Formulation, characterization and clinical evaluation of propranolol hydrochloride gel for transdermal treatment of superficial infantile hemangioma.

http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–11 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.931968

RESEARCH ARTICLE

Formulation, characterization and clinical evaluation of propranolol hydrochloride gel for transdermal treatment of superficial infantile hemangioma Drug Dev Ind Pharm Downloaded from informahealthcare.com by Queen's University on 01/01/15 For personal use only.

Wenhu Zhou1, Shiying He1,2, Yijun Yang1, Dan Jian3, Xiang Chen3, and Jinsong Ding1 1

Department of Pharmaceutics, School of Pharmaceutical Sciences, Central South University, Changsha, Hunan Province, China, 2Shenzhen Zhijun Pharmaceutical Co., Ltd., Shenzhen, Guangdong Province, China, and 3Department of Dermatology, Xiang Ya Hospital, Central South University, Changsha, Hunan Province, China Abstract

Keywords

The objective of the present study is to formulate and characterize propranolol hydrochloride (PPL HCl) gel, and to evaluate the efficacy of this formulation in transdermal treatment for superficial infantile hemangioma (IH). The transdermal PPL HCl gel was prepared by a direct swelling method, which chose hydroxypropyl methylcellulose (HPMC) as the matrix and used terpenes plus alcohols as permeation enhancer. Permeation studies of PPL HCl were carried out with modified Franz diffusion cells through piglet skin. Our results pointed to that among all studied permeation enhancers, farnesol plus isopropanol was the most effective combination (Q24, 6027.4 ± 563.1 mg/cm2, ER, 6.8), which was significantly higher than that of control gel (p50.05). High percutaneous penetration with related lower plasma drug level of PPL HCl gel was confirmed by microdialysis technique in rats using the homemade PPL HCl oral solution as a control. Clinical studies also confirmed the excellent therapeutic response and few side effects of the PPL HCl gel. These results suggest that transdermal application of the PPL HCl gel is an effective and safe formulation in treating superficial IH.

Clinical evaluation, microdialysis, percutaneous permeation, propranolol hydrochloride gel, superficial infantile hemangioma

Introduction Infantile hemangiomas (IHs) are the most common tumors of infancy, affecting 2–3% of newborns and occurring in 4–10% of children within the first year of life. Most hemangioma lesions are relatively small in size, sporadic, localized in the skin, and pose only minor clinical problems1. However, approximately 20% of patients have multiple lesions and approximately 10% of the tumors grow rapidly to a significant size. In these cases, the tumors can be problematic, and some hemangioma lesions may be extremely disfiguring and destructive to normal tissue, and even life-threatening2. One of the most common problematic hemangioma is superficial hemangioma, which could locate in the face, ear, orbit, etc3. Current treatment options for hemangiomas include systemic or intralesional corticosteroids, chemotherapeutic agents (vincristine, alpha-interferon), laser endoscopic resection, or a combination of these therapies. Unfortunately, the

Address for correspondence: Professor Jinsong Ding, Department of Pharmaceutics, School of Pharmaceutical Sciences, Central South University, Tongzipo Road #172, Changsha 410013, Hunan Province, China. Tel: + 86 731 82650250. Fax: + 86 731 82650442. E-mail: [email protected] or Professor Xiang Chen, Department of Dermatology, Xiang Ya Hospital, Central South University, Xiang Ya Road #87, Changsha 410008, Hunan Province, China. Tel: +86 731 432 7128. Fax: +86 731 432 8478. E-mail: [email protected]

History Received 4 March 2014 Revised 28 May 2014 Accepted 2 June 2014 Published online 25 August 2014

existing treatment approaches possess numerous side effects and show limited success. In 2008, a small number of cases of infants with hemangiomas documented a dramatic improvement following treatment with oral propranolol, a known non-selective beta-blocker4. Since then, numerous reports have confirmed the therapeutic effect of propranolol for IHs, though the underlying mechanisms are not understood. In the meantime, some limitations of propranolol in treating IHs have been found. First, propranolol has a short biological half-life and is subjected to extensive hepatic firstpass metabolism. Second, severe side effects may occur at high doses of propranolol for long term treatment5. Finally, the side effect profile of propranolol in children remains largely unknown. Transdermal administration is a potent alternative route that can overcome undesirable shortcomings of oral administration. Transdermal delivery can offer inherent advantages as follows: (a) bypassing the first pass metabolism; (b) enabling control of input; and (c) avoiding problems of stomach emptying, pH effects and enzymatic deactivation associated with gastrointestinal tract passage. Actually, compared to other available treatments for superficial IH, transdermal propranolol displays the same efficacy but with a better safety profile6. A number of transdermal drug delivery systems, different in their formulations such as transdermal solution6 or ointment7, have been developed to assess clinical efficacy and safety for transdermal delivery of propranolol.

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Although local application of propranolol can directly deliver drug to the disease site and maximize local efficacy with minimal potential side effects, the stratum corneum (SC) of intact skin can prevent percutaneous absorption of drugs and compromise efficacy of transdermal delivery. To solve this problem, numerous active or passive methods for penetration enhancement have been tested over the years. One long-standing approach for improving transdermal drug delivery is to exploit penetration enhancers, which can decrease the barrier resistance and promote drug flux through interaction with skin. To date numerous chemicals have been evaluated as potential penetration enhancers. Among them, terpenes appear to be promising candidates as clinically acceptable enhancers. They were obtained from natural sources with good safety profile and high percutaneous enhancement ability at low concentrations (1–5%)8. For example, they do not cause skin toxicity or if any, only mild irritation, no lasting erythema9,10, and their safety (GRAS) was approved by the FDA of USA11. Therefore, in the present study, terpenes were selected as permeation enhancers to explore their effect to assist PPL HCl to across piglet skin in vitro. An efficient PPL HCl gel was developed using HPMC as a gel matrix and terpenes as the chemical permeability enhancer. In order to optimize the penetration capability of PPL HCl, terpenes combined with alcohols were employed as binary permeation enhancers. In addition, these optimized gel formulations were characterized by in vitro quality evaluation, in vivo safety, microdialysis and pharmacokinetics. At last, the efficacy and safety profile for these formulations was evaluated by a small-scale clinical trial in infants.

Materials and methods Materials Limonene, 1,4-cineole, borneol, limonene, anethole, L-menthol, nerolidol, farnesol, tetrahydrogeraniol, geraniol were obtained from Julongtang Biochemical Co., Inc. (Hubei, China). Propranolol hydrochloride was purchased from Yabang Pharma Co., Ltd. (Jiangsu, China). HPMC and Tween 60 were obtained from Xilong Chemical Manufacturing Co., Ltd. (Guangdong, China). Glycerol, propanediol, isopropanol and ethylparaben were purchased from Erkang Pharmaceutical Co., Ltd. (Hunan, China). Acetonitrile and methanol used were of HPLC grade and supplied by Tedia Company, Inc. (Fairfield, OH). All other reagents used were of analytical pure grade or chromatographic pure grade and were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Piglets, 18 ± 2 kg and 4 ± 1 weeks old, were obtained from Hunan Provincial Center for Disease Control and Prevention (Hunan CDC) (No.SCXK (Hunan) 2011-0004). New Zealand white rabbits, 1.8–2.5 kg, were provided by the Laboratory Animal Center of Central South University (No.SCXK (Hunan) 2011-0012). SD rats, 180 ± 20 g were obtained from Changsha Tianqin biological technology co., LTD (No.SCXK (Hunan) 2009-0012). Formulation of PPL HCl gel The PPL HCl gel was prepared by a direct swelling method, similar to previously described12. For the preparation of the gels, 2.5 g HPMC was dispersed in 15-ml glycerin with continuous stirring, and water was added to get swelling for 24 h. Afterwards, 5 g PPL HCl was dissolved in water, and ethylparaben was dissolved in ethanol (few drops) to mix with terpene and tween 60. Then both solutions were added to the above gel with continuous stirring and water was added to make up 100 g of gel. Control gels were prepared by the same procedure but omitting PPL HCl.

Drug Dev Ind Pharm, Early Online: 1–11

In vitro skin permeation study Skin preparation Piglets were bought from Hunan CDC, and the abdominal skin was used for the experiment. The full thickness skin was excised and fat adhering to the dermis side was cleaned using a blunt scalpel, followed by repeatedly washed with saline. Care was taken to avoid damaging skin. The skins were sealed in aluminum foil paper at 20 C and used within 2 weeks. Effect of terpenes on the permeation of PPL HCl The PPL HCl gels containing 1%, 3% or 5% (w/w) terpene were prepared by the method previously described. The terpenes used here include limonene, 1, 4-eucalyptus, borneol, anethole, L-menthol, nerolidol, farnesol, tetrahydrogeraniol, geraniol. Furthermore, the effect of binary permeation enhancers which contained both terpenes and alcohols (isopropanol or propanediol) was also investigated. Frozen skins were slowly thawed and mounted on Franz diffusion cells with epidermis facing the donor chamber and the area available for permeation was 0.785 cm2. The skin was equilibrated for 1 h with phosphate buffered saline (pH 7.4) in the receptor chamber. The receptor solution was magnetically stirred throughout the experiment at thermostatically maintained temperature (37 ± 1 C). A total of 200 mg of PPL HCl gel was placed on each skin and all donor cells were occluded with parafilm. Several holes were poked on the parafilm to keep an open condition. Receptor samples (2 ml) were taken at predetermined time intervals (1, 2, 4, 8, 12, and 24 h) and stored at 4 C prior to HPLC analysis (LC-2010 C Shimadzu). Following each receptor sample withdrawal, the same volume was replenished immediately with receptor buffer. The amount of drug withdrawn was corrected in the subsequent calculations of cumulative amount of the drug penetrated and accumulated permeation amount was calculated by the following equation: P Ct V þ i¼n1 C i Vi i¼1 ð1Þ Q¼ A Q—the accumulated permeation amount per unit area (cm2) within time t, Cn—the measured concentration value within time t, V—the total volume of solution in receptor chamber, Vi—the sampling volume per time point, Ci—the measured value of concentration within time i, A—the area available for permeation (0.785 cm2). Effect of drug loading on the permeation of PPL HCl To determine drug dosage and specification of gel in clinical application, 2.5%, 5% and 7.5% PPL HCl gels were prepared, and in vitro permeation studies were performed using the same method described above. Permeation data analysis The permeation of PPL HCl from gel was measured over 24 h, and plots were constructed of the cumulative corrected amounts of PPL HCl (mg cm2) against time (h). The x-intercept of the extrapolated linear region gives the lag time. The slope of this linear portion of the graph provides the maximum flux values at steady state (mg cm2 h1). The permeation-enhancing activities were expressed as enhancement ratios of flux (ER) which were calculated by Equation (2): ER ð%Þ ¼

PPL flux with enhancer in gel 100: PPL flux with no enhancer in gel

ð2Þ

DOI: 10.3109/03639045.2014.931968

The data were presented as mean ± SD. Statistical analysis was made using analysis of variance (ANOVA). p Values50.05 were considered as statistically significant.

Propranolol gel for superficial infantile hemangioma

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throughout the experiment by intraperitoneal injection (i.p.) urethane (1 g/kg). Microdialysis probe calibration

Characterization of the PPL HCl gel Physical examination Physical examinations were carried out by inspecting visually for their color, appearance and consistency. Measurement of pH To measure the pH of gel formulations, 1 g of gel was dissolved in 10-ml distilled water and stirred for 5 min, and the pH value was determined by a digital pH meter (pHs-3C, TianHeng Scientific Co., Ltd., Hunan, China).


Viscosity The measurement of viscosity of the prepared gel was performed with a Brookfield Viscometer (DV-III, Brookfield, Middleboro, MA) using spindle # CPE40 at 25.0 ± 0.1 C12.

To determine the in vitro relative recovery of PPL HCl, microdialysis probes were placed in a well-stirred isotonic PBS (pH 7.4) containing different concentrations of PPL HCl (i.e. 100, 10, and 0.5 mg/ml) at 37 C. The probes were perfused with blank PBS (pH 7.4) at 1 ml/min and the dialysate samples were collected at 30 min intervals. Drug concentrations were analyzed by HPLC to give the relative in vitro recovery. Similarly, for in vitro delivery, microdialysis probes were placed in a wellstirred isotonic phosphate buffer at 37 C. PBS buffers with different drug concentrations (i.e. 100, 10, and 0.5 mg/ml) were perfused through probe. The in vivo delivery was calculated by perfusing drug solutions into a microdialysis probe which was inserted into rat subcutaneous tissue. The probe relative recovery, R, and delivery, D, are defined by the following equations:

Consistency The measurement of consistency was carried out on a cone penetrometer (ZHA-5A, Tianda Tianfa Technology Co., Ltd., Tianjin, China)13. Briefly, a cone was used to dig into the center of a cup filled with the gel from a fix distance of 10 cm. The distance between the surface of the gel and the tip of the cone inside the gel was recorded after 5 s, and the unit was 0.1 mm. Drug content A total of 200 mg of gel was dissolved in 25-ml distilled water and shaken for 2 h on a mechanical shaker in order to fully dissolve the drug, followed by analyzing on a Shimadzu LC-2010 C HPLC instrument. Stability studies The prepared gels were packed in aluminum collapsible tubes and subjected to stability studies using a thermostated container (LRH-250-Y, Taihong Medical Equipment Ltd, Jiangxi, China) at 5 C and room temperature for 3 months or 40 C for 1 month. Samples were withdrawn at appropriate time intervals and evaluated for physical appearance, pH, drug content and centrifugal stability. Skin irritation evaluation Eight rabbits (body weight 1.8–2.5 kg) of either sex were used in this study. An area of 3 cm 3 cm was marked on both sides of dorsal skin with hair shaved. A 5% PPL HCl gel was applied to one side while the other side was used as the control. Gel was applied twice a day for 14 d and the site was monitored for any sensitivity reaction graded from 0 to 4 for no reaction, slight patchy erythema (barely perceptible-light pink), moderate patchy erythema(dark pink), moderate to severe (light red) and severe erythema (extreme redness), respectively. The skin surface was observed for any visible change such as erythema (redness) or edema after 1, 24, 48, and 72 h of the formulation application. In vivo drug permeation and pharmacokinetics

Cdialysis 100% Cout

ð3Þ

Cin Cdialysis 100%: Cin

ð4Þ

R ð%Þ ¼

D ð%Þ ¼

where Cdialysis, Cout, and Cin represent the drug concentration in dialysate, sample and perfusate, respectively. The in vivo recovery, therefore, was calculated according to the following equation:14 R R in vivo ¼ in vitro ð5Þ D D In vivo studies The day before experiment, the abdomen skins of rats were shaved carefully with an electrical animal hair clipper. On the day of experiment, the rats were anesthetized with urethane (1 g/kg, i.p.). A microdialysis probe was implanted under abdomen skin as superficially as possible, using an 18 G 1.5 in. needle as a guide. The actual depth of the probe was measured by Doppler imagining. The skins were recovered from the insertion trauma in 1 h. Afterwards, the probe was connected to the pump via a teflon tubing and perfused at flow rate of 1 ml/min. Then, PPL HCl was orally (dissolution in PBS) or transdermal administered. For oral PPL HCl, rats underwent intragastric administration of drug solution with a dosage of 60 mg/kg PPL HCl. For transdermal administration, 2.5% PPL HCl gel (30 mg/kg) or 5% PPL HCl gel (60 mg/kg) was applied to the abdomen skin at a round area of 4.9 cm2 at the point where the microdialysis probe was implanted. Microdialysis samples were collected every 30 min for 13 h, and analyzed without any further processing. To determination of plasma concentration of PPL HCl, about 150-ml blood was collected immediately before and at predetermined time points after administration (set the time point at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12 13 for oral administration and 0, 1, 2.5, 4, 5.5, 7, 8.5, 10, 11.5, 13 for PPL HCl gel). Blood was collected from the caudal vein and centrifuged at 3500 rpm at 4 C for 10 min. Blood samples were analyzed using a validated LC-MS/MS method (API3000, AB SCIEX).

Animals Sprague–Dawley rats (160–200 g) were used. The study was carried out in accordance with the Principles of Laboratory Animal Care (NIH publication). The rats were anaesthetized

Evaluation of clinical efficacy in infants A small-scale clinical trial was conducted to evaluate the efficacy of the developed 2.5% and 5% PPL HCl gels in comparison to

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placebo (control gels). A double-blind and randomized clinical trial was conducted at Xiangya hospital, Hunan, China. Protocol of the study was approved by the ethical committee of the Xiangya Hospital.

reduction of the size or even increase; grade II, improve, cessation in growth and color fading of IH and the reduction of the size less than 60%; grade III, effective, the reduction of the size between 60% and 89%; grade IV, clinical cure, the reduction of the size 489%.

Inclusion criteria To be eligible for participation in the study, patients have to meet the following criteria: (1) Age41 month, and definite diagnosis of superficial hemangioma; (2) no propranolol contraindications such as sinus bradycardia, atrioventricular block, tracheal bronchial asthma, bronchitis and pneumonitis; (3) no congenital heart disease or other systemic diseases; (4) no previous intervention; and (5) signing the informed consent by their parents or guardians.


General data Thirty-six superficial IH children (20 males and 16 females) meeting the above criteria were accepted in our out-patient clinic. Their ages varied from 2 to 24 months. The size of the tumor varied from 0.7 cm 0.4 cm to 3.8 cm 2.2 cm. The distributions of the 36 IHs were as follows: head and neck (18/36, 50%), trunk and limbs (10/36, 27.8%), genitals (4/36, 11.1%), ears (3/36, 8.3%) and orbital (1/36, 2.8%). Drugs and administration 2.5% and 5% PPL HCl gels were prepared as described above and each group of parents were advised to apply the gel in a thin layer (about 2 mg/cm2 for 2.5% PPL HCl gel and about 4 mg/cm2 for 5% PPL HCl gel, respectively), according to the previous trials15. All parents were instructed to gently spread the gel onto the surface of the IH twice daily for 2 weeks. Drug was withdrawn under the following conditions: (1) IH faded away without recurrence after withdrawing the gel; (2) adverse response or other situations requiring ceasing this therapy. Data collection During the initial 2 d of treatment, vital signs of the children were measured before and 2 h after the drug administration. Any changes in tumor volume and color were recorded by specially-assigned persons at each follow-up time point. Safety parameters (adverse events, thyroid function, standard hematology and biochemistry profiles, vital signs, color Doppler and electrocardiogram) were assessed before and after treatment. Outcome evaluation After the treatment, the scores for the clinical efficacy were assessed by the following standards: grad I, non-effective, no

Statistical analysis Curative effects of different groups were analyzed using Ridit analysis. p50.05 was considered statistically significant.

Results The effect of terpenes on the skin penetration of PPL HCl The effects of terpenes on the permeation profile of PPL HCl were summarized in Table 1, and the rank order of enhancement effect for PPL HCl is farnesol4L-menthol4nerolidol4tetrahydrogeraniol41,4cineole4geraniol4limonene4anethole4 borneo. The absorption of PPL HCl was enhanced significantly by the addition of farnesol, L-menthol and nerolidol (Table 1, p50.05). In particular, farnesol provided an approximately 3.9-fold increase in PPL HCl flux followed by L-menthol, with a 3.7-fold increase, and nerolidol with a 3.4-fold increase. To study the effect of terpene concentration on the transdermal permeation of PPL HCl, the three terpenes with related better penetration enhancement abilities were used at different concentrations (1, 3, and 5%, w/w). As displayed in Figure 1, terpene concentration played an important role in ER, but no linear relationship existed between terpene concentration and the permeation rate. The accumulated permeation amount of PPL HCl increased significantly in the 3% terpene for all permeation enhancers comparing with the control (Figure 1D, p50.05). However, at a higher terpenes concentration (5%), an abnormally decreased of drug penetration amount was observed. So, as a general rule, the highest permeation rate was achieved with the gel containing 3% of terpenes. Effect of binary permeation enhancers on the skin penetration of PPL HCl The effect of the three terpenes (farnesol, L-menthol and nerolidol) combined with short-chain alcohols on PPL HCl transdermal delivery was examined to find the stable formulation with the best transdermal efficacy16. The alcohols tested in this study include isopropanol and propanediol which are relatively tolerated by the skin. The transdermal delivery of PPL HCl with the two alcohols showed different trends (Figure 2). The PPL HCl gel containing isopropanol exhibited higher transdermal PPL HCl delivery compared to the control (alcohol-free) PPL HCl gel, and the ER increased in the order is isopropanol + farnesol4isopropanol + L-menthol4isopropanol + nerolidol. Among the tested terpenes and alcohols, the

Table 1. The effect of terpenes enhancers on permeation parameters of PPL HCl gel (n ¼ 5, mean ± SD). Terpene Control Limonene 1,4-cineole Borneol Anethole L-menthol Nerolidol Farnesol Tetrahydrogeraniol Geraniol

Q24 (mg cm2)

Linear equation

R2

Flux (mg cm2 h1)

ERflux

889.1 ± 76.9 2834.2 ± 140.1 3178.7 ± 400.6 1599.5 ± 948.5 1921.5 ± 452.0 4192.7 ± 988.3* 4075.0 ± 924.7* 4531.9 ± 723.3* 3556.5 ± 657.0 3111.6 ± 469.6

Q ¼ 51.6t 175.3 Q ¼ 132.9t 373.5 Q ¼ 138.9t 161.0 Q ¼ 75.9t 232.5 Q ¼ 91.0t 285.0 Q ¼ 190.8t 354.1 Q ¼ 176.2t 94.4 Q ¼ 199.3t 210.2 Q ¼ 167.5t 480.4 Q ¼ 144.9t 381.3

0.9967 0.9985 0.9983 0.9983 0.9967 0.9982 0.9952 0.9979 0.9988 0.9988

51.6 132.9 138.9 75.9 91.0 190.8 176.2 199.3 167.5 144.9

1.0 2.6 2.7 1.5 1.8 3.7 3.4 3.9 3.3 2.8

*p50.05, comparing with control.


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Figure 1. The effect of terpenes concentration on percutaneous penetration of PPL HCl gel (Means ± SD, n ¼ 5). (A) farnesol, (B) L-menthol, (C) nerolidol and (D) Q24 for each formulations.

combination of farnesol with isopropanol showed the highest transdermal delivery efficiency, with the ER 1.5-fold higher than that of the farnesol. However, decreased ER was observed in the presence of propanediol compared to the propanediol-free PPL HCl gel. When propanediol was added to the PPL HCl gel, the ER was 1.54 (farnesol), 1.25 (L-menthol) and 1.3 (nerolidol) fold less than their PPL HCl gels without propanediol, respectively. Effect of drug loading on the skin penetration of PPL HCl To study the effect of drug loading on the skin penetration of PPL HCl, a series of gel with different drug loading were prepared. When the drug loading of the PPL HCl in gel was increased from 2.5% to 5%, the Q24 increased significantly from (1689.9 ± 862.5) to (6027.4 ± 563.1) mg cm2 (Figure 3 inset, p50.05). However, a slight decrease in Q24 was observed when drug loading increased from 5% to 7.5%. Since saturated Q24 was observed when drug loading was switched to 7.5%, 2.5% and 5% of the PPL HCl gels were selected for further studies. Characterization of the PPL HCl gel The developed PPL HCl gels were evaluated for physicochemical tests including pH, viscosity, consistency, drug content and stability as shown in Table 2. The prepared PPL HCl gel formulations were milky white viscous with a smooth and homogeneous appearance. They were easily spreadable, and no discoloration or crystallization was

observed. The pH values of gels were determined to be in the range of 6.87 ± 0.24 which is favorable for transdermal application. Because pH of all the formulations fell in the range of normal physiological pH on the skin, and the formulations did not produce any irritation to the skin17. Determination of viscosity of gel matrix, as reported by Chaudhary18, is important since viscosity plays a major role in controlling the release of drug from gel matrix. Viscosity of all the gels was in the range of 63.2 ± 4.1 Pa S, which was suitable for drug release. Another criterion for a gel to meet the ideal quality is good consistency, which represents that gel readily spreads on skin. The therapeutic efficacy of a formulation also depends upon its spreading value. Consistencies of all the gels were in the range of 192.7 ± 6.9, making them easy to spread on the skin. Drug content of gels in a range of 100.1 ± 0.4% means that the drug was dispersed homogeneously throughout the gels with good content uniformity. The PPL HCl gels were stable over 30 d of accelerated testing (40 C/75% RH) with little variation or decrease of drug content as shown in Table 2. Furthermore, the PPL HCl gels subjected to storage for 3 months exhibited respectable stability with respect to drug content and physico-chemical properties (data not shown). Skin irritation test Following 14 d application of the gel, the results of the skin irritation test were recorded, and no allergic symptoms like erythema or edema appeared for up to 14 d. The irritation score


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Figure 2. The effect of binary permeation enhancers on percutaneous penetration of PPL HCl gel (Means ± SD, n ¼ 5). (A) farnesol combined with alcohols, (B) L-menthol combined with alcohols, (C) nerolidol combined with alcohols and (D) Q24 for each formulations (*p50.05).

Figure 3. The effect of drug loading on percutaneous penetration of PPL HCl gel (Means ± SD, n ¼ 5). Insert: Q24 for each formulations (*p50.05).


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for PPL HCl gel was zero, indicating the safety and acceptability for transdermal administration. In vivo drug permeation and pharmacokinetics


Probe recovery The in vitro recovery of PPL HCl was independent of its concentration with the value of 68.55 ± 1.94%, 58.79 ± 0.65% and 69.17 ± 5.09% for drug concentration at 0.5, 10, and 100 mg/ml, respectively. The mean in vitro recovery was 65.5%. Similarly, in vitro delivery from the standard solutions in linear probe was 56.12 ± 1.48%, 64.88 ± 2.02% and 58.65 ± 3.28%, respectively. And the mean in vitro delivery turned out to be 59.9%. For in vivo delivery, the probe was implanted in the skin with a depth of 0.07 ± 0.02 cm measured by Doppler ultrasound scanning (Voluson E8, General Electric, Milwaukee, WI). The in vivo delivery turned out to be 45.72 ± 5.93%, 36.74 ± 7.44% and 37.11 ± 3.10% with the concentration of 0.5, 10, and 100 mg/ml, respectively. The mean recovery was 39.9%, which was less than that of in vitro experiment. Therefore, the in vivo recovery ratio was calculated to be 43.9%.

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2.4 ± 0.8 mg/ml h, which was approximately 324 and 520 times lower than by the topical route at the same dosage (Figure 4). After oral administration of 60 mg/kg PPL HCl solution, drug was rapidly absorbed and reached a Cmax of 1916.7 ± 447.2 ng/ml at a median Tmax of 0.5 h. AUC0t was 14535.5 ± 2376.8 ng/ml h in plasma (Figure 5). However, after the application of PPL HCl gels, PPL concentration in plasma rose much more slowly and reached a plateau at about 7 h, with AUC0t 1695.6 ± 734.2 (for 2.5% PPL HCl gel) and 3128.3 ± 641.6 ng/ml h for (5% PPL HCl gel). Therefore, the absolute PPL HCl for a total expose of 60 mg/kg oral solution was much higher compared with the PPL HCl gel at the same dosage, with AUC0t being approximately 4.6-fold higher. Clinical studies Of all the potential participants, 36 met the inclusion criteria and were randomly assigned to a study group, including 15 in the 5% PPL HCl gel group, 15 in the 2.5% PPL HCl gel group, and 6 in the placebo group. Results of the clinical study showed some marked reduction in the size of IH lesions followed by softening and faded color in PPL HCl gel groups. Moderate to excellent

PPL HCl concentrations in dialysate and plasma The in vivo concentration–time profiles for PPL HCl in the skin and plasma after oral and transdermal administration are shown in Figures 4 and 5. PPL HCl was absorbed into the skin after transdermal administration of the 2.5% and 5% PPL HCl gel, with Cmax of 156.6 ± 17.2 and 259.1 ± 23.8 mg/ml, and AUC0t of 868.1 ± 45.2 and 1248.3 ± 87.6 mg/ml h, respectively. While in oral route, the Cmax and AUC0t were 0.8 ± 0.2 mg/ml and Table 2. Physico-chemical evaluation tests applied to PPL HCl gel. Batch Parameters pH Viscosity (Pa S) Consistency (0.1 mm) Drug content (%) Stability (%)

No.1

No.2

No.3

6.88 ± 0.19 62.4 ± 2.3 189.8 ± 5.2 100.5 ± 0.7 101.2 ± 2.2

6.80 ± 0.20 61.8 ± 4.9 186.8 ± 6.4 100.0 ± 0.3 101.5 ± 1.9

6.82 ± 0.28 65.4 ± 5.0 195.9 ± 6.7 99.8 ± 0.4 101.6 ± 2.4

Figure 4. Mean skin drug concentration–time curve of PPL HCl after oral and transdermal administration in SD rats (Means ± SD, n ¼ 4).

Figure 5. Mean plasma concentration–time curve of PPL HCl after oral and transdermal administration in SD rats (Means ± SD, n ¼ 4).

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improvement in the disease symptoms after treatment with the PPL HCl gels were graded II, III and IV, respectively (Tables 3 and 4 and Figure 6). For those patients graded in III and IV, within 7 d after the initiation of the PPL HCl gel treatment, change in color from intense red to purple, associated with softening of the lesion was observed. After the dramatic initial response, IHs continued to improve progressively with respect to both color and size. Transdermal application of 5% PPL HCl gel was well tolerated in all children, and the treatment did not affect blood pressure, heart rate or other safety parameters. No local irritation was observed in any patient. Data for curative effect of different groups are summarized in Table 3, and typical cases are summarized in Table 4. The effect of the PPL HCl gel was observed at every follow-up period. IHs growth stopped for almost all patients who were treated with the PPL HCl gel. However, no significant differences in the response of IHs between two dosages were found using Ridit analysis (p40.05).

Discussion Transdermal drug delivery is a promising alternative to oral dosage, especially for drugs that are subjected to the first-pass elimination such as PPL HCl. The main barrier or rate-limiting step in transdermal drug delivery of polar, water-soluble drug, such as PPL HCl, is the lipophilic part of SC, in which ceramides are arranged in a bilayer. In present study, to achieve successful transdermal drug delivery through the skin, penetration enhancers were employed to circumvent this natural barrier. The major objective of this study was to explore whether PPL HCl was able to penetrate the skin at a high rate by utilizing different skin permeation enhancers such as terpenes and alcohols. Terpenes have a good safety profile and can produce a high enhancement at low concentrations for both hydrophilic and lipophilic drugs10. A proposed mechanism for terpenes to improve drug skin permeation is an increase in drug diffusivity in the skin, due to modified intercellular lipid packing to disrupt their highly ordered structures19,20. In addition, the molecular mechanism might be also related to the preferential hydrogen bonding of oxygencontaining terpenes with ceramide head groups thereby breaking the lateral/transverse hydrogen bond network of the lipid bilayer21. For hydrophilic drugs such as PPL HCl, terpenes have been found to increase the permeation mainly by enhancing diffusion through the intercellular lipids22. Only hydrophilic Table 3. The relationship between dosage and treatment efficacy (number of cases [%]). Dosage

n

I (%)

II (%)

III (%)

IV (%)

Blank control 6 5 (83.3%) 1 (16.7%) 0 0 2.5% PPLHCL gel* 15 0 6 (40.0%) 8 (53.3%) 1 (6.7%) 5% PPLHCL gel* 15 1 (6.7%) 3 (20.0%) 9 (60.0%) 2 (13.3%) *p50.05, comparing with blank control.

terpenes were used in this study since the published studies suggested that hydrophilic terpenes are more effective in enhancing the permeation of hydrophilic drugs11,23. In the series of terpenes, farnesol, L-menthol and nerolidol provided the best enhancement activity for PPL HCl permeation. This was attributed to that farnesol and nerolidol possess an amphiphile-like structure that was appropriate for the disruption of the lipid packing of the SC. Since alcoholic hydroxyl groups can accept or donate hydrogen bonds, they lead to disruption of existing hydrogen bonding between SC, thereby facilitating the permeation of PPL. In addition, as a chain molecule, farnesol shows greater permeation-enhancing properties than cyclic terpenes for hydrophilic drugs, due to its lower vaporization energy24. Except for farnesol, L-menthol provided a higher PPL HCl permeation amount than the rest of the terpenes. This could be explained for the following reasons. First, with a low boiling point, L-menthol has relatively weaker intermolecular cohesive forces or self-association, which means that the oxygen of the functional ether and carbonyl group is mostly free. Therefore, the energy required for competitive hydrogen bonding in skin ceramide is relatively low25. Second, compared with other terpenes, L-menthol has a lower molecular-weight with higher vapor pressure, enabling a rapid transport of the drug to the dermis site26,27. Finally, it has been reported that the () enantiomer of menthol (L-menthol) has a better stereospecific effects in interaction with the skin than other terpenes9. In this study, three terpenes (farnesol, L-menthol and nerolidol) were used to investigate the effect of terpene concentration on permeation enhancement. The result showed that terpenes have a complex concentration-dependent effect on ER. As the concentration of terpenes increased from 1% to 3%, the cumulative penetration amounts of drug increased drastically (Figure 1D). However, the permeation of PPL HCl did not further escalate as the terpene concentration increasing from 3 to 5%. The highest ER was obtained from the gel containing 3% farnesol. These results suggest the effect of terpene is saturated at concentration up to 5%, which is consistent with a previous report28. The increase in ER with the enhancer concentrations increase from 1% to 3% is normally attributed to the ability of the terpene to penetrate the skin and increase skin permeability. While the reduction of drug transport rate at higher terpene concentrations may be due to the interaction between terpene and the drug. Solubilization of the drug by terpene was achieved at high terpene concentrations. Thus, the thermodynamic activity of the drug decreases, resulting in a decrease of the driving force for the drug absorption into skin29. It is also probable that at higher terpene levels the biological membrane tends to dehydrate and reduce drug permeation across the skin. In the present study, an attempt was made to combine terpene with alcohols to further enhance permeation. The result indicated a synergistic effect for permeation efficacy when isopropanol was used as combined permeation enhancer. After combination with isopropanol, enhance efficacies for farnesol, L-menthol and nerolidol rose approximately 1.5-fold (Figure 2D). This might

Table 4. Details of typical clinical cases. Number

Gender

Age (month)

Location

Dosage

Size before treatment (cm2)

Size after treatment (cm2)

Grade

1 (Figure 6A) 2 (Figure 6B) 3 (Figure 6C) 4 (Figure 6D) 5 (Figure 6E) 6 (Figure 6F) 7 (Figure 6G)

Female Female Female Male Female Male Male

3 7 7 2 4 11 2

Neck Belly Neck Eyelid Back Head Head

2.5% 2.5% 2.5% 5% 5% 5% Control

0.7 0.4 1.8 1.0 0.9 0.8 1.3 0.7 2.5 1.1 0.6 0.3 2.3 3.7

0.4 0.3 1.2 0.6 0.3 0.2 clinical cure 1.6 0.8 clinical cure 2.4 3.8

II III IV IV II IV I


DOI: 10.3109/03639045.2014.931968

Figure 6. Photographs of superficial HI patients before (left) and after (right) treatment with PPLHCl gel. (A–C) treatment with 2.5% PPL HCl gel; (D–F) treatment with 5% PPL HCl gel and (G) treatment with control gel. More information was available in Table 4.

be related to that permeation of isopropanol into the SC can alter the solubility parameter value of the skin, making it more close to the terpenes with a consequent improvement for terpenes partitioning into the membrane30. Additionally, it is also feasible that the rapid permeation of isopropanol, or evaporative loss of this volatile solvent would modify the thermodynamic activity of


9

the drug within the formulation. As isopropanol is lost, the drug concentration can increase beyond saturated solubility, providing a supersaturated state with a greater driving force for permeation31. At last, isopropanol as a volatile solvent may extract some of the lipids from the SC when used at high concentration for a prolonged time. Though not an ‘‘enhancing’’ effect, such a mechanism would clearly improve drug flux through skin. However, when terpenes combined with propanediol, ER was found to be lower than the propanediol-free gel. One possible explanation for this is that the small-polar structure of propanediol would reduce the solubility of hydrophilic terpenes in gel and weaken their permeation enhancement. At last, effect of drug loading on the skin penetration of PPL HCl was studied. As shown in Figure 3, with an increase of the drug loading from 2.5% to 5%, the Q24 value increased drastically (p50.05). However, when drug loading was further increased to 7.5%, Q24 decreased rather than increased. The initial increase may be due to a higher concentration of PPL HCl with stronger diffusion dynamics. On the contrary, the decrease of Q24 by 7.5% PPL HCl gel was mainly attributed to the low pH condition in gel with more PPL HCl. Since the HPMC matrix is pH sensitive, the gel viscosity would increase at lower pH condition, resulting in slowing down the drug release rate from the gel and concomitantly a decrease of the Q24. Microdialysis is a main technique for evaluating drug absorption after transdermal administration. Actually, many reports have described in vivo penetration study of transdermal formulations using dermal microdialysis in rats32, pigs33, and even humans34. Microdialysis calibration is important when a quantitative fluid concentration of a drug is desired. Among the various methods, retrodialysis is the most common, which predicts the relative recovery of drug from the extracellular fluid to dialysate. In theory, drug recovery from tissue to perfusate is the same as the drug loss from perfusate to tissue across the probe membrane. However, in present study, in vitro recovery was slightly higher than delivery. This result indicates that there is some directional bias for drug in crossing the dialysis membrane. Therefore, in vivo recovery is calculated by scale conversion to eliminate this variation. Using the microdialysis method, drug concentration in the dermis was determined after transdermal and oral administration. For transdermal gel, drug penetrated into the skin and accumulated gradually, reaching peak concentrations at approximately 6.5 h after dosing. While after oral administration, drug accumulation in dermis was very low, and was eliminated from the target site quickly (Figure 4). The Cmax of PPL HCl in the skin after application of PPL HCl gel was 324 times higher than that with the oral route at the same dosage, but that in plasma was only approximately one fourth. These occurred because the orally administered PPL HCl reaches skin tissues via the systemic circulation, whereas the transdermally administered PPL diffuses directly into target tissues. Since most superficial IHs are located no deeper than the papillary dermis, the drug accumulation in dermis is crucial for their treatment. Although the mechanism of PPL in halting the proliferation of IH is unknown, dermis is speculated to be the site of action. After penetrating to dermis, PPL can downregulate angiogenic factors, and upregulate capillary endothelial cell apoptosis, which are responsible for reducing the size of the hemangiomas35. In this study, the PPL HCl gel formulation showed high concentrations of the drug in dermis with a low concentration in plasma, indicating a high efficacy for superficial IHs without any potential systemic side effects. However, for oral administration, drug concentrations in skin are much lower than that in plasma. Therefore, this transdermal gel should be a promising alternative route for PPL HCl application in the treatment of IH. In this study, it was also


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W. Zhou et al.

observed that the plasma AUC0–t of 2.5% PPL HCl gel was approximately 2 times lower than that of 5% PPL HCl gel, which means this 2.5% PPL HCl gel would be an alternate for patients who are prone to adverse reactions of PPL HCl in clinic. Due to the limited literature and research available on the impact of transdermal therapy of PPL HCl on superficial IHs, a precise protocol for its clinical use does not yet exist. In this study, the parents were advised to apply the gel twice a day because this is easy for medication and perhaps can decrease the risk of hypoglycemia. After treatment with the PPL HCl gel for superficial IHs, the vast majority (over 96%) of lesions respond to transdermal gel and for at least 21 of the 30 patients the treatment was clinically effective or completely healed the IH. Besides, the blank gel did not show apparent therapeutic effects at our experimental time points. Thus, the outcome of the clinical studies clearly demonstrated the impressive effects of the PPL HCl gel applied transdermally in the treatment of superficial IHs, which is in agreement with the observation from the study of Bonifazi et al.36 To study the dose–effect relationship of PPL HCl gel in patients, we applied transdermal PPL HCl at drug concentration of 2.5% and 5% to IHs. The response of superficial HIs treated with 5% PPL HCl gel was better than that of 2.5% gel, but no significant differences were found (Table 3, p40.05). Thus, the preliminarily recommended dose would be 2.5% PPL HCl gels, but more cases are needed for further evaluation. As compared with pretreatment, the size of IH was markedly reduced and the skin color changed and softened after treatment. Moreover, this treatment was well tolerated, with no adverse effects. This preliminary study shows that transdermal propranolol therapy, as an alternate or complimentary treatment tool, appears to have a beneficial effect on superficial IHs. In conclusion, the PPL HCl gel is an effective treatment for IHs and, at least in this study, it has a good safety profile, although large, long-term studies are needed to further understand the risk.

Conclusions In summary, a PPL HCl gel was developed as a transdermal drug delivery for treatment of superficial IHs. The transdermal PPL HCl gel was prepared by the direct swelling method with HPMC as the matrix and farnesol combined with isopropanol as the permeation enhancer. These permeation enhancers successfully increased the cumulative penetration of PPL HCl. After formulation screening and process optimization, PPL HCl gels were prepared with stable characteristics and quality. The in vivo dermal microdialysis technique was utilized to confirm that the drug penetrated into the skin from the PPL HCl gel efficiently without a high plasma drug level compared to oral solution. Clinical findings indicated that the PPL HCl gel appears to be a valuable and effective treatment option for superficial IHs in terms of the wonderful scores for the clinical efficacy without side effects. More comparative, randomized studies with a greater number of patients are still needed to confirm the safety and efficacy of the PPL HCl gel.

Declaration of interest This study was supported by the National Natural Science Foundation of China (No.81071290, X. Chen) and the National Outstanding Youth Fund (X. Chen). The authors report no declarations of interest.

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