JP3586194B2 - Infrared thermography - Google Patents

Description

実施例８
細胞表面レセプターに対するペプチドの効果；酵素反応の測定
血管内皮細胞増殖因子（ＶＥＧＦ）は、血管内皮細胞のキナーゼドメインレセプター（ＫＤＲ）への結合と活性化を通し血管の発生の多くをコントロールする２量体ホルモンである。ＶＥＧＦへの反応では、細胞はマイトージェン活性化タンパク質キナーゼ（例えばＭＡＰＫＫＫ、Ｓ６キナーゼ（ｐ９０ｒｓｋ））のようなシグナル伝達分子のカスケードを迅速に活性化する。ＶＥＧＦはまた心筋細胞では転写因子２のリン酸化と活性化も引き起こす。（Ｆｅｒｒａｎａ， Ｎ．， Ｄａｖｉｓ−Ｓｍｙｔｈ Ｔ， Ｅｎｄｒｏｃｒ Ｒｅｖ １９９７ Ｆｅｂｌ１８（１）：４−２５）。現在利用可能なこの経路を解析する方法ではＶＥＧＦに対する急性反応をキャプチャーできない。ＶＥＧＦの活性化は急性キナーゼ／フォスファターゼ活性を有し、その結果最終的には結合の加水分解と形成を伴うことから、使用する装置の感度が十分であればこれら酵素反応より生じた熱は測定可能であるかもしれない。この仮説を検証するため、赤外サーモグラフィーを用い、ＶＥＧＦで処理されたヒト血管上皮細胞（ＨＵＶＥＣ）により生じた熱を測定した。図１０Ｆに示すように、ＶＥＧＦはＨＵＶＥＣ細胞に産熱を生じせしめたことから、赤外サーモグラフィーは培養細胞忠の酵素反応（例えばキナーゼ／フォスファターゼ活性）のモニターに利用できるだろう。即ち、赤外サーモグラフィーは、酵素反応への化合物の効果及び有効性の評価に利用できる。
実施例９
化学反応（例えばリガンド／結合−パートナー相互作用）のモニタリング
結合の形成と加水分解は、その想定リガンドへのレセプター結合固有のプロセスである。このプロセスは通常は発熱反応を伴い、そして現時点ではマイクロカロリーメトリーを利用してのみ測定される（Ｋｏｅｎｉｇｂａｕｅｒ， Ｐｈａｒｍ． Ｒｅｓ． Ｊｕｎ；１１（６）７７７−７８３（１９９４））。これら測定はレセプター／薬物相互作用に関する等温結合を確立するためのベースである。酸及び塩基の混合は結合プロセスから生じる発熱反応を惹起するため、酸／塩基滴定を利用し赤外イメージングが分子内／分子間の結合動態の測定に利用できるか決定した。図１１に示すように０．２５ｍＭのＮａＯＨを各種濃度のＨＣｌと混合し赤外サーモグラフィーにより測定したところ、産熱反応を用量依存的に阻害した。これらデータはリガンド（例えば薬物）と結合パートナー（例えばレセプター）との相互作用のような分子事象の測定に於ける赤外サーモグラフィーの有用性を実証している。別の実施例は、赤外サーモグラフィーを用いた薬物−レセプター、タンパク質−タンパク質、タンパク質−ＤＮＡ、ＤＮＡ−ＤＮＡ、ＤＮＡ−ＲＮＡ及びタンパク質−炭水化物相互作用のモニターを含む。
【００５４】
実施例１０
不活性固体表面上での触媒のモニタリング（例えばコンビケム（Ｃｏｍｂｉ−Ｃｈｅｍ）ビーズ）
本発明に示すような赤外サーモグラフィーは高度に規定された無細胞系での産熱の測定にも利用できる。触媒剤はコンビケムビーズ（Ｂｏｒｍａｎ， Ｃｈｅｍ． Ｅｎｇ． Ｎｅｗｓ ７４：３７（１９９６））のような不活性固相表面への結合により固定され特徴付けられることが多い。そのような事情より、本発明を利用しコンビケムビーズ上での触媒反応の熱分析を行った。触媒的に活性なコンビケムビーズは、２５ｍＬのビーカー又は９６ウエルのマイクロタイタープレート内の溶媒に浸しながら分析された。図１２は産熱がビーズを含むビーカー領域に局在して出力される（図１２Ａ、矢印）（温度差０．３℃）又は活性なビーズを含むマイクロタイタープレートのウエルに局在するが不活性なビーズを含むウエルには局在しないこと（図１２Ｂ）を示している。即ち、赤外サーモグラフィーは非侵襲的及び非破壊的方法による迅速触媒活性の測定に利用できる。
【００５５】
実施例１１
エアゾール誘導冷却のサーモグラフィー分析
薬物供給に利用される吸入剤投薬量測定のためのエアゾールシステムでは、供与忠に装置チャンバー内の温度が低下が起こる。この影響は薬物供給の効率性に関し大きな障害である。チャンバー温度の低下はしばしばＭＤＩチャンバー及びステム内に於ける薬物結晶化を引き起こし、その結果薬物供給は非効率的になる。赤外熱イメージングは薬物供給忠の温度消失のモニター、ならびにこの問題を軽減又は改善した改良型装置の試験に利用できる。即ち、赤外サーモグラフィーをＭＤＩの作動誘導チャンバー冷却の測定法として試験した。図１３Ａは、０、１又は５回連続作動した場合の、ＭＤＩの迅速熱プロフィールを示す。熱イメージは以下の３領域に於ける温度変動について解析された；領域１−バルブステム／エクスパンジョンチャンバーの表面；領域２−キャニスターの中央部表面；領域３−測定チャンバーがあるキャニスターヘッド内部。領域温度の経時的変化を図示した図１３Ｂは、動作依存的に温度の低下が特にバルブステム／エクスパンジョンチャンバーで起こっていることを示している。動作毎に温度は繰り返し低下する。
【００５６】
更に、動物及びヒトに於ける吸入剤の生体利用度と生物活性を測定すること、及び定量化することは困難なことが多い。吸入剤の生体利用度を測定することができるアッセイには、選択された組織内（例えば肺）への放射性標識吸入剤の取り込みを測定するものが含まれる。赤外サーモグラフィーは吸入化合物の生体利用度／生物活性を測定するための非侵襲的方法を提供する。即ち、赤外サーモグラフィーを、ヌードマウスの胸部域内の吸入剤により誘導された熱活性の測定に利用した。図１３Ｃは、附形剤またはアルブテロールを含む吸入剤で処理されたヌードマウスの熱プロフィールを示す。胴部温度の図示例からは、吸入剤を投与してから２．５分後には胴部温度が上昇することがわかる（図１３Ｄ）。まとめると、これらの結果は赤外サーモグラフィーが装置内の吸入剤供給、及び動物モデルに於ける吸入剤の生体利用度／生物活性の非侵襲的測定に利用できることを示している。
【００５７】
実施例１２
赤外サーモグラフィーはβ_３−ＡＲ−アゴニストで処理されたマウスに於けるＩＢＡＴ産熱の測定に利用できる。
【００５８】
培養脂肪細胞及びＣＨＯ細胞に於ける産熱に対するβ_３ＡＲ−アゴニストの刺激能については、上記実施例３及び６、及び図５、８及び９の中に示し、考察された。糖尿病及び肥満の治療には異化作用物質（例えばβ_３ＡＲ−アゴニスト）の利用に適した潜在的臨床応用があることから、赤外サーモグラフィーが動物体に於いてβ_３ＡＲ−アゴニスト誘導効果を測定できるか示すことは重要である。図１４Ａ及び１４Ｂは、赤外サーモグラフィーがβ_３ＡＲ−アゴニストを投与された動物に於ける、肩胛骨間褐色脂肪組織域（ＩＢＡＴ）の用量依存的及び時間依存的な産熱増加の測定に利用できることを示している。処理された動物に於ける血清グリセロールを直接測定することで、β_３ＡＲアゴニスト誘導産熱は異化作用性の活性の増加を反映するという仮説を検証した。処理マウスでは、β_３ＡＲアゴニスト誘導産熱は血清グリセロールの増加と相関していた（相関係数＝０．９２）。即ち、これは動物に於ける異化作用薬物のｉｎ ｖｉｖｏ効果をモニターする赤外サーモグラフィーの利用を実証している。
【００５９】
実施例１３
各種時間及び薬物濃度のモノアミン再取り込み阻害剤で処理されたｏｂ／ｏｂマウスのサーモグラフィー分析
モノアミン再取り込み阻害剤は異化作用活性を刺激する薬物群である（Ｓｔｏｃｋ， Ｉｎｔ． Ｊ． Ｏｂｅｓｉｔｙ ２１：５２５−２９（１９９７））。代表的モノアミン再取り込み阻害剤であるＧＷ４７３５５９Ａの効果を、処理したマウスに赤外サーモグラフィーを使いモニターした。図１５は、赤外サーモグラフィーがＧＷ４７３５５９Ａで処理されたｏｂ／ｏｂマウスに於けるＩＢＡＴ産熱の用量依存的（図１５Ａ）及び時間依存的（図１５Ｂ）増加を測定することを示している。即ち、赤外サーモグラフィーは、化合物の生体利用度、及び活性に関する非侵襲性の高感度かつ強力な代替アッセイを提供する。
【００６０】
実施例１４
体重変化を予測する代替アッセイとしてのサーモグラフィー分析
糖尿病または肥満治療のためのβ_３ＡＲ−アゴニスト処理は、長期間（数週間から数ヶ月）にわたる治療を必要とする。β_３ＡＲ−アゴニスト治療に所望される結果は体重の減少である。図１６に示すデータは、赤外サーモグラフィーを利用することで、薬物治療の結果としての体重減少を予測できることを示している。高脂肪色を与えられたＡＫＲマウスに２週間にわたりプラセボまたはβ_３ＡＲアゴニスト（１日２回）を投与した。試験期間中、産熱活性は赤外サーモグラフィーにより肩胛骨間域を測定し、同時に体重の減少を測定した。２週間後の体重減少は、肩胛骨間域の産熱と相関していた（ｒ＝０．９７）。即ち、赤外サーモグラフィーは、化合物選択、及び効果及び有効性の評価に関して、臨床前及び臨床応用の両方に適した非侵襲的代替アッセイを提供できる。
【００６１】
糖尿病および肥満患者での体重減少を目的とした薬物治療は長期の治療になることが多い。赤外サーモグラフィーは、薬物が投与された場合には、薬物により誘導された代謝変化の変化を検出する。動物をモノアミン再取り込み阻害剤で処理した後の赤外サーモグラフィーにより記録されたＩＢＡＴ産熱の変化は、急性（治療＝２時間；ｒ＝０．９２）及び慢性（治療＝１４日、投薬後２時間；ｒ＝０．９４）治療プロトコールの両方について、体重増加の％減少に関し有意に相関していた。赤外サーモグラフィーは、体重減少を推測するための非侵襲代替アッセイを提供し、それにより労力のかかる、長期かつコストのかかる体重減少試験を排除する。
【００６２】
実施例１５
食事誘導産熱に及ぼす遺伝的影響
代謝は遺伝的及び環境的因子の影響を受けることが多くの証拠より示されている。糖尿病、脂肪代謝異常または肥満の遺伝的素因を持つ個人について、代謝速度を変化させ環境因子の一つは食事である。サーモグラフィー使い、様々な遺伝子型の動物に於ける薬物誘導熱産生の変化に対する食事の効果を測定することができるかは不明である。そのために、３種類の近郊系マウスＡＫＲ／Ｊ、Ｃ５７ＢＬ／６Ｊ及びＳＷＲ／Ｊに、β_３−アドレノレセプターアゴニストＢＲＬ３７３４４で処理する前１４週間、高脂肪食及び低脂肪食を与え続けた。処置前と処置６０分後に赤外サーモグラフィーを使って、肩胛骨間域で生じた熱を測定した。図１７に示すように、肥満マウスＡＫＲ／Ｊは高脂肪食を取った場合にＢＲＬ３７３４４に対してより大きな産熱反応を示した。これに対し、肥満耐性マウスであるＳＷＲ／Ｊは、低脂肪食を与えた時により大きな産熱反応を示した。Ｃ５７ＢＬ／６Ｊでは高または低脂肪食間に産熱反応の違いは殆どなかった。これらの結果は、熱発生に於ける薬物誘導変化に対する遺伝子型と環境変化（例えば食事）の効果の測定に赤外サーモグラフィーが利用できることを実証した。即ち、本技術は特定の環境または遺伝的背景について、所望の特性を持つ化合物の選択、及び効果と有効性の等級付けに利用できる。同ように、本技術は特定薬物候補に関し、所望特性を持つ動物及び環境因子の同定、等級付け、及び選択に利用できる。
【００６３】
実施例１６
ヒトに於ける食物誘導産熱の赤外サーモグラフィー
カロリー摂取はヒトの代謝活性に劇的かつ急速な影響を与える。従って、ヒト試験者背面の産熱出力は、食物摂取の日時と試験者のパターンの関数として変動するだろう。このことは、食事前後に赤外サーモグラフィーを使って背温度をモニターすることで患者プロフィール中に提示される。図１８Ａは、昼食前後の時点に関するサーモグラフィープロフィールの定量分析を示す。図１８Ｂは、３回別々にの昼食前後に２名の男性被験者及び１例の女性被験者について行われた同様の測定（裸身洞部デルタＴ）をまとめた図を示す。このデータセットは一致しており、かつ再現性を有しており、ヒト赤外サーモグラフィーが産熱のモニタリングに利用できることを示している。この方法は、ヒト患者に適用可能な多くの状況、例えば食事調整、薬物治療、薬物／薬物及び薬物／環境相互作用のモニターに有用であろう。赤外サーモグラフィーを使った結果に基づき、食物及び環境の変化ならびに薬物使用を規定することができる。
【００６４】
実施例１７
ヒトに於ける薬物誘導産熱の赤外サーモグラフィー
交感神経作用薬であるエピネフィリンは、齧歯類で強力な発熱及び抗肥満特性を有すると報告されている（Ａｓｔｒｕｐ ｅｔ ａｌ．， Ａｍ． Ｊ． Ｃｌｉｎ． Ｎｕｔｒ． ４２：１８３−９４（１９８５））。体重減少及び体組成の測定は、肥満に関する薬物治療の効果の決定に最初に利用されるマーカーである。しかし、これらのマーカーを利用する研究は、時間がかかり、大規模でコスト高の傾向がある。これら問題を回避するために、代替マーカーが開発されてきた。間接カロリメトリーは、静止状態の代謝速度の決定に利用されるが、その複雑さより広くは利用されていない。グルコース、グリセロール、非エステル化脂肪酸、トリグリセリドのような生化学マーカーが利用されているが、侵襲性である。しかし産熱イメージングはヒトに於けるエンドＦｅリンの特性測定には利用されていない。
【００６５】
２例のヒト被験者での産熱に対するエンドフェリンの効果を、０．６−０．７ｍｇ／ｋｇの投与量のデフェドリン処理６０分後に赤外イメージングし、検出した。図１９は被験者Ａ及びＢでのエフェドリンにより誘導された産熱反応を決定する熱イメージである（それぞれデルタＴ℃＝０．６３及び０．４６）。従って、赤外サーモグラフィーは臨床試験に於ける薬物の効果、有効性、薬物動態、薬力学を評価する非侵襲的代替アッセイとして利用することができる。
【００６６】
実施例１８
ｄｂ／ｄｂマウスに於ける薬物−薬物相互作用の赤外サーモグラフィー
薬力学的な薬物−薬物相互作用の結果は様々であり、生命に大きな脅威を与える状態から救命治療の範囲に及ぶだろう。このようなことから、薬物相互作用の迅速評価に適した方法を特定する必要性は大きい。複数の薬物投与は代謝速度を変えることから、培養細胞及び／又は動物に於ける熱産生に対しては、各種薬物の相加的、減法的及び／または相乗的効果が存在することが予想される。従って、赤外サーモグラフィーを利用しｉｎ ｖｉｖｏの産熱に及ぼす各種薬剤を混合することの影響を分析できるか決定することは興味深い。更に赤外サーモグラフィーを利用することで、前臨床及び臨床試験において併用治療の有効性、有用性及び毒性を特定し、等級付けすることができる。
【００６７】
ＧＷ１９２９はリガンド活性化核レセプターＰＰＡＲγの転写活性を活性化することで糖尿病動物に於ける血糖コントロールを改善する作用物質である。ＣＧＰ１２１７７Ａは、細胞表面β_３−アドレナリンレセプターの刺激を介し作用する、肥満治療薬である（Ｋｅｎａｌｉｎ， Ｌｅｎｈａｒｄ ａｎｄ Ｐａｕｌｉｋ， ＣＵｒｒ Ｐｒｏｔ Ｐｈａｒｍ；１（ｕｎｉｔ４．６）：１−３６（１９９８））。多くの糖尿病患者は肥満でることから、これら２種類の作用物質（即ちＧＷ１９２９及びＣＧＰ１２１７７Ａ）に何れかの薬力学的相互作用があるか決めることは興味深い。即ち、ｄｂ／ｄｂマウスを２週間にわたりＧＷ１９２９にて、あるいはＧＷ１９２９無しに治療され、肩甲骨間域で生じた熱を赤外サーモグラフィーを利用し測定した。図２０に示すように、ＧＷ１９２９及びＣＧＰ１２１７７Ａの両方で治療された動物は、いずれか単独で治療された動物に比べより産熱反応が大きかった。即ち、これら結果は、熱産生を変化させる薬力学的薬物−薬物相互作用の強さの測定への赤外サーモグラフィーの利用を実証した。
【００６８】
実施例１９
ＶＥＧＦによる組織血管形成のサーモグラフィー分析
腫瘍は、腫瘍増殖力を増強する局所の血管形成により支持された活発に増殖し、代謝する組織領域である。サーモグラフィーは、組織血管形成に伴うものを含む腫瘍に随伴する産熱上昇を検出できる。血管上皮増殖因子（ＶＥＧＦ）は腫瘍組織中に発見され、その存在はそれが局在する組織内での産熱増加に関連する。即ち、上皮血管構造が露出しているヌードマウスを利用し、腫瘍検出及び血管形成に於けるサーモグラフィーの有用性を示した。ヌードマウスにＶＥＧＦペプチド又はコントロールを注射し、続いて両注射部位のサーモグラフィーイメージングを行った。図２１は、サーモグラフィーイメージがＶＥＧＦ注射局所部位で産熱が増加するが、コントロール注射局所部位では増加しないことを示していることを表す。即ち、赤外サーモグラフィーを利用し、組織血管形成を変化させる作用物質の効果をモニターできる。
【００６９】
実施例２０
赤外サーモグラフィーによる腫瘍温度モニタリング
腫瘍温度は、腫瘍に於ける代謝速度の指標である。腫瘍は最大代謝活性に関してはＶＥＧＦの存在に依存しており、また腫瘍温度はＶＥＧＦの有用性の変化を反映する。この関連性は、抗ＶＥＧＦ抗体又は非特異的抗ＩｇＧ抗体のいずれかで処理された腫瘍保有マウスをサーモグラフィー分析することで示される。図２２は、ＶＥＧＦが抗ＶＥＧＦ抗体の存在により中和されると腫瘍温度が低下することを示す定量的サーモグラフィー分析を表す。即ち、赤外イメージングは、癌治療の効果のモニター、及び抗ガン剤開発に於ける補助として利用できる。
【００７０】
実施例２１
赤外サーモグラフィーによる禿げ（脱毛）のモニタリング
脱毛は各種治療（例えば癌患者放射線治療、手術等）の望ましくない副作用の結果生じ、また加齢とともに自然に起こるが、手術又は薬学的介入により髪の増殖を快復することができる。髪は熱消失に対する断熱作用を提供し、一定体温の維持に役立つことから、赤外サーモグラフィーが脱毛測定に利用できるか決定することは興味深い。化学療法により誘導される禿げの治療及び予防には進歩が認められない理由の一つは、再現性のある動物モデルならびに脱毛を定量的に測定する方法がないことに拠る。赤外イメージングが化学療法により誘導される脱毛を定量的に測定できるか決定するために、子宮内において既知発癌剤、エトポシドで処理された新生児ラットを脱毛に関し熱プロファイル分析した。図２３は、脱毛の結果として全面部及び背部両方で熱活性が増加していることを示す熱イメージ（図２３Ａ）及び定量分析（図２３Ｂ）を表している。即ち、これらの結果赤外サーモグラフィーが、一般に禿げ、及び脱毛の治療法の特定を支援する、又は副作用として脱毛を起こす薬物を特定する方法（例えば毒性スクリーニング）として脱毛を測定できることを実証している。
【００７１】
実施例２２
男性勃起機能不全に適した薬物治療後のサーモグラフィー
男性勃起機能不全（ＭＥＤ）は、生殖器への血流を増加する薬物により治療される。局所産熱の増加には局所血流量の増加が伴う。この様式で作用し、ＭＥＤを治療する薬物の一つはピナシディル（Ｐｉｎａｃｉｄｉｌ）である。図２４は、赤外サーモグラフィーは、３．０または０．３ｍｇピナシディル／ｋｇを何れか投与した２時間後に、ピナシディルがラット生殖器の産熱を増加させること検出することを示している。即ち、赤外サーモグラフィーは、ＭＥＤを適用とする薬物候補の特定と評価、ならびに副作用として勃起を起こす候補を特定するのに適した定量かつ非侵襲的方法を提供する。
【００７２】
実施例２３
サーモグラフィーによる抗炎症剤の効果モニタリング
関節炎は関節の炎症を特徴とする病気であり、抗炎症剤により治療できる。炎症反応には反応部位に於ける産熱の増加を伴うことから、関節炎及び関節炎治療薬の効果はサーモグラフィーによりモニターできる。この応用を示すために、正常動物の片足にペプチドグリカンポリサッカライド（ＰＧＰＳ）を２週間注射し関節炎モデルを確立した。もう一方の脚は未治療とした。関節炎誘導処置後の赤外サーモグラフィーは、未処置脚に比べると処置した脚での産熱のレベルが高いことを示し（図２５Ａ）、赤外サーモグラフィーが炎症を起こす作用物質の能力のモニターに利用できることが示唆された。続く抗炎症剤、プレニソロンによる処置は産熱の上昇を元に戻し、処置後両足のサーモグラフィープロフィールは近似した（図２５Ｂ）。即ち、赤外サーモグラフィーは炎症反応及び抗炎症剤の効果のモニタリング、ならびに関節炎適用例の治療に関する薬物候補の効果の評価に適した有用な道具である。
【００７３】
上記の全ての引用文献は参照され、その全体がここに取り込まれる。
【００７４】
当業者は本開示を読むことで、本発明の真の範囲から逸脱することなしに形状及び詳細について各種変更が可能であることを認識するだろう。
【図面の簡単な説明】
【図１】培養細胞中の赤外産熱のイメージングへの利用に好適な装置の略図。１＝赤外線カメラ；２＝細胞培養インキュベーター（３７＋／−．０２℃）；３＝等温チャンバー；４＝プレートホルダー；５＝コンピューターインターフェイス。
【図２】生動物に於ける産熱イメージングへの利用に好適な装置の略図。１＝赤外線カメラ；２＝等温チャンバー；３＝加温パッド（３７℃）；４＝コンピューターインターフェイス；５＝肩甲骨間褐色脂肪組織（ＩＢＡＴ）。
【図３Ａ】グレーのスケールインデックス又は図示された数値段階により示された、ロテノン及びＦＣＣＰで処理された酵母（図３Ａ；３８４−ウエルプレート）の赤外サーモグラフィーのデータ（ロテノン＝−●−；ＦＣＣＰ＝−▲−）。
【図３Ｂ】グレーのスケールインデックス又は図示された数値段階により示された、ヒト脂肪細胞培養体（Ｆｉｇ３Ｂ；９６−ウエルプレート）の赤外サーモグラフィーのデータ（ロテノン＝−●−；ＦＣＣＰ＝−▲−）。
【図４Ａ】脱共役タンパク質２（ＵＣＰ２）を発現する酵母細胞のサーモグラフィー分析（図４Ａ）の例。
【図４Ｂ】脱共役タンパク質２（ＵＣＰ２）を発現する酵母細胞のサーモグラフィー分析（図４Ｂ）の例。
【図４Ｃ】脱共役タンパク質２（ＵＣＰ２）を発現する酵母細胞内のＵＣＰ２発現の分子解析（図４Ｃ）の例。
【図５】フォルスコリン（−■−）又はイソプロテレノール（−●−）存在下にβ_３−ＡＲレセプターを過剰発現しているチャイニーズハムスター卵巣細胞（ＣＨＯ）内の産熱の赤外サーモグラフィー分析（ブランク＝−▲−）。
【図６】脂肪細胞に於けるトリグリセリド蓄積（−●−）及び熱産生（−▲−）の分析。
【図７】インシュリン及び９−シスレチノイン酸存在下に、複数のパーオキシゾーム増殖因子活性化レセプターγ（ＰＰＡＲγ）に関する用量反応曲線を示す分化中脂肪細胞の赤外サーモグラフィーイメージ。
【図８Ａ】ＰＰＡＲγアゴニストであるＧＷ１９２９で処理されたｏｂ／ｏｂマウスの肩甲骨内域の赤外サーモグラフィー分析の要約（図８Ａ）。
【図８Ｂ】ＰＰＡＲγアゴニストであるＧＷ１９２９で処理されたｏｂ／ｏｂマウスに於けるサーモグラフィックシグナルと、各種ＰＰＡＲγアゴニストの最少有効量（ＭＥＤ）との相関性（図８Ｂ）（Ｒ２＝０．９５４）の例示。
【図９】脂肪細胞に於けるβ−ＡＲアゴニスト（５０ｎＭ）−誘導産熱のロテノン感受性を示す例。
【図１０】血管内皮増殖因子（ＶＥＧＦ）存在下（灰色実線）及び非存在下（破線）に於ける初代ヒト上皮細胞（ＨＵＶＥＣ細胞）１０分時の赤外サーモグラフィー分析の例。
【図１１】ＨＣｌへＮａＯＨを添加した後の化学反応の赤外サーモグラフィー分析。
【図１２Ａ】２５ｍｌビーカー中（図１２Ａ）（１＝バルク溶媒）；２＝活性ビーズ；３＝Ｏ−環）又は９６ウエルマイクロタイタープレート（図１２Ｂ；不活性及び活性ビーズ）中のコンビ−ケムビーズ上に於ける触媒反応の赤外サーモグラフィー分析。
【図１２Ｂ】２５ｍｌビーカー中（図１２Ａ）（１＝バルク溶媒）；２＝活性ビーズ；３＝Ｏ−環）又は９６ウエルマイクロタイタープレート（図１２Ｂ；不活性及び活性ビーズ）中のコンビ−ケムビーズ上に於ける触媒反応の赤外サーモグラフィー分析。
【図１３Ａ】５連続作動中及び後の計量型投与吸引（ＭＤＩ）装置（図１３Ａ；作動０、１及び５）の赤外サーモグラフィー分析（図１３Ｂ）。吸引剤、アルブテロールで処理されたヌードマウスの赤外サーモグラフィー分析（図１３Ｃ）、及びそれに続くアルブテロール活性の動態を示す胸部域定量分析（図１３Ｄ）の例。
【図１３Ｂ】５連続作動中及び後の計量型投与吸引（ＭＤＩ）装置（図１３Ａ；作動０、１及び５）の赤外サーモグラフィー分析（図１３Ｂ）。吸引剤、アルブテロールで処理されたヌードマウスの赤外サーモグラフィー分析（図１３Ｃ）、及びそれに続くアルブテロール活性の動態を示す胸部域定量分析（図１３Ｄ）の例。
【図１３Ｃ】５連続作動中及び後の計量型投与吸引（ＭＤＩ）装置（図１３Ａ；作動０、１及び５）の赤外サーモグラフィー分析（図１３Ｂ）。吸引剤、アルブテロールで処理されたヌードマウスの赤外サーモグラフィー分析（図１３Ｃ）、及びそれに続くアルブテロール活性の動態を示す胸部域定量分析（図１３Ｄ）の例。
【図１３Ｄ】５連続作動中及び後の計量型投与吸引（ＭＤＩ）装置（図１３Ａ；作動０、１及び５）の赤外サーモグラフィー分析（図１３Ｂ）。吸引剤、アルブテロールで処理されたヌードマウスの赤外サーモグラフィー分析（図１３Ｃ）、及びそれに続くアルブテロール活性の動態を示す胸部域定量分析（図１３Ｄ）の例。
【図１４Ａ】β_３−ＡＲアゴニスト（１．０ｍｇ／ｋｇ＝−●−；０．１ｍｇ／ｋｇ＝−■−；０．０１ｍｇ／ｋｇ＝−▲−）で処理された後の用量−反応曲線ならびに動態データを示す、マウスの肩甲骨内産熱の赤外サーモグラフィー分析の例。
【図１４Ｂ】β_３−ＡＲアゴニスト（１．０ｍｇ／ｋｇ＝−●−；０．１ｍｇ／ｋｇ＝−■−；０．０１ｍｇ／ｋｇ＝−▲−）で処理された後の用量−反応曲線ならびに動態データを示す、マウスの肩甲骨内産熱の赤外サーモグラフィー分析の例。
【図１５Ａ】ｏｂ／ｏｂマウスの肩甲骨間産熱の咳ガイサーモグラフィー分析−−モノアミン再取り込み阻害剤ＧＷ７３５５９Ａ処理後の用量−反応（図１５Ａ）及び動態データ（図１５Ｂ）（１０．０ｍｇ−●−；５．０ｍｇ／ｋｇ＝−■−；１．０ｍｇ／ｋｇ＝−▲−）。
【図１５Ｂ】ｏｂ／ｏｂマウスの肩甲骨間産熱の咳ガイサーモグラフィー分析−−モノアミン再取り込み阻害剤ＧＷ７３５５９Ａ処理後の用量−反応（図１５Ａ）及び動態データ（図１５Ｂ）（１０．０ｍｇ−●−；５．０ｍｇ／ｋｇ＝−■−；１．０ｍｇ／ｋｇ＝−▲−）。
【図１６】β_３−ＡＲアゴニスト処理マウスに於ける肩甲骨間産熱と体重減少の相関性を示すグラフ（ＩＢＡＴ産熱＝棒、２−週間体重減少＝−●−）（Ｒ２＝０．９７）
【図１７】１９週齢齧歯類動物モデルのβ_３−伝達産熱に及ぼす遺伝的背景及び食物の影響を特徴付ける赤外サーモグラフィーの利用を示すグラフ（低脂肪食＝実線灰色棒；高脂肪食＝斜線棒（１４週間給餌した動物）。
【図１８Ａ】ヒトに於ける食事誘導産熱の赤外サーモグラフィー分析。図１８Ａは背温度の経時変化を示す。
【図１８Ｂ】ヒトに於ける食事誘導産熱の赤外サーモグラフィー分析。図１８Ｂは昼食前後の男性２名と女性２名の背温度の平均差を示す（平均値＝３−５日／被験者）。
【図１９】エフェドリン（左＝０分；右＝６０分）（用量＝０．６−０．７ｍｇ／ｋｇ−デルタＴ℃（背全面＝被験者Ａは０．６３、被験者Ｂは０．４６））処理されたヒト（被験者Ａ及び試験者Ｂ）に於ける薬物誘導産熱の赤外サーモグラフィー分析の例。
【図２０】ｏｂ／ｏｂマウスの肩甲骨間産熱に影響する、２種類の薬剤、β_３−ＡＲアゴニストＣＧＰ１２１７７（１ｍｇ／ｋｇ−腹腔内注射）及びＰＰＡＲγアゴニストＧＷ１９２９間相互作用の赤外サーモグラフィーによる特徴付け（１＝肩甲骨間領域）。
【図２１】ヌードマウスに於けるＶＥＧＦペプチド誘導産熱活性の赤外サーモグラフィー分析（１＝ＶＥＧＦ；２＝コントロール）。
【図２２】腫瘍温度に及ぼす抗−ＶＥＧＦ抗体の影響に関する赤外サーモグラフィー分析。
【図２３Ａ】新生児マウスの無毛に及ぼす、子宮内エトポシド処置（６ｍｇ／ｋｇ）の効果に関する赤外サーモグラフィー分析。図２３Ａは、０日、３日及び５日目に得たサーモグラフィーのイメージを示す。
【図２３Ｂ】新生児マウスの無毛に及ぼす、子宮内エトポシド処置（６ｍｇ／ｋｇ）の効果に関する赤外サーモグラフィー分析。図２３Ｂは、投薬後のトルソデルタＴ℃の経時変化を示す（背＝白地の棒、前＝実線、灰色の棒）。
【図２４】ラットの生殖器産熱のピナシジル（Ｐｉｎａｃｉｄｉｌ）誘導変化の赤外サーモグラフィー分析（ＰＯ投与後２時間）
【図２５Ａ】プロテオグリカンポリサッカライド（ＰＧＰＳ）により誘導された（図２５Ａ；１＝非−関節脚部；２＝関節脚部）及び６ｍｇ／ｋｇ経口プレドニゾロンにより誘導された（図２５Ｂ）（賦形剤＝ＨＰＭＣ＋０．１％ＴＷ８０；経口）脚部炎症の赤外サーモグラフィー分析。
【図２５Ｂ】プロテオグリカンポリサッカライド（ＰＧＰＳ）により誘導された（図２５Ａ；１＝非−関節脚部；２＝関節脚部）及び６ｍｇ／ｋｇ経口プレドニゾロンにより誘導された（図２５Ｂ）（賦形剤＝ＨＰＭＣ＋０．１％ＴＷ８０；経口）脚部炎症の赤外サーモグラフィー分析。[0001]
This application claims priority to US Provisional Application No. 60 / 085,736, filed May 15, 1998, the entire disclosure of which is hereby incorporated by reference. Invite to
[0002]
【Technical field】
The present invention relates generally to thermography, and more particularly, to monitoring physiological and molecular events that trigger an exothermic response in animals (including humans), plants, tissues, cells and cell-free systems utilizing infrared thermography. About the method. The method can be used to screen, identify, and rank drug candidates for multiple diseases, disorders, and conditions.
[0003]
【background】
Thermodynamics is the science of the link between work and heat. In fact, the occurrence of any chemical reaction or physiological process in an animal or cell involves the absorption or generation of heat, and the heat absorbed or generated by the system is proportional to the work done. Thus, measurements of heat generation (ie, heat production) can be used to calculate the energy used or generated for chemical reactions and physiological processes.
[0004]
Various methods are available for detecting the expression of proteins that control heat production in cells (eg, uncoupling proteins, UCPs) (eg, Northern or Western blotting), but these methods require a great deal of effort. And does not directly measure the activity of the protein. Guanosine-5'-diphosphate (GDP) -binding assays and fluorescent dyes (eg, JC-1 or rhodamine derivatives) provide a direct measure of UCP activity (Nedergarrd and Cannon, Am. J. Physiol. 248 (3pt1). ): C365-C371 (1985); (Peers et al, Biochemistry 30: 4480-4486 (1991)) However, the GDP-binding assay requires protein purification, and the use of dyes requires non-selective staining and cytotoxicity. Limited by the metabolism of the dye by the cells, and more importantly, all of these techniques cannot directly measure real-time fluctuations during heat production and are invasive.
[0005]
Bomb calorimeters and microcalorimeters are used to quantitatively measure the heat generated or consumed by cultured cells (Bottcher and Furst, J. Biochem. Biophys. Methods, 32: 191-194 (1996)) or chemical reactions. To provide a suitable method. However, despite recent advances in multi-channel calorimeters, there is no method for quickly analyzing thermal changes in multiple (≧ 60) simultaneous reactions. In addition, temperature gradients on fixed surface areas, such as cell culture plates or skin surfaces, cannot be measured with a calorimeter.
[0006]
Infrared thermometers have been developed that can measure the amount of infrared energy emitted from a specific body part (for example, the ear canal). However, these devices cannot be used to measure the heat production or chemical reaction of the separated cells and tissues, nor can they measure the heat generation from multiple samples immediately over a long period of time. Further, these devices do not provide large surface area images.
[0007]
Infrared interactance devices have also been developed. Unlike infrared thermometers, these devices emit near radiation at wavelengths <1000 nm. Because these devices measure near infrared absorption, they do not provide accurate heat production measurements.
[0008]
The present invention provides a rapid, non-invasive method of instantaneously measuring heat production in isolated vesicles, including animals, plants, tissues and cultured cells. The invention is also applicable to molecular interactions such as receptors / ligands that alter heat generation, enzyme catalysis and other chemical reactions. Using the present method based on the use of infrared thermography, drug candidates for treating various diseases, disorders and conditions can be screened and identified.
[0009]
Summary of the Invention
The present invention relates generally to methods for monitoring physiological changes and molecular interactions utilizing infrared thermography. Infrared thermography offers a non-invasive approach to analyzing the effects of various agents on heat production in chemical reactions in animals, plants, cultured cells and cell-free systems. The invention allows for the screening of compounds for their ability to alter heat dissipation and allows for the identification of compounds that have application in the treatment of various diseases, disorders and conditions.
[0010]
The objects and advantages of the present invention will become apparent from the following description.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a method of using infrared thermography as a tool to monitor temperature changes that occur during molecular interactions, including those that occur in isolated cells, tissues, plants, animals (eg, humans). I do. Infrared thermography is used to analyze the effects of various agents on heat production in various cell, tissue, plant and animal types during enzyme catalysis and more generally during ligand interaction with binding partners. Available. That is, infrared thermography can be used to identify agents suitable for the treatment of various diseases, disorders and conditions with altered thermogenic responses.
[0012]
Generally, "infrared radiation" refers to a frequency having a wavelength between about 2.5 to about 50 microns, or in other words, a frequency of about 200 to about 4000 per cm (cm @ -1 or "wave number"). Means having electromagnetic radiation. As those familiar with infrared (IR) radiation and the IR spectrum understand, the frequency of electromagnetic radiation, which is generally characterized as infrared, is emitted or absorbed by vibrating molecules, and Such vibrations generally correspond to the thermal state of the material in relation to the surroundings of the material. All solids whose temperatures are above 0 ° absolutely irradiate some infrared energy, and at temperatures below about 3500 ° K (3227 ° C., 5840 ° F.) this heat radiation is mainly due to the infrared region of the electromagnetic spectrum. Happens in. Thus, there is a linear correlation between body temperature and the emitted infrared radiation. In the present invention, it is preferred to monitor radiation in the range of 3-100 microns, more preferably 3-15 microns, and most preferably 3-12 microns (e.g. 6-12 microns).
[0013]
Furthermore, as those familiar with electromagnetic radiation understand, 2.5 cm ^-1 The following wavelengths are considered the "near IR" region of the electromagnetic spectrum and represent harmonic oscillations and low-level electronic term transitions. As used herein, "infrared" is not intended to encompass the "near IR" region of the electromagnetic spectrum.
[0014]
According to the present invention, an infrared imaging system, preferably a high resolution infrared imaging system, utilizes the images provided by the central processing unit for data analysis, for example, to generate heat output from cultured cells or tissues or laboratory animals. Used for immediate monitoring (see FIGS. 1 and 2 and examples below). Examples of suitable systems are those made by the FLIR infrared system (AGEMA900 or QWIP SC3000). Similarly, the temperature can be measured using a device such as a non-contact infrared thermometer (C-1600MP) manufactured by Linear Laboratories. To date, neither AGEMA 900 nor C-1600 MP has been typically utilized in the practice of the method of the present invention. However, these devices are adapted by techniques known to those skilled in the art, for example, from a temperature of 5.0 ° C. or higher to 0.000001 ° C. or lower, preferably 1.0 ° C. to 0.00001 ° C., or 0.5 ° C. to 0.0001 ° C., more preferably 0.3 ° C. to 0.0005 ° C. or 0.25 ° C. to 0.001 ° C., most preferably 0.2 ° C. to 0.002 ° C. (FIG. 1 and FIG. (See Examples) can be measured for temperature changes.
[0015]
In one embodiment, the present invention relates to a method of monitoring the effect of an agent on heat production in isolated cells, tissues or animals (including primates (eg, humans)). The method comprises the steps of: i) using infrared thermography to measure the heat produced by a given surface area of the cell, tissue or animal before exposure to the agent; ii) (eg, maintaining / proliferating the cell or tissue) Exposing cells, tissues or animals to the agent (either by adding the agent to the culture medium or treating the animal with the agent using standard donating techniques), iii) using infrared thermography Measuring the heat produced by the cells, tissues or animals during and / or after treatment with the substance, and iv) comparing the measurements obtained in steps i) and (iii), In some cases, an agent that causes a decrease in the temperature of a cell, tissue or animal is a thermogenesis inhibitor, and an agent that causes an increase in temperature is a thermogenesis enhancer.
[0016]
Cells that can be monitored according to the present invention include isolated naturally occurring cells (including primary cultures and established cell lines) and processed cells (eg, isolated processed cells). The cells can be attached to the solid support in suspension or in either a single layer or multiple layers. Examples of suitable supports include plastic or glass plates, dishes or slides, membranes and filters, flasks, tubes, beads and other related containers. Preferably, a plastic multi-well plate is used, with 96-well and 384-well microtiter plates being preferred. Preferred cell titers are between 100 and 100,000 cells / cm for adherent cells ² And 100-1,000 cells / μl in suspension culture, but potentially any cell number / concentration can be used.
[0017]
Naturally occurring isolated cells that can be monitored by the methods of the present invention include eukaryotic cells, preferably mammalian cells. Primary culture cells, established cell lines, and hybridomas (such as those available from the American Type Culture Collection) can be used. Specific examples include fat (eg, adipocytes and their precursor cells), muscle (myotubes, myoblasts, myocytes), liver (eg, hepatocytes, Kupfer cells), digestive system (eg, intestinal epithelium, salivary glands). Pancreas (eg, α and β-cells), bone marrow (eg, osteoblasts, osteoclasts and their progenitors), blood (eg, lymphocytes, fibroblasts, reticulocytes, hematopoietic progenitor cells), skin (eg, keratinocytes) Cells, melanocytes), amniotic fluid or placenta (eg, chorionic villi), tumor (eg, cancer, sarcoma, lymphoma, leukemia), brain (eg, neurons, hypothalamus, adrenal gland and pituitary gland), respiratory system (eg, lung) , Respiratory tract), connective tissue (eg, chondrocytes), eyes, kidneys, heart, bladder, pancreas, thymus, gonads, thyroid, and other organs involved in endocrine control. There are no restrictions on the cells that can be used. The invention is applicable to cells from plants, fungi, protozoa and Monera (eg bacteria). Cells can be cultured using established culture techniques, and optimizing culture conditions can properly ensure viability, growth and / or differentiation.
[0018]
Processed cells that can be monitored by this method include proteins directly or jointly involved in temperature control, energy balance and energy source utilization, growth and differentiation, and other aspects of physiology or metabolism that alter the heat produced by the cells. Includes cells that have been engineered to produce or overproduce. These cells are engineered prokaryotic cells (Monera kingdom: E. coli), engineered higher or lower eukaryotic cells, or cells that are present in or isolated from a transgenic animal. Examples of higher eukaryotic cells (eg, from the plant and animal kingdoms) include cell lines (eg, CV-1, COS-2, C3H10T1 /) available from the American Type Culture Collection. 2, HeLa and SF9). Examples of lower eukaryotic cells include fungi (eg, yeast) and protozoa (eg, slime molds and ciliates). Cells or transgenic animals can be engineered to express various proteins, including nuclear receptors and transcription factors (eg, retinoid receptors, PPARs, CCAAT-enhancer-binding proteins (CEBPs), polymerases), cell surface receptors ( For example, transmembrane and non-transmembrane receptors, G protein-coupled receptors, kinase-coupled receptors), membrane transporters and channels (eg, non-coupled proteins, sugar transporters, ion channels), signaling proteins (eg, phosphodiesters) These include, but are not limited to, esterases, cyclases, kinases, phosphatases) and viruses (eg, AIDS, herpes, hepatitis, adeno). Engineered cells can be produced by introducing into a host cell a construct comprising a sequence encoding the protein to be expressed and an operably linked promoter. Appropriate vectors and promoters can be selected based on the desired host, and the introduction of the construct into the host can be performed using any of a variety of standard transfection / transduction techniques (molecular biology, laboratory manual ( Molecular Biology, A Laboratory Manual), 2nd edition, J. Sambrook, EF Fritsh and T. Maniatis, Cold Spring Harbor Press, 1989). The cells thus produced can be cultured using established culture techniques, and the expression of the introduced protein coding sequence can be guaranteed by optimizing the culture conditions.
[0019]
The method of the present invention is based on the efficacy, selectivity, efficacy, pharmacokinetics and pharmacodynamics of active agents in a variety of cell-free, cell, tissue, plant, animal and human based thermogenic assays. It can be used to identify, characterize, grade, and select agents (eg, drugs or drug candidates) suitable for use in treating a disease, disorder or condition. For example, a test substance can be screened for its ability as a catabolic or anabolic drug by utilizing infrared thermography. Cultured cells (eg, primary cells such as adipocytes or yeast, or established cells such as C3H10T1 / 2 mesenchymal stem cells, osteoblasts or adipocytes, plants, animals or humans (patients in clinical trials in drug development) After treatment with a test substance, the change in thermal properties can be measured by infrared thermography. Agents that increase heat production (heat production by cells) are potentially useful as catabolic drugs, Drugs that suppress thermogenesis are potentially useful as anabolic drugs.
[0020]
In addition to changes in metabolism, changes in thermogenesis can reflect changes in growth and differentiation. That is, the method of the present invention provides for the identification of agents (e.g., drugs or drug candidates) suitable for use in treating diseases, disorders or conditions involving changes in metabolism, toxicity, cell proliferation, organ development and / or differentiation. Available for characterization, grading, and selection.
[0021]
Examples of pathophysiologies that are potentially susceptible to treatment with anabolic drugs identified by infrared thermography include anorexia, hair loss, autoimmunity, cachexia, cancer, catabolism with aging, diabetes, transplantation Includes rejection, growth retardation, osteoporosis, fever, bacterial and viral infections. Diseases, disorders or conditions that are potentially susceptible to the treatment of catabolic drugs identified by infrared thermography include those associated with obesity, such as hypertension, dyslipidemia and cardiovascular disease. As well as diseases, disorders or conditions associated with growth promotion (eg, cancer, giantosis, certain viral infections). Pathophysiologies that are susceptible to treatment using agents identified by infrared thermography generally involve, but are not limited to, catabolic or altered anabolic effects (eg, metabolic disease). The method is also applicable to male erectile dysfunction (MED) and other diseases, disorders and conditions, including inflammation, hypertension, gastrointestinal tract disease, behavioral injury (CNS disease), as well as diseases involving altered blood flow. In pharmaceutical research and development, there is no limitation on the pathophysiology that can be analyzed by the present invention (eg, analysis of drug efficacy, efficacy, toxicity, pharmacokinetics and pharmacodynamics).
[0022]
According to the present invention, binding of a ligand (protein or non-protein (eg, nucleic acid)) to a binding partner (protein or non-protein (eg, nucleic acid)) whose binding induces a thermogenic reaction is monitored by use of infrared thermography. it can. The ligand and / or binding partner can be in a cell or in a cell-free environment (eg, a solution). The ligand and / or binding partner can be a synthetic chemical entity that is not normally present in nature, or the ligand and / or binding partner can be a naturally occurring protein, nucleic acid, polysaccharide, lipid, hormone, or other natural entity. It may be an entity found in nature, such as a substance or a cell that occurs in an organism. The effect of a test substance (eg, a potential ligand) on the heat generated by its binding partner can be measured using infrared thermography. One suitable method is: i) measuring the heat generated by the binding partner, ii) adding the test substance to the binding partner, and iii) binding the potential ligand (test substance) with the binding partner. And iv) comparing the measurements of (i) and (ii), wherein the agent that alters the calorific value is the ligand for the binding partner. In addition, test substances can be screened for their ability to alter the thermogenic response resulting from ligand binding to its binding partner. Such agents are allosteric regulators, agonists or antagonists of the ligand and / or binding partner. Such a screen comprises: i) measuring the heat generated by adding the first member of the binding pair (ligand or binding partner) to the second member of the binding pair using infrared thermography; ii) The heat generated upon addition of the first member of the binding pair, the second member of the binding pair and the test substance is measured and iii) comparing the measurements of (i) and (ii), wherein An agent that changes the amount of heat generated when a ligand is added to a binding partner is, for example, a modulator of interaction due to binding to either member or both members of the binding pair.
[0023]
Further in accordance with the present invention, agents can be screened for their ability to modulate the catalytic rate of a particular enzyme. The method measures the heat generated when the enzyme is added to the substrate using infrared thermography, measures the heat generated by the addition of the test substance, the enzyme and its substrate, and compares the results Including. Agents that alter thermogenesis are enzyme inhibitors or activators. Controls that can be performed according to these methods include measuring the heat generated by adding the enzyme to the test compound (without the substrate) and the heat generated by adding the substrate to the test compound (without the enzyme). By such control, the heat production for each addition can be determined. By utilizing such a method, a test substance can be screened for its ability to act as a substrate. Such agents can increase thermogenesis when mixed with enzymes in the absence of any other known substrate.
[0024]
In another embodiment, the invention relates to an agent identified using the above assay. An agent identified by the above assay can be formulated as a pharmaceutical composition. Such compositions comprise the agent, and a pharmaceutically acceptable diluent, or carrier. The agent can be present in dosage unit form (eg, tablets or capsules) or as a solution, but is preferably sterile, especially when administered by injection. Dosage and administration will vary depending upon, for example, the patient, the agent, and the effect desired. Optimal dosages and methods can be readily determined by one skilled in the art.
[0025]
In another embodiment, the invention relates to a method of monitoring the effects of environmental changes (eg, food) and / or genetic background on heat production in various organisms (animals, plants, tissues, and cells). The method includes: i) heat generated by the organism under various environmental conditions (eg, feeding different diets; high or low fat, protein, or carbohydrate diets), or different genetic backgrounds (eg, syngeneic animals, populations) ) Measuring the heat generated by the organism having the) using infrared thermography; and ii) exposing the organism to various agents (eg, placebo or thermogenic agents; including untreated controls); iii) measuring the heat generated by the organism treated with the agent using infrared thermography and comparing iv) the measurements of steps (i) and (iii) with environmental changes and genetic changes. Determining the effect of the background.
[0026]
The methods of the invention identify, infer, characterize, grade, and determine how different environments (eg, diet) or genotypes affect basal or agent-induced heat production. And can be used for choosing. There are no restrictions on the population, species, strain, sex, or age, or food or environmental conditions that can be used. The organism can be naturally occurring (eg, a mouse), syngeneic (eg, an AKR / J mouse) or created (a transgenic mouse). The method uses the heat generated by altering diet, circadian rhythm, light rhythm, altitude, barometric pressure, humidity, temperature, noise, sleep state, physical or mental stress, cellular or biological damage. It can include measuring by thermography. The meal may be loose (eg, a cafeteria meal) or well-characterized (eg, a laboratory meal). Organisms may be eaten as intended or as free. Agents that alter heat production can be naturally occurring, synthetic, known or unknown, safe or toxic, and anabolic, catabolic, or ineffective. Environmental changes (diet or other), genotypes, or agents that enhance thermogenesis (body temperature production) are potentially useful in identifying catabolic states. Environmental changes (diet or other), genotypes, or agents that suppress thermogenesis (body temperature production) are potentially beneficial in identifying anabolic states.
[0027]
In another embodiment, the present invention relates to a method for monitoring drug-drug interactions in various organisms (humans, animals, plants, tissues and cells). The method includes: i) measuring the heat produced by an organism (such as cells) using infrared thermography prior to exposure to the agent; and ii) combining the organism (such as cells) with a single agent. And exposure to multiple agents (eg, added to the medium or administered by injection, gastrointestinal nutrition, topical application, etc.); and iii) after treatment with a single agent, and multiple agents. Measuring the heat produced by the organism (such as cells) using infrared thermography after the treatment with (iv), and iv) determining the difference in heat generated in steps (i) and (iii), Comparing the heat generated after exposure to one agent to the heat generated after exposure to multiple agents. The difference in heat generated after exposure to multiple agents (as opposed to a single agent) indicates that the agent interacts with or induces a thermogenic response.
[0028]
As noted above, it is suggested that agents that produce a change in thermogenesis when used in combination as compared to when used alone are involved in pharmacodynamic drug-drug interactions. Such interactions are potentially harmful or beneficial to an organism, tissue or cell. In such cases, infrared thermography can still be used to identify, predict, characterize, grade, and / or select which types of agents (eg, drugs) interact with each other. There are no restrictions on the type and number of agents available, on cells, tissues and organisms. The agent can be naturally occurring, synthetic, agonist, antagonist, inhibitor, activator, safe, toxic, anabolic, catabolic, known or unknown. Cells, tissues and organisms can be obtained from plants, animals (eg, humans), fungi, protozoa, or the kingdom Monera. Infrared thermography involves changes in the pharmacokinetic and pharmacodynamic parameters of cells, tissues and / or organisms, including changes in the duration of exposure, the concentration of active agents and pharmaceutical compositions, and the concentrations of active agents used. Can be used to measure the heat produced by
[0029]
In a further embodiment, the present invention relates to a method of monitoring hair loss (balding) and regeneration. In this case, infrared thermography can be used to identify, predict, characterize, grade, and / or select various treatments or environmental stimuli that alter hair growth. There is no limitation on the type of treatment or stimulus that can alter hair loss or growth. The type of treatment may include diet, exercise, pharmacological, radioactive, or surgical intervention, and may be invasive or non-invasive. The stimulus to alter hair growth may be naturally occurring in the environment (eg, radon gas) or may be the result of environmental pollution (contamination of things like pesticides). Thus, infrared thermography can be used to monitor the safety, efficacy and efficacy of various treatments against hair loss and growth.
[0030]
In another embodiment, the invention is directed to a method for assessing the safety profile of a medicament. According to this embodiment, various types of proteins (eg, cytochrome P450s, etc.), intracellular organs (eg, microsomes, etc.), cells, tissues, and organs that are targeted by the active substance are separated and treated with various concentrations of the active substance. The generated heat can be monitored using infrared thermography. The method comprises the steps of: i) potency and efficacy of the agent on promoting or inhibiting thermogenesis in a desired target (eg, a protein, intracellular organ, cell, tissue, or organ involved in the therapeutic effect of the agent). Determining the sex and ii) the agent's effect on promoting or inhibiting heat production in an unwanted target (eg, a protein, intracellular organ, cell, tissue, or organ involved in the toxic effect of the agent). Determining potency and efficacy and iii) determining the selectivity of the agent by comparing steps (i) and (ii). Pharmaceutical agents that exhibit increased selectivity between various targets (eg, proteins, intracellular organs, cells, tissues, and / or organs) can be expected to have improved safety profiles. In this embodiment, the effect on the heat generated by the binding partner and / or the enzyme catalyst when the concentration of the active substance is changed can be used to determine the selectivity and safety profile for multiple targets. Optimal selectivity between desired and unwanted targets (eg, cell type, binding partner, or enzyme) can be readily determined by one skilled in the art.
[0031]
In another embodiment, the invention is directed to a method for assessing the physical state and / or amount of a compound. According to this embodiment, the physical state of the compound can be determined. Because it changes the physical state from solid (ie, frozen liquid) to liquid (ie, melting), liquid to solid (ie, crystallization), liquid to gas (ie, vaporization, evaporation), and solid to gas (ie, sublimation). This is because it relates to a compound that changes its physical properties as it changes. This embodiment is applicable to, but not limited to, compounds in open containers, closed systems, and pressurized containers (ie, inhalants). The amount of liquid can be measured using the present invention. According to this embodiment, the various amounts of the test substance give rise to a specific thermal profile, whereby their specific thermal properties can be used to determine the amount of active substance present.
[0032]
From the following description, it will be appreciated that the invention has applicability in practice associated with animals or animal tissues. For example, the method is applicable to mammals, including primates (eg, humans) and animals commonly used in research (eg, rats, mice, hamsters, guinea pigs and rabbits), birds, amphibians and reptiles, and insects. . Certain aspects of the present invention are described in more detail in the following non-limiting examples.
[0033]
【Example】
The experimental protocols and details below are referred in whole or in part to the following non-limiting examples.
[0034]
Fat cells:
Human subcutaneous fat cells were purchased from Zen-Bio, Inc. (Research Triangle Park, NC). C3H10T1 / 2 clone 8 fibroblasts were obtained as described above (Lenhard et al., Biochem. Pharmacol. 54: 801-808 (1997), Paulik and Lenhard, Cell Tissue Res. 290: 79-87 (1997)). To differentiate into adipocytes. After 7 days in culture, the accumulation of triglycerides was determined using lipoprotein lipase and GPO-Trinder reagent (assay kit 337-B, Sigma Diagnostics, St. Louis, MO (Sigma Diagnostics, St. Louis, MO)). 50 μl / cm ² ) And the lysates were incubated at 37 ° C. for 2 hours and determined. Optical density was measured using a spectrophotometer set at a wavelength of 540 nm. Lipolysis was measured as previously described (Lenhard et al., Biochem. Pharmacol. 54: 801-808 (1997)).
[0035]
Cloning of UCP2 and yeast transformants:
Human skeletal muscle DNA (# 7175-1) was purchased from Clontech (Palo Alto, Calif.) The UCP2-specific sequence was 5 'and 3' of the published sequence (GenBank U82819). 'PCR amplified from sample using oligonucleotide primers fitted to the ends. ₄ Vent polymerase (New England Biolabs, New England Biolabs, Berlyly, Mass.) In a standard reaction mixture containing 5% DMSO. The cycle parameters were 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 1 minute, and this was repeated 29 times. Samples were passed through an S-400 spin column (Pharmacia, Piscataway, NJ) before ligation with a vector for transforming E. coli. The identity of the clone was confirmed by DNA sequence analysis. In yeast experiments, sequence EieieieieieishishishishijijieitishijiAATTTCATGGTTGGGTTCAAGGCCA (SEQ ID NO: 1) (sense) and ShieititijititishishititieititishieijititieishitiCGAGTTAGAAGGGAGCCTCTCGGGA (SEQ ID NO: 2) (antisense) PCR using a primer having a continues sequence TitieieishiGTCAAGGAGAAAAAACCCCGGATCG (SEQ ID NO: 3) (sense) and JieieieijijieiAAAACGTTCATTGTTCCTTATTCAG (SEQ ID NO: 4) The UCP2 coding sequence was amplified in the second PCR using the primer having (antisense). The PCR product was prepared using the homologous recombination in yeast strain W303 (a / a homozygotes for ade2-1, his3-1, 15leu2-3, 112, trp1-1, ura3-1) using pYX233 (R & D). Systems). Yeast transformants were selected on BSM-trp agar (Bio 101, Vista, CA (Bio 101, Vista, CA)). The correct UCP2 sequence was confirmed by sequencing the plasmid back-extracted from yeast into E. coli.
[0036]
For analysis of UCP2 expression and thermogenesis, the yeast containing the expression plasmid was subcultured in BSM-trp broth for 24 hours, washed once, 2% DL-lactic acid, pH 4.5, 1% ethanol, 0.1% A in a YP containing 5% casamino acid and 40 mg / ml adenine. ₆₀₀ = 0.001 was taken. After 16 hours, glucose was added to 0.4% (w / v) to induce cultures. To confirm the expression of UCP2, yeast (A ₆₀₀ = 0.1) was dissolved in NuPAGE sample buffer containing 5% β-mercaptoethanol (Novex, San Diego, Calif. (Novex, San Diego, Calif.)) And the soluble proteins were dissolved in a 10% NuPAGE gel (Novex, San Diego, CA). Separation on California (Novex, San Diego, CA). A synthetic peptide utilizing the UCP2 amino acid sequence LKRALMAAYQSREAPF (SEQ ID NO: 5) was synthesized and used to generate antibodies (Zeneca, Wilmington, DE (Zeneca, Wilmington, DE)). Western blot analysis was performed according to published methods (Paulik and Lenhard, Cell Tissue Res. 290: 79-87 (1997)).
[0037]
Experimental animal protocol:
Age and weight matched male + ob / + ob mice (Jackson Labs, Bar Harbor, ME) at 72 ° F, 50% relative humidity, 12 hour light / dark cycle, solid feed (NIH R & M / Auto 6F) -Ovals 5K67, PMIFeds (R), Richmond, Indiana (Richmond, Indiana) at 5 animals / cage. To a 41-day-old animal, 0.05 M N-methylglucamine (control) and a 0.05 M N-methylglucamine solution of GW1929 at a concentration of 5 mg / kg once a day (8:00 to 9:00 am) were given. Gavage was administered orally. Two weeks after the administration, the animals were administered 1 mg / kg of CGP12177A (intraperitoneal) and the animals were anesthetized with isofluorane. Infrared thermographic images were obtained and temperature calculated for 6 or more animals per treatment group. This study was conducted in accordance with the principles of laboratory animal care (NIH Publication No. 86-23, Rev. 1985) and company policies regarding animal care and use, and relevant codes of practice.
[0038]
Male AKR / J, C57BL / 6J and SWR / J mice were purchased from Jackson Laboratories, (Bar Harbor, ME), including Surwit et al. (Metabolism44 (5): 645). -651 (1995)), a high-fat and low-fat diet containing high sucrose content as defined by the standard.After feeding this diet for 14 weeks, the interscapular hair was shaved and the animals received 1 mg / kg BRL 37344. Dosing (intraperitoneal) and the animals were anesthetized with isofluorane Nude mice (BALB / C) were 6-8 weeks old and were dosed twice by inhalation before performing temperature profiling. Used both Lewis rat strains (200-250 g). Outer thermographic image and temperature calculations were recorded using Age Ma Thermo Vision (Agema Thermovision) 900 infrared system.
[0039]
Infrared thermography:
The exotherm was measured using a Sterling Cool Door Agemas Thermovision 900 Infrared System AB (Marietta, GA) equipped with a SW scanner and a 40 ° × 25 ° lens detecting a spectral response of 2-5.4 microns. Was. The scanner was equipped with an internal calibration system with an accuracy of 0.08 ° C. The focal length was 6 cm. Images were captured using an iterative function set to 16 or an averaging function set to 32. Data were analyzed using the OS-9 Advanced System and ERIKA 2.00 software according to the manufacturer's instructions (FLIR Infrared Systems AB, Danderyd, Sweden). Thermography of adipocytes was performed using a Queue Systems Inc. (Parkersburg, WV) (Parkersburg, WV) incubator, model QWJ500SABA, at room temperature of the cultured cells of 37 ° C ± 0. It was performed by maintaining at 02 ° C. Yeast spectral analysis was performed at 30 ± 0.02 ° C. using the same incubator system. For microplate applications, the cells were treated with an experimental agent (eg, rotenone) and the temperature was equilibrated in an incubator for 10 minutes before the thermogenesis was measured immediately. Various color scales of visible wavelength were used to depict the temperature variations of the recorded images. The temperature scale is constant, but the color scale image is variable and can be adjusted using the level and span controls.
[0040]
Various infrared detection system parameters were optimized using both yeast and cultured adipocytes. In the cell titer experiment using yeast, the lower detection limit was A ₆₀₀ = 0.01 and the maximum thermogenic reaction is A ₆₀₀ = 0.1-0.2. The temperature activity of the C3H10T1 / 2 cell suspension is 8 × 10 ³ The maximum signal detected from cells / well is 1 × 10 ⁵ Obtained in cells / well. As expected, a maximal thermogenic response of adherent adipocytes was observed when using confluent cells. Comparison of the microtiter plates indicated that the 384-well format was optimal for measuring the thermogenicity of the yeast suspension, whereas the 96-well format was optimal for the culture of adherent adipocytes. Wells outside the culture plate were excluded from the detection system due to increased heat transfer at the plate edge. Wells with larger diameters (ie> 1 cm) were less satisfactory because of the observed meniscus effect and non-uniform heat conduction. Furthermore, the amount of medium per well is important because too much decreases the signal, while too little causes a meniscus and increases background noise. Optimum results were obtained when utilizing 50 μl / well for both 96-well plates containing adherent adipocytes and 384-well plates containing yeast suspension. To minimize the temperature and reflectivity (ie, thermal noise) generated by airflow and light, the detection system and the object to be profiled need to be contained. Materials with low reflective properties (eg, alumite), especially those with low reflective properties from an infrared detector standpoint, are ideal for minimizing exogenous thermal noise during thermal profiling (eg, chamber plates, etc.). Finally, extending the temperature equilibration time (ie> 10 min) improved the signal to noise ratio.
[0041]
Example 1
Design of a device for infrared thermography.
The method of the present invention utilizes an apparatus conveniently composed of a central processing unit with a high resolution infrared imaging system and software suitable for data analysis. One example of a suitable device is a device from FLIR Infrared Systems (AGEMA900) or a non-contact infrared thermometer from Linear Laboratories (C-1600MP). FIG. 1 shows a schematic of these devices for infrared thermography of cell-free systems or cell culture. An isothermal chamber made of a non-reflective material (eg, alumite) that provides a heat sink to buffer temperature fluctuations minimizes thermal noise (eg, reflections and airflow) from the culture plate and surrounding environment. To maintain thermal uniformity within the plate, a plate holder can be placed in the isothermal chamber. The use of an incubator also prevents ambient temperature fluctuations and improves cell response and viability. In the diagram shown in FIG. 1, the camera instantly monitors the heat generated from the cells in culture, the image is recorded by a central processing unit for data capture, and further analyzed by software analysis. FIG. 2 shows a schematic of a device designed for infrared thermography of the mouse interscapular region. This device has features in common with the device of FIG. 1 and includes an infrared camera, an isothermal chamber and a computer interface. The device also includes an infrared screening platform with an anesthesia manifold to maintain the animal's state of anesthesia, such as a tightly controlled heating block made of anodized aluminum, and an isothermal chamber to minimize airflow. .
[0042]
Example 2
Infrared thermography in cultured cell systems (eg, adipocytes and yeast): Effect of small molecules on thermogenic activity.
[0043]
As part of the initial work to verify the use of infrared thermography to monitor mitochondrial thermogenesis, human adipocytes cultured in 96-well microtiter plates and suspended in 384-well microtiter plates Yeasts were treated with rotenone or carbonyl cyanide, p- (trifluoromethoxy) phenylhydrazone (FCCP). Rotenone is an inhibitor of mitochondrial electron transport (Ahmad-Juan et al., Biochem. Soc. Trans. 22: 237-241 (1994)), while FCCP is an uncoupler of mitochondrial respiration. (Terada, Biochem. Biochem. Biophys. Acta 639: 225-242 (1981)). Next, the heat generated from the cells was measured using an Agema Thermovision 900 infrared imaging system (FIG. 1). As shown in the dose response assay shown in FIG. 3, rotenone treatment inhibited the thermogenesis of yeast (FIG. 3A) and human adipocytes (FIG. 3B). In contrast, FCCP promoted thermogenesis in both cells compared to untreated cells. Kinetic analysis showed that a change in thermogenesis was detectable after 10 treatment of the cells with either agent. These results demonstrate the applicability of infrared thermography to measure the dose-dependent thermogenic effects of small molecules (eg, FCCP, rotenone) on cultured cells or cell-free systems.
[0044]
Example 3
In rodents, interscapular brown adipose tissue (IBAT) is an important site for adaptive thermogenesis (Himms-Hagen, Proc. Soc. Exp. Biol. Med. 208: 159-169 (1995)). And is localized within the interscapular region of rodents. This tissue is rich in mitochondria that express an anion transporter, an uncoupling protein (UCP1, previously known as UCP) (Ricquier et al., FASEB J. 5: 2237-2242 (1991)). ). UCP1 uncoupled oxidative phosphorylation by respiration within the IBAT results in the production of heat in place of ATP. Although UCP1 is not abundant in humans, UCP2 (Fleury et al., Nat. Genet. 15: 269-272 (1997)) is abundantly expressed in humans. UCP2 mRNA is widely expressed, and its expression is altered in obesity (Enerback et al., Nature 387: 90-94 (1997)). Furthermore, UCP2 plays an important role in the regulation of energy balance, probably because the antidiabetic agent thiazolidinedione (eg troglitazone) increases UCP2 expression, probably by activating the nuclear receptor PPARγ. (Shimabukuro et al., Biochem. Biophys. Res. Commun. 237: 359-361 (1997)).
[0045]
Although UCP1 regulates mitochondrial-mediated heat production in rodents, there is no direct evidence that UCP2 plays a similar role. To assess the role of UCP2 in thermogenesis and to validate the use of infrared thermography to detect physiological changes in cells engineered to express foreign proteins, the UCP2 gene was transformed into a human cDNA. Cloned from the library and expressed in yeast using a galactose-induced expression system. As shown in FIG. 4A, expression of UCP2 in yeast resulted in increased heat production compared to cells lacking UCP2. As expected, cell treatment with rotenone inhibited UCP2-mediated thermogenesis (FIGS. 4A and 4B). For example, Western blot analysis confirmed that UCP2 was expressed in these cells (FIG. 4C). Maximal expression and thermogenesis were observed 2 hours after induction of UCP2 synthesis with galactose. This result indicates that various molecules (eg, a reporter such as UCP2) are involved in mitochondria-mediated heat production in cell models (eg, fungi (eg, yeast), SF9, CHO, Neurospora, etc.) engineered to express foreign proteins. Demonstrating the applicability of infrared thermography as a tool to measure the effect of genes.
[0046]
To further validate the use of infrared thermography to detect physiological changes in cells engineered to overexpress foreign proteins, the beta 3 adrenergic receptor receptor (β ₃ AR) was cloned from a human cDNA library and expressed in Chinese hamster ovary (CHO) cells. β ₃ Engineered CHO cells overexpressing AR were temperature-profiled for reactivity to isoproterenol, a well-characterized β-AR agonist (FIG. 5). As shown in FIG. 5, CHO cells respond to isoproterenol in a dose-dependent manner and infrared thermography scans cell surface receptors (eg, β ₃ AR) can be used for evaluation, identification and grading of ligands. These results also provide a noninvasive tool that can be used for grading, selecting and identifying compounds suitable for drug discovery using engineered cell models that overexpress foreign proteins, or that can be extended to antisense expression. The use of infrared thermography is also being verified. In addition, infrared imaging is also used to monitor the activity of intracellular kinase activity as exemplified by the dose-dependent response of CHO cells upon administration of the well-characterized protein kinase A (PKA) agonist, phosphocholine. Yes (Figure 5). That is, infrared thermography can be used to monitor agents that affect intracellular enzymes.
[0047]
Example 4
Infrared analysis of cells treated with PPARγ and β-AR agonists: effects on cell proliferation and differentiation
It is interesting to know if infrared thermography can be used to analyze the medicinal effects of drugs affecting energy metabolism. Troglitazone is an antidiabetic agent that increases anabolism (eg, adipogenesis and mitochondrial volume) in C3H10T1 / 2 cells and reduces catabolism (eg, base lipolysis and aerobic respiration) (Lenhard). et al., Biochem. Pharmacol. 54: 801-808 (1997)). The effect of troglitazone on these cells is a result of the activation of the transcription factor PPARγ / RXR, which induces the differentiation of stem cells into adipocytes (Lenhard et al., Biochem. Pharmacol. 54: 801-808 ( 1997), Paulik and Lenhard, Cell Tissue Res. 290: 79-87 (1997), Lehmann et al., J. Biol. Chem. 270: 12953-12956 (1995)). Thus, infrared thermography could be used to test the effects of troglitazone and five structure-related agonists on thermogenesis in C3H10T1 / 2 cells; the effects of these agents on cellular triglyceride accumulation, as well as markers of adipogenesis. (Figures 6 and 7). As shown in FIG. 6, troglitazone treatment increased triglyceride accumulation in these cells, consistent with the observation that this drug promotes adipogenesis (Lenhard et al., Biochem. Pharmacol. 54: 801-). 808 (1997), Lehmann et al., J. Biol. Chem. 270: 12953-12956 (1995)). In contrast, cells treated with higher concentrations of troglitazone, or a number of related PPARγ agonists, have reduced thermogenesis (FIG. 7), with reduced heat production as these cells differentiate into adipocytes. It was suggested that In addition, the potency rating of the various PPARγ agonists tested in the thermogenesis and adipogenesis assays was BRP49653 (EC ₅₀ (ΜM) = 0.063)-GW1929 (EC ₅₀ (ΜM) = 0.052)> troglitazone (EC ₅₀ (ΜM) = 0.316) -Pioglitazone (EC ₅₀ (ΜM) = 0.389)> ciglitazone (EC ₅₀ (ΜM) = 0.123)> Englitazone (EC ₅₀ (ΜM) => 10.0) (FIG. 7). Since UCP expression increases with the differentiation of stem cells into adipocytes (Paulik and Lenhard, Cell Tissue Res. 290: 79-87 (1997), Tai et al., J. Biol. Chem. 271: 29909-29914. (1996)), these observations suggest that increased UCP expression is not sufficient for stimulation of thermogenesis in adipocytes. This view is consistent with the proposal that promotion of adipocyte thermogenesis requires an additional signal (eg, β-AR stimulation) in addition to increasing UCP expression (Lenhard et al., Biochem. Pharmacol. 54: 801-808 (1997), Paulik and Lenhard, Cell Tissue Res. 290: 79-87 (1997)). In addition, these results indicate that infrared thermography has a pharmacological effect (ie, action, potency, kinetics, etc.) on thermogenesis of agents that affect cell growth and / or differentiation, such as troglitazone and other nuclear receptor ligands. It can be used for research.
[0048]
Example 5
Relationship between interscapular heat production and minimum effective dose in ob / ob mice treated with PPARγ agonists
Reagents that activate the transcription factor PPARγ / RXR have drug potential as antidiabetic agents. Infrared thermography using the methods described herein can be used to monitor the effects of these drugs on animal model systems. This application is crucial for drug development and testing.
[0049]
Genotype ob / ob mice were used as an animal model for diabetes. These animals can be used for testing the activity of the diabetes therapeutic agent PPARγ agonist GW1929x. FIG. 8A shows the thermogenic effect of GW1929x treatment of a group of ob / ob mice. Control mice were treated in the same way as the experimental group except that they were treated with a drug vehicle lacking the drug. As expected, treatment with the antidiabetic drug for 1-2 weeks prior to the assay resulted in reduced IBAT thermogenesis in treated mice compared to control animals (FIG. 8A). This animal thermogenesis assay also has quantitative value for determining drug efficacy. As detected by infrared thermography in cell cultures, the minimum effective dose (MED) of the PPARγ agonist group in animals (Henke et al., J. Med Chem. 41, 5020-5036 (1998)) Suppress heat production (FIG. 8B and FIG. 7 described in Example 4). That is, the grade of potency of a PPARγ agonist tested in cell culture plates (BRL49653-GW1919>troglitazone-pioglitazone>ciglitazone> englitazone) corresponded to the MED grade in animal studies (R ² = 0.95). These results indicate that infrared thermography can be used to study, identify, grade, and select compounds based on their ability to alter heat production or heat consumption in animals.
[0050]
Example 6
β ₃ -AR-mediated catabolism and thermogenesis: Measurement of metabolic mediated thermal activity
Catecholamines are thought to regulate body temperature and components, possibly by controlling UCP expression (Rehnmark et al., J. Biol. Chem. 265: 16464-16471 (1990)) or activity (Blaak et al. Int. J. Obes.Relat.Metab.Disorder.17 Suppl3: S78-S81 (1993)). In adipocytes, catecholamines activate β-adrenoceptors (β-ARs) and stimulate the intracellular cAMP pathway (Lafontan and Berlan, J. Lipid Res. 34.1057-1091 (1993)). Stimulation of the β-AR pathway by injection of norepinephrine into animals mimics the cold induction of UCP1 mRNA in IBAT (Silva, Mol. Endocrinol. 2: 706-713 (1998)). Similarly, norepinephrine (Rehnmark et al., Exp. Cell Res. 182: 75-83 (1989)) and β ₃ -AR-agonists (Champigny and Ricquier, J. Lipid Res. 37: 1907-1914 (1996)) directly stimulate UCP1 expression in cultured cell cultures. That is, catecholamine and β ₃ -It is often assumed that the thermogenic effect of AR-agonists is mediated by increased UCP expression. However, β ₃ It has not yet been determined whether AR-agonists can induce thermogenesis without increased UCP expression.
[0051]
β ₃ -AR-agonists are suitable therapeutic candidates for the treatment of diabetes and obesity. The mechanism of action of these drugs is thought to be related to an increase in the metabolic rate (Scarspace, Ann. NY Acad. Sci. 813: 111-116 (1997)). -Agonists have also been found to increase fever by adipocytes. In response, it was determined whether infrared imaging could be used to monitor the effects of β-AR-agonists on thermogenesis in cultured adipocytes. The heat production in C3H10T1 / 2 adipocytes is selective β ₃ CL316243, an AR-agonist, and non-selective β ₃ -Stimulated by treatment with the AR-agonist isoproterenol. β ₁ The AR agonist, RO363, and the β-2AR-agonist, albuterol, are less effective at promoting thermogenesis in these cells than CL316243 or isoproterenol, indicating that multiple ligands are more sensitive to selectivity and efficacy. It was shown that thermography was available. Contrary to the β-AR-agonist, rotenone, a mitochondrial electron transfer inhibitor, inhibited the heat production of cells treated with 50 nM of various β-AR-agonists (FIG. 9). Cycloheximide (100 μM), an inhibitor of protein synthesis, is responsible for ββ ₃ -AR-did not affect mediated heat production. Furthermore, the heat production at 15 minutes after CL316243 treatment was greater than after 18 hours (CL316243 had a dose-dependent thermogenic effect (use dose range 0.8-100 nM)). Therefore, β ₃ -AR-induced thermogenesis may be an acute reaction that does not require increased protein (e.g., UCP) synthesis. However, as others suggest, these results indicate that β ₃ -Does not exclude the role of AR (Rehnmark et al., J. Biol. Chem. 265: 16464-16471 (1990); Lafontan and Berlan, J. Lipid Res. 34: 1057-1091 (1993); Silva. Endocrinol. 2: 706-713 (1988)).
[0052]
As a control, the effect of β-AR-agonist on lipolysis was also measured. Dose response analysis showed that the various β-AR-agonists had similar EC50s in both thermogenic and lipolytic assays (Table 1). When the effectiveness of various β-AR-agonists was compared in both assays, a correlation coefficient of 0.99 was observed. Therefore, the same β-AR signal pathway transmits lipolysis and heat production in adipocytes. It was suggested that Taken together, these results demonstrate the utility of infrared thermography to monitor ligand-altered metabolic activity in cells. Generally, this is also extended to the measurement of thermogenic activity via both anabolic and catabolic effects of potential drug candidates.
[0053]

Example 8
Effect of peptide on cell surface receptor; measurement of enzymatic reaction
Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of the development of blood vessels through the binding and activation of vascular endothelial cells to kinase domain receptors (KDRs). In response to VEGF, cells rapidly activate a cascade of signaling molecules such as mitogen-activated protein kinases (eg, MAPKKK, S6 kinase (p90rsk)). VEGF also causes phosphorylation and activation of transcription factor 2 in cardiomyocytes. (Ferana, N., Davis-Smyth T, Endrocr Rev 1997 Febl 18 (1): 4-25). Currently available methods for analyzing this pathway do not capture the acute response to VEGF. Since the activation of VEGF has an acute kinase / phosphatase activity, which ultimately involves the hydrolysis and formation of bonds, the heat generated from these enzymatic reactions can be measured if the equipment used is sufficiently sensitive. It may be possible. To test this hypothesis, infrared thermography was used to measure the heat generated by VEGF-treated human vascular epithelial cells (HUVEC). As shown in FIG. 10F, VEGF produced heat in HUVEC cells, so infrared thermography could be used to monitor enzymatic reactions (eg, kinase / phosphatase activity) in cultured cells. That is, infrared thermography can be used to evaluate the effect and effectiveness of a compound on an enzymatic reaction.
Example 9
Monitoring of chemical reactions (eg, ligand / binding-partner interactions)
Bond formation and hydrolysis are processes inherent in receptor binding to its putative ligand. This process usually involves an exothermic reaction and is currently measured only using microcalorimetry (Koenigbauer, Pharm. Res. Jun; 11 (6) 777-783 (1994)). These measurements are the basis for establishing isothermal binding for receptor / drug interactions. Since acid and base mixing can cause an exothermic reaction resulting from the binding process, acid / base titration was used to determine if infrared imaging could be used to measure intra / intermolecular binding kinetics. As shown in FIG. 11, 0.25 mM NaOH was mixed with various concentrations of HCl and measured by infrared thermography. As a result, the thermogenic reaction was inhibited in a dose-dependent manner. These data demonstrate the usefulness of infrared thermography in measuring molecular events such as the interaction of a ligand (eg, a drug) with a binding partner (eg, a receptor). Another example includes monitoring drug-receptor, protein-protein, protein-DNA, DNA-DNA, DNA-RNA and protein-carbohydrate interactions using infrared thermography.
[0054]
Example 10
Monitoring of catalysts on inert solid surfaces (eg, Combi-Chem beads)
Infrared thermography as described in the present invention can also be used to measure heat production in a highly defined cell-free system. Catalytic agents are often immobilized and characterized by binding to an inert solid surface such as CombiChem beads (Borman, Chem. Eng. News 74:37 (1996)). Under such circumstances, the present invention was used to carry out a thermal analysis of a catalytic reaction on combichem beads. Catalytically active combichem beads were analyzed while immersed in solvent in a 25 mL beaker or 96-well microtiter plate. FIG. 12 shows that the heat production is localized and output in the beaker region containing the beads (FIG. 12A, arrow) (temperature difference 0.3 ° C.) or localized in the wells of the microtiter plate containing the active beads, No localization in wells containing active beads (FIG. 12B). Thus, infrared thermography can be used to measure rapid catalytic activity by non-invasive and non-destructive methods.
[0055]
Example 11
Thermographic analysis of aerosol induced cooling
In an aerosol system for measuring an inhalant dosage used for drug supply, the temperature in a device chamber is lowered in a delivery manner. This effect is a major obstacle to the efficiency of drug delivery. Lowering the chamber temperature often causes drug crystallization in the MDI chamber and stem, resulting in inefficient drug delivery. Infrared thermal imaging can be used to monitor the temperature loss of drug delivery, as well as to test improved devices that reduce or ameliorate this problem. That is, infrared thermography was tested as a measure of MDI actuation induction chamber cooling. FIG. 13A shows the rapid thermal profile of the MDI when operating 0, 1, or 5 times continuously. Thermal images were analyzed for temperature fluctuations in the following three regions: region 1-the surface of the valve stem / expansion chamber; region 2-the central surface of the canister; region 3-the interior of the canister head with the measurement chamber. FIG. 13B, which illustrates the change over time in the region temperature, shows that the temperature drop is operation dependent, especially in the valve stem / expansion chamber. The temperature repeatedly decreases with each operation.
[0056]
Furthermore, it is often difficult to measure and quantify the bioavailability and biological activity of inhalants in animals and humans. Assays that can measure the bioavailability of an inhalant include those that measure the uptake of radiolabeled inhalant into selected tissues (eg, lungs). Infrared thermography provides a non-invasive method for measuring the bioavailability / bioactivity of inhaled compounds. That is, infrared thermography was used to measure inhalant-induced thermal activity in the thoracic region of nude mice. FIG. 13C shows the thermal profile of nude mice treated with excipients or inhalants containing albuterol. The illustrated example of the torso temperature shows that the torso temperature increases 2.5 minutes after administration of the inhalant (FIG. 13D). Taken together, these results indicate that infrared thermography can be used for inhalation delivery in devices and for non-invasive measurement of inhalation bioavailability / bioactivity in animal models.
[0057]
Example 12
Infrared thermography is β ₃ -Can be used to measure IBAT thermogenesis in mice treated with AR-agonists.
[0058]
Β for heat production in cultured adipocytes and CHO cells ₃ The stimulating ability of AR-agonists was shown and discussed in Examples 3 and 6 above and in FIGS. 5, 8 and 9. Catabolic agents (eg, β ₃ (AR-agonists), there are potential clinical applications suitable for the use of infrared thermography in animals. ₃ It is important to show if the AR-agonist inducing effect can be measured. 14A and 14B show that the infrared thermography is β ₃ It shows that it can be used to measure the dose-dependent and time-dependent increase in heat production of the interscapular brown adipose tissue area (IBAT) in animals to which an AR-agonist has been administered. By directly measuring serum glycerol in treated animals, β ₃ We tested the hypothesis that AR agonist-induced thermogenesis reflects increased catabolic activity. In treated mice, β ₃ AR agonist-induced thermogenesis correlated with increased serum glycerol (correlation coefficient = 0.92). Thus, it demonstrates the use of infrared thermography to monitor the in vivo effects of catabolic drugs in animals.
[0059]
Example 13
Thermographic analysis of ob / ob mice treated with monoamine reuptake inhibitors at various times and drug concentrations
Monoamine reuptake inhibitors are a class of drugs that stimulate catabolic activity (Stock, Int. J. Obesity 21: 525-29 (1997)). The effect of GW473559A, a representative monoamine reuptake inhibitor, was monitored on treated mice using infrared thermography. FIG. 15 shows that infrared thermography measures the dose-dependent (FIG. 15A) and time-dependent (FIG. 15B) increase in IBAT thermogenesis in ob / ob mice treated with GW473559A. Thus, infrared thermography provides a non-invasive, sensitive and powerful alternative assay for compound bioavailability and activity.
[0060]
Example 14
Thermographic analysis as an alternative assay to predict weight change
Β for the treatment of diabetes or obesity ₃ AR-agonist treatment requires long-term treatment (weeks to months). β ₃ The desired result for AR-agonist treatment is weight loss. The data shown in FIG. 16 shows that infrared thermography can be used to predict weight loss as a result of drug treatment. Placebo or β for 2 weeks in AKR mice given high fat color ₃ An AR agonist (twice daily) was administered. During the test period, thermogenic activity was measured by infrared thermography in the interscapular area, and at the same time weight loss was measured. Weight loss at 2 weeks was correlated with interscapular interstitial heat production (r = 0.97). That is, infrared thermography can provide a non-invasive alternative assay suitable for both preclinical and clinical applications with respect to compound selection and evaluation of efficacy and efficacy.
[0061]
Drug treatment for weight loss in diabetic and obese patients is often a long-term treatment. Infrared thermography detects drug-induced changes in metabolic changes when the drug is administered. Changes in IBAT thermogenesis recorded by infrared thermography following treatment of animals with monoamine reuptake inhibitors were acute (treatment = 2 hours; r = 0.92) and chronic (treatment = 14 days, 2 Time; r = 0.94) Both treatment protocols were significantly correlated with% reduction in weight gain. Infrared thermography provides a non-invasive alternative assay for estimating weight loss, thereby eliminating laborious, long-term and costly weight loss tests.
[0062]
Example 15
Genetic effects on diet-induced fever
Much evidence has shown that metabolism is affected by genetic and environmental factors. For individuals with a genetic predisposition to diabetes, fat metabolism or obesity, diet is one of the environmental factors that alters metabolic rate. It is unclear whether thermography can be used to determine the effect of diet on changes in drug-induced thermogenesis in animals of various genotypes. Therefore, three types of suburban mice, AKR / J, C57BL / 6J and SWR / J, ₃ -Continued on a high and low fat diet for 14 weeks prior to treatment with the adreno receptor agonist BRL37344. The heat generated in the interscapular region was measured using infrared thermography before and 60 minutes after the treatment. As shown in FIG. 17, the obese mouse AKR / J showed a greater thermogenic response to BRL37344 when consuming a high fat diet. In contrast, SWR / J, an obese resistant mouse, showed a greater thermogenic response when fed a low fat diet. C57BL / 6J had little difference in thermogenic response between high or low fat diets. These results demonstrate that infrared thermography can be used to measure the effects of genotype and environmental changes (eg, diet) on drug-induced changes in thermogenesis. That is, the technology can be used to select compounds with desired properties, and for grading efficacy and efficacy, for a particular environment or genetic background. Similarly, the technology can be used to identify, grade, and select animal and environmental factors with desired properties for a particular drug candidate.
[0063]
Example 16
Infrared thermography of food-induced heat production in humans
Caloric intake has a dramatic and rapid effect on human metabolic activity. Thus, the heat production output behind the human tester will vary as a function of the date and time of food intake and the tester's pattern. This is indicated in the patient profile by monitoring the back temperature using infrared thermography before and after a meal. FIG. 18A shows a quantitative analysis of thermographic profiles for time points before and after lunch. FIG. 18B shows a summary of similar measurements (naked sinus delta T) made on two male subjects and one female subject before and after three separate lunches. This data set is consistent and reproducible, indicating that human infrared thermography can be used to monitor heat production. This method would be useful in many situations applicable to human patients, such as dietary adjustment, drug treatment, monitoring of drug / drug and drug / environment interactions. Based on the results using infrared thermography, changes in food and environment and drug use can be defined.
[0064]
Example 17
Infrared thermography of drug-induced thermogenesis in humans
Epinephrine, a sympathomimetic, has been reported to have potent fever and anti-obesity properties in rodents (Astrup et al., Am. J. Clin. Nutr. 42: 183-94 (1985)). . Measurement of weight loss and body composition are the first markers used to determine the efficacy of drug treatments for obesity. However, studies utilizing these markers tend to be time consuming, large and costly. To avoid these problems, alternative markers have been developed. Indirect calorimetry is used to determine resting metabolic rates, but is not widely used due to its complexity. Biochemical markers such as glucose, glycerol, non-esterified fatty acids, and triglycerides have been used, but are invasive. However, thermal imaging has not been used to measure the properties of endo-Fe phosphorus in humans.
[0065]
The effect of endoferrin on heat production in two human subjects was detected by infrared imaging 60 minutes after treatment with dephedrine at a dose of 0.6-0.7 mg / kg. FIG. 19 is a thermal image that determines the thermogenic response induced by ephedrine in subjects A and B (Delta T ° C. = 0.63 and 0.46, respectively). Thus, infrared thermography can be used as a non-invasive alternative assay to evaluate drug efficacy, efficacy, pharmacokinetics, and pharmacodynamics in clinical trials.
[0066]
Example 18
Infrared thermography of drug-drug interaction in db / db mice
The consequences of pharmacodynamic drug-drug interactions can vary, and range from life threatening conditions to life-saving therapies. Therefore, there is a great need to identify a method suitable for rapid evaluation of drug interactions. Since the administration of multiple drugs alters the metabolic rate, it is anticipated that additive, subtractive and / or synergistic effects of various drugs will exist on heat production in cultured cells and / or animals. You. It is therefore interesting to determine whether infrared thermography can be used to analyze the effect of mixing various drugs on in vivo heat production. In addition, the use of infrared thermography can identify and grade the efficacy, usefulness, and toxicity of the combination treatment in preclinical and clinical trials.
[0067]
GW1929 is an agent that improves the glycemic control in diabetic animals by activating the transcriptional activity of the ligand-activated nuclear receptor PPARγ. CGP12177A is a cell surface β ₃ -It is a therapeutic agent for obesity that acts through stimulation of adrenergic receptors (Kenalin, Lenhard and Paulik, CUrr Prot Pharm; 1 (unit 4.6): 1-36 (1998)). Because many diabetics are obese, it is interesting to determine which of these two agents (ie, GW1929 and CGP12177A) has any pharmacodynamic interaction. That is, db / db mice were treated for 2 weeks with or without GW1929, and the heat generated in the interscapular region was measured using infrared thermography. As shown in FIG. 20, animals treated with both GW1929 and CGP12177A had a greater thermogenic response than animals treated with either alone. Thus, these results demonstrated the use of infrared thermography to measure the strength of pharmacodynamic drug-drug interactions that alter thermogenesis.
[0068]
Example 19
Thermographic analysis of tissue angiogenesis with VEGF
Tumors are areas of actively proliferating and metabolizing tissue supported by local angiogenesis that enhance tumor growth. Thermography can detect increased heat production associated with tumors, including those associated with tissue angiogenesis. Vascular epidermal growth factor (VEGF) is found in tumor tissue and its presence is associated with increased thermogenesis in the tissue where it is localized. That is, the usefulness of thermography in tumor detection and angiogenesis was demonstrated using nude mice with exposed epithelial vasculature. Nude mice were injected with VEGF peptide or control, followed by thermographic imaging of both injection sites. FIG. 21 shows that the thermographic images show that heat production increases at the VEGF injection local site but not at the control injection local site. That is, the effects of agents that alter tissue angiogenesis can be monitored using infrared thermography.
[0069]
Example 20
Tumor temperature monitoring by infrared thermography
Tumor temperature is an indicator of the rate of metabolism in a tumor. Tumors depend on the presence of VEGF for maximal metabolic activity, and tumor temperature reflects changes in VEGF availability. This association is shown by thermographic analysis of tumor-bearing mice treated with either anti-VEGF antibodies or non-specific anti-IgG antibodies. FIG. 22 depicts a quantitative thermographic analysis showing that tumor temperature decreases when VEGF is neutralized by the presence of anti-VEGF antibodies. That is, infrared imaging can be used to monitor the effects of cancer treatment and as an aid in developing anticancer drugs.
[0070]
Example 21
Monitoring baldness (hair loss) by infrared thermography
Hair loss can be the result of undesirable side effects of various treatments (eg, cancer patient radiation therapy, surgery, etc.) and occurs spontaneously with aging, but can be restored to hair growth by surgery or pharmacological intervention. It is interesting to determine if infrared thermography can be used to measure hair loss, as hair provides adiabatic action against heat loss and helps maintain constant body temperature. One of the reasons for the lack of progress in treating and preventing baldness induced by chemotherapy is due to the lack of reproducible animal models and methods for quantitatively measuring hair loss. To determine whether infrared imaging can quantitatively measure chemotherapy-induced hair loss, neonatal rats treated with a known carcinogen, etoposide, in utero were subjected to thermal profile analysis for hair loss. FIG. 23 shows a thermal image (FIG. 23A) and a quantitative analysis (FIG. 23B) showing that the thermal activity is increased both on the whole surface and on the back as a result of hair loss. Thus, these results demonstrate that infrared thermography can generally help identify hair loss and hair loss treatments or measure hair loss as a way to identify drugs that cause hair loss as a side effect (eg, toxicity screening). .
[0071]
Example 22
Thermography after drug treatment suitable for male erectile dysfunction
Male erectile dysfunction (MED) is treated with drugs that increase blood flow to the genitals. Increased local heat production is accompanied by increased local blood flow. One drug that works in this manner and treats MED is Pinacidil. FIG. 24 shows that infrared thermography detects that pinacidil increases rat reproductive tract heat production 2 hours after either 3.0 or 0.3 mg pinacidil / kg was administered. That is, infrared thermography provides a quantitative and non-invasive method suitable for identifying and evaluating drug candidates to which MED is applied, and for identifying candidates that cause erection as a side effect.
[0072]
Example 23
Monitoring the effect of anti-inflammatory drugs by thermography
Arthritis is a disease characterized by joint inflammation and can be treated with anti-inflammatory agents. Since the inflammatory response is accompanied by an increase in heat production at the reaction site, the effects of arthritis and therapeutic agents for arthritis can be monitored by thermography. To demonstrate this application, a normal animal was injected into one leg with peptidoglycan polysaccharide (PGPS) for two weeks to establish an arthritis model. The other leg was untreated. Infrared thermography after arthritis-induced treatment showed higher levels of heat production in the treated leg compared to the untreated leg (FIG. 25A), and infrared thermography was used to monitor the ability of the agent to cause inflammation. It was suggested that it could be done. Subsequent treatment with the anti-inflammatory agent, prenisolone, reversed the increase in thermogenesis, and the thermographic profiles of both feet were similar after treatment (FIG. 25B). Thus, infrared thermography is a useful tool suitable for monitoring inflammatory responses and the effects of anti-inflammatory agents, and for evaluating the effects of drug candidates on the treatment of arthritis applications.
[0073]
All references cited above are referenced and incorporated herein in their entirety.
[0074]
Those skilled in the art, upon reading this disclosure, will recognize that various changes in shape and detail can be made without departing from the true scope of the invention.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of an apparatus suitable for use in imaging infrared thermogenesis in cultured cells. 1 = infrared camera; 2 = cell culture incubator (37 +/- 0.02 ° C); 3 = isothermal chamber; 4 = plate holder; 5 = computer interface.
FIG. 2 is a schematic diagram of an apparatus suitable for use in thermogenetic imaging in live animals. 1 = infrared camera; 2 = isothermal chamber; 3 = heating pad (37 ° C); 4 = computer interface; 5 = interscapular brown adipose tissue (IBAT).
FIG. 3A. Infrared thermographic data of rotenone and FCCP treated yeast (FIG. 3A; 384-well plate) as indicated by the gray scale index or the numerical steps shown (rotenone = − ● −; FCCP). =-▲-).
FIG. 3B. Infrared thermographic data of human adipocyte cultures (FIG. 3B; 96-well plate) (rotenone = − ● −; FCCP = − ▲ −) as indicated by the gray scale index or the numerical steps shown. ).
FIG. 4A shows an example of a thermographic analysis of yeast cells expressing uncoupling protein 2 (UCP2) (FIG. 4A).
FIG. 4B. Example of thermographic analysis of yeast cells expressing uncoupling protein 2 (UCP2) (FIG. 4B).
FIG. 4C is an example of molecular analysis of UCP2 expression in yeast cells expressing uncoupling protein 2 (UCP2) (FIG. 4C).
FIG. 5. β in the presence of forskolin (-■-) or isoproterenol (-●-) ₃ -Infrared thermographic analysis of thermogenesis in Chinese hamster ovary cells (CHO) overexpressing the AR receptor (blank =--).
FIG. 6: Analysis of triglyceride accumulation (− ● −) and heat production (− ▲ −) in adipocytes.
FIG. 7 is an infrared thermographic image of differentiating adipocytes showing a dose response curve for multiple peroxisome proliferator-activated receptors γ (PPARγ) in the presence of insulin and 9-cis retinoic acid.
FIG. 8A. Summary of infrared thermographic analysis of the inner scapula of ob / ob mice treated with GW1929, a PPARγ agonist (FIG. 8A).
8B: Correlation between thermographic signal in ob / ob mice treated with GW1929 which is a PPARγ agonist and the minimum effective dose (MED) of various PPARγ agonists (FIG. 8B) (R2 = 0.954) Illustration.
FIG. 9 shows examples of the rotenone sensitivity of β-AR agonist (50 nM) -induced thermogenesis in adipocytes.
FIG. 10 shows an example of infrared thermographic analysis of primary human epithelial cells (HUVEC cells) at 10 minutes in the presence (solid gray line) and absence (dashed line) of vascular endothelial growth factor (VEGF).
FIG. 11 is an infrared thermographic analysis of the chemical reaction after addition of NaOH to HCl.
FIG. 12A. Combi-Chem beads in a 25 ml beaker (FIG. 12A) (1 = bulk solvent); 2 = active beads; 3 = O-ring) or 96-well microtiter plate (FIG. 12B; inert and active beads). Infrared thermographic analysis of the catalytic reaction above.
FIG. 12B: Combi-Chem beads in a 25 ml beaker (FIG. 12A) (1 = bulk solvent); 2 = active beads; 3 = O-ring) or 96-well microtiter plate (FIG. 12B; inert and active beads). Infrared thermographic analysis of the catalytic reaction above.
FIG. 13A. Infrared thermographic analysis (FIG. 13B) of a metered dose aspiration (MDI) device (FIG. 13A; runs 0, 1, and 5) during and after 5 consecutive runs. Example of infrared thermographic analysis of nude mice treated with the inhalant, albuterol (FIG. 13C), followed by quantitative thoracic area analysis of the kinetics of albuterol activity (FIG. 13D).
FIG. 13B. Infrared thermographic analysis (FIG. 13B) of a metered dose aspiration (MDI) device (FIG. 13A; runs 0, 1, and 5) during and after 5 consecutive runs. Example of infrared thermographic analysis of nude mice treated with the inhalant, albuterol (FIG. 13C), followed by quantitative thoracic area analysis of the kinetics of albuterol activity (FIG. 13D).
FIG. 13C. Infrared thermographic analysis (FIG. 13B) of a metered dose aspiration (MDI) device (FIG. 13A; runs 0, 1, and 5) during and after 5 consecutive runs. Example of infrared thermographic analysis of nude mice treated with the inhalant, albuterol (FIG. 13C), followed by quantitative thoracic area analysis of the kinetics of albuterol activity (FIG. 13D).
FIG. 13D. Infrared thermographic analysis (FIG. 13B) of a metered dose aspiration (MDI) device (FIG. 13A; runs 0, 1, and 5) during and after 5 consecutive runs. Example of infrared thermographic analysis of nude mice treated with the inhalant, albuterol (FIG. 13C), followed by quantitative thoracic area analysis of the kinetics of albuterol activity (FIG. 13D).
FIG. 14A shows β ₃ FIG. 10 shows dose-response curves and kinetic data after treatment with an AR agonist (1.0 mg / kg =-●-; 0.1 mg / kg =-■-; 0.01 mg / kg =-▲-); Infrared thermographic analysis of heat production in the scapula of mice.
FIG. 14B shows β ₃ FIG. 10 shows dose-response curves and kinetic data after treatment with an AR agonist (1.0 mg / kg =-●-; 0.1 mg / kg =-■-; 0.01 mg / kg =-▲-); Example of infrared thermographic analysis of heat production in the scapula of mice.
FIG. 15A. Cough guy thermographic analysis of interscapular fever in ob / ob mice--dose-response (FIG. 15A) and kinetic data (FIG. 15B) (10.0 mg- ●) after treatment with GW73559A, a monoamine reuptake inhibitor. −; 5.0 mg / kg = − ■ −; 1.0 mg / kg = − ▲ −).
FIG. 15B. Cough guy thermographic analysis of interscapular fever in ob / ob mice--dose-response (FIG. 15A) and kinetic data (FIG. 15B) (FIG. 15B) after treatment with GW73559A, a monoamine reuptake inhibitor. −; 5.0 mg / kg = − ■ −; 1.0 mg / kg = − ▲ −).
FIG. 16 ₃ -Graph showing the correlation between interscapular heat production and weight loss in AR agonist-treated mice (IBAT fever = bar, 2-week weight loss =-●-) (R2 = 0.97)
FIG. 17. β in a 19-week-old rodent model ₃ -Graph showing the use of infrared thermography to characterize the genetic background and food effects on transmitted thermogenesis (low fat diet = solid gray bar; high fat diet = hatched bar (animal fed 14 weeks).
FIG. 18A. Infrared thermographic analysis of diet-induced thermogenesis in humans. FIG. 18A shows the change over time in the back temperature.
FIG. 18B. Infrared thermographic analysis of diet-induced thermogenesis in humans. FIG. 18B shows the average difference in back temperature between two men and two women before and after lunch (mean value = 3-5 days / subject).
FIG. 19: Ephedrine (left = 0 min; right = 60 min) (dose = 0.6-0.7 mg / kg-delta T ° C. (entire back = 0.63 for subject A, 0.46 for subject B) ) Example of infrared thermographic analysis of drug-induced thermogenesis in treated humans (Subject A and Tester B).
FIG. 20. Two drugs, β, affect interscapular fever in ob / ob mice ₃ Characterization by infrared thermography of the interaction between the AR agonist CGP12177 (1 mg / kg-ip injection) and the PPARγ agonist GW1929 (1 = interscapular region).
FIG. 21. Infrared thermographic analysis of VEGF peptide-induced thermogenic activity in nude mice (1 = VEGF; 2 = control).
FIG. 22. Infrared thermographic analysis of the effect of anti-VEGF antibody on tumor temperature.
FIG. 23A. Infrared thermographic analysis of the effect of intrauterine etoposide treatment (6 mg / kg) on hair loss in neonatal mice. FIG. 23A shows thermographic images obtained on days 0, 3, and 5.
FIG. 23B. Infrared thermographic analysis of the effect of intrauterine etoposide treatment (6 mg / kg) on hair loss in neonatal mice. FIG. 23B shows the time course of torsodelta T ° C. after dosing (back = white bar, front = solid line, gray bar).
FIG. 24. Infrared thermographic analysis of Pinacidil-induced changes in genital fever of rats (2 hours after PO administration).
FIG. 25A Proteoglycan polysaccharide (PGPS) induced (FIG. 25A; 1 = non-articular leg; 2 = articular leg) and 6 mg / kg oral prednisolone (FIG. 25B) (vehicle = HPMC + 0.1% TW80; oral) Infrared thermographic analysis of leg inflammation.
FIG. 25B Proteoglycan polysaccharide (PGPS) induced (FIG. 25A; 1 = non-articular leg; 2 = articular leg) and 6 mg / kg oral prednisolone (FIG. 25B) (vehicle = HPMC + 0.1% TW80; oral) Infrared thermographic analysis of leg inflammation.

Claims (7)

A drug discovery method for screening test substances for their ability to produce a thermodynamic change in a cell-free sample,
i) measuring the temperature of the sample using infrared thermography;
ii) contacting the test substance with the sample;
iii) utilizing infrared thermography to measure the temperature of the sample resulting from step ii);
iv) comparing the temperature obtained in step (i) with the temperature obtained in step (iii),
A method wherein the temperature difference between the temperature obtained in step (i) and the temperature obtained in step (iii) indicates that the test substance causes a thermodynamic change in the sample,
The sample comprises an organic compound and a thermodynamic change occurs in the sample when the test substance binds to the organic compound, wherein the measuring steps (i) and (iii) are in the range of 3-15 microns. A method performed using infrared thermography at a specific wavelength or within a specific wavelength range. 2. The method according to claim 1, wherein the organic compound is a protein, carbohydrate, lipid or nucleic acid. 3. The method according to claim 2, wherein said organic compound is a protein. 4. The method according to claim 3, wherein said protein is a receptor. Step (iii) comprises measuring the temperature of the sample resulting from step (ii) at a plurality of time points, step (iv) comprising the temperature obtained in step (i) and the temperature obtained in step (iii). Comparing the temperature at each of the time points, wherein the difference between the temperature obtained in step (i) and the temperature obtained in step (iii) in at least one of the time points causes the test substance to be in the sample. 2. The method of claim 1, wherein the method is shown to cause a thermodynamic change. The method according to claim 1, wherein the infrared thermography is an infrared imaging thermography. A method of screening a test substance for its ability to produce a thermodynamic change in a cell-free sample,
i) measuring the temperature of the sample using infrared thermography;
ii) contacting the test substance with the sample;
iii) utilizing infrared thermography to measure the temperature of the sample resulting from step ii);
iv) comparing the temperature obtained in step (i) with the temperature obtained in step (iii),
A method wherein the temperature difference between the temperature obtained in step (i) and the temperature obtained in step (iii) indicates that the test substance causes a thermodynamic change in the sample,
The sample resulting from step (ii) contains both the ligand and the binding partner of the binding pair, and when the test substance inhibits the binding of the member of the binding pair, it does not contain the test substance and thus the member of the binding pair A screening method that produces a difference between the temperature obtained in step (i) and the temperature obtained in step (iii) as compared to a control that binds to each other .

Images