Synthesis and biological characterization of spiro[2H-(1,3)-benzoxazine-2,4'-piperidine] based histone deacetylase inhibitors.

Histone Deacetylases (HDACs) have become important targets for the treatment of cancer and other diseases. In previous studies we described the development of novel spirocyclic HDAC inhibitors based on the combination of privileged structures with hydroxamic acid moieties as zinc binding group. Herein, we report further explorations, which resulted in the discovery of a new class of spiro[2H-(1,3)-benzoxazine-2,4'-piperidine] derivatives. Several compounds showed good potency of around 100 nM and less in the HDAC inhibition assays, submicromolar IC50 values when tested against tumour cell lines and a remarkable stability in human and mouse microsomes. Two representative examples exhibited a good pharmacokinetic profile with an oral bioavailability equal or higher than 35% and one of them studied in an HCT116 murine xenograft model showing a robust tumour growth inhibition. In addition, the two benzoxazines were found to have a minor affinity for the hERG potassium channel compared to their corresponding ketone analogues.

Desacyl-ghrelin and synthetic GH-secretagogues modulate the production of inflammatory cytokines in mouse microglia cells stimulated by beta-amyloid fibrils.

Data from Alzheimer's disease (AD) patients and AD animal models demonstrate the accumulation of inflammatory microglia at sites of insoluble fibrillar beta-amyloid protein (fAbeta) deposition. It is known that fAbeta binds to CD36, a type B scavenger receptor also involved in internalization of oxidized low-density lipoprotein (LDL), and initiate a signaling cascade that regulates microglial recruitment, activation, and secretion of inflammatory mediators leading to neuronal dysfunction and death. The recent demonstration of a binding site for the growth hormone secretagogues (GHS) on CD36 prompted us to ascertain whether ghrelin and synthetic GHS could modulate the synthesis of inflammatory cytokines in fAbeta-activated microglia cells. We demonstrate that N9 microglia cells express the CD36 and are a suitable model to study the activation of inflammatory cytokines synthesis. In fact, in N9 cells exposed to fAbeta(25-35) for 24 hr, the expression of interleukin (IL)-1beta and IL-6 mRNA significantly increased. Interestingly, 10(-7) M desacyl-ghrelin, hexarelin, and EP80317 in the nanomolar range effectively counteracted fAbeta(25-35) stimulation of IL-6 mRNA levels, whereas ghrelin was ineffective. Similarly, the effects of fAbeta(25-35) on IL-1beta mRNA levels were attenuated by desacyl-ghrelin, hexarelin, and EP80317, but not ghrelin. Because we have observed that the specific GHS receptor GHS-R1a is not expressed in N9 cells, the actions of GHS should be mediated by different receptors. Reportedly, hexarelin and EP80317 are capable of binding the CD36 in mouse macrophages and reducing atherosclerotic plaque deposition in mice. We conclude that desacyl-ghrelin, hexarelin, and EP80317 might interfere with fAbeta activation of CD36 in microglia cells.

Ghrelin inhibits inflammatory pain in rats: involvement of the opioid system.

This study examined the effects of intracerebroventricular (i.c.v.), intraperitoneal (i.p.) or intraplantar (i.pl.) administration of ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, in the development of hyperalgesia and edema induced by intraplantar injection of carrageenan in rats. Central ghrelin (4 ng to 4 microg/rat) given 5 min before carrageenan produced a dose-related reversal of carrageenan-induced mechanical hyperalgesia measured by Randall-Selitto test with an ED50 of 81.7 ng/rat. Ghrelin at the dose of 4 microg/rat i.c.v. was also effective in inhibiting edema. When ghrelin (4 microg/rat i.c.v.) was administered 150 min after carrageenan, it failed to modify either hyperalgesia or the paw volume. Given i.p., 30 min before carrageenan, ghrelin (20-160 microg/kg) induced a significant dose-dependent inhibition of hyperalgesia with an ED50 of 77 microg/kg and a slight reduction of edema. Intraplantar ghrelin (40 ng to 12 microg/rat) did not significantly modify both the hyperalgesic and edematous activities of carrageenan. The anti-hyperalgesic and anti-edematous effects of ghrelin (4 microg/rat i.c.v.) were reversed by naloxone (10 microg/rat i.c.v.). Systemic administration of the peripheral selective opioid antagonist, naloxone methiodide (3 mg/kg s.c.), did not antagonize antinociception elicited by i.p. ghrelin. Overall these data indicate that ghrelin exerts an inhibitory role on inflammatory pain through an interaction with the central opioid system.

Endocrine effects of centrally injected nociceptin in the rat.

In the present study we investigated the mechanisms involved in the endocrine effect of nociceptin/orphanin FQ (OFQ) in the rat and the possible interaction between OFQ and morphine in the control of growth hormone (GH) secretion. The intracerebroventricular administration of OFQ (2.3 or 23 microg/rat, i.c.v.) in freely moving male rats caused an increase in the secretion of both GH and prolactin (PRL). The possible involvement of the catecholaminergic (CA) system was studied by administering OFQ to CA-depleted rats (rats given 200 mg/kg of alpha-methyl-p-tyrosine subcutaneously 2 h before the i.c.v. dose of OFQ). In these CA-depleted rats, administration of OFQ (23 microg/rat, i.c.v.) did not stimulate GH secretion, whereas it significantly enhanced PRL secretion. In rats anesthetized with ketamine, which induces a significant increase of GH, PRL and corticosterone secretion by activating the sympathetic tone, OFQ (23 microg/rat, i.c.v.) did not modify GH and corticosterone levels, whereas again it significantly potentiated PRL secretion. Overall these results indicate that CA system is involved in the stimulatory action of OFQ on GH but not on PRL secretion. In fact the stimulation of PRL, but not that of GH, was still evident after impairment of the CA system. Pretreatment with OFQ (23 microg/rat, i.c.v.) attenuated the GH secretion induced by morphine (1 mg/kg, given by intra-arterial injection), thus showing a negative interaction between OFQ and morphine in the control of GH secretion.