The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body weight reduction. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compound 48 for development as a clinical candidate for the treatment of obesity.

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Competitive antagonists of the antidiuretic (ADH) activity of vasopressin were first described some six years ago. When studied in vitro, ADH antagonists displace vasopressin from specific renal binding sites and antagonize, in a competitive fashion, vasopressin stimulation of adenylate cyclase and transepithelial water, salt, and urea fluxes. When studied in vivo, the ADH antagonists increase renal water excretion and antagonize, in a competitive fashion, the ADH activity of vasopressin. Marked species heterogeneity is apparent with ADH antagonists in vivo, and inconsistencies between in vitro and in vivo findings within the same species are reported. Other renal responses associated with administration of ADH antagonists include changes in renal hemodynamics and renal salt and urea excretion. The effects on salt excretion appear to be limited to those species in which vasopressin stimulation of epithelial salt reabsorption has been demonstrated. In summary, the role of vasopressin as the principal factor regulating renal water handling is supported by experience with ADH receptor antagonists. However, that experience also indicates the emerging significance of autocoids, and other synergistic factors, to affect ADH receptor/effector mechanisms and to modulate renal ADH responses.

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The addition of ascorbate to the culture medium of early log-phase mouse fibroblasts (L-929 cells) resulted in a 5-fold increase of prolyl hydroxylase activity. Maximal activity was reached within 2 hr after addition of 5 jM ascorbate. The total amount of enzyme-related antigen did not change on treatment with ascorbate and the activation was shown to be independent of RNA and protein synthesis. The increase in activity caused by ascorbate is therefore due to the activation of a preformed precursor. Enzyme (molecular weight 260,000-300,000) and putative precursor (molecular weight 85,000-105,000) were separated by chromatography on DEAE-Sephadex. Treatment of intact cells with dithiothreitol resulted in an almost quantitative conversion of the enzyme to the smaller inactive protein. When these cells were treated with ascorbate or incubated overnight in fresh medium the enzyme reappeared and precursor concentrations decreased proportionately. Ascorbate may act by bringing about aggregation of enzymatically inactive subunits.Green and Goldberg (1) observed that significant hydroxylation of proline did not occur in fibroblast cultures until late log-phase was reached. Gribble et al. (2) then showed that the appearance of hydroxyproline at the end of the logarithmic growth of L-929 mouse fibroblasts occurred along with a sharp increase in prolyl hydroxylase* activity. In early logphase cells a similar increase of enzyme activity could be induced by concentration of the cells to a high density (3), or by incubation of the cells at 370 in the presence of lactate (4).The fact that this activation is independent of RNA and protein synthesis suggested the presence of an inactive precursor of the enzyme. More direct evidence for this hypothesis was provided by the observation that during lactate treatment or cell crowding the amount of protein that crossreacts with a monospecific antibody against the enzyme (crossreacting protein), remained virtually constant, while the enzyme activity increased several-fold (5). Subsequently, McGee and Udenfriend (6) isolated an enzymatically inactive protein from early log-phase, L929 fibroblasts that crossreacts with antibody to the enzyme and obtained evidence that it may be an enzymatically inactive precursor of prolyl hydroxylase.There have been many reports that the amount of hydroxyproline formed by cultured fibroblasts and osteoblasts is increased by exposure to ascorbate (7-14). Peck et al. (8) reported that hydroxylation of radioactive proline, which had previously been incorporated into the protein of cultured bone cells, was stimulated by ascorbate even in the presence of puromycin or cycloheximide. These findings indicate that ascorbate stimulates the hydroxylation of a prolinerich peptide. Recently, Peterkofsky (14) reported that ascorbate deteriorates rapidly in tissue culture medium and that by maintaining adequate concentrations of ascorbate, hydroxyproline formation was markedly increased in early log-phase cells, while collagen polypeptide formation was unaff...