A moldable sustained release bupivacaine formulation for tailored treatment of postoperative dental pain

A moldable and biodegradable dental material was designed for customized placement and sustained delivery of bupivacaine (BP) within an extraction cavity. Microparticles comprising poly(lactic-co-glycolic acid) (PLGA) containing BP were generated via solvent-evaporation and combined with absorbable hemostat Gelfoam®. Kinetics of drug release were evaluated by in vitro dialysis assays, showing higher release within the first 24 hours, with subsequent tapering of release kinetics. Formulations of Gelfoam® and BP-PLGA microparticles (GelBP), with three targeted dosing profiles (0.25, 0.5, and 1 mg/kg/day), were evaluated alongside acute subcutaneous BP injections (2 mg/kg) to determine analgesic efficacy in a rat model of tooth extraction pain. Molar extraction resulted in mechanical and thermal cold hyperalgesia in male and female rats. GelBP outperformed acute BP in blocking post-surgical dental pain, with the 0.25 mg/kg GelBP dose preventing hypersensitivity to mechanical (p < 0.01) and thermal cold stimuli (p = 0.05). Molar extraction also resulted in decreased food consumption and weight. Males receiving acute BP and 0.25 mg/kg GelBP maintained normal food consumption (p < 0.002) and weight (p < 0.0001) throughout 7 days. Females, receiving 0.25 mg/kg GelBP maintained weight on days 5–7 (p < 0.04). Customized, sustained release formulation of anesthetic within a tooth extraction cavity holds potential to eliminate post-operative dental pain over several days.

(e.g., NaCl content). TGA measurements were performed with a TA Q50 instrument using a nitrogen gas atmosphere at a ramp rate of 10°C/min from 25°C to 600°C. All TGA measurement were performed in triplicate.
Scanning electron microscopy (SEM). SEM images were acquired to examine morphology of the particles and formulations. Samples were deposited on aluminum stubs, and sputter coated with a layer of gold/palladium, to create a charged surface. The particles were then examined using an FEI-brand SEM with an accelerating voltage between 5-20 kV under high vacuum.

UHPLC analysis
Chromatographic analysis was performed on a Waters ACQUITY UHPLC system equipped with a PDA detector set to 205 nm to quantify BP. A 0.10 mol/L phosphate buffer was prepared by dissolution of sodium dihydrogen phosphate monohydrate salt (98+%, ACS Reagent), and the pH adjusted to 4.00 by addition of 1% HCl solution (concentrated HCl, Trace Metals Grade, Fisher Scientific). ACN (≥99.9%, Sigma Aldrich) was used as the second eluent. Calibration standards were prepared by dissolution of solid BP acid and base forms to produce stock solutions, which were then further diluted into ACN to prepare the analytical standards at concentrations ranging from 0.68 -55.5 µg/mL. The acid form of BP was chromatographically separated on a Waters ACQUITY BEH C18 column (1.7 µm, 2.1 x 50 mm) by isocratic elution with 70% phosphate buffer/30% ACN at a flow rate of 0.60 mL/min. The base form of BP was chromatographically separated on a Waters ACQUITY UPLC HSS C18 SB column (1.8 µm, 2.1 x 50 mm) by isocratic elution with 55% phosphate buffer/45% ACN at a flow rate of 0.60 ml/min. Calibration standards were analyzed and peaks corresponding to BP were integrated, plotted against the concentration, and the calibration curve was calculated by linear least-squares regression without weighting. Calibration standards were periodically reanalyzed to serve as quality control samples and monitor continuing system performance.

In vivo Gelfoam formulations
All formulations were supplied as a lyophilized complex of Gelfoam  + PLGA microparticles, with enough material for 4 animals, by RTI International to Duke investigators in numericallycoded tubes. All samples were stored at 4C until preparation prior to use in 600 ul 0.9% NaCl.
The respective complexes were gently mixed with a plastic spatula, rolled into a ball between gloved fingers, and divided into four equal parts. Each ¼ was applied to a different animal in the same group.

Mechanical behavioral phenotyping
In brief, animals were loosely restrained by placing them in a regular housing cage, divided down the middle, where escape was prevented by setting the lid loosely on the cage, allowing access to the vibrissal pads. Mechanical hyperalgesia was assessed using a 1.494 g von Frey filament applied to the vibrissal pad 10 times for a duration of 1 second, with an interstimulus interval of 1 second.

Supplemental Tables
Supplemental Table 1. Ratio of BP-PLGA microparticles to drug-free PLGA microparticles that were then combined with Gelfoam for use in the GelBP formulations for controlled release of BP in vitro studies Supplemental  Supplemental Figure 4. Release profile of unencapsulated BP from matrix material. The majority of BP is released in the first 24 hours. 1mg of free BP-HCl in 0.9 wt/vol% NaCl was combined with Gelfoam® (at either 50 mg or 100 mg of matrix).
Supplemental Figure 5. Effect of GelBP.5 and GelBP1 on post-surgical dental pain. Following tooth extraction, rats in the GelVeh control group exhibited mechanical hyperalgesia (a) and cold hyperalgesia (b), that peaked on day 1 following surgery. Administration of BP+GelVeh, BP+GelBP.5, or BP+GelBP1 did not prevent hypersensitivity to mechanical and thermal stimuli. No differences in contralateral mechanical (c) or cold sensitivity were observed between groups at any time point (d). Data are expressed as mean  SEM. N= 8 (4 males + 4 females) per group.  Figure 6. Effect of GelBP.5 and GelBP1 on food consumption and weight following tooth extraction. Male (a) and female (b) rats receiving only GelVeh prior to tooth extraction exhibited decreased food consumption. Males receiving BP+GelVeh, but not BP+GelBP.5 or BP+GelBP1, maintained normal food consumption throughout the 7-day testing paradigm, though significant group differences were not observed in females. Among males, those receiving BP+GelBP.5 or BP+GelVeh maintained normal weight throughout the 7-day period (c). Among females, no group differences were observed (d). Data are expressed as mean  SEM. N= 4 males or 4 females per group. *P ≤ 0.05, **P ≤ 0.01 and ***P < 0.001 different from GelVeh.