Substrate Of OCTN2 Research Articles

In Vitro and In Vivo Assessment of Pharmacokinetic Profile of Peramivir in the Context of Inhalation Therapy

Objective: The aim was to evaluate the pharmacokinetics and underlying mechanisms of peramivir, a clinically approved antiviral agent for severe influenza, subsequent to airway inhalation in rats, thereby surmounting the constraints associated with the sole currently available intravenous formulation. Methods: Pharmacokinetic and tissue distribution investigations of peramivir were carried out in rats following both intravenous and inhaled administration. In vitro cell models were verified to investigate peramivir’s transmembrane transport and cellular uptake across diverse cell systems. Results: In vivo, peramivir exhibited restricted permeability, predominantly localizing within the alveolar epithelial lining fluid and lung tissue after inhalation, accompanied by minimal systemic dissemination. In vitro, it manifested low permeability across cell models, with no participation of efflux transporters. Despite the low rate of A549 uptake, the underlying uptake transport mechanism was still revealed. Peramivir was verified as an OCTN2 substrate. A robust correlation was observed between the in vitro and in vivo findings. Conclusions: A preclinical pharmacokinetic platform applicable to inhaled medications was established. Inhalation of peramivir augments exposure at the target site while diminishing systemic exposure, presenting potential therapeutic benefits in terms of efficacy and safety and suggesting it as a favorable alternative administration pathway.

Absorption mechanism of dragon's blood phenolic extracts in Caco-2 cells

The purpose of this study was to study the absorption characteristics of eight main components from dragon's blood phenolic extracts in Caco-2 cells based on the humancolon cancer cell Caco-2 model, and to clarify the oral absorption mechanism of such phenolic extracts. UPLC-MS/MS was used in this study to determine the content of 8 active ingredients including thevetiaflavone, 7,4'-dihydroxyflavone, 7,4'-dihydroxy-5-methoxyhomoisoflavanone, 7,4'-dihydroxyhomoisoflavanone, loureirin C, loureirin A, loureirin B and pterostilbene from dragon's blood phenolic extracts, and Caco-2 cells were used to investigate the effects of incubation time, concentration, temperature, P-gp inhibitor, MRP inhibitor, OCTN1 inhibitor and OCTN2 inhibitor on the absorption of each component. In addition, the transport experiment was conducted to measure the apparent permeability coefficient P_(app) and transport rate of the eight main components to predict the oral absorption mechanism of dragon's blood phenolic extracts. The experimental results showed that the cell uptake of the eight main components in dragon's blood phenolic extracts was time-dependent and concentration dependent, and the uptake of each component did not need to consume energy, which was consistent with the passive diffusion process. P-gp inhibitor, MRP inhibitor and OCTN1 inhibitor had no effect on the cell uptake of each component, only the addition of OCTN2 inhibitor significantly reduced the uptake of pterostilbene(P<0.05). In the transport results, the ER values of the outflow rates of the eight components were all less than 1.5. The above results show that the absorption mechanism of the eight components in Draconis resina phenolic extract may be passive diffusion, and pterostilbene may be the substrate of OCTN2.

Memantine transport by a proton-coupled organic cation antiporter in hCMEC/D3 cells, an in vitro human blood-brain barrier model

Memantine is clinically used for the treatment of patients with Alzheimer's disease and is highly distributed to the brain. The aim of this study is to characterize memantine transport at the blood-brain barrier (BBB) using hCMEC/D3 cells, a human BBB model. The initial uptake velocity of memantine in hCMEC/D3 cells was concentration-dependent, and was reduced by metabolic inhibitors, but was independent of extracellular sodium ion and membrane potential. Intracellular alkalization and intracellular acidification markedly reduced and enhanced the uptake, respectively. The uptake was strongly inhibited by quinidine, pyrilamine and verapamil, and was moderately inhibited by TEA (substrate of OCTs and OCTNs) and l-carnitine (substrate of OCTN2), but was not inhibited by MPP(+) (substrate of OCTs and PMAT) or ergothioneine (substrate of OCTN1). Although relatively abundant expression of OCTN2 gene has been observed in hCMEC/D3 cells, knockdown of OCTN2 with siRNA did not decrease memantine uptake. Memantine and diphenhydramine each showed inhibition of the other's uptake in a competitive manner. Thus, proton-coupled organic cation antiporter(s) appears to be involved in the transport of memantine in hCMEC/D3 cells, at least in part. Our results indicate that the in vivo BBB permeability of memantine in humans can be predicted from the in vitro uptake clearance in hCMEC/D3 cells.

L-Carnitine ester of prednisolone: Pharmacokinetic and pharmacodynamic evaluation of a type I prodrug

To evaluate whether PDSC, an L-carnitine ester derivative of prednisolone and OCTN2 substrate, could provide a targeted delivery of the corticosteroid into the lung tissues of an asthmatic guinea pig model. PRED (prednisolone) and PDC (an L-carnitine prodrug of prednisolone not recognized by OCTN2) served as controls. Water solubility and logP values were determined, and PDSC and PDC in vivo were quantified by LC-MS/MS. Unlike PRED, the intra-tracheal instillation of PDSC resulted in effective and prolonged accumulation of prednisolone in the lung tissues, leading to 3.8-fold higher reduction in inflammatory cell count in the bronchoalveolar fluid, and less severe lung and bronchial lesions in the asthmatic guinea pig. PDC showed similar pharmacokinetic profile to PRED, but exhibited higher efficiency (1.7-fold higher) at reducing the inflammatory cell count and the severity of lung histopathology, possibly due to the release of L-carnitine in vivo. The collective data suggest that PDSC has the potential to be an effective prodrug for the treatment of asthma with concomitant reduction in systemic side effects, and that novel prodrugs produced by L-carnitine conjugation can have useful applications in the targeted accumulation of drugs in the lungs.

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.

A Substrate Pharmacophore for the Human Organic Cation/Carnitine Transporter Identifies Compounds Associated with Rhabdomyolysis

The human organic cation/carnitine transporter (hOCTN2) is a high affinity cation/carnitine transporter expressed widely in human tissues and is physiologically important for the homeostasis of L-carnitine. The objective of this study was to elucidate the substrate requirements of this transporter via computational modeling based on published in vitro data. Nine published substrates of hOCTN2 were used to create a common feature pharmacophore that was validated by mapping other known OCTN2 substrates. The pharmacophore was used to search a drug database and retrieved molecules that were then used as search queries in PubMed for instances of a side effect (rhabdomyolysis) associated with interference with L-carnitine transport. The substrate pharmacophore was composed of two hydrogen bond acceptors, a positive ionizable feature and ten excluded volumes. The substrate pharmacophore also mapped 6 out of 7 known substrate molecules used as a test set. After searching a database of ~800 known drugs, thirty drugs were predicted to map to the substrate pharmacophore with L-carnitine shape restriction. At least 16 of these molecules had case reports documenting an association with rhabdomyolysis and represent a set for prioritizing for future testing as OCTN2 substrates or inhibitors. This computational OCTN2 substrate pharmacophore derived from published data partially overlaps a previous OCTN2 inhibitor pharmacophore and is also able to select compounds that demonstrate rhabdomyolysis, further confirming the possible linkage between this side effect and hOCTN2.

Genetic deficiency of carnitine/organic cation transporter 2 (slc22a5) is associated with altered tissue distribution of its substrate pyrilamine in mice

Carnitine/organic cation transporter 2 (OCTN2) recognizes various cationic compounds as substrates in vitro, but information on its pharmacokinetic role in vivo is quite limited. This paper demonstrates altered tissue distribution of the OCTN2 substrate pyrilamine in juvenile visceral steatosis (jvs) mice, which have a hereditary defect of the octn2 gene. At 30 min after intravenous injection of pyrilamine, the tissue-to-plasma concentration ratio (K(p)) in the heart and pancreas was higher, whereas the K(p) in kidney and testis was lower in jvs mice compared with wild-type mice. Pyrilamine transport studies in isolated heart slices confirmed higher accumulation, together with lower efflux, of pyrilamine in the heart of jvs mice. The higher accumulation in heart slices of jvs mice was abolished by lowering the temperature, by increasing the substrate concentration, and in the presence of other H(1) antagonists or another OCTN2 substrate, carnitine, suggesting that OCTN2 extrudes pyrilamine from heart tissue. On the other hand, the lower distribution to the kidney of jvs mice was probably due to down-regulation of a basolateral transporter coupled with OCTN2, because, in jvs mice, (i) the K(p) of pyrilamine in kidney assessed immediately after intravenous injection (approximately 1 min) was also lower, (ii) the urinary excretion of pyrilamine was lower, and (iii) the uptake of pyrilamine in kidney slices was lower. The renal uptake of pyrilamine was saturable (K(m) approximately 236 microM) and was strongly inhibited by cyproheptadine, astemizole, ebastine and terfenadine. The present study thus indicates that genetic deficiency of octn2 alters pyrilamine disposition tissue-dependently.

PDZK1 Regulates Two Intestinal Solute Carriers (Slc15a1 and Slc22a5) in Mice

Gastrointestinal (GI) absorption of certain therapeutic agents is thought to be mediated by solute carrier (SLC) transporters, although minimal in vivo evidence has been reported. Here, we show key roles of postsynaptic density 95/disk-large/ZO-1 (PDZ) domain-containing protein, PDZK1, as a regulatory mechanism of two solute carriers, Slc15a1 (oligopeptide transporter PEPT1) and Slc22a5 (carnitine/organic cation transporter OCTN2) in mouse small intestine by using pdzk1 gene knockout (pdzk1(-/-)) mice. GI absorption of cephalexin, a substrate of PEPT1, after p.o. administration was delayed in pdzk1(-/-) mice compared with wild-type mice. Absorption of carnitine, a substrate of OCTN2, was also decreased in pdzk1(-/-) mice. Immunohistochemical analysis revealed the localization of both PEPT1 and OCTN2 at apical membrane of small intestinal epithelial cells in wild-type mice, whereas such apical localization was reduced in pdzk1(-/-) mice, with a concomitant decrease in their protein levels assessed by Western blotting in intestinal brush-border membranes. Electron microscopy revealed localization of PEPT1 in intracellular vesicular structures in pdzk1(-/-) mice. In addition, we first identified interaction between PEPT1 and PDZK1 in mouse small intestine and found that PDZK1 stimulates transport activity of PEPT1 by increasing its expression level in human embryonic kidney 293 cells. Taken together, the present findings provide direct evidence that PDZK1 regulates two intestinal SLC transporters in vivo as an adaptor protein for these transporters and affects oral absorption of their substrates. These findings also raise the possibility that intestinal absorption of the substrate drugs for PEPT1 and OCTN2 is governed by the protein network of these transporters and their adaptor PDZK1.

Involvement of Carnitine/organic Cation Transporter OCTN2 (SLC22A5) in Distribution of its Substrate Carnitine to the Heart

This study was designed to clarify the pharmacological role of carnitine/organic cation transporter (Octn) family members in mouse heart. Immunohistochemical analysis revealed that Octn1 was exclusively expressed on endothelial cells in blood vessels. Octn2 was detected on the plasma membrane of cardiac muscle cells by immunoelectron microscopy. Octn3 was not detected in the heart. Integration plot analysis showed that coadministration of unlabeled L-carnitine reduced distribution of L-[3H]carnitine to the heart. L-[3H]Carnitine uptake in heart slices was reduced by carnitine analogs and various Octn2 substrates. L-[3H]Carnitine uptake by heart slices from juvenile visceral steatosis (jvs) mice, which have a hereditary octn2 gene deficiency, was negligible. Distribution of [3H]quinidine, another Octn2 substrate, to the heart was not reduced by L-carnitine, and [3H]quinidine uptake in heart slices was Na(-)-independent and inhibited by cationic drugs, but not carnitine analogs. [3H]Quinidine uptake by heart slices from jvs mice was similar to that of wild-type mice. These results demonstrate that OCTN2 is functionally expressed on the plasma membrane of muscle cells and is involved in distribution of carnitine to the heart. Some mechanism(s) other than OCTN2 is involved in the distribution of quinidine to the heart.

Organic cation transporter mRNA and function in the rat superior cervical ganglion.

Reuptake of extracellular noradrenaline (NA) into superior cervical ganglion (SCG) neurones is mediated by means of the noradrenaline transporter (NAT, uptake 1). We now demonstrate by single-cell RT-PCR that mRNA of the organic cation transporter 3 (OCT3, uptake 2) occurs in rat SCG neurones as well. Furthermore, our RT-PCR analyses reveal the presence of mRNA for novel organic cation transporters 1 and 2 (OCTN1 and OCTN2), but not for OCT1 or OCT2 in the ganglion. Making use of the NAT as a powerful, neurone-specific transporter system, we loaded[3H]-N-methyl-4-phenylpyridinium ([3H]-MPP+) into cultured rat SCG neurones. The ensuing radioactive outflow from these cultures was enhanced by desipramine and reserpine, but reduced (in the presence of desipramine) by the OCT3 inhibitors cyanine 863, oestradiol and corticosterone. In contrast, cyanine 863 enhanced the radioactive outflow from cultures preloaded with [3H]-NA. Two observations suggest that a depletion of storage vesicles by cyanine 863 accounts for the latter phenomenon: first, the primary radioactive product isolated from supernatants of cultures loaded with [3H]-NA was the metabolite [3H]-DHPG; and second, inhibition of MAO significantly reduced the radioactive outflow in response to cyanine 863. The outflow of [3H]-MPP+ was significantly enhanced by MPP+, guanidine, choline and amantadine as potential substrates for OCT-related transmembrane transporters. However, desipramine at a low concentration essentially blocked the radioactive outflow induced by all of these substances with the exception of MPP+, indicating the NAT and not an OCT as their primary site of action. The MPP+-induced release of [3H]-MPP+ was fully prevented by a combined application of desipramine and cyanine 863. No trans-stimulation of [3H]-MPP+ outflow was observed by the OCTN1 and OCTN2 substrate carnitine at 100 microM. Our observations indicate an OCT-mediated transmembrane transport of [3H]-MPP+. Amongst the three OCTs expressed in the SCG, OCT3 best fits the profile of substrates and antagonists that cause trans-stimulation and trans-inhibition, respectively, of [3H]-MPP+ release.

Activities of Cytochrome P450 Enzymes in Liver and Kidney Microsomes from Systemic Carnitine Deficiency Mice with a Gene Mutation of Carnitine/Organic Cation Transporter

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.