Use of ultrasound to enhance percutaneous absorption of benzydamine.

Use of Ultrasound to Enhance Percutaneous Absorption of Benzydamine Phonophoresis is the movement of drugs through intact skin into soft tissue by ultrasonic perturbation. [1] The technique involves placing the topical formulation on the skin over the area to be treated and massaging the area with an ultrasonic source.

Phonophoresis was first reported by Fellinger and Schmid in 1954 when they administered ultrasound in conjunction with hydrocortisone ointment in the treatment of polyarthritis of the digital joints of the hand. [2] Griffin and Touchstone reported in vitro research on swine tissue demonstrating that cortisol could be driven into skeletal muscle and paravertebral nerve by ultrasound. [3,4] Griffin et al have compared ultrasonically driven hydrocortisone with a placebo in 102 patients. [5] In their study, 68% of those patients receiving hydrocortisone in conjunction with ultrasound exhibited a marked decrease in pain and a significant increase in range of motion, whereas only 28% of those patients receiving the placebo and ultrasound showed a marked decrease in pain and an increase in ROM. Kleinkort and Wood conducted a retrospective study of 285 patients treated with ultrasonically driven hydrocortisone that was applied as a 10% ointment or a 1% cream and concluded that the phonophoretically administered 10% hydrocortisone ointment provided an effective alternative for delivery of anti-inflammatory agents, thereby avoiding the discomfort of percutaneous injections. [6]

Over the past 30 years, numerous other clinical reports have been published concerning ultrasonic treatment in conjunction with the following drugs: phenylbutazone, [7] lidocaine, [8] Carbocaine [R], [9] tetracycline, [10] and interferon. [11] Because of poor study design, however, the currently available literature does not allow the assessment of the adequacy of the controls used, the sensitivity of the analytical methods, or the suitability of the procedures used for clinical evaluation. [1] Despite its use for over 30 years, few pharmacokinetic data are available quantifying the efficacy of phonophoresis.

McElnay and co-workers recently reported that ultrasound treatment led to enhanced percutaneous absorption of fluocinolone in healthy volunteer subjects in a double-blind, placebo-controlled, cross-over clinical trial. [12] This finding was in contrast to an earlier study that showed ultrasound did not significantly increase the percutaneous absorption of lidocaine. [13]

Because the technique has been alleged to enhance percutaneous penetration of numerous drugs, further controlled clinical studies are required into the effect of ultrasound on percutaneous absorption. The aim of this study was to investigate the effect of both pulsed- and continuous-output ultrasound on the percutaneous absorption of benzydamine from a gel base in healthy volunteer subjects in a double-blind, placebo-controlled clinical trial. Benzydamine is a nonsteroidal anti-inflammatory drug that has been shown to be active after both topical [14] and systemic administration [15] and to be relatively devoid of both local and systemic side effects. [16] Dell'Orto and Cristalle reported that a high concentration of benzydamine is maintained in inflamed tissues for a much longer period of time when the drug is administered topically as opposed to orally. [17] Benzydamine is widely used in the form of Difflam [R] cream (*1) (3% weight per weight [w/w] benzydamine hydrochloride) for the relief of symptoms associated with painful inflammatory conditions of the musculoskeletal system. [18] Because of properties of the drug and the widespread application of ultrasound therapy and topical benzydamine, both separately and in combination, by physical therapists and physicians, we chose benzydamine for this investigation.

This investigation was based on the hypothesis that if ultrasound did promote percutaneous absorption of benzydamine, there would be a significant change in the percentage of benzydamine absorbed after treatment with ultrasound. In addition, the depth of ultrasound penetration is related to frequency [19]; therefore, by including a range of ultrasound frequencies in the experimental protocol, this phenomenon can be investigated.

Method

Subjects

Ten healthy volunteer subjects (4 male, 6 female; aged 20-33 years) participated in the study. All subjects gave their written informed consent, and the study was approved by the University Ethics Committee of The Queen's University of Belfast.

Equipment and Materials

The ultrasound apparatus used in this study, Sonacel Multiphone II, (*2) has a range of frequencies (0.75, 1.5, and 3.0 MHz) and intensities (0.3-3.3 W/cm.sup.2.). Each frequency is produced by a separate ceramic transducer housed in an aluminum-alloy screw cap, which constitutes the ultrasound applicator. The ultrasound instrument provides both a continuous output and a 1:1 pulsed were (2 msec on, 2 msec off.)

We used a gel preparation (*1) that contained 3% w/w benzydamine hydrochloride. The transmission of ultrasound energy through the gel preparation was measured using a Medisonics precision power meter. (*1) Percentages of transmission relative to deionized, degassed water recorded for each frequency were 97% (0.75 MHz), 87% (1.5 MHz), and 139% (3.0 MHz). These data indicate that the gel provide a good coupling agent, allowing good transmission of ultrasonic energy between the apparatus and the subject's skin.

Pilot Study

Initial experimentation was carried out to develop a suitable experimental protocol, in particular to standardize the ultrasonic frequency-intensity combinations to be used, the duration of application of the ultrasound, and the contact time of the anti-inflammatory gel with the skin. A five-minute contact time of the gel on the forearm before the application of ultrasound was chosen for use in this study in an attempt to allow the drug to penetrate the outer layers of the skin before ultrasound application. The surface recovery method was used to assess percutaneous absorption of benzydamine. [20] A method designed to estimate back diffusion of absorbed drug, and therefore give an indication of the depth of drug penetration, was also developed.

Initial experimentation indicated that application of ultrasound at an intensity of 1.5 [W/cm.sup.2] was the maximum output that could be tolerated without discomfort by some volunteers. In investigating the effect of pulsed-output ultrasound, an intensity of 1.0 [W/cm.sup.2] was used because it was the maximum output obtainable from the generator at a frequency of 3.0 MHz (1:1 pulsed output).

Based on the initial results, the protocol shown below, involving investigation of both continuous- and pulsed-output ultrasound, was developed. The trial consisted of three treatment sessions, which were conducted seven days apart in a double-blind crossover fashion with the subjects acting as their own controls. Both forearms of each of the subjects were treated during each session. The treatments were fully randomized so that each subject received each of the six ultrasound treatment combinations (ie, 0.0, 0.75, 1.5, and 3.0 MHz continuous output at 1.5 W/cm.sup.2.; 0.0 and 3.0 MHz 1:1 pulsed output at 1.0 W/cm.sup.2.) over the three sessions.

Continuous-Output Ultrasound

Procedure

The two treatment periods for investigation of continuous-output ultrasound were as follows:

Session 1. The subjects washed their forearms with soap and water and then rinsed them well so that no residue of soap remained. A site on each forearm of each subject was marked using a circular stencil (5 cm diameter) and a ball-point pen, the ink of which was shown not to interfere with the benzydamine assay procedure. A portion of gel (approximately 2 g, but weighed accurately) was applied to the treatment site on the right forearm and left for a contact period of five minutes. The ultrasound applicator was then used to massage the area with a circular motion for a further five minutes. Treatment with the ultrasound head was randomized, with the ultrasonic generator being set to one of a range of four frequencies (0.0, 0.75, 1.5, or 3.0 MHz) at an intensity of 1.5 [W/cm.sup.2]. During the treatment periods, neither the administrator (RH) nor the subjects knew at which frequency-intensity combination the generator had been set. After the ultrasound treatment on the right arm, the remaining gel was carefully removed from the arm. It was weighed immediately together with the detachable ultrasound head using an Oertling LA164 balance (*3) (accurate to 1 mg). This weighing was performed to account for any gel adhering to the surface of the ultrasound head. A sample of the recovered gel (approximately 200 mg) was weighed accurately and dissolved in 10 mL of a solvent mixture (1:1 acetonitrile-to-water solution). Meanwhile, the subject rinsed the right forearm in water, and a 5-cm diameter petri dish containing 10 mL of isotonic phosphate buffer (pH 7.4) was placed over the treatment site and held in position using crepe bandage. The petri dish was left in place for 30 minutes to allow back diffusion of the absorbed drug and therefore give an indication of the depth of drug penetration.

The complete procedure was repeated with the left forearm using a different ultrasound frequency-intensity combination. Both gel and buffer samples were analyzed for benzydamine content using a high-performance liquid chromatography (HPLC) assay. [21]

Session 2. The procedure was identical to that for Session 1; however, each subject received treatment with each of the four frequencies.

Pulsed-Output Ultrasound

Procedure

The protocol for investigation of pulsed-output ultrasound (Session 3) was identical to the protocol for Session 1; however, pulsed-output ultrasound was used at an intensity of either 1.0 [W/cm.sup.2] (1:1 pulsed outout and a frequency of 3.0 MHz) of 0.0 [W/cm.sup.2] (control). Treatment was randomized with each subject receiving one treatment on each arm. The subjects and the administrator of the ultrasound were again unaware of the frequency-intensity combinations being used.

High-performance Liquid

Chromatography Analysis of

Benzydamine

The surface recovery method was used to assess percutaneous absorption of benzydamine. [20] The residual amount of drug in the formulation base after treatment and in the buffer used for estimation of back diffusion were determined using reversed-phase HPLC with ultraviolet (UV) detection at 305 nm. The equipment used was as follows: an Altex 110A HPLC pump, (*4) a Kontron MS1660 autosampler, (*5) a Spectroflow 773 variable-wavelength UV detector (*6) set to 305 nm (the measured wavelength of maximum UV absorbance [[lambda] max] of benzydamine hydrochloride in water), and a Shimadzu Chromatopac C-R3A data recorder-processor. (*7) The analytical column was a Novopak C.sub.18 (*8) (5-[micrometer] particle size) with 150- X 3.9-mm internal dimensions. Both the analytical column and the guard column (5 cm; packed with Pellicular ODS C.sub.18 (*9) were enclosed in a block heater (*4) and maintained at 30[degrees]C. The mobile phase (consisting of 62% acetonitrile, 37.5% water, 0.5% acetic acid, and 5 mmol sodium dodecyl sulphate) was filtered through a 0.5-[micrometer] membrane filter (*9) before use; the flow rate of the mobile phase was 0.9 mL/min. Indomethacin was used as the internal standard. Benzydamine gel samples were dissolved in 10mL of a solvent mixture (1:1 acetonitrile-to-water solution) and injected directly onto the column. An aliquot (0.1 mL) of 1 mg/mL indomethacin solution was added to each of the 10-mL buffer samples before injection directly onto the column. Under the assay conditions described, indomethacin and benzydamine had elution times of 3.2 and 5.0 minutes, respectively (Figure). The amount of benzydamine absorbed was expressed as a percentage of the amount applied, and the back-diffused drug was calculated as a percentage of the amount of drug absorbed (Tabs. 1, 2).

Data Analysis

An analysis of variance (ANOVA) was used to compare data for the continuous-output frequency-intensity combinations, and a paired t test was used to compare the results during use of pulsed-output ultrasound.

Results

The procedures were well tolerated by all subjects, and informal questioning indicated that the subjects and the physician (RH) who administered the ultrasound were indeed unaware as to which ultrasound frequency-intensity combination was being used. Statistical comparison of the results for continuous-output ultrasound treatment using an ANOVA revealed no significant differences (p [is greater than] .05) in absorption or back diffusion between control data and those for ultrasound use at differing frequencies (Tab. 1). Statistical comparison of the results for the 1:1 pulsed-output ultrasound treatment using the paired t test also revealed no significant differences (p [is greater than] 0.5) in drug absorption or back diffusion between control data and data obtained following ultrasound treatment (Tab. 2).

Discussion

The experimental protocol was developed specifically for this investigation based on previous studies [12, 13] and on initial experimentation. The aim of the five-minute contact time of the gel before the application of ultrasound was to help saturate the outer layers of the skin (stratum corneum) with benzydamine before ultrasound application. We anticipated that the ultrasound source should then be more effective at driving the drug through the stratum corneum to the underlying tissues. In actual phonophoretic treatment of patients with anti-inflammatory products, presoak periods are not used, although this procedure would make good biopharmaceutical sense. Further investigations are currently underway to determine the effect on phonophoresis of increasing the contact time before ultrasound application. It is possible that in multiple-application therapy on different days, ultrasound may drive in drug present in the outer layers of skin remaining from the previous application, especially where the drug is retained in the stratum corneum, as is the case with corticosteroids. [22]

The original hypothesis on which this investigation was based was that if ultrasound did promote percutaneous absorption of benzydamine, there would be a significant change in the percentage of benzydamine absorbed and recovered for treatments using ultrasound. The depth of ultrasonic penetration is related to frequency, with the proportion of the incident ultrasonic energy absorbed by tissue increasing as the frequency of the ultrasound is increased. [19] A low treatment frequency, therefore, results in ultrasound being absorbed over a relatively long distance, and the "sonic" energy will be deep penetrating. [19] We envisaged, if phonophoresis was successful, that application of continuous-output ultrasound at a frequency of 0.75 MHz would have resulted in higher drug absorption but in a low percentage of recovery (because of deep penetration), whereas treatment with a frequency of 3.0 MHz was expected to promote percutaneous absorption and give a high percentage of recovery (because of shallow penetration). The results, however, showed no significant differences between data for continuous-output ultrasound treatments versus no ultrasound treatment (p [is greater than] .05; Tab. 1).

The back-diffusion technique, used for the first time in this study, was found to be an inefficient method of estimating penetration of benzydamine, because less than 2% of the absorbed drug was recovered from the site of application. Further studies are required before this back-partitioning method could be recommended as a test of percutaneous absorption.

Heat production by the ultrasound source was noted in the form of a circle of hyperemia after treatment. It has been postulated that the thermal effects of ultrasound alone may account for any enhancement of percutaneous absorption that takes place. [23]

It has been shown by differential scanning calorimeter (DSC) studies that the mechanism of action of skin permeation enhancers (eg, azone and decylmethyl sulphoxide) is via disruption of the stratum corneum lipid structure. [24, 25] Investigations using a DSC to determine whether ultrasound increases lipid fluidity within the stratum corneum also are currently being undertaken.

The fact that ultrasound did not enhance the percutaneous absorption of benzydamine under these experimental conditions was supported by the results obtained for pulsed-output ultrasound. Although these results indicated a trend toward increased drug absorption in the presence of ultrasound, the data were not statistically (p [is greater than] .05; paired t test; Tab. 2) or drug recovery (p [is greater than] .05; Tab. 2).

Conclusions

Although there are several theoretical mechanisms by which ultrasound may influence percutaneous absorption, clearly the absorption of benzydamine was not affected in this study. The data in this study could be explained by the following hypotheses:

1. Ultrasound energy does not influence percutaneous absorption; that is, phonophoresis does not work.

2. Benzydamine in the present formulation has physical and chemical properties that make it unsuitable for enhanced percutaneous absorption by the phonophoretic effect.

Because phonophoresis has been alleged to enhance percutaneous penetration of numerous drugs, further controlled clinical studies are required into the effect of ultrasound on percutaneous absorption.

Acknowledgments

We acknowledge the assistance of Jane Whiteman. Thanks are also due to all the volunteers who took part in this investigation.

References

[1] Skauen KM, Zentner GM: Phonophoresis. International Journal of Pharmaceutics 20:235-245, 1984

[2] Fellinger K, Schmid J: Klinik und Therapie des Chronischen Gelenkhuematismus. Vienna, Austria, Maudrich, 1954, pp 549-552 (Austrian)

[3] Griffin JE, Touchstone JC: Ultrasonic movement of cortisol into pig tissues: I. Movement into skeletal muscle. Am J Phys Med 42:77-85, 1963

[4] Griffin JE, Touchstone JC: Low-intensity phonophoresis of cortisol in swine. Phys Ther 48:1336-1344, 1968

[5] Griffin JE, Echternach JL, Price RE, et al: Patients treated with ultrasonic driven hydrocortisone and with ultrasound alone. Phys Ther 47:594-601, 1967

[6] Kleinkort JA, Wood F: Phonophoresis with 1 percent versus 10 percent hydrocortisone. Phys Ther 55:1320-1324, 1975

[7] Brondolo W: Phenylbutazone with ultrasonics in some cases of arthrosynovitis of the knee. Archives of Orthopedics 73:532-540, 1960

[8] Novak EJ: Experimental transmission of lidocaine through intact skin by ultrasound. Arch Phys Med Rehabil 45:231-232, 1964

[9] Cameroy BM: Ultrasound enhanced local aneurnal of Orthopedics 8: 47, 1966

[10] Parikov VA: Injection of tetracycline into the tissues of cow udders by ultrasonics. Veterinariia 43:88-91, 1966

[11] Marmur RK, Shpak TN: Histologic and histochemic changes in the cornea in induced herpetic keratitis and its experimental therapy. Oftalmol Zh 35:170-174, 1980

[12] McElnay JC, Kennedy TA, Harland R: The influence of ultrasound on the percutaneous absorption of fluocinolone acetonide. International Journal of Pharmaceutics 40:105-110, 1987

[13] McElnay JC, Matthews MP, Harland R, et al: The effect of ultrasound on the percutaneous absorption of lignocaine. Br J Clin Pharmacol 20:412-424, 1985

[14] Catanese B, Facchini Y, Barillari G, et al: Serum levels of benzydamine following the topical use of this drug in gynaecology. Clin Exp Obstet Gynecol 7:84-88, 1980

[15] Schlag G, Kopera H, Stulemeijer SM et al: The anti-inflammatory effect of benzydamine hydrochloride demonstrated with a new clinical pharmacological method. Arzeimittelforschung 20:1725-1728, 1970

[16] Silverstrine B, Cioli V: Rassenga critica della proprieta farmacologiche della benzidamina ed osservazioni sui suoi impieghi ginecologici, Conv. Ginecologico Terme di Saturnia (GR), Italia 1977. Clinica Europea 17:9-22, 1968 (Italian)

[17] Dell'Orto R, Cristalle V: Clinical studies with benzydamine ointment in orthopaedics and traumatology. Clinica Europea 7:296-303, 1968 (translated from Italian)

[18] British National Formulary, Number 14, 1987

[19] Ter Haar G: Basic physics of therapeutic ultrasound. Physiotherapy 64:100-103, 1978

[20] Webster RC, Maibach HI: In vivo methods for percutaneous absorption measurements. In Bronaugh RL, Maibach HI (eds): Percutaneous Absorption. New York, NY, Marcel Dekker Inc, 1985, pp 245-250

[21] Benson HAE, McElnay JC: A high-performance liquid chromatography assay for the measurement of benzydamine hydrochloride in topical pharmaceutical preparations. J Chromatogr 394:395-399, 1987

[22] Vickers CFW: Existence of reservoir in the stratum corneum. Arch Dermatol 88:21-23, 1963

[23] Coakley WT: Biophysical effects of ultrasound at therapeutic intensities. Physiotherapy 64:166-169, 1978

[24] Goodman M, Barry BW: Differential scanning calorimetry (DSC) of human stratum corneum: Effect of zone. J Pharm Pharmacol 37(Suppl): 80P, 1985

[25] Goodman M, Barry BW: Action of skin permeation enhancers azone, oleic acid and decylmethyl sulphoxide: Permeation and DSC studies. J Pharm Pharmacol 38(Supply): 71P, 1986

(*1) Riker Laboratories, Loughborough, England, United Kingdom.

(*2 SCI Instruments Ltd, Hertfordshire, England, United Kingdom.

(*3) Oertling, Orpington, Kent, England, United Kingdom.

(*4) Anachem Ltd, Luton, Bedfordshire, England, United Kingdom.

(*5) Kontron Electrolab, London, England, United Kingdom.

(*6) Kratos Analytical Instruments, Urmstone, Manchester, England, United Kingdom.

(*7) Dyson Instruments Ltd, Tyne and Wear, England, United Kingdom.

(*8) Waters Associate, Millipore Corp, 34 Maple St, Milford, Ma 01757.

(*9) Millipore Ltd, Middlesex, England, United Kingdom.

H Benson, BSc, is a postgraduate research student, Department of Pharmacy, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn Rd, Belfast, BT9 7BL, Northern Ireland, United Kingdom.

J McElnay, PhD, MPSNI, is a lecturer in biopharmacy, Department of Pharmacy, The Queen's University of Belfast.

R Harland, MB, is Chief Medical Officer, University Health Service, The Queen's University of Belfast, 25 University Square, Belfast, BT7 1PB, Northern Ireland, United Kingdom.

Address correspondence to Dr McElnay at Department of Pharmacy, The Queen's University of Belfast, Medical Biology Centre, 97 Lisburn rd, Belfast, BT9 7BL, Northern Ireland, United Kingdom.

This article was adapted from a presentation at the 1986 British Pharmaceutical Conference, Jersey, Channel Islands, United Kingdom, September 22-24, 1986.

This article was submitted May 29, 1987; was with the authors for revision for 12 weeks; and was accepted June 8, 1988.