Journal of Pharmaceutics & Drug Delivery Research ISSN: 2325-9604

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Research Article, J Pharm Drug Deliv Res Vol: 1 Issue: 3

Therapeutic Effects of Sinomenine Microemulsion- Based Hydrogel on Adjuvant- Induced Arthritis in Rats

Youjun Zhou1, Shuangying Gui1,2*, Jian Wang3,4 , Shanshan Qian1 and Enyuan Pan1
1Department of Pharmacy, Anhui University of Traditional Chinese Medicine, Shihe Road, Hefei, 230031, P.R,China
2Anhui Engineering Research Center for Chinese Medicine Preparation, Shihe Road, Hefei, 230031, P.R, China
3Anhui Key Laboratory of Modern Chinese Medicine & Materia, Shihe Road, Hefei, 230031, P.R, China
4Anhui“115”Xin’an Traditional Chinese Medicine Research & Development Innovation Team, 45 Shihe Road, Hefei, 230031, P.R, China
Corresponding author : Shuangying Gui
Department of Pharmacy, Anhui University of Traditional Chinese Medicine, Shihe Road, Hefei, 230031, P.R, China
Tel: 13856002612; Fax: 0551-5169044
E-mail: [email protected]
Received: October 01, 2012 Accepted: December 12, 2012 Published: December 17, 2012
Citation: Zhou Y, Gui S, Wang J, Qian S, Pan E (2012) Therapeutic Effects of Sinomenine Microemulsion-Based Hydrogel on Adjuvant-Induced Arthritis in Rats. J Pharm Drug Deliv Res 1:3. doi:10.4172/2325-9604.1000110

Abstract

Therapeutic Effects of Sinomenine Microemulsion- Based Hydrogel on Adjuvant-Induced Arthritis in Rats

Sinomenine is an alkaloid isolated from the traditional Chinese medicinal herb Sinomenium acutum, which has been used for the treatment of rheumatoid arthritis in China. The objective of this work was to investigate the effect of sinomenine microemulsion-based hydrogel (SMBH) on Freund’s complete adjuvant-induced arthritis(AA) in Wistar rats. The SMBH was formulated as follows: 4%sinomenine, 2% oleic acid, 16.7% Tween-20, 41% distilled water,33.3% absolute alcohol, 3% menthol, and 2% carbomer (w/w).Treatment was started on the seventh day after AA was induced. All groups were daily applied to the hind feet skin with SMBH or sinomenine gel (SG) for 14 days.

Keywords: Sinomenium; Microemulsion-based hydrogel;Rheumatoid arthritis

Keywords

Sinomenium; Microemulsion-based hydrogel; Rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by cytokine-mediated inflammation of the synovial lining of the joints and destruction of cartilage and bone. The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α) might be important in the processes, leading to cartilage and bone destruction, and in limiting mechanisms involved in cartilage repair [1,2]. TNF-α activates synoviocytes to mediate synovial hyperplasia, and to produce extracellular matrix-degradative enzymes and chemokines for the development of arthritic reaction. Prostaglandin E2 (PGE2) is an important molecule in the pathogenesis of rheumatoid arthritis, which is secreted by a lot of cells, including macrophage cells and synovial fibroblast [3]. Therefore, its effect on the proliferation and apoptosis of synoviocytes should not be neglected. Sinomenine (7,8 -didehydro-4-hydroxy-3,7- dimethoxy- 17-methylmorphinan-6-one), an active alkaloid of the climbing plant Sinomenium acutum, has been used for the treatment of RA [4]. It can significantly suppress the production of RA-related inflammatory mediators of IL-1, IL-6, MMP-2 and MMP-9 in rats [5].
Microemulsions have several advantages such as enhanced drug solubility, good thermodynamic stability [6], and better transdermal ability than conventional formulations [7]. It consists of oil, surfactant, co-surfactant and water. Various mechanisms for skin permeation enhancement of permeants by microemulsions have been proposed. Firstly, microemulsions act as drug is released from the inner pseudophase to the outer pseudophase and finally further into the outer. Secondly, microemulsion droplets might breakdown on the surface of stratum corneum and then release their content into skin. Thirdly, skin permeation of loaded drug occurs directly from the microemulsion fusion at the stratum corneum. The last mechanism is caused by the nano-sized droplets dispersed in continuous phase which can move easily into the stratum corneum and carry the drug through the skin barrier.
Our previous study had shown that the optimum formulation of sinomenine microemulsion had good transdermal delivery ability and good release performance with some sustained release characteristics in vitro [8,9]. As most of the microemulsions possessed a very low viscosity, some gelling agents such as Carbomer 940 and carrageenan had been used to increase the viscosity of microemulsion and to form microemulsion-based hydrogel (MBH) which are more suitable for topical application [10]. In vivo pharmacokinetic studies showed transdermal administration of sinomenine MBH (SMBH) could provide sustained plasma concentration [11].
In RA animal models, at present, adjuvant arthritis (AA) and collagen-induced arthritis (CIA) are widely used because of similarity with people RA clinical features. In clinical manifestation, laboratory index and pathological mechanism of AA model are very close to people RA. Due to the AA rat model is simple and cost lower than CIA. In this study, we selected AA rat model as RA animal model for research the therapeutic effects of SMBH or sinomentine gel (SG) on RA.

Materials and Methods

Materials
Oleic acid and Tween 20 were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjing, China). Sinomenine was obtained from Shanxi Taikang Biological Technology Co., Ltd. (Shanxi, China). Menthol was purchased from Guangzhou Chemical Reagent Company (Guangzhou, China). Carbopol 940 was purchased from Beijing Haidian Huiyou Fine Chemical Factory (Beijing, China). Freund’s complete adjuvant was purchased from Sigma (St. Louis, MO, USA). ELISA kits for IL-1, TNF-α and PGE2 were purchased from R&D Systems (Minneapolis, MN, USA). Fluocinonide Ointment was purchased from Tianjin Pacific Pharmaceutical Co., Ltd. (Tianjin, China). Other chemicals are of HPLC or analytical grade.
Preparation of SG
Sinomenine was solubilized in absolute alcohol in 4% proportion, and mixed with 2% Carbopol 940 solution. Triethanolamine was added under moderate magnetic stirring (ETS-D4 stirrer, IKA, Germany) to adjust pH value to 7.0.
Preparation of SMBH
The preparation process of SMBH was described in a previous paper [11]. In brief, the formulation was as follows: 4% sinomenine, 2% oleic acid, 16.7% Tween20, 41% distilled water, 33.3% absolute alcohol, 3% menthol, and 2% Carbopol 940 (w/w). The Tween20 and absolute alcohol was mixed with distilled water, and then dropped into oleic acid contained sinomenine, under moderate magnetic stirring. After equilibration, the microemulsion was mixed with Carbopol 940 solution, and added triethanolamine to adjust pH value to 7.0.
Characterization of sinomenine-loaded microemulsion
The morphology of sinomenine-loaded microemulsion was observed using a transmission electron microscope (JEM-1230, Jeol, Japan). One drop of sinomenine-loaded microemulsion was deposited on a film-coated copper specimen grid and allowed to stand for 10 min, after which any excess fluid was removed with filter paper. The grid was later stained with one drop of 2% phosphotungstic acid. The average diameter of droplets and polydispersivity index of the microemulsion were determined at 25°C by Zetasizer 3000 photon correlation spectroscopy (Worcestershire, UK). The electro conductivity of the microemulsion was measured by conductivity meter (Shanghai hongyi instrument Co. Ltd, Nanjing, China). The refractive index of the microemulsion was determined by automatic refractometer (Xiguang industrial Co. Ltd, Shanghai, China).
Characterization of MBH
The pH values were evaluated at 25°C by pH digital acidimeter (PHREX-1, Shanghai, China). The viscosity was determined at 25°C using viscometer (NDJ-1, shanghai, China) according to Chinese Pharmacopoeia Appendix (2010 Edition). The stability of SMBH was investigated from respects of clarity, diameter of droplets, phase separation observation and the content of sinomenine at 4°C and 25°C up to 3 months. The centrifuge test was performed to assess the physical stability with centrifuging rate at 10,000 rpm (LG16-G, Beijing, China) for 10 min, and HPLC was used to analyse the sinomenine content of SMBH.
Experimental animals
Healthy Wistar rats (equal number of males and females, 200-240 g,) were purchased from Animal Center of Anhui Medical University. All animals were housed under controlled conditions (22-23°C, 12 h light, 12 h dark) for 7 days before experiment, and had free access to food and water.
Treatment of arthritis in rats
The rats were immunized by subcutaneous injection on left-hind paw with 0.1 ml Freund’s complete adjuvant (FCA). Arthritis usually developed 7 days later after immunization. The appearance of significant redness and/or swelling in digits or other parts of paw were considered to be arthritis. The animals were assigned to eight groups randomly, and respectively applied to the hind feet skin with SMBH at dose of 30 mg/kg, 60 mg/kg, and 120 mg/kg, with SG at dose of 60 mg/kg, and 120 mg/kg, with Fluocinonide Ointment at dose of 60 mg/kg as positive control, with the same dosage of microemulsion-based hydrogel without sinomenine as negative control and model group. All groups were treated daily for 14 days [12]. Thereafter, the rats were anesthetized by 25% urethane, and the hind paws and sera were collected for further analysis.
Evaluation of arthritis activity
Arthritis index was used to evaluate arthritis activity. The signs of arthritis were daily observed, and the swelling degrees of the non arthritis foot were determined at 14, 19, 24 and 29 days. After 14 days of treatment, the paws were collected for histopathological evaluation.
ELISA for inflammatory cytokines of IL-1, TNF-α and PGE2 in blood
The blood were collected after anaesthetization, and stored in -20°C for next tests. TNF-α, IL-1 and PGE2 content in the samples were measured using enzyme linked immunoassay kit according to the manufacturer’s protocol.
Histology
Whole ankle joints were fixed in 10% formalin at 4°C for 12 h. Subsequently, joints were processed for histology by routine paraffin embedding, sectioning, hematoxylin and eosin (H&E) staining, and microscopic observation.
Statistical analysis
The data were expressed as the mean ± S.D. Differences between treatment groups were tested by one-way ANOVA and two-tailed unpaired Student’s t test. Analysis of variance of repeated data measured with SPSS 10.0 and P values less than 0.05 at 95% confidence level was considered significant.

Results

Characterization of microemulsion
The viscosity of sinomenine microemulsion is 2.03 Pa·s. The electro conductivity was 137.2 μΩ. The refractive index was 1.45. The mean diameter of droplets in sinomenine microemulsion was 32.0 nm. In general, the sinomenine-microemulsion was transparent with visible sky-blue opalescence, and the microemulsion droplets were round, uniform, and well-dispersed.
Characterization of MBH
The pH value of MBH was 6.80, which would result in less stimulation to skin than microemulsion. The viscosity (3.26 Pa·s) of MBH increased significantly compared to that of microemulsion, which led more suitable topical administration. The centrifuged tests indicated that MBH had good physical stability. MBH were stable at 4°C and 25°C. There was no significant change of particle size, phase separation and degradation of sinomenine observed up to 3 months.
Effect of SMBH on paw inflammation in AA rats
All groups treated with SMBH or SG showed alleviated paw swelling compared to models (P<0.01) (Table 1).
Table 1: Effect on paw swelling in AA rats (± S.D, n=8).
Effect of SMBH on polyarthritis index in AA rats
Table 2 indicated that the groups treated with SMBH (120, 60, and 30 mg/kg) significantly induced secondary arthritis inflammatory response and decreased the polyarthritis index in AA rats compared to models (P<0.01).
Table 2: Effect on polyarthritis index in AA rats (± S.D, n=8).
Effect of SMBH on TNF-α, IL-1 and PGE2 in AA rats
ELISA analysis showed that the level of TNF-α, IL-1 and PGE2 in blood of AA rats were significantly reduced by SMBH, with comparison to that of SG (Table 3).
Table 3: Effect on IL-1, TNF-α, PGE2 in blood of AA rats (± S.D., n=8).
Effect of SMBH or SG on synovial inflammation
With observation of H&E staining sections (Olympus optical microscopy, Japan), the ankle joints of normal control rats showed no inflammation and destruction at all (Figure 1a), while the AA rats exhibit hyperplastic synovium, increased synovial cells, pannus formation, and inflammatory cells infiltration into synovium (Figure 1b). After administration of SMBH, SG and Fluocinonide Ointment, hyperplastic synovium, inflammatory cells infiltration, and pannus formation were significantly reduced in AA rats (Figures 1c-1e).
Figure 1: Changes of the histological morphology of rats ankle joints by H&E staining. A) Normal group. The arrow showed monolayer synovial cell. B) Model group. The rrow showed hyperplastic synovial cell and pannus. C) Sinomenine MBH (120 mg·kg-1). The arrow showed synovial cell was less and synovium was nearly monolayered. D) Sinomenine hydrogel (120 mg·kg-1). The arrow showed monolayer or double layer thin synovium. E) Positive control group. The arrow showed synovial cell was less and synovium was nearly monolayered.

Discussion

Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by cytokine-mediated inflammation of the synovial lining of the joints and destruction of cartilage and bone. And the major medicine treating RA is COX-2 selective inhibitor. Non-steroidal anti-inflammatory drugs (NSAIDs), which represent an effective therapy for treating RA, elicit their effects by inhibiting cyclooxygenase (COX) activity and blocking the downstream production of prostaglandin [13]. However, this kind of medicine has side-effects such as cardiovascular effects, which have limited its use.
Sinomenine is a kind of alkaloid isolated from Sinomenium scutum, which has been used for the treatment of rheumatoid arthritis, chronic nephritis, resisting addiction of morphine and anti-heart rate abnormal treatment in China for over one thousand years. Sinomenine was found to downregulate the mRNA expression of inflammatory cytokines TNF-α and IL-1 by inhibiting the NF-кB binding activity and cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats [14].
The proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α) are not only critical for host defense against microbial agents but also play important roles in joint inflammation and cartilage destruction in various forms of arthritis. For example, both IL-1 and TNF-α are expressed in the synovial lining cells and are present in the synovial fluid from patients with RA or osteoarthritis (OA). The pivotal roles of IL-1 and TNF-α in RA are further demonstrated by the efficacy achieved with biological antagonists of IL-1 and TNF-α, these agents demonstrate significant disease modifying activity in human patients with moderate or severe RA [15].
Several lines of evidence suggest that at least some of the proinflammatory aspects of this disease are mediated by PGE2. Specifically, PGE2 has been associated with the edema and the erosion of cartilage and juxta-articular bone commonly found in RA. PGE2 is also produced in response to proinflammatory cytokines, which in turn negatively regulates both IL-17 and TNF-α expression and TNF/IL-1-induced activation of fibroblast-like synoviocytes through EP2/EP4 receptors, resulting in the modulation of proinflammatory cascades [16]. It is an autocrine factor in rheumatoid arthritis, so its effect on the proliferation and apoptosis of synoviocytes should not be neglected.
Therefore, IL-1, TNF-α and PGE2 are better value to assess the degree of the arthritis.
Treatment experiments indicated that the SMBH has therapeutical effect on AA rats. SMBH (120, 60, and 30 mg/kg) significantly reduced paw swelling and decreased the polyarthritis index in AA rats. The results of study showed that the level of TNF-α, IL-1 and PGE2 in blood of AA rats were significantly reduced by SMBH.
For skin care and the topical treatment of dermatological disease, a wide choice of vehicles ranging from solids to semisolids and liquid preparations is available to clinicians and patients. Within the major group of semisolid preparations, the use of transparent gels has expanded both in cosmetics and in pharmaceuticals. The result of our precious in vivo pharmacokinetic studies showed transdermal administration of SMBH could provide sustained plasma concentration.
The multiple favorable reports of cutaneous drug delivery potential of microemulsions, have, however, mainly been performed in vitro. Only few reports have confirmed this potential in vivo- presumably because of the novelty of the vehicles for topical administration and the lack of appropriate in vivo techniques to assess skin absorption. Sinomenine is used in the therapy of RA and has clear and definite therapeutic effects, but the therapeutic mechanism is unclear. The therapeutic mechanism will be studied in our future research.

Conclusion

The results of this study indicated that SMBH and SG both have therapeutical effect on AA rats, but the treatment effect of SMBH is superior to that of SG, which may be related to the reduction of IL-1 and TNF-2.

Acknowledgements

The work was supported by the Natural Science Foundation of Anhui Province (No. 11040606M219) and the Natural Science Foundation of the Anhui Higher Education Institutions of China (No. KJ2012A184) and the National Natural Science Foundation of China; Contract grant number: (No.81274099).

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