This paper reports a multi-throughput multi-organ-on-a-chip system formed on a pneumatic pressure-driven medium circulation platform with a microplate-sized format as a novel type of ...microphysiological system. The pneumatic pressure-driven platform enabled parallelized multi-organ experiments (i.e. simultaneous operation of multiple multi-organ culture units) and pipette-friendly liquid handling for various conventional cell culture experiments, including cell seeding, medium change, live/dead staining, cell growth analysis, gene expression analysis of collected cells, and liquid chromatography-mass spectrometry analysis of chemical compounds in the culture medium. An eight-throughput two-organ system and a four-throughput four-organ system were constructed on a common platform, with different microfluidic plates. The two-organ system, composed of liver and cancer models, was used to demonstrate the effect of an anticancer prodrug, capecitabine (CAP), whose metabolite 5-fluorouracil (5-FU) after metabolism by HepaRG hepatic cells inhibited the proliferation of HCT-116 cancer cells. The four-organ system, composed of intestine, liver, cancer, and connective tissue models, was used to demonstrate evaluation of the effects of 5-FU and two prodrugs of 5-FU (CAP and tegafur) on multiple organ models, including cancer and connective tissue.
This paper reports a multi-throughput multi-organ-on-a-chip system formed on a pneumatic pressure-driven medium circulation platform with a microplate-sized format as a novel type of ...microphysiological system. The pneumatic pressure-driven platform enabled parallelized multi-organ experiments (
i.e.
simultaneous operation of multiple multi-organ culture units) and pipette-friendly liquid handling for various conventional cell culture experiments, including cell seeding, medium change, live/dead staining, cell growth analysis, gene expression analysis of collected cells, and liquid chromatography-mass spectrometry analysis of chemical compounds in the culture medium. An eight-throughput two-organ system and a four-throughput four-organ system were constructed on a common platform, with different microfluidic plates. The two-organ system, composed of liver and cancer models, was used to demonstrate the effect of an anticancer prodrug, capecitabine (CAP), whose metabolite 5-fluorouracil (5-FU) after metabolism by HepaRG hepatic cells inhibited the proliferation of HCT-116 cancer cells. The four-organ system, composed of intestine, liver, cancer, and connective tissue models, was used to demonstrate evaluation of the effects of 5-FU and two prodrugs of 5-FU (CAP and tegafur) on multiple organ models, including cancer and connective tissue.
A multi-throughput multi-organ-on-a-chip system was formed on a pneumatic pressure-driven medium circulation platform as a novel type of microphysiological system.
The aim of this study was to assess the cardiovascular effects of a selective phosphodiesterase 5 inhibitor ER-118585, ...4-(3-chloro-4-methoxybenzyl)amino-1-(2-hydroxy-7-azaspiro3.5non-7-yl)-6-phthalazinecarbonitrile monohydrochloride. The present results indicated that 1) ER-118585 significantly inhibited the human ether-a-go-go related gene (HERG) tail current at 10 nM and above with an IC50 value of 40.7 nM in human embryonic kidney 293 cells transfected with HERG cDNA; 2) ER-118585 at 100 and 1000 nM significantly increased the action potential duration (APD) at 50% and 90% repolarization in isolated papillary muscles of guinea pig; and 3) intravenous infusion of ER-118585 at 10 μg/kg/min significantly prolonged the QT interval by 10.5±1.6% from 281±2 ms to 311±6 ms in six anesthetized dogs subjected to atrial pacing. In consideration of both the plasma concentration of ER-118585 (984±78 nM, n=3) and its protein binding fraction (99.0±0.1%, n=5), the free plasma concentration was estimated at 9.8±0.8 nM, which is consistent with the minimum concentration of HERG current inhibition. In conclusion, these evaluation methods demonstrated that ER-118585 could prolong the QT interval via APD prolongation, attributable to the inhibition of the HERG potassium current.
1. The pharmacokinetics and disposition of E4177, an angiotensin II (Ang II) type 1 receptor antagonist, were studied in the beagle dog and cynomologus monkey following intravenous (i.v.) and oral ...(p.o.) administration. The relationship between the plasma concentrations of E4177 and Ang II receptor blockade were investigated in both species. 2. Single 0.3 mg/kg i.v. doses of E4177 in dog and monkey were eliminated rapidly. The elimination half-lives were 1.9 and 2.0 h, and the systematic plasma clearance values were 9.1 and 12.9 ml/min/kg respectively. 3. The oral bioavailability of single doses of 0.3-3 mg/kg of E4177 was > 60% in both dog and monkey. The absorption by both species was rapid, with peak plasma levels observed within 1 h, and the areas under the concentration versus time curve to infinity were proportional to the dose. 4. The apparent volumes of distribution at the steady-state were 1.0 and 1.2 l/kg in dog and monkey respectively. Tissue penetration was probably limited by the relatively high binding to plasma proteins (approximately 92.0 and 98.6% in the dog and monkey respectively). 5. Faecal excretion was the major elimination pathway for radioactivity (approximately 90% of the dose) in both species after 1 mg/kg p.o. administration of 14C-E4177. Unchanged E4177 was the major radioactive component in the urine and faeces (0-24 h) of both species, accounting for approximately 85% of dose. In monkey, a minor metabolite in excreta and plasma was identified as the phase 1 metabolite M1, which is produced from E4177 by methyl-hydroxylation. M1 was not detected in dog. 6. The unbound plasma concentration versus blockade of the exogenous Ang II-induced vasopressor response was also determined after an i.v. administration of E4177 to dog and monkey. The relationship between the unbound E4177 concentration and the effect was highly significant in both species. The IC50 of the dog and monkey were not significantly different: 2.6 and 2.7 ng/ml respectively.
1. The disposition of radioactivity of a non-peptide angiotensin II type 1 receptor antagonist (E4177) has been studied in groups of male rats after a single oral 1 mg/kg dose of 14C-E4177 was ...administered by gavage. We have also used light-microscopic autoradiography to investigate the localization of radioactivity in the target tissues for this angiotensin II receptor antagonist. 2. The radioactivity was absorbed quickly, and the maximum blood levels (Cmax) were reached at 0·38 ± 0·14 h after dosing. The concentrations then declined bi-exponentially with a mean apparent half-life for the first phase (t α) of 0·46 ± 0·07 h and a terminal half life (t β) of 6·22 ± 1·08 h. By 24 h, the levels had decreased to 2·7 ± 1·5% Cmax. The blood beta max levels radioactivity at 48 h after administration were below the limit of quantification. 3. Radioactivity was distributed throughout the body at 15 min after administration. Tissues inwhich radioactivity was present at higher levels thaninplasma were the liver and kidney. Radioactivity was rapidly eliminated from the tissues and was not retained in any individual organ. 4. The major route of excretion was via the bile. Since > 90% of the administered radioactivity was recovered by 24 h after administration, the excretion was relatively rapid. The major metabolite in bile was a glucuronide of E4177 biphenylcarboxylic acid (E4177- Glu). 5. Light-microscopic autoradiographic observations revealed a strong localization of radioactivity throughout the surface cells of the adrenal glomerulosa, the blood vessels in kidney and the surface of the aortic smooth muscle cells, which are all rich in angiotensin II type 1 (AT1) receptors.
The gastroepiploic artery, used widely as a conduit in coronary artery bypass grafting, has high vasospasticity. The aims of this study were to examine the vasorelaxant effects of three ...phosphodiesterase 3 (PDE3) inhibitors, olprinone, milrinone and amrinone, on isolated gastroepiploic arterial preparations in comparison with a calcium channel blocker diltiazem, and to confirm the mRNA expression of PDE3A isoenzyme using reverse transcription–polymerase chain reaction (RT-PCR) in the human gastroepiploic artery isolated from stomach removed in cancer surgery. In endothelium-denuded gastroepiploic arterial preparations, phenylephrine (100 μM) produced spontaneous, rhythmical changes in tension consisting of repeated contraction and relaxation. Olprinone at a concentration of 10 μM (
n
=
6) significantly inhibited the frequency (2.7
±
1.1 times/30 min vs. 6.2
±
0.7 times/30 min in the vehicle group), maximum tension (1.7
±
0.6 g vs. 3.6
±
0.6 g in the vehicle group) and minimum tension (0.6
±
0.2 g vs. 1.7
±
0.3 g in the vehicle group) of rhythmical changes. Such potency is comparable to that of diltiazem, but is stronger than milrinone and amrinone. RT-PCR using PDE3A- or PDE3B-specific oligonucleotide primer demonstrated the existence of PDE3A sequence in the gastroepiploic artery. These results suggest that olprinone, a potent PDE3A inhibitor, would be suitable for protecting against perioperative spasm during coronary artery bypass graft surgery.
1. (5R)-3-2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl-5-methoxymethyl2-oxazolidinone (E2011) has two chiral centers in its structure. In vivo optical inversion of the hydroxy group at one of ...the chiral centers converts E2011 to a diastereoisomer (ER-20593). Pharmacokinetic parameters of E2011 and ER-20593 were determined after administration of E2011 to rat at 10 mg/kg, and the plasma concentration ratios of E2011 to ER-20593 were almost constant after Tmax of the plasma concentrations. 2. E2011 and ER-20593 were separately administered orally to six species in addition to rat, and the species differences in both directions of epimerization (i.e. from E2011 to ER-20593 and from ER-20593 to E2011) were studied by measuring the plasma concentrations of both compounds. In mouse, guinea pig, dog, and squirrel monkey, the epimerization of E2011 to ER-20593 did not occur, but the epimerization of ER-20593 to E2011 did. In rat, pig and rhesus monkey, the inversion of E2011 to ER-20593 occurred, but the ratios of this inversion were smaller than those for the inversion in the opposite direction. E2011 underwent about 15% inversion to ER-20593inrat,which was the largest inversion in the seven species examined. 3. To study the mechanism of the epimerization, deuterium-labelled E2011 and ER20593 (created by substituting the proton at the chiral center of the parent compounds for deuterium) were orally administered (separately) to rat and dog, and the concentration ratios and molecular weights of E2011 and ER-20593 in the plasma were determined by hplc and FAB(+)-mass spectrometry respectively. The results indicated that the major mechanism of the epimerization was oxidation to the carbonyl form followed by reduction to the original epimer and or the other epimer. 4. The carbonyl form of E2011 (CO-E2011) was reduced to E2011 and ER-20593 (alcohol forms) by liver cytosol and microsomes from rat and dog in vitro with NADH or NADPH. The resultant epimeric ratios (E2011:ER-20593) were consistent with the in vivo results in rat and dog. 5. In conclusion, species differences in the epimerization of E2011 would result from product stereoselectivity of the reductase activity with the carbonyl intermediate.
Organ and cellular distribution and expression constancy of microsomal cytochrome P450 (CYP) 2C and 3A in humans were studied with new polyclonal antibodies to CYP2C (MP-1) and 3A (NF-2) active in ...formalin-fixed, paraffin-embedded tissues. Antibodies were raised against purified human CYP2C9 and CYP3A4. On western blotting, MP-1 reacted with 2C8, 2C9, 2C18 and 2C19, and NF-2 with 3A4. In both frozen and paraffin sections, hepatocytes showed diffuse immunoreactivity with MP-1 and centrilobular staining with NF-2. In-paraffin sections of 40 kinds of nonneoplastic tissues, epithelium of the small and large intestine, bile duct, nasal mucosa, kidney and adrenal cortex stained positively with both MP-1 and NF-2 antibodies. Epithelium of gastric fundic glands, salivary glands, tracheobronchial glands, Brunner's glands, the prostate, uterine cervix and nasopharynx showed definite reactivity with MP-1. Epithelium of the gastric mucosa with intestinal metaplasia, duodenum, gallbladder and intercalated ducts of the pancreas and chief cells of the parathyroid and the corpus luteum of the ovary reacted with NF-2. Among the neoplastic tissues, MP-1 reacted with pleomorphic adenoma of the salivary gland and carcinomas of six different organs, and NF-2 with those of 7 different organs. These results indicate that CYP2C and CYP3A are distributed widely and organ specifically, as well as being variably expressed in neoplastic and normal states.
1. Six metabolites present in rat urine after the oral administration of E2011 ((5R)3-2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl-5-methoxymethyl-2-oxazolidinone) were isolated with an ...Amberlite XAD-4 column and hplc, and termed HPM-1, HPM-2, HPM-31, HPM-32, HPM-33 and HPM-4. 2. To determine the correspondence of the findings of the metabolites between tlc (which was used in our previous study) and hplc, the six metabolites were isolated from rat urine after the administration of 14C-labelled E2011 with an Amberlite XAD-4 column and hplc, and then analysed by tlc. HPM-1, HPM-2, HPM-31, HPM-32, HPM-33 and HPM4 were identified as IM7, IM3, IM4, IM2, IM1 and E2011, respectively. 3. The structures of the metabolites were identified with nmr and mass spectrometry. One of the compounds identified, HPM-4, was the unchanged drug, E2011, and HPM-2 was O-desmethyl-E2011. Another metabolite (HPM-33), the main metabolite in the urine, was identified as (4S)-hydroxy-E2011, and the others were (4S)-hydroxy-O-desmethylE2011 (HPM-1), 2′′-hydroxy-E2011 (HPM-31) and (4R)-hydroxy-E2011 (HPM-32). 4. In conclusion, the main metabolic pathway of E2011 in the rat consisted of O-demethylation and hydroxylation.
1. After oral administration of 14C-labelled (5R)-3-\2-((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl-5-methoxymethyl-2-oxazolidinone (E2011) at a dose of 1 mg/kg, the blood level of radioactivity ...reached a maximum concentration (Cmax) of 0.545 μg eq./ml after 0.25 h in the rat and of 0.900 μg eq./ml after 0.5 h in the dog. In dog plasma, Cmax for radioactivity and unchanged E2011 were 0.862 μg eq./ml and 0.650 μg/ml respectively with corresponding Tmax (time at Cmax) of 0.75 and 0.25 h. The unchanged drug in dog plasma was below the detection limit (5 ng/ml plasma) after 24 h. 2. The tissue levels of radioactivity were measured at 0.25 (Tmax), 6, 24, and 168 h after max administration to the rat and at 0.5 (Tmax), 24, and 168 h in the dog. The radioactivity was max distributed in all tissues examined at Tmax in the rat and dog. The radioactivity levels of the cerebral cortex in the rat and dog were 26 and 36% of the plasma level at Tmax. The radioactivity in tissues decreased at almost the same rate as that in plasma. Plasma protein binding of the unchanged drug in the rat in vitro were about 70% in the range of 0.1-10 μg/ml, and those in the dog were about 45% in the same concentration range. 3. Cumulative excretion of radioactivity in the rat was 74.5% in urine and 22.5% in faeces after 7 days. In the dog, 55.5 and 36.5% of the radioactivity administered were excreted in urine and faeces respectively after 7 days. The biliary excretion of radioactivity in the cannulated rat was 23.0% within 48 h. 4. In tlc analysis of plasma and tissues of the rat and dog, the radioactivity for the unchanged drug was much higher than metabolites. In tlc analysis of urine, the same metabolites were detected in the rat and dog, and the radioactivity of a metabolite, IM1, was the highest in the both animals. Eight metabolites were detected in the plasma, tissues and excreta of the rat, and four metabolites in the dog. 5. In conclusion, the absorption, distribution, metabolism and excretion of 14C-labelled E2011 in the rat and dog have been established, and only minor differences were observed between these species.
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