Formulation and Evaluation of Nelfinavir Extended Release Trilayer Matrix Tablets by Design of Experiment

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1 Rangu and Gande: Formulation and Evaluation of Nelfinavir Extended Release Trilayer Matrix Tablets by Design of International Journal of Pharmaceutical Sciences and Nanotechnology Research Paper Formulation and Evaluation of Nelfinavir Extended Release Trilayer Matrix Tablets by Design of Experiment Rangu Nirmala* and Gande Suresh Department of Pharmaceutics, Mewar University, NH-79, Chittorgarh , Rajasthan, India. Received July 23, 2018; accepted August 10, 2018 Volume 11 Issue 5 September October 2018 MS ID: IJPSN NIRMALA ABSTRACT The present study was aimed to develop once-daily controlled release trilayer matrix tablets of nelfinavir to achieve zero-order drug release for sustained plasma concentration. Nelfinavir trilayer matrix tablets were prepared by direct compression method and consisted of middle active layer with different grades of hydroxypropyl methylcellulose (HPMC), PVP (Polyvinyl Pyrrolidine) K-30 and MCC (Micro Crystalline Cellulose). Barrier layers were prepared with Polyox WSR-303, Xanthan gum, microcrystalline cellulose and magnesium stearate. Based on the evaluation parameters, drug dissolution profile and release drug kinetics DF8 were found to be optimized formulation. The developed drug delivery system provided prolonged drug release rates over a period of 24 h. The release profile of the optimized formulation (DF8) was described by the zero-order and best fitted to Higuchi model. FT-IR studies confirmed that there were no chemical interactions between drug and excipients used in the formulation. These results indicate that the approach used could lead to a successful development of a controlled release formulation of nelfinavir in the management of AIDS. KEYWORDS: Nelfinavir; HPMC; Polyox WSR-303; Xanthan gum; Geomatrix Technology. Introduction Oral ingestion has long been the most convenient and commonly employed route of drug delivery due to its ease of administration and flexibility in the design of the dosage form. There are many ways to design modified release dosage forms for oral administration and one of them is multi layered matrix tablet. One to three layer matrix tablets is a drug delivery device, which comprises a matrix core containing the active solute and one or more barriers incorporated during tableting process (Colombo et al., 1990). The barrier layers delay the interaction of active solute with dissolution medium, by limiting the surface available for the solute release and at the same time controlling solvent penetration rate (Conte et al., 1993; Qui et al., 1998). Hydrophilic polymers have been given considerable attention in the formulation of controlled release drug delivery systems for various drugs. HPMC K4M, HPMC K15M are a few representative examples of the hydrophilic polymers that have been extensively used in the formulation of controlled release systems (Gohel et al., 2007). Guar gum is soluble in water, it swells in gastric fluid to produce a highly viscous layer around the tablet through which the drug can slowly diffuse (Tobyn et al., 1996) and is used for the fabrication of matrices with uniform drug release characteristics (Talukdar et al., 1998; Talukdar et al., 1993). Regarding geomatrix technology, there have been different approaches to achieve zero-order drug release from dosage forms for sustained plasma concentration. Among different approaches to achieve zero-order release from hydrophilic matrix technologies, multilayer matrices have been widely evaluated and developed for commercial products under the trade name of Geomatrix. The technology makes use of bilayer or trilayer tablets to modulate the release and to achieve constant release (Wen and Park 2010). Nelfinavir mesylate (NFV) is an anti-viral drug, used in the treatment of Acquired Immunodeficiency Syndrome (AIDS). Poor oral bioavailability and shorter half-life (3.5 5 h) remain a major clinical limitation of nelfinavir leading to unpredictable drug bioavailability and frequent dosing. In this context, the objective of the present study was to formulate nelfinavir control release trilayer tablets by Geomatrix technology which can increase the oral bioavailability of sustained release of the drug. Materials and Methods Materials and Reagents Nelfinavir pure drug was generous gift from Optimus Generics Ltd., Hyderabad, India. PVP K 30, Microcrystalline cellulose, HPMC K 4 M, HPMC K 15 M was obtained from Rubicon labs, Mumbai. Polyox WSR 303 was obtained from Aurobindo Pharma Ltd., Hyderabad and Xanthan Gum was gifted from MSN Labs Ltd. Hyderabad. All other chemicals used were of analytical grade. 4259

2 4260 Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018 Formulation and Evaluation Methods Formulation of Controlled Release Nelfinavir Trilayer Matrix Tablets The trilayered matrix tablets of Nelfinavir were prepared by direct compression method. The first step in the formulation was to develop the middle active layer so as to give at least 90% drug release during 12 hours. The release profile of this layer might not be of constant rate type but would be preferably of constantly falling rate type. This layer would then be sandwiched between barrier layers (Upper & Lower layers) so as to continue the drug release for 24 h (Derakhshandeh and Soleymani, 2010). Preparation of Middle Active Layer of Nelfinavir Trilayered Tablets Twenty-seven formulations (F1-F27) for active layer were prepared by direct compression method using 3 3 Response surface method (3 variables and 3 levels of polymers) by using Design of experiment software with polymers like different HPMC grades and Guar gum. All the formulations were varied in concentration of polymers, magnesium stearate constituted in all the formulations. These materials were screened through #60 and mixed together in motor by using pestle. Final mixtures were compressed by using 12 mm diameter flat punches on a sixteen-station rotary tablet press. Formulation trials of active layer were depicted in Table 1. The prepared tablets were subjected to dissolution studies (Potturi et al., 2015). TABLE 1 Formulation trials of middle active layer of nelfinavir. F. CODE Nelfinavir HPMC K4M HPMC K15M Guar Gum PVP K-30 MCC Mg Stearate TOTAL F F F F F F F F F F F F F F F F F F F F F F F F F F F Preparation of Upper and Lower Layers of Nelfinavir Trilayered Tablets The barrier layers were formulated employing hydrophobic swellable polymer polyox WSR 303 the swelling erosion modeling fillers which include water soluble MCC, EC and Xanthan gum. The procedure adopted to make the compacts was via direct compressions. For the first procedure the Polyox, xanthan gum and the filler were mixed in mortar and lubricated with magnesium stearate. Formulation of upper and lower layers was depicted in Table 2 (Wadher et al., 2011). TABLE 2 Composition of nelfinavir trilayered matrix tablet. INGREDIENTS AF8 BF8 CF8 DF8 EF8 FF8 GF8 HF8 MIDDILE ACTIVE LAYER (F8) (750 mg) Nelfinavir HPMC K 4 M HPMC K 15 M Guar Gum PVP K MCC Cellulose Magnesium stearate UPPER AND LOWER LAYER (100 mg) Polyox WSR Xanthan gum Ethyl cellulose MCC Magnesium stearate Talc Formulation of Extended Release Tryilayered Tablets of Nelfinavir The powder mixtures required for active and barrier layers were weighed accurately and thoroughly mixed using mortar and pestle for about 20 minutes. Initially, the volume of die cavity (12 mm, round) was adjusted equivalence to the weight of trilayered matrix tablets (950 mg). Then the pre-weighed amount of powder equivalent to bottom layer (100 mg) was taken and placed in the die cavity and slightly compressed for uniform spreading. The upper punch was lifted up and the granules equivalent to 750 mg of the drug was placed over the bottom layer in the die cavity and again slightly compressed. The remaining volume of the die cavity was filled with pre-weighed (100 mg) amount of powder equivalent to top layer and compressed with the full force of compression on rotary tablets press to obtain trilayered tablets (Table 2). Tri-layered matrix tablets of each composition were compressed and tested for their friability, Hardness, drug content and drug release characteristics with a suitable number of tablets for each test (Jalpar and Patel, 2011). Evaluation of Trilayer Matrix Tablets of Nelfinavir Hardness, Thickness, Friability, Weight variation: Hardness of ten randomly picked tablets was determined using Monsanto hardness tester.

3 Rangu and Gande: Formulation and Evaluation of Nelfinavir Extended Release Trilayer Matrix Tablets by Design of Thickness: The thickness of the tablets was measured using Vernier calipers. Friability: A sample of twenty randomly selected tablets were accurately weighed and placed in a Roche friabilator. The friabilator was operated for 4 min at a speed of 25 rpm. The tablets were removed from the friabilator, de-dusted and reweighed. The percent loss in weight due to abrasion and impact was calculated as, %Friability= (Loss in weight/ Initial weight) 100. Weight variation: The weight variation test was performed as per the I.P. guidelines. Twenty randomly taken tablets were weighed together and the average weight was determined. Each tablet was then weighed individually and deviation from average weight was calculated. Content uniformity: 20 tablets were randomly selected, and average weight was calculated. Tablets were powdered in a glass mortar. Powder equivalent to 10 mg was weighed and dissolved in 100mL of Phosphate buffer ph 6.8 filtered and drug content analyzed spectrophotometrically in UV spectrophotometer at 254 nm. In Vitro swelling studies: The degree of swelling of polymer is an important factor affecting adhesion. For conducting the study, a tablet was weighed and placed in a petri dish containing 5 ml of phosphate buffer ph 6.8 in 12 h at regular intervals of time (1, 2, 4, 8, 10 and 12 h), the tablet was taken carefully by using filter paper. The swelling index was calculated using the following formula Swelling Index (S.I) = (Wt Wo)/Wo 100 Where S.I = swelling index, Wt = weight of tablet after swollen at time t, Wo = weight of the initial tablet. In-vitro drug release profile: In vitro drug release studies for developed trilayer matrix tablets were carried out by using dissolution apparatus II paddle type (Electrolab TDL-08L). The drug release profile was studied in 900mL Phosphate buffer ph 6.8 at 37 ± 0.5 o C temperature. The amount of drug release was determined at different intervals up to 24h by UV visible spectrophotometer (Shimadzu UV 1800) at 254nm. Drug release kinetics: To describe the kinetics of the drug release from matrix tablet, mathematical models such as Zero-order, First order and Higuchi, models were used. The criterion for selecting the most appropriate model was chosen on the basis of the goodness-or fit test. Drug-Excipient Compatibility Studies Fourier Transform Infrared Spectroscopy (FTIR): FTIR spectra for pure drug, physical mixture and optimized formulations were recorded using a Fourier transform Infrared spectrophotometer. The analysis was carried out in Shimadzu-IR Affinity 1 Spectrophotometer. The samples were dispersed in KBr and compressed into disc/pellet by application of pressure. The pellets were placed in the light path for recording the IR spectra. The scanning range was cm 1 and the resolution was 1 cm 1. SEM studies: The surface and shape characteristics of Tablets were determined by scanning electron microscopy (SEM) (HITACHI, S-3700N). Photographs were taken and recorded at suitable magnification. Stability studies: The stability study of the formulated Nelfinavir trilayer tablets were carried out under different conditions according to ICH guidelines using stability chamber (REMI make). Accelerated Stability studies were carried out at 40 0 C / 75 % RH for the best formulations for 6 months. The tablets were characterized for the hardness, friability, drug content and cumulative % drug released during the stability study period. Results and Discussion Preparation of Middle Active Layer The matrix tablets of Nelfinavir were prepared without the barrier layers. All the formulation trails were subjected to in vitro dissolution to determine the release profiles. From the above results, among all the formulations the formulation F8 was decided as optimized formulation for active layer based on the highest drug release i.e ± 1.86 within 12hrs when compared with other preparations (Figure 1-4). Formulation F8 was choosen as active layer for further studies. The prepared trilayer tablets were shown in Figure 5 and the tablets were evaluated for different parameters and found to be within the limits and the results are summarized in Table 3. The drug content of all formulation is in between %; drug content depends on angle of repose because if the angle of repose was good then drug content is also uniform because if the flow property is good then the drug is evenly distributed in the formulation (Table 3). Fig. 1. In vitro dissolution studies of nelfinavir middle active layer tablets F1-F7.

4 4262 Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018 Fig. 2. In vitro dissolution studies of nelfinavir middle active layer tablets F8-F13. Fig. 4. In vitro dissolution studies of nelfinavir middle active layer tablets F21-F27. Fig. 3. In vitro dissolution studies of nelfinavir middle active layer tablets F14-F20. TABLE 3 Physico-chemical evaluation properties of Nelfinavir trilayered tablets. F. CODE *Weight variation (mg) # Thickness (mm) #Hardness (Kg/Cm 2 ) #Friability (%) Fig. 5. Preparation of trilayer matrix tablets of nelfinavir. # Content uniformity (%) Swelling index (%) AF ± ± ± ± ± ± 0.76 BF ± ± ± ± ± ± 0.72 CF ± ± ± ± ± ± 0.64 DF ± ± ± ± ± ± 0.81 EF ± ± ± ± ± ± 1.03 FF ± ± ± ± ± ± 0.84 GF ± ± ± ± ± ± 0.72 HF ± ± ± ± ± ± 0.79 *Values are expressed in mean ± SD : (n=20) #Values are expressed in mean ± SD : (n=3) TABLE 4 In vitro Drug Release Profile for Prepared Extended release trilayered Tablet of Nelfinavir (AF8-HF8). Time (h) AF8 BF8 CF8 DF8 EF8 FF8 GF8 HF8 Marketed Product 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.18 TABLE 4 Contd

5 Rangu and Gande: Formulation and Evaluation of Nelfinavir Extendedd Release Trilayer Matrix Tablets by Design of Time (h) AF8 BF8 CF8 DF8 EF8 FF8 GF8 HF ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.35 Marketed Product ± ± ± ± ± ± 1.52 Fig. 7. First order plots for the optimized formulation of nelfinavir trilayered tablets. Fig. 8. Higuchi plots for the optimized formulation of nelfinavir trilayered tablets. Fig. 6. Zero order plots for the optimized formulation of nelfinavir trilayered tablets. Fig. 9. Korsmeyer-Peppas plots for the optimized formulation of nelfinavir trilayered tablets. Evaluation of Trilayer Matrix Tablets of Nelfinavir The Swelling study of trilayered matrix tablet of Nelfinavir was given in Table 8 showed that the swelling index of the tablet increases with increase in time up to 12 hours, this may be attributed to the fact that the erosion of biodegradable polymer Xanthan gum. This indicates that the drug will remain in intestinal region till drug is released completely from the delivery system and promotes evacuation after its release (Table 3). In vitro dissolution studies of trilayer matrix tablets of Nelfinavir: The release of Nelfinavir from different formulations was carried out and the results are depicted in Table 4. The trilayer tablets extended the drug release up to 24 hrs. The highest drug release was found in the formulation DF8 i.e 98.72% within 24 h. DF8 was found to be optimized formulation based on the dissolution and other evaluation parameters. The marketed product drug release was found to be 93.78% up to 24h. The results are shown in Table 4. Release Order Kinetics Release order kinetics for optimized (DF8) formulation: From the above results it is apparent that the regression coefficient value closer to unity in case of zero order plot i.e indicates that the drug release follows a zeroa lesser order mechanism (Table 5). This data indicates amount of linearity when plotted by the first order equation. Hence it can be concluded that the major mechanism of drug release follows zero order kinetics. Further, the translation of the data from the dissolution studies suggested possibility of under- the data in to various mathematical modelling such as standing the mechanism of drug release by configuring Higuchi and Korsmeyer-Peppas plots. Further the n value obtained from the Korsmeyer-Peppas plots i.e indicating non Fickian (anomalous) transport thus it projected that delivered its active ingredient by coupled diffusion and erosion (Figure 6-9). In vitro drug release order kinetics for marketed product: From the above results it is apparent that the regression coefficient value closer to unity in case of First order plot i.e indicates that the drug release follows a first order mechanism (Table No 5). This data indicates a lesser amount of linearity when plotted by the zero-

6 4264 Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018 order equation. Hence it can be concluded that the major mechanism of drug release follows first order kinetics. TABLE 5 Release order kinetics of optimized and marketed product. S. No. Formulation Zero order First order Higuchi Model Korsmeyer- Peppas n R 2 R 2 R 2 Model R 2 1 DF Marketed Product Further, the translation of the data from the dissolution studies suggested possibility of understanding the mechanism of drug release by configuring the data in to various mathematical modelling such as Higuchi and Korsmeyer-Peppas plots. The n value obtained from the Korsemeyer-Peppas plots i.e indicating non-fickian (anomalous) transport thus it projected that delivered its active ingredient by coupled diffusion and erosion (Figure 10-13). Fig. 10. Zero order plots for the marketed product. Fig. 11. First order plot for the marketed product. Fig. 12. Higuchi plot for the marketed product. Fig. 13. Korsmeyer-Peppas plot for the marketed product. Design of experiment: This method is mainly used to explain the effect of one factor on other factor, whether this effect is significant or not, if significant how it influences the response. In this present work the effect of one factor (Guar Gum) on other two factors (HPMC K 4M, HPMC K 15M) is explained (Figure 14). In the above graph the effect of Guar Gum on % cumulative drug release is examined, and it clearly indicates that there is a very significant effect of Guar Gum on % cumulative drug release. The formulations with all 3 factors shown % cumulative drug release in between but when Guar Gum is removed from the formulations the maximum % CDR is near This is the effect of factor (Guar Gum) on response (Figure 15). There is a negligible effect on Swelling Index of formulations because all formulations have excellent Swelling property and there is slight influence on Swelling Index by Guar Gum. Characterization FT-IR: Overall there was no alteration in peaks of Nelfinavir pure drug (Figure 16) and optimized formulation (Figure 18), suggesting that there was no interaction between drug & excipients. FTIR spectrum of pure drug and other polymers are shown in (Figure 17). There is additional peak appearing or disappearing hence no significant changes in peaks of optimized formulation was observed when compared to pure drug indicating absence of any interaction. SEM studies: SEM further confirmed both diffusion and erosion mechanisms to be operative during drug release from the optimized formulation (DF8). Initially, tablet matrix showed swelling with pore formation that is clearly visible from SEM image. At the end of 24 h, the matrix was intact, and pores had formed through it. SEM images also show the formation of gel structure indicating swelling and pore formation on the tablet surface (Figure 19). Stability studies: Optimized formulation DF8 was selected for stability studies on the basis of high cumulative % drug release. Stability studies were conducted for 6 months according to ICH guidelines. From these results it was concluded that, optimized formulation is stable and retained their original properties with minor differences.

7 Rangu and Gande: Formulation and Evaluation of Nelfinavir Extended Release Trilayer Matrix Tablets by Design of Fig. 14. Response surface plot showing the influence of amount of polymer on the release profile of nelfinavir for percent cumulative drug release. Fig. 15. Response surface plot showing the influence of amount of polymer on swelling index of nelfinavir. Fig. 16. FT-IR spectrum of pure drug nelfinavir.

8 4266 Fig. 17. FT-IR spectrum of nelfinavir physical mixture. Fig. 18. FT-IR spectrum of optimized formulation DF8. Fig. 19. SEM studies for optimised DF8. Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018

9 Rangu and Gande: Formulation and Evaluation of Nelfinavir Extended Release Trilayer Matrix Tablets by Design of Conclusions It was concluded that trilayer matrix tablets of Nelfinavir could be successfully prepared by direct compression technique using different polymers combination. Based on the evaluation parameters, drug dissolution profile and release drug kinetics DF8 was found to be optimized formulation. Nelfinavir tablets were prepared by direct compression and consist of middle active layer with different grades of HPMC, MCC and PVP K30, upper and lower layers were prepared with Polyox WSR 303, Xanthan gum, microcrystalline cellulose, ethyl cellulose and Magnesium stearate. The tablets were also evaluated for physicochemical characteristics and release kinetics. The physicochemical characteristics of the prepared tablets were satisfactory. The developed drug delivery systems showed prolonged drug release rates over a period of 24 h. The release profile of the optimized formulation (DF8) was described by the zero-order and Higuchi model. The results indicate that the approach used could lead to a successful development of an extended release formulation of the drug. These results also demonstrated the suitability of three-layered tablet formulation of Nelfinavir to provide controlled release for prolonged period of time and improved linearity for Nelfinavir in comparison to marketed product in the effective management of AIDS with patient compliance. References Abdu S and Poddar SS (2004). A flexible technology for modified release of drugs multi layered tablets. J Control Rel 97: Colombo P, Conte U, Gazzaniga A, Maggi L, Sangalli M. E and Peppas N (1990). Drug release modulation by physical restrictions of matrix swelling. Int J Pharm 63: Conte U, Maggi L, Colombo P and La Manna A (1993). Multi-layered hydrophilic matrices as constant release devices. J Control Rel 26: Derakhshandeh K and Soleymani M (2010). Formulation and in vitro evaluation of nifedipine controlled release tablet: Influence of combination of hydrophilic and hydrophobic matrix forms. Asian J Pharmaceutics 4(4): Hong Wen and Kinam Park (2010). Oral controlled release formulation design and drug delivery. Theory to practice edited by Wiley publication, New Jersey Gohel MC, Parikh RK, Padshala MN, Sarvaiya KG and Jena DG (2007). Formulation and optimization of directly compressible isoniazid modified release matrix tablet. Ind J Pharm Sci, 69(5): Jalpar R and Patel Bhavik A (2011). Formulation and evaluation of oral controlled drug delivery system for a model anti diabetic drug Metformin. Int J Curr Pharma Res 3(2): Potturi PV and Sudhakar Y (2015). Formulation and in vitro/ in vivo evaluation of controlled release Entacapone trilayer matrix tablets by geomatrix. Int J Drug Delivery 7: Sinko P.J (2007). Martin s Physical Pharmacy and Pharmaceutical Sciences. Published by Wolters Kluwer Health Pvt. Ltd, New Delhi 5: Talukdar MM, Mooter VD, Augustijns P, Maga TT, Verbeke N and Kinget R (1998). In vitro evaluation of xanthan gum as potential excipients for oral controlled release matrix tablet formulation. Int J Pharm 169: Talukdar MM and Vercammen JP (1993). Evaluation of xanthan gum as a hydrophilic matrix for controlled release dosage forms. Drug Dev Ind Pharm 19: Tobyn MJ, Stani forth JN and Baichwal AR (1996). Prediction of physical properties of a novel polysaccharide-controlled release system. Int J Pharm 128: Wadher KJ, Kakde RB and Umekar M (2011). Formulation of sustained release metformin hydrochloride matrix tablets: Influence of hydrophilic polymers on the release rate and in vitro evaluation. Inter J Res Control Rel 1(1): Yihong Q, Chidambaram N and Kolette F (1998). Design and evaluation of layered diffusional matrices for zero order sustained-release tablets. J Control Rel 51: Address correspondence to: Rangu Nirmala, Associate Professor, Surabhi Dayakar Rao College of Pharmacy, Rimmanaguda (Vill), Gajwel (Mdl), Siddipet District, Gajwel, Telangana , India. Mob: rangunirmala@gmail.com

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