O objetivo do presente estudo foi determinar os parâmetros farmacocinéticos da ropivacaína encapsulada em lipossomas após anestesia local em 14 voluntários sadios. Neste estudo randomizado, cruzado e duplo-cego, os voluntários receberam anestesia infiltrativa na maxila de ropivacaína a 0,5% encapsulada em lipossomas e ropivacaína 0,5% com epinefrina a 1:200.000 em duas sessões distintas. Amostras de sangue foram coletadas antes e após (de 15 a 1440 min) a administração das formulações de ropivacaína. A quantificação da concentração plasmática de ropivacaína foi feita por meio de HPLC com detecção por UV. Os parâmetros farmacocinéticos AUC 0-24 (área sob a curva de concentração × tempo do tempo 0 até 24 horas) , AUC 0-∞ (área sob a curva de concentração x tempo do tempo 0 até o infinito), C max (concentração máxima da droga), CL (clearance renal), T max (tempo em que ocorre a concentração máxima); t 1/2 (meia vida de eliminação) e V d (volume de distribuição) foram analisados pelo teste de Wilcoxon. Nenhuma diferença (p > 0,05) foi observada entre as duas formulações em cada parâmetro farmacocinético avaliado e as concentrações plasmáticas de ropivacaína, considerando cada período de tempo. Ambas as formulações apresentaram perfil farmacocinético semelhante, indicando que a formulação lipossomal poderia ser uma opção mais segura para o uso deste anestésico local, devido à ausência de vasoconstritor. The aim of this study was to determine the pharmacokinetic parameters of liposomal ropivacaine after dental anesthesia in 14 healthy volunteers. In this randomized, double-blind and crossover study, the volunteers received maxillary infiltration of liposome-encapsulated 0.5% ropivacaine and, 0.5% ropivacaine with 1:200,000 epinephrine in two different sessions. Blood samples were collected before and after (from 15 to 1440 min) the administration of either ropivacaine formulation. HPLC with UV detection was used to quantify plasma ropivacaine concentrations. The pharmacokinetic parameters AUC 0-24 (area under the plasma concentration × time curve from baseline to 24 h), AUC 0-∞ (area under the plasma concentration-time curve from baseline to infinity), C max (maximum drug concentration), CL (renal clearance), T max (maximum drug concentration time), t 1/2 (elimination half-life) and Vd (volume of distribution) were analyzed using the Wilcoxon signed-rank test. No differences (p > 0.05) were observed between both formulations for any of the pharmacokinetic parameters evaluated and plasma ropivacaine concentrations, considering each period of time. Both formulations showed similar pharmacokinetic profiles, indicating that the liposomal formulation could be a safer option for use of this local anesthetic, due to the absence of a vasoconstrictor. Keywords: ropivacaine, local anesthesia, dentistry, liposome, pharmacokinetic Pharmacokinetic Profile of Liposome-encapsulated Ropivacaine after Maxillary Infiltration Anaesthesia J. Braz. Chem. Soc. 1946 Introduction Prolonged-action local anesthetic is required when postoperative pain and discomfort are expected, especially after major surgical procedures. 1 In many countries, bupivacaine, the racemic mixture of S-and D-bupivacaine, is the only long-acting local anesthetic available in dental practice. Ropivacaine (RVC), another long-acting local anesthetic of the cyclic aminoamide family, is synthesized in the S-enantiomer form and presents lower toxicity to the cardiovascular and central nervous systems, compared to bupivacaine. 2 Traditionally, most of local anesthetic formulations are administered together with a vasoconstrictor in order to increase anesthesia duration and to reduce systemic absorption rate. 3 It was recently demonstrated that the use of these formulations increased, especially those containing epinephrine 1:100.000. 5 Alternative drug delivery systems, such as liposomes, have been used to prolong the duration of action of many drugs, including local anesthetics. 6-10 These vesicles are nontoxic and nonimmunogenic because their components (phosphatidyl choline and cholesterol) are also found in biological membranes. 17 Previous authors have shown that liposomal encapsulation of bupivacaine altered its pharmacokinetic profile after extradural injection in rabbits, resulting in lower concentrations of the drug in plasma, liver and myocardium. 18 Grant and co-workers 16 observed that when encapsulated in liposomes, bupivacaine remained at the injection site for a significantly longer period of time after subcutaneous injection in mice. Attempting to simulate an accidental intravascular injection of a local anesthetic, Boogaerts and coworkers 8 assessed the acute CNS (central nervous system) and cardiac toxicities induced by intravenous infusion in rabbits of 0.25% bupivacaine, with and without epinephrine (1:200,000), compared to liposomeencapsulated bupivacaine. They demonstrated a reduction of CNS and cardiac toxicities using liposome-encapsulated bupivacaine. The addition of epinephrine to the plain solution did not decrease the CNS and cardiac toxicities induced by bupivacaine. It was recently demonstrated in animal studies, which used sciatic and infraorbital nerve blockades, that encapsulation of ropivacaine into unilamellar vesicles increased the duration and the intensity of analgesic effects. 6 Although long-acting local anesthetics are normally used in low doses in dentistry, high doses may be required for removal of four impacted third molars in a single session. The present study is the first attempt to measure the pharmacokinetic parameters of ropivacaine after maxillary infiltration anesthesia using a liposome-encapsulated formulation in healthy volunteers. The pharmacokinetic parameters of an RVC formulation containing epinephrine (vasoconstrictor) were also assessed for comparison. Experimental Materials RVC hydrochloride was donated by Cristalia Prod. Quim. Farm. Ltda (Itapira, SP, Brazil). Egg phosphatidylcholine (EPC), cholesterol (Ch) and α-tocopherol (α-T) were purchased from Sigma Chemical Company (St Louis, MO). All other reagents used were of analytical grade. RVC-liposome formulation The liposomal RVC formulations were prepared as described by Araújo and co-workers. 10 The following liposomal characterization was previously determined by Araújo and co-workers. Subjects This research was approved by the Ethical Committee of Piracicaba Dental School, University of Campinas (approval #164/2006). Fourteen healthy volunteers (seven males and seven females) aged 24 (± 3.1) years old were selected, and signed a written informed consent. The volunteers presented no systemic or oral disorders, had no history of allergy to any of the local anesthetics used, and were not taking any medication, as determined by oral questioning and by their documented health histories. Prior to the beginning and right after the end of the study, all the subjects were submitted to laboratory tests to confirm that they were in good health and that the females were not pregnant. The same tests were performed at the end of the study to ensure that all results previously obtained were not altered by the anesthetic formulations tested. These tests included cross-reactive protein, blood-hemoglobin, lymphocyte count, platelet count, erythrocyte sedimentation rate, serum (S)-sodium, S-potassium, S-chloride, S-albumin, S-alkaline phosphate, S-gamma-glutamyl-transferase, S-aspartate transaminase, S-alanine transaminase, S-creatine, plasma glucose, urea, cholinesterase, total protein, bilirubin, uric acid, urine glucose, urine leukocyte count, urine protein, and urine hemoglobin. Serology tests for human immunodeficiency virus and hepatitis B and C were also performed. Female subjects underwent a urine bHCG pregnancy test. Ambulatory procedures Anesthetic procedures In this double-blind and crossover study, the volunteers randomly received 1.8 mL of 0.5% ropivacaine with 1:200,000 epinephrine, and liposome-encapsulated 0.5% ropivacaine, for infiltration anesthesia at the apex of the right maxillary canine, in two different sessions spaced one week apart. Ropivacaine with 1:200,000 epinephrine was obtained by simple dilution of the commercially available solution of ropivacaine (Naropin ® 10 mg mL -1 , AstraZeneca, São Paulo, Brazil) immediately before application. Under sterile conditions, 5 mL of 1% ropivacaine was diluted with 5 mL of 1:100,000 (v/v) epinephrine (Drenalin ® , Ariston Ind. Quim. Farm. Ltda, São Paulo, SP, Brazil). The local anesthetics (1.8 mL) were placed into coded sterile 3 mL Luer-Lok syringes (Becton Dickinson, Curitiba, Brazil) fitted with disposable needles (30G, one-inch, Becton-Dickinson Company, Franklin Lakes, NJ, USA). After topical anesthesia on the injection site with 20% benzocaine, the formulations were injected at the maxillary buccal fold of the right-canine region at an injection rate of 1 mL min -1 . The same operator performed the maxillary infiltration anesthesia in all the subjects. Blood sampling and drug analysis Blood samples (4.5 mL) from a forearm vein were collected with a heparinized cannula before and 15, Pharmacokinetic and statistical analyses The following pharmacokinetic parameters were evaluated by computer software (PK Solutions, noncompartmental pharmacokinetics data analysis, 2001; Summit Research Services, Montrose, CO, USA): C max (maximum drug concentration); T max (maximum drug concentration time); AUC 0-24 , (area under the plasma concentration-time curve from baseline to 24 h); AUC 0-∞ (area under the plasma concentration-time curve from baseline to infinity); CL (renal clearance); t 1/2 (elimination half-life) and Vd (volume of distribution). Statistical analysis was performed using the Student's t-test in order to compare the ropivacaine concentrations between the groups at each time interval. Pharmacokinetic parameters were compared using the Wilcoxon signed-rank test. The significance level was set at 5%, and the tests were performed with BioEstat 5.0 (Fundação Mamirauá, Belém, PA, Brazil) Results and Discussion In an earlier study, Araújo and co-workers 6 demonstrated that the size distribution of liposomal formulations containing RVC presented two modes, one with a maximum at 371 nm (85%), and another with a peak at 128 nm (15%). The efficiency of encapsulation was around 24%, which was sufficient to modify the release profile of the pharmaceutical, with a reduction of the release rate over a one-hour period from 76 to 58%. In the same study it was also shown that, compared to RVC alone, the liposomal RVC formulation increased the duration and intensity of analgesic effects in sciatic and infraorbital nerve blocking experiments. Extending the earlier work of Araújo and co-workers 6 here we report on the first attempt to assess the pharmacokinetic parameters of ropivacaine after maxillary infiltration anesthesia using a liposome-encapsulated ropivacaine formulation in healthy volunteers, comparing the results with a commercial RVC formulation containing epinephrine vasoconstrictor. The calibration curve for determination of plasma ropivacaine ( 22 The detection limit for ropivacaine observed in our study (30 ng mL -1 ) was close to the limit observed by those authors (25 ng mL -1 )
