Laying performance, digestibility and plasma hormones in laying hens exposed to chronic heat stress as affected by betaine, vitamin C, and/or vitamin E supplementation.

Heat stress had a negative effect on laying hens' performance, thus this research was to study the influences of betaine (Bet, 1000 mg/kg betaine), vitamin C (VC, 200 mg/kg ascorbic acid), and vitamin E (VE, 150 mg/kg α-Tocopherol acetate) and their possible combinations on egg production, digestibility of nutrients, plasma hormones and reproductive organs of dual-purpose hens exposed to chronic heat stress. Two hundred and eighty eight hens and thirty-six cocks from 32 to 48 weeks of age were divided into nine treatment groups of four replicates, each containing eight hens and one cock. One group was kept under thermo-natural condition and the eight others were kept under chronic heat stress (CHS). One of these eight was used as a negative control, while the others were supplemented with VC, VE and/or betaine and their possible combinations. Body weights, laying rate, feed intake, and feed conversion ratio in hens reared under CHS rooster without any supplementation during 32 to 48 weeks of impairment (P = 0.0052) were recorded. Hens reared under heat stress and fed a diet supplemented with either Bet, VC, VE or combination of the supplements increased production traits. However, hens supplemented with VC showed the greatest production traits. Plasma glucose, estradiol-17 (E2), progesterone (P4), tri-iodothyronine (T3) and thyroxine (T4) decreased in hens reared under CHS and fed a diet with no supplementation compared to the other treatments (P = 0.001). Liver weights, spleen weights, thyroid gland weights, ovary weights, oviduct weights and oviduct lengths were lowest in hens reared under CHS and fed a diet with no supplementation (P = 0.0480). In conclusion, dual purpose hens reared under CHS and supplemented with VC at 200 mg/kg diet and Bet at 1000 mg/kg enhanced the laying performance and combated CHS.

Disposition of CP-671, 305, a selective phosphodiesterase 4 inhibitor in preclinical species.

1. The disposition of (+)-2-[4-({[2-(benzo[1,3] dioxol-5-yloxy)-pyridine-3-carbonyl]-amino)-methyl)-3-fluoro-phenoxyl-propionic acid (CP-671,305), a potent and selective inhibitor of phosphodiesterase 4 (subtype D), was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 2. CP-671,305 demonstrates generally favourable pharmacokinetic properties in all species examined. Systemic plasma clearance after intravenous administration was low in Sprague-Dawley rats (9.60+/-1.16 ml min(-1) kg(-1)), beagle dogs (2.90+/-0.81 ml min(-1) kg(-1)) and cynomolgus monkeys (2.94+/-0.87ml min(-1) kg(-1)) resulting in plasma half-lives > 5 h. Moderate to high bioavailability in rats (43-80%), dogs (45%) and monkeys (26%) was observed after oral dosing. In rats, oral pharmacokinetics were dose dependent over the dose range studied (10 and 25 mgkg(-1)). 3. CP-671,305 was > 97% bound to plasma proteins in rat, dog, monkey and human. 4. The principal route of clearance of CP-671,305 in rats and dogs was by renal and biliary excretion of unchanged drug. This finding was consistent with CP-671,305 resistance towards metabolism in hepatocytes and NADPH-supplemented liver microsomes from preclinical species and human. 5. CP-671,305 did not exhibit competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, 2C9, 2C19, 2D6 and 3A4 (IC50's > 50 microM). Likewise, no time-dependent inactivation of the five major cytochrome P450 enzymes was discernible with CP-671,305. 6. Overall, the results indicate that the absorption, distribution, metabolism and excretion (ADME) profile of CP-671,305 is relatively consistent across preclinical species and predict potentially favourable pharmacokinetic properties in humans, supporting its selection for toxicity/safety assessment studies and possible investigations in humans.

Pharmacokinetics and metabolism of a cysteinyl leukotriene-1 receptor antagonist from the heterocyclic chromanol series in rats: in vitro-in vivo correlation, gender-related differences, isoform identification, and comparison with metabolism in human hepatic tissue.

CP-199,331 is a potent antagonist of the cysteinyl leukotriene-1 (LT(1)) receptor, targeted for the treatment of asthma. The pharmacokinetic/metabolism properties of CP-199,331 were studied in rats and compared with those in human liver microsomes/hepatocytes. In vitro biotransformation of CP-199,331 in rat and human hepatocytes was similar, consisting primarily of CP-199,331 O-demethylation. Marked sex-related differences in plasma clearance (CL(p)) of CP-199,331 were observed in rats: 51 and 1.2 ml/min/kg in males and females, respectively. This difference in CL(p) was attributed to gender differences in metabolizing capacity because V(max) and K(m) values for CP-199,331 metabolism were 30-fold higher and 8-fold lower, respectively, in male rat liver microsomes compared with female microsomes. Scale-up of the in vitro microsomal data predicted hepatic clearance (CL(h)) of 64 and 2.5 ml/min/kg in male and female rats, respectively. These values were in close agreement with the in vivo CL(p), suggesting that CP-199,331 CL(p) in male and female rats was entirely due to hepatic metabolism. Studies with rat recombinant cytochromes P450 and anti-rat cytochrome P450 (CYP) antibodies revealed the involvement of male rat-specific CYP2C11 in the metabolism of CP-199,331. In contrast, CP-199,331 metabolism in human liver microsomes was principally mediated by CYP3A4. The projected human clearance in liver microsomes and hepatocytes varied 6-fold from low to moderate, depending on CYP3A4 activity. Considering that O-demethylation is the major route of elimination in humans, the in vivo clearance of CP-199,331 may exhibit moderate variability, depending on CYP3A4 abundance in the human population.

Discovery of CP-199,330 and CP-199,331: two potent and orally efficacious cysteinyl LT1 receptor antagonists devoid of liver toxicity.

CP-199,330 (3) and CP-199,331 (4) are cysLT1 receptor antagonists that are equipotent to marketed cysLT1 receptor antagonists zafirlukast and pranlukast, show good pharmacokinetics in rats and monkeys, and are devoid of liver toxicity in monkeys as seen in CP-85,958 (1).

Development of new chromanol antagonists of leukotriene D4.

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

N-carbamoyl analogs of Zafirlukast: potent receptor antagonists of leukotriene D4.

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.

Development of 2,2-dimethylchromanol cysteinyl LT1 receptor antagonists.

A new series of cysLT1 receptor antagonists represented by CP-288,886 (7) and CP-265,298 (8) were developed which are equipotent to clinical cysLT1 receptor antagonists Zafirlukast (1) and Pranlukast (2).

Biarylcarboxylic acids and -amides: inhibition of phosphodiesterase type IV versus [3H]rolipram binding activity and their relationship to emetic behavior in the ferret.

In addition to having desirable inhibitory effects on inflammation, anaphylaxis, and smooth muscle contraction, PDE-IV inhibitors also produce undesirable side effects including nausea and vomiting. In general, compounds that inhibit PDE-IV also potently displace [3H]rolipram from a high-affinity binding site in rat cortex. While this binding site has not been identified, it has been proposed to be an allosteric binding site on the PDE-IV enzyme. Preliminary studies have suggested that the emetic potency of PDE-IV inhibitors is correlated with affinity for the brain rolipram binding site rather than potency at inhibiting PDE-IV enzyme activity. Efforts to eliminate the emetic potential of PDE-IV inhibitors were directed toward developing compounds with decreased [3H]rolipram binding affinity while retaining PDE-IV potency. Thus, a novel series of 4-(3-alkoxy-4-methoxyphenyl)benzoic acids and their corresponding carboxamides were prepared and evaluated for their PDE-IV inhibitory and rolipram binding site properties. Modification of the catechol ether moiety led to phenylbutoxy and phenylpentoxy analogues that provided the desired activity profile. Specifically, 4-[3-(5-phenylpentoxy)-4-methoxyphenyl]-2-methylbenzoic acid, 18, was found to exhibit potent PDE-IV inhibitory activity (IC50 0.41 microM) and possessed 400 times weaker activity than rolipram for the [3H]rolipram binding site. In vivo, compound 18 was efficacious in the guinea pig aerosolized antigen induced airway obstruction assay (ED50 8.8 mg/kg, po) and demonstrated a significant reduction in emetic side effects (ferret, 20% emesis at 30 mg/kg, po).

Ampiroxicam, an anti-inflammatory agent which is a prodrug of piroxicam.

Ampiroxicam is a nonacidic ether carbonate prodrug of piroxicam. Our results demonstrate that, in contrast to piroxicam, ampiroxicam does not possess detectable prostaglandin synthesis inhibitory activity in vitro. Ampiroxicam, however, has similar in vivo potency to piroxicam in suppressing paw swelling in rat adjuvant arthritis. In an acute model of paw inflammation in rats, ampiroxicam is less potent than piroxicam itself: the ED50's of ampiroxicam are 9- and 3.5-fold higher than those of piroxicam following a single or multiple (5) daily oral doses, respectively. Using the phenylbenzoquinone stretching test as a method of evaluating acute analgetic activity, the ED50 for ampiroxicam is about 3-fold higher than that of piroxicam. These tests of activity share the property of being partially prostaglandin-dependent. Ampiroxicam itself is not observed in plasma after oral dosing to man, nor in the rat, dog, and monkey as reported here. Bioavailability studies show that conversion to piroxicam is about 100%, 90%, 70%, and 50% in these four species, respectively. These results indicate that ampiroxicam's anti-inflammatory activity is produced in vivo by conversion to piroxicam and support its credentials as an efficacious prodrug of piroxicam.

Role of protein kinases in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists. Differential effects of a novel protein kinase inhibitor.

Isoquinoline sulfonamides have recently been shown to exert novel inhibitory effects on mammalian protein kinases by competitively binding to the ATP substrate site (Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki, 1984, Biochemistry, 23: 5036-5041). We synthesized a unique analog of the previously reported compounds, 1-(5-isoquinolinesulfonyl) piperazine (C-I), in order to assess the role of protein kinases in modulating the agonist-stimulated oxidative burst of human polymorphonuclear leukocytes (PMN). Compound C-I, at micromolar concentration, markedly inhibited the release of superoxide anion from human PMN stimulated with phorbol myristate acetate or the synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol. These data are consonant with previously reported data which indicate that the calcium and phospholipid-dependent protein kinase, protein kinase C, serves as the intracellular receptor for these agonists. In contrast, superoxide anion production stimulated by the complement anaphylatoxin peptide C5a or the synthetic chemotaxin formyl-methionyl-leucyl-phenylalanine were not inhibited by C-I. These data suggest that parallel pathways exist for the agonist-stimulated respiratory burst of human neutrophils, only one of which utilizes the calcium and phospholipid-dependent protein kinase.

Radioimmunoassay for leukotriene B4.

A rabbit immunized with leukotriene B(4) [LTB(4); (5S,12R)-6, 14-cis-8, 10-trans-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (K(a)) for [14,15-(3)H]LTB(4) of 3.2 x 10(9) M(-1) at 37 degrees C and in a concentration of 0.37 mug/ml of the immune plasma. When 10 mul of anti-LTB(4) and 3.9 nCi of [14,15-(3)H]LTB(4) (28 Ci/mmol; 1 Ci = 3.7 x 10(10) becquerels) were incubated in a volume of 250 mul, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB(4) and with 1.95 ng of (5S,12S)-6-trans-8-cis-LTB(4). The sulfidopeptide leukotrienes, LTC(4) and LTD(4), displaced the radioligand from this antibody with less than 1/100th the activity of LTB(4), and the diastereoisomers of 6-trans-LTB(4), 5-L-hydroxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB(4) and quantitatively correlated with the physical measurement by integrated A(269) in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB(4) generated averaged 4.6 ng per 10(6) rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB(4), a highly potent chemotactic factor, to unfractionated biologic fluids.

Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis.

A rabbit immunized with a conjugate of leukotriene D4 (LTD4) and bovine albumin via the icosanoid carboxyl produced antibodies with comparable affinities for leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4) and their 11-trans stereoisomers. The antibodies bound 3H-labeled 11-trans-LTC4 and 11-trans-LTC4 with the same average association constant (Ka) of 2.8 x 10(9) M-1 at 37 degrees C and were present at a concentration of 0.32 microgram/ml of the immune rabbit plasma. When 9.5 microliter of anti-LTD4 and 108 pmol of 11-trans-[3H]LTC4 (40 Ci/mmol) were incubated in a volume of 300 microliter with LTC4, LTD4, LTE4, or their 11-trans stereoisomers, 50% inhibition of 11-trans-[3H]LTC4 binding was achieved at levels varying between 0.3 and 0.7 ng. As assessed with synthetic analogs of the natural leukotrienes, the antibodies recognized neither those changes within the 6-sulfidopeptide unit of LTD4 produced by deamination or modest peptide lengthening nor the specific stereochemistry of the delta 14-cis double bond. However, the antibodies did recognize the triene lipid domain and the position and spatial orientation of the glutathione or cysteinylglycine function. Binding of 11-trans-[3H]LTC4 by anti-LTD4 was not inhibited by glutathione, cystinylbisglycine, arachidonic acid, or 5-hydroxy-6,8,11,14-icosatetraenoic acid, and leukotriene B4 (LTB4) was about 1/1000th as active as LTC4, LTD4, or LTE4. Mouse lymphoma (WEHI-5) and rat basophil leukemia (RBL-1) cells, when stimulated with calcium ionophore A23187, each produced immunoreactive leukotrienes; and LTC4, LTD4, and LTE4 from RBL-1 cells were individually quantitated by radioimmunoassay after resolution by high-performance liquid chromatography.

Contractile activities of structural analogs of leukotrienes C and D: role of the polar substituents.

Twenty-three structural analogs of the leukotriene components of slow reacting substance of anaphylaxis (SRS-A), in which the polar regions of the leukotriene were systematically modified, were tested for their contractile activities on guinea pig pulmonary parenchymal strips and guinea pig ileum. The structural modifications allowed evaluation of the separate contributions of the four polar units in the C-1 to C-6 region of the SRS-A leukotrienes to smooth muscle spasmogenic activity. The free NH2-terminal amino group of the S-linked peptide was necessary for full activity, and its deletion or substitution reduced activity by more than one but less than two orders of magnitude. A similar level of importance was apparent for the free glycine carboxyl group. In contrast, a free eicosanoid carboxyl at C-1 is not required for full activity on the airway and for substantial activity on the ileum. A role for the C-5 hydroxyl is indicated by the inactivity of the one available 5-desoxy analog. Nucleophilic, divalent sulfur is not critical to leukotriene D (LTD) activity, in that one sulfoxide had substantial function. The conformational relationship between the eicosanoid and peptide moieties of LTD is of considerable importance in that epimers at the C-5 or C-6 position were less active than LTD by more than two orders of magnitude. Several lines of evidence suggest that the relative geometrical arrangement of the C20 chain and the peptide unit is important to activity, consistent with the existence of a true receptor for LTD.

Contractile activities of structural analogs of leukotrienes C and D: necessity of a hydrophobic region.

Sixteen structural analogs of leukotrienes C and D were tested for their contractile activities on guinea pig pulmonary parenchymal strip and ileum. The analogs differed from the native structures in the position of either the thioether-linked peptide side chain or the hydroxyl group (or both) or in the number and positions of ethylenic bonds. Analogs in which the thioether-linked peptide chain was attached other than at the C-6 position had substantial reductions in activity on both smooth muscle preparations, whereas analogs in which the various ethylenic bonds were saturated retained substantial contractile activity in both assays. These observations demonstrate that, although a hydrophobic region of the eicosinoid is necessary for contractile activity, the length of this segment is more critical than its precise stereochemistry. Analogs of leukotrienes based on the possibility of parallel biosynthetic routes deriving from 8-, 11-, and 15-hydroperoxyeicosatetraenoic acid as precursors were found to effect a comparatively weak contractile response so that their role as biological agents in this respect seems unlikely.

Leukotriene A. Isolation from human polymorphonuclear leukocytes.

Leukotriene A, an unstable intermediate in the conversion of arachidonic acid to stable leukotrienes, was isolated from human polymorphonuclear leukocytes. The allylic epoxide intermediate is rapidly hydrolyzed under acidic conditions. A method was therefore developed for esterification and extraction of the intermediate as the methyl ester from an alkaline aqueous phase, into an aprotic solvent. This was achieved by addition of methanol and an excess of diazomethane in ether to the incubatio mixture, followed by addition of water, and phase separation. The identity of the isolated compound with the previously synthesized methyl ester of 5 (S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicostatetraenoic acid (leukotriene A), was established by comparing chromatographic and chemical properties of the isolated compound and synthetic leukotriene A.

Comparative airway and vascular activities of leukotrienes C-1 and D in vivo and in vitro.

The pharmacologic activities of leukotrienes C-1 and D(LTC-1 and LTD), constituents of slow reacting substance of anaphylaxis (SRS-A), were evaluated in vitro on airway contractile tissues and in vivo on pulmonary mechanical function, mean systemic arterial pressure, and cutaneous microcirculation. In vitro both LTC-1 and LTD were potent and selective peripheral airway agonists, being more active than histamine; furthermore, LTD was active on peripheral airways at concentrations 1/100th those of LTC-1. The concentration-effect relationship for LTD and the profile of antagonism by FPL 55712 are consistent with the activity of this molecule at two separate peripheral airway receptors. In vivo, LTC-1 and LTD were nearly equally active in their effects on pulmonary mechanics, and the pattern of alterations was consistent with the predominant site of action being in the lung periphery. Furthermore, both agents had a direct systemic arterial hypotensive effect and were vasoactive on the cutaneous microcirculation. Thus, these compounds are likely to be major mediators of the pathologic alterations in immediate type hypersensitivity reactions in which peripheral airway constriction and hypotension are prominent features.

Slow reacting substances of anaphylaxis: identification of leukotrienes C-1 and D from human and rat sources.

Slow reacting substance(s) of anaphylaxis (SRS-A) was isolated from both human (lung) and rat sources and compared with three synthetic SRS-As of known structure-leukotrienes (LTs) C-1, C-2, and D. Reversed-phase liquid chromatography was used both as a final purification step and a means of comparison of biologically derived and synthetic substances. Two major peaks of SRS-A activity of both rat and human origin corresponded chromatographically with LTC-1 and LTD, respectively, and had equivalent specific activities on the guinea pig ileum. With guinea pig ileum, the specific activities (units/pmol) for synthetic leukotrienes and anaphylactic peaks were (mean +/- SEM): synthetic LTC-1, 1.93 +/- 0.13; SRS-A(rat) peak I, 1.69 +/- 0.43; synthetic LTD, 6.10 +/- 1.15; SRS-A(rat) peak II, 7.14 +/- 0.51; and SRS-A(hu) peak II, 1.90. Both synthetic LTC-1 and LTD and their SRS-A natural counterparts had a preferential contractile activity on guinea pig peripheral airway compared to central airways and were at least 200 times more active than histamine on peripheral airways on a molar basis. Leukotriene D is the major SRS-A of human lung and accounts for almost all of the biological activity. It likely is formed from leukotriene C-1 in vivo by an enzymic process of the well-known gamma-glutamyltransferase type.