Design,synthesis, and biological evaluation of novel,centrally-acting thyrotropin-releasing hormone analogues

Novel, metabolically stable and centrally acting TRH analogues with substituted pyridinium moieties replacing the [His(2)] residue of the endogenous peptide were prepared by solid-phase Zincke reaction. The 1,4-dihydropyridine prodrugs of these analogues obtained after reducing the pyridinium moiety were able to reach the brain and maintain a sustained concentration of the charged, degradation-resistant analogues formed after enzymatic oxidation of the prodrug, as manifested by the analeptic action measured in mice. Among the four analogues reported, compound 2a showed the highest potency and longest duration of action in reducing the pentobarbital-induced sleeping time compared to the parent TRH. No binding to the endocrine TRH-receptor was measured for 2a; thus, this compound emerged as a potent, centrally acting TRH analogue.     

Hepatic effects of phenobarbitone in female mice fed sublethal levels of dietary cyanide

Female albino mice were fed sublethal doses of KCN (approx. 10 micrograms/mouse/day) for 7 days, injected intraperitoneally with phenobarbitone (50 mg/kg body wt/day) in the subsequent 3 days, and sacrificed 24 hr after the last injection. Phenobarbitone sleeping time was increasingly shortened (16-27%) daily in cyanide-fed mice in comparison with cyanide-free controls. Both compounds administered singly or simultaneously increased the liver weight/body weight ratios by not more than 10%. Aniline hydroxylase, glucose-6-phosphatase, NADPH- and NADH-cytochrome c reductase activities were similarly increased. Aniline hydroxylase activity was most markedly increased (by a factor of 4). The toxicological implications of these results are discussed.     

Neuropharmacological evaluation of RX 77368--a stabilised analogue of thyrotropin-releasing hormone (TRH)

L-Pyroglutamyl-L-histidyl-L-2,3-dimethylprolineamide (Pyr-His-Dmp . NH2; RX 77368) a stabilised analogue of thyrotropin-releasing hormone (TRH) has been examined for neuropharmacological effects in animal tests. The compound was more potent than either TRH or clinically established drugs in four animal tests of antidepressant potential (reserpine reversal, clonidine antagonism, tremorine reversal and learned immobility). RX 77368 also antagonised barbiturate sleeping time. Given by itself to rats the peptide produced arousal as characterised by EEG and EMG measurements and delayed the onset of sleep. The arousal induced was not accompanied by increases in locomotor activity. The profile of pharmacological activity for RX 77368 did not correspond to the profiles of tricyclic antidepressants, psychic-stimulants or analeptic drugs. The possible clinical uses for such a molecule are discussed.     

Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).     

Effect of thyrotropin-releasing hormone (TRH) on local cerebral glucose utilization, by the autoradiographic 2-deoxy[14C]glucose method, in conscious and pentobarbitalized rats

Effects of TRH and pentobarbital alone, and in combination, on local cerebral glucose utilization of rats were studied by the autoradiographic 2-deoxy[14C]glucose method. TRH (5 mg/kg i.v.) reduced the rate of cerebral glucose utilization slightly in the whole brain. Locally, significant depression was observed in the following structures: frontal and visual cortices, hippocampus Ammon's horn and dentate gyrus, medial and lateral geniculate bodies, nucleus accumbens, caudate-putamen, substantia nigra, pontine gray matter, superior colliculus, superior olivary nucleus, vestibular nucleus, lateral lemniscus and cerebellar cortex. Pentobarbital (30 mg/kg i.v.) produced a marked and diffuse reduction in the rate of glucose utilization throughout the brain. TRH given 15 min after the administration of pentobarbital markedly shortened the pentobarbital sleeping time and caused some reversal of the depression in local cerebral glucose utilization produced by pentobarbital. These effects were almost completely abolished by pretreatment with intracerebroventricular injection of atropine methyl bromide (20 microgram/rat). These results indicate that although TRH acts to cause a reduction in the rate of cerebral glucose utilization, it reverses the depression induced by pentobarbital, via a cholinergic mechanism, in a number of structures, some of which are related to monoaminergic systems and the reticulo-thalamo-cortical activating system.     

Conformational analysis of pGlu-His-Amph and pGlu-His-Pro-Amph related to their central nervous system activity

The compounds pGlu-His-Pro-Amph and pGlu-His-Amph obtained from the condensation of TRH or a fragment of TRH with amphetamine show activities which are different regarding the parent compounds. Although the two derivatives exhibit about the same low toxicity they differ in several pharmacological properties. Physicochemical analysis by 1H-NMR and CD spectroscopy was carried out in order to detect in the two compounds conformational differences that might explain their different activities. The results show that in the proline containing peptide the amphetamine has a hindered rotation in comparison with the compounds devoid of proline. This, together with the occurrence of a cis conformer having different properties than the trans conformer could be the origin of the biological difference observed between the two hybrid compounds.     

A survey of the response of different strains of mice to substances metabolised by microsomal oxidation; hexobarbitone, zoxazolamine and warfarin.

Sixteen strains of mice were compared with respect to their hexobarbitone sleeping time and their zoxazolamine paralysis time. The strains were A2G, CBA, CE, C3H, C57BL, C57L, DBA, F/st, ICFW, NMRI, NZB, Schneider, Simpson, SM, TO and 129/rr. All the strains except 129 Rr were also tested for survival on a diet containing 0.05% racemic Warfarin. There was highly significant interstrain correlation between hexobarbitone sleeping time and zoxazolamine paralysis time (r = 0.72) and between hexobarbitone sleeping time Warfarin survival (r = 0.68). There was a significant correlation between zoxazolamine paralysis time and Warfarin survival (r = 0.56). The correlations can be explained if: (1) there is a genetically determined interstrain variable which is some common component of the microsomal mixed-function oxidase systems involved in the hydroxylation of the three substances; (2) the anticoagulant action of Warfarin is caused more by a hydroxylated metabolite of Warfarin than by Warfarin itself. Phenobarbitone pretreatment shortened hexobarbitone sleeping times and zoxazolamine paralysis times, but its effect was greater in those strains with longer initial hexobarbitone sleeping times and zoxazolamine paralysis times. Piperonyl butoxide pretreatment lengthened hexobarbitone sleeping times, but had no effect on zoxazolamine paralysis times. Warfarin survival was unaltered by pretreatment with either phenobarbital or piperonyl butoxide.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.     

Effects of thyrotropin-releasing hormone (TRH) and MK-771 on schedule-controlled behavior of squirrel monkeys, rabbits and pigeons

The effects of TRH (0.001-10.0 mg/kg) and a more potent TRH analog, MK-771 (0.001-5.6 mg/kg), were studied on comparable schedule-controlled performances of squirrel monkeys, rabbits and pigeons. Responding was maintained in the presence of different stimuli by a multiple fixed-ratio (FR), fixed-interval (FI) schedule of food presentation (monkeys and pigeons) or 0.25% saccharin solution (rabbits). Generally, TRH and MK-771 produced decreases in responding under both schedules and in all three species. TRH and MK-771 were roughly equipotent in the squirrel monkey, whereas in the pigeon and rabbit MK-771 was approximately 20 times more potent than TRH in decreasing responding to 50 percent of control levels. The duration of action of doses of TRH and MK-771 that reduced responding to 50 percent of control was approximately 3 hr in the squirrel monkey; recovery of performance occurred twice as fast under the FR schedules. With the pigeon, TRH effects that produced 50 percent decreases in responding lasted over 6 hours, whereas behaviorally comparable doses of MK-771 lasted about 4 hours. With few exceptions, TRH and MK-771 appear to produce similar effects of schedule-controlled behavioral performances of the squirrel monkey, rabbit and pigeon. Compared to the effects of other behaviorally-active substances under these procedures, TRH and MK-771 exert a distinctive array of effects.     

Brain receptor binding characteristics and pharmacokinetics of JTP-2942, a novel thyrotropin-releasing hormone (TRH) analogue

JTP-2942 competed with [3H]-Me-TRH for the binding sites in rat brain in vitro, and its inhibitory effect was approximately 17 times less potent than TRH, as shown by Ki values of 673 and 39.7 nM, respectively. Both JTP-2942 and TRH significantly increased apparent dissociation constant (Kd values) for brain [3H]-Me-TRH binding. Intravenous injection of JTP-2942 (0.3-3 mg/kg) and TRH (3 and 10 mg/kg) produced a significant reduction of [3H]-Me-TRH binding sites (Bmax values) in rat brain. Although the decrease by TRH was maximal 10 min after the injection and declined rapidly with time, the decrease by JTP-2942 (1 and 3 mg/kg) tended to be maximal at 30 min later and it lasted until 120 min. The intravenous injection of JTP-2942 was at least 3 times more potent than that of TRH in decreasing Bmax values for brain [3H]-Me-TRH binding. Plasma concentration of JTP-2942 (0.3-3 mg/kg) after intravenous injection in rats rose with the increase of dose, and it peaked immediately after the injection, thereafter decreasing with t1/2 of 19.3-29.9 min. It is concluded that JTP-2942, compared to TRH, may exert fairly potent and sustained occupation of brain TRH receptors under in vivo condition. Thus, JTP-2942 could be clinically useful for the treatment of CNS disorders.     

Distribution and characterization of the TRH receptors in the CNS of ataxic mutant mouse

The distribution of TRH receptors in the membrane fraction of the CNS in ataxic mutant mice (C3Hf/Nem-rol and C57BL/6j-tg) was studied. TRH binding sites in cerebellum and frontal lobe of the ataxic form and the non-ataxic heterozygotes of Rolling Mouse Nagoya were decreased in comparison with the controls, whereas those in the spinal cord of Rolling Mouse Nagoya and cerebellum of Tottering Mouse were increased in the ataxic mice over the controls. Kinetic studies were performed on cerebrum and cerebellum of the different ataxic mutant mice. Such species differences in the distribution of the TRH receptors have to be considered in the action of TRH in individual ataxia cases.     

Hypnotic activity of N3-benzylthymidine on mice

The pharmacological effect in mice of N3-benzylthymidine (N3-ByTd) was examined by two routes of administration; intravenous (i.v.) and intracerebroventricular (i.c.v.), and compared with the effect of administration of N3-benzyluridine (N3-ByUd) previously reported. Hypnotic activity, pentobarbital (PB)-induced sleeping time, motor incoordination and spontaneous activity were used as indices of pharmacological effects. N3-ByTd (0.5-2.0 mumol/mouse, i.c.v.) and N3-ByUd (1.5-3.0 mumol/mouse, i.c.v.) were found to possess dose-dependent hypnotic activity, and N3-ByTd had more potent hypnotic activity than N3-ByUd. Both N3-ByTd and N3-ByUd (0.5 and 1.0 mumol/mouse, i.c.v., respectively) showed a synergistic effect on PB-induced sleep, although their parent compounds, thymidine (Td) and uridine (Ud), did not potentiate the activity at each dose. In motor incoordination, the effect of N3-ByTd (0.5 mumol/mouse) continued for 6 hr after i.c.v. injection. All compounds decreased the spontaneous activity of mice by i.c.v. administration. Furthermore, both N3-ByTd and N3-ByUd decreased the activity, when they were administered by i.v. These results reveal that both N3-benzylpyrimidine nucleosides have more direct depressant effects on the central nervous system (CNS) than the parent compounds. Among the pyrimidine nucleoside derivatives tested, N3-ByTd was found to be the most potent hypnotic substance.     

Sleeping habits of tamarins, Saguinus mystax and Saguinus fuscicollis (Mammalia; Primates; Callitrichidae), in north-eastern Peru

The sleeping habits of moustached tamarins, Saguinus mystax , and saddle-back tamarins, Saguinus fuscicollis , were studied in northeastern Peru. Five types of sleeping sites were distinguished: 1) Jessenia bataua palms; 2) tree hollows; 3) dense tangles of vegetation; 4) crotches; 5) open horizontal branches. Both tamarin species used Jesseniu-palms most frequently. Tree hollows ranked second in the saddle-back tamarins, but were never used by moustached tamarins. Sleeping sites of moustached tamarins were located significantly higher than those of saddle-back tamarins. Jessenia -palms used by moustached tamarins were significantly higher than palms from a random transect sample, but this was not the case for Jessenia -palms used by saddle-back tamarins. For both species, concealment seems to be more important than height above ground. The maximum number of subsequent nights spent in the same sleeping site was two in moustached tamarins and six in saddle-back tamarins. The two tamarin species did not compete for sleeping sites. While the general pattern of sleeping site selection conforms to hypotheses predicting safety from predators as a major factor, differences between the two tamarin species reflect general niche differences between them. Most sleeping sites are located in exclusively used parts of the home range. Moustached tamarins generally use sleeping sites that are close to the last feeding site of the afternoon. The distance between simultaneously used sleeping sites of moustached and saddle-back tamarins are generally close together, which helps to minimize time spent out of interspecific association.     

Induction of wet-dog shaking in rats by analogues and metabolites of thyrotrophin-releasing hormone (TRH)

The ability of thyrotrophin-releasing hormone (TRH), its metabolites and several analogues to induce wet-dog shaking (WDS) was tested by their injection into the periaqueductal grey region of male rats. TRH and its metabolite deamido-TRH (TRH-OH) both stimulated WDS, though TRH-OH gave a longer duration of response; other TRH metabolites were inactive. Of the TRH analogues studied, RX77368 (pGlu-His-3,3'-dimethyl-ProNH2) was the most potent in this behavioural test system. Both CG3509 and CG3703 were also very active in inducing WDS, as were their deamidated metabolites. The relative stability of the TRH analogues to enzymic degradation in the brain may be related to their enhanced behavioural activity over TRH. The production from these analogues of biologically-active metabolites may also explain the increased activity in stimulating WDS of the parent peptides.     

N,N'-diallylpentobarbital: antagonism of barbital in mice and rats

N,N'-Diallylpentobarbital (DAPB) antagonized barbital (B)-induced sleep in mice and rats. DAPB [80 mg/kg, intraperitoneal (i.p.)] reduced the barbital (350 mg/kg, i.p.)-induced sleeping time to 40% of the control in mice. Twenty, 40 and 80 mg/kg, i.p. of DAPB reduced barbital-induced sleeping time when administered 60 min prior to injection of barbital. DAPB (80 mg/kg, i.p.) also shortened barbital (250 mg/kg, i.p.)-induced sleeping time to about 70% of the control in rats. The result indicates that DAPB is an antagonist against hypnotic activity of barbital.     

Effects of dietary cholesterol on pyroglutamyl aminopeptidase activity in mouse frontal cortex, pituitary, and adrenal glands.

Pyroglutamyl aminopeptidase (pGluAP) is an omega peptidase that hydrolyzes biologically active peptides, such as thyrotropin-releasing hormone (TRH), with neuronal and extraendocrine functions. We analyzed the effects of a cholesterol-enriched diet on soluble and membrane-bound pGluAP activity in frontal cortex, pituitary and adrenal glands of male and female mice using fluorimetric assays. Significant increases were observed in soluble pGluAP activity in the frontal cortex and adrenal glands in males and in the pituitary in females. Membrane-bound pGluAP activity was increased in the frontal cortex and pituitary of males and females after the mice were fed a cholesterol-enriched diet. These increases may produce changes in the metabolism of endogenous substrates, including TRH, which may be related to alterations in its neuromodulator functions and to the possible relationship between TRH and other neurotransmitter systems.     

Increased bone mass in male and female mice following tamoxifen administration

Tamoxifen is capable of preserving bone mass in gonadectomized rodents as well as intact female mice; however, a detailed 3D quantitative analysis of the structural changes produced in the growing skeleton of intact mice of both genders by this agent is lacking. Employing quantitative microcomputed tomography (muCT), we assessed the effects of 4-hydroxytamoxifen (OHT) on the femora of C57BL/6J mice administered this agent either for 12 (males and females) or 2 (females) weeks. In mice of either gender, but especially in females, 12 weeks of OHT exposure led to dramatic increases in both cortical and trabecular bone. Females exposed to OHT for either 2 or 12 weeks demonstrated significantly increased cortical wall thickness, trabecular bone volume, connectivity, and number, as well as decreased trabecular separation. Significant increases in several of these parameters were also evident in males after 12 weeks of OHT administration. In view of the expanding use of OHT to induce Cre-mediated recombination events, our findings suggest that care should be exercised when interpreting the skeletal phenotypes of mice exposed this agent, particularly in situations where the effects of OHT might synergize with the phenotypic outcome of a specific genetic alteration.     

Effects of the dopamine agonist cabergoline on the pulsatile and TRH-induced secretion of prolactin, LH, and testosterone in male beagle dogs

In the present study, the pulsatile serum profiles of prolactin, LH and testosterone were investigated in eight clinically healthy fertile male beagles of one to six years of age. Serum hormone concentrations were determined in blood samples collected at 15 min intervals over a period of 6 h before (control) and six days before the end of a four weeks treatment with the dopamine agonist cabergoline (5 microg kg(-1) bodyweight/day). In addition, the effect of cabergoline administration was investigated on thyrotropin-releasing hormone (TRH)-induced changes in the serum concentrations of these hormones. In all eight dogs, the serum prolactin concentrations (mean 3.0 +/- 0.3 ng ml(-1)) were on a relatively constant level not showing any pulsatility, while the secretion patterns of LH and testosterone were characterised by several hormone pulses. Cabergoline administration caused a minor but significant reduction of the mean prolactin concentration (2.9 +/- 0.2 ng ml(-1), p < 0.05) and did not affect the secretion of LH (mean 4.6 +/- 1.3 ng ml(-1) versus 4.4 +/- 1.7 ng ml(-1)) or testosterone (2.5 +/- 0.9 ng ml(-1) versus 2.4 +/- 1.2 ng ml(-1)). Under control conditions, a significant prolactin release was induced by intravenous TRH administration (before TRH: 3.8 +/- 0.9 ng ml(-1), 20 min after TRH: 9.1 +/- 5.9 ng ml(-1)) demonstrating the role of TRH as potent prolactin releasing factor. This prolactin increase was almost completely suppressed under cabergoline medication (before TRH: 3.0 +/- 0.2 ng ml(-1), 20 min after TRH: 3.3 +/- 0.5 ng ml(-1)). The concentrations of LH and testosterone were not affected by TRH administration. The results of these studies suggest that dopamine agonists mainly affect suprabasal secretion of prolactin in the dog.     

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxyl or counterpart hydroxyl with histidine 524 of the human estrogen receptor alpha

We investigated the role of H524 of the human estrogen receptor alpha (ERalpha) for the binding of various estrogens [estradiol (E(2)), 3-deoxyestradiol (3-dE(2)), and 17beta-deoxyestradiol (17beta-dE(2))] and antiestrogens [4-hydroxytamoxifen (OHT), RU 39 411 (RU), and raloxifene (Ral)], which possess the 17beta-hydroxyl or counterpart hydroxyl (designated: 17beta/c-OH), with the exception of 17beta-dE(2) and OHT. The work involved a comparison of the binding affinities of these ligands for wild-type and H524 mutant ERs, modified or not with diethyl pyrocarbonate (DEPC), a selective histidine reagent. Alanine substitution of H524 did not significantly change the association affinity constant (relative to OHT) of 17beta-dE(2), whereas those of RU, Ral, E(2), and 3-dE(2) were decreased 3-fold, 14-fold, 24-fold, and 49-fold, respectively. Values of the two ligands available in radiolabeled form (E(2) and OHT) were correlated with the dissociation rate constants, which were increased 250-fold and 2-fold, respectively. The action of DEPC on wild-type ER led to a homogeneous ER population which still bound antiestrogens and 17beta-dE(2) with practically unchanged affinities (less than 4-fold decreases in relative affinity constants), while E(2) and 3-dE(2) displayed markedly decreased affinities (56-fold decrease for E(2)). Conversely, DEPC treatment of H524A mutant ER did not induce marked decreases in the relative affinities of any of the checked compounds (decreases wild-type ER) and very weakly protected H524A ER. Molecular modeling was tentatively used to interpret the biochemical results.     

Cardiovascular responses of heart transplant patients to exercise training

Orthotopic heart transplantation (OHT) represents an effective alternative for individuals with end-stage heart disease. The current literature reports only the responses of OHT patients to greater than or equal to 4 mo of exercise training (ET) and frequently lacks adequate controls. Most programs currently treating OHT patients usually provide 6-12 wk of ET. This study describes the effects of a 10-wk supervised ET program in 12 male OHT patients and 5 other male OHT patients who served as a comparison group. Graded exercise tests were performed before and after ET. After ET, maximal O2 consumption was significantly greater for the ET group than the comparison group (P less than 0.05) and the mean increase in peak heart rate was 18 +/- 4 and 6 +/- 4 (SE) min-1 for ET and comparison groups, respectively (P less than 0.05). Maximal ventilation was also significantly greater for the ET group at after ET, while resting heart rate and blood pressure and peak blood pressure, O2 pulse, respiratory rate, and ventilatory equivalents for O2 and CO2 were not significantly changed. We conclude that after OHT a 10-wk ET program improves maximal O2 consumption and, by improving peak heart rate, improves O2 delivery.