Gastrointestial absorption of pimozide is enhanced by inhibition of P-glycoprotein

Following the death due to cardiac arrest of a patient taking pimozide, sertraline and aripiprazole antipsychotic/antidepressant combination therapy, a role of drug-drug interaction was suggested. Here, we investigated P-glycoprotein (P-gp)-mediated interaction among the three drugs using in vitro methods. Sertraline or aripiprazole significantly increased the permeability of pimozide in Caco-2 cell monolayers. ATPase assay indicated that pimozide is a P-gp substrate, and might act as a P-gp inhibitor at higher concentrations. The values of the kinetic parameters of carrier-mediated efflux, calculated from the concentration dependence of pimozide efflux from LLC-GA5-COL150 cells expressing human P-gp, were as follows: maximum transport rate (Jmax) = 84.9 ± 8.9 pmol/min/mg protein, half-saturation concentration (Kt) = 10.6 ± 4.7 μM, first-order rate constant (kd) = 0.67 ± 0.14 pmol/min/mg protein. Further, the efflux ratio of pimozide in LLC-GA5-COL150 cells was significantly decreased in the presence of sertraline or aripiprazole. These results indicate that pimozide is a substrate of P-gp, and its efflux is inhibited by sertraline and aripiprazole. Thus, P-gp inhibition by sertraline and/or aripiprazole may alter the gastrointestinal permeability of co-administered pimozide, resulting in an increased blood concentration of pimozide, which may increase the likelihood of pimozide’s known life-threatening side effect of QT prolongation.

prolonged. This suggests that a drug interaction mechanism(s) other than inhibition of CYP3A4 74 and/or CYP2D6 is involved. We speculated that the drug transporter P-glycoprotein (P-gp) might 75 be an interaction site. 76 P-gp is a member of the ATP-binding cassette superfamily, and is mainly localized at 77 the intestine, blood-brain barrier, adrenal gland, enterocytes, hepatocytes, placenta and renal 78 proximal tubules in humans [9]. P-gp is responsible for the efflux of many xenobiotics and plays 79 major roles in drug absorption, distribution and excretion. In the intestine, P-gp mediates the 80 efflux of its substrates, restricting the absorption of many xenobiotics, including drugs [10][11][12][13]. 81 Recent studies show that sertraline, aripiprazole, and several of their metabolites have a P-gp-82 inhibitory effect [14][15][16], whereas pimozide is not known to be a P-gp substrate. The 83 bioavailability of pimozide is limited (about 50%) [17]. Therefore, if pimozide is a P-gp substrate, 84 there is a possibility that its absorption could be increased when it is used in combination with P-85 gp inhibitors. In order to test this idea, we examined whether pimozide is a P-gp substrate, and 86 whether its gastrointestinal permeability might be influenced by sertraline and/or aripiprazole by 87 means of a series of in vitro studies.      Where X0 is pimozide concentration per protein amount before incubation and X1 is that after 173 incubation. Therefore, X0 -X1 is the net transport of pimozide from LLC-GA5-COL150 cells.

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Kinetic analysis 176 To estimate the kinetic parameters of carrier-mediated transport in the efflux assays, the 177 transport rate (J) was fitted to the following equation (1), containing saturable and nonsaturable-linear 178 terms, by using the nonlinear least-square regression analysis program, MULTI, as previously reported

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(1) J = J max × S/(K t + S) + k d × S Where Jmax is the maximum transport rate for the carrier-mediated transport, S is the substrate 182 concentration, Kt is the half-saturation concentration, and kd is the first-order rate constant.

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Transport study across Caco-2 cell monolayers 202 The Papp of pimozide in the A-to-B direction in the presence of sertraline was 5.9-fold higher 203 than that in the absence of sertraline. The Papp in the B-to-A direction was decreased by sertraline,

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aripiprazole and verapamil, and sertraline had the greatest effect ( Fig 1A). Overall, the addition of 205 sertraline, aripiprazole and verapamil decreased the efflux ratio to 2.9 %, 60.6 % and 43.9 % 206 respectively, compared with pimozide alone (Fig 1B). ATPase assay for P-gp substrate 218 We investigated the pimozide concentration-sensitive ATPase activity of P-gp (Fig 2).

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Concentration-dependent elevation of ATPase activity was initially observed, though concentrations 220 of pimozide over about 10 M decreased the ATPase activity. decreased significantly and the permeability was increased when sertraline or aripiprazole was co-257 administered with pimozide, compared with the ER of pimozide alone (Fig 1B). Moreover, the ATPase 258 activity of P-gp-expressing inverted membrane vesicles was elevated in a pimozide-concentration-259 dependent manner (Fig 2). However, the ATPase activity was reduced in the high concentration range,

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suggesting that pimozide is both a P-gp substrate and an inhibitor at higher concentrations. Futhermore, 261 the efflux of pimozide from P-gpoverexpressing LLC-GA5-COL150 cells had both saturable and non-262 saturable linear components (Fig 3).  Fig 4). In the gastrointestinal tract, the effective concentration 271 of pimozide is estimated to be at least 9 μM and its Kt for P-gp was 10.6 μM, suggesting that P-gp-272 mediated drug interactions could occur. Importantly, because the experimental concentrations of 273 sertraline and aripiprazole were set based upon the clinically used doses, it is likely that P-gp-mediated 274 drug interaction in the gastrointestinal tract could occur in clinical situations when these drugs are co-275 administered with pimozide.

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In conclusion, pimozide is a substrate of P-gp, and its absorption is increased by sertraline and