Primary axillary hyperhidrosis is a chronic, idiopathic disorder . This condition is characterized by visible, excessive, bilateral, and relatively symmetrical sweating without apparent cause . It is often distressing; interfering with patient's daily activities . A variety of treatment modalities have been used in the management of this disorder. Topical agents include aluminum chloride, anticholinergics, boric acid, 2–5% tannic acid solutions, resorcinol, potassium permanganate, formaldehyde, glutaraldehyde, and methenamine. All of these agents are limited by staining, contact sensitization, irritation, or limited effectiveness [4,5]. Surgical options of treatment include the removal of axillary tissue by minor skin excision and subcutaneous curettage, liposuction curettage, and endoscopic transthoracic sympathectomy [6–8]. Subdermal laser-assisted axillary hyperhidrosis treatment using a 1064-nm Nd-YAG laser was performed resulting in significant clinical improvement with minimal side effects; however, all surgical procedures are invasive and potential risks of complications and side effects are common . Recent trials have found injections of Botulinum toxin type A and type B (BoNTA and BoNTB) to be an effective treatment for primary axillary hyperhidrosis [10,11]. Resistance to type A is estimated at 5–10% of injected patients and the side effects associated with the use of type B include dry mouth, headache, and sensory motor symptoms of the hand , suggesting the need for a long-acting treatment with a strong safety profile. Photodynamic therapy (PDT) involves the use of photochemical reactions mediated through the interaction of photosensitizing agents, light, and oxygen for the treatment of malignant or benign diseases . PDT is a two-step procedure. In the first step, the photosensitizer is administered to the patient by one of several routes (topical, oral, or intravenous), and it is allowed to be taken up by the target cells. The second step involves the activation of the photosensitizer in the presence of oxygen with a specific wavelength of light directed toward the target tissue . As the photosensitizer is preferentially absorbed by hyperproliferative tissue and the light source is directly targeted on lesional tissue, PDT achieves dual selectivity, minimizing damage to adjacent healthy structures [12,13]. Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein and used in lipstick manufacture and it is Food and Drug Administration approved . Von Tappeimer and Jesionek have been mentioned to be the pioneers in the field of PDT, and in 1904 they used topical eosin (5%) and light to treat skin cancer, lupus vulgaris, and condylomata . Eosin acted as potent photosensitizer inside cells when activated by light in the presence of oxygen [15,16]. The intense pulsed light (IPL) that emits light of a broadband spectrum in the range of 400–1200 nm has been used earlier to activate 5-aminolevulinic acid in treatment of acne [17,18]. Application of 400 nm cutoff filter of IPL allows shorter wavelengths (below the indicated filter) to be filtered and only longer wavelengths to pass through . Therefore, IPL was used in this study to activate the eosin that has an optimum absorption peak at 517 nm according to our laboratory studies. The purpose of this study was to evaluate the efficacy and safety of liposomal eosin hydrogel that is activated by IPL in the reduction of primary axillary hyperhidrosis.
Materials and methods
Liposomal eosin hydrogel was provided as a gift from the pharmaceutical technology laboratory at the National Institute of Laser Enhanced Sciences, Cairo University. Liposomal eosin was prepared from phosphatidylcholine from soy bean, cholesterol, sodium deoxycholate, eosin Y, chloroform analar grade, and phosphate buffer saline (PBS) and was purchased from Sigma Chemical Co (St. Louis, USA). Gel formulations were prepared from carboxymethyl cellulose (CMC; Al Nasr grade, USP 25), purchased from Normest Co. For Scientific Development (53-fourth stage, 10th of Ramadan City, Egypt).
Preparation of liposomal eosin hydrogel
Liposomal eosin hydrogel and free eosin (0.018%) in 5% sodium CMC (NaCMC) hydrogel has been prepared by sprinkling the weighed amount of NaCMC and the preservative mixture of 0.1% methylparaben and 0.01% Propylparaben gradually into the vortex of a 100 ml vesicle suspension, placed in a 200 ml aluminum foil covered beaker, and stirred with a mechanical stirrer for 2 h under ambient conditions to obtain a homogeneous mixture. The gel was left overnight for air removal in a vacuum desiccator. Plain and 0.02% free eosin gel formulation were also prepared following the same procedure. Gel formulations were examined visually for color, texture, and eosin release from hydrogel.
In-vitro eosin release from hydrogel
The release of eosin from free and liposomal gel preparations was studied using a dialysis bag (Visking 20/32, made of cuproban) earlier washed with PBS (pH 7.4.). The dissolution medium used in this study was PBS (pH 5.5, skin pH). The dialysis bags containing 1 gm of the free eosin and liposomal eosin vesicle gel were immersed in a 50-ml beaker containing 40 ml of the dissolution medium. Samples from the dissolution medium were removed at preset time intervals (10, 20, 30, 40, 50, 60, 70, and 80 min) and the absorbance of eosin was recorded at λ max 517 nm in a 1 cm quartz cell on a Shimadzu UV-1650 UV–VIS double beam spectrophotometer (Shimadzu, Japan). The concentration was estimated from a previously prepared standard calibration curve. The experiments were conducted independently in triplicate.
Skin irritation and penetration studies in rat
Twenty-four rats with average weight were used and divided into three equal groups; A, B, and C. Both free eosin and liposome containing gel were applied separately (500 mg/rat) on the back of the animals of groups A and B, respectively. Two hours after shaving and division into two groups, the site of the first group was observed for any sensitivity reaction. The second group was irradiated after 2 h of application with 48 J/cm2 at 550 nm, 80 mw light-emitting diode (LED, Photon Scientific, PHOTON Scientific Equipments, Cairo, Egypt) and autopsy samples were taken 24 h after irradiation, fixed and mounted in paraffin and stained with hematoxylin and eosin for histological study. Group C received plain NaCMC gel for control study. Animals were treated according to the standard regulations of international institutional guidelines and by guidelines set forth by the Guider.
Patients and methods
This was a single-blinded, placebo-controlled, and intrapatient comparative study. Twenty patients, minimum age of 18 years, were eligible to participate in this study. The inclusion criteria were based on the characteristics of primary axillary hyperhidrosis  defined by the following criteria: persistent bilateral and relatively symmetric excessive sweating with cessation of focal sweating during sleep lasting at least 6 months in duration and without apparent cause. An onset age of less than 25 years and the frequency of hyperhidrosis was elicited at least once per week, which impaired daily activities. Hyperhidrosis Disease Severity Scale (HDSS) score 3 or 4 (Table 1) and positive minor starch–iodine test were part of the inclusion criteria. Patients were excluded from the study if pregnant, lactating, or had a skin disease such as intertrigo, fungal, or bacterial infections at the treatment sites. An informed written consent was obtained from all patients after full explanation of the procedure.
Minor's starch–iodine test was used to assess the size of hyperhidrotic surface area of both axillae and to identify the sites to be treated. A 5% iodine solution is applied to axilla and allowed to dry, and then starch is brushed on the area. The starch–iodine combination turns into a dark blue color wherever there is excess sweat. The area of demarcation was marked, measured, and photographed before treatment. Eosin hydrogel was applied to the right axilla for 1 h and the placebo gel, which served as a control, was applied to the left. Patients were subjected to repeated sessions using IPL (EPI-C PLUS, Espansione group, Espansione Marketing Spa, Orefici, Italy) with a 400 nm filter, 2.5×4.5 spot area, total area 11.25 cm2, 20 ms pulse duration, and 25 J/cm2 fluence, once weekly, for four sessions. A 400 nm filter allows only wavelengths between 400 and 1200 nm to pass through, preventing emissions of wavelengths shorter than 400 nm.
Assessment of Hyperhidrosis Severity Scale
Efficacy was measured by HDSS, which is a disease-specific scale that provides a qualitative measure of severity of the patient's condition based on how it affects their daily activities. A score of 3 or 4 indicates severe hyperhidrosis, whereas a score of 1 or 2 indicates mild or moderate primary hyperhidrosis, respectively . A 1-point improvement in HDSS score was associated with 50% reduction in sweat production and a 2-point improvement with 80% reduction sweating . HDSS were evaluated at baseline, at every session, and at the end of treatment. The primary efficacy endpoint was the proportion of treatment responders, defined as patients who reported at least 1-point improvement from baseline HDSS score after two sessions.
Assessment of sweating area reduction
The minor starch–iodine test was carried out before treatment and at the end of treatment for the right axilla (treated) and the left axilla (placebo). The blue dark area, which represented the hyperhidrotic area, was measured to assess the change in size.
Data were coded and entered using the ‘MS Excel XP’ and ‘Origin 7.5’ Software (Microsoft corporation, NewYork, USA). Data were summarized using mean±standard deviation for quantitative variables and percentages for qualitative variables. Comparisons between two groups were made using Student's t-test. P value of less than or equal to 0.05 was considered significant at 95% confidence level and P value of less than or equal to 0.01 was considered highly significant at 99% confidence level. Analysis of variance test was carried out for comparative purposes between mean reductions of hyperhidrotic surface area at baseline, after treatment and at 8-months follow up.
Patients self assessment
Patients were questioned about any adverse effects throughout the trial. Patients' satisfaction was evaluated as satisfied, partially satisfied, or dissatisfied and were asked whether they would repeat the same procedure again if necessary.
Hydrogel in-vitro studies
All gel formulae were elegant in shape (transparent and nongritty). The free eosin gel formulation had a bright reddish brown color, whereas the formula containing the eosin-loaded liposomes exhibited a color change from reddish brown to light orange indicating the entrapment of eosin inside liposomal vesicles. Figure 1 represents drug released from the two gel formulae containing free eosin and gel containing eosin loaded in liposomes at different time intervals. From Fig. 1 and the statistical data, there was a significant difference in drug release between the two formulations within the first 10 min. Drug released from free eosin gel was significantly higher (P<0.05) than that from liposomal eosin gel at all time intervals starting from 10 min, up to 90 min. Observation of rat skin for 2 h did not show any signs of skin irritation (redness). The histological sections of mice skin showed normal sweat gland and sweat duct in skin treated with free eosin gel (Fig. 2a) compared with the sections of irradiated skin treated with gel containing liposomal eosin that showed damaged and necrotic sweat glands and sweat ducts located in the deep dermal layer (Fig. 2b).
The mean age of patients was 34.4±6.5 years (range 23–44 years). There were 11 women (55%) and nine men (45%). Fifteen patients (75%) were of HDSS score 4 and five patients (25%) were of HDSS score 3. Before treatment, the mean area of sweating as determined by a minor starch–iodine test was 65±28.2 cm2 for the right axilla (treated site) and 64.3±27.7 cm2 for the left axilla (placebo site) (Table 2). This slight difference between right and left axilla is considered a normal physiological variation and it was obviously elicited in 18 (90%) patients, who reported slightly more sweating in the right axilla compared with their left, at baseline. Each patient received four sessions, once weekly. Patients experienced varying degrees of reduction in axillary sweating in the treated sites as determined by both the subjective method (HDSS) and objectively, by measuring the reduction in surface area of sweating compared with the baseline.
Results of assessment of Hyperhidrosis Disease Severity Scale
It is noteworthy, is that 1 week after the second session, patients reported an improvement of 1-point in HDSS score which means 50% reduction in sweating, one patient reported an 80% reduction in sweating as indicated by a 2-point improvement in HDSS (score 4 to score 2). One week after the last sessions, a 3-point improvement on the 4-point (HDSS) was reported in 7 patients (35%), a 2-point improvement was reported in 9 patients (45%), and only 1-point improvement was elicited in 4 patients (20%) (Fig. 3). Three patients reported improvement at the placebo site after the second session, which worsened thereafter and 3 patients (15%) reported a 1-point improvement from HDSS score 4 to score 3 at the placebo site (Fig. 4). No irritation or burning sensations were experienced by any of the patients. Finally, no recurrence occurred during the 8-month follow-up period.
Reduction of the hyperhidrotic surface area
On measurement of the mean area of hyperhidrosis before and after treatment (right axilla) using the minor starch–iodine test, it was found that there was a highly significant reduction in size from 65±28.2 cm2 at baseline to 5.7±5.2 cm2 (Table 2) at the end of treatment (P<0.01) (Fig. 5a and b). No significant difference was found in the mean area of hyperhidrosis (64.3±27.7 cm2) before treatment in placebo site (left axilla) and after treatment (63.95±28.8 cm2) (P=0.72). A 90.1% reduction in the area of hyperhidrosis of the right axilla was obtained versus 2.2% reduction in the placebo site (Fig. 6a and b). A highly significant difference (P<0.01) was found in the mean area of hyperhidrosis between the right axilla (treated site) and left axilla (placebo site) at baseline, at the end of treatment and at 8-month follow-up (Table 3).
Nineteen patients were satisfied and asked for treatment of the placebo site by the same technique as used for the right axilla. One patient who improved by 1-point on HDSS was partially satisfied.
Axillary hyperhidrosis is the second most common type of primary hyperhidrosis. The disease negatively affects professional and personal life . A variety of treatment modalities such as topical, surgical, and botox injections have been used in the management of primary axillary hyperhidrosis but no ideal treatment has been discovered yet. Recurrence and side effects of these modalities suggested the need for a long-acting treatment with a strong safety profile . The results proved that the application of the liposomal eosin hydrogel (photosensitizer) and subsequent photoactivation with IPL is an effective method in reduction of hyperhidrosis in patients with primary axillary hyperhidrosis. Both, quantity of axillary sweat production and patients' symptoms, were markedly improved and efficacy was sustained, for the duration of the trial and the 8-month follow-up. The mechanism of action of PDT in treatment of hyperhidrosis is not fully understood. However, we can explain the elicited improvement in reduction of sweating on the same basis as in documented studies that discuss the effect of PDT on sebaceous glands during the treatment of acne using 5-aminolevulinic acid as a photosensitizer, in which reduction in the number of gland and secretion of sebum was elicited [16,18]. Use of IPL with cut off filter of 400 nm to photoactivate the liposomal eosin hydrogel that has a peak of absorption at 517 nm leads to singlet oxygen production, and subsequent destruction of sweat glands, reducing their number and the secretion of sweat. This explanation was proved by the histopathological findings of the irradiated skin of mice that were treated with liposomal eosin hydrogel. The histological section showed the presence of eosin hydrogel inside the sweat glands (Fig. 2), the damaged, necrotic sweat glands and sweat ducts located in deep dermal layer in the irradiated sections. The hydrophobic characteristic of the prepared liposomal eosin hydrogel facilitates the migration of eosin into skin layers such that there was no eosin accumulation in the epidermis. Moreover, irradiation of liposomes with laser or coherent light changes the packing properties of phospholipids membrane, leading to a decrease in transition temperature at which membrane fluidity changes from gel state to liquid crystalline state (permeable phase) [22,23] and subsequently an increased amount of eosin is delivered into skin cells, which is observed in necrotic sweat glands in the dermal layers. It is noteworthy that the onset of action was rapid; 1 week after the second session, patients reported a 1-point improvement on the 4-point (HDSS), which means 50% reduction in sweating. On evaluation of patients 1 week after the last session, a 3-point improvement on the 4-point (HDSS) was reported in seven patients (35%), a 2-point improvement was reported in nine patients (45%), and only 1-point improvement was elicited in four patients (20%). The subjective reduction in patients' sweating was higher throughout the trial for every patient than the reduction in hyperhidrotic area measured by the starch–iodine test. There were some instances in which patients had a positive starch–iodine test despite being symptomatically free of sweating. In these cases, the patients' subjective reduction of sweating may potentially be the more significant outcome, as a reduction in patients' symptoms is the ultimate goal of treatment . Naumann and Lowe  found that BoNTA treatment significantly reduced daily activity limitations 4 weeks after injection. A 2-point improvement on the 4-point (HDSS) was reported in 75% of their patients in the 75-U and 50-U BoNTA groups and in 25% of the placebo group; the improvements in HDSS scores were corroborated by gravimetric results. The median duration of the BoNTA effect was 197 days, 205 days, and 96 days in the 75-U, 50-U, and placebo groups, respectively however, the effect of total surface area involvement on treatment efficacy was not evaluated . The result of our study cannot be directly compared with their trial; they have measured sweat production by gravimetry as opposed to hyperhidrotic area used in this trial. Any light source, either laser or nonlaser, with suitable spectral characteristics and a high output at an absorption maximum of the photosensitizer can be used for PDT. In contrast, lasers in PDT are not without limitations . The lasers are relatively expensive, they require special maintenance and, when coupled with fiberoptics, they may be used only in small skin lesions. In the treatment of large skin lesions, noncoherent light sources are superior to laser systems because of their large illumination fields . Comparing the IPL with low-emitting diode laser (LED), the IPL is much faster as the irradiation required 3 min for one axilla whereas the LED required 20 min . Other advantages of noncoherent light sources are their low cost, small size of the equipments, and ready availability. In addition, polychromatic light sources allow the use of different photosensitizers with different peaks of absorption . Given the right dose of drug and light, noncoherent light sources seem to be as effective as laser sources . IPL was thus suitable for photoactivation of eosin gel with its matching absorption peak. PDT using liposomal eosin hydrogel is noninvasive, safe, with no risk of multiple frequent injections, and not time-consuming. It is a cheap method of treatment when compared with botox, as both liposomal eosin hydrogel gel and IPL have low running cost. In this study, follow-up for 8 months after the end of treatment showed no recurrence but because of seasonal variation there was normal physiological increase of sweating during hot weather. Nineteen patients were satisfied with the treatment procedure and the results; one patient was partially satisfied because of the high expectation as he had 50% sweat reduction. To our knowledge, this is the first single-blinded, placebo-controlled intrapatients comparative study to assess the efficacy of PDT in the management of axillary hyperhidrosis.
PDT using eosin gel, which is photoactivated by IPL, is a noninvasive, safe, and effective method for management of primary axillary hyperhidrosis with no risk of multiple frequent injections and is not time-consuming. It is painless compared with painful botulinum injection or surgical excisions, which require long postoperative care. It is a cheap method of treatment when compared with botox as both eosin gel and IPL have low running cost. The degree of reduction of hyperhidrosis was satisfactory for all patients except one, because of the high expectations of having lower grade of HDSS. Further studies and long-term follow-up are needed to judge the longevity of this technique.
The authors thank Dr Maha A. Fadel, assistant professor of pharmaceutical sciences, and Dr Shahira Almenshawy, lecturer of pharmaceutical sciences, for providing us with the eosin gel preparation and performing the in-vitro study. Liposomal eosin gel was provided as a gift from the pharmaceutical technology laboratory at National Institute of Laser Enhanced Sciences, Cairo University.
There is no conflict of interest.
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Keywords:© 2011 Egyptian Women's Dermatologic Society
hyperhidrosis; intense pulsed light; photodynamic therapy