Stability of alkoxycarbonylamidine prodrugs.

PURPOSE: Alkoxycarbonylamidine prodrug modification was used to mask the positively-charged amidine moiety of an Arg-Gly-Asp peptidomimetic and enhance oral bioavailability. The aqueous stability of ethoxycarbonylamidine (ECA), ethanethiocarbonylamidine (ETCA) and phenoxycarbonylamidine (PCA) prodrugs was examined. METHODS: Degradation was followed by RP-HPLC and rate constants were determined from a degradation scheme defined by product analysis. RESULTS: ECA gave a pH of maximum stability at pH approximately 7 and was independent of pH below pH approximately 4. A novel degradation pathway of ECA, conversion to ethoxycarbonyl- aminocarbonyl, was observed below pH 7. The relative rates below pH 7 were ECA approximately ETCA < PCA, in the same order of decreasing pKa of the conjugate acid of the substituted amidino group. Base-catalyzed cleavage of ECA to yield the amidine derivative gave the relative rates ECA < ETCA < PCA, in agreement with the decreasing pKa of the leaving groups. CONCLUSIONS: The observed rate constants at all pHs were small enough that only 5-30% (depending on the substituent) undesirable degradation is predicted during transit time of the gut. The spontaneous post-absorptive conversion to the amidine drugs at neutral pH is predicted to be 6x greater for the PCA than the ECA prodrugs.

Stress response and drug metabolism in mice.

A modified stress model was studied to investigate the relationship between the stress response and drug metabolism in mice. Stress was induced in male CD-1 mice by a daily i.p. injection of hypertonic (1.5 M) saline for up to 3 days, whereas control animals received isotonic (0.15 M) saline. Two hours after receiving the saline injection on the first, second, and third day, animals were euthanized, and serum corticosterone (CORT) and liver aminopyrine N-demethylase (AD) and aniline hydroxylase (AH) activities were determined. To detect any effect of osmotic stimulation, a second control group was given 1.5 M saline as drinking water. There was no difference in CORT levels, AD activity, or AH activity between untreated animals and 0.15 M saline treatment. Intra-peritoneal injection of 1.5 M saline markedly increased serum CORT concentrations compared to 0.15 M saline regardless of the duration of the treatment. Injection of 1.5 M saline also decreased both hepatic enzyme activities each time point. Osmotic stimulation alone by hypertonic drinking water had no significant effect on CORT levels, AD activity, or AH activity. In another series of experiments, intact, sham-operated, and adrenalectomized mice were exposed to the stress model. Injected hypertonic saline decreased AD and AH activities in intact and sham-operated animals compared to isotonic saline-treated animals but both enzyme activities were reduced after adrenalectomy regardless of saline treatment used. In conclusion, a suitable model was established to study the interactions between the stress response and the hepatic drug metabolism in mice.

Estrogen production by bovine binucleate and mononucleate trophoblastic cells in vitro.

The bovine placenta has long been known as a source of steroid hormones. We performed three experiments to compare production of estrogens by bovine mononucleate and binucleate trophoblastic cells and examined effects of cortisol, progesterone, pregnenolone, testosterone, and androstenedione. In the first experiment, binucleate trophoblastic cells were purified by unit gravity sedimentation from six enzymatically dispersed placentas between 150 and 180 days of gestation. Cells (8 x 10(5)/ml) were incubated first at 37 degrees C for 6 h with Medium 199 alone (M199/6h) or with 10(-7) M cortisol (cortisol/6h). Medium then was replaced with 10(-7) M progesterone, 10(-7) M pregnenolone, 10(-7) M testosterone, or M199, and a second incubation was conducted for 4 h. Estradiol production did not differ between cells incubated for the first 6 h in M199 vs. cortisol and was not affected by progesterone or pregnenolone. Testosterone increased (p < 0.05) estradiol production. Estrone production did not differ between cells incubated for the first 6 h in M199 vs. cortisol; estrone production was not affected by either progesterone, pregnenolone, or testosterone. Mononucleate as well as binucleate cells were purified from placentas between 165 and 180 days of gestation and used in two other experiments. In the first of these, enriched populations of binucleate and mononucleate cells were incubated first for 6 h with Medium 199 (M199) or 10(-7) M cortisol. Medium then was replaced with 10(-7) M testosterone, 10(-7) M androstenedione, or M199 and incubation continued for 4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of hemolysis and storage on quantification of hormones in blood samples from dogs, cattle, and horses.

Veterinary diagnostic endocrinology laboratories frequently receive hemolyzed plasma, serum, or blood samples for hormone analyses. However, except for the previously reported harm done by hemolysis to canine insulin, effects of hemolysis on quantification of other clinically important hormones are unknown. Therefore, these studies were designed to evaluate effects of hemolysis on radioimmunoassay of thyroxine, 3,5,3'-triiodothyronine, progesterone, testosterone, estradiol, cortisol, and insulin in equine, bovine, and canine plasma. In the first experiment, hormones were measured in plasma obtained from hemolyzed blood that had been stored for 18 hours. Blood samples were drawn from pregnant cows, male and diestrous female dogs, and male and pregnant female horses. Each sample was divided into 2 equal portions. One portion was ejected 4 times with a syringe through a 20-gauge (dogs, horses) or 22-gauge (cows) hypodermic needle to induce variable degrees of hemolysis. Two subsamples of the blood were taken before the first and after the first, second, and fourth ejections. One subsample of each pair was stored at 2 to 4 C and the other was stored at 20 to 22 C for 18 to 22 hours before plasma was recovered and stored at -20 C. The second portion of blood from each animal was centrifuged after collection; plasma was recovered and treated similarly as was blood. Concentrations of thyroxine in equine plasma, of 3,5,3'-triiodothyronine, estradiol, and testosterone in equine and canine plasma, and of cortisol in equine plasma were not affected by hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)