Goto Norio, Morita Yutaka, Terada Katsuhide
Honjo Reserch Section Drug Development Technology Center, Customer Joy Department Eisai Japan, Eisai Co., Ltd.
Chem Pharm Bull (Tokyo). 2016;64(8):1092-8. doi: 10.1248/cpb.c15-00784.
The transfer of urea from a urea formulation to the stratum corneum varies with the formulation base and form, and impacts the formulation's therapeutic effect. Consequently, determining the amount of urea transferred is essential for developing efficient formulations. This study assessed a simple method for measuring the amount of urea accumulated in the stratum corneum. Conventional methods rely on labeling urea used in the formulation with radiocarbon ((14)C) or other radioactive isotopes (RIs), retrieving the transferred urea from the stratum corneum by tape stripping, then quantitating the urea. The handling and use of RIs, however, is subject to legal regulation and can only be performed in sanctioned facilities, so methods employing RIs are neither simple nor convenient. We therefore developed a non-radiolabel method "tape stripping-colorimetry (T-C)" that combines tape stripping with colorimetry (urease-glutamate dehydrogenase (GLDH)) for the quantitative measurement of urea. Urea in the stratum corneum is collected by tape stripping and measured using urease-GLDH, which is commonly used to measure urea nitrogen in blood tests. The results indicate that accurate urea measurement by the T-C method requires the application of 1400 mg (on hairless rats) of a 20% urea solution on a 50 cm(2) (5×10 cm) area. Further, we determined the amount of urea accumulated in the stratum corneum using formulations with different urea concentrations, and the time course of urea accumulation from formulations differing in the rate of urea crystallization. We demonstrate that the T-C method is simple and convenient, with no need for (14)C or other RIs.
尿素从尿素制剂转移至角质层的情况会因制剂基质和形态的不同而有所变化，进而影响制剂的治疗效果。因此，确定尿素的转移量对于开发高效制剂至关重要。本研究评估了一种测量角质层中累积尿素量的简单方法。传统方法依赖于用放射性碳（¹⁴C）或其他放射性同位素（RI）标记制剂中使用的尿素，通过胶带剥离从角质层中回收转移的尿素，然后对尿素进行定量。然而，RI的处理和使用受到法律法规的限制，且只能在经批准的设施中进行，因此采用RI的方法既不简单也不方便。我们因此开发了一种非放射性标记方法“胶带剥离比色法（T-C）”，该方法将胶带剥离与比色法（脲酶-谷氨酸脱氢酶（GLDH））相结合用于尿素的定量测量。角质层中的尿素通过胶带剥离收集，并使用脲酶-GLDH进行测量，脲酶-GLDH常用于血液检测中测量尿素氮。结果表明，通过T-C方法准确测量尿素需要在50 cm²（5×10 cm）的面积上对无毛大鼠涂抹1400 mg的20%尿素溶液。此外,我们使用不同尿素浓度的制剂确定了角质层中累积的尿素量，以及不同尿素结晶速率的制剂中尿素累积的时间进程。我们证明T-C方法简单方便，无需¹⁴C或其他RI。
