Development and pharmacological evaluation of drug-delivery systems for cyclic amino-amide local anesthetics: bupivacaine, mepivacaine and ropivacaine

Author(s)

Daniele Ribeiro de Araujo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2005-10-06

Examining board members

Eneida de Paula; Gloria Maria Braga Poterio; Silvia S Guterres; Pietro Ciancaglini; Francesco Langone

• Advisor: Eneida de Paula; Angélica de Fátima de Assunção Braga

Abstract

Local anesthetics (LA) are among the different classes of pharmacological compounds used to attenuate or to eliminate pain. These drugs, which are able to reversibly block the excitation/transmission of the nerve impulse in axons have a relatively short action and a significant toxicity to the Central Nervous and Cardiovascular systems. In order to prolong the time of action and to reduce the systemic toxicity of LA, many investigations have been carried out with LA in drug-delivery systems. These novel formulations, called long-acting local anesthetics, allow the controlled release - avoiding high plasmatic concentrations to be reached ¿ in such a way that they prolong analgesia as well as they reduce LA¿s intrinsic toxicity. Our aim was to prepare, to characterize (the encapsulation, stability and drug-release) and to evaluate - both in vitro (cellular toxicity) and in vivo (latency, intensity and duration of anesthesia as well as local toxicity) the pharmacological activity of new drug-delivery systems for LA, in comparison to the commercially available anesthetics. We have chosen the cyclic amino-amide anesthetics Bupivacaine (BVC), Mepivacaine (MVC) and Ropivacaine (RVC), since they are largely used in medicine and dentistry. The drug-carrier systems used were large unilamellar liposomes (400 nm LUV, composed by phosphathidylcholine and cholesterol) and cyclodextrins: ß-CD and hydroxypropyl (HPß-CD). Stability tests showed that liposomes were able to keep their content and that LA incorporation did not change their permeability at different times and temperatures. Partition coefficients were measured and indicated the profile expected from the substitution degree, i.e. MVC < RVC < BVC (P values = 93, 132 and 136, respectively). Chemical stability was evaluated through oxidative tests and no changes in the lipid oxidation levels were observed up to 4 months after preparation and storage at 4ºC, with and without LA. Nevertheless, light-scattering measurements revealed an increase in the vesicles size after 30 days, restraining the use of the liposome formulations for a longer time. LA complexation with CDs was evidenced by Differential Scanning Calorimetry, which runs detected changes in the fusion temperature of the pure LA, as well as loss of the characteristic endothermic peak of dehydration of the cyclodextrin¿s (ß-CD and HPß-CD) cavity following complexation with BVC and RVC. Electron microscopy images revealed that LA:CD complexation leads to disappearance of the typical crystal structures of pure LA and CD, what was not noticed with juxtaposition (or physical mixture) of the compounds. Kinetic studies showed that complexation equilibrium is reached in a few hours, while phase-solubility tests detected an enhance in the water solubility of BVC and RVC in the presence of increasing CD concentrations, also allowing calculation of the association constants between LA:ß-CD (8.9 and 7.9 M-1) and LA:HPß-CD (14.7 and 10.0 M-1) for BVC and RVC, respectively. In vitro dialysis equilibrium tests disclosed a decrease in the release of BVC and RVC after complexation with HPß-CD or ß-CD as well as for liposomal RVC, when compared to free LA molecules, in a clear demonstration that the interaction with the carriers changed LA permeation through the membranes. Toxicity tests in vitro, including fibroblast cells culture and human erythrocyte hemolysis, have registered a decrease in the LA toxicity after complexation with both CDs (BVC and RVC) or encapsulation in liposomes (MVC and RVC). In vivo tests have shown, using the infraorbital nerve blockade test in rats, that liposomal formulations (MVCLUV and RVCLUV) prolonged the action and enhanced the intensity of the analgesia effect of free LA, as well as of vasoconstrictor associated-LA preparations (for MVC), suggesting that liposomal LA formulations could possible replace vasoconstrictors use in long-lasting surgical procedures or at the post-operatory period, specially for dentistry purposes. Motor blockade tests revealed that all the samples used: free, CD-complexed or liposome-encapsulated LA changed in a dose-dependent manner the motor function of the animals after caudal (intrathecal) or sciatic (infiltrative) injection, leading to the reversible loss of motor answer in all animals studied. None of the LA formulations tested (with ß-CD, HPß-CD or LUV), were able to prolong or to intensify the motor blockade induced by BVC and RVC. However, both BVCHPß-CD and RVCHPß-CD systems significantly reduced the latency for the motor blockade, indicating a fast onset of action, without changing the overall motor blockade, what is highly desirable for a LA. Evaluation of the sensorial blockade in the sciatic nerve of mice demonstrated that BVCHPß-CD and RVCHPß-CD, as well as RVCLUV formulation increased the intensity and duration of the analgesia effect, when compared to free or ß-CD-complexed LA, revealing that those systems can be of great interest at the post-operatory period. Intrathecal injection of 0.5% BVCHPß-CD enhanced the animals¿ nonciceptive limiar in relation to BVCß-CD and free BVC, revealing an increase in the analgesia potency and duration of action. This result allowed us to suggest that the enhance in the nonciceptive effect (ca 1.5 times) observed with the new LA formulations can be attributed, not only to the increase in total LA concentration available to the nerve fibers, but also to the lower protein-binding of the LA molecules, as well as to their lower clearance; altogether, these mechanisms keep the LA for a longer time in contact with the neuronal membrane. Morphological analysis of nervous¿ system cells disclosed neurotoxic signals in part of the animals treated with 0.5% BVCHPß-CD that were not observed if HPß-CD or BVC, alone, were used. The rise in the BVC toxic effect observed after complexation with HPß-CD can be explained by the enhanced availability of the LA molecules in contact with the neuronal membrane, what lead us to suggest that employment of the complex, at lower BVC dosages (0.25% for instance) could reduce the neurotoxic effect without changing the anesthetic potency. Besides, nephro-histological images did not detect any sign of morpho-functional alterations after intrathecal treatment of the animals with HPß-CD, BVC or BVCHPß-CD substantiating the potentiality of the clinical use of that LA formulation (AU)