Enhanced aqueous solubility and bioavailability of ...

Analgesics · Anti-inflammatories · Antiphlogistics · Antirheumatic Drugs

Enhanced aqueous solubility and bioavailability of capsaicin by the preparation of an inclusion complex Xiaoyu Chen1, Xun Sun1, Ke Ren1, Xiaoning Zhang2, Zhirong Zhang1, Tao Gong1 1

2

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, P. R. China School of Medicine, Tsinghua University, Beijing, P. R. China

Correspondence to: Tao Gong, Key Laboratory of Drug Targeting and Drug Delivery Systems, Sichuan University, No. 17, Section 3, Renmin South Road, Chengdu, Sichuan 610041, P. R. China; e-mail: [email protected]

Abstract tion with HP-b-CD, due to its solubilizing activity. The absorption behavior and bioavailability of capsaicin and its complexation were evaluated after their subcutaneous (s. c.) injection in rats. The absorption rates of capsaicin across subcutaneous tissues and its bioavailability were significantly enhanced by the formation of an inclusion complex with HP-b-CD. The present results indicate the potential of utilizing HP-b-CD to improve the subcutaneous absorption and bioavailability of capsaicin.

1. Introduction Capsaicin (8-methyl N-vanillyl-6-nonenamide, CAS 40486-4) (Fig. 1) a major pungent agent present in various species of capsicum fruits, is used to treat various diseases such as rheumatoid arthritis, osteoarthritis, diabetic neuropathy, postherpetic neuralgia and trifacial neuralgia [1]. To date, the most reported dosage forms of capsaicin were gel and ointment for topical application [2 – 3]. However, high concentrations of capsaicin were found in bone, not in brain and spinal cord, after topical application [4], which has limited its therapeutic effects on the nervous systems. Moreover, it has been established that high concentrations of capsaicin were found in brain and spinal cord by intravenous and subcutaneous administrations [5]. Therefore, to improve the curative effect of capsaicin on the nervous system, intravenous and subcutaneous injections are administered. But the aqueous solubility of capsaicin is quite poor, which can be only dissolved in 0.9 % NaCl with 10 % ethanol and 10 % Tween 80 [5]. However, the content of Tween 80 in injections should be limited from 0.5 % to 4 % according to the pharmacopoeia of People’s Republic of China 2005 (CP2005). Moreover, both ethanol and Tween 80 are stimulants. Therefore, it was neArzneimittelforschung 2010;60(9):571–574 © ECV · Editio Cantor Verlag, Aulendorf (Germany)

Key words n

n n

Capsaicin, bioavailability, inclusion complex, pharmacokinetics, subcutaneous absorption CAS 404-86-4 Hydroxypropyl-b-cyclodextrin

Arzneimittelforschung 2010;60(9):571–574

cessary and urgent to improve the dissolubility and develop a safe injectable formulation for capsaicin. Currently, several approaches such as salt formation [6], crystal engineering [7], nanosizing [8], lipid formulation [9], cyclodextrin complexation [10] and prodrug strategies [11] have emerged to improve the aqueous solubility of drug. Among these, we focus on the strategy of cyclodextrin complexation. Cyclodextrins (CDs) are cyclic oligosaccharides, containing six, seven, or eight D(+)-glucopyranose units (a-, b-, and c-cyclodextrin) attached by a-1,4-linkage. CDs can trap the lipophilic drug as guest in a cage-like meshwork, thereby enhancing its solubility and dissolution rate [12 – 14]. b-Cyclodextrin appears to be the best natural cyclodextrin due to its efficient drug complexation and availability in pure form [15], and natural b-cyclodextrin can be modified to im-

Fig. 1: Chemical structure of capsaicin.

Chen et al. – Capsaicin

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For private or internal corporate use only

To increase the aqueous solubility and poor bioavailability of capsaicin (CAS 404-86-4), in this paper, the effects of hydroxypropyl-b-cyclodextrin (HP-b-CD) on the aqueous solubility and the pharmacokinetic characteristics of capsaicin were investigated. The corresponding inclusion complex of capsaicin/HP-b-CD at the molar ratio of 1 : 1 was obtained by the method of saturated aqueous solution and characterized by differential scanning calorimetry and X-ray diffractometry. The dissolution rate of capsaicin was significantly increased by the complexa-



prove the low aqueous solubility (18.5 g l–1 at 25 'C). One of the pharmaceutically important b-cyclodextrin derivatives is hydroxypropyl-b-cyclodextrin. Among the CDs, hydroxypropyl-b-cyclodextrin (HP-b-CD) has higher water solubility and greater solubilizing capacity and complexing property than the parent compound and improved safety feature [16, 17]. More and more studies also applied HP-b-CD to improve the solubility and dissolution rate, physical and chemical stability and the bioavailability of drugs, especially when the rate-limiting step in drug absorption is poor dissolution [14, 18]. Therefore, HP-b-CD was employed to enhance the aqueous solubility and bioavailability of capsaicin in this study. The main objective of the present study was to evaluate the effect of HP-b-CD on the solubility, dissolution rate and pharmacokinetics by subcutaneous injection of capsaicin. We prepared complexation of capsaicin with HP-b-CD by the method of saturated aqueous solution. Dissolution rates and pharmacokinetic analysis of capsaicin in rats were also examined and comparatively evaluated.

2. Method 2.1 Materials Capsaicin (95.63 % C18H27NO3, MW = 305) was purchased from Shanghai Beixiang Biotechnology Co., Ltd, and capsaicin standard (batch number: 110839-200403) was purchased from the National Institute for the Control of Biological and Pharmaceutical Drugs (P. R. China). HP-b-CD (average MW = 1429), was purchased from Xian Lide Biology and Chemical Engineering Co., Ltd. Methanol (Fisher) was of high-pressure liquid chromatography (HPLC) grade. All other reagents and solvents used in this experiment were of the highest purity commercially available. The water used was purified by using the Aquapro (model: ABW-0501-U) water purification system.

2.2 Animals Healthy male Sprague-Dawley rats (180 – 200 g) were purchased from the Laboratory Animal Center of Sichuan University, Sichuan (P. R. China). Prior to the experiments, the rats were housed in a temperature and humidity controlled room (23 'C, 55 % air humidity) with free access to water and standard rat chow. The rats were acclimated for at least 5 days and fasted overnight but supplied with water ad libitum before the experiments. All experiments were approved by the Institutional Animal Care and Use Committee of Sichuan University.

2.3 Determination of capsaicin Concentrations of capsaicin were determined by HPLC. The HPLC measurements were carried out by using a Shimadzu assembly equipped with a LC-10AT model pump, a HypersilODS2 column (150 mm66.0 mm, 5 lm, Dalian Institute of Chemical Physics, P. R. China), a RF-10AXL model fluorescence detector at an excitation wavelength of 275 nm and an emission wavelength of 313 nm. The mobile phase used was methanol:water (64 : 36) which was delivered at a flow rate of 1.0 ml min–1. The injection volume was 20 ll. The mobile phase was filtered through a type HA, 0.45 lm membrane filter (Millipore Corporation) and deaerated under reduced pressure.

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2.4 Preparation of the solid complexes Inclusion complex: The solid capsaicin/ HP-b-CD inclusion complex was prepared by the method of saturated aqueous solution with the weights taken for 1.00 g of capsaicin and 4.69 g of HP-b-CD (i. e. molar ratio of 1 : 1). Capsaicin, dissolved in a small amount of ethanol, was dropped in saturated aqueous solution of HP-b-CD slowly, and agitated for 1 h at 50 'C and 500 rpm. Then, the aqueous solution was filtered through 0.45 lm Millipore filter. The filtrate was frozen and dehydrated (Thermosavant model: VLP200) to dryness.

2.5 Characterization of the solid complexes and phase solubility studies The prepared inclusion complexes were validated and characterized by DSC, XRD and phase solubility studies as previously reported [19].

2.6 Dissolution studies In vitro dissolution studies were conducted according to CP2005 in deionized water at 37 ± 0.5 'C by the paddle method at a rotation speed of 100 ± 2 rpm using a six-vessel dissolution apparatus (ZRS-4, Tianjin University). Powdered samples containing 5 mg of capsaicin or its equivalent in the inclusion complex or physical mixture were added to the dissolution medium (250 ml). At predetermined time intervals (5, 10, 15, 20, 30, 40, 50, and 60 min), aliquots (5 ml) were withdrawn for HPLC determination of capsaicin concentration following filtration (0.2 lm) and replaced by an equal volume of the same dissolution medium kept at 37 ± 0.5 'C. Samples should be withdrawn from a zone roughly midway between the surface of dissolution medium and the top of the rotating blade.

2.7 In vivo pharmacokinetic analysis in rats 2.7.1 Subcutaneous administration Ten rats were divided into 2 groups at random. Uncomplexed capsaicin, serving as a control, was dissolved in 0.9 % NaCl with 10 % ethanol and 10 % Tween 80 to obtain a final concentration of 2 mg/ml [20]. Capsaicin at a dose of 10 mg/kg was injected into the dorsal subcutaneous tissue of Sprague Dawley rats. The inclusion complex was diluted by 0.9 % NaCl to achieve the capsaicin concentration of 2 mg/ml. Then, the inclusion complex (corresponding to the dose of 10 mg/kg capsaicin) was subcutaneously injected into Sprague Dawley rats. After injection, blood samples from all rats were obtained from the end of tail. The blood samples were withdrawn at the desired time point into heparinized centrifuge tubes and immediately centrifuged at 9006g for 4 min.

2.7.2 Plasma analytical procedure of capsaicin A 100 ll aliquot of plasma was pipetted into a centrifuge tube. The centrifuge tube was coated with 50 ll of p-phenylphenol solution at a concentration of 43.6 ng/ml. The solution was then extracted with 1 ml of acetic ether for capsaicin and then followed by mechanical shaking for 3 min. After centrifugation for 10 min at 11826g, 1 ml of acetic ether phase was transferred to another tube and evaporated to dryness by nitrogen (N2). The residue was redissolved in 75 ll of methanol and 20 ll of this solution was injected into the HPLC directly.

2.7.3 Data analysis Pharmacokinetic parameters for capsaicin in rats were measured using DASver2.0 software. The relative bioavailability was calculated by the following equation: (Frel) = 100 %6(AUCIC6Dfree)/(AUCfree6DIC). Arzneimittelforschung 2010;60(9):571–574 © ECV · Editio Cantor Verlag, Aulendorf (Germany)


3.2 Dissolution studies

2.7.4 Statistical analysis The results were expressed as the mean – S. D. of at least five independent experiments, unless otherwise stated. Statistical analysis was performed using the Student’s t-test to compare two data sets. A p-value < 0.05 was considered statistically significant.

3.3 In vivo pharmacokinetic analysis in rats

3. Results and discussion 3.1 Determination of capsaicin Content determination of capsaicin in ethanol for the in vitro study was carried out by HPLC with a linear range from 5 to 50 mg/L (A = 0.0107C-0.003, r = 0.9997). Based on the concentrations of capsaicin at 20, 40, and 50 mg/L, the intra-day precision of this assay was 0.76 %, 0.58 %, and 0.17 % with a mean recovery of 97.35 %, 96.24 %, 99.36 %, and inter-day precision was 0.29 %, 0.89 %, and 0.97 %, respectively (n = 3). Concentrations of capsaicin in the plasma were determined by HPLC with a linear range from 0.1 to 2 mg/L (As/Ai = 1.2544C-0.2029, r = 0.9965; As was the peak area of capsaicin, and Ai was the peak area of internal standard). Based on the concentration of capsaicin at 0.2, 0.5, and 2 mg/L, the assay intra-day precision was 3.01 %, 3.57 %, and 2.76 % with a mean recovery of 99.29 %, 99.06 %, and 93.14 %, and inter-day precision was 4.93 %, 4.69 %, and 4.15 %, respectively (n = 5).

In the current study, subcutaneous administration was chosen for the application of capsaicin/ HP-b-CD complex. Capsaicin is a highly toxic compound, the toxicity of capsaicin after intravenous injection is the highest among different routes of administration, and the LD50 value after subcutaneous administration is higher than that after intramuscular and intraperitoneal administration [22]. Therefore, subcutaneous injection is a relatively safer administration route. For the purpose of studying the biopharmaceutical aspects of capsaicin, one prerequisite was that the pharmacokinetic parameter of the subcutaneous administration should be known to establish the basic kinetic knowledge. The plasma drug concentration-time curves for capsaicin after single s.c. application of 10 mg/kg of capsaicin are shown in Fig. 3. The plasma levels of uncomplexed and complexed capsaicin were adequately described using a non-compartment model. A summary of the pharmacokinetic parameters of free and complexed capsaicin after s. c. administration are shown in Table 1.

Fig. 3: Plasma concentration versus time profiles of (s) complexed and (n) free capsaicin after s.c. administration in rats. Arzneimittelforschung 2010;60(9):571–574 © ECV · Editio Cantor Verlag, Aulendorf (Germany)


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Fig. 2: Dissolution profiles of (u) free capsaicin, (n) physical mixture, and (s) inclusion complex.

The dissolution data are shown in Fig. 2. The dissolution efficiency parameter was evaluated by the mean percentage of dissolved capsaicin at 60 min according to the regulations of CP2005. As could be seen from the figure, it was evident that the HP-b-CD complex exhibited faster dissolution than the corresponding PM and the free capsaicin, being immediately dispersed and completely dissolved within 15 min. The uncomplexed capsaicin dissolved only to the extent of 10.5 % at the end of 60 min. The percentage of capsaicin dissolved from the PM and the HP-b-CD complex was 32.8 % and 96 %, respectively. These facts were consistent with the results of the solubilizing activity of HP-b-CD, suggesting that the enhancing effect of HP-b-CD on the dissolution rate of capsaicin could be explained from the enhanced aqueous solubility of capsaicin after the formation of complexation. In addition, according to literature, a decrease in crystallinity of the drug might be another factor in the enhanced dissolution by the complex besides an increase in solubility [21].


Table 1: Pharmacokinetic parameters of free and complexed capsaicin after s. c. administration. Parameter


AUC(0 – t) AUC(0 – 8) Cmax Tmax

Unit

Free capsaicin

Complexed capsaicin

mg/L × min mg/L × min mg/L min

41.073 ± 2.589 44.976 ± 4.353 0.813 ± 0.024 100 ± 1.79

97.224 ± 2.589 103.26 ± 4.353 1.706 ± 0.059 30.15 ± 2.96

The absorption rates of capsaicin across subcutaneous tissues were enhanced significantly by the formation of the inclusion complex with HP-b-CD. After s. c. injection, uncomplexed capsaicin was not detected until 80 min (0.1154 mg/L), and complexed capsaicin could be detected at 10 min (0.4900 mg/L), which was due to the dissolution rate of capsaicin which was significantly increased by the complexation with HP-b-CD. As a result, the relative bioavailability of the inclusion complex was also 2.36 fold higher than that of capsaicin. Further pharmacokinetic studies on rats are under investigation to provide more useful information with respect to the application of capsaicin and its complexation. In conclusion, the dissolution rate of the capsaicin/ HP-b-CD complex was much faster than that of capsaicin alone as a consequence of the increased solubility and decrease in crystallinity caused by complexation. It was also demonstrated that the permeability of capsaicin through subcutaneous tissues was enhanced via complexation with HP-b-CD by in vivo model studies. The present results suggest the potential use of HP-bCD for improving the subcutaneous absorption of capsaicin as injections. Acknowledgements The present work was funded by the 973 Program of the Chinese government (No. 2009CB930300) and supported by the National Science Foundation of P. R. China (No. 30873165).

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Arzneimittelforschung 2010;60(9):571–574 © ECV · Editio Cantor Verlag, Aulendorf (Germany)

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