Significant Reduction In Clearance Research Articles

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.

Effects of fluconazole on the pharmacokinetics and pharmacodynamics of antimony in cutaneous leishmaniasis-infected hamsters

Pentavalent antimony (Sb V) compounds are the drugs of choice for the treatment of all forms of leishmaniasis. For 20 years there has been an interest in antifungal azoles for treating leishmaniasis, with variable success. In the current study, we examined the effects of co-administration of fluconazole (FLZ) on the pharmacokinetics and pharmacodynamics of Sb V in cutaneous leishmaniasis-infected hamsters. Hamsters were divided into four groups. All hamsters were injected with 0.1 mL of 1 × 10 8 promastigotes/mL into the right foot on Day 1. Treatment was started 5 days after the infection. The antimony group received 80 mg/kg/day of Pentostam ® intramuscularly whilst the FLZ group received FLZ 20 mg/kg/day orally for 14 days. The combination group received both Pentostam ® and FLZ at the above mentioned doses for 14 days. Animals in the control group received no treatment. The infected footpads were measured on Days 1 and 14. A pharmacokinetic study was conducted on Days 1 and 14 of treatment, representing single- and multiple-dose pharmacokinetics, respectively. Blood samples were collected at different time intervals up to 24 h. Sb V was determined using flameless atomic absorption spectrophotometry. Pharmacokinetic parameters were calculated using a non-compartmental analysis. In the single-dose study, there was no statistically significant difference in any of the pharmacokinetic parameters of Sb V when given alone or with FLZ. However, on Day 14 a significant increase in peak plasma concentration ( C max) (three-fold) and area under the concentration–time curve (AUC) (four-fold) of antimony was observed when Sb V was co-administered with FLZ. A statistically significant prolongation of the terminal half-life from 1.63 to 8.67 h ( P < 0.05) was also observed. A significant reduction in clearance was detected. However, FLZ had no effect on the pharmacodynamics of Sb V as measured by footpad sizes. In conclusion, FLZ did not improve the therapeutic effect of Sb V when given concomitantly despite the significant increase in blood concentration and prolongation of the elimination half-life of Sb V.

The cerebral and systemic kinetics of thiopentone and propofol in halothane anaesthetized sheep.

The cerebral and systemic kinetics of intravenous thiopentone (250 mg over 2 minutes, n=5) and propofol (100 mg over 2 minutes, n=6) were determined in sheep anaesthetized with halothane (2.0%) and mechanically ventilated to an end-expired carbon dioxide tension of 40 mmHg. The sheep were previously instrumented with arterial and sagittal sinus (effluent from the brain) blood sampling catheters. Systemic kinetics were inferred from the time-course of the arterial blood concentrations, and cerebral kinetics from the time-course of the arterio-sagittal sinus concentration difference across the brain. Under halothane anaesthesia, the peak arterial concentrations of each drug occurred at the end of the two-minute infusion, and was 42.3 mg/l and 12.3 mg/l for thiopentone and propofol, respectively. Propofol had a significantly larger systemic clearance (3.19 l/min) than thiopentone (0.99 l/min). The brain concentrations of propofol equilibrated more slowly with the arterial concentrations than those of thiopentone. The extraction ratio across the brain near the end of the infusions (1.5 min) were 0.85 and 0.46 respectively. These data were also compared to analogous previously published data for initially conscious sheep. The systemic kinetics of thiopentone were little affected by halothane anaesthesia. For propofol, halothane anaesthesia was associated with a statistically significant reduction in clearance (50% of awake), a slower initial half-life (247% of awake), and the emergence of a second slower half-life in some sheep. The cerebral kinetics of both drugs were subtly altered by halothane anaesthesia.

The absolute bioavailability and pharmacokinetics of butorphanol nasal spray in patients with hepatic impairment

The objective of the study was to investigate the effects of hepatic impairment on the absolute transnasal bioavailability and pharmacokinetics of butorphanol. Twelve (eight men and four women) healthy subjects and 12 (eight men and four women) patients with hepatic impairment received a 1 mg dose of butorphanol by intravenous or transnasal administration on two separate occasions. Hepatic function was assessed by antipyrine and indocyanine green clearance tests. Serial blood and urine samples were collected after each dose. Plasma samples were analyzed for butorphanol, and urine samples were analyzed for butorphanol and its metabolites. No statistical difference in maximum plasma concentration (Cmax) for butorphanol was observed between the two groups of volunteers after transnasal administration. However, total plasma clearance (CL), steady-state volume of distribution, area under the concentration-time curve [AUC(0-infinity)], and elimination half-life of butorphanol in patients with hepatic impairment were significantly altered (approximately twofold to threefold). The absolute transnasal bioavailability of butorphanol was significantly higher (approximately 20%) in patients with hepatic impairment. A greater fraction of the administered dose was recovered from the urine in hepatically impaired patients compared to that in healthy subjects (23% to 31% versus 10% to 11%). There was a significant reduction in CL of indocyanine green and antipyrine in hepatically impaired patients. The percentage of reduction in butorphanol CL was highly correlated to the estimated degree of portosystemic shunts in the patients with hepatic impairment. Based on the comparable Cmax but the increased AUC in patients with liver dysfunction, the initial dose of butorphanol nasal spray may not need to be adjusted. However, the subsequent dosing intervals for butorphanol should be prolonged.

Clinical significance of the trimethadione tolerance test in chronic hepatitis: A useful indicator of hepatic drug metabolizing capacity

Trimethadione (TMO) was chosen as an indicator of quantitative hepatic microsomal function, and its pharmacokinetics were studied in 52 patients with chronic hepatitis. Findings in these patients were compared with those for 26 healthy subjects and 13 patients with renal failure. Patients with chronic hepatitis, but not those with renal failure, showed significant reduction in clearance (CL) and prolongation of half-life (t1/2), and the extent of abnormalities was found to reflect the severity of histologic changes in liver tissue. The serum dimethadione (DMO)/TMO ratio 4 h after the administration of TMO altered in parallel with the CL and t1/2 of TMO, and abnormalities in this simple ratio were also related to the histologic severity of changes in the liver tissue. A low DMO/TMO ratio (< 0.4) was associated with advanced histologic changes (chronic active hepatitis with bridging or chronic active hepatitis with cirrhosis), whereas a high DMO/TMO ratio (> 0.4) was associated with mild histologic changes (chronic persistent hepatitis or chronic active hepatitis) (sensitivity, 0.81; specificity, 0.86). These results indicate that the DMO/TMO ratio, which can be obtained from a single blood sampling, reflects the histologic severity of changes in tissue liver, and that the TMO tolerance test is a useful indicator of quantitative liver function.

Clinical pharmacokinetics of drugs in obesity. An update.

Obesity is common enough to constitute a serious medical and public health problem. Drug prescription for obese patients is difficult since dosages based on pharmacokinetic data obtained in normal-weight individuals could induce errors. In obese patients, physiopathological modifications are likely to affect drug tissue distribution and elimination. Body constitution is characterised by a higher percentage of fat and a lower percentage of lean tissue and water. Although the cardiac output and total blood volume are increased, the blood flow per gram of fat is less than in nonobese individuals. Histological hepatic alterations are commonly reported in morbidly obese individuals. A higher glomerular filtration rate is also observed. Most of the pharmacokinetic information concerning obesity deals with distribution. Published data concerning molecules with moderate and weak lipophilicity are homogeneous. In obese compared with normal weight individuals, the total volume of distribution (Vd) is moderately increased (aminoglycosides, caffeine) or similar (H2-blockers, neuromuscular blockers), but the Vd corrected by kilogram of actual bodyweight is significantly smaller. These drugs distribute to a limited extent in excess bodyweight. For highly lipophilic drugs, despite this common characteristic, discrepancies in distribution in obesity exist between drugs belonging to different pharmacological classes. Some drugs show a clear augmentation of Vd and elimination half-life (benzodiazepines, carbamazepine, trazodone, verapamil, sufentanil), indicating a marked distribution into adipose tissue. For others, Vd and Vd/kg are decreased (cyclosporin, propranolol), suggesting that factors other than lipid solubility intervene in tissue distribution. As a general trend, the total clearance (CL) of drugs metabolised by oxidation, conjugation or reduction, and also of drugs with flow-dependent hepatic clearance, is not diminished in obesity. Usually CL is identical in obese and nonobese individuals, sometimes it is increased in obesity (enflurane, halothane, prednisolone, some benzodiazepines). With some drugs a significant reduction in CL is observed in obese individuals (methylprednisolone, propranolol). Renal clearance of aminoglycosides and cimetidine increases in obese individuals. Practical guidelines for dosage adjustment are proposed. For drugs with distribution restricted to lean tissues, the loading dose should be based on the ideal bodyweight of patients. For drugs markedly distributed into fat tissue the loading dose is based on total bodyweight. Adjustment of the maintenance dose depends on possible changes in CL. In some cases (atracurium, prednisolone) dosage adjustment does not follow these recommendations, owing to pharmacodynamic data.

Lithium Clearance Measurements During Recombinant Interleukin 2 Treatment: Tubular Dysfunction in Man

Renal tubular function was evaluated in nine patients undergoing recombinant interleukin 2 (rIL2) treatment for metastatic colorectal carcinoma. A lithium clearance technique was used and the activities of the lysosomal enzyme N N-acetyl-beta-D-glucosaminidase were also measured in the patients' urine, before treatment, during treatment, and then 2 days and 23 days after rIL2 therapy had finished. Significant reductions in clearances of creatinine, sodium, and lithium were observed. The fractional excretions of sodium and lithium were also reduced. Twenty-three days following cessation of rIL2 treatment, there was still a significant reduction in creatinine clearance compared with pretreatment values (p < .01). The clearances of sodium and lithium were also reduced compared with pretreatment values although this did not achieve significance. The fractional reabsorption of sodium and water by the proximal nephron increased during rIL2 treatment, from 0.707 +/- 0.030 (pretreatment) to 0.793 +/- 0.043. This increased reabsorption of sodium and water persisted, rising to 0.849 +/- 0.029, 2 days following cessation of treatment (p < .001, means +/- SEM). Twenty-three days later this had returned toward the pretreatment value, being 0.781 +/- 0.036. The fractional reabsorption of sodium by the distal nephron was also significantly elevated, both during and 2 days after completing rIL2 treatment. Twenty-three days after cessation of rIL2, this value had returned to the pretreatment value. However, in contrast, the fractional reabsorption of water by the distal nephron demonstrated no change during rIL2 treatment, but 2 days posttreatment was significantly reduced and remained low for a further 3 weeks.

Effects of large nongrazable particles on clearance and swimming behaviour of zooplankton

Latex beads (diameter 45 μm) were used to mimic pelagic particle composition dominated by large nongrazable particles and to examine their effect on clearance and swimming behaviour of a tintinnid, Favella ehrenbergii, a rotifer, Brachionus plicatilis, a gastropod veliger, Philine aperta, and copepodites of the neritic copepod Acartia tonsa. Clearance was measured as a function of bead concentration and compared with swimming patterns and grazing behaviour. The ciliates and the rotifers were not sensitive to mechanical obstruction by the polystyrene beads. The gastropod veligers and the copepodites, however, change swimming behaviour by increasing and jumping frequencies accompanied by a significant reduction in clearance. These observations imply nthat laboratory grazing rates may overestimate in situ grazing activity if larger, noningestible particles are present in the water coloumn.

Changes in lignocaine disposition during long-term infusion in patients with acute ventricular arrhythmias.

Lignocaine disposition was studied in 30 patients with acute ventricular arrhythmias. Serum concentrations of lignocaine, its metabolites Monoethylglycine xylidide (MEGX) and glycine xylidide (GX), and alpha 1-acid glycoprotein (AAG) were analyzed during and after a 48-h lignocaine infusion. AAG concentrations tended to rise in patients with acute myocardial infarction (AMI), leading to binding of the drug in plasma. Lignocaine clearance was estimated at various times during the infusion using a Bayesian parameter estimation program and was found to decline over the course of the infusion. There was a significant reduction in clearance based on estimates obtained at the end of the infusion compared with estimates obtained during the first 0-5 h. Clearance was reduced both in patients who had an AMI and those who did not. Multiple linear regression analysis of the clearance data revealed that these changes could be described by a linear function of time and AAG concentration. These findings suggest that other factors in addition to protein binding changes may influence lignocaine disposition during long-term infusion.

Propranolol interactions with diazepam, lorazepam, and alprazolam.

Healthy subjects received single doses of diazepam (5 to 10 mg iv), lorazepam (2 mg iv), or alprazolam (1 mg orally) on two occasions, once in the control state and once with propranolol, 80 mg three times a day. Compliance with the propranolol regimen was verified by measurement of serum propranolol levels (overall means: 100 ng/ml) and by reduction in sensitivity to intravenous metaproterenol to one-thirtieth that of the control value. Propranolol prolonged diazepam elimination t1/2 (58 and 49 hr), reduced its clearance (0.20 and 0.24 ml/min/kg), and increased the 168-hr AUC for desmethyldiazepam, the major metabolite of diazepam (5.63 and 4.81 micrograms/ml . hr). Propranolol had no significant effect on lorazepam t1/2 (13.2 and 12.7 hr) or clearance (1.33 and 1.36 ml/min/kg), nor on alprazolam t1/2 (15.9 and 18.5 hr) or clearance (1.1 and 0.8 ml/min/kg). Thus propranolol induces a small but significant reduction in clearance of diazepam, biotransformed mainly by the oxidative reaction of N-demethylation. Propranolol does not impair lorazepam clearance by glucuronide conjugation nor that of alprazolam by aliphatic hydroxylation.