In vitro-based prediction of human plasma concentrations of food-related compounds.

Efforts have been made to replace animal experiments in safety evaluations, including in vitro-based predictions of human internal exposures, such as predicting peak plasma concentration (Cmax) values for xenobiotics and comparing these values with in vitro-based toxicity endpoints. Herein, the authors predicted the Cmax values of food-related compounds in humans based on existing and novel in vitro techniques. In this study, 20 food-related compounds, which have been previously reported in human pharmacokinetic or toxicokinetic studies, were evaluated. Human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIEC) and Caco-2 cells, HepaRG cells, equilibrium dialysis of human plasma, and LLC-PK1 cell monolayer were used to assess intestinal absorption and availability, hepatic metabolism, unbound plasma fraction, and secretion and reabsorption in renal tubular cells, respectively. After conversion of these parameters into human kinetic parameters, the plasma concentration profiles of these compounds were predicted using in silico methods, and the obtained Cmax values were found to be between 0.017 and 183 times the reported Cmax values. When the in silico-predicted parameters were modified with in vitro data, the predicted Cmax values came within 0.1-10 times the reported values because the metabolic activities of hiPSC-SIECs, such as uridine 5'-diphospho-glucuronosyl transferase, are more similar to those of human primary enterocytes. Thus, combining in vitro test results with the plasma concentration simulations resulted in more accurate and transparent predictions of Cmax values of food-related compounds than those obtained using in silico-derived predictions alone. This method facilitates accurate safety evaluation without the need for animal experiments.

Simultaneous Evaluation of Membrane Permeability and UDP-Glucuronosyltransferase-Mediated Metabolism of Food-Derived Compounds Using Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.

Simultaneous detection of eight species of tree nut in foods using two tetraplex polymerase chain reaction assays.

Tree nuts comprise a category of food allergens that must be included in the food labels in several countries. We developed a polymerase chain reaction (PCR) method using eight specific primer pairs to detect eight representative tree nuts (almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, and walnut) under the same experimental conditions. The specificity of the eight primer pairs was confirmed by PCR testing against a variety of plant and animal samples. The detection limit of the method ranged from 1 fg to 1 pg DNA of individual tree nuts. The method detected tree nut DNA in processed and unprocessed food. In addition, the primer pairs could be combined into two sets of tetraplex PCR system. The developed method is specific, sensitive, and efficient, making it useful for detecting trace amounts of eight species of tree nut in foods.

Activation of post-synaptic dopamine D1 receptors promotes the release of tissue plasminogen activator in the nucleus accumbens via PKA signaling.

We have previously demonstrated that tissue plasminogen activator (tPA) plays an important role through the conversion of plasminogen to plasmin in the release of dopamine in the nucleus accumbens (NAc) evoked by depolarization or the systemic administration of drugs of abuse such as morphine and nicotine. In the present study, we examined the mechanisms by which drugs of abuse increase extracellular tPA activity in the NAc in vivo using in situ zymography. The dopamine D(1) receptor (D(1) R) agonist SKF38393, but not D(2) receptor agonist quinpirole, significantly increased extracellular tPA activity in the NAc. The effect of SKF38393 was blocked by pre-treatment with the dopamine D(1) R antagonist SCH23390. Microinjection of Rp-cAMPs, a protein kinase A inhibitor, into the NAc completely blocked the effect of SKF38393. Systemic administration of morphine and methamphetamine increased extracellular tPA activity in the NAc, and these effects were completely blocked by pre-treatment with SCH23390 and raclopride. The results suggest that activation of post-synaptic dopamine D(1) Rs in the NAc leads to an increase in extracellular tPA activity via protein kinase A signaling. Furthermore, dopamine D(2) receptors are also involved in the release of tPA induced by morphine and methamphetamine.

Possible involvement of protease-activated receptor-1 in the regulation of morphine-induced dopamine release and hyperlocomotion by the tissue plasminogen activator-plasmin system.

We have previously demonstrated that tissue plasminogen activator (tPA)-plasmin system participates in the rewarding effect of morphine, by regulating dopamine release in the nucleus accumbens (NAc). However, it is unclear how plasmin increases the morphine-induced release of dopamine and hyperlocomotion. In the present study we investigated whether protease activated receptor-1 (PAR-1) is involved in the regulation of acute morphine-induced dopamine release by the tPA-plasmin system. Morphine significantly but transiently increased extracellular tPA activity in the NAc, which was completely blocked by naloxone. Microinjection of a PAR-1 antagonist, (tyr(-1))-thrombin receptor activating peptide 7, into the NAc significantly reduced morphine-induced dopamine release in the NAc and hyperlocomotion although the treatment had no effect on basal dopamine release and spontaneous locomotor activity. Furthermore, the PAR-1 antagonist blocked the ameliorating effect of plasmin on the defect of morphine-induced dopamine release in the NAc of tPA-deficient mice. In contrast, intracerebroventricular injection of the PAR-1 antagonist had no effect on the antinociceptive effects of morphine in mice. These results suggest that PAR-1 is a target for the tPA-plasmin system in the regulation of acute morphine-induced dopamine release in the NAc.

The rewards of nicotine: regulation by tissue plasminogen activator-plasmin system through protease activated receptor-1.

Nicotine, a primary component of tobacco, is one of the most abused drugs worldwide. Approximately four million people die each year because of diseases associated with tobacco smoking. Mesolimbic dopaminergic neurons mediate the rewarding effects of abused drugs, including nicotine. Here we show that the tissue plasminogen activator (tPA)-plasmin system regulates nicotine-induced reward and dopamine release by activating protease activated receptor-1 (PAR1). In vivo microdialysis revealed that microinjection of either tPA or plasmin into the nucleus accumbens (NAc) significantly potentiated whereas plasminogen activator inhibitor-1 reduced the nicotine-induced dopamine release in the NAc in a dose-dependent manner. Nicotine-induced dopamine release was markedly diminished in tPA-deficient (tPA-/-) mice, and the defect of dopamine release in tPA-/- mice was restored by microinjection of either exogenous tPA or plasmin into the NAc. Nicotine increased tPA protein levels and promoted the release of tPA into the extracellular space in the NAc. Immunohistochemistry revealed that PAR1 immunoreactivity was localized to the nerve terminals positive for tyrosine hydroxylase in the NAc. Furthermore, we demonstrated that plasmin activated PAR1 and that nicotine-induced place preference and dopamine release were diminished in PAR1-deficient (PAR1-/-) mice. Targeting the tPA-plasmin-PAR1 system would provide new therapeutic approaches to the treatment of nicotine dependence.

Involvement of tissue plasminogen activator-plasmin system in depolarization-evoked dopamine release in the nucleus accumbens of mice.

Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin. In the present study, we investigated the role of the tPA-plasmin system in depolarization-evoked dopamine (DA) and acetylcholine (ACh) release in the nucleus accumbens (NAc) and hippocampus, respectively, of mice, by using in vivo microdialysis. Microinjection of either tPA or plasmin significantly potentiated 40 mM KCl-induced DA release without affecting basal DA levels. In contrast, plasminogen activator inhibitor-1 dose-dependently reduced 60 mM KCl-induced DA release. The 60 mM KCl-evoked DA release in the NAc was markedly diminished in tPA-deficient (tPA-/-) mice compared with wild-type mice, although basal DA levels did not differ between the two groups. Microinjections of either exogenous tPA (100 ng) or plasmin (100 ng) into the NAc of tPA-/-mice restored 60 mM KCl-induced DA release, as observed in wild-type mice. In contrast, there was no difference in either basal or 60 mM KCl-induced ACh release in the hippocampus between wild-type and tPA-/-mice. Our findings suggest that the tPA-plasmin system is involved in the regulation of depolarization-evoked DA release in the NAc.

Modification by the tissue plasminogen activator-plasmin system of morphine-induced dopamine release and hyperlocomotion, but not anti-nociceptive effect in mice.

The extracellular serine protease tissue plasminogen activator (tPA) that converts plasminogen into plasmin is abundantly expressed throughout the central nervous system. We have recently demonstrated that the tPA-plasmin system participates in the rewarding and locomotor-stimulating effects of morphine by acutely regulating morphine-induced dopamine release in the nucleus accumbens (NAc). In the present study, we examined the effects of microinjections of plasminogen activator inhibitor-1 (PAI-1), tPA or plasmin into the NAc on morphine-induced dopamine release, hyperlocomotion and anti-nociceptive effects in ICR mice. A single morphine treatment resulted in an increase in protein levels of PAI-1 in the NAc. Microinjection of PAI-1 into the NAc dose-dependently reduced morphine-induced dopamine release and hyperlocomotion. In contrast, microinjection of tPA into the NAc significantly potentiated morphine-induced dopamine release and hyperlocomotion without affecting basal levels. Furthermore, microinjection of plasmin enhanced morphine-induced dopamine release, but did not modify the hyperlocomotion induced by morphine. The intracerebroventricular injection of PAI-1, tPA and plasmin at high doses had no effect on the anti-nociceptive effects of morphine. These results suggest that the tPA-plasmin system is involved in the regulation of morphine-induced dopamine release and dopamine-dependent behaviors but not the anti-nociceptive effects of morphine.

The role of tissue plasminogen activator in methamphetamine-related reward and sensitization.

In the central nervous system, tissue plasminogen activator (tPA) plays a role in synaptic plasticity and remodeling. Our recent study has suggested that tPA participates in the rewarding effects of morphine by regulating dopamine release. In this study, we investigated the role of tPA in methamphetamine (METH)-related reward and sensitization. Repeated METH treatment dose-dependently induced tPA mRNA expression in the frontal cortex, nucleus accumbens, striatum and hippocampus, whereas single METH treatment did not affect tPA mRNA expression in these brain areas. The METH-induced increase in tPA mRNA expression in the nucleus accumbens was completely inhibited by pre-treatment with R(+)-SCH23390 and raclopride, dopamine D1 and D2 receptor antagonists, respectively. In addition, repeated METH treatment increased tPA activity in the nucleus accumbens. There was no difference in METH-induced hyperlocomotion between wild-type and tPA-deficient (tPA-/-) mice. On the other hand, METH-induced conditioned place preference and behavioral sensitization after repeated METH treatment were significantly reduced in tPA-/- mice compared with wild-type mice. The defect of behavioral sensitization in tPA-/- mice was reversed by microinjections of exogenous tPA into the nucleus accumbens. Our findings suggest that tPA is involved in the rewarding effects as well as the sensitization of the locomotor-stimulating effect of METH.