David T.-W. Lau

Applying Bailer's Method for AUC Confidence Intervals to Sparse Sampling

Bailer (1) developed a method for constructing confidence intervals for areas under the concentration-vs-time curve (AUCs) with only one sample per subject but with multiple subjects sampled at each of several time points post dose. We have modified this method to account for estimation of the variances. How the need to estimate variances affects study design is discussed. An extension of Bailers method is proposed where variances are modeled as a function of the means, in order to get more precise estimates of variances. The modified and extended methods are applied to a rat toxicokinetic study with only two rats per time point per treatment group.

Effect of Current Magnitude and Drug Concentration on lontophoretic Delivery of Octreotide Acetate (Sandostatin®) in the Rabbit

The effect of current magnitude and drug concentration on transdermal iontophoretic delivery of octreotide acetate (Sandostatin®) was examined in the rabbit. Plasma samples were collected over 24 hours and octreotide concentrations were determined by a radioimmunoassay. Without an electrical current, negligible plasma concentrations of octreotide were obtained. Following initiation of iontophoresis, plasma concentrations of octreotide increased rapidly, although did not sustain at a plateau level during the dosing period. Octreotide concentrations declined rapidly after removal of the device. Increasing the electrical current from 50 µA/cm2 to 150 µA/cm2 yielded a proportional increase in the delivery. Increasing the drug concentration in the device from 2.5 mg/mL to 5 mg/mL resulted in approximately proportional increase in plasma octreotide concentrations; however, further increase in plasma concentrations was not observed for drug concentrations beyond 5 mg/mL. lontophoretic delivery at the conditions which yielded the highest octreotide concentrations in this study (5 mg/mL solution at 150 µA/cm2 for 8 hours) yielded an apparent bioavailability (which represents an underestimate of the absolute bioavailability determined when the patches are run to exhaustion) of approximately 8%.

Glutathione S-Transferase-Mediated Metabolism of Glyceryl Trinitrate in Subcellular Fractions of Bovine Coronary Arteries

The possible role of glutathione S-transferases (GTSs) in vascular glyceryl trinitrate (GTN) metabolism was investigated. GTN degradation to form its dinitrate metabolites (GDNs) in the 9000g (9k) supernatant fraction of bovine coronary arteries (BCA) was examined. BCAs were homogenized with a 3x volume of phosphate buffer, and the 9k fraction was obtained by centrifugation. GTN (40 ng/ml; 1.76 x 10−7M) was incubated for 2 hr in the 9k fraction of BCA in the presence of reduced glutathione (2 x 10−3M). Samples were taken at 10, 20, 40, 60, and 120 min. GTN was observed to degrade readily, exhibiting a half-life of 26 min in the incubate. While both 1,2- and 1,3-GDNs were generated from GTN, formation of 1,3-GDN was predominant (GDN ratio, as 1,2/1,3-GDN, = 0.7−0.8). Coincubation with 2 x 10−5Mconcentrations of two GST inhibitors, sulfobromophthalein (SBP) and ethacrynic acid (ECA), decreased the rate of GTN loss. The GTN half-lives in SBP- and ECA-treated incubations were 66 and 84 min, respectively. In addition, the pattern of GDN formation was also altered. The resultant GDN ratios exceeded unity in the presence of these inhibitors, indicating that 1,3-GDN formation was attenuated to a greater extent than that of 1,2-GDN. These data suggest that vascular GTN metabolism in BCA is carried out by cytosolic GST isozymes which possess a preference for C-2 denitration of GTN.

Cutaneous Metabolism of Nitroglycerin in Vitro. II. Effects of Skin Condition and Penetration Enhancement

The effects of skin storage, skin preparation, skin pretreatment with a penetration enhancer, and skin barrier removal by adhesive tape-stripping on the concurrent cutaneous transport and metabolism of nitroglycerin (GTN) have been studied in vitro using hairless mouse skin. Storing the skin for 10 days at 4°C did not alter barrier function to total nitrate flux [GTN + 1,2-glyceryl dinitrate (1,2-GDN) + 1,3-glyceryl dinitrate (1,3-GDN)]. However, metabolic function was significantly impaired and suggested at least fivefold loss of enzyme activity. Heating skin to 100°C for 5 min appreciably damaged hairless mouse skin barrier function. The ability to hydrolyze GTN was still present, however, and remained constant over the 10-hr experimental period, in contrast to the “control,” which showed progressively decreasing enzymatic function with time. Pretreatment of hairless mouse skin in vivo (prior to animal sacrifice, tissue excision, and in vitro transport/metabolism studies) with 1-dodecylazacyclo-heptan-2-one (Azone), a putative penetration enhancer, significantly lowered the skin barrier to nitrate flux (relative to the appropriate control). Again, barrier perturbation resulted in essentially constant metabolic activity over the observation period. The ratio of metabolites formed (1,2-GDN/1,3-GDN) was increased from less than unity to slightly above 1 by the Azone treatment. Adhesive tape-stripping gradually destroyed skin barrier function by removal of the stratum corneum. The effects of 15 tape-strips were identical to those of Azone pretreatment: a greatly enhanced flux, a constant percentage formation of metabolites over 10 hr (once again), and an increase in the 1,2-GDN/1,3 GDN ratio. Overall, the experiments caution that, for transdermal drug delivery candidates susceptible to skin metabolism, the status of barrier function (enhancer pretreated, skin damage or disease, etc.) may significantly affect systemic availability.

Cutaneous Metabolism of Nitroglycerin in Vitro. I. Homogenized Versus Intact Skin

The metabolism of nitroglycerin (GTN) to 1,2- and 1,3-glyceryl dinitrate (GDN) by hairless mouse skin in vitro has been measured. In the first set of experiments, GTN was incubated with the 9000g supernatant of fresh, homogenized tissue in the presence and absence of glutathione (GSH), a cofactor for glutathione-S-transferase. After 2 hr of incubation with GSH, 30% of the initially present GTN had been converted to 1,2- and 1,3-GDN; without GSH, less than 5% of the GTN was metabolized. The ratio of 1,2-GDN to 1,3-GDN produced by the homogenate was 1.8– 2.1. In the second series of studies, GTN was administered topically to freshly excised, intact hairless mouse skin in conventional in vitro diffusion cells. The concurrent transport and metabolism of GTN was then monitored by sequential analysis of the receptor phase perfusing the dermal side of the tissue. Three topical formulations were used: a low concentration (1 mg/ml) aqueous solution, a 2% ointment, and a transder-mal delivery system. Delivery of total nitrates (GTN + 1,2-GDN + 1,3-GDN) into the receptor phase was similar for ointment and patch formulations and much greater than that from the solution. The percentage metabolites formed, however, was greatest for the solution (61% and 2 hr, compared to 49% for the patch and 35% for the ointment). As has been noted before, therefore, the relative level of skin metabolism is likely to be greatest when the transepidermal flux is small. Distinct from the homogenate experiments, the 1,2/1,3-GDN ratios in the penetration studies were in the range 0.7– 0.9. It would appear that homogenization of the skin permits GTN to be exposed to a different distribution of enzymes than that encountered during passive skin permeation.

Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites

A sensitive, specific capillary gas chromatographic-electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs greater than 76% and for GTN and the GDNs greater than 95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1.3-GDN maximum concentrations (Cmax) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the Cmax ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN Cmax ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.

Pharmacokinetics of intranasally-administered dihydroergotamine in the rat.

Intranasal dosing of dihydroergotamine (DHE) allows convenient self-administration and provides an alternate route of administration for the treatment of migraine in addition to the existing parenteral dosage forms. In this study, the pharmacokinetics of 3H-DHE were investigated following intravenous and intranasal dosing (0.343 mg DHE/animal) in the rat. Intranasal administration of DHE resulted in rapid absorption. The extent of absorption of the radiolabeled dose was approximately 45%–60%. Absolute bioavailability of the parent drug was 35%–40%, as determined by deconvolution and by the ratios of AUC0−∞ following intranasal and intravenous dosing. Due to the limited capacity of the nostrils, approximately half of the intranasal dose was swallowed into the gastrointestinal tract. Biliary excretion was found to be the predominant pathway of radioactivity excretion following both routes of administration. The results from this study suggest that intranasal administration provides a viable means of delivering DHE into the systemic circulation.

Variable glyceryl dinitrate formation following infusions of glyceryl trinitrate at different vascular sites in the rat

The availability of glyceryl trinitrate (GTN) and the differential formation of dinitrate metabolites (GDNs) in various organs as a function of routes of administration were investigated in the rat. GTN was infused at 2.0 µg/min via the left femoral vein (LFV), left external jugular vein (LJV), left femoral artery (LFA), and hepatic portal vein (HPV). Blood concentrations of GTN and GDNs were measured in femoral arterial samples. Different infusions yielded GTN steady-state concentrations in the following rank order: LJV ≥ LFV > LFA ≥ HPV. Furthermore, the GDN formation ratios (1,2-GDN/1,3-GDN) are different: LFV ≥ LJV > LFA > HPV. The availabilities of GTN through the leg, vein, and liver were derived. GTN is significantly extracted and metabolized in these organs, and the leg and the vein prefer 1,2-GDN formation, while the liver forms 1,3-GDN predominantly.