The Usefulness of Determining Plasma and Tissue Concentrations of Phosphorodiamidate Morpholino Oligonucleotides to Estimate Their Efficacy in Duchenne Muscular Dystrophy Patients.

Currently, four kinds of phosphorodiamidate morpholino oligomers (PMOs), such as viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD-model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD-model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD-model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. SIGNIFICANCE STATEMENT: We evaluated the relationship between the plasma or tissue concentrations and the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in the skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetic/pharmacodynamic (PK/PD) analysis. The results suggest that PK/PD analysis can be conducted by using the human PK profile or tissue (muscle)/plasma concentration ratios and skipping efficiency in DMD-model mice.

Prediction of Human Pharmacokinetics of Phosphorodiamidate Morpholino Oligonucleotides in Duchenne Muscular Dystrophy Patients Using Viltolarsen.

Several modified antisense oligonucleotides (ASOs) have recently been approved for clinical use. Some are phosphorodiamidate morpholino oligomers (PMOs), which, unlike other nucleic acids, are not negatively charged. Thus, PMOs differ from other ASOs in their pharmacokinetic (PK) properties. Drugs with a PMO backbone have been administered to Duchenne muscular dystrophy pediatric patients; however, appropriate methodologies are not currently available to predict their human PK from nonclinical data. In this study, we used viltolarsen as a representative PMO to investigate the applicability of the allometric scaling approach to human PK prediction. We first summarized the nonclinical and clinical PK data for viltolarsen as showing high total clearance, low serum protein binding, metabolic resistance, and urinary excretion as the unchanged drug in both animals and humans. We then investigate the PK of viltolarsen in mice, rats, cynomolgus monkeys, and dogs and used the results, with body weight, to extrapolate to humans by several methods. The estimate of human total clearance obtained from cynomolgus monkeys was the best, and body weight may be the key factor in accurately predicting human total clearance. In contrast, all of the well-known prediction methods for the volume of distribution at steady state gave underestimates. However, the human PK profiles predicted from the PK parameters in cynomolgus monkeys fit the observed human plasma concentrations well. These results are expected to contribute to the further development of PMOs. SIGNIFICANCE STATEMENT: We investigated how to predict the human PK of phosphorodiamidate morpholino oligomers from nonclinical data. The estimates of human PK parameters and profiles determined from cynomolgus monkeys by an allometric scaling approach were the most suitable, and the cynomolgus monkey body weight may be the key factor in accurately predicting human total clearance.

Improved In Vitro-In Vivo Correlation by Using the Unbound-Fraction-Adjusted IC50 for Breast Cancer Resistance Protein Inhibition.

PURPOSE: One function of the blood-brain barrier (BBB) is the efflux of xenobiotics by breast cancer resistance protein (BCRP), and inhibition of BCRP can cause unexpected central nervous system toxicity. Despite the importance of BCRP inhibition and the associated risk of BBB penetration in vivo, there has been little investigation of it to date. In this study, inhibition of BCRP-mediated transport was assessed by in vitro assay in the presence of bovine serum albumin (BSA) to change the unbound inhibitor concentrations, and the in vitro-in vivo correlation (IVIVC) at the BBB was evaluated. METHODS AND RESULTS: The IC50 values of BCRP inhibitors were determined in vitro with and without BSA and the inhibitors were categorized into two groups. One group of compounds had little risk of inhibiting BCRP because of their low unbound concentrations. In contrast, the other group has the potential to facilitate BBB penetration by inhibiting BCRP. In the IVIVC approach, brain concentrations and the brain-to-plasma ratio were better correlated with the ratio of the unbound plasma concentration at steady-state to the unbound-fraction-adjusted IC50. CONCLUSION: We have found a way to obtain a better in vitro-in vivo correlation for BCRP-mediated transport.

Cellular internalization, transcellular transport, and cellular effects of silver nanoparticles in polarized Caco-2 cells following apical or basolateral exposure.

When considering the safety of ingested nanomaterials, it is important to quantitate their transfer across intestinal cells; however, little information exists about the effects of nanomaterial size or exposure side (apical versus basolateral epithelial surface) on nanomaterial transfer. Here, we examined cellular internalization and transcellular transport, and the effects of nanomaterials on Caco-2 monolayers after apical or basolateral exposure to Ag or Au nanoparticles with various sizes. After apical treatment, both internalization and transfer to the basolateral side of the monolayers were greater for smaller Ag nanoparticles than for larger Ag nanoparticles. In contrast, after basolateral treatment, larger Ag nanoparticles were more internalized than smaller Ag nanoparticles, but the transfer to the apical side was greater for smaller Ag nanoparticles. Au nanoparticles showed different rules of internalization and transcellular transport compared with Ag nanoparticles. Furthermore, the paracellular permeability of the Caco-2 monolayers was temporarily increased by Ag nanoparticles (5 µg/mL; diameters, ≤10 nm) following basolateral but not apical exposure. We conclude that the internalization, transfer, and effects of nanomaterials in epithelial cell monolayers depend on the size and composition of nanomaterials, and the exposure side.

A phase 1/2 study of NS-87/CPX-351 (cytarabine and daunorubicin liposome) in Japanese patients with high-risk acute myeloid leukemia.

OBJECTIVES: NS-87/CPX-351 is a dual-drug liposomal encapsulation of cytarabine and daunorubicin. NS-87/CPX-351 exerts antileukemic action by maintaining a synergistic molar ratio of cytarabine to daunorubicin of 5:1 within the liposome while in circulation. Patients with high-risk acute myeloid leukemia (AML), which includes therapy-related AML and AML with myelodysplasia-related changes (AML-MRC), have poorer outcomes than those with other AML. METHODOLOGY: This open-label phase 1/2 (P1/2) study was conducted in 47 Japanese patients aged 60-75 years with newly diagnosed high-risk AML to evaluate the pharmacokinetics, safety, and efficacy of NS-87/CPX-351. RESULTS: In the 6 patients enrolled in the P1 portion, no dose-limiting toxicities (DLTs) were reported, and 100 units/m2 during the induction cycle was found to be acceptable. Cytarabine and daunorubicin had a long half-life in the terminal phase (32.8 and 28.7 h, respectively). In the 35 patients enrolled in the P2 portion, composite complete remission (CRc; defined as complete remission [CR] or CR with incomplete hematologic recovery [CRi]) was achieved in 60.0% (90% CI: 44.7-74.0) of the patients. Adverse events due to NS-87/CPX-351 were well tolerated. OUTCOMES: NS-87/CPX-351 can be considered as a frontline treatment option for Japanese patients with high-risk AML.

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum.

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.

Contribution of Human Liver and Intestinal Carboxylesterases to the Hydrolysis of Selexipag In Vitro.

In liver microsomes, selexipag (NS-304; ACT-293987) mainly undergoes hydrolytic removal of the sulfonamide moiety by carboxylesterase 1 (CES1) to yield the pharmacologically active metabolite MRE-269 (ACT-333679). However, it is not known how much CES in the liver and intestine contributes to the hydrolysis of selexipag or how selexipag is metabolized in the intestine, including by hydrolysis. To obtain a better understanding of selexipag metabolism in humans, we determined the percentage contribution of CES1 and carboxylesterase 2 (CES2) to the hydrolysis of selexipag and 7 of its analogs with different sulfonamide moieties and evaluated its nonhydrolytic metabolism in human liver microsomes and human intestinal microsomes (HIMS). For selexipag, the percentage contributions of CES1 and CES2 in human liver microsomes were 77.0% and 9.99%, respectively, while the percentage contribution of CES2 in HIMS was 100%. In HIMS, the rate of hydrolysis of selexipag was the lowest among the compounds tested, and no difference between the presence and absence of nicotinamide adenine dinucleotide phosphate was noted. We infer from these results that selexipag is likely to be hydrolyzed by CES2 as well as CES1, and only selexipag itself and the MRE-269 produced by hydrolysis in the intestine would be absorbed after oral administration.

Gene expression differences in the duodenum of 129/Sv and DBA/2 mice compared with that of C57BL/6J mice.

We compared the cadmium (Cd) concentration in the liver and kidney of different strains of mice after exposure to 50 ppm Cd for 30 days via drinking water. Cd concentration in the liver and kidney of C57BL/6J mice were higher than those of 129/Sv and DBA/2 mice. Since orally ingested heavy metals are absorbed in the small intestine and then widely distributed to target tissues, microarray analyses were performed to compare the expression levels of transport-related genes in the duodenum between C57BL/6J mice and 129/Sv or DBA/2 mice. The expression levels of 9 and 11 genes were elevated more than 2.0-fold and 13 and 12 genes were reduced less than 0.5-fold in 129/Sv mice and DBA/2 mice, respectively. Among these low expressed genes, 10 genes (Slc2a2, Slc5a1, Slc16a2, Slc22a13, Slc22a18, Slc25a11, Slc36a1, Slco6c1, Abca3 and Abcd1) were common between the two types of strains. These results suggest that some of those genes might be involved in Cd absorption and its toxicity.

Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro.

Because of their useful chemical and physical properties, nanomaterials are widely used around the world - for example, as additives in food and medicines - and such uses are expected to become more prevalent in the future. Therefore, collecting information about the effects of nanomaterials on metabolic enzymes is important. Here, we examined the effects of amorphous silica particles with various sizes and surface modifications on cytochrome P450 3A4 (CYP3A4) activity by means of two different in vitro assays. Silica nanoparticles with diameters of 30 and 70 nm (nSP30 and nSP70, respectively) tended to inhibit CYP3A4 activity in human liver microsomes (HLMs), but the inhibitory activity of both types of nanoparticles was decreased by carboxyl modification. In contrast, amine-modified nSP70 activated CYP3A4 activity. In HepG2 cells, nSP30 inhibited CYP3A4 activity more strongly than the larger silica particles did. Taken together, these results suggest that the size and surface characteristics of the silica particles determined their effects on CYP3A4 activity and that it may be possible to develop silica particles that do not have undesirable effects on metabolic enzymes by altering their size and surface characteristics.

DNA microarray analysis of hepatic gene expression in mice exposed to cadmium for 30 days.

Although cadmium causes hepatotoxicity, its molecular mechanism is unclear. In the present study, transcriptional responses in the liver of C57BL/6J mice given 50 ppm cadmium as a drinking water for 30 days were evaluated with DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were not elevated following the administration of cadmium. Cadmium increased the expressions of 2 genes and reduced those of 15 genes in the liver of mice before the leading to hepatotoxicity.