Improving synthetic efficiency using the computational prediction of biological activity.

A process has been developed whereby libraries of compounds for lead optimization can be synthesized and screened with greater efficiency using computational tools. In this method, analogues of a lead chemical structure are considered in the form of a virtual library. Less than 1/3 of the library is selected as a training set by clustering the compounds and choosing the centroid of each cluster. This training set is then used to generate a model using PLS regression upon the experimental values from that assay using 1D/2D descriptors. The model is applied to the remaining compounds (the test set) for which assay values are predicted and a rank ordering established. An example of this was a set of 169 PDE4 inhibitors. A predictive model was achieved using a training set of 52 compounds. When applied to the remaining 117 compounds this model allowed a rank ordering of these compounds for synthesis and testing. Selecting the top 33 compounds of the test set gives 78% of the compounds with the desired activity (hits) by synthesizing only 50% of the library, including the training set. Selecting the top 59 of the test set gives 97% of the hits from only 67% of the library. This process succeeds by avoiding two principal weaknesses of 2D descriptors: lack of interpretation and lack of extrapolation. Two principal assumptions of QSAR are shown to be unnecessary; removing descriptor redundancy does not improve fit and a predictive r2 greater than 0.5 is not necessary if rank-ordering is desired.

Variation between sheep in renal excretion of [14C]allantoin.

The objectives of the present study were to investigate the recovery of [14C]allantoin in urine of sheep dosed intravenously and degradation of allantoin by rumen micro-organisms. The recovery of [14C]allantoin in the urine of eight sheep was measured during three periods in two experiments. Individual values of [14C]allantoin recovery varied from 66 to 95 % (mean value 83 (se 1.6) %). The recovery of [14C]allantoin showed no relation to the level of feed intake. There was some evidence that glomerular filtration rate was an important factor affecting the amount of urinary allantoin recovered in one experiment. Incomplete recovery of plasma [14C]allantoin in the urine indicated losses of plasma [14C]allantoin via non-renal routes. This is supported by the disappearance of 14C from rumen contents incubated in vitro with [14C]allantoin for 48 h (88 %) and the presence of 14C in saliva in vivo from sheep sampled after dosing with [14C]allantoin. However, the amount of 14C activity in the saliva was very low (equivalent to only 1.5 % of the total dose in sheep producing saliva at a rate of 15 litres/d). The proportion of renal and non-renal excretion of purine derivatives was found to be unpredictable both between and within individual animals. The factors responsible for this variability need to be identified, and existing models of excretion of purine derivatives may need to be modified accordingly to improve their accuracy of prediction. A single intravenous injection of [4,5-14C]allantoin provides a simple alternative to infusion methods used to measure the proportion of plasma allantoin excreted in the urine of sheep. Using this method it may be feasible to validate PD excretion models in other ruminant livestock.

Temporal changes in reproductive hormones and conceptus-endometrial interactions during embryonic diapause and reactivation of the blastocyst in European roe deer (Capreolus capreolus).

The roe deer blastocyst is in diapause between August and December, after which time it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 +/- 0.4 pg ml(-1)) and expansion (1.2 +/- 0.4 pg ml(-1)) but increased by 30 times at trophoblast elongation (49.17 +/- 0.37 pg ml(-1)). Prolactin remained at basal concentrations (4.69 +/- 0.86 ng ml(-1)) and increased after implantation (12.34 +/- 2.71 ng ml(-1)). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 +/- 1.97 ng ml(-1); luteal progesterone: 6.72 +/- 0.81 ng mg(-1) protein). Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions increased 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation.

In vitro genotoxicity testing of ARASCO and DHASCO oils.

ARASCO and DHASCO oils are microbially-derived triglycerides rich in arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acids, respectively. Both oils were tested for mutagenic activity in three different in vitro mutagenesis assays. All assays were conducted with and without metabolic activation. Neither ARASCO nor DHASCO oil was mutagenic in the Ames reverse mutation assay using five different Salmonella histidine auxotroph tester strains, nor were the oils mutagenic in the mouse lymphoma TK(+/-) forward mutation assay. The oils showed no clastogenic activity in chromosomal aberration assays performed with Chinese hamster ovary cells. Based on these assays, neither ARASCO nor DHASCO oils appear to have any genotoxic potential.

A developmental safety study in rats using DHA- and ARA-rich single-cell oils.

The long-chain omega-3 and omega-6 fatty acids, docosahexaenoic and arachidonic acids, are important in fetal development, but may be depleted from the mother during pregnancy as she transfers reserves to the developing fetus in utero and later to the infant through her breast milk. Pregnant women can increase their dietary intake of these nutrients to maintain adequate maternal reserves and ensure an optimal infant supply. DHASCO(R) and ARASCO(R) oils, concentrated sources of docosahexaenoic and arachidonic acids, respectively, have been tested in acute and subchronic studies without toxic effects. The present developmental toxicity study was undertaken to test for potential teratogenic activity of these oils to ensure their safe use during pregnancy. DHASCO and ARASCO oils were administered by oral gavage to pregnant rats at doses up to 1250 and 2500 mg/kg body weight/day, respectively, during the period of organogenesis. Caesarean sections and necropsies were performed on day 20 of gestation. Maternal reproductive outcomes were analyzed, and fetal external, soft and skeletal tissue were examined. Treatment with these oils did not produce overt maternal toxicity, nor did either oil result in changes in pre- or postimplantation losses, resorptions, live births or sex ratios. Neither oil caused fetal malformations. Increased frequencies of renal variations in development occurred in a non-dose-dependent manner and were not toxicologically significant. We conclude that these oils are not teratogenic at doses that represent a 100-fold safety factor over expected use levels.

A combined subchronic (90-day) toxicity and neurotoxicity study of a single-cell source of docosahexaenoic acid triglyceride (DHASCO oil).

Docosahexaenoic acid (DHA), a 22-carbon long-chain polyunsaturated fatty acid of the omega-3 family, is a major structural component of neural membranes and is a particularly important nutrient during infant development. New safe and well-defined sources of DHA are required for infant formula fortification and dietary supplementation. DHASCO oil is an algal-derived triglyceride containing 40-50% DHA. Previous studies have shown that DHASCO oil is neither mutagenic nor toxic in acute or 28-day subchronic tests. To further establish the safety of this oil, a 90-day subchronic toxicity study in rats which included haematology, clinical chemistry, pathology and ophthalmologic, neurobehavioural and neuropathological assessments, using doses of 0.5 and 1.25g/kg body weight/day was performed. There were no treatment-related adverse effects in any of the parameters measured at either dose. Based on these results, the no-adverse-effect level (NOAEL) for DHASCO oil under the conditions of this study corresponds to the highest dose level. The DHA in the DHASCO oil was bioavailable, resulting in significant elevations in the levels of this fatty acid in liver, heart and brain after 90 days of administration. In conclusion, this 90-day subchronic toxicity study provides additional evidence that DHASCO oil is a safe and bioavailable source of dietary DHA.

High-affinity binding of peptide agonists to the human B1 bradykinin receptor depends on interaction between the peptide N-terminal L-lysine and the fourth extracellular domain of the receptor.

The aim of this study was to identify the location of the N terminus of peptide agonist ligands when bound to the human B1 bradykinin (BK) receptor. To reach this aim, we exploited the fact that high-affinity binding of kinin peptides to the human B1 receptor subtype requires a peptide N-terminal L-Lys, whereas high-affinity binding to the B2 receptor subtype does not require this residue. This was done by comparing the affinities of BK, a B2 receptor-selective peptide, and kallidin or Lys-BK, a less receptor-selective peptide, for chimeric proteins in which each B1 receptor domain had been substituted in the human B2 receptor and expressed in HEK293 cells. Individual substitution of transmembrane domains 1-7 (TM-I-VII) and extracellular domains 1-4 (EC-I-IV) of the B1 receptor in the B2 receptor influenced the affinities of BK and Lys-BK approximately equally. In contrast, substitution of B1 EC-IV dramatically reduced the affinity and potency of BK, whereas these parameters for Lys-BK were essentially unaltered. Substitution of either the N- or C-terminal half of B1 EC-IV in the B2 receptor only had a limited effect on the peptide binding constants, indicating the involvement of multiple residues throughout this domain. Complementary mutations of the N-terminal residue in Lys-BK revealed that both the positive charge and the proper spatial orientation of this residue were required for interaction with B1 EC-IV. Thus, the N-terminal residue of peptide agonists when bound to the human B1 receptor is positioned extracellularly and interacts with EC-IV.

Oral antinociception and oedema inhibition produced by NPC 18884, a non-peptidic bradykinin B2 receptor antagonist.

This study investigates the antinociceptive and the oedema inhibition properties of the novel non-peptide bradykinin (BK) B2 receptor antagonist, NPC 18884. Given by i.p. or p.o. routes NPC 18884 produced graded and long-lasting (at least 2.5h and 5.0h, respectively, for i.p. and p.o. administration) inhibition of acetic acid-induced abdominal constrictions in mice, with mean ID50 values of 8.3 nmol/kg and 439.9 nmol/kg. NPC 18884 also inhibited kaolin-induced abdominal constrictions (44+/-9% and 48+/-3% of inhibition, for i.p. and p.o. routes, respectively). Given by i.p. or p.o. routes NPC 18884 attenuated both phases of formalin-induced licking, as well as formalin-induced oedema formation. At similar doses NPC 18884 produced significant inhibition of capsaicin-induced nociception. NPC 18884, like HOE 140 given i.p., prevented the nociception caused by BK with mean ID50 values of 0.85 nmol/kg and 0.44 nmol/kg, respectively. Given orally NPC 18884, but not HOE 140, caused graded inhibition of BK-induced nociception (mean ID50 value of 50 nmol/kg). In rats, NPC 18884 given i.p. prevented BK and carrageenan-induced hyperalgesia (mean ID50 values of 6 nmol/kg and 13 nmol/kg), without affecting the hyperalgesia induced by des-Arg9-bradykinin (DABK) or by prostaglandin E2 (PGE2). NPC 18884 given i.p. inhibited the mouse paw oedema induced by tyrosine8-bradykinin or by carrageenan, but had no effect on DABK-induced oedema in mice pre-treated with Escherichia coli endotoxin, or that induced by PGE2. Thus, the novel non-peptide BK B2 receptor antagonist NPC 18884 produces rapid onset, potent and relatively long-lasting oral antinociceptive and oedema inhibition properties. The anti-BK actions of NPC 18884 are quite selective towards the BK B2 receptor-mediated responses.

Effect of glucose supply on fasting nitrogen excretion and effect of level and type of volatile fatty acid infusion on response to protein infusion in cattle.

Two experiments were carried out on cattle nourished entirely by intragastric infusion, to determine the extent to which glucose or a glucose precursor determines the response to protein infusion in energy-undernourished animals. In order to determine the requirement for glucose in 1-year-old fasting cattle, glucose was infused at increments to supply 0, 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5 g/kg metabolic body weight (W0.75) and the effects on plasma beta-hydroxybutyrate and N excretion were measured. At 5.5 g glucose/kg W0.75 plasma beta-hydroxybutyrate was reduced to a basal level of 1.65 mmol/l and fasting N excretion reduced from 529 to 280 mg N/kg W0.75. No further reduction was observed with the higher level of 6.5 g glucose/kg W0.75. In the second trial, three steers were used in a 3 x 3 Latin square design and infused with a volatile fatty acid mixture of 65, 27 and 8 mol acetic, propionic and butyric acids respectively/100 mol, either at an estimated maintenance energy level of 450 kJ/kg W0.75 and supplying a calculated glucose equivalent level of 13.0 g/kg W0.75 (M1A), or at 1.5 x maintenance supplying a glucose equivalent of 20 g/kg W0.75 (M1.5A). Another mixture infused at the maintenance energy level contained 49, 43 and 8 mol acetic, propionic and butyric acids respectively/100 mol but with a glucose equivalent of 20 g/kg W0.75 (M1P). Casein was infused at each of these energy treatments to supply 0, 200, 400, 800, 1600 and 2500 mg N/kg W0.75 daily, and N balance and blood metabolites were measured. N retention increased linearly (r 0.98) with casein infusion. The coefficients for N retention were 0.55, 0.57 and 0.64 for M1A, M1.5A and M1P respectively. The mean efficiency of N utilization was 0.58. The results suggest that provided the glucose need is met there is no relationship between energy supply and efficiency and level of protein retention. However, the results also indicate that glucose requirement in cattle may be higher than that previously observed in sheep.

Determination of 15N isotopic enrichment and concentrations of allantoin and uric acid in urine by gas chromatography/mass spectrometry.

A method for the determination of 15N enrichment and concentration of allantoin and uric acid simultaneously in urine using gas chromatography/mass spectrometry (GC/MS) is described. The urine samples contained [1,3-15N2] uric acid and its oxidation product allantoin. The uric acid and allantoin were isolated using an AG1-X8 (Cl-form) anion-exchange column and heated with a mixture containing 1:1 dimethylformamide and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). The tert-butyldimethylsilyl (TBDMS) derivatives of allantoin and uric acid formed were injected into a gas chromatograph interfaced with a mass spectrometer operated under electron impact ionization conditions. Isotope ratio measurements were made from the abundance of the M-57 ions at m/z 398, 399 and 400 for allantoin and at m/z 567 and 569 for uric acid. 15N2 allantoin (99 at.%) was produced from [1,3-15N2] uric acid by treatment with uricase and used as a standard. Quantitation of allantoin and uric acid was based on isotopic dilution by spiking the urine sample with known quantities of 99 at.% [15N] uric acid and allantoin internal standards. The observed isotope ratio measurements from the prepared standards matched the theoretical values. Coefficients of variation in measurements of isotope ratio and concentration were 0.2 and 0.5%, respectively. The method was applied in a study to measure the urinary recovery of [1,3-15N2] uric acid continuously infused for 8-10 h into the blood of four sheep each on two occasions. Within 24 h, 65.9 +/- 9.1% of the tracer was excreted in the urine unchanged. Little was converted into allantoin (approximately 7% of the dose). The total recovery (5 days) of the infused tracer averaged 69.5 +/- 7.6% as uric acid and 76.8 +/- 9.3% as the sum of uric acid and allantoin. Uricase activities in plasma, liver and kidney of sheep were also measured using [1,3-15N2] uric acid as a substrate. Uricase activity was estimated to be 0.6 mU g-1 wet tissue in the liver and there appeared to be none in plasma and kidney. The low uricase activities in sheep tissues appeared to explain the limited conversion of the intravenously administered [15N] uric acid to allantoin but did not explain the large quantities of allantoin excreted in urine (8.96 +/- 0.86 and 1.36 +/- 0.25 mmol d-1 for allantoin and uric acid, respectively). The GC/MS method for the determination of 15N enrichment and concentration of allantoin and uric acid in urine is accurate and precise and provides a useful tool for studies on uric acid and allantoin metabolism.

Oral anti-inflammatory action of NPC 18884, a novel bradykinin B2 receptor antagonist.

This study describes the anti-inflammatory actions of NPC 18884, a non-peptide bradykinin B2 receptor antagonist in bradykinin and carrageenan-induced inflammation in the mouse model of pleurisy. The selectivity of NPC 18884 was assessed in the pleurisy caused by histamine, substance P and des-Arg9-bradykinin. NPC 18884 given intraperitoneally or orally inhibited bradykinin-induced leukocytes influx (ID50 value of 63 nmol/kg and 141 nmol/kg, respectively). The NPC 18884 also inhibited the exudation induced by bradykinin (P < 0.05). NPC 18884 given either intraperitoneally or orally caused dose-dependent inhibition of the exudation and total and differential cell content caused by intrapleural injection of carrageenan (1%, assessed 4 h after), with mean ID50, values of 132 and 295 nmol/kg, respectively. The NPC 18884 actions installs rapidly (0.5 h), lasted for up to 4 h and were selective for the bradykinin B2 receptors; at similar doses it had no significant effect against the inflammatory responses induced by des-Arg9-bradykinin, histamine or substance P. These results indicate that the novel non-peptide bradykinin B2 receptor antagonist, NPC 18884, exhibited selective intraperitoneal and oral anti-inflammatory properties when assessed in the inflammatory reaction induced by bradykinin and carrageenan in the mice model of pleurisy.

The effects of acute and chronic protein depletion and accretion on plasma concentrations of insulin-like growth factor-1, fibronectin and total protein for ruminants nourished by intragastric infusion of nutrients.

The relationships of N input or protein status and the concentrations of serum insulin-like growth factor-1 (IGF-1), plasma fibronectin (FN) and total protein (TP) were examined in three experiments with steers and sheep nourished by intragastric infusion of nutrients. In Expt 1, three steers (340 kg live weight) were infused with three levels of volatile fatty acids (0, 300 and 600 kJ/kg metabolic weight (W0.75) per d) and six levels of casein (0, 200, 400, 650, 1500 and 2500 mg N/kg W0.75 per d). Each N treatment was imposed for 5 d. In Expts 2 and 3, five groups of sheep (about 35 kg live weight) were infused with casein at 500 mg N/kg W0.75 per d for 2 weeks followed by 1500, 500 or 50 mg N/kg W0.75 per d in Expt 2, and in Expt 3, with 100 mg N/kg W0.75 per d for 6 weeks or 10 mg N/kg W0.75 per d for 4 weeks. Non-protein energy was maintained constant at 500 kJ/kg W0.75 per d throughout. Daily N balance and total body N content at the end were measured, and protein status was defined as a percentage of cumulative N accretion or depletion in relation to the total body N content at maintenance. It was found that IGF-1 and FN responded rapidly and substantially to altered N input, and that when daily N input was maintained constantly at sub-maintenance, their continuous declines were related closely to progressive protein depletion in the sheep. Plasma TP concentration was independent of N input when N input was altered acutely in the steers, but declined significantly and gradually with severe, chronic body protein depletion in the sheep. Plasma content of TP in the sheep however reduced acutely with a reduction in N input. Plasma volume fell substantially over the first 2 weeks of protein depletion, compensating for the declines in TP content and maintaining TP concentration plateau. The possible implications of the changes in TP concentration and content (concentration x volume) to body protein loss in sheep are discussed.

Stable expression of the human kinin B1 receptor in Chinese hamster ovary cells. Characterization of ligand binding and effector pathways.

To delineate ligand binding and functional characteristics of the human B1 kinin receptor, a stable clone of Chinese hamster ovary cells expressing a single class of binding sites for [3H]des-Arg10-lysylbradykinin with a Kd of 0.3 nM and a Bmax of 38 fmol/mg protein ( approximately 40,000 receptors/cell) was isolated. Studies with peptide analogs showed that a lysine residue at position 1 (based on the lysylbradykinin sequence) of ligands was essential for high affinity binding to the human B1 receptor. In marked contrast to cloned Chinese hamster ovary cells expressing the human kinin B2 receptor, which internalized approximately 80% of the ligand within 5 min upon exposure to 2 nM [3H]bradykinin, exposure of cells expressing the B1 receptor to 1 nM [3H]des-Arg10-lysylbradykinin resulted in minimal ligand internalization. Stimulation of the B1 receptor led to inositol phosphate generation and transient increases in intracellular calcium, confirming coupling to phospholipase C, while immunoprecipitation of photoaffinity-labeled G-proteins from membranes indicated specific coupling of the receptor to Galphaq/11 and Galphai1,2. The B1, unlike the B2, receptor does not desensitize (as demonstrated by continuous phosphoinositide hydrolysis), enhancing the potential role of this receptor during inflammatory events.

Antioedematogenic and antinociceptive actions of NPC 18521, a novel bradykinin B2 receptor antagonist.

The novel pseudopeptide bradykinin B2 receptor antagonist containing the 1,3,8-triazaspiro[4,5]decan-4-one ring system, NPC 18521 (D-Arg-Arg-[1,3-phenyl,8-triazaspiro[4,5]-decane-4-one-3-acetyl]-S er-D -tetrahydroisoquinolinyl-octahydroindolinyl-Arg) (10 and 30 nmol/kg, i.p.), given 30 min prior, produced significant and long-lasting inhibition of rat paw oedema induced by bradykinin (3 nmol/paw) and carrageenan (300 micrograms/paw), without affecting the oedema induced by the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin, in rats pretreated with Escherichia coli endotoxin. In contrast, when injected locally into the rat or mouse hindpaw, NPC 18521 (1-100 nmol) elicited dose-related oedema formation. This effect was almost completely blocked by cyproheptadine (20 mg/kg, i.p.) or by compound 48/80 (12 micrograms/paw), but was unaffected by Hoe 140 (D-Arg-[Hyp5,Thi5,Tic7,Oic8]bradykinin). NPC 18521 (0.3-10 nmol/kg, i.p.) produced significant inhibition of acetic acid, acetylcholine and kaolin- but not zymosan-induced abdominal constrictions in mice. The calculated mean ID50 values for these effects were 0.84, 0.46 and 0.55 nmol/kg, respectively. The antinociceptive action of NPC 18521 (3 nmol/kg, i.p.) had a rapid onset (15 min) and lasted for up to 120 min. Given topically (0.01-0.3 nmol), NPC 18521 produced significant attenuation of both the early and the late phase of the formalin-induced licking, as well as formalin-induced oedema formation. In addition, NPC 18521 given both systemically or topically, produced significant inhibition of the neurogenic nociception caused by topical injection of capsaicin. Given topically in the rat paw, NPC 18521 (10 nmol) caused marked hyperalgesia, an effect which was completely prevented by cyproheptadine (20 mg/kg, i.p.), but was unaffected by Hoe 140 (3 nmol/kg, i.p.). Given intraperitoneally, 30 min prior, NPC 18521 (3-30 nmol/kg) like Hoe 140 (1-10 nmol/kg) prevented, in a dose-dependent manner, bradykinin (3 nmol/paw)-induced hyperalgesia with mean ID50 values of 13.16 and 1.36 nmol/kg, respectively. Thus, the novel pseudopeptide bradykinin B2 receptor antagonist, NPC 18521, has an effect with rapid onset, and produces potent and relatively long-lasting antioedematogenic and antinociceptive properties. However, in contrast to Hoe 140, given locally into the hindpaw, NPC 18521 elicited marked oedema formation and hyperalgesia, an effect which seems to be secondary to mast cell degranulation and histamine and/or serotonin release. Finally, the anti-bradykinin actions of NPC 18521 are quite selective towards the bradykinin B2 receptor-mediated responses.

The N terminus of bradykinin when bound to the human bradykinin B2 receptor is adjacent to extracellular Cys20 and Cys277 in the receptor.

Chemical cross-linking combined with site-directed mutagenesis was used to evaluate the role of extracellular cysteines and their positions relative to the binding site for the agonist bradykinin (BK) in the human BK B2 receptor. All extracellular cysteines, Cys20, Cys103, Cys184, and Cys277, in the receptor were mutated to serines, and single and double mutants were transfected into COS-7 cells. The Ser20 and Ser277 single mutants and the Ser20/Ser277 double mutant bound [3H]BK and the antagonist [3H]NPC17731 with pharmacological profiles identical to the wild-type B2 receptor. In contrast, the Ser103 and Ser184 single mutants were unable to bind either of the two radioligands. However, these mutants were still expressed as determined by immunoblotting with anti-B2 receptor antibodies. Previous studies on the bovine B2 receptor showed that bifunctional reagents, which are reactive to amines at one end and to free sulfhydryls in the opposite end, cross-link the N terminus of receptor-bound BK to a sulfhydryl in the receptor (Herzig, M. C. S., and Leeb-Lundberg, L. M. F. (1995) J. Biol. Chem. 270, 20591-20598). Here, we show that m-maleimidobenzoyl-N-hydroxysuccinimide ester and 1,5-difluoro-2, 4-dinitrobenzene cross-linked BK to the wild-type human B2 receptor and the Ser20 and Ser277 single mutant receptors, whereas these reagents were unable to cross-link BK to the Ser20/Ser277 double mutant. These results show that Cys103 and Cys184 are both required for expression of high affinity agonist and antagonist binding sites in the human B2 receptor, while Cys20 and Cys277 are not required. Furthermore, the results provide direct biochemical evidence that the N terminus of BK, when bound to the B2 receptor, is adjacent to Cys277 in extracellular domain 4 and Cys20 in extracellular domain 1 of the receptor.

Synthesis and characterization of pseudopeptide bradykinin B2 receptor antagonists containing the 1,3,8-triazaspiro[4.5]decan-4-one ring system.

A series of pseudopeptides containing alkyl-, cycloalkyl-, aryl-, and aralkyl-substituted 1,3,8-triazaspiro[4.5]decan-4-one-3-acetic acids as amino acid surrogates to replace the Pro2-Pro3-Gly4-Phe5 section of the peptide bradykinin B2 receptor antagonist [Pro3, Phe5]HOE 140 (D-Arg0-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-D-Tic7+ ++-Oic8-Arg9) were prepared. These psuedopeptides were examined in vitro for their B2 receptor affinities as well as for their ability to block bradykinin mediated actions in vivo. Two compounds in particular, NPC 18521 (I) and NPC 18688 (V) were quite potent in these latter assays, indicating that a significant portion of this prototypical second generation decapeptide antagonist can be replaced with a more compact nonpeptide molecule.

Preclinical evaluation of single-cell oils that are highly enriched with arachidonic acid and docosahexaenoic acid.

Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are important in human brain and retina development, and there is growing evidence showing the importance of these fatty acids in infant nutrition. Triglyceride oils, highly enriched in ARA (ARASCO) and DHA (DHASCO), were evaluated using very high dose acute (20 g/kg) and 4-wk subchronic gavage feedings in weanling Sprague-Dawley rats. The combination of these oils, Formulaid, was also tested in the 4-wk subchronic study, ARASCO, DHASCO and Formulaid were found to have a no-observable-adverse-effect level of more than 2.5 g/ kg/day, 1.25 g/kg/day and 3.75 g/kg/day, respectively. This represents a 50-fold safety margin over the intended use of Formulaid in infant formula. Survival, clinical signs, body weight gain, food consumption, haematology, clinical chemistry and histopathological evaluations failed to show any significant differences in animals administered ARASCO, DHASCO or Formulaid compared with that in control animals administered equal amounts of high oleic sunflower oil. The bioavailability of ARASCO, DHASCO and Formulaid was verified by increases in DHA and ARA levels in heart and liver tissues in these animals. Because these oils are enriched in only a single bioactive fatty acid, and they have been shown to be safe, they may offer a new source of these fatty acids in speciality foods such as infant formula.