Nanosuspension formulations of poorly water-soluble compounds for intravenous administration in exploratory toxicity studies: in vitro and in vivo evaluation.

This study was conducted to investigate the use of a nanosuspension for intravenous injection into dogs to increase exposure without toxic additives for preclinical studies in the discovery stage. Nanosuspensions were prepared with a mixer mill and zirconia beads with a vehicle of 2% (w/v) poloxamer 338, which was confirmed to lead to no histamine release in dogs. Sterilized nanosuspensions of poorly water-soluble compounds, cilostazol (Cil), spironolactone (Spi) and probucol (Pro), at 10 mg ml(-1) were obtained by milling for 30 min, followed by autoclaving for 20 min at 121 °C and milling for 30 min (mill-autoclave-mill method). The particle sizes (d50) of Cil, Spi and Pro were 0.554, 0.484 and 0.377 µm, respectively, and the percentages of the nominal concentration were 79.1%, 99.6% and 75.4%, respectively. In chromatographic data, no extra peaks were observed. The particle size of Cil was 0.564 µm after storage for 16 days at 2-8 °C. Cil in nanosuspension, but not in microsuspension, rapidly dissolved in dog plasma. Cil nanosuspension at 0.4 mg kg(-1) and Cil saline solution at 0.03 mg kg(-1) , around the saturation solubility, were intravenously administered to dogs. Nanosuspension increased exposure. The versatility of the mill-autoclave-mill method was checked for 15 compounds, and the particle size of 12 compounds was in the nano range. The nanosuspension optimized in this study may be useful for intravenous toxicological and pharmacological studies in the early stage of drug development. Copyright © 2016 John Wiley & Sons, Ltd.

Practical method for preparing nanosuspension formulations for toxicology studies in the discovery stage: formulation optimization and in vitro/in vivo evaluation of nanosized poorly water-soluble compounds.

The present study aimed to develop a practical method for preparing nanosuspension formulations of poorly water-soluble compounds for enhancing oral absorption in toxicology studies in the discovery stage. To obtain a suitable nanosuspension formulation for the intended purpose, formulations were optimized with a focus on the following characteristics: i) containing a high drug concentration, ii) consisting of commonly used excipient types in proper quantities for toxicology studies, iii) having long-term stability, and iv) having versatility for use with diverse compounds. Test compounds were milled with various excipients by wet media milling methods using a mixer mill (10 mg/batch) and a rotation/revolution mixer (0.5 g/batch). As a result, 100 mg/mL nanosuspensions of all 11 test compounds could be prepared with an optimized dispersing agent, 0.5% hydroxypropyl methylcellulose (HPMC) (3 cP)-0.5% Tween 80. Notably, it was found that the molecular weight of HPMC influenced not only particle size but also the stability of nanosuspensions and they were stable for 4 weeks at 5°C. The nanosuspensions increased in vitro dissolution rates and provided 3.9 and 3.0 times higher Cmax and 4.4 and 1.6 times higher area under the concentration-time curve from 0-24 h (AUC0-24 h) in rats (oral dose of 300 mg/kg) for cilostazol and danazol, respectively. In conclusion, applying a wet media milling method with the combination of HPMC of a small molecular weight and Tween 80 as a dispersing agent, nanosuspensions can be practically prepared and conveniently utilized for enhancing the oral absorption of poorly water-soluble compounds in toxicology studies in the discovery stage.

Hematological and morphological investigation of thrombogenic mechanisms in the lungs of phenylhydrazine-treated rats.

Abnormality in hematological condition including hemolytic disorders has been suggested one of the risk factor of pulmonary thrombosis. We previously reported that phenylhydrazine (PHZ) could induce acute thrombosis in the rat lung. In this study, time-related hematological and histopathological changes were evaluated in PHZ-treated rats to reveal the pathogenesis of pulmonary thrombosis in hemolytic condition. Male Sprague-Dawley rats were administered PHZ at 40 mg/kg/day daily for up to 4 days (n=6). At 24 h after the last administration (i.e. on days 1, 2, 3, or 4), animals were euthanized and samples were subjected to hematology, light microscopy, and electron microscopy. PHZ-treated rats developed severe anemia on day 1 or later. On day 2 and after, congestion in the alveolar septa corresponding to accumulation of deformed/ghost erythrocytes in the alveolar capillaries was observed, which was the earliest change that preceded thrombus formation. Focal fibrin deposition in the alveolar septa was noted on day 3 and it expanded widely by day 4, while endothelial injury were minimally noted just on day 4. These congestive/thrombotic changes were predominant in the pulmonary capillaries. Changes in hemostatic parameters were noted only on day 4; which were prolonged prothrombin time and activated partial thromboplastin time, greatly increased plasma thrombin-antithrombin complex levels with statistical significance, and slightly decreased fibrinogen levels. In conclusion, the trigger of acute pulmonary thrombosis in PHZ-treated rats was considered to be regional stasis resulting from blockage caused by the deformed erythrocytes, and subsequent systemic hemostatic disruption.

The suitability of rat hepatoma cell line H4IIE for evaluating the potentials of compounds to induce CYP3A23 expression.

To investigate the suitability of H4IIE cells for detecting cytochrome P450 (CYP) induction in vitro, we compared CYP induction by typical CYP inducers in H4IIE cells and rat primary hepatocytes by examining gene expression and enzyme activity, and by immunocytochemistry. The cells were preincubated with 0.1 µM of dexamethasone (DEX) for 24 h, followed by 48 h of exposure to 10 µM of beta-naphthoflavone (bNF), 100 µM of phenobarbital (PB) and 10 µM of DEX. Cyp1a1, Cyp2b1/2 and Cyp3a23/3a1 (Cyp3a23) expressions in H4IIE cells were up-regulated 280-, 1.5- and 65-fold relative to those in vehicle-treated cells, respectively. The fold inductions of those expressions in rat primary hepatocytes were 80-, 33- and 152-fold, respectively. Comprehensive gene expression analysis using DNA microarrays showed that Cyp3a23, Gsta2, Ugt2b12, Udpgt and Sult2a1 expressions were up-regulated in H4IIE cells exposed to 10 µM of DEX. CYP3A activity was not increased, but some H4IIE cells exposed to DEX were stained strongly with anti-CYP3A antibody. We cloned these cells and obtained cloned H4IIE (cH4IIE) cells with expression level of Cyp3a23 higher than those of vehicle-treated cells. It was confirmed that preincubation with 0.1 µM of DEX increased pregnane X receptor (Pxr) expression level and enhanced the Cyp3a23 induction effects of test compounds significantly. Retrospective examination of in vitro CYP induction assay using cH4IIE cells resulted in 80% correlation with the data from in vivo rat toxicity studies. These results suggested that cH4IIE cells are suitable for evaluating the potentials of a compound to induce CYP3A23 expression.

In vitro assay for drug-induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis.

To evaluate new drugs' potential for hepatosteatosis, we developed a cell-based assay using a fluorescent fatty acid analog: BODIPY558/568 C12. Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C12. The formation of fluorscecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C12 was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down-regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up-regulated by peroxisome proliferator-activated receptor-alpha agonists: CFR and VPA. From these findings, we concluded that the cell-based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis.

Cell-based fluorescence assay for evaluation of new-drugs potential for phospholipidosis in an early stage of drug development.

To evaluate new-drugs potential for phospholipidosis (PL), we developed a cell-based fluorescence assay using a fluorescent-labeled phospholipid analogue (NBD-PE). CHL/IU cells derived from newborn hamster lung were exposed to positive reference compounds (amiodarone, imipramine, chloroquine, propranolol, chlorpromazine and amantadine) in the presence of NBD-PE, and the level of PL, as indicated by accumulation of fluorescent inclusions in the cytoplasm, was evaluated using fluorescence microscopy and fluorometry. All positive reference compounds induced accumulation of fluorescent inclusions in a concentration-dependent manner with an increase in fluorescence intensity. Fluorescence microscopically, the positive dose of test compound was determined as the concentration with a grade equivalent to or above that of 3.13 microM of amiodarone. Based on this criterion, 8 of 20 test compounds including PL-positive or -negative compounds were judged positive that were concurrent with the pathological results from rat toxicity studies. Furthermore, a positive criterion for fluorometry was decided as equivalent to or above 25% of maximum intensity induced by 1.56-25.0 microM amiodarone. In comparison of fluorometry methods with fluorescence microscopy method, 19 of 20 compounds were judged same. From these findings, we concluded that the assay developed in this study is a rapid and reliable method to predict new-drugs potential for PL at an early stage of drug development.

Gene expression profiling in streptozotocin treated mouse liver using DNA microarray.

Streptozotocin (SZ) is known to exert toxic effects not only on pancreatic islet beta cells but also on other organs including liver. For analyzing changes in genes expression associated with SZ toxicity, we performed DNA microarray analyses on the liver obtained from SZ-treated mice. Eight-week-old male ICR mice were treated i.p. with 200 mg/kg of SZ, and the blood and liver were taken at 6, 24 and 48 h after the treatment. Labeled cRNA prepared from total RNA of the liver was hybridized to the GeneChip Murine Genome U74A V.2 (Affymetrix). The number of the probe sets, which were clearly up-regulated or down-regulated, were over 100 at 6 and 24h after the SZ-treatment, and it decreased at 48 h after the treatment. Many of the up-regulated genes were categorized into cell cycle/apoptosis related genes, immune/allergy related genes and stress response/xenobiotic metabolism related genes. On the other hand, genes related to glucose, lipid and protein metabolisms were down-regulated. These changes started prior to the elevation of the serum glucose levels, indicating the direct action of SZ on the liver rather than the secondary effect of diabetes. This may be related with the previously reported hepatic changes such as lipid peroxidation, mitochondrial swelling and inhibition of hepatocyte proliferation observed before the development of hyperglycemia.

Morphological and gene expression analysis in mouse primary cultured hepatocytes exposed to streptozotocin.

Streptozotocin (SZ) is known to exert toxic effects not only on pancreatic islet beta cells but also on other organs including the liver. For analyzing direct effects of SZ on hepatocytes, we performed morphological analysis and DNA microarray analysis on mouse primary cultured hepatocytes. Hepatocytes were taken from non-treated Crj:CD-1(ICR) mice. The primary cultured hepatocytes were treated with SZ at concentrations of 0, 1, 3, 10, 30 and 100 mM. After the treatment for about 6 or 24h, cell survival assay using tetrazolium salt (WST-1), light microscopic/electron microscopic analysis and gene expression analysis were performed. For the gene expression analysis, target (labeled cRNA) prepared from total RNA of the hepatocytes was hybridized to the GeneChip Murine Genome U74A V.2 (Affymetrix). The signal intensity calculation and scaling were performed using Microarray Suite Software Ver 5.0. IC50 of the cell survival assay was around 62 mM at 6 h exposure and 7 mM at 24 h exposure. Marked chromatin margination was observed in nuclei of the hepatocytes treated with SZ at concentrations of 3 or 10mM. Gene expression analysis revealed similar expression changes to those of in vivo, i.e. up-regulation in cell proliferation/ apoptosis related genes, and down-regulation of lipid metabolism related genes. These results potently supported the hypothesis that many of the hepatic alteration including histopathological and gene expression changes are induced by direct effect of SZ rather than by the secondary effect of the hyperglycemia or hypoinsulinemia.

Hepatic changes in the acute phase of streptozotocin (SZ)-induced diabetes in mice.

We have reported the streptozotocin (SZ)-induced hepatic lesions in the subacute phase (4 to 12 weeks after the treatment), which are characterized by appearance of oncocytic hepatocytes, cytomegalic hepatocytes and bile duct hyperplasia. In this study, we focused on the acute phase (6 to 48 hours after the treatment) of the SZ-induced hepatic lesions of mice to clarify the onset of the hepatic alterations, especially before the induction of hyperglycemia. Livers were taken from 8-week-old Crj:CD-1 (ICR) male mice at 6, 12, 24, 36 and 48 hours after the 200 mg/kg b.w. of SZ-injection. SZ-induced hyperglycemia was noted at 36 and 48 hours after the treatment, but the hepatic changes including lipid peroxidation, mitochondrial swelling, peroxisome proliferation and inhibition of hepatocyte proliferation occurred before the elevation of the serum glucose levels. The present findings indicate the direct effects of SZ on hepatocytes rather than the secondary effects of diabetes, and certain correlations between the hepatocytic changes in the acute phase and those in the subacute one. In addition, ulcer and submucosal edema of the gallbladder were observed at 36 or 48 hours after the SZ-treatment, which can be a novel finding in SZ-treated animal.

The novel dominant mutation Dspd leads to a severe spermiogenesis defect in mice.

Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.

Na+/Ca2+ exchanger-deficient mice have disorganized myofibrils and swollen mitochondria in cardiomyocytes.

The Na(+)/Ca(2+) exchanger (NCX1) plays a key role in maintaining Ca(2+) homeostasis in cardiomyocytes. Disruption of Ncx1 gene in mice results in embryonic lethality between embryonic day 9 and 10, with the mice lacking spontaneous heartbeats. We examined the mechanism of lack of heartbeats in Ncx1-deficient mice. Ultrastructual analysis demonstrated that Ncx1-deficient mice showed severe disorganization of myofibrils, a lack of Z-lines and swelling of mitochondria in cardiomyocytes. However, the expressions of cardiac-specific genes including transcription factor genes and contractile protein genes were not changed in Ncx1-deficient mice. Abnormal Ca(2+) handling itself or the lack of heartbeats due to the inactivation of Ncx1 gene may cause the disorganization of myofibrillogenesis. Although NCX1 protein levels were decreased in heterozygous mice, there were no changes in NCX2 and NCX3 protein levels between wild type and heterozygous mice.

Tissue-specific regulation of expression and activity of P-glycoprotein in adjuvant arthritis rats.

Cyclosporine A and steroids are effective against rheumatoid arthritis and also known as substrates of P-glycoprotein (P-gp). We investigated the effect of arthritis on the hepatic and intestinal P-gp activity in rats, and substantiated the expression level of the hepatic P-gp. Doxorubicin was used as a P-gp substrate. Cumulative biliary excretion and intestinal exsorption of doxorubicin following intravenous administration were compared between adjuvant arthritis (AA) and normal rats. Intestinal P-gp activity was also investigated by intestinal everted sac method, and hepatic P-gp was detected by FITC-labeled antibody and visualized using a confocal laser microscope system. Biliary clearance of doxorubicin in AA rats was significantly decreased from that in normal rats. The expression level of the hepatic P-gp in AA rats was very low compared to normal rats, indicating down-regulation. Intestinal exsorption clearance was not different between AA and normal rats. Permeability of doxorubicin across intestinal everted sac was comparable between AA and normal rats, corresponding to in vivo study. In AA rats, hepatic P-gp activity was decreased due to the reduction of expression level, but intestinal P-gp activity was not changed. Different regulation systems may be involved in liver and intestine.