximum of three decimal places. : Below the limit of quantitation (<244 ng equivalents [14C] vaniprevir/g). : Not detectable (sample shape not discernible from background). One or more samples were above the upper limit of quantitation (> ng equivalents [14C] vaniprevir/g). 25

59 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 2 after a single oral administration of [ 14C] vaniprevir to male pigmented rats (Group 2, 60 mg/kg) Matrix Adrenal gland Aorta Bile Blood Bone Bone marrow Cecum Cecum contents Cerebellum Cerebrum Choroid plexus Diaphragm Epididymis Esophageal contents Esophagus Exorbital lacrimal gland ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) B06862 B06863 (24 hr) (168 hr) B06861 (4 hr) B06864 (672 hr)

60 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 2 (Cont.) after a single oral administration of [14C] vaniprevir to male pigmented rats (Group 2, 60 mg/kg) Matrix Eye Eye (lens) Fat (abdominal) Fat (brown) Harderian gland Intraorbital lacrimal gland Kidney Large intestinal contents Large intestine Liver Lung Lymph nodes Medulla Muscle Myocardium Nasal turbinates ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) B06862 B06863 (24 hr) (168 hr) B06861 (4 hr) B06864 (672 hr)

61 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 2 (Cont.) after a single oral administration of [ 14C] vaniprevir to male pigmented rats (Group 2, 60 mg/kg) Matrix Olfactory lobe Pancreas Pituitary gland Preputial gland Prostate Renal cortex Renal medulla Salivary gland Seminal vesicle Skin (nonpigmented) Skin (pigmented) Small intestinal contents Small intestine Spinal cord Spleen Stomach ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) B06862 B06863 (24 hr) (168 hr) 4680 B06861 (4 hr) NR B06864 (672 hr)

62 Test Article: [資料 : PK020] Concentrations of radioactivity in blood and tissues determined by wholebody autoradiography at specified times 組織分布 続き Table 2 (Cont.) after a single oral administration of [ 14C] vaniprevir to male pigmented rats (Group 2, 60 mg/kg) ng Equivalents [ 14C] vaniprevir/g Animal Number (Sacrifice Time) Matrix B06861 B06862 B06863 B06864 (4 hr) (24 hr) (168 hr) (672 hr) Stomach contents Testis 279 Thymus 818 Thyroid 1260 Urinary bladder NR Urine NR Uveal tract 339 ng Equivalent/g values are reported to three significant figures with a maximum of three decimal places. : Below the limit of quantitation (<244 ng equivalents [14C] vaniprevir/g). : Not detectable (sample shape not discernible from background). NR: Not represented (tissue not present in section) One or more samples were above the upper limit of quantitation (> ng equivalents [14C] vaniprevir/g). 29

63 Test Article: [資料 : PK020] 組織分布 続き Additional Information: is not widely distributed following oral administration of [ 14C] vaniprevir to male Sprague Dawley rats. [ 14C] derived radioactivity was not measurable at any collection time point in cerebellum, cerebrum, eye lens, olfactory lobe, and spinal cord. The number of analyzed matrices containing quantifiable levels of radioactivity decreased markedly with time, and by the final collection time point, 336 hours postdose, radioactivity was not quantifiable in any of the analyzed matrices. In SD rats, the highest maximum levels of radioactivity after oral administration were observed in GI tract contents, where the highest values ranged from 875,500 (cecum contents) to 2,600,000 ng equivalents/g (small intestinal contents). Bile also showed high levels of radioactivity, with a maximum of 492,000 ng equivalents/g. Other than the GI tract contents and bile, the matrices with the highest observed maximum concentrations of radioactivity with values (ng equivalents/g) and the corresponding sampling times were kidney (10,600; 4 hours), stomach (11,700; 2 hours), renal cortex (12,300; 4 hours), plasma (LSC) (13,800; 4 hours), cecum (17,000; 4 hours), small intestine (24,700; 4 hours), and liver (145,000; 2 hours). All other analyzed matrices contained less than 10,600 ng equivalents/g at all sampling times. Concentrations of radioactivity were measurable in the medulla of the brain at one sampling time, 4 hours postdose; all other lobes of the brain had no quantifiable radioactivity, suggesting that [ 14C] vaniprevirderived radioactivity has limited, if any, penetration of the blood:brain barrier. [14C] vaniprevirderived radioactivity for uveal tract in the eye of SD (albino) rats, was measurable at 2 and 4 hours postdose, with 1,140 and 1,570 ng equivalents/g, respectively. Radioactivity was not detectable in the uveal tract from 24 through 336 hours postdose. Additional Information: was not widely distributed following oral administration of [ 14C] vaniprevir to male Long Evans rats. [14C] vaniprevirderived radioactivity was not measurable at any collection time point in bone, cerebellum, cerebrum, eye, medulla, olfactory lobe, prostate, seminal vesicle, and spinal cord. The number of analyzed matrices containing quantifiable levels of radioactivity decreased markedly with time, and by the final collection time point, 672 hours postdose, radioactivity was not quantifiable in any of the analyzed matrices. In LE rats, the highest maximum levels of radioactivity after oral administration were observed in GI tract contents, where the highest values ranged from 330,000 (esophageal contents) to 910,000 ng equivalents/g (stomach contents). Bile also showed high levels of radioactivity, with a maximum of 232,000 ng equivalents/g. Other than the GI tract contents and bile, the matrices with the highest observed maximum concentrations of radioactivity with values (ng equivalents/g) and the corresponding sampling times were stomach (3,330; 4 hours), renal medulla (3,450; 4 hours), small intestine 30

64 Test Article: [資料 : PK020] 組織分布 続き Additional Information (Cont.): (3,590; 4 hours), kidney (3,770; 4 hours), renal cortex (4,270; 4 hours), cecum (4,500; 4 hours), blood analyzed by QWBA (9,400; 4 hours) and liver (140,000; 4 hours). All other analyzed matrices contained less than 3,330 ng equivalents/g at all sampling times. Concentrations of radioactivity were not quantifiable in brain, suggesting that [ 14 C] vaniprevirderived radioactivity did not cross the blood:brain barrier. [14C] vaniprevirderived radioactivity for uveal tract in the eye of LE rats, was measurable at one time point only, 4 hours postdose, with 339 ng equivalents/g. Radioactivity in the uveal tract was not detectable by 24 hours postdose. The amount of radioactive equivalents was similar and low in both pigmented and nonpigmented rats indicating the vaniprevir does not bind to melanin. 31

65 Test Article: [資料 : PK003] [資料 : PK014] [資料 : PK015] 肝への分布 Species: Gender / Number of Animals: Feeding Condition: Vehicle/Formulation: Method of Administration: Dose (mg/kg): Analyte: Assay: Sampling times: Table 1 Rat, Dog, Monkey and Chimpanzee M/3 rat, M/2 dog, monkey and chimpanzee Fasted PEG400 P.O. 5 (rat, dog and monkey) and 10 (chimpanzee) LCMS/MS 1, 2, 4, 6, 8, 12 and 24 hr Plasma and liver concentrations at specified times after a single oral administration of vaniprevir (5 mg/kg) Time Rat Liver Rat Plasma Dog Liver Dog Plasma Monkey Liver Monkey Chimpanzee Chimpanzee (hr) (nm) (nm) (nm) (nm) (nm) Plasma (nm) Liver (nm) Plasma (nm) 1 6, , , , ,903 < , <2 8 4,040 <7 < , < <2 The animals were dosed at 5 mg/kg P.O. in PEG400, with the exception of the chimpanzees that was dosed at 10 mg/kg in chocolate milk. The data represent the mean of n=3 in rat and n=2 in dog, monkey and chimpanzee. : Not applicable 32

66 Test Article: [資料 : PK003] [資料 : PK014] [資料 : PK015] 肝への分布 続き Species: Gender / Number of Animals: Feeding Condition: Vehicle/Formulation: Method of Administration: Dose (mg/kg): Analyte: Assay: Rat, Dog and Monkey M/3 rat, M/2 or 3 dog, and M/1 monkey Fasted Imwitor:Cremophor:Tween 80 (80:10:10), Imwitor:Tween 80 (50:50) and PEG400 P.O. Rat (5, 30 and 300), Dog (2 and 5) and (2 mg/kg + ritonavir), Monkey (5) LCMS/MS Table 2 Liver and plasma exposure in rat, dog and monkey following a single oral administration of vaniprevir Species P.O. Dose (mg/kg) Rat (n=3) 5 Rat (n=3) 30 Rat (n=3) 300 Dog (n=3) 2 Dog (n=3) 2 Dog (n=2) 5 Monkey (n=1) 5 Plus 5 mg/kg P.O. ritonavir Plasma AUC ( M hr) < ± ± Liver AUC ( M hr) ± ± Liver AUC/ Plasma AUC >

67 Test Article: [資料 : PK003] [資料 : PK014] [資料 : PK015] 肝への分布 続き Additional Information: demonstrated good liver exposure (333 M at 2 hrs) in rat, dog, and monkey following an oral dose of 5 mg/kg and in chimpanzee (31 M at 12 hr) following a 10 mg/kg oral dose. The rat, dog, and monkey liver AUC to plasma AUC ratio following a 5 mg/kg oral dose was >874, 102, and 455, respectively. At low doses, the liver exposure of vaniprevir is at least 100fold greater than the plasma exposures in preclinical species. In rats, the liver exposure increased more than dose proportionally between 5 and 30 mg/kg, but less than dose proportionally between 30 and 300 mg/kg. Also in rat, the liver to plasma concentration ratios decrease as the plasma concentrations increase ranging from a liver to plasma concentration ratio of ~4000 when the plasma concentration is M to 16 when the plasma concentration is 14 M [資料 : PK015]. The dog liver exposure was more than dose proportional between 2 and 5 mg/kg. 34

68 Test Article: [資料 : TT 7230] 妊娠動物での検討 Placental Transfer Species: Rat Rat Gestation Day / Number of Animals: 20 days/4 20 days/4 Vehicle/Formulation: 10% Tween 80 10% Tween 80 Method of Administration: P.O. P.O. Dose (mg/kg/day): Analyte: Assay: LCMS/MS LCMS/MS Concentration (nm) : Sampling Time (hr) Day 20/8hr Day 20/24hr Maternal Plasma 15.2 ± ± Fetal Plasma ± ± Ratio (Fetal/Maternal) ± ± Mean ± SEM, Values are the mean ± SE of the individual fetal plasma concentration divided by the corresponding maternal plasma concentration.. 35

69 Test Article: [資料 : TT 7220] [資料 : TT 妊娠動物での検討 続き 7230] Placental Transfer Species: Rabbit Rabbit Gestation day/number of Animals: 20 days/4 20 days/4 Vehicle/Formulation: 10% Tween 80 10% Tween 80 Method of Administration: P.O. P.O. Dose (mg/kg/day): Analyte: Assay: LCMS/MS LCMS/MS Concentration (nm) : Sampling Time (hr) Day 20/2hr Day 20/24hr Day 20/2hr Day 20/24hr Maternal Plasma 1.64 ± ± ± ± Fetal Plasma ± ± ± ± Ratio (Fetal/Maternal) ± ± ± Mean ± SEM, Values are the mean ± SE of the individual fetal plasma concentration divided by the corresponding maternal plasma concentration. Value obtained from only one ratio >0. Additional Information: Placental transfer of vaniprevir was investigated in pregnant female rats (n=4) by measuring the concentrations of unchanged drug in maternal and fetal plasma on gestation day (GD) 20/21 at 8 and 24 hours post dose following 120 mg/kg/day oral dose in rats on GD 6 through GD 20. In rats, the mean fetal to maternal plasma concentration ratio was at 8 hours and 7.6 at 24 hours. The placental transfer of vaniprevir was also studied in pregnant female rabbits (n=25) split into two dose groups. The rabbits received 120 or 240 mg/kg/day of vaniprevir orally on GD 7 through GD 20. Maternal and fetal plasma was collected on GD 20 from four nonfasted rabbits per group at 2 and 24 hours post dose. In rabbits, the mean fetal to maternal plasma concentration ratio was and at 2 and 24 hours, respectively, in the 120 mg/kg/day dose group and and at 2 and 24 hours, respectively, in the 240 mg/kg/day dose group. The results suggest that vaniprevir readily crosses the placenta in rats and rabbits. 36

70 Test Article: [資料 : PK001] [資料 : PK006] [資料 : PK008] [資料 : PK017] [資料 : PK027] 血漿蛋白への可逆的結合 Study System: In vitro Mouse, rat, rabbit, dog, monkey, chimpanzee and human. equilibrium dialysis and liquid scintillation counting Test System and Method: Radionuclide: [3H]/[ 14C] Analyte: Total radioactivity/lcms/ms Percent of Bound Radioactivity to Plasma: [3 H]/[ 14 C] vaniprevir Mouse Rat Rabbit Dog Monkey Chimpanzee Human Concentration (µm) ± ± ± ± ± ± ± ± ± ± ± ± ± 0.4 Mean ± SD (n=3), : Not determined Additional Information: The percent bound of vaniprevir in plasma was determined to be across all species. 37

71 Test Article: [資料 : PK001] [資料 : PK006] [資料 : PK008] [資料 : PK017] [資料 : PK027] 血漿蛋白への可逆的結合 続き Study System: In vitro Human/Equilibrium dialysis and LCMS/MS Test System and Method: Analyte: Percent of Bound to Human Serum Albumin and α 1Acid Glycoprotein (Mean ± SD): Mean Percent Bound Percent Bound Protein Percent Bound (1 and 10 µm) (10 µm vaniprevir) (1 µm vaniprevir) (N=3) (N=3) (N=3) Human Serum Albumin 94.8 ± ± (40 mg/ml) α1 Acid Glycoprotein 66.6 ± ± (1mg/mL) Additional Information: The analysis was conducted at 1 and 10 µm vaniprevir and for both human serum albumin (HAS) and α1 acid glycoprotein (AAG) and no concentration dependent binding was observed, indicating that vaniprevir does not saturate binding to either HSA or AAG at concentrations up to 10 µm vaniprevir. Also, the data show that vaniprevir binds to both HSA and AAG. 38

72 Test Article: [資料 : PK001] [資料 : PK006] [資料 : PK008] [資料 : PK017] [資料 : PK027] 血漿蛋白への可逆的結合 続き Study System: In vitro Mild, moderate and severe hepatic insufficient human subjects/equilibrium dialysis and LCMS/MS Test System and Method: Analyte: Percent of Bound to Plasma (Mean ± SD): Mild hepatic insufficient Moderate hepatic insufficient Severe hepatic insufficient Healthy Concentration (µm) (n=9) (n=11) (n=2) (n=24) ± ± ± 0.9 Additional Information: The percent bound of vaniprevir in plasma was determined to be across human subjects with mild, moderate and severe hepatic insufficiency as well as in healthy subjects. 39

73 Test Article: [資料 : PK001] 血球移行性 Study System: In vitro Blood and plasma from rat, dog, and human. equilibrium dialysis and liquid scintillation counting Test System and Method: Radionuclide: [3H] Analyte: Total radioactivity BloodtoPlasma Concentration Ratio: Concentration Rat Dog Human 0.1 µm 0.7 ± ± µm 0.7 ± ± ± µm 0.8 ± ± ± 0.0 Mean ± SD (n=3) Additional Information: The equilibrium bloodtoplasma ratios for [3H] vaniprevir in rat, dog and human blood were 0. 7, 0.7, and 0.8, respectively, and were constant over the concentration range of 0.1 to 10 µm. 40

74 Test Article: [資料 : PK006] マウスにおける in Vivo 代謝 Species/Gender/Number of Animals: Ras transgenic CB6F1 mouse/m/10 Vehicle/Formulation: 10%Tween/90%H2O Method of Administration: P.O. Dose/Feeding Condition: 300 mg/kg / Fasted Radionuclide/Specific activity, μci/mg: [3H] / 5.19 Biological Samples: Plasma, urine, feces Analytes: and metabolites Assays: HPLC with radiometric detection, microbeta counter, LCMS/MS, liquid scintillation counting The Presence of [3 H] vaniprevir and its Metabolites in Plasma and/or Excreta in Mice: Biological Fluid Plasma (Pl) Bile (B) Urine (U) Feces (F) NS NS Metabolites Present in Plasma and Excreta M6 NS NS M7 NS NS M8 NS NS M9 NS NS M10 NS NS : Detected by LCMS/MS and radiometric detection, NS: Not studied Additional Information: In plasma and feces, the majority of the radioactivity was in the form vaniprevir with minor amounts of oxidative metabolites (M6, M7, M8, M9 and M10) also detected. The data indicate that oxidative metabolism contributes to the clearance of vaniprevir in mice. 41

75 Test Article: [資料 : PK007] ウサギにおける in Vivo 代謝 Species/Gender/Number of Animals: Rabbit/F/3 Vehicle/Formulation: 10%Tween80/90%H 2O Method of Administration: P.O. Dose/Feeding Condition: 240 mg/kg / Fasted Radionuclide/Radioactivity per Subject ( Ci): [3H] / Analyte: Total radioactivity Assays: Combustion and/or liquid scintillation counting Percent of Administered Dose (Mean ± SD) Dose Collection Period Urine Feces Bile Total 240 mg/kg P.O. 072 hr 0.5 ± ± 14.1 NS 67.3 ± 14.1 The Presence of [3 H] vaniprevir and its Metabolites in Plasma and/or Excreta in Rabbit: Biological Fluid Plasma Bile Urine Feces NS NS NS Metabolites Present in Plasma and Excreta M6 NS NS NS M7 NS NS NS M8 NS NS NS M9 NS NS NS M10 NS NS NS : Detected by LCMS/MS and radiometric detection, NS: Not studied Additional Information: The data indicated that has reasonable absorption and that oxidative metabolism contributes its clearance in rabbit. 42

76 Test Article: [資料 : PK002] [資料 : PK009] ラットにおける in Vivo 代謝 Species/Gender/Number of Animals: Vehicle/Formulation: Method of Administration/ Dose/Feeding Condition: Radionuclide/Radioactivity per Subject ( Ci): Analytes/Assays: Percent of Administered Dose (Mean ± SD) Dose Collection Period 2 mg/kg IV 072 hr 60 mg/kg P.O hr Percent of Dose Excreted in Urine and Feces Following a Interval (hr) Total : Bileduct cannulated, NS: Not studied Rat /M/2 Rat/M/3 DMSO 10%Tween80/90%H 2O IV/2 mg/kg/fasted P.O./60 mg/kg/fasted [3H]/50 [14C]/400 Total radioactivity/combustion and/or liquid scintillation counting Urine Feces Bile ± ± 6.7 NS mg/kg Oral Dose of [ C]vaniprevir to SpragueDawley Rats: Rat 1 Rat 2 Urine Feces Urine Feces Urine Total ± 6.3 Rat 3 Feces

77 Test Article: [資料 : PK002] [資料 : PK009] ラットにおける in Vivo 代謝 続き [ 14C]vaniprevir Metabolites as Percentage of Dose in Rat Feces (024 hour) Following a 60 mg/kg Oral Dose to SpragueDawley Rats: Analytes Rat 1 Rat 2 Rat 3 Mean SD M M M M M M Total Additional Information: Following the 2 mg/kg IV dose, the mean recovery in 72 hours was 71.7% with 15.6%, 52.1% and 4.2% recovered in urine, bile and feces, respectively. The radioactivity recovered in urine was in the form of oxidative metabolites and the radioactivity in bile was in the form of oxidative metabolites (~67%) and (~33%). Several oxidative metabolites, M3, M6, M7, M7a, M8, M9 and M10 were identified in rat bile. Biliary clearance of vaniprevir both as metabolites and vaniprevir is a major clearance pathway in rat. Following a 60 mg/kg oral dose to intact rats, urine and feces were collected for 244 hours and the recoveries were % in urine and % in feces with a total recovery of %. The majority of the radioactivity (74 11%) was recovered in feces in the first 24 hours. The radioactivity recovered in rat feces was in the form of oxidative metabolites (~50%) and (~50%). The oxidative metabolites detected in rat feces were M6, M7, M8, M9 and M10. A single direct gluthatione adduct, M13, was also detected in rat feces. Overall, about 33% of the administered oral dose was excreted into feces as oxidative metabolites indicating that the absorption of vaniprevir in rat is at least moderate. The IV BDC study showed that vaniprevir is eliminated into bile as vaniprevir in addition to metabolites, such that the actual absorption of vaniprevir in rats is likely greater than 36%. The plasma of rats following a 114 mg/kg oral dose of [3H]vaniprevir was profiled and minor amounts of the oxidative metabolites M6, M7, M8, M9, M10 and M11 were detected [資 料 : PK004]. 44

78 Test Article: [資料 : PK002] [資料 : PK012] [資料 : PK021] イヌにおける in Vivo 代謝 Species/Gender/Number of Animals: Vehicle/Formulation: Method of Administration/ Dose/Feeding Condition: Radionuclide/Radioactivity ( Ci): Analytes/Assays: Percent of Administered Dose (Mean ± SD) Dose Collection Period 2 mg/kg IV 072 hr 5 mg/kg P.O hr Percent of Dose Excreted in Urine and Feces Following a 5 Interval (hr) Dog/M/3 Dog/M/3 DMSO 10%Tween80/90%H 2 O IV/2 mg/kg/fasted P.O./5 mg/kg/fasted [3 H] / 150 [14 C] / 85 Total radioactivity/combustion and/or liquid scintillation counting Urine Feces 3.5 ± ± ± ± 6.4 mg/kg Oral Dose of [14C] to Dogs: Dog H08453 Urine Total 1.21 : Study conducted at Covance, : Included cage wash and debris, NS: Not studied Dog H08454 Dog H08455 Feces Urine Feces Urine Feces NS NS NS NS NS NS NS Total 68.5 ± ± 4.3 Bile 53.0 ± 3.3 NS

79 Test Article: [資料 : PK002] [資料 : PK012] [資料 : PK021] イヌにおける in Vivo 代謝 続き Percent Metabolite in Feces Following a 5 mg/kg Oral Dose of [14C] to Dogs: Dog H08453 Dog H08454 Dog H08455 Analytes (824 hr) (024 hr) (824 hr) M M M M M M5a M M M7a M M M M Total Mean SD

80 Test Article: [資料 : PK002] [資料 : PK012] [資料 : PK021] イヌにおける in Vivo 代謝 続き Concentrations of and [14C] Equivalents in Dog Plasma Following a 5 mg/kg Oral Dose of [ 14C]: Time (hr) ( M) [14C] equivalents ( M) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 : Blow the lower limit of quantitation 47

81 Test Article: [資料 : PK002] [資料 : PK012] [資料 : PK021] イヌにおける in Vivo 代謝 続き Additional Information: The in vivo metabolism was studied in BDC dogs following a 2 mg/kg IV dose of [ 3H]vaniprevir (n=3) and in intact dogs following a 5 mg/kg oral dose of [ 14C]vaniprevir (n=3). Following the 2 mg/kg IV dose, the mean recovery in 72 hours was 69% with 3.5%, 53.0% and 12% recovered in urine, bile and feces, respectively. The amount of radioactivity recovered in the urine was too low to profile. The radioactivity recovered in the bile was in the form of oxidative metabolites (~70%) and vaniprevir (~30%). Several oxidative metabolites, M6, M7, M8, M9 and M10, were detected in dog bile. The radioactivity in dog feces was not profiled. Similar to rat, metabolism is the major elimination pathway of vaniprevir in dog. The recovery of radioactivity in the feces following an IV dose to BDC dogs also indicates a potential for intestinal secretion of vaniprevir and/or metabolites. Following a 5 mg/kg oral dose of [ 14C]vaniprevir to intact dogs, urine, and feces were collected for 172 hours and the mean overall radioactive recovery was % with %, % and 3.4% recovered in urine, feces, and cage wash/debris, respectively. The majority of the radioactive dose (74 13%) was recovered in feces in the first 24 hours. The radioactivity recovered in feces was in the form of numerous oxidative metabolites (M1, M2, M3, M4, M5, M5a, M6, M7, M7a, M8, M9 and M10), a net hydrolysis product (M14) most likely resulting from bacterial gut reduction of the amide bond, and vaniprevir. Quantitatively, the radioactivity in feces was comprised of ~71% oxidative metabolite, ~27% vaniprevir and ~2% of the net hydrolysis product, M14. The large amount of oxidative metabolites recovered in feces following an oral dose of vaniprevir to intact dogs indicates that vaniprevir is wellabsorbed in dogs. It also indicates that oxidative metabolism is the predominant clearance mechanism in dog. is the major circulating component in dog since the plasma concentrations of vaniprevir as determined by LC/MS/MS agrees well with the concentration of [14C]vaniprevir radioactive equivalents. The total radioactivity in dog plasma was too low to profile at timepoints other than one hour. In a one hour plasma profile, vaniprevir is the only detectable radioactive component; no metabolites are detected in dog plasma. 48

82 Test Article: [資料 : PK013] [資料 : PK022] ヒトにおける in Vivo 代謝 Species/Gender/Number of Animals: Vehicle/Formulation: Method of Administration/ Dose/Feeding Condition: Radionuclide/Radioactivity per Subject ( Ci): Analytes/Assays: Percent of Administered Dose (Mean ± SD) Dose Collection Period 575 mg P.O hr Human/M/6 Capsule P.O./575 mg/fasted [14 C] / 204 Total radioactivity/combustion and/or liquid scintillation counting Urine Feces Bile Total 0.40 ± ± 1.2 NS 96.0 ± Cumulative (0168 hr) Excretion of Radioactivity in Urine, Feces and Toilet Tissue Following a 575 mg Oral Dose of [ C] to Humans: Subject Number Urine Feces Toilet Tissue Total Mean SD Study conducted at Covance, NS: Not studied 49

83 Test Article: [資料 : PK013] [資料 : PK022] ヒトにおける in Vivo 代謝 続き Percent of Dose Excreted in Urine and Feces Following a 575 mg Oral Dose of [ 14C] to Humans: Interval (hr) Urine Feces Urine Feces Urine Feces Urine Feces Urine Feces NS 11.1 NS NS Total : Blow the lower limit of quantitation, NS: Not sampled Urine Feces NS

84 Test Article: [資料 : PK013] [資料 : PK022] ヒトにおける in Vivo 代謝 続き Percent Metabolite in Feces (096 hr) Following a 575 mg Oral Dose of [ 14C] to Humans: Subject M M M M M M5a M M M7a M M M M Total Mean SD

85 Test Article: [資料 : PK013] ヒトにおける in Vivo 代謝 続き Percent Metabolite (Mean Standard Deviation) in Rat (60 mg/kg P.O.), Dog (5 mg/kg P.O.) and Human (575 mg P.O.) Feces: Species Rat (024 hr) Dog (024 hr) Human (096 hr) M M M M M M5a M M M7a M M M M M Total : Not detected Additional Information: The metabolism and excretion of [ 14C]vaniprevir were evaluated in six healthy male subjects following a 575 mg (204 Ci) oral dose. The mean total recovery in the excreta was %, with , and % of the dose recovered in urine, feces and toilet tissue, respectively. The majority ( %) of the radioactive dose was recovered in feces in the first 96 hrs. 52

86 M1, M2 M3 M14 M6, M7a M13 M7 M11 M10 M9 M8

87 Test Article: [資料 : PK001] [資料 : PK006] [資料 : PK008] In Vitro 代謝 Type of Studies: Source of Materials: Methods: Assays: Experiments were conducted to examine the in vitro metabolism of [ 3H]/[ 14C] vaniprevir in liver microsomes (S9 or microsomes supplemented with DPH) from mouse, rabbit, rat, dog, and human, and hepatocytes from mouse, rat, dog, and human. Mouse, rabbit, rat, dog, and human liver microsomes were prepared at MRL or purchased from Xenotech. Mouse, rat, dog, and human cryopreserved hepatocytes were prepared at MRL or purchased from Celsius/In Vitro Technologies (IVT). [3H] / [ 14C] (10 μm) was incubated with human, rabbit, rat, dog, and rash2 mouse liver microsomes at 37 C in a final volume of ml of M potassium phosphate buffer (ph 7.4) containing mg/ml microsomal protein, and 1 mm DPH. The total amount of organic solvent was 0.5%. Reactions were terminated by the addition of equal volumes of acetonitrile. Samples were then vortexed, centrifuged, and the supernatants were removed and evaporated to dryness and reconstituted in 30% acetonitrile or analyzed directly. [3H] (1, 10, and 50 μm) was incubated with rat, dog, and human hepatocytes at 37 C for 4 hr with cells/ml in HBSS with 10 mm HEPES. The total amount of organic solvent was 0.5%. Reactions were terminated by the addition equal volumes of acetonitrile. Samples were then vortexed, centrifuged, and the supernatants were analyzed. LCMS/MS with radiochemical detection. 54

88 Test Article: [資料 : PK001] [資料 : PK006] [資料 : PK008] In Vitro 代謝 続き Metabolites of Observed in Liver Preparations (S9 or Microsomes Supplemented with DPH) and Hepatocytes: S9 Hepatocytes Metabolite Rat Dog Rabbit Mouse Human Mouse Rat Dog Human M1 M2 M3 M4 M5 M6 M7 M7a M8 M9 M10 Microsomes supplemented with DPH, : Detected, : Not detected Additional Information: In in vitro liver preparations (S9 or microsomes supplemented with DPH or hepatocytes), vaniprevir undergoes minor amounts of oxidative metabolism in mouse, rats, rabbits, dogs, rhesus monkey and human. In human liver S9, four minor metabolites (M7, M8, M9, and M10) were observed. In rat liver S9 only a minor amount of M10 was formed and in dog liver S9 no metabolites were observed. In female rabbit S9, mouse liver microsomes and mouse hepatocytes seven minor oxidative metabolites (M3, M6, M7a, M7, M8, M9, and M10) were observed. formed metabolites M9 and M10 when incubated with human hepatocytes and M8 when incubated with dog hepatocytes. No metabolites were observed after incubating vaniprevir with rat hepatocytes. The metabolic pathways observed in vitro are consistent with those observed in vivo and indicate that vaniprevir forms predominantly oxidative metabolites. 55

89 Test Article: [資料 : PK002] [資料 : PK007] [資料 : PK009] [資料 : PK021] [資料 : PK022] ラット イヌ ウサギ及びヒトにおけるマスバランス Excretion of Radioactivity in Rats, Dogs, Human and Rabbit After IV or Oral Admistration of [3 H or 14C] : Species Dose Collection Period Percent of Administered Dose (Mean ± SD) (hr) Urine Feces Bile Total Rat 2 mg/kg IV mg/kg P.O ± ± 6.7 NS 81.8 ± 6.3 Dog 2 mg/kg IV ± ± ± ± mg/kg P.O ± ± 6.43 NS 91.5 ± 4.26 Rabbit 240 mg/kg P.O ± ± 14.1 NS 67.3 ± 14.1 Human 575 mg P.O ± ± 1.2 NS 96.0 ± 1.1 Study conducted at Covance, Includes % from cage wash/debris, NS: Not studied 56

90 Test Article: [資料 : PK002] [資料 : PK006] [資料 : PK007] [資料 : PK009] [資料 : PK012] [資料 : PK013] [資料 : PK022] ラット イヌ ウサギ及びヒトにおけるマスバランス 続き Additional Information: Following an IV administration of [3H]vaniprevir (2 mg/kg; n=2) to BDC rats, the total radioactivity recovered in the excreta (072 hr) represented 71.7% of the dose, with 15.6% excreted into urine, 4.2% excreted into feces and 52.1% excreted into bile. After oral dosing of [14C]vaniprevir (60 mg/kg; n=3) to intact rats, 0.5% and 81.3% of the dose were recovered in the urine and feces, respectively. The majority of the dose was excreted in the first 24 hours. The recovery of radioacvitiy in the feces of BDC rats following an IV dose indicated a potential for intestinal secretion. Following IV administration (2 mg/kg; n=3) of [3H]vaniprevir to BDC dogs, recovery of total radioactivity over the 072 hr collection period was 68.5%, with 3.5%, 12.0% and 53.0% of the dose recovered in urine, feces, and bile, respectively. The recovery of radioactivity in dog feces following IV dosing to BDC animals indicates a potential for intestinal secretion. After oral dosing of [14C]vaniprevir to intact dogs (5 mg/kg P.O.; n=3) the total recovery was good (91.5%), with 0.78% and 87.3% of the dose was recovered in urine and feces, respectively. Biliary secretion represented the predominant route for excretion of radioactivity. Excretion data were obtained in intact female rabbits following a 240 mg/kg oral dose of [ 3H]vaniprevir (n=3). Urine and feces were collected for 72 hours. Sixtyseven percent of the total dose was recovered primarily in feces, with 0.5% in urine. Excretion data were obtained in six healthy male subjects following a 575 mg oral dose of [ 14C]vaniprevir formulated as a liquid filled capsule. Urine, feces, and toilet tissue were collected for 168 hours post dose. The mean overall recovery was 96% and individual subject recoveries ranged from %. In the first 96 hours, the mean overall recovery was 94%, with 93.3% of the dose excreted into feces, 0.4% of the dose excreted into urine and 0.1% of the dose on toilet tissue. Overall, there was complete recovery of the radioactive dose in human. The low recovery in urine and high recovery of radioactivity in feces indicated that the predominant route for the vaniprevir excretion in human is through biliary secretion. 57

91 Test Article: [資料 : TT 7230] ラットにおける乳汁排泄 Species: Rat Gestation Day/Number of Animals: 20 days/4 Vehicle/Formulation: 10% Tween 80 Method of Administration: Oral Analyte: Assay: LCMS/MS Mean Maternal Plasma and Milk Concentrations and Lactational Transfer on LD 14: Matrix and Sampling Point 180 mg /kg/day Maternal Plasma Concentation at 8 hrs ( M) Maternal Milk Concentation at 8 hrs ( M) Maternal Milk/Plasma Concentation Ratio at 8 hrs Mean SEM Additional Information: The excretion of vaniprevir into the milk of lactating rats (n=6) was investigated by measuring concentrations of the parent compound in maternal plasma and milk on lactation day (LD) 14 following oral administration of vaniprevir at 180 mg/kg/day from GD 6 to LD 14. Maternal plasma and milk concentrations were determined in samples collected on LD 14 from four nonfasted rats at 8 hours post dose. The mean maternal milk to plasma vaniprevir concentration ratio was 0.147, indicating lactational transfer of vaniprevir in rats. 58

92 Test Article: [資料 : PK001] CYP 基質としてのバニプレビルの評価 Types of Studies: Source of Materials: Methods: Assay: Additional Information: Experiments were carried out to evaluate if vaniprevir is a substrate of cytochrome P450 Pooled human liver microsomes and anticyp antibodies were obtained from Merck Research Laboratories, West Point, PA. Immunoinhibition experiments were performed using selective inhibitory monoclonal antibodies (MAbs) for anticyp2c, anticyp2d6, and anticyp3a. Reaction mixtures contained 100 mm potassium phosphate buffer mixed with 1 mg/ml pooled human liver microsomes and MAbs (anticyp2c9, anticyp2d6, anticyp3a, or control antiascites). Samples were preincubated at 37 C in a shaking waterbath for 5 min before the addition of [3 H] vaniprevir, at 1, 10, and 50 M final concentrations. The reaction was initiated by the addition of 1 mm DPH and the samples were further incubated in the water bath at 37 C for 60 min. The reaction was stopped by the addition of an equal volume of acetonitrile. LCMS/MS and radiochemical detection The oxidative metabolism of vaniprevir is mediated predominantly via CYP3A with no significant contribution from CYP2D6 or CYP2C. 59

93 Test Article: [資料 : PK001] [資料 : PK010] [資料 : PK018] ヒト肝ミクロソーム中の CYP 及び UGT1A1の阻害 Types of Studies: Source of Materials: Methods: Assay: Experiments were carried out to evaluate the potential for vaniprevir to inhibit human liver microsomal cytochrome P450 and UGT (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, CYP3A, and UGT1A1) activities. Pooled human liver microsomes The K m value for each CYP marker substrate (testosterone and midazolam for 3A4, dextromethorphan for 2D6, diclofenac for 2C9, phenacetin for 1A2, mephenytoin for 2C19, and amodiaquine for 2C8) was determined in pooled human liver microsomes. To evaluate IC50 (concentration of inhibitor required to decrease activity by 50%), a single concentration of the marker substrate (~Km ) and varying concentrations of vaniprevir or a positive control inhibitor were employed. The incubation mixtures contained 0.25 or 0.5 mg/ml microsomal protein and an assay solution containing deionized water, 0.5 M KPO4 buffer, and 1000 units/ml glucose6phosphate dehydrogenase. The reaction was initiated with a cofactor solution consisting of 20 mg/ml βdp, 13.3 mg/ml MgCl2, and 20 mg/ml glucose6phosphate. The reaction was allowed to proceed for 10 to 45 minutes at 37 C in a shaking water bath. The reactions were terminated with the addition of a 5% acetonitrile/3% formic acid solution plus the appropriate internal standard. Pooled human liver microsomes (0.5 mg/ml) was incubated with 20 M estradiol a substrate for UGT1A1, 5 mm UDPGA and 25 g/ml alamethicin in 200 L of 81 mm HEPES and 9 mm MgCl 2 buffer, ph 7.0 at 37 C for 20 minutes. Estradiol glucuronidation was quantified in the absence (solvent control) and presence of increasing concentrations ( M) of either vaniprevir or nicardipine a known inhibitor of UGT1A1. LCMS/MS 60

94 Test Article: [資料 : PK001] [資料 : PK010] [資料 : PK018] ヒト肝ミクロソーム中の CYP 及び UGT1A1の阻害 続き Evaluation of as a Reversible Inhibitor of Cytochrome P450s and UGT1A1 in Human Liver Microsomes: Enzyme Involved CYP1A2 Reaction (Substrate concentration) Phenacetin Odeethylation (40 M) CYP2C8 Amodiaquine Ndeethylation (4 M) CYP2C9 Diclofenac 4 hydroxylation (10 M) CYP2C19 (S)Mephenytoin 4 hydroxylation (20 M) CYP2D6 Dextromethorphan Odemethylation (20 M) CYP2B6 Bupropion Hydroxylation (180 M) CYP3A Midazolam 1 hydroxylation (20 M) Compound Tested furafylline vaniprevir ketoconazole vaniprevir sulphaphenazole vaniprevir tranylcypromine vaniprevir quinidine vaniprevir N( methylbenzyl)1aminobenzotriazole vaniprevir Conc. Range ( M) vaniprevir Testosterone 6ßhydroxylation (75 M) UGT1A1 Estradiol (20 M) 0 50 vaniprevir nicardipine vaniprevir IC 50 ( M) 6.06 > > > >

95 Test Article: [資料 : PK001] [資料 : PK010] [資料 : PK018] ヒト肝ミクロソーム中の CYP 及び UGT1A1の阻害 続き Additional Information: The nonpreincubation dependent inhibitory potential of vaniprevir towards CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, CYP3A, and UGT1A1 activities was investigate in human liver microsomes. does not reversibly inhibit CYP1A2, CYP2C8, CYP2C19, or CYP2D6 (IC 50 >50 µm) and only slightly inhibits CYP3A (IC 50 = 19 µm), CYP2C9 (IC 50 = 26 µm), CYP2B6 (IC 50 = 51 M) and UGT1A1 (IC 50 = 19 3 µm) in comparison to positive control inhibitors. is a weak timedependent inhibitor of CYP3A (Ki = 42 M, Kinact = 0.14 min 1 ). 62

96 Test Article: [資料 : PK001] [資料 : PK023] ヒト CYP 3A 2B6及び1A2 の誘導 Types of Studies: Source of Materials: Methods: Assay: Experiments were carried out to evaluate the in vitro induction potential of vaniprevir on human cytochrome P450 3A4, 2B6, and 1A2. Cultures of primary human hepatocytes (n=3 donors) Hepatocytes were plated and cultured for 24 hours in InVitroGROTM CP plating medium. Twentyfour hours after plating, the hepatocytes were dosed for 48 hours with vehicle control, vaniprevir, and the positive control inducers Rifampicin and Phenobarbital prepared in InVitroGRO TM HI incubation medium. The dosing solutions were replaced every 24 hours. The freshly prepared hepatocytes were cultured and treated using the same procedure except for an additional 24 hour recovery period prior to dosing solutions being applied. At the end of the 48 hour incubation, changes in CYP3A4 gene expression were determined by measuring mr and testosterone 6βhydroxylation. Changes in CYP3A expression following treatment with vaniprevir were reported as fold DMSO control and as percent of the changes observed with the positive control Rifampicin. CYP2B6 and CYP1A2 induction studies were conducted in accordance with applicable BD Biosciences Discovery Labware SOPs and the study protocol. Hepatocytes from three donors were thawed, plated in collagen I coated 24well plates, and cultured for approximately 24 hours in culture medium prior to initiation of the study. Following the adaptation period, the hepatocytes were then treated for approximately 48 hours with vehicle control 0.1% (v/v) DMSO and media, test article, and the positive control inducers phenobarbital (1000 µm) or omeprazole (50 µm), prepared in incubation medium, with all solutions being replaced with fresh solutions every 24 hours. At the end of the 48 hour incubation period, whole cellbased CYP2B6 and CYP1A2 enzyme changes were evaluated using bupropion hydroxylation and phenacetin Odeethylation, respectively, measured by LCMS/MS detection. Total R was isolated for quantitative PCR analysis of CYP2B6 and CYP1A2 mr expression. LCMS/MS and quantitative PCR 63

97 Test Article: [資料 : PK001] [資料 : PK023] ヒト CYP 3A 2B6及び1A2 の誘導 続き Additional Information: showed a low potential for induction of CYP3A4 R expression in cultured human hepatocytes (at 20 µm vaniprevir the mean induction was 18% that of rifampicin at a concentration of 10 µm); however, no increase in CYP3Adependent testosterone 6βhydroxylase activity was observed. The lack of increase in CYP3A activity could result from the weak vaniprevir reversible/timedependent inhibition of CYP3A. As expected, the positive control rifampicin (10 µm) induced CYP3A4 R expression (3865 fold) and enzyme activity (516 fold). exhibited no induction of mr or enzyme activity for CYP2B6 or CYP1A2 when it was incubated with human hepatocytes at concentrations up to 20 µm. The positive control inducer phenobarbital (1000 µm) induced CYP2B6 mr 824 fold and enzyme activity 8 11 fold and the positive control inducer omeprazole (50 µm) induced CYP1A2 mr from fold and enzyme activity fold. 64

98 Test Article: [資料 : PK001] [資料 : PK025] Pgp OATP1B1 OATP1B3及び BCRP を介した輸送 Types of Studies: Source of Materials: Methods: To evaluate the uptake into primary hepatocytes and if vaniprevir is a substrate for Pglycoproteins, OATP1B1, OATP1B3, and BCRP mediated transporters Isolated cryopreserved human hepatocytes were obtained from InVitro Technologies (Baltimore, MD). Human MDR1, mouse Mdr1a, rat Mdr1a, dog MDR1transfected LLCPK1, control LLCPK1 cells, OATP1B1 and OATP1B3 stably transfected MDCKII cells (MDCKIIOATP1B1 and MDKCIIOATP1B3) and BCRP in membrane vesicles and stably transfected MDCKII cells (MDCKIIhBCRP). Hepatocytes were suspended at 2.5 x 106 cells/ml with Hanks balanced salt solution (HBSS) plus 10 mm HEPES and 0.1% BSA. To determine vaniprevir uptake at 37 C, cell suspensions were preincubated for 3 minutes at 37 C before adding dosing solution containing [ 3H] vaniprevir. Final concentration of vaniprevir was 0.1 μm. Uptake reaction was terminated by separating the cells from the medium with centrifugal filtration technique. Aliquots of were placed into 0.4mL microcentrifuge tubes containing 3M KOH and covered a silicone and mineral oil mixture (density at 1.015). The samples were then centrifuged for 2 minutes at 13,500 rpm. After the cells pelleted in 3M KOH dissolved, the tube was sliced off with a tube cutter, and dissolved cell pellets and supernatant were transferred into liquid scintillation vials separately. An aliquot of 2N HCl was added to the vials containing dissolved cell pellets, followed by the addition of liquid scintillation cocktail (Ready Safe, Beckman Coulter). Total radioactivity was counted on a liquid scintillation counter and cellular uptake (pmol/10 6 cells) was calculated based on the total radioactivity. Human MDR1, mouse Mdr1a, rat Mdr1a, dog MDR1transfected LLCPK1, and control LLCPK1 cells were plated at a density of 9.0 x 104 cells/0.15 ml/well on porous (0.4 m) matrigelcoated polycarbonate membrane filters (MultiScreen Caco2 96well filter plates, Millipore, Bedford, MA) in a feeder tray with 30 ml of medium. Cells were supplemented with fresh medium on the third day and used for the transport studies on the fifth day after plating. One hour before the start of the transport experiments, the medium was aspirated and the cell culture inserts were transferred to 96well MultiScreen Caco2 Transport Analysis Plates (Millipore), and the cells were washed with transport buffer (0.1% BSA containing HBSS with 10 mm HEPES) added to 65

99 Test Article: [資料 : PK001] [資料 : PK025] Pgp OATP1B1 OATP1B3及び BCRP を介した輸送 続き Methods (Cont.): both cell culture insert and reservoir sides. The transport experiment was then initiated by replacing the medium in each compartment with 0.15 ml of transport buffer with 1 M [3H] vaniprevir in the dosing compartment and transport buffer without the compound to the receiver compartment. To inhibit endogenous activity of breast cancer resistant protein (BCRP; ABCG2) in LLCPK1 cells, a specific inhibitor of BCRP, Ko143 (Leadgenex, Taejeon, Korea), at a final concentration of 1 M, was added to transport buffer and used for a parallel experiment. Transcellular transport of 5 M [ 3H] ritonavir and 1 M [ 3H] verapamil were evaluated in parallel as positive controls. After a 3 hour incubation period in a CO2 incubator, aliquots were taken from both receiver and donor compartments and transferred to vials for liquid scintillation counting. OATP2 (OATP1B1, SLCO1B1) and OATP8 (OATP1B3, SLCO1B3) cds were amplified from human liver cd libraries (Notebook/Page: 17328/151 A. Demartis, IRBM and R. Stocco, Merck Frosst, Montreal). OATP2 was cloned into the expression vector pcd3.1hygro (Invitrogen, Carlsbad, CA) and stably transfected into MDCKII cells (originally provided by Dr. P. Borst, The Netherlands Cancer Institute cells). OATP8 was cloned into the pcd5/frt vector (Invitrogen) and stably transfected into FlpIn MDCKII cells (MDCKII cells stably transfected with Flp Recombinase Target site). OATP2 and OATP8 mediated uptake was determined in MDCKII cells stably transfected with OATP2 or OATP8 cds. Prior to the experiment, cells were treated with 10 mm sodium butyrate to increase OATP expression. Cells were dislodged with trypsin EDTA and resuspended in HBSS plus 10 mm HEPES. Cells were then suspended in 96 deep well plates at a density of 0.6 x 10 6 cells/well. Uptake was initiated by the addition of the indicated concentrations of [3 H] vaniprevir or the positive control substrates [3H]E2 17βG (for OATP2 studies) or [3H]CCK8 (for OATP8 studies) in HBSS. Cells were then incubated for the indicated time at 37 C and uptake was stopped by the addition of 0.1% w/v BSA in ice cold phosphate buffered saline (PBS), followed by immediate centrifugation at 1800 x g at 4ºC, and washing of the cell pellets with of 0.1% w/v BSA in PBS three times. Cell pellets were resuspended in 50% acetonitrile, and scintillation fluid was added before measuring the radioactivity. 66

100 Test Article: [資料 : PK001] [資料 : PK025] Pgp OATP1B1 OATP1B3及び BCRP を介した輸送 続き Methods (Cont.): The time and ATPdependent uptake of vaniprevir into membrane vesicles isolated from baculovirus infected Spodoptera frugiperda (Sf9) cells containing human BCRP (ABCG2) (Invitrogen Life Technologies (Carlsbad, CA)) was measured. Methotrexate (100 µm, Moravek (Brea, CA)) was used as the positive control substrate, all reagents were commercially obtained. Briefly, 20 μl test compound or Methotrexate, dissolved in transport buffer (0.25 M sucrose, 10 mm TrisHCl buffer (ph 7.4), 10 mm MgCl2 ) was added to 10 µl (2 mg protein/ml) of BCRP or control vesicles and preincubated for 3 minutes at 37 C. Uptake was initiated by the addition of 20 µl ATP regenerating reagent or AMP reagent (final concentration of 5 mm ATP or AMP, 10 mm creatine phosphate and 100 µg/ml creatine phosphokinase in transport buffer), followed by incubation at 37 C for the indicated time. Uptake was stopped by the addition of 200 µl icecold stop buffer (0.25 M sucrose, 0.1 M NaCl, 10 mm TrisHCl buffer (ph 7.4)), followed by transfer of the reaction mixture to prewetted 96well glass fiber type B filter plate (1.0 µm) (Millipore, Billerica, MA), and application of vacuum. Filters containing the membrane vesicles were washed with 200 µl icecold stop buffer, six times. Compound in vesicles retained on the filter, were extracted by adding 3 times, 100μL of 80% acetonitrile to the filter, and collecting the filtrate into a 96deep well plate. Filtrate was diluted 1:1 with 80% acetonitrile containing 0.4uM labetalol as internal standard. The experiment was performed in triplicate. Samples were analyzed quantitatively by LC/MS/MS using an Applied Biosystems SCIEX API 5000 triple quadruple mass spectrometer (Concord, ON, Canada) with a TurboIonSpray ion source in the positive ion mode, coupled to a Thermo Scientific Transcend LX2 system (Franklin, MA.), with a flow rate of 800 μl/min to direct sample into the mass spectrometer. A Waters ACQUITY UPLC C18 (2x30 mm, 1.7 μm; Milford, MA.) column was used for sample analysis. Mobile phase A consisted of 0.1% formic acid in water. Mobile phase B was acetonitrile with 0.1% formic acid. A gradient elution program was utilized where the solvent composition was held at 5% B for 0.3 min, changed from 5% B to 95% B at 0.5 min, and followed by holding at 95% B for 0.3 min, then, went back to 5% B at 1.1 min. The column was then reequilibrated at the original solvent composition. Mass spectrometric detection was 67

101 Test Article: [資料 : PK001] [資料 : PK025] Pgp OATP1B1 OATP1B3及び BCRP を介した輸送 続き Methods (Cont.): accomplished by multiple reaction monitoring (MRM) of transitions unique to each compound. The transitions of the protonated precursor ions to the selected product ions of MK7009 and internal standardlabetalol were m/z and , respectively. Methotrexate (100 μm) was used as the positive control. The transition of the protonated precursor ion to the selected product ion of methotrexate was m/z The bidirectional transport of vaniprevir was evaluated in MDCKII cells and MDCKII cells stably expressing human BCRP (MDCKIIhBCRP) provided under license agreement by Solvo Biotechnology (Budapest, Hungary). MDCKII derived cell lines can form a tight monolayer and therefore can be used to assess vectorial transport of compounds from basolateral to apical (B A) and from apical to basolateral (A B). MDCKII and MDCKIIhBCRP cell lines were seeded onto 96well transwell culture plates (Millipore, Billerica, MA) at a density of x 10 6 cells/well and cultured at 37 C /5% CO 2 for two days. 24 hours prior to the experiment, cells were treated with 10 mm sodium butyrate (SigmaAldrich, St. Louis, MO) to increase hbcrp expression. Before the experiment, cells were washed with Hank s Balanced Salt Solution (HBSS) with 10 mm HEPES, ph 7.4, three times. (final concentration 1 μm) containing either 0, 2, or 5 μm of Ko143 (( ) Medicinal Chemistry Department, MRL, Rahway, NJ) were prepared in HBSS with 10 mm HEPES. Ko143 is a specific inhibitor of BCRP. Substrate solution (150 μl) was added to either the apical (A) or the basolateral (B) compartment of the culture plate, and buffer (150 μl) containing 0, 2, or 5 μm of Ko143 was added to the compartment opposite to that containing the test compound. At t = 3 hr, 50 μl samples were removed from both sides of monolayers dosed with test compound and placed in 96 well plates, internal standard (50 μl 1 μm labetolol) was added to the samples. Samples were analyzed quantitatively as stated above for the BCRP membrane vesicle experiments with the exception that prazosin (5 μm) was used as the positive control. The transition of the protonated precursor ion to the selected product ion of prazosin was m/z The experiment was performed in triplicate. 68

102 Test Article: [資料 : PK001] Pgp OATP1B1 OATP1B3及び BCRP を介した輸送 続き Assay: Liquid scintillation counter and LC/MS/MS Additional Information: The uptake kinetics showed a saturable (Km = 1 M) transportermediated component and a passive nonsaturable component for vaniprevir uptake into human hepatocytes. Additional in vitro transporter experiments indicated that vaniprevir is a substrate for human liver uptake transporters OATP1B1 and OATP1B3, and the human liver efflux transporters Pglycoprotein (Pgp) but not human breast cancer resistant protein (BCRP). 69

103 Pgp OATP1B1 OATP1B3 BSEP BCRP MRP2 MRP3及び MRP4を介し た輸送への影響 Types of Studies: Source of Materials: Methods: Test Article: [資料 : PK011] [資料 : PK016] [資料 : PK019] [資料 : PK024] Experiments were carried out to evaluate vaniprevir as a potential inhibitor of MDR1 Pglycoprotein, OATP1B1, OATP1B3, BSEP, BCRP, MRP2, MRP3, and MRP4mediated transport activity. OATP1B1 and OATP1B3 stably transfected MDCKII cells and membrane vesicles isolated from baculovirus infected Spodoptera frugiperda (Sf9) cells containing human BSEP, BCRP, MRP2, MRP3 or MRP4 and LLCMDR1 and LLCPK1 cell lines. Inhibitory effect of vaniprevir on OATP1B1mediated [3H] pitavastatin (0.1 µm) uptake and OATP1B3mediated [3H] Sulfobromophthalein sodium salt (BSP) (0.1 µm) was conducted in OATP1B1 and OATP1B3 stably transfected MDCKII cells (MDCKIIOATP1B1 and MDKCIIOATP1B3), respectively. Briefly, 24 hours prior to the experiment, cells were treated with 10 mm sodium butyrate (SigmaAldrich, St Louis, MO) to increase OATP1B1 and OATP1B3 expression. Cells were dislodged with trypsin EDTA and resuspended in Hanks' Balanced Salt Solution (HBSS) with 10 mm Hepes, ph 7.4. Cells were then suspended in 96deep well plates at a density of 0.4 x 10 6 cells/well. Uptake was initiated by the addition of 0.1 µm [ 3H] pitavastatin or 0.1 µm BSP containing various concentrations of vaniprevir dissolved in HBSS buffer. Cells were then incubated for 10 minutes at 37 C and uptake was stopped by the addition of ice cold phosphate buffered saline (PBS), immediate centrifugation at 3000 rpm at 4 C, followed by washing of the cell pellets with PBS for three times. Cell pellets were resuspended in 50% acetonitrile and scintillation fluid was added. The experiment was performed in triplicate. The inhibitory effect of the test compound on ATPdependent [ 3H] Taurocholic acid (TCA) (1 μm, Perkin Elmer (Boston, MA)) uptake was conducted in membrane vesicles isolated from baculovirus infected Sf9 cells containing human BSEP (ABCB11) (Invitrogen Life Technologies, Carlsbad, CA). All reagents were commercially obtained. Briefly, 20 µl of [3H] TCA, with or without various concentrations of test compound or 100 μm atorvastatin (prototypic BSEP inhibitor), dissolved in transport buffer (0.25 M sucrose, 10 mm TrisHCl buffer (ph 7.4), 10 mm MgCl2) was added to 10 μl (2 mg protein/ml) of BSEP vesicles and preincubated for 5 minutes at 37 C. Uptake was initiated by the addition of 20 μl ATP regenerating reagent or AMP reagent (final concentration of 5 mm ATP or AMP, 10 mm creatine phosphate and 100 μg/ml creatine phosphokinase in transport buffer, ph 7.4), followed by incubation at 37 C for the indicated time. Uptake was 70

104 Pgp OATP1B1 OATP1B3 BSEP BCRP MRP2 MRP3及び MRP4を介し た輸送への影響 続き Methods (Cont.): Test Article: [資料 : PK011] [資料 : PK016] [資料 : PK019] [資料 : PK024] stopped by the addition of 200 μl icecold stop buffer (0.25 M sucrose, 0.1 M NaCl, 10 mm TrisHCl buffer (ph 7.4)), followed by transfer of the reaction mixture to prewetted 96well glass fiber type B filter plate (1.0 μm) (Millipore, Billerica, MA), and application of vacuum. Filters containing the membrane vesicles were washed with 200 μl icecold stop buffer, six times. The filter plate was dried and 50 μl scintillation fluid (Optiphase Supermix, Perkin Elmer, Boston, MA) was added to each sample. Radioactivity was determined by liquid scintillation counting in a MicroBeta Wallac Trilux Scintillation Counter (Perkin Elmer, Boston, MA). The experiment was performed in triplicate. ATPdependent (BSEP mediated) [ 3H] TCA uptake was calculated by subtracting the uptake in the absence of ATP from that in the presence and data were then normalized to % control, where uptake in the absence of test compound was 100%. The inhibitory effect of vaniprevir on ATPdependent [ 3H]Methotrexate (MTX) (10μM, MT701, Movarek, CA) uptake was conducted in membrane vesicles isolated from baculovirus infected Sf9 cells containing human BCRP (Invitrogen Life Technologies, Carlsbad, CA). All reagents were commercially obtained. Briefly, 20 μl of [3H]MTX, with or without various concentrations of vaniprevir or 2 μm Ko143 (prototypic BCRP inhibitor), dissolved in transport buffer was added to 10 μl (2 mg protein/ml) of BCRP vesicles and preincubated for 5 minutes at 37 C. Uptake was initiated by the addition of 20 μl ATP regenerating reagent or AMP reagent, followed by incubation at 37 C for the indicated time. Uptake was stopped by the addition of 200 μl icecold stop buffer followed by transfer of the reaction mixture to prewetted 96well glass fiber type B filter plate and application of vacuum. Filters containing the membrane vesicles were washed with 200 μl icecold stop buffer, six times. The filter plate was dried and 50 μl scintillation fluid was added to each sample. The inhibitory effect of vaniprevir on ATPdependent [ 14C]Ethacrynic acidglutathione conjugate (EASG) (1.0 μm, EASG) uptake was conducted in membrane vesicles isolated from baculovirus infected Sf9 cells containing human MRP2 (ABCC2) (Invitrogen LifeTechnologies, Carlsbad, CA). All reagents were commercially obtained. Briefly, 20 μl of [14C]EASG, with or without various concentrations of vaniprevir or 100 μm BSP (prototypic MRP2 inhibitor), dissolved in transport buffer (0.25 M sucrose, 10 mm TrisHCl buffer (ph 7.4), 10 mm MgCl2 ) was added to 10 μl (2 mg protein/ml) of MRP2 vesicles 71

105 Pgp OATP1B1 OATP1B3 BSEP BCRP MRP2 MRP3及び MRP4を介し た輸送への影響 続き Methods (Cont.): Test Article: [資料 : PK011] [資料 : PK016] [資料 : PK019] [資料 : PK024] and preincubated for 5 minutes at 37 C. Uptake was initiated by the addition of 20 μl ATP regenerating reagent or AMP reagent (final concentration of 5 mm ATP or AMP, 10 mm creatine phosphate and 100 μg/ml creatine phosphokinase in transport buffer), followed by incubation at 37 C for the indicated time. Uptake was stopped by the addition of 200 μl icecold stop buffer (0.25 M sucrose, 0.1 M NaCl, 10 mm TrisHCl buffer (ph 7.4)), followed by transfer of the reaction mixture to prewetted 96well glass fiber type B filter plate (1.0 μm) and application of vacuum. Filters containing the membrane vesicles were washed with 200 μl icecold stop buffer, six times. The filter plate was dried and 50 μl scintillation fluid was added to each sample. Radioactivity was determined by liquid scintillation counting in a MicroBeta Wallac Trilux Scintillation Counter. The experiment was performed in triplicate. ATP dependent [ 14C]EASG uptake in hmrp2 containing vesicles was calculated by subtracting the uptake in the absence of ATP from that in the presence and data were then normalized to % control, where uptake in the absence of vaniprevir was 100%. The inhibitory effect of the test compound on ATPdependent [ 3H] Estradiol17βD Glucuronide (EG) (1 μm, Perkin Elmer (Boston, MA)) uptake was conducted in membrane vesicles isolated from baculovirus infected Sf9 cells containing human MRP3 (ABCC3) (Invitrogen Life Technologies, Carlsbad, CA). All reagents were commercially obtained. Briefly, 20 µl of [3H] EG, with or without various concentrations of test compound or 100 μm BSP (prototypic MRP3 inhibitor), dissolved in transport buffer (0.25 M sucrose, 10 mm TrisHCl buffer (ph 7.4), 10 mm MgCl2) was added to 10 μl (2 mg protein/ml) of MRP3 vesicles and preincubated for 5 minutes at 37 C. Uptake was initiated by the addition of 20 μl ATP regenerating reagent or AMP reagent (final concentration of 5 mm ATP or AMP, 10 mm creatine phosphate and 100 μg/ml creatine phosphokinase in transport buffer, ph 7.4), followed by incubation at 37 C for the indicated time. Uptake was stopped by the addition of 200 μl icecold stop buffer (0.25 M sucrose, 0.1 M NaCl, 10 mm TrisHCl buffer (ph 7.4)), followed by transfer of the reaction mixture to prewetted 96well glass fiber type B filter plate (1.0 μm) (Millipore, Billerica, MA), and application of vacuum. Filters containing the membrane vesicles were washed with 200 μl icecold stop buffer, six times. The filter plate was dried and 50 μl scintillation fluid (Optiphase Supermix, Perkin Elmer, Boston, MA) was added to each sample. 72

106 Pgp OATP1B1 OATP1B3 BSEP BCRP MRP2 MRP3及び MRP4を介し た輸送への影響 続き Methods (Cont.): Test Article: [資料 : PK011] [資料 : PK016] [資料 : PK019] [資料 : PK024] Radioactivity was determined by liquid scintillation counting in a MicroBeta Wallac Trilux Scintillation Counter (Perkin Elmer, Boston, MA). The experiment was performed in triplicate. ATPdependent (MRP3mediated) [3H] EG uptake was calculated by subtracting the uptake in the absence of ATP from that in the presence and data were then normalized to % control, where uptake in the absence of test compound was 100%. The inhibitory effect of the test compound on ATPdependent [ 3H] Folic acid (FA) (10 μm, Moravek (Brea, CA)) uptake was conducted in membrane vesicles isolated from baculovirus infected Sf9 cells containing human MRP4 (ABCC4) (Invitrogen Life Technologies, Carlsbad, CA). All reagents were commercially obtained. Briefly, 20μL of [3H] FA, with or without various concentrations of test compound or 100 μm BSP (prototypic MRP4 inhibitor), dissolved in transport buffer (0.25 M sucrose, 10 mm TrisHCl buffer (ph 7.4), 10 mm MgCl2 ) was added to 10 μl (2 mg protein/ml) of MRP4 vesicles and preincubated for 5 minutes at 37 C. Uptake was initiated by the addition of 20 μl ATP regenerating reagent or AMP reagent (final concentration of 5 mm ATP or AMP, 10 mm creatine phosphate and 100 μg/ml creatine phosphokinase in transport buffer), followed by incubation at 37 C for the indicated time. Uptake was stopped by the addition of 200 μl icecold stop buffer (0.25 M sucrose, 0.1 M NaCl, 10 mm TrisHCl buffer (ph 7.4)), followed by transfer of the reaction mixture to prewetted 96well glass fiber type B filter plate (1.0 μm) (Millipore, Billerica, MA), and application of vacuum. Filters containing the membrane vesicles were washed with 200 μl icecold stop buffer, six times. The filter plate was dried and 50 μl scintillation fluid (Optiphase Supermix, Perkin Elmer, Boston, MA) was added to each sample. Radioactivity was determined by liquid scintillation counting in a MicroBeta Wallac Trilux Scintillation Counter (Perkin Elmer, Boston, MA). The experiment was performed in triplicate. ATPdependent (MRP4mediated) [3H] FA uptake was calculated by subtracting the uptake in the absence of ATP from that in the presence and data were then normalized to % control, where uptake in the absence of test compound was 100%. 73

107 Pgp OATP1B1 OATP1B3 BSEP BCRP MRP2 MRP3及び MRP4を介し た輸送への影響 続き Methods (Cont.): Test Article: [資料 : PK011] [資料 : PK016] [資料 : PK019] [資料 : PK024] The LLCMDR1 and LLCPK1 cell lines were cultured in 24well transwell culture plates (Millipore, Billerica, MA). [3H]Digoxin (0.1 μm) and the inhibitors were prepared in transport buffer (Hanks buffer with 10 mm HEPES, ph 7.4). vaniprevir concentrations tested were 0, 5, 10, 25, 100, and 500 μm. Prior to the transport study, cells were washed three times with transport buffer and preincubated with varying concentrations of inhibitor at 37 C for 20 min on both apical (A) and basolateral (B) compartments. Substrate solution (500 μl) was added to either the A or B compartment of the culture plate, and buffer (500 μl) was added to the compartment opposite to that containing the compound. The inhibitor at various concentrations was added to both compartments. At t=3 hr, 50 μl of sample was taken out from both sides and scintillant (200 μl, Scintisafe Econo II, Fisher Chemicals, NJ) was added). The experiment was performed in triplicate. Assay: Liquid scintillation counter Additional Information: inhibited Pgpmediated transport of digoxin in MDR1 transfected LLCPK1 cells with an IC 50 = 38 ± 17 μm. also inhibited OATP1B1mediated pitavastatin uptake into MDCKIIOATP1B1 cells with an IC 50 value of 0.30 ± 0.01 μm and inhibited sulfobromophtalein uptake into MDCKIIOATP1B3 cells with an IC 50 value of 0.30 ± 0.02 μm. inhibited BSEPmediated transport of taurocholic acid with an IC 50 = 1.3 ± 0.2 μm, BCRPmediated methotrexate transport with an IC 50 of 13.0 ± 2.5 μm, MRP2mediated ethacrynic acid gluthathione conjugate transport with an IC 50 of 42.2 ± 5.8 μm, MRP3mediated transport of estradiol17βdglucuronide with an IC50 of 9.9 ± 1.8 μm and MRP4mediated transport of folic acid with an IC 50 of 8.0 ± 1.0 μm. 74