Anal. Chem. 1992, 64, 2661-2663
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Human in Vivo Percutaneous Absotptiometry Using the Laser-Photoacoustic Method Ryuichi Takamoto;*+ Ryujiro Namba,t Masahiro Matsuoka,t and Tsuguo Sawadat Safety & Analytical Research Center, Shiseido Company, Ltd., 1050 Nippa-cho, Kohoku-ku, Yokohama 223,Japan, and Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
INTRODUCTION The percutaneous absorptiometry by the PA method using an open-ended PA cell has been expected to be an easy and simple method for in vivo percutaneous absorption measurements. In particular, one advantage of this method is its applicability to humans in vivo. Thus, this advantageous capability has been studied in obtaining detailed information.lt2 However, conventional open-ended PA cells13 were not favorable for SIN and sensitivity measurements since a weak powder lamp such as Xe or Hg has been used as the light source. Further, PA signals were amplified by the Helmholtz resonator, which involves a question of usefulness when applied to human skin in vivo. This is because the PA cell itself is comparatively large on account of its inside structure. Therefore, it was difficult to detect small changes in the drug amount followed by percutaneous absorption at an optional site on skin. The site for applying these PA cells on human skin in vivo was determined but subject to the spot where the air in the cell is tightly sealed and hardly influenced by pulse. It is rather difficult to measure a t an optional site on skin with high sensitivity. We have tried to develop an open-ended PA cell that is capable of measuring at any optional site on human skin in vivo with ease and high sensitivity. The following points are carefully considered for achieving this goal. In order to improve the SIN and sensitivity, a laser was used as the light source and three functions of adjusting resonance, differentiation, and volume were provided. A new structure for the PA cell was designed by considering two points. One was that the functions worked efficiently to improve SIN with easy operation, while another was that its dimensions were as compact as possible. Furthermore, the cell body was made in a form that could be easily used. As a result of studying these points, a stick type portable double open-ended PA cell was developed. This PA cell was equipped with an optical fiber which guided the laser beam light source into the PA cell. At first, we developed the in vitro percutaneous absorptiometry combining the open-ended PA cell with a longitudinal diffusion cell. This system is able to measure the reduced amount of the drug on extracted skin using the PA cell and to simultaneously measure the amount of drug passed through the skin with absorbance in real time. It was directly identified that a transmission phenomenon such as percutaneous absorption was able to be evaluated by applying this newly developed open-ended PA cell.4 We tried showing this capability with human in vivo percutaneous absorptiometry using the portable double open-ended PA cell.
EXPERIMENTAL SECTION Figure 1 shows the construction of the portable double openended PA cell. It consists of the MC with an optical fiber and Shiseido Co., LM. The University of Tokyo. (1) Kolmel, K.; Sennhenn, B.; Giese, K. J.SOC.Cosmet. Chem. 1986, .?7. - . , R7.5-.1R.5. - . - - - -. (2) Giese, K.; Nicolaus, A,; Sennhenn, B.; Kolmel, K. Can. J. Phys. 1986,64, 1139-1142. (3) Poulet, P.; Chambron, J. J . Photoacoustics 1983, 1 (3), 329-346. +
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Diagram of a portable and double open-ended PA cell for in VIVO measurement. Figure 1.
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Diagram of In VIVOpercutaneous absorptlometry uslng the
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the RC. A microphone is screwed therein to control the volume in both P A cells. Volume and thickness of the air layer in the samplechamber in the PA cell are 0.1 cm3and 0.2 cm, respectively. The length of pipe connecting the sample chamber and the microprone chamber is 12 cm. These values were determined to cause a resonance at a frequency of several kilohertz which is hardly influenced by pulsation from skin and environmental noises. Before measuring, the microphone chamber in the MC was shifted to get the maximum amplification by resonance of the P A signal. The differential system is employed to minimize the influence of noise that causes the degeneracy of S/N and reproducibility. This means that SIN is improved by subtracting noise detected by the RC from the PA signal and noise detected by the MC which is irradiated with the laser beam. Controlling the capacity in the RC by shifting the microphone chamber then makes it possible to remove noise at the maximum. This is because the noise level detected by the RC approaches the MC noise level by manual controlling. Figure 2 shows an in vivo percutaneous absorptiometrysystem using the portable double open-ended PA cell. The Ar+ laser beam (wavelength488 nm, Spectra Physics, Model-164) used as the light source was modulated at 3.3 kHz, which is a resonance frequency to the PA cell, using the signal generator (NF, 1925) and the light modulator (Intra Action Corp., AOM-40). Next, it was led to the optical fiber (Mihubishi Rayon, SK-40, 1 mm in diameter X 1 m). The intensity of the light source was determined to have an output from the optical fiber mounted MC of 10mW (belowthe maximum permissibleexposure (MPE) on human skin). The P A signal detected by the double PA cell (MC and RC) was analyzed using a lock-in amplifier (NF, 5610A) and recorded using a chart recorder (Rika Denki Kogyo, NP0393). Shikonin ((S)-5,8-dihydroxy-2-(l-hydroxy-4-methyl-3-pentenyl)-l,4-naphthalenedione;C16H1605, Wako Pure Chemical, Reagent class, >95% ) that absorbs light of 488-nm wavelength) (4) Takamoto, R.;Namba, R.; Nakata, 0.; Sawada, T. Anal. Chem.
1990,62, 674-677.
0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
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Flgure 3. Comparison of SIN when applying the functions of this PA cell: (a) PA signal using only the MC applied the resonance model is used; (b) application of the resonance and the differential mode; (c) application of the resonance and the differential mode with adjusting the volume of the PA cell.
was used as the model sample. It has the effect of accelerating the healing of a wound. Shikonin ointment (3 % ) was prepared using a hydrocarbon base (Taisho Pharmaceutical, Plastibase) as the vehicle. Shikonin was dissolved in isopropyl alcohol (IPA) and mixed with the vehicle.
Figure 4. Photographshowing the measurement of in vivo percutaneous absorption of a human by the open-ended PA cell.
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RESULTS AND DISCUSSION S/Nof Portable Double Open-Ended PA Cell. The measurement of humans in vivo was performed by the apparatus using the double open-ended PA cell as shown in Figure 2. Direct data obtained by this method are shown in Figure 3. While the background signal amplitude was 4.5 pV with unirradiated skin, the P A signal detected from human skin was 35.5 pV. Using only the MC, the noise level was 3.4 pV and S/N was 10.4 by resonance (shown in (a) of Figure 3). Next, by applying the double P A cell, the noise level became 1.7 pV and S/N was 20.9 (shown in (b) of Figure 3). The S/N was about 2 times that of the former by means of differentiation. Additionally, the noise level detected by the RC was allowed to come close to that of the MC by controlling the capacity of the RC. The result indicated excellent values since noise was less than 0.3 pV and S/N was 118 (shown in (c) of Figure 3). It was found that the S/N was 10 times greater than that using only the MC. Further, measuring shikonin ointment applied to the forearm of a human in vivo using this laser-PA method made detection possible at a S/N of 12 with a 15% coefficient of variation, for 1.3 pg/cm2 of shikonin in ointment. These results suggested that the portable double open-ended PA cell was applicable for human in vivo percutaneous absorptiometry. Trial of Human in Vivo Percutaneous Absorptiometry. In vivo absorptiometry was attempted using 3% shikonin ointment applied to a human forearm. Figure 4 shows a photograph of the measurements. These measurements using the double P A cell were done every 10 min to avoid the acceleration of percutaneous absorption owing to thermal influence by irradiation with the laser. The time of measurement took 30 s by taking into account the functions of the lock-in amplifier as a signal analyzer. The S/N obtained under such conditionswas about 60. Figure 5 showsthe results repeated five times. When the PA signals obtained by applying only the vehicle were compared with that of 3% shikonin ointment measured under the same conditions, the former was nearly constant with time, while the latter indicated a 25% reduction in the initial PA signal after 100 min. It was confirmed that the reduction in the PA signal is not a phenomenon due to a chemical change or destruction of shikonin by irradiation. Such a change in the PA signal is considered to accompany the percutaneous absorption of shikonin. In addition, this result suggested that this method
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Time [min.] Flgure 5. PA signal versus time when measuring human In vivo percutaneousabsorption: (a) sample 3 % shikoninointment; (b) sample vehicle.
is available for simple human in vivo percutaneous absorptiometry. For the percutaneous absorptiometric evaluation, in vivo methods using a radioisotope (RI) and in vitro methods using a diffusion cell have generally been applied on animal skin. However, the most important informationwe require is human in vivo percutaneous absorption. Therefore, studying the difference in percutaneous absorption between human and animal is very important. We tried comparing human in vivo percutaneous absorption with that of hairless mice using this PA method. Under the same conditions as that of human beings, in vivo percutaneous absorptiometry was applied to hairless mouse. Figure 6 shows the time courses of reduced shikonin on the skin of a human and hairless mouse. The amount of shikonin reduced on skin after t hours is obtained from the difference between the initial PA signal (Q(0))and that after t hours ( Q ( t ) )which , is expressed by (Q(0)- Q(t)).For the case where shikonin was applied, these results showed that the in vivo absorbability of a hairless mouse is higher than that of a human. In addition, it is noteworthy that the rate of absorption of human in vivo advanced after 70 min. The significance of this interesting phenomenon is now clarified by studying the pharmaceutical approach.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
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Time [min. I Flguro 6. Comparlson of the percutaneous absorptlon of a human with that of a halrless mouse.
As a result, it was demonstrated that this laser-PA method is able to measure human percutaneous absorption in vivo.
CONCLUSION A portable double open-ended PA cell that is capable of measuring percutaneous absorption in vivo was developed. This open-ended PA cell, which is a resonance type, has the function of adjusting the volume of the PA cells (MC and RC). By operation of this function and differentiation of the PA signal from noise, a sensitivity of this PA method with 1order of magnitude superiority to that of the conventional open-ended PA cell was achieved. Shikonin ointment (37%)
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as the model sample was applied to humans and hairless mice, and the in vivo percutaneous absorptiometry was performed. As a result, the PA signal that seems to be related to percutaneous absorption of shikonin was obtained, which suggested that human in vivo percutaneous absorptiometry using this laser-PA method is feasible. A difference in percutaneous absorption of a human and a hairless mouse was demonstrated by this method. It is worth noting that these data obtained by treating the drug without a label and measuring under the same conditions were compared. In general, almost all methods of percutaneous absorptiometry have been used on animal skins, and it is expected that important information is obtained for research and development of percutaneous absorptiometry that more closely reflects the clinical conditions for humans in vivo. By using a laser beam, a highly sensitive analysis is feasible; also the improvement in resolving power can be expected during the depthwise analysis. A practical problem, yet unsolved with regard to using a laser beam as the light source, is the limitation in wavelength despite its high sensitivity, which restricts the number of drugs amenable to this technique. However, by using oscillating multiple Ar+ laser beams and using a nonlinear optical crystal wavelength conversion unit (Ascal, UVA-U, CW-UV lights of several wavelengths are attainable. Needless to say, for the case of applying a UV laser to human skin in vivo, safety must be seriously considered. RECEIVEDfor review July 6, 1992. Accepted July 23, 1992.
