JP2004529147A - hCG prescription - Google Patents

Abstract

A pharmaceutical composition of water-soluble and physiologically active proteins and polypeptides such as hCG hormone, which is useful for treating various diseases.
The pharmaceutical composition comprises a physiologically active protein or a peptide formulated in the form of an oil-in-water emulsion, and optionally a muramyl peptide. More specifically, it relates to human chorionic gonadotropin (gonadotropin) (hCG) and related hormones.

Description

実施例１
【００５７】
本発明の組成物は当業者に公知の任意の方法により製造することができる。種々の用途に要求されるＨＬＢ値は、油中水乳化剤（例えば、アルラセル＋スクアレン）の場合は３−６；湿潤剤の場合は７−９；水中油乳化剤の場合は８−１３；洗浄剤の場合は１３−１５；可溶化剤（例えば、アルミニウム又はマグネシウム・ステアレート）の場合は１５−１８である。これらの値は、特定の目的のための乳化剤の選択における目安として文献に広く引用されている。これらは非イオン性乳化剤との使用のために設計される。アナログシステムがアニオン又はカチオン乳化剤のために開発されているが、これらは非イオン性乳化剤についてのものと比較して有用性が小さい。ＨＬＢ値が多くの非イオン性乳化剤について文献に記載されている。この特別の使用において、油相が約0.5ないし55％、乳化剤が約0.1ないし15％、非イオン性界面活性剤が約0.05ないし5％、治療薬が0.00001ないし10％、更に水性連続相を有する水中油エマルジョンが最も適当である。この組成物における乳化剤は、ホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジルグリセロール、ホスファチジン酸、スフィンゴミエリンおよびカルジオリピンからなる群から選択されるホスホリピド化合物又はホスホリピドの混合物である。乳化剤の実施例は、Arlacel Aである。界面活性剤は、脂肪アルコール、脂肪酸のポリエチレングリコールエステル、ポリエトキシル化脂肪酸エステル、ポリエトキシル化脂肪アルコールエーテル、1又はそれ以上の水酸基を有する有機化合物のアルキレンオキシド縮合物およびポリエトキシル化アルキルフェニルエーテルからなる群から通常、選択される。界面活性剤は天然の生物学的に相容性を有する界面活性剤、例えばレシチン又は薬理学的に許容し得る人工界面活性剤、例えばTWEEN-80であってもよい。レシチンに関係する天然成分の適当なものとしては、例えば、EPICURON 120（Lucas Meyer,ドイツ国）（これは約70％のホスファチジルコリンと、12％のホスファチジルエタノールアミンと、約15％の他の種類のホスホリピドとの混合物）；OVOTHIN 160（Lucas Meyer,ドイツ国）（これは約60％のホスファチジルコリンと、18％のホスファチジルエタノールアミンと、約12％の他の種類のホスホリピドとの混合物）；精製ホスホリピド混合物-LIPOID E-75又はLIPOID E-80（Lipoid,ドイツ国）（これは約80％のホスファチジルコリンと、8％のホスファチジルエタノールアミンと、約3.6％の非極性リピドと、約２％のスフィンゴミエリンとの混合物）を挙げることができる。精製卵黄ホスホリピド、大豆油ホスホリピド又は他の精製ホスホリピド混合物もこの成分として有用である。この列挙したものは代表例であり、これに限定することを意図したものではない。なぜならば、当業者に公知の他の種類のホスホリピドも使用することができるからである。界面活性剤の他の例として、ひまし油などの原料からの脂肪酸のポリエチレングリコールエステル（EMULFOR）；ポリエトキシル化脂肪酸、例えばステアリン酸（SIMULSOL M-53）；NONIDET；ポリエトキシル化イソオクチルフェノール/ホルムアルデヒドポリマー（TYLOXAPOL）；ポリオキシエチレン脂肪アルコールエーテル（BRIJ）；ポリオキシエチレン・ノンフェニルエーテル（TRITON N）；ポリオキシルエチレン・イソオクチルフェニルエーテル（TRITON X）などを挙げることができる。他の例において、エマルジョンを1又はそれ以上の共界面活性剤を実質的に含まずに形成、安定化させることができる。この場合の共界面活性剤の例としては、非ハロゲン化脂肪族C3-C6アルコール、遊離脂肪酸、モノ‐又はジ‐グリセリド、ポリグリセロール脂肪酸エステル、又はリソホスファチジルコリンを挙げることができる。しかし、これらとは別の組成物も等しく好適に使用可能であり、当業者にとって容易に製造することができよう。例えば、この組成物には、5.0％のPLURONIC、10％のスクアレン、0．4％のTween-80、qsホスフェート緩衝塩液（pH＝7.4）を含むものであってもよい。これらの成分は試験管に添加され、ミルキーエマルジョンが得られるまで渦流混合される。この組成物は投与直前に製造され、４℃で冷凍される。組成物２は例えば、5.0％のTETRONIC 1501、10％のスクアラン、0．4％のTween-80、qsホスフェート緩衝塩液（pH＝7.4）を含むものである。これら組成物に対して、固体N-アセチルムラミル-L-トレオニル-D-イソグルタミン（Thr-MDP）が500ug/mLの濃度まで添加され、“濃縮物”が得られる。この濃縮物はついで、抗原の2x濃度溶液（塩水中、hCG；1mg／mL）と混合され、本発明の処方が得られる。このhCGは好ましくは、この組成物の約0.00001ないし10重量％、好ましくは約0.0001ないし5重量％、最も好ましくは約0.001ないし1重量％の範囲で含有される。更に、ｐHはhCGの安定性に適した範囲とすべきである。この組成物の連続相は水性であり、これに更に、塩、糖、抗酸化剤、保存剤、殺菌剤、緩衝塩液、浸透圧剤、凍結防止剤、他の薬理学的に有用な添加物又は溶質を含有させてもよい。典型的な保存剤の例としては、チメロサール、クロルブタノールおよびメチル、エチル、プロピル又はブチルパラベンなどを挙げることができる。典型的な浸透圧調整剤の例としては、グリセロールおよびマンニトールが含まれ、特にグリセロールが好ましい。好ましい油相抗酸化剤の例としては、アルファ-トコフェロール又はアルファ-トコフェロール・スクシネートを挙げることができる。上記水相にはポリアミンカルボン酸の抗酸化剤、例えば、エチレンジアミノテトラ酢酸又はその薬理学的許容塩などを含んでいてもよい。
実施例２
【００５８】
この組成物は2つの部分からなる。部分Iは、N-アセチルムラミル-L-トレオニル-D-イソグルタミン（Thr-MDP）、すなわち、ミコバクテリウム細胞壁成分の誘導体である。部分IIは、最終濃度が5％のスクアラン、1．25％のプルロン酸および0.2％のTWEEN 80（ビヒクル）のものを含むホスフェート緩衝塩液からなるものである。実際的目的のため、hCGの所望量をミクロ流動化ビヒクル（部分II）と混合させて均質エマルジョンを得る。ついで、MDPを添加し、簡単に混合する。この混合物中のMDPの濃度を調節し、臨床応答を得るための最適な濃度を決定する。佐剤対照として、生産者のマニュアル（Piece Chemicla, Rockford, I11.）に従った明礬又は完全フロイントアジュバント（CFA）と混合した可溶性hCGをマウスに注射する。
【００５９】
当業者は、このようなモデルマウスは、均等の経験および治療がヒト、家畜又は農業用動物においても同様の臨床的応答を生じさせることを示すということを認識されるであろう。所望の臨床結果を生じさせるのに有効な書法およびhCGの量は、過大な実験を要することなく、当業者に周知の常套の手法により経験的に決定し得るであろう。このような混合物での治療による副作用を最小にするため、ヒト、家畜又は農業用動物にとって効果的な応答を得、それにより所望の臨床的結果を得るのに必要な最小投与量を決定することも当業者にとって可能であろう。通常の使用の場合、この混合物は標準的手法の任意の1つにより注入することができよう。しかし、特に好ましい方法は、皮下注射又は筋肉内注射を、このエマルジョンが数日又は数週間、安定に留まるような部位に施すことである。
実施例３
【００６０】
hCGの水性懸濁液はホモジナイザーで攪拌しながら、室温で油相中に添加する。この水相の添加は、油相5−6部に対し、hCGの水性懸濁液が3−5の割合に達したときに停止する。攪拌は水相の小滴サイズが約0.05−0.5ミクロンになるまで継続する。この油相は、93.6％のMarcol 52(白色パラフィンEsso oil)と；6.0％のアルラセルA又はアルラセル80又はSpan 80(マンニド・モノオレート)；0.4％のTween 80(ポリオキシエチレン20ソルビタン・モノオレート)を含有する。この油相の化合物は別途、オートクレーブ中で110℃まで加熱したり、又は混合物として滅菌ろ過させてもよい。このエマルジョンの安定性は、（1）乳化後に小滴を直接、ピペットで採取し、水面に落し、その小滴が広がらずにそのままに維持されるか否かを知ることにより、更に、（2）37℃で4週間貯蔵し、それでも水相が形成されないということを知ることにより判定することができる。このようにして製造されたエマルジョンの最終濃度は、薬10％のhCGを含み、この調剤は患者に対して筋肉内又は皮下を介して0.5ｍLの投与量で使用される。
実施例４
【００６１】
上記実施例に限定されることなく、ダイマーhCGを含む本発明の組成物は、安定性を更に向上させるため、2種類の乳化剤を同時に含有するものでもよい。この組成物において、第1の乳化剤は、ポリオキシプロピレン・ポリオキシエチレン・ブロックコポリマー、グリセロール・モノオレート、グリセロール・ジオレート、ソルビタン・セスキオレート、Brij 93ポリオキシエチレン・アルコール、ソルビタン・モノオレートおよびこれらの混合物からなる群から選択される。第２の乳化剤は、ポリオキシエチレン脂肪酸エステル、プルロン酸F68エチレンオキシド、卵レシチンおよびこれらの混合物からなる群から選択される。この組成物における油分は、トリカプロイン、トリカプリリン、トリパルミチンおよびこれらの混合物からなる群から選択されるトリグリセリドである。他の適当な油相の例には、植物油および動物油が含まれる。適当な植物油の例としては、オリーブ油、ベニバナ油、ゴマ油、大豆油などである。動物油の例としては、グリセロールエステルを含有するものなどである。好ましい鉱油の例としては、No.40ホワイトオイル、カーネーション・ライトオイルおよびクレアロール・ライトオイルなどである。これらのオイルの混合物も等しく使用することができる。
【００６２】
鉱油を使用する第1次エマルジョンの場合、約1ないし50容量％、好ましくは約2ないし40容量％の水性hCG溶液；約8ないし58容量％、好ましくは約14ないし25容量％の鉱油；約2ないし30容量％、好ましくは約5ないし15容量％の第1の乳化剤が混合される。水性hCG溶液は好ましくは、例えば磁気攪拌機を使用した激しい攪拌下で、油相に徐々に添加され、この攪拌が約15分ないし60分間、好ましくは約25分ないし35分間、継続される。この混合された第1次エマルジョンに対し、ついで、マイクロ流動化装置を用いて高剪断乳化が行われ、この場合、剪断速度は約100，000ないし5,000,000/秒、好ましくは約500，000ないし1,000,000/秒、圧力降下は約1,000ないし10,000psi、好ましくは約1,000ないし3,000psi、最も好ましくは約1,800ないし2,000psiとする。このマイクロ流動化装置についてのより完全な情報は米国特許、No.4,533,254に記述されている。このマイクロ流動化装置は短時間（1秒足らず）の高い剪断速度を与えることができ、hCGなどのたんぱく質の変質を生じさせない。この第1次エマルジョンは、5ミクロン親水性ポリビニリデン・ジフルオリド・フィルター（Duropore,Millipore社）を用いてろ過される。乳化工程の前に、hCG溶液に対し、アルブミンを約1ないし5g％、好ましくは約2ないし3g％の割合で添加し、多重エマルジョンサイズ分布を更に安定化させるようにしてもよい。水性hCGを鉱油又は固定油に分散させた第1エマルジョンは本発明の液状多重エマルジョンの製造に適している。これによって、第1次エマルジョン小滴の径を5ミクロン未満、好ましくは3ミクロン未満にさせることができるからである。
実施例５
【００６３】
0.25％のコポリマーP123を有する50％v/vの塩類の外側水相を含む水中油中水（水/油/水）多重エマルジョンが作られる。残りの50％v/vは、72％の塩水と、18％のSpan 80と、エマルジョン0.5mL当り32mgのコポリマーL310および0.5mgのTNP-HEAとからなる油中水相である。このエマルジョンはエマルジョン0.5mL当り1mgのhCGを含有する。このエマルジョンの顕微鏡的外観は油中水型エマルジョンの粒径が1.0ないし20ミクロンのものである。子の組成物のための他の適当な油中水型乳化剤は、SF 1328、アルラセルP135、DC 3225C、DC 5200、Abil EM-90およびAbil WE-09である。幾つかの他の油中水型乳化剤を上記の成分の代わりに使用し得ることは当業者にとって明らかであろう。
実施例６
【００６４】
hCGを含むリポソームが当業者に公知の標準的手法により作られる（例えば、米国特許No.6,110,492）。以下のような成分を使用することができる。すなわち、DLPCジラウロイル・ホスファチジル・コリン；DMPCジミリストイル・ホスファチジル・コリン；DPPCジパリトイル・ホスファチジル・コリン；DSPCジステアロイル・ホスファチジル・コリン；DOPCジオレイル・ホスファチジル・コリン；DLｎPCジリノレオイル・ホスファチジル・コリン；DMPGジミリストイル・ホスファチジル・グリセロール；CHOLコレステロール；LAリピドAで或る。典型的製造において、多層リポソームがDMPC：DMPG：CHOL：LA(モル比＝９：１：7.5：0.011)から作られる。リピド（脂質）Aは佐剤として含有させる。この脂質混合物は回転蒸発される。つまり、梨型フラスコ中のクロロホルム溶液から約４０℃、真空中で蒸発されて乾燥した薄膜に形成される。有機溶媒を完全に除去させるため、乾燥器内でフラスコを非常に低い真空下（約0.05mmHg）、室温で一晩乾燥させる。乾燥後、脂質を脱イオン化・滅菌・無発熱原水中にて渦攪拌させながら注意深く膨潤させる。得られた懸濁液を-55℃で凍結させ、Virtis Unitop 800SL Freeze Mobileを用い、-20℃で一晩、ついで翌日、0−10℃で凍結乾燥する。この凍結乾燥された脂質を、カプセル化されるべき物質、すなわちhCGの存在下で再構築させ、この物質を含む多層リポソームを得る。適当な再構築緩衝液はホスフェート緩衝塩液（PBS）又はトリス-グリシン/NaClである。この再構築緩衝液中のリポソーム・ホスホリピド濃度は10−200mMである。カプセル化されなかったhCGは除去される。つまり、リポソームを、0.15M NaClを用い、27000xgで10分間、10℃で3回洗浄することによりhCGが除去される。得られたリポソームは0.15M NaCl中、又は適当な等張性緩衝液中に懸濁させ、最終ホスホリピド濃度が10−200mMのものを得る。別の方法として、この洗浄工程を省略し、調剤中にカプセル化されないhCGおよびカプセル化されたhCGの双方を残すようにしてもよい。
実施例７
【００６５】
この実施例はhCGをMDPに結合させるものである。この手法は第１の反応をチオール化合物で冷却する必要がある。この反応は2-[N-モルホリノ]エタンスルホン酸（MES）（pH：4.5−5.0）中で行われる。凍結乾燥され、MES（0.5mL）（pH：4.5−5.0）中に再懸濁されたMDP(10mg)と、MES（pH：4.5−5.0）中に溶解された0.5mLのEDC(0.5mg又は約2mM)とが一緒にされ、室温で15分間、反応に供せられる。2-メルカプトエタノール（20mMの最終濃度）が添加されEDCが冷却され、遠心分離により分離される。この反応混合物が1回、MESで洗浄され、ついで0.5mLのMES（pH：4.5−5.0）中に再懸濁される。MESに溶解させたhCGが活性化MDPに対し、約2：1のモル比で添加される。この反応混合物のｐHを、MES（0.5M,pH=8.5）の添加により15分に亘り徐々に8.5まで増加させ、室温で2時間反応させる。MDPに添加されたhCGの濃度は、MDPの末端カルボキシル基の定量分析から計算され、mgMDP当りのモルとして表される。この反応はヒドロキシアミンを10mMの最終濃度まで添加することにより冷却される。この冷却方法は、未反応のMDP活性化部位を加水分解させ、当初のカルボキシルを再生させる。他の冷却の手段には、20-50mMのトリス、リシン、グリシン又はエタノールアミンの添加が含まれる。その他の生物学的応答改質剤（例えば、IL2）は当業者にとって周知であり、hCGに加えて、この目的のために使用することができる。分別は遠心分離,洗浄工程,選択された緩衝液への再懸濁により行うことができる。
実施例８
【００６６】
本発明の組成物は、クリームとして使用することができ、これを局所的に適用することにより皮膚中に浸透させることができる。この局所用組成物の1例として、2％のグリセロール・ソルビタン・オレオステアレート（アルラセル481）；6％のポリエトキシ化脂肪酸（アルラセル989）；15％のデシルオレート（セチオールC）；8％の枝分かれ鎖パラフィン（アルラモールHD）；1％のマイクロワックス；1％のMDP;4％のグリセリン；0.7％のマグネシウム・スルフェート・7水和物；0.2%のソルビン酸ナトリウム塩；1％のhCG；0.1％のジアンモニウム・ヒドロゲノシトレート；クエン酸（pHを4−5に調節するため）；100％となる量の脱ミネラル水からなるものである。
実施例９
【００６７】
18歳を超えた男性および妊娠していない女性を、ウエスターン・ブロット法(Western Blot)に記載されているHIV感染の血清診断に基づき、AIDS確定基準、臨床的徴候を有する者、および検査前30日以内でCD4 Tリンパ球数値が＜300細胞/ULの者を選択する。標準的臨床および実験パラメータを用いて薬効を評価する。この臨床パラメータには、日和見感染性の変化、体重の変化、胃腸機能の変化（便通の規則性、頻度）、エネルギーレベルの変化、食欲、体力および忍耐力の変化、および生活の質の総体的変化が含まれる。実験パラメータには、CD4およびCD8リンパ球数、選択されたヘマトロジー、血液化学および尿検査、更に可能な場合、PCRによるウイルス性RNAの測定によるウイルス負荷の変化が含まれる。
【００６８】
本発明の趣旨に従ってhCG処方の使用により、HIV陽性患者が臨床的に改善され、日和見感染性の減少、体重の増加、胃腸機能の変化（下痢の軽減を含む）、エネルギーレベルの増加、食欲増大、愛欲の増大、体力および忍耐力の改善、生活の質の総体的改善が認められた。実験パラメータにおいては、CD4およびCD8リンパ球数の増加、ヘマトロジー、血液化学および尿検査の数値の改善、PCRによるウイルス性RNAの測定によるウイルス負荷の減少、TCIDにより測定したときの伝染力の減少など、多くの改善が認められた。hCG処方の使用により、若干の副作用が見られたが、それは軽い発熱、悪寒、頭痛、筋肉痛に限定されたものである。
実施例１０
【００６９】
C3H経歴（H2k/k,Harlan Sprague Dawley）のメスのマウスをこれら検査に使用する。これら動物は“Guide for the Care and Use of Laboratory Animals（実験動物の取り扱い注意ガイド）"(DHHS,NIH)に従って維持され、食事および水が無制限に与えられる。腫瘍細胞ラインHOPE2がin vitroでの連続的通過により維持される。腫瘍は、150,000のin vitro通過細胞の皮下注射により先天性C3Hマウスに開始される。腫瘍は2週間おきに2垂直方向で測定される。各治療グループは、治療を受けていない対照グループと比較される。治療はHOPE2細胞の接種後10日から、すなわち腫瘍の多くが触知可能（略50−75mm^３）となった時点から開始される。治療は、MDP混合物又はAlumアジュバントに可溶性hCGたんぱく質を添加したものをマウスに注射すること（合計0.2mLを皮下注射）により開始される。接種直前に、hCGをHanksバランスド塩溶液（HBSS）中のMDPと60秒間混合し、各マウスが、ヒトに相当するところの、2,000又は5,000IUのhCGたんぱく質を0.2mL、投与されるようにする。生産者の指示に従ってAlum(Pierce Chemical社)をhCGと混合し、各マウスが、ヒトに等価の、5,000IUのhCGを0.2mL、投与されるようにする。この実施例において、腫瘍細胞接種後、41日目において、可溶性hCGの単一の注射を受けたマウス（夫々2,000又は5,000IU）の内の5/8および4/8が多少の腫瘍を示すに過ぎなかった。対照的に、Alumに添加したhCGを用いて注射したマウスの全て（8/8）が著しく成長しつつある腫瘍を示した。更に、腫瘍成長の可なりの抑制がMDPにhCGを添加したものを用いて注射したグループにおいてのみ認められ、これは対照（治療しない）グループ又はAlumで治療したグループとは、その差が全く対照的である。腫瘍成長の抑制又は腫瘍緩解向上は、Alumに添加したhCGを用いて単一注射したマウスにおいては認められなかった。
【００７０】
従って、本発明は、ダイマーhCGおよびムラミルペプチドを含む薬剤組成物によって特徴づけられる。その他、本発明は、（a）リポソームと関連させたダイマーhCGの治療的有効量と；（ｂ）該リポソームを囲む油を含む水中油エマルジョンとを含有し；該エマルジョンが、該エマルジョンが存在しないときのhCGおよびリポソームとの関連で治療応答を増大させるのに十分な量を以って含有されている薬剤組成物によって特徴づけられる。更に、本発明は、ＨＩＶ感染者を治療するための方法であって、該患者に有効量のhCGを投与することからなり、該hCGが水中油型エマルジョンの形で投与され、該エマルジョンを、該エマルジョンが存在しないときのhCGおよびリポソームとの関連で治療応答を増大させるのに十分な量で含む方法を提供する。更に、本発明は、がん患者を治療するための方法であって、該患者に有効量のhCGを投与することからなり、該hCGが水中油型エマルジョンの形で投与され、該エマルジョンを、該エマルジョンが存在しないときのhCGおよびリポソームとの関連で治療応答を増大させるのに十分な量で含む方法を提供する。本発明は更に、連続的水相と非連続的油相とを有すると共に、ダイマーhCGを少なくとも0.01重量％含む水中油型エマルジョンを含有する薬剤組成物を提供する。本発明は更に、薬剤処方を製造するための方法であって、hCGを含む水性懸濁液、少なくとも1つの油性成分および十分な量の少なくとも1種の乳化剤を混合し、水中油型エマルジョンとして該処方を得る方法を提供する。本発明は更に、水中油型エマルジョンと、少なくとも１種の天然又は組換え水溶性生物学的活性たんぱく質と、任意の少なくとも１種のムラミルペプチドと含む薬剤組成物を提供する。本発明は更に、薬剤組成物の即座の製造のためのキットであって、（１）エマルジョンを収容する第1の容器であって、該エマルジョンがスクアラン又はスクアレンと、アルラセルと、水性溶液とを有してなるものと；（2）水性溶液に溶かした、又は粉体としての治療効果量のダイマーhCGを収容した第2の容器とを具備してなり；各容器中の成分の濃度が、双方の容器中の成分を組み合わせたときに、１ないし３０％のアルラセル、１ないし３０％のスクアラン又はスクアレン、０．０００１ないし３０％のムラミルペプチド、０．０１ないし９９％のhCGの処方を生じさせるものであるものを提供する。
【００７１】
以上、本発明を現在、最も実際的で、好ましい実施例と思われるものとの関連で詳細に説明したが、ここに記載した原理および概念から逸脱することなく、多く変形し得ることは当業者に明らかであろう。従って、本発明の正しい範囲は付記した請求の範囲の最も広い解釈により判定されるべきであり、上述のような変形例を全て含むと共に、明細書に記載した均等の関係のあるものも全て包含するものと解されるべきである。【Technical field】
[0001]
The present invention relates generally to pharmaceutical formulations of water-soluble, physiologically active proteins and polypeptides, such as the hCG hormone, which are useful for treating various diseases. In particular, virus-induced diseases, immune diseases and malignancies are suitable for treatment with the compositions of the present invention. More specifically, treatment for diseases caused by viruses such as the human immunodeficiency virus (HIV), which gives rise to acquired immunodeficiency syndrome (AIDS) and related cancers in humans, is expected.
[Background Art]
[0002]
Human chorionic gonadotropin (gonadotropin) (hCG) belongs to a class of glycoprotein hormones, including luteinizing hormones (lutropine, LH), follitropin (FSH) and thyrotropin (TSH). included. Each of these hormones consists of two dissimilar, non-covalently associated subunits, alpha and beta. These hormones share a common alpha subunit, and the beta subunits differ in length and amino acid sequence and are unique to each hormone. The closest hormones are hCG and LH, which are 85% identical except for the amino acid sequence at the carboxy terminus of hCG. hCG and LH act through a common receptor. It is traditionally believed that hCG and related hormones play a role in normal human physiology. For example, hCG is already established as a gestational hormone and plays an important role in reproductive physiology during pregnancy. Extensive background information on hCG is provided in a review article by the present inventors, which is incorporated herein by reference to Herna F. Acevedo, "Human chorionic gonadotropin (hCG): The hormone of life and death, A review." ing.
[0003]
Recently, it has been proposed that the hCG hormone is useful for treating HIV / AIDS and cancer. No. 5,700,781; No. 5,811,390; No. 5,851,997; No. 5,997,871; 5,877,148; 5,677,275 or reverse transcriptase in HIV-1 infected by lymphocytes and mononuclear leukocytes by Bourinbaiar AS, Nagorny R. Effect of human chorionic gonadotropin (hCG) on activity. FEMS Microbiol Lett. September 1, 1992; 75 (1): 27-30 and Bourinbaiar AS, Nagorny R. "HIV from lymphocytes to trophoblasts" Inhibitory Effect of Human Chorionic Gonadotropin (hCG) on Transmission "FEMS Lett. Aug. 31, 1992; 309 (1): 82-4, the contents of which are incorporated herein by reference. However, some researchers have argued that contaminants found in commercial hCG preparations affect anti-HIV and anti-cancer activity and refute the idea that hCG acts on anti-viral and cancer activity. . See, for example, Lee-Huang S, Huang PL, Sun Y, Huang PL, Kung HF, Blithe DL, Chen HC. "Lysozyme and NRase as anti-HIV components in beta-core formulations of human chorionic gonadotropin" Proc Natl Acad Sci USA.March 16, 1999; 96 (6): 2678-81; Sairam MR, Antakly T. DebunkinghCG.Nat Biotechno.November 1997 (12): 1228; Albini A, Paglieri I, Orengo G, Carlone S, Aluigi MG, DeMarchi R, Matteucci C Mantovani A, Carozzi F, Donini S, Benelli R. "A beta-core fragment of human chorionic gonadotropin is responsible for Kaposi's sarcoma-inducing cells and a novel immortalized Kaposi's sarcoma cell line. AIDS. March 1997; 11 (6): 713-21; Lunardi-Iskandar Y, Bryant JL, Blattner WA, Hung CL, Flamand L, Gill P, Hermans P, Birken S, Gallo RC. "Effects of Urine Factors from Women During Early Pregnancy on HIV, SIV and Related Diseases" Nat Med. April 1998; 4 (4): 428-34; Sispas NV, Aroni K, Tsavaris N, Mavrag. ani K, Paikos S, Kordossis T. “AIDS-related cutaneous Kaposi's sarcoma: failure to treat with human chorionic gonadotropin” J Chemother. 1999 Feb; 11 (1): 78-9; Masood R, McGravey ME , Zheng T, Cai J, Arora N, Smith DL, Sloane N, Gill PS. "Antineoplastic urinary protein suppresses Kaposi's sarcoma and angiogenesis in vitro and in vivo" Blood. February 1, 1999 ; 93 (3): 1038-44; Griffiths SJ, Adams DJ, Talbot SJ. Ribonuclease inhibits Kaposi's sarcoma. Nature. Dec. 11, 1997; 390 (6660): 568; Darzynkiewicz Z. "Butler Achieved: Investigation of Killer of Kaposi's Sarcoma Cells in Preparation of Human Chorionic Gonadotropin" J Natl Cancer Inst. 1999 Jan 20; 91 (2): 104-6; and Noonan D, Albini A. Anti-KS activity still a mystery. Nat Med. July 1998; 4 (7): 748, the abstract of which is for reference. , Incorporated herein. Because of the confounding results, some authors have suggested that commercial preparations of urine and crude clinical grade hCG from pregnant women include anti-Kaposi's sarcoma and various other factors associated with anti-HIV activity . Researchers have found, among other things, urinary factors such as eosinophil-induced neurotoxin (EDN), anti-tumorigenic urinary protein (ANUP), inhibin, activin A and vascular growth inhibitors. In addition, some authors have warned openly that hCG has the opposite effect and rather promotes carcinogenesis, substantially denying the utility of hCG as an anticancer or antiviral agent. I have. For example, Simonart et al. (Simonart T, Hermans P, Van Vooren JP, Meuris S. Paradoxical prokaposi sarcoma activity of human chorionic gonadotropin preparations, Blood. July 1999; 94 (1): 376-7).
[0004]
While there is still considerable debate in the prior art as to whether hCG or certain contaminants have antiviral / anticancer activity, this hormone or contaminant in commercially available hCG preparations has a practical perspective. It is inconvenient. Like many other water-soluble glycoprotein hormones, hCG is rapidly metabolized in the body, and therefore has a relatively short half-life, so it is often used or administered with very high amounts of hCG In need. Either of them is impractical and expensive. See, for example, U.S. Patent No. 5,700,781, "Methods for treating Kaposi's sarcoma and HIV infection"; Harris, incorporated herein by reference.
[0005]
Variants of hCG release as so-called "slow release" or "sustained release" forms of hCG release have been previously proposed, but none have been successfully implemented. This is because there is no specific implementation guidance for this hormone specific form. See, for example, US Patent No. 5,851,997, "Use of human chorionic gonadotropin as an immunopotentiating antiviral agent"; Harris. In this patent, Harris refers to various types of slow release methods for hCG. In particular, Harris teaches a sustained release form of hCG as a transdermal hCG patch. This is similar to DURAGESIC®, in which the transdermal release of a protein such as hCG is effected iontophoretically or electroosmically, ie under the influence of an electric field. Another sustained release form of hCG designed by Harris is the implanted hCG release system. Like the NORPLANT® Levonorgestrel implant, which is a representative type of device in this category, this depolarizes hCG via a non-biodegradable rate limiting membrane member consisting of, for example, a hydrogel or microporous polymer. Is to be released. Another type of other hCG transplant designed by Harris is one that incorporates a pump function to administer the hormone. This pump function is driven osmotically or activated by the patient. Thus, Harris teaches various alternative modes of hCG administration. However, the mode of release contemplated is through certain sustained release drug delivery devices, ie, transdermal patches and various types of implants or pumps. Nothing is taught about the reliability of drug administration as well as other forms of hCG administration as a practical mode.
[0006]
It has been reported by the above inventors that muramyl peptide alone can suppress retrovirus replication (Acevedo HF, Raikow RB, Acevedo HO, Delgado TF, Pardo M., CGP 11637, by synthetic muramyl dipeptide analog Prevention of carcinogenic virus infection in mice. Antimicrob Agents Chemother. Nov 1985; 28 (5): 589-96; Lazdins JK, Woods-Cook K, Walker M, Alteri E., Lipophilic muramyl peptide MTP- PE is an effective inhibitor of HIV replication in macrophages. AIDS Res Hum Retroviruses. Oct 1990; 6 (10): 1157-61; Masihi KN, Lange W, Rohde-Schulz B, Chedid L., Muramil Dipeptides suppress human immunodeficiency virus replication in vitro. AIDS Res Hum Retroviruses. March 1990; 6 (3): 393-9).
[0007]
However, the combination of hCG and muramyl peptide is contrary to the current thinking of those skilled in the art. This is because muramyl peptides are most often used as vaccine adjuvants to boost the immune response to antigens present in vaccine preparations, ie hCG (eg, US Patent Nos. 5,840,313 or 5,876,724). Thus, this combination may effectively inactivate hCG, and may even worsen the course of the disease. In view of certain investigators' consideration that certain muramyl peptides do actually enhance HIV replication, the prediction that clinical use of muramyl peptides will exacerbate HIV disease is particularly worrisome (Schreck R , Bevec D, Dukor P, Baeuerle PA, Chedid L, Bahr GM., Selection of muramyl peptides based on lack of activation of nuclear factor-kappa B as potential adjuvants for AIDS vaccines. Clin Exp Immunol. 1992 Nov; 90 (2): 188-93; Masihi KN, Lange W, Rohde-Schulz B., Bacterial immune modulator exacerbates human immunodeficiency virus infection in promononuclear cells. J Acquir Immune Defic Syndr. 1990; 3 (3): 200-5). To date, both MDP isolated from natural sources and synthetic MDP have been associated with significant toxicity when administered to mammals. This toxicity has severely limited the clinical use of MDP. Occasionally, muramyl peptides are taught to be useful when combined with physiologically active proteins. For example, US Pat. No. 5,932,208 discloses a composition combining a muramyl peptide with cytokin, but does not teach the combination with hCG.
[0008]
However, combinations of hCG variants and muramyl peptides are known in the prior art mainly as ovulation-inducing vaccines or cancer vaccines. These vaccines contain exclusively the beta chain of hCG or the carboxy terminal peptide of the beta chain of hCG, and also contain, if not all, dimeric hCG (e.g., U.S. Patent Nos. 4,313,871; No.4,256,629; No.4,310,455). No.6,146,633; No.6,143,305; No.6,096,318; No.6,039,948; No.5,891,992; No.5,817,753; No.5,698,201; No.5,106,619; No.5,006,334; No.4,855,285; No.4,791,062; No. .4,762,913). The preferential preference for beta-chain hCG or its peptides is that vaccination with dimer or whole hCG causes undesired cross-reactivity to the relevant glycoprotein hormones such as LH, FSH and TSH, This was primarily due to the harmful consequences of the host. To date, the conventional literature does not disclose any combination of hCG dimer with muramyl peptide.
[0009]
Contrary to the prior belief widely recognized in the prior art, the present inventors have surprisingly found that the combination of whole or dimeric hCG molecules with muramyl peptides reduces the anti-HIV and anti-cancer activity of hCG in vivo. It has been found that they increase synergistically and do not cause harmful effects on the host. Furthermore, it has surprisingly been found that hCG formulated with or without muramyl peptide in the form of an oil-in-water emulsion also has a similar effective clinical effect.
[Patent Document 1]
U.S. Patent No.5,700,781
[Patent Document 2]
U.S. Patent No. 5,811,390
[Patent Document 3]
U.S. Patent No.5,851,997
[Patent Document 4]
U.S. Patent No. 5,997,871
[Patent Document 5]
U.S. Patent No. 5,877,148
[Patent Document 6]
U.S. Patent No. 5,677,275
[Patent Document 7]
U.S. Patent No. 5,840,313
[Patent Document 8]
U.S. Patent No. 5,876,724
[Patent Document 9]
U.S. Patent No. 5,932,208
[Patent Document 10]
U.S. Patent No. 4,313,871
[Patent Document 11]
U.S. Patent No. 4,256,629
[Patent Document 12]
U.S. Patent No. 4,310,455
[Patent Document 13]
U.S. Patent No. 6,146,633
[Patent Document 14]
U.S. Patent No. 6,143,305
[Patent Document 15]
U.S. Patent No. 6,096,318
[Patent Document 16]
U.S. Patent No. 6,039,948
[Patent Document 17]
U.S. Patent No. 5,891,992
[Patent Document 18]
U.S. Patent No. 5,817,753
[Patent Document 19]
U.S. Patent No.5,698,201
[Patent Document 20]
U.S. Patent No. 5,106,619
[Patent Document 21]
U.S. Patent No. 5,006,334
[Patent Document 22]
U.S. Patent No. 4,855,285
[Patent Document 23]
U.S. Patent No. 4,791,062
[Patent Document 24]
U.S. Patent No. 4,767,842
[Patent Document 25]
U.S. Patent No. 4,762,913
[Patent Document 26]
U.S. Patent No. 5,709,879
[Patent Document 27]
U.S. Patent No. 5,506,204
[Patent Document 28]
U.S. Patent No. 5,534,492
[Patent Document 29]
U.S. Patent No.4,158,052
[Patent Document 30]
U.S. Patent No. 4,220,637
[Patent Document 31]
U.S. Patent No. 4,323,559
[Patent Document 32]
U.S. Patent No. 4,323,560
[Patent Document 33]
U.S. Patent No. 4,409,209
[Patent Document 34]
U.S. Patent No. 4,423,038
[Patent Document 35]
U.S. Patent No. 4,185,089
[Patent Document 36]
U.S. Patent No. 4,406,889
[Patent Document 37]
U.S. Patent No. 4,082,735
[Patent Document 38]
U.S. Patent No. 4,082,736
[Patent Document 39]
U.S. Patent No. 4,427,659
[Patent Document 40]
U.S. Patent No. 4,461,761
[Patent Document 41]
U.S. Patent No. 4,314,998
[Patent Document 42]
U.S. Patent No. 4,101,536
[Patent Document 43]
U.S. Patent No. 4,369,178
[Patent Document 44]
U.S. Patent No.5,075,287
[Patent Document 45]
U.S. Patent No. 5,376,369
[Patent Document 46]
U.S. Patent No. 5,264,431
[Patent Document 47]
U.S. Patent No. 5,709,879
[Patent Document 48]
U.S. Patent No. 5,643,605
[Patent Document 49]
U.S. Patent No.5,075,109
[Patent Document 50]
U.S. Patent No. 4,293,539
[Patent Document 51]
U.S. Patent No. 4,919,929
[Patent Document 52]
U.S. Patent No. 4,767,628
[Patent Document 53]
U.S. Patent No. 4,962,091
[Patent Document 54]
U.S. Patent No. 4,849,228
[Patent Document 55]
U.S. Patent No. 4,728,721
[Patent Document 56]
U.S. Patent No. 4,902,515
[Patent Document 57]
U.S. Patent No. 4,719,246
[Patent Document 58]
U.S. Patent No.4,990,336
[Patent Document 59]
U.S. Patent No. 4,897,268
[Patent Document 60]
U.S. Patent No. 4,533,254
[Patent Document 61]
U.S. Patent No. 6,110,492
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0010]
Accordingly, it is an object of the present invention to provide new compositions that can overcome the above and other disadvantages in the art. That is, the present invention has actually enabled the use of water-soluble hormones specially formulated and useful for treating viral diseases and cancer. In view of these and other considerations, the administration of hCG with muramyl peptide results in a better clinical effect or response compared to currently available non-denatured forms of commercially available hCG. Was found by the present inventors. Further, it is expected that hCG can be formulated in the form of an oil-in-water emulsion regardless of the use of the muramyl peptide. Other forms of formulation such as liposome encapsulated release are equally effective. Accordingly, the present invention also contemplates compositions having a novel, therapeutically effective form of the dimeric hCG.
[0011]
It is another object of the present invention to provide a method of making a composition that can overcome the disadvantages of the prior art.
[0012]
The present invention further contemplates administering to a patient a formulation comprising a clinically effective amount of hCG for the treatment or prevention of a patient affected or afflicted with a viral infection.
[0013]
As another aspect of the present invention, there is provided a method of treating animal cancer by administering a composition of the present invention at a dose effective to eliminate or prevent the growth of cancer in animals, including humans. Is done.
[0014]
Potentially preferred uses by the present inventors include the treatment of diseases such as AIDS, Kaposi's sarcoma (both endemic and HIV related), multiple myeloma, lymphoma, melanoma, other types of cancer Tumors and leukemias.
[0015]
Although not intended to be particularly limiting, it is preferred that these pharmaceutical compositions are formed as a solution, whereby parenteral administration is performed, particularly subcutaneous, intramuscular, intravenous or infusion. To do. The compositions of the present invention may also be advantageously administered alone or in the form of a crude drug ensuring their protection, for example encapsulated in capsules or microspheres or liposomes, and permeating the gastric wall.
BEST MODE FOR CARRYING OUT THE INVENTION
[0016]
The present invention is based on the fact that muramyl dipeptides (MDPs) are used in vivo, despite the fact that muramyl dipeptides (MDPs) are commonly used as components of vaccine formulations to elicit immunity against the antigenic determinants of vaccines. It is based on the discovery that it can actually act as an adjuvant rather than antagonizing hCG activity. Thus, despite the teachings of the prior art described above, muramyl peptide compounds in combination with hCG are useful in the treatment, prevention or management of HIV infection and other life-threatening diseases such as cancer.
[0017]
As used below, the term "sustained release device" refers to a particular device, such as a transdermal patch, subcutaneous implant and pump containing a drug.
[0018]
As used below, the term "slow release formulation" refers to forms of drug release other than sustained release devices and includes hCG formulations not taught in the prior art related to hCG formulations.
[0019]
What is defined by the term "adjuvant" refers to a substance that is incorporated into an antigen and is co-injected with the antigen. This adjuvant non-specifically enhances the immune response to the antigen. The primary purpose of using immunotherapeutic adjuvants is to use a lower dose of antigen at a lower dose than that achieved by administering an equivalent aqueous antigen, resulting in a more persistent and higher level of fluid. Or to achieve cell-mediated immunity. Adjuvants are used in combination with abiotic drugs (instead of living microorganisms) for the manufacture of vaccines. Adjuvants further increase the effective immune response to low or non-immunogenic tumor cells or cells that are already present in the body and are infected with an intracellular pathogen that is not adequately arrested by the naturally manifested immune response.
[0020]
As used below, the term "emulsifier" is intended to refer to non-ionic surfactant compounds derived from alkylene oxides and / or hexahydric alcohols and / or higher natural fatty acids such as esters or ester-ethers. .
[0021]
The term "complete Freund's adjuvant (CFA)" refers to a potent immunostimulant that has been successfully used with many antigens on an experimental basis. The CFA consists of mineral oil, an emulsifying / stabilizing agent such as Arlacel A, and inactivated mycobacteria such as Mycobacterium tuberculosis. An aqueous antigen / protein solution is mixed with these components to form a water-in-oil emulsion. However, this CFA has severe side effects, such as pain, abscess, fever, and cannot be used in human or veterinary vaccines. This side effect is mainly due to the patient's response to the mycobacterial component of CFA.
[0022]
"Incomplete Freund's adjuvant (IFA)" is similar to CFA but has no bacterial components. Although not approved for use in the United States, IFA is useful for some types of vaccines in other countries. IFA has been successfully used in humans as several animal vaccines, such as influenza and polio vaccines, rabies, dog distemper, and foot and mouth disease. Experiments have shown that the oils and emulsifiers used in this IFA cause tumors in mice, and indicate that for human use, the use of another adjuvant is desirable.
[0023]
Muramyl dipeptide (MDP) represents the smallest unit of the mycobacterial cell wall complex, which has the adjuvant activity observed with CFA. Many synthetic analogs of this MDP have been developed, which have a wide range of adjuvant potencies and side effects. Among them, mention may be made of three analogs particularly useful as vaccine adjuvants, threonyl derivatives of MDP, n-butyl derivatives of MDP and lipophilic derivatives of muramyl tripeptide (see US Pat. No. 5,709,879). These compounds effectively stimulate humoral, cell-mediated immunity and have low toxicity. The muramyl peptide has a phospholipid tail, which allows the hydrophobic portion of the molecule to bind to the lipid environment, while the muramyl peptide portion binds to the aqueous environment. Thus, MTP-PE itself acts as an emulsifier, and a stable oil-in-water emulsion is formed.
[0024]
The term "muramyl peptide" will be clear to the skilled person. In particular, it is a compound comprising one or more sugar residues, wherein at least one of the sugar residues (often muramic acid residues) is at least one or more (usually two or more) amino acid residues Has been replaced by The muramyl peptide compound can improve the cellular antigenic response in mammals, and may also be peptidoglycan which is also prototype muramyl dipeptide (MDP) or an analog or derivative thereof.
[0025]
A group of muramyl peptide compounds has previously been disclosed as being useful for treating or preventing progressive sepsis, septic shock, and having immunopotentiating antibacterial activity. However, muramyl peptides are only effective in immunocompetent hosts. Representative examples of muramyl peptides are described in U.S. Patent Nos. 5,506,204 and 5,534,492, which are incorporated herein by reference.
[0026]
Various analogs of the prototype muramyl dipeptide are known, some of which have been proposed as therapeutics for restoring immune function or for non-specific stimulation of the immune system. These analogs and the prototype MDP itself are commonly referred to as "muramyl peptides".
[0027]
According to a first aspect of the present invention, there is provided a composition comprising a combination of a muramyl peptide compound and a water-soluble physiologically active protein (eg, hCG).
[0028]
Not only hCG but also other therapeutic agents can be used, for example, insulin, glucagon, calcitonin, atrial natriuretic peptide, secretin, cholecystokinin, thyroid stimulating hormone releasing thymopentin, adrenocorticotropic hormone, growth hormone releasing factor , Enkephalin, oxytocin, vasopressin, luteinizing hormone-releasing hormone and the like. These compositions may also be formulated in a matrix material. Examples of matrix materials in this case include chitosan, algin, saturated polysaccharide-degrading glycerides, glycerol palmitostearate, saturated C12 to C22 fatty acid esters of polyalcohols, glyceryl and polyethylene glycol behenate, polyethylene oxide, ammonium glycite. Luridinate and the like.
[0029]
Adjuvants include monophosphoryl lipid A, lipid A, keyhole himpet hemocyanin, histidine-tag, alum, Freund's adjuvant, beta-gal, palmitic acid, saponin, lipopolysaccharide, BCG cell wall skeletone, trehalose monomyco Rate, trehalose dimycolate, lipid X, isoprinosine, lysosperman (A, B or C), muramyl dipeptide (MDP) lipid conjugate and the like. The lipid may be a saturated or unsaturated phospholipid or glycolipid. Examples of preferred lipids include 1,2-dimyristoylphosphatidylcholine, 1,2-dipalmitoylphosphatidylcholine, 1,2-dimyristoylphosphatidylglycerol, cholesterol, and combinations thereof.
[0030]
Examples of solid lipids suitable for preparing the compositions of the present invention include triglycerides consisting of natural even, unbranched fatty acids having a chain length of C10-C18, microcrystalline glycerol of naturally occurring saturated, even, unbranched fatty acids. Triesters such as tricaprin, trilaurin, trimyristin, tripalmitin, tristearin and the like. In general, any lipid component or mixture of lipid components that is a solid phase at room temperature (25 ° C.) when measured in bulk can be suitably used as the lipid core.
[0031]
Examples of preferred phospholipids that can be used to make the compositions of the present invention include natural phospholipids such as soy lecithin, egg lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidyl-ethanolamine, phosphatidic acid, sphingomyelin , Diphosphatidylglycerol, phosphatidylsulin, phosphatidyl-choline, cardiolipin and the like; or synthetic phospholipids such as dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, monophosphoryl lipid A, diphosphoryl lipid A and the like; and other examples include hydrogenated or partially hydrogenated lectins and phospholipids.
[0032]
Non-natural surfactants and detergents can be added to the composition of the present invention in any proportion as appropriate. As used herein, the term "surfactant" or "detergent" is capable of forming micelles in an aqueous solution and includes various artificial molecules having both a lipophilic domain and a hydrophilic domain. Generally, emulsifiers include sorbitan esters, polyoxyethylene sorbitan mono-, di- or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers and combinations thereof. Examples of non-natural surfactants include, but are not limited to, polysorbate ("TWEEN" or sorbitan monooleate), sodium dodecyl sulfate (SDS), polyethoxylated castor oil ("CREMOPHOR"), NP-40, and Many other synthetic molecules can be mentioned. Additionally, some other synthetic surfactants, such as dimethyldioctadecyl ammonium bromide ("DDA") and certain linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers (trademarks of PLURONIC) Commercially available) have been reported as having adjuvant (adjuvant) activity. Of these, poloxamer 401, Pluronic L62LF, Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501, Tetronic 150R1, Tetronic 701, Tetronic 901, Tetronic 1301, Atmos 300, Tween 20, Tween 40, Tween 60, Tween 80, and Tetronic 130R1 is particularly preferred. Emulsifiers further include Arlacel A or Arlacel 80 or Span 80 (so-called mannide monooleate). These surfactants can be used in various doses. For example, the preferred range for Arlacel A, Arlacel 80 or Span 80 in the composition of the present invention is about 2-15% by weight, and the range for Tween 80 is for example 0.2-4% by weight. Generally, the surfactant may comprise less than 30%, preferably less than 25%, more preferably less than 10%, and most preferably less than 5% of the total weight of the composition.
[0033]
Examples of functional equivalents of MDP include, but are not limited to: That is, muramyl di- or tripeptide-like N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine (GMDP), N-acetyl-D-glucosaminyl (beta-1-4) -N -Acetyl-muramyl-L-alanyl-D-isoglutamine, N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamic acid (GMDP-A), muramyl dipeptide-phosphatidyl-ethanolamine, muramyl・ Tripeptide-phosphatidyl-ethanolamine, muramyl ・ Tripeptide-phosphatidyl-ethanolamine, CGP11637 (nor-MDP), alpha (N-acetyl-muramil-L-alanyl-D-isoglutamine) beta, gamma-dipalmitoyl- sn-glycerol, alpha (N-acetyl-muramyl-D-alanyl-D-isoglutamine) Gamma-dipalmitoyl-sn-glycerol, alpha (N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine) beta, gamma-dipalmitoyl-sn-glycerol, alpha (N-acetyl-muramyl- (D-alanyl-D-isoglutaminyl-L-alanine) beta, gamma-dipalmitoyl-sn-glycerol, N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1,2-dipalmitoyl -Sn-glycero-3-hydroxyphosphoryloxy) ethylamide, glucoasminylmuramyl peptide, muramethide, murabutide, muradimethide, miramistin, N-acetyl-muramyl-L-threonyl-D-isoglutamine, N-acetyl-muramyl-L -Alpha-aminobutyryl-D-isoglutamine, 6-O-ste Royl-N-acetyl-muramyl-L-alpha-aminobutyryl-D-isoglutamine, N-acetyl-muramyl-L-valyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-isoglutamine, N -Acetyl-desmethylmuramyl-L-alanyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-glutamine butyl ester, N-acetyl-muramyl-L-seryl-D-isoglutamine, N -Butyryl-muramyl-L-alpha-aminobutyryl-D-isoglutamine, N-acetyl-muramyl-L-threonyl-D-isoglutamine, bis (6-O-muramyl dipeptide) O-palmitoyl taltronate, muramylt Ripeptide-phosphatidyl-ethanolamine, N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L- Alanine-2- (1,2-dipalmitoyl-sn-glycero-3- (hydroxyphosphoryloxy)) ethylamide, N-acetyl-muramyl-L-alanyl-D-isoglutamine butyl ester, N-acetyl-muramyl- L-alanyl-D-isoglutaminyl-L-alanine-2--1,2-dipalmitoyl-sn-glycero-3- (hydroxyphosphoryloxy)) ethylamide (MTP-PE), cholesteryl-MDP, N-acetyl-muramyl- Beta-butyl glycoside of L-alanyl-D-isoglutamine, 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-[(R) -1- (methoxycarbonyl) ethyl]- Alpha-D-glucopyranose, (beta-butyl-MDP, MTPO-26, beta-cholesteryl-MDP), saponin (Taurosid I), N-acetylnor-muramyl-LN-methylara Ru-D-isoglutamine octylamide, UDP-N-acetyl-muramyl-pentapeptide, L4-MDP-ONB, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, 1,6-anhydro Muramyl dipeptide, N-acetylglucosamine-beta-1,4-N-acetyl-muramyl-pentapeptide-pyrophosphoryl-undecaprenol, 3-O- [N-acetyl-muramyl-D-isoglutaminyl] -1, 2-di-O-dipalmitoyl-sn-glycerol, L-threonyl-MDP-GDP, L-allothreonyl-MDP-GDP, trehalose 6,6-diester, muramyl dipeptide, B30-muramyl dipeptide and muramyl dipeptide Lysine and the like can be mentioned.
[0034]
It should be understood that the aminoacyl residue may be any aminoacyl component selected from the following groups. That is, alanyl group, valyl group, leucyl group, isoleucyl group, alpha-aminobutyryl group, threonyl group, methionyl group, cysteinyl group, glutamyl group, isoglutamyl group, glutaminyl group, isoglutaminyl group, aspartyl group, phenylalanyl group, tyrosyl Group, tryptophanyl group, lysyl group, ornithinyl group, arginyl group, histidyl group, asparinginyl group, prolyl group, hydroxypropyl group, seryl group, glycyl group and the like.
[0035]
Other muramyl peptide derivatives include those obtained by substituting an L-alanyl residue of a muramyl peptide group with an L-threonyl residue. Furthermore, any adjuvant muramyl peptide having a substituent at the 1,4 or 6 position of the saccharide group can be used, provided that it has the same advantageous effects as the preferred muramyl peptide described above. It becomes. No. 4,158,052; No. 4,220,637; No. 4,323,559; No. 4,323,560; No. 4,409,209; No. 4,423,038; No. 4, 185, 089; No. 4, 406, 889; No. 4, 082, 735; No. 4, 082,736 No.4,427,659; No.4,461,761; No.4,314,998; No.4,101,536; No.4,369,178; No.5,075,287; No.5,376,369; No.5,264,431; No.5,709,879 (incorporated herein by reference) What is described is equally useful in the present invention.
[0036]
In the above composition, other useful additives include: N-cyclohexanoylarginine; a mixture of N-cyclohexanoyltyrosine and N-cyclohexanoylleucine; N-phenylsulfonylvaline, N-phenylsulfonylleucine, N-phenylsulfonylphenylalanine, A mixture of -phenylsulfonyllysine and N-phenylsulfonylarginine; N-benzoylvaline, N-benzoylleucine, N-benzoylphenylalanine, N-benzoyllysine, N-benzoylarginine, sodium 2-cyclohexyl Examples thereof include a mixture with a stabilizer such as butyrate.
[0037]
In addition to the above-mentioned components, ovalbumin, cholera toxin, an acylated amino acid or a salt thereof, a polyamino acid containing at least one acylated amino acid or a salt thereof, a sulfonated amino acid or a salt thereof, or a combination thereof can be used. It will be clear to the trader. The composition may include microspheres.
[0038]
Polymer matrices for forming microspheres are known. For example, semipermeable microspheres containing enzymes, hormones, vaccines and other biologics are disclosed in U.S. Patent No. 5,643,605. Another U.S. Patent No. 5,075,109 discloses administering a mixture of at least two microsphere solids comprising a bioactive agent, wherein one of the microsphere solids is about 1-10 microns in size. To improve the immune response. U.S. Pat. No. 4,293,539 discloses a formulation for controlled release of an active ingredient from a copolymer obtained from about 60 to 95% by weight lactic acid and about 40 to 4% by weight glycolic acid. U.S. Pat. No. 4,919,929 discloses the administration of a molded article of a biocompatible matrix material containing an antigenic material. U.S. Pat.No. 4,767,628 discloses a composition comprising active, acid-stable polypeptides and polylactides which, when placed in an aqueous physiological environment, has a substantially constant rate and essentially a single phase state. Which release the polypeptide. U.S. Patent No. 4,962,091 discloses a microsuspension of a water-soluble polymeric polypeptide suspended in a polyactide matrix. U.S. Pat.Nos. 4,849,228 and 4,728,721 disclose biodegradable high molecular weight polymers whose content of water-soluble low molecular weight compound, calculated on the assumption that it is a monobasic acid, is high. The molecular weight is less than 0.01 mol per 100 g of the polymer. U.S. Patent Nos. 4,902,515 and 4,719,246 disclose polylactide compositions comprising segments of poly (R-lactide) combined with segments of poly (S-lactide). U.S. Pat.No. 4,990,336 discloses a multiphase sustained release system having an allergen extract encapsulated in microspheres of bioerodible encapsulated polymer, which allows for sustained multiphase release of the allergen. I have. The system contains a first part of an extract of the allergen, which when injected suppresses initial allergenicity and produces a gradual local response similar to that normally observed with conventional low allergen doses. Released to produce a second portion of the extract of the allergen, which, apart from the release of the first portion of the extract of the allergen, results in substantially higher levels of the allergen extract in the administration. To provide something that can cause significant reactions in the patient. U.S. Patent No. 4,897,268 discloses a microcapsule release system in which each component is encapsulated in various molar ratios of a biodegradable copolymer excipient, and the release of each component is extended over time. It is performed at a constant speed. Thus, various means for producing and using microspheres are known and can be readily applied for the purposes of the present invention.
[0039]
The microspheres of the present invention may further include liposomes. Compositions suitable for forming liposomes are known from the numerous technical literature in this regard. Preferred lipid compositions are those that activate phospholipids, such as phosphatidylcholine (fatty acid derivatives consisting of 12-20 carbon atoms) (especially those consisting of 16-20 carbon atoms), phosphatidylglycerin, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol. And phosphatidic acid. These phospholipids can be used alone or in combination. Furthermore, it is particularly advantageous to use a mixture of synthetic or natural distearyl-phosphatidylcholine (DSPC) or phosphatidylcholine, a mixture of phosphatidylglycerin (PS), or a mixture of phosphatidylglycerol (PG). Preferably, the liposome is formed from a mixture comprising the last-mentioned phospholipid, DSP and phosphatidylcholine (PC) in a proportion of 1 to 10, preferably 3 parts by volume of DSPC or phosphatidylcholine (PC). preferable. The biological activity of these liposomes containing the derivatives of the present invention has been found to be equally effective whether the liposomes are monolayer or multi-layer. Preferably, the liposomes have a particle size of at least 0.1 micron, for example, 1 to 10 microns.
[0040]
The compositions of the present invention are useful as modulators of nonspecific antimicrobial resistance for systemic enhancement of immune response and nonspecific immunity.
[0041]
Thus, for example, in therapeutic treatment and supportive care of a condition of diminished immune response (i.e., with a specific or supporting form of further treatment), in particular, the condition of diminished cellular and humoral immune response and the condition of delayed delayed hypersensitivity response It is also useful in the treatment of common conditions where modulation of the immune response is desired.
[0042]
In addition, it is useful in the treatment or supportive care of types of immunodeficiency associated with idiopathic immunodeficiency or elderly patients or patients suffering from severe burns or systemic infections.
[0043]
Furthermore, it is also useful for the treatment or supportive treatment of viral infections such as sporadic herpes and sporadic varicella infections, HIV infections, malignant tumors and the like.
[0044]
Slow release of hCG is also contemplated for use in RNA and DNA viruses. Examples of these include, but are not limited to, hepatitis virus, herpes virus, cold virus, raftbury fever virus.
[0045]
It will be apparent to those skilled in the art that the exact dosage of the active ingredient required to achieve a given effect can be selected as appropriate depending on the particular compound, size, age and condition of the patient to be treated. There will be. These dosages will be readily determined by routine methods known to those skilled in the art.
[0046]
The adjuvant compositions and vaccines of the invention are generally administered by injection. However, easier modes of administration, such as oral administration, can also be created. When the composition of the present invention is used directly for clinical treatment and prophylaxis, it can be in the form of an oral or parenteral preparation. The term "parenteral" includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pump, infusion and the like. Although not intended to be limiting, the composition may be intra-articular, intra-synovial, endothelial, periosteal, intratumoral, intratumoral, intralesional, intralesional, sublingual, sublingual, buccal, transdermal, topical Administration can also be via target or via inhalation. The composition can also be administered via a dressing for wounds, lesions. However, this composition is particularly preferred for oral administration. For oral administration, the compositions of the present invention, alone or in combination with pharmacologically acceptable carriers, can be used in pharmacological preparations, such as capsules, pills, troches, tablets, dragees, sassy, tea bags. , Granules, powders, coated tablets, sugar-coated tablets, wafers, sugar cubes, gels, hydrogels (eg, hydrophilic hygroscopic polysaccharides, foams, suppositories, inhalants, juices, shakes, chewing gums, toothpastes, toothpastes, gargles) It can be used in the form of medicines, candies, emulsions and the like.
[0047]
Examples of suitable pharmacological carriers include fillers (e.g., lactose, sucrose, mannitol, glucose, starch, sorbitol, glycine, calcium phosphate, microcrystalline cellulose); binders (e.g., starch, casein, gelatin, gum arabic) Alginate gel; glucose, sucrose, sorbitol, mannitol, gum tragacanth, hydroxypropylcellulose, hydroxypropoxymethylcellulose, carboxymethylcellulose, 2-methyl-5-vinylpyridine / methyl methacrylate / ethyl acrylate copolymer, polyvinylpyrrolidone, sodium alginate); ; Lubricants (eg, stearic acid, hardened oil, magnesium stearate, calcium stearate, polyoxyethylene monostearate, talc, oxidized Disintegrating agents (eg, potato starch, starch containing a surfactant); wetting agents (eg, sodium lauryl sulfate).
[0048]
The compositions of the present invention can also be administered in the form of liposomes. As is known, liposomes or artificial lipid vesicles can generally be obtained from phospholipids or other lipid substances. In addition, they may contain muramyl peptides, metabolizable oils and, if desired, further emulsifiers. Liposomes can be formed from mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. A typical process for preparing a liposome preparation comprising liposomes containing the encapsulated composition of the present invention comprises hydrating the lipid or liposome with a solution of the substance to be encapsulated; And hydrating each of the plurality of portions with a solution comprising the substance to be encapsulated; combining the plurality of portions with each other to form a single liposome preparation, whereby the capsule To form a liposome preparation containing liposomes containing the immobilized substance. Non-toxic, physiologically acceptable and metabolizable lipids capable of forming liposomes can be used. This composition in liposome form may contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. Examples of preferred lipids are phospholipids and phosphatidylcholine (lecithin), which may be natural or synthetic. Methods for forming liposomes are known. For example, a cocolate containing a biologically relevant molecular component, a negatively charged lipid component, and a divalent cation component. The shelf life of this cocolate is long even in a dry state. Conveniently, the cocolate can be ingested.
[0049]
For the above-mentioned indications, the dosage used will, of course, be dependent on the nature and severity of the condition being treated, the mode of administration and the form of compound used. For the average patient, a suitable dosage is about 100 IU to 20,000 IU, which may be used as a single dose or as separate doses. 1 to 4 times daily, or once every 3 days, once every week, once every week, once every two weeks, once a month, once every March May be. As a unit dosage form, for repeated administration, the compounds of the present invention can be used in an amount of about 100 IU to 10,000 IU, preferably about 1,000 IU to 5,000 IU per injection. If a single dose is desired for treatment, the dose can be increased to about 20,000 IU. It will be easy for those skilled in the art to convert the IU units of hCG to III weight units, or vice versa, based on the product description provided in the commercial preparation of hCG.
[0050]
The compositions of the present invention can be used in various other ways unrelated to the therapeutic purpose. For example, the composition can be used as a labeled or unlabeled reagent for detecting hCG or detecting regression to hCG in various immunoassays, biological assays, and the like. Examples of preferred labels include radioisotopes, enzymes, fluorescent molecules, chemiluminescent labels, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and the like. Such a known reagent can be used for various known assays, for example, a radioimmunoassay, an enzyme immunoassay, for example, an ELISA, a fluorescent immunoassay and the like.
[0051]
The present invention encompasses the use of the compositions of the present invention with drugs or compounds conventionally used conventionally in various disease categories. Among these compounds, mention may be made of active agents of the following categories and / or members of the categories. That is, corticosteroids, corticosteroids, aldosterone antagonists, amino acids, anabolic agents, androgens, anti-AIDS drugs, anthelmintics, anti-acne agents, anti-adrenergic agents, anti-allergic agents, anti-amoebic agents, anti-androgens, anti- Anemia, anti-angina, anti-arthritic, anti-asthmatic, anti-atherosclerotic, anti-bacterial, anti-gallstone, anti-gallstone agent, anticholinergic, anticoagulant, anticocculant, Antidiabetic, antidiarrheal, antidiuretic, antidote, antiestrogenic, antifibrinolytic, antifungal, antiglaucoma, antihemophilic, antibleeding, antihistamine, antihyperlipidemic Agent, anti-hyperlipoproteinemic agent, anti-hypertensive agent, anti-hypertensive agent, anti-infective agent, anti-local infectious agent, anti-inflammatory agent, anti-keratinizing agent, anti-malarial agent, antibacterial agent, anti- Mitotic agents, antifungal agents, antitumor agents, antineutropenia Anti-parasitic agent, anti-parasitic agent, anti-reverse peristaltic agent, anti-pneumocystis agent, anti-prostatic hypertrophy agent, protozoan animal agent, anti-pruritic agent, anti-psoriasis agent, anti-rheumatic agent, anti-schistosomiasis agent, Antiseborrheic agent, antisecretory agent, anticonvulsant, antithrombotic agent, cough suppressant, antiulcer agent, antiurinosis, antiviral agent, appetite suppressant, benign prostatic hypertrophy, osteolysis Agents, bronchodilators, carbonic anhydrase inhibitors, cardiosuppressants, cardioprotectives, inotropic agents, cardiovascular agents, cholagogues, cholinergic agents, cholinergic agonists, cholinesterase inactivators, coccidiosis Sedatives, diagnostic aids, diuretics, ectoparasitic insecticides, enzyme inhibitors, estrogens, fibrinolytics, free oxygen radical scavengers, glucocorticoids, gonad irritants, hair growth stimulants, hemostats, hormones, low Cholesterol treatment, hypoglycemic, low Lipidemia drugs, hypotension drugs, immunizing agents, immune modulators, immunomodulators, immunostimulants, immunosuppressants, impotence treatment adjuvants, inhibitors, keratolytics, LHRH agonists, liver failure drugs , Luteolicin, mucopolysaccharide degrading enzyme, mydriatic, nasal decongestant, neuromuscular blocking agent, non-hormonal sterol derivative, labor-promoting agent, plasminogen activator, platelet activating factor antagonist, platelet aggregation inhibitor Agents, potentiators, progestins, prostaglandins, prostate growth inhibitors, prothyrotropins, pulmonary surfactant, radioactive agents, regulators, relaxants, dispensing agents, anti-scabies drugs, sclerosis agents, selective adenosine Al antagonists , Steroids, depressants, symptomatic multiple sclerosis drugs, concomitant drugs, thyroid hormones, thyroid depressants, thyroid-like drugs, amyotrophic lateral sclerosis drugs, osteoarthritis drugs, anxiety Angina treatment, uric acid excretion promoting agent, vasoconstrictor drugs, vasodilators, wound medicine, trauma treatment, and the like can be mentioned xanthine oxidase inhibitor.
[0052]
The compounds useful in the present invention can be administered in the form of a drug cocktail. The cocktail is a mixture of any of the above compounds in combination with the composition of the present invention. The mixture need not necessarily be physically mixed, and the agents may be administered separately, sequentially, or simultaneously.
[0053]
In addition to AIDS, a drug of particular interest is an anticancer agent, which can be administered with the compositions of the present invention. Examples of the antitumor agent include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, AcrQnine, adzelesin, aldesleukin, altrethamine, ambomycin, amethanthrone acetate, aminoglutethimide, ansacrine, anastrozole, Anthramycin, asparaginase, asperuline, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafidimesilate, viserecin, bleomycin sulfate, brequinal sodium, bropyrimine, busulfan, ccactinomycin, carsterone , Carassemide, carbetimer, carboplatin, carmustine, carbicin hydrochloride, calzelesin, sedefingo , Chlorambucil, sirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexolmaplatin, dezaguanine, dezaguanine mesylate, diaziquon, dodetaxel, doxorubicin , Doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, juazomycin, edatrexate, ephlomitin hydrochloride, elesamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, elbrozole , Esorbicin hydrochloride, estramustine, estramustine phosphate sodium, Etanidazole, Ethiodized Oil I 131, Etoposide, Edposide Phosphate, Etopurine, Fadrozole Hydrochloride, Fazarabine, Fenretinide, Floxyuridine, Fludarabine Phosphate, Fluorouracil, Fluorocitabine, Fosquidone, Fostriesin Sodium, Gemcitabine, Gemcitabine Hydro Chloride, Gold AU 198, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosin, interferon alpha-2a, interferon alpha-2b, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib , Iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, Rozole hydrochloride, lometrexol sodium, lomustine, loxoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melephalan, menogallil, mercaptopulin, methotrexate, methotrexate sodium, Metopurine, methourendepa, mitindamide, mitocalcin, metoclomine, mitogillin, mitomasine, mitomycin, mitospar, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogaramycin, ormaplatin, oxyslan, paclitaxel, pegaspargum, periomycin, periomycin Puromycin sulfate, perfosfamide, pipov Roman, Piposulfan, Pyroxantrone hydrochloride, Plicamycin, Promestan, Porfimer sodium, Porphyromycin, Prednistin, Procarbazine hydrochloride, Puromycin, Puromycin hydrochloride, Pyrazofarine, Ribopurine, Logretimide, Sahumgol, Saphingol hydrochloride, Semustine, Simtrazen, Sparfosate sodium, Sparsomycin, Spirogermanium hydrochloride, Spiromustine, Spiroplatin, Streptonigrin, Streptozocin, Strontium chloride Sr 89, Sulofenul, Talisomycin, Taxane, Taxoid, Tecogalane sodium, Tegafa, Teloxantrone hydrochloride, Temoporfin, Teniposide, Teloxy , Test lactone, thiamipurine, thioguanine, thiotepa, thiazofurin, tirapazamine, topotecan hydrochloride, toremifene citrate, trestron acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptrelin, tubrozole hydrochloride , Uracil mustard, uredepa, vapleotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinbricinate sulfate, vinleulosin sulfate, vinorelbine tartrate, vinrosi Ginsulfate, vinzolidine sulfate, borozol, zeniplatin, dinostatin, sorbicin hydrochloride Can be mentioned.
[0054]
Examples of other anti-tumor compounds include 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adesipenol, adzeresin, aldesleukin, an ALL-TK antagonist, altretamine, ambamustine, amidox , Amifostine, aminolevulinic acid, anrubicin, atlusacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, anti- Androgen, prostate carcinoma, anti-estrogen, anti-neoplastic, antisense oligonucleotide, aphidicolin glycinate, apoptosis gene modulator, apoptosis regulator, aprinoic acid, ara-CDP-DL-PTBA, argini Deaminase, asraculin, atamestan, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivative, balanol, batimastat, BCR / ABL antagonist, benzocholine, benzoyl stauros Porin, beta-lactam derivative, beta-alethin, beta-chlamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisadiridinyl spermine, bisnafide, bisstraten A, visellesin, bleflate, bropirimine, butidotitanium, buthionine sulfoximine, Calcipotriol, calphostin C, camptothecin derivative, canaripox IL-2, capecitabine, carboxamide-amino-triazole, carboxamide Azole, CaRest M3, CARN 700, cartilage induction inhibitor, calzeresin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, Clomiphene analog, clotrimazole, chorismycin A, chorismycin B, combretastatin A4, combretastatin analog, conagenin, clambesidine 816, crisnatol, cryptophysin 8, cryptophycin A derivative, clacin A, cyclopentantra Quinone, cycloplatam, cypemycin, cytarabine octophosphate, cytolytic factor, cytostatic, daclyximab, decitabine, dehydrodideminin B, deslorelin, dexphosphamide, dexrazoxane, Kusveramil, diaziquinone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol 9-, diosamycin, diphenylspiromustine, docosanol, dolasetron, doxyfluridine, droloxifene, dronabinol, zuokanaisin SA, ebselen, ecomustine Analogs, edolfosin, edrecolomag, eflonitin, elemen, emmiteur, epirubicin, epristeride, estramustine analog, estrogen agonist, estrogen antagonist, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgratin, filgratin , Flavopiridol, frenzelastine, fluasterone, fludarabi , Fluorodaunornisin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium tolate, gallocitabine, ganyrelix, geratinase inhibitor, gemcitabine, glutathione inhibitor, hepsulfam, heregselamide, hexamethylene samide , Hypericin, ibandronic acid, idarubicin, idoxifen, idramantone, ilmofosin, ilomastat, imidazoacridone, imiquimod, immunostimulatory peptide, insulin-like growth factor-1 receptor inhibitor, interferon agonist, interferon, interleukin, iobenguan, io Dodoxorubicin, ipomeanol 4-, irinotecan, iropract, irsogladine, i Sobenazole, isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitor, leukocyte alpha interferon, leuprolide + Estrogen + progesterone, leuprolorelin, levamisole, liarozole, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compound, lysoclinamide 7, lobaplatin, romblysin, lometrexol, lonidamine, rosoxantrone, lovastatin, loxoribine, lutexateline, llutetexantine , Lysophilin, cytolytic peptide, maytansine, mannostatin A, marimastat Masoprocol, Maspin, Matrilysin inhibitor, Matrix metalloproteinase inhibitor, Menogalil, Mervalon, Metereline, Methioninase, Metoclopramide, MIF inhibitor, Mifepristone, Miltefosin, Millimostim, Mismatched double-stranded RNA, Mitoglazone, Mithractor, Mitomycin-like Body, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mopharotene, molgramostim, monoclonal antibody, human chorionic gonadotrophin, monophospholipid A + myobacterial cell wall sk, mopidamole, multiple drug resistance genie inhibitor, multiple tumor suppression Therapeutic agent based on agent 1, mustard anticancer agent, micaperoxide B, mycobacterial cell wall extract, myriapolone, N-acetyldinaline, N-substituted benzua De, nafarelin, nagres chip, naloxone + pentazocine, napavin, naphterpine, nalgrastim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisamicin, nitric oxide modulator, nitroxide antioxidant, nitritol, O6-benzylguanine, Octreotide, Oxenone, Oligonucleotides, Onapristone, Ondansetron, Olasin, Oral Cytokin Inducer, Ormaplatin, Osaterone, Oxaliplatin, Oxaunomycin, Paclitaxel Analog, Paclitaxel Derivative, Parauamine, Palmitoyl Hizoxin, Pamidron Acid, panaxitriol, panomifene, parabactin, pazeliptin, pegaspargase, perdesine, pentosan Sodium polysulfate, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate, phosphatase inhibitor, picibanil, pilocarpine hydrochloride, pirarubicin, pyrithrexime, placenta A, pracetin B, plasminol Gen / activator inhibitor, platinum complex, platinum compound, platinum triamine complex, porfimer sodium, porphyromycin, propyl bis-acridone, prostaglandin J2, proteasome inhibitor, protein A-based immunomodulator, protein kinase C suppression Drug, protein kinase C inhibitor microalgal, protein tyrosine phosphatase inhibitor, purine nucleoside phosphorylase inhibitor, purpurin, Lazoloacridine, pyridoxylated hemoglobin / polyoxyethylene conjugate, raf antagonist, raltitrexide, ramosetron, ras farnesyl protein transferase inhibitor, ras inhibitor, ras-GAP inhibitor, demethylated retelliptin, rhenium Re186 etidronate, rhizoxin, Riboenzyme, RII retinamide, logretimide, rohitkin, romultide, roquinimex, rubiginone B1, ruboxil, safingol, sintopin, SarCNU, sarcophytol A, salgramostim, Sdi 1 mimetix, semustine, senescence induction inhibitor 1, sense oligonileo Signal transduction inhibitor, signal transduction regulator, single-chain antigen-binding protein, schizophyllan, sobuzoxane, sodium borocaptate, phenylacetic acid Thorium, sorberol, somatomedin-binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipamide, stromelysin inhibitor, sulfomosine, super Active vasoactive intestinal peptide antagonist, sulista, suramin, swainsonine, synthetic glycosaminoglycan, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalane sodium, tegafar, terrapyrilium, telomerase inhibitor, temoporfin, temozolomide, Teniposide, tetrachlorodecaoxide, terazomin, taliblastine, talidomide, thiocoraline, thromboprotein, pseudothromboprotein Thymalfasin, thymoprotein receptor agonist, thymotrinan, thyroid stimulating hormone, tin ethyl ethiopurpurulin, tirapazamine, titanocene dichloride, topotecan, topsetin, toremifene, totipotent stem cell factor, translation inhibitor, tretinoin, triacetyluridine, triciribine, Trimetrexate, Triptrelin, Tropisetron, Tulsteride, Tyrosine kinase inhibitor, Tyrphostin, UBC inhibitor, Ubenimex, Urogenital synth-induced growth inhibitor, Urokinase receptor antagonist, Bapleotide, Variolin B, Vector system, Erythrocyte gene therapeutic , Verasol, veramin, berzine, verteporfin, vinorelbine, vincartine, vitaxin, borozol, zanoterone, zeniplatin , Mention may be made of Jirasukorubu, and zinostatin-Suchimarama.
[0055]
It is also an object of the present invention to produce a formulation containing other biologically active water-soluble proteins and peptides instead of hCG. Such proteins include growth hormone or somatotropins such as human growth hormone (HGH), bovine growth hormone (BGH or BST), porcine growth hormone (PGH or PST), analogs and derivatives thereof, epidermal growth factor (FGF) and Its analogs. Other possible proteins are interleukins, interleukin receptors, interleukin receptor agonists, chemokines and interferons. For example, interferon alpha-2a, interferon alpha-2b, interferon alpha-N1, interferon alpha-n3, interferon beta, interferon beta-1al, interferon beta-1b, interferon gamma-Ia, interferon Gamma-Ib, interferon omega, interferon tau, interleukin-1, interleukin-1 alpha, interleukin-1 beta, interleukin-10, interleukin-11, interleukin-12, interleukin-15, Interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, MIP-1 alpha and beta, RANTES, etc. . In addition, the protein can be selected from blood cell growth stimulating factors and precursors, erythropoiesis promoting factor (EPO) and the like. Other equally preferred proteins are parathyroid hormone (PTH), selenoprotein P, cystatin B and its hepatic thiol protease inhibitor analogs, endotoxin neutralizing protein, lymphocyte migration inhibitory factor (LIF), mast cell growth Factor (MGF), megakaryocyte mimicking factor (MGDF), granular leukocyte macrophage colony mimicking factor (GM-CSF), genofibrate, alpha-calcitonin, beta-calcitonin, tumor necrosis factor (TNF), tumor invasion inhibitor, These include TGF-beta cytokines, transacting regulatory proteins (TAT) of HIV and other retroviruses, protease inhibitors, BPC157, fat-lowering hormone, analogs and variants of these proteins, and the like. Further conceivable are insulin, glucagon, gastrin, angiotensin, secretin, lactotropin, thyotropic hormone, melanocyte stimulating hormone, luteinizing hormone (LH), vesicle stimulating hormone (FSH), thyroid stimulating hormone (TSH). ), Thrombopoietin (TPO), luteinizing hormone stimulating hormone, human climacteric disorders gonadotropin, vasopressin, oxytocin, protirelin, corticotropin, SOD, urokinase and lysozyme. Other cytokines are known and can be used equally well in the compositions of the present invention. Not limited to the biologically active proteins mentioned above, but other polypeptides such as G-CSF, M-CSF, LIF, inhibin A, inhibin B, activin A, activin B, NAP-1, MCP-1 , MIP-1 alpha, MIP-1 beta, MIP-2, SIS beta, SIS delta, SIS epsilon, PF4, PBP, gamma IP-10, MGSA, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD -ECGF, INS, IGF-I, IGF-II, NGF-beta, GRO / MGSA, PF4, PBP / CTAP / beta TG, IP-10, KC, 9E3, MCAF, ACT-2 / PAT 744 / G26, LD -78 / PAT 464, RANTES, G26, I309, JE, TCA3, ICAM-1, ICAM-2, LFA-1, LFA-3, CD72, CTAPIII, ENA-78, GRO, I-309, PF-4, LD-78 can be mentioned.
[0056]
The protein is not limited to the above-mentioned proteins, and proteins encoded by genes as shown in Table 1 below can also be used in the composition of the present invention.
Table 1

Example 1
[0057]
The composition of the present invention can be manufactured by any method known to those skilled in the art. HLB values required for various applications are 3-6 for water-in-oil emulsifiers (eg, Arlacel + squalene); 7-9 for wetting agents; 8-13 for oil-in-water emulsifiers; 13-15 in the case of; 15-18 in the case of a solubilizer (eg, aluminum or magnesium stearate). These values are widely cited in the literature as a guide in selecting an emulsifier for a particular purpose. These are designed for use with nonionic emulsifiers. Although analog systems have been developed for anionic or cationic emulsifiers, they have less utility than for nonionic emulsifiers. HLB values have been described in the literature for many nonionic emulsifiers. In this particular use, the oil phase has about 0.5 to 55%, the emulsifier about 0.1 to 15%, the nonionic surfactant about 0.05 to 5%, the therapeutic agent 0.00001 to 10%, and an aqueous continuous phase. Oil-in-water emulsions are most suitable. The emulsifier in this composition is a mixture of a phospholipid compound or a phospholipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. An example of an emulsifier is Arlacel A. Surfactants include fatty alcohols, polyethylene glycol esters of fatty acids, polyethoxylated fatty acid esters, polyethoxylated fatty alcohol ethers, alkylene oxide condensates of organic compounds having one or more hydroxyl groups and polyethoxylated alkyl phenyl ethers. Usually selected from the group consisting of: The surfactant may be a natural biologically compatible surfactant such as lecithin or a pharmaceutically acceptable artificial surfactant such as TWEEN-80. Suitable natural components related to lecithin include, for example, EPICURON 120 (Lucas Meyer, Germany) (which comprises about 70% phosphatidylcholine, 12% phosphatidylethanolamine, and about 15% other types of OVOTHIN 160 (Lucas Meyer, Germany) (this is a mixture of about 60% phosphatidylcholine, 18% phosphatidylethanolamine, and about 12% other types of phospholipids); purified phospholipid mixture -LIPOID E-75 or LIPOID E-80 (Lipoid, Germany) (which comprises about 80% phosphatidylcholine, 8% phosphatidylethanolamine, about 3.6% non-polar lipids and about 2% sphingomyelin Mixtures). Purified egg yolk phospholipids, soybean oil phospholipids or other purified phospholipid mixtures are also useful as this component. This listing is a representative example and is not intended to be limiting. This is because other types of phospholipids known to those skilled in the art can also be used. Other examples of surfactants include polyethylene glycol esters of fatty acids from raw materials such as castor oil (EMULFOR); polyethoxylated fatty acids such as stearic acid (SIMULSOL M-53); NONIDET; polyethoxylated isooctylphenol / formaldehyde polymer ( TYLOXAPOL); polyoxyethylene fatty alcohol ether (BRIJ); polyoxyethylene non-phenyl ether (TRITON N); polyoxyl ethylene isooctyl phenyl ether (TRITON X). In another example, the emulsion can be formed and stabilized substantially free of one or more co-surfactants. Examples of co-surfactants in this case include non-halogenated aliphatic C3-C6 alcohols, free fatty acids, mono- or di-glycerides, polyglycerol fatty acid esters, or lysophosphatidylcholine. However, other compositions may equally well be used and will be readily manufactured by those skilled in the art. For example, the composition may include 5.0% PLURONIC, 10% squalene, 0.4% Tween-80, qs phosphate buffered saline (pH = 7.4). These components are added to the test tube and vortex mixed until a milky emulsion is obtained. This composition is prepared immediately before administration and frozen at 4 ° C. Composition 2 contains, for example, 5.0% TETRONIC 1501, 10% squalane, 0.4% Tween-80, qs phosphate buffered saline (pH = 7.4). To these compositions, solid N-acetylmuramyl-L-threonyl-D-isoglutamine (Thr-MDP) is added to a concentration of 500 ug / mL to obtain a "concentrate". This concentrate is then mixed with a 2 × concentration solution of the antigen (in saline, hCG; 1 mg / mL) to obtain the formulation of the invention. The hCG preferably comprises from about 0.00001 to 10%, preferably from about 0.0001 to 5%, most preferably from about 0.001 to 1% by weight of the composition. Further, the pH should be in a range suitable for hCG stability. The continuous phase of the composition is aqueous, and further includes salts, sugars, antioxidants, preservatives, bactericides, buffered saline, osmotic agents, antifreeze agents, and other pharmacologically useful additives. A substance or a solute may be contained. Examples of typical preservatives include thimerosal, chlorbutanol and methyl, ethyl, propyl or butyl paraben. Examples of typical osmotic agents include glycerol and mannitol, with glycerol being particularly preferred. Examples of preferred oil phase antioxidants include alpha-tocopherol or alpha-tocopherol succinate. The aqueous phase may contain an antioxidant of polyamine carboxylic acid, for example, ethylenediaminotetraacetic acid or a pharmaceutically acceptable salt thereof.
Example 2
[0058]
This composition consists of two parts. Part I is N-acetylmuramyl-L-threonyl-D-isoglutamine (Thr-MDP), a derivative of the mycobacterium cell wall component. Part II consists of a phosphate buffered saline containing a final concentration of 5% squalane, 1.25% pluronic acid and 0.2% of TWEEN 80 (vehicle). For practical purposes, the desired amount of hCG is mixed with the microfluidizing vehicle (Part II) to obtain a homogeneous emulsion. Then add the MDP and mix briefly. The concentration of MDP in this mixture is adjusted to determine the optimal concentration for obtaining a clinical response. As an adjuvant control, mice are injected with soluble hCG mixed with alum or complete Freund's adjuvant (CFA) according to the manufacturer's manual (Piece Chemicla, Rockford, Ill.).
[0059]
One of skill in the art will recognize that such model mice show that equal experience and treatment produces a similar clinical response in humans, livestock or agricultural animals. The effective writing and amount of hCG to produce the desired clinical result will be empirically determined by routine procedures well known to those skilled in the art without undue experimentation. To minimize the side effects of treatment with such mixtures, obtain an effective response for humans, livestock or agricultural animals and thereby determine the minimum dose required to achieve the desired clinical result. Would be possible for those skilled in the art. For normal use, this mixture could be injected by any one of the standard techniques. However, a particularly preferred method is to give a subcutaneous or intramuscular injection at a site where the emulsion remains stable for days or weeks.
Example 3
[0060]
The hCG aqueous suspension is added to the oil phase at room temperature while stirring with a homogenizer. The addition of the aqueous phase is stopped when the aqueous suspension of hCG reaches 3-5 parts relative to 5-6 parts of oil phase. Stirring is continued until the droplet size of the aqueous phase is about 0.05-0.5 microns. This oil phase contains 93.6% Marcol 52 (white paraffin Esso oil); 6.0% Arlacel A or Arlacel 80 or Span 80 (mannide monooleate); 0.4% Tween 80 (polyoxyethylene 20 sorbitan monooleate). contains. The oil phase compound may be separately heated to 110 ° C. in an autoclave or sterile filtered as a mixture. The stability of this emulsion can be further determined by (1) pipetting small droplets directly after emulsification, dropping them on the water surface, and knowing whether the droplets remain unspread and (2) ) Storage at 37 ° C. for 4 weeks and can be determined by knowing that no aqueous phase is still formed. The final concentration of the emulsion thus prepared contains the drug 10% hCG, and this preparation is used for the patient intramuscularly or subcutaneously in a dose of 0.5 mL.
Example 4
[0061]
Without being limited to the above examples, the composition of the present invention containing dimer hCG may contain two kinds of emulsifiers simultaneously in order to further improve the stability. In this composition, the first emulsifier is a polyoxypropylene polyoxyethylene block copolymer, glycerol monooleate, glycerol diolate, sorbitan sesquiolate, Brij 93 polyoxyethylene alcohol, sorbitan monooleate and mixtures thereof. Selected from the group consisting of: The second emulsifier is selected from the group consisting of polyoxyethylene fatty acid esters, pluronic acid F68 ethylene oxide, egg lecithin and mixtures thereof. The oil component in the composition is a triglyceride selected from the group consisting of tricaproin, tricaprylin, tripalmitin, and mixtures thereof. Examples of other suitable oil phases include vegetable and animal oils. Examples of suitable vegetable oils are olive oil, safflower oil, sesame oil, soybean oil and the like. Examples of animal oils include those containing glycerol esters. Examples of preferred mineral oils include No. 40 white oil, carnation light oil and clearol light oil. Mixtures of these oils can be used equally.
[0062]
In the case of a primary emulsion using mineral oil, about 1 to 50% by volume, preferably about 2 to 40% by volume of the aqueous hCG solution; about 8 to 58% by volume, preferably about 14 to 25% by volume of mineral oil; 2 to 30% by volume, preferably about 5 to 15% by volume, of the first emulsifier is mixed. The aqueous hCG solution is preferably slowly added to the oil phase, for example under vigorous stirring using a magnetic stirrer, and this stirring is continued for about 15 to 60 minutes, preferably for about 25 to 35 minutes. The mixed primary emulsion is then subjected to high shear emulsification using a microfluidizer, wherein the shear rate is about 100,000 to 5,000,000 / sec, preferably about 500,000 to 1,000,000. Per second, with a pressure drop of about 1,000 to 10,000 psi, preferably about 1,000 to 3,000 psi, and most preferably about 1,800 to 2,000 psi. More complete information on this microfluidizer is described in U.S. Patent No. 4,533,254. The microfluidizer can provide high shear rates in a short time (less than one second) and does not alter proteins such as hCG. This primary emulsion is filtered using a 5 micron hydrophilic polyvinylidene difluoride filter (Duropore, Millipore). Prior to the emulsification step, albumin may be added to the hCG solution at a rate of about 1 to 5 g%, preferably about 2 to 3 g%, to further stabilize the multiple emulsion size distribution. The first emulsion in which aqueous hCG is dispersed in mineral oil or fixed oil is suitable for producing the liquid multiple emulsion of the present invention. Thereby, the diameter of the primary emulsion droplets can be made smaller than 5 microns, preferably smaller than 3 microns.
Example 5
[0063]
A water-in-oil-in-water (water / oil / water) multiple emulsion is made comprising an outer aqueous phase of 50% v / v salt with 0.25% copolymer P123. The remaining 50% v / v is an aqueous-in-oil phase consisting of 72% saline, 18% Span 80, 32 mg copolymer L310 and 0.5 mg TNP-HEA per 0.5 mL emulsion. This emulsion contains 1 mg of hCG per 0.5 mL of emulsion. The microscopic appearance of this emulsion is that of a water-in-oil emulsion with a particle size of 1.0 to 20 microns. Other suitable water-in-oil emulsifiers for child compositions are SF 1328, Arlacel P135, DC 3225C, DC 5200, Abil EM-90 and Abil WE-09. It will be apparent to those skilled in the art that several other water-in-oil emulsifiers may be used in place of the above components.
Example 6
[0064]
Liposomes containing hCG are made by standard techniques known to those skilled in the art (eg, US Pat. No. 6,110,492). The following components can be used: DLPC dilauroyl phosphatidyl choline; DMPC dimyristoyl phosphatidyl choline; DPPC dipalitoyl phosphatidyl choline; DSPC distearoyl phosphatidyl choline; DOPC dioleyl phosphatidyl choline; DLnPC dilinoleoyl phosphatidyl choline; Some are phosphatidyl glycerol; CHOL cholesterol; LA lipid A. In a typical production, multilamellar liposomes are made from DMPC: DMPG: CHOL: LA (molar ratio = 9: 1: 7.5: 0.011). Lipid (lipid) A is included as an adjuvant. This lipid mixture is rotary evaporated. That is, it is formed into a dry thin film by evaporating from a chloroform solution in a pear-shaped flask at about 40 ° C. in a vacuum. The flask is dried in a desiccator under very low vacuum (about 0.05 mm Hg) at room temperature overnight in order to completely remove the organic solvent. After drying, the lipids are carefully swollen in a deionized, sterilized, non-pyrogenic raw water while vortexing. The obtained suspension is frozen at −55 ° C. and freeze-dried at −20 ° C. overnight at −20 ° C. and then the next day using a Virtis Unitop 800SL Freeze Mobile. The lyophilized lipid is reconstituted in the presence of the substance to be encapsulated, ie, hCG, to give a multilamellar liposome containing this substance. Suitable reconstitution buffers are phosphate buffered saline (PBS) or Tris-glycine / NaCl. The liposome phospholipid concentration in this reconstitution buffer is 10-200 mM. Unencapsulated hCG is removed. That is, hCG is removed by washing the liposome three times at 27 ° C. for 10 minutes at 10 ° C. using 0.15 M NaCl. The resulting liposomes are suspended in 0.15 M NaCl or in a suitable isotonic buffer to give a final phospholipid concentration of 10-200 mM. Alternatively, this washing step may be omitted, leaving both unencapsulated hCG and encapsulated hCG in the formulation.
Example 7
[0065]
This example binds hCG to MDP. This approach requires that the first reaction be cooled with a thiol compound. This reaction is performed in 2- [N-morpholino] ethanesulfonic acid (MES) (pH: 4.5-5.0). MDP (10 mg) lyophilized and resuspended in MES (0.5 mL) (pH 4.5-5.0), and 0.5 mL EDC (0.5 mg or 0.5 mg) dissolved in MES (pH 4.5-5.0) About 2 mM) and allowed to react for 15 minutes at room temperature. 2-Mercaptoethanol (final concentration of 20 mM) is added and the EDC is cooled and separated by centrifugation. The reaction mixture is washed once with MES and then resuspended in 0.5 mL MES (pH 4.5-5.0). HCG dissolved in MES is added in a molar ratio of about 2: 1 to activated MDP. The pH of the reaction mixture is gradually increased to 8.5 over 15 minutes by the addition of MES (0.5 M, pH = 8.5) and allowed to react at room temperature for 2 hours. The concentration of hCG added to MDP was calculated from the quantitative analysis of the terminal carboxyl groups of MDP and is expressed as moles per mg MDP. The reaction is cooled by adding hydroxyamine to a final concentration of 10 mM. This cooling method hydrolyzes unreacted MDP activation sites to regenerate the original carboxyl. Other means of cooling include the addition of 20-50 mM Tris, lysine, glycine or ethanolamine. Other biological response modifiers (eg, IL2) are well known to those of skill in the art and can be used for this purpose in addition to hCG. Fractionation can be performed by centrifugation, washing steps, and resuspension in a selected buffer.
Example 8
[0066]
The composition of the present invention can be used as a cream and can be penetrated into the skin by topical application. One example of this topical composition is 2% glycerol sorbitan oleostearate (Allacel 481); 6% polyethoxylated fatty acid (Allacel 989); 15% decyl oleate (Cetiol C); 8% branched chain Paraffin (Arlamol HD); 1% microwax; 1% MDP; 4% glycerin; 0.7% magnesium sulfate heptahydrate; 0.2% sodium sorbate; 1% hCG; Diammonium hydrogen citrate; citric acid (to adjust the pH to 4-5); consisting of 100% demineralized water.
Example 9
[0067]
Men over 18 years of age and non-pregnant women were tested for AIDS, based on serodiagnosis of HIV infection as described in the Western Blot, with clinical signs, and before testing. Select those with a CD4 T lymphocyte count <300 cells / UL within 30 days. Evaluate efficacy using standard clinical and experimental parameters. These clinical parameters include changes in opportunistic infectivity, changes in body weight, changes in gastrointestinal function (regulation and frequency of bowel movements), changes in energy levels, changes in appetite, physical fitness and perseverance, and overall quality of life. Includes change. Experimental parameters include CD4 and CD8 lymphocyte counts, selected hematology, blood chemistry and urinalysis, and, where possible, changes in viral load by measuring viral RNA by PCR.
[0068]
The use of the hCG formulation in accordance with the spirit of the present invention clinically improves HIV-positive patients, reduces opportunistic infectivity, gains weight, alters gastrointestinal function (including reducing diarrhea), increases energy levels, increases appetite Increased lust, improved physical fitness and perseverance, and improved overall quality of life. Experimental parameters include increased CD4 and CD8 lymphocyte counts, improved values in hematology, blood chemistry and urinalysis, decreased viral load by measuring viral RNA by PCR, and decreased infectivity as measured by TCID. Many improvements were observed. Some side effects were seen with the use of the hCG prescription, but were limited to mild fever, chills, headache, and muscle pain.
Example 10
[0069]
Female mice with a C3H background (H2k / k, Harlan Sprague Dawley) are used for these tests. These animals are maintained according to the "Guide for the Care and Use of Laboratory Animals" (DHHS, NIH) and are provided with unlimited access to food and water. The tumor cell line HOPE2 is maintained by continuous passage in vitro. Tumors are initiated in congenital C3H mice by subcutaneous injection of 150,000 in vitro passaged cells. Tumors are measured every two weeks in two vertical directions. Each treatment group is compared to an untreated control group. Treatment begins 10 days after inoculation of HOPE2 cells, ie, many of the tumors are palpable ³ ). Treatment is initiated by injecting mice with MDP mixture or Alum adjuvant plus soluble hCG protein (0.2 mL subcutaneous injection). Immediately before inoculation, hCG was mixed with MDP in Hanks Balanced Salt Solution (HBSS) for 60 seconds so that each mouse received 0.2 mL of 2,000 or 5,000 IU of hCG protein, equivalent to a human. I do. Alum (Pierce Chemical) is mixed with hCG according to the manufacturer's instructions so that each mouse receives 0.2 mL of 5,000 IU of hCG, equivalent to a human. In this example, at day 41 after tumor cell inoculation, 5/8 and 4/8 of mice (2,000 or 5,000 IU, respectively) that received a single injection of soluble hCG showed some tumor. It was not too much. In contrast, all (8/8) of the mice injected with hCG added to Alum showed markedly growing tumors. Furthermore, considerable suppression of tumor growth was only observed in the group injected with MCG plus hCG, which was completely different from the control (untreated) group or the Alum-treated group. It is a target. No suppression of tumor growth or improvement in tumor remission was observed in mice injected alone with hCG added to Alum.
[0070]
Accordingly, the invention is characterized by a pharmaceutical composition comprising the dimer hCG and the muramyl peptide. Additionally, the invention comprises (a) a therapeutically effective amount of a dimeric hCG associated with a liposome; and (b) an oil-in-water emulsion comprising an oil surrounding the liposome; wherein the emulsion is absent. Sometimes characterized by a pharmaceutical composition contained in an amount sufficient to increase the therapeutic response in the context of hCG and liposomes. Further, the present invention is a method for treating a person infected with HIV, comprising administering to the patient an effective amount of hCG, wherein the hCG is administered in the form of an oil-in-water emulsion, the emulsion comprising: Methods are provided that include an amount sufficient to increase a therapeutic response in the context of hCG and liposomes when the emulsion is not present. Further, the invention is a method for treating a cancer patient, comprising administering to the patient an effective amount of hCG, wherein the hCG is administered in the form of an oil-in-water emulsion, the emulsion comprising: Methods are provided that include an amount sufficient to increase a therapeutic response in the context of hCG and liposomes when the emulsion is not present. The present invention further provides a pharmaceutical composition comprising an oil-in-water emulsion having a continuous aqueous phase and a discontinuous oil phase and comprising at least 0.01% by weight of dimer hCG. The present invention is further directed to a method for manufacturing a pharmaceutical formulation, comprising mixing an aqueous suspension comprising hCG, at least one oily component and a sufficient amount of at least one emulsifier to form an oil-in-water emulsion. A method for obtaining a prescription is provided. The invention further provides a pharmaceutical composition comprising an oil-in-water emulsion, at least one natural or recombinant water-soluble biologically active protein, and any at least one muramyl peptide. The present invention further provides a kit for the ready production of a pharmaceutical composition, comprising: (1) a first container containing an emulsion, wherein the emulsion comprises squalane or squalene, aluracell, and an aqueous solution. And (2) a second container containing a therapeutically effective amount of the dimer hCG dissolved in an aqueous solution or as a powder; wherein the concentration of the components in each container is: When the ingredients in both containers are combined, a formulation of 1-30% Arlacel, 1-30% squalane or squalene, 0.0001-30% muramyl peptide, 0.01-99% hCG Provide what is causing it.
[0071]
While the present invention has been described in detail in connection with what is presently considered to be the most practical and preferred embodiment, those skilled in the art will recognize that many modifications may be made without departing from the principles and concepts described herein. It will be obvious. Therefore, the proper scope of the present invention should be determined by the broadest interpretation of the appended claims, and includes all the above-described modifications and all equivalents described in the specification. Should be understood.

Claims (33)

A pharmaceutical composition comprising dimer hCG and muramyl peptide. The pharmaceutical composition according to claim 1, further comprising an emulsifier. 3. The pharmaceutical composition according to claim 2, further comprising a surfactant. 4. The pharmaceutical composition according to claim 3, further comprising an oil. The pharmaceutical composition according to claim 1, wherein the content of the dimer hCG is at least 0.01% by weight. (A) a therapeutically effective amount of a dimeric hCG associated with the liposome; and (b) an oil-in-water emulsion comprising an oil surrounding the liposome; the emulsion comprising hCG and the liposome in the absence of the emulsion. A pharmaceutical composition, characterized in that it is contained in an amount sufficient to increase the therapeutic response in the context of 7. The pharmaceutical composition according to claim 6, wherein said oil is squalene. 7. The pharmaceutical composition according to claim 6, wherein said hCG comprises at least 0.1% by volume of said oil. 7. The pharmaceutical composition according to claim 6, further comprising an emulsifier. 10. The additional emulsifier is selected from the group consisting of sorbitan esters, polyoxyethylene sorbitan mono-, di- or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers and combinations thereof. A pharmaceutical composition as described. 7. The pharmaceutical composition according to claim 6, further comprising an adjuvant. The adjuvants are N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine (GMDP), N-acetyl-D-glucosaminyl (beta-1-4) -N-acetyl-muramyl-L -Alanyl-D-isoglutamine, N-acetyl-glucosaminyl-N-acetyl-muramyl-L-alanyl-D-isoglutamic acid (GMDP-A), muramyl dipeptide-phosphatidyl-ethanolamine, muramyl tripeptide-phosphatidyl- Ethanolamine, muramyl tripeptide-phosphatidyl-ethanolamine, CGP11637 (nor-MDP), alpha (N-acetyl-muramyl-L-alanyl-D-isoglutamine) beta, gamma-dipalmitoyl-sn-glycerol, alpha ( N-acetyl-muramyl-D-alanyl-D-isoglutamine) beta, gamma-dipalmitoyl-sn-g Lycerol, alpha (N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine) beta, gamma-dipalmitoyl-sn-glycerol, alpha (N-acetyl-muramyl-D-alanyl-D-isoglutaminyl-L -Alanine) beta, gamma-dipalmitoyl-sn-glycerol, N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl (Oxy) ethylamide, glucoasminylmuramyl peptide, muramethide, murabutide, muradimethide, miramistin, N-acetyl-muramyl-L-threonyl-D-isoglutamine, N-acetyl-muramyl-L-alpha-aminobutyryl-D-isoglutamine , 6-O-stearoyl-N-acetyl-muramyl-L- Alpha-aminobutyryl-D-isoglutamine, N-acetyl-muramyl-L-valyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-isoglutamine, N-acetyl-desmethylmuramyl-L- Alanyl-D-isoglutamine, N-acetyl-muramyl-L-alanyl-D-glutamine butyl ester, N-acetyl-muramyl-L-seryl-D-isoglutamine, N-butyryl-muramyl-L-alpha-aminobutyryl -D-isoglutamine, N-acetyl-muramyl-L-threonyl-D-isoglutamine, bis (6-O-muramyl dipeptide) O-palmitoyl taltronate, muramyl tripeptide-phosphatidyl-ethanolamine, N-acetyl -Muramil-L-alanyl-D-isoglutaminyl-L-alanine-2- (1,2-dipalmitoyl-s n-glycero-3- (hydroxyphosphoryloxy)) ethylamide, N-acetyl-muramyl-L-alanyl-D-glutamine butyl ester, N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2 Beta-butyl glycoside of -1,2-dipalmitoyl-sn-glycero-3- (hydroxyphosphoryloxy)) ethylamide (MTP-PE), cholesteryl-MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine , 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-[(R) -1- (methoxycarbonyl) ethyl] -alpha-D-glucopyranose, (beta-butyl-MDP , MTPO-26, beta-cholesteryl-MDP), saponin (Taurosid I), N-acetylnor-muramyl-LN-methylalanyl-D-isoglutamine / octylamide , UDP-N-acetyl-muramyl-pentapeptide, L4-MDP-ONB, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, 1,6-anhydro muramyl dipeptide, N-acetylglucosamine- Beta-1,4-N-acetyl-muramyl-pentapeptide-pyrophosphoryl-undecaprenol, 3-O- [N-acetyl-muramyl-D-isoglutaminyl] -1,2-di-O-dipalmitoyl- selected from the group consisting of sn-glycerol, L-threonyl-MDP-GDP, L-allothreonyl-MDP-GDP, trehalose 6,6-diester, muramyl dipeptide, B30-muramil dipeptide and muramyl dipeptide-lysine The pharmaceutical composition according to claim 11, which is A method for treating an HIV-infected person, comprising administering to the patient an effective amount of hCG, wherein the hCG is administered in the form of an oil-in-water emulsion, and wherein the emulsion is not present. In the context of hCG and liposomes in an amount sufficient to increase a therapeutic response. 14. The method according to claim 13, further comprising an effective amount of the muramyl peptide, wherein the muramyl peptide is administered in an amount of 1-500 μg / kg body weight. 14. The method of claim 13, wherein said hCG is provided in association with a liposome. A method for treating a cancer patient, comprising administering to said patient an effective amount of hCG, wherein said hCG is administered in the form of an oil-in-water emulsion, wherein said emulsion is present in the absence of said emulsion. A sufficient amount to increase the therapeutic response in the context of the hCG and the liposomes. 17. The method of claim 16, further comprising an effective amount of the muramyl peptide, wherein the muramyl peptide is administered in an amount of 1-500 [mu] g / kg body weight. 17. The method of claim 16, wherein said hCG is provided in association with a liposome. 17. The method according to claim 16, wherein the hCG is administered via intravenous, intraperitoneal, intraenvironmental, intramuscular, subcutaneous, intradermal, buccal, oral, nasal, intralesional, and topical. A pharmaceutical composition comprising an oil-in-water emulsion having a continuous aqueous phase and a discontinuous oil phase and containing at least 0.01% by weight of dimer hCG. 21. The pharmaceutical composition according to claim 20, further comprising a therapeutically effective amount of a muramyl peptide. 21. The pharmaceutical composition according to claim 20, wherein said oil phase comprises mineral oil, squalene, peanut oil, vegetable oil or silicone oil. 21. The emulsion comprising a surfactant selected from the group consisting of sorbitan esters, polyoxyethylene sorbitan mono-, di- or triesters, polyoxyethylene fatty acids, polyoxyethylene fatty acid ethers and combinations thereof. A pharmaceutical composition as described. The surfactant may be polysorbate ("TWEEN" or sorbitan monooleate), sodium dodecyl sulfate (SDS), polyethoxylated castor oil ("CREMOPHOR"), NP-40, dimethyldioctadecyl ammonium bromide ("DDA"), Linear polyoxypropylene-polyoxyethylene (POP-POE) block polymer, poloxamer 401, Pluronic L62LF, Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501, Tetronic 150R1, Tetronic 701, Tetronic 901, Tetronic 1301, Atmos 300, Tween 24. The pharmaceutical composition according to claim 23 comprising 20, Tween 40, Tween 60, Tween 80, and Tetronic 130R1, Arlacel A, Arlacel 80, Span 80 and mannide monooleate. 21. The pharmaceutical composition according to claim 20, wherein said aqueous phase comprises water itself, phosphate buffered saline, saline, Ringer's solution, or a combination thereof. A method for manufacturing a pharmaceutical formulation, comprising mixing an aqueous suspension comprising hCG, at least one oily component and a sufficient amount of at least one emulsifier to obtain the formulation as an oil-in-water emulsion. Features method. 27. The method of claim 26, further comprising the step of adding a therapeutically effective amount of a muramyl peptide to the oil-in-water emulsion. A pharmaceutical composition comprising an oil-in-water emulsion, at least one natural or recombinant water-soluble biologically active protein, and optionally at least one muramyl peptide. The biologically active protein is somatotropin, human growth hormone (HGH), bovine growth hormone (BGH or BST), porcine growth hormone (PGH or PST), epidermal growth factor (FGF), interferon alpha, interferon alpha- 2a, interferon alpha-2b, interferon alpha-N1, interferon alpha-n3, interferon beta, interferon beta-1 al, interferon beta-1b, interferon gamma-Ia, interferon gamma-Ib, interferon Omega, interferon-tau, interleukin-1, interleukin-1 alpha, interleukin-1 beta, interleukin-10, interleukin-11, interleukin-12, interleukin-15, interleukin Kin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, erythropoiesis factor (EPO), parathyroid hormone (PTH), selenoprotein P, cystatin B, endotoxin-neutralizing protein, megakaryocyte mimicking factor (MGDF), granular leukocyte macrophage colony mimicking factor (GM-CSF), cytokin synthesis inhibitor factor (CSIF), genofibrate, alpha-calcitonin, beta-calcitonin, Tumor necrosis factor (TNF), lymphocyte migration inhibitory factor (LIF), tumor invasion inhibitory factor, transacting regulatory protein (TAT), protease inhibitor, BPC157, fat-lowering hormone, insulin, glucagon, gastrin, angiotensin, secretin, lacto Tropic hormone, thyotropic hormone, melanocyte stimulating hormone, Body forming hormone (LH), vesicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), thrombopoietin (TPO), luteinizing hormone stimulating hormone, human climacteric disorder gonadotropin, vasopressin, oxytocin, protirelin, corticotropin, SOD, Urokinase, lysozyme, G-CSF, M-CSF, LIF, inhibin A, inhibin B, activin A, activin B, NAP-1, MCP-1, MIP-1 alpha, MIP-1 beta, MIP-2, SIS beta , SIS delta, SIS epsilon, PF4, PBP, gamma-IP-10, MGSA, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGF, INS, IGF-I, IGF-II, NGF-beta, GRO / MGSA, PF4, PBP / CTAP / Beta TG, IP-10, KC, 9E3, MCP-1 / MCAF, MCAF, ACT-2 / PAT 744 / G26, LD-78 / PAT 464, RANTES, G26, I309 , JE, TCA3, B7.1, B7.2, ICAM-1, ICAM-2, LFA-1, LFA-3, CD72, CTAPIII, ENA-78, GRO, I-309, PF-4, LD-78 , Eosinophil-induced neurotoxin (EDN) Antitumorigenic urinary protein (Anup), ribonuclease, angiostatin, pharmaceutical composition according to claim 28 wherein is selected from the group consisting of analogs and variants. 29. The pharmaceutical composition according to claim 28, wherein said biologically active protein is selected from those listed in Table 1. A kit for the immediate manufacture of a pharmaceutical composition, comprising: (1) a first container containing an emulsion, the emulsion comprising squalane or squalene, allacel, and an aqueous solution. And (2) a second container containing a therapeutically effective amount of dimer hCG dissolved in an aqueous solution or as a powder; wherein the concentration of the components in each container is The combination of the ingredients results in a formulation of 1-30% Arlacel, 1-30% squalane or squalene, 0.0001-30% muramyl peptide, 0.01-99% hCG. A kit comprising: 32. The kit of claim 31, further comprising a therapeutically effective amount of a muramyl peptide added to the first or second container. 32. The kit of claim 31, further comprising a therapeutically effective amount of a muramyl peptide contained in another container.