Margaret A. Cascieri

Cloning and Expression of a Novel Neuropeptide Y Receptor

The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

Peroxisome Proliferator-Activated Receptors γ and α Mediate in Vivo Regulation of Uncoupling Protein (UCP-1, UCP-2, UCP-3) Gene Expression

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Molecular cloning and characterization of a new receptor for galanin

Galanin (GAL) is a widely distributed neuropeptide with diverse biological effects including modulation of hormone release, antinociception and modification of feeding behavior. Its effects are mediated through G‐protein‐coupled receptors (GPCR) for which only a single type has been cloned, GAL receptor 1 (GALR1). We describe the cloning of a second galanin receptor type, GALR2, from rat hypothalamus. The GALR2 amino acid sequence is 38% identical to GALR1 and is pharmacologically similar to GALR1 when expressed in COS‐7 cells. GALR2 is encoded by a single gene containing at least one intron and expressed in a diverse range of tissues.

Tachykinin NK1 receptor antagonists act centrally to inhibit emesis induced by the chemotherapeutic agent cisplatin in ferrets

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.

Identification, localization and receptor characterization of novel mammalian substance P-like peptides

Hemokinin-1 (HK-1) is a novel substance P (SP)-like peptide that is encoded by the preprotachykinin C (PPT-C) gene recently identified in mouse B cells and shown to be a potentially important regulator of B cell development (Nat. Immunol. 1 (2000) 392). We have now isolated and characterized the human and rat orthologs of PPT-C and examined activities of human and mouse HK-1 on the three tachykinin receptors, neurokinin-1-3 (NK1-3). The rat PPT-C polypeptide is highly homologous to mouse PPT-C and contains the same processing sites to generate predicted HK-1. The human PPT-C polypeptide is also homologous to mouse PPT-C, however, it contains two potential monobasic cleavage sites rather than a single dibasic cleavage site at the amino-terminal end of the predicted HK-1 peptide. Thus, human PPT-C has the potential to generate full length predicted HK-1 as well as a truncated version (HK-1(4-11)). Polymerase chain reaction analysis revealed that both human and mouse PPT-C were expressed in a variety of tissues with strong signals detected in the skin of both species and in the mouse brain. Binding and functional analysis indicated that human and mouse HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that PPT-C encodes another high affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor.

Human β3-adrenergic receptor agonists containing 1,2,3-triazole-substituted benzenesulfonamides

Compounds containing a 1,2,3-triazole-substituted benzenesulfonamide were prepared and found to be potent and selective human beta3-adrenergic receptor agonists. The most interesting compound, trifluoromethylbenzyl analogue 12e (beta3 EC50 = 3.1 nM with >1500-fold selectivity over binding to both beta1- and beta2 receptors), stimulates lipolysis in the rhesus monkey (ED50 = 0.36 mg/kg) and is 25% orally bioavailable in the dog.

Potent, orally absorbed glucagon receptor antagonists.

The SAR of 2-pyridyl-3,5-diaryl pyrroles, ligands of the human glucagon receptor and inhibitors of p38 kinase, were investigated. This effort resulted in the identification of 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrr ole 49 (L-168,049), a potent (Kb = 25 nM), selective antagonist of glucagon.

The novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets

The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK-0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.

Visualization of rat brain receptors for the neuropeptide, substance P

Biochemical analysis of the binding of [125I]Bolton-Hunter coupled substance P [( 125I]BH-SP) to slide-mounted sections of rat brain demonstrated that [125I]BH-SP labels a binding site with a structure-activity profile characteristic of a substance P receptor. Under optimized preincubation and incubation conditions, the locations of substance P (SP) receptors were visualized by film and emulsion autoradiography. Receptor densities were quantified by computer-assisted densitometry. SP receptors are widely but discretely distributed throughout sensory, limbic and cortical areas of rat brain, though several motor areas also possess SP receptors. No receptors were detected in the substantia nigra and interpeduncular nucleus, which are innervated by SPergic nerves; these regions of the brain may possess a low affinity SP receptor not detectable with this assay. Findings are discussed in the framework of an overall notion of the role of neuropeptides in the biochemistry of emotion.

In vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists

The ability of tachykinin NK1 receptor antagonists to inhibit GR73632 (D-Ala-[L-Pro9,Me-Leu8]substance P-(7-11))-induced foot tapping in gerbils was employed as an indirect measure of brain penetration and this was compared with their ability to prevent acute emesis induced by cisplatin in ferrets. (+)-GR203040 ((2S,3S and 2R,3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin- 3-yl)-amine), CP-99,994 ((2S,3S)-cis-3-(2-methoxybenzylamino)-2-phenyl piperidine) dihydrochloride), and L-742,694 (2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-4-(5-(3-oxo-1,2, 4-triazolo)methylmorpholine) potently inhibited GR73632-induced foot tapping (ID50 < or = 0.85 mg/kg), and acute retching induced by cisplatin (ID50 < or = 0.18 mg/kg). RPR100893 ((3aS,4S,7aS)-7,7-diphenyl-4-(2-methoxyphenyl)-2-[(S)-2-(2-m ethoxyphenyl)proprionyl] perhydroisoindol-4-ol) was not a potent antagonist of retching (ID50 4.1 mg/kg) or foot tapping (ID50 > 10 mg/kg). High doses (3-10 mg/kg) of CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-N-[(4-quinolinyl)methyl] -4-piperineamine) dihydrochloride), FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-propyl]-N-methy l-N-phenylmethyl-L-3-(2-naphthyl)-alaninamide), and LY303870 ((R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(pi peridinyl)piperidin-1-yl)acetyl)amino]propane) were required to inhibit foot tapping; these agents were not anti-emetic in this dose range. SR140333 ((S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane; 3-10 mg/kg) failed to inhibit foot tapping or emesis. Affinities for the human and ferret tachykinin NK1 receptor were highly correlated (r = 0.93, P = 0.0008). Inhibition of foot tapping in gerbils, but not NK1 receptor binding affinity, predicted anti-emetic activity in ferrets (r = 0.75, P < 0.01). These findings confirm that the anti-emetic activity of tachykinin NK1 receptor antagonists is dependent on brain penetration.