Safety in the preparation of cytotoxic drugs: How to integrate gravimetric control in the quality assurance policy?

PURPOSE: We present the way to integrate gravimetric control (GC) in a centralized preparation of cytotoxic drugs unit. Two different modalities are described. In the first strategy, the balance is located inside the isolator, whereas in the second, it is located outside in order to remove many technical and ergonomic constraints. These two modalities are compared in terms of benefits and limits. METHODS: GC consists in comparing the observed weight variation with the expected weight variation using a precision balance. According to the B-in strategy, this variation is directly attributable to the weight of the cytotoxic solution injected, whereas with the B-out strategy, the weight of various additional components must be taken into account. RESULTS: Five hundred and seventy-seven preparations have been weighed. For "B-in" strategy, the 95% confidence interval is [1.02-1.14%] and every preparation is below the threshold of 5%. For "B-out" strategy, the 95% confidence interval is [2.34-2.63%] and 94% of preparations are below the threshold of 5%. B-in strategy is distinctly more precise than B-out strategy and can be applied to all preparations. However, B-out strategy is a feasible option in practice and enables the detection of an important mistake. All in all, results obtained from B-out strategy can be considered as a quality indicator in the production line. CONCLUSION: Results of GC are helpful in the final step of release, which the pharmacist is responsible for. Many contributions in the quality assurance policy could justify using of GC in every unit.

Optimization of a model of red blood cells for the study of anti-oxidant drugs, in terms of concentration of oxidant and phosphate buffer.

The aggression of erythrocytes by an oxidative stress induces hemolysis. This paper aims to valid a model of erythrocytes in terms of composition of the phosphate buffer solution and of concentration of a well-known oxidant, AAPH. Three compositions of phosphate buffer solution are mixed with three concentrations of oxidant. The influence of these two parameters on hemolysis is independently studied by a variance analysis and a Kruskal-Wallis test when ANOVA is not available. The hemolysis rate increases with time at fixed oxidant concentration, but is not influenced by the composition of the buffer solution. The highest hemolysis rate, 90%, was only measured within 2 h with the highest oxidant concentration. If we retain this concentration of oxidant, the lower concentration of the buffer can by eliminated by a significant less hemolysis and the highest concentration of the buffer can by chosen in regard of the better precision for a similar hemolysis compared to the mean buffer. We hope to study the effect of anti-oxidant agent with such a model of erythrocytes.

In vivo and in vitro antioxidant properties of furosemide.

The aim of this study was to investigate in vivo and in vitro antioxidant properties of furosemide. In vitro, human red blood cells were submitted to oxidative stress (AAPH), in absence or in presence of different concentrations of furosemide. Potassium efflux was measured in order to quantify the oxidative stress after the action of AAPH on red blood cells. Allophycocyanin assay was also used to investigate antioxidant capacities of furosemide. For the in vivo experiment, male Wistar rats were used. A control group (n = 5) was treated by a daily intraperitoneal injection of saline solution (0.2 ml); 2 other groups (J0 and J+) were treated for 7 days by one daily intraperitoneal injection of furosemide (0.10 mg/kg/day). In the J+group, the injection of furosemide was done one hour before the experiment, while in the J0 group the last injection of furosemide was done on the 6th day and an injection of saline was performed one hour before the experiment. On the day of experiment, a laparotomy was performed under general anesthesia and blood was collected from abdominal aorta. Oxidative stress and antioxidant capacities were evaluated on Wistar rat red blood cells and plasma. In vitro results (oxidative challenge with AAPH) showed that oxidative stress was decreased in presence of furosemide. This was due to a potent free radical scavenging effect of furosemide. In vivo studies confirmed that furosemide had antioxidant properties. These data may be of great relevance in clinical practice, considering the use of large doses of furosemide in patients presenting pathology involving the production of free radicals.

Effect of exercise training on metallothionein levels of hypertensive rats.

PURPOSE: Because oxidative stress may be involved in arterial hypertension by affecting the balance between relaxing and contracting factors of vascular smooth muscle, the training-induced adaptation of antioxidant defenses could be implicated in the antihypertensive effect of chronic exercise. It has been suggested that metallothionein (MT), a metal-binding protein, plays an antioxidant role in mammals. The aim of this experiment was to study whether chronic exercise (swimming) influences both the development of arterial hypertension in spontaneously hypertensive rats (SHR) and the modification of MT levels. METHOD: Male SHR and Wistar Kyoto (WKY) rats as control were trained to swim 1 h.d-1 5 d.wk-1 for 8 wk and sacrificed 72 h after the last exercise period. MT and total thiol levels were then measured. RESULTS: Exercise training 1) reduced systolic blood pressure and heart rate in both SHR WKY rats, and 2) was associated with a decrease in hepatic and cardiac MT levels; there was an increase in the aortic MT amounts in exercised SHR only. No modifications were noted in the gastrocnemius muscle or kidneys. In exercised animals, total thiols were lower in the liver but not in kidneys. CONCLUSION: Chronic exercise induced a reduction in arterial hypertension development in SHR rats and an adaptation of the MT levels in cardiac, hepatic, and aortic tissues. Further experiments are needed to pinpoint the role of the MT in these two cases in which oxidative stress occurs.

Lidocaine inhibits potassium efflux and hemolysis in erythrocytes during oxidative stress in vitro.

Lidocaine is a widely used local anesthetic agent. The aim of this work was to study the action of lidocaine on human red blood cells exposed to an oxidative stress in vitro. Blood was obtained from healthy volunteers. After separation from plasma, the erythrocytes were suspended in phosphate buffer. Oxidative stress was induced by incubation with a free radical generator, the 2,2' azobis (2-amidinopropane) hydrochloride (AAPH). Erythrocytes were incubated with or without lidocaine at two concentrations (36.93 and 73.85 microM) and with or without AAPH (20 mM). Electron paramagnetic resonance (EPR) spectroscopy was performed to identify the free radical species generated by AAPH using the spin trap 5-5'-dimethyl-L-pyroline-N-oxide (DMPO). Different sets of experiments were run. Potassium efflux was measured by flame photometry in each group at time 0 min and every 30 min of the experiment for 2 h. Hemolysis was studied by the Drabkin method at increasing concentrations of AAPH (20, 50, and 100 mM) and with or without lidocaine (36.93 microM). The oxygen radical absorbance capacity (ORAC) was measured by using allophycocyanin (APC) as a fluorescent indicator protein, and the antioxidant capacity of lidocaine (36.93 microM) was studied by the analysis of fluorescence of the APC. AAPH was shown to produce alkoxyl free radicals. Oxidative stress induced a marked increase in the potassium efflux and the hemolysis that was AAPH dose-dependent. Lidocaine inhibited the potassium efflux and delayed the occurrence of hemolysis. Lidocaine did not show any antioxidant properties for the free radical species generated by AAPH. In this model, lidocaine protects erythrocytes against oxidative stress. This effect is not explained by a free radical scavenging property. The results may be of great interest in clinical practice such as intravenous regional anesthesia or the prevention of ischemia-reperfusion injury.

Alteration in plasma antioxidant capacities in chronic renal failure and hemodialysis patients: a possible explanation for the increased cardiovascular risk in these patients.

OBJECTIVE: The high incidence of cardiovascular diseases in chronic renal failure (CRF) and hemodialyzed (HD) patients is now well established and the involvement of oxidative stress has been hypothesized in these phenomena. The aim of our study was to evaluate the level of oxidative stress in healthy controls (CTL) compared with CRF and HD patients before (pre-HD) and after (post-HD) the dialysis session, carried out on a high biocompatible polyacrylonitrile membrane AN69. METHODS: Several indicators of the extracellular redox status were evaluated in plasma. The ascorbyl free radical (AFR) was directly measured using electron spin resonance spectroscopy (ESR) and expressed with respect to the vitamin C level to obtain a direct index of oxidative stress. Indirect plasma parameters such as vitamin E, thiol and uric acid levels were also quantified. The plasma antioxidant status (PAS) was evaluated by the allophycocyanin test. Nitric oxide (NO) stable-end metabolites: nitrites and nitrates (NO(x)), were measured in plasma. RESULTS: In CRF patients, vitamin C and thiol levels were low, and the AFR/vitamin C ratio high compared with the CTL. On the other hand, PAS and uric acid levels were shown to be higher in CRF patients. After the dialysis session, vitamin C level decreased and AFR/vitamin C ratio increased. The thiol levels were shown to be increased, in return PAS and uric acid levels were significantly lower after the dialysis session. NO(x) levels rose during CRF, but were significantly decreased after the dialysis procedure. No differences in vitamin E status were observed between CTL, CRF and HD patients. CONCLUSION: Our study demonstrates that profound disturbances in the extracellular redox system occur during the course of chronic renal failure and hemodialysis, and may provide an explanation for the cardiovascular complications in these patients.

Ascorbyl free radical release in diabetic patients.

The objective of this study was to verify the presence of oxidative stress in type 1 and type 2 diabetic patients and to provide evidences for an use of ascorbyl free radical (AFR)/vitamin C ratios as tools exploring the level of oxidative stress in diabetic patients. TBARS levels, oxygen-radical absorbing capacity assay and AFR release assessed by electron paramagnetic resonance (EPR) were used to explore the existence of oxidative stress in diabetes. Endogenous antioxidants (alpha-tocopherol, vitamin C and uric acid) were also measured. Scavenging capacities of plasma were decreased in diabetic patients. A significant decrease of plasma vitamin C and an increase of AFR/vitamin C ratios were noted in type 2 diabetic patients. Uricemia was decreased in type 1 diabetic patients. Our results suggest a possible use of AFR/vitamin C ratios as indicators of oxidative stress in diabetes mellitus.

Effects of bupivacaine on human erythrocytes submitted to stress and evidence for an interaction between bupivacaine and flumazenil.

AIMS: To examine the effects of bupivacaine on erythrocytes submitted to an oxidative stress (AAPH) and to provide evidence for an in vitro interaction between bupivacaine and flumazenil. METHODS: Human erythrocytes were studied with or without AAPH in the presence of different concentrations of bupivacaine (0.15, 0.3, 0.9 and 1.8 mmol l-1 ), or flumazenil (0.16 mmol l-1 ) and with the association of flumazenil and two doses of bupivacaine (0.15 and 0.3 mmol l-1 ). Potassium efflux was measured by flame photometry at T0, and every 30 min for 2 h. RESULTS: In the absence of AAPH, extracellular potassium remained unchanged. Oxidative stress induced a significant increase in extracellular potassium, which was not modified by incubation with flumazenil. Bupivacaine significantly lowered the increase in extracellular potassium in a dose-related fashion. The association with flumazenil blunted the effects of bupivacaine. Discussion In this model, bupivacaine proved effective in protecting erythrocytes against oxidative stress. Flumazenil interacted with bupivacaine and blunted its protective effects.