Intracerebroventricular administration of TRH Agonist, RX-77368 alleviates visceral pain induced by colorectal distension in rats.

Thyrotropin-releasing hormone (TRH) acts centrally to exert pleiotropic actions independently from its endocrine function, including antinociceptive effects against somatic pain in rodents. Whether exogenous or endogenous activation of TRH signaling in the brain modulates visceral pain is unknown. Adult male Sprague-Dawley rats received an intracerebroventricular (ICV) injection of the stable TRH analog, RX-77368 (10, 30 and 100 ng/rat) or saline (5 µl) or were semi-restrained and exposed to cold (4°C) for 45 min. The visceromotor response (VMR) to graded phasic colorectal distensions (CRD) was monitored using non-invasive intracolonic pressure manometry. Naloxone (1 mg/kg) was injected subcutaneously 10 min before ICV RX-77368 or saline. Fecal pellet output was monitored for 1 h after ICV injection. RX-77368 ICV (10, 30 and 100 ng/rat) reduced significantly the VMR by 56.7%, 67.1% and 81.1% at 40 mmHg and by 30.3%, 58.9% and 87.4% at 60 mmHg respectively vs ICV saline. Naloxone reduced RX-77368 (30 and 100 ng, ICV) analgesic response by 51% and 28% at 40 mmHg and by 30% and 33% at 60 mmHg respectively, but had no effect per se. The visceral analgesia was mimicked by the acute exposure to cold. At the doses of 30 and 100 ng, ICV RX-77368 induced defecation within 30 min. These data established the antinociceptive action of RX-77368 injected ICV in a model of visceral pain induced by colonic distension through recruitment of both opioid and non-opioid dependent mechanisms.

Pharmacokinetic Drug Interactions of an Orally Available TRH Analog (Rovatirelin) With a CYP3A4/5 and P-Glycoprotein Inhibitor (Itraconazole).

The effects of itraconazole on the pharmacokinetics of rovatirelin were investigated in an open-label, single-sequence drug-drug interaction study in 16 healthy subjects. Subjects were administered a single oral dose of rovatirelin (1.6 mg) on day 1 and day 15. From day 8 through 16, subjects received daily oral doses of itraconazole (200 mg/day). Concentrations of rovatirelin and (thiazolylalanyl)methylpyrrolidine (TAMP), the major metabolite of rovatirelin formed by cytochrome P450 (CYP) 3A4/5, were determined in plasma and urine. Pharmacokinetic parameters were used to evaluate the drug-drug interaction potential of rovatirelin as a victim. With coadministration, maximum concentration (Cmax ) and area under the concentration-time curve extrapolated to infinity (AUCinf ) of rovatirelin increased 3.05-fold and 2.82-fold, respectively, and the 90% confidence intervals of the ratios for Cmax (2.64-3.52) and AUCinf (2.47-3.23) did not fall within the 0.8-1.25 boundaries. Urinary excretion of rovatirelin increased at almost the same ratio as the AUCinf ratio with coadministration; however, renal clearance did not change. Cmax , AUCinf , and urinary excretion of TAMP were decreased by coadministration. Itraconazole has the potential to inhibit drug transport via intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP); therefore, substrate assessments of rovatirelin for the 2 transporters were evaluated using Caco-2 cell monolayers. In vitro studies showed that rovatirelin is a substrate for P-gp but not for BCRP. The current study shows that itraconazole's effect on rovatirelin pharmacokinetics is mediated through inhibition of CYP3A4/5 and intestinal P-gp.

Evaluation of the effects of chemotherapy-induced fatigue and pharmacological interventions in multiple mouse behavioral assays.

Fatigue is a common symptom in many diseases and disorders and can reduce quality of life, yet lacks an adequate pharmacological intervention. To identify and develop such interventions, and to better understand fatigue, additional preclinical research is necessary. However, despite numerous mouse behavioral assays reportedly detecting fatigue-like behavior, the assumption that fatigue-like behavior is detected in many assays has not been validated through a cross-assay study. Thus, we modeled fatigue in mice by administering 5-fluorouracil, a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in mice, then evaluated its effects via voluntary wheel running activity (VWRA), locomotor activity in the open field test (OFT), immobility in the forced swim test (FST), and distance run in the treadmill fatigue test (TFT) and treadmill exercise capacity test. Additionally, taltirelin or methylphenidate was administered to alleviate fatigue-like behavior. As a result of 5-fluorouracil treatment, VWRA and the TFT were markedly reduced, indicating fatigue. The OFT, FST, and treadmill exercise capacity test, however, failed to detect fatigue-like behavior. Interestingly, both taltirelin and methylphenidate alleviated fatigue-like behavior in TFT. These data suggest that, of the current assays, only the TFT and VWRA should be expected to detect fatigue-like behavior. Moreover, this study provides additional evidence that taltirelin may provide a novel treatment for chemotherapy-induced fatigue and warrants further evaluation as an anti-fatigue therapeutic.

Abdominal surgery induced gastric ileus and activation of M1-like macrophages in the gastric myenteric plexus: prevention by central vagal activation in rats.

Inflammation plays a role in abdominal surgery (AS)-induced intestinal ileus that is alleviated by electrical vagal stimulation. Intracisternal injection of RX-77368, the stable thyrotropin-releasing hormone agonist, activates dorsal motor nucleus neurons and gastric vagal efferent discharges. We investigated the gastric inflammation induced by AS and the modulation by intracisternal RX-77368 in rats. RX-77368 (50 ng/rat) or saline was injected followed, 1 h later, by laparotomy and small intestinal/cecal manipulation. The sham group had anesthesia alone. After 6 h, gastric emptying (GE) and the inflammation in gastric corpus were determined. AS inhibited GE by 72% vs. control and doubled the number of M1-like macrophage immunoreactive for major histocompatibility complex class II (MHCII; M1 marker) but not for cluster of differentiation 206 (CD206; M2 marker) (MHCII+/CD206-) while there was no change in M2-like macrophages (MHCII-/CD206+). AS increased mRNA levels of interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) by 1.7- and 1.5-fold, respectively, in the gastric submucosa plus muscle layers and the infiltration of neutrophils labeled by myeloperoxidase by 9.5-fold in the muscularis externa. RX-77368 inhibited AS-related gastric changes while not altering these parameters in the sham group. There was a significant negative correlation between GE and IL-1ß (r = -0.46), TNF-α (r = -0.44), M1 macrophage (r = -0.82), and neutrophils (r = -0.91). The M2-like macrophages and IL-10 expression were unchanged by AS with intracisternal saline or RX-77368. These data indicate that AS activates gastric M1 macrophages and increases proinflammatory cytokines expression, which are prevented by central vagal activation and may contribute to the correlated dampening of postoperative gastric ileus.NEW & NOTEWORTHY MHCII+/CD206- (M1) and MHCII-/CD206+ (M2) constitute two distinct populations of macrophages that are in close apposition to the cholinergic neurons in the rat gastric myenteric plexus (MP). Abdominal surgery (6 h) activates M1 macrophage leading to inflammation in the gastric MP correlated with the delayed gastric emptying, which was abolished by central vagal stimulation via intracisternal injection of RX-77368. Vagal stimulation linked with the cephalic phase may have potential beneficial effects to curtail postoperative gastric ileus.

Taltirelin alleviates fatigue-like behavior in mouse models of cancer-related fatigue.

Fatigue affects most cancer patients and has numerous potential causes, including cancer itself and cancer treatment. Cancer-related fatigue (CRF) is not relieved by rest, can decrease quality of life, and has no FDA-approved therapy. Thyrotropin-releasing hormone (TRH) has been proposed as a potential novel treatment for CRF, but its efficacy against CRF remains largely untested. Thus, we tested the TRH analog, taltirelin (TAL), in mouse models of CRF. To model fatigue, we used a mouse model of chemotherapy, a mouse model of radiation therapy, and mice bearing colon 26 carcinoma tumors. We used the treadmill fatigue test to assess fatigue-like behavior after treatment with TAL. Additionally, we used wild-type and TRH receptor knockout mice to determine which TRH receptor was necessary for the actions of TAL. Tumor-bearing mice displayed muscle wasting and all models caused fatigue-like behavior, with mice running a shorter distance in the treadmill fatigue test than controls. TAL reversed fatigue-like behavior in all three models and the mouse TRH1 receptor was necessary for the effects of TAL. These data suggest that TAL may be useful in alleviating fatigue in all cancer patients and provide further support for evaluating TAL as a potential therapy for CRF in humans.

Effect of L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 against in-vitro and in-vivo models of cerebral ischemia and associated neurological disorders.

Central nervous system plays a vital role in regulation of most of biological functions which are abnormally affected in various disorders including cerebral ischemia, Alzheimer's and Parkinson's (AD and PD) worldwide. Cerebral stroke is an extremely fatal and one of the least comprehensible neurological disorders due to limited availability of prospective clinical approaches and therapeutics. Since, some endogenous peptides like thyrotropin-releasing hormone have shown substantial neuroprotective potential, hence present study evaluates the newer thyrotropin-releasing hormone (TRH) analogue L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 for its neuroprotective effects against oxygen glucose deprivation (OGD), glutamate and H2O2 induced injury in pheochromocytoma cell lines (PC-12 cells) and in-vivo ischemic injury in mice. Additionally, the treatment was further analyzed with respect to models of AD and PD in mice. Cerebral ischemia was induced by clamping both bilateral common carotid arteries for ten minutes. Treatment was administered to the mice five minute after restoration of blood supply to brain. Consequential changes in neurobehavioural, biochemical and histological parameters were assessed after a week. L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 showed significant reduction in glutamate, H2O2 and OGD -induced cell death in concentration and time dependent manner. Moreover, L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 resulted in a substantial reduction in CA1 (Cornus Ammonis 1) hippocampal neuronal cell death, inflammatory cytokines, TNF-α, IL-6 and oxidative stress in hippocampus. In addition, L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 was found to be protective in two acute models of AD and PD as well these findings demonstrate the neuroprotective potential of L-pGlu-(1-benzyl)-l-His-l-Pro-NH2 in cerebral ischemia and other diseases, which may be mediated through reduction of excitotoxicity, oxidative stress and inflammation.

Effect of rovatirelin, a novel thyrotropin-releasing hormone analog, on the central noradrenergic system.

Rovatirelin ([1-[-[(4S,5S)-(5-methyl-2-oxo oxazolidin-4-yl) carbonyl]-3-(thiazol-4-yl)-l-alanyl]-(2R)-2-methylpyrrolidine) is a novel synthetic agent that mimics the actions of thyrotropin-releasing hormone (TRH). The aim of this study was to investigate the electrophysiological and pharmacological effects of rovatirelin on the central noradrenergic system and to compare the results with those of another TRH mimetic agent, taltirelin, which is approved for the treatment of spinocerebellar degeneration (SCD) in Japan. Rovatirelin binds to the human TRH receptor with higher affinity (Ki=702nM) than taltirelin (Ki=3877nM). Rovatirelin increased the spontaneous firing of action potentials in the acutely isolated noradrenergic neurons of rat locus coeruleus (LC). The facilitatory action of rovatirelin on the firing rate in the LC neurons was inhibited by the TRH receptor antagonist, chlordiazepoxide. Reduction of the extracellular pH increased the spontaneous firing of LC neurons and rovatirelin failed to increase the firing frequency further, indicating an involvement of acid-sensitive K+ channels in the rovatirelin action. In in vivo studies, oral administration of rovatirelin increased both c-Fos expression in the LC and extracellular levels of noradrenaline (NA) in the medial prefrontal cortex (mPFC) of rats. Furthermore, rovatirelin increased locomotor activity. The increase in NA level and locomotor activity by rovatirelin was more potent and longer acting than those by taltirelin. These results indicate that rovatirelin exerts a central nervous system (CNS)-mediated action through the central noradrenergic system, which is more potent than taltirelin. Thus, rovatirelin may have an orally effective therapeutic potential in patients with SCD.

Synthesis and neuromodulatory effects of TRH-related peptides: inhibitory activity on catecholamine release in vitro.

BACKGROUND: A detailed comprehension of central mechanisms underlying feeding behavior holds considerable promise for the treatment of alimentary disorders. METHODS: In order to elucidate the tight interrelationships occurring at the hypothalamic neuronal endings between aminergic neurotransmitters and co-localized appetite modulators, we initially studied the effects of two anorexigenic peptides structurally related to thyrotropin-releasing hormone (TRH, 1), namely cyclo(His-Pro) (CHP, 2) and pGlu-His-Gly-OH (3), on [(3)H]-norepinephrine and [(3)H]-dopamine release from perfused rat hypothalamic synaptosomes. Furthermore, a number of TRH and CHP analogues were synthesized and tested for their ability to influence neurotransmitter release in the selected neuronal model. RESULTS: Peptide 3 showed only a slight inhibitory activity on norepinephrine release, whereas no effect was observed for compound 2. TRH analogue 8, metabolically stabilized by the replacement of pyroglutamate with the pyrohomocysteic acid (pHcs), was found to be inactive. Conversely, a significant inhibitory effect on dopamine and norepinephrine release was observed for the CHP-related diketopiperazines cyclo(Leu-Pro) (11) and cyclo(His-Gly) (14). CONCLUSIONS: These results suggest a potential role for cyclo-dipeptides 11 and 14 in the hypothalamic modulation of appetite suppressant circuitry.

Tripeptide amide L-pyroglutamyl-histidyl-L-prolineamide (L-PHP-thyrotropin-releasing hormone, TRH) promotes insulin-producing cell proliferation.

A very small tripeptide amide L-pyroglutamyl-L-histidyl-L-prolineamide (L-PHP, Thyrotropin-Releasing Hormone, TRH), was first identified in the brain hypothalamus area. Further studies found that L-PHP was expressed in pancreas. The biological role of pancreatic L-PHP is still not clear. Growing evidence indicates that L-PHP expression in the pancreas may play a pivotal role for pancreatic development in the early prenatal period. However, the role of L-PHP in adult pancreas still needs to be explored. L-PHP activation of pancreatic ß cell Ca2+ flow and stimulation of ß-cell insulin synthesis and release suggest that L-PHP involved in glucose metabolism may directly act on the ß cell separate from any effects via the central nervous system (CNS). Knockout L-PHP animal models have shown that loss of L-PHP expression causes hyperglycemia, which cannot be reversed by administration of thyroid hormone, suggesting that the absence of L-PHP itself is the cause. L-PHP receptor type-1 has been identified in pancreas which provides a possibility for L-PHP autocrine and paracrine regulation in pancreatic function. During pancreatic damage in adult pancreas, L-PHP may protect beta cell from apoptosis and initiate its regeneration through signal pathways of growth hormone in ß cells. L-PHP has recently been discovered to affect a broad array of gene expression in the pancreas including growth factor genes. Signal pathways linked between L-PHP and EGF receptor phosphorylation suggest that L-PHP may be an important factor for adult ß-cell regeneration, which could involve adult stem cell differentiation. These effects suggest that L-PHP may benefit pancreatic ß cells and diabetic therapy in clinic.

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 µM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

Protective effects of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia.

In the present study, the newly synthesized TRH analog (L-pGlu-(2-propyl)-L-His-l-ProNH(2); NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H(2)O(2)-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6h. Exposure of NP-647 was given before and during OGD. In glutamate and H(2)O(2) induced injury, exposure of NP-647 was given 1, 6 and 24h prior to exposure of glutamate and H(2)O(2) exposure. NP-647, per se found to be non-toxic in 1-100µM concentrations. NP-647 showed protection against OGD at the 1 and 10µM. The concentration-dependent protection was observed in H(2)O(2)- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.

Brain-gut interactions between central vagal activation and abdominal surgery to influence gastric myenteric ganglia Fos expression in rats.

We previously showed that medullary thyrotropin-releasing hormone (TRH) or the stable TRH agonist, RX-77368 administered intracisternally induces vagal-dependent activation of gastric myenteric neurons and prevents post surgery-induced delayed gastric emptying in rats. We investigated whether abdominal surgery alters intracisternal (ic) RX-77368 (50 ng)-induced gastric myenteric neuron activation. Under 10 min enflurane anesthesia, rats underwent an ic injection of saline or RX-77368 followed by a laparotomy and a 1-min cecal palpation, or no surgery and were euthanized 90 min later. Longitudinal muscle/myenteric plexus whole-mount preparations of gastric corpus and antrum were processed for immunohistochemical detection of Fos alone or double labeled with protein gene-product 9.5 (PGP 9.5) and vesicular acetylcholine transporter (VAChT). In the non surgery groups, ic RX-77368 induced a 17 fold increase in Fos-expression in both gastric antrum and corpus myenteric neurons compared to saline injected rats. PGP 9.5 ascertained the neuronal identity of myenteric cells expressing Fos. In the abdominal surgery groups, ic RX-77368 induced a significant increase in Fos-expression in both the corpus and antrum myenteric ganglia compared with ic saline injected rats which has no Fos in the gastric myenteric ganglia. However, the response was reduced by 73-78% compared with that induced by ic RX 77368 without surgery. Abundant VAChT positive nerve fibers were present around Fos positive neurons. These results indicate a bidirectional interaction between central vagal stimulation of gastric myenteric neurons and abdominal surgery. The modulation of gastric vagus-myenteric neuron activity could play an important role in the recovery phase of postoperative gastric ileus.

TRH-like peptides.

TRH-like peptides are characterized by substitution of basic amino acid histidine (related to authentic TRH) with neutral or acidic amino acid, like glutamic acid, phenylalanine, glutamine, tyrosine, leucin, valin, aspartic acid and asparagine. The presence of extrahypothalamic TRH-like peptides was reported in peripheral tissues including gastrointestinal tract, placenta, neural tissues, male reproductive system and certain endocrine tissues. Work deals with the biological function of TRH-like peptides in different parts of organisms where various mechanisms may serve for realisation of biological function of TRH-like peptides as negative feedback to the pituitary exerted by the TRH-like peptides, the role of pEEPam such as fertilization-promoting peptide, the mechanism influencing the proliferative ability of prostatic tissues, the neuroprotective and antidepressant function of TRH-like peptides in brain and the regulation of thyroid status by TRH-like peptides.

Protective effects of TRH and its analogues against various cytotoxic agents in retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells.

TRH (thyroliberin) and its analogues were reported to possess neuroprotective effects in cellular and animal experimental models of acute and chronic neurodegenerative diseases. In the present study we evaluated effects of TRH and its three stable analogues, montirelin (CG-3703), RGH-2202 and Z-TRH (N-(carbobenzyloxy)-pGlutamyl-Histydyl-Proline) on the neuronally differentiated human neuroblastoma SH-SY5Y cell line, which is widely accepted for studying potential neuroprotectants. We found that TRH and all the tested analogues at concentrations 0.1-50 µM attenuated cell damage induced by MPP(+) (2 mM), 3-nitropropionate (10 mM), hydrogen peroxide (0.5 mM), homocysteine (250 µM) and beta-amyloid (20µM) in retinoic acid differentiated SH-SY5Y cells. Furthermore, we demonstrated that TRH and its analogues decreased the staurosporine (0.5 µM)-induced LDH release, caspase-3 activity and DNA fragmentation, which indicate the anti-apoptotic proprieties of these peptides. The neuroprotective effects of TRH (10 µM) and RGH-2202 (10 µM) on St-induced cell death was attenuated by inhibitors of PI3-K pathway (wortmannin and LY294002), but not MAPK/ERK1/2 (PD98059 and U0126). Moreover, TRH and its analogues at neuroprotective concentrations (1 and 10 µM) increased expression of Bcl-2 protein, as confirmed by Western blot analysis. All in all, these results extend data on neuroprotective properties of TRH and its analogues and provide evidence that mechanism of anti-apoptotic effects of these peptides in SH-SY5Y cell line involves induction of PI3K/Akt pathway and Bcl-2. Furthermore, the data obtained on human cell line with a dopaminergic phenotype suggest potential utility of TRH and its analogues in the treatment of some neurodegenerative diseases including Parkinson's disease.

Cold ambient temperature reverses abdominal surgery-induced delayed gastric emptying and decreased plasma ghrelin levels in rats.

We investigated whether acute cold-induced vagal activation through brainstem thyrotropin-releasing hormone (TRH) signaling influences abdominal surgery-induced delayed gastric emptying (GE) in fasted rats. Laparotomy and cecal palpation or sham (short anesthesia alone) was performed 10 min before or 30 min after cold exposure (4-6°C) lasting 90 min. Non-nutrient GE was assessed during 70-90 min of cold exposure. Control groups remained at room temperature (RT). The stable TRH analog, RX-77368 (50 ng/rat) was injected intracisternally immediately before surgery and GE monitored 30-50 min postsurgery in rats maintained at RT. Plasma acyl (AG) and total ghrelin levels were assessed using the new RAPID blood processing method and radioimmunoassays. Desacyl ghrelin (DAG) was derived from total minus AG. In rats maintained at RT, abdominal surgery decreased GE by 60% compared to sham. Cold before or after surgery or RX-77368 normalized the delayed GE. In non-fasted rats, cold exposure increased plasma AG and DAG levels at 2 h (2.4- and 2.7-times, respectively) and 4 h (2.2- and 2.0-times, respectively) compared to values in rats maintained at RT. In fasted rats, abdominal surgery decreased AG and DAG levels by 2.4- and 2.1-times, respectively, at 90 min. Cold for 90 min after surgery normalized AG and DAG levels to those observed in sham-treated animals kept at RT. These data indicate that endogenous (cold exposure) and exogenous (TRH analog) activation of medullary TRH vagal signaling prevent abdominal surgery-induced delayed GE. The restoration of circulating AG levels inhibited by abdominal surgery may contribute to alleviate postoperative gastric ileus.

Effects of TRH and its analogues on primary cortical neuronal cell damage induced by various excitotoxic, necrotic and apoptotic agents.

The tripeptide thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) has been shown to possess neuroprotective activity in in vitro and in vivo models. Since its potential utility is limited by relatively rapid metabolism, metabolically stabilized analogues have been constructed. In the present study we investigated the influence of TRH and its three stable analogues: Montirelin (MON, CG-3703), RGH-2202 (L-6-keto-piperidine-2carbonyl-l-leucyl-l-prolinamide) and Z-TRH (N-carbobenzyloxy-pGlutamyl-Histydyl-Proline) in various models of mouse cortical neuronal cell injury. Twenty four hour pre-treatment with TRH and its analogues in low micromolar concentrations attenuated the neuronal cell death evoked by excitatory amino acids (EAAs: glutamate, NMDA, kainate, quisqualate) and hydrogen peroxide. All the peptides showed neuroprotective action on staurosporine (St)-evoked apoptotic neuronal cell death, but this effect was caspase-3 independent. Interestingly, in mixed neuronal-glial cell preparations only MON decreased St- and glutamate-evoked neurotoxicity. None of the peptides inhibited the doxorubicin- and lactacystin-induced neuronal cortical cell death, agents acting via activation of death receptor (FAS) or inhibition of proteasome function, respectively. Furthermore, we found that neither inhibitors of PI3-K (wortmannin, LY 294002) nor MAPK/ERK1/2 (PD 098059, U 0126) were able to inhibit neuroprotective properties of TRH and MON in St model of apoptosis. The protection mediated by TRH and MON it that model was also not connected with influence of peptides on the pro-apoptotic GSK-3beta and JNK protein kinase expression and activity. Further studies showed that calpains, calcium-activated proteases were induced by Glu, but not by St in cortical neurons. Moreover, the Glu-evoked increase in spectrin alpha II cleavage product induced by calpains was blocked by TRH. The obtained data showed that the potency of TRH and its analogues in inhibiting EAAs- and H(2)O(2)-induced neuronal cell death from the highest to lowest activity was: MON>TRH>Z-TRH>RHG. Interestingly, all peptides were active against St-induced apoptosis, however, on concentration basis MON was far more potent than the other peptides. None of the peptides inhibited Dox- and LC-evoked apoptotic cell death. Additionally, the data exclude potential role of pro-survival (PI3-K/Akt and MAPK/ERK1/2) and pro-apoptotic (GSK-3beta and JNK) pathways in neuroprotective effects of TRH and its analogues on St-induced neuronal apoptosis. Moreover, the results point to involvement of the inhibition of calpains in the TRH neuroprotective effect in Glu model of neuronal cell death.

A novel TRH analog, Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds to [3H][3-Me-His2]TRH-labelled sites in rat hippocampus and cortex but not pituitary or heterologous cells expressing TRHR1 or TRHR2.

Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is a novel synthetic peptide that mimics and amplifies central actions of thyrotropin-releasing hormone (TRH) in rat without releasing TSH. The aim of this study was to compare the binding properties of this pentapeptide and its all-L counterpart (Glp-Asn-Pro-Tyr-TrpNH(2)) to TRH receptors in native rat brain tissue and cells expressing the two TRH receptor subtypes identified in rat to date, namely TRHR1 and TRHR2. Radioligand binding studies were carried out using [(3)H][3-Me-His(2)]TRH to label receptors in hippocampal, cortical and pituitary tissue, GH4 pituitary cells, as well as CHO cells expressing TRHR1 and/or TRHR2. In situ hybridization studies suggest that cortex expresses primarily TRHR2 mRNA, hippocampus primarily TRHR1 mRNA and pituitary exclusively TRHR1 mRNA. Competition experiments showed [3-Me-His(2)]TRH potently displaced [(3)H][3-Me-His(2)]TRH binding from all tissues/cells investigated. Glp-Asn-Pro-D-Tyr-D-TrpNH(2) in concentrations up to 10(-5)M did not displace [(3)H][3-Me-His(2)]TRH binding to membranes derived from GH4 cells or CHO-TRHR1 cells, consistent with its lack of binding to pituitary membranes and TSH-releasing activity. Similar results were obtained for the corresponding all-L peptide. In contrast, both pentapeptides displaced binding from rat hippocampal membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.7+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2), analogous to cortical membranes (pIC(50) Glp-Asn-Pro-D-Tyr-D-TrpNH(2): 7.8+/-0.2; pIC(50) Glp-Asn-Pro-Tyr-TrpNH(2): 6.6+/-0.2). Neither peptide, however, displaced [(3)H][3-Me-His(2)]TRH binding to CHO-TRHR2. Thus, this study reveals for the first time significant differences in the binding properties of native and heterologously expressed TRH receptors. Also, the results raise the possibility that Glp-Asn-Pro-D-Tyr-D-TrpNH(2) is not displacing [(3)H][3-Me-His(2)]TRH from a known TRH receptor in rat cortex, but rather a hitherto unidentified TRH receptor.

Exploratory neuropharmacological evaluation of a conformationally constrained thyrotropin-releasing hormone analogue.

A conformationally constrained peptidomimetic derived from the endocrine and neuroactive tripeptide thyrotropin-releasing hormone (pGlu-His-Pro-NH(2)) was synthesized by convenient solid-phase organic chemistry and evaluated as a potential central nervous system agent. While this ethylene-bridged peptide analogue has been reported to lack the hormonal effect of the native peptide, we have shown in animal models that it possesses central nervous system activity characteristic of thyrotropin-releasing hormone. Compared to control, the peptidomimetic showed significant analeptic and antidepressant-like potencies. Moreover, an enhanced selectivity in antidepressant-like effect was measured when compared to that of the native peptide. Immobilized artificial membrane chromatography and in vitro metabolic stability studies also revealed that this constrained peptidomimetic has higher affinity to the blood-brain barrier than the native peptide and is metabolically stable. Consequently, this structure may be used as a template to design centrally selective and metabolically stable thyrotropin-releasing hormone analogues as potential neuropharmaceutical agents.

[History of clinical studies on hypothalamic hormone analogs in Mexico]. / Historia de los estudios clínicos con los análogos de las hormonas hipotalámicas en México.

Early clinical trials in Mexico with analogs of luteinizing hormone-releasing hormone (LH-RH) also known as gonadotropin releasing hormone (Gn-RH), were reviewed. Extensive clinical studies were carried out at IMSS with agonists of LH-RH, both in men and woman. All subjects responded to LH-RH agonists with a release of LH and FSH, but repeated administration of these analogs, initially aimed at stimulation of fertility (thought to stimulate fertility), was later shown to result in inhibition due to desensitization of pituitary gland and downregulation of LH-RH receptors. Various clinical investigations with LH-RH antagonists were also carried out. This included the first demonstration that LH-RHantagonists can suppress LH and FSH and sex steroid secretion in men and women. Various studies in Mexico with early LH-RH antagonists aimed at the development of new contraceptive methods were reviewed. Modern LH-RH antagonist Cetrorelix was shown to be effective in men and women and useful in treatment of uterine leiomyomas and benign prostatic hyperplasia. Major oncological studies were also carried out with agonist D-Trp6-LH-RH and antagonist Cetrorelix in men with prostate cancer, which demonstrated therapeutic efficacy of both types of analogs. Some endocrine studies with early analogs of somatostatin were also cited and a clinical trial with somatostatin analog vapreotide in patients with relapsed prostate cancer was reviewed. All these studies played a major role in introducing analogs of hypothalamic-releasing hormones into clinical medicine.

Brainstem thyrotropin-releasing hormone regulates food intake through vagal-dependent cholinergic stimulation of ghrelin secretion.

The brainstem is essential for mediating energetic response to starvation. Brain stem TRH is synthesized in caudal raphe nuclei innervating brainstem and spinal vagal and sympathetic motor neurons. Intracisternal injection (ic) of a stable TRH analog RX77368 (7.5-25 ng) dose-dependently stimulated solid food intake by 2.4- to 3-fold in freely fed rats, an effect that lasted for 3 h. By contrast, RX77368 at 25 ng injected into the lateral ventricle induced a delayed and insignificant orexigenic effect only in the first hour. In pentobarbital-anesthetized rats, RX77368 (50 ng) ic induced a significant bipeak increase in serum total ghrelin levels from the basal of 8.7+/-1.7 ng/ml to 13.4+/-2.4 ng/ml at 30 min and 14.5+/-2.0 ng/ml at 90 min, which was prevented by either bilateral vagotomy (-60 min) or atropine pretreatment (2 mg/kg, -30 min) but magnified by bilateral adrenalectomy (-60 min). TRH analog ic-induced food intake in freely fed rats was abolished by either peripheral atropine or ghrelin receptor antagonist (D-Lys-3)-GHRP-6 (10 micromol/kg) or ic Y1 receptor antagonist 122PU91 (10 nmol/5 microl). Brain stem TRH mRNA and TRH receptor 1 mRNA increased by 57-58 and 33-35% in 24- and 48-h fasted rats and returned to the fed levels after a 3-h refeeding. Natural food intake in overnight fasted rats was significantly reduced by ic TRH antibody, ic Y1 antagonist, and peripheral atropine. These data establish a physiological role of brainstem TRH in vagal-ghrelin-mediated stimulation of food intake, which involves interaction with brainstem Y1 receptors.