Masaaki Odomi

Chronic corticosteroid administration causes mitochondrial dysfunction in skeletal muscle.

Abstract Corticosteroid myopathy is a major clinical problem in patients undergoing chronic corticosteroid treatment and shows insidious and progressive muscle atrophy in proximal limbs. Although several mechanisms underlying the pathophysiology of muscle injury have been postulated, precise pathogenesis is still not clear. We evaluated the mitochondrial functions in patients receiving corticosteroids compared with those in healthy controls or patients not receiving corticosteroids. The serum levels and total production of lactate were investigated by an aerobic exercise test using a bicycle ergometer. Mitochondrial respiratory activities and oxidative damage in biopsied skeletal muscles were also studied. The results of aerobic exercise tests revealed a significant overproduction of lactate in patients treated with corticosteroids (p < 0.005), which was positively correlated with total corticosteroid doses administered (p < 0.0001). In these patients, mitochondrial enzyme activity in complex I was significantly decreased (p < 0.05) and oxidative damage of biopsied skeletal muscle was remarkable both in mitochondrial and nuclear DNAs (p < 0.001). The results suggest that chronic corticosteroid administration induces mitochondrial dysfunction and oxidative damage in skeletal muscles, which may be the pathogenesis, at least in part, of corticosteroid-induced myopathy.

In vitro-in vivo correlation for wet-milled tablet of poorly water-soluble cilostazol.

The purpose of the present study was to investigate oral bioavailability of an immediate release tablet containing wet-milled crystals of a poorly water-soluble drug, cilostazol, and to establish in vitro-in vivo correlation. Sub-micron sized cilostazol (median diameter: 0.26 microm) was successfully prepared using a beads-mill in water in the presence of a hydrophilic polymer and an anionic surfactant. The milled suspension was solidified with a sugar alcohol as a water-soluble carrier by spray-drying method. The co-precipitate was compressed into an immediate release tablet with common excipients. Oral bioavailability of the wet-milled cilostazol tablet in male beagle dogs was 13-fold higher than the hammer-milled commercial tablet in fasted condition. Food did not increase the oral bioavailability of the wet-milled tablet, while 4-fold increase was found for the commercial tablet. Irrespective to the bioavailability enhancement, in vitro dissolution rate of the wet-milled tablet was even slower than the commercial tablet by the compendial method (USP Apparatus 2). On the other hand, a good correlation was found between the dissolution profiles obtained by a flow-through cell method (USP Apparatus 4, closed-loop system without outlet filter) using a large volume of water and sodium lauryl sulfate (SLS) solution at the concentration lower than the critical micellar concentration (cmc) as dissolution media corresponding to the fasted and fed conditions, respectively.

Involvement of human cytochrome P450 3A4 in reduced haloperidol oxidation

AbstractObjective: The present study was conducted to identify in vitro the cytochrome P450(CYP) isoform involved in the metabolic conversion of reduced haloperidol to haloperidol using microsomes derived from human AHH-1 TK +/− cells expressing human cytochrome P450s. The inhibitory and/or stimulatory effects of reduced haloperidol or haloperidol on CYP2D6-catalyzed carteolol 8-hydroxylase activity were also investigated. Results: The CYP isoform involved in the oxidation of reduced haloperidol to haloperidol was CYP3A4. CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 2E1 were not involved in the oxidation. The kM value for the CYP3A4 expressed in the cells was 69.7 μmol · l−1, and the Vmax was 4.87 pmol · min−1 · pmol−1 P450. Troleandomycin, a relatively selective probe for CYP3A enzymes, inhibited the CYP3A4-mediated oxidation of reduced haloperidol in a dose-dependent manner. Quinidine and sparteine competitively inhibited the oxidative reaction with a ki value of 24.9 and 1390 μmol · l−1, respectively. Carteolol 8-hydroxylase activity, which is a selective reaction probe for CYP2D6 activity, was inhibited by reduced haloperidol with a ki value of 4.3 μmol · l−1. Haloperidol stimulated the CYP2D6-mediated carteolol 8-hydroxylase activity with an optimum concentration of 1 μmol · l−1, whereas higher concentrations of the compound (>10 μmol · l−1) inhibited the hydroxylase activity. Conclusion: It was concluded that CYP3A4, not CYP2D6, is the principal isoform of cytochrome P450 involved in the metabolic conversion of reduced haloperidol to haloperidol. It was further found that reduced haloperidol is a substrate of CYP3A4 and an inhibitor of CYP2D6, and that haloperidol has both stimulatory and inhibitory effects on CYP2D6 activity.

Metabolism of carteolol by cDNA-expressed human cytochrome P450

AbstractObjectives: To determine human cytochrome P450 isoform(s) (CYPs) involved in the metabolism of carteolol, the biotransformation of the compound was investigated in vitro using ten isoforms of human cytochrome P450 expressed in human AHH-1 TK ± cell lines. In addition, the inhibitory effects of carteolol on the activities of important CYP isoforms, namely, CYP1A2, 2C9, 2C19, 2E1, and 3A4, were examined. Results: Carteolol was metabolised to 8-hydroxycarteolol by CYP 2D6 with KM and Vmax values of 183 μmoles · l−1 and 26.09 pmol · min−1 · pmol−1 P450, respectively. CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4 were not involved in the metabolism of the compound. CYP2D6-mediated carteolol 8-hydroxylase activity was inhibited by quinidine, propranolol, nortriptyline, dextromethorphan, sparteine, bufuralol, and biperiden. Biperiden competitively inhibited the catalytic reaction with a Ki value of 0.45 μmoles · l−1. Carteolol did not affect the following catalytic reactions:␣CYP1A2-mediated (R)-warfarin 6-hydroxylation, CYP2C9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated (S)-mephenytoin 4-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6β-hydroxylation. Conclusion: 8-Hydroxylation is the only cytochrome P450-catalyzed metabolic reaction of carteolol by its expressed microsomes, and CYP2D6 is the principal isoform of the enzyme involved in the catalytic reaction. Carteolol has neither stimulative nor inhibitory effects on CYP1A2, 2C9, 2C19, 2E1, and 3A4 activities.

Peptide Derivation of Poorly Absorbable Drug Allows Intestinal Absorption Via Peptide Transporter

The purpose of the present study was to examine whether the intestinal absorption of low-permeability drugs could be improved by utilization of the intestinal influx transporter PEPT1. We investigated whether peptide derivatives of poorly absorbable nonamino acid-like drugs might be substrates of PEPT1, using rebamipide (Reb) as a model drug. We synthesized several peptide derivatives of rebamipide and examined their inhibitory effect on the uptake of [(3)H]Gly-Sar by PEPT1-expressing HeLa cells. Some of the peptide derivatives inhibited PEPT1-mediated uptake of [(3)H]Gly-Sar. Next, uptake of the inhibitory peptide derivatives was evaluated in PEPT1-expressing Xenopus oocytes and HeLa cells. Ser(Reb)-Gly exhibited significantly increased uptake by PEPT1-expressing cells in comparison with that by mock cells. The permeability of Ser(Reb)-Gly across a Caco-2 cell monolayer was significantly higher than that of rebamipide itself, and the transport was decreased in the presence of PEPT1 substrates. Further, a rat intestinal perfusion study revealed increased absorption of Ser(Reb)-Gly compared with rebamipide. These results demonstrate that the addition of a dipeptide moiety to a poorly absorbable nonpeptide/nonamino acid-like drug can result in absorption via the intestinal transporter PEPT1, though there is some selectivity as regards the structure of the added peptide moiety.

Mitochondrial damage in patients with long-term corticosteroid therapy: development of oculoskeletal symptoms similar to mitochondrial disease

Abstract. Two patients with long-term corticosteroid administration sporadically developed limb muscle wasting followed by ophthalmoplegia, and the skeletal muscle pathology revealed ragged-red fibers (RRFs) with abnormal mitochondria, in addition to the findings of corticosteroid myopathy. The oculoskeletal symptoms of the present cases resemble those of chronic progressive external ophthalmoplegia, a type of mitochondrial disease. The ocular muscles have more RRFs than limb muscles, and large multiple deletions of mitochondrial DNA was detected in ocular and limb muscles of the two patients by PCR but not by Southern blotting. Immunohistochemistry demonstrated that 8-hydroxy-deoxyguanosine (8-OH-dG) and 4-hydroxy-2-nonenal were intensely stained in skeletal muscles of these patients particularly in RRFs. High-performance liquid chromatography with electrochemical detection analysis revealed an increase in 8-OH-dG from mitochondrial DNA. These findings may suggest that long-term corticosteroid administration potentially induces oxidative stress-mediated mitochondrial damage, resulting in the development of the oculoskeletal symptoms in some patients.

Novel oral absorption system containing polyamines and bile salts enhances drug transport via both transcellular and paracellular pathways across Caco-2 cell monolayers

The combinatorial use of spermine (SPM), a typical polyamine, and sodium taurocholate (STC), a typical bile salt, was found to be a promising safe preparation for improving the oral absorption of poorly water-soluble and/or poorly absorbable drug in our previous studies utilizing rats and dogs. To clarify the mechanisms behind the synergistic enhancement effect of the polyamine and bile salt, the transport of rebamipide, which is classified into Biopharmaceutics Classification System Class IV, was investigated in Caco-2 cell monolayers. The synergistic enhancement of rebamipide transport by SPM and STC was certainly observed in Caco-2 cells as well, while the separate use of either SPM or STC did not significantly improve the transport of rebamipide. The combinatorial use of SPM and STC significantly decreased the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers, suggesting that the opening of paracellular pathway. On the other hand, it was also confirmed that the decrease in TEER was transient and reversible after removal of SPM and STC and that cell viability was maintained. Voltage-clamp study clearly showed that their combinatorial use improved rebamipide transport via both paracellular and transcellular pathways, and that the contribution of transcellular route could be larger than paracellular route.

Ocular distribution of carteolol after single and repeated ocular instillation in pigmented rabbits

Abstract. To investigate the distribution and elimination of carteolol in pigmented rabbits, 14C‐carteolol eye drops were instilled singly and repeatedly. After single ocular instillation, the radioactivity in the iris and ciliary body reached maximum levels at 24 h. The elimination rate of pigmented tissues decreased at a half‐life of approximately 15 days. The concentration of radioacitivy in pigmented tissues increased markedly by repeating the ocular instillation and reached a maximum after the 8oth repeated instillation. The concentration of radioactivity at 1 h after 80th instillation was 63.7 times that in the iris, 61.1 times that in the ciliary body and 17.2 times that in the retina & choroid after single instillation. No accumulation of radioactivity was found in other ocular tissues.

Stereoselective pharmacokinetics and interconversions of flosequinan enantiomers containing chiral sulphoxide in rat

1. In order to study the pharmacokinetics of flosequinan enantiomers ((+-)-7-fluoro-1-methyl-3-methylsulphinyl-4-quinolone) containing chiral sulphur, plasma levels of (+)-(R)- and (-)-(S)-flosequinan (R-FSO and S-FSO) and two metabolites (flosequinan sulphide (FS) and flosequinan sulphone (FSO2)) were measured after oral and i.v. administration of racemic flosequinan (rac-FSO), R-FSO and S-FSO in male rat. 2. The pharmacokinetic parameters of the enantiomers were different after oral and i.v. administration of R-FSO and S-FSO. The plasma clearance of R-FSO was higher than S-FSO. 3. The major metabolites of boh R-FSO and S-FSO was FSO2. A minor metabolite, FS, was also detected in plasma. 4. Interconversions occurred after the oral and i.v. administration of R-FSO and S-FSO. The amount of interconversion from S-FSO and R-FSO was greater than that from R-FSO to S-FSO. The rate of interconversion after oral administration was higher than that after i.v. administration. 5. After i.v. administration of FS, R-FSO and S-FSO were detected in plasma, suggesting that the interconversion occurred via formation of FS. 6. The pharmacokinetic parameters of R-FSO after administration of rac-FSO differed from that after administration of R-FSO, indicating the interaction between each enantiomer.

Simple and rapid quantitative assay of 13C-labelled urea in human serum using liquid chromatography—atmospheric pressure chemical ionization mass spectrometry

A simple and rapid quantitative method for 13C-labelled urea ([13C]urea) in human serum was developed by using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS). This method is used to establish and normalize the [13C]urea breath test, which is considered as an effective diagnostic method for Helicobacter pylori infection. HPLC-APCI-MS, involving a simple pretreatment process such as diluting serum with water, was shown to be able to discriminate the extrinsic [13C]urea from intrinsic urea present at high concentration in serum. In addition, a 13C nuclear magnetic resonance spectroscopic quantitative method for [13C]urea in human urine is also described. The precision and accuracy of measured concentrations in these two methods were found to be within the acceptable limit. An application of these methods to investigate the pharmacokinetic profile of orally administered [13C]urea in human serum and urine is also presented.