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EXAMPLE 2 In Vivo Enhancement of Skin Permeability The effect of ultrasound energy on skin penetration of D-.sup.3 H-mannitol and .sup.3 H-inulin in rats and mice in vivo was determined. Sprague-Doweley (CD) male rats (weighing 200-250 g each) and hairless mice were used in this example. The conditions were chosen so as to minimize external skin damage. An ultrasonic therapy unit, Burdick UT420A, approved for use in the treatment of human and animal tissue to facilitate healing through a combination of mechanical and thermal effects, was used for the in vivo studies. Test solutions of 5 to 20 microliter D-.sup.3 H-mannitol were applied to a shaven site in the upper back of the rats, followed by an aquasonic gel. Surface changes in skin from both in vivo and in vitro experiments were investigated by electromicroscopy technique. For scanning electron microscopy (SEM) examination, pieces of dorsal skin from the mice and rats were fixed with 2% glutaraldehyde, followed by 1% osmium tetroxide, dehydrated in a series of ascending alcohol concentrations, followed by critical point drying and examined with the electron microscope. Measurement of the amount of drug excreted in the urine was determinative of the rate of permeation in in vivo studies. Urine was collected via a catheter inserted into the bladder. The incision was stitched closed and the rat put in a restrainer. Test solutions of microl. .sup.3 H-D-mannitol and 12.5-50 microl. .sup.3 H-inulin were applied to the skin, followed by ultrasound application. Urine was collected every 15-30 minutes for the first 2 hours and at several time points afterwards. Volume and radioactivity of each urine sample was measured. Urine samples of ultrasound treated and control rats were compared by thin layer chromatography on silica plates developed in t-butanol:pyridine:water (6:4:3) visualized by phosphomolybdic acid reagent. The developed silica gel plate was cut into several sections according to the distance migrated by the spots relative to each other (R.sub.f) and the pieces placed in scintillation vials. One milliliter of distilled water was added to each vial to dissolve the drug. 10 ml of Aquasol.TM. (New England Nuclear, Boston, MA) scintillation liquid was added, and the radioactivity measured. FIG. 3 compares excretion of .sup.3 H-D-mannitol after topical application of 20 microliter radiolabeled .sup.3 H-D-mannitol, followed immediately by ultrasound treatment at 1.5-2.0 W/cm.sup.2 for 2-3 minutes, with excretion by control rats (no ultrasound). The ultrasound treated rats had 20 times more radioactivity in their urine during the first two hours than did the control rats. The effect of the ultrasound decreased with time. After 10 hours the radioactivity in the urine of the two groups was indistinguishable. The difference in release rate of .sup.3 H-D-mannitol was observed only if the ultrasound treatment was applied immediately after drug application. When the rats were treated with ultrasound, 10 or more hours after drug application, no change in the release rate was observed. Mannitol is totally and rapidly eliminated from the body and therefore appears completely in the urine. The effect of ultrasound on the permeating molecules as evaluated by thin layer chromatography indicated no decomposition of the mannitol by the ultrasonic energy. FIG. 4 compares the percentage of excreted [.sup.3 H] at a specific time interval to total amount of radioactivity excreted over 10 hours following topical application of both .sup.3 H-D-mannitol and .sup.3 H-inulin in rats. This was used as a measure of the relative drug penetration. In the ultrasound treated rats the percentages of [.sup.3 H] excreted during the first two hours were 20% to 30% for .sup.3 H-D-mannitol and 50% for .sup.3 H-inulin as compared to 2-5% for .sup.3 H-D-mannitol and 10% for .sup.3 H-inulin in the respective controls. As shown by Table I, the ratio of relative drug permeation, R, is defined as: ##EQU1## where p.sub.us is the percentage of [.sup.3 H] in urine of ultrasound treated rats at a time, t, divided by the total amount of [.sup.3 H] excreted in the urine over 10 hours. p.sub.ct is the percentage of [.sup.3 H] in the urine of untreated rats at a time, t, divided by the total amount of [.sup.3 H] excreted by the rats over a period of ten hours. As can be seen from Table I, skin permeation for both drugs was 6 to 20 fold greater in ultrasound treated rats during the first 2 hours than that of the corresponding control group. TABLE I ______________________________________ Ratio of relative drug permeation in rats time D-.sup.3 H--mannitol .sup.3 H--inulin (hours) 20 mCi 10 mCi 50 mCi ______________________________________ 2 19 19 6 5 2.5 1.5 1.5 10 1 1 1 ______________________________________ FIG. 5 shows the relative D-mannitol permeation in mice after the application of 5 microl. .sup.3 H-D-mannitol followed immediately by ultrasound treatment at 1-1.5 W/cm.sup.2 for 1 min. The results are expressed as the percentage of [.sup.3 H] excreted at a specified time divided by the total [3H] excreted over ten hours. There was no change in the percentage of excreted radioactivity when the ultrasound treatment was applied 10 hours after drug application. These examples demonstrate that the transdermal permeation of .sup.3 H-D-mannitol and .sup.3 H-inulin in rats is increased 6-20 fold within the first two hours of ultrasound treatment. This increase was observed only during the initial phase of the transdermal permeation, before establishment of a steady state. The fact that the ultrasound energy shortened the lag time for transdermal permeation of D-mannitol and inulin in rats, and did not change the drug release rate during the steady-state phase, suggests that the ultrasound treatment enhanced the diffusion of the administered drugs through an alternative pathway such as the hair follicles in the rat's skin, rather than diffusion through the bulk of the stratum coreum. Intensities of less than 5 W/cm.sup.2 of ultrasound at a frequency of 1 MHz do not result in a significant temperature rise nor have any destructive effect on viable mouse or rat skin. Skin temperature did not increase more than 1.degree. C. when ultrasound energy was applied for less than 3 minutes to rats at less than 3 W/cm.sup.2 or 1 minute to mice at 1.5 W/cm.sup.2. Longer treatment sometimes caused skin burns. In summary, the examples using skin from two species, both in vivo and in vitro, demonstrate that ultrasound is an effective means of enhancing the passage of molecules through skin and into the circulatory system. Frequencies, power and time of application can be optimized to suit the individual situation, both with respect to the type of skin and the molecules to be diffused. Although this invention has been described with reference to specific embodiments, it is understood that modifications and variations of the method for using ultrasound energy to enhance passage of molecules into and through skin may occur to those skilled in the art. It is intended that all such modifications and variations be included within the scope of the appended claims. |
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