Accelerated Communication Mode of Administration-Dependent Brain Uptake of Indomethacin: Sustained Systemic Input Increases Brain Influx

Nonsteroidal anti-inflammatory drugs, including indomethacin, have been found in both epidemiological and clinical studies to reduce the prevalence and severity of Alzheimer’s disease. However, long-term use of indomethacin is limited by significant gastrointestinal and renal toxicities. An indomethacin prodrug that delivers low and continuous blood levels of the drug showed a superior safety profile and similar efficacy in comparison to an equivalent dose of free indomethacin because of limited systemic exposure and preferred brain uptake. The purpose of the present investigation was to evaluate whether sustained systemic input causes an increased brain influx in comparison to rapid input of the drug. Oral indomethacin, indomethacin prodrug, or intravenous indomethacin infusion was administered to rats. The infusion was designed to mimic the plasma indomethacin levels resulting from the prodrug. The resultant blood levels and brain indomethacin uptake were evaluated. The brain indomethacin concentrations 8 h following indomethacin administration were 0.45, 0.3, and 0.31 g/g after the oral indomethacin, oral prodrug, and intravenous infusion, respectively. The corresponding plasma concentrations were 14.1, 4.1, and 4 g/ml. Therefore, brain versus plasma indomethacin level ratios were 2.5-fold higher after slow systemic input of indomethacin in comparison to rapid drug input. In conclusion, indomethacin brain uptake was found to be mode of administration-dependent, and a sustained input function increases the drug brain uptake. Thus, these unique results indicate that an appropriate indomethacin controlled release delivery system may induce the desirable brain-related pharmacodynamic effects, while avoiding the concentration-dependent adverse effects. These findings may contribute to improved therapy in Alzheimer’s disease. Nonsteroidal anti-inflammatory drugs, including indomethacin, have been found in both epidemiological and clinical studies to reduce the prevalence and severity of Alzheimer’s disease (Veld et al., 2001). Indomethacin inhibits amyloid plaque formation via -secretase inhibition, which is a cyclooxygenase-independent process (Weggen et al., 2001). In addition, nonsteroidal anti-inflammatory drugs have cyclooxygenase-dependent anti-inflammatory and neuroprotective effects (Halliday et al., 2000; Weggen et al., 2001). However, long-term use of indomethacin for Alzheimer’s disease is limited by significant gastrointestinal and renal toxicities that are concentration-dependent (Tabet and Feldman, 2002). In a previous study, we reported on a novel oral prodrug of indomethacin, comprising the drug attached to the sn-2 position of a phospholipid that exhibited a superior safety profile and similar efficacy to an equimolar dose of free indomethacin (Dvir et al., 2006). This unique result was derived from the pharmacokinetic properties of the prodrug which, after oral administration, resulted in a sustained release profile of the drug in the plasma, with slower absorption rate having a half-life value of 23.5 h in comparison to free indomethacin (10.5 h). The amount of indomethacin that was absorbed after the administration of an equimolar dose of the prodrug decreased 2-fold, Cmax decreased 4-fold, and tmax was delayed 2-fold in comparison to oral administration of the free drug. The unique pharmacokinetics of the prodrug was also related to the disposition of indomethacin to the brain where, despite the lower systemic drug concentrations, elevated brain indomethacin uptake was obtained after the administration of the prodrug to rats in comparison to administration of the free drug. Up to a 4-fold higher brain to plasma concentration ratio of indomethacin was found after oral administration of the prodrug in comparison to oral administration of free indomethacin. Hence, even with the lower systemic indomethacin concentrations, the prodrug did not cause significant reduction in indomethacin brain levels, and resulted in equivalent brain-related pharmacodynamic activities. The purpose of the present investigation was to investigate the factors that caused this unique phenomenon of indomethacin disposition to the brain: specifically, to evaluate whether the preferred indomethacin brain uptake after the administration of the prodrug in comparison to the free drug was due to the phospholipid complex, or This work is a part of A.D.’s Ph.D. dissertation. A.H. is affiliated with the David R. Bloom Center of Pharmacy (Jerusalem, Israel). Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.011817. ABBREVIATIONS: HPLC, high performance liquid chromatography; VPA, valproic acid. 0090-9556/07/3502-321–324$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 11817/3177199 DMD 35:321–324, 2007 Printed in U.S.A. 321 at A PE T Jornals on Jne 2, 2017 dm d.aspurnals.org D ow nladed from was due to pharmacokinetic reasons, i.e., the input function of indomethacin to the systemic circulation. Therefore, we administered an intravenous infusion in a manner that delivers low and sustained indomethacin plasma concentrations, mimicking the systemic indomethacin profile resulting from oral administration of the prodrug, and evaluated the resultant blood levels and brain indomethacin concentrations. Materials and Methods Materials. The indomethacin-phospholipid conjugate was supplied by DPharm Ltd. (Rehovot, Israel). Indomethacin, ibuprofen, formic acid, and ammonium acetate were purchased from Sigma Chemical Co. (St. Louis, MO). Saline was obtained from Teva Medical (Ashdod, Israel). Ethanol, methanol, acetonitrile, water, and ethyl acetate (J.T. Baker, Deventer, Holland) were high performance liquid chromatography (HPLC) grade. All other chemicals were of analytical reagent grade. Experimental Procedures. All surgical and experimental procedures were reviewed and approved by the Animal Experimentation Ethics Committee of the Hebrew University Hadassah Medical School (Jerusalem, Israel). Male Wistar rats (Harlan Israel, Jerusalem, Israel), 275 to 300 g in weight, were used for all surgical procedures. One day before the pharmacokinetic experiment, an indwelling cannula was placed in the right jugular vein of the animals, by a method described before (Hoffman and Levy, 1989). The cannula was tunneled beneath the skin and exteriorized at the dorsal part of the neck. After completion of cannula implantation, the animals were transferred to metabolic cages to recover overnight. During this recovery period and throughout the experiment, food, but not water, was deprived. Animals were randomly assigned to the different experimental groups. Two groups of animals (n 4 in each group) were administered an equimolar oral dose (0.01 mmol) of free indomethacin or indomethacinphospholipid prodrug in the same vehicle and volume (1 ml/kg) by oral gavage. An additional group of rats (n 4) was administered an intravenous infusion of a commercially available indomethacin i.v. injection (Merck and Co. Inc., Darmstadt, Germany). The indomethacin solution was infused through the jugular vein cannula by an automatic infusion pump (PHD 2000 Syringe Pump; Harvard Apparatus Inc., Holliston, MA). Systemic blood samples (400 l) were taken at 5 min predose, and 1, 2, 4, and 8 h postdose. To prevent dehydration, equal volumes of physiological solution were introduced to the rats after each withdrawal of blood sample. Eight hours after the pharmacokinetic experiment began, the animals were anesthetized with ether, a systemic blood sample was withdrawn, the animals were sacrificed, and the whole brain was obtained and stripped of its external vasculature and meninges. The brain samples were divided into two pieces and accurately weighed (0.75–1 g/brain sample) to perform duplicate analysis. Analytical Methods. An HPLC system (Waters 2695 separation module; Waters, Milford, MA) with a photodiode array UV detector (Waters 2996) was used for determining the amount of indomethacin in plasma and brain, by a method described before with some modifications (Ioffe et al., 2002). To determine brain levels of indomethacin, the brain samples were spiked with 40 l of internal standard solution (ibuprofen, 250 g/ml), followed by extraction (Polytron tissue homogenizer, 25,000 rpm) into 5 ml of ethyl acetate. After homogenizing, samples were centrifuged, and supernatant was transferred, evaporated to dryness, and redissolved in 80 l of diluent comprising 0.07% ammonium acetate in methanol/acetonitrile/water (88:11:1% v/v, respectively). Then, 20 l of the resulting solution were injected into the HPLC system. The HPLC conditions were as follows: LiChrospher RP-18 column (Merck), an isocratic mobile phase, and 0.1% formic acid in methanol/acetonitrile/water (68:12:20% v/v), at a flow rate of 1 ml/min at room temperature. Duplicate analyses were performed to all brain samples. Separate standard curves were carried out for brain and plasma samples (r 0.999). The minimum quantifiable concentrations for indomethacin plasma and brain samples were 100 ng/ml and 200 ng/g, respectively. The interand intraday coefficients of variation were 1.0 and 0.5%, respectively. Pharmacokinetic Analysis. Plasma concentration versus time curves for indomethacin in individual rats were analyzed by means of the noncompartmental analysis model. To achieve the desired indomethacin concentrations in the i.v. infused animals, the rate of the intravenous indomethacin infusion was calculated using the following equation: R CpVdk/(1 e ), where the plasma concentration (Cp) at any time (t) can be achieved at a constant infusion rate (R) if the volume of distribution (Vd) and elimination constant (k) are known. Statistical Analysis. All values are expressed as mean standard deviation (S.D.). To determine statistically significantly differences among the experimental groups, the nonparametric Kruskal-Wallis test was used for multiple comparisons, and the two-tailed nonparametric Mann-Whitney U test for two-group comparison was used when appropriate. A p value of less than 0.05 was termed significant.