Tissue distribution of renadirsen sodium, a dystrophin exon-skipping antisense oligonucleotide, in heart and diaphragm after subcutaneous administration to cynomolgus monkeys.

The pharmacokinetics and tissue distribution of renadirsen sodium, a dystrophin exon-skipping phosphorothioate-modified antisense oligonucleotide with 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), after subcutaneous or intravenous administration to cynomolgus monkeys were investigated. The plasma concentration of renadirsen after subcutaneous administration at 1, 3, and 10 mg/kg increased with the dose. The absolute bioavailability at 3 mg/kg after subcutaneous administration was calculated as 88.6%, and the time to reach maximum plasma concentration of renadirsen was within 4 h, indicating the efficient and rapid absorption following subcutaneous administration. The exposure of muscle tissues to renadirsen was found to increase with repeated dosing at 6 mg/kg, and higher exposure was observed in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen achieved more exon 45-skipped dystrophin mRNA in the diaphragm and heart than in the quadriceps femoris and anterior tibialis muscles. Renadirsen also showed a cumulative skipping effect in a repeated-dose study. The findings on exon 45-skipped dystrophin mRNA in these muscle tissues were consistent with the concentration of renadirsen in these tissues. Because it is not feasible to directly evaluate drug concentration and exon skipping in the heart and diaphragm in humans, the pharmacokinetics and pharmacodynamics of renadirsen in these tissues in monkeys are crucial for the design and interpretation of clinical settings.

Methemoglobinemia induced by 1,2-dichloro-4-nitrobenzene in mice with a disrupted glutathione S-transferase Mu 1 gene.

A specific substrate to Mu class glutathione S-transferase (GST), 1,2-dichloro-4-nitrobenzene (DCNB), was administered to mice with a disrupted GST Mu 1 gene (Gstm1-null mice) to investigate the in vivo role of murine Gstm1 in toxicological responses to DCNB. A single oral administration of DCNB at doses of 500 and 1000 mg/kg demonstrated a marked increase in blood methemoglobin (MetHB) in Gstm1-null mice but not in wild-type mice. Therefore, Gstm1-null mice were considered to be more predisposed to methemoglobinemia induced by a single dosing of DCNB. In contrast, 14-day repeated-dose studies of DCNB at doses up to 600 mg/kg demonstrated a marked increase in blood MetHB in both wild-type and Gstm1-null mice. However, marked increases in the blood reticulocyte count, relative spleen weight, and extramedullary hematopoiesis in the spleen were observed in Gstm1-null mice compared with wild-type mice. In addition, microarray and quantitative reverse transcription-polymerase chain reaction analyses in the spleen showed exclusive up-regulation of hematopoiesis-related genes in Gstm1-null mice. These changes were considered to be adaptive responses to methemoglobinemia and attenuated the higher predisposition to methemoglobinemia observed in Gstm1-null mice in the single-dose study. In toxicokinetics monitoring, DCNB concentrations in plasma and blood cells were higher in Gstm1-null mice than those in wild-type mice, resulting from the Gstm1 disruption. In conclusion, it is suggested that the higher exposure to DCNB due to Gstm1 disruption was reflected in methemoglobinemia in the single-dose study and in adaptive responses in the 14-day repeated-dose study.

Effect of the metabolic capacity in rat liver S9 on the positive results of in vitro micronucleus tests.

A high incidence of positive results is obtained with in vitro genotoxicity tests, which do not correlate with the in vivo negative results in many cases. To address this issue, the metabolic profile of rat liver 9000 × g supernatant fraction (S9) pretreated with phenobarbital (PB) and 5,6-benzoflavone (BNF) was characterized. Furthermore, the in vitro micronucleus tests of 10 compounds were performed with PB-BNF-induced rat S9. PB-BNF increased cytochrome P450 (CYP) activity and CYP1A1, CYP1A2, CYP2B1/2, CYP2C6, CYP3A1, and CYP3A2 expression in rat S9, whereas it decreased CYP2C11 and CYP2E1 expression. PB-BNF-induced S9 enhanced the micronucleus induction (MI) of benzo[a]pyrene (BaP), cyclophosphamide (CPA), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP), which are metabolized by CYP1A1, CYP2C6, and CYP1A2, respectively. In contrast, coumarin and chlorpheniramine showed MI with PB-BNF-induced S9 despite the fact that they show negative results in the in vivo studies. Furthermore, diclofenac, piroxicam, lansoprazole, and caffeine showed MI regardless of the enzyme induction by PB-BNF, whereas phenacetin did not show MI. These results indicate that PB-BNF-induced rat S9 is effective in detecting the genotoxic potential of promutagens, such as BaP, CPA, and PhIP, but not of coumarin and chlorpheniramine, probably due to the differences in the in vitro and in vivo metabolic profile and its exposure levels of the drugs.

A novel model of continuous depletion of glutathione in mice treated with L-buthionine (S,R)-sulfoximine.

L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, was administered to mice via drinking water for 14 days in order to establish an animal model with continuously depleted levels of GSH. No toxicity was observed at 20 mM BSO, even though a significant decrease in liver weight was observed at 30 mM BSO. GSH levels in the liver, kidney, brain, lung, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, plasma and blood cells from mice given 20 mM of BSO were all less than those from the control mice continuously throughout a 24-hr period. The ratios of the GSH levels to that of the control were 46.4% and 16.7% in the liver and kidney, respectively, suggesting a decrease in GSH conjugation activity in vivo by GSH depletion. Liver cytochrome P450 content and UDP-glucuronosyltransferase activity to p-nitrophenol were not influenced by the BSO dosing. To confirm the adequacy of this GSH-depletion model, 0.125 or 0.25% of acetaminophen (APAP) was administered via diet to this model for 14 days. Nine out of the ten mice given both 20 mM BSO and 0.25% APAP died on Day 2, and remarkable necrosis was observed in the hepatocytes and renal tubular epithelium. Moreover, focal necrosis of hepatocytes with proliferation of fibroblasts was observed on Day 15 in some mice coadministered 20 mM BSO and 0.125% APAP. However, no toxicity was observed in mice given APAP alone. Based on these results, a mouse given 20 mM of BSO via drinking water for 14 days was concluded to be an animal model with continuously depleted levels of GSH in various organs without toxicity. This model shows high susceptibility to toxicity induced by chemicals which are metabolized to electrophilic and reactive metabolite(s), such as APAP.

Toxicokintic and toxicodynamic analysis of clofibrate based on free drug concentrations in nagase analbuminemia rats (NAR).

Toxicokinetics (TK) is usually performed by measurement of the total drug concentrations in plasma. However, free drug concentrations in plasma are considered to correlate directly with toxicodynamics (TD). In the present study, to evaluate the applicability of TK/TD analysis based on free drug concentrations, we investigated the TK/TD of clofibrate, which binds to albumin with a higher ratio, using an albumin-deficient mutant strain, Nagase analbuminemia rats (NAR). TK, blood chemistry, histopathology, drug and fatty acid metabolizing enzymes and microarray analysis in the liver were examined after a 4-day oral administration of clofibrate. Compared to Sprague-Dawley (SD) rats, the parent strain of NAR, 4.1-fold higher AUC(0-24hr) based on free drug concentrations (3445 versus 844 microg.hr/ml) was observed in NAR when both rats showed the same level of AUC(0-24hr) based on the total drug concentrations (4436 versus 4237microg.hr/ml). Additionally, more severe hepatocellular hypertrophy, increase in aspartate transaminase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), decrease in total cholesterol (T.CHO), phospholipid (PL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increase in the mRNA levels of fatty acid metabolizing enzymes (FAOS, CAT, and CPT) were observed in NAR at the same dose. These results demonstrated that NAR developed more severe toxicities and pharmacological effects than SD rats correlating with the higher AUC of the free drug concentrations. The results also suggested that TK/TD analysis based on the free drug concentration is appropriate to interpret the relationship between exposure and toxicity in cases of protein binding saturation including protein decrease or species differences on protein binding, especially when drugs showing a higher protein binding ratio are dosed.

Expression of the theta class GST isozyme, YdfYdf, in low GST dogs.

We have reported the existence of low glutathione S-transferase (GST) dogs whose GST activity to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activity) is quite low, and have also reported significant individual differences in dog liver GST-D activity. The dogs were classified as "low", "middle", or "high" GST dogs based on their GST-D activity. In the present study, in order to investigate the causes of quite low GST-D activity in low GST dogs and the individual differences in dog GST-D activity, glutathione (GSH) conjugation of DCNB was kinetically analyzed. Moreover, liver cytosolic proteins whose expression levels were significantly lower in low GST dogs than in high GST dogs were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and LC tandem mass spectrometry (LC/MS/MS). Interestingly, Vmax values for this reaction well reflected their GST-D activities in all groups, i.e. they were 3.8, 80.6, and 169.2 nmol/min/mg protein in the low, middle, and high GST dogs, respectively. However, Km values were almost the same (260.0-283.7 microM) among these groups. These suggest that GSH conjugation of DCNB should be catalyzed by the same enzyme in all the dogs, and individual differences in the GST-D activity should be the result of individual differences in the expression level of the GST isozyme, which catalyzes conjugation of DCNB. In 2D-DIGE, the expression levels of the two protein spots were significantly lower in the low GST dogs than in the high GST dogs. Positive good correlation (r > 0.800) was observed between GST-D activity and expression levels of these two protein spots. Moreover, expression levels were quite low in low GST dogs. These two proteins were both identified as the theta class GST isozyme, YdfYdf, which specifically catalyzes GSH conjugation of DCNB in dog livers. In the present study, we present two novel findings based on an enzyme kinetic study and protein-expression analysis: (1) GSTYdfYdf is expressed at quite a low level in the liver of low GST dogs, and (2) individual differences in dog liver GST-D activity would be due to individual differences in the expression level of GSTYdfYdf. Considering these findings, low GST dogs might have high susceptibility, including an unexpected toxicity at abnormal exposure to chemicals metabolized by GSTYdfYdf.

Characterization of phenotypes in Gstm1-null mice by cytosolic and in vivo metabolic studies using 1,2-dichloro-4-nitrobenzene.

Glutathione S-transferase Mu 1 (GSTM1) has been regarded as one of the key enzymes involved in phase II reactions in the liver, because of its high expression level. In this study, we generated mice with disrupted glutathione S-transferase Mu 1 gene (Gstm1-null mice) by gene targeting, and characterized the phenotypes by cytosolic and in vivo studies. The resulting Gstm1-null mice appeared to be normal and were fertile. Expression analyses for the Gstm1-null mice revealed a deletion of Gstm1 mRNA and a small decrease in glutathione S-transferase alpha 3 mRNA. In the enzymatic study, GST activities toward 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB) in the liver and kidney cytosols were markedly lower in Gstm1-null mice than in the wild-type control. Gstm1-null mice had GST activities of only 6.1 to 21.0% of the wild-type control to DCNB and 26.0 to 78.6% of the wild-type control to CDNB. After a single oral administration of DCNB to Gstm1-null mice, the plasma concentration of DCNB showed larger AUC0-24 (5.1-5.3 times, versus the wild-type control) and higher Cmax (2.1-2.2 times, versus the wild-type control), with a correspondingly lower level of glutathione-related metabolite (AUC0-24, 9.4-17.9%; and Cmax, 9.7-15.6% of the wild-type control). In conclusion, Gstm1-null mice showed markedly low ability for glutathione conjugation to DCNB in the cytosol and in vivo and would be useful as a deficient model of GSTM1 for absorption, distribution, metabolism, and excretion/toxicology studies.