In vitro evaluation of dissolution behavior for a colon-specific drug delivery system (CODES) in multi-pH media using United States Pharmacopeia apparatus II and III

United States Pharmacopeia dissolution apparatus II (paddle) and III (reciprocating cylinder) coupled with automatic sampling devices and software were used to develop a testing procedure for acquiring release profiles of colon-specific drug delivery system (CODES^™) drug formulations in multi-pH media using acetaminophen (APAP) as a model drug. System suitability was examined. Several important instrument parameters and formulation variables were evaluated. Release profiles in artificial gastric fluid (pH 1.2), intestinal fluid (pH 6.8), and pH 5.0 buffer were determined. As expected, the percent release of APAP from coated core tablets was highly pH dependent. A release profile exhibiting a negligible release in pH 1.2 and 6.8 buffers followed by a rapid release in pH 5.0 buffer was established. The drug release in pH 5.0 buffer increased significantly with the increase in the dip or paddle speed but was inversely related to the screen mesh observed at lower dip speeds. It was interesting to note that there was a close similarity (f ₂=80.6) between the release profiles at dip speed 5 dpm and paddle speed 100 rpm. In addition, the release rate was reduced significantly with the increase in acid-soluble Eudragit E coating levels, but lactulose loading showed only a negligible effect. In conclusion, the established reciprocating cylinder method at lower agitation rates can give release profiles equivalent to those for the paddle procedure for CODES^™ drug pH-gradient release testing. Apparatus III was demonstrated to be more convenient and efficient than apparatus II by providing various programmable options in sampling times, agitation rates, and medium changes, which suggested that the apparatus II approach has better potential for in vitro evaluation of colon-specific drug delivery systems.     

The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N′-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1 mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5–3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5–3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5–3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.     

Determination of p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C₁₈ column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2 → 120.2 and 152.1 → 110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1–500 μg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.     

Calcium channel blocking agents protect against acetaminophen-induced cytotoxicity in rat hepatocytes

The effect on acetaminophen-induced cytotoxicity of three calcium channel blocking agents-diltiazem, verapamil and gallopamil-was studied in primary cultures of rat hepatocytes and compared with the chelating agent EGTA. Using the measurement of cytosolic lactate dehydrogenase (LDH) as an index of cytotoxicity, it was demonstrated that a 1-hr pretreatment with calcium channel blocking agents protected cells against acetaminophen cytotoxicity, but were less effective than EGTA. These data suggest that influx of extracellular Ca²⁺ into the cells could have a role in the genesis of hepatocyte injury by acetaminophen.     

Acetaminophen attenuates glomerulosclerosis in obese Zucker rats via reactive oxygen species/p38MAPK signaling pathways

Focal segmental glomerulosclerosis is a critical pathological lesion in metabolic syndrome-associated kidney disease that, if allowed to proceed unchecked, can lead to renal failure. However, the exact mechanisms underlying glomerulosclerosis remain unclear, and effective prevention strategies against glomerulosclerosis are currently limited. Herein, we demonstrate that chronic low-dose ingestion of acetaminophen (30 mg/kg/day for 6 months) attenuates proteinuria, glomerulosclerosis, podocyte injury, and inflammation in the obese Zucker rat model of metabolic syndrome. Moreover, acetaminophen treatment attenuated renal fibrosis and the expression of profibrotic factors (fibronectin, connective tissue growth factor, transforming growth factor β), reduced inflammatory cell infiltration into the glomeruli, and decreased the expression of monocyte chemoattractant protein, glutathione (GSH) reductase, and nuclear factor erythroid 2-related factor 2, but increased the level of GSH synthetase in obese animals. Further in vivo and in vitro studies using human renal mesangial cells exposed to high glucose or hydrogen peroxide suggested that the renoprotective effects of acetaminophen are characterized by diminished renal oxidative stress and p38MAPK hyperphosphorylation.     

Catalytic oxidation of acetaminophen by tyrosinase in the presence of L-proline: a kinetic study

A kinetic study of acetaminophen oxidation by tyrosinase in the presence of a physiological nucleophilic agent such as the amino acid L-proline is performed in the present paper. The o-quinone product of the catalytic activity, 4-acetamido-o-benzoquinone, becomes unstable through the chemical addition of L-proline, in competition with the nucleophilic addition of hydroxide ion from water. In both cases, the catechol intermediate, 3(')-hydroxyacetaminophen, is generated, as can be demonstrated by liquid chromatography. When the effect of the presence of the nucleophilic agent on the time course of the enzymatic reaction was kinetically analyzed, it was seen to decrease the duration of the lag period and increase the steady-state rate. Rate constants for the reaction of 4-acetamido-o-benzoquinone with water and L-proline were also determined. The results obtained in this paper open a new possibility to acetaminophen toxicity, that has been attributed hitherto to its corresponding p-quinone, N-acetyl-p-benzoquinone imine.     

High-performance liquid chromatographic assay for acetaminophen glucuronide in human liver microsomes

A rapid and specific high-performance liquid chromatographic assay was developed for the determination of acetaminophen glucuronide formed by human liver microsomes. In addition, incubation conditions were systematically evaluated. Conditions that yielded the optimal rate of acetaminophen glucuronide formation over various concentrations of acetaminophen (0.15–30 mM) consisted of the following: 0.1 M potassium phosphate buffer, 1 mM magnesium chloride, 30 μg/mg alamethicin, 4 mM uridine 5′-diphosphoglucuronic acid at a pH of 7.1. Alamethicin produced higher and more consistent APAPG formation rates compared to Brij-58. Adding saccharolactone to the incubation medium reduced the velocity of the reaction. Acetaminophen glucuronide, acetaminophen, and the internal standard (paraxanthine), were analyzed on a C₁₈ column with UV detection at 250 nm. The mean correlation coefficient (r²) of the standard curves for acetaminophen glucuronide was >0.99 over the range of 0.1–25 nmol. The intra- and inter-day coefficients of variation were <4%. This method is suitable for in vitro studies using acetaminophen glucuronide formation as an index reaction for UGT activity.