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vol.58 issue1A comparative study of 0.125% racemic bupivacaine (S50-R50) and 0.125% and 0.25% 50% enantiomeric excess bupivacaine (S75-R25) in epidural anesthesia for labor analgesiaTopical anesthesia associated with sedation for phacoemulsification: experience with 312 patients author indexsubject indexarticles search
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Revista Brasileira de Anestesiologia

Print version ISSN 0034-7094On-line version ISSN 1806-907X

Rev. Bras. Anestesiol. vol.58 no.1 Campinas Jan./Feb. 2008 



Preparation, characterization and in vitro evaluation of 50% enantiomeric excess bupivacaine (S75-R25)-loaded microspheres*


Preparación, caracterización y evaluación in vitro de microesferas de bupivacaina en exceso enantiomérico de 50% (S75-R25)



Pedro Paulo Tanaka, TSAI; Jean Pierre EstèbeII; Richard CamposIII; François ChevanneIV; Pascal Le CorreIV; Sérgio Bernardo Tenório, TSAI; Maria Fernanda TorresV

IProfessor Adjunto da Disciplina de Anestesiologia da Universidade Federal do Paraná, Visiting Associate Professor (Anesthesia Departament) Stanford University School of Medicine
IIMédico do Service d'Anesthésie et Réanimation, Hôpital Hôtel Dieu, Rennes, França
IIIMestrando do Departamento de Cirurgia da UFPr, Anestesiologista Hospital Nossa Senhora da Graças
IVFaculté des Sciences Pharmaceutiques et Biologiques, Rennes, França
VProfessora Adjunta de Anátomo-Fisiologia da Unicenp

Correspondence to




BACKGROUND AND OBJECTIVES: Microspheres can be used as a controlled delivery system to prolong the duration of action of local anesthetics. The objective of this study was the preparation, characterization and analysis of the in vitro release of 50% enantiomeric excess bupivacaine (S75-R25)-loaded microspheres.
METHODS: Microspheres were prepared using the copolymer of polylactide-co-glycolic acid by the spray-dryed method.
RESULTS: Characterization of microspheres regarding their size and content were similar to the theoretical values. The in vitro release demonstrated a biphasic pattern.
CONCLUSIONS: Manufacturing of 50% enantiomeric excess bupivacaine-loaded microspheres by the spray-dryed method with results similar to bupivacaine-loaded microspheres can be done.

Key Words: ANESTHETICS, Local: bupivacaine.


JUSTIFICATIVA Y OBJETIVOS: Las micro esferas pueden ser utilizadas como un sistema de liberación controlada para prolongar la acción de anestésicos locales. Este estudio tuvo como objetivo la preparación, caracterización y el análisis de la liberación in vitro de micro esferas de bupivacaina en exceso enantiomérico de 50% (S75-R25).
MÉTODO: Las micro partículas fueron preparadas utilizando el copolímero de ácido poliláctico-co-glicólico con bupivacaina en exceso enantiomérico de un 50% por el método spray-dryed.
RESULTADOS: La caracterización de las micro esferas con relación a su tamaño y contenido fueron similares a los valores teóricos. La liberación in vitro presentó un estándar bifásico.
CONCLUSIONES: El proceso de fabricación de micro esferas con bupivacaina en exceso enantiomérico de 50% por el método spray-dryed se puede realizar con resultados semejantes a los encontrados con micro esferas de bupivacaina.




Local anesthetics, both for their peripheral action and when applied on the neuro axis, are an important component of the multimodal management of acute pain and selected chronic conditions. Besides providing excellent analgesia at rest and during activities, their use is associated with a reduction in the consumption of opioids and, consequently, their collateral effects 1. However, in the absence of a continuous mode of administration, such as patient controlled analgesia or through a catheter, the benefits of local anesthetics do not last more than 6 hours when administered subcutaneously, or more than 20 hours in the case of the femoral nerve block 2. Attempts to prolong the blockade using adjuvant or increasing the concentration or the volume of the local anesthetics have limited benefits 3. Regional administration of local anesthetics can be improved by incorporating slow-releasing systems, such as implants 4, liposomes 5, making complexes with cyclodextrin 6 or using microspheres7. Among those systems, the use of microspheres is more interesting because their prolonged release rate and smaller size allow the local administration through a needle. Enantiomeric excess bupivacaine-loaded microspheres could provide prolonged release of the drug, increasing the duration of action and decreasing their transference to the systemic circulation, therefore avoiding high plasma concentrations.

The objective of this study was to prepare, characterize and analyze the in vitro release of 50% enantiomeric excess bupivacaine (S75-R25)-loaded microspheres.



This study was approved by the Pharmaceutics and Biopharmaceutics Laboratory of the University of Rennes, France.

Fifty percent enantiomeric excess bupivacaine was encapsulated as a base obtained by the precipitation in an alkaline medium (ammonia hydroxide) of an aqueous solution saturated with 50% enantiomeric excess bupivacaine. The purity of the resulting base was analyzed by high-performance liquid chromatography, comparing it with 50% enantiomeric excess bupivacaine chloride. Microspheres were prepared dissolving the 50% enantiomeric excess bupivacaine base and polymer (RG 503H, Boehringer Ingelheim). The relationship of the weight of 50% enantiomeric excess bupivacaine polymers used was 40-60 (weight %). The solution was processed by the spray-dryed method with a laboratorial Mini Büchi B-191 (Figure 1) using a 0.7 mm tip. The parameters of the process were defined as follows: entrance temperature (50°C); exhaust temperature (43°C); aspirator (100%); pump (2.5 mL.min-1); and spray flow (600 nL.h-1). Microspheres were stored in vacuum at a temperature of 4°C until characterization.



Characterization of microspheres


Microspheres were dispersed in 5 mL of an aqueous solution of 0.05% Tween 20, ultracentrifuged by 10 seconds and then dispersed in 75 mL of distilled water. After dispersion, size distribution was evaluated by laser cuts using the Malvern Mastersizer S (Figure 2). Distribution parameters of the sizes were mass diameter D (v; 0.5) for 50% of the sample, mass mean diameter D (4.3), and amplitude: [D(v; 0.9) D(v; 0.1)] – D(v; 0.5), Each bath was measured three times.



Drug content

Weight samples of 50% enantiomeric excess bupivacaine-loaded microspheres (S75-R25; approximately 20 mg) were dissolved in methylene chloride (1 mL). The drug was then extracted in 0.1 N sulfuric acid (5 mL) covered with ethydocaine hydrochloride as the internal control. After mixing (5 minutes) and centrifuging (3.000 rpm, 10 minutes), 20 µL of the aqueous phase were diluted in 2 mL of the mobile phase, and 20 µL of this diluted solution were injected into the chromatograph. The conditions of the analysis of high-performance liquid chromatography have already been described 8. The high-performance liquid chromatography system was composed of a model 6000 water pump equipped with a Wisp 717 automatic water injector, and a Milton Roy model 3100 spectrum LCD monitor, detector of wave length placed at 205 nm and a Delsi Enica 21 integrator. Analysis were determined by using a 125 ´ 3 Merck Lichrospher RP-B column maintained at 30°C. The mobile phase was 22:78 (v/v) a mixture of acetonitrile and 0.01 M aqueous solution of KH2PO4 acidified with 0.1% H3PO4, at a flow rate of 0.5 mL.min-1.

Study of in vitro release

A Distek model 5100A dissolution test and a rotator arm (100 rpm) was used for the release studies (Figure 3). The means of release was an aqueous solution of NaCl (900 mL) adjusted to a pH of 2.0 with HCl and with the thermostat set at 37°C. Samples of 50% enantiomeric excess bupivacaine-loaded microspheres with known weight (approximately 20 mg) were suspended in 1 mL of an aqueous solution containing 2.5% mannitol, 0.75% sodium carboxymethylcellulose, and 0.05% Tween 20. the cumulative percentage of the release of 50% enantiomeric excess bupivacaine was measured continuously at 205 nm using the Uvikon Kontron Model 992 spectrophotometer (Figure 4). Each microsphere bath was analyzed three times and the data processed by the data system software Icalis IDIS EE. The mean time of dissolution (Td) was derived from percentage charts of time of release using the Weibull equation with the Simed software package SIPHAR. The same process was repeated for a pH of 7.35 after the addition of a phosphate buffer.






The mean size of the microspheres was 10.74 µm (Table I).



The experimental contents of the microspheres were very close to the theoretical values, according to Table II.



The composition of each vial containing 50% enantiomeric excess bupivacaine-loaded microspheres to be suspended in 20 milliliters of distilled water is: Polimer RG 503H – 750 mg; bupivacaine – 500 mg; mannitol 1 g; Tween 20 – 10 mg. The theoretical content after resuspension will be 3.175% (m/v) of a solution containing 50% enantiomeric excess bupivacaine chloride.

Table III and Figure 5 show the kinetic profile of the in vitro release of the microspheres.






The choice of polymer is an important factor since it should be biocompatible and have storage capacity for fair amount of the drug to be used. The copolymer of polylactide-co-glycolic acid (PLGA) was chosen because each gram can store up to 0.8 g of bupivacaine and it is biodegradable 9. Unlike the polyactide acid (PLA) polymer that can store only 22% of local anesthetic, which can make its production in industrial scale impracticable 7.

The ideal rate between the local anesthetic and the polymer is between 55% and 75%, because a concentration above this level is not associated with more prolonged blockade 9. In the present study, the relationship between 50% enantiomeric excess bupivacaine and PLGA was 40-60, respecting the formulation values found in the literature. The amount of local anesthetic released from the microspheres is critical to assure an adequate neural block, requiring that the slow release maintain the therapeutic concentration before its elimination 10.

The amount of local anesthetic incorporated to the microspheres in the present study was very close to the theoretical values, indicating that encapsulation by the spray-dryed method was efficient, in contrast with the low efficiency of encapsulation by methods that use evaporation or solvent extraction with some polymers 7.

The kinetic profile of the in vitro release of 50% enantiomeric excess bupivacaine loaded microspheres followed a biphasic pattern, i.e., an initial increase followed by a phase of slow release. This profile was analyzed in two different pHs, an acid, which would help the release of the contents of the microspheres and the other mimicking the human pH through the addition of a phosphate buffer to the solution. Comparing to bupivacaine-loaded microspheres prepared in a similar fashion, the initial increase was not significant, which can be justified by the small number (3) of samples analyzed in this process 11. Despite of this, the results of the in vitro evaluation of the present study are in line with a prior study that predicted the release of 20% of the total content of bupivacaine in the first 24 hours, followed by the release of 7% of the content a day until the tenth day 9. Microspheres are degraded in monomers that are metabolized through the Krebs cycle 10.

It is possible to conclude that the manufacture of local anesthetic-loaded microspheres by emulsifying 50% enantiomeric excess bupivacaine with a polymer (PLGA), with elimination of the solvent through the spray-dryed method and lyophilization of the same is possible and it shows results similar to those of bupivacaine.

ACKNOWLEDGEMENTS: Laboratorio Cristalia for providing 50% enantimeric excess bupivacaine.



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Correspondence to:
Dr. Pedro Paulo Tanaka
435 Sheridan Avenue #306
94306 Palo Alto, CA EUA

Submitted em 3 de janeiro de 2007
Accepted para publicação em 25 de setembro de 2007



* Received from CET/SBA do Hospital de Clínicas da Universidade Federal do Paraná, Curitiba, PR

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