INTRODUCTION:

IVIVC is an attempt to link in vitro drug performance to in vivo drug product biopharmaceutic-pharmacokinetic performance¹. When a relationship has been established between the in vitro dissolution characteristics and the in vivo performance of the batches, it is described as an in vitro/in vivo correlation (IVIVC)^{2, 3}. IVIVC models are divided into four categories^{4, 5}known as levels A, B, C, and D. Level A applies to a model that can predict the entire in vivo time course from the in vitro data. The other levels use a number of tablet/capsule formulations and relate summary statistics for the in vitro and in vivo behavior of the drug. Because levels B, C, and D are based on summary statistics that do not uniquely reflect the time dependence of the data, they are not considered to be as useful as level A. Level– A correlation provide a way of verifying dissolution methods by showing the similarity of the entire (hypothetical) in vivo dissolution profile with the entire in vitro dissolution profile. The in vivo concentration profile can be analyzed by a model independent procedure called deconvolution. Level – A correlation may provide information on the transit of solid dosage forms⁶. An in vitro- in vivo correlation (IVIVC) is most commonly established by developing formulations with different release rates, collecting data on the in vitro dissolution rates and In vitro dissolution of a drug is of relevance to predict accurately in vivo performance of that formulation. Dissolution test is the only in vitro quality control test available till date, which can provide an insight to predict in vivo behavior of the drug product.

It serves as a tool to distinguish between “acceptable and unacceptable” (bioequivalent or bioinequivalent) drug products. The value of the dissolution test as a quality control tool is significantly enhanced if an IVIVC is established⁷.

MATERIALS AND METHODS:

Standard nimesulide and its internal standard tolbutamide for HPLC analysis were supplied by Panacea Biotec Ltd., Lalru (Punjab). Analytical grade sodium di hydrogen phosphate, sodium tetra borate, chloroform, dichloromethane, HPLC grade methanol, acetonitrile, water were purchased from Qualigens fine chemical (Mumbai) and commercial tablets of nimesulide plain (Nimulid 100mg) were provided by Panacea Biotec Ltd., Lalru.

IN VIVO STUDIES

Clinical protocol:

Eight healthy male volunteers, aged 20 to 24 years, weighing 58 to 75 kg and within 15% of their ideal body weight, participated in the studies. All volunteers have given the written consent and the clinical protocol was approved by the REB of Guru Jambheshwar University of Science and Technology. At the time of studies the volunteers were not suffering from any diseases or mental disorder. Prior to pharmacokinetic studies the volunteers were checked for any sort of hypersensitivity reactions to the drug. 10 mg of Nimesulide was given to all the volunteers at the same time. During 24 hours they were observed for any hypersensitivity reactions. No hypersensitivity was observed in any of the volunteers.

The study was performed as a single-dose, randomized, crossover design in 8 healthy volunteers with a 7 days washout period between doses. The samples were taken under medical supervision. Volunteers remained under clinical observation until collection of the last blood sample. During study, participants were provided with standardized meals. The consumption of alcohol or caffeine or any other drug was not allowed during this time. Preparations were administered with approximately 200 ml of water.

Drug Analysis:

Blood samples (3ml) from an antecubital vein were collected into EDTA containing tubes at 0, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hrs following nimesulide administration. The blood samples were centrifuged at 4000 rpm and clear plasma was separated, transferred into sealable tubes and stored at -20’C. Plasma concentration was measured by reverse phase ion pair HPLC with UV absorbance detection using the sample preparation method described below.

To 1ml of plasma sample (containing nimesulide), 100µl of internal standard (Tolbutamide, 10µg/ml) and 1ml of 10mM sodium tetraborate buffer were added. After mixing, 6ml of chloroform were added and Vortexed for 2 minutes. The tubes were centrifuged (4000 rpm for 10min) and 2ml of organic layer separated. Evaporated it upto dryness then, Reconstituted with 0.5ml methanol. Filter the sample through syringe filter and 20µl aliquot of this sample was injected to HPLC.

A reverse phase C18 column (Waters spherisorb 250 × 4.6, average particle size 5µ) was used. The mobile phase (Disodium hydrogen phosphate: methanol: acetonitrile=50:30:20 v/v) was delivered 0.7ml/min, and nimesulide and tolbutamide peaks were detected at 439nm.The nimesulide / tolbutamide peak height ratio were plotted against the standard nimesulide values. Plasma sample concentration was derived from this linear regression plot.

Fig .1 Plasma Nimesulide concentrations Vs time curve obtained in 8 healthy male volunteers after single administration (100mg).

IN VITRO STUDIES:

Dissolution study was carried out with 100 mg Nimesulide Tablet (Nimulid). The study was carried out using Dissolution Medium Alkaline Borate Buffer (pH 8.4), 900 ml, Dissolution apparatus type II (Paddle type) at 50rpm with 37 ± 0.5⁰C Temp. and samples were analyzed by double beam UV-Spectrophotometer at 439 nm λ_{max .}

Fig.2 In vitro release profile of nimesulide (100 mg)

RESULTS AND DISCUSSION:

Dissolution study was carried out with 100 mg Nimesulide Tablet for assessing the in vitro data for the correlation. In vivo studies was performed on the healthy male volunteers show that nimesulide is well absorbed after oral administration and maximum concentration are attain between 0.42 to 2.64 µg/ml being reached within 0.5 to 3 h and was found to be extensively bound to albumin upto 99%. Same studies was performed for nimesulide MD 100mg that shows maximum absorption in mouth and Cmax was 2.72 µg/ml being reach within 2h. In in vivo studies we have determined the pharmacokinetic profile of the commercial tablet of nimesulide plain and nimesulide MD, correlate these pharmacokinetic parameter with in vitro studies data to establish the IVIVC of two different marketed formulation of nimesulide.

No adverse effects were reported by any volunteer. Laboratory test result were within the reference values at end of the clinical protocol. The following drug concentrations were obtained at different time interval after In vitro and In vivo studies:

Table 1.In vitro and In vivo drug concentration of Nimesulide 100 mg

Vitro (x1) µg/ml	Vivo (x2) µg/ml	(X1)²	(X2)²
38.30	0.42	1466.89	0.176
47.20	0.73	2227.84	0.533
70.00	1.80	4900.00	3.240
87.77	2.64	7703.57	6.970
89.04	2.34	7928.12	5.476
89.22	2.0	7960.21	4.000

For IVIVC we perform t-test for two individual data (Plasma data v/s dissolution data) for Nimesulide after completing the experimental work as showing in following table:

Table 2. Nimesulide 100 mg (mean time parameters using statistics)

Parameters	Formulation
Standard deviation (S.D.)	49.26
t- test	2.40 ± 2.228

Table 3. Application of t-test on Nimesulide 100 mg tablet

Formulation(F) + plasma data (PD)	t-test	Values (p<0.05±2.228, D.F.- 10)
F+PD	T1	2.40

We found the result at particular level, at particular degree of freedom like (5% level on 10 degree of freedom) found ± 2.228 tabulated value. This value is the standard value. When we do the experimental work on the nimesulide plain to perform t-test we got the value (2.40), at the same experimental condition which described above. It shows correlation between plasma data and dissolution data. Development of In vitro/In vivo correlation shown by following Figure:

Fig.-3 Depicting the development of IVIVC

CONCLUSION:

IVIVC can impart In vivo meaning to the in vitro dissolution test and can be useful as surrogate for bioequivalence. Further, IVIVC can also allow setting of more meaningful dissolution specifications. From the results of all experimental works we can conclude that IVIVC is possible in the nimesulide formulation and it achieved the correlation of level A because t-test value found within the limit.

REFERENCES:

1. Polli, JE, John, R, Crison and Gordon, L. Amidon. Novel approach to the analysis of in vitro in vivo relationships. J. Pharm. Sci. 1996; 85: 753-760.

2. Food and Drug Administration. 1997. Guidance for industry, extended release oral dosage forms; development, evaluation and application of in vitro/in vivo correlations. Rockville, MD: Food and Drug Administration.

3. Young, D, Devane, JG, Butler, J. Advances in experimental medicine and biology, vol. 423, In vitro-in vivo correlations. New York: Plenum Press, 1997.

4. Skelly, JP, Shiu, GF. In vitro/In vivo correlations in Biopharmaceutics: scientific and regulatory implications. Eur J Drug Metab Pharmacokinet. 1993; 18:121–129.

5. United States Pharmacopeia. 1993. In Vitro and in vivo evaluation of dosage forms. Pharmacopeial Forum 19:5366–5379.

6. Johannes Kramer, IVIVC: A perspective from the work bench. Oral drug absorption edited by Jennifer B. Dressman, H. Lennam, 1996, 307-322.

7. Shishoo C.J, Savale S.S., Shah S.A., Rathod I.S. In vitro in vivo correlation of oral drug formulation: an over view. Ind. J. Pharm Sci., 2000, 62 (3) 153- 157.

Received on 20.10.2009 Modified on 30.11.2009

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 257-259