International Journal of Drug Discovery and Medical Research

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International Journal of Drug Discovery and Medical Research URL: www. ijddmr.org Email : editor@ijddmr.com, VOL.

Seth Department of Pharmacy, Sumandeep Vidyapeeth, Pipariya (Gujarat) *Corresponding author: Jaimin D patel jaimin.dmpatel@gmail.

1 International Journal of Drug Discovery and Medical Research URL: www. ijddmr.org editor@ijddmr.com, VOL. 1; 2;2012 ISSN FORMULATION & EVALUATION OF METHOTREXATE LOADED NANOPARTICLE Jaimin D patel *, Sachin P Chauhan, A. K. Seth Department of Pharmacy, Sumandeep Vidyapeeth, Pipariya (Gujarat) *Corresponding author: Jaimin D patel jaimin.dmpatel@gmail.com ABSTRACT Nanoparticles represent a particulate system, which can be produced with an established technique. Methotrexate loaded ethyl cellulose nanoparticles was prepared using solvent evaporation method with PVA as dispersion medium. Nine batches of nanoparticles with varying drug: polymer ratio and dispersion medium were prepared by using 3 2 factorial designs. Prepared nanoparticles were evaluated with respect to particle size, drug entrapment efficiency and in vitro release study. In-vitro release pattern of Methotrexate from Nanoparticles in the phosphate buffer of ph 7.4, showed a biphasic pattern with an initial burst effect and prolonged release over 24hrs. Furthermore, the excipients used in the thesis are well accepted worldwide in the pharmaceutical field. Among the nine formulations, formulation 8 (F8) has been selected as optimized formulation as it proved to be mean particles 267 nm, entrapment efficiency % and in-vitro release %. Stability studies showed that similar drug content and closest in vitro release profile to initial data when the sample stored at 25 0 C. Key words: Methotrexate, Nanoparticle, Solvent evaporation technique, In-vitro release. INTRODUCTION Nanoparticles (NPs) [1] Over the past two decades there has been a variety of controlled drug delivery strategies used to enhance the efficacy of the drug, such as developing soluble macromolecular carriers, micellar carriers, and colloidal micro- and nanoparticulate carriers. Those approaches, especially micro- and nanoparticulate carriers, have become extremely valuable in altering the pharmacokinetic and pharmacodynamics properties of the drug. Nanoparticles are solid, colloidal particles consisting of macromolecular substances that vary in size from 10 nm to 1000 nm. The drug of interest is dissolved, entrapped, adsorbed, attached or encapsulated into the nanoparticle matrix. Depending on the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained with different properties and release characteristics for the encapsulated therapeutic agent. [2] NPs can be further distinguished as nanospheres and nanocapsules. The illustration can be seen in Figure 1. In nanospheres, drugs are dispersed in the matrix structure as a uniform dispersion, whereas nanocapsules are vesicular systems in which the drug can be dissolved or entrapped in the inner core or adsorbed onto the surface of inner vesicles. Criteria of ideal NPs [3] As a drug carrier system, a nanoparticle (NP) formulation should meet the main criterion, in which the matrix should be biocompatible and biodegradable. The matrix must be non-toxic and must not exhibit any antigenic behaviour to the body. Moreover, the NPs should be able to load the drug, which is either hydrophilic or hydrophobic in nature, in a sufficient amount and maintain the integrity of the drug-matrix before release of the drug at the target site. The matrix, should therefore, protect the drug until it reaches the target site. Finally, the system should provide reliable and reproducible release kinetics for the drug in a controlled fashion. In terms of drug-carrier production, the cost-effective and reproducibility of the manufacturing of the drugcarrier complex should also be taken into consideration. Proposed advantages of NPs actually include: [4] Possible controlled drug release and drug targeting, Better drug stability, High drug payload,incorporation of lipophilic and hydrophilic drugs, Reducing the toxicity of the carrier. Diminished problems with respect to large scale production and sterilization processes,the drug incorporated is protected from biochemical degradation, Can be lyophilized and spray dried, Surface modification can be easily performed, Reduction of drug toxicity Reduction of side effects Ex 5-fluorouracil,Improve bioavailability Ex : Vincamycin, For prolong drug action Ex : Insulin, For controlled release of the drug Ex : Ibuprofen, Indomethacin. NPs is a better carrier than the emulsion if a prolonged and a sustained delivery of the drug is desired Limitation of NPs [4, 5] Much research has been conducted on nanoparticulate system; however it is still in an ongoing process to establish the formulation which is pharmaceutically and clinically accepted. Controlling the particle size has always been a challenge in developing NPs formulations. Although nano-sized particles can be easily made by various methods of NPs preparation, the physical stability of NPs, mainly the particle size, sometimes cannot be maintained due to the potential risk of particle aggregation.nps can load either hydrophilic or hydrophobic drugs; however, the level of loading appears to be low, compared to microparticles. Materials and Methods Materials Procurement of drug and excipients: 56

2 The drug, excipients and chemicals/ reagents used for research project work are enlisted as follows: List of materials used- Methotrexate, Ethyl cellulose, Dichloromethane, methanol, polyvinyl alcohol All other chemicals and reagents used were of analytical reagents (AR) grad Methods Preformulation Studies [6] Preformulation is defined as the phase of research and development process where physical, chemical and mechanical properties of a new drug substance are characterized alone and when combined with excipients, in order to develop stable, safe and effective dosage form. A thorough understanding of physicochemical properties may ultimately provide a rationale for formulation design, or support the need for molecular modification or merely confirm that there are no significant barriers to the formulation development. The goals of the program therefore are: To establish the necessary physicochemical characteristic of a new drug substance. To establish its compatibility with different excipients. Hence, Preformulation studies on the obtained sample of drug include solubility analysis, melting point determination and compatibility studies. Identification Tests [7, 8] IR Spectroscopy: The FT-IR spectrum of the obtained sample of the drug was compared with the standard FT-IR spectra of the pure drug by using FTIR Spectrophotometer (Shimadzu). Solubility analysis: Solubility analysis was carried out select a suitable solvent system to dissolve the drug and also to test its solubility in the dissolution medium which was to be used. It was checked by solubilising fixed amount of drug in 5 ml of each solvent analysed. Melting point determination: Melting point determination of the obtained drug sample was done by open capillary method. Drug was taken in glass capillary whose one end was sealed by flame. The capillary containing drug was dipped in liquid paraffin inside the melting point apparatus. Melting point is a good first indication of purity of the sample since the presence of relatively small amount of impurity can be detected by a lowering as well as widening in the melting point range. Spectroscopic Studies [9, 10] Determination of λmax (UV Spectroscopy): Stock solution (1000 µg/ml) of methotrexate was prepared in phosphate buffer solution ph 7.4. This solution was appropriately diluted with same buffer solution to obtain suitable diluted solution. The resultant solution was scanned in the range of nm on Shimadzu double beam UV Spectrophotometer. It showed a λmax at 302 nm in phosphate buffer solution ph 7.4. Preparation of standard calibration curve of methotrexate [11] Accurately weighed 50 mg methotrexate was dissolved in 50ml of phosphate buffer solution ph 7.4, which gives stock solution (S-I) of 1000µg/ml. From this stock solution withdraw 10ml and diluted to 100 ml with same fresh phosphate buffer solution, which gives second stock solution (S-II) having 100µg/ml concentration. From this solution (S-II) aliquots of 0.4ml, 0.8ml, 1.2ml, 1.6ml, and 2ml, were pipetted out into a series of 10 ml volumetric flasks and make up to mark with phosphate buffer solution ph 7.4 in order to get concentrations in the range of 4 to 20 µg/ml which is within the Beer s range. The absorbance of the resulting solution was then measured at 302 nm using UV spectrophotometer against respective parent solvent as blank. The standard curve was obtained by plotting Absorbance vs. Concentration in µg/ml. Compatibility studies by IR-Spectroscopy FT-IR spectroscopy was carried out to check the compatibility between drug and polymer. The FT-IR spectra of drug with polymers were compared with the standard FT-IR spectrum of the pure drug. Method of formulation Amongst the many formulations techniques solvent evaporation technique solvent evaporation techniques has been selected as it is highly feasible at lab scale. In present study, the effect of two different parameters such as drug: polymer ratio and concentration of dispersion medium was studied as it is highly influencing the preparation of NPs. So, here total 9 formulations have been formulated and evaluated by using 3 2 full factorial design. Solvent evaporation method [12] Nanoparticles formulations were prepared by solvent evaporation method. The various different amount of ethyl cellulose was dissolved in solvent mixture of methanol (2 ml) and dichloromethane (8 ml) very slowly on a magnetic stirrer and methotrexate (25 mg) was added to it and the contents were allowed to stand at room temperature for 30 to 45 minutes with occasional vortexing to allow complete solubilization of drug and polymer. This solution was poured into 5 ml of each different concentration aqueous polyvinyl alcohol solution. The resulting solution was homogenized by using high pressure homogenizer for 3 minutes to form o/w emulsion. This emulsion was immediately added drop wise to 125 ml of aqueous PVA solution. The contents were stirred for 6 hours at room temperature with a magnetic stirrer to evaporate organic volatile solvent, allowing the formation of a turbid nanoparticulate suspension. The suspension was filtered through membrane filter. The filtrate was centrifuged (1000 rpm for 10 minutes) and supernatant was collected. Further the ultracentrifugation (35000 rpm for 1 hour) was carried for supernatants. Following ultracentrifugation, the pellet was washed and collected two times with deionized water to remove adsorbed drug and was suspended in deionized water to prevent clumping on storage RESULTS AND DISCUSSION The current investigation deals with the optimization of nanoparticles of methotrexate. The concentration of methotrexate was kept constant at 25 mg. Characterization of methotrexate. Melting point The melting point was found to be in the range of 194º-196ºC which was in good agreement with the reported values. Spectroscopic studies UV spectroscopic studies (determination of λmax) The λmax of methotrexate was found to be at 302 nm in phosphate buffer ph 7.4 as shown in figure no 9. Standard graph of methotrexate in phosphate buffer ph 7.4 is shown in figure no 10. Good linearity was observed with the plot. The r 2 value in phosphate 57

3 buffer ph 7.4 was are shown in table no 10, which were very nearer to 1 and hence obeyed Beer-Lambert law. Determination of infrared absorption spectrum The FT-IR spectrum of methotrexate is shown in figure no 10. Spectral assignments for methotrexate IR spectrum indicated characteristic peaks belonging to functional group. From the FTIR spectrum it is concluded that there is no interaction between the methotrexate and used excipients. Characterization and Evaluation Percentage Yield: The obtained percentage yield of each formulations are shown in the Table no 11. It was obtained in between the range from 41 to 56 %. From the data it was concluded that the F2 and F8 had the highest percentage yield. Entrapment Efficiency: The obtained entrapment efficiency of each formulations are shown in the Table no 11. It was obtained in between the range from 16 to 65 %. The results revealed that, the formulation F8 gave better encapsulation and hence better entrapment efficiency. In-vitro Release Studies: Formulations F1 to F9 were subjected to in-vitro release studies. The release studies were performed in a phosphate buffer ph 7.4. The results obtained in the range of 59 to 96 %. Formulation F8 showed higher in vitro release i.e, % in a span of 24 hrs of study. Release kinetics The dissolution data were examined for models first order, zero order and Higuchi shown in table. The derived correlation coefficient (r 2 ) indicated good fit of Higuchi model suggesting that diffusion is the predominant mechanism limiting drug release. Scanning Electron Microscopy (SEM): Scanning Electron Microscopy (SEM) studies were carried out for the selected formulation (F8), the pictures revealed that the methotrexate nanoparticles were smooth and spherical. Particle Size Analysis: The particle size analysis was done for drug loaded nanoparticles in order to find the diameter of the particles. The mean particle size of the drug loaded nanoparticles was found to be 267 nm. Stability Studies for the Formulations: The selected formulation F8 was subjected to stability studies for a period of four weeks at 25 o C± 2 o C/60% ± 5% RH. Both physical and chemical changes were observed during the study. Physical stability was analyzed by morphological appearance and chemical stability was analyzed by the change in the drug contents. The results revealed that no much change in morphological appearance as well as in the drug content were observed. As a result of which the formulation was found to be stable at 25 o C± 2 o C/60% ± 5% RH. The present study has been satisfactorily attempted to formulate methotrexate loaded nanoparticle system for treatment of cancer using ethyl cellulose as polymer and polyvinyl alcohol as dispersion medium. The method used for the formulation was solvent evaporation method using 3 2 full factorial design. From the experimental results it can be summarised that, The IR spectra revealed that there was no interaction between polymers and drug, hence they are compatible. Practical yield and Percentage entrapment efficiency were higher for ethyl cellulose based nanoparticles. From the in vitro release profiles, it was concluded that dissolution behaviour of methotrexate was increased by using nanoparticles. As the concentration of dispersion medium and drug: polymer ratio increased, the percentage cumulative drug release also increased. From all formulations, F8 showed higher percentage drug release. For the mechanism of kinetics release, all the formulations showed higuchi release. Formulation F8 follows the first order kinetics followed by higuchi model which indicates that release profile may be diffusion control. From all above study like practical percentage yield, entrapment efficiency, in vitro release study formulation F8 was selected as optimised batch. Further study was evaluated for F8 formulation like surface morphology, particle size analysis and stability study. SEM analysis of the nanoparticles revealed that the optimized formulation was Smooth and spherical with ideal surface morphology. The particle size analysis revealed that formulation gave particles in the range of 250 to 400 nm which is suitable for intravenous administration of formulation. Stability studies for 4 weeks revealed that the formulations were stable 25 o C ± 2 o C and 60% ± 5% RH. It should be stored in a cool and dry place.thus, the formulated nanoparticles seem to be a potential candidate as controlled drug delivery system for symptomatic therapy of cancer treatment. REFERENCES 1. Kreuter J. Nanoparticles, in Encyclopaedia of Pharmaceutical Technology, Swarbrick J, Editor. 1994, Marcel Dekker Inc.: New York, USA. p Vauthier C, Dubernet C, Chauvierre C, Brigger I, Couvreur P, Drug delivery to resistant tumors: the potential of poly(alkyl cyanoacrylate) nanoparticles. J. Controlled Release, (2): p Barratt GM. Therapeutic applications of colloidal drug carriers. Pharm. Sci. Technol. To., : p Couvreur P, Dubernet C, Puisieux F. Controlled drug delivery with nanoparticles: current possibilities and future trends long-circulating and target-specific nanoparticles: theory to practice. Pharmacol. Rev., (2): p : p Guiot P, Couvreur P. editors. Polymeric nanoparticles and microspheres. Boca Raton: CRC Press; Wadke PA, Jacobson S. Preformulation Testing. In: Liberman HA, Lachman L,editors. Pharmaceutical Dosage Forms-Tablets.Vol-I. New York: Marcel Dekkar Inc.; Florey K, Al-badr AA, Brenner GS. Analytical profiles of drug substances. Vol. 16, New York: Academic Press Inc. 1987; p Dibbern HW, Muller RM, Wirbitzki E. UV and IR spectra: Pharmaceutical substances (UV and IR) and pharmaceutical and cosmetic excipients (IR). Aulendorf: Editio Cantor Verlag; Sghirlanzoni A, Pareyson D, Benvenuti C, Cei G, Cosi V, Lombardi M et al. Efficacy of intranasal administration of neostigmine in myasthenic patients. J Neurol 1992;239: Vyas SP, Khar RK. Controlled drug delivery concepts and advances. 1st ed. New Delhi: Vallabh Prakashan; p , Yeh M. Degradation Kinetics of methotrexate in Solution. Drug Dev Ind Pharm 2000;26(11):

4 12.El-Bagory IM, Hosny EA, Al-Suwayeh SA, Mahrous GM, Al-Jenoobi FI. Effects of sphere size, polymer to drug ratio and plasticizer concentration on the release of theophylline from ethylcellulose microspheres. Saudi Pharm J 2007 July- Oct;15(3-4): TABLE 1: EVALUTION OF NANOPARTICLES Formulat Percentage Yield Entrapment ion code efficiency in % F ± ± 0.96 F ± ± 1.32 F ± ± 1.9 F ± ± 2.12 F ± ± 1.19 F ± ± 2.5 F ± ± 1.08 F ± ± 3.08 F ± ± 2.56 TABLE 3: % DRUG RELEASE FOR BATCHES F4 TO F6 TIME F4 F5 F ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.65 TABLE 2: % DRUG RELEASE FOR BATCHES F1 TO F3 TIME F1 F2 F ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.76 TABLE 4: % DRUG RELEASE FOR BATCHES F7 TO F9 TIM E F7 F8 F ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.54 n=3 TABLE 5: STABILITY STUDY OF NANPARTICLES: Temperature and relative humidity, Appearance and % CDR OF OPTIMIZED BATCH (F8) Formulation code Temperature and relative humidity Appearance Weeks % CDR Weeks F8 25 o C± 2 o C/60% ± 5% RH No change TABLE 5: STABILITY STUDY OF NANPARTICLES PARTICLE SIZE AND ZETA POTENTIAL ANALYSIS OF OPTIMIZED BATCH (F8 ) Properties F8 Particle size (nm) ± 3.12 Polydispersity index 0.33 ± Zeta Potential (mv) ± 0.3

5 Figure-1Scanning Electron microscopy: 60

8. FORMULATION OF LANSOPRAZOLE NANOPARTICLES

8. FORMULATION OF LANSOPRAZOLE NANOPARTICLES FORMULATION OF LANSOPRAZOLE NANOPARTICLES Preparation of capsule of modified solubility to protect the drug from degradation To protect the drug from degradation