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. The animals were kept unrestrained during the entire study. At 0.25, 0.5, 1, 2, 4 and 6?h after the dose, animals were anesthetized with ether and a blood sample withdrawn from the retro-orbital sinus vein into a heparinized ISRIB clinical trial polypropylene tube. Animals were then decapitated and tissue samples (whole brain, lungs, heart, liver, kidney and spleen) were collected. Plasma was separated by centrifuging blood at 13,000?rpm for 10?min and tissue samples were washed with normal saline. All samples were frozen at ?80?��C until drug analysis. The reversed-phase HPLC method described in Section 2.6 was slightly modified and used for the determination of indinavir concentration in biological samples. All solutions were prepared in water:acetonitrile (50:50?v/v). Stock solutions (1?mg/mL) of indinavir were prepared and diluted to give standard solutions of concentration 0.1, 0.25, 0.5, 1, 2.5 and 5??g/mL. A stock solution (1?mg/mL) of verapamil hydrochloride as internal standard (IS) was diluted to give a working 5??g/mL IS solution. To 500??L tissue homogenate (either sample or control obtained from drug free animals) prepared in ice cold saline [0.2?g tissue per mL prepared using a tissue homogenizer Metformin concentration (Remi Instruments Ltd., Mumbai, India) at 6000?rpm], 100??L perchloric acid (60%) was added and, after vortex-mixing for 5?min, the resulting suspension was centrifuged at 13,000?rpm for 10?min (Biofuge, Heraeus, Germany). To 300??L plasma or tissue homogenate supernatant, 100??L standard indinavir solution, 100??L IS working solution, 1?mL 4?M KOH solution and 5?mL diethyl ether were added and the resulting mixture vortexed for 15?min and then centrifuged at 6500?rpm for 10?min. The organic layer was separated and evaporated to dryness under vacuum (GL 66, Toshniwal, Mumbai, India) after which the residue was reconstituted in 100??L 100??M phosphoric acid and 20??L of the resulting solution was injected to HPLC. Linear regression of calibration curves gave with the following equations and correlation coefficients: Plasma Y=0.263X+0.001, CYTH4 R2=0.998; brain Y=0.172X+0.001, R2=0.997; lung Y=0.208X+0.026, R2=0.992; heart Y=0.220X+0.017, R2=0.990; liver Y=0.281X+0.003, R2=0.996; kidney Y=0.258X+0.009, R2=0.996; and spleen Y=0.204X+0.008, R2=0.996. Pharmacokinetic parameters (Cmax, Tmax, AUC0�C6h, t1/2, MRT) and therapeutic availability (TA) were calculated using Kinetica software (version 5.0) and are expressed as mean��standard deviation (SD). Comparison between groups was done using the paired students t-test with P