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Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [(3)H]-labeled E(2) or E(1), and with [(14)C]-E(1) in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E(2) right harpoon over left harpoon E(1)) by trophoblast varied with development; it favored E(2) in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [(3)H](2)O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E(2) in both free and sulfate fractions. It was concluded that local metabolism of E(2) is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.

Design and Preparation of 2-Benzamido-pyrimidines as Inhibitors of IKK. -Optimization of the lead compound (Ia) through a parallel synthetic and classical medicinal chemistry effort results in the identification of compounds (Ib) and (Ic) as potent inhibitors of IKK2. -(WAELCHLI*, R.; et al.; Bioorg. Med. Chem. Lett. 16 (2006) 1, 108-112; Novartis Inst. Biomed. Res., CH-4002 Basel, Switz.; Eng.) -D. Singer 13-160

During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulationZday 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (nZ55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a major capsule-associated protein that migrated at w10 kDa was identified by N-terminal sequencing as equine b2 microglobulin (b2M). During fixation, b2M in the capsule underwent limited proteolysis to an w8 kDa form lacking nine amino acids from the N terminus, and was subsequently degraded. Expression of b2M mRNA was detected in the yolk-sac wall tissues and endometrium between days 13.5 and 17.5. During this period, b2M in the capsule was evidently not part of a complex with major histocompatibility complex class 1 heavy a chain bands because these were undetectable in the capsule and uterine lavage. Uterocalin (p19) was detected in uterine lavage and capsule throughout fixation, but in yolk-sac fluid only before fixation. These studies indicate that intact b2M is a major protein associated with the embryonic capsule before fixation, after which it undergoes limited proteolysis to a truncated w8 kDa form that remains in the capsule after the conceptus is immobilized.

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The JAK kinases JAK1, JAK2, JAK3, and TYK2 play key roles in cytokine signaling. Activation of the JAK/STAT pathways is linked to many diseases involving the immune system, including atopic dermatitis. As systemic JAK inhibitor pharmacology is associated with side effects, topical administration to the skin has been considered to locally restrict the site of action. Several orally bioavailable JAK inhibitors repurposed for topical use have been recently approved or are in clinical development. Here, we disclose our clinical candidate CEE321, which is a potent pan JAK inhibitor in enzyme and cellular assays. In contrast to repurposed oral drugs, CEE321 does not display high potency in blood and has a high clearance in vivo. Therefore, we consider CEE321 to be a “soft drug”. When applied topically to human skin that was stimulated with the cytokines IL4 and IL13 ex vivo, CEE321 potently inhibited biomarkers relevant to atopic dermatitis.

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16-17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F(2 alpha) (PGF(2 alpha)). Uterocalin and beta(2)-microglobulin (beta(2)M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF(2 alpha). Two forms of uteroglobin were distinguished by partial amino acid sequences of approximately 6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF(2 alpha) one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.

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