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tripolyphosphate microspheres for the intra-articular delivery of lornoxicam: Optimization and in vivo evaluation
Accepted Manuscript Title: Injectable long acting chitosan/tripolyphosphate microspheres for the intra-articular delivery of lornoxicam: optimization and in vivo evaluation. Author: Hend Abd-Allah Amany O. Kamel Omaima A. Sammour PII: DOI: Reference:
S0144-8617(16)30473-8 http://dx.doi.org/doi:10.1016/j.carbpol.2016.04.096 CARP 11036
To appear in: Received date: Revised date: Accepted date:
18-2-2016 20-4-2016 22-4-2016
Please cite this article as: Abd-Allah, Hend., Kamel, Amany O., & Sammour, Omaima A., Injectable long acting chitosan/tripolyphosphate microspheres for the intra-articular delivery of lornoxicam: optimization and in vivo evaluation.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.04.096 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Injectable long acting chitosan/tripolyphosphate microspheres for the intraarticular delivery of lornoxicam: optimization and in vivo evaluation. Hend Abd-Allah, Amany O. Kamel*, Omaima A. Sammour
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt Address: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University. African organization Unity Street, Cairo, Egypt, P.O. box 11566. *
Correspondence: Amany Kamel Email [email protected]
Address : Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Monazzamet El Wehda El Afrikeya Street, Abbasseya, Cairo, Egypt Tel.: +201001429211 ; fax: +202 24051106.
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Lornoxicam loaded chitosan/TPP microspheres prepared with ionotropic gelation for intra-articular delivery Optimization of formulation variables was studied using full factorial design experiment to achieve the highest lornoxicam encapsulation efficiency In-vivo anti-inflammatory activity of the optimized microspheres was tested in a mono-iodoacetate induced osteoarthritic model in rats
The present study could be beneficial in managing chronic pain and inflammation following various orthopedic joint related surgeries and decreasing long term joint diseases as osteoarthritis
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Abstract Chitosan microspheres were formulated for the intra-articular delivery of lornoxicam in knee osteoarthritis, to minimize associated side-effects after prolonged oral administration. Ionotropic-gelation technique was employed using tripolyphosphate as anionic cross-linker. Fullfactorial design experiment was conducted to optimize lornoxicam entrapment-efficiency%. Formulations were assessed for their particle size, in-vitro drug release, Scanning electron microscopy, Differential-scanning-calorimetry and Fourier transform infra-red spectroscopy studies. Changing independent variables, chitosan pH, TPP pH and lornoxicam concentration resulted in different values of entrapment-efficiency% ranging from 13.5%±0.35 to 59.5%±2.2. Particle size ranged from 3.57µm±0.02 to 6.12µm±0.00 and lornoxicam %release was prolonged for up to 8 days. SEM results showed spherical shape of the microspheres. FTIR and DSC studies confirmed the crosslinking of chitosan with tripolyphosphate. In-vivo therapeutic effect of lornoxicam microspheres was investigated using Monosodiumiodoacetate (MIA) induced osteoarthritis model in rats. Optimized formula showed long-term in-vivo anti-inflammatory effect relative to lornoxicam solution injected intra-articularly with significant reduction of histological, inflammatory and biochemical parameters of osteoarthritis. Key
words:
Lornoxicam,
Chitosan,
Tripolyphosphate,
Monoiodoacetate.
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Microspheres,
Intra-articular,
1- Introduction Knee osteoarthritis, is among the big challenges causing pain and disability especially in elderly patients. First choice treatment option is oral administration of non-steroidal antiinflammatory drugs (NSAIDs) for relieving both inflammation and symptomatic pain (Yoshimi, et.al., 2010). However, Oral administration of NSAIDs is hampered by numerous side effects as GIT problems. Therefore, localized NSAIDs intra-articular administration has emerged as a promising strategy for management of osteoarthritis. The major limitation of the intra-articular administration of drug solutions is their low retention times in the intra-articular spaces, which emphasis the need to develop drug delivery systems that would function as depot systems for NSAIDs, slowly releasing the active drugs and providing localized sustained action. For this application chitosan has come to be a particularly interesting polymer. It possesses a number of beneficial properties that makes it unique among polymers used for intra-articular delivery. Chitosan is non-toxic, biodegradable, biocompatible, hydrophilic and because of its cationic nature it has very good mucoadhesive, antibacterial and membrane permeability properties. Interestingly; chitosan has been used in articular cartilage engineering as a scaffolding material and was found to increase chondrocyte proliferation when injected intra-articularly in rats. Together with some structural similarity with various glycosaminoglycanes found in articular cartilage (Bedouet, et.al., 2014). Chitosan microspheres display several advantages over hydrophobic polyester based particles for the intra-articular delivery. They reduce the amount of foreign materials injected in joint cavity, also hydrophilic surfaces are known to inhibit monocyte adhesion and lymphocytes proliferation (MacEwan, Brodbeck, Matsuda and Anderson, 2005). 4
Numerous publications describe the use of biodegradable microparticles as means for intra-articular delivery. Flubiprofen gelatin microspheres were able to prolong the drug release and increase targeting efficiency in the knee joints (Lu, Zhang, Sun and Zhong, 2007). Also, diclofenac sodium loaded albumin microspheres showed successful reduction of arthritis after their intra-articular administration in rabbit knee joints (Tuncay, et. al., 2000). Chitosan microspheres were able to extend the residence time of celecoxib in the rats knee joint after intraarticular injection (Thakkar, Sharma, Mishra, Chuttani and Murthy, 2004). Ionotropic gelation for chitosan microspheres preparation; is a very simple mild process producing complete hydrophilic environment of chitosan particles by avoiding the use of organic solvents (Tiyaboonchai, 2013). It is based on the electrostatic interaction between chitosan positively charged amino groups and negatively charged groups from various anions. The most popular polyanion investigated is tripolyphosphate (TPP) due to its non-toxic properties and quick gelling ability (Mattu, Li and Ciardelli, 2013). Lornoxicam is a weakly acidic NSAID of oxicam class with analgesic and antiinflammatory properties. Lornoxicam inhibits cyclooxygenase (COX), key enzyme of arachidonic acid pathway, thus inhibiting prostaglandin synthesis (Zhang, Bi, Li and Huang, 2011). Lornoxicam is used orally in cases of acute pain and inflammation in rheumatoid arthritis, osteoarthritis and post-operative pain related to orthopedic surgeries in multiple divided doses (8mg, 3 to 4 times daily) due to its short half-life (2-5hrs) (Kidd and Frenzel, 1996). However, lornoxicam with its low aqueous solubility and poor dissolution in upper GIT causes GIT irritation and formulation problems that limit its therapeutic applications by delaying rate of absorption (Hamza and Aburahma, 2009). Targeting local site of osteoarthritis by the intraarticular injection to maintain a sustained drug effect is a valuable goal to improve therapeutic 5
efficacy of lornoxicam and avoid systemic complications. However, its poor aqueous solubility constitutes a problem in development of intra-articular injection. Hence, the aim of this study was to investigate some key factors to optimize lornoxicam entrapment efficiency (EE%) in chitosan/TPP microspheres for safer intra-articular delivery. This was a challenge due to the hydrophobic nature of lornoxicam and the solely hydrophilic environment of the system. To the best of our knowledge lornoxicam loaded chitosan/TPP microspheres for intra-articular treatment of osteoarthritis has not been reported yet.
Optimization of formulation variables by full
factorial design was done to achieve highest lornoxicam EE% in chitosan/TPP microspheres with acceptable size for the intra-articular delivery route. In-vivo sequential alterations in inflammatory cytokines that parallel pathological changes in osteoarthritis were tested in monoiodoacetate (MIA) induced osteoarthritis rat model. 2-Materials and methods 2.1- Materials Lornoxicam (LOR): kindly supplied by El Obour Pharmaceutical Co., cairo, Egypt. Chitosan
medium
molecular
weight
(viscosity
200000cps,
75-85%
deacetylated),
Tripolyphosphate (TPP), Monosodiumiodoacetate (MIA), Chloral hydrate and glacial acetic acid were purchased from Sigma-aldrich,UK. Sodium hydroxide, Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, hydrochloric acid and isopropanol were purchased from El Nasr Pharmaceuticals, cairo, Egypt. 2.2- Preparation of lornoxicam loaded chitosan/TPP microspheres Lornoxicam loaded chitosan/TPP microspheres were prepared by ionotropic gelation technique following a preliminary study that optimized the preparation parameters of plain
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microspheres with particle size suitable for intra-articular delivery (Cho, Chun, Kim and Park, 2014). Briefly, an amount of 2ml TPP (0.25%w/v) containing different concentrations of lornoxicam (1%, 1.5% and 2%w/w) was added to 5ml of chitosan solution (0.1%w/v) in 0.1%v/v acetic acid, under magnetic stirring at room temperature for 30mins (IKAMAG®, model C-MAG HS7, Germany). The formed microspheres suspension was then centrifuged at 4930xg for 5mins at 4°C (Cooling centrifuge model Z216 MK, Germany). The microspheres were washed two times with deionized water (Zhang, Tang, Xu and Li, 2013). The formed pellets were then freeze dried (Christ alpha, 1-2 LD plus, Germany). A full factorial design experiment was built up to study the effect of three independent variables viz. drug concentration at -1 (1%), 0 (1.5%) and 1 (2%) levels, chitosan solution pH at -1 (4) and +1 (4.5) levels and TPP solution pH at -1 (8), 0 (8.5) and +1 (9) levels on the studied response, lornoxicam EE% as shown in table 1. Design of the experiments and generation of a response surface methodology and data analysis were performed using Design-Expert® program v.9.0.4. (Stat-Ease, Inc., Minneapolis, USA). Response surface methodology was tested at an alpha of 0.0001 for proper modeling of EE% response. Analysis of variance (ANOVA) was used to evaluate experimental data with various descriptive statistical analyses such as R2, Adjusted R2 and predicted R2 to reflect statistical significance of the developed mathematical model. 2.3- Check point analysis for the factorial model Check point analysis was performed to validate model reliability in predicting microspheres behavior (Ricci, et. al., 2006). Central point’s chosen according to the contour plots obtained at different levels of factors corresponding to a total of three check points then chitosan microspheres were prepared, EE% was measured, and %bias was calculated to estimate agreement between actual and predicted EE% according to the following equation: 7
Bias% = Difference between actual and predicted values/Actual value X 100. 2.4- Characterization of lornoxicam chitosan/TPP microspheres 2.4.1- Particle size analysis Microspheres were suspended in isopropanol (where no swelling was expected) and sonicated for 30secs (Jalalipour, Tajerzadeh, Najafabadi & Barghi, 2008; Elmoafy, Osman, Elshamy and Awad, 2014 and Osman, et. al., 2013). Particle size expressed as volume mean diameter (D 4,3) was measured using laser diffraction particle size analyzer (Mastersizer X, Malvern Instruments Ltd., UK). 2.4.2.-Determination of lornoxicam entrapment efficiency% Accurately weighed amount of particles were suspended in 0.1N HCL for 24 hrs. Digested microspheres were filtered using 0.22µm membrane filter (Calderon, et. al., 2013). Lornoxicam was determined spectrophotometrically at 376nm (spectrophotometer, Jenway-6800, Japan). EE% was calculated using the following equation. EE%=Wa/Wt X 100 Wa= weight of actual entrapped lornoxicam Wt= weight of theoretical formulated lornoxicam 2.4.3- Microspheres morphology
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Surface characteristics of microspheres were examined by scanning electron microscopy (SEM, JSM 5500, Joel, Japan). Samples mounted onto aluminum stubs and made electrically conductive by coating with a thin layer of gold. 2.4.4- In-vitro drug release study Weighed amount of microspheres corresponding to 2mg lornoxicam were dispersed in phosphate buffer saline (PBS7.4) in a dialysis bag and was placed in vials containing 28ml PBS 7.4 with 1%tween 80. The vials were placed in a shaking water bath shaken at 50 rpm and 37±0.5ºC (GFL, Germany) (Zhang, Bi, Li and Huang, 2011). Aliquots were taken at different time intervals and replenished with same volume of buffer. Samples were assayed spectrophotometrically at 376nm for lornoxicam content. Obtained data were kinetically treated to determine order of release using zero, first and Higuchi diffusion models. 2.4.5- Differential scanning calorimetry (DSC) Thermal properties of lornoxicam, chitosan, TPP and selected lornoxicam loaded chitosan microspheres as well as the physical mixture of their components were investigated using DSC (Shimadzu-DSC 60, Kyoto, Japan). Samples were sealed in aluminum pans and heated at a rate of 10ºC/min. till 300ºC, using dry nitrogen as carrier gas with a flow rate of 25ml/min. 2.4.6-Fourier transform infrared (FT-IR) spectroscopy FTIR spectra of lornoxicam, chitosan, TPP and selected lornoxicam loaded chitosan microspheres were recorded by FTIR spectrophotometer using KBr disc method (Shimadzu, Kyoto, Japan) (Mattu, Li and Ciardelli, 2013). All spectra were recorded at a resolution of 4 cm1
from 4000 to 400cm-1. 9
2.5-Anti-inflammatory activity in rats 2.5.1- Induction of Knee joint osteoarthritis The in vivo effectiveness of the prepared microspheres were assessed using monoiodoacetate (MIA) induced arthritis model in rats. Sixty-six male albino rats weighing 190250gm were divided into four groups. Group I (n=6 rats): served as normal control, Group II (n=24 rats): Osteoarthritic control group non-treated, Group III (n=18 rats): rats treated with intra-articular lornoxicam solution in normal saline (4mg lornoxicam /kg) (Zhang and Huang, 2012), Group IV (n=18 rats): rats treated with intra-articular lornoxicam chitosan/TPP microspheres (L9) (4mg lornoxicam /kg). Under 10% chloral hydrate (4ml/kg) anesthesia, rats of groups II, III and IV were intra-articularly injected with 3mg/50µl MIA in right knee joints using 27gauge needle. One day after MIA injection, substantial inflammation of knee joints was observed and drug treatment was intra-articularly administered (Ikeuchi, et.al., 2014) to groups III and IV.
At different intervals, viz., 7, 14 and 21 days, 6 rats were sacrificed. For
osteoarthritic control group II another 6 rats were sacrificed after 1 day of MIA induction to monitor full development of osteoarthritis in the rat knees before drug admistartion (Al-Saffar, Ganabadi, Yaakub and Fakurazi, 2009). The protocol of this study was reviewed and approved by the ethics committee of faculty of pharmacy, Ain Shams University, Cairo, Egypt. 2.5.2-Clinical assessment of osteoarthritic rats Effects of lornoxicam intra-articular treatment on rats were monitored by analyzing; knee joint diameter, cytokine (Interleukin 6, IL6) measurement and histological evaluation of knee joints. Assessments were done at different time intervals throughout the study i.e. 0, 1, 7, 14 and 21 days.
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2.5.2.1-Joint swelling measurement Knee joint diameter, was measured using Vernier caliper (Cole-Parmer Instrument co.), before MIA joint induction. Anti-inflammatory effect of intra-articularly injected formulae, as reduction of knee joint swelling was determined. %knee joint swelling inhibition was calculated by measuring difference between average values of knee joint diameters between treated animals and control group II at each time interval using the following equation (Bishnoi, Jain, Hurkat and Jain, 2014). % inhibition of knee swelling= (VC-VT)/VC x100 Where VC= knee diameter of control group II, VT= knee diameter of each treated group 2.5.2.2- Measurement of biological marker of inflammation (Interleukin 6, IL6) Rats from different animal groups were sacrificed by cervical dislocation. Rats knee joints were amputated and cut by bone cutter, pulverized in liquid nitrogen bath using a mortar and pestle to obtain fine powder. Powder was transferred to 50ml tubes and suspended in normal saline. The suspension was homogenized at 4°C using high-speed homogenizer (Ultra Turrax T25; IKA, Germany). Supernatant was separated by centrifugation (5000rpm, 10mins, 4°C) and stored at -80°C until further analysis (Yoshimi, et. al., 2010). Cytokine (interleukin 6, IL6) was analyzed using commercially available ELIZA kit (Ray Bio® Rat IL-6, Ray Biotech., USA) according to the operation instruction of the kit for quantitative determination. Data were expressed as pg cytokine/gm tissue. 2.5.2.3-Histopathologic analysis
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Rats were sacrificed after different time intervals from MIA induction. Knee joints were dissected and fixed in 10% formalin. Decalcification was performed using 10%formic acid followed by serial dilutions with alcohol for dehydration. Obtained tissue sections were stained by hematoxylin and eosin stain for examination by light microscope (Bar-yehudaet, et. al., 2009). 2.6- Statistical analysis One-way analysis of variance (ANOVA) followed by Tukey Kramer test were done using Graph Pad Instat® software. Statistical significance level (P) value was set at ≤ 0.05. 3-Results and discussion Changing independent variables, chitosan pH, TPP pH and lornoxicam concentration resulted in different values of EE% ranging from 13.5%±0.35 to 59.5%±2.2 (table 1). Particle size of lornoxicam loaded chitosan/TPP microspheres ranged from 3.57µm±0.02 to 6.12µm±0.00 (table 1), which lies within the accepted range for intra-articular drug delivery (510µm), in terms of injectability and phagocytosis by synovial macrophages to increase drug residence time in joints. (Thakkar, Sharma, Mishra, Chuttani and Murthy, 2004). By fitting the data for entrapment efficiency % response to various models, the best fitted model for the variables was found to be the two factor interaction (2FI) model (P<0.0001). The generated equation was as follows:
Entrapment Efficiency= -217.897 + 56.7167 * chitosan pH + 0.283333 *TPP pH + 218.733 * Drug conc. -0.433333 * chitosan pH * TPP pH -20.6 * chitosan pH * Drug conc. -13.4 * TPP pH * Drug conc.
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The model showed R2=0.9741, adjusted R2 =0.9600 and predicted R2=0.9259. Adequate precision measured a signal to noise ratio to ensure that the model can be used to navigate the design space, its value was 29.673. Ratio greater than 4 is usually desirable (Abdel Hafez, Hathout and Sammour, 2014). A good agreement between experimental and predicted values for EE% response was noticed, suggesting statistical validity and significance of the model figure 1A. Box-Cox plot for power transformation figure 1B was generated by plotting power response transformation (symbolized by λ) against natural logarithm of residual sum of squares (Ln residual sum of squares). It showed that the current point lied inside the 95% confidence interval suggesting no recommended transformation and thus first order regression model was used. 3.1-Check point analysis Check points are useful determination for prediction power of the regression factorial model obtained. A good agreement between actual and predicted responses was obtained; the max estimated bias% was 3.7% (figure 2). A value of 10% bias was considered to be a low value (Ricci, et. al., 2006). This finding confirmed the predictability of the model, suggesting possibility to utilize this model in optimization of EE% of drugs under similar experimental conditions. Assessment of the main effects and interactions by the experimental study showed that the EE% of lornoxicam in chitosan/TPP microspheres was affected by all the studied factors. All the terms except two factor interaction AB were significant at p<0.05 (table not shown). Concerning the effect of chitosan solution pH on lornoxicam EE%, results of the three dimensional plots (figure 3A) show a significant increase in EE% (P<0.001) upon increasing
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chitosan solution pH from 4 to 4.5. pH of the chitosan solution has a great impact on the particle size, so it was varied in this study to explore its influence on the lornoxicam EE%. The increase in EE% could be ascribed to two different effects: first, being a weak acid with pKa of 4.7, lornoxicam molecules gain negative surface charge as the pH of the chitosan solution increases thus favoring interactions with cationic charge of chitosan which resulted in higher EE%. Moreover; increase in particle size observed at higher pH also improved lornoxicam EE% (Mattu, Li and Ciardelli, 2013). Regarding the effect of TPP solution pH, (figure 3B) shows a significant (p<0.001) increase in EE% among all formulae with decrease in TPP solution pH. Environmental pH can only be varied within a restricted range, since acidic pH will lower the charge density of TPP, decreasing its cross-linking capability, while pH larger than 6 would lower charge density of chitosan, decreasing not only its capability to cross-link but also its solubility and thus promoting its aggregation. Lornoxicam shows distinct pH dependent solubility characterized by very poor solubility in acidic conditions. Improvement of lornoxicam EE% by decreasing pH of TPP solution from its original alkaline pH (8.9-9) is due to inhibition of lornoxicam loss from the microspheres due to decreased drug solubility in aqueous phase as TPP pH decreased (Sezer and Akbuga, 1995). As for the effect of lornoxicam concentration on EE%, (figure 3C) shows a significant increase in the EE% (p<0.001) by the increase in the lornoxicam concentration. This could be attributed to the increase in particle size of chitosan/TPP microspheres as larger particles have large cores which allow more drug to be encapsulated. The increase in the EE% could also be explained by the fact that chitosan/TPP microspheres carry a positive charge which can electrostatically interact with negatively charged drugs through a combination of ionic and 14
hydrogen bonding interactions. At high drug concentration, molecular abundance (crowding) between drug molecules within chitosan droplets occurs leading to steric hindrance at the escaping sites, hindering escape of drug molecules out of droplets and thus increasing EE% (El Gindy, El Khodairy, Molokhia and Elzoghby, 2011). From factorial design experiment it was concluded that microspheres formulation (L9) showed the highest EE% of 59.5% ±2.2 and particle size of 5.4µm±0.03, which is suitable for intra-articular delivery as previously reported in other studies involving the intra-articular delivery route. Thakkar, Sharma, Mishra, Chuttani and Murthy, 2004, studied the effect of celecoxib loaded chitosan microspheres prepared by emulsion chemical cross linking technique, having particle size of 8 µm for intraarticular delivery, results showed that the anti-arthritic effect and duration was enhanced compared to celecoxib alone, as the microspheres were in the desired size for phagocytosis by synovial macrophages thereby prolonging its local therapeutic effect. Conclusively, it was reported by Butoescu, Jordan and Doelker, 2009, that by analyzing the diameters of all types of microparticles tested for intra-articular delivery of drugs, it was found that the most suitable size is between 5-10 µm. This size range ensures the capture of the microparticles by the joint macrophages leading to sustainment of the drugs local effect in the joints. 3.2-Microspheres morphology Chitosan microspheres had spherical shape and rough surface. Indentations appearing on the surface of microspheres could be due to rapid evaporation of solvent and formation of crimple structure (figure 4) (Calderon, et.al., 2013). The hydrophobicity of lornoxicam, remaining as insoluble particles on the surface pores can also cause surface roughness (Cho, Chun, Kim and Park, 2014). 3.3-In-vitro release study 15
Another desirable property for intra articular drug delivery systems is sustained drug release. Effects of drug loading and pHs of chitosan and TPP solutions on lornoxicam release from chitosan/TPP microspheres were studied (figure 5 A, B and C). Sustained release profiles were achieved over approximately 8days compared to lornoxicam suspension in PBS7.4 which was released rapidly from the dialysis membrane within the first 6hrs. This emphasizes that entrapment of lornoxicam in chitosan/TPP microspheres can effectively sustain its release as a consequence of chitosan hydration and swelling which is responsible for prolonged release. An initial burst release followed by a second stage of slow drug release, is probably a consequence of rapid dissolution of surface bound drug (Mattu, Li and Ciardelli, 2013). By comparing the results of different formulae at day 4 (96 hrs.) it was found that the release rate was significantly (p< 0.05) increased with the increase of drug amount from 63%±1(L13) to 67.4±2(L11) and then to 71.6±1(L5) for lornoxicam concentration 1%, 1.5% to 2%, respectively. These results suggest that lornoxicam release behavior is related to lornoxicam concentration, where higher lornoxicam loading caused significant enlargement of particle size (p<0.05), thereby increasing total particle surface area available for release from single particle. Drug release rate increased with increasing microparticles diameter as large microparticles become more porous during drug release than small microparticles (Kang, Ko, Kim and Im, 2014). Increasing the chitosan and TPP solution pH led to quicker drug release showing values after 4 days of 56.7%±5.8(L9), 64.2%±3.2(L15) and 71.6%±1(L5) for TPP pH 8, 8.5 and 9, respectively and 71.6%±1(L5) and 64.7%±2(L6) for chitosan pH 4.5 and 4, respectively. Ioniccrosslinking density of chitosan/TPP microparticles was improved by using TPP and chitosan solutions at low pH values. Ionization degree of TPP and chitosan is high with quick gelling of 16
chitosan due to complete cross linking without deprotonation i.e. crosslinking sites increased, chitosan microspheres with tighter structure were formed and diffusion rate of lornoxicam decreased correspondingly (Ma and Lu, 2010). Pore structure of chitosan microspheres was modified by the change of pH of the TPP solution, at pH 8.9, open porous structure was observed; which is more degradable than high density compact structure leading to high release rate (Mi, Shyu, Lee and Wong, 1999). Kinetic analysis of in-vitro release profile of lornoxicam chitosan microspheres was done to ascertain release order. Since the dtermination coefficient (R2) for Higuchi model was nearer to unity, this indicates that diffusion through matrices was the main factor controlling lornoxicam release from chitosan/TPP microspheres. 3.4-Differential scanning calorimetry study (DSC) Thermal behavior of drug loaded microspheres was compared to those of original powders (lornoxicam, chitosan, TPP and their physical mixture). DSC curves figure6A shows that lornoxicam exhibited a sharp exothermic peak at 229ºC corresponding to its melting and decomposition (Hamza and Abu Rahma, 2009). Chitosan polymer showed a characteristic endothermic peak at 86ºC due to loss of bound water (Zhang, Yang, Tang, Hu and Zou, 2008). Polysaccharides usually have strong affinity for water and in solid state these macromolecules may have disordered structure that can be easily hydrated. Thermogram of the physical mixture revealed the existence of lornoxicam exothermic peak at 233ºC, small shift indicates the absence of interaction between the drug and excipients. Noticeable broadening of the peak was observed which could be due to the dilution effect caused by presence of formulation excipients. Lornoxicam peak disappeared in L9 indicating its presence as molecular dispersion within
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polymer matrix. An endothermic peak at 165ºC was observed which suggests that addition of TPP to chitosan promotes structural change due to ionic cross linking in the microparticles. 3.5-Fourier transform infrared spectroscopy (FTIR) FTIR spectra of lornoxicam, chitosan, TPP and L9 lornoxicam loaded chitosan/TPP microspheres are shown in figure6B. Lornoxicam spectra showed characteristic peaks of drug at 3066 cm-1 corresponding to –NH stretching vibration. Sharp peaks were observed at 1644, 1596 and 1554.5 cm-1 due to stretching vibration of C=O group in the primary amide and bending vibration of –NH group of the secondary amide, respectively. Absorption peaks at 1385.6, 1334 and 1155cm-1 are assigned to stretching vibrations of O=S=O group. Peak at 828cm-1 corresponds to –CH aromatic ring bending. Peak at 768.4cm-1 could be related to C-CL bending (Tadros and Fahmy, 2014). Chitosan showed broad band at around 3431cm-1which could be attributed to stretching vibration of –NH2 and –OH, related to extra-molecular hydrogen bonding of molecules. Characteristic absorption bands appearing at 1657cm-1 (Amide I), 1599cm-1 (-NH2 bending) and 1320cm-1 (Amide III) were observed. Peaks at 1084 and 1034cm-1 were assigned to the secondary hydroxyl group and primary hydroxyl group (Vaezifar, et.al., 2013). In the FTIR spectrum of (L9), –OH peak at 3431cm-1 was shifted to 3400cm-1 and the band became less wide, indicating reduced hydrogen bonding. This is due to more open structure resulting from crosslinking with TPP. –NH2 Bending vibration was observed at 1628cm-1 instead of 1599cm-1 due to interaction of TPP ions with –NH3+ ions of chitosan. Peak at 1215cm-1 is assigned to P=O groups of TPP. Shifting of this peak to 1209cm-1 and decrease in its intensity is another evidence of electrostatic interaction between negatively charged phosphate groups of TPP and positively charged amino groups in chitosan (Li and Huang, 2012). Disappearance of some peaks of
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lornoxicam and shift of other peaks confirmed its entrapment inside the chitosan/TPP microspheres. 3.6-Anti-inflammatory activity in rats 3.6.1-Joint swelling measurement Anti-inflammatory effect of lornoxicam solution, lornoxicam loaded chitosan/TPP microspheres (L9) were assessed by %inhibition in knee joint swelling after MIA induced osteoarthritis in rat’s knees. The %inhibition of knee joint swelling after day 7 of MIA induction for groups (III and IV) was statistically higher than non-treated osteoarthritic control group II (p<0.05) (figure7A) with values of 20.5±2%, 23.3±1% and 13.3±1%, respectively. Reduction in knee joint swelling was attributed mainly to the intra-articular injection of lornoxicam, a powerful non-selective COX inhibitor reducing prostaglandin production, thereby initiating rapid relief of inflammation and edema relative to group II. Inhibition achieved in group II without any treatment is due to the impact of natural body’s response to reduce inflammation (Elron-Gross, Glucksam, Biton and Margalit, 2009). The %inhibition of knee swelling for group III treated with intra-articular injection of lornoxicam solution after day 14 did not significantly change from that of day 7 showing values of 21.2±1.5% and 20.5±2%, respectively (p>0.05). However, there was a continued increase in %inhibition in group IV having value of 33.3±1% after 14 days of MIA induction. This is attributed to the rapid elimination of lornoxicam solution from joint cavity after intra-articular administration. Lornoxicam solution was reported to have a short residence time (<48 hrs.) in joint. Free drugs with high concentration instilled in joint cavity also, could endanger local cells viability. Encapsulation of lornoxicam in chitosan/TPP microspheres provided an advantage of 19
preserving cells viability (Erdemli, et.al., 2014). Prolongation of lornoxicam residence time inside the joint led to sustainment of its action to cover the whole period of the study i.e. 21 days, reaching %inhibition of joint swelling of 36.7±0.7% for rats of group IV. 3.6.2-Measurement of biological marker of inflammation (Interleukin 6, IL6) Inflammatory cytokines play a crucial role in the process of cartilage degeneration in osteoarthritis. They are synthesized abundantly by osteoarthritic synovium and increased levels of IL6 were observed in osteoarthritic synovial fluid (Hashizume and Mihara, 2009). This study gives an insight into the proinflammatory activities of IL6 in an established model of MIA induced osteoarthritis in rats. Development of osteoarthritis was associated with increased levels of IL6 in knee joint after 1 day of MIA intra-articular injection (figure 7B) with mean value of 434.3±8.4pg/gm which was significantly higher than IL6 values in knee joints of normal rats of group I showing mean value of 56.7±24pg/gm (p<0.05). Intra-articular administration of lornoxicam in osteoarthritic rat knee joints in groups III and IV, showed a significant reduction in IL6 values if compared to values of group II day 7 with values of 385.5±19.2, 218.32±2 and 286±74pg/gm tissue for groups II, III and IV, respectively. Marked effect seen with lornoxicam inhibition of IL6 production may present a favorable additional anti-inflammatory and analgesic property to lornoxicam, since IL6 exhibits, a plethora of pro-inflammatory effects and its levels have been correlated with inflammatory diseases (Zhang, Bi, Li and Huang, 2011). Animals treated with intra-articular injection of free lornoxicam solution (group III), seemed to have short term effect where reduction of IL6 levels at days 14 and 21 post MIA induction were not statistically significant from their effect after 7 days (p>0.05) with values of
20
255±45, 251±63 and 218.32±45pg/gm, respectively. This confirms the escape of lornoxicam solution from joint cavity where local drug concentration falls and thereby decreases its antiinflammatory therapeutic activity. Animals of group IV treated with an intra-articular injection of (L9) showed better results than those of group III treated with lornoxicam solution from day 14 with a stepwise decrease in the IL6 values in the rast knee joints from 7 days to 14 and then finally 21 days, showing values of 286±74, 213±22.6 and 154.212pg/gm, respectively. This stepwise decrease in pro-inflammatory cytokine (IL6), suggests sustained effect of lornoxicam when entrapped in chitosan microspheres relative to free lornoxicam solution, thus maintaining high local therapeutic concentrations of lornoxicam over longer period and fewer side effects than oral lornoxicam. 3.6.3-Histopathologic analysis Histopathological examination of knee joints of animals of group I (normal control) (figure 8) shows normal histological structure of articular cartilaginous surface, synovial membrane and periarticular tissues. Neither inflammatory cells infiltrations nor edema were observed. Chondrocytes are normally distributed in parallel rows in articular cartilage. Control group II histological manifestations on day 1 shows necrobiotic changes in superficial layer of articular cartilage. Thickening and edema with inflammatory cells were observed in synovial membrane. After 7 days of induction, progression of osteoarthritis was obvious with complete loss of synovial lining, hyperplasia, necrosis with inflammatory cells in synovial membrane and decrease in chondrocytes number. On day 14, proliferation in fibrous connective tissues with fibrosis i.e. osteoarthritis chronicity was observed. Complete loss of chondrocytes, articular cartilage surface resorption with hypertrophy and hyperplasia of synovial membrane was observed. Reaching day 21, fibrosis and hyperplasia in synovial membrane was evident. Cluster 21
formation of chondrocytes with complete loss of articular cartilage and adipose tissue deposition in functionless spaces has occurred. Time course histological features in rats of group III treated with lornoxicam solution, on day 7 of MIA induction showed focal resorption in articular cartilage and inflammatory cells infiltration with decrease in chondrocytes number. At day 14, resorption in articular cartilage was enhanced, together with fibrosis and hyperplasia in the synovial membrane and the beginning of chondrocytes cluster formation. At the end of the study (day 21), increased severity of histological picture was evident with signs of chronic osteoarthritis as periarticular fibrosis. These results supported the fact for rapid drug solution clearance when injected intra-articularly from knee joints and preventing its therapeutic effect from reversing histopathologic manifestations in osteoarthritic knee joints. As for rats of group IV treated with L9, at day 7 of MIA induction, histopathology of knee joints was quite similar to the picture in rats of group III after 7 days, however, these conditions gradually resolved by day 14 showing normal chondrocytes distribution with fewer articular resorptions and nearly intact synovial membrane. Moving on to day 21 of the study, apparently intact cartilaginous structures with few focal resorption and disappearance of inflammatory cells was observed. The histopathological picture mirrored the results of knee joint swelling and IL6 levels in joint tissues, where gradual decrease in knee swelling and IL6 levels was accompanied by significant reduction in histopathological manifestations in rat knee joints. Conclusion Chitosan/TPP microspheres (L9) is a promising carrier that can be exploited for local intra-articular delivery of lornoxicam to the site of osteoarthritis relative to lornoxicam drug
22
solution. It showed better reduction of inflammatory conditions in a MIA induced osteoarthritic rat model than lornoxicam solution with prolonged pharmacological effect. This formula can be used as a good option in other inflammatory processes encountered in daily orthopedic practice like tendinitis and tensosynovitis, which often cause considerable discomfort and impaired functions of the joints Declaration of interest The authors reports no conflict of interest
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Figure 1 :(A) Predicted versus actual values of EE% (B) Box-Cox plot for power transformations. Figure 2: Contour plot generated at (A): the medium levels of XA (chitosan pH) and XB (TPP pH) showing the predicted values of central points at medium level of XC (lornoxicam conc. 1.5%w/w). (B): medium levels of XA (chitosan pH) and XB (TPP pH) showing the predicted values of central points at low level of XC (lornoxicam conc. 1%w/w). (C): medium level of XA (chitosan pH) and low level of XB (TPP pH) showing the predicted values of central points at low level of XC (lornoxicam conc. 1%w/w)
Figure 3: Three dimensional surface plot of the effect of (A): chitosan pH (XA) and TPP pH (XB) at the minimum level of lornoxicam concentration (1%w/w) (XC). (B): chitosan pH (XA) and TPP pH (XB) at the maximum level of lornoxicam concentration (2%w/w) (XC). (C): chitosan pH (XA) and TPP pH (XB) at the medium level of lornoxicam concentration (1.5%w/w)
Figure 4: Scanning electron micrographs of lornoxicam loaded chitosan/TPP microspheres (L9) at magnification 6500X, 3500X,5000X.
Figure 5: Effect of (A): lornoxicam concentration, (B): chitosan solution pH and (C): TPP solution pH on lornoxicam release rate in PBS 7.4 at 37˚C.
Figure 6 (A). DSC thermogram of lornoxicam, chitosan, TPP, physical mixture, and drug loaded chitosan/TPP microspheres(L9). (B): FTIR spectra of lornoxicam, chitosan, TPP and lornoxicam loaded microspheres L9
Figure 7: A. Percentage inhibition of Rat knee joint swelling in MIA induced arthritis at various time intervals. B. IL6 concentration in rat knee joints in MIA induced arthritis at various time intervals
Figure 8: Histological evaluation of rat knee joints from different groups (x40 H&E stain): (A) group II 1 day after osteoarthritis induction; (B) group I; (C) group II 7 days after induction; (D) group II 14 days after induction; (E) group II 21 days after induction; (F) group III 7 days after induction; (G) group III 14 days after induction; (H) group III 21 29
days after induction; (I) group IV 7 days after induction; (J) group IV 14 days after induction; (K) group IV 21 days after induction
30
Table 1: Composition of prepared lornoxicam loaded chitosan/TPP microspheres according to the factorial design experiment and their measured responses. Particle size ±S.D.
Formula code
XA
XB
XC
EE%±S.D.
L1
1
0
-1
33.7±1.0
5.18±0.00
L2
1
-1
0
50.5±1.4
4.07±0.02
L3
-1
0
0
34.5±0.0
5.23±0.03
L4
1
0
0
40.0±0.1
5.30±0.02
L5
1
1
1
32.3±1.9
6.12±0.00
L6
-1
1
1
25.6±1.3
6.10±0.10
L7
-1
-1
0
42.2±2.0
4.24±0.02
L8
-1
1
-1
13.5±0.3
5.28±0.00
L9
1
-1
1
59.5±2.2
5.40±0.03
L10
1
-1
-1
45.9±0.2
4.02±0.00
L11
1
1
0
30.5±0.5
5.99±0.00
L12
-1
0
1
34.5±0.0
5.28±0.04
L13
1
1
-1
27.8±0.8
5.24±0.00
L14
-1
0
-1
17.1±1.1
4.35±0.01
L15
1
0
1
52.0±0.7
6.00±0.02
L16
-1
-1
-1
24.8±5.0
3.57±0.02
L17
-1
1
0
17.8±1.5
5.05±0.01
L18
-1
-1
1
47.7±1.1
5.50±0.00
(µm)
* XA: chitosan pH 4 (-1); 4.5 (1) XB: TPP pH 8 (-1); 8.5 (0); 9 (1) XC: Lornoxicam conc. 1% (-1); 1.5% (0); 2% (1).
31
S0144-8617(16)30473-8 http://dx.doi.org/doi:10.1016/j.carbpol.2016.04.096 CARP 11036
To appear in: Received date: Revised date: Accepted date:
18-2-2016 20-4-2016 22-4-2016
Please cite this article as: Abd-Allah, Hend., Kamel, Amany O., & Sammour, Omaima A., Injectable long acting chitosan/tripolyphosphate microspheres for the intra-articular delivery of lornoxicam: optimization and in vivo evaluation.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.04.096 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Injectable long acting chitosan/tripolyphosphate microspheres for the intraarticular delivery of lornoxicam: optimization and in vivo evaluation. Hend Abd-Allah, Amany O. Kamel*, Omaima A. Sammour
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt Address: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University. African organization Unity Street, Cairo, Egypt, P.O. box 11566. *
Correspondence: Amany Kamel Email [email protected]
Address : Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Monazzamet El Wehda El Afrikeya Street, Abbasseya, Cairo, Egypt Tel.: +201001429211 ; fax: +202 24051106.
1
Lornoxicam loaded chitosan/TPP microspheres prepared with ionotropic gelation for intra-articular delivery Optimization of formulation variables was studied using full factorial design experiment to achieve the highest lornoxicam encapsulation efficiency In-vivo anti-inflammatory activity of the optimized microspheres was tested in a mono-iodoacetate induced osteoarthritic model in rats
The present study could be beneficial in managing chronic pain and inflammation following various orthopedic joint related surgeries and decreasing long term joint diseases as osteoarthritis
2
Abstract Chitosan microspheres were formulated for the intra-articular delivery of lornoxicam in knee osteoarthritis, to minimize associated side-effects after prolonged oral administration. Ionotropic-gelation technique was employed using tripolyphosphate as anionic cross-linker. Fullfactorial design experiment was conducted to optimize lornoxicam entrapment-efficiency%. Formulations were assessed for their particle size, in-vitro drug release, Scanning electron microscopy, Differential-scanning-calorimetry and Fourier transform infra-red spectroscopy studies. Changing independent variables, chitosan pH, TPP pH and lornoxicam concentration resulted in different values of entrapment-efficiency% ranging from 13.5%±0.35 to 59.5%±2.2. Particle size ranged from 3.57µm±0.02 to 6.12µm±0.00 and lornoxicam %release was prolonged for up to 8 days. SEM results showed spherical shape of the microspheres. FTIR and DSC studies confirmed the crosslinking of chitosan with tripolyphosphate. In-vivo therapeutic effect of lornoxicam microspheres was investigated using Monosodiumiodoacetate (MIA) induced osteoarthritis model in rats. Optimized formula showed long-term in-vivo anti-inflammatory effect relative to lornoxicam solution injected intra-articularly with significant reduction of histological, inflammatory and biochemical parameters of osteoarthritis. Key
words:
Lornoxicam,
Chitosan,
Tripolyphosphate,
Monoiodoacetate.
3
Microspheres,
Intra-articular,
1- Introduction Knee osteoarthritis, is among the big challenges causing pain and disability especially in elderly patients. First choice treatment option is oral administration of non-steroidal antiinflammatory drugs (NSAIDs) for relieving both inflammation and symptomatic pain (Yoshimi, et.al., 2010). However, Oral administration of NSAIDs is hampered by numerous side effects as GIT problems. Therefore, localized NSAIDs intra-articular administration has emerged as a promising strategy for management of osteoarthritis. The major limitation of the intra-articular administration of drug solutions is their low retention times in the intra-articular spaces, which emphasis the need to develop drug delivery systems that would function as depot systems for NSAIDs, slowly releasing the active drugs and providing localized sustained action. For this application chitosan has come to be a particularly interesting polymer. It possesses a number of beneficial properties that makes it unique among polymers used for intra-articular delivery. Chitosan is non-toxic, biodegradable, biocompatible, hydrophilic and because of its cationic nature it has very good mucoadhesive, antibacterial and membrane permeability properties. Interestingly; chitosan has been used in articular cartilage engineering as a scaffolding material and was found to increase chondrocyte proliferation when injected intra-articularly in rats. Together with some structural similarity with various glycosaminoglycanes found in articular cartilage (Bedouet, et.al., 2014). Chitosan microspheres display several advantages over hydrophobic polyester based particles for the intra-articular delivery. They reduce the amount of foreign materials injected in joint cavity, also hydrophilic surfaces are known to inhibit monocyte adhesion and lymphocytes proliferation (MacEwan, Brodbeck, Matsuda and Anderson, 2005). 4
Numerous publications describe the use of biodegradable microparticles as means for intra-articular delivery. Flubiprofen gelatin microspheres were able to prolong the drug release and increase targeting efficiency in the knee joints (Lu, Zhang, Sun and Zhong, 2007). Also, diclofenac sodium loaded albumin microspheres showed successful reduction of arthritis after their intra-articular administration in rabbit knee joints (Tuncay, et. al., 2000). Chitosan microspheres were able to extend the residence time of celecoxib in the rats knee joint after intraarticular injection (Thakkar, Sharma, Mishra, Chuttani and Murthy, 2004). Ionotropic gelation for chitosan microspheres preparation; is a very simple mild process producing complete hydrophilic environment of chitosan particles by avoiding the use of organic solvents (Tiyaboonchai, 2013). It is based on the electrostatic interaction between chitosan positively charged amino groups and negatively charged groups from various anions. The most popular polyanion investigated is tripolyphosphate (TPP) due to its non-toxic properties and quick gelling ability (Mattu, Li and Ciardelli, 2013). Lornoxicam is a weakly acidic NSAID of oxicam class with analgesic and antiinflammatory properties. Lornoxicam inhibits cyclooxygenase (COX), key enzyme of arachidonic acid pathway, thus inhibiting prostaglandin synthesis (Zhang, Bi, Li and Huang, 2011). Lornoxicam is used orally in cases of acute pain and inflammation in rheumatoid arthritis, osteoarthritis and post-operative pain related to orthopedic surgeries in multiple divided doses (8mg, 3 to 4 times daily) due to its short half-life (2-5hrs) (Kidd and Frenzel, 1996). However, lornoxicam with its low aqueous solubility and poor dissolution in upper GIT causes GIT irritation and formulation problems that limit its therapeutic applications by delaying rate of absorption (Hamza and Aburahma, 2009). Targeting local site of osteoarthritis by the intraarticular injection to maintain a sustained drug effect is a valuable goal to improve therapeutic 5
efficacy of lornoxicam and avoid systemic complications. However, its poor aqueous solubility constitutes a problem in development of intra-articular injection. Hence, the aim of this study was to investigate some key factors to optimize lornoxicam entrapment efficiency (EE%) in chitosan/TPP microspheres for safer intra-articular delivery. This was a challenge due to the hydrophobic nature of lornoxicam and the solely hydrophilic environment of the system. To the best of our knowledge lornoxicam loaded chitosan/TPP microspheres for intra-articular treatment of osteoarthritis has not been reported yet.
Optimization of formulation variables by full
factorial design was done to achieve highest lornoxicam EE% in chitosan/TPP microspheres with acceptable size for the intra-articular delivery route. In-vivo sequential alterations in inflammatory cytokines that parallel pathological changes in osteoarthritis were tested in monoiodoacetate (MIA) induced osteoarthritis rat model. 2-Materials and methods 2.1- Materials Lornoxicam (LOR): kindly supplied by El Obour Pharmaceutical Co., cairo, Egypt. Chitosan
medium
molecular
weight
(viscosity
200000cps,
75-85%
deacetylated),
Tripolyphosphate (TPP), Monosodiumiodoacetate (MIA), Chloral hydrate and glacial acetic acid were purchased from Sigma-aldrich,UK. Sodium hydroxide, Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, hydrochloric acid and isopropanol were purchased from El Nasr Pharmaceuticals, cairo, Egypt. 2.2- Preparation of lornoxicam loaded chitosan/TPP microspheres Lornoxicam loaded chitosan/TPP microspheres were prepared by ionotropic gelation technique following a preliminary study that optimized the preparation parameters of plain
6
microspheres with particle size suitable for intra-articular delivery (Cho, Chun, Kim and Park, 2014). Briefly, an amount of 2ml TPP (0.25%w/v) containing different concentrations of lornoxicam (1%, 1.5% and 2%w/w) was added to 5ml of chitosan solution (0.1%w/v) in 0.1%v/v acetic acid, under magnetic stirring at room temperature for 30mins (IKAMAG®, model C-MAG HS7, Germany). The formed microspheres suspension was then centrifuged at 4930xg for 5mins at 4°C (Cooling centrifuge model Z216 MK, Germany). The microspheres were washed two times with deionized water (Zhang, Tang, Xu and Li, 2013). The formed pellets were then freeze dried (Christ alpha, 1-2 LD plus, Germany). A full factorial design experiment was built up to study the effect of three independent variables viz. drug concentration at -1 (1%), 0 (1.5%) and 1 (2%) levels, chitosan solution pH at -1 (4) and +1 (4.5) levels and TPP solution pH at -1 (8), 0 (8.5) and +1 (9) levels on the studied response, lornoxicam EE% as shown in table 1. Design of the experiments and generation of a response surface methodology and data analysis were performed using Design-Expert® program v.9.0.4. (Stat-Ease, Inc., Minneapolis, USA). Response surface methodology was tested at an alpha of 0.0001 for proper modeling of EE% response. Analysis of variance (ANOVA) was used to evaluate experimental data with various descriptive statistical analyses such as R2, Adjusted R2 and predicted R2 to reflect statistical significance of the developed mathematical model. 2.3- Check point analysis for the factorial model Check point analysis was performed to validate model reliability in predicting microspheres behavior (Ricci, et. al., 2006). Central point’s chosen according to the contour plots obtained at different levels of factors corresponding to a total of three check points then chitosan microspheres were prepared, EE% was measured, and %bias was calculated to estimate agreement between actual and predicted EE% according to the following equation: 7
Bias% = Difference between actual and predicted values/Actual value X 100. 2.4- Characterization of lornoxicam chitosan/TPP microspheres 2.4.1- Particle size analysis Microspheres were suspended in isopropanol (where no swelling was expected) and sonicated for 30secs (Jalalipour, Tajerzadeh, Najafabadi & Barghi, 2008; Elmoafy, Osman, Elshamy and Awad, 2014 and Osman, et. al., 2013). Particle size expressed as volume mean diameter (D 4,3) was measured using laser diffraction particle size analyzer (Mastersizer X, Malvern Instruments Ltd., UK). 2.4.2.-Determination of lornoxicam entrapment efficiency% Accurately weighed amount of particles were suspended in 0.1N HCL for 24 hrs. Digested microspheres were filtered using 0.22µm membrane filter (Calderon, et. al., 2013). Lornoxicam was determined spectrophotometrically at 376nm (spectrophotometer, Jenway-6800, Japan). EE% was calculated using the following equation. EE%=Wa/Wt X 100 Wa= weight of actual entrapped lornoxicam Wt= weight of theoretical formulated lornoxicam 2.4.3- Microspheres morphology
8
Surface characteristics of microspheres were examined by scanning electron microscopy (SEM, JSM 5500, Joel, Japan). Samples mounted onto aluminum stubs and made electrically conductive by coating with a thin layer of gold. 2.4.4- In-vitro drug release study Weighed amount of microspheres corresponding to 2mg lornoxicam were dispersed in phosphate buffer saline (PBS7.4) in a dialysis bag and was placed in vials containing 28ml PBS 7.4 with 1%tween 80. The vials were placed in a shaking water bath shaken at 50 rpm and 37±0.5ºC (GFL, Germany) (Zhang, Bi, Li and Huang, 2011). Aliquots were taken at different time intervals and replenished with same volume of buffer. Samples were assayed spectrophotometrically at 376nm for lornoxicam content. Obtained data were kinetically treated to determine order of release using zero, first and Higuchi diffusion models. 2.4.5- Differential scanning calorimetry (DSC) Thermal properties of lornoxicam, chitosan, TPP and selected lornoxicam loaded chitosan microspheres as well as the physical mixture of their components were investigated using DSC (Shimadzu-DSC 60, Kyoto, Japan). Samples were sealed in aluminum pans and heated at a rate of 10ºC/min. till 300ºC, using dry nitrogen as carrier gas with a flow rate of 25ml/min. 2.4.6-Fourier transform infrared (FT-IR) spectroscopy FTIR spectra of lornoxicam, chitosan, TPP and selected lornoxicam loaded chitosan microspheres were recorded by FTIR spectrophotometer using KBr disc method (Shimadzu, Kyoto, Japan) (Mattu, Li and Ciardelli, 2013). All spectra were recorded at a resolution of 4 cm1
from 4000 to 400cm-1. 9
2.5-Anti-inflammatory activity in rats 2.5.1- Induction of Knee joint osteoarthritis The in vivo effectiveness of the prepared microspheres were assessed using monoiodoacetate (MIA) induced arthritis model in rats. Sixty-six male albino rats weighing 190250gm were divided into four groups. Group I (n=6 rats): served as normal control, Group II (n=24 rats): Osteoarthritic control group non-treated, Group III (n=18 rats): rats treated with intra-articular lornoxicam solution in normal saline (4mg lornoxicam /kg) (Zhang and Huang, 2012), Group IV (n=18 rats): rats treated with intra-articular lornoxicam chitosan/TPP microspheres (L9) (4mg lornoxicam /kg). Under 10% chloral hydrate (4ml/kg) anesthesia, rats of groups II, III and IV were intra-articularly injected with 3mg/50µl MIA in right knee joints using 27gauge needle. One day after MIA injection, substantial inflammation of knee joints was observed and drug treatment was intra-articularly administered (Ikeuchi, et.al., 2014) to groups III and IV.
At different intervals, viz., 7, 14 and 21 days, 6 rats were sacrificed. For
osteoarthritic control group II another 6 rats were sacrificed after 1 day of MIA induction to monitor full development of osteoarthritis in the rat knees before drug admistartion (Al-Saffar, Ganabadi, Yaakub and Fakurazi, 2009). The protocol of this study was reviewed and approved by the ethics committee of faculty of pharmacy, Ain Shams University, Cairo, Egypt. 2.5.2-Clinical assessment of osteoarthritic rats Effects of lornoxicam intra-articular treatment on rats were monitored by analyzing; knee joint diameter, cytokine (Interleukin 6, IL6) measurement and histological evaluation of knee joints. Assessments were done at different time intervals throughout the study i.e. 0, 1, 7, 14 and 21 days.
10
2.5.2.1-Joint swelling measurement Knee joint diameter, was measured using Vernier caliper (Cole-Parmer Instrument co.), before MIA joint induction. Anti-inflammatory effect of intra-articularly injected formulae, as reduction of knee joint swelling was determined. %knee joint swelling inhibition was calculated by measuring difference between average values of knee joint diameters between treated animals and control group II at each time interval using the following equation (Bishnoi, Jain, Hurkat and Jain, 2014). % inhibition of knee swelling= (VC-VT)/VC x100 Where VC= knee diameter of control group II, VT= knee diameter of each treated group 2.5.2.2- Measurement of biological marker of inflammation (Interleukin 6, IL6) Rats from different animal groups were sacrificed by cervical dislocation. Rats knee joints were amputated and cut by bone cutter, pulverized in liquid nitrogen bath using a mortar and pestle to obtain fine powder. Powder was transferred to 50ml tubes and suspended in normal saline. The suspension was homogenized at 4°C using high-speed homogenizer (Ultra Turrax T25; IKA, Germany). Supernatant was separated by centrifugation (5000rpm, 10mins, 4°C) and stored at -80°C until further analysis (Yoshimi, et. al., 2010). Cytokine (interleukin 6, IL6) was analyzed using commercially available ELIZA kit (Ray Bio® Rat IL-6, Ray Biotech., USA) according to the operation instruction of the kit for quantitative determination. Data were expressed as pg cytokine/gm tissue. 2.5.2.3-Histopathologic analysis
11
Rats were sacrificed after different time intervals from MIA induction. Knee joints were dissected and fixed in 10% formalin. Decalcification was performed using 10%formic acid followed by serial dilutions with alcohol for dehydration. Obtained tissue sections were stained by hematoxylin and eosin stain for examination by light microscope (Bar-yehudaet, et. al., 2009). 2.6- Statistical analysis One-way analysis of variance (ANOVA) followed by Tukey Kramer test were done using Graph Pad Instat® software. Statistical significance level (P) value was set at ≤ 0.05. 3-Results and discussion Changing independent variables, chitosan pH, TPP pH and lornoxicam concentration resulted in different values of EE% ranging from 13.5%±0.35 to 59.5%±2.2 (table 1). Particle size of lornoxicam loaded chitosan/TPP microspheres ranged from 3.57µm±0.02 to 6.12µm±0.00 (table 1), which lies within the accepted range for intra-articular drug delivery (510µm), in terms of injectability and phagocytosis by synovial macrophages to increase drug residence time in joints. (Thakkar, Sharma, Mishra, Chuttani and Murthy, 2004). By fitting the data for entrapment efficiency % response to various models, the best fitted model for the variables was found to be the two factor interaction (2FI) model (P<0.0001). The generated equation was as follows:
Entrapment Efficiency= -217.897 + 56.7167 * chitosan pH + 0.283333 *TPP pH + 218.733 * Drug conc. -0.433333 * chitosan pH * TPP pH -20.6 * chitosan pH * Drug conc. -13.4 * TPP pH * Drug conc.
12
The model showed R2=0.9741, adjusted R2 =0.9600 and predicted R2=0.9259. Adequate precision measured a signal to noise ratio to ensure that the model can be used to navigate the design space, its value was 29.673. Ratio greater than 4 is usually desirable (Abdel Hafez, Hathout and Sammour, 2014). A good agreement between experimental and predicted values for EE% response was noticed, suggesting statistical validity and significance of the model figure 1A. Box-Cox plot for power transformation figure 1B was generated by plotting power response transformation (symbolized by λ) against natural logarithm of residual sum of squares (Ln residual sum of squares). It showed that the current point lied inside the 95% confidence interval suggesting no recommended transformation and thus first order regression model was used. 3.1-Check point analysis Check points are useful determination for prediction power of the regression factorial model obtained. A good agreement between actual and predicted responses was obtained; the max estimated bias% was 3.7% (figure 2). A value of 10% bias was considered to be a low value (Ricci, et. al., 2006). This finding confirmed the predictability of the model, suggesting possibility to utilize this model in optimization of EE% of drugs under similar experimental conditions. Assessment of the main effects and interactions by the experimental study showed that the EE% of lornoxicam in chitosan/TPP microspheres was affected by all the studied factors. All the terms except two factor interaction AB were significant at p<0.05 (table not shown). Concerning the effect of chitosan solution pH on lornoxicam EE%, results of the three dimensional plots (figure 3A) show a significant increase in EE% (P<0.001) upon increasing
13
chitosan solution pH from 4 to 4.5. pH of the chitosan solution has a great impact on the particle size, so it was varied in this study to explore its influence on the lornoxicam EE%. The increase in EE% could be ascribed to two different effects: first, being a weak acid with pKa of 4.7, lornoxicam molecules gain negative surface charge as the pH of the chitosan solution increases thus favoring interactions with cationic charge of chitosan which resulted in higher EE%. Moreover; increase in particle size observed at higher pH also improved lornoxicam EE% (Mattu, Li and Ciardelli, 2013). Regarding the effect of TPP solution pH, (figure 3B) shows a significant (p<0.001) increase in EE% among all formulae with decrease in TPP solution pH. Environmental pH can only be varied within a restricted range, since acidic pH will lower the charge density of TPP, decreasing its cross-linking capability, while pH larger than 6 would lower charge density of chitosan, decreasing not only its capability to cross-link but also its solubility and thus promoting its aggregation. Lornoxicam shows distinct pH dependent solubility characterized by very poor solubility in acidic conditions. Improvement of lornoxicam EE% by decreasing pH of TPP solution from its original alkaline pH (8.9-9) is due to inhibition of lornoxicam loss from the microspheres due to decreased drug solubility in aqueous phase as TPP pH decreased (Sezer and Akbuga, 1995). As for the effect of lornoxicam concentration on EE%, (figure 3C) shows a significant increase in the EE% (p<0.001) by the increase in the lornoxicam concentration. This could be attributed to the increase in particle size of chitosan/TPP microspheres as larger particles have large cores which allow more drug to be encapsulated. The increase in the EE% could also be explained by the fact that chitosan/TPP microspheres carry a positive charge which can electrostatically interact with negatively charged drugs through a combination of ionic and 14
hydrogen bonding interactions. At high drug concentration, molecular abundance (crowding) between drug molecules within chitosan droplets occurs leading to steric hindrance at the escaping sites, hindering escape of drug molecules out of droplets and thus increasing EE% (El Gindy, El Khodairy, Molokhia and Elzoghby, 2011). From factorial design experiment it was concluded that microspheres formulation (L9) showed the highest EE% of 59.5% ±2.2 and particle size of 5.4µm±0.03, which is suitable for intra-articular delivery as previously reported in other studies involving the intra-articular delivery route. Thakkar, Sharma, Mishra, Chuttani and Murthy, 2004, studied the effect of celecoxib loaded chitosan microspheres prepared by emulsion chemical cross linking technique, having particle size of 8 µm for intraarticular delivery, results showed that the anti-arthritic effect and duration was enhanced compared to celecoxib alone, as the microspheres were in the desired size for phagocytosis by synovial macrophages thereby prolonging its local therapeutic effect. Conclusively, it was reported by Butoescu, Jordan and Doelker, 2009, that by analyzing the diameters of all types of microparticles tested for intra-articular delivery of drugs, it was found that the most suitable size is between 5-10 µm. This size range ensures the capture of the microparticles by the joint macrophages leading to sustainment of the drugs local effect in the joints. 3.2-Microspheres morphology Chitosan microspheres had spherical shape and rough surface. Indentations appearing on the surface of microspheres could be due to rapid evaporation of solvent and formation of crimple structure (figure 4) (Calderon, et.al., 2013). The hydrophobicity of lornoxicam, remaining as insoluble particles on the surface pores can also cause surface roughness (Cho, Chun, Kim and Park, 2014). 3.3-In-vitro release study 15
Another desirable property for intra articular drug delivery systems is sustained drug release. Effects of drug loading and pHs of chitosan and TPP solutions on lornoxicam release from chitosan/TPP microspheres were studied (figure 5 A, B and C). Sustained release profiles were achieved over approximately 8days compared to lornoxicam suspension in PBS7.4 which was released rapidly from the dialysis membrane within the first 6hrs. This emphasizes that entrapment of lornoxicam in chitosan/TPP microspheres can effectively sustain its release as a consequence of chitosan hydration and swelling which is responsible for prolonged release. An initial burst release followed by a second stage of slow drug release, is probably a consequence of rapid dissolution of surface bound drug (Mattu, Li and Ciardelli, 2013). By comparing the results of different formulae at day 4 (96 hrs.) it was found that the release rate was significantly (p< 0.05) increased with the increase of drug amount from 63%±1(L13) to 67.4±2(L11) and then to 71.6±1(L5) for lornoxicam concentration 1%, 1.5% to 2%, respectively. These results suggest that lornoxicam release behavior is related to lornoxicam concentration, where higher lornoxicam loading caused significant enlargement of particle size (p<0.05), thereby increasing total particle surface area available for release from single particle. Drug release rate increased with increasing microparticles diameter as large microparticles become more porous during drug release than small microparticles (Kang, Ko, Kim and Im, 2014). Increasing the chitosan and TPP solution pH led to quicker drug release showing values after 4 days of 56.7%±5.8(L9), 64.2%±3.2(L15) and 71.6%±1(L5) for TPP pH 8, 8.5 and 9, respectively and 71.6%±1(L5) and 64.7%±2(L6) for chitosan pH 4.5 and 4, respectively. Ioniccrosslinking density of chitosan/TPP microparticles was improved by using TPP and chitosan solutions at low pH values. Ionization degree of TPP and chitosan is high with quick gelling of 16
chitosan due to complete cross linking without deprotonation i.e. crosslinking sites increased, chitosan microspheres with tighter structure were formed and diffusion rate of lornoxicam decreased correspondingly (Ma and Lu, 2010). Pore structure of chitosan microspheres was modified by the change of pH of the TPP solution, at pH 8.9, open porous structure was observed; which is more degradable than high density compact structure leading to high release rate (Mi, Shyu, Lee and Wong, 1999). Kinetic analysis of in-vitro release profile of lornoxicam chitosan microspheres was done to ascertain release order. Since the dtermination coefficient (R2) for Higuchi model was nearer to unity, this indicates that diffusion through matrices was the main factor controlling lornoxicam release from chitosan/TPP microspheres. 3.4-Differential scanning calorimetry study (DSC) Thermal behavior of drug loaded microspheres was compared to those of original powders (lornoxicam, chitosan, TPP and their physical mixture). DSC curves figure6A shows that lornoxicam exhibited a sharp exothermic peak at 229ºC corresponding to its melting and decomposition (Hamza and Abu Rahma, 2009). Chitosan polymer showed a characteristic endothermic peak at 86ºC due to loss of bound water (Zhang, Yang, Tang, Hu and Zou, 2008). Polysaccharides usually have strong affinity for water and in solid state these macromolecules may have disordered structure that can be easily hydrated. Thermogram of the physical mixture revealed the existence of lornoxicam exothermic peak at 233ºC, small shift indicates the absence of interaction between the drug and excipients. Noticeable broadening of the peak was observed which could be due to the dilution effect caused by presence of formulation excipients. Lornoxicam peak disappeared in L9 indicating its presence as molecular dispersion within
17
polymer matrix. An endothermic peak at 165ºC was observed which suggests that addition of TPP to chitosan promotes structural change due to ionic cross linking in the microparticles. 3.5-Fourier transform infrared spectroscopy (FTIR) FTIR spectra of lornoxicam, chitosan, TPP and L9 lornoxicam loaded chitosan/TPP microspheres are shown in figure6B. Lornoxicam spectra showed characteristic peaks of drug at 3066 cm-1 corresponding to –NH stretching vibration. Sharp peaks were observed at 1644, 1596 and 1554.5 cm-1 due to stretching vibration of C=O group in the primary amide and bending vibration of –NH group of the secondary amide, respectively. Absorption peaks at 1385.6, 1334 and 1155cm-1 are assigned to stretching vibrations of O=S=O group. Peak at 828cm-1 corresponds to –CH aromatic ring bending. Peak at 768.4cm-1 could be related to C-CL bending (Tadros and Fahmy, 2014). Chitosan showed broad band at around 3431cm-1which could be attributed to stretching vibration of –NH2 and –OH, related to extra-molecular hydrogen bonding of molecules. Characteristic absorption bands appearing at 1657cm-1 (Amide I), 1599cm-1 (-NH2 bending) and 1320cm-1 (Amide III) were observed. Peaks at 1084 and 1034cm-1 were assigned to the secondary hydroxyl group and primary hydroxyl group (Vaezifar, et.al., 2013). In the FTIR spectrum of (L9), –OH peak at 3431cm-1 was shifted to 3400cm-1 and the band became less wide, indicating reduced hydrogen bonding. This is due to more open structure resulting from crosslinking with TPP. –NH2 Bending vibration was observed at 1628cm-1 instead of 1599cm-1 due to interaction of TPP ions with –NH3+ ions of chitosan. Peak at 1215cm-1 is assigned to P=O groups of TPP. Shifting of this peak to 1209cm-1 and decrease in its intensity is another evidence of electrostatic interaction between negatively charged phosphate groups of TPP and positively charged amino groups in chitosan (Li and Huang, 2012). Disappearance of some peaks of
18
lornoxicam and shift of other peaks confirmed its entrapment inside the chitosan/TPP microspheres. 3.6-Anti-inflammatory activity in rats 3.6.1-Joint swelling measurement Anti-inflammatory effect of lornoxicam solution, lornoxicam loaded chitosan/TPP microspheres (L9) were assessed by %inhibition in knee joint swelling after MIA induced osteoarthritis in rat’s knees. The %inhibition of knee joint swelling after day 7 of MIA induction for groups (III and IV) was statistically higher than non-treated osteoarthritic control group II (p<0.05) (figure7A) with values of 20.5±2%, 23.3±1% and 13.3±1%, respectively. Reduction in knee joint swelling was attributed mainly to the intra-articular injection of lornoxicam, a powerful non-selective COX inhibitor reducing prostaglandin production, thereby initiating rapid relief of inflammation and edema relative to group II. Inhibition achieved in group II without any treatment is due to the impact of natural body’s response to reduce inflammation (Elron-Gross, Glucksam, Biton and Margalit, 2009). The %inhibition of knee swelling for group III treated with intra-articular injection of lornoxicam solution after day 14 did not significantly change from that of day 7 showing values of 21.2±1.5% and 20.5±2%, respectively (p>0.05). However, there was a continued increase in %inhibition in group IV having value of 33.3±1% after 14 days of MIA induction. This is attributed to the rapid elimination of lornoxicam solution from joint cavity after intra-articular administration. Lornoxicam solution was reported to have a short residence time (<48 hrs.) in joint. Free drugs with high concentration instilled in joint cavity also, could endanger local cells viability. Encapsulation of lornoxicam in chitosan/TPP microspheres provided an advantage of 19
preserving cells viability (Erdemli, et.al., 2014). Prolongation of lornoxicam residence time inside the joint led to sustainment of its action to cover the whole period of the study i.e. 21 days, reaching %inhibition of joint swelling of 36.7±0.7% for rats of group IV. 3.6.2-Measurement of biological marker of inflammation (Interleukin 6, IL6) Inflammatory cytokines play a crucial role in the process of cartilage degeneration in osteoarthritis. They are synthesized abundantly by osteoarthritic synovium and increased levels of IL6 were observed in osteoarthritic synovial fluid (Hashizume and Mihara, 2009). This study gives an insight into the proinflammatory activities of IL6 in an established model of MIA induced osteoarthritis in rats. Development of osteoarthritis was associated with increased levels of IL6 in knee joint after 1 day of MIA intra-articular injection (figure 7B) with mean value of 434.3±8.4pg/gm which was significantly higher than IL6 values in knee joints of normal rats of group I showing mean value of 56.7±24pg/gm (p<0.05). Intra-articular administration of lornoxicam in osteoarthritic rat knee joints in groups III and IV, showed a significant reduction in IL6 values if compared to values of group II day 7 with values of 385.5±19.2, 218.32±2 and 286±74pg/gm tissue for groups II, III and IV, respectively. Marked effect seen with lornoxicam inhibition of IL6 production may present a favorable additional anti-inflammatory and analgesic property to lornoxicam, since IL6 exhibits, a plethora of pro-inflammatory effects and its levels have been correlated with inflammatory diseases (Zhang, Bi, Li and Huang, 2011). Animals treated with intra-articular injection of free lornoxicam solution (group III), seemed to have short term effect where reduction of IL6 levels at days 14 and 21 post MIA induction were not statistically significant from their effect after 7 days (p>0.05) with values of
20
255±45, 251±63 and 218.32±45pg/gm, respectively. This confirms the escape of lornoxicam solution from joint cavity where local drug concentration falls and thereby decreases its antiinflammatory therapeutic activity. Animals of group IV treated with an intra-articular injection of (L9) showed better results than those of group III treated with lornoxicam solution from day 14 with a stepwise decrease in the IL6 values in the rast knee joints from 7 days to 14 and then finally 21 days, showing values of 286±74, 213±22.6 and 154.212pg/gm, respectively. This stepwise decrease in pro-inflammatory cytokine (IL6), suggests sustained effect of lornoxicam when entrapped in chitosan microspheres relative to free lornoxicam solution, thus maintaining high local therapeutic concentrations of lornoxicam over longer period and fewer side effects than oral lornoxicam. 3.6.3-Histopathologic analysis Histopathological examination of knee joints of animals of group I (normal control) (figure 8) shows normal histological structure of articular cartilaginous surface, synovial membrane and periarticular tissues. Neither inflammatory cells infiltrations nor edema were observed. Chondrocytes are normally distributed in parallel rows in articular cartilage. Control group II histological manifestations on day 1 shows necrobiotic changes in superficial layer of articular cartilage. Thickening and edema with inflammatory cells were observed in synovial membrane. After 7 days of induction, progression of osteoarthritis was obvious with complete loss of synovial lining, hyperplasia, necrosis with inflammatory cells in synovial membrane and decrease in chondrocytes number. On day 14, proliferation in fibrous connective tissues with fibrosis i.e. osteoarthritis chronicity was observed. Complete loss of chondrocytes, articular cartilage surface resorption with hypertrophy and hyperplasia of synovial membrane was observed. Reaching day 21, fibrosis and hyperplasia in synovial membrane was evident. Cluster 21
formation of chondrocytes with complete loss of articular cartilage and adipose tissue deposition in functionless spaces has occurred. Time course histological features in rats of group III treated with lornoxicam solution, on day 7 of MIA induction showed focal resorption in articular cartilage and inflammatory cells infiltration with decrease in chondrocytes number. At day 14, resorption in articular cartilage was enhanced, together with fibrosis and hyperplasia in the synovial membrane and the beginning of chondrocytes cluster formation. At the end of the study (day 21), increased severity of histological picture was evident with signs of chronic osteoarthritis as periarticular fibrosis. These results supported the fact for rapid drug solution clearance when injected intra-articularly from knee joints and preventing its therapeutic effect from reversing histopathologic manifestations in osteoarthritic knee joints. As for rats of group IV treated with L9, at day 7 of MIA induction, histopathology of knee joints was quite similar to the picture in rats of group III after 7 days, however, these conditions gradually resolved by day 14 showing normal chondrocytes distribution with fewer articular resorptions and nearly intact synovial membrane. Moving on to day 21 of the study, apparently intact cartilaginous structures with few focal resorption and disappearance of inflammatory cells was observed. The histopathological picture mirrored the results of knee joint swelling and IL6 levels in joint tissues, where gradual decrease in knee swelling and IL6 levels was accompanied by significant reduction in histopathological manifestations in rat knee joints. Conclusion Chitosan/TPP microspheres (L9) is a promising carrier that can be exploited for local intra-articular delivery of lornoxicam to the site of osteoarthritis relative to lornoxicam drug
22
solution. It showed better reduction of inflammatory conditions in a MIA induced osteoarthritic rat model than lornoxicam solution with prolonged pharmacological effect. This formula can be used as a good option in other inflammatory processes encountered in daily orthopedic practice like tendinitis and tensosynovitis, which often cause considerable discomfort and impaired functions of the joints Declaration of interest The authors reports no conflict of interest
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Zhang,Z., Bi,X., Li,H., & Huang,G.(2011). Enhanced targeting efficiency of PLGA microspheres loaded with Lornoxicam for intra-articular administration. Drug delivery, 18(7), 536-544. Zhang,Z., & Huang,G.(2012). Intra-articular lornoxicam loaded PLGA microspheres: enhanced therapeutic efficiency and decreased systemic toxicity in the treatment of osteoarthritis. Drug delivery, 19(5), 255-263. Zhang, J., Tang, Q., Xu, X., & Li, N. (2013). Development and evaluation of a novel phytosome-loaded chitosan microsphere system for curcumin delivery. International journal of pharmaceutics, 448(1), 168-174.
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Figure 1 :(A) Predicted versus actual values of EE% (B) Box-Cox plot for power transformations. Figure 2: Contour plot generated at (A): the medium levels of XA (chitosan pH) and XB (TPP pH) showing the predicted values of central points at medium level of XC (lornoxicam conc. 1.5%w/w). (B): medium levels of XA (chitosan pH) and XB (TPP pH) showing the predicted values of central points at low level of XC (lornoxicam conc. 1%w/w). (C): medium level of XA (chitosan pH) and low level of XB (TPP pH) showing the predicted values of central points at low level of XC (lornoxicam conc. 1%w/w)
Figure 3: Three dimensional surface plot of the effect of (A): chitosan pH (XA) and TPP pH (XB) at the minimum level of lornoxicam concentration (1%w/w) (XC). (B): chitosan pH (XA) and TPP pH (XB) at the maximum level of lornoxicam concentration (2%w/w) (XC). (C): chitosan pH (XA) and TPP pH (XB) at the medium level of lornoxicam concentration (1.5%w/w)
Figure 4: Scanning electron micrographs of lornoxicam loaded chitosan/TPP microspheres (L9) at magnification 6500X, 3500X,5000X.
Figure 5: Effect of (A): lornoxicam concentration, (B): chitosan solution pH and (C): TPP solution pH on lornoxicam release rate in PBS 7.4 at 37˚C.
Figure 6 (A). DSC thermogram of lornoxicam, chitosan, TPP, physical mixture, and drug loaded chitosan/TPP microspheres(L9). (B): FTIR spectra of lornoxicam, chitosan, TPP and lornoxicam loaded microspheres L9
Figure 7: A. Percentage inhibition of Rat knee joint swelling in MIA induced arthritis at various time intervals. B. IL6 concentration in rat knee joints in MIA induced arthritis at various time intervals
Figure 8: Histological evaluation of rat knee joints from different groups (x40 H&E stain): (A) group II 1 day after osteoarthritis induction; (B) group I; (C) group II 7 days after induction; (D) group II 14 days after induction; (E) group II 21 days after induction; (F) group III 7 days after induction; (G) group III 14 days after induction; (H) group III 21 29
days after induction; (I) group IV 7 days after induction; (J) group IV 14 days after induction; (K) group IV 21 days after induction
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Table 1: Composition of prepared lornoxicam loaded chitosan/TPP microspheres according to the factorial design experiment and their measured responses. Particle size ±S.D.
Formula code
XA
XB
XC
EE%±S.D.
L1
1
0
-1
33.7±1.0
5.18±0.00
L2
1
-1
0
50.5±1.4
4.07±0.02
L3
-1
0
0
34.5±0.0
5.23±0.03
L4
1
0
0
40.0±0.1
5.30±0.02
L5
1
1
1
32.3±1.9
6.12±0.00
L6
-1
1
1
25.6±1.3
6.10±0.10
L7
-1
-1
0
42.2±2.0
4.24±0.02
L8
-1
1
-1
13.5±0.3
5.28±0.00
L9
1
-1
1
59.5±2.2
5.40±0.03
L10
1
-1
-1
45.9±0.2
4.02±0.00
L11
1
1
0
30.5±0.5
5.99±0.00
L12
-1
0
1
34.5±0.0
5.28±0.04
L13
1
1
-1
27.8±0.8
5.24±0.00
L14
-1
0
-1
17.1±1.1
4.35±0.01
L15
1
0
1
52.0±0.7
6.00±0.02
L16
-1
-1
-1
24.8±5.0
3.57±0.02
L17
-1
1
0
17.8±1.5
5.05±0.01
L18
-1
-1
1
47.7±1.1
5.50±0.00
(µm)
* XA: chitosan pH 4 (-1); 4.5 (1) XB: TPP pH 8 (-1); 8.5 (0); 9 (1) XC: Lornoxicam conc. 1% (-1); 1.5% (0); 2% (1).
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