IN VITRO AND IN VIVO EVALUATION OF SUSTAINED RELEASE KETOPROFEN-LOADED NANOPARTICLES

The purpose of this study is to i) fabricate a biodegradable nanoparticle formulation of Ketoprofen, ii) evaluate its characteristics, iii) investigate its in vitro dissolution and in vivo pharmaceutical property. The nanoparticle formulation was prepared by spray drying method using Eudragit L100 as the matrix polymer. Size and morphology of drug-loaded nanoparticles were characterized with the electron microscopes (TEM, SEM). These successfully prepared nanoparticles by spray drying method are spherical in shape and quite homologous with diameter size of 100 – 200 nm. The in vitro dissolution studies were conducted at pH 1.2 and 7.4. The results indicated that there is a significant increase in Keto concentration at pH 7.4 compared to pH 1.2. For the in vivo assessment, our Keto-loaded nanoparticles and referential Profenid were administered by oral gavages to rabbits. The results implied that Keto-loadednanoparticles remarkably increased AUC compared to Profenid.


INTRODUCTION
Ketoprofen is analgesics drug, classified into non-steroidal anti-inflammatory group.It is commonly used to treat rheumatism and arthritis.However, the conventional capsule formulation of Ketoprofen has several disadvantages such as the short half-life, low bioavailability and the side effects [1,2].
To overcome these disadvantages, during the last few decades, several new approaches using polymer for preparing Ketoprofen nanoparticles have been studied.Besides thesustained release ability [4][5][6][7], polymeric nanoparticle formulation presents the drug in very fine nanodroplets offering very high surface area for absorption.This helps with quick absorption of the drug, thus improves oral bioavailability.
Moreover, poor permeability is also one of the major factors that limit oral bioavailability of several drugs.
Owing to low bioavailability, some drugs have to be administered at significantly higher doses, whereas the improvement in bioavailability can be translated into reduction in the drug dose and dose-related side effects of many hydrophobic drugs [8].Most of the current methods described for preparation of polymeric studies.Finally, anin vivo assessment of pharmaceutical properties was also conducted.

Materials
The pharmaceutical drug, Ketoprofen (3- Aerosil -a pharmaceutical excipient used in this research was purchased from GmbH (Germany).
All other chemicals usedwere procured from Merck (Germany).

Preparation of Particles
Nanoparticles containing Ketoprofen and Eudragit L100 were prepared by spray drying method.Briefly,the drug-polymer mixture was prepared by separately dissolving the Eudragit L100 and drug into acetone, using a magnetic stirrer and then mixing the solutions.The ratio of drug and polymer in prepared drug-polymer mixture was 1:3.The continuous process to

Characterization of Particles
The particle size and morphological examination of the nanoparticles were performed with TEM (JEM 1400, Japan) and SEM (JSM 6480LV-JEOL, Japan) measurements.The samples were placed on carbon-coated copper grids for viewing by TEM.For SEM, the samples from dry powder particles were prepared by gently dipping copper grids into the dry nanoparticles.

2.4.Quantifying Ketoprofen by Highperformance liquid chromatography
Ketoprofen concentration in unlnown samples were also determined by highperformance liquid chromatography (HPLC) using a Nucleosil ODS column (250  4,6 mm; 5 m particle size) at ambient temperature.The mobile phase consisted of 40% Acetonitrile and 60% water containing 1% acid acetic and 0.3% triethylamine.The system was run isocratically at a flow rate of 1.2mL/min.

In vitro dissolution studies
The ultimate aim of this work was to develop sustained release drug delivery system of Ketoprofen.To evaluate this ability, the release tests were performed at both pH conditions in stomach and small intestine.The pH of stomach is acidic, ranging from 1.3 to 5 depending on the fed-fasted conditions.The small intestine has a significantly higher pH level ranging from 6.5 to 7.

CONCLUSION
We used Eudragit L100 as a matrix polymer to prepare nanoparticle formulation of Ketoprofen.On the basis of the results of the investigations presented, it can be concluded that this formulation allows prolonged drug release.Due to its solubility, this polymer has the ability to prevent release drug from particles in the acidic medium, whereas, in pH condition of intestine, it is rapidly dissolved and shows a complete releasing the encapsulated material., 39, 173-191 (1993).
T, The Spray drying of pharmaceuticals,

Manufacturing
of suspensions are drug leakage from the particles into water phase, drug degradation, microbiological problems and physical changes such as aggregate formation in the course of time [10-13].To increase the physical and chemical stability of the nanoparticles, dry powders would be desirable [11, 14-16].Spray drying technique to produce nanoparticle dry powders have been studied.Spray-drying involves the conversion of a solution droplet into a dry particle by evaporation of the solvent in a one-step process [17-19].Compared with lyophilized powder obtained from an aqueous suspension, spray dried powders can be prepared without the problems of drug leakage to another phase, and thus, the recovery of drug in the particles is quantitative [17, 20].In this work, our laboratory has used successfully spray drying method for preparation of Ketoprofen nanoparticles with Eudragit L100, and the resultant dry powder was studied some characteristics.We pointed out several important chemical and physical properties of nanoparticles with Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM).An evaluation of the ability to release the drug at the condition of pH = 1.2 and pH = 7.4 for in vitro 5 [21].At each stage, an amount of 100 mg dry powder was weighed and filled into a gelatin capsule.Round-bottomed cylindrical glass vessels having a total volume of 900 mL were used as released chambers.The solutions were kept in a water bath at 37ºC ± 0.5 0 C and stirred at a speed of 75 rpm.For the acid stage, 900 mL of HCl 0.1N was used as the release medium.Aliquot (10 mL) was withdrawn at appropriate times.Immediately after each sampling, the aliquot was filtered with a membrane filter (0.45 μm in pore diameter) and the same volume fresh fluid at 37 0 C was supplemented to the test medium.The amount of ketoprofen released was determined as section 2.4.The measurements were performed three times; the values reported are mean values.The repeatability of the method was evaluated by analyzing three parallel samples.After two hours, we continued for the base stage.In this stage, a buffer solution at pH 7.4 was used as the release medium.The test was performed similarly for further four hours.The percentage of Ketoprofen released was determined from the following equation: gavages to rabbits.All rabbits were made to fast 18 hours prior to the dose administration and remained fasting until 4 hours after dose administration.The 100 mg of Keto-EUD containing 20 % of Keto was filled in gelatin capsule and then administered to rabbits by oral gavages.Blood samples of 2.5 mL were collected into vacutainer tubes containing EDTA prior and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 12.0, 24.0 and 36.0 hours after administration.After this collection, the blood samples were centrifuged at approximately 3500 rpm at 2-8 ºC for about 15 min.Each plasma specimen was collected and stored at -20 ºC until analysis.For the comparison, Profenid containing 32.4 % of Keto was also administered to rabbits by oral gavages following the similar procedures.The amount of Keto for oral administration was 50 mg for both Keto-EUD and Profenid.Preparation of plasma samples for determination of Keto in rabbit plasma by HPLC Keto in the plasma samples was determined by HPLC.Piroxicam was used as internal standard.A solution of internal standard was prepared with methanol at 50 μg.mL - .Samples were prepared as follows: 50 μL of plasma were extracted with 1 mL of internal standard solution in polypropylene tubes containing 100 μL of H 3 PO 4 and 3 mL of tertbutyl methyl ether.Samples were then sonicated for 5 min, sequent vortexed for 5 min and then centrifuged at 4,500 rpm for 5 min.Supernatants were transferred into glass test tubes.A blank (50 μL of blank plasma extracted with internal standard) and double blank (50 μL of blank plasma extracted with blank methanol) were also prepared.Samples were dried under nitrogen at 40 ºC, reconstituted with 500 μL of methanol, and transferred into a glass insert in an autosampler vial for HPLC assay.
L100 NPs by spray drying method.The obtained products were white dry powder samples, smooth and homogenous.Their TEM images were shown in Figure 1 which presents the successfully prepared nanoparticles.These exemplary TEM images showed solid, quite homogenous particles with size about 100 -200 nm.Grain boundaries or crystals were not detected, therefore, it was concluded that these nanoparticles had a matrix-type structure.The particles produced are amorphous due to rapid TAÏ P CHÍ PHAÙ T TRIEÅ N KH&CN, TAÄ P 16, SOÁ K1-2013 Trang 19 evaporation of the solvent from the droplets [21].The amorphous state has higher internal energy, larger free volume and greater molecular mobility in comparison to the crystalline state [23].These properties of the amorphous state lead to greater solubility.Amorphous solid have been used to achieve faster dissolution rates of drugs and to modify drug release [23, 24].For morphological examinations, the SEM photographs of drug loaded nanoparticles are shown in Figure 2. It can be clearly observed from these photographs that the nanoparticles made of polymer Eudragit L100 were spherical and smooth surface.

Figure 1 .
Figure 1.Exemplary TEM images of the particles fabricated.

Figure 2 .
Figure 2. Exemplary SEM Images of particles fabricated 3.2.Drug release from particles The results of in vitro release study in acidic and basic medium of our Keto-EUD nanoparticles (N20), referential Profenidwere shown in Table 2 and plotted in Figure 4.The percentages of Keto released from Profenid were almost negligible (less than 5 %) after two hours.On the other hand, for the first 30 min, the percentages of Keto released from N20 were 12% and then gradually increased with the increasing time.However, the percentages of Keto released were non-linear function of time.It implies that the Keto released from Keto-EUD nanoparticles due to the diffusion of Keto from the outer shells of nanoparticles or/and due to the partial dissolution of EUD L100 in acidic medium.The diffusion of drugs from the outer shells of nanoparticles was confirmed in the previous publications [25, 26].Keto in the outer shells

Figure 3 .
Figure 3.In vitro release of Keto-EUD nanoparticles, Profenid and pure Ketoin the acidic medium (in the first two hours) and in the basic medium (the continuous time) The in vivo assessment of oral administration of Keto-EUD nanoparticles was tested in rabbits with the aim to investigate the absorption ability of Keto-EUD nanoparticles, the maximum Keto concentration in plasma (C max ), time of maximum concentration (T max ) and the enhancement ratio of Keto-EUD nanoparticles.Keto-EUD nanoparticles and Profenid were administered by oral gavages to rabbits with the amount of Keto of 50 mg for each rabbit.The oral administration procedures and the preparation of plasma were stated in section 2.6.The plasma concentration-time profiles of Keto after oral administration in rabbits were shown in Figure 4.A comparison of T max between N20 and Profenid indicates that the concentration of Keto in plasma increased rapidly in both cases and peaks were observed after 2 hours.These results imply that Keto released in intestine from Keto-EUD nanoparticles and Profenid penetrated through intestine into blood stream of rabbits and the T max of Keto-EUD nanoperticles and Profenid were almost the same.

Figure 4 .
Figure 4. Plasma concentration-time profiles of Keto after oral administration in rabbits of N25 and Profenid.

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vitro release of N20 and Profenid in the acidic medium (in the first two hours) and in the basic medium (the continuous time) a a

Table 2 .
Pharmacokinetic parameters of Keto following oral administration of Keto-EUD nanoparticles and Profenid a .
a 50 mg of dose of Keto for each rabbit.b C max denotes maximum drug concentration.c T max denotes time of maximum concentration.d AUC area under the plasma concentration-time curve.e Determined by the equation: the ratio = the corresponding AUC/AUC of Profenid.