CHF6001 II: a novel phosphodiesterase 4 inhibitor, suitable for topical pulmonary administration--in vivo preclinical pharmacology profile defines a potent anti-inflammatory compound with a wide therapeutic window.

CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide] is a novel phosphodiesterase 4 (PDE4) inhibitor designed for use in pulmonary diseases by inhaled administration. Intratracheal administration of CHF6001 to ovalbumin-sensitized Brown-Norway rats suppressed the antigen-induced decline of lung functions (ED50 = 0.1 µmol/kg) and antigen-induced eosinophilia (ED50 = 0.03 µmol/kg) when administered (0.09 µmol/kg) up to 24 hours before antigen challenge, in agreement with CHF6001-sustained lung concentrations up to 72 hours after intratracheal treatment (mean residence time 26 hours). Intranasal, once daily administration of CHF6001 inhibited neutrophil infiltration observed after 11 days of tobacco smoke exposure in mice, both upon prophylactic (0.15-0.45 µmol/kg per day) or interventional (0.045-0.45 µmol/kg per day) treatment. CHF6001 was ineffective in reversing ketamine/xylazine-induced anesthesia (a surrogate of emesis in rat) up to 5 µmol/kg administered intratracheally, a dose 50- to 150-fold higher than anti-inflammatory ED50 observed in rats. When given topically to ferrets, no emesis and nausea were evident up to 10 to 20 µmol/kg, respectively, whereas the PDE4 inhibitor GSK-256066 (6-[3-(dimethylcarbamoyl)phenyl]sulfonyl-4-(3-methoxyanilino)-8-methylquinoline-3-carboxamide) induced nausea at 1 µmol/kg intratracheally. A 14-day inhalation toxicology study in rats showed a no-observed-adverse-effect level dose of 4.4 µmol/kg per day for CHF6001, lower than the 0.015 µmol/kg per day for GSK-256066. CHF6001 was found effective and extremely well tolerated upon topical administration in relevant animal models, and may represent a step forward in PDE4 inhibition for the treatment of asthma and chronic obstructive respiratory disease.

Discovery of diaryl imidazolidin-2-one derivatives, a novel class of muscarinic M3 selective antagonists (Part 1).

Pharmacophore-based structural identification, synthesis, and structure-activity relationships of a new class of muscarinic M3 receptor antagonists, the diaryl imidazolidin-2-one derivatives, are described. The versatility of the discovered scaffold allowed for several structural modifications that resulted in the discovery of two distinct classes of compounds, specifically a class of tertiary amine derivatives (potentially useful for the treatment of overactive bladder by oral administration) and a class of quaternary ammonium salt derivatives (potentially useful for the treatment of respiratory diseases by the inhalation route of administration). In this paper, we describe the synthesis and biological activity of tertiary amine derivatives. For these compounds, selectivity for the M3 receptor toward the M2 receptor was crucial, because the M2 receptor subtype is mainly responsible for adverse systemic side effects of currently marketed muscarinic antagonists. Compound 50 showed the highest selectivity versus M2 receptor, with binding affinity for M3 receptor Ki = 4.8 nM and for M2 receptor Ki = 1141 nM. Functional in vitro studies on selected compounds confirmed the antagonist activity toward the M3 receptor and functional selectivity toward the M2 receptor.

Discovery of diaryl imidazolidin-2-one derivatives, a novel class of muscarinic M3 selective antagonists (Part 2).

Synthesis and biological activity of a novel class of quaternary ammonium salt muscarinic M3 receptor antagonists, showing high selectivity versus the M2 receptor, are described. Selected compounds exhibited potent anticholinergic properties, in isolated guinea-pig trachea, and good functional selectivity for trachea over atria. In vivo, the same compounds potently inhibited acetylcholine-induced bronchoconstriction after intratracheal administration in the guinea pig.

Pharmacological assessment of the duration of action of glycopyrrolate vs tiotropium and ipratropium in guinea-pig and human airways.

1. Our study was aimed at investigating the duration of the bronchodilator action of the antimuscarinc drug glycopyrrolate compared to tiotropium and ipratropium. In the guinea-pig isolated trachea, the time (t1/2) necessary for a contractile response to carbachol (0.3 microM) to return to 50% recovery after washout of the antagonist was studied. The offset of the antagonist effect of glycopyrrolate, tiotropium and ipratropium (10 nM each) was t1/2 = 4.0 +/- 0.5, > 4.5 and 0.5 +/- 0.1 h, respectively. At 4.5 h from the washout of the antagonist, the recovery of the response to carbachol was 50 +/- 8, 10 +/- 4 and 70 +/- 7%, respectively. 2. In the human isolated bronchus, the offset of the bronchodilator effects of glycopyrrolate (3 nM), tiotropium (1 nM) and ipratropium (10 nM) was t1/2 = 3.7 +/- 0.2; > 6 and 3.0 +/- 0.2 h, respectively. At 6.0 h from the washout of the antagonist, the recovery of the response to carbachol (1 microM) was 101 +/- 10, 27 +/- 3 and 110 +/- 10%, respectively. 4. In anaesthetized guinea-pigs, acetylcholine-induced bronchoconstriction was markedly reduced by intratracheal instillation of glycopyrrolate (3 nmol kg(-1); 88.1 +/- 4% inhibition), tiotropium (1.3 nmol kg(-1); 86.2 +/- 5% inhibition) or ipratropium (1.45 nmol kg(-1); 88.1 +/- 10% inhibition). These inhibitory effects assessed 3 or 24 h after antagonist administration were reduced to 69.9 +/- 5 and 29.7 +/- 6%; 28.3 +/- 5 and 14.2 +/- 5% for glycopyrrolate and ipratropium, respectively, whereas they remained stable (83.5 +/- 4; 70.6 +/- 6) for tiotropium. The residual inhibitory effect of glycopyrrolate was also assessed at 16 h from administration, and proved to be as low as that found at 24 h (31.2 +/- 10 vs 29.7 +/- 6%, respectively). 5. In conclusion, glycopyrrolate-induced bronchodilation has a longer duration than that of ipratropium, but less than that of tiotropium. The efficacy of a possible glycopyrrolate-based therapy for asthma or chronic obstructive pulmonary disease given once-a-day is not guaranteed by the present investigation.

CHF3381, a novel antinociceptive agent, attenuates capsaicin-induced pain in rats.

Here, we have examined the effect of the novel antinociceptive agent CHF3381 on the development of nocifensive behaviour as well as secondary mechanical allodynia and hyperalgesia induced by intraplantar injection of capsaicin in rats. Vehicle, CHF3381 or gabapentin were orally administered 1 h before capsaicin injection. The duration of nocifensive behaviour was measured during the first 5 min after capsaicin injection. Secondary mechanical allodynia and hyperalgesia were measured at 5 and 15 min after capsaicin injection, respectively. CHF3381 produced a significant suppression of nocifensive behaviour and completely blocked the development of mechanical allodynia and hyperalgesia at 100 and 200 mg/kg. Gabapentin weakly inhibited the development of nocifensive behaviour and mechanical allodynia. On the contrary, gabapentin (100 mg/kg) completely prevented the development of mechanical hyperalgesia. In conclusion, CHF3381 had full efficacy for all the capsaicin-induced pain parameters tested, suggesting that CHF3381 may have a therapeutic utility in the management of pain states involving central sensitisation.

Mechanisms of action of CHF3381 in the forebrain.

(1) Aim of this study was to gain insight into the mechanism of action of CHF3381, a novel putative antiepileptic and neuroprotective drug. (2) CHF3381 blocked NMDA currents in primary cultures of cortical neurons: maximal effect was nearly -80% of the NMDA-evoked current, with EC(50) of approximately 5 micro M. This effect was selective, reversible, use-dependent and elicited at the concentrations reached in the rodent brain after peripheral administration of therapeutic doses. (3) CHF3381 also inhibited voltage-gated Na(+) currents in an apparently voltage-dependent manner. However, this effect could be obtained only at relatively high concentrations (100 micro M). (4) Consistent with the mild effects on voltage-gated Na(+) channels, CHF3381 (100 micro M) failed to affect electrical stimulation-evoked glutamate overflow in hippocampal slices. In contrast, the anti-convulsant agent and Na(+) channel blocker lamotrigine (100 micro M) inhibited stimulation-evoked glutamate overflow by approximately 50%. (5) CHF3381 reduced kindled seizure-induced c-fos mRNA levels within the same brain regions, and to a similar level, as the selective NMDA receptor antagonist MK801, providing circumstantial evidence to the idea that CHF3381 blocks NMDA receptors in vivo. (6) The present mechanistic studies suggest that the primary mechanism of action of CHF3381 in the forebrain is blockade of NMDA receptors. On this basis, this compound may have a potential use in other diseases caused by or associated with a pathologically high level of NMDA receptor activation.

Neuroprotective activity of CHF3381, a putative N-methyl-D-aspartate receptor antagonist.

The aim of this study was to evaluate the neuroprotective effect of CHF3381, a novel putative NMDA antagonist characterized by a good therapeutic index. We have compared the effects of CHF3381 on kainate seizure-induced neurodegeneration with those produced by the non competitive NMDA receptor antagonist MK-801 and by the Na channel blocker lamotrigine. All compounds have been employed at doses incapable of preventing or attenuating seizures. The fluorescent marker Fluoro-Jade B has been used to identify degenerating cells. Animals pretreated with lamotrigine presented the same degree of cell damage as the controls. As for the controls, a clear correlation was also observed between seizure severity and neurodegeneration. In contrast, MK-801 and CHF3381 completely prevented cell damage. These data indicate that CHF3381 may be successfully utilized in neurological disorders characterized by or associated with neurodegenerative excitotoxicity.

Formoterol and beclomethasone dipropionate interact positively in antagonising bronchoconstriction and inflammation in the lung.

These studies were designed to assess the pharmacodynamic interaction between formoterol and beclomethasone dipropionate (BDP) in controlling the bronchoconstriction and inflammatory response induced by various challenges in guinea-pigs and rats. In anaesthetised guinea-pigs, superfusion of the formoterol/BDP combination into the tracheal lumen had significantly more effect than the single components in antagonising the bronchoconstricting and inflammatory responses to acetylcholine or ovalbumin in a standard model of airway hyper-responsiveness. After ovalbumin challenge, the combination completely protected animals from death at doses lower than those effective when given separately. The combination, at doses ineffective individually, even counteracted the development of lung oedema induced by sephadex in the rat. Finally, in tracheal strips from ovalbumin-sensitised guinea-pigs pre-treatment with BDP (30 mg kg(-1) i.m.) completely reversed the rightward shift of the formoterol dose-response curve due to beta(2)-receptor desensitisation. In conclusion, these results indicate that formoterol and BDP together induce a favourable pharmacodynamic interaction which can be considered more than additive, at least in these experimental settings.

Synthesis and anticonvulsant activity of a class of 2-amino 3-hydroxypropanamide and 2-aminoacetamide derivatives.

Several studies have demonstrated that N-substituted amino acid derivatives exhibit weak anticonvulsant activities in vivo. In the present study, a series of amides of aminoacids structurally related to aminoacetamide have been synthesised and investigated for anticonvulsant activity. Among the molecules investigated, those containing a bicyclic (tetralinyl, indanyl) group linked to the aminoacetamide chain (40, 47 and 59) were among the most active as anticonvulsants (ED50 > 10, <100 mg/kg after oral administration) against tonic seizures in the mouse maximal electroshock, bicuculline and picrotoxin tests at doses devoid of neurotoxic activity. Altogether, these results suggest the described compounds as a class of orally available anticonvulsants. The ability of these compounds to partially block veratridine-induced aspartate efflux from rat cortical synaptosomes suggests that their anticonvulsant activity may be only partly the consequence of an interaction with neuronal voltage-dependent sodium channels. Some of the most potent compounds appear worthy of a further investigation aimed at assessing their anticonvulsant activity in other models and at elucidating the underlying mechanism of action.

Antinociceptive activity of the N-methyl-D-aspartate receptor antagonist N-(2-Indanyl)-glycinamide hydrochloride (CHF3381) in experimental models of inflammatory and neuropathic pain.

N-(2-Indanyl)-glycinamide hydrochloride (CHF3381) is a novel low-affinity, noncompetitive N-methyl-d-aspartate receptor antagonist. The current study compared the antinociceptive effects of CHF3381 with those of gabapentin and memantine in in vitro and in vivo models of pain. In isolated rat spinal cord, CHF3381 and memantine, but not gabapentin, produced similar inhibition of the wind-up phenomenon. CHF3381 suppressed the maintenance of carrageenan-induced thermal and mechanical hyperalgesia in the rat with a minimum significantly effective dose (MED) of 30 mg/kg p.o. Memantine produced a partial reversal of both thermal and mechanical hyperalgesia (MED = 10 and 15 mg/kg i.p., respectively). Gabapentin reversed mechanical hyperalgesia (MED = 10 mg/kg s.c.), but did not affect thermal hyperalgesia. In the mouse formalin test, CHF3381 and memantine preferentially inhibited the late phase (MED = 30 and 20 mg/kg i.p., respectively); gabapentin inhibited only the late phase (MED = 30 mg/kg s.c.). Unlike morphine, CHF3381 chronic administration was not accompanied by the development of tolerance in the formalin test. Furthermore, morphine tolerance did not cross-generalize to CHF3381. In rats with a sciatic nerve injury, CHF3381 relieved both cold and mechanical allodynia (MED = 100 mg/kg p.o.). In contrast, memantine was inactive. Gabapentin blocked cold allodynia (MED = 30 mg/kg s.c.), but had marginal effects on mechanical allodynia. In diabetic neuropathy, CHF3381 reversed mechanical hyperalgesia (MED = 50 mg/kg p.o.). Memantine (15 mg/kg i.p.) produced an antinociceptive effect, whereas gabapentin (100 mg/kg p.o.) had no significant effect. Thus, CHF3381 may be useful for the therapy of peripheral painful neuropathies.

Taxas de serviço, concepção e prenhez de vacas nelore tratadas com gonadotrofina coriônica humana e 17?-estradiol após a inseminação artificial em tempo fixo / Service, conception and pregnancy rates of nelore cows treated with human corionic gonadotropin and estradiol-17? after timed artificial insemination

Avaliaram-se estratégias para reduzir a mortalidade embrionária em bovinos. Vacas Nelore (Bos taurus indicus) foram inseminadas em tempo fixo (IATF - D0) após o protocolo GnRH/PGF2a/GnRH e divididas nos grupos: Controle (Gc; n=55), hCG (GhCG; n=55) recebeu 3000 UI da hCG no D5 , E2 (GE2; n=55) recebeu 5 mg de 17?-Estradiol (E2) no D12 e hCG/E2 (GhCG/E2; n=55) recebeu 3000 UI da hCG no D5 e 5mg do E2 no D12. A IATF foi o 1o dia da estação de reprodução de 64 dias. Usou-se o teste do c2 para comparar as taxas de serviço (TS), concepção (TC) e prenhez à IATF (TPIATF), às IAs de repasse e acumuladas. A TS e a TC das IAs de repasse não diferiu (P>0,10). Houve diferenças nas TPIATF (P<0,05), pois a TPIATF do GE2 (5,4%) foi a mais baixa e a do Gc (34,5%) e GhCG (36,5%) tenderam (P<0,10) ser maiores que a do GhCG/E2 (18,2%). Não houve efeito da hCG sobre as taxas de prenhez. O E2 reduziu a TPIATF e ao final da estação as vacas que receberam o E2 (GE2 + GhCG/E2) tiveram TPacum (66,4%) mais baixa (P<0,05) de que aquelas (80,9%) que não receberam (GC + GhCG). Em conclusão, as estratégias adotadas de manipulação luteínica ou folicular não aumentaram as taxas de prenhez em vacas Nelore após uma IATF.

Strategies to reduce embryo mortality in cattle have been evaluated. Nelore (Bos taurus indicus) cows were submitted to timed artificial insemination (TAI - D0) after a GnRH/PGF2a/GnRH protocol. Cows were divided in the following groups: Control (Gc; n=55), hCG (GhCG; n=55) received 3000 IU of hCG on D5 , E2 (GE2; n=55) received 5 mg of Estradiol-17b (E2) on D12 and hCG/E2 (GhCG/E2; n=55) received hCG on D5 and E2 on D12. TAI represented the 1st day of a 64 day breeding season. The c2 test was applied to compare mating rate (MR), conception rate (CR) and pregnancy rate (PR) to TAI and AIs after natural estrus within the season as well as their accumulated figures at the end of the season. MR and CR for AIs within season did not differ (P>.10). PR to TAI differed (P<.05) among groups. GE2 showed the lowest rate (5,4%). Gc (34.5%) and GhCG (36.5%) tended (P<.10) to be higher than GhCG/E2 (18.2%). There was no effect of hCG on pregnancy rates. E2 reduced PR to TAI and at the end of season which means that cows receiving E2 (GE2 + GhCG/E2) had lower (66.4%; P<.05) PR than cows not receiving E2 (GC + GhCG; 80.9%). In conlusion, the strategies to manipulate luteal and follicular function herein tested did not increase pregnancy rates in Nelore cows submitted to a TAI.