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In vitro investigations of drug release and penetration — enhancing effect of ultrasound on transmembrane transport of flufenamic acid
Exp Toxic Patho11998; SO: 450-452 Gustav Fischer Verlag
IDepartment of Clinical Pharmacology and 2Department of Physiotherapy, Friedrich Schiller University Jena, Germany
In vitro investigations of drug release and penetration - enhancing effect of ultrasound on transmembrane transport of flufenamic acid * M. HIPPIUS', U. SMOLENSKI 2, CH UHLEMANN1, U. SCHREIBER1, and A. HOFFMANN i With 3 figures Address for correspondence: PD Dr. rer. nat. habil. Marion Hippius, Klinikum der Friedrich-Schiller-Universitat, Jena, Institut fiir Klinische Pharmakologie, D - 07740 Jena, Germany. Key words: Drug release; Drug penetration; Phonophoresis; Ultrasound, effect on transmembrane transport; Flufenamic acid.
Summary Percutaneous absorption studies are performed in various
in vitro models to determine the rate of drug absorption via
the skin. We designed an phonophoretic drug delivery system to investigate the influence of ultrasound on transmembrane transport of different drugs. Phonophoresis is defined as the migration of drug molecules, contained in a contact agent, through the skin under the influence of ultrasound. We investigated the absorption offlufenamic acid in a buffer medium in dependence of ultrasound energy and application time. For evaluating membrane penetration of flufenamic acid, the concentration range of buffer solution was measured. Flufenamic acid was determined by using a fluorimetric method. Ultrasound energy was supplied for between 5 and 30 min at a range of intensities (0; 0.3; 0.6; 0.9; 1.2; 1.5 W/cm 2). energy levels commonly used for therapeutic purpose. The pronounced effect of ultrasound on the transmembrane absorption of the drug was observed at all ultrasound energy level studied. The time of application was found to play an important role in delivery and transport of drug. Dependent on time, we observed an arise of temperature up to 4.5 o. It appears that there was no difference between an intensity of 0.3 and 1.5 W/cm2 and the measured drug concentrations in solution. The highest penetration was observed at an intensity of 1.0 W/cm 1 after 30 min. These results were not significantly different from concentration in measurements after 30 min and 0.5 and 1.5 W/cm2 • It seems that the arise of drug concentration is caused by effects of temperature and by variation of membrane delivery in dependence of temperature.
Introduction Topical application of drugs has focused attention on their skin permeability and on chemical permeability enhancers with decrease the barrier function of the stratum corneum. The major barrier to the delivery of transcutaneous drugs is the skin. Pharmaceutical companies are continually involved in research to try to find new ways to enhance the delivery of topical drugs. Although complex chemical enhancers have been integrated into some transdermal delivery systems, physical agents such as electricity and ultrasound are becoming increasingly popular as enhancers. The use of ultrasound as an enhancer is referred to as phonophoresis or sonophoresis [1]. Phonophoresis is defined as the migration of drug molecules, contained in a coupling or a contact agent, through the skin under the influence of ultrasound. Enhanced drug penetration is thought to result from the thermal, mechanical, and chemical alterations of biological tissues by ultrasonic waves. These waves penetrate up to 5 cm below the skin [2]. Although physical therapists and physicians often treat patients with topical antiinflammatory drugs enhanced with ultrasound, there is a paucity of research confirming that phonophoresis significantly enhances drug diffusion [3]. The aim of the study was to test an phonophore tic drug delivery system to investigate the influence of ultrasound on transmembrane transport of flufenamic acid.
Investigations and results
* Dedicated to Prof. Dr. WOLFGANG KLINGER on occasion of his 65th birthday on July 3, 1998. 450
Exp Toxic Pathol SO (1998) 4-6
Phonophore sis is administered in the same manner as ultrasound, except that medication is used in the coupling
agent. Phonophoresis is often applied via continuous ultrasound with an intensity between 1.0 and 2.0 W/cm2 , at a frequency in a range of 2-3 MHz, and with a treatment duration of 5-20 minutes. Ultrasound energy was supplied for between 5 and 30 min at a range of intensities (0; 0.3; 0.6; 0.9; 1.2; 1.5 W/cm 2), energy levels commonly used for therapeutic purpose. The duration of treatment, output frequency, and power level required for phonophoretic drug transport was investigated. 1. Influence of ultrasound intensity on temperature The pronounced effect of ultrasound on the transmembrane absorption of the drug was observed at all ultrasound energy levels studied (fig. 1).
5
The time of application was found to play an important role in delivery and transport of drug. Dependent on time, we observed an arise of temperature up to 4.5 o. 2. Influence of ultrasound intensity on the absorption of flufenamic acid It appears that there was no difference between an intensity of 0.2 and 1.5 W/cm 2 and the measured drug concentrations in solution (fig. 2). The highest penetration was observed at an intensity of 1.0 W/cm2 after 30 min. These results were not significantly different from concentration measurements after 30 min and 0.5 and 1.5 W/cm 2.
temperature difference [0C]
4
3
2
o
o
10
5
15
25
20
30
time [min]
Fig. 1. Influence of ultrasoud intensity on temperature
1.0.0 W/cm> ..0.2 W/cm' +(J.5 W/cm> +0.7 W/cm'
1.0 W/cm> -1 .5 W/cm>
ng/ml (thousand)
4
3
2
a Fig. 2. Influence of ultrasound intensity on the absorption of flufenamic acid
a
5
10
15
20
25
time [min] .0 W/cm' 0).5 W/cm' .1 .0 W/cm'
1.5 W/cm'
30
I
Exp Toxic Pathol 50 (1998) 4-6
451
4000
ng / ml
3500 3000 2500 2000 1500 1000 500 0 0.0
0.1
0.2
W/cm 2
0.3
0.4
1.....0 min +5 min ...10 min ..15 min *20 min +25 min +30 min
3. Influence oftime on the absorption of flufenamic acid We observed also an increase of drug concentration of flufenamic acid without ultrasonic treatment in dependence of application (fig. 3). It seemd that the arise of drug concentration was caused by effects of temperature and by variation of membrane delivery in dependence of temperature. The highest penetration of drug was noted after 30 minutes in case of the intensity of ultrasound of I.S W/cm2 •
Discussion The purpose oft this study was to determine the best conditions for enhancement induced by therapeutical ultrasound in an in vitro model. Percutaneous absorption studies are performed in various in vitro models to determine the rate of drug absorption via the skin. We designed an phonophoretic drug delivery system to investigate the influence of ultrasound on transmembrane transport of different drugs [4]. Drug application and ultrasound therapy each can act independently to control symptoms and aid recovery. Phonophoresis had been shown to enhance transdermal transport of various drugs [S]. Our study was performed to research into a better selection of ultrasoud of parameters to enhancement of transdermal flufenamic acid transport. The results of those model experiments will be investigated in men.
Experimental 1. In vitro phonophore sis experiments
We investigated the absorption of flufenamic acid through a nitrocellulose membrane (Serva) into a buffer 452
Exp Toxic Pathol50 (1998) 4-6
0.5
Fig. 3. Influence on time on the absorption of flufenamic acid
medium. (Tyrode's salts) in dependence of ultrasound energy and application time. Method and solution conditions of drug delivery system were as reported in our previous paper [4]. Flufenamic acid in a concentration of 10 % was incorporated in an ointment of Vaselinum album and Paraffinum subliquidum in equal parts. For evaluating membrane penetration of flufenamic acid, the concentration range of buffer solution was measured. Flufenamic acid was determined by using a modified fluorimetric method [6, 7]. Ultrasound energy was supplied for between Sand 30 min at a range of intensities (0; 0.3; 0.6; 0.9; 1.2; I.S W/cm2) energy levels commonly used for therapeutic purpose.
Literature 1. TYLE P, AGRAWALA P: Drug delivery by phonophoresis.
Ph arm Res 1989; 6: 355-361 2. QUILLIN WS: Phonophoresis: a review of the literature and technique. Athletic Training 1980; 15: 109-110 3. BENSON HAF, McELNAY JC, HARLAND R, HADGRAFf J: Influence of ultrasound on the percutaneous absorption of nicotinate esters. Pharmaceutical Research 1991; 8: 204-208 4. HIPPIUS M, UHLEMANN CH, SMOLENSKI U, et al.: Phonophorese von Salicylsaure in der Pharmakotherapie rheumati scher Erkrankungen. in: N. RIETBROCK: Die Haut als Transportorgan fur Arzneistoffe. Steinkopffverlag Darmstadt, 1990, S. 47-52 5. MENON, GK, BOMMANNAN DB, ELIAS PM: High-frequency sonophoresis: Permeation pathways and structural basis for enhanced permeability. Skin Pharmacol. 1994; 7: 130-139 6. DELL H-D, FIEDLER J, JACOBI H, WASCHE B: Zur Biochemie und Pharmakokinetik von Etofenamat, Arzneim.Forsch.lDrug Res. 1977; 27(1): 1322-1325 7. KHIER AA, EI-SADEK M, BARAKA M:Spectrophotometric method for determination of flufenamic and mefenamic acid. Analyst 1987; 112: 1399-1403
IDepartment of Clinical Pharmacology and 2Department of Physiotherapy, Friedrich Schiller University Jena, Germany
In vitro investigations of drug release and penetration - enhancing effect of ultrasound on transmembrane transport of flufenamic acid * M. HIPPIUS', U. SMOLENSKI 2, CH UHLEMANN1, U. SCHREIBER1, and A. HOFFMANN i With 3 figures Address for correspondence: PD Dr. rer. nat. habil. Marion Hippius, Klinikum der Friedrich-Schiller-Universitat, Jena, Institut fiir Klinische Pharmakologie, D - 07740 Jena, Germany. Key words: Drug release; Drug penetration; Phonophoresis; Ultrasound, effect on transmembrane transport; Flufenamic acid.
Summary Percutaneous absorption studies are performed in various
in vitro models to determine the rate of drug absorption via
the skin. We designed an phonophoretic drug delivery system to investigate the influence of ultrasound on transmembrane transport of different drugs. Phonophoresis is defined as the migration of drug molecules, contained in a contact agent, through the skin under the influence of ultrasound. We investigated the absorption offlufenamic acid in a buffer medium in dependence of ultrasound energy and application time. For evaluating membrane penetration of flufenamic acid, the concentration range of buffer solution was measured. Flufenamic acid was determined by using a fluorimetric method. Ultrasound energy was supplied for between 5 and 30 min at a range of intensities (0; 0.3; 0.6; 0.9; 1.2; 1.5 W/cm 2). energy levels commonly used for therapeutic purpose. The pronounced effect of ultrasound on the transmembrane absorption of the drug was observed at all ultrasound energy level studied. The time of application was found to play an important role in delivery and transport of drug. Dependent on time, we observed an arise of temperature up to 4.5 o. It appears that there was no difference between an intensity of 0.3 and 1.5 W/cm2 and the measured drug concentrations in solution. The highest penetration was observed at an intensity of 1.0 W/cm 1 after 30 min. These results were not significantly different from concentration in measurements after 30 min and 0.5 and 1.5 W/cm2 • It seems that the arise of drug concentration is caused by effects of temperature and by variation of membrane delivery in dependence of temperature.
Introduction Topical application of drugs has focused attention on their skin permeability and on chemical permeability enhancers with decrease the barrier function of the stratum corneum. The major barrier to the delivery of transcutaneous drugs is the skin. Pharmaceutical companies are continually involved in research to try to find new ways to enhance the delivery of topical drugs. Although complex chemical enhancers have been integrated into some transdermal delivery systems, physical agents such as electricity and ultrasound are becoming increasingly popular as enhancers. The use of ultrasound as an enhancer is referred to as phonophoresis or sonophoresis [1]. Phonophoresis is defined as the migration of drug molecules, contained in a coupling or a contact agent, through the skin under the influence of ultrasound. Enhanced drug penetration is thought to result from the thermal, mechanical, and chemical alterations of biological tissues by ultrasonic waves. These waves penetrate up to 5 cm below the skin [2]. Although physical therapists and physicians often treat patients with topical antiinflammatory drugs enhanced with ultrasound, there is a paucity of research confirming that phonophoresis significantly enhances drug diffusion [3]. The aim of the study was to test an phonophore tic drug delivery system to investigate the influence of ultrasound on transmembrane transport of flufenamic acid.
Investigations and results
* Dedicated to Prof. Dr. WOLFGANG KLINGER on occasion of his 65th birthday on July 3, 1998. 450
Exp Toxic Pathol SO (1998) 4-6
Phonophore sis is administered in the same manner as ultrasound, except that medication is used in the coupling
agent. Phonophoresis is often applied via continuous ultrasound with an intensity between 1.0 and 2.0 W/cm2 , at a frequency in a range of 2-3 MHz, and with a treatment duration of 5-20 minutes. Ultrasound energy was supplied for between 5 and 30 min at a range of intensities (0; 0.3; 0.6; 0.9; 1.2; 1.5 W/cm 2), energy levels commonly used for therapeutic purpose. The duration of treatment, output frequency, and power level required for phonophoretic drug transport was investigated. 1. Influence of ultrasound intensity on temperature The pronounced effect of ultrasound on the transmembrane absorption of the drug was observed at all ultrasound energy levels studied (fig. 1).
5
The time of application was found to play an important role in delivery and transport of drug. Dependent on time, we observed an arise of temperature up to 4.5 o. 2. Influence of ultrasound intensity on the absorption of flufenamic acid It appears that there was no difference between an intensity of 0.2 and 1.5 W/cm 2 and the measured drug concentrations in solution (fig. 2). The highest penetration was observed at an intensity of 1.0 W/cm2 after 30 min. These results were not significantly different from concentration measurements after 30 min and 0.5 and 1.5 W/cm 2.
temperature difference [0C]
4
3
2
o
o
10
5
15
25
20
30
time [min]
Fig. 1. Influence of ultrasoud intensity on temperature
1.0.0 W/cm> ..0.2 W/cm' +(J.5 W/cm> +0.7 W/cm'
1.0 W/cm> -1 .5 W/cm>
ng/ml (thousand)
4
3
2
a Fig. 2. Influence of ultrasound intensity on the absorption of flufenamic acid
a
5
10
15
20
25
time [min] .0 W/cm' 0).5 W/cm' .1 .0 W/cm'
1.5 W/cm'
30
I
Exp Toxic Pathol 50 (1998) 4-6
451
4000
ng / ml
3500 3000 2500 2000 1500 1000 500 0 0.0
0.1
0.2
W/cm 2
0.3
0.4
1.....0 min +5 min ...10 min ..15 min *20 min +25 min +30 min
3. Influence oftime on the absorption of flufenamic acid We observed also an increase of drug concentration of flufenamic acid without ultrasonic treatment in dependence of application (fig. 3). It seemd that the arise of drug concentration was caused by effects of temperature and by variation of membrane delivery in dependence of temperature. The highest penetration of drug was noted after 30 minutes in case of the intensity of ultrasound of I.S W/cm2 •
Discussion The purpose oft this study was to determine the best conditions for enhancement induced by therapeutical ultrasound in an in vitro model. Percutaneous absorption studies are performed in various in vitro models to determine the rate of drug absorption via the skin. We designed an phonophoretic drug delivery system to investigate the influence of ultrasound on transmembrane transport of different drugs [4]. Drug application and ultrasound therapy each can act independently to control symptoms and aid recovery. Phonophoresis had been shown to enhance transdermal transport of various drugs [S]. Our study was performed to research into a better selection of ultrasoud of parameters to enhancement of transdermal flufenamic acid transport. The results of those model experiments will be investigated in men.
Experimental 1. In vitro phonophore sis experiments
We investigated the absorption of flufenamic acid through a nitrocellulose membrane (Serva) into a buffer 452
Exp Toxic Pathol50 (1998) 4-6
0.5
Fig. 3. Influence on time on the absorption of flufenamic acid
medium. (Tyrode's salts) in dependence of ultrasound energy and application time. Method and solution conditions of drug delivery system were as reported in our previous paper [4]. Flufenamic acid in a concentration of 10 % was incorporated in an ointment of Vaselinum album and Paraffinum subliquidum in equal parts. For evaluating membrane penetration of flufenamic acid, the concentration range of buffer solution was measured. Flufenamic acid was determined by using a modified fluorimetric method [6, 7]. Ultrasound energy was supplied for between Sand 30 min at a range of intensities (0; 0.3; 0.6; 0.9; 1.2; I.S W/cm2) energy levels commonly used for therapeutic purpose.
Literature 1. TYLE P, AGRAWALA P: Drug delivery by phonophoresis.
Ph arm Res 1989; 6: 355-361 2. QUILLIN WS: Phonophoresis: a review of the literature and technique. Athletic Training 1980; 15: 109-110 3. BENSON HAF, McELNAY JC, HARLAND R, HADGRAFf J: Influence of ultrasound on the percutaneous absorption of nicotinate esters. Pharmaceutical Research 1991; 8: 204-208 4. HIPPIUS M, UHLEMANN CH, SMOLENSKI U, et al.: Phonophorese von Salicylsaure in der Pharmakotherapie rheumati scher Erkrankungen. in: N. RIETBROCK: Die Haut als Transportorgan fur Arzneistoffe. Steinkopffverlag Darmstadt, 1990, S. 47-52 5. MENON, GK, BOMMANNAN DB, ELIAS PM: High-frequency sonophoresis: Permeation pathways and structural basis for enhanced permeability. Skin Pharmacol. 1994; 7: 130-139 6. DELL H-D, FIEDLER J, JACOBI H, WASCHE B: Zur Biochemie und Pharmakokinetik von Etofenamat, Arzneim.Forsch.lDrug Res. 1977; 27(1): 1322-1325 7. KHIER AA, EI-SADEK M, BARAKA M:Spectrophotometric method for determination of flufenamic and mefenamic acid. Analyst 1987; 112: 1399-1403