Thyrotropin-releasing hormone (TRH) analogues show enhanced CNS selectivity because of increased biological stability

TRH exerts both endocrinological and neuropharmacological actions. Two analogues of TRH, Pyr-His-Mep . NH2 (L-trans-3-methylprolineamide) and Pyr-His-Dmp . NH2 (L-3,3-dimethylprolineamide) have been examined for their neuropharmacological and endocrinological effects. Comparisons of their ability to provoke hyperthermia in rabbits demonstrated that both analogues were more potent than TRH, but like the parent peptide had only a limited ability to cross the blood brain barrier. This conclusion was confirmed by whole body autoradiographical studies. In contrast both analogues had a similar potency to TRH with respect to the ability to provoke TSH release. It is concluded that the increased potency in neuropharmacological tests results from enhanced bioavailability to CNS sites and that a similar rationale can be used to explain the CNS selectively claimed in the literature for other analogues of TRH.     

A comparison of the locomotor effects induced by centrally injected TRH and TRH analogues

Thyrotrophin-releasing hormone (TRH) and its stable analogues CG3509 and RX77368 were injected directly into the nucleus accumbens, septum and striatum of the rat and locomotor activity was recorded. TRH (5-20 micrograms) caused a dose-dependent increase in locomotor activity when injected into the nucleus accumbens. TRH (20 micrograms) also increased locomotor activity after administration into the septum but not when put into the striatum. Both the TRH analogues (0.1 and 1.0 microgram) produced closely related increases in activity when injected into either the nucleus accumbens or septum but CG3509 was more potent with a longer lasting effect. Also, in contrast with TRH (20 micrograms), both TRH analogues stimulated locomotor activity when injected into the striatum at a dose of 1 microgram but the effect was less marked and delayed in onset compared to the nucleus accumbens and septum response. Dopamine (100 micrograms) injected into the accumbens or septum also produced significant increases in locomotor activity. The locomotor effects of the peptides are discussed in relation to a possible dopamine-mediated mechanism which contrasts with the actions of TRH and the analogues on barbiturate anaesthesia.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Thyrotrophin-releasing hormone inactivation by human postmortem brain

The mechanisms of inactivation of thyrotrophin-releasing hormone (TRH) by peptidases in several areas of normal human postmortem brain have been investigated by radioimmunoassay and high-performance liquid chromatography. Of the several brain regions studied, the cerebral cortex (Brodman's area, BA10) had the highest TRH-degrading activity in both subcellular fractions. Deamidated-TRH (TRH-OH) was the only product formed by the soluble fraction whereas the histidyl-proline diketopiperazine, cyclo(His-Pro), and a small amount of TRH-OH were formed by the particulate fraction. Several centrally acting TRH analogues showed varying degrees of resistance to degradation by the peptidases in the two fractions, the most stable analogue being RX77368 (pGlu-His-3,3'-dimethyl(ProNH2]. Areas of human postmortem brain appear to contain two of the enzymes capable of degrading TRH, a proline endopeptidase forming TRH-OH and a pyroglutamyl aminopeptidase forming cyclo(His-Pro). The use of the assay procedures in further studies on the inactivation of TRH by peptidases from brain areas of patients with neurological disorders may provide complementary information on the dynamics of TRH in these disorders. The stability of the centrally acting TRH analogues may prove useful in examining their therapeutic potential.     

Phencyclidine and Analogues: Effects on Brain Protein Synthesis

Abstract: Phencyclidine and four analogues were tested for their capacity to inhibit total protein synthesis in a brain homogenate. At 1.0 m M they were all found to be potent inhibitors with values ranging from 36% to 96% inhibition. At this high concentration two of the analogues were equal to or more effective than the classic protein synthesis inhibitors cycloheximide and emetine. At lower concentrations the inhibition dropped off sharply to 18% at 1.0 × 10⁻⁴ M and 9% at 1.0 × 10⁻⁵ M for phencyclidine. If the inhibition observed in the brain homogenate occurs in vivo it may account for the high incidence of memory loss reported with phencyclidine use.     

Mechanisms of brain inactivation of centrally-acting thyrotrophin-releasing hormone (TRH) analogues: a high-performance liquid chromatography study

High-performance liquid chromatography (HPLC) was used to investigate the degradation in vitro of several centrally-acting analogues of thyrotrophin-releasing hormone (TRH) by two subcellular fractions prepared from different areas of rat brain. Of the seven analogues studied, RX77368 (pGlu-His-(3,3'-dimethyl)-ProNH2) was the most stable analogue, showing only a small amount of degradation by the particulate fraction containing a pyroglutamyl aminopeptidase, whereas the other analogues (RX74355, CG3509, CG3703, [3MeHis]TRH, PGHPA and MK771) showed varying degrees of resistance to degradation by this enzyme and the proline endopeptidase in the soluble fraction. However, TRH was rapidly inactivated to its deamidated form, TRH-OH and the histidyl-proline diketopiperazine by both fractions. The relative stability of these TRH analogues to enzyme action may provide some explanation for their enhanced biological activity in vivo.     

Two novel thioamide analogues of TRH with selective activity on CNS.

TRH analogues containing C-terminal tioamide group and norvaline ([Nva2, Prot3] TRH) or norleucine ([Nle2, Prot3] TRH) in position 2 were synthesized and tested for hormonal and central nervous system (CNS) activities. Receptor binding studies revealed that the analogues neither bind to pituitary nor to brain TRH receptors. Accordingly, no TSH releasing activity was recorded. However, both analogues significantly affected sleeping time and breathing frequency. Dissociation of endocrine effects from those on the CNS of [Prot3] TRH was achieved with the replacement of histidine2 by aliphatic amino acids. The presence of central histidine is not essential for the analogues to be active on the CNS.     

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.     

Further Observations on the Effect of Synthetic Thyrotrophin-releasing Hormone in Man

Synthetic thyrotrophin-releasing hormone (TRH) given intravenously in doses of 50 μg or more causes a significant rise in serum thyroid-stimulating hormone (TSH) levels but has no effect on serum growth hormone, plasma luteinizing hormone, or plasma 11-hydroxycorticosteroids under carefully controlled basal conditions.The peak TSH response to intravenous TRH occurs at 20 minutes. The mild and transient side effects, which occur only after intravenous TRH, include nausea, a flushing sensation, a desire to micturate, a peculiar taste, and tightness in the chest. There is considerable variability in response to a given dose of TRH in the same subject on different occasions and in different subjects. Oral administration of TRH in doses of 1 mg and above causes a rise in serum TSH, maximal at two hours, a consistent response being obtained at doses of 20 mg and above. A rise in serum protein-bound iodine (P.B.I.) follows that of TSH, a consistent response being observed at 40-mg doses of TRH orally. Measurements of serum TSH after intravenous administration of TRH or of serum TSH or serum P.B.I. after oral TRH should prove useful tests of pituitary TSH reserve.     

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.     

Barbiturate competition for TRH receptors in mouse brain: neuromodulation of anesthesia

In vitro thyrotropin releasing hormone (TRH) radioligand binding assays were performed using purified presynaptic and postsynaptic membranes derived from various regions of mouse brain. These studies revealed the pattern of central distribution of specific TRH binding sites. The highest concentrations of both types of membrane receptors were localized in the limbic forebrain. The brain stem contained a high density of only presynaptic receptors, and the cerebral cortex contained a moderate-high level of only postsynaptic receptors. Barbiturate analogues effectively competed for all forebrain and brain stem, but not cortical, TRH receptors, thus implicating these specific receptors in the neuromodulation of barbiturate anesthesia. The results of in vivo radioligand binding assays for [3H] TRH disposition after central infusions concomitant with barbiturate vs. saline challenges further support this viewpoint.     

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.     

Thyrotrophin-Releasing Hormone Analogues Increase Dopamine Release from Slices of Rat Brain

Abstract: Rat brain slices were incubated with a high concentration of K⁺, thyrotrophin-releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K⁺ (30 m M ) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (10⁵–10⁻³ M ) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose-dependent fashion, whereas RX 77368 (10⁻⁴ M ) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.     

Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors

The initial rates of Na(+)-dependent D-aspartate and D-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggesting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6-24 h from resuspension, however this final 'next day' uptake activity was too low to be of practical use in kinetic studies. Freezing aliquots of rabbit jejunal vesicles in liquid N2 until the time of assay resulted in complete stabilization of D-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the 'next day' stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the 'next day' specific activities of D-glucose uptake in rat jejunal brush border vesicles and D-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na(+)-dependent D-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N2 or the modified media and allowed to stabilize overnight, revealed a single transport system with a Km of 0.31-0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of D-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than the rapid freezing protocol, it is shown that the modified homogenate should however be preferred when dealing with slowly permeant ions like choline since it provides in this case the only alternative to reliable measurement of uptake rates across a stable and equilibrated vesicle preparation.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

The effect of dexamethasone on TSH and prolactin secretion after TRH stimulation

Serum thyrotropin (TSH) and prolactin levels were measured after intravenous administration of 400 μg of synthetic thyrotropin-releasing hormone (TRH) in 13 normal subjects and six hypothyroid patients before and after three days of administration of dexamethasone 2 mg per day. In the normal subjects dexamethasone suppressed baseline serum levels and secretion of TSH after TRH stimulation. On the other hand, it had no effect on the hypothyroid patients. In the control group dexamethasone also suppressed baseline serum levels but not secretion of prolactin after TRH stimulation. Dexamethasone had no effect on prolactin levels in the hypothyroid group. It is concluded that in normal patients short-term administration of dexamethasone has an inhibitory effect on TSH secretion at the pituitary level. As for prolactin, our results could indicate that TRH is a more potent stimulator of prolactin secretion than of TSH secretion, or that TSH and prolactin pituitary thresholds for TRH are different.     

Neuroprotective effect and brain receptor binding of taltirelin,a novel thyrotropin-releasing hormone (TRH) analogue,in transient forebrain ischemia of C57BL/6J mice

Thyrotropin-releasing hormone (TRH) and some of its stable analogues have been shown to improve neurologic dysfunctions such as brain trauma in both animals and humans. Our previous study revealed that taltirelin, a novel orally active TRH analogue, binds to rat brain TRH receptors in vivo. The present study was undertaken to investigate whether taltirelin has neuroprotective effects in transient brain ischemia of C57BL/6J mice induced by bilateral carotid artery occlusion (2VO). Neuronal cell density in the hippocampal CA1 region of C57BL/6J mice was significantly (39.9%) decreased 1 week after 2VO-reperfusion, compared to the case of the sham group, and this reduction of hippocampal neuronal density was significantly suppressed by an intravenous (i.v.) injection of taltirelin (0.3 mg/kg). The i.v. injection of taltirelin at this dosage produced a significant increase in the dissociation constant (Kd) of specific [3H]MeTRH binding in sham and 2VO-reperfusion groups (33.6 and 51.4%, respectively) compared with the vehicle-treated group. These results indicate that the intravenously injected taltirelin bound to TRH receptors in the ischemic brain. There was little difference in the brain-to-plasma concentration ratio (Kp) of [14C]sucrose between the sham and 2VO groups of C57BL/6J mice, indicating that the tight junction of the blood-brain barrier may be intact in the ischemic brain.In conclusion, the study has shown that taltirelin may have a significant neuroprotective effect on the ischemic brain.     

A radioimmunoassay for a highly active luteinizing hormone-releasing hormone analogue and relation between the serum level of the analogue and that of gonadotropin

Antisera to an LH-RH analogue, des-Gly10-[D-Leu6]-LH-RH-ethylamide (TAP-144) were produced in 10 rabbits. By using these antisera, a specific and sensitive radioimmunoassay for TAP-144 was established. Sensitivity of the radioimmunoassay ranged from 5 to 100 per assay tube with these antisera. Lh, FSH, TRH, LH-RH, and the 1-6 fragment of TAP-144 did not practically cross-react with LH-RH analogues, though the degree of the cross-reactivity differed among individual sera. The average cross-reactivity of the 10 antisera showed low specificities to TAP-144 analogues which are altered at positions 2, 3, 4, 5, and 6, but showed high specificities to those altered at positions 8 and 9. The antisera also showed low cross-reactivities to LH-RH analogues replaced at position 6 by D-amino acids and those altered at position 10 by alkylamines, but they showed fairly high cross-reactivities to analogues which are altered simultaneously at both positions 6 and 10. When TAP-144 was administered intraperitoneally to rats on the diestrous day, serum concentrations of TAP-144 increased dose-dependently but maximal serum concentrations of both LH and FSH were attained in response to higher doses of TAP-144. The peak LH and FSH concentrations appeared 70 to 110 min after the peak TAP-144 concentration had been reached. Similar delays in reaching the peak LH and FSH levels were also observed when TAP-144 was administered intravenously, subcutaneously and intramuscularly. When TAP-144 was administered intravaginally, a low but constant serum level of TAP-144 was maintained from 5 to 300 min after the administration, but serum LH and FSH levels declined to a low level from 180 min after the TAP-144 administration.     

Effects of acute CS2 intoxication on protein metabolism in rat brain

The effect of acute CS₂ exposure on the rat brain protein metabolism was studied with control and phenobarbitone pretreated adult male rats 1, 4 and 46 h after exposure. Increased activity of acid proteinase was detected in both test groups 1 and 4 h after exposure and it was accompanied by changes in ¹⁴C-labelled leucine turnover as well as in RNA content. The changes were more conspicuous in cerebellum than in brain in both test groups while phenobarbitone pretreatment modified the brain response towards intoxication. This modification probably represents inherent effects of barbiturate on brain protein metabolism as well as altered metabolism of CS₂.The activities of creatine kinase and nonspecific cholinesterase displayed only subtle changes as assayed in cerebral homogenate and serum. Thus a single acute CS₂ intoxication apparently causes definitive transient changes in brain protein metabolism; serum enzyme determinations may not reflect the magnitude of these changes.