Title: Intradermal drug delivery by low-frequency sonophoresis (25 kHz)
Authors: Pietro SantoiannI, Massimiliano Nino, and Gabriella Calabro
Affiliations: Department of  Dermatology, University "Federico II" of Naples, Italy
Keywords: alopecia areata, melasma, solar lentigo, intradermal delivery, low-frequency sonophoresis (LFS), ultrasound. 
Citation: Dermatology Online Journal 10 (2): 24

Abstract: Ultrasound waves (US) have been proposed to facilitate the absorption of active compounds (transdermal delivery) stimulating some disaggregation of the horny layer and promoting convective movements within the epidermis. Drugs used for alopecia areata, melasma, and lentigo, although proved effective, have limited effects resulting from only partial penetration into the skin. This study has evaluated the efficacy of low-frequency sonophoresis (LFS) at 25 kHz produced by a sonicator apparatus for treatment of alopecia areata, melasma, and solar lentigo. Thirty patients affected by alopecia areata were treated by application of methylprednisolone or cyclosporine solution followed by LFS. In a case-control study 48 women with melasma and 48 with solar lentigo were also treated by depigmenting emulsion and LFS application. For alopecia areata after 36 applications with LFS and 3-month treatment, the results were 57 percent partial regrowth and 29 percent total with methylprednisolone, and 33 percent partial regrowth and 34 percent total when cyclosporine was used. For melasma and solar lentigines the results when the drug application was followed by LFS, were (after  twice-a-week application for 3 months) 75 percent complete depigmentation and 25 percent partial for melasma, 43 percent total regression and 57 percent partial for solar lentigo. <br><br><I>Conclusion:</I> This is the first report of sonophoresis at a frequency of 25 kHz in dermatocosmetology. The study shows that LFS, a not aggressive technique, enhance penetration of topic agents obtaining effects at the level of the epidermis, dermis and appendages (intradermal delivery), giving better results in the treatment of some cosmetic skin disorders.
Body: 
I: Introduction
Corticosteroids are used successfully for the treatment of alopecia areata. Azelaic and kojic acids have been shown to have some depigmenting activity for melasma and solar lentigo. Biodisposability of most topical formulations is extremely low (about 1-5 % of applied dose) and, ineffective drug delivery into the skin may lead to poor results.
Experimental studies have shown that ultrasound waves (US)  bring about a  disaggregation of the horny layer and stimulate convective movements within the epidermis, facilitating the absorption of the active compounds (transdermal delivery) [1, 2]. To potentiate the epidermal penetration of the drugs used the study aimed to prove the efficacy of an enhancing delivery system with a sonicator apparatus using low-frequency sonophoresis (LFS) at 25 kHz to obtain intradermal delivery.

I: Methods
US waves (25 kHz) were obtained by a sonicator apparatus with a power intensity ranging from 50 to 100 mW/cm<sup><small>2</small></sup>.  
	A:  Alopecia areata
Methylprednisolone ointment and cyclosporine solution were used for treatment of alopecia areata.
Thirty patients (median age, 23.6 years) were divided into two subgroups: group A treated by methylprednisolone plus LFS; group B by cyclosporine plus LFS. The drug was applied three times a week for a 3-month period. Prior to drug application a superficial peeling by US waves was performed for 10 minutes, and methylprednisolone in a water-in-oil vehicle was applied (at 0.05 mg/cm<sup><small>2</small></sup>); cyclosporine dissolved in seed-oil was applied at 5 mg/cm<sup><small>2</small></sup>. At the beginning and at the end of treatment usual laboratory tests were performed. Cyclosporinemia was evaluated at day 0, and at days 30 and 60 and at the end of the treatment. Arterial pressure was monitored, and HPLC analysis showed that US waves treatment does not induce cyclosporine degradation or structural modifications.
	A:  Melasma and solar lentigo
A depigmenting emulsion containing ascorbic, azelaic, and kojic acids was used for the treatment of 48 women with melasma and for 48 with solar lentigines. Each group of patient was divided into three subgroups: group A treated by emulsion plus LFS; group B by depigmenting emulsion only; group C by placebo plus LFS. 
Patients underwent two weekly treatments for five consecutive weeks (average ten  applications). The skin color was converted into numeric code (by a spectrocolorimeter X-Rite 968) and results measured by an increase in the luminosity index (L).
I: Results 

	A:  Alopecia areata
	
	
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Results of patients treated with Methylprednisolone  (Group A).

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Treatment by  methylprednisolone plus application of US waves: Before treatment (Fig. 2). After 2 months treatment (Fig. 3).


figure 4: 4.gif
after 3-months treatment
	
	
Both methylprednisolone and cyclosporine in combination with US have shown remarkable hair regrowth. The results obtained by methylprednisolone plus LFS were as follows: 57 percent partial regrowth and 29 percent total (Fig. 1). Figures 2-4 show clinical results in a patient with partial regrowth after 2 months' and total after 3-months' treatment (36 applications). Results by cyclosporine plus LFS were 33 percent partial regrowth and 34 percent total (Fig. 5); Figures 6-8 show clinical results in a patient with total regrowth after 3 months. No patient showed arterial pressure modifications or blood-test  alterations. Only two patients presented a limited increase of cyclosporinemia (220 ng/ml -N. V. 20-200 ng/ml). 

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Results of patients treated with cyclosporine (Group B). 


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Treatment by  cyclosporine plus application of US waves: results in a patient.  Before treatment (Fig. 6). after 1 month of treatment (Fig. 7).

figure 8: 8.jpg
After 3 months' treatment.


	A:  Melasma and solar lentigo

The results showed for melasma and lentigo a good degree of depigmentation, compared to controls (group B and C). The more remarkable results were obtained for melasma treated with emulsion and LFS (group A): three cases out of four obtained complete depigmentation (Figs. 9, 10), and one only partial. Depigmentation occurred to a lesser extent in group B (total in 25 percent and partial in 75 percent), and in group C (total depigmentation in 81 percent and partial in 19 percent). 

figures 9 and 10: 9.jpg, 10.jpg
Treatment by emulsion  plus US waves: results in a patient with melasma (group A). Before treatment (Fig. 9). After 5 weeks' treatment (Fig. 10). 


For solar lentigo, in group A total remission was obtained for 43 percent patients and partial regression for 57 percent (Figs. 11, 12). In group B, total depigmentation occurred in 12 percent of cases, partial in 50 percent; in the remaining 38 percent no result was achieved. Fifty percent of patients in group C experienced a very limited regression; no benefit was achieved for the other 50 percent. 

figures 11 and 12: 11.jpg, 12.jpg
Treatment by emulsion  plus US waves: results in a patient with solar lentigo (group A). Before treatment (Fig. 11). After 5 weeks' treatment (Figure 12). 


I: Conclusion
Effects induced by US at 25 kHz include micromechanical, thermic, and cavitation effects. The micromechanical effect with microvibrations induced by US waves at 25 kHz stimulate the disaggregation of the horny layer of the epidermis [3], and the  molecular movements of the solute increase their kinetic power and temperature (thermic Joule effect) [4]. Moreover, the increased temperature with pressure modifications increase the permeability of membranes (pushing  function). The movements produced by US and transmitted to the vehicle applied on skin induce pressure modifications resulting its transformation to vapor state as microbubbles that break into surface (cavitation effect) [5]. Wave-induced depression and pressure bring about deformation and explosion of microbubbles, generating a very high push between liquid and skin surface;  explosion increases horny layer disaggregation (peeling effect).
This study shows that US, with a nonaggressive technique, enhance penetration of topic agents giving better results in the treatment of some cosmetic skin disorders. Combination of drugs in a suitable vehicle with LFS allows good penetration also of high-molecular-weight compounds.

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