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.

Straight-chain naltrexone ester prodrugs: diffusion and concurrent esterase biotransformation in human skin.

Naltrexone (NTX) is an opioid antagonist used for treatment of narcotic dependence and alcoholism. Transdermal naltrexone delivery is desirable to help improve patient compliance. The purpose of this study was to increase the delivery rate of NTX across human skin by using lipophilic alkyl ester prodrugs. Straight-chain naltrexone-3-alkyl ester prodrugs of 2-7 carbons in chain length were synthesized and evaluated. In vitro human skin permeation rates were measured using a flow-through diffusion cell system. The melting points, solubilities, and skin disposition of the drugs were determined. The prodrugs were almost completely hydrolyzed on passing through the skin and appeared as NTX in the receiver compartment. The mean NTX flux from the prodrug-saturated solutions exceeded the flux of NTX base by approximately 2-7-fold. The amount of drug detected in the skin was significantly greater after treatment with the prodrug solutions compared with treatment with NTX base. The extent of parent drug (NTX) regeneration in the intact skin ranged from 28 to 91%. Higher NTX regeneration percentages in skin appeared to correlate with increased drug delivery rates. Definitively, the highly oil-soluble prodrugs provide a higher NTX flux across human skin in vitro and undergo significant metabolic conversion in the skin.

Purification of human monocyte-specific esterase (MSE): molecular and kinetic characteristics.

Human monocyte-specific esterase (MSE) derived from leukaemic AMoL-M5 blast cells was purified to homogeneity by the sequential application of anion-exchange, hydrophobic interaction, affinity and gel filtration chromatographic procedures. The resulting enzymatically active MSE primarily existed as an apparent trimer which, under both reducing and non-reducing conditions, dissociated to an inactive 63.4 kD glycoprotein monomer. Electrophoretic studies further confirmed that purified MSE comprised a narrow series of pI (5.5-6.1) forms and one main charge species. Neuraminidase failed to modify observed pI values for individual MSE isoenzymes, and endoglycosidase H treatment revealed that the deglycosylated form of MSE had an apparent molecular weight of 60.1 kD. In support of the known cytochemical characteristics of human MSE, substrate kinetic studies demonstrated that purified enzyme hydrolysed esters of higher acyl chain length (butyrate > propionate > acetate) but did not show peptidase activity. Amino acid sequencing of the MSE N-terminus further revealed that there was almost complete identity with human alveolar macrophage esterase and close similarities with rat and rabbit liver carboxylesterases. These kinetic and molecular studies are particularly important in elucidating the biological and functional role(s) of one of the few haemopoietic cell enzymes that can be considered truly lineage-specific.

Biochemical characterization of flestolol esterase.

The blood esterase that mediates the metabolism of flestolol, an ultra short-acting beta blocker, was characterized. Esterase activity occurred in plasma of human, dog, rat, and guinea pig and not in erythrocytes of the same species. The esterase activity was greatest in humans and guinea pigs followed by dogs and rats. Purified human serum cholinesterase was very active against flestolol while human serum albumin was slightly active. Human and bovine erythrocyte membrane acetylcholinesterases, electric eel acetylcholinesterase, human hemoglobin, dog, rat, chicken, and bovine serum albumin were all inactive. Esterase activity with flestolol was inhibited in human, dog, and rat blood by echothiophate, eserine, and sodium fluoride. Guinea pig blood esterase activity was inhibited by echothiophate and sodium fluoride, but not by eserine. Metabolic interaction studies indicated that succinylcholine, procaine, and chloroprocaine interfere with the metabolism of flestolol in human blood. Succinylcholine prolonged the in vitro half-life of flestolol in dog blood, but acetylcholine, procaine, and chloroprocaine had no effect. Flestolol did not affect the metabolism of procaine or chloroprocaine in human and dog blood. The metabolism rate of flestolol decreased in individuals with atypical, fluoride-resistant and silent forms of serum cholinesterase.

Substituted coumarins as esterase-sensitive prodrug moieties with improved release rates.

Our laboratory has recently reported a coumarin-based prodrug system for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. However, the release from this prodrug system was undesirably slow for some drug moieties. In this report, we describe the synthesis and evaluation of several substituted coumarin-based prodrugs of model amines with significantly increased release rates.

Serum paraoxonase status: a major factor in determining resistance to organophosphates.

A number of lines of evidence suggest that serum paraoxonase is protective against poisoning by organophosphorus substrates of this enzyme. Birds that have very low levels of paraoxon hydrolyzing activity in their sera are very susceptible to parathion poisoning. Rabbits, which have a sevenfold higher enzyme level compared with rats, have a fourfold higher resistance to paraoxon poisoning than rats. Rabbit paraoxonase hydrolyzes chlorpyrifos-oxon with a much higher turnover number than does rat paraoxonase, resulting in a very high resistance of rabbits to chlorpyrifos toxicity. Direct tests of paraoxonase protection have been carried out by injecting purified rabbit enzyme into rats. The protection achieved was higher for chlorpyrifos-oxon than for paraoxon, probably due to the high hydrolytic activity of the rabbit enzyme for chlorpyrifos-oxon. In humans, a substrate-dependent polymorphism of serum paraoxonase is observed, where one isoform of paraoxonase has a high turnover number for paraoxon and the other a low turnover number. Both isoforms appear to hydrolyze chlorpyrifos-oxon and phenylacetate at the same rate. Cloning and sequencing of the human paraoxonase cDNAs has elucidated the molecular basis of the polymorphism. Arginine at position 192 determines high paraoxonase activity, and glutamine at this position, low paraoxonase activity. In addition to the polymorphism, a 13-fold variation in serum enzyme levels within a given genetic class is seen. The experiments reported here demonstrate that rabbit paraoxonase injected into mice provides protection against the parent insecticide chlorpyrifos as well as the toxic oxon. These results suggest that serum paraoxonase status may serve as a biomarker for insecticide susceptibility in humans.

Protection of organophosphate-inactivated esterases with phosphotriesterase.

The protective effect of phosphotriesterase (PTE) on cholinesterase (ChE) and carboxylesterase (CaE) activities was studied in mice. The PTE pretreatment (120 U/g body wt, 9.6 micrograms/g body wt) given i.v. 10 min before diisopropyl fluorophosphate, sarin, or soman variably prevented ChE inhibition in erythrocytes and plasma and CaE in plasma. PTE also protected the brain and lung ChEs against inactivation by organophosphates (OPs). The recovery of the enzymes was dependent on the OP used. Postexposure therapy with PTE, given 1.5 hr after paraoxon, also prevented ChE inhibition in erythrocytes, brain, and lung 24 hr after exposure. The distribution studies with [125I]PTE showed that PTE does not markedly gain access into the central nervous system.