- Email: [email protected]
Skin penetration-enhancing effect of drugs by phonophoresis
journal of
controlled release
ELSEVIER
Journal of Controlled Release 37 (1995) 291-297
Skin penetration-enhancing effect of drugs by phonophoresis Hideo Ueda a, Kenji Sugibayashi a,b, Yasunori Morimoto a,b,. a Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-02, Japan b Life Science Research Center, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-02, Japan
Received 22 April 1994; accepted 13 June 1995
Abstract The skin penetration enhancement of nine drugs by phonophoresis was analyzed using ultrasonic irradiation at 150 kHz in in vitro skin permeation experiments conducted to elucidate the flux and permeability coefficient of drugs, and hydrodynamic parameters of skin. The flux of lipophilic drugs after sonication was similar to that before sonication, whereas that for hydrophilic drugs after sonication was increased 6.88-7.43-fold. Permeability coefficients of hydrophilic drugs through full thickness skin with ultrasound were closer to that through stripped skin without ultrasound, while that of lipophilic drugs was only slightly changed. A comparison of hydrodynamic parameters of skin with and without ultrasonication indicated an increase in the aqueous region of the stratum comeum and a constant pore size. Thus, ultrasonication has a great effect on the skin permeation of hydrophilic drugs which usually have low permeability. Keywords: Phonophoresis;Ultrasonication; Skin permeability; Hydrodynamicparameter, skin; Ultrasound; Transdermal
1. Introduction Phonophoresis has been identified as a skin penetration-enhancing method of drugs across the viable epidermis into the underlying tissues [ 1 ]. Phonophoresis has been used to deliver drugs into the systemic circulation, as have various chemical enhancers [ 2 - 4 ] and iontophoresis [5,6], and may be advantageous in pulsatile drug delivery. Although ultrasound has been used as a clinical treatment and in diagnosis [ 7 - 9 ] , the detailed mechanism for skin penetration enhancement is still unclear. Minimal information is available on the effects o f ultrasonic frequency, power, duty cycle and period of application on transdermal drug penetration. Recently, Bommannan et al. [ 10] reported the usefulness of high frequency ( 10 and 16 M H z ) irradiation to concentrate ultrasonic energy on the stratum cor* Corresponding author. 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10168-3659(95)00087-9
neum, the main barrier to whole skin permeation of drugs, in vivo. Electron microscopy indicated that the intercellular route of the stratum corneum was influenced by ultrasonication [ 11 ]. In the present study, the influence of ultrasound on skin was investigated in vitro. Flux and permeability coefficients of drugs, and hydrodynamic parameters of skin were determined using excised hairless rat skin. Nine drugs with different polarities were used in these experiments.
2. Materials and methods 2.1. E q u i p m e n t
A continuous output ultrasound generator (Dai-Ichi High-Frequency Co., Ltd., Tokyo, Japan) connected
292
H. Ueda et al. / Journal of Controlled Release 37 (1995) 291-297
generator ultrasoundtransducer-~=,,-~
I%'~mm donorcompartmer~~~/~
hairlessrat skin ----=,
-
--
~
star-headmagnet
~
synchronousmotor
Fig. 1. Schematic diagram of experimental apparatus.
to an ultrasonic transducer with 150 kHz frequency and an effective radiation area of 3.14 cm 2 was used. The power (2 W / c m z) from the generator decreased to 111 mW/cm 2 on skin [ 12]. Assembly of the apparatus is shown in Fig. 1.
2.2. Materials Ketoprofen (KP) and flurbiprofen (FP), isosorbide dinitrate (ISDN), and ibuprofen (IP) were supplied by Kaken Pharmaceutical Industries Co., Ltd. (Tokyo, Japan), Toko Pharmaceutical Industries Co., Ltd. (Tokyo), and Nisshin Chemical Co., Ltd. (Tokyo), respectively. Aminopyrine (AMP) and lidocaine (LC) were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan), antipyrine (ANP), cyclobarbital ( CB ) and 5-fl uorouracil ( 5-FU) from Tokyo Kasei Industries Co., Ltd. (Tokyo), deuterium oxide (D20) from Merck Co., Ltd. (Darmstadt, Germany), and FITC-dextran from Sigma Chemical Co., Ltd. (St. Louis, USA). Other chemicals and solvents were of reagent grade and obtained commercially.
2.3. Skin membrane preparation Full thickness hairless rat skin was freshly excised from the abdomen of male WBN/ILA-Ht strain rats weighing 160-180 g (7-8 weeks old, Life Science Research Center, Josai University, Saitama, Japan). Stripped skin (skin tape stripped 20 times to remove the stratum corneum) was also used for comparison.
2.4. Skin permeation experiment Excised abdominal hairless rat skin was mounted on a vertical diffusion cell (donor and receiver cell volume, 5 and 12.5 ml; effective diffusion area, 4.91 cm2). Although a drug suspension is necessary to maintain unit thermodynamic activity in the donor compartment, the drug concentration was adjusted to 50% of solubility in water to prevent absorption and reflection of ultrasound by drug particles. The receiver compartment was filled with water and stirred using a star-head magnetic bar driven by a constant speed motor (MC-301, Scinics, Tokyo) at 1200 rpm. The drug solution in the donor compartment was replaced every hour to minimize decreases in drug thermodynamic activity. The transducer tip of the ultrasound assembly was positioned 1 mm above the skin surface. The permeation experiment was performed at 32°C, but the temperature of donor solution rose to 35-36°C within 3 min during ultrasonic irradiation (data not shown). An adequate sample volume (0.5 or 5 ml) was withdrawn at predetermined times from the receiver compartment to measure drug concentrations; the same volume of fresh water was replaced after sampling to keep the volume constant. After reaching pseudo steady-state permeation (about 3-6 h after beginning the experiment), ultrasound was applied to the donor compartment for 1 h. After terminating the ultrasound, permeability was again measured, therefore, determination of permeation flux of drugs before, during and after ultrasonic irradiation was possible.
2.5. Determination of hydrodynamic parameters [13] Excised full thickness skin was mounted on the vertical diffusion cell at 32°C as in the skin permeation
293
H. Ueda et al. /Journal of Controlled Release 37 (1995) 291-297
experiment. 5 ml of ANP solution in D 2 0 ( 1 mg/ml) was placed into donor compartment, and an FITC-dextran (average molecular weight, 71 200) solution in water (20 ng/ml) added to the receiver compartment as a volume marker. The osmotic pressure was adjusted to 3.08, 0, and - 3 . 0 8 Osmol/l by addition of NaCI either to the donor or receiver compartment. Ultrasound was applied to the donor compartment for 1 h and the experimental setup was maintained for 12 h to assure the steady-state permeation of ANP. The donor and receiver solutions were then replaced with fresh solutions, and the permeation experiment without ultrasound was continued over 6 h. Finally, a sample was withdrawn from the receiver and assayed for ANP, D 2 0 , and FITC-dextran. Hydrodynamic parameters were measured as described previously [ 13].
2.6. Analytical methods
The concentrations of 5-FU, ANP, AMP, CB, 1SDN, LC, KP, IP and FP were analyzed by a high-performance liquid chromatography system equipped with a pump (LC-6A, Shimadzu, Kyoto, Japan), an ultraviolet spectrophotometric detector (SPD-6A, Shimadzu), a 4.6 mm × 250 mm stainless-steel column packed with Nucleosil 5C~8 (Macherey Nagel, Germany) and an integrator (C-R 6A, Shimadzu). Details were given previously [ 14]. DzO was quantified from the intensity of the O-D stretching vibrational band at 2512 cm -~ [15]. The absorbance of the sample in a calcium fluoride cell (0.025 mm thick) was determined with an infrared spectrophotometer (260-30, Hitachi, Tokyo). FITC-dextran was quantitated by fluorescence spectroscopy. Excitation was at 495 nm and the fluorescence intensity was measured at 515 nm (emission wavelength).
2.7. Statistical analysis
The fluxes with and without ultrasound irradiation were compared using the Student's t-test. Deviated values in Fig. 5 were rejected using the Smirnov test.
3. Results
3.1. Effect of ultrasound on the in vitro skin permeation of drugs Table 1 lists the physicochemical properties of drugs used in this experiment. All drugs were of low molecular weight with octanol/water partition coefficients (Kow) varying over 5 orders of magnitude [ 14]. Drugs with log Kow>-0 were designated lipophilic, those with log Kow < 0, hydrophilic. Table 2 shows the flux of drugs through hairless rat skin before, during and after ultrasonic irradiation. The flux during irradiation was higher than that before for every drug. Fig. 2a and b shows the time course of flux of a typical lipophilic drug, ISDN and a hydrophilic drug, ANP, respectively. The ISDN flux was significantly increased by ultrasonic irradiation (p <0.05, between 3 and 4 h), and the flux after irradiation recovered to a similar level as before irradiation (not significant, p = 0.05, between Table 1 Physicochemical properties of drugs used in this study
5-Fluorouracil (5-FU) Antipyrine (ANP) Aminopyrine (AMP) Cyclobarbital ( CB ) Isosorbide dinitrate (ISDN) Lidocaine (LC) Ketoprofen (KP) lbuprofen (IP) Flurbiprofen (FP)
MW ~ Log K~,,w
Cw ( m g / m l ) ~
130.1 188.2 236.1 236.3 236.1 234.3 254.3 206.3 244.3
12.2 634 62.1 3.07 0.972 3.03 0.17 0.051 0.027
- 1.70 -0.69 0.38 0.87 1.20 2.30 3.11 3.80 3.86
"Molecular weight. bLog octanol/water partition coefficient at 32°C. CSolubility in water at 32°C. Table 2 Fluxes of drugs through hairless rat skin before, during and after ultrasonic irradiation
at]' ~l~x
5-FU ANP AMP CB
ISDN LC
1.04 7.3 7.27
2.67 11.2 5.42
22.1 7.37 186 52.7 152 51.3
0.56 8.43 4.74
KP
IP
FP
21.7 0.83 1.42 0.65 66.9 2.64 3.23 1.40 43.8 2.12 1.55 0.99
"Average flux before irradiation. bMaximum flux during irradiation (/zg/cm 2 per h). CAverage flux after irradiation.
H. Ueda et al. / Journal of Controlled Release 37 (1995) 291-297
294
(a) ISDN
(b) ANP
20
300 OFF
OFF
OFF
OFF
15
"E
0) 10155 fl/ ,T 00 -- 2
200100
4 Time
(h)
8I
6
0
--
I
2
4 Time
(h)
6
8
Fig. 2. Effect of ultrasound on the flux of ANP and ISDN: (a) typical lipophilic drug ISDN and (b) hydrophilic drug ANP. Shadowed area shows the duration of ultrasonic irradiation. Each data point represents the mean + SE of 3 experiments.
3 and 8 h). The ANP flux was also increased by ultrasonic irradiation (p < 0.05, between 3 and 4 h), however, the flux after irradiation remained higher than that before irradiation (p < 0.05, between 3 and 8 h). Thus the extent of recovery of ANP was lower than ISDN. Comparison of flux recovery for each drug suggests that it depends on the hydrophilicity or lipophilicity of the drug. These results also suggest that the effect of ultrasound may be both reversible and irreversible. The enhancing ratios during and after ultrasound irradiation were defined as Emax (the maximum enhancing ratio) and Eirr (the irreversible enhancing ratio), respectively, and were calculated according to the following equations: Emax = J m a x / J l
( 1)
Eirr=J2/Ji
(2)
shows the E .... and E ~ for each drug. The Eir~and Ema x values of hydrophilic drugs were greater than for lipophilic drugs. The E .... of lipophilic drugs, however, was slightly higher or comparable to the Ein-, whereas the Emax of hydrophilic drugs was similar to the E~rr. In order to evaluate the irreversible effect, the permeability coefficients before and after irradiation were calculated by the following general equation:
P=J/Cv
(3)
where Cv is the concentration of drug in the donor solution and P is the permeability coefficient of the drug. As shown in Fig. 4, as drug lipophilicity increased, the permeability coefficients before and after ultrasound application became progressively more similar. At the hydrophilic end of the range the permeability after ultrasound was increased relative to that before ultrasound compared to lipophilic drugs. The permeability after ultrasound was thus closer to that of stripped skin compared to that before ultrasound.
where J~ and J2 are average flux before and after ultrasound irradiation, respectively, and Jmax is the maximum flux during ultrasound irradiation. Fig. 3a and b
(b) E i r r
(a) E m a x
10
1 .o
5 - FU ANP AMP CS ISDN LC
KP
IP
FP
0
5 - FU ANP AMP
CBISDNLC KP IP FP
Fig. 3. Enhancing ratios of drugs during and after ultrasonic irradiation.
295
H. Ueda et al. /Journal of Controlled Release 37 (1995) 291-297
Table 3 Hydrodynamic parameters and pore size of hairless rat skin
3.2. Measurement o f hydrodynamic parameters o f skin
Jo2o (/.d/h) It was assumed that drug permeation was influenced by the convective flow of solvent [ 16]. Permeation clearance of a drug (CLDrug, /zl/h) can then be expressed by the following equation [ 13]: (4)
CLDrug = CLL + CLr, + ( 1 - 8) Jsolvent
where C L L and CLp are the mean drug clearance through the lipid and pore domains, and Jsolvent and 8 are the solvent flux through the pore domain (/.~l/h) and the reflection coefficient of drug, respectively. In this experiment, CLANP and JD2o were calculated using A N P and D20 fluxes, and the 8 value was elucidated from the correlation between CLANP and JD~o to estimate the pore size of the stratum corneum. Jo~o, an index of aqueous region of the stratum corneum, was determined from the average JD~o under three osmotic
O
~-5 E ~-6
o
A
ZX
q-7
-9
i
i
i
i
i
i
5.300+ 0.558 0.274+ 0.221 0--0.627 22.427_+2.096 0.311+_0.218 04).660
us, ultrasonic irradiation. Each data represents the mean _+SE shown in Fig. 5. pressures. Fig. 5a and b shows the correlation between CLAN p and JD2O with and without ultrasonication, respectively. The pore size was calculated by the Levitt equation [ 17] using the slope in Fig. 5, ( 1 - 8) value. On average, JD20 increased about 4-fold with ultrasound irradiation with no significant difference observed in the pore size (Table 3). These results indicate that the barrier function of stratum corneum especially against hydrophilic compounds is markedly reduced by ultrasound.
4. D i s c u s s i o n
AA
i
Pore size (nm)
O©
O
ooo
Without US With US
1- 3
i
- 4 - 3 - 2 - 1 0 1 2 3 4 Log Kow
Fig. 4. Relationship between log P of drugs through hairless rat skin and log Kow. O, before ultrasonic irradiation; A, after ultrasonic irradiation; O, stripped skin.
The objective of the present study was to analyze the effect of ultrasound on skin permeation of drugs using physicochemical parameters of skin (i.e., permeation and hydrodynamics). Comparison of flux during and after ultrasonication showed that the effect of ultrasound was reversible in the permeation of lipophilic drugs, whereas it was irreversible in that of hydrophilic ones. The Eir~ (index of irreversible effect) of hydrophilic drugs was higher than that of lipophilic drugs. Ultrasound can increase skin permeability of drugs by
(a) with ultrasonic irradiation
(b) without ultrasonic irradiation 15
50 r
CLANP= 0.311 Jo2o+ 0.525
40 f
i
r = 0,755
30
,
20
t~
lO o
CLANp= 0.274 Jo2o+0.601 r = 0.653
12
6
3 0
10
20 30 Jo=o (pl/h)
40
50
o
0
I 3
I 6
I 9
I 12
I 15
JD2O(BI/h)
Fig. 5. Relationship between permeation clearance of ANP (CLANP) and flux of D20 (JDzo). Lines and equations were obtained by linear regression analyses. Q, 3.08 Osmol/l; A, 00smol/1; I , -3.08 Osmol/l.
296
14. Ueda et al. / Journal of Controlled Release 37 (1995) 291-297
way of either lowering the barrier properties of the stratum corneum or temperature increase, or both. Since the stratum corneum normally serves as a great barrier to hydrophilic compounds, a large Ei~ of hydrophilic drugs suggests that the barrier properties of stratum corneum decrease due to ultrasound. The thermal effect of ultrasound might be related to the increase in skin permeability of lipophilic drugs, so the Ei~r was low. It was found that the P value of hydrophilic drugs was much increased by ultrasonication compared to those of lipophilic drugs (Fig. 4). This figure may suggest an increase in the aqueous region in sonicated skin compared to that before sonication. Permeability of drugs used in the present study through full thickness skin without ultrasound was dependent on the Kow value, while the permeability through stripped skin was practically constant. If a part of the barrier function of the stratum corneum which consists of about 20 cornified cell layers is removed by ultrasound, the sonicated skin behaves like stripped skin. Thus, it would be considered that the barrier function of a few cornified layers in the stratum corneum disappeared with ultrasound, so that skin permeability of hydrophilic drugs was increased by ultrasound while that of lipophilic drugs was little increased. In order to confirm an increase in the aqueous region of skin, the hydrodynamic parameters of skin with and without ultrasound irradiation were compared. No significant difference was observed in the calculated pore size, whereas the JD_,O value was increased about 4-fold with ultrasound irradiation (Fig, 5). These results support that the barrier function of the stratum corneum was lowered by increases in the aqueous regions of skin by ultrasound. B o m m a n n a n et al. [ 1 1 ] reported that high frequency ultrasound with low intensity facilitated skin permeation of colloidal particles of lanthanum hydroxide via the intercellular route in the stratum corneum. Ultrasound used in the present study may also act on the intercellular region, especially stratum corneum lipids, and facilitate skin permeation of hydrophilic compounds. Detailed information for stratum corneum using some techniques such as IR, DSC and other studies could make it possible to discuss the mechanism of enhancement by ultrasound. Ultrasound increases temperature by cavitational and mechanical effects. These effects are essential components of ultrasonic energy [ 18,19 ]. Skin permeabil-
ity is increased by increases in temperature [20]. The ultrasonic apparatus used in this study raised the temperature in the donor compartment 3--4°C during ultrasonication (data not shown). This may account for the reversible effect accompanying ultrasound. It has also been reported that cavitational and mechanical effects are destructive of biological tissues and cells [21-23 ]. If so, this may account for the irreversible effect (i.e., a decrease in barrier function of stratum corneum).
Acknowledgements The authors would like to thank Dai-Ichi High-Frequency Co. Ltd. for supplying the ultrasonic equipment.
References [ 1] D.M. Skauenand G.M. Zentner,Phonophoresis,Int. J. Pharm. 20 (1984) 235-245. [2] B.W. Barry,Mode of actionof penetrationenhancersin human skin, J. Control. Release 6 (1987) 85-97. [3] K. Sugibayashi,K. Hosoya, Y. Morimoto and W.I. Higuchi, Effect of the absorptionenhancer,Azone, on the transport of 5-fluorouracilacross hairlessrat skin,J. Pharm. Pharmacol. 37 (1985) 578-580. [4] B.W. Barry and S.L. Bennet,Effect of penetrationenhancers on the permeation of mannitol, hydrocortisone and progesterone through human skin, J. Pharm. Pharmacol. 39 (1987) 535. [5] P. Tyle, Iontophoreticdevice for drug delivery,Pharm. Res. 3 (1986) 318-326. [6] A.K. Bangaand Y.W. Chien, Iontophoreticdeliveryof drugs: Fundamentals,developmentsand biochemicalapplication,J. Control. Release 7 (1988) 1-24. [7] H.A.E. Benson,J.C. McElnayand R. Harland,Phonophoresis of lignocaineand prilocainefrom Emla cream, Int. J. Pharm. 44 (1988) 65~59. [8] H.A.E. Benson, J.C. McElnay, R. Harland and J. Hadgraft, Influence of ultrasound on the percutaneous absorption of nicotinateesters, Pharm. Res. 8 ( 1991) 204-209. [9] R. Bruck, M. Nanavaty, D. Jung and F. Siegel, Effect of ultrasound on the in vitro penetration of ibuprofen through human epidermis, Pharm. Res. 8 (1989) 697-701. [10] D. Bommannan,H. Okuyama, P. Stanffer and R.H. Guy, Sonophoresis. I. The use of high-frequency ultrasound to enhancetransdermaldrug delivery,Pharm.Res. 9 (1992) 559564. [ 11] D. Bommannan,G.K. Menon, H. Okuyama, P.M. Elias and R.H. Guy,Sonophoresis.II. Examinationof the mechanism(s) of ultrasoundenhancedtransdermaldrugdelivery,Pharm.Res. 9 (1992) 1043-1047.
H. Ueda et al. /Journal of Controlled Release 37 (1995) 291-297
[ 12] Standards of Electronic Industries Association of Japan, AM29 ( 1987). [ 13 ] T. Hatanaka, E. Manabe, K. Sugibayashi and Y. Morimoto, An application of the hydrodynamic pore theory to percutaneous absorption of drugs, Pharm. Res. 11 (1994) 6 5 4 ~ 5 8 . [ 14] T. Hatanaka, M. Inuma, K. Sugibayashi and Y. Morimoto, Prediction of skin permeability of drugs. I. Comparison with artificial membrane, Chem. Pharm. Bull. 79 (1990) 34523459. [ 1 5 ] V . Thornton and F.E. Condon, Infrared spectrometric determination of deuterium oxide in water, Anal. Chem. 22 (1959) 690-691. [ 16] O. Kedem and A. Katchalsky, A physical interpretation of phenomenological coefficients of membrane permeability, J. Gen. Physiol. 45 ( 1961 ) 143-179. [ 17 ] D.G. Levitt, General continuum analysis of transport through pores, Biophys. J. 15 (1975) 533-551. [ 18 ] W.T. Coakley, Biophysical effects of ultrasound at therapeutic intensities, Physiotherapy 64 (1978) 166-169.
297
[ 19] L.A. Frizzell, Biological effects of acoustic cavitation, in: K.S. Suslick (Ed.), Ultrasound. Its Chemical, Physical, and Biological Effects, VCH, New York, 1988, pp. 287-303. [20] I.H. Blank, R.J. Scheuplein and D.J. McFarlane, Mechanism of percutaneous absorption: III. The effect of temperature on the transport of non-electrolytes across the skin, J. Invest. Dermatol. 49 (1967) 582-589. [21 ] P.R. Clarke and C.R. Hill, Physical and chemical aspects of ultrasonic disruption of cells, J. Acoust. Soc. Am. 47 (1970) 649~553. [ 22 ] P.G. Sacks, M.W. Miller and C.C. Church, The exposure vessel as a factor in ultrasonically induced mammalian cell lysis. I. Comparison of tube and chamber systems, Ultrasound Med. Biol. 8 (1982) 289-298. [23] C.C. Church, F.G. Flynn, M.W. Miller and P.G. Sacks, The exposure vessel as a factor in ultrasonically induced mammalian cell lysis. II. An explanation of the need to rotate exposure tube, Ultrasound Med. Biol. 8 (1982) 299-309.
controlled release
ELSEVIER
Journal of Controlled Release 37 (1995) 291-297
Skin penetration-enhancing effect of drugs by phonophoresis Hideo Ueda a, Kenji Sugibayashi a,b, Yasunori Morimoto a,b,. a Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-02, Japan b Life Science Research Center, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-02, Japan
Received 22 April 1994; accepted 13 June 1995
Abstract The skin penetration enhancement of nine drugs by phonophoresis was analyzed using ultrasonic irradiation at 150 kHz in in vitro skin permeation experiments conducted to elucidate the flux and permeability coefficient of drugs, and hydrodynamic parameters of skin. The flux of lipophilic drugs after sonication was similar to that before sonication, whereas that for hydrophilic drugs after sonication was increased 6.88-7.43-fold. Permeability coefficients of hydrophilic drugs through full thickness skin with ultrasound were closer to that through stripped skin without ultrasound, while that of lipophilic drugs was only slightly changed. A comparison of hydrodynamic parameters of skin with and without ultrasonication indicated an increase in the aqueous region of the stratum comeum and a constant pore size. Thus, ultrasonication has a great effect on the skin permeation of hydrophilic drugs which usually have low permeability. Keywords: Phonophoresis;Ultrasonication; Skin permeability; Hydrodynamicparameter, skin; Ultrasound; Transdermal
1. Introduction Phonophoresis has been identified as a skin penetration-enhancing method of drugs across the viable epidermis into the underlying tissues [ 1 ]. Phonophoresis has been used to deliver drugs into the systemic circulation, as have various chemical enhancers [ 2 - 4 ] and iontophoresis [5,6], and may be advantageous in pulsatile drug delivery. Although ultrasound has been used as a clinical treatment and in diagnosis [ 7 - 9 ] , the detailed mechanism for skin penetration enhancement is still unclear. Minimal information is available on the effects o f ultrasonic frequency, power, duty cycle and period of application on transdermal drug penetration. Recently, Bommannan et al. [ 10] reported the usefulness of high frequency ( 10 and 16 M H z ) irradiation to concentrate ultrasonic energy on the stratum cor* Corresponding author. 0168-3659/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD10168-3659(95)00087-9
neum, the main barrier to whole skin permeation of drugs, in vivo. Electron microscopy indicated that the intercellular route of the stratum corneum was influenced by ultrasonication [ 11 ]. In the present study, the influence of ultrasound on skin was investigated in vitro. Flux and permeability coefficients of drugs, and hydrodynamic parameters of skin were determined using excised hairless rat skin. Nine drugs with different polarities were used in these experiments.
2. Materials and methods 2.1. E q u i p m e n t
A continuous output ultrasound generator (Dai-Ichi High-Frequency Co., Ltd., Tokyo, Japan) connected
292
H. Ueda et al. / Journal of Controlled Release 37 (1995) 291-297
generator ultrasoundtransducer-~=,,-~
I%'~mm donorcompartmer~~~/~
hairlessrat skin ----=,
-
--
~
star-headmagnet
~
synchronousmotor
Fig. 1. Schematic diagram of experimental apparatus.
to an ultrasonic transducer with 150 kHz frequency and an effective radiation area of 3.14 cm 2 was used. The power (2 W / c m z) from the generator decreased to 111 mW/cm 2 on skin [ 12]. Assembly of the apparatus is shown in Fig. 1.
2.2. Materials Ketoprofen (KP) and flurbiprofen (FP), isosorbide dinitrate (ISDN), and ibuprofen (IP) were supplied by Kaken Pharmaceutical Industries Co., Ltd. (Tokyo, Japan), Toko Pharmaceutical Industries Co., Ltd. (Tokyo), and Nisshin Chemical Co., Ltd. (Tokyo), respectively. Aminopyrine (AMP) and lidocaine (LC) were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan), antipyrine (ANP), cyclobarbital ( CB ) and 5-fl uorouracil ( 5-FU) from Tokyo Kasei Industries Co., Ltd. (Tokyo), deuterium oxide (D20) from Merck Co., Ltd. (Darmstadt, Germany), and FITC-dextran from Sigma Chemical Co., Ltd. (St. Louis, USA). Other chemicals and solvents were of reagent grade and obtained commercially.
2.3. Skin membrane preparation Full thickness hairless rat skin was freshly excised from the abdomen of male WBN/ILA-Ht strain rats weighing 160-180 g (7-8 weeks old, Life Science Research Center, Josai University, Saitama, Japan). Stripped skin (skin tape stripped 20 times to remove the stratum corneum) was also used for comparison.
2.4. Skin permeation experiment Excised abdominal hairless rat skin was mounted on a vertical diffusion cell (donor and receiver cell volume, 5 and 12.5 ml; effective diffusion area, 4.91 cm2). Although a drug suspension is necessary to maintain unit thermodynamic activity in the donor compartment, the drug concentration was adjusted to 50% of solubility in water to prevent absorption and reflection of ultrasound by drug particles. The receiver compartment was filled with water and stirred using a star-head magnetic bar driven by a constant speed motor (MC-301, Scinics, Tokyo) at 1200 rpm. The drug solution in the donor compartment was replaced every hour to minimize decreases in drug thermodynamic activity. The transducer tip of the ultrasound assembly was positioned 1 mm above the skin surface. The permeation experiment was performed at 32°C, but the temperature of donor solution rose to 35-36°C within 3 min during ultrasonic irradiation (data not shown). An adequate sample volume (0.5 or 5 ml) was withdrawn at predetermined times from the receiver compartment to measure drug concentrations; the same volume of fresh water was replaced after sampling to keep the volume constant. After reaching pseudo steady-state permeation (about 3-6 h after beginning the experiment), ultrasound was applied to the donor compartment for 1 h. After terminating the ultrasound, permeability was again measured, therefore, determination of permeation flux of drugs before, during and after ultrasonic irradiation was possible.
2.5. Determination of hydrodynamic parameters [13] Excised full thickness skin was mounted on the vertical diffusion cell at 32°C as in the skin permeation
293
H. Ueda et al. /Journal of Controlled Release 37 (1995) 291-297
experiment. 5 ml of ANP solution in D 2 0 ( 1 mg/ml) was placed into donor compartment, and an FITC-dextran (average molecular weight, 71 200) solution in water (20 ng/ml) added to the receiver compartment as a volume marker. The osmotic pressure was adjusted to 3.08, 0, and - 3 . 0 8 Osmol/l by addition of NaCI either to the donor or receiver compartment. Ultrasound was applied to the donor compartment for 1 h and the experimental setup was maintained for 12 h to assure the steady-state permeation of ANP. The donor and receiver solutions were then replaced with fresh solutions, and the permeation experiment without ultrasound was continued over 6 h. Finally, a sample was withdrawn from the receiver and assayed for ANP, D 2 0 , and FITC-dextran. Hydrodynamic parameters were measured as described previously [ 13].
2.6. Analytical methods
The concentrations of 5-FU, ANP, AMP, CB, 1SDN, LC, KP, IP and FP were analyzed by a high-performance liquid chromatography system equipped with a pump (LC-6A, Shimadzu, Kyoto, Japan), an ultraviolet spectrophotometric detector (SPD-6A, Shimadzu), a 4.6 mm × 250 mm stainless-steel column packed with Nucleosil 5C~8 (Macherey Nagel, Germany) and an integrator (C-R 6A, Shimadzu). Details were given previously [ 14]. DzO was quantified from the intensity of the O-D stretching vibrational band at 2512 cm -~ [15]. The absorbance of the sample in a calcium fluoride cell (0.025 mm thick) was determined with an infrared spectrophotometer (260-30, Hitachi, Tokyo). FITC-dextran was quantitated by fluorescence spectroscopy. Excitation was at 495 nm and the fluorescence intensity was measured at 515 nm (emission wavelength).
2.7. Statistical analysis
The fluxes with and without ultrasound irradiation were compared using the Student's t-test. Deviated values in Fig. 5 were rejected using the Smirnov test.
3. Results
3.1. Effect of ultrasound on the in vitro skin permeation of drugs Table 1 lists the physicochemical properties of drugs used in this experiment. All drugs were of low molecular weight with octanol/water partition coefficients (Kow) varying over 5 orders of magnitude [ 14]. Drugs with log Kow>-0 were designated lipophilic, those with log Kow < 0, hydrophilic. Table 2 shows the flux of drugs through hairless rat skin before, during and after ultrasonic irradiation. The flux during irradiation was higher than that before for every drug. Fig. 2a and b shows the time course of flux of a typical lipophilic drug, ISDN and a hydrophilic drug, ANP, respectively. The ISDN flux was significantly increased by ultrasonic irradiation (p <0.05, between 3 and 4 h), and the flux after irradiation recovered to a similar level as before irradiation (not significant, p = 0.05, between Table 1 Physicochemical properties of drugs used in this study
5-Fluorouracil (5-FU) Antipyrine (ANP) Aminopyrine (AMP) Cyclobarbital ( CB ) Isosorbide dinitrate (ISDN) Lidocaine (LC) Ketoprofen (KP) lbuprofen (IP) Flurbiprofen (FP)
MW ~ Log K~,,w
Cw ( m g / m l ) ~
130.1 188.2 236.1 236.3 236.1 234.3 254.3 206.3 244.3
12.2 634 62.1 3.07 0.972 3.03 0.17 0.051 0.027
- 1.70 -0.69 0.38 0.87 1.20 2.30 3.11 3.80 3.86
"Molecular weight. bLog octanol/water partition coefficient at 32°C. CSolubility in water at 32°C. Table 2 Fluxes of drugs through hairless rat skin before, during and after ultrasonic irradiation
at]' ~l~x
5-FU ANP AMP CB
ISDN LC
1.04 7.3 7.27
2.67 11.2 5.42
22.1 7.37 186 52.7 152 51.3
0.56 8.43 4.74
KP
IP
FP
21.7 0.83 1.42 0.65 66.9 2.64 3.23 1.40 43.8 2.12 1.55 0.99
"Average flux before irradiation. bMaximum flux during irradiation (/zg/cm 2 per h). CAverage flux after irradiation.
H. Ueda et al. / Journal of Controlled Release 37 (1995) 291-297
294
(a) ISDN
(b) ANP
20
300 OFF
OFF
OFF
OFF
15
"E
0) 10155 fl/ ,T 00 -- 2
200100
4 Time
(h)
8I
6
0
--
I
2
4 Time
(h)
6
8
Fig. 2. Effect of ultrasound on the flux of ANP and ISDN: (a) typical lipophilic drug ISDN and (b) hydrophilic drug ANP. Shadowed area shows the duration of ultrasonic irradiation. Each data point represents the mean + SE of 3 experiments.
3 and 8 h). The ANP flux was also increased by ultrasonic irradiation (p < 0.05, between 3 and 4 h), however, the flux after irradiation remained higher than that before irradiation (p < 0.05, between 3 and 8 h). Thus the extent of recovery of ANP was lower than ISDN. Comparison of flux recovery for each drug suggests that it depends on the hydrophilicity or lipophilicity of the drug. These results also suggest that the effect of ultrasound may be both reversible and irreversible. The enhancing ratios during and after ultrasound irradiation were defined as Emax (the maximum enhancing ratio) and Eirr (the irreversible enhancing ratio), respectively, and were calculated according to the following equations: Emax = J m a x / J l
( 1)
Eirr=J2/Ji
(2)
shows the E .... and E ~ for each drug. The Eir~and Ema x values of hydrophilic drugs were greater than for lipophilic drugs. The E .... of lipophilic drugs, however, was slightly higher or comparable to the Ein-, whereas the Emax of hydrophilic drugs was similar to the E~rr. In order to evaluate the irreversible effect, the permeability coefficients before and after irradiation were calculated by the following general equation:
P=J/Cv
(3)
where Cv is the concentration of drug in the donor solution and P is the permeability coefficient of the drug. As shown in Fig. 4, as drug lipophilicity increased, the permeability coefficients before and after ultrasound application became progressively more similar. At the hydrophilic end of the range the permeability after ultrasound was increased relative to that before ultrasound compared to lipophilic drugs. The permeability after ultrasound was thus closer to that of stripped skin compared to that before ultrasound.
where J~ and J2 are average flux before and after ultrasound irradiation, respectively, and Jmax is the maximum flux during ultrasound irradiation. Fig. 3a and b
(b) E i r r
(a) E m a x
10
1 .o
5 - FU ANP AMP CS ISDN LC
KP
IP
FP
0
5 - FU ANP AMP
CBISDNLC KP IP FP
Fig. 3. Enhancing ratios of drugs during and after ultrasonic irradiation.
295
H. Ueda et al. /Journal of Controlled Release 37 (1995) 291-297
Table 3 Hydrodynamic parameters and pore size of hairless rat skin
3.2. Measurement o f hydrodynamic parameters o f skin
Jo2o (/.d/h) It was assumed that drug permeation was influenced by the convective flow of solvent [ 16]. Permeation clearance of a drug (CLDrug, /zl/h) can then be expressed by the following equation [ 13]: (4)
CLDrug = CLL + CLr, + ( 1 - 8) Jsolvent
where C L L and CLp are the mean drug clearance through the lipid and pore domains, and Jsolvent and 8 are the solvent flux through the pore domain (/.~l/h) and the reflection coefficient of drug, respectively. In this experiment, CLANP and JD2o were calculated using A N P and D20 fluxes, and the 8 value was elucidated from the correlation between CLANP and JD~o to estimate the pore size of the stratum corneum. Jo~o, an index of aqueous region of the stratum corneum, was determined from the average JD~o under three osmotic
O
~-5 E ~-6
o
A
ZX
q-7
-9
i
i
i
i
i
i
5.300+ 0.558 0.274+ 0.221 0--0.627 22.427_+2.096 0.311+_0.218 04).660
us, ultrasonic irradiation. Each data represents the mean _+SE shown in Fig. 5. pressures. Fig. 5a and b shows the correlation between CLAN p and JD2O with and without ultrasonication, respectively. The pore size was calculated by the Levitt equation [ 17] using the slope in Fig. 5, ( 1 - 8) value. On average, JD20 increased about 4-fold with ultrasound irradiation with no significant difference observed in the pore size (Table 3). These results indicate that the barrier function of stratum corneum especially against hydrophilic compounds is markedly reduced by ultrasound.
4. D i s c u s s i o n
AA
i
Pore size (nm)
O©
O
ooo
Without US With US
1- 3
i
- 4 - 3 - 2 - 1 0 1 2 3 4 Log Kow
Fig. 4. Relationship between log P of drugs through hairless rat skin and log Kow. O, before ultrasonic irradiation; A, after ultrasonic irradiation; O, stripped skin.
The objective of the present study was to analyze the effect of ultrasound on skin permeation of drugs using physicochemical parameters of skin (i.e., permeation and hydrodynamics). Comparison of flux during and after ultrasonication showed that the effect of ultrasound was reversible in the permeation of lipophilic drugs, whereas it was irreversible in that of hydrophilic ones. The Eir~ (index of irreversible effect) of hydrophilic drugs was higher than that of lipophilic drugs. Ultrasound can increase skin permeability of drugs by
(a) with ultrasonic irradiation
(b) without ultrasonic irradiation 15
50 r
CLANP= 0.311 Jo2o+ 0.525
40 f
i
r = 0,755
30
,
20
t~
lO o
CLANp= 0.274 Jo2o+0.601 r = 0.653
12
6
3 0
10
20 30 Jo=o (pl/h)
40
50
o
0
I 3
I 6
I 9
I 12
I 15
JD2O(BI/h)
Fig. 5. Relationship between permeation clearance of ANP (CLANP) and flux of D20 (JDzo). Lines and equations were obtained by linear regression analyses. Q, 3.08 Osmol/l; A, 00smol/1; I , -3.08 Osmol/l.
296
14. Ueda et al. / Journal of Controlled Release 37 (1995) 291-297
way of either lowering the barrier properties of the stratum corneum or temperature increase, or both. Since the stratum corneum normally serves as a great barrier to hydrophilic compounds, a large Ei~ of hydrophilic drugs suggests that the barrier properties of stratum corneum decrease due to ultrasound. The thermal effect of ultrasound might be related to the increase in skin permeability of lipophilic drugs, so the Ei~r was low. It was found that the P value of hydrophilic drugs was much increased by ultrasonication compared to those of lipophilic drugs (Fig. 4). This figure may suggest an increase in the aqueous region in sonicated skin compared to that before sonication. Permeability of drugs used in the present study through full thickness skin without ultrasound was dependent on the Kow value, while the permeability through stripped skin was practically constant. If a part of the barrier function of the stratum corneum which consists of about 20 cornified cell layers is removed by ultrasound, the sonicated skin behaves like stripped skin. Thus, it would be considered that the barrier function of a few cornified layers in the stratum corneum disappeared with ultrasound, so that skin permeability of hydrophilic drugs was increased by ultrasound while that of lipophilic drugs was little increased. In order to confirm an increase in the aqueous region of skin, the hydrodynamic parameters of skin with and without ultrasound irradiation were compared. No significant difference was observed in the calculated pore size, whereas the JD_,O value was increased about 4-fold with ultrasound irradiation (Fig, 5). These results support that the barrier function of the stratum corneum was lowered by increases in the aqueous regions of skin by ultrasound. B o m m a n n a n et al. [ 1 1 ] reported that high frequency ultrasound with low intensity facilitated skin permeation of colloidal particles of lanthanum hydroxide via the intercellular route in the stratum corneum. Ultrasound used in the present study may also act on the intercellular region, especially stratum corneum lipids, and facilitate skin permeation of hydrophilic compounds. Detailed information for stratum corneum using some techniques such as IR, DSC and other studies could make it possible to discuss the mechanism of enhancement by ultrasound. Ultrasound increases temperature by cavitational and mechanical effects. These effects are essential components of ultrasonic energy [ 18,19 ]. Skin permeabil-
ity is increased by increases in temperature [20]. The ultrasonic apparatus used in this study raised the temperature in the donor compartment 3--4°C during ultrasonication (data not shown). This may account for the reversible effect accompanying ultrasound. It has also been reported that cavitational and mechanical effects are destructive of biological tissues and cells [21-23 ]. If so, this may account for the irreversible effect (i.e., a decrease in barrier function of stratum corneum).
Acknowledgements The authors would like to thank Dai-Ichi High-Frequency Co. Ltd. for supplying the ultrasonic equipment.
References [ 1] D.M. Skauenand G.M. Zentner,Phonophoresis,Int. J. Pharm. 20 (1984) 235-245. [2] B.W. Barry,Mode of actionof penetrationenhancersin human skin, J. Control. Release 6 (1987) 85-97. [3] K. Sugibayashi,K. Hosoya, Y. Morimoto and W.I. Higuchi, Effect of the absorptionenhancer,Azone, on the transport of 5-fluorouracilacross hairlessrat skin,J. Pharm. Pharmacol. 37 (1985) 578-580. [4] B.W. Barry and S.L. Bennet,Effect of penetrationenhancers on the permeation of mannitol, hydrocortisone and progesterone through human skin, J. Pharm. Pharmacol. 39 (1987) 535. [5] P. Tyle, Iontophoreticdevice for drug delivery,Pharm. Res. 3 (1986) 318-326. [6] A.K. Bangaand Y.W. Chien, Iontophoreticdeliveryof drugs: Fundamentals,developmentsand biochemicalapplication,J. Control. Release 7 (1988) 1-24. [7] H.A.E. Benson,J.C. McElnayand R. Harland,Phonophoresis of lignocaineand prilocainefrom Emla cream, Int. J. Pharm. 44 (1988) 65~59. [8] H.A.E. Benson, J.C. McElnay, R. Harland and J. Hadgraft, Influence of ultrasound on the percutaneous absorption of nicotinateesters, Pharm. Res. 8 ( 1991) 204-209. [9] R. Bruck, M. Nanavaty, D. Jung and F. Siegel, Effect of ultrasound on the in vitro penetration of ibuprofen through human epidermis, Pharm. Res. 8 (1989) 697-701. [10] D. Bommannan,H. Okuyama, P. Stanffer and R.H. Guy, Sonophoresis. I. The use of high-frequency ultrasound to enhancetransdermaldrug delivery,Pharm.Res. 9 (1992) 559564. [ 11] D. Bommannan,G.K. Menon, H. Okuyama, P.M. Elias and R.H. Guy,Sonophoresis.II. Examinationof the mechanism(s) of ultrasoundenhancedtransdermaldrugdelivery,Pharm.Res. 9 (1992) 1043-1047.
H. Ueda et al. /Journal of Controlled Release 37 (1995) 291-297
[ 12] Standards of Electronic Industries Association of Japan, AM29 ( 1987). [ 13 ] T. Hatanaka, E. Manabe, K. Sugibayashi and Y. Morimoto, An application of the hydrodynamic pore theory to percutaneous absorption of drugs, Pharm. Res. 11 (1994) 6 5 4 ~ 5 8 . [ 14] T. Hatanaka, M. Inuma, K. Sugibayashi and Y. Morimoto, Prediction of skin permeability of drugs. I. Comparison with artificial membrane, Chem. Pharm. Bull. 79 (1990) 34523459. [ 1 5 ] V . Thornton and F.E. Condon, Infrared spectrometric determination of deuterium oxide in water, Anal. Chem. 22 (1959) 690-691. [ 16] O. Kedem and A. Katchalsky, A physical interpretation of phenomenological coefficients of membrane permeability, J. Gen. Physiol. 45 ( 1961 ) 143-179. [ 17 ] D.G. Levitt, General continuum analysis of transport through pores, Biophys. J. 15 (1975) 533-551. [ 18 ] W.T. Coakley, Biophysical effects of ultrasound at therapeutic intensities, Physiotherapy 64 (1978) 166-169.
297
[ 19] L.A. Frizzell, Biological effects of acoustic cavitation, in: K.S. Suslick (Ed.), Ultrasound. Its Chemical, Physical, and Biological Effects, VCH, New York, 1988, pp. 287-303. [20] I.H. Blank, R.J. Scheuplein and D.J. McFarlane, Mechanism of percutaneous absorption: III. The effect of temperature on the transport of non-electrolytes across the skin, J. Invest. Dermatol. 49 (1967) 582-589. [21 ] P.R. Clarke and C.R. Hill, Physical and chemical aspects of ultrasonic disruption of cells, J. Acoust. Soc. Am. 47 (1970) 649~553. [ 22 ] P.G. Sacks, M.W. Miller and C.C. Church, The exposure vessel as a factor in ultrasonically induced mammalian cell lysis. I. Comparison of tube and chamber systems, Ultrasound Med. Biol. 8 (1982) 289-298. [23] C.C. Church, F.G. Flynn, M.W. Miller and P.G. Sacks, The exposure vessel as a factor in ultrasonically induced mammalian cell lysis. II. An explanation of the need to rotate exposure tube, Ultrasound Med. Biol. 8 (1982) 299-309.