In vivo evidence of organic cation transporter-mediated tracheal accumulation of the anticholinergic agent ipratropium in mice.

Ipratropium bromide (IPR) is an anticholinergic used to treat chronic obstructive pulmonary disease (COPD), and is a substrate of organic cation transporters. The present study aimed to assess the contribution of organic cation transporters to tracheobronchial absorption of IPR in vivo by directly injecting [(3) H]IPR into the tracheal lumen of mice and measuring its accumulation in tracheal tissue. RT-PCR and immunohistochemical analysis showed that Octn1, Octn2, and Oct2 were localized at epithelial cells in the respiratory tract. Electron-microscopic immunohistochemistry indicated that Octn1 and Octn2 were localized at the apical portions of ciliated epithelial cells of trachea. In vitro uptake studies in HEK293 cells expressing these transporters demonstrated that IPR is a preferred substrate of Octn2. Inhibition of mouse tracheal accumulation of [(3) H]IPR by carnitine was concentration-dependent, reaching a maximum of 42% at 1 mM, whereas inhibition by 0.1 mM MPP(+) amounted to 62%. Tracheal accumulation of [(3) H]IPR was unchanged when mice were simultaneously injected with Octn1 substrate ergothioneine and organic anion transporter substrate estrone sulfate. These results suggest that Octn2 is involved in membrane permeation of IPR in the respiratory tract in vivo. Targeting organic cation transporters may be an effective strategy for delivery of cationic anti-COPD drugs to patients.

Organic cation transporter-mediated renal secretion of ipratropium and tiotropium in rats and humans.

Ipratropium bromide (ipratropium) and tiotropium bromide (tiotropium), anticholinergic agents with bronchodilating properties, are used to treat patients with chronic obstructive pulmonary disease. Because they are actively secreted into urine, the interaction of these agents with organic cation transporters (OCTs/Octs) was examined in rat kidney slices and in cultured cells expressing rat Oct (rOct) or human OCT (hOCT). Uptake of radiolabeled ipratropium in rat kidney slices was significantly inhibited by OCT/Oct substrates including cimetidine, imipramine, and quinidine, but not by organic anion transporter substrates (e.g., p-aminohippuric acid and estrone-3-sulfate). [(3)H]Tiotropium uptake showed similar characteristics. Reverse transcription-polymerase chain reaction showed that, in rat kidney, mRNA expression of rOct2 was the highest, followed by rOct1, but little rOct3 was detected. In vitro, rOct1 and rOct2 transported both anticholinergics, but rOct3 accepted only ipratropium. Ipratropium uptake by rat kidney slices consisted of two components with K(m) values of 0.114 ± 0.06 and 24.5 ± 2.21 µM. The K(m) value of rOct2-mediated ipratropium uptake (0.143 ± 0.03 µM) was consistent with that of the high-affinity component. The OCT/Oct inhibitor corticosterone, at a concentration of 1 µM (IC(50), 1.11 ± 0.20 µM for rOct2-mediated ipratropium transport), inhibited ipratropium by 18.4%, suggesting that rOct2 is involved in renal secretion of ipratropium. In a similar manner, ipratropium and tiotropium were taken up by cultured cells expressing hOCT1 and hOCT2 but not hOCT3. We conclude that OCT2/Oct2 plays a role in renal secretion of both anticholinergics in these species. Coadministration of these anticholinergics with cationic drugs recognized by OCT2/Oct2 may decrease renal clearance, resulting in increased systemic exposure.

Transport of ipratropium, an anti-chronic obstructive pulmonary disease drug, is mediated by organic cation/carnitine transporters in human bronchial epithelial cells: implications for carrier-mediated pulmonary absorption.

Ipratropium bromide, an anticholinergic drug used for the treatment of asthma and chronic obstructive pulmonary disease, has low oral bioavailability, but systemic exposure, superior to oral administration, can be achieved by inhalation. Therefore, we investigated the pulmonary absorption mechanism of ipratropium using human bronchial epithelial BEAS-2B cells. [3H]Ipratropium uptake by BEAS-2B cells was temperature-dependent and saturable, with a K(m) value of 78.0 microM, suggesting involvement of carrier-mediated uptake. An RT-PCR study showed that organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in BEAS-2B cells, but organic cation transporters (OCTs) are not. Uptake of [3H]ipratropium by HEK293 cells expressing OCTN1 (HEK293/OCTN1) and OCTN2 (HEK293/OCTN2) was significantly increased, compared with mock-transfected cells, and the estimated K(m) values were 444 microM and 53.0 microM, respectively. Finally, the contributions of OCTN1 and OCTN2 to ipratropium uptake were evaluated by measuring [3H]ipratropium uptake by BEAS-2B cells in which OCTN1 or OCTN2 gene expression had been silenced. Knock-down of OCTN1 or OCTN2 suppressed the uptake of [3H]ipratropium to 78.2% and 14.8% of that by control BEAS-2B cells, respectively. In addition, another anticholinergic, tiotropium, was also taken up by both HEK293/OCTN1 and HEK293/OCTN2 cells. Therefore, ipratropium and tiotropium are taken up primarily by OCTN2, and to a lesser extent by OCTN1, in bronchial epithelial cells. These findings are consistent with the pharmacological activity of the drugs after administration via inhalation.