Investigation of (Oxodioxolenyl)methyl carbamates as nonchiral bioreversible prodrug moieties for chiral amines.

The preparation of (oxodioxolenyl)methyl carbamates and their evaluation as novel nonchiral prodrug moieties for chiral primary and secondary amino functional drugs are described. 4-(Carbamoylmethyl)-2-oxo-1,3-dioxolene derivatives of 3,4-dimethoxyphenethylamine with 5-methyl, 5-phenyl, and 5-anisyl substitution (5a, 5b, and 5c) on the dioxolenone ring were prepared as model amine prodrugs by a one step process involving displacement of p-nitrophenol from appropriately substituted ring opening of these carbamates led to a cascade reaction resulting in the rapid and quantitative regeneration of the parent amine drug. Aryl substitution did not significantly alter the hydrolysis rates of these dioxolenone carbamates in buffers at pH 1 and 7.4 or in rat plasma, although the hydrolysis rates of 5-phenyl- (1b) and 5-anisyl- 4-methyl-1,3-dioxol-4-en-2-one (1c) in pH 7.4 phosphate buffer were 2-3 fold faster than that of the 5-methyl-substituted analog (1a). Application of this prodrug strategy to the chiral fibrinogen receptor antagonist L-734,217 resulted in a prodrug that gave quantitative reconversion in rat and dog plasma in vitro and oral bioavailability of 23 +/- 6% in dogs for the parent drug.

Degradation of O6-benzylguanine in aqueous polyethylene glycol 400 (PEG 400) solutions: concerns with formaldehyde in PEG 400.

The degradation of O6-benzylguanine (BG) in aqueous polyethylene glycol (PEG) 400 solution at room temperature had been investigated using chromatographic and spectrometric methods. The degradation of BG in this solvent appeared to arise from a reaction between BG and formaldehyde. The formaldehyde was present as an impurity in PEG 400 and probably formed through air oxidation of PEG 400. The major product of this reaction was believed to be a methylene-bridged compound containing two BG molecules. This was probably produced via an intermediate imine, a schiff base between one BG molecule and formaldehyde. This degradation reaction was the only observable reaction in the 40% PEG/water solvent (pH 8.0) i.e. degradation of the drug via hydrolysis was minimal under these conditions.

Urinary and biliary disposition of the lactone and carboxylate forms of 20(S)-camptothecin in rats.

Recently, analytical methods have become available for determination of both the lactone (active form) and the carboxylate (inactive form) forms of 20(S)-camptothecin in biological fluids. Studies in our laboratory have shown that there are significant differences in the in vivo behavior of the two forms of camptothecin and that much higher plasma levels of the lactone form are present in rats after dosing with camptothecin (lactone) than after dosing with the sodium salt of the ring-opened camptothecin (carboxylate form). The present studies show that there are significant differences in the urinary and biliary elimination of the two forms and that the urinary excretion of the carboxylate form appears to be pH dependent. This apparent pH dependence of the urinary elimination of the carboxylate form may provide a method of reducing the bladder toxicity associated with the use of camptothecin. After administration of a 1 mg/kg iv dose of camptothecin (lactone) to rats, 10.1 +/- 4.2% of the dose was excreted into the urine and 7.5 +/- 4.2% of the dose was excreted into the bile. Following an equivalent intravenous dose of the carboxylate form, 39.5 +/- 10.4% of the dose was excreted into the urine and 26.4 +/- 8.9% of the dose was excreted into the bile.

Plasma pharmacokinetics of lactone and carboxylate forms of 20(S)-camptothecin in anesthetized rats.

20(S)-Camptothecin exists in equilibrium between its lactone (CPT) and its carboxylate forms (Na-CPT) under stimulated physiological conditions, with the equilibrium favoring the carboxylate form. The rates of lactone hydrolysis were studied in plasma, serum albumin, and blood and were found to be faster than in aqueous buffers at equivalent pH values. From mechanistic information and in vivo activity data, the lactone appears to be the active form of the drug. It has been argued, therefore, that if an equilibrium existed between the lactone and the carboxylate, Na-CPT could be used to deliver the lactone effectively. In the present study, plasma pharmacokinetics were performed in sodium pentobarbital-anesthetized rats treated with both CPT (lactone) and the sodium salt of camptothecin (carboxylate, Na-CPT) and the lactone and carboxylate, as well as the total drug, concentration versus time profiles were assessed. It was found that plasma concentrations and AUC values for the lactone were significantly higher after dosing with CPT than after dosing with Na-CPT. After i.v. administration, the ratio of plasma lactone to carboxylate was skewed by the apparent rapid and extensive uptake of the lactone into tissues and the rapid clearance of both species. From our results, it appears that the lower in vivo activity of Na-CPT compared to that from CPT administration might be attributed to the altered conversion of carboxylate into lactone in vivo compared to that predicted from in vitro data.

Towards continuous glucose monitoring: in vivo evaluation of a miniaturized glucose sensor implanted for several days in rat subcutaneous tissue.

A miniaturized amperometric, enzymatic, glucose sensor (outer diameter 0.45 mm) was evaluated after implantation in the subcutaneous tissue of normal rats. A simple experimental procedure was designed for the long-term assessment of the sensor's function which was performed by recording the current during an intraperitoneal glucose load. The sensor was calibrated by accounting for the increase in the current during the concomitant increase in plasma glucose concentration, determined in blood sampled at the tail vein. This made it possible to estimate the glucose concentration in subcutaneous tissue. During the glucose load, the change in subcutaneous glucose concentration followed that in blood with a lag time consistently shorter than 5 min. The estimations of subcutaneous glucose concentration during these tests were compared to the concomitant plasma glucose concentrations by using a grid analysis. Three days after implantation (n = 6 experiments), 79 estimations were considered accurate, except for five which were in the acceptable zone. Ten days after implantation (n = 5 experiments), 101 estimations were accurate, except for one value, which was still acceptable. The sensitivity was around 0.5 nA.mmol-1.l-1 on day 3 and day 10. A longitudinal study on seven sensors tested on different days demonstrated a relative stability of the sensor's sensitivity. Finally, histological examination of the zone around the implantation site revealed a fibrotic reaction containing neocapillaries, which could explain the fast response of the sensor to glucose observed in vivo, even on day 10. We conclude that this miniaturized glucose sensor, whose size makes it easily implanted, works for at least ten days after implantation into rat subcutaneous tissue.

Design and in vitro studies of a needle-type glucose sensor for subcutaneous monitoring.

A new miniaturized glucose oxidase based needle-type glucose microsensor has been developed for subcutaneous glucose monitoring. The sensor is equivalent in shape and size to a 26-guage needle (0.45-mm o.d.) and can be implanted with ease without any incision. The novel configuration greatly facilitates the deposition of enzyme and polymer films so that sensors with characteristics suitable for in vivo use (upper limit of linear range greater than 15 mM, response time less than 5 min, and sensitivity yielding a 5:1 signal-to-background ratio at normal basal glucose levels) can be prepared in high yield (greater than 60%). The sensor response is largely independent of oxygen tension in the normal physiological range. It also exhibits good selectivity against common interferences except for the exogenous drug acetaminophen.

In vitro and in vivo evaluation in dogs of a miniaturized glucose sensor.

A miniaturized glucose sensor was developed, consisting of a platinum wire entirely coated with teflon, except for a 1 mm section near its extremity where glucose oxidase is immobilized. The in vitro sensitivity to glucose of the sensors was 2.3 +/- 0.4 nA/mM, mean +/- SEM (n = 23). These sensors were implanted in the subcutaneous tissue of normal beagles. Two consecutive glucose infusions (15-30 mg/kg/min) were performed. The current generated by the sensor was used for calculation of the sensitivity coefficient (SC) (nA/mM), and the background current in the absence of glucose (lo) (nA). These parameters were used for determination of the apparent subcutaneous glucose concentration. The in vivo sensitivity was less than the in vitro sensitivity (0.5 +/- 0.1 vs. 2.2 +/- 0.2; n = 12 comparisons; p less than 0.01). Stability of sensor function was demonstrated by the absence in variation of SC and lo, calculated from the different plateaus obtained during the glucose infusions. This study provides a simple method for evaluating in vivo the function of a miniaturized sensor implanted in subcutaneous tissue.

Application of cell culture toxicity tests to the development of implantable biosensors.

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate buffer, and a non-toxic sensor was readily obtained.

Pulsed amperometric detection of glucose in biological fluids at a surface-modified gold electrode.

A nonenzymatic glucose sensor that utilizes permselective membranes to achieve the selectivity required for screening glucose in biological fluids has been described. Interference from endogenous oxidizable substances such as amino acids, urea, ascorbic acid, and uric acid, as well as the effect of chloride and proteins on glucose response, is studied by using flow injection analysis. A set of membranes made of Naflin perfluorinated membrane and collagen, when arranged in front of the working electrode (gold), result in significant improvement in the system selectivity. Even at physiological pH, which is far from being the optimum pH for pulsed amperometric detection of carbohydrates, the sensor shows a good limit of detection (4-5 micrograms of glucose injected).

Study and development of multilayer needle-type enzyme-based glucose microsensors.

Glucose oxidase (GOD) was covalently coupled to a cellulose acetate (CA) layer, using bovine serum albumin (BSA) and parabenzoquinone (PBQ) linkages. Prior to GOD coupling this CA layer was deposited on the platinum tip of a needle-type sensor and covered with an outer layer of polyurethane (PU). Such microsensors were found to be active, their GOD load reaching 1.6 to 3.0 micrograms mm-2 and their glucose response reaching 1 to 3 microA M-1 mm-2, even when the upper limit of their linear range reached 10-30 mM. Due to the multilayer structure and composition of these microsensors, small anions such as ascorbate were partially discriminated from neutral molecules such as hydrogen peroxide. When implanted subcutaneously in anaesthetized rats, sensor responses correlated correctly with blood glucose concentration but presented sensitivity coefficients significantly different to those determined in vitro: a 2 point calibration procedure was found necessary for in vivo experiments.