Print version ISSN 0034-7094
Rev. Bras. Anestesiol. vol.55 no.3 Campinas May/June 2005
Drug-delivery systems for racemic bupivacaine (S50-R50) and bupivacaine enantiomeric mixture (S75-R25): cyclodextrins complexation effects on sciatic nerve blockade in mice*
Sistemas de liberación controlada con bupivacaína racémica (S50-R50) y mescla enantiomérica de bupivacaína (S75-R25): efectos de la complexación con ciclodextrinas en el bloqueo del nervio ciático en ratones
Daniele Ribeiro de Araújo, M.D.I; Leonardo Fernandes Fraceto, M.D.II; Angélica de Fátima de Assunção Braga, TSA, M.D.III; Eneida de Paula, PhDIV
IDoutoranda em Biologia Funcional
e Molecular - Bioquímica da UNICAMP
IIProfessor Doutor da Faculdade de Farmácia e Bioquímica, Universidade de Sorocaba - UNISO
IIIProfessora Associada do Departamento de Anestesiologia - Faculdade de Ciências Médicas da UNICAMP
IVProfessora Associada do Departamento de Bioquímica, Instituto de Biologia da UNICAMP
BACKGROUND AND OBJECTIVES: Bupivacaine-induced
side effects have led to the search for new local anesthetics (LA) with similar
potency and lower toxicity, such as bupivacaine enantiomeric mixture (S75-R25).
Drug-delivery systems for LA in carriers, such as cyclodextrins (CD), have been
developed to improve anesthetic potency and therapeutic index of those drugs.
This study aimed at preparing, characterizing and evaluating the anesthetic
efficacy of inclusion complexes of bupivacaine enantiomeric mixture (S75-R25)
and racemic bupivacaine (S50-R50) with hydroxypropylb-
cyclodextrin (HPb-CD) comparing them to clinically
METHODS: Inclusion complexes were obtained by mixing appropriate volumes of HPb-CD and S50-R50 or S75-R25 to final 1:1 or 1:2 molar ratios and were characterized by phase solubility experiments. Affinity constants (K) were determined between HPb-CD and each LA. Motor and sensory blocks induced by plain or complexed LA formulations were evaluated in mice by sciatic nerve block. Three LA concentrations were used during the experiment: 0.125, 0.25 and 0.5%.
RESULTS: Solubility experiments results were indicative of S50-R50:HPb-CD and S75-R25:HPb-CD complexation, with similar affinity constant (K) values: 14.7 M-1 and 14,3 M-1, respectively. In vivo experiments have shown that complexation has enhanced differential nerve blockade induced by LA: i) motor blockade duration induced by S75-R25 was similar, to the induced by but less intense S50-R50 ( p < 0.001). S50-R50HPb-CD and S75-R25HPb-CD complexes have decreased onset (p < 0.01 and p < 0.05, respectively), without changing motor block intensity (Emax) as compared to plain drugs; ii) sensory block evaluation has revealed higher analgesic intensity with S50-R50HPb-CD (2-fold, p < 0.001) and S75-R25HPb-CD (1.5-1.8-fold, p < 0.01 and p < 0.001, respectively) with both molar ratios (1:1 and 1:2, LA:CD), in addition to prolonging analgesic effect as compared to S50-R50 and S75-R25.
CONCLUSIONS: More pronounced analgesic effects obtained by complexation with HPb-CD have shown that both formulations, S50-R50HPb-CD and S75-R25HPb-CD, are very useful for postoperative pain relief, requiring lower LA concentrations. Nevertheless, it is worth noticing that S75-R25 - being less toxic than racemic bupivacaine - is an interesting alternative for the development of more effective and safe drug-delivery systems as compared to racemic bupivacaine (S50-R50).
Key words: ANESTHETICS, Local: bupivacaine; ANIMALS, mices
JUSTIFICATIVA Y OBJETIVOS: Los efectos
adversos asociados al uso de bupivacaína llevaron a la búsqueda
por nuevos anestésicos locales (AL) con perfil de bloqueo semejante y
menos tóxicos, surgiendo nuevas preparaciones como la mezcla enantiomérica
de bupivacaína (S75-R25). Los sistemas de liberación controlada,
conteniendo AL en carreadores como ciclodextrinas (CD), tienen como objetivo
mejorar la eficacia anestésica y el índice terapéutico
de esas drogas. Este estudio visó la preparación, caracterización
y evaluación de la eficacia anestésica de los complejos de inclusión
de la mezcla enantiomérica de la bupivacaína (S75-R25) y de la
bupivacaína racémica (S50-R50) con hidroxipropilb-ciclodextrina
(HPb-CD) comparándolos con las preparaciones
actualmente utilizadas en la clínica.
MÉTODO: Los complejos de inclusión fueron preparados mezclándose cantidades apropiadas de HPb-CD y S50-R50 ó S75-R25 en las razones molares (1:1 y 1:2) y caracterizados por estudios de solubilidad de fases. Se determinaron las constantes de afinidad (K) de cada AL por la HPb-CD. Los bloqueos motor y sensorial inducidos por las drogas libres y complejadas fueron evaluados en ratones, a través del bloqueo del nervio ciático. Para la realización de los experimentos, se utilizaron tres concentraciones de AL: 0,125; 0,25 y 0,5%.
RESULTADOS: Los estudios de solubilidad indicaron la formación de complejos de inclusión de S50-R50 y S75-R25 con HPb-CD, con valores de constante de afinidad (K) análogos para los dos anestésicos: 14,7 M-1 (S50-R50: HP-bCD) y 14,3 M-1 (S75-R25: HP-bCD). Las pruebas en animales mostraron que la complejidad potenció el bloqueo nervioso diferencial inducido por los AL: i) la duración del bloqueo motor inducido por S75-R25 fue análogo al del S50-R50, pero menos intenso (p < 0,001). Ya los complejos, S50-R50HPb-CD y S75-R25 HPb-CD redujeron la latencia (p < 0,01 y p < 0,05, respectivamente) sin modificar la intensidad del bloqueo motor (Emáx), con relación a las drogas libres; ii) la evaluación del bloqueo sensorial mostró un aumento en la intensidad de la analgesia con los sistemas S50-R50 HPb-CD (2 veces, p < 0,001) y S75-R25 HPb-CD (1,5-1,8 veces; p < 0,01 y p < 0,001, respectivamente), en las dos proporciones molares (1:1 y 1:2, AL:CD), además del prolongamiento de la duración del efecto analgésico, cuando comparados a las formulaciones S50-R50 y S75-R25.
CONCLUSIONES: Los efectos analgésicos más pronunciados logrados después de la complexación con HPb-CD muestran que ambas formulaciones, S50-R50 HPb-CD y S75-R25 HPb-CD, son de grande interés para el alivio del dolor en el período postoperatorio, con la ventaja de ser administradas en menores concentraciones. Mientras, hay que resaltar que la mezcla enantiomérica de bupivacaína (S75-R25), por ser una droga menos tóxica, representa una alternativa en el desarrollo de formulaciones de liberación controlada más seguras y eficaces de que con la bupivacaína racémica (S50-R50).
Bupivacaine is the amino-amide local anesthetic (LA) more widely used in surgical procedures, especially for prolonged regional anesthesia. Bupivacaine-induced side effects (central nervous system and cardiovascular toxicity) have led to the search for new agents with similar blockade profile, but with lower cardiac toxicity 1. Results were LA such as ropivacaine and levobupivacaine, synthesized in the enantiomeric form S(-) with have shown that in addition to molecular changes, stereoselectivity is a major factor to decrease bupivacaine's cardiotoxicity. Recent studies have shown decreased arrhythmogenic ability and lower risk of levobupivacaine-induced seizures 2-3, as well as the maintenance of anesthetic properties similar to bupivacaine 4. Investigations on stereoselectivity have allowed for changes in bupivacaine stereoisomers R (+) and S(-) and for the synthesis of a new local anesthetic formulation with 25% R(+)-bupivacaine and 75% S(-)-bupivacaine, improving drug's anesthetic profile as compared to levobupivacaine and increasing its safety margin 5.
Although there are differences in enantiomers pharmacokinetic and pharmacodynamic profiles, physico-chemical properties such as water or lipid solubility and pKa are preserved 4, and they are factors potentially limiting therapeutic benefits of traditional systems. An alternative would be the development of drug delivery systems with LA in carriers such as cyclodextrins (CD) which allow the manipulation of some physico-chemical properties in addition to improving therapeutic effects, thus favoring their clinical use 6.
CDs are cyclic oligosaccharides made of glucose units (Figure 1) able to complex molecules of adequate size and polarity in their hydrophobic cavity forming inclusion complexes. Complexation changes physico-chemical properties, such as drug solubility, stability and bioavailability 7. In fact, this property makes CD very promising for LA complexation, and several studies with racemic bupivacaine have shown prolonged duration and significant therapeutic index increase 8-11. So, it is interesting to evaluate whether complexation with CD would also change the anesthetic profile of bupivacaine enantiomeric mixture, a potentially less toxic formulation, as compared to racemic bupivacaine. This study aimed at preparing, characterizing and evaluating in mice the anesthetic efficacy of inclusion complexes of bupivacaine enantiomeric mixture (S75-R25) and racemic bupivacaine (S50-R50) with hydroxypropylb-cyclodextrin (HPb-CD) comparing them to clinically available preparations.
Solid complexes were prepared by mixing adequate amounts of HPb-CD and S50-R50 or S75-R25 volumes in water to obtain molar ratios 1:1 or 1:2. The mixtures were kept shaking up during 24 hours at room temperature. Following, the solution was lyophilized and stored at -20 ºC. Solid complexes were weighed in sufficient amounts to obtain desired drug concentrations and then were dissolved in buffer (20 mM HEPES, pH 7.4, with 154 mM NaCl). S50-R50 and S75-R25 concentrations were: 0.125%; 0.25% e 0.5%.
Phase solubility studies were performed 12 to determine the affinity constants (K) between HPb-CD, and each LA, for physico-chemical characterization of formed complexes. Experiments were carried out to evaluate the solubility of both LA in pH 7.4 and - in the absence of HPb-CD - both S50-R50 and S75-R25 are insoluble in the concentration used (30 mM). In our study, increasing HPb-CD concentrations (0.5, 10 and 20 mM) were added to 30 mM of S50-R50 or S75-R25 and then dissolved in buffer solution (50 m M HEPES, pH 7.4, with 154 mM NaCl).
Samples were agitated at room temperature for 24 hours until balance was reached. Then, samples were filtered and analyzed by spectrophotometry (Beckman DU-70 spectrophotometer) at a wavelength of 260 nm 13, and LA concentration in the solution was calculated. All experiments were repeated three times. High molar ratio LA: HPb-CD (until 2:1) used in this experiment is justified by the fact that the affinity constant (K) is calculated using the linear portion of phase solubility diagram, according to the following equation 12:
Anesthetic Efficacy Evaluation
This experiment was performed after the approval by the Animal Experiment Ethics Committee, Instituto de Biologia, Universidade Estadual de Campinas (CEEA) - IB - UNICAMP, which follows the standards of the Brazilian College of Animal Experiment (COBEA). The study involved groups of 7 male albino mice of Swiss strain, with approximately 60 days of age and body weight varying from 30 to 35 grams. Animals supplied by the Lab Animals Facility, UNICAMP (CEMIB) were submitted to 12-hour dark/light cycle with free water and food, and were collectively lodged (5 animals per cage) and adapted to experiment site for at least 7 days.
Anesthetic efficacy experiments were performed by sciatic nerve block in mice 14. Formulations, including vehicle, were randomly administered by 0.1 mL infiltration in the popliteal space located on the posterior knee close to sciatic nerve. Motor and sensory blocks were simultaneously evaluated and all measures were obtained by the same investigator. Animals were observed for 24 hours after treatment to check the incidence of systemic (seizures and mortality) or local (failures in recovering normal movements of the injected paw) toxic effects. Experimental groups were distributed as follows:
- Control Group: HPb-CD
- Group S50-R50: racemic bupivacaine
- Group S75-R25: bupivacaine enantiomeric mixture
- Group S50-R50HPb-CD (1:1): racemic bupivacaine with hydroxypropylb-cyclodextrin in 1:1 molar ratio (S50-R50:HPb-CD).
- Group S50-R50HPb-CD (1:2): racemic bupivacaine with hydroxypropylb-cyclodextrin in 1:1 molar ratio (S50-R50:HPb-CD)
- Group S75-R25HPb-CD (1:1): enantiomeric bupivacaine with hydroxypropylb-cyclodextrin in 1:1 molar ratio (S75-R25:HPb-CD)
- Group S75-R25HPb-CD (1:2): enantiomeric bupivacaine with hydroxypropylb-cyclodextrin in 1:2 molar ratio (S75-R25:HPb-CD)
The ability of each mouse to normally walk with the four paws on a metal mesh (with 5 mm diameter holes) in normal and inverted position was measured before the experiment to evaluate motor block. Only animals fulfilling this requirement were submitted to the experiment. Motor block level was evaluated according to score values 0 (normal movements), 1 (inability to totally bend injected limb) and 2 (total injected limb paralysis) in the following moments: every minute during the first 5 minutes after administration and then in 10-minute intervals until total movements recovery 15. Onset (time elapsed between injection and motor block 1 or 2), time to reach highest score (Tmax), highest score value (maximum effect Emax), time to motor function recovery and total local anesthetic effect (estimated by the area under the effect-time curve, AUC) were evaluated.
Sensory block was measured by mechanical stimulation 16 with adequate equipment called analgesimeter, which generates gradual strength increase (in grams) on the dorsal surface of animal's paw. To prevent stress-induced analgesia, each animal was involved in a towel so that head and limbs with free. Paw withdrawal reflex was considered representative of pain threshold (Pain Withdrawal Threshold to Pressure - PWTP).
PWTP baseline was measured before experiments and animals presenting pain thresholds below or above 30 to 50 g were excluded from the experiment. Maximum cut-off pressure value of 150 g was considered representative of sensory block. After vehicle or drug administration, animals were measured for 5 hours at 15-minute intervals in the first hour, 30-minute intervals in the second and third hours and at 60-minute intervals for the last two hours. Analgesia was defined as increased tolerated pressure threshold of at least 50% above the group treated with vehicle. Analgesic end point was established as the absence of statistical difference between test and control groups 17.
Kruskal-Wallis test was used for statistical analysis of motor block (onset, Tmax, recovery time, Emax and AUC) and data were expressed in median (minimum limit and maximum limit). PWTP values during sensory block were expressed in mean ± standard deviation (SD) and were evaluated in individual times by Analysis of Variance (One-way ANOVA) with further Tukey-Kramer test. Statistical significance was defined as p < 0.05 18.
Characterization of Inclusion Complexes with Cyclodextrins
Complexation of S50-R50 and S75-R25 with HPb-CD at pH 7.4 and 25 ºC could be observed by phase solubility experiments (Figure 2). Systems phase solubility diagram indicates formation of soluble complexes with linear increase in solubility of S50-R50 and S75-R25 (approximately 1.3-fold) as a function of tested HPb-CD concentrations. Values obtained for affinity constants (K) were similar for both LA, being 14.7 M-1 for S50-R50:HP-bCD and 14.3 M-1 for S75-R25:HP-bCD.
Motor Block Evaluation
Plain and complexed LA injection has changed, in concentration-dependent manner, the motor function after sciatic nerve block with reversible loss of motor reflexes in all treated animals (Table I). The group treated with vehicle (HP-bCD) has not shown any sign of motor block.
Comparisons between plain drugs (S50-R50 and S75-R25) have shown that S75-R25 has induced shorter onset (p < 0.001) in all tested concentrations, although lower motor block intensity has been observed in 0.125% concentration (p < 0.001 as shown by Emax values) as compared to S50-R50. However, motor block recovery time and total effect (AUC) induced by both commercial preparations were similar.
Complexed formulations with S50-R50 have induced shorter motor block onset as compared to plain drug in all tested concentrations (p < 0.01 for inclusion complexes 1:1 and 1:2). In addition, statistical analysis has also revealed differences between R50HPb-CD (1:2)and S50-R50 for Tmax (p < 0.05), recovery time and AUC (p < 0.001 and p < 0.05, respectively) in 0.5% concentration.
With systems S75-R25HPb-CD (1:1 e 1:2), onset was significantly shorter (p < 0.05 and p < 0.01, respectively) as compared to plain formulation in different concentrations. AUC was significantly higher (p < 0.05) for the complex in 1:2 molar ratio as compared to 1:1 complex and plain formulation in 0.5% concentration.
Sensory Block Evaluation
Sensory block evaluation by PWTP test has shown concentration-dependent ratios for plain formulations and inclusion complexes of S50-R50 or S75-R25 with HPb-CD. Effects of treatment with HPb-CD, plain and complexed drugs were evaluated along time (Figures 3 and 4).
Vehicle infiltration in the sciatic nerve has not changed baseline values for pain threshold in mice. However, all formulations have increased treated animals pain threshold, which was statistically different as compared to control group (p < 0.001). In addition, complexes with HPb-CD have increased sensory block duration and intensity as compared to plain drugs (p < 0.001). However comparisons between complexes (1:1 and 1:2, in all concentrations) have shown similar sensory block profiles.
Complexed formulations, S50-R50HPb-CD(1:1 and 1:2) have changed animals response to mechanical stimulation at PWTP, increasing pain threshold and nociceptive effect duration. Complexes in 0.125% concentration have induced longer analgesia (up to 4 hours) and effect intensity (approximately 2-fold at 120 minutes, p < 0.001) as compared to plain formulation (Figure 3A). Complexed formulations in 0.25% concentration have also improved analgesic effect at 150 minutes as compared to S50-R50 (2-fold, p < 0.001) (Figure 3B). Complexed formulations at 0.5% have shown similar profile as intermediate concentration, being the analgesic intensity significantly higher (p < 0.001) as compared to plain drug (Figure 3C).
In groups treated with complexes S75-R25HPb-CD (1:1 e 1:2) in different concentrations, analgesic intensity was approximately 1.5 to 1.8-fold higher with complexed drugs (p < 0.001 in 0.125% and 0.25% concentrations) as compared to plain drug (240 to 300 minutes x 150 to 300 minutes, respectively) (Figures 4A and 4B). In 0.5% concentration, although analgesic duration with complexes has been similar to plain drug, post-complexes pain threshold was significantly higher (p < 0.01) (Figure 4C).
CD may be described as cylindrical capsules with internal hydrophobic cavity which, once filled with a molecule of compatible molecular size and polarity, generate inclusion complexes. For several conveniences (price, availability, cavity size) b-CD has become the most widely used CD for complexation and release systems of drugs. However, its low water solubility and nephrotoxicity have directed research to the synthesis of more soluble, less toxic and more adequate CD for parenteral use, such as hydroxypropilb-CD 7 (HPb-CD). Our study has used HPb-CD aiming at minimizing problems of CD pharmaceutical use, especially low water solubility and systemic toxicity.
Studies have shown that HPb-CD and some other CD derivatives (sulfated CD), have increased the solubility of LA such as etidocaine, lidocaine, prilocaine, mepivacaine and especially bupivacaine 19, in addition to improving their therapeutic index 8-11. In fact, this study has shown linear increase in S50-R50 and S75-R25 solubility, which were favored by inclusion complexes formation with HPb-CD. Similar physico-chemical characteristics of S50-R50 and S75-R25 have oriented complexation with HPb-CD determining similar affinity constant values and indicating favorable drug positioning in CD hydrophobic cavity.
The sciatic nerve blockade in mice was choosed because it is a well described experimental model to study LA 14,15,17,20,21.
Treatment with plain S50-R50 and S75-R25 (0.125%; 0.25% and 0.5%) formulations has shown that motor block induced by S75-R25 was similar but lower in intensity as compared to S50-R50. The administration of S75-R25 in mice has increased intensity and prolonged analgesia as compared to S50-R50, especially in lower concentrations (0.125% and 0.25%). Literature reports that S75-R25 provides satisfactory conditions for surgical procedures with adequate motor and sensory blocks 22-23, prolonging postoperative analgesia as compared to S50-R50 24. In addition, S75-R25 has shown to be safer than S50-R50 5,22-23, which has motivated investigations with complexed S75-R25 HPb-CD formulations.
Our results have shown that complexed formulations with HPb-CD have significantly shortened onset without changing motor block intensity (Emax) as compared to plain formulations. Other parameters, such as motor block duration and total LA effect (AUC) were only affected by complexes in 1:2 molar ratio (LA:CD). These effects were probably due to HPb-CD properties, such as higher water solubility, helping the access of hydrophobic anesthetics such as S50-R50 and S75-R25, to the site of action.
In comparing plain formulations and systems with HPb-CD for both anesthetics, it has been observed a benefit in analgesia intensity and duration in all tested concentrations, indicating differential nervous block potentiation due to interaction both of S50-R50 and S75-R25 with HPb-CD, thus increasing permanence time of LA in the site of action.
Changes in LA:CD molar ratio have shown that 1:2 complexes have prolonged motor block recovery time as compared to plain formulations in all concentrations (0.125%; 0.25% and 0.5% for S50-R50 and 0.5% for S75-R25), although 1:1 and 1:2 complexes were not different between them after comparing sensory block quality and/or duration. This LA pharmacodynamic change may be an inherent HPb-CD effect (since it was observed both with S50-R50 and S75-R25), due to factors such as differences in drug transfer ratio or complex dissociation in relation to HPb-CD 8.
LA release systems aim primarily at improving drug therapeutic index to treat acute postoperative pain, decreasing systemic toxicity and acting more on sensory than on motor fibers 8-11. More pronounced analgesic effects obtained after complexation with HPb-CD make S50-R50HPb-CD and S75-R25HPb-CD of great interest to relieve postoperative pain, with the advantage of being administered in lower concentrations as compared to commercially available LA. However, for being potentially less toxic, enantiomeric bupivacaine mixture (S75-R25) is an alternative for the development of more effective and safer drug release systems as compared to bupivacaine (S50-R50).
02. Ohmura S, Kawada M, Ohta T et al - Systemic toxicity and resuscitation in bupivacaine, levobupivacaine, or ropivacaine-infused rats. Anesth Analg, 2001;93:743-748. [ Links ]
Dra. Daniele Ribeiro de Araújo
Universidade Estadual de Campinas - UNICAMP
Address: Cidade Universitária Zeferino Vaz s/n
Depto. de Bioquímica - Instituto de Biologia
ZIP: 13083-970 City: Campinas, Brazil
Submitted for publication September 21, 2004
Accepted for publication January 31, 2005
* Received from Laboratório de Biomembranas e Sistemas Biomiméticos, Departamento de Bioquímica - Instituto de Biologia, Universidade Estadual de Campinas - UNICAMP