Design of hydrogels of 5-hydroxymethyl tolterodine and their studies on pharmacokinetics, pharmacodynamics and transdermal mechanism

European Journal of Pharmaceutical Sciences 96 (2017) 530–541

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Design of hydrogels of 5-hydroxymethyl tolterodine and their studies on pharmacokinetics, pharmacodynamics and transdermal mechanism Wenhua Liu, Lirong Teng, Kongtong Yu, Xiangshi Sun, Chunyu Fan, Chaoxing Long, Na Liu, Shuang Li, Bing Wu, Qingji Xu, Fengying Sun ⁎, Youxin Li ⁎ School of Life Sciences, Jilin University, Qianjin Street No.2699, Changchun, Jilin Province 130012, China

a r t i c l e

i n f o

Article history: Received 11 August 2016 Received in revised form 28 September 2016 Accepted 20 October 2016 Available online 24 October 2016 Keywords: 5-Hydroxymethyl tolterodine (5-HMT) Hydrogels Pharmacokinetics Pharmacodynamics Transdermal mechanism

a b s t r a c t development, single-factor experiments were employed to evaluate the effect of adding different matrix, enhancers, 5-HMT, ethanol and glycerol on drug skin development, single-factor experiments were employed to evaluate the effect of adding different matrix, enhancers, 5-HMT, ethanol and glycerol on drug skin permeation. Finally, Carbopol 940 was selected as the gel matrix with N-methyl pyrrolidone (NMP) chosen as the enhancer. The relationship between time and the steady accumulative percutaneous amount (Q, μg cm−2) of optimized 5HMT hydrogels was Q4–12 h = 1290.8 t1/2 − 1227.7. The absolute bioavailability of 5-HMT hydrogels was 20.7% showed in pharmacokinetic study. No skin irritation was observed in 5-HMT hydrogels skin irritation study. In the pharmacodynamic study, the overactive bladder model was induced by 150 μg/kg of pilocarpine in rats. The significant effects of 5-HMT hydrogels on the inhibition of urine output on rat model were last to 12 h. The optimized 5-HMT hydrogels displayed prolonged pharmacological responses. 5-HMT hydrogels effectively avoided the metabolism difference of enzyme in bodies compared with tolterodine tablets, provided one single active compound in plasma to reduce the variability of having two active compounds. To further elucidate the transdermal mechanism, fourier transform infrared (FTIR) spectroscopy, differential scanning calorimeter (DSC) and activation energy measurements were used to study the transdermal routes and changes of stratum corneum during drug release. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Tolterodine is widely used for the treatment of urinary incontinence and other overactive bladder (OAB) symptoms (Andersson et al., 1998). After oral administration, tolterodine is rapidly absorbed through the gastrointestinal tract and metabolized in the liver from 5-methyl group to 5-hydroxymethyl tolterodine (5-HMT) under the oxidation action of CYP2D6 in extensive metabolisers (EMs) (Dmochowski and Appell, 2000). 5-HMT is the major pharmacologically active metabolite of tolterodine and exhibits an anti-muscarinic activity similar to tolterodine (Abrams et al., 1998). Both tolterodine and the 5-HMT metabolite display a high specificity for muscarinic receptors. The 5-HMT concentration in human serum is 10 times higher than tolterodine among the EMs. However, approximately 7% of patients can be classified as poor metabolisers (PMs) because of the lack of CYP2D6 enzyme (Nilvebrant et al., 1997). Among the PMs, only a little part of tolterodine are metabolized to 5-HMT. The tolterodine concentration is 10-fold higher than that of 5-HMT, leading to a poor therapeutic efficacy

⁎ Corresponding authors. E-mail addresses: [email protected] (F. Sun), [email protected] (Y. Li).

http://dx.doi.org/10.1016/j.ejps.2016.10.024 0928-0987/© 2016 Elsevier B.V. All rights reserved.

(Pahlman and Gozzi, 1999). In addition, there are various equilibrium concentrations between tolterodine and 5-HMT, resulting in difference in therapeutic efficacy and side effects. The EMs/PMs difference may be eliminated through direct administration of 5-HMT. However, 5-HMT is not suitable for oral administration due to increasing hydrophilicity compared with tolterodine and reduced absorption in gastrointestinal tract. Meanwhile, both tolterodine and 5-HMT lack functional selectivity and have systemic side effects, which limit their therapeutic effects (Sun et al., 2010). Therefore, there is a great interest to develop a suitable dosage form of 5-HMT to provide relatively consistent drug levels at the application site and avoid the oral side effects. Currently, tolterodine patches and nanosized microemulsion have been reported. Due to the complicated technologies, their application was limited. A 24 h of transdermal administration of tolterodine shows a flatter serum concentration-time profile, accompanying by a low bioavailability (Jacobsen et al., 2003). However, few researches have been reported on the application of 5-HMT. Until recently, 5HMT-loaded PLGA microsphere has been developed that provides a sustained release of drug with a steady serum drug concentration (Teng et al., 2013). However, PLGA can biodegrade into water and carbon dioxide in vivo that changes the pH value of microenvironment and leads to inflammatory response (Wang et al., 2015). Hydrogels

W. Liu et al. / European Journal of Pharmaceutical Sciences 96 (2017) 530–541

have been frequently used in drug delivery due to its high water content and analogous natural living tissue more than any synthetic biomaterials (Liu et al., 2014). For instance, Gelnique™ (oxybutynin chloride) 10% gel was first approved by Food and Drug Administration (FDA) in 2009 for the treatment of OAB with the symptoms of urge urinary incontinence, urgency, and frequency (Staskin and Robinson, 2009). In this study, 5-HMT hydrogels were designed to provide steady plasma 5-HMT levels and pharmacokinetical and pharmacodynamical studies were conducted to evaluate their performance. Single-factor experiments were applied in ideal formulation screening to provide steady plasma drug levels with daily administration. Skin irritation experiment was conducted to evaluate biocompatibility of hydrogels. Fourier transform infrared (FTIR), differential scanning calorimeter (DSC) and activation energy measurement were used to elucidate transdermal mechanism such as transdermal routes and changes of stratum corneum during drug release (Lee et al., 2005; Narishetty and Panchagnula, 2004).

2. Materials and Methods 2.1. Materials 5-Hydroxymethyl tolterodine (5-HMT) was synthesized in our laboratory. Carbopol 934, 940 and 980 were purchased from Shanhe Pharmaceutical Excipients Co., Ltd. (Anhui, China). Pilocarpine was purchased from Sigma-Aldrich (St. Louis, MO, USA). All organic reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. All chemicals were of chromatographic grade. Healthy Kunming female mice weighing 18–22 g, healthy male Sprague Dawley (S.D.) rats weighing 180–220 g and healthy New Zealand male rabbits with weights ranging from 1.5 kg to 2.0 kg were provided by Experimental Animal Center of Jilin University, China. The animals were kept in plastic cages in a room at a temperature of 20 ± 4 °C, with a standard 12/12 light-dark cycle. Food and water were freely available. All experiments were performed under the Guidelines for Animal Experiments, Jilin University, China.

2.2. Synthesis and Characterization of 5-HMT 5-HMT was synthesized according to the method of (Teng et al., 2013). Carboxyl acid 1 was methyl esterified with a catalytic amount of H2SO4, then reduced to alcohol 3 by LiAlH4. After alcohol 3 was tosyl-protected, diisopropylamine 5 was obtained by substituting 4 with diisopropylamine. Compound 5 was converted to acid 6 via Grignard-reaction with CO2. Alcohol 8 was obtained by methyl esterification and reduction, similar to the process of preparing 3 from 1. Finally, the benzyl-protection group was removed by Raney-Ni/H2, and 5-HMT was prepared. The partition coefficient (P) of 5-HMT between epidermis and saline was determined with abdominal skin of mice. New excised abdominal skin was washed with saline solution, then placed in PBS (0.25% trypsin) and incubated for 6 h at 37 ± 1 °C. Then epidermal and dermal tissue was separated using scraper. The excised epidermal tissue was washed with deionized water repeatedly and dried in the air. The dried skin was dealt with 5% azone, oleic acid, linoleic acid and NMP for 2 h, respectively. Then the skin was rinsed with ethanol. 30 mg of handled skin was put in culture dish with the addition of 50 μg/mL of 5-HMT in saline. The culture dish was placed in constant temperature bath (37 ± 1) °C for 12 h. The drug content was analyzed by HPLC. The partition coefficient (P) was calculated as follow: content per mg in epidermis P ¼ drug drug content per mg in saline

531

Table 1 Formulations design of 5-HMT hydrogels (n = 6). Batch Carbopol 5-HMT type content (%)

Ethanol content (%)

Glycerol content (%)

Azone Oleic (%) acid (%)

NMP (%)

A B C D E F G H I J K L

20 20 20 20 20 20 10 30 20 20 20 20

10 10 10 10 10 10 10 10 5 15 10 10

– – – 5 – – – – – – – –

– – – – – 10 10 10 10 10 10 10

934 940 980 940 940 940 940 940 940 940 940 940

2 2 2 2 2 2 2 2 2 2 1 3

– – – – 5 – – – – – – –

2.3. Preparation of 5-HMT Hydrogels Hydrogels were prepared according to the formulas shown in Table 1. Carbopol at a concentration of 1.5% (w/w) was dispersed in deionized water and swelled overnight to realize sufficient hydration. Glycerol was added into the swelling hydrogels. 5-HMT with or without additive was dissolved in ethanol with the addition of penetration enhancers (10%, w/w), followed by adding into the water phase matrix. Triethanolamine was added to adjust pH value of hydrogels, and then deionized water was added to give a total weight of 10 g. The hydrogels were equilibrated at room temperature for 24 h prior to use.

2.3.1. Single-factor Experiment Carbopol 934, 940 or 980, with different dynamic viscosity, were used to investigate the effects of matrix on drug transdermal flux. Azone, oleic acid and N-methyl pyrrolidone (NMP), were used to investigate the kinds and different penetration mechanism of penetration enhancers on drug transdermal flux. Furthermore, formulation factors (the content of 5-HMT, ethanol and glycerol) were optimized by evaluating their influence on drug transdermal flux. Statistical analysis was performed on Design Expert software with an ANOVA test. The optimized gel formulation was based on the maximum skin penetration, which was selected for the transdermal mechanism studies and the in vivo studies.

Table 2 Cumulative penetration amounts of 5-HMT hydrogels through the skin (n = 6). Batch

2

Q (μg/cm ) A B C D E F G H I J K L

J (μg/cm2 h1/2)

Cumulative penetration

Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12 Q4–12

h h h h h h h h h h h h

= = = = = = = = = = = =

87.3 t1/2 − 58.04 117.2 t1/2 + 42.52 31.79 t1/2 + 442.4 855.7 t1/2 − 827.6 22.157 t1/2 + 38.817 914.8 t1/2 − 633.9 808.2 t1/2 − 803.7 719.4 t1/2 − 300 850.6 t1/2 − 745.2 818.9 t1/2 − 256.7 744.6 t1/2 − 897 1290.8 t1/2 − 1227.7

2

R

0.998 0.969 0.919 0.999 0.997 0.995 0.996 0.994 0.998 0.989 0.995 0.999

87.3 117.2 31.79 855.7 22.157 914.8 808.2 719.4 850.6 818.9 44.6 1290

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2.4. Hydrogels Characterization 2.4.1. Viscosity The viscosities of hydrogels were determined at 25 °C with a digital viscometer (SNB-1, Jingmi, Shanghai). 50 mL of hydrogels were placed in the test tub, and the dynamic viscosity was recorded with a 4 rotor at 6 rpm and 12 rpm, respectively. All measurements were repeated three times. 2.4.2. Stability The cold-resistant experiment, heat-resistant experiment and pH test were conducted to evaluate the stability of drug loaded hydrogels. 5-HMT hydrogels were packed into preservative film to avoid contacting with air, and placed at the atmosphere of (60 ± 2) °C and (− 15 ± 2) °C, respectively. After 24 h, the hydrogels were taken out and returned to room temperature. The drug loading and pH value were measured by HPLC and pH meter respectively after dissolution. The drug loading and pH variation were used to estimate the stability of hydrogels. The room temperature stability test was also carried out to evaluate the stability. 5-HMT hydrogels were packed into preservative film and placed at room temperature for 1, 2 and 3 months, respectively. Then hydrogels were taken out and the drug loading was determinated. The appearance and drug loading were used to estimate the stability of hydrogels. 2.5. Skin Preparation 2.5.1. Preparation of Full Thickness Skins The skin was prepared using a method reported earlier (Fang et al., 2008). Kunming mice (18–22 g) and S.D. rats (180–220 g) were sacrificed through cervical fracture, and full thickness abdominal skin was excised. Hairs on the abdominal skin of mice were removed using surgical scissors and then subcutaneous fat were carefully scraped using blades. The skin was scrubbed with isopropyl alcohol to remove residual fat and finally washed with saline solution. 2.5.2. Preparation of Split Thickness Skins Full abdominal skins were obtained as the above method. Epidermis was separated from the full skins after treatment with 1 M sodium bromide solution for 4 h. The other part was dermis. The stratum corneum was obtained by treating the epidermis with 0.1% (w/w) trypsin solution at (37 ± 1) °C for 12 h (Panchagnula et al., 2001). The freshly prepared epidermis, dermis and stratum corneum were cleaned with deionized water and kept at 4 °C until use.

283 nm. Cumulative release amount Q of 5-HMT was calculated as follow: Q¼

n X

5ci

i¼1

where ci represents 5-HMT concentration at time i. Cumulative release rate f was calculated as follow: f ¼

Q H

where H represents the amount of 5-HMT contained in 0.06 g gel. 2.7. Skin Irritation Evaluation Six New Zealand white rabbits were anesthetized through injection with pentobarbital sodium (30 mg/kg, i.m.) 24 h prior to experiment. Hairs on the backside area were removed by hair clippers (a circle area with a radius of 3 cm) and washed with deionized water. 5-HMT hydrogels (Table 1, batch L) were applied on the left side skin surface at the dosage of 20 mg/kg three times a day for 7 days. The response of the 5-HMT hydrogels applied on the left skin surface was observed in comparison with to the right side under the natural light. The primary irritation index (PII) was calculated (Table 3) and assessed according to response category table (Table 4). 2.8. In Vivo Pharmacokinetic Study In vivo pharmacokinetic study was conducted on New Zealand white rabbits, weighing 1.5–2.0 kg. Food and water were not restricted during the study (Sun et al., 2013). Hairs on the dorsal skins were gently removed by hair clippers without causing any skin damage, and the treated site was marked in a circle area with a radius of 4 cm. The rabbits were i.v. injected with 5-HMT aqueous solution at a dose of 20 mg/kg and administered onto the skin with 5-HMT hydrogels at a dose of 20 mg/kg (Table 1, batch L). Blood samples were collected from the ear vein at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h. The blood samples were collected into a tube containing 5 μL of heparin and centrifuged at 13,000 rpm for 10 min immediately. Subsequently, 200 μL of separated plasma, 100 μL of internal standard solution of diphenhydramine hydrochloride at the concentration of 1.5 ng/mL in methanol-water (1:1, v/v) together with 100 μL of Na2CO3 aqueous (0.1 M) were mixed into a tube.

2.6. In Vitro Skin Permeation In vitro skin permeation experiments were carried out using Franztype diffusion cells (Rightway, Huizhou). The skin was mounted between the receptor compartment and donor compartment with the stratum corneum side facing upwards into the donor compartment. The donor side was filled with 0.1 g of 5-HMT hydrogels, and the receiver side was filled with 5 mL of pH 7.4 PBS. The available diffusion area between two sides was 0.636 cm2. The incubation temperature was 32 ± 0.5 °C, and stirring rate was 200 rpm. At sampling times, 5 mL of the release medium was collected and replaced with the same volume of fresh PBS (Park et al., 2012). The concentrations of 5-HMT were determined by high performance liquid chromatography (HPLC, Waters, USA) equipment with a Waters 600 pump and a Waters 2487 Dual Absorbance Detector under the following conditions: Agilent XDB C18 column (250 × 4.6 mm, 5 mm) with a pH 3.0 acetate buffer-methanol (50:50, v/v) as the mobile phase at a flow-rate of 1 mL/min. The chromatography was carried out at 40 °C and the detection wavelength of

Table 3 Score standards on skin irritation reaction. Skin irritation reaction Erythema No erythema Very slight erythema (barely perceptible) Well-defined erythema Moderate to severe erythema Severe erythema (beet redness) to eschar formation Edema No edema Very slight edema (barely perceptible) Well-defined edema (edges of the area well defined by definite raising) Moderate edema (raising approximately 1 mm) Severe edema (raised N1 mm and extending beyond the area of exposure)

Primary irritation index (PII) 0 1 2 3 4

0 1 2 3 4

W. Liu et al. / European Journal of Pharmaceutical Sciences 96 (2017) 530–541 Table 4 Evaluation standards on skin irritation intensity. Category

Primary irritation index (PII)

Negligible Slight irritation Moderate irritation Severe irritation

0–0.4 0.5–1.9 2.0–4.9 5.0–8.0

The mixed solution was extracted with an n-hexane: methylene dichloride: isopropanol solvent (300:150:15, v/v/v) and vortexed for 3 min. After centrifugation at 13,000 rpm for 10 min, the organic layer was transferred into a new tube and dried under a nitrogen atmosphere. The residue was reconstituted in 150 μL mobile phase and kept at − 80 °C until analysis. The concentrations of 5-HMT were measured using a liquid chromatography tandem mass spectrometry (LC/MS) equipped with an HPLC (Agilent 1100, Agilent Technologies, USA) and an triple quadrupole mass spectrometer (Sciex API 4000, Applied Biosystems, Canada). 2.9. Pharmacodynamic Studies 2.9.1. Effects of Pilocarpine on Voided Volume The voided volume test was performed on pilocarpine-induced bladder overactivity in male rats (Sun et al., 2010). Two groups of male rats (n = 8) were i.p. injected with saline 75 μg/kg and 150 μg/kg pilocarpine before the experiment (Sun et al., 2013). The rats were given water (10 mL/kg) 15 min before the experiment. The voided volume and urinary output were recorded at 2, 4, 6, 8, 10 and 12 h. A little water was added into the collection bottles to reduce the experiment error resulting from the evaporation of urine. 2.9.2. Effects of 5-HMT Hydrogels on Pilocarpine-induced Bladder Overactivity Four groups of male rats (n = 8) were treatment with saline (i.p.)/ saline (p.o.), pilocarpine (150 μg/kg, i.p.)/saline (p.o.), pilocarpine (150 μg/kg, i.p.)/tolterodine tablets (0.4 mg/kg, p.o.), pilocarpine (100 μg/kg, i.p.)/5-HMT hydrogels (1.5 mg/kg, Table 1, batch L) with a 15 min interval between two injections. The rats were given water (10 mL/kg) 15 min before the experiment. Then the rats were placed into the cages with autonomic monitoring apparatus. The voided volume and urinary output were recorded at 2, 4, 6, 8, 10 and 12 h, respectively. Furthermore, the urine was collected and the Na+ and K+ contents in urine were determined by a semi-automatic biochemistry analyzer.

NMP and hydrogels for 24 h at 32 ± 0.5 °C, respectively. The stratum corneum was blot up with filter paper to attain 20–25% hydration. 100 mg of prepared samples were scanned on a DSC (DSC 1, Mettler Toledo, Switzerland). The temperature range and heating rate were controlled at 10 °C/min from 0 °C to 200 °C under nitrogen atmosphere (Shakeel et al., 2008). Each sample was measured in triplicate. 2.11.2. FTIR Spectral Analysis Fourier transform infrared (FTIR) spectroscopy was conducted to analyze the stratum corneum treatment with or without hydrogels. The stratum corneum was obtained and pre-treated with saline (0.01% w/v sodium azide) and soaked in it for three days. The stratum corneum was blot up with filter paper and the FTIR spectra were recorded on spectrophotometer (IR Prestige-21, Shimadzu, Japan). Then the stratum corneum was administrated with ethanol, triethanolamine, NMP and optimized hydrogels and proceeded permeation study, following by washing and drying. FTIR spectra of the stratum corneum treatment with hydrogels were recorded again. For sampling, the tissue fragments of stratum corneum were pressed with potassium bromide to make a pellet by applying a pressure of 300 kg/cm2. The spectra were detected in KBr disks over a range of 4000–400 cm− 1. 2.11.3. Activation Energy Measurement In vitro skin permeation study of 5-HMT hydrogels was carried out at 27 °C, 37 °C and 47 °C. The activation energy of 5-HMT was calculated as the method reported previously (Golden et al., 1986). 2.12. Statistical Analysis Each experiment was repeated for three times. All the data was represented by the mean value ± standard. Student's t-test was used to identify significant differences with a p b 0.05. 3. Results and Discussion 3.1. 5-HMT Characterization 5-HMT was synthesized in our laboratory and X-ray diffraction (XRD) has been used to identify its purity in our early reports (Teng et al., 2013). The partition coefficient (P) of 5-HMT between epidermis and saline was shown in Table S1. After dealing with 10% azone, the P of 5-HMT got smaller (p b 0.05) in epidermal tissues, which made it easy for 5-HMT to get through the epidermal tissues, and then enter into the body. On the contrary, 10% oleic acid and NMP (p b 0.05)

2.10. Histological Examination Histomorphology of rat bladder biopsies after treatment with or without hydrogels was performed to evaluate the biocompatibility of 5-HMT hydrogels. The rats were sacrificed after treatment for 24 h. The bladder sections were collected and fixed with 10% formaldehyde solution for 72 h. The samples were dehydrated, embedded in paraffin wax and stained with hematoxylin and eosin (H&E) for histological observation. 2.11. Transdermal Mechanism Studies 2.11.1. DSC Analysis The stratum corneum was obtained from mice and rats and equilibrated in oversaturated potassium sulphate solution for three days. Then the stratum corneum was treated with ethanol, triethanolamine,

533

Fig. 1. Chemical structure of 5-HMT.

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Fig. 2. Morphological observations of transparent 5-HMT hydrogels after room temperature stability test: batch D, batch H and batch L.

increased the P of 5-HMT in epidermal tissues, and thus promoted skin retention rates (Fig. 1). 3.2. Viscosity and Stability To investigate the mechanical properties of 5-HMT hydrogels, the viscosities of hydrogels were measured. Viscosity played an important role in the topical formulation. Low viscosity would increase skin adhesion difficulties (Onuki et al., 2005). All formulations displayed a suitable viscosity between 88.50 ± 0.26 and 92.30 ± 0.70 Pa s and moderate flow ability (Table S2). We randomly selected three batch (D, H and L) to investigate their stability in room temperature and extreme environment. The drug loading and pH value was monitored before and after the experiments (Table S3). The pH value of three batches was almost unchanged no matter in cold-resistant or heat-resistant experiments. However, in heat-resistant test, the drug loading significantly increased with a pvalue b 0.01 which might attribute to the evaporation of water and ethanol within hydrogels. After placing at room temperature for 3 month, the appearance of 5-HMT hydrogels was still transparent with no stratification and the drug loading was almost unchanged (Fig. 2 and Table S4). 3.3. Formulations Screening and In Vitro Percutaneous Permeation The in vitro transdermal permeation of 5-HMT hydrogels was measured over 12 h in mice (Changez et al., 2006). The relation of cumulative permeating amounts of 5-HMT (Q, μg cm − 2 ) against time (h) was shown in Fig. 3. Carbopol 934 based hydrogels showed a low release profile. Carbopol 980 based hydrogels displayed an initial burst release. Carbopol 940 based hydrogels revealed a relatively constant release. Results indicated that the gel matrix Carbopol 940 was a good candidate for the delivery of 5HMT, giving a more steady release than Carbopol 934 and 980 (p b 0.05) (Fig. 3a). The effects of 10% azone, oleic acid and NMP on the skin permeation of 5-HMT were evaluated in comparison with the hydrogels without enhancers. The azone and NMP groups increased transdermal absorption of 5-HMT compared with the 5-HMT hydrogels without enhancers (p b 0.001) (Fig. 3a) or oleic acid groups (p b 0.001) (Fig. 3b). Stratum corneum of skin was the greatest barrier against drug transdermal penetration. The polar molecule of NMP might competitively combine hydrogen bond, destruct ordered structure of stratum corneum, and thus reduce the diffusion resistance of drug in the skin (Wen et al., 2009).

Azone acted through destroying the ordered arrangement of lipid layers and increasing the liquidity of membrane, and thus promoted drug permeation (Xu and Zhu, 2007). Oleic acid would change the oil-water partition coefficients of drug and influence the permeation procedure (Touitou et al., 2002). The 5-HMT hydrogels with 10% NMP as enhancer displayed the maximum Q and permeated significantly. Specific transdermal mechanism would be discussed in the next section. Three other important factors, the content of drug, ethanol and glycerol, were optimized (Fig. 3c–e). Ethanol was used as solubilizer, anticorrosive agent and accelerant during transdermal absorption. Three ethanol contents (10%, 20% and 30%) were evaluated their effect on release profile. High or low ethanol content would bring relatively fast release. We inferred that high ethanol content increased the drug solubility. However, low ethanol content accelerated water evaporating, both of which would increase drug concentration and promote drug permeating. The median could accelerate drying of gel to form a skim, thereby maintained internal water content and hydration degree of skin. Glycerol as a common humectant was added into the hydrogels formulation to prevent moisture loss and keep the formulation soft (Gwon et al., 2010). Three additive amounts of glycerol (5%, 10% and 15%) were investigated for their influence on permeating amounts (Fig. 3d). Similarly, high glycerol content would hold back water evaporating, excessive hydration would prevent drug release. Low glycerol content would increase skin adhesion difficulties. Suitable glycerol content would adjust the viscosity of hydrogels, thus decreased diffusion resistance and promoted drug release. Without doubt, high drug loading could surely lead to a high diffusion concentration after administration. The hydrogels with high drug loading would attain higher transdermal delivery rate and amounts of drug (p b 0.01), which would reduce the administration area. However, excessive drug loading not only produced drug precipitation but also aggravated burst release degree. Statistical analysis of release profile was shown in Table 2. The drug release kinetics of twelve formulations followed zero-order release pattern, with R2 N 0.95. Batch L possessed the maximum Q and maximum steady state flux [J, μg/(cm2 h1/2)]. Thus, 1.5% Carbopol 940, 10% NMP, 20% ethanol, 10% glycerol and 3% 5-HMT were chosen as the optimized hydrogels formulation. 3.4. Skin Irritation Evaluation Erythema and edema were checked on test areas of three rabbits after applying with 5-HMT hydrogels at the seven days. And no erythema and edema were found on the three rabbits. Results indicated 5-HMT hydrogels had no skin irritation (Table 5). 3.5. In Vivo Pharmacokinetic Study The pharmacokinetic studies were carried out on New Zealand white rabbits (weight 1.5–2.0 kg) using 5-HMT hydrogels (batch F). The plasma concentration vs. time profiles of 5-HMT after administrations of 5-HMT (20 mg/kg, intravenous injection) and 5-HMT hydrogels (20 mg/kg, topical), respectively, were showed in Fig. 4. The corresponding pharmacokinetic parameters of rabbit plasma were summarized in Table 6. After i.v. injection of 5-HMT, the C max of 5-HMT was 2703 ± 625 ng/mL and the drugs were exhausted after 12 h. While, the C max for 5-HMT hydrogels was 45 ± 41 ng/mL and 5HMT released from hydrogels lasted over 48 h. It indicated that topical administration of 5-HMT prolonged the treatment time. Tolterodine could be metabolized to the pharmacologically metabolized 5-HMT via CYP2D6. Due to differences in metabolic capacity, the absolute bioavailability of tolterodine could vary a great deal. Compared with tolterodine hydrogels, 5-HMT hydrogels effectively avoided the metabolism difference of enzyme in bodies and

W. Liu et al. / European Journal of Pharmaceutical Sciences 96 (2017) 530–541

535

Fig. 3. Cumulative permeating amounts vs. time profiles of 5-HMT hydrogels with different Carbopol (a); enhancers (b); 5-HMT content (c); ethanol content (d) and glycerol content (e) in vitro (mean ± S.D., n = 3).

provided a single active compound in plasma, which might provide security for patients under controllable dose and thus lowered potential adverse effect of two active compounds. The absolute

3.6. In Vivo Pharmacodynamic Study

Table 5 Reaction on skin and score of 5-HMT hydrogels.

Erythema Edema

bioavailability for 5-HMT hydrogels reached 20.7%, which was almost 2-fold higher than that of tolterodine hydrogels reported previously (Sun et al., 2013).

1

2

3

X

0 0

0 0

0 0

0 0

Effects of pilocarpine on voided volume in rats were shown in Fig. 5. 150 μg/kg of pilocarpine increased appreciable overactivity of the rat urinary bladder compared with control group (p b 0.05), which explained rats modeling was successful.

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W. Liu et al. / European Journal of Pharmaceutical Sciences 96 (2017) 530–541

Fig. 4. Plasma concentration vs. time profiles for 5-HMT after administration of 5-HMT (20 mg/kg, intravenous) and 5-HMT hydrogels (20 mg/kg, topical). Each point represents mean ± S.D. (n = 3).

The in vivo pharmacological response vs. time profiles in rat treatment with 5-HMT and 5-HMT hydrogels was shown in Fig. 6. The cumulative urinary output (8.16 ± 1.57) within 12 h was higher than that of the control group (5.15 ± 0.83), which indicated that pilocarpine-induced bladder overactivity rat model was established successfully. After p.o. administration of tolterodine tablets (0.4 mg/kg), the contractions of the rat urinary bladder were improved. The cumulative urinary output was reduced from 6.08 ± 0.73 to 4.11 ± 1.00 (p b 0.05) within 8 h and from 8.16 ± 1.57 to 4.90 ± 0.92 (p b 0.05) within 12 h. After topical administration of 5-HMT hydrogels (Table 1, batch L) at a dose of 1.5 mg/kg, the cumulative urinary output was reduced to 1.99 ± 1.09 (p b 0.05) within 6 h and 3.12 ± 1.45 (p b 0.01) within 12 h. 5HMT hydrogels displayed better pharmacological responses than tolterodine tablets, which was identical with pharmacokinetic studies. Meanwhile, there was no significant difference in the contents of urinary sodium and potassium between the 5-HMT hydrogels group and the control group, either (p N 0.05).

Fig. 5. Effects of pilocarpine on voided volume in rats. Each point represents mean ± S.D. (n = 8). *Represents p b 0.05 and **represents p b 0.01 vs. control group.

cholesterol. The melting peaks of lipids in stratum corneum distributed from 90 °C to 120 °C (Figs. 9 and 10). Untreated stratum corneum revealed the sharpest melting peak. The melting peaks of stratum corneum treatment with ethanol, triethanolamine, NMP and 5-HMT hydrogels shifted to lower melting points and turned down. DSC results

3.7. Histological Examination The histological examination results of bladder were shown in Fig. 7. The normal bladder tissue contained 4–5 layers of urothelium cells, a layer of lamina propria mononuclear cells and smooth muscle cells full of longitudinal and transversal fibers. There were no pathological changes in bladder biopsies after treatment with and without hydrogels. The Na + and K + contents in urine were also measured and the results suggested that they decreased slightly (p N 0.05). 3.8. Transdermal Mechanism Study 3.8.1. DSC Analysis The skin structure of rats was shown in Fig. 8. The stratum corneum of mice and rats was mainly made up of free fatty acids, ceramides and

Table 6 Pharmacokinetic parameters of rabbit plasma (mean ± S.D., n = 3). Parameters

Tmax (h)

Cmax (ng/mL)

AUC0–48 h (ng h/mL)

Absolute BA (%)

5-HMT (i.v.) 5-HMT hydrogels

0.25 4.00

2703 ± 625 45 ± 41

4595 953

– 20.7

Fig. 6. Effects of 5-HMT and 5-HMT hydrogels (batch L) on the respective urinary output (a) and cumulative urinary output (b) in rats. Each point represents mean ± S.D. (n = 8). *Represents p b 0.05 and **represent p b 0.01 vs. pilocarpine group.

W. Liu et al. / European Journal of Pharmaceutical Sciences 96 (2017) 530–541

537

Fig. 7. Histological photomicrographs of rat bladder biopsies after treatment with and without hydrogels. Original magnification: 100×.

suggested that 5-HMT hydrogels increased transdermal flux of drug by effecting ceramides and cholesterol status in stratum corneum, whose melting points distributed N 100 °C. The ethanol, triethanolamine, NMP together influenced the permeation effect. The order effect of influence factors was 5-HMT hydrogels N NMP N triethanolamine N ethanol in the stratum corneum of both mice and rats. We speculated the alcohols such as ethanol and triethanolamine could extract the intercellular lipid in stratum corneum (Pillai et al., 2004). NMP, as a typical enhancer, acted the most. It might lower the orderliness of lipid in stratum corneum and thus increase the flowability of lipid. The intrinsic chemical bonds might be broke and new hydrogen bonds might form during percutaneous absorption process of drug (Jain et al., 2002). Furthermore, considering about the partition coefficient (P) of 5-HMT between epidermis and saline (azone b oleic acid b NMP) in Section 3.1 and in vitro permeation results (NMP N azone N oleic acid) in Section 3.3, we draw a conclusion that oil-water partition coefficients would not decide permeation effect of drug (Manganaro

Fig. 8. Skins structure of rats, including epidermis, dermis and subcutaneous tissue.

and Wertz, 1996; Nicolazzo et al., 2004). The drug permeation behaviour in skin was influenced by numerous factors such as skin structure, skin component, structure of enhancers and drug formulation (Nicolazzo et al., 2005). 3.8.2. FTIR Analysis The C\\H and O\\H stretching peaks of proteins and lipids in stratum corneum mainly appeared in the wave number of 3000– 2700 cm− 1. The peaks appearing between 1700 cm− 1 and 1500 cm− 1 belonged to amide I and amide II bands, respectively. It could be seen that there were obvious differences in the stratum corneum of mice before and after treatment with triethanolamine and 5-HMT hydrogels, which might be due to the change of hydration bonds of lipid during drug entering into the stratum corneum (Fig. 11). Moreover, little difference was observed in the wave number of 1700–1500 cm− 1. It indicated that ceramide changed a little. However, there was no significant variation between the untreated and treated stratum corneum of rats (Fig. 12). This might be attributed to the high degree of hydration of stratum corneum, which covered up other absorption peaks in infrared spectra. DSC together with the FTIR results suggested that 5-HMT hydrogels increased transdermal flux of drug through altering lipid status in stratum corneum. 3.8.3. Activation Energy Measurement Diffusional parameters about activation energy were shown in Table 7. Cd represented the 5-HMT concentration in diffusion cells. It could be seen that the steady-state flux (J) increased with the increasing of temperature. The activation energy was 68.64 kcal/mol (conversion into human epidermis 7.63 kcal/mol) for 5-HMT hydrogels (batch L), which was lower than activation energy for ion transport across human epidermis (10.7 kcal/mol) (Schneider et al., 2013; Pagano and Thompson, 1968). It proved that the rate of transdermal delivery depended on rate of delivery lipophilic part. The drug permeating

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Fig. 9. DSC thermograms: variation of lipids melting point of stratum corneum of mice treated with ethanol, triethanolamine, NMP, hydrogels and untreated.

might disturb the hydrogen bonds of tight ceramides structures, which was in accordance with DSC and FTIR results (Bouwstra et al., 2003). 4. Conclusion Carbopol 940 as gel matrix, 10% NMP as chemical enhancer, 20% ethanol, 10% glycerol as good solvent and 3% 5-HMT together formed the optimized hydrogels formulation with sustained percutaneous drug release profile showed in in vitro and in vivo studies. 5-HMT hydrogels significantly improved the therapeutic effect, avoided the metabolism difference of enzyme in bodies and provided single active compound

in plasma. Compared with tolterodine hydrogels in early report (Sun et al., 2013), it realized effective dose control and reduced the potential adverse effect from two active compounds. 5-HMT hydrogels appeared to be a promising new therapeutant for the treatment of overactive bladder.

Acknowledgements This work was supported by the Central Lab of General Biology of Jilin University (No.20140311072YY).

Fig. 10. DSC thermograms: variation of lipids melting point of stratum corneum of rat treated with ethanol, triethanolamine, NMP, hydrogels and untreated.

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Fig. 11. FTIR spectral analysis of stratum corneum of mice treatment without (a) and with ethanol (b), triethanolamine (c), NMP (d) and hydrogels (e).

539

540

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Fig. 12. FTIR spectral analysis of stratum corneum of rat treatment without (a) and with ethanol (b), triethanolamine (c), NMP (d) and hydrogels (e).

Table 7 Diffusional parameters of activation energy on mice skins. T (°C)

Js (μg cm−2 h−1)

D/h2 (×10−4 h−1)

Cd (×104 μg mL)

Ps (×10−4 cm h−1)

Ea (kcal/mol)

27 37 47

238.2 313.5 412.9

8.52 6.65 5.22

3 3 3

79.4 104.5 137.6

68.64

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