Effects of cysteine on the pharmacokinetics of intravenous clarithromycin in rats with protein-calorie malnutrition

Effects of cysteine on the pharmacokinetics of clarithromycin were investigated after intravenous administration of the drug at a dose of 20 mg/kg to control rats (4-week fed on 23% casein diet) and rats with PCM (protein-calorie malnutrition, 4-week fed on 5% casein diet) and PCMC (PCM treated with 250 mg/kg for oral cysteine twice daily during the fourth week). Clarithromycin has been reported to be metabolized via hepatic microsomal cytochrome P450 (CYP) 3A4 to 14-hydroxyclarithromycin (primary metabolite of clarithromycin) in human subjects. It has also been reported that in rats with PCM, CYP3A23 level decreased to 40-50% of control level, but decreased CYP3A23 level in rats with PCM completely returned to control level by oral cysteine supplementation (rats with PCMC). Human CYP3A4 and rat CYP3A23 proteins have 73% homology. In rats with PCM, the area under the plasma concentration-time curve from time zero to time infinity, AUC (567, 853 and 558 microg min/ml for control rats and rats with PCM and PCMC, respectively) and percentage of clarithromycin remaining after incubation with liver homogenate (69.6, 83.9 and 71.7%) were significantly greater than those in control rats and rats with PCMC. Moreover, in rats with PCM, the total body clearance, CL (35.3, 23.4 and 35.8 ml/min/kg), nonrenal clearance, CL(NR) (21.3, 15.2 and 24.1 ml/min/kg) and maximum velocity for the disappearance of clarithromycin after incubation with hepatic microsomal fraction, V(max) (351, 211 and 372 pmol/min/mg protein) were significantly slower than those in control rats and rats with PCMC. However, above mentioned each parameter was not significantly different between control rats and rats with PCMC. The above data suggested that metabolism of clarithromycin decreased significantly in rats with PCM as compared to control due to significantly decreased level of CYP3A23 in the rats. By cysteine supplementation (rats with PCMC), some pharmacokinetic parameters of clarithromycin (AUC, CL, CL(NR) and V(max)) were restored fully to control levels because CYP3A23 level was completely returned to control level in rats with PCMC.     

Influence of 4-week and 8-week exercise training on the pharmacokinetics and pharmacodynamics of intravenous and oral azosemide in rats

Cytochrome P450 expression was determined in the livers of control, 4-week exercised (4WE) and 8-week exercised (8WE) rats. Even though the 4-week and 8-week exercise training caused 53 and 25% increases, respectively, in total cytochrome P450 contents in the liver, exercise training did not cause any changes in the levels of P450 1A2 (which primarily metabolizes azosemide), 2E1 and 3A23 in the liver, as assessed by both Western and Northern blot analyses. Also, exercise training failed to alter the activity of NADPH-dependent cytochrome P450 reductase. The plasma concentrations of norepinephrine and epinephrine were significantly (2 to 3 folds) higher in 4WE rats than in controls, presumably due to physical stress, but the catecholamine levels in 8 WE rats returned to control levels. After intravenous administration (10 mg/kg of azosemide), the amount of unchanged azosemide excreted in 8-h urine (Ae(Azo, 0-8 h)) was significantly greater (46% increase) in 4WE rats than that in control rats. This resulted in a significantly faster (82% increase) renal clearance of azosemide. However, the nonrenal clearances were not significantly different between control and 4WE rats. The significantly greater Ae(Azo, 0-8 h) in 4WE rats was mainly due to a significant increase in intrinsic active secretion of azosemide in renal tubules and not due to a decrease in the metabolism of azosemide. After oral administration (20 mg/kg), Ae(Azo, 0-8 h) was also significantly greater (264%) in 4WE rats and this again was due to a significant increase in intrinsic active renal secretion of azosemide and not due to an increase in gastrointestinal absorption. After both intravenous and oral administration, the 8-h urine output was not significantly different between control and 4WE rats although Ae(Azo, 0-8 h) increased significantly in 4WE rats. This could be due to the fact that the urine output reached a plateau at 10 mg/kg after intravenous administration and 20 mg/kg after oral administration of azosemide to rats and possibly due to increase in plasma antidiuretic hormone levels and aldosterone production in 4WE rats.     

Effects of cysteine on amino acid concentrations and transsulfuration enzyme activities in rat liver with protein-calorie malnutrition

The changes in amino acid concentrations and transsulfuration enzyme activities in liver were investigated after 4-week fed on 23% casein diet (control group) and 5% casein diet without (protein-calorie malnutrition, PCM group) or with (PCMC group) oral administration of cysteine, 250 mg/kg (twice daily, starting from the fourth week) using rats as an animal model. By supplementation with cysteine in PCM rats (PCMC group), cysteine level was elevated almost close to the control level, and glutathione (GSH), aspartic acid and serine levels were restored greater than the control levels. The measurement of transsulfuration enzyme activities exhibited that gamma-glutamylcysteine ligase (gamma-GCL) activity was up-regulated in rats with protein restriction (PCM group), and cysteine supplementation (PCMC group) down-regulated to the control level. One-week supplementation of cysteine (PCMC group) significantly down-regulated the cysteine sulfinate decarboxylase activity. These results indicate that the availability of sulfur amino acid(s) especially cysteine appears to play a role in determining the flux of cysteine between cysteine catabolism and GSH synthesis.     

Effects of protein-calorie malnutrition during suckling and post-weaning periods on discontinuous cranial traits in rats

The influences of protein-calorie malnutrition (PCM), and sex during lactation and post-lactation on the frequencies of 25 discontinuous cranial traits (DCT), were investigated in Holtzman rats. Significant differences were observed in about 20% of the traits. Those traits were: the interfrontal fusion, the posterior incurvation of the palatine border, the double maxillary foramen, the double posterior palatine foramen, and the double frontal foramen. Total PCM was the nutritional factor which showed the greatest influence on the variability of the DCTs. It was followed, in decreasing order, by the PCM imposed during post-lactation and lactation. Sex had more influence than early PCM but less than late PCM. It is concluded that despite their apparent stability, a substantial number of DCTs were altered by both biological (like sex) and environmental factors (like nutritional deficiencies) imposed at different stages of postnatal development.     

Morphology of the mammotrophs and gonadotrophs in the anterior pituitary gland of rats with protein-calorie malnutrition

The structure of the anterior pituitary gland of rats with protein-calorie malnutrition was studied at the light and electron microscopic levels. Male Sprague-Dawley rats were fed a low-protein diet from 20 to 50 days of age. The hypophyses from these animals, along with those from age-matched controls, were then removed and fixed for light microscopic immunocytochemical analyses, using anti-rat PRL or anti-ovine LH beta serum, or for routine ultrastructural study. The overall number of mammotrophs and gonadotrophs appeared to be unaffected by the deficiency in dietary protein. However, the nuclei, in both cell types, were significantly smaller in the malnourished rats. Cytoplasmic volume was also apparently less than in control rats, leading to the "crowding" of adjacent cell types. Additionally, in the malnourished rats, the mammotrophs assumed a less polygonal profile, while the gonadotrophs, which were more irregularly shaped in the central zone, rarely displayed the negative image of a Golgi complex. Ultrastructurally, the mammotrophs in the experimental rats had a less extensively developed granular endoplasmic reticulum and Golgi complex. The number of secretary granules was not different; however, their size was markedly less and their shape was round to ovoid, rather than pleomorphic. Two types of gonadotrophs were distinguished, based on the classification of Kurosumi et al. ('76). The type-I cell (former "FSH" cell) had a smaller Golgi complex, less vesicular endoplasmic reticulum and smaller secretory granules. The major alteration in the type-II gonadotroph (former "LH" cell) was the smaller size of the secretory granules. The morphological changes found in this study support our previous physiological observations (Herbert, '80), which demonstrate that prolactin and gonadotrophin secretion are severely impaired in rats suffering from protein-calorie malnutrition.     

Effects of oral dosing paradigms (gavage versus diet) on pharmacokinetics and pharmacodynamics

In cancer chemopreventive studies, test agents are typically administered via diet, while the preclinical safety studies normally employ oral gavage dosing. Correspondence in pharmacokinetic and pharmacodynamic profiles between the two dosing approaches cannot be assumed a priori. Sulindac, a non-steroidal anti-inflammatory agent with potential chemopreventive activity, was used to assess effects of the two oral dosing paradigms on its pharmacokinetics and pharmacodynamics. Time-dependent concentrations of sulindac and its sulfone metabolite were determined in plasma and potential target organ, mammary gland. Prostaglandin E(2) was used as a pharmacodynamic biomarker and measured in mammary gland. An inverse linear relationship was detected between pharmacodynamic and pharmacokinetic markers, area under the curve for prostaglandin E(2) levels and sulindac sulfone concentrations, respectively, in the mammary tissue. Marked differences in pharmacokinetics and pharmacodynamics were observed after administration of sulindac by the two oral dosing paradigms. In general, oral gavage resulted in higher peak and lower trough concentrations of sulindac in plasma and mammary tissue, higher area under concentration-time curve in plasma and mammary tissue, and greater effect on prostaglandin E(2) levels than the corresponding diet dosing. This study illustrates potential pitfalls and limitations in trying to generalize based on data obtained with different oral dosing schemes and their extrapolation to potential efficacy and health risks in humans.     

Changes at the liver cell level in rats subjected to protein-calorie malnutrition

The effect of a low protein-calorie diet (restricted diet) on cellular growth and on RNA metabolism in Wistar rat liver has been studied. Experimentation was carried out over 30 days and the comparisons were made against well-nourished group (10% protein, controls). Liver weight and hepatic proteins dropped significantly in malnourished rats. Both rate of DNA and number of nuclei were unchanged. However, protein/DNA and liver weight/number of nuclei ratios decreased, which led to an atrophy phenomenon. DNase specific activity however, was not modified. Liver RNA content together with RNase activity dropped in deficient rats. Protein synthesis capacity (RNA/protein) did not change. These results suggest that restricted diet leads to a lower hepatocyte size with a decreased rat of RNA turnover.     

Ventricular remodeling and diastolic myocardial dysfunction in rats submitted to protein-calorie malnutrition

The effects of protein-calorie malnutrition (PCM) on heart structure and function are not completely understood. We studied heart morphometric, functional, and biochemical characteristics in undernourished young Wistar rats. They were submitted to PCM from birth (undernourished group, UG). After 10 wk, left ventricle function was studied using a Langendorff preparation. The results were compared with age-matched rats fed ad libitum (control group, CG). The UG rats achieved 47% of the body weight and 44% of the left ventricular weight (LVW) of the CG. LVW-to-ventricular volume ratio was smaller and myocardial hydroxyproline concentration was higher in the UG. Left ventricular systolic function was not affected by the PCM protocol. The myocardial stiffness constant was greater in the UG, whereas the end-diastolic pressure-volume relationship was not altered. In conclusion, the heart is not spared from the adverse effects of PCM. There is a geometric alteration in the left ventricle with preserved ventricular compliance despite the increased passive myocardial stiffness. The systolic function is preserved.     

Sexual behaviour of male rats rehabilitated after early protein-calorie malnutrition.

The effect of early protein-calorie malnutrition (from birth to the age of 55 days) followed by nutritional rehabilitation (from the 58th day) on sexual behaviour was studied in male rats aged about 125 days. The sexual stimulation conditions on the part of the oestrus female were made as optimal as possible and were fully controlled. About half the malnourished males displayed precopulatory and copulatory behaviour, while in the control group these values were almost 100%. When malnourished males copulated, their copulatory performance did not differ very greatly from that of the well-nourished controls. The other experimental males displayed no signs of sexual behaviour during the testing period. Exposure of the males to the scent of a female in the latter's absence greatly stimulated their interest in the odour (sniffing the floor of the experimental box) in both the control and the malnourished animals which afterwards copulated. The results indicate that the sexual behaviour of males subjected to protein-calorie deficiency in early ontogenesis is at the very least delayed, if not completely suppressed.     

Subperiosteal gain and endosteal loss in protein-calorie malnutrition

Subperiosteal and medullary cavity diameters of 91 Guatemalan boys hospitalized with a diagnosis of protein-calorie malnutrition show a slight but significant increase in total width but a marked reduction at the endosteal surface, and in cortical area and percent cortical area, indicative of continuing subperiosteal apposition and a dramatic excess of endosteal resorption.     

The effect of cysteine on the altered expression of class alpha and mu glutathione S-transferase genes in the rat liver during protein-calorie malnutrition

Protein-calorie malnutrition (PCM) represents a global health problem. The breakdown rate of glutathione S-transferase (GST) subunits determines their differential contents during protein depletion. Hepatic GST expression and the underlying mechanistic basis were investigated in PCM rats. PCM caused no change in rGSTA1/2 subunit. In contrast, rGSTA3/5 subunit was 2.4-fold induced during PCM, while the levels for rGSTM1 and M2 subunits were 30% and 70% suppressed. Increased GSTA3/5 expression was significantly prevented by cysteine or methionine treatment, although such treatment failed to restore the rGSTM2 level. In contrast to differential GST protein expression, PCM caused a 5-10-fold increase in rGSTA2/A3/A5 and M1 mRNAs, whereas rGSTM2 mRNA was 70% decreased. The elevations in rGSTA2/A3/A5 and M1 mRNAs were completely abolished by cysteine or methionine treatment during PCM, although the rGSTM2 mRNA level was not restored. PCM induced oxidative stress in the liver, as evidenced by protein carbonylation. Antioxidant response element (ARE)-binding activity of nuclear extracts from PCM rats was increased, which was immunodepleted with anti-Nrf-1/2 antibodies. Activation of nuclear ARE-binding proteins was inhibited by cysteine. Data showed that hepatic GSTs were differentially expressed during PCM, that certain GST mRNAs were increased with the ARE activation, and that cysteine was active in preventing increases in GST mRNAs and ARE activation.     

Influence of nicorandil on the pharmacodynamics and pharmacokinetics of gliclazide in rats and rabbits

Chronic diabetes precipitates ischaemic heart disease (IHD) and many other disorders. IHD inturn is shown in the form of angina initially. According to EUROPA study, the incidence of angina is high in type II diabetics. Gliclazide, a second generation sulphonylurea derivative is widely used in the treatment of type-II diabetes and is known to release insulin by K⁺ channel inhibition. Nicorandil, a newer antianginal drug widely used now a days acts by opening potassium channels in the cardiac muscle cell and also by releasing nitric oxide. However its action on pancreatic cell K⁺ channel is not known. Since there is possibility for drug interaction leading to decreased activity of gliclazide the present study was conducted to evaluate the effect of the combination.Studies in normal and alloxan induced diabetic rats were conducted with oral doses of 2 mg/kg bd. wt. of gliclazide, 1.8 mg/kg bd. wt. of nicorandil and their combination with adequate washout periods in between treatments. Studies in normal rabbits were conducted with 5.6 mg/1.5 kg bd. wt. of gliclazide, 1.4 mg/1.5 kg bd. wt. of nicorandil and their combination given orally. Blood samples were collected in rats from retro orbital puncture at 0, 1, 2, 3, 4, 6, 8, 10 and 12 h and by marginal ear vein puncture in rabbits at 0, 1, 2, 3, 4, 6, 8, 12, 16, 20 and 24 h. All the blood samples were analysed for glucose by GOD/POD method. The blood samples of rabbits were analysed by HPLC for gliclazide.Gliclazide produced hypoglycaemic/antidiabetic activity in normal and diabetic rats with peak activity at 1 h and 8 h and hypoglycaemic activity in normal rabbits at 3 h, while nicorandil alone produced significant hyperglycaemia at 4 h and reduced the effect of gliclazide with no significant change in pharmacokinetics when administered in combination. The interaction observed appears to be pharmacodynamic at the receptor level as expected.     

Effect of in vivo nicotine exposure on chlorpyrifos pharmacokinetics and pharmacodynamics in rats

Routine use of tobacco products may modify physiological and metabolic functions, including drug metabolizing enzymes, which may impact the pharmacokinetics of environmental contaminants. Chlorpyrifos is an organophosphorus (OP) insecticide that is bioactivated to chlorpyrifos-oxon, and manifests its neurotoxicity by inhibiting acetylcholinesterase (AChE). The objective of this study was to evaluate the impact of repeated nicotine exposure on the pharmacokinetics of chlorpyrifos (CPF) and its major metabolite, 3,5,6-trichloro-2-pyridinol (TCPy) in blood and urine and also to determine the impact on cholinesterase (ChE) activity in plasma and brain. Animals were exposed to 7-daily doses of either 1 mg nicotine/kg or saline, and to either a single oral dose of 35 mg CPF/kg or a repeated dose of 5 mg CPF/kg/day for 7 days. Groups of rats were then sacrificed at multiple time-points after receiving the last dose of CPF. Repeated nicotine and CPF exposures resulted in enhanced metabolism of CPF to TCPy, as evidenced by increases in the measured TCPy peak concentration and AUC in blood. However, there was no significant difference in the amount of TCPy (free or total) excreted in the urine within the first 24-h post last dose. The extent of brain acetylcholinesterase (AChE) inhibition was reduced due to nicotine co-exposure consistent with an increase in CYP450-mediated dearylation (detoxification) versus desulfuration. It was of interest to note that the impact of nicotine co-exposure was experimentally observed only after repeated CPF doses. A physiologically based pharmacokinetic model for CPF was used to simulate the effect of increasing the dearylation V_max based upon previously conducted in vitro metabolism studies. Predicted CPF-oxon concentrations in blood and brain were lower following the expected V_max increase in nicotine treated groups. These model results were consistent with the experimental data. The current study demonstrated that repeated nicotine exposure could alter CPF metabolism in vivo, resulting in altered brain AChE inhibition.     

Effects of glucose supplementation on the pharmacokinetics of intravenous chlorzoxazone in rats with water deprivation for 72 h

It was reported that in rats with water deprivation for 72 h with food (dehydration rat model), the expression of CYP2E1 was 3-fold induced with an increase in mRNA level and glucose supplementation instead of food during 72-h water deprivation (dehydration rat model with glucose supplementation) inhibited the CYP2E1 induction in dehydration rat model. It was also reported that chlorzoxazone (CZX) is metabolized to 6-hydroxychlorzoxazone (OH-CZX) mainly via CYP2E1 in rats. Hence, the effects of glucose supplementation on the pharmacokinetics of CZX and OH-CZX were investigated after intravenous administration of CZX at a dose of 25 mg/kg to control male Sprague-Dawley rats and dehydration rat model and dehydration rat model with glucose supplementation. Based on the above mentioned results of CYP2E1, it could be expected that increased formation of OH-CZX in dehydration rat model could decrease in dehydration rat model with glucose supplementation. This was proven by the following results. In dehydration rat model with glucose supplementation, the AUC of OH-CZX was significantly smaller (1900 versus 1050 microg min/ml), AUC(OH-CZX)/AUC(CZX) ratio was considerably smaller (105 versus 34.3%), C(max) was significantly lower (20.6 versus 8.08 microg/ml), total amount excreted in 24-h urine as unchanged OH-CZX was significantly smaller (62.3 versus 42.7% of intravenous dose of CZX), and in vitro V(max) (2.18 versus 1.20 nmol/min/mg protein) and CL(int) (0.0285 versus 0.0171 ml/min/mg protein) were significantly slower than those in dehydration rat model.