Design and in vivo evaluation of a microparticulate depot formulation of buprenorphine for veterinary use

Abstract

Buprenorphine is a frequently used analgetic agent in veterinary medicine. A major drawback, however, is the short duration of action requiring several daily administrations. We therefore designed a poly-lactic-co-glycolic acid (PLGA) based microparticulate drug formulation for sustained parenteral drug release. Particles were designed to allow for a fast onset of action and a duration of the analgesic effect of at least two days in laboratory mice. Microparticles were produced using a solvent evaporation technique. Release rate was dependent on polymer type and particle size. Spherical particles used for subsequent animal studies had a mean size of 50 µm and contained 4.5% of buprenorphine. Drug release was characterized by an initial burst release of 30% followed by complete release over seven days. In vivo pharmacokinetic experiments in female C57BL/6 J mice confirmed prolonged exposure in plasma and brain tissue and correlated with the pharmacological effect in the hot plate assay or after minor abdominal surgery. No adverse side effects with respect to food and water intake, body weight, local tolerability, or nesting behavior were observed. Our formulation is an attractive alternative to established immediate release formulations. A use for prolonged pain management in laboratory animals is proposed.

Figure 1

Effect of particle size and type of polymer on in vitro release of buprenorphine HCl loaded PLGA microspheres. Release was studied in 54 mM HEPES buffer at pH 7.4 with 1.5% SDS at 37 °C. Data is presented as mean ± SD (n ≥ 3).

Figure 2

Scanning electron microscopy pictures of buprenorphine loaded PLGA (RG 502 H) microspheres. Focused ion beam technique was used to examine inner structure through removal of top layer of polymer (b) and vertical sectioning of particles (c).

Figure 3

Plasma and regional brain concentration–time profiles. (a) Plasma concentration–time profile of RG 502 H-Big (BUP-Depot) and non-retard formulation. Unpaired t-test showed significant difference between formulations for 2 h (P = 0.0009) but not for 12 h (P = 0.051). (b) Regional brain concentrations of BUP-Depot in mid-and forebrain, medulla oblongata and pons, and cerebellum. (c) Brain-to-plasma ratio-time profiles of BUP-Depot. Brain concentrations are defined as sum of concentrations of mid- and forebrain, medulla oblongata and pons. (d) Specific binding of BUP-Depot and non-retard formulation in brain. Specific binding is defined as difference between combined concentrations of mid- and forebrain, medulla oblongata and pons and concentrations in cerebellum. Unpaired t-test showed significant difference at 2 h (P = 0.01) and 12 h post injection (P = 0.006) between both formulations. BUP-Depot (1.2 mg/kg) and non-retard formulation (0.1 mg/kg) were injected once subcutaneously. Dashed line in all graphs represents threshold of 1 ng/mL or 1 ng/g. Data expressed as mean ± SD. a: 24 h time point of non-retard formulation could not be statistically analyzed as only 2 out of 7 animals showed values higher than LLOQ for concentrations in plasma and cerebellum. Values below LLOQ were excluded, therefore no SD value is shown. (P < 0.05 *; P < 0.01 **).

Figure 4

Effect of sustained-release buprenorphine (BUP-Depot), non-retard formulation and saline on withdrawal response latency to thermal stimulus of 54 °C in mice and on delta mouse grimace score after sham-ovariectomy. (a) One way analysis of variance with post hoc Tukey test shows significant (*P < 0.05;**P < 0.01) increase in latency for BUP-Depot at 2 h, 12 h, 24 h time points compared to NaCl and compared to non-retard formulation at 24 h. Non-retard formulation shows only a significant increase in latency at the two hours’ time point compared to NaCl animals. (n ≥ 11 per time point and group). (b) Standard analgesic protocol was used after sham-ovariectomy, treatment groups received either one injection of BUP-Depot or two injections of non-retard formulation 6 h apart. No significant difference between delta mouse grimace scores could be shown for both groups 3–12 h post-surgery using Mann–Whitney test (α = 0.05). Data expressed as mean ± SD for all experiments. (n = 6 per group).

Figure 5

Effects of one injection of BUP-Depot compared to two injections of non-retard formulation after sham-ovariectomy on nest complexity score, body weight, food and water intake. (a) Nest scores during baseline measurements on day 1 and 2 and 24 h post-surgery show no significant differences between both groups at any time point (Day 1: P = 0.92; Day 2: P = 0.73; 24 h: P = 0.92). (b) Body weight change in percent 24 and 48 h post-surgery show no significant difference between BUP-Depot and non-retard formulation using unpaired two-tailed Student t-test (24 h: P = 0.91; 48 h: P = 0.62). Food intake (c) and water intake (d) in gram during baseline measurements, 24, and 48 h post-surgery. Baseline measurements consists of average data from day 1 and day 2 of procedure. No significant difference between non-retard formulation and BUP-Depot group could be found using unpaired two-tailed Student t-test ((c) Baseline: P = 0.69; 24 h: P = 0.34; 48 h: P = 0.78), (d) Baseline: P = 0.54; 24 h: P = 0.88). Only exception was 48 h post-surgery water intake, indicating significant difference between BUP-Depot and non-retard formulation group (P = 0.038). Data is expressed as mean ± SD. (n = 6 per group).