International Journal of PharmTech Research 
CODEN (USA): IJPRIF        ISSN : 0974-4304
                                                                                                           Vol.2, No.3, pp 1978-1984,       July-Sept 2010

Optimization and Formulation of Immediate Release Tablet of Nateglinide by Using
32 Factorial Designs
Pani Nihar Ranjan*, Nath Lila Kanta
Department of Pharmaceutical Sciences,  Dibrugarh University,
 Dibrugarh, 786004, Assam, India.
*Corres.Author: niharpani@gmail.com
Tel: +91-9435476382,Fax: +91-373 2370323

Abstract: The water insoluble anti-diabetic drug nateglinide may be slowly or incompletely dissolved in gastro intestinal tract. The rate of dissolution and bioavailability of the drug may be increased by using superdisintegrants in its immediate release tablets. In the present study the selection of proper superdisintegrants among sodium starch glycolate, cross povidone, starch 1500 and cross carmelose sodium (CCS) was carried out to develop immediate release tablets of nateglinide. The differential scanning calorimetry, isothermal stress testing and FT-IR studies were carried out for compatibility testing between drug and excipients used in tablets. A 32 full factorial design was used to investigate the joint influence of 2 independent variables: amount of selected superdisintegrants, CCS and hardness of the tablets. The results of multiple linear regression analysis revealed that the dependent variables; disintegration time and drug release at 0.5h values are strongly dependent on the selected independent variables. The sign of coefficient of polynomial equation signified that an increase in disintegration time on decreasing the hardness of the tablets. An increase in the value of drug release at time 30 min observed on increasing the concentration of CCS. A checkpoint batch was also prepared to prove the validity of the evolved mathematical model. Tablet (F-5) containing 4%w/w of CCS at 5 kg/cm2 met the required DT (as per USP limit) and DR0.5 (100%). The systematic formulation approach helped in understanding the effect of formulation processing variables. Stability studies of factorial batches indicated that no significant change in appearance of the tablets, disintegration time, and percentage drug release were observed.
Keywords: Factorial design. Cross carmelose sodium. Nateglinide. Immediate release tablets.

Introduction and Experimental
Despite of increasing interest in controlled release drug delivery systems, more attention has been paid to formulate the poorly soluble drugs as most common tablets which intended to be swallowed to disintegrate and release their medicaments rapidly in the Gastro Intestinal Tract for better bioavailability. Bioavailability of the poorly soluble drug from a solid oral dosage form depends on the release of the drug substance from the dosage form1-3 i.e., disintegration of the solid oral dosage form which will increase the wettability of the drug by increasing the surface area of the drug particles. This highlights the importance of proper choice of disintegrant/superdisintegrants(SD) i.e. cross carmelose sodium, sodium starch glycolate, 

cross povidone, starch-1500 etc. and its consistency of performance which are of critical importance to increase the rate of dissolution and hence its bioavailability4-5. There are various factors (hardness, concentration of binders, disintegrants etc.) affecting the disintegration time (DT) and rate of dissolution of the drug.
The objective of the present study was to optimize the concentration of superdisintegrants and hardness of the immediate release tablets of Nateglinide (NTG). NTG was chosen because of its poor solubility and less bioavailability. Meglitinide derivative NTG is used for the treatment of type-2 diabetes mellitus6-7. .
Materials and Methods
NTG was obtained as gift sample from Glenmark Pharmaceuticals, Nashik, India. Lactose, Cross carmelose sodium (CCS), sodium starch glycolate (SSG), Cross povidone (CP), Sarch-1500, Polyvinylpyrolidine (PVP), Aerosil, talc and ethanol were procured from SD fine chemicals, Mumbai and all other chemicals used were of analytical grade.

Preformulation Studies
1.	Differential scanning calorimetry
A differential scanning calorimeter (JADE DSC, Perkinelmer, USA) was used for thermal analysis of drug and drug-excipients mixtures8. Excipients that were expected to be used in the development of formulation and the maximum expected ratio were selected for the present study. Individual samples (drug and excipients) as well as physical mixtures of drug and selected excipients (all passed through 60-mesh sieve) were weighed directly in the DSC aluminum pan and scanned in the temperature range of 50-300 0C, under nitrogen atmosphere. Heating rate of 20 0C/min was used and thermograms obtained were observed for any interaction.

2.	Isothermal stress testing
For isothermal stress testing (IST) study9 drug and different excipients were weighed directly in to 4ml glass vials (n = 2) and mixed on a vortex mixer for 2 min. In each of the vials, 10% w/w water was added and the drug-excipients blend was further mixed with a glass capillary (both the ends of which were heat sealed). To prevent any loss of material, capillary was broken and left inside the vial. Each vial was sealed using a teflon-lined screw cap and stored at 50 0C (Hot air oven, Universal, Narang Scientific, India). These samples were periodically examined for any unusual color change. After 3 weeks of storage at the above conditions, samples were quantitatively analyzed using UV-Visible spectrophotometer. Drug-excipients blends without added water and stored in refrigerator served as controls.

3. FT-IR spectroscopy
FT-IR spectra of drug and drug-excipient blends were recorded on an IR spectrophotometer9 (FTIR-8400S, Shimadzu, Japan) in the range of 4000-500 cm-1 using potassium bromide discs.

Preparation of Immediate Release Tablets
NTG, half quantity of the CCS and Lactose were passed through sieve No. 80 and they were mixed in a poly bag for 20 min. Alcoholic solution of Povidone (10% w/v) was added to the mixture in a quantity just enough to bind the mass. The wet mass was screened in sieve No. 20 and kept for drying in a tray dryer at 450C. The dried granules were mixed with remaining half of CCS and lubricated with 2% w/w talc and 2% w/w aerosil. The granules ready for compression were converted into tablets using a single-punch tablet punching machine (Hardik, Ahmedabad, India). The composition of the preliminary and factorial design batches is shown in Tables 1 and 2 respectively.

Evaluation of Granules and Tablets
The prepared granules were evaluated for angle of repose, bulk density, tapped density, compressibility index and hausner ratio10. The prepared tablets were evaluated for weight variation, friability, hardness, thickness and DT11.
In-vitro drug release study was carried out in USP (United States Pharmacopeia) dissolution apparatus II (TDT-08L, Electrolab, India) by rotating paddle at 50 rpm in 1000 ml of 0.1 N HCl + 0.5 % Sodium lauryl sulphate as dissolution medium maintaining temperature at 37±0.5 0C12. An aliquot of 5 ml was withdrawn at different time intervals (5, 10, 20, 30, 45 min) and equal volume of fresh dissolution medium was replaced to maintain the original volume. The samples were suitably diluted and analyzed in UV-Visible spectrophotometer at 210 nm to determine the amount of drug release13.

Full factorial design
A 32 randomized full factorial design was used to optimize the variables in the present study. In this design 2 factors were evaluated, each at 3 levels, and experimental trials were performed at all 9 possible combinations14 -16. The percentage (2, 4 and 6) of CCS (X1), and hardness (3, 5 and 7 kg/cm2) of tablets (X2), were selected as independent variables. The DT and percentage of drug release at 0.5 h (DR0.5h) were selected as dependent variables.

Stability Study
The factorial batches were subjected for accelerated stability (40±2 0C/75±5% RH) testing. After specified period of time (1, 2, 3, 4, 5, 6 month), samples were withdrawn and subjected for assay and in-vitro dissolution study. Stability study was conducted as per ICH (International Conference on Harmonisation) guidelines.

Table. 1. Results of analysis of IST samples after 3 weeks of storage at stressed conditions.
Sample	Ratio (drug-excipient)	%Drug remaininga	

	Control samplesb	Stressed samplesc	
NTG	--	100.81 ± 0.72	99.74 ± 2.53	
NTG + Lactose	1:02	101.42 ± 1.34	100.27 ± 1.65	
NTG + CCS	1:01	100.73 ± 1.82	99.83 ± 1.82	
NTG + Povidone	2:01	101.8 ± 1.48	99.34 ± 1.86	
NTG + Talc	3:01	100.62 ± 2.68	99.27 ± 2.04	
NTG + Aerosil	3:01	100.53 ± 1.81	99.62 ± 1.18	
Values expressed as average ± standard deviation.
Drug excipient blends without added water and stored in refrigerator.
Drug excipient blends with 10% (w/w) added water and stored at 50 0C for 3 weeks.

Table. 2. Results of Preliminary Batches of Immediate release Nateglinide Tablets.
Formulation*	T-l	T-2	T-3	T-4	T-5	
Nateglinide (% w/w)	40	40	40	40	40	
Sodium starch glycolate (% w/w)T	-	6	-	-	-	
Starch 1500 (% w/w)T	-	-	6	-	-	
Cross PovidoneT	-	-	-	6	-	
Cross carmelose sodium (% w/w)T	-	-	-	-	6	
Lactose ….qs…to (% w/w)	100	100	100	100	100	
Disintegration time (seconds)	>3600	>3600	>3600	136	20	
Drug release at 0.5 h (% w/w)	3.2	37.7	7.2	62.6	100.6	
*All batches contained 10%w/w polyvinylpyrrolidone in ethyl alcohol as a binder, 2%w/w talc and 2%w/w Aerosil. Hardness of all batches was 5 kg/cm2, †Intragranular 50%w/w; extragranular 50%w/w.

Table. 3. 32 Full Factorial Design Layout*.
Batch Code†	Variable Levels in Coded Form	Dissolution at 0.5h	DT	

	Xt (%)	X2 (kg/cm2)	% (w/w) ± SD	DT ± SD	
F-l	-1	-1	67.83 ±0.35	158.67±3.05	
F-2	-1	0	61.7±1.01	179.33±2.52	
F-3	-1	1	54.9±0.82	193.33±4.5	
F-4	0	-1	100.33±0.76	33±2.66	
F-5	0	0	94.33±0.55	39.67±1.53	
F-6	0	1	91.4±1.21	53.67±3.05	
F-7	1	-1	99.97±0.4	17.331± 1.53	
F-8	1	0	100.5±0.66	19.67±1.52	
F-9	1	1	100.13±0.3	30±1.01	
Check Point	-0.5	0.5	77.83±0.72	100.37±3.28	
Codes values	Actual value			

	Xi (%)	X2 (kg/cm2)	
		
-1	2	3	
		
0	4	5	
		
1	6	7	

*X1, indicates amount of cross carmelose sodium (% w/w); X2, Hardness of the tablets (kg/cm2); SD, Standard deviation and DT, disintegration time.
†All batches contained 10%w/w polyvinylpyrrolidone in ethyl alcohol as a binder, 2%w/w talc and 2%w/w Aerosil.

Table. 4. Calculations for Testing the Model in Portions*.
	For % of dissolution at 0.5h	
	DF	SS	MS	F	R2	
Regression	
FM	5	8377	1675	1662	0.9969	
RM	4	8376	2094	2138	0.997	
Error			
FM	3	11.46	3.82	
		
RM	4	11.83	2.96	
		
	For disintegration time	
	DF	SS	MS	F	R2	
Regression	
FM	5	1.30E+05	26081	2763	0.9981	
RM	4	1.30E+05	32598	3402	0.998	
Error			
FM	3	77.59	25.86	
		
RM	4	90.11	22.53	

*DF indicates: degrees of freedom; SS, sum of squares; MS, mean of squares; F, Fischer's ratio; R2, regression coefficient; FM, full model; and RM, reduced model. Details of calculations are shown in Mendenhall W and Sincich T.19

Table. 5. Summary of Regression Analysis Results.
For % of dissolution at 0.5h	
Respons	bo	bi	b2	bn	b22	bi2	
FM	95.19	19.36	-3.62	-14.52	0.25	3.28	
RM	95.36	19.36	-3.62	-14.52		3.8	
For disintegration time	
Respons	bo	bi	b2	bn	b22	bi2	
FM	41.15	-77.39	11.33	57.61	1.44	-5.5	
RM	42.11	-77.39	11.33	57.61		-5.5	
*FM indicates full model; and RM, reduced model.

Fig. 1. Comparision of DSC thermogram of nateglinide with drug-excipients mixture.

Fig. 2. Response surface plot of % of drug release at 0.5h.

Fig.3. Response surface plot of Disintegration time (s).

Results and Discussion
Drug-excipient interaction study at an early stage of product development is an important exercise in the development of a stable dosage form. A sharp endothermic peak was observed at 142.81 0C in DSC thermogram of NTG. However, the endothermic peak of NTG was well preserved at 142.81±2 0C in the DSC thermogram of NTG-excipients mixtures (Fig.1). This result inferred that there was no interaction between drug and excipients8.
In IST study, it was observed that there was no physical change (colour and appearance) as well as drug content (Table-1) after storage of drug-excipient blends under stressed conditions which supported the result of DSC study on drug-excipient compatibility testing9.
There was no appearance or disappearance of peaks in the spectra of drug and excipients mixture when compared to the spectrum of pure drug in the FT-IR study. It was confirmed that excipients used for the formulation tablets are compatible with NTG9.
The flow properties of granules can be judged from the angle of repose, compressibility index and hausner ratio. The angle of repose (θ) <300 indicates free flowing material and >400 with poor flow properties10. The compressibility index <10% indicates excellent flow properties and >38% with poor flow properties11. The hausner ratio 1.00-1.11 indicates free flowing and >1.60 with poor flow properties11. Values for angle of repose (θ), compressibility index (%) and hausner ratio for all prepared granules were found to be in the range of 22.52 to 25.730, 7.14 to 7.71, and 1.07 to 1.08 respectively, which showed that the granules were free flowing and can be used for tablet compression. Percentage of weight variation was observed within the limit of ±7.5%w/w for all the prepared tablets, which is well accepted for uncoated tablets as per USP-NFXXIV. Friability test of the prepared tablet except batch F-1 to F-3 was passed (weight loss <1%w/w) which, assumed that tablets (F-4 to F-9) formulated at higher hardness have sufficient mechanical integrity and strength.
Lactose was selected as soluble diluent for water insoluble drug NTG by considering its advantages in terms of easy availability, cost-effectiveness, and relative moisture insensitivity. Povidone was used as binder considering its widespread applicability in industry. The preliminary trial batches were conducted arbitrarily without addition of superdisintegrants (T-1) and in addition with 6% w/w of SSG (T-2), Starch 1500 (T-3), CP (T-4), and CCS (T-5) at 5 kg/cm2 hardness for the selection of Superdisintegrants. It was observed from Table 1 that the DT of T-1, T-2, T-3 and T-4 were more than 2hr. The results (Table 2) obtained from preliminary trial that the batch containing CCS showed less DT and more percentage of drug release at 0.5 h (DR0.5h) than other superdisintegrants containing batches, hence CCS was considered for further investigation. Hardness of the tablets has impact on the DT as well as the amount of drug release from tablets so it needs to optimize. In order to investigate the influence of concentration of CCS and hardness of the tablet systematically, a 32 factorial design was employed in this investigation.
The amount of CCS (X1) in tablets and the hardness of the tablets (X2) were chosen as independent variables in a 32 full factorial design. A statistical model incorporating interactive and polynomial terms was used to evaluate the responses.
Y = b0 + b1 X1 + b2X2 + b12X1X2 + b11X12 + b22X22, (1)
Where, Y is the dependent variable, b0 is the arithmetic mean response of the 9 runs, and bi is the estimated coefficient for the factor Xi. The main effects (X1 and X2) represent the average result of changing one factor at a time from its low to high value. The interaction terms (X1X2) showed how the response changes when two factors are simultaneously changed. The polynomial terms (X12 and X22) are included to investigate nonlinearity. The DT and DR0.5h for the 9 batches (F1 to F9) showed a wide variation (i.e, 16 to 198 s and 54.7 to 101.1 % w/w respectively). The data clearly indicates that the DT and DR0.5h values are strongly dependent on the selected independent variables. The fitted equations (full and reduced) relating the responses DT and DR0.5h to the transformed factor are shown in Table 3. The polynomial equations can be used to draw conclusions after considering the magnitude of coefficient and the mathematical sign it carries (ie, positive or negative). Table 4 shows the results of the analysis of variance (ANOVA), which was performed to identify insignificant factors15. The high values of correlation coefficient for DT and percentage of drug release at 0.5h indicate a good fit.
The significance level of coefficient b22 was found to be P = 0.2625 and 0.5485 in full model for DT and DR0.5h respectively; hence it was omitted from the full model for both the cases to generate the reduced models. The results of statistical analysis are shown in Table 4. The coefficients b1, b2, b11, and b12 were found to be significant at P<0.05, hence they were retained in both the reduced model. The reduced model was tested in portions to determine whether the coefficient b22 contributes significant information for the prediction of DT and DR0.5h or not16. The results of multiple linear regression analysis (reduced model) reveal that, on increasing the concentration of CCS, a decrease in DT is observed; the coefficients b1 bear a negative sign. The coefficient b2 bear a positive sign signifies an increase in DT on decreasing the hardness (kg/cm2) of the tablets. An increase in the value of DR0.5h observed on increasing the concentration of CCS (b1 is positive) and on decreasing the hardness (b2 is negative) of the tablets.
The response surface plot of % of CCS (X1) and hardness of the tablets (X2) versus DT and that versus % of DR0.5h are shown in Fig. 2 and 3 respectively. The response plot showed that there is a significant effect of both factors on selected response.
The model predicts required DT (as per USP limit) and DR0.5 (100%) from point prediction, at 4 % CCS and 5 kg/cm2hardness (F-5). Though model also predicted the required response at lower concentration of CCS and hardness; the tablets did not get DT within 2hrs in lower CCS concentration and failed to pass the friability at lower hardness.
A checkpoint batch was prepared at X1 = -0.5 level and X2 = +0.5. From the reduced model, it is expected that the DR0.5h value of the checkpoint batch should be 77.83 and the value of disintegration time should be 102.25 seconds. Table 3 indicates that the results are as expected. Thus, we can conclude that the statistical model is mathematically significant.
Stability studies of factorial batches indicated that no significant change in appearance of the tablets, disintegration time, and percentage drug release were observed.
In this study, the dissolution of poorly soluble drug nateglinide was significantly enhanced by using CCS in compare with SSG, CP and starch 1500 as superdisintegrants in immediate release tablet. The results of a 32 full factorial design revealed that the amount of CCS and hardness of the tablets significantly affect the dependent variables, disintegration time, and percentage of DR0.5h from the tablets. It is concluded that by adopting a systematic formulation approach, an optimum point can be reached in the shortest time with minimum efforts. Tablet containing 4%w/w of CCS at 5 kg/cm2 met the required DT (as per USP limit) and DR0.5 (100%).

References
1-	Sekar V. and Chellan V. R., Immediate release tablets of Telmisartan using superdisintegrant formulation, evaluation and stability studies, Chem Pharm Bull., 2008, 56, 575-577.
2-	Mallick S., Pattnaik S., Swain K. and De P. K., Current prospectives of solubilization: potential for improved bioavailability, Drug Dev Ind Pharm., 2007, 33, 865-873.
3-	Moneghini M., Carcano A., Perssutti B. and Relbess F., Formulation design studies of Atenolol Tablets, Pharm Dev Tech., 2000, 5, 297-301.
4	Mahajan H. S., Kuchekar B.S. and Badhan A.C., Mouth dissolve tablets of sumatriptan succinate, Indian J Pharm Sci., 2004, 66, 238-240.
5	Zhao N., Larry L. A., Augsburger L.L., Functionality comparison of 3 classes of superdisintegrants in promoting aspirin tablet disintegration and dissolution, AAPS Pharm Sci Tech., 2005, 6, E634-E640.
6	McLeod J. F., Clinical Pharmacikuinetics of Nateglinide, Clin Pharmacokinet. 2004, 43, 97-120.
7	Ikenoue T., Okazaki K., Fujitani S., Tsuchiya Y., Akiyoshi M., Maki T. and Kondo N., Nateglinide suppresses postprandial hypertriglyceridemia in Zucker fatty rats and Goto-Kakizaki rats: comparison with Voglibose and Glibenclamide, Biol Pharm Bull., 1997, 20, 354-359.
8	Kandarapu R., Grover V., Chawla H. P. S. and Garg S., Evaluation of compatibility of Ketorolac tromethamine with selected polymers and common tablet excipients by thermal and isothermal stress testing, S T P Pharm Sci., 2001, 11, 449-457.
9	Verma R. K. and Garg S., Compatibility studies between isosorbide mononitrate and selected excipients used in the development of extended release formulations, J Pharm Biomed Anal., 2004, 35, 449-458.
10	Lachman L., Liberman H. A.and Kanig J.L., The Theory and Practice of Industrial Pharmacy. 3rd ed. Mumbai: Varghese Publishing House, 1987, 66-99.
11	Lindberg N., Palsson M., Pihl A., Freeman R., Freeman T., Zetzener H. and Enstad G., Drug Dev Ind Pharm., 2004, 30, 785-791.
12   FDA/CDER, Dissolution method – List of all drug in data base, US Food and administration website, available at http://www.accessdata.fda.gov/scripts/cder/ dissolution/dsp_SearchResults_Dissolutions.cfm. (accessed 22/12/2008).
13 Basu S. K. and Rajendran A., Studies in the Development of Nateglinide Loaded Calcium Alginate and Chitosan Coated Calcium Alginate Beads, Chem Pharm Bull., 2008, 56, 1077-1084.
14 Bolton S., Pharmaceutical Statistics. 2nd ed., New York: Marcel Decker Inc, 1990, 234.
15 Mendenhall W. and Sincich T., Multiple regression, 3rd ed., San Francisco, CA: Dellen Publishing Co., USA, !989, 141-226.
16 Franz R. M., Browne J. E. and Lewis A. R., Experiment design, modeling and optimization strategies for product and process development, In: Pharmaceutical Dosage Forms: Disperse Systems. 1st ed. Vol 1, New York: Marcel Decker Inc, 1988, 427-519.

*****