Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.     

Reduced beta-adrenergic sensitivity in patients with type 1 diabetes and hypoglycemia unawareness

OBJECTIVE: We tested the hypothesis that impaired tissue sensitivity to catecholamines contributes to hypoglycemia unawareness in subjects with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 21 subjects with type 1 diabetes underwent a standardized insulin infusion protocol to produce a stepwise decrease in plasma glucose to 45-min plateaus of 4.3, 3.6, 3.0, and 2.3 mmol/l. Glycemic thresholds, maximum responses for adrenergic and neuroglycopenic symptoms, and counterregulatory hormones were determined. Patients were classified as hypoglycemia unaware if the initiation of adrenergic symptoms occurred at a plasma glucose level 2 SD below that of nondiabetic volunteers. beta-Adrenergic sensitivity was measured as the dose of isoproterenol required to produce an increment in heart rate of 25 beats per minute above baseline (I25) in resting subjects. RESULTS: Subjects with type 1 diabetes and hypoglycemia unawareness experienced the onset of adrenergic symptoms at a lower plasma glucose level than did those with awareness (2.5+/-0.1 vs. 3.7+/-0.1 mmol/l, P < 0.001), whereas neuroglycopenic symptoms occurred at similar glucose levels (2.7+/-0.2 vs. 2.8+/- 0.1 mmol/l). The plasma glucose levels for counterregulatory hormone secretion (epinephrine 2.9+/-0.2 vs. 4.1+/-0.2 mmol/l; norepinephrine 2.7+/-0.1 vs. 3.2+/-0.2 mmol/l; cortisol 2.5+/-0.2 vs. 3.3+/-0.2 mmol/l, P < 0.01) were also lower in subjects with unawareness. The maximal epinephrine (1,954+/-486 vs. 5,332+/- 1,059 pmol/l, P < 0.01), norepinephrine (0.73 +/- 0.14 vs. 1.47+/-0.21 nmol/l, P = 0.04), and cortisol (276+/-110 vs. 579+/-83 nmol/l, P < 0.01) responses were reduced in the unaware group. I25 was greater in unaware subjects than in subjects without unawareness (1.5+/-0.3 vs. 0.8+/-0.2 microg), where I25 was not different from that of controls (0.8 +/-0.2 microg). CONCLUSIONS: We conclude that subjects with type 1 diabetes and hypoglycemia unawareness have reduced beta-adrenergic sensitivity, which may contribute to their impaired adrenergic warning symptoms during hypoglycemia.     

Differential item functioning in the Danish translation of the SF-36

Statistical analyses of Differential Item Functioning (DIF) can be used for rigorous translation evaluations. DIF techniques test whether each item functions in the same way, irrespective of the country, language, or culture of the respondents. For a given level of health, the score on any item should be independent of nationality. This requirement can be tested through contingency-table methods, which are efficient for analyzing all types of items. We investigated DIF in the Danish translation of the SF-36 Health Survey, using two general population samples (USA, n = 1,506; Denmark, n = 3,950). DIF was identified for 12 out of 35 items. These results agreed with independent ratings of translation quality, but the statistical techniques were more sensitive. When included in scales, the items exhibiting DIF had only a little impact on conclusions about cross-national differences in health in the general population. However, if used as single items, the DIF items could seriously bias results from cross-national comparisons. Also, the DIF items might have larger impact on cross-national comparison of groups with poorer health status. We conclude that analysis of DIF is useful for evaluating questionnaire translations.     

Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage

The relative contributions of protein tyrosine kinases (PTKs) and protein kinase C isoenzymes (PKCs), a family of serine/threonine kinases, in integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa) exposure are the subject of much controversy. In the present study we measured the effect of the PTK inhibitor herbimycin A and the PKC inhibitor bisindolylmaleimide I on 125I-fibrinogen binding to alpha(IIb)beta3 and on aggregation/secretion induced by different agonists. Dose-response studies showed complete inhibition of alpha(IIb)beta3 exposure by 30 micromol/L (ADP stimulation) and 35 to 40 micromol/L (alpha-thrombin stimulation) herbimycin A. In contrast, inhibition of exposure by bisindolylmaleimide I varied from none (for ADP and epinephrine), to 30% (for platelet-activating factor), and to approximately 80% (for alpha-thrombin). Studies with a submaximal dose of herbimycin A (approximately 50% inhibition of the ADP-response) and a maximal dose of bisindolylmaleimide I showed that optical aggregation had a similar sensitivity to the inhibitors as alpha(IIb)beta3 exposure with minimal interference by secreted ADP. Thus, the relative contributions of tyrosine and serine/threonine kinases in alpha(IIb)beta3 exposure and aggregation differ among the different agonists, with an exclusive role for PTKs in ADP- and epinephrine-induced responses and a role for both PTKs and PKCs in responses induced by platelet-activating factor and alpha-thrombin.     

Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate

Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4, 5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor's action.     

Human infection with Hymenolepis diminuta: case report from Spain

We report a case of Hymenolepis diminuta infection in a human. The patient was a 5-year-old girl referred to us through the onset of a cyanotic attack. Treatment with a single dose (10 mg/kg of body weight) of praziquantel was ineffective, but the parasite was eradicated after three treatment cycles with the same drug at dosages of 25 mg/kg/day for 5 days.     

A novel cartilage protein (CILP) present in the mid-zone of human articular cartilage increases with age

A novel, somewhat basic noncollagenous protein was purified from guanidine hydrochloride extracts of human articular cartilage using cesium chloride density gradient centrifugation, followed by ion-exchange chromatography at pH 5, and gel filtration on two serially coupled columns of Superose 6 and Superdex 200. The protein of 91.5 kDa contains a single polypeptide chain substituted with N-linked oligosaccharides. It appeared unique to cartilage as studied by enzyme-linked immunosorbent assay and immunoblots of various tissue extracts. Its concentration in articular cartilages showed some variability with age being lower in young individuals. It represents a chondrocyte product, since it is synthesized by articular chondrocytes in explant cultures. Interestingly, the distribution of the protein in the articular cartilage provides important information on the nature of chondrocytes at different compartments in the tissue. Thus, chondrocytes in the middle/deeper layers of the tissue in particular, appeared to have produced the protein and deposited it in the interterritorial matrix. The protein was neither seen in the superficial nor in the deepest regions of the articular cartilage. Based on its immunolocalization we have named this protein CILP (cartilage intermediate layer protein).     

Modifications of carbohydrate residues and ZP2 and ZP3 glycoproteins in the mouse zona pellucida after fertilization

Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.