Hyaluronan-tethered opioid depots: synthetic strategies and release kinetics in vitro and in vivo.

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.

Synthesis and evaluation of hydrolyzable hyaluronan-tethered bupivacaine delivery systems.

Local anesthetics are useful for reducing acute pain, but their short duration precludes them from use in solely managing postoperative pain. To prolong the duration of local anesthesia, we conjugated bupivacaine to native hyaluronan (HA) and divinyl sulfone cross-linked Hylan A (Hylan B particles) using a hydrolyzable linker incorporating an imide. Bupivacaine was prepared for conjugation to HA by forming the acryl imide derivative. Separately, the carboxyl group of HA was reacted with nipsylethylamine (NEA) using carbodiimide-mediated coupling to provide HA-NEA that was subsequently reduced with tris(2-carboxyethylphosphine) hydrochloride to yield HA carrying a free sulfhydryl (HA-SH). The HA-bupivacaine conjugate was assembled by reacting HA-SH with acrylbupivacaine. Characterization of the conjugates showed 22% degree of modification by 1 mol of carboxyl. In vitro release studies comparing bupivacaine admixed in HA with bupivacaine conjugated to HA showed half-lives of 0.4 +/- 0.1 h, and 16.9 +/- 0.2 h, respectively, and the bupivacaine was released chemically unaltered as confirmed by LC-MS. In vivo studies to assess the duration of anesthetic activity were performed in a rat sciatic nerve blockade model. For these studies, bupivacaine was conjugated to Hylan B following a similar procedure, and the degree of modification obtained was 14%. Free bupivacaine (3 and 16 mg/kg) and free bupivacaine (3 mg/kg) admixed with Hylan B particles showed nerve block over 4, 9, and 6 h, respectively. Free bupivacaine (3 mg/kg) admixed with bupivacaine (13 mg/kg) conjugated to Hylan B particles showed a four to 5-fold longer impairment of motor function over the free bupivacaine formulations with a total block time of 19 h. Bupivacaine conjugated to Hylan B particles has the potential to prolong the duration of local anesthesia.