Ruy Tchao

Effect of Processing Methods and Heat Treatment on the Formation of Wax Matrix Tablets for Sustained Drug Release

The objective of this study was to evaluate the effects of processing methods and heat treatment on matrix formation and subsequent drug release from wax matrix tablets for controlled release. Phenylpropanolamine hydrochloride (PPA) and Compritol® were processed with appropriate diluent(s) using either dry blending (DB), wet granulation (WG), partial melt granulation (PMG), or melt granulation (MG). Then the tablets were heat-treated at 80°C. Particle size distribution and compressibility, along with drug release, tablet micro-morphology, wettability, porosity, and tortuosity were investigated. The drug release was different for the four processing methods even though the tablet formulation was identical. Heat treatment further retarded drug release and its effect was related to the previous manufacturing processes. Scanning Electron Microscopy (SEM) showed that heat treatment redistributed the wax and formed a film-like structure covering drug and excipients. The contact angle of tablets made from DB, WG, and PMG methods increased after heat treatment, while that of tablets made from DB, WG, and PMG methods increased after heat treatment, while that of tablets made from MG remained constant. Tablet tortuosity calculated from drug release rate constants increased dramatically after heat treatment. Drug release from the wax tablets with or without heat treatment was best described by the Higuchi equation. Different processing methods produced different matrix structures that resulted in different drug release rates. Heat treatment retarded drug release mainly by increasing tortuosity of the matrix. Contact angle measurement and SEM analysis indicated that heat treatment caused the wax to melt, redistribute, coat the drug and diluents, and form a network structure.

Effect of antioxidants on para-aminophenol-induced toxicity in LLC-PK1 cells.

The present studies were designed to investigate the susceptibility of LLC-PK1 cells to cytotoxicity induced by para-aminophenol (PAP) and the ability of antioxidants to prevent PAP-induced cytotoxicity. LLC-PK1 cells were incubated for 4 h with varying concentrations of PAP (0-0.2 mM). Incubation was continued for 20 h and viability was monitored at 24 h after initial exposure to PAP. For coincubation experiments, cells were incubated for 4 h with various antioxidants [including ascorbate, glutathione (GSH), butylated hydroxytoluene (BHT), beta-nicotinamide adenine dinucleotide (NADH), or beta-nicotinamide adenine dinucleotide phosphate (NADPH)] in the absence or presence of 0.1 mM PAP. For preincubation experiments, cells were incubated for 1 h with ascorbate, GSH or NADPH. Antioxidants were removed and cells were exposed to 0 or 0.1 mM PAP for 4 h. Viability was determined 24 h following PAP exposure. LLC-PK1 cells displayed a steep concentration-response relationship for PAP; 0.1 mM PAP caused approximately 50% loss of viability. Coincubation with ascorbate, GSH and NADPH was without effect on cell viability in the absence of PAP and attenuated PAP-induced losses in viability. In contrast, NADH was ineffective in preventing PAP-induced cytotoxicity. BHT alone produced a significant loss of cell viability and was ineffective in preventing PAP cytotoxicity. Inability of NADH to prevent PAP-induced cytotoxicity was related to rapid degradation of NADH in aqueous solution. Preincubation of cells with ascorbate or GSH but not NADPH was associated with attenuation of PAP-induced cytotoxicity. These data suggest that (1) PAP is cytotoxic to LLC-PK1 cells, (2) a portion of PAP cytotoxicity is due to nonenzymatic oxidation that occurs in the incubation medium, and (3) a portion of PAP cytotoxicity is due to enzymatic or nonenzymatic oxidation that occurs within cells.

Nephrotoxic and hepatotoxic potential of imidazolidinedione-, oxazolidinedione- and thiazolidinedione-containing analogues of N-(3,5-dichlorophenyl)succinimide (NDPS) in Fischer 344 rats ☆

Nephrotoxicity of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) in rats is believed to involve metabolism on the succinimide ring. To further investigate this hypothesis, we synthesized and tested the following NDPS analogues, which contain other cyclic imide rings and may therefore be metabolized differently than NDPS: 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlorophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-dichlorophenyl)-1-methyl-2,4-imidazolidinedione (DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Male Fischer 344 rats were administered DCPO, DCPI, DCPM, DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS (0.6 mmol/kg, i.p. in corn oil) or corn oil (4 ml/kg). As evidenced by diuresis, proteinuria, elevated blood urea nitrogen levels, increased kidney weights and proximal tubular damage, NDPS produced severe nephrotoxicity in the rats. In contrast, DCPO, DCPI, DCPM and DCPT were mild nephrotoxicants. None of the compounds elevated serum alanine transferase activity or liver weights in the rats, however DCPT produced centrilobular necrosis. These experiments confirm that NDPS-induced nephrotoxicity is critically dependent on the presence of the succinimide ring. Furthermore, replacement of the succinimide ring with a thiazolidinedione ring produced a more pronounced effect on the liver than on the kidney. Liver damage has been reported in type II diabetic patients taking troglitazone, rosiglitazone and pioglitazone. Since these compounds also contain a thiazolidinedione ring, DCPT may be useful for investigating the role of this structural feature in hepatotoxicity.

The effect of aromatic fluorine substitution on the nephrotoxicity and metabolism of N-(3,5-dichlorophenyl)succinimide in fischer 344 rats

N-(3,5-Difluorophenyl)succinimide (DFPS) is a non-toxic analogue of the nephrotoxic fungicide N-(3,5-dichlorophenyl)succinimide (NDPS). Although NDPS must be metabolized to produce renal damage, the metabolic fate of DFPS is unknown. These studies were therefore designed to examine the nephrotoxic potential of putative DFPS metabolites and to determine if DFPS is metabolized differently from NDPS. Male Fischer-344 rats were administered (1.0 mmol/kg. i.p. in corn oil) DFPS, N-(3,5-difluorophenyl)succinamic acid (DFPSA), N-(3,5-difluorophenyl)-2-hydroxysuccinimide (DFHS), N-(3,5-difluorophenyl)-2- or -3-hydroxysuccinamic acids (2- and 3-DFHSA, respectively), N-(3,5-difluoro-4-hydroxyphenyl)succinimide (DFHPS). N-(3,5-difluoro-4-hydroxyphenyl) succinamic acid (DFHPSA) or corn oil only (1.2 ml/kg). Although some of the compounds produced changes in renal function and histology, these alterations were not indicative of irreversible kidney damage. DFPSA, 2-DFHSA, 3-DFHSA and DFHPSA were detected in the urine of rats 3 h after administration of 0.2 mmol/kg [14C]DFPS. The same metabolites were produced by isolated rat hepatocytes, but not by renal proximal tubule cells. Formation of the oxidative metabolites in vitro was prevented by the cytochrome P450 inhibitor 1-aminobenzotriazole. It appears that DFPS undergoes hepatic biotransformation similar to NDPS and that some of its metabolites have reversible effects on renal proximal tubules.

Effect of structural modifications on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats.

Glitazones, used for type II diabetes, have been associated with liver damage in humans. A structural feature known as a 2,4‐thiazolidinedione (TZD) ring may contribute to this toxicity. TZD rings are of interest since continued human exposure via the glitazones and various prototype drugs is possible. Previously, we found that 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT) was hepatotoxic in rats. To evaluate the importance of structure on DCPT toxicity, we therefore studied two series of analogs. The TZD ring was replaced with: a mercaptoacetic acid group {[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]acetic acid, DCTA}; a methylated TZD ring [3‐(3,5‐dichlorophenyl)‐5‐methyl‐2,4‐thiazolidinedione, DPMT]; and isomeric thiazolidinone rings [3‐(3,5‐dichlorophenyl)‐2‐ and 3‐(3,5‐dichlorophenyl)‐4‐thiazolidinone, 2‐DCTD and 4‐DCTD, respectively]. The following phenyl ring‐modified analogs were also tested: 3‐phenyl‐, 3‐(4‐chlorophenyl)‐, 3‐(3,5‐dimethylphenyl)‐ and 3‐[3,5‐bis(trifluoromethyl)phenyl]‐2,4‐thiazolidinedione (PTZD, CPTD, DMPT and DFMPT, respectively). Toxicity was assessed in male Fischer 344 rats 24 h after administration of the compounds. In the TZD series only DPMT produced liver damage, as evidenced by elevated serum alanine aminotransferase (ALT) activities at 0.6 and 1.0 mmol kg−1 (298.6 ± 176.1 and 327.3 ± 102.9 Sigma‐Frankel units ml−1, respectively) vs corn oil controls (36.0 ± 11.3) and morphological changes in liver sections. Among the phenyl analogs, hepatotoxicity was observed in rats administered PTZD, CPTD and DMPT; with ALT values of 1196.2 ± 133.6, 1622.5 ± 218.5 and 2071.9 ± 217.8, respectively (1.0 mmol kg−1 doses). Morphological examination revealed severe hepatic necrosis in these animals. Our results suggest that hepatotoxicity of these compounds is critically dependent on the presence of a TZD ring and also the phenyl substituents. Copyright

Effect of gender, dose, and time on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT)-induced hepatotoxicity in Fischer 344 rats.

1. The thiazolidinedione ring present in drugs available for type II diabetes can contribute to hepatic injury. Another thiazolidinedione ring-containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazoli-dinedione (DCPT), produces liver damage in rats. Accordingly, the effects of gender, dose, and time on DCPT hepatotoxicity were therefore evaluated. 2. Male rats were more sensitive to DCPT (0.4–1.0 mmol kg−1 by intraperitoneal administration) as shown by increased serum alanine aminotransferase levels and altered hepatic morphology 24 h post-dosing. Effects in both genders were dose dependent. In males, DCPT (0.6 mmol kg−1) produced elevations in alanine aminotransferases and changes in liver h after dosing that progressively worsened up to 12 h. DCPT-induced renal effects were mild. 3. It is concluded that male rats are more susceptible to DCPT hepatotoxicity and that damage occurs rapidly. DCPT primarily affects the liver and can be a useful compound to investigate the role of the thiazolidinedione ring in hepatic injury. However, the gender dependency and rapid onset of DCPT hepatotoxicity require further investigation.

Role of biotransformation in 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats

Cytochrome P450 (CYP)-mediated metabolism in the thiazolidinedione (TZD) ring may contribute to the hepatotoxicity of the insulin-sensitizing agents such as troglitazone. We were interested in determining if biotransformation could also be a factor in the liver damage associated with another TZD ring containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Therefore, hepatotoxic doses of DCPT (0.6 or 1.0 mmol/kg, i.p.) were administered to male Fischer 344 rats after pretreatment with vehicle, 1-aminobenzotriazole (ABT, non-selective CYP inhibitor) and troleandomycin (TAO, CYP3A inhibitor). Alternatively, rats were pretreated with vehicle or the CYP3A inducer dexamethasone (DEX) prior to a non-toxic DCPT dose (0.2 mmol/kg, i.p.). Vehicle-, ABT-, TAO- and DEX-only control groups were also run. Toxicity was assessed 24 h after DCPT administration. Both hepatotoxic doses of DCPT induced elevations in serum alanine aminotransferase (ALT) levels that were attenuated by ABT or TAO pretreatment. Liver sections from rats that received vehicle+DCPT revealed areas of gross necrosis and neutrophil invasion, whereas sections from ABT+DCPT and TAO+DCPT rats showed minor changes compared to controls. DEX pretreatment potentiated ALT levels associated with the non-toxic DCPT dose. Furthermore, DEX+DCPT rat liver sections exhibited hepatic injury when compared against rats that received vehicle+DCPT. Blood urea nitrogen levels, urinalysis and kidney morphology were not markedly altered by any combination of pretreatments or treatments. Enzyme activity and Western blotting experiments with rat liver microsomes confirmed the effects of the various pretreatments. Our results suggest that hepatic CYP3A isozymes may be involved in DCPT-induced liver damage in male rats. We believe this is the first report demonstrating that modulation of the biotransformation of a TZD ring-containing compound can alter hepatotoxicity in a common animal model.

Nephrotoxic potential of N-(3,5-dichlorophenyl)glutarimide and N-(3,5-dichlorophenyl)glutaramic acid in Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) produces kidney damage in rats. Although many NDPS analogues have been screened as possible nephrotoxicants, the one-carbon homologue, N-(3,5-dichlorophenyl)glutarimide (NDPG), has not been evaluated. This study examined the nephrotoxic potential of NDPG and a putative metabolite, N-(3,5-dichlorophenyl)glutaramic acid (NDPGA). Male Fischer 344 rats (N = 3-4 per group) were administered a single i.p. injection in corn oil of NDPG or NDPGA (0.4 or 1.0 mmol/kg), NDPS (0.4 mmol/kg), or corn oil alone. Renal function was monitored for 48 h. In contrast to NDPS, NDPG and NDPGA did not significantly alter renal function or kidney morphology when compared to corn oil-treated controls. These experiments show that replacement of the succinimide ring in NDPS with a glutarimide ring abolishes toxicity.

Epithelial cell interaction in air-liquid interface culture

SummaryA novel culture method has been developed to study the interaction of epithelial cells in the absence of a solid substratum. Starting with either a single cell suspension or aggregates, cells were floated at the interface of air and liquid culture medium. Two epithelial cell lines have been studied in this system: Madin-Darby canine kidney cells (MDCK), and a rat bladder tumor cell line (NBT-II). Starting with a single cell suspension of MDCK, the floating cells coalesced in 24 h into sheets of cells. The cells were morphologically polarized with the apical surface facing the liquid medium. Domes were observed regularly in these sheets of cells. NBT-II cells migrated actively from aggregates at the air-liquid interface. In this floating culture, NBT-II cells produced extensive cell processes similar to those seen in cells grown on a solid surface. Because cells at the air-liquid interface lack a solid substratum for adhesion, cell membrane processes such as lamellapodia, retraction fibers, pseudopods, and long, intercellular connections can only exert a tension equal to or less than the surface tension of the liquid. Dimethyl sulfoxide 2% stimulated desmosome formation in floating NBT-II cells, resulting in a cribriform pattern in the sheet of cells. This method of interface can lead to new understanding of morphogenesis of epithelial cells, and the mechanism, of cell motility and formation of cell processess.

LOCALIZATION OF CYP2D16 IN THE GUINEA PIG ADRENAL CORTEX BY IMMUNOHISTOCHEMISTRY AND IN SITU HYBRIDIZATION

Recent reports indicate that the cytochrome P450 isozyme, CYP2D16, is expressed at high levels in the inner regions of the guinea pig adrenal cortex and may contribute to xenobiotic and/or steroid metabolism in the gland. In the present studies, immunohistochemical and in situ hybridization techniques were employed to definitively establish the localization of CYP2D16 within the adrenal cortex. In male guinea pigs of various ages, CYP2D16 protein and mRNA were highly localized to the zona reticularis (ZR); none was detectable in the zona fasciculata (ZF), zona glomerulosa (ZG) or the medulla. In contrast, the steroidogenic P450 isozyme, CYP17, was distributed throughout the ZF and ZR. From the earliest stages of development of the ZR, CYP2D16 staining was intense. As guinea pigs aged, the ZR progressively enlarged and comprised a proportionately greater amount of the cortex. At all ages, CYP2D16 was uniformly distributed throughout only the ZR. Coinciding with the age-related growth of the ZR and increase in adrenal CYP2D16 content was an increase in adrenal xenobiotic-metabolizing activity. The results establish that CYP2D16 has an intraadrenal localization that is unique among P450 isozymes, suggesting novel regulatory mechanisms and indicating that CYP2D16 may serve as a specific marker for ZR cells. The increase in CYP2D16 expression with age probably accounts for increasing levels of xenobiotic metabolism and may also contribute to an increase in intraadrenal cortisol degradation in older animals.