수용성 파클리탁셀의 이화학적 특성

Abstract

파클리탁셀(Paclitaxel)은 난소암, 유방암, 폐암등의 치료효과가 매우 탁월하여 암의 화학요법제중 가장 주목받는 치료제로 알려져 있다. 그러나, 이러한 탁월한 효과에도 불구하고 탁솔은 물에 매우 난용성인 관계로 제제화에 많은 어려움이 있으며 주사제의 경우 신경병증, 근증, 근육통증 등 여러 종류의 부작용을 갖고 있다. 본 연구에서는 이러한 문제를 해결하고자 수용성 파클리탁셀(Paclitaxel) 프로드럭(prodrug) 개발을 위한 연구의 일부로 소수성 약물인 파클리탁셀(Paclitaxel)에 친수성 폴리에틸렌글리콜(Polyethyleneglycol)을 도입함으로써 수용성 파클리탁셀 프로드럭(PP7)을 성공적으로 합성하였다.(USPAT 6,703,417) 물에 대한 용해도는 폴리에틸렌글리콜(Polyethyleneglycol)을 약물에 붙임으로써 해결되었고 이러한 약물의 life time은 고분자와 약물사이에 self-immolating linker(L)과 같은 연결그룹을 도입하여 효소 접근시 파클리탁셀(Paclitaxel)의 구조적인 입체장애를 줄여 빠르게 가수분해가 일어날 수 있도록 하였다. 그런데 이러한 경우에 분자의 형태와 rigidity에 관계되는 용액 및 수송성질은 최적응용을 위해 아주 중요한 부분이다. 따라서, 용액에서 modified drug(PP7)의 거동과 한쪽 말단이 CH3이고 다른 한쪽은 self-immolating linker를 갖는 succinate로 capping된 mPEG와 mPEG-succinate의 거동을 비교하여 연구하였다. 이들의 mobility를 알아보기 위해 수용액(D2O)에서 자기확산의 농도, 온도 의존성을 PFG -NMR 실험으로서 접근하였고 diffusivity는 Einstein-Somcynski Eq.과 Stokes-Einstein Eq.에 의해 검토하였다. 소수성 분자와 친수성 분자의 결합은 용액에서 마이셀(micelle)과 같은 콜로이드의 회합 형태를 갖는 양친성(amphiphilic) 분자를 이룰 수 있는 가능성이 있다. 이의 검토를 위하여 PP7의 농도에 따른 표면장력을 조사하였으며 더불어 용액 점도를 측정하였다. 또한, 이성분계의 상평형 그림(phase diagram)에 관한 연구는 의학적 응용에 중요하다. 왜냐하면 상평형 그림(phase diagram)은 용해도, 상전이, 대응하는 조성에 관한 정보를 준다. 따라서, 여기에서는 PP7-5,000/물(H20) 계에 대해 20℃부터 DSC로 조사하였고 그 결과 전형적인 eutectic system의 거동을 보임을 확인하였다. 실제 이러한 연구들은 active drug system에 매우 중요하다. 이러한 실험으로부터 얻어지는 분자의 mobility와 상호작용 그리고 상거동에 관한 정보는 향후 인체에서 약물(drug)의 수송메카니즘 및 경구용제제를 연구하기 위해 아주 중요한 기초 자료가 되어질 것이다. 물론, 단순한 계에 관한 연구부터 시작하여 나중에는 인체에서 단백질과의 상호작용까지 고려하여 연구되어져야 한다고 생각된다. 또한, 본 연구에서는 앞서 언급한 실험 결과들을 뒷받침하여 주는 또 다른 접근의 하나로 진공상태와 물 분위기에서 PP7-5,000의 형태(conformation)를 조사하기 위하여 컴퓨터 시뮬레이션을 이용한 접근을 시도하였다. 다시 말해, 앞서 언급한 PP7-5,000이 마이셀(micelle) 형성과 같은 중요한 분자들의 응집(aggregation)을 보이지 않음을 뒷받침하고자 또 다른 측면, 즉 시뮬레이션을 통하여 고분자의 동적성질을 조사코자 하였다. 단, 진공상태가 아닌 물 분자의 존재 하에서는 PEG의 분자량이 5,000인 PP7-5,000 고분자에 대해서는 molecular dynamics(MD) 시뮬레이션의 한계로 인하여 조사가 어려워, PEG의 분자량이 550인 PP7-550에 대하여 실험하였고 이의 결과로부터 PP7-5,000의 형태 (conformation)를 간접적으로 유추 해석하였다. PP7-5,000의 경우, 진공상태 뿐만이 아니라 물분자 존재 하에서도 PEG 사슬의 부분이 paclitaxel 분자의 주위를 둘러싸는 구조를 가지리라 설명할 수 있다. 이는 hydrodynamic property의 결과와 잘 일치한다. 그리고, 파클리탁셀보다 훨씬 더 소수성 구조를 갖는 켐프토세신(camptothecin)이라는 또 다른 항암제에 대해서 같은 조건으로 시뮬레이션을 시도하여 비교 평가의 모델로 제시하였다. 이러한 결과들 또한 active drug system에 매우 중요하다.|Paclitaxel(Taxol) is a antineoplastic agent that is derived from the bark of the Pacific yew tree (Taxus brevifolia) (Wani et al., 1971). In contrast to Vinca alkaloids, the anticancer action of taxol is that it inhibits cellular growth by promoting and stabilizing microtubule assembly by non-covalent interaction with tubulin, thereby blocking cell replication in the late G2 mitotic phase of the cell cycle (Kumar, 1981; Manfredi et al.,1984). Because of its poor water solubility, paclitaxel is currently formulated in taxol with a mixture of polyoxyethyleneglycerol triricinoleate 35 (Cremophor EL) and dehydrated ethanol USP (1:1, v/v). Cremophor EL itself is toxic and produces vasodilation, laboured breathing, lethargy and hypotension when administered intravenously. The application of some pharmaceutically active substances such as the anti-cancer drug, paclitaxel, suffer from their low solubility in body fluids and serious side effects. It has been reported that the human toxicity of paclitaxel included myelosuppression, emesis, weight loss, hepatic dysfunction and increases in total plasma lipids, cholesterol and triglyceride and so on (Rowinsky et al., 1993). Paclitaxel has been used to ovarian carcinoma, breast carcinoma, leukemia, melanoma, prostate carcinoma and so on, and has become especially important for the management of ovarian and breast carcinoma (Rowinsky et al., 1990, McGuire et al., 1989, Sarosy et al., 1992, Holmes et al., 1991). Oral administration of the pure drug is problematic as it has a poor absorption due to its poor solubility in aqueous media.(Sparreboom et al., 1997). Despite several polar sites the molecule is only poorly soluble in aqueous media. Also, the pump function for paclitaxel of the multi-drug pump transporter P-glycoprotein of the gastrointestinal tract is poor. There were several ways to overcome this problem. One possible way is substitution at the reactive sites on the carbon 2' respectively 7. The first such derivatives of paclitaxel with poly(ethylene-glycol) PEG , however, were too stable so that a high percentage of the polymer drug was excreted before the drug could be made bio-available by esterases on-site of their intended interaction. This problem was solved by the development of the prodrug PP7 which connects the hydrophilic PEGS, a succinic acid mono-ester of poly(ethylene-glycol) monomethylether, with a self-immolating linker(L) at the carbon 7. This has been successfully done and the advantage of these modifications was that the stability of the modified drug was too high. A patented self-immolating link75)~78) between PEG and the drug which provides a hydrolytic stability of the drug-polymer link in the body liquid of only a few minutes, just sufficient to transport the major amount of the drug to the site where its activity is required. The term "prodrug" indicates that PP7 is not the finally active species. Enzymatic cleavage of the bond with the polymer substituent is necessary so that the paclitaxel molecule can interfere in the late phase of the G2 mitosis by stabilizing the microtubule through non-covalent interaction. To get a better understanding for oral administration of the PP7, solution behaviors of the PP7 were characterized by NMR, diffusivity and calorimetry etc.. The mobility of the functionalized polymer and of the amphiphilic drug modified-polymer in D2O was studied by the temperature and concentration dependence of the long-time self-diffusion coefficients of the components. The technique applied was pulse-gradient field NMR spectroscopy. The diffusivity of the molecules can be described in terms of the Einstein-Sutherland and the Stokes-Einstein equations. First results indicate that combining the drug with the polymer rather influences the rigidity parameter and the pair-interaction in solution than the shape and the hydrodynamic radius of the polymer coils. Also, the surface tension and solution viscosity were measured in water to support previous results. Unexpectedly, micelle-formation, although highly probable, was not observed within the concentration range investigated here. For any kind of medical application and administration the phase-diagram of the binary isobaric phase diagram is important since it gives information about solubility, phase transitions and the corresponding compositions. The system PP7+H2O was studied calorimetrically from 20°C and shows the typical behaviour of an eutectic system. The properties of the phase diagram are discussed in view of other solution properties of the system presented earlier. Finally, The knowledge of the phase behaviour of pharmaceutical systems is therefore of great importance far beyond just academic interest. The investigation of the phase diagram is one more step on the way to understand the mechanism of drug activity with respect to conformation, shape and size in solution, transport mechanisms, and interaction with other constituents of body fluids. In order to investigate the morphology of Paclitaxel and PP7 I used some organic solvents, and the samples prepared by simple solvent treatment were characterized by differential scanning calorimetry(DSC), infrared spectroscopy(IR) and X-ray diffraction(XRD). The results of this study show that the morphologies of Paclitaxel and PP7 (dihydrated, anhydrous and amorphous) can be controlled by conventional solvent treatments.56) In earlier studies, no superstructures like micelles were found in the prodrug investigated by us that far. However, the formation of supramolecular structures may be advantageous because they mean a higher local concentration and the circulation time of the prodrug could probably be enhanced. This is in particular desirable since synthesis and purification of the prodrug are rather costly. Therefore, studies of the behaviour of the prodrug are required to determine structure and interactions of the prepolymer in solution. I report here about recent results of molecular simulations which were executed of the prodrug in vacuum and in the presence of water. The present explanation of the experimental results is that the PEG-chain coils-up around the lipophilic core of the paclitaxel in a way comparable to the tertiary structure of proteins. The results are important for further engineering on active drug systems.