Hyaluronan-Tethered Opioid Depots: Synthetic Strategies and

Aug 22, 2008 - Drug and Biomaterial Research and Development, Genzyme Corporation, 153 Second Avenue, Waltham, Massachusetts 02451. Bioconjugate ...
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Bioconjugate Chem. 2008, 19, 1767–1774

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Hyaluronan-Tethered Opioid Depots: Synthetic Strategies and Release Kinetics In Vitro and In ViWo Diego A. Gianolio, Michael Philbrook, Luis Z. Avila, Lauren E. Young, Lars Plate, Michael R. Santos, Richard Bernasconi, Hanlan Liu, Sujin Ahn, Wei Sun, Peter K. Jarrett, and Robert J. Miller* Drug and Biomaterial Research and Development, Genzyme Corporation, 153 Second Avenue, Waltham, Massachusetts 02451. Received February 4, 2008; Revised Manuscript Received July 14, 2008

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.

INTRODUCTION Perioperative pain management has been shown to maintain the patient’s functional abilities, reduce hospital costs due to faster recovery times, and improve patient well-being due to decreased incidence of adverse events (1-3). Pain control may have a further benefit of improving clinical outcome by reducing postoperative complications such as myocardial infarction or ischemia, risk of tachycardia and dysrhythmia impaired wound healing, risk of atelectasis, thromboembolic events, peripheral vasoconstriction, and metabolic acidosis (4-9). Techniques used by anesthesiologists for perioperative pain control include epidural or intrathecal opioid analgesia, patientcontrolled analgesia with systemic opioids, and regional analgesic techniques, including but not limited to intercostal blocks, plexus blocks, and local anesthetic infiltration of incisions (10). Many hydrophilic opioids, such as morphine, have very short half-lives in the body (i.e., 2-3 h), and therefore require frequent bolus injections or continuous pump infusion to maintain therapeutic levels in the target tissues over time (11). Problems, however, remain in the modalities necessary to administer them. Frequent injections are inconvenient for patients and potentially expose the patient to inconsistent drug levels and an increased risk of infections (12). Issues associated with in-dwelling catheters providing continuous infusion include catheter dislodgment, infections, pump failure, fibrosis, and increased costs due to physician and nursing time (13, 14). To overcome the many challenges associated with the current modes of analgesic delivery, manipulation of the pharmacoki* Corresponding author. Tel: + 781-434-3645. Fax: + 781-672-5823. E-mail: [email protected].

netic properties of the drug, more specifically, the absorption of the opioid, may help to expand the management of perioperative pain. Extended-release formulations may reduce analgesic gaps associated with intermittent delivery, decrease the risk associated with repeated injections and in-dwelling catheters, as well as reduce the burden of frequent dose administration placed on health care providers. In addition, the use of extended delivery systems has demonstrated shorter lengths of hospital stay and enhanced patient functionality (15). Currently, there are extended-delivery systems for oral administration of the opioids morphine, oxycodone, and oxymorphone that are used for treating postoperative pain. In addition, a parenteral delivery system, DepoDur, uses a liposomal formulation of morphine that, when delivered by single epidural injection, provides superior pain control during the first 1-2 postoperative days compared with epidural or intravenous administration of unencapsulated morphine (16). Hyaluronan (HA) is a naturally occurring linear polysaccharide of alternating β-1,3-D-glucuronic acid and β-1,4-N-acetyl-D-glucosamine units found in the extracellular matrix and synovial fluid (17). HA is hydrophilic and forms gels of varying viscosity depending on its molecular weight and concentration. In addition, HA can be modified using a variety of cross-linkers generating materials with a range of biophysical properties (18-20). Prolonging morphine’s effect by delivering from viscous HA formulations was observed in rabbits whereby the duration of action depended directly on the HA formulation viscosity (21). In other studies, HA was used to control morphine delivery by ionic complexation with multivalent cations. A morphine-HA complex was prepared using calcium as the divalent cation (22). This complex was injected into rats and the morphine concen-

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tration in blood was monitored over time. The results showed that the HA-morphine complexation decreased the peak systemic drug concentration and prolonged the time of systemic exposure when compared to aqueous morphine sulfate control. In addition, increasing the calcium content was directly related to duration of pain control as measured by paw withdrawal response time in a rat incision pain model. We previously reported a method to prolong the release of the local anesthetic bupivacaine by conjugating to HA via a hydrolytically labile imide linkage (23). The aim of the current study was to investigate the release rate of morphine conjugated to insoluble cross-linked HA (hylan B gel particles) via a hydrolytically labile ester linkage. The morphine-conjugated gel particles would serve as a drug depot that could be administered to local tissues or spaces (e.g., intra-articular space (24)) and would release the drug slowly to provide prolonged, local analgesia. The morphine-conjugated gel particles could also be administered via intrathecal, subcutaneous, or intramuscular routes to provide prolonged exposure of morphine above its minimum effective analgesic concentration to the systemic circulation. We hypothesized that a reversible covalent bond may more effectively maintain the drug in the carrier for a longer period of time relative to a physical admixture. Thus, ester conjugates (1) of morphine (2) as well as its structural controls naloxone and codeine (3, 4) with HA were prepared. The conjugates of naloxone and codeine were prepared to evaluate relative stabilities of phenolic ester and alkyl ester groups, respectively.

Our study included a divinylsulfone (DVS) cross-linked form of HA, hylan B, that shows longer residence time at the implantation site compared to non-cross-linked HA (25). This report describes a method to prepare morphine tethered to HA and hylan B particles. The rate of release of morphine from the conjugates was determined in phosphate buffered saline (PBS) and plasma. The rate of absorption of locally administered hylan B-morphine conjugate was compared against free morphine and morphine/hylan B particles admixture formulations by measuring morphine concentrations in plasma following subcutaneous administration in a rat model.

EXPERIMENTAL PROCEDURES Materials and Methods. Morphine free base was a generous gift from Mallinckrodt Inc. (St. Louis, MO). Hyaluronan (1.3 MDa) was obtained by bacterial fermentation (Genzyme Corporation) and cross-linked with DVS (hylan B particles) following a previously published method (26). Anhydrous solvents and starting materials were purchased from SigmaAldrich Co. and used without further purification. Rat plasma was purchased from GeneTex, Inc. Protease inhibitor cocktail

Gianolio et al.

was purchased from Sigma-Aldrich Co. 1H and 13C NMR spectra were collected on a Varian spectrometer (400 MHz) against an internal standard of tetramethylsilane (δ ) 0.0 ppm). Mass spectra and high resolution mass spectrometry (HRMS) was performed on Applied Biosystems Qstar XL spectrometer with Turbolonspray source. Thin layer chromatography (TLC) was performed on Silica Gel 60 F254 precoated on aluminum sheets (EM Separation Technology) using CH3OH/CH2Cl2 (1:4 and 1:9). Flash chromatography was carried out using silica gel (60 Å, 35-70 µ, 230-400 mesh, Teledyne Isco) using a gradient of 5% methanol in dichloromethane. Phosphate buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH ) 7.3-7.5 was purchased as a 10-fold concentrate (EMD Chemicals, Inc.) and diluted to a working concentration with deionized water. HA-drug conjugates were purified by dialysis in PBS using membrane tubing (7 kDa MWCO, Spectrum). In Vitro release experiments in PBS were based on methodology reported in the literature (27, 28) using tangential flow dialysis cassettes (Slide-A-Lyzer, 10 kDa MWCO, Pierce). UV/visible measurements were obtained using a Varian Cary 50 Probe spectrometer. The molecular weight and degree of molar modification of HA conjugates were determined using size exclusion chromatography and a multiangle laser light scattering (SEC-MALLS) analyzer (model DAWN-EOS, Wyatt Technology). The SEC-MALLS analyzer was connected to refractive index and UV/visible (model UVIS205, Perkin-Elmer) detectors. Separation was performed using a size exclusion column (300 Å, 7.5 × 300 mm, Macrosphere, Alltech) and a mobile phase of 0.05 M Na2SO4 solution. HA degree of molar modification was defined as the molar percentage of conjugated repeating dimeric units in HA. The quantitation of drug purity and release rate in Vitro in PBS was determined by high performance liquid chromatography (HPLC model Waters 600) connected to a photodiode array detector (Waters 996 PDA) reading at 214 nm using a C18 reversephase column (80 Å, 4.6 × 150 mm, W Phenomenex Synergi 4 µm Max-RP) and mobile phase consisting of 90% 10 mM KH2PO4, 8% acetonitrile, and 2% methanol. The separation was performed at a column temperature of 25 °C ( 2 °C, flow rate of 1 mL/min, and under isocratic conditions. The quantification of morphine and morphine derivatives in Vitro in PBS and plasma was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). The separation was performed at 25 °C ( 2 °C using a C18 reverse-phase column (80 Å, 2.0 × 50 mm, Phenomenex Synergi 4 µm Hydro RP). Mobile phases (A ) 0.1% formic acid in water, B ) 0.1% formic acid in acetonitrile) were delivered with a binary pump (series 1100, Agilent Technologies) using a flow rate of 0.5 mL/min. A gradient was used: 5-15% B over 1 min, 15-50% B over 1 min, 50-90% B over 0.5 min, and 90% B for 0.5 min. The effluent was monitored by tandem mass spectrometry (MS/MS) using a mass spectrometer (API4000, Applied Biosystems/MDS Sciex) with an electrospray ionization (ESI) interface in positive multiple reaction monitoring (MRM) mode. Synthesis of Drugs Intermediates and HA Conjugates. Synthesis of Acryl Naloxone (5). Naloxone free base (800 mg, 2.44 mmol) was placed in a 250 mL round-bottom flask and anhydrous toluene (100 mL) was added under a nitrogen atmosphere. Triethylamine (408 µL, 2.93 mmol) was added and the solution was allowed to stir for 30 min at 50 °C. Acryloyl chloride (340 µL, 4.15 mmol) was added subsequently and the reaction mixture was allowed to stir at 50 °C overnight under nitrogen. Upon completion of reaction, as monitored by TLC, the mixture was cooled to room temperature and the excess reagent was quenched by the addition of MeOH (1 mL). The solvents were removed under reduced pressure and the crude product was dissolved in dichloromethane (100 mL), transferred

Hyaluronan-Tethered Opioid Depots

into a separatory funnel, and washed with water (150 mL). The organic phase was collected, dried with Na2SO4, hydroquinone (60 ppm) was added, and concentrated under reduced pressure. Acryl naloxone (5, 790 mg, 85% yield) was isolated by silica gel flash-chromatography (5% MeOH in CH2Cl2) to give a white solid. Rf (methanol/dichloromethane, 1/9): 0.68. mp: 85-87 °C. 1 H NMR (DMSOd6): δ ) 1.3 (d, 1H, J ) 10.4 Hz, CH), 1.4 (dt, 1H, J ) 3.3 Hz, 14.0 Hz, CH), 1.8 (d, 1H, J ) 12.0 Hz, CH), 2.0 (t, 1H, J ) 10.4 Hz, CH), 2.1 (td, 1H, J ) 3.2, 14 Hz, CH), 2.4 (dt, 1H, J ) 4.8 Hz, 12.6 Hz, CH), 2.6 (dt, 2H, J ) 4.7 Hz, 16.0 Hz, CH2), 2.9 (dt, 1H, J ) 5.2 Hz, 14.4 Hz, CH), 3.0 (d, 1H, J ) 4.2 Hz, CH), 3.1 (m, 1H, CH), 3.2 (s, 2H, CH2), 4.9 (s, 1H, CH), 5.2 (d, 1H, J ) 10.4 Hz, CH), 5.3 (d, 1H, J ) 17.2 Hz, CH), 5.9 (m, 1H, CH), 6.2 (d, 1H, J ) 10.0 Hz, CH acryl), 6.4 (m, 1H, CH acryl), 6.5 (dd, 1H, J ) 1.2 Hz, 17.2 Hz, CH acryl), 6.8 (d, 1H, J ) 8.4 Hz, CH aromatic), 6.9 (d, 1H, J ) 8.4 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 22.7, 29.8, 31.1, 35.6, 42.9, 50.2, 56.9, 61.4, 69.7, 90.2, 119.3, 122.5, 127.1, 130.3, 131.3, 131.4, 133.9, 147.2, 163.0, 207.5 ppm. HRMS calculated for C22H23NO5 (M + H+): 382.1649; found: 382.1584. Synthesis of Methacryl Naloxone (6). Compound 6 was prepared as described above using naloxone free base (800 mg, 2.44 mmol), triethylamine (408 µL, 2.93 mmol), and methacryloyl chloride (405 µL, 4.15 mmol). Methacryl naloxone (6, 790 mg, 82% yield) was isolated by silica gel flash chromatography (5% MeOH in CH2Cl2) to give a white solid. Rf (methanol/ dichloromethane, 1/9): 0.68. mp: 164-166 °C. 1H NMR (DMSOd6): δ ) 1.3 (d, 1H, J ) 11.2 Hz, CH), 1.5 (dt, 1H, J ) 3.2 Hz, 14.0 Hz, CH), 1.6 (m, 1H, CH), 1.8 (d, 1H, J ) 12.4 Hz, CH), 2.0 (s, 3H, CH3), 2.1 (d, 1H, J ) 14.0 Hz, CH), 2.4 (dt, 1H, J ) 5.0 Hz, 13.2 Hz, CH), 2.6 (dt, 2H, J ) 4.6 Hz, 16.4 Hz, CH2), 2.9 (dt, 1H, J ) 3.4 Hz, 14.2 Hz, CH), 3.0 (d, 1H, J ) 4.4 Hz, CH), 3.1 (m, 1H, CH), 3.2 (s, 2H, CH2), 4.9 (s, 1H, CH), 5.2 (d, 1H, J ) 10.0 Hz, CH), 5.3 (d, 1H, J ) 17.2 Hz, CH), 5.8 (m, 1H, CH), 5.9 (s, 1H, CH acryl), 6.3 (s, 1H, CH acryl), 6.8 (d, 1H, J ) 8.0 Hz, CH aromatic), 6.9 (d, 1H, J ) 8.4 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 18.0, 22.7, 29.8, 31.1, 35.6, 42.9, 50.1, 56.9, 61.4, 69.7, 90.1, 119.3, 122.5, 125.3, 128.2, 128.9, 130.2, 131.1, 131.7, 134.8, 147.3, 164.1, 207.5 ppm. HRMS calculated for C23H25NO5 (M + H+): 396.1806; found: 396.1801. Synthesis of Ethylacryl Naloxone (7). Compound 7 was prepared using the same method as compound 5 starting with naloxone free base (800 mg, 2.44 mmol), triethylamine (408 µL, 2.93 mmol), and ethylacryloyl chloride (440 µL, 4.15 mmol). Ethylacryl naloxone (7, 883 mg, 89% yield) was isolated by silica gel flash chromatography (5% MeOH in CH2Cl2) to give a white solid. Rf (methanol/dichloromethane, 1/9): 0.72. mp: 93-95 °C. 1H NMR (DMSOd6): δ ) 1.1 (t, 3H, J ) 7.4 Hz, CH3), 1.3 (d, 1H, J ) 12.0 Hz, CH), 1.5 (dt, 1H, J ) 3.3 Hz, 14.0 Hz, CH), 1.8 (m, 1H, CH), 2.0 (m, 1H, CH), 2.1 (d, 1H, J ) 14.8 Hz, CH), 2.3 (d, 1H, J ) 14.4 Hz, CH), 2.4 (q, 2H, J ) 7.4 Hz, CH2), 2.6 (m, 2H, CH2), 2.9 (dt, 1H, J ) 4.9 Hz, 14.6 Hz, CH), 3.0 (s, 1H, CH), 3.1 (m, 1H, CH), 3.2 (s, 2H, CH2), 4.9 (s, 1H, CH), 5.2 (d, 1H, J ) 9.2 Hz, CH), 5.3 (d, 1H, J ) 17.2 Hz, CH), 5.8 (s, 1H, CH acryl), 5.9 (m, 1H, CH), 6.3 (s, 1H, CH acryl), 6.8 (d, 1H, J ) 8.0 Hz, CH aromatic), 6.9 (d, 1H, J ) 8.0 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 12.6, 22.7, 24.5, 29.7, 31.0, 35.6, 42.9, 50.1, 56.8, 61.4, 69.7, 90.1, 119.3, 122.5, 126.2, 128.1, 128.8, 130.2, 131.0, 131.7, 140.6, 147.4, 164.0, 207.4 ppm. HRMS calculated for C24H27NO5 (M + H+): 410.1962; found: 410.1935. Synthesis of Propylacryl Naloxone (8). The preparation was identical to the method described for compound 5 using naloxone free base (800 mg, 2.44 mmol), triethylamine (408 µL, 2.93 mmol), and propylacryloyl chloride (493 µL, 3.78

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mmol). Propylacryl naloxone (8, 876 mg, 85% yield) was isolated by silica gel flash chromatography (5% MeOH in CH2Cl2) to give a white solid. Rf (methanol/dichloromethane, 1/9): 0.72. mp: 89-91 °C. 1H NMR (DMSOd6): δ ) 0.9 (t, 3H, CH3), 1.3 (d, 1H, J ) 10.8 Hz, CH), 1.4 (m, 1H, CH), 1.5 (sexlet, 2H, J ) 7.2 Hz, CH2), 1.8 (d, 1H, J ) 8.0 Hz, CH), 2.0 (t, 1H, J ) 10.8 Hz, CH), 2.1 (td, 1H, J ) 2.8 Hz, 14.0 Hz, CH), 2.3 (t, 1H, J ) 7.4 Hz, CH), 2.4 (dt, 1H, J ) 5.0 Hz, 12.4 Hz, CH), 2.6 (dt, 2H, J ) 5.0 Hz, 19.4 Hz, CH2), 2.9 (dt, 1H, J ) 5.1 Hz, 14.2 Hz, CH), 3.0 (d, 1H, J ) 4.0 Hz, CH), 3.1 (m, 1H, CH), 3.2 (s, 2H, CH2), 4.9 (s, 1H, CH), 5.2 (d, 1H, J ) 10.4 Hz, CH), 5.3 (d, 1H, J ) 17.2 Hz, CH), 5.8 (s, 1H, CH acryl), 5.9 (m, 1H, CH), 6.3 (s, 1H, CH acryl), 6.8 (d, 1H, J ) 8.0 Hz, CH aromatic), 6.9 (d, 1H, J ) 8.4 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 13.5, 21.0, 22.7, 29.8, 31.1, 33.4, 35.6, 42.9, 50.1, 56.9, 61.4, 69.7, 90.2, 119.2, 122.5, 127.3, 130.3, 131.2, 131.7, 139.0, 147.3, 164.0, 207.5 ppm. HRMS calculated for C25H29NO5 (M + H+): 424.2119; found: 424.2089. Synthesis of Acryl Codeine (9). Compound 9 was prepared using the method for compound 5 using codeine free base (800 mg, 2.67 mmol), triethylamine (895 µL, 6.41 mmol), dimethylaminopyridine (33 mg, 0.27 mmol), and acryloyl chloride (477 µL, 5.87 mmol). Acryl codeine (9, 504 mg, 54% yield) was obtained as an oily substance and used without further purification. Reversed-phase HPLC analysis: 97% AUC at tR ) 7.25 min. TLC Rf (methanol/dichloromethane, 1/9): 0.40. 1H NMR (DMSOd6): δ ) 1.6 (d, 1H, J ) 11.6 Hz, CH), 2.0 (dt, 1H, J ) 5.1 Hz, 12.4 Hz, CH), 2.2 (m, 2H, CH2), 2.3 (s, 3H, CH3), 2.5 (dd, 1H, J ) 4.0 Hz, 12.0 Hz, CH), 2.7 (s, 1H, CH), 2.9 (d, 1H, J ) 18.8 Hz, CH), 3.3 (m, 1H, CH), 3.7 (s, 3H, -OCH3), 5.0 (d, 1H, J ) 6.8 Hz, CH), 5.2 (m, 1H, CH), 5.5 (d, 1H, J ) 10.0 Hz, CH), 5.6 (d, 1H, J ) 9.6 Hz, CH), 6.0 (d, 1H, J ) 10.4 Hz, CH acryl), 6.2 (m, 1H, CH acryl), 6.4 (d, 1H, J ) 16.4 Hz, CH acryl), 6.5 (d, 1H, J ) 8.0 Hz, CH aromatic), 6.6 (d, 1H, J ) 8.4 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 19.9, 34.7, 42.1, 42.6, 46.0, 56.3, 58.0, 68.3, 87.6, 114.1, 119.0, 127.2, 127.9, 128.1, 130.1, 130.7, 131.9, 141.4, 146.2, 164.8 ppm. HRMS calculated for C21H23NO4 (M + H+): 354.1700; found: 354.1631. Synthesis of Methacryl Codeine (10). Compound 10 was prepared as described for compound 5 using codeine free base (800 mg, 2.67 mmol), triethylamine (895 µL, 6.41 mmol), dimethylaminopyridine (33 mg, 0.27 mmol), and methacryloyl chloride (573 µL, 5.87 mmol). Methacryl codeine (10, 627 mg, 64% yield) was obtained as an oily substance and used without further purification. Reversed-phase HPLC analysis: 94% AUC at tR ) 7.35 min. TLC Rf (methanol/dichloromethane, 1/9): 0.40. 1 H NMR (DMSOd6): δ ) 1.5 (d, 1H, J ) 19.2 Hz, CH), 1.9 (s, 3H, CH3), 2.0 (dt, 1H, J ) 4.8 Hz, 12.4 Hz, CH), 2.2 (m, 2H, CH2), 2.3 (s, 3H, CH3), 2.4 (m, 1H, CH), 2.7 (t, 1H, J ) 2.4 Hz, CH), 2.9 (d, 1H, J ) 18.8 Hz, CH), 3.3 (q, 1H, J ) 2.9 Hz, CH), 3.7 (s, 3H, -OCH3), 5.0 (d, 1H, J ) 6.4 Hz, CH), 5.2 (m, 1H, CH), 5.5 (d, 1H, J ) 9.6 Hz, CH), 5.6 (d, 1H, J ) 11.6 Hz, CH), 5.7 (s, 1H, CH acryl), 6.0 (s, 1H, CH acryl), 6.5 (d, 1H, J ) 8.0 Hz, CH aromatic), 6.6 (d, 1H, J ) 8.4 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 17.9, 20.9, 32.9, 37.7, 41.3, 43.5, 46.0, 56.4, 58.6, 67.7, 87.0, 114.6, 119.5, 124.7, 125.7, 126.1, 128.5, 128.8, 135.7, 137.1, 141.8, 146.1, 165.9 ppm. HRMS calculated for C22H25NO4 (M + H+): 368.1857; found: 368.1795. Synthesis of Mono-Acryl Morphine (11). Morphine free base (100 mg, 0.35 mmol) was placed in a 50 mL round-bottom flask and anhydrous pyridine (5 mL) was added under a nitrogen atmosphere and allowed to stir for 15 min. Acrylic anhydride (85 µL, 0.74 mmol) was added and the reaction mixture was stirred at room temperature overnight under nitrogen. After completion of the reaction, as monitored by TLC, MeOH (100

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µL) with hydroquinone (60 ppm) was added. The crude product was obtained by azeotropic removal of pyridine with toluene under reduced pressure. The desired product was subsequently purified by silica gel flash chromatography, using a gradient of methanol in dichloromethane (5%). Monoacryl morphine (11, 67 mg, 57% yield) was obtained as a white waxy solid. Reversed-phase HPLC analysis: 90% AUC at tR ) 6.54 min. TLC Rf (methanol/dichloromethane, 1/4): 0.56. 1H NMR (DMSOd6 + D2O): ) 1.5 (d, 1H, J ) 12.0 Hz, CH), 1.9 (dt, 1H, J ) 4.3 Hz, 12.4 Hz, CH), 2.1 (m, 2H, CH2), 2.2 (s, 3H, CH3), 2.3 (s, 1H, CH), 2.5 (dd, 1H, J ) 4.2 Hz, 12 Hz, CH), 2.8 (d, 1H, J ) 17.6 Hz, CH), 3.3 (s, 1H, CH), 4.0 (s, 1H, CH), 4.6 (d, 1H, J ) 6.0 Hz, CH), 5.1 (d, 1H, J ) 9.6 Hz, CH), 5.4 (d, 1H, J ) 9.6 Hz, CH), 5.6 (d, 1H, J ) 10.4 Hz, CH acryl), 6.0 (m, 1H, CH acryl), 6.3 (m, 1H, CH acryl), 6.4 (d, 1H, J ) 8.4 Hz, CH aromatic), 6.6 (d, 1H, J ) 8.0 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): ) 21.2, 34.7, 42.4, 43.0, 46.0, 58.3, 66.4, 92.9, 119.4, 121.8, 127.6, 128.3, 130.9, 131.1, 132.2, 132.9, 133.8, 150.1, 163.9 ppm. HRMS calculated for C20H21NO4 (M + H+): 340.1544; found: 340.1535. Synthesis of Di-Acryl Morphine (12). Compound 12 was prepared as described for compound 11 using morphine free base (1 g, 3.51 mmol), and acrylic anhydride (2.02 mL, 17.55 mmol). Diacryl morphine (12, 887 mg, 64% yield) was obtained as a white waxy solid. Reversed-phase HPLC analysis: 95% AUC at tR ) 8.55 min. TLC Rf (methanol/dichloromethane, 1/4): 0.73. 1H NMR (DMSOd6): δ ) 1.6 (d, 1H, J ) 11.6 Hz, CH), 2.1 (dt, 1H, J ) 4.5 Hz, 12.4 Hz, CH), 2.2 (t, 1H, J ) 12.0 Hz, CH), 2.3 (m, 1H, CH), 2.34 (s, 3H, CH3), 2.5 (m, 1H, CH), 2.8 (s, 1H, CH), 3.0 (d, 1H, J ) 18.8 Hz, CH), 3.3 (s, 1H, CH), 5.1 (d, 1H, J ) 6.8 Hz, CH), 5.2 (s, 1H, CH), 5.5 (d, 1H, J ) 10.0 Hz, CH), 5.6 (d, 1H, J ) 10.4 Hz, CH), 5.8 (d, 1H, J ) 9.6 Hz, CH acryl), 6.0 (d, 1H, J ) 10.4 Hz, CH acryl), 6.1 (m, 1H, CH acryl), 6.3 (m, 2H, CH acryl), 6.5 (d, 1H, J ) 17.2 Hz, CH acryl), 6.6 (d, 1H, J ) 8.0 Hz, CH aromatic), 6.8 (d, 1H, J ) 8.0 Hz, CH aromatic) ppm. 13C NMR (DMSOd6): δ ) 20.3, 34.3, 42.0, 42.5, 46.0, 58.0, 67.7, 88.1, 115.6, 119.3, 121.6, 127.2, 127.9, 129.8, 130.9, 131.5, 131.8, 132.7, 133.6, 148.8, 163.1, 164.7 ppm. HRMS calculated for C23H23NO5 (M + H+): 394.1649; found: 394.1632. Synthesis of HA-Drug Conjugates (14). The HA conjugates with naloxone, codeine, and morphine were prepared following a procedure described previously (23). The carboxyl group of HA (1.3 million MW) was reacted with 3-nitro-2-pyridinesulfenylethylamine (NEA) (29, 30) using carbodiimide-mediated coupling to provide HA-NEA with 10% degree of molar modification. To a solution of HA (1.5 g, 3.74 mmol) in water (375 mL) was added 1-hydroxybenzotriazole (1.0 g, 7.48 mmol) followed by NEA (1.5 g, 5.61 mmol). The pH of the reaction mixture was adjusted to 4.5 using 2 N HCl. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (2.15 g, 11.22 mmol) was added, and the reaction was allowed to stir at room temperature for 2 h. The resulting HA-NEA was purified by exhaustive dialysis using a 7 kDa MWCO membrane against PBS, followed by dialysis against water overnight. HA-NEA was recovered by lyophilization as yellow foam (95% yield). Molecular weight and degree of modification of carboxy-modified HA-NEA were determined by SECMALLS analysis with detection at 350 nm. HA carrying a free sulfhydryl (13, HA-SH) was obtained after dissolving HA-NEA (1.5 g, 3.54 mmol) in a 25 mM solution of tris-2-(carboxyethyl)phosphine hydrochloride (375 mL) followed by stirring at 4 °C for 4 h. The free sulfhydryl was purified by dialysis against succinate buffer at pH 4 followed by water. The retentate was lyophilized to give a white solid (80% yield). Each HA-drug conjugate was assembled by reacting 13 with the corresponding drug intermediate (5-12) illustrated in Figure 1. Thus, aliquots

Gianolio et al.

of 13 (1.0 g, 2.50 mmol) were dissolved in water (250 mL) followed by pH adjustment to 8 using triethanolamine. One molar equivalent of each intermediate 5-12, per molar equivalent of free sulfhydryl (0.25 mmol), dissolved in dimethylformamide was added and the reaction solution was allowed to stir at 4 °C overnight. The consumption of the free sulfhydryl groups was monitored colorimetrically (Ellman’s reagent) (31, 32). The HA-drug conjugates were isolated after dialysis (7 kDa MWCO membrane) against PBS followed by water, lyophilized, and recovered as white foam (95% yield). MW (1.3 million) and degree of molar modification (10%) was consistent for all conjugates. Degree of molar modification was determined by UV/visible spectroscopy with detection at 280 nm, using naloxone, codeine, and morphine, respectively, as standards. Synthesis of Hylan B-Morphine (15). Morphine was conjugated to hylan B as described above. The synthesis was performed starting from sterile hylan B particles and conducted under aseptic conditions. hylan B particles (371 mL, 5.4 mg/ mL in 0.9% NaCl solution) were functionalized with NEA, and reduced to the free sulfhydryl form. Degree of molar modification was 8% as determined colorimetrically using Ellman’s reagent (31, 32). Hylan B particles carrying free sulfhydryl groups were reacted with a half-molar equivalent of 12 per free sulfhydryl molar equivalent. The hylan B-morphine conjugate particles (15) were transferred into a 7 kDa MWCO membrane and dialyzed at 4 °C against PBS overnight, followed by dialysis against water for 6 h. The retentate was lyophilized to give a white solid product (2.0 g, 98% yield). Degree of molar modification was 8% as determined by UV/visible spectroscopy with detection at 280 nm, using morphine as a standard. In Vitro Studies, Release Rate of Drugs in PBS. The in Vitro release rates of drugs (infinite sink) from compounds 14 and 15 incubated in PBS were compared to admixtures of free drugs in a HA and hylan B solution, respectively. To prepare the test samples, compounds 14 and 15 were dissolved at 5% w/w in distilled water. The admixed control samples were prepared by dissolving one part naloxone, codeine, and morphine, respectively, in 99 parts of a 5% aqueous solution of HA (1.3 million MW) and Hylan B. Aliquots (approximately 1 to 1.5 g) of each solution were injected in triplicate into tangential flow dialysis cassettes (10 kDa MWCO). The cassettes were placed into separate screw-cap jars containing PBS (250 mL) at 37 °C with stirring. Samples of the receptor phase (1 mL of the elution medium) were removed over time and analyzed by HPLC. Release Rate of Morphine in Rat Plasma. The in Vitro release rate of morphine from compound 15 incubated in rat plasma was compared to the release rate in plasma in the presence of a cocktail of protease inhibitors with broad specificity for the inhibition of serine, cysteine, aspartic proteases, and aminopeptidases. To prepare the test samples, compound 15 was dissolved at 0.024% w/w in rat plasma, together with sodium azide (0.06% w/w). Protease inhibitors were added to an aliquot of the mixture and samples were incubated at 37 °C with gentle mixing. Fifty microliter aliquots were collected over time, 200 µL of acetonitrile containing 100 ng/mL naloxone as the internal standard were added, and samples were centrifuged. The supernatant was subjected to LC-MS/MS analysis of morphine, monoacryl morphine, and diacryl morphine. Pharmacokinetic (PK) Profile of Morphine in a Rat Surgical Model. A total of 32 male Sprague-Dawley rats weighing 250 to 300 g were acclimated with free access to food and water for a minimum of one week prior to the performance of the experimental procedures. Deionized water was available ad libitum throughout the study period. Rats were allocated to treatment groups (Table 1), 8 rats per group, based on body weights taken during the acclimation period. The mean weights for each group were matched to ensure that the mean values

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Figure 1. Synthesis of drug intermediates (5-12), HA-SH intermediate (13), HA-drug conjugates (14), and hylan B-morphine conjugate (15). Table 1. Amounts of Free Morphine and Morphine from Conjugate 15 Injected Subcutaneously in Each Rata group 1 2 3 4

sample

dose

morphine 5 mg/kg saline vehicle control 0 mg/kg Hylan B morphine/admix (nonconjugated) 5 mg/kg Hylan B-morphine conjugate (15) 146 mg/kg

dose volume 5 mL 5 mL 5 mL 5 mL

a N ) 8 for each group. Morphine amounts are expressed as morphine sulfate pentahydrate (FW ) 758.83).

and standard deviation satisfy the assumption of homogeneity. At time point 0, the animals were briefly sedated under inhalation anesthesia induced with 2-3% isoflurane in 100% oxygen, the dorsal area was shaved, a small skin incision was made, and blunt scissors were used to create a subcutaneous pocket. Five milliliters of either a control solution or a test formulation was injected into two sites (caudal and rostral from the incision), and the incision was closed with a wound clip. The experimenter was blinded as to which formulation was injected. At time points 1, 4, 8, 24, and 30 h, the animals were briefly anesthetized with inhalation anesthetic and approximately 250 µL of blood was collected via the retro-orbital sinus. The

blood was collected into tubes containing EDTA as anticoagulant and centrifuged. Following centrifugation, the plasma was collected and transferred into separate tubes. The concentration of morphine in the plasma over time was analyzed by LC-MS/ MS. Control animals were injected with 5 mg/kg morphine (i.e., morphine sulfate pentahydrate) either as a solution of free morphine or as an admixture of Hylan B gel particles and free morphine. This dose was based on previous experience that showed adequate levels of analgesia in this model without general sedation (33-35). The treatment group (Hylan Bmorphine conjugate) used a ca. 30-fold higher morphine loading (146 mg/kg) compared to the controls. Samples were analyzed by LC-MS/MS as described above.

RESULTS AND DISCUSSION Synthesis of HA-Drug Conjugates. Morphine possesses two functional groups (i.e., allylic and phenolic hydroxyl groups) that can be acrylated to form an ester. The hydrolytic cleavage of the ester bonds in the resulting HA-morphine conjugate (14) regenerates authentic morphine and releases it from the polymer at a slower rate compared to morphine admixed in HA. Further, it may be possible to modulate the rate of hydrolysis by

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Figure 2. Release profiles of free and HA-conjugated naloxone (14) into receptor phase. N ) 3, bars are SD. Receptor phase is defined as the elution medium (PBS) that is sampled to quantify the drug released. Conjugates were prepared using intermediates 5-8. Increasing size of the R group (H, Me, Et, Pr) vicinal to the ester bond resulted in slower release rates.

increasing the size of the alkyl functional groups (i.e., R ) Me, Et, and Pr) in vicinal position to the ester bond. With these possibilities in mind, we studied the effects of acylation site and vicinal R group on the rate of hydrolysis using HA-naloxone and HA-codeine conjugates as structural controls. Using the approach described in Figure 1, naloxone, codeine, and morphine were conjugated to HA (14) with a 10% degree of molar modification and an overall mass recovery ranging between 80% and 90%. To avoid premature hydrolysis during workup and consequent loss of naloxone, codeine, and morphine from the conjugates, PBS and water used for dialysis were chilled to 4 °C ((2 °C). Release Rate of Drugs in PBS. Figure 2 shows the in Vitro release profiles obtained from the naloxone conjugates. Naloxone hydrochloride admixed in HA quickly eluted into the receptor phase, with 100% of the drug eluting within 5 h. In contrast, the HA-naloxone R ) H conjugate (14), obtained using intermediate 5, showed more prolonged release with 100% of the naloxone eluting within 2 days. In agreement with our expectations, delivery of naloxone was prolonged even further when R ) Me, obtained using intermediate 6, with half of the drug eluting within 12 h and 100% of the drug eluting within 3 days. For R ) Et, obtained using intermediate 7, half of the drug eluted within 3-4 days and 100% of the drug eluted within 16 days. A parallel release profile was observed for R ) Pr, obtained using intermediate 8, indicative of comparable steric protection of the ester bond by Et and Pr groups. It is known that steric hindrance of the substituents around the tetrahedral transition state influence the rate of ester hydrolysis (36). In particular, less sterically hindered putative tetrahedral intermediates are more stable. This greater stability leads to a higher equilibrium concentration and, in turn, a faster hydrolysis rate. The release profiles followed approximately a first-order rate consistent with the expected pseudo-first-order hydrolysis reaction. Similar results were observed for the HA-codeine conjugates (see Figure 3). Codeine admixed in HA eluted into the receptor phase, with 100% of the drug eluting within 5 h. HA-codeine R ) H conjugate, obtained using intermediate 9, showed prolonged delivery with half the drug eluting within 14 days and 100% of the drug eluting within 55 days. Codeine delivery was significantly slower for R ) Me, obtained using intermediate 10, with only half of the drug eluted within 55 days. Figure 4 shows a comparison of the release profiles for the HA-naloxone and codeine conjugates described above and

Gianolio et al.

Figure 3. Release profiles of free and HA-conjugated codeine (14) into receptor phase. N ) 3, bars are SD. Conjugates were prepared using intermediates 9 and 10. Increasing size of the R group (H, Me) vicinal to the ester bond resulted in slower release rates.

Figure 4. Release profiles of HA conjugated naloxone, codeine, and morphine (14) into receptor phase. N ) 3, bars are SD. Conjugates were prepared using intermediates 5, 9, and 12, respectively. Allylic leaving group (codeine and morphine) resulted in a slower release rate relative to phenolic leaving group (naloxone). Table 2. Half-Lives (days) for HA-Naloxone, Codeine, and Morphine Conjugates (14) in PBS Determined by HPLC Using an Infinite Sink sample HA admixture conjugate (14), conjugate (14), conjugate (14), conjugate (14),

R R R R

) ) ) )

H Me Et Pr

naloxone

codeine

morphine

0.1 0.3 0.5 4.0 4.0

0.1 14.0 55.0 -

0.1 14.0 -

HA-morphine conjugate obtained using intermediates 5, 9, and 12, respectively. The naloxone phenolic was a much better leaving group than the codeine allylic group. HA-morphine conjugate possessed both phenolic and allylic ester groups, functioning also as HA cross-linker. However, the release profile of morphine approximately overlapped with the release profile of codeine, indicating that the rate-limiting step was the hydrolysis of the allylic ester. Half-life values in days observed for all HA conjugates are summarized in Table 2. PK Profile of Morphine in a Rat Surgical Model. Enzymatic and nonenzymatic release of steroid drugs conjugated through ester bonds to HA were reported in the literature (37, 38). Release rates in ViVo using healthy animals, and in biological fluids obtained from healthy humans, were reported to be consistent with the release rates observed in aqueous buffered

Hyaluronan-Tethered Opioid Depots

Figure 5. Pharmacokinetics in a rat surgical model. N ) 8, bars are SD. Conjugation resulted in prolonged release relative to free morphine and morphine hylan B admix. Group 1 ) free morphine (5 mg/kg), group 2 ) saline, group 3 ) morphine (5 mg/kg) and hylan B admix, group 4 ) morphine (146 mg/kg) conjugated to hylan B (15).

solutions. With this in mind, we performed an in ViVo study using a rat surgical model and the hylan B-morphine conjugate (15). Release rate of morphine from compound 15 was determined in plasma by LC-MS/MS after subcutaneous administration. The release rate was compared to a physical admixture of morphine in hylan B and bolus injection of morphine. The treatment group (compound 15) used a ca. 30fold higher morphine loading compared to the controls as the conjugate exhibited sustained release of morphine; a higher loading would be required to maintain the plasma morphine concentrations within the therapeutic window over time. The loading of morphine in the treatment group would also ensure that the plasma drug concentrations would be detectable by LCMS/MS over the course of the study. Figure 5 shows the release profiles obtained from this experiment. Rapid clearance from the rats was observed for morphine sulfate admixed with hylan B and bolus injection of morphine sulfate with minimal to no detectable levels of the drug within 5 h. In agreement with our expectations, compound 15 resulted in sustained release of morphine with a significant level of morphine (ca. 400 ng/mL) detected after 30 h. The rats were observed during the course of the PK study. Approximately 4 h post-dosing animals injected with compound 15 were hypothermic with body temperature of ca. 30 °C, and showed nonlocomotor, catatonic behavior. Heating pads were used to warm up the animals, and within one hour, their body temperature returned to normal (37.5 °C), and normal locomotor activity was observed. Twenty-four hours post-dosing, the rats performed continuous grooming activity and repetitive jaw movements. These observations were consistent with the effects of high-dose narcotic anesthesia reported in the literature (39-42). Enzymatic and Non-Enzymatic Release Rates of Morphine. Morphine overdosing was unexpected according to our calculations based on the release profiles obtained in PBS. An additional in Vitro study in enzymatic and non-enzymatic conditions was conducted to study the potential for nonspecific esterases to increase the release rate of morphine in ViVo from the hylan B-morphine conjugate (15) obtained using intermediate 12. Figure 6 shows a comparison of the release profiles for compound 15, in PBS as determined by HPLC and in rat plasma as determined by LC-MS/MS. Morphine was the only species detected in the plasma samples confirming that there was no monoacryl morphine (11) and diacryl morphine (12) remaining from the conjugation reaction. Morphine admixed in hylan B eluted into the receptor phase, with 100% of the drug eluting within 5 h. hylan B-morphine conjugate eluted in PBS showed prolonged delivery with half the drug eluting within 14 days

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Figure 6. Comparison of release profiles of free and hylan B conjugated morphine (15) into receptor phase (PBS and in the presence of plasma). N ) 3, bars are SD. Solid lines are the calculated slopes based on early data points. Conjugate was prepared using intermediate 12. Faster release rate of morphine was observed in the presence of plasma without protease inhibitors. Release rate in plasma in the presence of protease inhibitors is similar to the release rate observed in PBS only.

and 100% of the drug eluting within 45 days. These release profiles were consistent with those observed for compound 14, obtained using intermediate 12. Interestingly, the release of morphine from the same conjugate was faster in plasma than in PBS with half the drug eluting within 2.5 days and 100% of the drug eluting within 10 days. However, the release profile of morphine from the hylan B conjugate in plasma in the presence of protease inhibitors approximately overlapped with the release profile in PBS (see Figure 6), indicating that, in rat plasma samples, morphine release could be influenced by nonspecific esterase activity. The release rates of morphine observed from hylan B conjugate in plasma in Vitro, and during a pharmacokinetic study using rats, indicated that the system may be capable of providing sustained delivery of the drug in ViVo. However, after observing the behavior of the animals consistent with narcotic overdosing, it will be necessary to take into account the nonspecific esterase activity of biological fluids together with the optimum dosing range to conceive of a new drug conjugate formulation. The ability to obtain sustained release of morphine in Vitro and in ViVo shown in this work is encouraging. This study identified a potential additional utility of HA as a drug carrier and deserves further investigation.

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