JP2002524481A5 - - Google Patents

Description

従って、スクアラミンはＮＨＥを非アロステリック動力学で阻害する（即ち、非古典的アロステリック阻害）。追加の試験において、スクアラミン（１時間の前処理において）はウサギＮＨＥ３のＶ_ｍａｘを濃度依存的様式で減少させることが観察された（１、５および７μｇスクアラミン／ｍｌで各々１３％、４７％および５７％）。Ｖ_ｍａｘに対するこの観察されたスクアラミン効果は時間依存性であり、最大効果は暴露１時間で生じた。観察された効果は、培地から細胞を除去後３時間以内であれば完全に可逆的であった。
【００５８】
ＮＨＥ３に対するスクアラミンの効果に関連する試験結果の点から、ＮＨＥ３は非刺激細胞のホメオスタシスの維持に重要であると信じられる。さらに、スクアラミンによる細胞活性化、特に病理学的血管形成間の（腫瘍増殖間のような）新規血管の形成に関与する内皮細胞または前駆細胞の活性化の妨害は、スクアラミンが腫瘍増殖を阻害する機構であると信じられる。
【００５９】
さらに出願者はスクアラミンが内皮細胞の形を変化させることを観察している。このことは細胞容量および形を調節する輸送タンパク質がスクアラミンの標的であろうことを示唆している。
【００６０】
スクアラミンの追加の試験はスクアラミンが刷子縁膜ベシクル（ＢＢＭＶ）ＮＨＥを、組織がスクアラミンで前処理されていた場合にのみ阻害したことを示している（３０分暴露で５１％阻害）。エックスチェンジャー活性測定間の、ＰＳ１２０線維芽細胞へのスクアラミンの直接添加は何の影響も与えなかった。
Ｄ． スクアラミンの薬物動力学
スクアラミンの薬物動力学研究が体内でのスクアラミンの滞留時間を確認するために実施された。図６ａから６ｃはスクアラミンが皮下（５０ｍｇ／ｋｇ、図６ａ）、腹腔内（用量２４０μｇ；１０ｍｇ／ｋｇ、図６ｂ）および静脈内（１０ｍｇ／ｋｇ、図６ｃ）投与された場合の試験結果を示している。静脈内（図６ｃ）に投与された場合のスクアラミンは受容可能であったが（３５分）、腹腔内（図６ｂ、半減期＝１７２分）および皮下（図６ａ、半減期＝５．６時間）投与の場合は幾分高めであった。
【００６１】
これらのスクアラミン半減期試験に加えて、静脈内投与後のマウス中のスクアラミン分布を確認するために試験を行った。図７はｉ．ｖ．投与２時間後のマウス組織中のスクアラミン分布を示している。いくらかのスクアラミンはほとんどの組織に含まれており、ほとんどのスクアラミンは肝臓および小腸に濃縮されていた。図７に示された試験は良好なスクアラミン分布を示している。しかしながら、注目されるのは脳組織中にはスクアラミンが少ないことである。このことから、スクアラミンは多分血液脳関門を通れないであろうことが結論された。脳腫瘍の処置において、スクアラミンは脳の内皮細胞に作用すると信じられるので、この様式では血液脳関門を通過する必要はない。
【００６２】
以下の実施例は本発明の方法におけるスクアラミンの抗血管新生を試験するために使用されたより詳細な実験を説明している。
ＩＩ．治療的投与および組成物
スクアラミンの投与様式は特定の治療使用に適するように選択されるであろう。投与様式には一般的に経皮、筋肉内、腹腔内、皮下、鼻孔内、吸入、リンパ節内、病巣内および経口経路が含まれるが、それらに制限されるわけではない。スクアラミン化合物は通常の経路、例えば、注入または大量注射により、または上皮または粘膜皮膚ライニングを通した吸収により（例えば、口内粘膜、直腸および腸粘膜その他）投与され、および他の生物学的に活性な薬剤と一緒に投与してもよい。投与は局所的でもまたは全身的でもよい。
【００６３】
本発明はまた活性成分としてスクアラミンを含んでいる医薬組成物も提供する。そのような組成物は治療的に有効量のスクアラミンおよび医薬として受容可能な担体または賦形剤を含んでいる。そのような担体の例としては、食塩水、緩衝化食塩水、デキストロース、水、イントラリピッドのような水中油微小乳剤、グリセロールおよびエタノールまたはそれらの組み合わせなどが含まれるが、それらに制限されるわけではない。医薬組成物の処方は投与様式に適するように選択されなければならない。
【００６４】
必要に応じ、医薬組成物は有効量の湿潤剤または乳化剤またはｐＨ緩衝化剤を含んでいてもよい。医薬組成物は液体溶液、懸濁液、乳剤、錠剤、ピル剤、カプセル、徐放性処方または散剤のような任意の形であろう。組成物はまた、トリグリセリドのような伝統的結合剤および担体を含む座剤として処方されてもよい。経口処方は、医薬品等級のマンニトール、ラクトース、デンプン、ステアリン酸マグネシウム、サッカリンナトリウム、セルロース、炭酸マグネシウムその他のような標準担体を含んでいるであろう。
【００６５】
種々の送達系が知られており、本発明の治療化合物を投与するために使用されるであろう、例えば、リポソーム中へのカプセル化、微粒子、腸溶性被覆系、微小カプセルなど。
【００６６】
一つの態様において、医薬組成物はルーチン的方法に従って処方され、ヒトへの静脈内投与に適した組成物が提供される。典型的には、静脈内投与のための組成物は５％デキストロースおよび滅菌水またはインターリピッドの溶液である。必要な場合、医薬組成物は可溶化剤および注射部位の痛みを軽減するための局所麻酔薬も含んでいてもよい。一般に、医薬組成物の成分は、活性薬剤の量を示しているアンプルまたはサシェットのような密封容器中、例えば、凍結乾燥粉末または水を含んでいない溶液として別々にまたは一緒に混合されて単一剤形として供給される。医薬組成物が注入により投与されるべき場合、滅菌医薬用水、デキストロース、塩または他の医薬として受容可能な担体を含んでいる注入瓶に調合されるであろう。医薬組成物が注射により投与される場合、成分が投与に先立って混合されるように、注射のための滅菌水または塩溶液のアンプルが提供されるであろう。
【００６７】
特定の障害または状態の処置に有効であろう治療的化合物（即ち、活性成分）の量は、障害または状態の性質に依存するであろうし、当業者には既知の標準臨床技術により決定できる。処方で用いられるべき正確な用量もまた、投与経路および障害または状態の重度に依存するであろうから、実施者および患者の環境の判断に従って決定されるべきである。有効治療量はインビトロまたは動物モデル試験系から誘導される用量−応答曲線の外挿から推定されるであろう。
【００６８】
静脈内投与に適した用量は一般的に、体重キログラム当たり約１マイクログラムから４０ミリグラムの活性化合物である。鼻孔内投与に適した用量は一般的に、約０．０１ｍｇ／ｋｇ体重から２０ｍｇ／ｋｇ体重である。経口投与に適した用量は一般的に、体重キログラム当たり約５００マイクログラムから８００ミリグラムであり、好適には約１−２００ｍｇ／ｋｇ体重である。坐剤は一般的に、活性成分として０．５から１０重量％のスクアラミンを含んでいる。経口処方は好適には１０％から９５％の活性成分を含んでいる。
【００６９】
抗血管新生または細胞傷害性薬剤としてまたは癌治療におけるスクアラミンの使用に対しては、例示的用量は約０．０１ｍｇ／ｋｇ体重から約１００ｍｇ／ｋｇ体重である。好適な用量は０．１から４０ｍｇ／ｋｇ体重である。
【００７０】
本発明はまた、本発明の医薬組成物で満たされた一つまたはそれ以上の容器を含んでいる医薬パックまたはキットも含んでいる。そのような容器に付随するものは製造を取り締まっている政府機関により規定された形の注意書き、医薬または生物学的生産物の使用または販売（その注意書きは製造元の代理店による認証を反映する）、ヒト投与のための使用または販売であろう。
【００７１】
本発明に従って使用される通常の細胞傷害性化学品および抗ホルモン化合物は、当業者には既知である任意の適した形で存在する。これらの化学化合物もまた、当業者には既知である任意の適した手段で投与されるであろう（経口、皮下、静脈内、腹腔内、リンパ節内および筋肉内）。
【００７２】
本発明の説明において、出願者は本発明が働く様式においてどのようにおよびなぜ本発明が働いているのかの説明においてある種の理論を述べてきた。これらの理論は情報を伝える目的でのみ示されたものである。出願者は特定の化学的または物理的機構または効能の理論に縛られてはいない。
【００７３】
本発明は種々の特定の好適な態様および特定の実施例に関して説明されてきたが、当業者は付随する請求の範囲に定義されているような本発明の精神および範囲から離れることなく種々の変更および変形が可能であることを認識するであろう。本明細書を通して参照されたすべての特許、出版物および参考文献は、全文のまま本明細書において援用される。
実施例１ウサギ角膜マイクロポケットアッセイ化合物が抗血管形成性であるか否かを調べる場合、ウサギ角膜マイクロポケットアッセイは容認された標準試験である。この試験では、切開はウサギの１眼に行い、刺激物は切開部位に置く。該刺激物は、普段は無血管の角膜部分での血管形成誘導に使用する。例として、重合体マトリックス中の充実性腫瘍を刺激物として角膜内に置くことができる。なぜなら、該腫瘍は新規毛細血管形成を刺激する多数の血管形成促進因子を放出するであろうからである。腫瘍‐由来血管形成促進因子は眼の角膜縁において内皮細胞を刺激し、刺激物に向かって血管形成を開始する。第二の重合体ペレット（例えば、エチレン／酢酸ビニル共重合体）は角膜縁と刺激物との間に置く。この重合体ペレットは空（陰性対照試験ペレット）であるか、または抗血管形成性について試験する化合物を含んでいる。該重合体ペレットは、試験物質を調節放出させるために使用する。ウサギ角膜における無血管角膜バックグラウンドのため、結果を視覚的に定性的に評価できる。さらに、血管数を計数でき、そしてそれらの長さなどを測定できるため、結果のより定量的な評価を提供できる。
【００７４】
ＶＸ２ウサギ癌腫を血管形成刺激物として作用するように、普段は無血管部位の２６のウサギ眼に移植した。スクアラミンを調節放出性エチレン／酢酸ビニル共重合体（重量で、２０％スクアラミンおよび８０％重合体）に取り込ませた。負荷した重合体ペレットを１３の角膜に置き、スクアラミンの持続性局所放出剤とした。重合体ブランクを対照として残りの１３眼に提供した。このように、それぞれのウサギの１眼をスクアラミン試験眼として用い、同じウサギの他の眼を対照眼として使用した。それらの眼は腫瘍移植後３週間、細隙灯立体顕微鏡を使用して毎週検査し、血管形成指数（“ＡＩ”）を計算した（この計算は図８と関連して、以下に詳細に記載されるであろう）。スクアラミン負荷した重合体は、ｉｎ ｖｉｔｒｏで処理期間中、活性スクアラミンを放出することが認められた。試験後、角膜を組織学的に検査した。
この試験を使用して、スクアラミンが腫瘍誘発毛細血管形成の強力な阻害剤であることが認められた。対照角膜と比較して、スクアラミン処理した角膜で認められた血管数はより少なく、これらの血管は一般に対照角膜の血管と比較して短かった。
次に角膜のいくつかを切り分け、腫瘍細胞自体へのスクアラミンの作用を観察した。非処理対照角膜には、腫瘍内および腫瘍に隣接して多くの血管が見られた。スクアラミン処理した角膜中の腫瘍は依然として生存可能であった（すなわち、腫瘍は死んでいなかった）が、腫瘍と関連した血管系は本質的に見られなかった。従って、スクアラミン‐処理腫瘍では対応する対照腫瘍切片と比べ、血管分布の著しい減少が見られた。これらの発見はスクアラミンが血管に対し作用し、腫瘍自体に作用しないことを示唆した。
【００７５】
図８はウサギ角膜マイクロポケットアッセイの結果をグラフに示す。定量的評価を提供するために、それぞれの眼の血管形成指数（“ＡＩ”）を決定した。血管形成指数を決定するために、初めに１眼の血管密度（“Ｄ_{ｖｅｓｓｅｌ}”）を以下のように０〜３等級に評価した：
【００７６】
【表２】

次にそれぞれの角膜の血管長（“Ｌ_{ｖｅｓｓｅｌ}”）を測定した。血管長は、角膜縁から最も成長した血管の遠位縁まで測定した最長血管の長さである。次いで、これらの測定値から以下の方程式：ＡＩ＝Ｄ_{ｖｅｓｓｅｌ}ｘＬ_{ｖｅｓｓｅｌ}に従って血管形成指数を決定した。
図８にウサギ角膜マイクロポケットアッセイにおけるそれぞれの角膜群（スクアラミン処理および非処理）の平均血管形成指数を示す。図に示すように、スクアラミンは新規血管成長を強く阻害した。スクアラミン処理眼は、非処理眼に比べ著しく低いＡＩ値を示した（１４日目で３７％低下（ｐ＝０．０５，Ｗｉｌｃｏｘｏｎ ランクサムテスト（ｒａｎｋ ｓｕｍ ｔｅｓｔ）および２１日目で４３％低下（ｐ＜０．０１））。このデータはスクアラミンが長期間にわたり、新規血管あるいは毛細血管形成を阻害することを説明する。より具体的には、スクアラミンは３週間後でさえ強い抗血管形成活性を示す。
実施例２
スクアラミンは炎症を起こさない
ウサギ角膜マイクロポケットアッセイ試験において、もしウサギ角膜が炎症を起こすと、この炎症は角膜の新規血管形成を誘導することができる。かかる炎症は試験結果をゆがめるであろう。従って、スクアラミンが角膜における炎症反応に、本質的におよびそれ自体関与するか否かを調べるために試験を行った。いくつかの非‐生物吸収性エチレン／酢酸ビニル共重合体ペレットを異なる濃度のスクアラミン、すなわち、重量で２％、１０％、および２０％のスクアラミンで負荷した。次に、これらのペレットを血管形成刺激物を含んでいないウサギ角膜に置いた。スクアラミンはこれらの濃度のいずれにおいても炎症を誘導しなかった。従って、スクアラミンは角膜に炎症を起こすことにより新規血管形成を誘導しない。
実施例３
脳腫瘍処置におけるスクアラミンの使用
ウサギ角膜マイクロポケットアッセイ試験の結果は、スクアラミンが血管新生を阻害する強力な抗血管形成薬であってもよいことを出願者に示唆した。脳における充実性腫瘍の急激な成長は血管新生に依存することを認識して、出願者は動物モデルにおける脳の充実性腫瘍の成長に対するスクアラミンの活性を評価した。
【００７７】
充実性脳腫瘍の中で、悪性グリオームは癌性腫瘍の最も一般的な形態である。これらの腫瘍は１５歳から３４歳までの青少年における３番目の主要な死因である。普段は静止性の脳および／またはＣＮＳ内皮細胞を増殖性および浸潤性の強い状態に誘導する能力により、悪性グリオームの性状を決定する。該グリオームは、血管形成誘導因子（“ＶＥＧＦ”）およびパラクリン的にＣＮＳ内皮細胞の誘導受容体を刺激する他の誘導因子を発現する（すなわち、ＶＥＧＦは腫瘍細胞から生じ、内皮細胞を刺激する）。続いてＣＮＳ内皮細胞は血管形成性浸潤を開始し、グリオームに栄養物を提供する。出願者は、グリオームに対するスクアラミンの抗血管形成活性を（１）内皮細胞のＶＥＧＦ‐媒介刺激を選択的に阻害する能力および（２）実験的マウスグリオームに対する作用により、試験した。
【００７８】
初めに、スクアラミンが内皮細胞に特異的に作用することを確かめるためにｉｎ ｖｉｔｒｏ試験を行った。図２に関連して記載したように、内皮細胞が血管形成の初期段階に関与することから、出願者はかかる細胞を使用した。具体的に言うと、腫瘍血管形成は連続し、重複した一連の段階である。初めに、内皮細胞は活性化され、増殖する。次に、蛋白分解酵素が生成され、該細胞が遊走する。続いて新規基底膜が生成される。このようにして、新規血管が生成され腫瘍サイズが増大する。
【００７９】
このｉｎ ｖｉｔｒｏ解析には、以下の細胞系：（ａ）ウシ網膜内皮細胞；（ｂ）９ＬおよびＣ６ラットグリオーム細胞；（ｃ）ヒトＨ８０グリオーム細胞；および（ｄ） ＶＸ２ウサギ癌腫細胞（先に記載したウサギ角膜マイクロポケットアッセイ試験で移植した腫瘍と同じ型である）を使用した。この解析で使用した内皮マイトジェンは２０ｎｇ／ｍｌのＶＥＧＦであった。
【００８０】
該細胞は最適増殖培地を含む組織培養平板に一晩付着させた。付着後、該細胞を溶媒のみ、または濃度を変えた（０，１０，２０，３０，６０，および９０μｇスクアラミン／ｍｌ）スクアラミンに暴露した。細胞増殖は、３日間毎日コールターカウンター（Ｃｏｕｌｔｅｒ Ｃｏｕｎｔｅｒ）を使用して計数した。１ウェルにつき合計１０，０００細胞を平板培養し、それぞれの実験濃度の四重試験を行った。その後、結果を平均した。ウシ網膜内皮細胞は増殖させ、スクアラミン処置前にヒト組み換え型ＶＥＧＦ２０ｎｇ／ｍｌを添加して細胞増殖を測定すること以外は、他の細胞系と同様の方法で処置した。
【００８１】
３０μｇ／ｍｌまでのスクアラミンに２４および４８時間暴露後のすべての腫瘍細胞系および、ＶＥＧＦで処置しない内皮細胞による細胞増殖は統計的に変化が見られなかった。しかしＶＥＧＦ‐刺激内皮細胞の増殖は、上記と同じ時間のスクアラミン暴露により濃度依存的に有意に低下した。内皮細胞増殖阻害百分率（％Ｉ）は以下の方程式：（対照サンプルの細胞数―実験サンプルの細胞数）ｘ１００＝％Ｉ（対照サンプルの細胞数）
により決定した。
以下の表はＶＥＧＦ‐刺激内皮細胞系の４８時間における結果を示す。
【００８２】
【表３】

付加的なデータは図９で説明する。この図は、３０μｇ／ｍｌスクアラミンのｉｎ ｖｉｔｒｏ適用後、１，２，および３日目の対照群増殖の百分率としての各種細胞系の増殖を示す。図９に示すように、増殖はＶＥＧＦ‐刺激内皮細胞で特に低下するが、他の細胞系（Ｈ８０，Ｃ６，およびＶＸ２）の増殖は著しい影響を受けなかった。
【００８３】
この情報に基づき、出願者はスクアラミンがｉｎ ｖｉｔｒｏで劇的および特異的に内皮細胞のＶＥＧＦ‐刺激増殖を阻害すると結論した。従って、スクアラミンは腫瘍誘発血管形成の強力な阻害剤であり、そしてこの作用はＶＥＧＦに誘導される内皮細胞増殖の特異的阻害により促進されると思われる。従って、腫瘍特異的抗血管形成療法のための、腫瘍誘導血管新生の減少または縮小にスクアラミンは十分適していると考えられる。
【００８４】
内皮細胞のＶＥＧＦ‐刺激増殖の阻害に加え、スクアラミンはまた、ｂ‐ＦＧＦ、ＰＤＧＦ_ｂｂ、細胞分散因子（ＨＧＦまたは肝細胞増殖因子）、ならし腫瘍培地、およびヒト脳嚢胞液により誘導されたヒト脳毛細血管内皮細胞の増殖刺激を妨害することが認められてきた。従って、腫瘍は種々の異なる増殖因子を産出し、スクアラミンはそれらのいくつかに阻害作用を持っている。
これらの試験結果から、出願者は脳腫瘍の動物モデルにおいてスクアラミンを試験した。脳に局在する腫瘍に対するスクアラミンの作用を試験するために、ラットグリオームが維持されているラット側腹から生存グリオーム小切片（１ｍｍ^３）を採り、２群のラットの脳に移植した。従って、このモデルにおいて、ラット脳に置かれた時腫瘍は生存している。移植３日後、およびいくつかの血管系の発生後に、１群のラットにスクアラミン２０ｍｇ／ｋｇ／日（ｉ．ｐ．）を開始した。対照動物群（図１０の“ベヒクル対照”）にはキャリアベヒクルのみ（無スクアラミン）を与え、他の動物群はスクアラミンで処置した（図１０の“スクアラミン”）。図に示すように、スクアラミン処理動物は平均生存時間が３８％増加した（ｘ＝２４．９日対ｘ＝１８．０日）。図１０は、この動物モデルでスクアラミン処置ラットは一般に生存時間が増加することをさらに説明する。
【００８５】
スクアラミン毒性試験は別の動物モデルにおいて行った。慣用の細胞毒性化合物はかなり毒性である。例えば、慣用の化学療法剤である、ＢＣＮＵは累積的毒性効果を持つ。この理由から、該化合物は患者に１回だけ適用する。ＢＣＮＵの使用に関しては、先にＣａｌａｂｒｅｓｉ ｉｎ Ｍｅｄｉｃａｌ Ｏｎｃｏｌｏｇｙ３０４ページおよび３０５ページに記載されている。スクアラミンの毒性を試験するために、１群のラットにスクアラミン２０ｍｇ／ｋｇ／日（ｉ．ｐ．）を毎日、３０日以上投与し、投与後２００日まで飼育した。この研究に使用した動物は健康であり続けた。この結果は、スクアラミンがほとんどまたは全く毒性がないことを示唆する。
実施例４
スクアラミンと慣用の癌療法との使用
先に記載したように、スクアラミンは増殖因子との相互作用後の内皮細胞活性化を阻害することによる、血管形成過程の上流阻害剤である。血管形成阻害作用を持つため、スクアラミンは増殖に血管新生を必要とする充実性腫瘍の処置に効果的であることが示されてきた。出願者は、スクアラミン（上流血管形成阻害剤）処置とアルキル化剤を使用した慣用の癌療法とを組み合わせることにより腫瘍を処置した場合に有益な結果が得られるか否かを検討した。
ａ．スクアラミン９Ｌグリオーム側腹試験
４群のラット（全体で２０匹のＦｉｓｈｅｒ３４４ラット、２００ｇ）に９Ｌ神経膠肉腫（９Ｌグリオーム）１ｍｍ^３の移植片を０日目に皮下適用した。該腫瘍は、十分なスクアラミンの脳レベルによる合併症を避けるためにラット側腹に移植した。
ランダム化および処置は以下のスキームに従って５日目に開始した：
【００８６】
【表４】

^１本明細書で使用する“Ｂ．Ｉ．Ｄ．”なる用語は成分を１日２回適用する（それぞれの日の異なる２回の時間に１０ｍｇ／ｋｇを投与する）ことを表す。
【００８７】
腫瘍移植後２５または２６日目に、腫瘍サイズを直接測定した。腫瘍サイズ（すなわち、容積“Ｖ”）は測定した腫瘍の長さ（“Ｌ”）、幅（“Ｗ”）、および高さ（“Ｈ”）から決定する容積計算に基づいて推測した（Ｖ_{ｔｕｍｏｒ ｓｐｈｅｒｏｉｄ}＝０．５ｘＬｘＷｘＨ）。表５はその結果をまとめたものである。表５に示した腫瘍容積は、実験終了まで生存した動物の各処置群の平均腫瘍容積を表す。
【００８８】
【表５】

表５はスクアラミンとニトロソウレアＢＣＮＵの組み合わせ（４群）により腫瘍を処置したときに得られた都合のよい結果を説明する。この群ではスクアラミンおよびＢＣＮＵで共に処置したとき、平均腫瘍サイズの９９．８％縮小が観察された。表５はさらにスクアラミン単独（３群）が腫瘍の処置に効果的であることを示す。腫瘍サイズは対照群に比べ、３群では８１．７％縮小した。
【００８９】
出願者は、慣用の細胞毒性化合物と組み合わせてスクアラミンを使用すると脳腫瘍の広がりを遅らせるまたは停止させることができると結論する。腫瘍自体は縮小または壊死する。スクアラミンと細胞毒性化合物との組み合わせ処置は生存を延ばすであろうと期待される。従って、この処置は潜在的に脳腫瘍の処置に有効であろう。
ｂ．乳房腫瘍処置におけるスクアラミンの使用
ヒトＭＸ‐１乳癌細胞系は以前、単独または組み合わせて使用するシクロホスファミドおよび他の細胞毒性化学療法剤のｉｎ ｖｉｖｏ活性の証拠を提供するために使用してきた（Ｔ．Ｋｕｂｏｔａ，ｅｔ ａｌ．，Ｇａｎｎ ７４，４３７‐４４４（１９８３）；Ｅ．Ｋｏｂａｙａｓｉ，ｅｔ ａｌ．，Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ ５４，２４０４‐２４１０（１９９４）；Ｍ．‐Ｃ．Ｂｉｓｓｅｒｙ，ｅｔ ａｌ．，Ｓｅｍｉｎａｒｓ ｉｎ Ｏｎｃｏｌｏｇｙ ２２（Ｎｏ．， Ｓｕｐｐｌ．１３），３‐１６（１９９５））。これらの参考文献はそれぞれ本明細書中に参照としてそのまま援用する。シクロホスファミド２００ｍｇ／ｋｇの単回投与に続く補助的療法としてスクアラミンを検討した。シクロホスファミドは腫瘍移植後１４日目の腫瘍が６５〜１２５μｌになった時に注射した。シクロホスファミドはすべての動物に部分退行を起こし、ごく一部の動物に完全な退行を起こした。次に、動物を３つの処置群にランダム化した（それぞれｎ＝２７）：１週間に５回、ベヒクル（Ｉｎｔｒａｌｉｐｉｄ）のみ投与；Ｉｎｔｒａｌｉｐｉｄ中のスクアラミン１０ｍｇ／ｋｇ／日投与；およびＩｎｔｒａｌｉｐｉｄ中のスクアラミン２０ｍｇ／ｋｇ／日投与。実験中どの時点でも腫瘍が２グラムを越えた動物は安楽死させた。実験は、長期治療を経験したマウスのみがなお生存するということを確実にするために９０日間継続した。高用量のスクアラミン投与は、動物の体重低下および潜在毒性の懸念から中断したため、これらの動物は実験の終わりの８週間はスクアラミン投与を受けなかった。低用量のスクアラミン処置は、調べたすべての時点で乳房腫瘍の進行速度を著しく（Ｐ＜０．０１）阻害した（図１１）。高用量のスクアラミン処置は、開始後３０日でのみ、乳房腫瘍進行を著しく（Ｐ＜０．０５）遅延させた（すなわち、スクアラミンをなお投与している時のみ）が、高用量スクアラミンはまたシクロホスファミドのみを投与した対照に比べ、これらの動物の長期治療速度を２倍にした（図１１）。シクロホスファミドおよび高用量のスクアラミンを投与した長期治療動物の病歴の検討から、スクアラミンの相加作用はスクアラミン処置開始後２週間以内に明らかになることが判明した。
ｃ．肺腫瘍処置におけるスクアラミンの使用
成長速度が異なるいくつかのヒト肺癌系を使用して肺癌のヌードマウス異種移植片モデルでの研究を行ってきた。集めたデータは、スクアラミンがシスプラチンとの組み合わせにより相乗活性を持つことを示す（例えば、図１２）。実験肺癌モデルデザインは、５ｘ１０^６腫瘍細胞の皮下注射とそれに続く３または４日目の化学療法剤の単回注射からなる。いくつかのマウス群に、ベヒクルとして２０％Ｉｎｔｒａｌｉｐｉｄを含むスクアラミンの毎日の腹腔内注入をその翌日から開始し、７〜１４日後に実験が終了するまで継続した。スクアラミンのみを投与するいくつかのマウス群に、化学療法剤との組み合わせ群のアミノステロール処置として、同じ日にアミノステロール投与を開始した。その後、実験終了時に腫瘍容積を測定し、比較した。攻撃的に増殖するＨ４６０ヒト肺腺癌系およびよりゆっくり増殖するＣａｌｕ‐６ヒト肺腺癌系に対し、単独療法剤としてスクアラミン投与を４または５日後に開始すると、スクアラミンは腫瘍増殖にわずかな作用しか見られないが、１日目に投与開始すれば増殖阻害に関与できることが明らかになった。しかし、シスプラチンと組み合わせて、４または５日目に最大耐容量またはそれに近い用量のスクアラミン投与を開始すると、スクアラミンはＨ４６０およびＣａｌｕ‐６系の両方に用量依存的様式でシスプラチン単独よりまさる、著しく、再現可能な腫瘍増殖阻害作用を示した。
ｄ．転移性肺癌におけるスクアラミンの使用
マウスＬｅｗｉｓ肺腺癌を雄Ｃ５７ＢＬ／６マウス後脚の皮下に移植し、１週間増殖させた。いくつかのマウス群を非処置、またはスクアラミン（２０ｍｇ／ｋｇ／日、ｓ．ｃ．）、シクロホスファミド（７，９および１１日目に１２５ｍｇ／ｋｇ，ｉ．ｐ．）、シスプラチン（７日目に１０ｍｇ／ｋｇ、ｉ．ｐ．）スクアラミンおよびシクロホスファミドの組み合わせ、またはスクアラミンおよびシスプラチンの組み合わせのいずれかで処置した。２０日目に、動物を屠殺し、各群における肺転移の平均数を測定した。すべての処置は転移の数を減少させた；しかし、最も効果的な処置はスクアラミンと細胞毒性化合物のいずれかの組み合わせであった（図１３）。
実施例５
ヒト前立腺細胞増殖に対するスクアラミンのｉｎ ｖｉｔｒｏでの研究
ｉｎ ｖｉｖｏでの研究に先立って、出願者はヒト前立腺癌細胞に対するスクアラミンの治療効果をｉｎ ｖｉｔｒｏで評価した。
【００９０】
スクアラミンの抗増殖作用は、組織培養したＬＮＣａＰ／Ｃ４‐２ヒト前立腺癌進行モデルに対するクリスタルバイオレット染色および［^３Ｈ］−チミジン取り込みの両方により評価した。出願者の研究室により開発された、この直系（ｌｉｎｅａｇｅ）由来細胞系はヒト腫瘍性前立腺‐疾患の進行をアンドロジェン依存性で、ほとんど転移性でない状態（ＬＮＣａＰ細胞）から、アンドロジェン非依存性（去勢宿主において増殖可能であると定義される）で非常に攻撃的な状態（Ｃ４‐２亜系）に発生反復する。ＬＮＣａＰ／Ｃ４‐２進行モデルは、前立腺癌治療薬の効力スクリーニングのための有効なモデルであることが以前示されている。
【００９１】
スクアラミンそれ自体は親ＬＮＣａＰまたは直系由来Ｃ４‐２細胞のいずれかの増殖にわずかな作用しか示さなかった。しかし、図１４に示すように外因性ＶＥＧＦ（Ｇｅｎｉｔｅｃｈ Ｃｏｒｐｏｒａｔｉｏｎ，Ｓｏｕｔｈ Ｓａｎ Ｆｒａｎｃｉｓｃｏ，ＣＡにより提供された）の添加により、ＬＮＣａＰ（図１４ａ）および、Ｃ４‐２増殖（図１４ｂ）の大部分は阻害された。見た目には、結果は増殖が抑制されたというよりむしろ、細胞破壊の結果に思えた。その後の研究（データは示していない）は、この作用が時間、および用量依存的であることを示唆し、ＶＥＧＦ量およびＶＥＧＦへの暴露増加に伴って増強された結果が得られた。さらにこの相乗作用は、初めのＶＥＧＦへの暴露と続くスクアラミンへの暴露に基づいているらしく、逆では作用はみられなかった（データは示していない）。
実施例６
ヒト前立腺癌−マウス異種移植モデルでの前立腺腫瘍増殖および散在に対するスクアラミンの治療効果に対するｉｎ ｖｉｖｏでの研究
Ｍａｔｒｉｇｅｌ^ＴＭと混合したＬＮＣａＰ細胞を無胸腺マウス５２匹の皮下に移植した。測定可能な腫瘍を発生し、血清前立腺特異的抗原（“ＰＳＡ”）（例えばＡｂｂｏｔｔ Ｄｉａｇｎｏｓｔｉｃｓ（Ｓｔ．Ｌｏｕｉｓ，ＭＯ）から市販されている標準キットを使用して測定した）量が十分上昇したマウスに精巣摘出を行った。去勢に続き、これらの動物を以下に概説する４群（１〜４）：と去勢してもスクアラミンで処置しない別の対照マウスコホートに分けた。
【００９２】
動物の大部分では、去勢時にＰＳＡレベルはゼロに低下した。３週までには、それらはリバウンドを始め、動物が死ぬか、または屠殺されるまで定常的に上昇を続けた。これら同じマウスにおいて、ＰＳＡリバウンドに対応して腫瘍サイズが増加し始める前、去勢時のサイズはプラトーになる傾向があった。この上記対照コホートの２匹の血清ＰＳＡカーブおよび腫瘍容積測定値をそれぞれ図１５ａおよびｂで説明する。
【００９３】
去勢後、ＰＳＡ低下に付随して１０匹のマウスにスクアラミン処置を開始した（１群）。これらのうち、３匹は精巣摘出後ＰＳＡ底（ｎａｄｉｒ）を一度も経験せず、１匹は病因不明の早期死亡に至った。６匹は、しかし、６〜１０週間の範囲でスクアラミン（２０ｍｇ／ｋｇ／日）を処置した。これらの動物は去勢時、または精巣摘出後のＰＳＡ低下中にスクアラミン処置を開始した。これらの動物はすべて１ｎｇ／ｍＬ以下の精巣摘出後のＰＳＡ底を経験し、スクアラミン療法中、このごく少量のレベルを維持した。腫瘍塊は、対照動物の反応とは反対に、縮小し、やがては消失する傾向にあった。１群の代表的な２匹の血清ＰＳＡレベルおよび腫瘍容積測定値を、それぞれ図１６ａおよびｂで説明する。ＰＳＡリバウンドの出現後にスクアラミン処置した動物の反応（２〜４群）は対照の、スクアラミン非処理コホートと同様（図１５）であるため、グラフには示さない。
実施例７
ホルモン除去と組み合わせたスクアラミン活性
ヒトアンドロジェン依存的ＬＮＣａＰ細胞（両側腹に２ｘ１０^６細胞／側腹）を等しい容積のＭａｔｒｉｇｅｌ^ＴＭと共に４０匹の無胸腺マウスの皮下に移植した。これらの動物は、血清ヒトＰＳＡレベルが１００ｎｇ／ｍＬを超え、腫瘍容積測定値が１０００ｍｍ^３を超えるまで周期的繁殖をさせた（移植後およそ６週間）。その後、すべての動物を外科的に去勢し、１群が８〜１０匹の５つの処置群にランダム化した。１群の動物はそれ以上処置をせず（対照）、２群には直ちにスクアラミン２０ｍｇ／ｋｇ／日（１週間に５日処理し、２日休止）の毎日の腹腔内注射を開始した。３群には、血清ヒトＰＳＡレベルが４‐１０ｎｇ／ｍＬに達したときに、スクアラミン（２０ｍｇ／ｋｇ／日スクアラミン、１週間に５日処理し、２日休止）処置を開始した。４群のマウスには血清ヒトＰＳＡ値が２０‐４０ｎｇ／ｍＬに達したときに、スクアラミン（２０ｍｇ／ｋｇ／日スクアラミン、１週間に５日処理し、２日休止）処置を開始し、５群は血清ヒトＰＳＡレベルが１００ｎｇ／ｍＬに達したときに、スクアラミン処置を開始した。
【００９４】
ヒト腫瘍異種移植片は、去勢後、腫瘍が再び増殖を開始する前２〜４週間、腫瘍増殖を低下または停止させることにより、去勢に反応することが明らかになった。同様に、血清ヒトＰＳＡレベルは去勢後２週間で底をもつ非検出レベルに低下したが、３週目までには血清ヒトＰＳＡレベルはすべての対照動物で上昇し始めた。ＰＳＡリバウンドの出現後にスクアラミン処置した動物では、それらの反応は対照のスクアラミン非処理コホートと同様の反応であった。去勢後にヒトＰＳＡ低下と付随してスクアラミン処置した動物では、１０匹のうち、６匹の動物を６〜１０週間処置した。これらの群の中で３匹は精巣摘出後ＰＳＡ底を全く経験せず、１匹の動物は病因不明の早期死亡に至った。この群の残り６匹の動物は１ｎｇ／ｍＬ以下の精巣摘出後のＰＳＡ底を経験し、スクアラミン処置中このごく微量レベルを維持した。腫瘍塊は時間とともに縮小し、６匹の動物すべてにおいて、やがては消失した。
【００９５】
これら組織中のインテグリン分布を調べるために腫瘍塊（またはスクアラミン処置が成功した動物の場合には、腫瘍移植部位）の組織学的検討を行った。注目に値する一知見は、成功したスクアラミン処置がインテグリンα_ｖβ_３の発現低下および、α_６β_４発現の増加の所見と一致するというものであった。インテグリンα_ｖβ_３は、腫瘍の血管形成増大および腫瘍の増殖ならびに転移の強い傾向と関連していた（例えば、Ｂ．Ｐ．Ｅｌｉｃｅｉｒｉ ａｎｄ Ｄ．Ａ．Ｃｈｅｒｅｓｈ，Ｊ．Ｃｌｉｎ．Ｉｎｖｅｓｔ．１０３，１２２７‐１２３０（１９９９）を参照されたい）。従って、スクアラミンが首尾よくＬＮＣａＰ腫瘍増殖を変化させる機序の一部は、腫瘍攻撃性と関連した細胞インテグリン発現を妨害することである。
【００９６】
ｉｎ ｖｉｔｒｏ実験もＬＮＣａＰ細胞系で行い、［３Ｈ］‐チミジン取り込みにより細胞増殖を測定した。ＬＮＣａＰ細胞は以前記載した（Ｊ．Ｔ．Ｈｓｉｅｈ ｅｔ ａｌ．，Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ ５３，２８５２‐２８５７（１９９３）を参照されたい）ように維持した。細胞は２０ｎｇ／ｍｌ血管形成誘導因子（ＶＥＧＦ）、２０μｇ／ｍＬスクアラミンまたはＶＥＧＦとスクアラミンとの組み合わせに暴露した。ＶＥＧＦまたはスクアラミン単独では血清を含む培地での細胞増殖にほとんど作用がないが、ＶＥＧＦとスクアラミンの組み合わせでは２４時間暴露後に［３Ｈ］‐チミジン取り込みは９８％以上低下した（表６）。結果として、ｉｎ ｖｉｖｏにおけるＬＮＣａＰ腫瘍のスクアラミン処置有効性の基になる機序は、腫瘍細胞成長の直接阻害およびまたは腫瘍細胞死の誘導に関与する。
【００９７】
【表６】

実施例８
カルボプラチンと組み合わせたスクアラミン活性
２つのヒト肺腫瘍系、Ｈ４６０（速やかに増殖する大細胞癌細胞系、ＡＴＣＣＨＴＢ‐１７７）およびＣａｌｕ‐６（未分化癌、ＡＴＣＣＨＴＢ‐５６）由来の約５ｘ１０^６細胞をヌードＢＡＬＢｃマウスの右前脚の皮下に接種した。腫瘍が平均容積約５０〜８０ｍｍ^３で肉眼視できる（３〜５日）ようになったとき、６０ｍｇ／ｋｇカルボプラチン（単回用量）もしくは２０ｍｇ／ｋｇスクアラミン（１日１回、５日間）、またはカルボプラチンとスクアラミンの組み合わせをマウスに腹腔内処置した。１群のマウスは８匹であった。腫瘍増殖阻害は、対照腫瘍が１．０グラムのサイズに達したときの腫瘍サイズを定量することおよび、平均処置腫瘍サイズと平均対照腫瘍サイズの比（Ｔ／Ｃ）を求めることにより評点した。従って、腫瘍増殖阻害は１００％ｘ［１―（Ｔ／Ｃ）］として評点した。別の抗腫瘍活性変数、腫瘍増殖遅延は処置群の平均腫瘍サイズが５００ｍｍ^３に到達するまでに要する時間を測定することにより評点した（Ｂ．Ａ．Ｔｅｉｃｈｅｒ ｅｔ ａｌ．，Ａｎｔｉｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ １８，２５６７‐２５７４ （１９９８））。カルボプラチン＋スクアラミンで見られた腫瘍増殖阻害はカルボプラチンまたはスクアラミン単独で見られた阻害より大きかった。このアッセイエンドポイントによる単独薬としてのスクアラミンの相対的不活性は、スクアラミンがこれら２種のヒト肺腫瘍異種移植片の阻害においてカルボプラチンと相乗作用をするという概念を支持する。同様に、スクアラミンはカルボプラチンにより引き起こされた腫瘍増殖遅延を＞２．３の強化因子により強化することが明らかになった（表７）。
【００９８】
【表７】

実施例９
カルボプラチンに加えパクリタキセルと組み合わせたスクアラミン活性のｉｎ ｖｉｖｏ評価
体重およそ２０グラムのメスのスプレーグ・ドーリー（Ｓｐｒａｇｕｅ Ｄａｗｌｅｙ）ｎｕ／ｎｕマウスに、ヌードマウス宿主において皮下増殖した腫瘍から採取したＭＶ−５２２ヒト肺腫瘍の断片を、トロカールにより、皮下移植した。腫瘍がおよそ５ ｘ ５ ｍｍに達したとき、動物を処置およびコントロール群に対形成した。腫瘍を持つマウス１０匹を含む各群を、実験中、個々に追跡した。薬剤（スクアラミン、パクリタキセルに加えカルボプラチン、またはパクリタキセルおよびカルボプラチンと一緒のスクアラミン）またはビヒクルの投与は、動物を対形成した日（第１日）に始めた。薬剤用量および計画は、マウスにおけるパクリタキセルにカルボプラチン加えた場合の最大耐容用量の以前の測定に基づき、選択した。静脈投薬の許容しうる代用物として、薬剤の腹腔（ｉ．ｐ．）投薬を選択した。
【００９９】
マウスは、第１日に始まり、週２回体重測定し、そして腫瘍測定は、カリパスにより週２回行った。これらの腫瘍測定値を、周知の公式、Ｌ^２ ｘ ２ Ｗ＝腫瘍重量により、ｍｇ腫瘍に変換した。実験は、コントロール腫瘍が、ｉｎ ｖｉｖｏ腫瘍測定に基づき、概算平均腫瘍サイズ１グラムに達したとき、終了した。終了に際し、すべてのマウスの重量を測定し、屠殺し、そしてその腫瘍を切除した。切除した腫瘍を、１０％ホルマリン中で固定する前に重量測定し、そして各処置群に関し、第１日の値からの平均切除腫瘍重量増加を、予定された終了時の屠殺まで生存したすべての動物に関し、計算した。この異種移植モデルにおいて、（平均切除処置腫瘍重量増加を平均切除コントロール腫瘍重量増加で割ったもの（Ｔ／Ｃ）） ｘ １００％を、１００％から減じ、各群に関する腫瘍増殖阻害（ＴＧＩ）値を得た。最初の腫瘍サイズに比べ、実験終了時の腫瘍サイズが減少していたマウスは、ＴＧＩの計算に含まれず、そしてその結果、ＴＧＩ値は、化学療法に対する腫瘍反応の最小の概算を示す可能性がある。
【０１００】
薬剤の組み合わせのいくつかは、ＭＶ−５２２腫瘍異種移植モデルにおいて、いくつかの腫瘍の縮小を引き起こした。これらの組み合わせでは、既定の腫瘍の最終重量（第３１日）を、対応するマウスに関し、第１日の処置開始時のそれ自体の計算された重量から減じた。この相違を最初の腫瘍重量で割り、１００％を乗じたのが、縮小パーセントであった。その後、グループ中で１匹より多いマウスが腫瘍サイズの減少を経験していた場合、平均腫瘍縮小パーセントをデータから計算した。
【０１０１】
研究中いかなる時点でも、腫瘍サイズの減少が＞５０％である場合、腫瘍は、部分的後退を経験したとみなされた。マウスにおいて、腫瘍が完全に消失した場合、これは完全反応または完全腫瘍縮小とみなされ、その後、組織学的検査によりこれを確認した。
【０１０２】
切除腫瘍重量変化の統計解析には、一方向分散分析（ＡＮＯＶＡ）に続くＢｏｎｆｅｒｒｏｎｉのまたはＴｕｋｅｙのｔ検定による対比較を用いた（Ｒ．Ｒ． ＳｏｋａｌおよびＦ．Ｊ． Ｒｏｈｌｆ， Ｂｉｏｍｅｔｒｙ， Ｗ．Ｈ．Ｆｒｅｅｍａｎ ａｎｄ Ｃｏｍｐａｎｙ，サンフランシスコ（１９８１）； Ｄ．Ｇ． Ａｌｔｍａｎ， Ｐｒａｃｔｉｃａｌ Ｓｔａｔｉｓｔｉｃｓ ｆｏｒ Ｍｅｄｉｃａｌ Ｒｅｓｅａｒｃｈ， ＣｈａｐｍａｎおよびＨａｌｌ，ニューヨーク（１９９１））。分散の均一性は、Ｌｅｖｅｎｅの試験を用いて評価し、そして非均一（ｈｅｔｅｒｏｌｏｇｏｕｓ）分散のデータは、ＡＮＯＶＡに適用する前に、適切にｌｏｇ変換した。データは平均±Ｓ．Ｅ．Ｍ．としてまたはビヒクルコントロールのパーセントとして示した。非正規分布（Ｋｏｌｍｏｇｏｒｏｖ−Ｓｍｉｒｎｏｖ試験により決定される）および／または等しくない分散を有する腫瘍重量の非母数分析もまた、Ｋｒｕｓｋａｌ−Ｗａｌｌｉｓ統計検定を用いて行い、対比較にはＭａｎｎ−Ｗｈｉｔｎｅｙ順位和検定を用いた。適切な平均または順位の間の相違は、０．０５以下のｐ値を生じた場合、有意とみなした。スクアラミンを含む療法（ｒｅｇｉｍｅｎ）に関するｐ値は、スクアラミンを含まない同じ療法に比較し、計算する。
【０１０３】
予備実験により、２０ｍｇ／ｋｇ、ｑｄ ｘ ５、ｉ．ｐ．投与のカルボプラチンと組み合わせた１０ｍｇ／ｋｇ、ｑｄ ｘ ５、ｉ．ｐ．投与のパクリタキセルの使用は、ヌードマウスに耐容され、そして毒性でない最大組み合わせ投薬に相当した。これらの用量およびｉｎ ｖｉｖｏ ＭＶ−５２２ヒト肺腫瘍異種移植モデルにおけるこの投薬療法でのパクリタキセルおよびカルボプラチンの組み合わせは、第３１日の３６５．９±６６．０ｍｇの平均切除腫瘍サイズまたは６０．３％のＴＧＩを生じ（ｐ＜０．０１）、この処置群において、いかなる動物に関しても腫瘍縮小の徴候はなかった。対照的に、スクアラミン、パクリタキセルおよびカルボプラチンを用いた三重組み合わせ化学療法は、−０．１±１９．３ｍｇの平均切除腫瘍増殖（ｐ≦０．００１）を生じ、または最終屠殺前の三週間、化学療法を行わなかったにもかかわらず、動物の三重療法組み合わせ群に関し、正味腫瘍静止を生じた。本研究のスクアラミン／パクリタキセル／カルボプラチン・アーム（ａｒｍ）に関するＴＧＩ指数は９６．１％であった。腫瘍サイズに対する化学療法の利益は、第７日までに明らかになり、そして本研究の２つのパクリタキセルおよびカルボプラチン含有アームの間の平均腫瘍サイズの相違は、その後増加した。最終屠殺時にスコア化されたスクアラミン処置動物における腫瘍は、パクリタキセルおよびカルボプラチンのみを投与された動物において見られるものより、有意に小さかった。パクリタキセルおよびカルボプラチンのみを投与されたマウスでは、実験終了時に評価されるように、腫瘍縮小を経験したものはなかったが、三重組み合わせ化学療法コーホートにおける４匹のマウスは、実験の経過に渡り、腫瘍縮小を示し、これらの動物に関する腫瘍サイズの平均減少は５４・８％であった。パクリタキセル／カルボプラチンおよび／またはスクアラミンを投与されたいかなる処置群においても、完全な腫瘍後退はなく、そして三重組み合わせ化学療法群においてのみ、部分的後退があった（７匹の生存動物中、６匹）。腫瘍サイズの一過性減少は、スクアラミンにシスプラチンを加えた場合に見られるように、化学療法の第１４日に最大であった。
【０１０４】
スクアラミン用量の最初の選択および組み合わせ化学療法の投薬療法は、げっ歯類におけるスクアラミンの最大耐容１日用量およびスクアラミン薬物動態学での以前での経験に基づいた。より低いスクアラミン用量で、パクリタキセルに加えカルボプラチンと一緒のスクアラミンが維持される可能性を別個に調べた。ＭＶ−５２２肺腫瘍異種移植モデルで、パクリタキセルおよびカルボプラチンと共に、第１−５日および第８−９日に、０．２−２０ｍｇ／ｋｇの一日用量のスクアラミンを用い、用量範囲決定実験を行った。本実験は、第３２日に終了し、そして最終切除腫瘍重量およびＴＧＩ値パーセントを決定し、これらの観察に基づき、平均値を計算した（表７）。最終屠殺時、平均切除コントロール腫瘍増殖は、６７４．３±７５．８ｍｇであり、一方、パクリタキセルおよびカルボプラチンのみで処置した動物における切除腫瘍増殖は、２２３．４±８２．５ｍｇであった。これは、パクリタキセルにカルボプラチンを加えた場合のＴＧＩ、６６．９％に相当し、これはこの二重薬剤処置に関し最初の実験で見られたＴＧＩ、６０．３％と類似である（上記を参照されたい）。スクアラミン処置群の平均切除腫瘍増殖値は、０．２ｍｇ／ｋｇの１日スクアラミン用量で１３２．０±４６．４ｍｇ（ＴＧＩ＝７４．２％）であり（ｐ＞０．０５）、そして２．０、１０．０および２０．０ｍｇ／ｋｇ／日スクアラミンでは７７．０ｍｇ未満（ＴＧＩ≧７８．８％）であった（表７）。多数の１日用量のスクアラミンで見られた最大亢進は、２．０ｍｇ／ｋｇ／日の用量で起こり；この用量では平均切除腫瘍増殖は、２２．９±２５．０ｍｇであり、９２．１％のＴＧＩに相当した（ｐ＝０．０１７）。統計的に有意な結果は、２．０（ｐ＝０．０１７）および１０．０（ｐ＝０．０３５）ｍｇ／ｋｇ／日スクアラミンで観察されたのみであり、そして２０ｍｇ／ｋｇ／日（ｐ＞０．０５）スクアラミンでは観察されなかった。しかし、本実験および以前の実験に渡るメタアナリシスは、２０ｍｇ／ｋｇ／日スクアラミンが、パクリタキセルにカルボプラチンを加えた場合の効果を有意に亢進することを示す（ｐ＜０．００１）。ＭＶ−５２２腫瘍のパクリタキセルおよびカルボプラチン処置にスクアラミンを添加する利点は、第５日までにすべての用量であきらかであり、そして実験の経過中、持続された。
【０１０５】
パクリタキセルおよびカルボプラチンのみを投与されたマウスで、腫瘍縮小を有したものはなかったが、実験の経過に渡り腫瘍サイズが減少したマウスは、各スクアラミン処置群に少なくとも２匹あり、２または１０ｍｇ／ｋｇ／日スクアラミンを投与された各群で、第３２日に、腫瘍縮小を示したマウスが１０匹中５匹だったのが最大であった。いかなる処置群においても、完全な腫瘍縮小が観察されたマウスはなかったが、すべての処置群（パクリタキセル／カルボプラチンのみを含む）で有意に部分的な腫瘍後退が見られ、化学療法開始後の第１５日でもっとも明らかであった。最大腫瘍反応を示した２つのスクアラミン処置群（２および１０ｍｇ／ｋｇ／日スクアラミン）において、２０匹のマウスのうち、１９匹が、第１５日に、少なくとも５０％の腫瘍負荷の減少を有した。
【０１０６】
スクアラミンの最終投薬３週間以上後も、パクリタキセルおよびカルボプラチンと組み合わせたスクアラミンの、腫瘍増殖に対する効果が持続することから、その期間にスクアラミン投与が行われることが重要である、必要な期間の研究を行った。先の段落に記載される実験の別個のアームは、パクリタキセルおよびカルボプラチンと共に１日用量０．２−４０ｍｇ／ｋｇとしてスクアラミンを１日２回投与された動物群に関し腫瘍増殖を考慮した。１日用量のスクアラミンに関し、パクリタキセルおよびカルボプラチンの腫瘍増殖阻害の、スクアラミン用量関連亢進があった。最良の反応は、１日スクアラミン用量１０および２０ｍｇ／ｋｇで見られた。これらの用量では、第３２日までに、平均切除腫瘍増殖は、１０ｍｇ／ｋｇで１１３．１ｍｇ、そして２０ｍｇ／ｋｇスクアラミンで９６．６ｍｇであり、コントロール腫瘍に関する平均切除腫瘍値、６７４．３ｍｇおよびパクリタキセルおよびカルボプラチン処置腫瘍の２２３．４ｍｇに比較し、好ましい値であった。しかし、いかなるスクアラミン用量も、パクリタキセルおよびカルボプラチンのみと比較した際、統計的有意に達しなかった。実験中、２−４匹のスクアラミン処置マウスで、部分的腫瘍後退が見られ、そして実験終了時に評価した際、各スクアラミン処置群において、２−３の動物において、永続的な部分的腫瘍縮小が見られたが、パクリタキセルおよびカルボプラチンのみで処置したいかなるマウスでも、腫瘍サイズの減少は見られなかった。また、パクリタキセルおよびカルボプラチンと共に第５日に２０ｍｇ／ｋｇスクアラミンを投与されたマウスにおいて、１つの完全後退が観察された。腫瘍後退を示したスクアラミン処置動物における平均腫瘍縮小パーセントは、２１．０−５０．４％の範囲内だった。
【０１０７】
実施例１０
放射線療法と組み合わせたスクアラミン活性
同系乳癌ＭＣＡ−４腫瘍細胞（５ ｘ １０^５） を、Ｃ３Ｈｆ／Ｋａｍオスマウス（４ヶ月齢）の右足に筋内注射した。腫瘍が直径６ｍｍに達したとき、マウスに無作為に：（ａ）処置なし、（ｂ）スクアラミンのみ（１１−１４日間、２０ｍｇ／ｋｇ／日 ｂ．ｉ．ｄ．）、（ｃ）１５Ｇｙのガンマ線照射、または（ｄ）スクアラミンに加え１５Ｇｙのガンマ線を投与するよう割り当てた。腫瘍が少なくとも直径１２ｍｍに達するまで、腫瘍増殖を追跡した。腫瘍放射線反応に対するスクアラミンの効果は、絶対および規準化腫瘍増殖遅延（ＡＧＤおよびＮＧＤ）により、決定した。ＡＧＤは、放射線照射（またはスクアラミン）処置した腫瘍が、８ないし１２ｍｍに増殖するための日数から、未処置腫瘍が８ないし１２ｍｍに増殖するための日数を減じたものとして定義される。ＮＧＤは、スクアラミンおよび放射線照射両方で処置した腫瘍が、８ないし１２ｍｍに増殖するための日数から、スクアラミンのみで処置した腫瘍が８ないし１２ｍｍに増殖するための日数を減じたものとして定義される。亢進因子（ＥＦ）は、ＡＧＤに対するＮＧＤの比として定義された。腫瘍増殖に関する観察時間から計算される、１５Ｇｙガンマ線と一緒のスクアラミンに関するＥＦ（表８）は、＞１．５である。
【０１０８】
【表８】

実施例１１
卵巣癌に関する、シスプラチンと組み合わせたスクアラミン活性
ヒト卵巣腫瘍細胞を、メスのスイスヌードマウスの背部中央領域に皮下注射した（動物当たり５ ｘ １０^７細胞）。ヒト卵巣腫瘍は、親２００８細胞株（ｃｆ． Ｐｉｅｔｒａｓら， Ｏｎｃｏｇｅｎｅ ９， １８２９−１８３８（１９９４））またはＨｅｒ−２／ｎｅｕ受容体を過剰発現しているトランスフェクション変異体いずれかであった。Ｈｅｒ−２／ｎｅｕ受容体の過剰発現は、生じた異種移植腫瘍をより血管形成性にし、そして化学療法により反応しにくくするとみなされる。１週間後、動物を、処置を受けない（コントロール）、単回用量のシスプラチン（４ｍｇ／ｋｇ）、スクアラミンでの毎日の処置（２ｍｇ／ｋｇ、第１−１０日）、またはシスプラチンに加えスクアラミンの処置を受ける群に、無作為化した。その後、化学療法の開始に続き、第２８日に腫瘍増殖をスコア化した。結果は、親２００８腫瘍およびＨｅｒ−２／ｎｅｕ受容体を過剰発現しているトランスフェクション変異体で非常に似ていた。親２００８細胞株での典型的な結果を表９に示す。これらの結果は、各処置群に関し、腫瘍増殖阻害指数を計算することにより、定量化することが可能である。この異種移植モデルにおいて、（第２８日の平均処置腫瘍重量増加を第２８日の平均コントロール腫瘍重量増加で割ったもの（Ｔ／Ｃ）） ｘ １００％を１００％から減じ、各群に関する腫瘍増殖阻害（ＴＧＩ）値を得た。シスプラチンは、本モデルにおいて２００８腫瘍細胞増殖に対し最小限の効果しかもたなかったが、スクアラミンはコントロールに比べ、第２８日の平均腫瘍サイズを有意に減少させた。シスプラチンにスクアラミンを加えた組み合わせは、腫瘍増殖を遅らせるのに非常に有効であった。
【０１０９】
【表９】

【図面の簡単な説明】
【図１】 図１はスクアラミンの一般構造式を示している。
【図２】 図２は血管新生過程の一般的大要を示している。
【図３】 図３はナトリウム水素エックスチェンジャー（ＮＨＥ）過程を例示するために使用された図である。
【図４】 哺乳動物ＮＨＥの種々のイソ形の阻害に対する通常のアミロリドの影響を示している。
【図５】 ５ａおよび５ｂは各々ＮＨＥイソ形３（ＮＨＥ３）およびＮＨＥ１阻害におけるスクアラミンの影響を示している。
【図６】 図６ａ、６ｂおよび６ｃはスクアラミンに関連する薬物動力学の結果を示している。
【図７】 図７はｉ．ｖ．投与後の種々の組織におけるスクアラミン分布を示している。
【図８】 図８はウサギ角膜マイクロポケットアッセイで決定されたスクアラミンを用いる血管新生指標を示している。
【図９】 図９は腫瘍細胞株と比較された、内皮細胞の増殖に対するスクアラミンの阻害効果を示している。
【図１０】 図１０は健康ラット脳内へ導入されたラット９Ｌ神経膠腫を用いた、神経膠腫致死率研究におけるスクアラミンの阻害効果を示している。
【図１１】 図１１はヒトＭＸ−１乳房腫瘍異種移植片を運び、スクアラミンで処置され、続いてシクロフォスファミド処置されたマウスの生存率を示している。
【図１２】 図１２はスクアラミンおよびシスプラチンでのマウス異種移植片−組み合わせ療法研究におけるヒト肺腺癌（Ｈ４６０）の阻害を示している。
【図１３】 図１３はルイス肺癌腫を皮下に移植されたマウスにおける、種々の化学療法処置法後の肺転移の数を示している。
【図１４】 図１４ａおよび１４ｂはＬＮＣａＰヒト前立腺細胞増殖に対する、スクアラミン、ＶＥＧＦおよびＶＥＧＦおよびスクアラミンの組み合わせ処置の効果を示している；図１４ｂはＣ４−２ヒト前立腺細胞増殖に対する、スクアラミン、ＶＥＧＦおよびＶＥＧＦおよびスクアラミンの組み合わせ処置の効果を示している。両方とも［^３Ｈ］−チミジン取り込みアッセイが利用された。
【図１５】 図１５ａおよび１５ｂは睾丸摘徐後の対照無胸腺マウスにおけるＰＳＡレベル（ａ）および腫瘍増殖（ｂ）に対するスクアラミンの効果を示している。
【図１６】 図１６ａおよび１６ｂは睾丸摘徐を行い、続いてスクアラミンで処置した無胸腺マウスにおけるＰＳＡレベル（ａ）および腫瘍増殖（ｂ）に対するスクアラミンの効果を示している。 [Document Name] Statement
Title of the Invention Other Anticancer Agent orStyleOf carcinoma using squalamine in combination with
[Claims]
    1. A first treatment processForAn effective amount of at least one cellScarHarmful chemical compoundsA first container containing; And the second course of treatmentForAn effective amount of squalamineA second container comprising:For treating tumorskit.
    2. CellScarHarmful chemical compounds include: nitrosoureas, cyclophosphamide, doxorubicin, 5-fluorouracil, paclitaxel and its derivatives, cisplatin, carboplatin, iproplatin, epirubicin, methotrexate, melphalan, thiotepa, mitoxantrone, vincristine, vinblastine, etoposide, The member according to claim 1, which is a member selected from the group consisting of teniposide, ifosfamide, bleomycin, procarbazine, chlorambucil, fludarabine, mitomycin C, vinorelbine, topotecan, irinotecan, carmustine, estramustine and gemcitabine.kit.
    3. CellScar3. The method according to claim 2, wherein the harmful chemical compound is BCNU.kit.
    4. A cellScar3. The method according to claim 2, wherein the harmful chemical compound is cyclophosphamide.kit.
    5. CellScar3. The method according to claim 2, wherein the harmful chemical compound is cisplatin.kit.
    6. CellsScar3. The method according to claim 2, wherein the harmful chemical compound is carboplatin.kit.
    7. A cellScar3. The method of claim 2, wherein the harmful chemical compound is a combination of carboplatin and paclitaxel.kit.
    8. A cellScar3. The method according to claim 2, wherein the harmful chemical compound is a combination of cisplatin and etoposide.kit.
    9. The method of claim 1, wherein the cells are in the first treatment step.Scar2. The method of claim 1, wherein the harmful chemical compound is administered intravenously.kit.
    10. The method according to claim 9, wherein squalamine is administered subcutaneously in the second course of treatment.kit.
    11. The method of claim 9, wherein squalamine is administered intraperitoneally in the second course of treatment.kit.
    12. The method of claim 9, wherein squalamine is administered intravenously in the second course of treatment.kit.
    13. The method of claim 9, wherein the first treatment step is performed prior to the second treatment step.kit.
    14. The method according to claim 1, wherein the first treatment step is completed before the start of the second treatment step.kit.
    15. The method according to claim 1, wherein the first treatment step is a cell.Scar2. The method according to claim 1, which is a single injection of the harmful chemical compound.kit.
    16. A cellScar16. The method of claim 15, wherein the harmful chemical compound is BCNU.kit.
    17. A cellScar16. The method according to claim 15, wherein the harmful chemical compound is cyclophosphamide.kit.
    18. A cellScar16. The method according to claim 15, wherein the harmful chemical compound is cisplatin.kit.
    19. A cellScar16. The method according to claim 15, wherein the harmful chemical compound is carboplatin.kit.
    20. The method according to claim 15, wherein in the second course of treatment, squalamine is administered subcutaneously.kit.
    21. The method according to claim 15, wherein in the second course of treatment, squalamine is administered orally.kit.
    22. The method of claim 15, wherein in the second course of treatment, squalamine is administered intravenously.kit.
    23. The method of claim 15, wherein squalamine is administered subcutaneously in the second course of treatment.kit.
    24. The method of claim 15, wherein in the second course of treatment, squalamine is administered intraperitoneally.kit.
    25. The method according to claim 1, wherein the tumor is a CNS tumor.kit.
    26. The method according to claim 1, wherein the tumor is a breast tumor.kit.
    27. The method according to claim 1, wherein the tumor is a lung tumor.kit.
    28. The method according to claim 1, wherein the tumor is an ovarian tumor.kit.
    29. The method according to claim 1, wherein the tumor is hepatocellular carcinoma.kit.
    30. The method according to claim 1, wherein the tumor is a neuroblastoma.kit.
    31. The method according to claim 1, wherein the tumor is a prostate tumor.kit.
    32. First treatment stepForradiationA kit for treating a tumor in combination with a therapy,Second procedureForEffective amount of squalamineKit comprising a container containing.
    33. The method according to claim 32, wherein squalamine is administered subcutaneously in the second course of treatment.kit.
    34. The method of claim 32, wherein in the second course of treatment, squalamine is administered orally.kit.
    35. The method of claim 32, wherein in the second course of treatment, squalamine is administered intravenously.kit.
    36. The method of claim 32, wherein in the second course of treatment, squalamine is administered intraperitoneally.kit.
    37. The method according to claim 32, wherein the tumor is a CNS tumor.kit.
    38. The method according to claim 32, wherein the tumor is a breast tumor.kit.
    39. The method according to claim 32, wherein the tumor is a lung tumor.kit.
    40. The method according to claim 32, wherein the tumor is an ovarian tumor.kit.
    41. The method according to claim 32, wherein the tumor is a prostate tumor.kit.
    42. The method according to claim 32, wherein the tumor is a neuroblastoma.kit.
    43. Third treatment stepForAn effective amount of at least one cellScarHarmful chemical compoundsContaining container33. The method of claim 32, further comprising:kit.
    44. A cellScarHarmful chemical compounds include: nitrosoureas, cyclophosphamide, doxorubicin, 5-fluorouracil, paclitaxel and its derivatives, cisplatin, carboplatin, iproplatin, epirubicin, methotrexate, melphalan, thiotepa, mitoxantrone, vincristine, vinblastine, etoposide, The member according to claim 43, which is a member selected from the group consisting of teniposide, ifosfamide, bleomycin, procarbazine, chlorambucil, fludarabine, mitomycin C, vinorelbine, topotecan, irinotecan, carmustine, estramustine and gemcitabine.kit.
    45. First treatment stepForAn effective amount of at least one antihormonal agentFirst container including; And the second course of treatmentForAn effective amount of squalamineA second container comprising:For treating tumorskit.
    46. The method according to claim 45, wherein the antihormonal agent is a member selected from the group consisting of an LHRH agonist and an antiandrogen.kit.
    47. The method according to claim 46, wherein the antiandrogen is selected from the group consisting of flutamide, biclutamide, nilutamide and luprolide.kit.
    48. First treatment stepForPhysical castrationA kit for treating a tumor in combination withSecond procedureForAn effective amount of squalamineKit comprising a container containing.
    49. First treatment stepForAn effective amount of a cytostaticFirst container including; And the second course of treatmentForAn effective amount of squalamineComprising a second container comprising:For treating tumorskit.
    50. The method according to claim 49, wherein the cytostatic agent is carboxamidotriazole.kit.
    51. The method of claim 49, wherein the cytostatic chemical compound is administered intravenously during the first course of treatment.kit.
    52. The method of claim 49, wherein the cytostatic chemical compound is administered subcutaneously during the first course of treatment.kit.
    53. The method of claim 49, wherein the cytostatic chemical compound is administered intraperitoneally during the first course of treatment.kit.
    54. The method of claim 49, wherein the cytostatic chemical compound is administered orally during the first course of treatment.kit.
    55. The method of claim 49, wherein in the second course of treatment, squalamine is administered intraperitoneally.kit.
    56. The method according to claim 49, wherein the tumor is a CNS tumor.kit.
    57. The method of claim 49, wherein the tumor is a breast tumor.kit.
    58. The method according to claim 49, wherein the tumor is a lung tumor.kit.
    59. The tumor is an ovarian tumor,Fallopian tube50. The method of claim 49, wherein the tumor is selected from the group consisting of a tumor and a peritoneal tumor.kit.
    60. The method of claim 59, wherein the tumor is an ovarian tumor.kit.
    61. The method of claim 49, wherein the tumor is a hepatocellular carcinoma.kit.
    62. The method of claim 49, wherein the tumor is a neuroblastoma.kit.
    63. The method of claim 49, wherein the tumor is a prostate tumor.kit.
    64. The method of claim 32, further comprising administering an effective amount of at least one cytostatic chemical compound in the third course of treatment.kit.
    65. The method according to claim 64, wherein the cytostatic chemical compound is carboxamidotriazole.kit.
    66. The method of claim 64, wherein the cytostatic chemical compound is administered intravenously during the first course of treatment.kit.
    67. The method of claim 64, wherein the cytostatic chemical compound is administered subcutaneously during the first course of treatment.kit.
    68. The method of claim 64, wherein the cytostatic chemical compound is administered intraperitoneally during the first course of treatment.kit.
    69. The method of claim 64, wherein the cytostatic chemical compound is administered orally during the first course of treatment.kit.
    70. The method of claim 64, wherein in the second course of treatment, squalamine is administered intraperitoneally.kit.
    71. The method according to claim 64, wherein the tumor is a CNS tumor.kit.
    72. The method of claim 64, wherein the tumor is a breast tumor.kit.
    73. The method of claim 64, wherein the tumor is a lung tumor.kit.
    74. The tumor is an ovarian tumor,Fallopian tube65. The method of claim 64, wherein the tumor is selected from the group consisting of a tumor and a peritoneal tumor.kit.
    75. The method of claim 74, wherein the tumor is an ovarian tumor.kit.
    76. The method according to claim 64, wherein the tumor is a hepatocellular carcinoma.kit.
    77. The method according to claim 64, wherein the tumor is a neuroblastoma.kit.
    78. The method of claim 64, wherein the tumor is a prostate tumor.kit.
    Claim 79 50. The kit according to any one of claims 1, 32, 45 or 49, applied in combination with a third course of treatment, wherein said third course of treatment comprises:cellScarHarmful chemicals, radiation, antihormones and cytostaticsApplication ofSelected from the group consisting of,kit.
    80. The method according to any one of claims 1, 32, 45 or 49, wherein the first and second treatments are given simultaneously or as a single composition.kit.
    81. The method of any one of claims 1, 32, 45 or 49, wherein the effective amount is a synergistically effective amount.kit.
DETAILED DESCRIPTION OF THE INVENTION
    [0001]
Background art
I. Information on traditional squalamine applications
  This application is a continuation-in-part of Ser. No. 09 / 150,724 filed Sep. 10, 1998,U.S. Patent Law119Based on the article,Provisional Application No. 60 / 016,387 filed on Apr. 26, 1996Claim profitNo. 08 / 840,706 filed on Apr. 25, 1997. All of these applications are incorporated herein in their entirety.
    [0002]
  The present invention relates to various methods using squalamine. Squalamine having the structure shown in FIG. 1 is an aminosterol isolated from the liver of a tiger shark (Squalas acantia). This aminosterol is the subject of U.S. Pat. No. 5,192,756 to Zasloff et al., Which is incorporated herein in its entirety. A method for synthesizing squalamine has already been devised as described in WO 94/19366 (filed September 1, 1994). This PCT application is incorporated herein in its entirety. This PCT application is also related to US patent application Ser. No. 08 / 023,347, filed Feb. 26, 1993, which is hereby incorporated by reference in its entirety. Additional methods of synthesizing squalamine are also described in US patent application Ser. No. 08 / 985,876, filed Dec. 5, 1997, which is hereby incorporated by reference in its entirety.
    [0003]
  U.S. Patent Nos. 5,733,899 and 5,721,226 disclose the use of squalamine as an anti-angiogenic agent. These US patents are incorporated herein by reference in their entirety. The use of additional squalamine (eg, as a sodium / proton exchanger (isoform 3) or NHE3 inhibitor and as an agent to inhibit endothelial cell proliferation) and squalamine synthesis techniques are disclosed in US Pat. No. 5,792,635. Have been. This patent is also incorporated herein in its entirety.
II. Information on the present invention
  About 50,000 cases of CNS (central nervous system) tumors are diagnosed each year. Of these, about 35,000 are metastatic tumors (eg, lung cancer, breast cancer, melanoma) and about 15,000 are primary tumors (mostly astrocytomas). Among malignant gliomas (ie, brain cancers), astrocytomas are the third leading cause of cancer death in patients between the ages of 15 and 34.
    [0004]
  Treatment options for patients with CNS tumors are very limited. Currently, surgery is the treatment of choice. Surgery provides a definitive diagnosis, removal of large tumor masses, and prolonged patient survival. The only post-operative adjunct treatment known to have an effect on the CNS is radiation, which can prolong survival. However, radiation treatment has many unwanted side effects. It damages the patient's normal tissues, including brain tissue. Irradiation can also cause nausea (nausea) in the patient and / or temporary hair loss.
    [0005]
  Chemotherapy, another common postoperative adjuvant cancer treatment, is relatively ineffective on CNS tumors. In particular, chemotherapy of CNS tumors with nitrosoureas does not work. Although many other cancer treatments have been studied and tested, they generally have little effect on prolonging survival. In view of these limited treatment options, patients with CNS tumors have a poor prognosis. The average survival time of patients with malignant astrocytoma who have undergone surgery and do not receive adjuvant treatment is about 14 weeks. Radiation therapy increases the average post-operative survival time to about 36 weeks. For all forms of treatment, the current two-year survival rate is less than 10%.
    [0006]
  To maximize survival, it is critical to begin treatment at an early stage of CNS tumor development. Typically, the degree of tumor angiogenesis (ie, the formation of blood vessels) correlates with patient survival. CNS tumors are the most neovascular of all human tumors. However, when the tumor is small, it is in the "avascular" phase, and its growth is restricted by a diffusion mechanism (ie, the cell receives its nutrients and the like by diffusion into the cell). At this stage, the tumor is viable but does not grow and cannot spread. However, thereafter, angiogenesis begins and the tumor converts to the "vascular" phase. At this stage, blood flow spreads as a growth mechanismTakeTeensRatherTumor growth is exponential (ie, the tumor has its own blood vessels to supply nutrients and the like). Mitotic cell clusters and metastases around the new blood vessels occur during the vascular phase (ie, tumors can spread to other areas of the body). Therefore, early treatment of the tumor (before reaching the vascular phase) can be expected to inhibit metastatic spread as well as control the primary tumor.
    [0007]
  Other types of cancer are also difficult to overcome with known cancer treatments. Lung cancer kills more Americans each year than combined with the next four most frequently diagnosed neoplasms. Estimates for 1994 show more than 170,000 new cases of lung cancer and about 150,000 deaths (Boring et al .; CA Cancer J. Clin. 1994, 44: 7-26). Approximately 80% of primary lung tumors are non-small cell tumors, including squamous cell and large cell carcinomas and adenocarcinoma.
    [0008]
  singleStyleTreatment is considered appropriate in most cases of early and late stage non-small cell lung cancer (NSCLC). Early stage tumors are potentially treatable by surgery, chemotherapy or radiation therapy, and late stage patients usually receive chemotherapy or the best supportive care. Intermediate or locally advanced NSCLC (25% to 30% of all NSCLC cases) is more typically moreStyleTo be treated. This stage is the stage of tumor development, and angiogenesis is a very important factor. New blood vessels are needed to support further tumor growth and metastatic progression. Thus, this stage can be treated with anti-angiogenic agents to prevent the development of new blood vessels. The efficacy of this therapy is,Further improvements can be achieved by combining with cytotoxic chemotherapy or radiation therapy to remove existing tumors.
    [0009]
  Breast cancer is also difficult to treat using known drugs. Breast cancer incidence in the United States has risen at a rate of about 2% per year since 1980, and the American Cancer Society reported in 1996infiltrationIt has been estimated that 182,000 cases of primary breast cancer have been diagnosed. Breast cancer is usually treated with surgery, radiation therapy, chemotherapy, hormonal therapy or a combination of various methods. Like other solid tumors, when breast cancer exceeds a certain size, it requires the development of new blood vessels to support its growth, and at this stage of development it can be treated with anti-angiogenic agents.
    [0010]
  The primary reason for failure of cancer chemotherapy in breast cancer is the development of resistance to cytotoxic drugs. Combination therapy using agents with different mechanisms of action is an accepted treatment, which prevents the development of resistance by the treated tumor. Anti-angiogenic agents act on normal host tissues but do not act on tumors and should not develop resistance, and are particularly useful in combination therapy.
    [0011]
  Prostate cancer is another cancer for which new treatments are needed. Despite the prevalence of prostate cancer, which is most often diagnosed as malignant among American men, little is known about the incidence of prostate cancer. Many factors involved in the development, growth and metastasis of human prostate cancer, and their relationships and interactions with each other, exacerbate the difficulty of treatment.
    [0012]
  Both prostate tumor cell growth and metastasis require sufficient metabolic support, as well as vascular contact, and thus rely on angiogenesis. Prostate cancer extracellular matrix (ECM) / stromal interactions are also important in the growth and spread of human prostate cancer. Of the many growth factors present in the ECM, b-FGF (basic fibroblast growth factor, also known as FGF-2) and VEGF (vascular endothelial cell growth factor) both have a malignant phenotype. It is outstanding because it is involved in inducing and promoting and maintaining the angiogenic process. Ultimately, the outcome of patients with prostate cancer depends on the tumor's ability to grow unhindered,infiltrationAnd the establishment of distal metastases. Thus, anti-angiogenic agents will effectively inhibit the growth and metastasis of such tumors.
    [0013]
  Current treatment for prostate cancer is androgenfactorFocus on the inhibition. Radiation, like in many cancers, is the first treatment, often followed by hormonal therapy. Such hormonal therapy seeks to specifically inhibit androgenic drugs. Inhibition produces the same result as surgical castration or chemical castration with agents such as LHRH (luteinizing hormone releasing hormone) agonists and antiandrogens (such as flutamide, vicultamide, nilutamide and luprolide). However, these treatments have failed in most patients with hormone-refractory lesions. Currently, men with hormone-refractory prostate cancer have few treatment options, none of which can be used. In this regard, tumor growth is accelerated and life expectancy rarely exceeds six months to over one year. In fact, 70% of these patients will eventually die of hormone-resistant disease.
    [0014]
  In such patients (under antihormonal therapy), the remaining prostate tumor cells appear to undergo renewed growth. Thus, anti-angiogenic agents are most effective at this stage (especially those that are most potent on sprouting, young blood vessels), thereby preventing new blood vessels and inhibiting further tumor growth and metastasis.
    [0015]
  Ovarian cancer is the most serious gynecological tumor type. Ovarian cancer contributes to more than 50% of all cancer-related gynecological causes of death, 80-90% of which are epithelial cell-derived tumors. In 1997, there were 26,700 new cases of ovarian cancer and 14,More than 000 people have died. There is a clear genetic component for ovarian cancer. The newly developed detection method has shown a strong correlation between breast cancer, ovarian cancer and genetic markers (including mutant oncogenes such as BRCA1 or erbB-2 or abnormal forms of c-Myc). Another good marker of ovarian cancer that has clinical utility is the circulating analyte CA125, whose serum levels generally reflect the status of cancer progression. CA125 is often monitored in ovarian cancer, but it is not a confirmed marker. Hormonal effects also exist on the cancer risk of ovarian cancer, as observed with breast cancer.
    [0016]
  About one third of ovarian cancer patientsLocalityIndicates a disease. Early-stage ovarian cancer can be treated with some combination of surgery, radiation, and chemotherapy;LocalityThe five-year survival rate for ovarian cancer is over 80%. However, the 5-year survival rate for metastatic stage III or IV ovarian cancer is less than 20%. In these advanced patients (the ovarian tumor escapes from the ovarian capsule andinfiltrationThe most aggressive treatment that only benefits the margins with chemotherapy is needed. The first chemotherapeutic treatment of ovarian cancer has been the use of platinum-based regimens for over a decade. With the advent of newer drugs such as taxanes (paclitaxel, docetaxel), gensitabine, newer vinca alkaloids (vinolebuvin) and topoisomerase inhibitors (topotecan, irinotecan), combination chemotherapy is more widely sought in patients with advanced ovarian cancer Was. The combination of a taxane and a platinum agent (sequentially or simultaneously) is a standard primary treatment in advanced ovarian cancer patients. However, despite the aggressive use of chemotherapy and surgery (with or without radiation therapy), the prognosis for these patients is poor, and the addition of additional non-cytotoxic drugs such as angiogenesis inhibitors is beneficial. Would.
    [0017]
  Worldwide, death from liver cancer is 10 per year⁶Or there is a presumption that it is more. Hepatocellular carcinoma (HCC) or hepatome is the most common liver cancer tumor andType BliverFlameOrC typeliverFeeling of fireIt is a tumor type closely related to staining. The high frequency of hepatitis virus infections observed in Asia and Africa makes these areas the most prevalent places for liver cancer, most of which are HCC. Although viral infection is considered an essential predisposition to HCC, infection alone is not a factor contributing to hepatocyte transformation and proliferation. By comparison, 13,500 people died from liver cancer in the United States in 1992, and two-thirds were HCC.
    [0018]
  Prognosis of liver cancer is generally poor, but localIs only invasive toTumors and cancers that can be conveniently removed surgically are a few exceptions. For locally advanced disease, only limited efficacy has been observed with interferon treatment, polyprenoic acid (a vitamin A derivative), 5-fluorouracil or cryoablation. To date, no meaningful combination chemotherapy has been demonstrated in human trials for HCC, but experimental evaluations of various therapies continue due to urgent clinical needs. A recent report (LX Qin et. Al., Ann Acad Med Singapore 28, 147-51 (1999)) shows that angiogenesis inhibitors can inhibit hepatoma tumor growth in a mouse xenograft tumor model; It suggests that it is a candidate drug to control metastasis. Therefore, angiogenesis inhibitors and cells such as α-interferon, 5-fluorouracil in HCC patients who are not suitable for therapeutic surgeryScarIt is reasonable to consider an active second therapeutic combination treatment, such as a noxious agent, or a vitamin A derivative.
    [0019]
  Childhood cancer is a less common tumor in oncology practice. In 1994, 7,700 children under the age of 15 were reported to have cancer. Although this is about 1% of all cancers, more attention is being paid by this oncology issue and the social benefits of treating children in cancer. The most common solid tumor in affected children is neuroblastoma, which is of neuroendocrine origin. Other solid tumors in children are Wilms tumor, rhabdomyosarcoma and retinoblastoma. Neuroblastoma accounts for 9% of all childhood cancers but 15% of childhood cancer deaths. Many childhood tumors have a genetic basis; for example, perhaps 20% of neuroblastomasEssentiallyHereditybyIt is estimated that.
    [0020]
  Neuroblastoma is most commonly found in the abdomen, many of which have been observed in the adrenal glands. The average age of diagnosis of a neuroblastoma patient is 2 years. Although early stages of neuroblastoma can be treated by surgery, most children have metastatic disease. Relapses have been observed in more than half of all patients, even in children with minimal residual disease after surgery. Clinical trials have emphasized the benefits of using postoperative chemotherapy (with or without radiation therapy). Although nonadjuvant chemotherapy is also commonly used in neuroblastoma patients, chemotherapy in an adjuvant setting has become more common. Neuroblastoma is particularly difficult to treat, and platinum-based regimens are often used in primary or secondary treatment. Chemotherapy plans in pediatric cancer patients are highly aggressive, with 4-6 cellsScarIntensive treatment with harmful drugs is included. Neuroblastoma tumors are an interesting opportunity for anti-angiogenic therapy because neuroblastomas are highly vascular, grow rapidly and metastasize rapidly. Cytostatic drugs are expected to show minimal additional toxic side effects, and the cells in which they are formulatedScarAntiangiogenic treatments for neuroblastoma allow for novel aggressive combination chemotherapy that includes cytostatic drugs, as they cannot attenuate the efficacy of harmful drugs.
Summary of the Invention  It is an object of the present invention to provide a method for treating malignant and cancerous tumors using squalamine in combination with other conventional cancer treatments. Although the invention can be applied to any responsive tumor, in a particularly preferred embodiment of the invention the tumor to be treated is found in the CNS, lung, breast, ovary, liver, neuroendocrine and prostate tissue.
    [0021]
  According to one method of the present invention, squalamine is used in combination with conventional cancer treatments to treat tumors. Such normal cancer treatments require cellsScarIt includes the use of harmful drugs as well as antihormones. In one embodiment, an effective amount of cellsScarCells by administering harmful chemical compounds or during the first course of treatmentScarThe tumor is treated by a combination of a second course of treatment in which the noxious compound and an effective amount of squalamine are administered.
    [0022]
  In this method, the cells used in the first course of treatmentScarHarmful chemical compounds are common cancer treatments. Suitable agents include nitrosoureas, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, topotecan and irinotecan, carmustine, estramustine, paclitaxel and its derivatives, and cisplatin, carboplatin, iproplatin and related platinum compounds. . These conventional cancer treatments are well known to those skilled in the art. M. C. Wiemann and Paul Calabresi, "Pharmacology of Antineoplastic Agents,"Medical Oncology, Chapter 10, Ed., Paul Calabresi et al., McMillan Publishing (1985).Medical OncologyIs incorporated herein in its entirety. One particularly preferred nitrosourea is BCNU, which is also known as carmustine. Another suitable cellScarHarmful agents are platinum compounds such as carboplatin, iproplatin or cisplatin, as are cyclophosphamide.Medical OncologyOther normal cells as described inScarHarmful chemical compounds can also be used without departing from the present invention.
    [0023]
  In another embodiment, the tumor is treated by first inhibiting the hormone affecting the tumor and then administering an effective amount of squalamine in a second course of treatment. In one embodiment, the hormone is specifically inhibited. If the tumor is the prostate, hormonal inhibition is by orchiectomy (ie, removal of one or both testes). Orchiectomy is by surgery or by administration of chemical agents such as LHRH (luteinizing hormone releasing hormone) agonists and / or antiandrogens (such as flutamide, vicultamide, nilutamide and luprolide).
    [0024]
  Cells administered in the first treatment stepScarThe noxious and antihormonal chemical compounds may be administered by any conventional technique used in the art (ie, oral, subcutaneous, intralymphatic, intraperitoneal, intravenous or intramuscular). In one embodiment of the present invention, a cellScarThe noxious chemical compound (preferably BCNU, cisplatin or cyclophosphamide) is administered intravenously. Similarly, squalamine can be administered by any of the methods known in the art as described above. In one embodiment of the invention, subcutaneous injections of squalamine are performed once or twice daily. In another aspect of the invention, intravenous administration of squalamine is performed once or twice daily.
    [0025]
  cellScarThe first course of treatment with the noxious chemical or antihormonal compound occurs before the second course of treatment (using squalamine), after the second course of treatment, or simultaneously with the second course of treatment. Further, the first course of treatment may be completed before the second course of treatment is started, and vice versa. In one embodiment of the invention, the first course of treatment is a cellScarA single intravenous administration of a noxious chemical or antihormonal compound (ie, BCNU, cisplatin or cyclophosphamide), and the second course of treatment involves daily subcutaneous injection of squalamine.
    [0026]
  In addition, the invention relates to cellsScarAlso included is the use of squalamine together with a noxious or antihormonal compound or the use of squalamine with two or more of these compounds. The present invention also relates to the use of squalamine together with cytostatics, or the use of these two treatment therapies with cells.ScarIt also includes the use of harmful compounds. Cytostatics are chemical compounds that stop the growth of tumor cells or normal stromal cells in a tumor, but are not toxic at pharmaceutically active concentrations. The pharmaceutically active concentration of the cytostatic used in the first course of treatment may be that of any known cell growth regulator, but is preferably the calcium pump inhibitor carboxamide triazole.
    [0027]
  In a second method for treating a tumor according to the present invention, the first course of treatment is radiation treatment, one or more conventional radiation treatments, a conventional radiation treatment regimen known to those skilled in the art. Use The tumor is exposed to radiation during this first course of treatment. The timing of the appropriate course of radiation treatment for a squalamine treatment regime can be determined by one of ordinary skill in the art through routine experimentation to provide effective tumor treatment.
    [0028]
  In addition to radiation and squalamine treatment, the tumor may also undergo one or more cells during the third course of treatment.ScarTreated with harmful or antihormonal compounds. In addition, in addition to radiation and squalamine treatment, the tumor is also treated with one or more cytostatic chemical compounds in a third course of treatment. The cytostatic used in the third course of treatment may be any known cell growth regulator that is not cytotoxic, but is preferably the calcium pump inhibitor carboxamide triazole. Tumors that have been treated with radiation, squalamine and a cell growth modulator may have one or more cells in the fourth course of treatment.ScarIt is further envisioned in the examples of the present invention that one may also be treated with a noxious chemical compound.
    [0029]
  Of course, the present invention also relates to the use of cells in addition to radiation or any other treatment.ScarIt also relates to the use of both harmful compounds and / or antihormonal compounds.
DETAILED DESCRIPTION OF THE INVENTION
  Squalamine has been recognized to have angiogenesis inhibitory activity, ie, squalamine inhibits blood vessel formation. It is therefore believed that squalamine as an anti-angiogenic agent would be effective in the treatment of certain diseases or mild or chronic diseases that depend on neovascularization. For example, squalamine is used to treat different conditions such as solid tumor carcinoma, macular degeneration, diabetic retinopathy, psoriasis or rheumatoid arthritis, all requiring another new blood flow.
    [0030]
  In addition, squalamine can selectively inhibit certain sodium / proton exchangers (also referred to herein as "NHE" or "proton pumps"). It is known that several different isoforms of NHE exist in mammals (ie, NHE1, NHE2, NHE3, NHE4 and NHE5). It has been observed that squalamine specifically inhibits NHE3, but not NHE1 or NHE2. Therefore, squalamine is not used for cancer, viral diseases and ischemia.IrrigationIt will be used to treat proliferation or activation depending on conditions that rely on the function of NHE3, such as flow injury.
    [0031]
  Further studies on squalamine and NHE have shown that squalamine acts on a very specific part of NHE3, the 76 carboxyl terminal amino acids of the molecule. If this portion of the NHE3 molecule is removed, squalamine has virtually no effect on the activity of the molecule, however, the molecule is still active as a sodium / proton exchanger.
    [0032]
  Applicants have discovered further uses of squalamine. In particular, the applicant uses conventional cancer treatment agents (cellsScarIt has been discovered that squalamine in combination with (harmful and antihormonal compounds) and radiation treatment will reduce tumor size and growth. Even more significant is that the combination reduces the rate of growth of highly proliferative CNS, lung, breast, ovarian, liver, neuroendocrine and prostate tumors and increases survival benefit. Discovered that can be given.
    [0033]
  In the practice of this aspect of the invention, the cellsScarHarmful chemical compounds and antihormones are used in the first course of tumor treatment, and squalamine is used in the second course of tumor treatment. The first and second treatments may be performed in any timeline or even simultaneously. In another embodiment, two or more cellsScarThe noxious compound and / or antihormonal agent may be administered simultaneously or sequentially as in the first course of treatment.
    [0034]
  Cells used in the first course of treatmentScarThe harmful chemical compound may be a conventional drug, but one of the following drugs is preferred: nitrosoureas, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, topotecan, irinotecan, carmustine, estramustine, paclitaxel And its derivatives, and cisplatin, carboplatin, iproplatin and related platinum compounds. These substances areMedical OncologyAs indicated in the above references, they are common cancer treatment agents known to those skilled in the art. One particularly preferred nitrosourea is BCNU, which is also known as "carmustine" or "1,3-bis (2-chloroethyl) -1-nitrosourea". Cyclophosphamide is also known as N, N-bis- (2-chloroethyl) -N '-(3-hydroxypropyl) phosphorodiamidic acid cyclic ester monohydrate. Doxorubicin is also known as adriamycin.
    [0035]
  Well-known topoisomerase inhibitors include irinotecan [7-ethyl-10- [4- (1-piperidino) -1-piperidino] carbonyloxycamptothecin], also known as CPT11, and topotecan [9- Dimethylaminomethyl-10-hydroxy-camptothecin].
    [0036]
  Paclitaxel is available under the trade name "Taxol". Various derivatives of paclitaxel, such as taxotere or other related taxanes, will be used in accordance with the present invention. Another cell used according to the inventionScarThe harmful chemical compound cisplatin is also known as cis-diaminedichloroplatinum. Well-known analogs of cisplatin are carboplatin and iproplatin (also known as CHIP cis-dichloro-trans-dihydroxo-bis [isopropylamine] platinum IV). Those skilled in the art can use specific cells that can be used in the process of the present invention.ScarHarmful drugs will be familiar.
    [0037]
  The antihormonal drug used in the first course of treatment is any conventional drug, but androgen inhibitors are preferred. Suitable androgen inhibitors are LHRH (luteinizing hormone releasing hormone) agonists and anti-androgens such as flutamide, vicultamide, nilutamide and luprolide. While these agents are suitable for treating prostate cancer, other antihormonal agents will be used for other tumors, as will be appreciated by those skilled in the art.
    [0038]
  In addition, the present invention relates to cells with antihormonal agents.ScarIt involves the use of harmful compounds or the use of two or more of these compounds.
Furthermore, there are no restrictions on the chemotherapeutic agents that can be used in the present invention. Other conventional chemotherapeutic agents that can be used with squalamine in the method of the present invention include methotrexate, melphalan, thiotepa, mitoxantrone, vincristine, etoposide, teniposide, ifosfamide, bleomycin, procarbazine, chlorambucil, fludarabine, mitomycin C, vinorelbine And gemcitabine.
    [0039]
  The first and / or second treatment may be oral, suitable such as "sq", "ip", "im", "il" or "iv". The technique is administered. As used herein, the terms “sq.”, “Ip”, “im”, “il” and “iv” are each a subcutaneous subcutaneous of squalamine or other substance. Administration, intraperitoneal administration of squalamine or other substance, intramuscular administration of squalamine or other substance, intralymphatic administration of squalamine or other substance, and intravenous administration of squalamine or other substance.
    [0040]
  In one embodiment, BCNU is first delivered to the patient as a single intravenous dose, after which squalamine is administered twice daily to s. q. Injected. In another embodiment, the cyclophosphamide is a cellScarIt is a harmful drug. In another embodiment, the cisplatin is a cellScarIt is a harmful drug. In yet another embodiment, carboplatin is used in combination with paclitaxel. Cells as neededScarThe noxious chemical compound and squalamine are delivered simultaneously by a common pharmaceutical carrier (ie, squalamine and cellScarOne injection containing both hazardous chemical compounds). Other suitable combinations of administration techniques may be used without departing from the invention. Those skilled in the art will be able toScarDepending on the noxious compound, the volume, etc., a suitable treatment regime will be ascertainable.
    [0041]
  The squalamine treatment procedure according to the invention is also used with radiation (ie cobalt or X-ray treatment) as a first treatment procedure. In this aspect of the invention, the first course of treatment is radiation treatment and the second course of treatment is squalamine administration. Radiation treatment can proceed on a schedule in combination with squalamine treatment to produce optimal results. Scheduling of such a course of treatment can be ascertained by those skilled in the art through routine experimentation.Medical OncologyAs described in (ibid), any radiation treatment may be used without departing from the present invention. In addition to radiation and squalamine treatment, the tumor may also undergo one or more cells during a third course of treatment.ScarThe treatment may be with a noxious compound or an antihormonal compound.
    [0042]
  The present invention is described below in terms of various specific examples and preferred embodiments. These examples and embodiments should be considered as illustrative of the present invention and not limiting of the present invention.
I. Physiological properties of squalamine
A. Anti-angiogenic activity
  Squalamine has been shown to be useful as an anti-angiogenic agent (ie, squalamine inhibits angiogenesis). Angiogenesis (the process by which new blood vessels are formed) occurs in many basic and physiological processes, such as embryogenesis, ovulation and wound healing. Angiogenesis is also essential for the development of many pathological processes such as diabetic retinopathy, inflammation and malignancy (tumor development). In view of its anti-angiogenic properties, squalamine will be used to treat many diseases that depend on angiogenesis as defined above.
    [0043]
  Angiogenesis is a multi-step process schematically shown in FIG. First, endothelial cells must be activated, for example, by binding a growth factor such as vascular endothelial cell growth factor ("VEGF") or basic fibroblast growth factor ("b-FGF"). No. The cells then migrate, divide, and digest their way through the extracellular matrix into adjacent tissues. The cells then come together to form capillaries and lie on a new basement membrane. This angiogenic process is shown at the top of FIG. Each of these developmental stages of angiogenesis is important and is affected by anti-angiogenic agents.
    [0044]
  Certain compounds which are believed to be anti-angiogenic compounds (ie, matrix metalloproteinase inhibitors such as minocycline, SU101 or marimistat) act at a later stage in this multi-step angiogenic process. These compounds will be referred to as "downstream" angiogenesis inhibitors. For a discussion of matrix metalloproteinase inhibitors, see Techer,Critical Reviews in Oncology / Hematology, Vol. 20 (1995) pp. See 9-39. This article is incorporated herein in its entirety. In contrast to these known anti-angiogenic compounds, squalamine acts at a very early stage of the process by inhibiting growth factor cell activation, ie, squalamine is an "upstream" angiogenesis inhibitor. is there. As shown in FIG. 2 (down), squalamine is normally active and inhibits the sodium-proton pump activated by growth factors. Inhibition of the proton pump leaves the cells quiescent, preventing capillary formation and angiogenesis in this manner. In fact, growth factor signals are interrupted in the presence of squalamine.
B. Capillary retraction activity
  In addition to its anti-angiogenic properties, squalamine has been shown to have a capillary regression effect on newly formed capillaries. A single dose (100 ng) of squalamine was added to the capillary bed of 2-3 day old young chick embryos. After 5 minutes, this dose of squalamine appeared to have little effect on the capillary bed. However, after 20 minutes, the capillary bed appeared to have disappeared (ie, the blood vessels appeared to be closed). After 40 minutes, capillary retraction was observed.
    [0045]
  Capillary beds were also observed after 60 minutes. At this point, some capillaries were seen to reappear, but only the larger vessels appeared to reappear. Small vessels did not reappear at that time. Four to five days after a single treatment with squalamine, the effects of squalamine were no longer apparent, but the newly formed capillaries in the embryos remained squalamine-induced regression for a limited time as long as they were newly formed. Sensitivity remained.
    [0046]
  From this study, it was concluded that squalamine-induced capillary retraction was reversible, at least for certain capillaries. It was also concluded that squalamine was more effective against small microvessels (ie, microvascular beds) compared to large vessels. Proximal examination of chicken microvessels exposed to squalamine revealed that vaso-occlusion was due to contraction of endothelial cell volume in cells wrapped around the vessel lumen. It has been hypothesized that occlusion or regression of small blood vessels by squalamine contributes significantly to the ability of squalamine to block the influx of nutrients and growth factors into the tumor, thereby slowing or preventing the growth rate of the tumor.
C. NHE inhibitory activity of squalamine
  Cell proliferation and division are essential for blood vessel and capillary growth and formation. Capillary tube formation requires a specific extracellular matrix. The cellular NHE alternating transport system is linked to the extracellular matrix. Activation of the NHE antiporter is necessary to induce cell proliferation, and disruption of the NHE antiporter disrupts matrix signals and disrupts cell growth. When endothelial cell proliferation is interrupted, capillary growth is impeded.
    [0047]
  The NHE antiporter of the cell will be activated in different ways. For example, insoluble fibronectin is independent of cell type, integrin α_vβ_lActivates the NHE antitransport mechanism by assembling and immobilizing (anchorage-dependent cell growth requires both soluble mitogens and insoluble matrix molecules). In addition, binding of the stimulus to the extracellular matrix or cell binding involving the virus also activates the NHE antiporter.
    [0048]
  When activated, the NHE antiporter induces cell growth by adjusting the pH of the cell. As shown in FIG. 3, chloride-bicarbonate exchanger and NHE are complementary pH regulating mechanisms in cells. Chloride-bicarbonate exchangers make cells more alkaline, while NHE contributes to the regulation of hydrogen ion concentration in cells. If NHE is inhibited, cells become acidic (lower pH) and growth stops. This does not mean that the cell dies; it only means that the cell has entered a quiescent state (ie, the cell does not divide). If the cells return to normal pH, growth is resumed. When NHE is activated, the cells become more alkaline (higher pH), excrete protons and growth proceeds. Interaction of various regulators (ie, serum components, second messengers, etc.) with a portion of the NHE cytoplasmic region activates the anti-portion mechanism, while interaction with another portion activates the anti-portion mechanism. Inhibit. These parts of NHE are described in Tse, et. al. , "The Mammalian Na⁺/ H⁺  Exchanger Gene Family-Initial Structure / Function Studios "J. Am. Soc. Nephr. , Vol4 (1993), pg. 969,et, seq. It is described in. This article is incorporated herein in its entirety.
    [0049]
  The sodium-proton pump (NHE) responds to different growth stimuli that activate the pump. As noted in connection with FIG. 2, proton pumps are activated by binding of growth factors (eg, VEGF and b-FGF) to cells. In addition, as shown in FIG. 3, other stimuli such as virus binding, addition of various mitogens, sperm binding to eggs, and the like, can also cause NHE activation and cell alkalinization. Binding of these stimuli to the extracellular matrix activates the cell's NHE antiporter and induces cell proliferation.
    [0050]
  There are at least five different mammalian NHE isoforms, each with a different tissue distribution. Nevertheless, they all work in the same manner. NHE1 is an alternating transport mechanism observed in all tissues. NHE2 and NHE3 have a more restricted tissue distribution.
    [0051]
  The effect of squalamine on NHE activity was measured to determine which isoforms of NHE were affected by squalamine. NHE activity can be determined in a variety of different cell conditions. Acid addition activates all of the alternating transport mechanisms and allows measurement of NHE. NHE activity can also be measured after growth factor stimulation of cells. In addition, NHE activity can be measured in unstimulated cells, since the antiporter is continuously functioning at a slow but non-zero rate, even if unstimulated. In each of these cellular states, NHE activity is usually measured in the absence of bicarbonate.
    [0052]
  A classic inhibitor of the activated NHE alternating transport mechanism, which binds to NHE⁺Amiloride, a direct competitive inhibitor of ions, does not abolish the antiporter activity in unstimulated cells. As shown in FIG. 4, amiloride and amiloride analogs act more specifically on NHE1 than NHE2 or NHE3. NHE3 is relatively resistant, especially to inhibition by amiloride. In contrast to amiloride, squalamine was observed to significantly down-regulate the activity of the antiporter when NHE1 activity was measured in unstimulated melanoma cells.
    [0053]
  The following describes the test used to determine that squalamine inhibits NHE3 but not NHE1 or NHE2. NHE-deficient fibroblasts (PS120) transfected with individual human NHE genes were supplemented with the pH-sensitive dye 2'7'-bis (2-carboxyethyl) -5,6-carboxyfluorescein (BCECE). NHE activity was determined by fluorometry using this dye and by the amiloride sensitive isotope.²²Na⁺Measured by cell uptake. Cells were acidified by exposing the cells to ammonium chloride in the absence of sodium to remove sodium and inactivate the proton pump. Ammonium chloride was washed away by exposing the cells to a bicarbonate ion-free medium containing tetramethylammonium chloride. The cells were eventually acidified, but the NHE ion pump was not activated in the absence of sodium. For this test, 7 μg / ml of squalamine was added to the cells in each case, as shown in FIGS. 5a and 5b. Next, sodium was added back at various concentrations (see abscissas in FIGS. 5a and 5b) to drive the anti-transport mechanism (FIG. 5a for human NHE3 and FIG. 5b for human NHE1). The alternating transport mechanism was driven at different rates depending on the amount of sodium added, as evidenced by the rate of cell pH change. As shown in FIG. 5a, when the effect of squalamine on human NHE3 was measured, the rate of pH change in the squalamine-treated cells was slower than that in the control group (no squalamine). This indicates that squalamine inhibits human NHE3. However, in FIG. 5b, there was no significant difference in the rate of pH change between the squalamine-treated sample and the control when measuring the human NHE1 alternating transport mechanism. From these studies, it was concluded that squalamine inhibits human NHE3 but not human NHE1. In addition, in a similar test, it was observed that rabbit NHE1 and NHE2 were not affected by squalamine, whereas rabbit NHE3 was inhibited by squalamine treatment.
    [0054]
  It took at least 30 minutes for the squalamine-induced NHE3 inhibitory effect to be observed in the transfected cells used in this study. Thus, squalamine was not acting like the classic NHE inhibitor amiloride or an analog of amiloride, which are direct competitive inhibitors of sodium and therefore act rapidly as NHE inhibitors. . Furthermore, the NHE inhibitory effect of squalamine was observed to occur in the absence of lactase dehydrogenase (LDH) leakage from cells. Because LDH leakage is a non-specific marker of cytotoxicity, squalamine is a common cellScarIt was concluded that it had no harmful effects.
    [0055]
  This NHE3 inhibitory effect of squalamine has been mapped to the 76 C-terminal amino acid on NHE3. If the 76 C-terminal amino acid of rabbit NHE3 was removed from the molecule, squalamine was observed to have virtually no effect on the activity of the molecule, but the molecule became active as a sodium / proton exchanger. Have maintained. Therefore, the 76 C-terminal amino acid of NHE3 is the site of inhibition by squalamine. While applicants do not wish to be bound by a particular theory of action, it is believed that the squalamine effect of these NHE3s on ancillary proteins has been linked to an inhibitory effect on tyrosine kinase-dependent activity.
    [0056]
  As indicated above, it was concluded that squalamine inhibits NHE3 but not NHE1. However, it was observed that this inhibitory effect of squalamine works in a different manner than classic and known NHE3 inhibitors. In contrast to squalamine, other inhibitors of NHE3 (eg, amiloride, amiloride analogs, genestein, calmodulin and protein kinase C) also inhibit NHE1. Such inhibitors only affect the absolute number of protons that can be secreted from the cell in view of the kinetic properties of the inhibition (ie, "V_max)) On the other hand, squalamine is V_maxNot only inhibits_mThe cells are also forced to lower pH as evidenced by the decrease in value. Note Table 1 below relating to the data collected in the test of FIG. 5a.
    [0057]
[Table 1]

Thus, squalamine inhibits NHE with non-allosteric kinetics (ie, non-classical allosteric inhibition). In an additional test, squalamine (in a 1 hour pretreatment) was compared to rabbit NHE3 V_maxIn a concentration-dependent manner (13%, 47% and 57% at 1, 5 and 7 μg squalamine / ml, respectively). V_maxThe observed squalamine effect on was time-dependent, with the maximum effect occurring at 1 hour of exposure. The observed effect was completely reversible within 3 hours after removing the cells from the medium.
    [0058]
  In view of the test results relating to the effect of squalamine on NHE3, it is believed that NHE3 is important in maintaining homeostasis of unstimulated cells. In addition, blocking squalamine inhibits cell activation, particularly the activation of endothelial cells or progenitor cells involved in the formation of new blood vessels during pathological angiogenesis (such as during tumor growth). Believed to be a mechanism.
    [0059]
  Applicants have further observed that squalamine alters endothelial cell shape. This suggests that transport proteins that regulate cell volume and shape may be targets for squalamine.
    [0060]
  Additional studies of squalamine show that squalamine inhibited brush border membrane vesicles (BBMV) NHE only when tissues were pretreated with squalamine (51% inhibition at 30 min exposure). Direct addition of squalamine to PS120 fibroblasts during the measurement of exchanger activity had no effect.
D. Pharmacokinetics of squalamine
  A squalamine pharmacokinetic study was performed to determine the residence time of squalamine in the body. 6a to 6c show the test results when squalamine was administered subcutaneously (50 mg / kg, FIG. 6a), intraperitoneally (dose 240 μg; 10 mg / kg, FIG. 6b) and intravenously (10 mg / kg, FIG. 6c). ing. Squalamine when administered intravenously (FIG. 6c) was acceptable (35 minutes), but intraperitoneal (FIG. 6b, half-life = 172 minutes) and subcutaneous (FIG. 6a, half-life = 5.6 hours). ) Administration was somewhat higher.
    [0061]
  In addition to these squalamine half-life tests, a test was performed to confirm squalamine distribution in mice after intravenous administration. FIG. v. 2 shows squalamine distribution in mouse tissues 2 hours after administration. Some squalamine was found in most tissues, and most squalamine was concentrated in the liver and small intestine. The test shown in FIG. 7 shows good squalamine distribution. However, it is noted that squalamine is low in brain tissue. From this, squalamine is probablyBlood-brain barrierIt was concluded that they would not pass through. In the treatment of brain tumors, squalamine is believed to act on endothelial cells of the brain, so this modeBlood-brain barrierYou do not need to go through.
    [0062]
  The following example illustrates a more detailed experiment used to test the anti-angiogenesis of squalamine in the method of the present invention.
II. Therapeutic administration and composition
  The mode of administration of squalamine will be selected to suit a particular therapeutic use. Modes of administration generally include, but are not limited to, transdermal, intramuscular, intraperitoneal, subcutaneous, intranasal, inhalation, intralymphatic, intralesional and oral routes. The squalamine compounds are administered by conventional routes, for example, by injection or bolus injection, or by absorption through epithelial or mucosal skin linings (eg, oral mucosa, rectal and intestinal mucosa, etc.), and other biologically active It may be administered together with the drug. Administration can be local or systemic.
    [0063]
  The present invention also provides a pharmaceutical composition comprising squalamine as an active ingredient. Such compositions comprise a therapeutically effective amount of squalamine and a pharmaceutically acceptable carrier or excipient. Examples of such carriers include, but are not limited to, saline, buffered saline, dextrose, water, oil-in-water microemulsions such as intralipids, glycerol and ethanol or combinations thereof. is not. The formulation of the pharmaceutical composition must be chosen to suit the mode of administration.
    [0064]
  Where necessary, the pharmaceutical composition may also include an effective amount of a wetting or emulsifying agent or pH buffering agent. The pharmaceutical composition may be in any form, such as a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition may also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations will include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
    [0065]
  Various delivery systems are known and will be used to administer the therapeutic compounds of the invention, for example, encapsulation in liposomes, microparticles, enteric coating systems, microcapsules and the like.
    [0066]
  In one embodiment, the pharmaceutical compositions are formulated according to routine methods to provide compositions suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions of 5% dextrose and sterile water or interlipid. Where necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the components of the pharmaceutical composition are combined separately in a sealed container, such as an ampoule or sachet, indicating the amount of the active agent, e.g., as a lyophilized powder or a water-free solution, or mixed together. Supplied as a dosage form. If the pharmaceutical composition is to be administered by infusion, it will be dispensed with an infusion bottle containing sterile pharmaceutical water, dextrose, salt or other pharmaceutically acceptable carrier. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water or saline for injection will be provided so that the ingredients may be mixed prior to administration.
    [0067]
  The amount of a therapeutic compound (ie, active ingredient) that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known to those skilled in the art. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disorder or condition, and should be decided according to the judgment of the practitioner and the circumstances of the patient. Effective therapeutic doses will be extrapolated from extrapolation of dose-response curves derived from in vitro or animal model test systems.
    [0068]
  Suitable doses for intravenous administration are generally about 1 microgram to 40 milligrams of active compound per kilogram of body weight. Suitable doses for intranasal administration are generally about 0.01 mg / kg to 20 mg / kg body weight. Suitable doses for oral administration are generally about 500 micrograms to 800 milligrams per kilogram of body weight, preferably about 1-200 mg / kg of body weight.SittingThe agents generally contain as active ingredient 0.5 to 10% by weight of squalamine. Oral formulations preferably contain 10% to 95% of the active ingredient.
    [0069]
  Anti-angiogenesis or cellsScarFor use of squalamine as a noxious agent or in the treatment of cancer, an exemplary dose is from about 0.01 mg / kg body weight to about 100 mg / kg body weight. A preferred dose is between 0.1 and 40 mg / kg body weight.
    [0070]
The present invention also includes a pharmaceutical pack or kit comprising one or more containers filled with the pharmaceutical composition of the present invention. Associated with such containers are notices in the form prescribed by the governmental authority regulating the use, use or sale of pharmaceutical or biological products, the notices reflecting certification by the manufacturer's agent. ), Would be use or sale for human administration.
    [0071]
  Normal cells used according to the inventionScarHarmful chemicals and antihormonal compounds exist in any suitable form known to those skilled in the art. These chemical compounds will also be administered by any suitable means known to those skilled in the art (oral, subcutaneous, intravenous, intraperitoneal, intralymphatic and intramuscular).
    [0072]
  In describing the present invention, applicants have stated certain theories in explaining how and why the present invention works in the manner in which the present invention works. These theories have been presented for informational purposes only. Applicants are not bound by any particular chemical or physical mechanism or theory of efficacy.
    [0073]
  Although the present invention has been described in terms of various specific preferred embodiments and specific embodiments, those skilled in the art will appreciate that various modifications can be made without departing from the spirit and scope of the invention as defined in the appended claims. And that variations are possible. All patents, publications and references cited throughout this specification are hereby incorporated by reference in their entirety.
Example 1 Rabbit Corneal Micropocket Assay To determine if a compound is anti-angiogenic, the rabbit corneal micropocket assay is an accepted standard test. In this test, an incision is made in one eye of the rabbit and the irritant is placed at the incision site. The stimulus is usually used for inducing angiogenesis in the avascular cornea. As an example, a solid tumor in a polymer matrix can be placed in the cornea as a stimulus. This is because the tumor will release a number of pro-angiogenic factors that stimulate new capillary formation. Tumor-derived angiogenic factors stimulate endothelial cells at the limbus of the eye and initiate angiogenesis towards the irritant. A second polymer pellet (eg, ethylene / vinyl acetate copolymer) is placed between the limbus and the irritant. The polymer pellet is empty (negative control test pellet) or contains the compound to be tested for anti-angiogenic properties. The polymer pellets are used for controlled release of the test substance. Due to the avascular corneal background in the rabbit cornea, the results can be evaluated qualitatively visually. Furthermore, the number of blood vessels can be counted and their length etc. can be measured, thus providing a more quantitative evaluation of the results.
    [0074]
  VX2 rabbit carcinoma was implanted into 26 rabbit eyes, usually at an avascular site, to act as an angiogenic stimulant. Squalamine was incorporated into a controlled release ethylene / vinyl acetate copolymer (20% squalamine and 80% polymer by weight). The loaded polymer pellets were placed on 13 corneas to provide a sustained local release of squalamine. A polymer blank was provided to the remaining 13 eyes as a control. Thus, one eye of each rabbit was used as a squalamine test eye and the other eye of the same rabbit was used as a control eye. The eyes were examined weekly using a slit lamp stereomicroscope three weeks after tumor implantation and the angiogenesis index ("AI") was calculated (this calculation is described in detail below in connection with FIG. 8). Will be). The squalamine-loaded polymer was found to release active squalamine during the treatment period in vitro. After the test, the cornea was examined histologically.
Using this test, squalamine was found to be a potent inhibitor of tumor-induced capillary formation. Fewer vessels were observed in squalamine-treated corneas compared to control corneas, and these vessels were generally shorter compared to control corneal vessels.
Next, some of the corneas were cut out and the effect of squalamine on the tumor cells themselves was observed. Untreated control corneas showed many blood vessels within and adjacent to the tumor. The tumor in the squalamine-treated cornea was still viable (ie, the tumor was not dead), but essentially no vasculature associated with the tumor was found. Thus, there was a marked decrease in vascularity in squalamine-treated tumors compared to the corresponding control tumor sections. These findings suggested that squalamine acts on blood vessels and not on the tumor itself.
    [0075]
  FIG. 8 graphically shows the results of the rabbit corneal micropocket assay. To provide a quantitative assessment, the angiogenesis index ("AI") of each eye was determined. To determine the angiogenesis index, the vascular density ("D_vessel") Was rated on a 0-3 scale as follows:
    [0076]
[Table 2]

  Next, the blood vessel length of each cornea (“L_vessel") Was measured. Vessel length is the length of the longest vessel measured from the limbus to the distal margin of the most mature vessel. Then, from these measurements, the following equation: AI = D_vesselxL_vesselThe angiogenesis index was determined according to
FIG. 8 shows the average angiogenesis index of each corneal group (squalamine-treated and untreated) in the rabbit corneal micropocket assay. As shown in the figure, squalamine strongly inhibited new blood vessel growth. Squalamine-treated eyes showed significantly lower AI values than untreated eyes (37% reduction on day 14 (p = 0.05, Wilcoxon rank sum test) and 43% reduction on day 21 ( p <0.01)) This data illustrates that squalamine inhibits new blood vessel or capillary formation for a long period of time, more specifically, squalamine has strong anti-angiogenic activity even after 3 weeks. Show.
Example 2
Squalamine does not cause inflammation
In the rabbit corneal micropocket assay test, if the rabbit cornea becomes inflamed, this inflammation can induce corneal neovascularization. Such inflammation will distort the test results. Therefore, tests were performed to determine whether squalamine is intrinsically and itself involved in the inflammatory response in the cornea. Some non-bioabsorbable ethylene / vinyl acetate copolymer pellets were loaded with different concentrations of squalamine, ie, 2%, 10%, and 20% by weight. These pellets were then placed on rabbit corneas without angiogenic stimuli. Squalamine did not induce inflammation at any of these concentrations. Therefore, squalamine does not induce new blood vessel formation by inflaming the cornea.
Example 3
Use of squalamine in the treatment of brain tumors
  The results of the rabbit corneal micropocket assay test suggested to applicants that squalamine may be a potent anti-angiogenic agent that inhibits angiogenesis. Recognizing that the rapid growth of solid tumors in the brain depends on angiogenesis, Applicants assessed the activity of squalamine on the growth of solid tumors of the brain in animal models.
    [0077]
  Among solid brain tumors, malignant gliomas are the most common form of cancerous tumors. These tumors are the third leading cause of death in adolescents between the ages of 15 and 34. Produces normally quiescent brain and / or CNS endothelial cellsinfiltrationThe ability to induce a strong sexual state determines the nature of the malignant glioma. The glioma expresses angiogenesis inducer ("VEGF") and other inducers that paracrinely stimulate the induced receptors of CNS endothelial cells (ie, VEGF arises from tumor cells and stimulates endothelial cells). . Subsequently, CNS endothelial cells are angiogenicinfiltrationStart and provide nutrition to the glioma. Applicants tested the anti-angiogenic activity of squalamine on gliomas by (1) the ability to selectively inhibit VEGF-mediated stimulation of endothelial cells and (2) the effect on experimental mouse gliomas.
    [0078]
  First, an in vitro test was performed to confirm that squalamine acts specifically on endothelial cells. As described in connection with FIG. 2, applicant used endothelial cells because they are involved in the early stages of angiogenesis. Specifically, tumor angiogenesis is a continuous, overlapping series of stages. Initially, endothelial cells are activated and proliferate. Next, proteolytic enzymes are produced and the cells migrate. Subsequently, a new basement membrane is generated. In this way, new blood vessels are created and tumor size increases.
    [0079]
  The following cell lines were used for this in vitro analysis: (a) bovine retinal endothelial cells; (b) 9L and C6 rat glioma cells; (c) human H80 glioma cells; and (d) VX2 rabbit carcinoma cells (previously The same type of tumor implanted in the rabbit corneal micropocket assay test described) was used. The endothelial mitogen used in this analysis was 20 ng / ml VEGF.
    [0080]
  The cells were allowed to adhere overnight to a tissue culture plate containing optimal growth media. After attachment, the cells were exposed to squalamine alone or at varying concentrations (0, 10, 20, 30, 60, and 90 μg squalamine / ml). Cell proliferation was counted using a Coulter Counter every day for 3 days. A total of 10,000 cells were plated per well and a quadruplicate test of each experimental concentration was performed. Thereafter, the results were averaged. Bovine retinal endothelial cells were expanded and treated in the same manner as the other cell lines except that cell proliferation was measured by adding 20 ng / ml human recombinant VEGF prior to squalamine treatment.
    [0081]
  Cell growth by all tumor cell lines and endothelial cells not treated with VEGF after exposure to squalamine for up to 30 μg / ml for 24 and 48 hours showed no statistical change. However, VEGF-stimulated endothelial cell proliferation was significantly reduced in a concentration-dependent manner by exposure to squalamine for the same time as above. The percentage of endothelial cell growth inhibition (% I) is given by the following equation:(Cell number of control sample−cell number of experimental sample) × 100=% I (cell number of control sample)
Determined by
The table below shows the results of the VEGF-stimulated endothelial cell line at 48 hours.
    [0082]
[Table 3]

Additional data is described in FIG. This figure shows the growth of various cell lines as a percentage of control growth on days 1, 2, and 3 after in vitro application of 30 μg / ml squalamine. As shown in FIG. 9, proliferation was particularly reduced in VEGF-stimulated endothelial cells, while proliferation of other cell lines (H80, C6, and VX2) was not significantly affected.
    [0083]
  Based on this information, Applicants concluded that squalamine dramatically and specifically inhibited VEGF-stimulated proliferation of endothelial cells in vitro. Thus, squalamine is a potent inhibitor of tumor-induced angiogenesis, and this effect appears to be facilitated by the specific inhibition of VEGF-induced endothelial cell proliferation. Thus, squalamine appears to be well-suited for reducing or reducing tumor-induced angiogenesis for tumor-specific anti-angiogenic therapy.
    [0084]
  In addition to inhibiting VEGF-stimulated proliferation of endothelial cells, squalamine also inhibits b-FGF, PDGF_bbIt has been found to interfere with the proliferation stimulation of human brain capillary endothelial cells induced by cell scatter factor (HGF or hepatocyte growth factor), conditioned tumor medium, and human cerebral cyst fluid. Thus, tumors produce a variety of different growth factors, and squalamine has an inhibitory effect on some of them.
From these test results, Applicants tested squalamine in an animal model of brain tumor. To test the effect of squalamine on tumors localized in the brain, small sections of viable glioma (1 mm) were collected from rat flank where rat glioma was maintained.³) Were implanted into the brains of two groups of rats. Thus, in this model, the tumor is alive when placed in the rat brain. Three days after transplantation and after development of some vasculature, one group of rats was started on squalamine 20 mg / kg / day (ip). A group of control animals ("vehicle control" in FIG. 10) received only the carrier vehicle (no squalamine) and the other group of animals was treated with squalamine ("squalamine" in FIG. 10). As shown, the squalamine treated animals had a 38% increase in mean survival time (x = 24.9 days vs. x = 18.0 days). FIG. 10 further illustrates that squalamine-treated rats generally have increased survival time in this animal model.
    [0085]
  The squalamine toxicity test was performed in another animal model. Conventional cytotoxic compounds are quite toxic. For example, the conventional chemotherapeutic agent, BCNU, has a cumulative toxic effect. For this reason, the compounds are applied only once to the patient. The use of BCNU has been previously described on pages 304 and 305 of Calabresi in Medical Oncology. To test the toxicity of squalamine, one group of rats received 20 mg / kg / day (ip) of squalamine daily for 30 days or more and were bred up to 200 days after administration. The animals used in this study remained healthy. This result suggests that squalamine has little or no toxicity.
Example 4
Use of squalamine with conventional cancer therapy
As described above, squalamine is an upstream inhibitor of the angiogenic process by inhibiting endothelial cell activation following interaction with growth factors. Due to its anti-angiogenic activity, squalamine has been shown to be effective in treating solid tumors that require angiogenesis for growth. Applicants have examined whether combining squalamine (an upstream angiogenesis inhibitor) treatment with conventional cancer therapies using alkylating agents would yield beneficial results when treating tumors.
a. Squalamine 9L glioma flank test
  Four groups of rats (total of 20 Fisher 344 rats, 200 g) received 1 mm of 9L gliosarcoma (9 L glioma).³Was applied subcutaneously on day 0. The tumor was implanted in the rat flank to avoid complications due to sufficient brain levels of squalamine.
Randomization and treatment started on day 5 according to the following scheme:
    [0086]
[Table 4]

¹The term "BID" as used herein refers to the application of the ingredient twice daily (administer 10 mg / kg at two different times each day).
    [0087]
  Tumor size was measured directly 25 or 26 days after tumor implantation. Tumor size (ie, volume “V”) was estimated based on volume calculations determined from the measured tumor length (“L”), width (“W”), and height (“H”) (V_{tumor spheroid}= 0.5xLxWxH). Table 5 summarizes the results. The tumor volumes shown in Table 5 represent the average tumor volume of each treatment group of animals that survived to the end of the experiment.
    [0088]
[Table 5]

  Table 5 illustrates the favorable results obtained when treating tumors with the combination of squalamine and nitrosourea BCNU (Group 4). In this group a 99.8% reduction in mean tumor size was observed when treated with squalamine and BCNU together. Table 5 further shows that squalamine alone (Group 3) is effective in treating tumors. Tumor size was reduced by 81.7% in the three groups compared to the control group.
    [0089]
  Applicants conclude that the use of squalamine in combination with conventional cytotoxic compounds can slow or stop the spread of brain tumors. The tumor itself shrinks or necroses. It is expected that combination treatment of squalamine and a cytotoxic compound will prolong survival. Therefore, this treatment would potentially be effective in treating brain tumors.
b. Use of squalamine in the treatment of breast tumors
  The human MX-1 breast cancer cell line has previously been used to provide evidence of the in vivo activity of cyclophosphamide and other cytotoxic chemotherapeutic agents used alone or in combination (T. Kubota, et al. ,Gann 74E., 437-444 (1983); Kobayashi, et al. ,Cancer Research 54, 2404-2410 (1994); -C. Bissery, et al. ,Seminars in Oncology 22(No., Suppl. 13), 3-16 (1995)). Each of these references is incorporated herein by reference in its entirety. Squalamine was studied as adjunctive therapy following a single dose of cyclophosphamide 200 mg / kg. Cyclophosphamide was injected on day 14 after tumor implantation when the tumor had reached 65-125 μl. Cyclophosphamide caused partial regression in all animals and only a few caused complete regression. The animals were then randomized into 3 treatment groups (n = 27 each): 5 times per week, vehicle (Intralipid) only; 10 mg / kg / day squalamine in Intralipid; and 20 mg squalamine in Intralipid / Kg / day administration. Animals with tumors greater than 2 grams at any time during the experiment were euthanized. The experiment was continued for 90 days to ensure that only mice that underwent long-term treatment were still alive. These animals did not receive squalamine administration during the last eight weeks of the experiment because high dose squalamine administration was discontinued due to animal weight loss and potential toxicity concerns. Low dose squalamine treatment significantly (P <0.01) inhibited the rate of progression of breast tumors at all time points examined (FIG. 11). High-dose squalamine treatment significantly (P <0.05) delayed breast tumor progression only 30 days after initiation (ie, only when squalamine was still administered), but high-dose squalamine also The long-term treatment rate of these animals was doubled compared to controls receiving phosphamide alone (FIG. 11). Examination of the medical history of long-term treated animals receiving cyclophosphamide and high doses of squalamine revealed that the additive effect of squalamine became evident within two weeks after the start of squalamine treatment.
c. Use of squalamine in the treatment of lung tumors
  Several human lung cancer lines with different growth rates have been studied in nude mouse xenograft models of lung cancer. The data collected indicate that squalamine has synergistic activity in combination with cisplatin (eg, FIG. 12). Experimental lung cancer model design is 5x10⁶It consists of a subcutaneous injection of tumor cells followed by a single injection of the chemotherapeutic agent on day 3 or 4. In some groups of mice, daily intraperitoneal injections of squalamine containing 20% Intralipid as vehicle were started the next day and continued until the end of the experiment 7-14 days later. Several groups of mice receiving squalamine alone started aminosterol treatment on the same day as aminosterol treatment in the combination group with chemotherapeutic agents. Thereafter, the tumor volume was measured at the end of the experiment and compared. For the aggressively growing H460 human lung adenocarcinoma line and the slower growing Calu-6 human lung adenocarcinoma line, when squalamine administration as monotherapy is started 4 or 5 days later, squalamine has a small effect on tumor growth However, it was clarified that if administration was started on the first day, it could be involved in growth inhibition. However, when initiating squalamine at or near maximum tolerated dose on day 4 or 5 in combination with cisplatin, squalamine is superior to cisplatin alone in a dose dependent manner for both H460 and Calu-6 systems, It showed a reproducible tumor growth inhibitory effect.
d. Use of squalamine in metastatic lung cancer
  Mouse Lewis lung adenocarcinoma was implanted subcutaneously in the hind leg of male C57BL / 6 mice and allowed to grow for one week. Some groups of mice were untreated or squalamine (20 mg / kg / day, sc), cyclophosphamide (125 mg / kg, ip on days 7, 9 and 11), cisplatin (7 On day 10 mg / kg, ip) treatment with either a combination of squalamine and cyclophosphamide or a combination of squalamine and cisplatin. On day 20, animals were sacrificed and the average number of lung metastases in each group was determined. All treatments reduced the number of metastases; however, the most effective treatment was any combination of squalamine and a cytotoxic compound (FIG. 13).
Example 5
In vitro study of squalamine on human prostate cell proliferation
  Prior to in vivo studies, applicants assessed the therapeutic effect of squalamine on human prostate cancer cells in vitro.
    [0090]
  The antiproliferative effect of squalamine was demonstrated by crystal violet staining and [V] on tissue-cultured LNCaP / C4-2 human prostate cancer progression model.³H] -thymidine incorporation was assessed. This lineage-derived cell line, developed by the applicant's lab, has been shown to progress human neoplastic prostate-disease progression from an androgen-dependent and less metastatic state (LNCaP cells) to an androgen-independent state. (Defined as capable of multiplication in a castrated host) and repetitively develop into a highly aggressive state (C4-2 subline). The LNCaP / C4-2 progression model has previously been shown to be an effective model for efficacy screening of prostate cancer therapeutics.
    [0091]
  Squalamine itself had little effect on the growth of either parental LNCaP or lineage-derived C4-2 cells. However, as shown in FIG. 14, the addition of exogenous VEGF (provided by Genetic Corporation, South San Francisco, Calif.) Inhibited most of LNCaP (FIG. 14a) and C4-2 growth (FIG. 14b). Was. In appearance, the results seemed to be the result of cell destruction, rather than suppression of growth. Subsequent studies (data not shown) suggested that this effect was time- and dose-dependent, with enhanced results with increasing amounts of VEGF and exposure to VEGF. Furthermore, this synergistic effect appeared to be based on initial exposure to VEGF followed by exposure to squalamine, and conversely no effect was seen (data not shown).
Example 6
In vivo study on the therapeutic effect of squalamine on prostate tumor growth and dissemination in a human prostate cancer-mouse xenograft model
  Matrigel^TMLNCaP cells were implanted subcutaneously in 52 athymic mice. Mice that develop measurable tumors and have elevated serum prostate specific antigen ("PSA") levels (e.g., measured using a standard kit commercially available from Abbott Diagnostics (St. Louis, MO)) Orchiectomy was performed. Following castration, these animals were divided into four groups (1-4) outlined below: and another control mouse cohort that was not castrated but treated with squalamine.
    [0092]
  In most of the animals, PSA levels dropped to zero upon castration. By three weeks they had started to rebound and continued to rise steadily until the animals died or were sacrificed. In these same mice, the size at castration tended to plateau before the tumor size began to increase in response to PSA rebound. The serum PSA curves and tumor volume measurements of the two animals in this control cohort are illustrated in FIGS. 15a and b, respectively.
    [0093]
  After castration, 10 mice were started on squalamine treatment, concomitant with PSA reduction (group 1). Of these, three were PSA after orchiectomybottomNo (nadir) had ever been experienced, and one had premature death of unknown etiology. Six, however, were treated with squalamine (20 mg / kg / day) in the range of 6-10 weeks. These animals began squalamine treatment at castration or during PSA decline after orchiectomy. All of these animals had PSA after orchiectomy of 1 ng / mL or less.bottomAnd maintained this tiny level during squalamine therapy. Tumor masses tended to shrink and eventually disappear, contrary to the response of control animals. Serum PSA levels and tumor volume measurements of a representative group of two animals are illustrated in FIGS. 16a and b, respectively. The response of squalamine-treated animals following the appearance of PSA rebound (Groups 2-4) is similar to the control, non-squalamine-treated cohort (Figure 15) and is not shown on the graph.
Example 7
Squalamine activity combined with hormone removal
Human androgen-dependent LNCaP cells (2 × 10⁶Cells / flank) with an equal volume of Matrigel^TMWere implanted subcutaneously in 40 athymic mice. These animals have serum human PSA levels above 100 ng / mL and tumor volume measurements of 1000 mm.³Breeding was carried out (approximately 6 weeks after transplantation). Thereafter, all animals were surgically castrated and randomized into 5 treatment groups, one group of 8-10 animals. One group of animals received no further treatment (control) and two groups immediately began daily intraperitoneal injections of squalamine 20 mg / kg / day (treated 5 days a week, 2 days off). Group 3 started squalamine (20 mg / kg / day squalamine, 5 days a week, 2 days rest) treatment when serum human PSA levels reached 4-10 ng / mL. Seramin (20 mg / kg / day squalamine, treated 5 days a week, 2 days rest) treatment was started when the serum human PSA level reached 20-40 ng / mL in group 4 mice. Started squalamine treatment when serum human PSA levels reached 100 ng / mL.
    [0094]
  Human tumor xenografts have been shown to respond to castration by reducing or stopping tumor growth 2-4 weeks after castration, before the tumors begin to grow again. Similarly, serum human PSA levels were two weeks after castration.bottom, But by week 3 serum human PSA levels began to increase in all control animals. In animals treated with squalamine after the appearance of PSA rebound, their responses were similar to the control squalamine untreated cohort. For animals that were squalamine-treated following castration with concomitant reduction in human PSA, 6 out of 10 animals were treated for 6-10 weeks. Three of these groups had PSA after orchiectomy.bottomOne animal did not experience any illness, leading to premature death of unknown etiology. The remaining six animals in this group were PSA after orchiectomy of 1 ng / mL or less.bottomAnd maintained this trace level during squalamine treatment. The tumor mass shrunk over time and eventually disappeared in all six animals.
    [0095]
  To examine the integrin distribution in these tissues, histological examination of the tumor mass (or, in the case of animals successfully treated with squalamine, the site of tumor implantation) was performed. One noteworthy finding is that successful squalamine treatment is associated with integrin alpha_vβ₃Expression of α and α₆β₄This was consistent with the finding of increased expression. Integrin alpha_vβ₃Has been associated with increased tumor angiogenesis and a strong tendency for tumor growth and metastasis (see, for example, BP Ericeiri and DA Cheresh,J. Clin. Invest. 103, 1227-1230 (1999)). Thus, part of the mechanism by which squalamine successfully alters LNCaP tumor growth is by interfering with cellular integrin expression associated with tumor aggression.
    [0096]
  In vitro experiments were also performed on the LNCaP cell line to measure cell proliferation by [3H] -thymidine incorporation. LNCaP cells were described previously (JT Hsieh et al.,Cancer Research 53, 2852-2857 (1993)). Cells were exposed to 20 ng / ml angiogenesis inducer (VEGF), 20 μg / mL squalamine or a combination of VEGF and squalamine. VEGF or squalamine alone had little effect on cell growth in media containing serum, whereas the combination of VEGF and squalamine reduced [3H] -thymidine incorporation by 98% or more after 24 hours of exposure (Table 6). Consequently, the mechanism underlying the efficacy of squalamine treatment of LNCaP tumors in vivo involves direct inhibition of tumor cell growth and / or induction of tumor cell death.
    [0097]
[Table 6]

Example 8
Squalamine activity in combination with carboplatin
Approximately 5 × 10 5 from two human lung tumor lines, H460 (a fast growing large cell carcinoma cell line, ATCCHTB-177) and Calu-6 (undifferentiated carcinoma, ATCCHTB-56)⁶Cells were inoculated subcutaneously into the right forelimb of nude BALBc mice. Tumor has an average volume of about 50-80 mm³When the mice became visible (3-5 days), 60 mg / kg carboplatin (single dose) or 20 mg / kg squalamine (once daily, for 5 days), or a combination of carboplatin and squalamine was intraperitoneally administered to the mice. Was treated internally. There were eight mice in one group. Tumor growth inhibition was scored by quantifying tumor size when control tumors reached 1.0 gram size and determining the ratio of mean treated tumor size to mean control tumor size (T / C). Therefore, tumor growth inhibition was scored as 100% x [1- (T / C)]. Another anti-tumor activity variable, tumor growth delay, was that the average tumor size of the treatment group was 500 mm³Were scored by measuring the time required to reach (BA Teicher et al.,Anticancer Research 18, 2567-1574 (1998)). Tumor growth inhibition seen with carboplatin plus squalamine was greater than that seen with carboplatin or squalamine alone. The relative inactivity of squalamine as a single agent by this assay endpoint supports the notion that squalamine synergizes with carboplatin in inhibiting these two human lung tumor xenografts. Similarly, squalamine was found to enhance carboplatin-induced tumor growth delay by potentiators of> 2.3 (Table 7).
    [0098]
[Table 7]

Example 9
  In vivo evaluation of squalamine activity in combination with carboplatin and paclitaxel
  Female Sprague Dawley nu / nu mice weighing approximately 20 grams were implanted subcutaneously by trocar with a fragment of MV-522 human lung tumor taken from a tumor grown subcutaneously in a nude mouse host. When tumors reached approximately 5 x 5 mm, animals were paired to treatment and control groups. Each group containing 10 tumor-bearing mice was individually followed during the experiment. Drug (squalamine, paclitaxel plus carboplatin, or squalamine with paclitaxel and carboplatin) or vehicle administration began on the day the animals were paired (day 1). Drug doses and schedules were selected based on previous measurements of the maximum tolerated dose of carboplatin plus paclitaxel in mice. Intraperitoneal (ip) dosing of the drug was chosen as an acceptable surrogate for intravenous dosing.
    [0099]
  Mice were weighed twice weekly, beginning on day 1, and tumor measurements were performed twice weekly with calipers. These tumor measurements were calculated using the well-known formula, L²  x 2 W = converted to mg tumor by tumor weight. The experiment was terminated when control tumors reached an estimated average tumor size of 1 gram based on in vivo tumor measurements. Upon termination, all mice were weighed, sacrificed, and their tumors excised. The resected tumors were weighed before fixation in 10% formalin, and for each treatment group the average resected tumor weight gain from the value on day 1 was calculated for all survivors until the scheduled termination sacrifice. Calculated for animals. In this xenograft model, (mean resection treated tumor weight gain divided by mean resection control tumor weight gain (T / C)) x 100% was subtracted from 100%, and tumor growth inhibition (TGI) values for each group I got Mice that had reduced tumor size at the end of the experiment compared to the initial tumor size were not included in the calculation of TGI, and as a result, TGI values may represent a minimal estimate of tumor response to chemotherapy. is there.
    [0100]
  Some of the drug combinations caused some tumor shrinkage in the MV-522 tumor xenograft model. In these combinations, the final weight of the defined tumor (day 31) was subtracted from its own calculated weight at the start of day 1 treatment for the corresponding mouse. The difference was divided by the initial tumor weight and multiplied by 100% to give the percent reduction. Thereafter, if more than one mouse in the group had experienced a decrease in tumor size, the average percent tumor shrinkage was calculated from the data.
    [0101]
  At any time during the study, a tumor was considered to have experienced a partial regression if the reduction in tumor size was> 50%. If the tumor completely disappeared in the mouse, this was considered a complete response or complete tumor shrinkage, which was subsequently confirmed by histological examination.
    [0102]
  Statistical analysis of resected tumor weight changes used a one-way analysis of variance (ANOVA) followed by a pairwise comparison by Bonferroni's or Tukey's t-test (RR Sokal and FJ Rohlf,Biometry, W.S. H. Freeman and Company, San Francisco (1981); G. FIG. Altman,Practical Statistics for Medical Research, Chapman and Hall, New York (1991)). Dispersion uniformity was assessed using the Levene test, and heterologous dispersion data were log transformed appropriately before applying to ANOVA. Data are means ± SEM. E. FIG. M. Or as a percentage of vehicle control. Nonparametric analysis of tumor weights with non-normal distribution (as determined by the Kolmogorov-Smirnov test) and / or unequal variances was also performed using the Kruskal-Wallis statistical test, with the Mann-Whitney rank sum for pairwise comparisons. The test was used. Differences between the appropriate means or ranks were considered significant if they resulted in a p-value of 0.05 or less. The p-value for a regimen containing squalamine is calculated relative to the same therapy without squalamine.
    [0103]
  Preliminary experiments showed that 20 mg / kg, qd x 5, i. p. 10 mg / kg, qd x 5, i.v. in combination with carboplatin for administration. p. The use of dosing paclitaxel was tolerated by nude mice and represented the maximum non-toxic combination dose. The combination of paclitaxel and carboplatin at this dose and these doses in an in vivo MV-522 human lung tumor xenograft model in vivo MV-522 resulted in an average resected tumor size of 365.9 ± 66.0 mg on day 31 or 60.3% TGI occurred (p <0.01) and there were no signs of tumor shrinkage for any of the animals in this treatment group. In contrast, triple combination chemotherapy with squalamine, paclitaxel and carboplatin resulted in a mean resected tumor growth of −0.1 ± 19.3 mg (p ≦ 0.001) or three weeks before final sacrifice. Despite no therapy, net tumor stasis occurred for the triple therapy combination group of animals. The TGI index for the squalamine / paclitaxel / carboplatin arm (arm) in this study was 96.1%. The benefit of chemotherapy on tumor size was evident by day 7, and the difference in average tumor size between the two paclitaxel and carboplatin-containing arms of the study increased thereafter. Tumors in squalamine-treated animals scored at final sacrifice were significantly smaller than those seen in animals receiving paclitaxel and carboplatin alone. None of the mice receiving paclitaxel and carboplatin alone underwent tumor shrinkage, as assessed at the end of the experiment, while four mice in the triple combination chemotherapy cohort showed tumor growth over the course of the experiment. A reduction was noted, with a mean reduction in tumor size for these animals of 54.8%. There was no complete tumor regression in any of the treatment groups receiving paclitaxel / carboplatin and / or squalamine and partial regression only in the triple combination chemotherapy group (6 of 7 surviving animals). . The transient reduction in tumor size was greatest at day 14 of chemotherapy, as seen with squalamine plus cisplatin.
    [0104]
  Initial selection of squalamine doses and dosing of combination chemotherapy were based on the maximum tolerated daily dose of squalamine in rodents and previous experience with squalamine pharmacokinetics. The possibility of maintaining squalamine with carboplatin in addition to paclitaxel at lower squalamine doses was separately investigated. Dose-ranging experiments were performed in a MV-522 lung tumor xenograft model with a daily dose of 0.2-20 mg / kg squalamine on days 1-5 and 8-9 along with paclitaxel and carboplatin. Was. The experiment ended on day 32 and the final resected tumor weight and percent TGI value were determined and based on these observations a mean value was calculated (Table 7). At final sacrifice, the mean resected control tumor growth was 674.3 ± 75.8 mg, while the resected tumor growth in animals treated with paclitaxel and carboplatin alone was 223.4 ± 82.5 mg. This corresponds to 66.9% TGI with paclitaxel plus carboplatin, which is similar to the TGI, 60.3% found in the first experiment for this dual drug treatment (see above). I want to do that). The mean resected tumor growth value of the squalamine treatment group was 132.0 ± 46.4 mg (TGI = 74.2%) at a daily squalamine dose of 0.2 mg / kg (p> 0.05), and 2. At 0, 10.0 and 20.0 mg / kg / day squalamine, it was less than 77.0 mg (TGI ≧ 78.8%) (Table 7). The maximal enhancement seen with multiple daily doses of squalamine occurred at a dose of 2.0 mg / kg / day; at this dose the mean resected tumor growth was 22.9 ± 25.0 mg, 92.1% (P = 0.017). Statistically significant results were only observed at 2.0 (p = 0.017) and 10.0 (p = 0.035) mg / kg / day squalamine, and at 20 mg / kg / day ( p> 0.05) was not observed with squalamine. However, meta-analysis across this and previous experiments shows that 20 mg / kg / day squalamine significantly enhances the effect of adding carboplatin to paclitaxel (p <0.001). The benefit of adding squalamine to paclitaxel and carboplatin treatment of MV-522 tumors was apparent at all doses by day 5 and was sustained throughout the course of the experiment.
    [0105]
  None of the mice receiving paclitaxel and carboplatin alone had tumor shrinkage, but at least two mice in each squalamine treatment group had a reduction in tumor size over the course of the experiment, with 2 or 10 mg / kg In each group receiving squalamine / day, on day 32, 5 out of 10 mice that showed tumor shrinkage were maximal. No mice showed complete tumor shrinkage in any of the treatment groups, but significant partial tumor regression was seen in all treatment groups (only containing paclitaxel / carboplatin), indicating that It was most apparent on the 15th. In the two squalamine treatment groups that showed the greatest tumor response (2 and 10 mg / kg / day squalamine), of the 20 mice, 19 had at least a 50% reduction in tumor load on day 15 .
    [0106]
  The study was conducted for the required period of time, in which squalamine in combination with paclitaxel and carboplatin had a sustained effect on tumor growth even more than 3 weeks after the last dose of squalamine, and it is important that squalamine is given during that period. Was. A separate arm of the experiment described in the previous paragraph considered tumor growth for a group of animals that received squalamine twice daily with paclitaxel and carboplatin at a daily dose of 0.2-40 mg / kg. With the daily dose of squalamine, there was a squalamine dose-related enhancement of paclitaxel and carboplatin tumor growth inhibition. The best response was seen at daily squalamine doses of 10 and 20 mg / kg. At these doses, by day 32, the mean resected tumor growth was 113.1 mg at 10 mg / kg and 96.6 mg at 20 mg / kg squalamine, with a mean resected tumor value for control tumors of 674.3 mg and paclitaxel And 223.4 mg of carboplatin-treated tumor. However, none of the squalamine doses reached statistical significance when compared to paclitaxel and carboplatin alone. During the experiment, 2-4 squalamine-treated mice showed partial tumor regression, and as assessed at the end of the experiment, a permanent partial tumor shrinkage was observed in 2-3 animals in each squalamine-treated group. Although seen, there was no reduction in tumor size in any of the mice treated with paclitaxel and carboplatin alone. Also, one complete regression was observed in mice receiving 20 mg / kg squalamine on day 5 with paclitaxel and carboplatin. The average percent tumor shrinkage in squalamine-treated animals that showed tumor regression was in the range of 21.0-50.4%.
    [0107]
  Example 10
  Squalamine activity combined with radiation therapy
  Syngeneic breast cancer MCA-4 tumor cells (5 x 10⁵) Was intramuscularly injected into the right leg of C3Hf / Kam male mice (4 months old). When tumors reached 6 mm in diameter, mice were randomized: (a) no treatment, (b) squalamine alone (11-14 days, 20 mg / kg / day bid), (c) 15 Gy. Assigned to gamma irradiation or (d) 15 Gy of gamma radiation in addition to squalamine. Tumor growth was followed until the tumor reached at least 12 mm in diameter. The effect of squalamine on tumor radiation response was determined by absolute and normalized tumor growth delay (AGD and NGD). AGD is defined as the number of days for an irradiated (or squalamine) treated tumor to grow to 8-12 mm minus the number of days for an untreated tumor to grow to 8-12 mm. NGD is defined as the number of days for tumors treated with both squalamine and radiation to grow to 8-12 mm minus the number of days for tumors treated with squalamine alone to grow to 8-12 mm. Enhancement factor (EF) was defined as the ratio of NGD to AGD. The EF for squalamine with 15 Gy gamma rays (Table 8), calculated from the observation time for tumor growth, is> 1.5.
    [0108]
[Table 8]

  Example 11
  Squalamine activity in combination with cisplatin for ovarian cancer
  Human ovarian tumor cells were injected subcutaneously into the mid-back area of female Swiss nude mice (5 × 10 5 per animal).⁷cell). Human ovarian tumors were derived from the parent 2008 cell line (cf. Pietras et al.Oncogene  9, 1829-1838 (1994)) or a transfection mutant overexpressing the Her-2 / neu receptor. Overexpression of the Her-2 / neu receptor is considered to render the resulting xenograft tumors more angiogenic and less responsive to chemotherapy. One week later, animals were treated with no treatment (control), a single dose of cisplatin (4 mg / kg), daily treatment with squalamine (2 mg / kg, days 1-10), or squalamine plus cisplatin. Groups were randomized to receive treatment. Thereafter, tumor growth was scored on day 28 following the start of chemotherapy. The results were very similar for the parent 2008 tumor and the transfection mutant overexpressing the Her-2 / neu receptor. Typical results for the parent 2008 cell line are shown in Table 9. These results can be quantified by calculating a tumor growth inhibition index for each treatment group. In this xenograft model, (mean treated tumor weight gain on day 28 divided by mean control tumor weight gain on day 28 (T / C)) x 100% was subtracted from 100% and tumor growth for each group Inhibition (TGI) values were obtained. Cisplatin had a minimal effect on 2008 tumor cell growth in this model, while squalamine significantly reduced the average tumor size on day 28 compared to controls. The combination of cisplatin plus squalamine was very effective at slowing tumor growth.
    [0109]
[Table 9]

[Brief description of the drawings]
FIG. 1 shows the general structural formula of squalamine.
FIG. 2 shows a general overview of the angiogenic process.
FIG. 3 is a diagram used to illustrate a sodium hydrogen exchanger (NHE) process.
FIG. 4 shows the effect of normal amiloride on inhibition of various isoforms of mammalian NHE.
FIGS. 5a and 5b show the effect of squalamine on NHE isoform 3 (NHE3) and NHE1 inhibition, respectively.
FIGS. 6a, 6b and 6c show the pharmacokinetic results associated with squalamine.
FIG. 7 shows i. v. Fig. 3 shows squalamine distribution in various tissues after administration.
FIG. 8 shows an angiogenesis index using squalamine determined in a rabbit corneal micropocket assay.
FIG. 9 shows the inhibitory effect of squalamine on endothelial cell proliferation compared to tumor cell lines.
FIG. 10 shows the inhibitory effect of squalamine in a glioma mortality study using rat 9L glioma introduced into healthy rat brain.
FIG. 11 shows the survival of mice carrying a human MX-1 breast tumor xenograft, treated with squalamine, and subsequently treated with cyclophosphamide.
FIG. 12 shows inhibition of human lung adenocarcinoma (H460) in a mouse xenograft-combination therapy study with squalamine and cisplatin.
FIG. 13 shows the number of lung metastases in mice implanted subcutaneously with Lewis lung carcinoma after various chemotherapy treatments.
14a and 14b show the effect of squalamine, VEGF and a combination treatment of VEGF and squalamine on LNCaP human prostate cell proliferation; FIG. 14b shows squalamine, VEGF and VEGF on C4-2 human prostate cell proliferation. And shows the effect of the combination treatment of squalamine. both[³[H] -thymidine incorporation assay was utilized.
FIGS. 15a and 15b show the effect of squalamine on PSA levels (a) and tumor growth (b) in control athymic mice after orchiectomy.
FIGS. 16a and 16b show the effect of squalamine on PSA levels (a) and tumor growth (b) in athymic mice undergoing orchiectomy followed by treatment with squalamine.