Laurence Attolini

Rapid Simultaneous Determination of Lidocaine, Bupivacaine, and Their Two Main Metabolites Using Capillary Gas-Liquid Chromatography with Nitrogen Phosphorus Detector

A gas-liquid chromatographic method for the simultaneous measurement in serum of bupivacaine, lidocaine, and their main metabolites, 2,6-pipecolylxylidide (PPX) and monoethylglycine xylidide (MEGX), respectively, is described. The procedure involves a one-step extraction and injection of the extract into a gas chromatograph equipped with a capillary column and nitrogen phosphorus detector under constant temperature conditions. Recovery of all components averaged 94%, with the lowest detection limit of 15 ng/ml for the four drugs. The precision within-series coefficients of variation ranged from 7.7% for bupivacaine, 8.6% for lidocaine, 10.2% for MEGX, and 15.8% for PPX. The interday coefficients ranged from 0.7 to 6.5%. Concomitant use of caffeine and carbamazepine may interfere with MEGX and bupivacaine determination, respectively. For this reason, in patients receiving one of these two drugs (or ingesting foods and beverages containing caffeine), the described method is not recommended.

Daily rhythms of heart rate, temperature and locomotor activity are modified by anaesthetics in rats: a telemetric study

Abstract The aim of this study was to evaluate the effect of anaesthesia (ether or ketamine) on daily rhythms of temperature (T), heart rate (H) and locomotor activity (A) in unrestrained rats by using implanted radio-telemetry transmitters.T, H and A were measured every 10min, in Wistar male rats, and analysed using Cosinor. The mean±SEM days needed, after surgical implantation, to detect a daily rhythm in H, T and A were also assessed. Six rats were anaesthetized for about 50min either by ketamine or ether in a 3 by 3 cross-over design. Mesors, amplitudes and acrophases of T, H and A were calculated three days before (D-3; D-2; D-1), the day of anaesthesia (D0) as well as the three following days (D1; D2; D3). ANOVA was performed in order to detect, firstly a possible effect due to the order of application of anaesthesia, secondly a significant difference between ether or ketamine-induced anaesthesia and finally a modification of the mesors, amplitudes and acrophases of T, H and A, induced by each anaesthesia, for D0, D1, D2 and D3 when compared to D-1.Our results indicate: (1) Alterations of the acrophases, mesors and amplitudes, except for the amplitude of A, of the daily rhythms of T, H and A on D0 of ketamine anaesthesia while regarding ether anaesthesia only amplitude of T and H and acrophase of A were modified on D0. Some of these modifications were still observed on the days following anaesthesia. A significant difference between ether and ketamine-induced anaesthesia was also observed. (2) A non-detection of T, H and A daily rhythms after surgical implantation, which was not observed after injection of either ether or ketamine alone. Almost 10 days were needed to detect a significant daily rhythm for T, H and A.The authors suggest that, the general anaesthetic agent was responsible for a perturbation of the mesors, amplitudes and acrophases of the daily rhythms of H, T and A while the non-detection of these rhythms after implantation was more due to the surgical aggression.

Kinetics of bupivacaine after clonidine pretreatment in mice

Previous studies have reported that clonidine pretreatment causes an increase in the local anaesthetic activity of bupivacaine. This study was designed to document possible changes in the pharmacokinetic behaviour of bupivacaine and its main metabolite, desbutylbupivacaine, PPX, in mice after a single, 0.1 mg · kg−1, injection of clonidine. Kinetic variables of bupivacaine were determined after a single 20 mg · kg−1 ip dose of bupivacaine in controls (Group I) and in clonidine (0.1 mg · kg−1 ip) pretreated mice (Group 2). The maximal concentration in serum (Cmax, 2.553 ± 0.862 μg · ml−1 versus 0.962 ± 0.141 μg · ml−1 for. Groups 2 and 1, respectively, P = 0.01) and the area under the concentration curve (AUC, 3.530 ± 0.330 μ · ml−1·−1 versus 1.755 ± 0.252 Hg · ml−1 · hr−1 for Groups 2 and 1, respectively, P < 0.01) of bupivacaine were higher in clonidine pretreated mice while the Clearance (Cl) was decreased in clonidine pretreated animals (0.603 ± 0.054 μ · ml−1 versus 1.264 ± 0.447 μg · ml−1 for Groups 2 and 1, respectively, P < 0.01). The ratio of AUC PPX/AUC bupivacaine (which may partially indicate the rate of metabolism) was lower in presence of clonidine (0.220 ± 0.019 against 0.425 ± 0.033 for Groups 2 and 1, respectively, P < 0.01). Our data indicate decreased metabolism in the clonidine-treated mice which suggests altered hepatic metabolism of bupivacaine by clonidine. This may explain the previously reported enhanced anaesthetic activity of bupivacaine in the presence of clonidine.RésuméDe précédents résultats nous ont permis de mettre en évidence une augmentation significative de l’activité anesthésique locale de la bupivacaine chez la souris après traitement par la clonidine. Le présent travail a pour but de rechercher si l’association de clonidine (injection unique de 0.1 mg · fgl−1 de clonidine) à la bupivacaine peut modifier la pharmacocinétique de l’anesthesique local et de son principal métabolite, la desbutylbupivacaine (PPX). Les paramètres pharmacocinétiques de la bupivacaine ont été déterminés après injection d’une dose unique de 20 mg · ml−1 ip de cet anesthésique local chez des animaux témoins (groupe 1) ou des animaux ayant reçu une injection préalable de clonidine (0.1 mg · ml−1, groupe 2). Nos résultats ont mis en évidence une augmentation significative du pic de concentration (Cmax, 2.553 ± 0.862 μg · ml−1 contre 0.962 ± 0.141 μg · ml−1 respectivement pour les groupes 2 et 1, P = 0.01) et de la surface sous courbe (AUC, 3.530 ± 0.330 μg · ml−1 · hr−1 contre 1.755 ± 0.252 μg · ml−1 · hr−1 respectivement pour les groupes 2 et 1, P<0.01) ainsi qu’une diminution significative de la clairance chez les animaux traités par clonidine (0.603 ± 0.054 contre 1.264 ± 0.447 respectivement pour les groupes 2 et 1, P< 0.01). En ce qui concerne la cinétique du métabolite, le PPX, le rapport AUC PPX/AUC bupivacaine (qui est un reflet partiel du taux de métabolisme) a été significativement abaissé en presence de clonidine (0.220 ± 0.019 contre 0.425 ± 0.033 respectivement pour les groupes 2 et 1, P< 0.01). Nos résultats montrent done une diminution de métabolisme de la bupivacaine par la clonidine pouvant expliquer l’augmentation de l’activité anesthésique locale constatee au cours de cette interaction.

Potassium channel agonists modify the local anaesthetic activity of bupivacaine in mice

PurposeThe mechanisms of action of local anaesthetics and potassium channel agonists (PCAs) may interfere by acting in a direct or indirect manner on the same ion channels. In a previously reported study, the bupivacaine-induced mortality was shown to be modified in different ways by four PCAs tested (diazoxide (D), levcromakalim (L), nicorandil (N) and pinacidil (P)) since bupivacaine-induced mortality was increased by high doses of P and L, decreased by N and stayed unchanged by D. The present study was designed to document the changes in bupivacaine (B) local anaesthetic activity in mice after a single injection of one of the four PCAs (D, L, N and P).MethodsEach PCA was tested at three different dosages. Controls received saline. The local anaesthetic activity was evaluated using sciatic nerve blockade. After injection of bupivacaine in the region of the sciatic nerve, the local anaesthetic activity was estimated as the loss of motor control of the injected limb.ResultsPCA treatment increased (P = 0.0001) the time needed for recovery from bupivacaine-induced local anaesthesia. The area under the effect vs time curve, assessing the total anaesthetic effect, was greater for N (P = 0.0016) and P (P = 0.038) but not for L (P = 0.11). Compared with controls, the maximal effect (Emax) was less for D (P = 0.009) and N (P = 0.038) but not for L (P = 0.185) or P (P = 0.45) treated groups. The injection of the PCA in the region of the sciatic nerve of the right hindlimb did not induce any alteration of the motor activity of the injected limb.ConclusionThe four PCAs decreased the maximal local anaesthetic effect and increased the duration of action of bupivacaine.RésuméBut de l’étudeLes mécanismes d’action des anesthésiques locaux et des activateurs des canaux potassiques (ACP) peuvent interférer en agissant d’une manière directe ou indirecte sur les mêmes canaux ioniques. Une étude récemment publiée montre que la mortalité induite par la bupivacaïne a été modifiée par quatre ACP testés (diazoxide (D), levcromakalim (L), nicorandil (N) et pinacidil (P)) comme suit: elle a été augmentée par de fortes doses en P et L, diminuée par N et demeure inchangée par D. La présente étude a pour but de rechercher les variations de l’activité anesthésique locale de la bupivacaine (B) chez la souris après injection unique de l’un des quatre ACP suivants: D, L, N et P.MéthodologieChaque ACP a été utilisé à trois doses différentes. Les témoins ont reçu une injection de sérum physiologique. L’activité anesthésique locale a été évaluée par une méthode de blocage du nerf sciatique. Après injection de la bupivacaine dans la région du nerf sciatique, l’activité anesthésique locale a été évaluée par la perte de l’activité motrice de la patte injectée.RésultatsLe traitement par ACP a augmenté (P = 0.0001) le temps nécessaire à la récupération après l’anesthésie locale induite par la bupivacaine. L’effet anesthésique local global, représenté par l’aire sous courbe de l’effet en fonction du temps a été plus élevé pour N (P = 0.0016) et P (P = 0.038) mais pas pour L (P = 0.11). L’effet anesthésique local maximal (Emax) a été plus bas pour D (P = 0.009) et N (P = 0.038) par rapport aux témoins mais pas pour L (P = 0.185) ou P (P = 0.45). Pour les quatre ACP testés, l’injection du produit lui-même dans la région du nerf sciatique n’a entraîné aucune modification de l’activité motrice de la patte injectée.ConclusionCe travail montre une diminution de l’activité anesthésique locale maximale de la bupivacaïne et une prolongation de sa durée d’action après traitement par ACP.

Cigarette smoke increases bupivacaine metabolism in rats

This study was designed to document possible changes in bupivacaine kinetics in rats after exposure to cigarette smoke. Rats (n = 15) were exposed to cigarette smoke (Borgwaldt type Hamburg II) for ten minutes per day during four days (C) or eight days (B); controls (A) were used simultaneously without exposure to cigarette smoke. After bupivacaine 20 mg · kg−1 ip at day 4 (C) or day 8 (B), blood was sampled (0.5 ml of blood collected by puncture at the retro-orbital sinus 0.25, 0.5, 1, 2, 4, 6 and 8 hours after administration) and bupivacaine and its main metabolite i.e., desbutylbupivacaine (PPX) were determined by gas liquid chromatography. The sensitivity of the method was 15 ng · ml−1 and the reproductibility was <6%. Serum bupivacaine concentrations were plotted against time and the pharmacokinetic variables were determined assuming a two compartment open model: Cmax, Tmax were derived directly from individual data. The β phase elimination halflives (T1/2β), the area under the serum concentration curve (AUC0∞), the total plasma clearance (Cl) and the total volume of distribution (Vd) were calculated. These variables were assessed according to non-linear fitting method. Cigarette smoking exposure did not change the pharmacokinetic variables of bupivacaine. However, the pharmacokinetic parameters of PPX, Cmax (0.175 ± 0.007 μg · ml−1, 0.119 ± 0.014 μg · ml−1 and 0.312 ± 0.023 μg · ml−1, for groups A, B and C, respectively), AUC (0.170 ± 0.006 μg · ml−1 · hr−1, 0.104 ± 0.013 μg · ml−1 · hr−1 and 0.433 ± 0.017 μgv· ml−1 · hr−1 for groups A, B and C, respectively) and the ratio AUC PPX/ AUC bupivacaine (0.306 ± 0.042, 0.153 ± 0.021 and 0.660 ± 0.054 for groups A, B and C, respectively) were higher (P < 0.0001) for group C. These results indicate an enzymatic induction after only short exposure to cigarette smoking and justify further studies to document possible variations of the metabolism of bupivacaine induced by exposure to cigarette smoke in humans.RésuméLe but de ce travail était de rechercher l’influence éventuelle du tabac sur la cinétique de la bupivacaïne chez le rat. Les animaux (n = 15) ont été exposés à la fumée de cigarette durant dix minutes par jour, soit pendant quatre jours (C), soit pendant huit jours (B), à l’aide d’une machine à fumer (Borgwaldt type Hamburg II); les témoins (A, non exposés à la fumée de cigarette) ont été utilisés simultanément et ont reçu une dose unique de 20 mg · kg−1 ip de bupivacaïne, ainsi que les animaux des groupes C au quatrième jour d’intoxication et ceux du groupe B au huitième jour. Les prélèvements de sang ont été faits au sinus rétro-orbitairé à 0,25, 0,5, 1, 2, 4, 6 et 8 heures après l’administration et la bupivacaïne, ainsi que son principal métabolite, la desbutylbupivacaïne (PPX), ont été déterminés par chromatographie en phase gazeuse. La sensibilité de la méthode était de 15 ng · ml−1 et la reproductibilité était de <6%. Les paramètres pharmacocinétiques ont été déterminés selon un modèle ouvert a deux compartiments: Cmax et Tmax ont été déterminés par inspection. La β demivie d’élimination (T1/2β), l’aire sous la courbe des concentrations (AUC0∞, la clairance plasmatique totale (Cl) et le volume de distribution (Vd) ont été calculés selon une méthode d’ajustement non linéaire. L’exposition des animaux à la fumée de cigarette n’a pas modifié les paramètres pharmacocinétiques de la bupivacaïne. A l’inverse, en ce qui concerne le PPX, le Cmax (0,175 ± 0,007 μg · ml−1, 0,119 ± 0,014 μg · ml−1 et 0,312 ± 0,023 μg · ml−1, respectivement pour les groupes A, B et C), l’AUC (0,170 ± 0,006 μg · ml−1 · hr−1, 0,104 ± 0,013 μg · ml−1 · hr−1 et 0,433 ± 0,017 μg · ml−1 · hr−1 respectivement pour les groupes A, B et C) et le rapport AUC PPX/ AUC bupivacaine (0,306 ± 0,042, 0,153 ± 0,021 et 0,660 ± 0,054 respectivement pour les groupes A, B et C) ont été plus élèvé (P < 0,0001) après exposition au tabac durant quatre jours (C) mais pas durant huit jours (B). Ces résultats seraient donc en faveur d’une induction enzymatique après exposition durant quatre jours et justifient d’autres études pour préciser les modifications du métabolisme de la bupivacaïne après exposition a la fumée de tabac chez l’homme.

Nicorandil affects diurnal rhythms of body temperature, heart rate and locomotor activity in rats.

The effects of nicorandil, a K+ channel opener with a potent vasodilator action, on diurnal rhythms of body temperature, heart rate and locomotor activity were assessed in rats. Transmitters were intraperitoneally implanted under ether anaesthesia. After recovery from surgery, body temperature, heart rate and locomotor activity were recorded during control, saline or nicorandil (10 mg x kg(-1) administered orally) treatment and for 5 days after treatment. For each period, Fourier analysis determined the predominant rhythmicity for body temperature, heart rate and locomotor activity while cosinor analysis assessed the corresponding mesors, acrophases and amplitudes and maxima and minima were directly plotted from raw data. The results indicated: (1) loss of the diurnal rhythmicity for all three rhythms after implantation; (2) stress-induced modifications of almost all the characteristics of the three rhythms after saline and (3) a loss of diurnal rhythmicity of heart rate after nicorandil, an effect that was not observed after saline and which was reversed when nicorandil administration was stopped. In conclusion, nicorandil perturbed the diurnal rhythmicity of heart rate while the rhythmicity of body temperature and locomotor activity was not affected.

Effects of Clonidine Pretreatment on the Local Anaesthetic Activity of Bupivacaine in Mice

This study was designed to document possible changes in bupivacaine local anaesthetic activity in mice after a single injection of clonidine (0.1 or 1 mgkg−1, i.p.). The local anaesthetic activity was evaluated during 60 min, according to a previously reported technique, using sciatic nerve blockade by injection of bupivacaine in the area of the sciatic nerve.

Bupivacaine kinetics during hyperthermia in rats

The aim of this study was to document possible alterations of bupivacaine pharmacokinetic behaviour in rats during hyperthermia. Two groups of Wistar AF IOPS male rats (Group A = normothermic controls, Group B = hyperthermia-induced animals) received a single 20 mg · kg−1 ip dose of bupivacaine. Two other groups (Group C = normothermic controls without bupivacaine, Group D = hyperthermia-induced animals without bupivacaine) received, under the same experimental conditions, an equivalent volume of saline. Hyperthermia-induced animals (Groups B and D) were placed in a water-bath at 40° C. Bupivacaine or saline were administered (Group B and D)four hours after the beginning of the experiment and blood samples were obtained by retro-orbital sinus puncture 0.25, 0.5, 1, 2, 4 and 8 hr after administration. Bupivacaine and its main metabolite, 2,6 desbutylbupivacaine (PPX) were assayed according to a gas liquid chromatographic method. The Cmax, Tmax, t1/2, Cl, Vd and AUC were determined according to a two compartment open model. Our data have demonstrated a decrease in clearance of bupivacaine (5.85 ± 0.23 ml · hr−1 and 4.59 ± 0.35 ml · hr−1 for groups A and B, respectively, P < 0.05, and, Tmax of PPX during hyperthermia (0.24 ± 0.03 hr and 0.15 ± 0.0 hr for Groups A and B, respectively, P < 0.05). In conclusion, hyperthermia induces a decrease in bupivacaine clearance in rats which may be of importance in clinical practice.RésuméCe travail a eu pour but de rechercher l’existence éventuelle de modifications de la cinétique de la bupivacaïne chez le rat au cours d’une hyperthermie induite. Deux groupes de rats Wistar A F IOPS mâles (groupe A = témoins normothermiques, groupe B = animaux «hyperthermiques») ont reçu une dose unique de 20 mg · kg−1 ip de bupivacaïne. Deux autre groupes (groupe C = témoins normothermiques ne recevant pas de bupivacaïne, groupe D = animaux «hyperthermiques» ne recevant pas de bupivacaïne) ont été injectés dans les mêmes conditions avec du serum physiologique. Les animaux «hyperthermiques» (group B and D) ont été placés dans un bain-marie à 40° C. La bupivacaïne ou le sérum physiologique ont été administrés (group B and D) quatre heures après le début de l’expérimentation et le sang a été prélevé par ponction au sinus rétroorbitaire 0,25, 0,5, 1, 2, 4 et 8 heures après l’administration. La bupivacaïne et son principal métabolite, la 2,6 desbutylbupivacaine (PPX), ont été déterminés par chromatographie gaz-liquide. Le Cmax, Tmax, t1/2, Cl, Vd et l’AUC ont été déterminés selon un modèle ouvert à deux compartiments. Les résultats obtenus ont montré une diminution statistiquement significative de la clairance de la bupivacaïne (5,85 ± 0,23 ml · hr−1 et 4,59 ± 0,35 ml · h−1 pour les groupes A et B respectivement, P < 0.05) et du Tmax de la PPX (0,24 ± 0,03 h et 0,15 ± 0,0 h pour les groupes A et B respectivement, P < 0.05) chez les animaux hyperthermiques. En conclusion, l’hyperthermie entraîne une diminution de la clairance de la bupivacaïne chez le rat qui si elle se vérifie chez l’homme, pourrait avoir des conséquences cliniques pratiques.

Effects of Calcium Antagonists on Binding of Local Anesthetics to Plasma Proteins and Erythrocytes in Mice

This study was designed to document possible changes in the binding of bupivacaine (BV), lidocaine (LC) and their main metabolites desbutylbupivacaine (PPX) and monoethylglycinexylydide (MEGX), respectively, to plasma proteins and erythrocytes in mice after acute treatment with the calcium antagonists diltiazem (DZ), nicardipine (NP) and verapamil (VP). A significant plasma protein binding of BV, LC and PPX was measured, whereas no binding could be detected for MEGX. The binding of BV was not modified by DZ, NP and VP, however, the total plasma level was increased in the presence of VP. For PPX a significant increase in total and free plasma levels and a decrease in protein binding were demonstrated after DZ and VP treatment. Concerning LC, a significant increase in total and free plasma levels was documented for DZ, NP and VP suggesting an inhibition of the metabolism of LC by the calcium antagonists. An increased penetration of LC into erythrocytes was also demonstrated which is consistent with the calcium antagonist-induced increase in LC free plasma levels. These effects may contribute in part to the previously observed increase in toxicity of BV by calcium antagonists, but are not likely to be the sole mechanism.

Kinetics of Bupivacaine After Nicorandil Treatment in Mice

Previous studies have reported interactions between potassium‐channel agonists and bupivacaine. This study was designed to document possible changes in the pharmacokinetic behaviour of bupivacaine and its main metabolite, N‐desbutylbupivacaine, in mice after a single 1 mg kg−1 intraperitoneal injection of nicorandil.