JP2023526069A - 長時間作用アシル化インスリン化合物 - Google Patents
長時間作用アシル化インスリン化合物 Download PDFInfo
- Publication number
- JP2023526069A JP2023526069A JP2022569032A JP2022569032A JP2023526069A JP 2023526069 A JP2023526069 A JP 2023526069A JP 2022569032 A JP2022569032 A JP 2022569032A JP 2022569032 A JP2022569032 A JP 2022569032A JP 2023526069 A JP2023526069 A JP 2023526069A
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- JP
- Japan
- Prior art keywords
- compound
- γglu
- pharmaceutically acceptable
- acceptable salt
- insulin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical class N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title abstract description 77
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- 101001034652 Homo sapiens Insulin-like growth factor 1 receptor Proteins 0.000 description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 7
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- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 6
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 6
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- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 6
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Abstract
Description
A14位の天然のチロシンアミノ酸残基がグルタミン酸アミノ酸残基に変異している、配列番号1のA鎖配列、及びB16位の天然のチロシンアミノ酸残基がヒスチジンアミノ酸残基に変異しており、B25位の天然のフェニルアラニンアミノ酸残基がヒスチジンアミノ酸残基に変異しており、B30位の天然のスレオニンアミノ酸残基が欠失している、配列番号2のB鎖配列(ここで、配列番号1の6位のシステインと配列番号1の11位のシステインとの間にジスルフィド結合が存在し、配列番号1の7位のシステインと配列番号2の7位のシステインとの間にジスルフィド結合が存在し、かつ配列番号1の20位のシステインと配列番号2の19位のシステインとの間にジスルフィド結合が存在し、
B29位のリジンアミノ酸残基は、リジン側鎖のエプシロン-アミノ基とHO2C-(CH2)18-CO-γGlu-γGlu-γGlu-Lys-Gly-、HO2C-(CH2)18-CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-アミノエトキシ)エトキシ]酢酸)-、又はHO2C-(CH2)18-CO-γGlu-γGlu-γGlu-εLys-Gly-とのコンジュゲーションによって化学修飾されている)、又はその薬学的に許容される塩。
実施例1
1)単鎖プレプロインスリン構築物(SCI)のE.coli発現及び精製:これは、好ましいアシル化方法に使用され得るか、又は成熟二鎖インスリン類似体(TCI、A14E、B16H、B25H、desB30-BHI)に酵素的に処理される;
2)リンカー脂肪酸中間体(化合物A)の合成;並びに
3a)TCIのアシル化、脱保護、又は3b)SCIのアシル化、脱保護、及び酵素消化、その後の精製及び塩交換により、実施例1を得る。
化合物A(C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OHは、固相ペプチド合成によって生成される。Fmoc-Lys(Boc)-OH(769mg、1.6mmol、樹脂に対して1.5eq.)を、PyBop(849mg、1.6mmol、樹脂に対して1.49eq.)、及び10mLのDMF中のDIEA(1520ul、8.7mmol、8eq.)と2分間混合し、DCMで事前に膨潤し、DMFで事前に洗浄したH-Gly-2-クロロトリチル-クロリド樹脂(1.1g、0.99mmol/g、1.1mmol;Peptides International RHG-1160-PI)を含む反応槽に移した。スラリーを1.5時間混合し、濾過し、次いで樹脂をDMFでよく洗浄する(カイザーテストは陰性である)。樹脂を20%ピペリジン/DMF(10mL、30分)で処理することにより、Fmoc保護基を除去する。樹脂(40mL)のDMF洗浄後、カイザーテストは陽性であり、最終生成物は、H-Lys(Boc)-Gly-2-クロロトリチル-クロリド樹脂(理論上は1.1mmol)である。
アシル化:化合物A((C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OH)(60.8mg;0.0437mmol;1.2eq.;上に記載の合成)及びTSTU(12.04mg 0.040mmol;1.1eq.)を、200μLのジメチルスルホキシド(DMSO)に溶解する。ジイソプロピルエチルアミン(DIEA、25.3μL;0.145mmol;4eq.)を、この溶液に添加し、得られた混合物を室温で30分間インキュベートして、DMSO中に化合物A-OSuエステルを生成し、直接使用する。
組換えhIR-A、hIR-B、又はhIGF-1Rを過剰発現する、安定してトランスフェクトされた293HEK細胞からの分画遠心分離工程を使用して調製された膜を使用して、実施例1、2、及び3、並びに対照化合物(生合成ヒトインスリン(BHI)、及びインスリン様成長因子1(IGF-1))を、ヒトインスリン受容体(hIR)及びヒトIGF-1受容体(hIGF-1R)シンチレーション近接アッセイ(SPA)競合的放射性リガンド結合アッセイにおいて試験する。
幾何平均Ki=10(Log Ki値の加算平均)
誤差はデルタ法を使用して計算され、ここで、SEM=GeoMean×((Log10 Ki値の標準偏差)/(nの平方根))×ln10である
インスリン受容体には、リガンド結合時に自身のチロシン残基を自己リン酸化して、インスリンシグナル伝達経路を誘導するように作用するアダプタータンパク質の動員を可能にする細胞内チロシンキナーゼドメインが含まれる。チロシン残基の受容体自己リン酸化を刺激するための機能的な細胞活性は、hIR-A、hIR-B、又はhIGF-1Rを過剰発現するHEK293細胞を、それぞれC末端C9エピトープ(TETSQVAPA、配列番号6)でリガンド処理した後に決定される。
0.1%カゼインの存在下でのIRA及びIRBリン酸化に対する実施例の効果を測定するために、hIRA-HEK293-zeo又はhIRB-C9-HEK293-G418細胞を、最初に、高グルコースを含み、0.1%BSAを含むピルビン酸ナトリウム(Gibco 11965-084)を含まないDMEM中の96ウェルのポリ-D-リジンコーティングプレート(Biocoat 354461)に60,000細胞/ウェルでプレーティングする。次に、細胞を組織培養インキュベーターで、37℃で一晩インキュベートする。96ウェルELISAプレートはまた、20mM炭酸ナトリウム緩衝液(pH9.6)中の3.5μg/mLの抗インスリン受容体抗体83-14(IRAアッセイ用)又は2.0μg/mLの抗C9抗体(IRBアッセイ用)でコーティングされ、4℃で一晩インキュベートされる。
実施例1、2、及び3のグルコース低下及び薬物動態を、ストレプトゾトシン(STZ)処置ラット糖尿病モデルで調査する。体重400~425グラムのオスのスプラーグ-ドーリーラットを、Envigo,Indianapolis,Indianaから入手する。約1週間順化させた後、ラットをイソフルランで麻酔し、Zanosar(89256、Teva Parenteral Medicines、40mg/kg、IV)を1回注射する。ラットをZanosarの注射から3日後に研究に使用する。非空腹時血中グルコースが400~550mg/dLである動物のみをこれらの研究に使用する。
この研究の目的は、3.6nmol/kgの単回皮下投与後の糖尿病ユカタンミニブタにおける実施例のグルコース低下及び薬物動態を調査することである。血液試料を168時間にわたって収集する。
配列番号1 修飾インスリンA鎖(A14E);式IのA鎖
GIVEQCCTSICSLEQLENYCN
FVNQHLCGSHLVEALHLVCGERGFHYTPK
ここで、29位のLysは、Lys側鎖のエプシロン-アミノ基とHO2C-(CH2)18-CO-γGlu-γGlu-γGlu-X-とのコンジュゲーションによって化学修飾され、Xは、-Lys-Gly-、-Lys-(2-[2-(2-アミノエトキシ)エトキシ]酢酸)-、又は-εLys-Gly-である。
FVNQHLCGSHLVEALHLVCGERGFHYTPK
ここで、29位のLysは、Lys側鎖のエプシロン-アミノ基とHO2C-(CH2)18-CO-γGlu-γGlu-γGlu-Lys-Gly-とのコンジュゲーションによって化学修飾される。
FVNQHLCGSHLVEALHLVCGERGFHYTPK
ここで、29位のLysは、Lys側鎖のエプシロン-アミノ基とHO2C-(CH2)18-CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-アミノエトキシ)エトキシ]酢酸)-とのコンジュゲーションによって化学修飾される。
FVNQHLCGSHLVEALHLVCGERGFHYTPK
ここで、29位のLysは、Lys側鎖のエプシロン-アミノ基とHO2C-(CH2)18-CO-γGlu-γGlu-γGlu-εLys-Gly-とのコンジュゲーションによって化学修飾される。
TETSQVAPA
MHHHHHHQAIFVLQGSLDQDPEFENLYFQIEGGRFVNQHLCGSHLVEALHLVCGERGFHYTPKRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQRGIVEQCCTSICSLEQLENYCN
FVNQHLCGSHLVEALHLVCGERGFHYTPK
Claims (17)
- Xが、-Lys-Gly-である、請求項1に記載の化合物、又はその薬学的に許容される塩。
- Xが、-Lys-(2-[2-(2-アミノエトキシ)エトキシ]酢酸)-である、請求項1記載の化合物、又はその薬学的に許容される塩。
- Xが、-εLys-Gly-である、請求項1に記載の化合物、又はその薬学的に許容される塩。
- 請求項1~7のいずれか一項に記載の化合物、又はその薬学的に許容される塩と、1つ以上の薬学的に許容される賦形剤と、を含む薬学的組成物。
- 患者のI型及び/又はII型糖尿病を治療する方法であって、それを必要とする患者に、有効量の請求項1~7のいずれか一項に記載の化合物、若しくはその薬学的に許容される塩、又は請求項8に記載の薬学的組成物を投与することを含む、方法。
- 患者の高血糖症を治療する方法であって、それを必要とする患者に、有効量の請求項1~7のいずれか一項に記載の化合物、若しくはその薬学的に許容される塩、又は請求項8に記載の薬学的組成物を投与することを含む、方法。
- 治療に使用される、請求項1~7のいずれか一項に記載の化合物、又はその薬学的に許容される塩。
- I型及び/又はII型糖尿病の治療に使用される、請求項1~7のいずれか一項に記載の化合物、又はその薬学的に許容される塩。
- 高血糖症の治療に使用される、請求項1~7のいずれか一項に記載の化合物、又はその薬学的に許容される塩。
- 請求項14に記載の群から選択される化合物を使用して、式Iの化合物又はその薬学的に許容される塩を調製するための、プロセス。
- 糖尿病の治療のための薬物の製造における、請求項1~7のいずれか一項に記載の化合物、又はその薬学的に許容される塩の使用。
- 高血糖症の治療のための薬物の製造における、請求項1~7のいずれか一項に記載の化合物、又はその薬学的に許容される塩の使用。
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