Bourré Ludovic, Giuntini Francesca, Eggleston Ian M, Wilson Michael, MacRobert Alexander J
National Medical Laser Centre, Division of Surgical and Interventional Sciences, Royal Free and University College Medical School, University College London, United Kingdom.
Mol Cancer Ther. 2008 Jun;7(6):1720-9. doi: 10.1158/1535-7163.MCT-08-0092.
Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (ALA) has been widely used in photodynamic therapy for a range of malignant and nonmalignant lesions. However, ALA is relatively hydrophilic and lacks stability at physiologic pH, limiting its bioavailability. We have investigated more lipophilic, uncharged ALA-peptide prodrugs based on phenylalanyl-ALA conjugates, which are water soluble and chemically stable for improving ALA delivery. Pharmacokinetics of the induced protoporphyrin IX (PpIX) were studied in transformed PAM212 keratinocyte cells and pig skin explants. The intracellular porphyrin production was substantially increased with Ac-L-Phe-ALA-Me (compound 1) and Ac-L-Phe-ALA (compound 3) compared with equimolar ALA: after 6-h incubation, the PpIX fluorescence measured using 0.01 mmol/L of compound 1 was enhanced by a factor of 5 compared with ALA. Phototoxicity results showed good correlation with PpIX levels, giving a LD(50) (2.5 J/cm(2)) of 25 micromol/L for ALA, 6 micromol/L for 5-aminolaevulinic hexyl ester, and 2.6 micromol/L for compound 1, which exhibited the highest phototoxicity. However, these results were stereospecific because the corresponding D-enantiomer, Ac-D-Phe-ALA-Me (compound 2), induced neither porphyrin synthesis nor phototoxicity. PpIX levels were considerably reduced when cells were incubated with compound 1 at low temperatures, consistent with active transport. In pig skin explants, compound 1 induced higher porphyrin fluorescence than ALA by a factor of 3. These results show that water-soluble peptide prodrugs of ALA can greatly increase its cellular uptake, generating more intracellular PpIX and improved tumor cell photosensitization. The derivatives are comparable in efficacy with 5-aminolaevulinic hexyl ester but less toxic and more stable at physiologic pH.
5-氨基乙酰丙酸（ALA）给药后细胞内卟啉生成已广泛应用于一系列恶性和非恶性病变的光动力治疗。然而，ALA相对亲水，在生理pH下缺乏稳定性，限制了其生物利用度。我们研究了基于苯丙氨酰-ALA共轭物的亲脂性、不带电荷的ALA-肽前药，它们水溶性好且化学稳定，可改善ALA递送。在转化的PAM212角质形成细胞和猪皮肤外植体中研究了诱导的原卟啉IX（PpIX）的药代动力学。与等摩尔的ALA相比，Ac-L-Phe-ALA-Me（化合物1）和Ac-L-Phe-ALA（化合物3）使细胞内卟啉生成显著增加：孵育6小时后，使用0.01 mmol/L化合物1测得的PpIX荧光比ALA增强了5倍。光毒性结果与PpIX水平显示出良好的相关性，ALA的半数致死剂量（LD50）（2.5 J/cm²）为25 μmol/L，5-氨基乙酰丙酸己酯为6 μmol/L，化合物1为2.6 μmol/L，化合物1表现出最高的光毒性。然而，这些结果具有立体特异性，因为相应的D-对映体Ac-D-Phe-ALA-Me（化合物2）既不诱导卟啉合成也不诱导光毒性。当细胞在低温下与化合物1孵育时，PpIX水平显著降低，这与主动转运一致。在猪皮肤外植体中，化合物1诱导的卟啉荧光比ALA高3倍。这些结果表明，ALA的水溶性肽前药可大大增加其细胞摄取，产生更多的细胞内PpIX并改善肿瘤细胞的光致敏作用。这些衍生物在疗效上与5-氨基乙酰丙酸己酯相当，但毒性较小且在生理pH下更稳定。
