32-Ascomycinyloxyacetic acid derived immunosuppressants. Independence of immunophilin binding and immunosuppressive potency.

The potent immunosuppressant ascomycin (1b) was selectively alkylated at the C-32 carbinol, thus providing esters and amides of 32-ascomycinyloxyacetic acid (4, AOAA). These compounds present structural variation at the FKBP/calcineurin interface. While the native carboxylic acid 4 shows no activity in vitro, esters and simple amides of 4 exhibit potent immunosuppression in the human MLR assay. Moreover, amides show inhibitory activity in the rat popliteal lymph node hyperplasia assay. Surprisingly, FKBP binding was weakened by several orders of magnitude when secondary hydrophobic aryl amides of 4 were tested, while maintaining potent immunosuppressive efficacy in vitro.

Discovery of ascomycin analogs with potent topical but weak systemic activity for treatment of inflammatory skin diseases.

Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid pharmacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FK506) despite a severalfold lower potency for inhibiting swine T cells in vitro, consistent with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30-fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FK506 in all three species. The potent topical activity and reduced systemic exposure of ABT-281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.

Changes in ovarian norepinephrine synthesis throughout the follicular and luteal phases.

The aims of this study were to determine norepinephrine (NE) synthesis in follicle-dominated and luteal-dominated ovaries as compared to oviducts, and to correlate NE synthesis with NE content and turnover rates. Rats were injected with pregnant mare's serum gonadotropin (PMSG) on Day 28. Ovaries and oviducts were removed during the follicular (Days 28-30) and luteal (Days 31-40) phases and incubated for 2 h with [3H] tyrosine. Tritiated and endogenous NE were determined by high-performance liquid chromatography. Ovarian NE synthesis from [3H] tyrosine was reduced by more than 50% within 24 h after PMSG injection, with a second 50% reduction on Day 30, concomitant with the endogenous gonadotropin surge. The lowest NE synthesis (15% of control values) was observed in the luteinized ovary on Day 33. Ovarian NE synthesis from [3H] L-dihydroxyphenylalanine (DOPA) was similar in control and PMSG-injected rats on selected days during the follicular and luteal phases. Oviductal NE synthesis decreased after PMSG injection, but was similar to control values during the luteal phase. Ovarian NE content was modestly reduced between Days 30 and 35, whereas oviductal NE content was not altered. After an injection of alpha-methyl-p-tyrosine on Day 33, ovarian and oviductal NE content decreased exponentially over a period of 10 h. The NE turnover rates were similar in control and PMSG-injected rats in both tissues. The following conclusions were reached: Circulating gonadotropins appear to suppress ovarian NE synthesis during the follicular phase. The low NE synthesis by the luteinized ovary is consistent with previous reports that follicles, but not corpora lutea (CL), contain catecholamine elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine in the rat ovary: ontogeny and de novo synthesis.

The aims of this study were to characterize the ontogeny of catecholamines (CA) in the rat ovary, to determine the ability of the immature ovary to synthetize norepinephrine (NE) in vitro, and to correlate between ovarian CA and plasma pituitary hormones. Ovaries, spleen (as a control tissue not subjected to endocrine regulation), and trunk blood were collected at 5-day intervals between days 5 and 40. Ovarian NE concentration increased markedly between days 20 and 35 of life, whereas the major rise in splenic NE concentration occurred between days 10 and 15. Ovarian and splenic tissues from neonatal females were capable of de novo synthesis of NE from tritiated tyrosine without an appreciable accumulation of L-Dopa and dopamine. The rate of NE synthesis by ovarian tissue taken from 20-day-old rats was significantly lower than that from 30- and 40-day-old rats, whereas NE production by splenic tissue from 20-, 30-, and 40-day-old rats were similar. Plasma FSH concentration was significantly elevated between days 10 and 20, whereas the major rise in plasma LH and PRL occurred between days 25 and 40. The following conclusions were reached. The delayed elevation of ovarian NE, compared to splenic NE, is attributable to a decreased production of NE by the ovary on day 20 and may involve a suppression or a delay in development of the activity of a key CA biosynthetic enzyme such as tyrosine hydroxylase. Given the temporal relationship between plasma gonadotropins, in particular FSH, and changes in ovarian NE, it is postulated that ovarian CA during neonatal development are subjected to regulation by circulating pituitary hormones.

Plasma catecholamines in the chronically cannulated sheep fetus: predominance of L-dihydroxyphenylalanine.

Dihydroxyphenylalanine (Dopa) and catecholamines (CA) concentrations were determined in plasma collected from chronically cannulated fetal and maternal sheep from days 125-140 of gestation. Dopa was measured by a radioenzymatic assay, followed by ion exchange chromatography and high performance liquid chromatography. The assay was linear to 2.5 ng Dopa, and its sensitivity was 35-45 pg. Dopamine (DA), norepinephrine, and epinephrine were determined simultaneously by the same radioenzymatic incubation procedure, followed by solvent extraction and two-dimensional thin layer chromatography. The Dopa level in the fetal circulation was 10-25 times higher than that of DA, 5-10 times higher than that of norepinephrine, and 100 times higher than that of epinephrine. Furthermore, Dopa was the only CA that was significantly (P less than 0.05) higher in fetal (3.5-4.5 ng/ml) than in maternal plasma (1-1.5 ng/ml). The data indicate that Dopa is the predominant circulating CA in the sheep fetus. While the physiological importance of this observation is unknown at the present time, fetal Dopa might serve as the source of free DA in fetal urine and/or amniotic fluid.