Biomedical subjects
Publications and source records attributed to D E MacIntyre.
The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.
Pyridyloxypropanolamines L-749,372 (8, beta 3 EC50 = 3.6 nM) and L-750,355 (29, beta 3 EC50 = 13 nM) are selective partial agonists of the human receptor, with 33% and 49% activation, respectively. Both stimulate lipolysis in rhesus monkeys (ED50 = 2 and 0.8 mg/kg, respectively), with minimal effects on heart rate. Oral bioavailability in dogs, 41% for L-749,372 and 47% for L-750,355, is improved relative to phenol analogs.
Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK1, NK2, and NK3. The NK1 subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK1 antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK1 receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK1 antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK1 antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK1 antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK1 antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK1 receptors, modulate photic responses of the SCN pacemaker.
Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.
A procedure is described for gram-scale refolding of Escherichia coli-derived human leptin inclusion bodies. Refolding was achieved by gradually reducing denaturant using a diafiltration method. Refolded leptin is characterized by in vivo modulation of food intake, reduction in body weight, and lowering of insulin and glucose levels in ob/ob mice. In addition, refolded leptin is characterized by radioimmunoassay (RIA) and activation of the leptin receptor in a cell-based assay. For comparison we also refolded leptin by a simple dilution method and produced periplasmic derived leptin, which did not require ex vivo folding. Leptin produced by these three methods and leptin obtained from commercial sources were compared using the RIA and the cell-based assay and appeared to be of comparable quality and potency.
The beta3-adrenergic receptor is an integral membrane protein consisting of seven transmembrane domains. Unlike the beta1 and beta2 receptors, this subtype lacks the consensus phosphorylation sites required for desensitization by serine kinases. Using the rodent specific beta3 agonist BRL 35135, our initial data indicated that beta3 receptor-mediated glycerol levels progressively decreased following daily oral doses of 5 mg/kg. Therefore, we initiated studies designed to delineate the possible mechanism(s) for this decreased response. Within 3 hours following a single oral dose of BRL 35135, serum glycerol levels and UCP (uncoupling protein) RNA levels were significantly increased whereas beta3 RNA levels were significantly decreased. Rats were dosed daily for 5 days with either vehicle or BRL 35135 (5 mg/kg, p.o.) and blood samples were collected for glycerol analysis. Adipose tissue was excised for lipolysis and adenyl cyclase measurements. In addition, UCP and beta3 receptor RNA levels were assessed. No effect on adipocyte BRL 37344-stimulated adenylyl cyclase activity was observed 3 hours following the initial dose of BRL 35135. Although a slight decrease (approximately 25%) in adenylyl cyclase activity could be observed 24 hours following the initial dose, it wasn't until day 4 of dosing that a significant decrease (50%) was observed. In contrast, beta3- stimulated lipolysis in adipocytes from BRL 35135-treated rats was decreased 85% within 24 hours and this decrease persisted through four days of treatment. These data indicate that the lipolytic response to beta3 receptor activation is decreased after only a single oral dose of BRL 35135, whereas receptor-mediated adenylyl cyclase activation, although initially unaffected, also desensitizes by day four of treatment.
Melanocortinergic neurons are believed to play a role in the control of food intake. Melanocortin receptor agonists and antagonists modulate feeding in several mouse models of chemically and genetically induced hyperphagia. To date, little information is available describing the role of this neurological system in the control of the natural feeding cycle in genetically intact rats. To evaluate the involvement of melanocortins in spontaneous nocturnal feeding, the synthetic melanocortin receptor agonist, MTII and the antagonist, SHU9119 were administered ICV (third ventricle) alone and in combination. Dose-dependent inhibition or stimulation of food intake was observed with MTII or SHU9119, respectively. Co-injections containing equal concentrations of MTII and SHU9119 resulted in food intake that was indistinguishable from controls. Food intake patterns observed in studies in which various dose combinations of MTII and SHU9119 were co-injected are consistent with the concept that both affect feeding by acting on similar melanocortin receptors. The hypothesis that effects of melanocortins on feeding may be mediated via an NPY related pathway was tested by co-injecting MTII and NPY in a 2-h satiated food intake paradigm. MTII inhibited food intake induced by 5.0 microg hNPY in a dose dependent manner with the highest dose tested abolishing the NPY feeding response. The studies suggest that melanocortins act via specific receptors to control food intake in rats, possibly via an NPY related pathway. If similar neurochemical processes operate in humans, selectively modulating specific melanocortin receptor signaling may be an approach to the treatment of human obesity.
The treatment of obesity requires modulation of both energy intake and energy expenditure, and pharmaceutical treatments are being developed to complement the traditional means of dietary restriction and exercise. The recent discovery of the protein leptin, which modulates both food intake and energy expenditure, has provided a new tool with which to define and analyze potential pathways for pharmacological intervention. Neurotransmitters, such as neuropeptide Y (NPY) and norepinephrine, act downstream of leptin to modulate energy homeostasis. Specific subtypes of the receptors for these neurotransmitters represent promising molecular targets for the discovery of new drugs for the treatment of obesity.
The preparation of a series of N-heteroarylpiperidine ether-based human NK1 antagonists is described. Two of the compounds 3-[-(2S,3S)-3-(((3,5-bis(trifluoromethyl)phenyl)methyl)oxy)- 2-phenylpiperidino}methyl]-1,2,4-triazole (11) and 5-[¿(2S,3S)-3-(((3,5-bis(trifluoromethyl)-phenyl)methyl)oxy)-2- phenylpiperidino}methyl]-3-oxo-1,2,4-triazolone (12)), in particular, are orally bioavailable and exhibited significant improvements in potency, both in vitro and in vivo, over the lead (carboxamidomethyl)piperidine ether 1. Rat liver microsome studies on a selected number of compounds from this series show the triazolone heterocycle to be considerably more stable than the others. Furthermore, both 11 and 12 have been profiled in a number of assays that may be predictive of the clinical utility of substance P antagonists.
Explore the source record for details and available documents.
These studies have compared the pharmacological profile of two non-peptide human type neurokinin1 (hNK1) receptor selective antagonists, L-741,671 and a quaternised compound L-743,310. In radioligand binding studies L-741,671 and L-743,310 had high affinity for ferret and cloned hNK1 receptors [Ki (nM) ferret 0.7 and 0.1; human 0.03 and 0.06, respectively] but low affinity for rodent NK1 receptors [Ki (nM) 64 and 17, respectively] suggesting that ferret receptors have hNK1-like binding pharmacology. Studies in vivo showed that L-741,671 and L-743,310 had equivalent functional activity in the periphery (ID50s of 1.6 and 2 micrograms/kg i.v., respectively) as measured by inhibition of plasma protein extravasation evoked in the oesophagus of guinea pigs by resiniferatoxin (7 nmol/kg i.v.). Using an in situ brain perfusion technique in anaesthetised rats, L-741,671 was shown to be much more brain penetrant than the quaternary compound L-743,310 which had an entry rate similar to the poorly brain penetrant plasma marker inulin. These compounds thus provided an opportunity to compare the anti-emetic effects of equi-active hNK1 receptor antagonists with and without brain penetration to central NK1 receptor sites. When tested against cisplatin-induced emesis in ferrets, L-741,671 (0.3, 1 and 3 mg/kg i.v.) produced marked dose-dependent inhibition of retching and vomiting but L-743,310 was inactive at 3 and 10 micrograms/kg i.v. In contrast, direct central injection of L-741,671 and L-743,310 (30 micrograms) into the vicinity of the nucleus tractus solitarius or L-743,310 (200 micrograms) intracisternally was shown to inhibit retching and vomiting induced by i.v. cisplatin. L-741,671 and L-743,310 had equivalent functional activity, at the same dose, against cisplatin-induced emesis when injected centrally. These observations indicated that had L-743,310 penetrated into the brain after systemic administration it would have been active in the cisplatin-induced emesis assay and so show that brain penetration is essential for the anti-emetic action of systemically administered NK1 receptor antagonists.
The synthesis and in vitro and in vivo evaluation of a series of 3-(benzyloxy)-1-azabicyclo-[2.2.2]octane NK1 antagonists are described. While a number of 3,5-disubstituted benzyl ethers afford high affinity, the 3,5-bis(trifluoromethyl)benzyl was found to combine high in vitro affinity with good oral activity. Detailed structure-activity relationship studies in conjunction with data from molecular modeling and mutagenesis work have allowed the construction of a model of the pharmacophore. Specific interactions that have been identified include an interaction between His-197 and one of the rings of the benzhydryl, a lipophilic pocket containing His-265 that the benzyl ether occupies, and a possible hydrogen bond between Gln-165 and the oxygen of the benzyl ether.
The 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan (3), which was derived from the screening lead N-ethyl-L-tryptophan benzyl ester, has been used as a starting point to identify high-affinity substance P receptor antagonists with improved in vivo activity. Altering the ester moiety to an amide or ether led to a substantial loss in binding affinity, but conversion to a ketone provided compounds with affinity comparable to the equivalent esters. A homochiral synthesis of the key intermediate amino ketone 15 was developed which allows its preparation on a large scale. From this intermediate a range of amine-containing acylamino derivatives were prepared with affinity optimized in the morpholinylbutyramide 161 which has an IC50 of 0.17 nM at the human NK1 receptor. In addition to improving affinity, the amino group also provided aqueous solubility for a number of these derivatives. When tested in vivo the quinuclidine derivative L-737,488 (16i) was found to be an orally active (ID50 = 1.8 mg/kg) inhibitor of substance P-induced dermal extravasation in the guinea pig.
A potent, reversible, tetrapeptide inhibitor of interleukin-1 beta converting enzyme (ICE), L-709,049, has been shown to suppress the in vitro production of mature IL-1 beta. We now report that this inhibitor also effectively suppresses the production of mature IL-1 beta in a murine model of endotoxic shock. Intraperitoneal administration of L-709,049 reduced the elevations of IL-1 beta in the plasma and peritoneal fluid of mice treated with LPS in a dose-related manner (ED50 = 2 +/- 0.9 mg/kg). LPS-induced elevations in IL-1 alpha and IL-6 in these mice were unaffected, indicating that the inhibitor specifically affected IL-1 beta production. Immunoblot analysis of plasma and peritoneal fluid indicated that L-709,049 suppressed the formation of mature IL-1 beta production in vivo. When mouse blood was incubated in vitro with LPS, IL-1 beta was released into the plasma. This assay was used to determine ex vivo the activity of an ICE inhibitor in the blood following its administration to mice. Blood obtained 15 minutes after ip administration of 10 mg/kg of L-709,049 to mice produced 80% less IL-1 beta than control blood, and IL-1 beta production returned to control levels in blood obtained 30 minutes after injection of this inhibitor. In addition, the capacity of the blood plasma obtained from these animals to prevent the cleavage of a synthetic substrate by ICE disappeared within 1 h of ip administration of 50 mg/kg of inhibitor.
The role of inducible nitric oxide synthase (iNOS) was examined in the hypotension and vascular hyporesponsiveness to norepinephrine (NE) invoked by lipopolysaccharide (LPS) in pentobarbital-anesthetized rats. Saline, dexamethasone (DEX), NG-monomethyl-L-arginine (LNMMA) or indomethacin (IND) were administered either pre-LPS (0.5 hr) or post-LPS (4.5 hr) treatment. Rats were then challenged with NE 10 min before LPS injection and 1, 4, and 5 hr after LPS. Administration of LPS produced a biphasic hypotension: an immediate hypotension, which partially recovered within 15 min and was unaffected by any of the pretreatments; and a secondary, more prolonged hypotension which was attenuated by DEX, LNMMA and IND. The NE-induced pressor effects were significantly attenuated 1, 4 and 5 hr post LPS. Pretreatment with LNMMA or DEX significantly attenuated the LPS-induced NE hyporesponsiveness 4 and 5 hr post LPS. LNMMA was the only post-LPS treatment able to reverse the NE hyporesponsiveness. The LPS-induced iNOS mRNA and protein expression was demonstrated in the liver, lung, spleen, heart, kidney and brain by Northern hybridization and Western blot analyses. Low levels of neuronal constitutive NOS mRNA and endothelial cell constitutive NOS mRNA were only detected in brain or myocardial tissue, respectively. Significant induction of iNOS mRNA and protein expression was also observed in the liver, lung and spleen of rats pretreated with DEX, LNMMA or IND. The continued expression of iNOS in the presence of a pharmacologically relevant dose of DEX suggests that DEX may not be an optimal pharmacological agent for defining the in vivo roles of iNOS.(ABSTRACT TRUNCATED AT 250 WORDS)
Equilibrium binding studies on canine mononuclear and granulocytic cells allow the identification of a single high affinity receptor for the human C-C chemokine RANTES (dissociation constant, 14 +/- 8 pM), that, in contrast to the human RANTES receptor, has no affinity for human macrophage inflammatory protein 1 alpha (hMIP-1 alpha). A single intradermal injection of hRANTES in dog resulted in eosinophil- and macrophage-rich inflammatory sites within 4 h. Cell infiltration peaked at 16-24 h after hRANTES injection. There was histological evidence of intravascular activation of eosinophils at 4 h, although eosinophils in the vasculature and interstitium contained apparently intact granules. Monocytes were the predominant cells adherent to venular endothelium at 16-24 h. Human MIP-1 alpha elicited no response in canine dermis, whereas monocyte chemoattractant protein 1 caused mild perivascular cuffing with monocytes. In contrast, human interleukin 8 induced a neutrophilic dermal infiltrate that was maximal by 4 h after challenge. This provides the first direct evidence in vivo that RANTES has significant proinflammatory activity and, in addition, could be a mediator in atopic pathologies characterized by eosinophilic and monocytic inflammatory responses.
Human polymorphonuclear leukocytes (PMNs) stimulated by N-formylmethionyl-leucylphenylalanine (fMLP), leukotriene B4 (LTB4) and platelet-activating factor (PAF) showed a similar sequence of ultrastructural changes. Exposure of unstirred PMNs to these soluble stimuli induced a rapid (within 10 s) plasma membrane ruffling followed by polarization of cells associated with formation of broad agranular lamellipodia (maximal by 30 s). Similar changes were observed in stirred PMN suspensions except that the polarized cells formed multicellular aggregates with the agranular lamellipodia extending outward from the clumps. In addition, these receptor-directed agonists induced concentration-dependent PMN aggregation that was rapid and reversible, and in cells pretreated with cytochalasin B the agonists induced a degranulatory response. The calcium ionophore ionomycin also activated PMNs but, in contrast to the other stimuli, ionomycin produced irreversible aggregation and evoked enzyme release in the absence of cytochalasin B. Ionomycin stimulated LTB4 release, whereas the response induced by PAF or fMLP was barely detectable. The possible role of LTB4 and PAF in mediating responses to other agonists was investigated pharmacologically using the 5-lipoxygenase inhibitor PF 5901 and a specific PAF antagonist, kadsurenone. PF 5901 blocked ionomycin-induced LTB4 release but did not inhibit aggregation or degranulation induced by ionomycin, PAF, fMLP, or LTB4. Kadsurenone blocked PAF-induced aggregation and degranulation but not responses to LTB4, ionomycin, or fMLP. We conclude that LTB4 and PAF are potent activators of human PMNs that act independently of one another and that these lipids appear not to be involved in the responses to the other agonists studied.
MK 287 (L-680,573), a tetrahydrofuran analog, potently inhibited [3H]C18-PAF binding to human platelet, polymorphonuclear leukocyte (PMN) and lung membranes with K1 values of 6.1 +/- 1.5, 3.2 +/- 0.7, and 5.49 +/- 2.3 nM, respectively. The inhibitory effects are stereospecific and competitive. The racemate, L-668,750 is less potent and the enantiomer, L-680,574 is 20-fold less potent than MK 287. Inhibition of the binding of [3H]C18-PAF to human PMN membranes by MK 287 was associated with the reduction of the affinity of the radioligand but not the number of the receptor sites. Binding of other radioligands (e.g., LTB4, LTC4, C5a, FMLP) to their specific receptors was unaltered at 1-10 microM MK 287. [3H]MK 287 bound to membranes from human platelets and PMNs: KD = 2.1 +/- 0.6 and 2.9 +/- 1.2 nM, respectively. When examined on isolated human cells, MK 287 potently and selectively inhibited PAF-induced aggregation of platelets in plasma (ED50 = 56 +/- 38 nM) or gel-filtered platelets (ED50 = 1.5 +/- 0.5 nM) and elastase release from PMNs (ED50 = 4.4 +/- 2.6 nM). In studies in vivo, MK 287 inhibited PAF-induced lethality in mice (ED50 = 0.8 mg/kg orally) and PAF-induced bronchoconstriction in guinea pigs (ED50 = 0.18 mg/kg intraduodenally and 0.19 mg/kg intravenously). Inhibition of PAF-induced bronchoconstriction was accompanied by parallel rightward shifts in concentration-response curves for PAF-induced platelet aggregation measured ex vivo.