Synthesis and Evaluation of in Vivo Anti-hypothermic Effect of the N- and C-Terminus Modified Thyrotropin-Releasing Hormone Mimetic: [(4S,5S)-(5-Methyl-2-oxooxazolidine-4-yl)carbonyl]-[3-(thiazol-4-yl)-L-alanyl]-L-prolinamide.

We explored orally effective thyrotropin-releasing hormone (TRH) mimetics, which show high central nervous system effects in structure-activity relationship studies based on in vivo antagonistic activity on reserpine-induced hypothermia (anti-hypothermic effect) in mice starting from TRH. This led us to the TRH mimetic: [(4S,5S)-(5-methyl-2-oxooxazolidine-4-yl)carbonyl]-[3-(thiazol-4-yl)-L-alanyl]-L-prolinamide 1, which shows a higher anti-hypothermic effect compared with that of TRH after oral administration. We next attempted further chemical modification of the N- and C-terminus of 1 to find more orally effective TRH mimetics. As a result, we obtained several N- and C-terminus modified TRH mimetics which showed high anti-hypothermic effects.

Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice.

Thyrotropin-releasing hormone (TRH)-like peptides were synthesized by replacing critical histidine and pGlu residues in the native peptide. The peptides were evaluated in vitro for receptor binding activity assay and in the cell functional assay; the peptides exhibit selective basal signaling agonist behavior toward TRH-R2. For example, peptides 8a, 8b, 8c, 8 f, 8 h, 8 l and 12 d activated TRH-R2 with potency (EC50) of 0.53 µM, 0.048 µM, 0.05 µM, 0.006 µM, 0.31 µM, 0.034 µM and 0.004 µM, respectively. In contrast for signaling activation of TRH-R1, the same peptide required higher concentration of 19.35 µM, 3.98 µM, 2.54 µM, 0.287 µM, 11.28 µM, 0.986 µM and 0.944 µM, respectively. The results showed that peptides were 36.5, 82.9, 50.8, 47.8, 36.3, 32.6 and 235-fold selective to TRH-R2 receptor subtype. The peptides were investigated for CNS activity at 10 µmol/kg in pentobarbital-induced sleep assay study. Peptides 8c (16.5 ± 1.4 min) and 8l (16.5 ± 2.1 min) displayed excellent CNS activity. In an in vivo study, peptide 8c did not cause significant change in the rat plasma TSH levels. The peptide 8c was further investigated for neuroprotective potential, and significantly reduced infracts volume and neurological score in the focal cerebral ischemia model in mice. Peptide 8c also significantly lowered MDA levels, indicating reduction of oxidative and enhanced percentage cell survival in CA1 region, when compared to ischemic brain.

Synthesis, receptor binding, and CNS pharmacological studies of new thyrotropin-releasing hormone (TRH) analogues.

As part of our search for selective and CNS-active thyrotropin-releasing hormone (TRH) analogues, we synthesized a set of 44 new analogues in which His and pGlu residues were modified or replaced. The analogues were evaluated as agonists at TRH-R1 and TRH-R2 in cells in vitro, and in vivo in mice for analeptic and anticonvulsant activities. Several analogues bound to TRH-R1 and TRH-R2 with good to moderate affinities, and are full agonists at both receptor subtypes. Specifically, analogue 21 a (R=CH3) exhibited binding affinities (Ki values) of 0.17 µM for TRH-R1 and 0.016 µM for TRH-R2; it is 10-fold less potent than TRH in binding to TRH-R1 and equipotent with TRH in binding to TRH-R2. Compound 21 a, the most selective agonist, activated TRH-R2 with a potency (EC50 value) of 0.0021 µM, but activated TRH-R1 at EC50=0.05 µM, and exhibited 24-fold selectivity for TRH-R2 over TRH-R1. The newly synthesized TRH analogues were also evaluated in vivo to assess their potencies in antagonism of barbiturate-induced sleeping time, and several analogues displayed potent analeptic activity. Specifically, analogues 21 a,b and 22 a,b decreased sleeping time by nearly 50% more than TRH. These analogues also displayed potent anticonvulsant activity and provided significant protection against PTZ-induced seizures, but failed to provide any protection in MES-induced seizures at 10 µmol kg(-1). The results of this study provide evidence that TRH analogues that show selectivity for TRH-R2 over TRH-R1 possess potent CNS activity.

Quantification of thyrotropin-releasing hormone by liquid chromatography-electrospray mass spectrometry.

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography-mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Synthesis of the phosphinic analogue of thyrotropin releasing hormone.

The synthesis of the phosphinic analogue of thyrotropin releasing hormone (TRH) GlpPsi[P(O)(OH)]HisProNH2, where the scissile peptide bond of TRH has been replaced by the hydrolytically stable phosphinic bond, has been achieved by a multistep synthetic strategy, providing thus one of the most potent synthetic inhibitors of pyroglutamyl peptidase II (PPII) reported to date (170 nM). The key synthetic step, an Ugi-type condensation reaction, produced directly the suitably protected for solid-phase peptide synthesis pseudodipeptidic block FmocGlu(OMe)Psi[P(O)(OH)]His(Tr)OH. Formation of the pyroglutamic ring was performed on solid phase, providing thus a general method for synthesizing pyroglutamyl phosphinic peptides on solid phase. Using this strategy, the phosphinic analogue of TRH has been synthesized for the first time.

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.

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.

Modifications of the pyroglutamic acid and histidine residues in thyrotropin-releasing hormone (TRH) yield analogs with selectivity for TRH receptor type 2 over type 1.

Thyrotropin-releasing hormone (TRH) analogs in which the N-1(tau) or the C-2 position of the imidazole ring of the histidine residue is substituted with various alkyl groups and the l-pyroglutamic acid (pGlu) is replaced with the l-pyro-2-aminoadipic acid (pAad) or (R)- and (S)-3-oxocyclopentane-1-carboxylic acid (Ocp) were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-R2). We observed that several analogs were selective agonists of TRH-R2 showing relatively less or no activation of TRH-R1. For example, the most selective agonist of the series 13, in which pGlu is replaced with the pAad and histidine residue is substituted at the N-1 position with an isopropyl group, was found to activate TRH-R2 with a potency (EC(50)=1.9microM) but did not activate TRH-R1 (potency>100 microM); that is, exhibited >51-fold greater selectivity for TRH-R2 versus TRH-R1. Analog 8, in which pGlu is replaced with pAad and histidine is substituted at the N-1(tau) position with a methyl group, exhibited a binding affinity (K(i)=0.0032 microM) to TRH-R1 that is similar to that of [Ntau(1)-Me-His]-TRH and displayed potent activation of TRH-R1 and TRH-R2 (EC(50)=0.0049 and 0.0024 microM, respectively). None of the analogs in which pGlu is replaced with the bioisosteric (R)- and (S)-(Ocp) and the imidazole ring is substituted at the N-1(tau) or C-2 position were found to bind or activate either TRH-R1 or TRH-R2 at the highest test dose of 100 microM.

Synthesis, receptor binding, and activation studies of N(1)-alkyl-L-histidine containing thyrotropin-releasing hormone (TRH) analogues.

Thyrotropin-releasing hormone (TRH) analogues in which the N(1)-position of the imidazole ring of the centrally placed histidine residue is substituted with various alkyl groups were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-R2). Analogue 3 (R=C2H5) exhibited binding affinity (Ki) of 0.012 microM to TRH-R1 that is about 1.1-fold higher than that of TRH. Several analogues were found to selectively activate TRH-R2 with greater potency than TRH-R1. The most selective agonist of the series 5 [R=CH(CH3)2] was found to activate TRH-R2 with a potency (EC50) of 0.018 microM but could only activate TRH-R1 at EC50 value of 1.6 microM; that is, exhibited 88-fold greater potency for TRH-R2 versus TRH-R1. The results of this study indicate that modulation of central histidine residue is important for designing analogues which were selective agonist at TRH receptor subtypes.

Thyrotropin-releasing hormone (TRH) analogues that exhibit selectivity to TRH receptor subtype 2.

Thyrotropin-releasing hormone (TRH) analogues in which the C-2 position of the imidazole ring of the centrally placed histidine residue is substituted with various alkyl groups were synthesized and studied as agonists for TRH receptor subtype 1 (TRH-R1) and subtype 2 (TRH-R2). Several analogues were found to be selective agonists for TRH-R2 exhibiting no activation of TRH-R1. For example, analogue 4 (R= c-C3H5) was found to activate TRH-R2 with a potency (EC50) of 0.41 microM but did not activate TRH-R1 (potency > 100 microM). This study describes the first discovery of TRH-R2-specific agonists and provides impetus to design predominately CNS-effective TRH peptides.

Centrally acting and metabolically stable thyrotropin-releasing hormone analogues by replacement of histidine with substituted pyridinium.

Metabolically stable and centrally acting thyrotropin-releasing hormone (TRH) analogues were designed by replacing the central histidine with substituted pyridinium moieties. Their analeptic and acetylcholine-releasing actions were evaluated to assess their potency as central nervous system (CNS) agents. A strong experimental connection between these two CNS-mediated actions of the TRH analogues was obtained in subject animals. The analogue 3-(aminocarbonyl)-1-(3-[2-(aminocarbonyl)pyrrolidin-1-yl]-3-oxo-2-[[(5-oxopyrrolidin-2-yl)carbonyl]amino]propyl)pyridinium (1a) showed the highest (TRH-equivalent) potency and longest, dose-dependent duration of action from a series of homologous compounds in antagonizing pentobarbital-induced narcosis when administered intravenously in its CNS-permeable prodrug form (2a) obtained via reduction of the pyridinium moiety to the nonionic dihydropyridine. The maximum change in hippocampal acetylcholine concentration upon perfusion of the pyridinium-containing tripeptides into the hippocampus of rats was also achieved with 1a. No binding to the endocrine TRH receptor was measured for the TRH analogues reported here; therefore, our design afforded a novel lead for centrally acting TRH analogues. We have also demonstrated the benefits of the prodrug approach on the pharmacokinetics and brain uptake/retention of pyridinium-containing TRH analogues (measured by in vivo microdialysis sampling) upon systemic administration.

A novel peptide synthesis using fluorous chemistry.

Three new fluorous supports for peptide synthesis, i.e., the trialkoxybenzhydryl-type (6), the Wang-type (7) and the tert-butyl-type support (8), were prepared. A bioactive peptide TRH was easily synthesized by an Fmoc strategy using the benzhydryl-type fluorous support with fluorous chemistry.

Prodrugs to enhance central nervous system effects of the TRH-like peptide pGlu-Glu-Pro-NH2.

Potential prodrugs for the TRH-like tripeptide pGlu-Glu-Pro-NH(2) were synthesized either by esterifying the Glu side-chain of the parent peptide in solution with alcohols in the presence of resin-bound dicyclohexylcarbodiimide or by solid-phase peptide chemistry. Affinities of these ester prodrugs to lipid membranes as predictors of the transport across the blood-brain barrier were compared by immobilized artificial membrane chromatography, and prodrug activation was tested in the brain tissue of experimental animals. Esters of pGlu-Glu-Pro-NH(2) with long-chain primary alcohols emerged as potentially useful prodrugs to improve the central nervous system activity of pGlu-Glu-Pro-NH(2) upon systemic administration, as revealed by the enhancement of analeptic activity in mice.

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.

Synthesis and biological evaluation of a novel pyroglutamyl-modified TRH analogue.

The TRH analogue 3, incorporating the (S)-isothiazolidine-1,1-dioxide-3-carboxylic acid (1) moiety in place of the native L-pyroglutamic acid (pGlu) residue, has been synthesized and fully characterized by 1H and 13C NMR. The effects of replacing pGlu with its sulphonamido counterpart on biological activity have been investigated. This peptide, which is significantly stabilized towards hydrolysis by pyroglutamyl peptidase type I (PP I, EC 3.4.19.3), has shown to maintain in vitro prolactin-releasing activity.

Synthesis and biology of new thyrotropin-releasing hormone (TRH) analogues.

We report synthesis and biological activities of several thyrotropin-releasing hormone (TRH) analogues in which the N-terminal pyroglutamic acid residue has been replaced with various carboxylic acids and the central histidine is modified with substituted-imidazole derivatives.

Brain-targeted chemical delivery of [Leu2, Pip3]-TRH: synthesis and biological evaluation.

A chemical targeting system for [Leu2, Pip3]-TRH (Gln,Leu,Pip) was synthesized in order to allow its specific delivery to the central nervous system (CNS). Sequential metabolism of the obtained 'packaged' chemical delivery system, (CDS), DHT-Pro-Pro-Gln-Leu-Pip-OCh, should yield a 'locked-in' precursor following the oxidative conversion of the dihydrotrigonellyl (DHT) to the trigonellyl (T+) moiety, followed by removal of the cholesteryl function and cleavage of the T+-Pro-Pro by prolyl endopeptidase. The antagonism of barbiturate-induced sleeping time was used to assess the activity of the CDS. The sleeping time after administration of vehicle and [Leu2]-TRH was 100.5 +/- 6.3 min, and 78.2 +/- 4.7 min, respectively. The [Leu2, Pip3]-TRH-CDS showed a significant decrease in sleeping time (58.2 +/- 3.4 min) compared to the vehicle or [Leu2]-TRH. These results indicate successful brain delivery of the precursor construct, and an effective release of the active GlnLeuPip in the brain.

Metabolism-based brain-targeting system for a thyrotropin-releasing hormone analogue.

Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormone (TRH) analogue [Leu(2)]TRH (pGlu-Leu-Pro-NH(2)), was covalently and bioreversibly modified on its N- and C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery of the parent peptide at the target site involve several enzymatic steps, including the oxidation of the 1,4-dihydropyridine moiety. Due to the lipid insolublity of the peptide pyridinium conjugate obtained after this reaction, one of the rudimentary steps of brain targeting (i.e., trapping in the central nervous system) can be accomplished. Our design also included spacer amino acid(s) inserted between the N-terminal residue of the progenitor sequence and the dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification. The release of the TRH analogue in the brain is orchestrated by a sequential metabolism utilizing esterase/lipase, peptidyl glycine alpha-amidating monooxygenase (PAM), peptidase cleavage, and glutaminyl cyclase. In addition to in vitro experiments to prove the designed mechanism of action, the efficacy of brain targeting for [Leu(2)]TRH administered in the form of chemical-targeting systems containing the embedded progenitor sequence was monitored by the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the activational effect on cholinergic neurons) in mice, and considerable improvement was achieved over the efficacy of the parent peptide upon using this paradigm.

Analogs of thyrotropin-releasing hormone using an aminoglycine-based template.

Novel thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) analogs, made by solid phase, were derived from the general scaffold pGlu-(D/L)Agl(X)-Pro-NH2 where Agl = aminoglycine. Analogs ranged from X being a proton to an acylating agent derived from substituted (aromatic heterocyclic rings) formic or acetic acids or an aminotriazolyl moiety (3'-amino-1H-1',2',4'-triazolyl) built on N(alpha) of aminoglycine or Nbeta of alpha,beta-diaminoproprionic acid (Dpr). X was expected to mimic the electronic and structural characteristics of the imidazole ring of histidine. Analogs were purified by HPLC, characterized by mass spectrometry and isolated as either diastereoisomeric mixtures or pure isomers. Analogs, tested for their binding affinity to mouse pituitary TRH receptors, have apparent equilibrium inhibitory constants > 1 microM.

Thyrotropin releasing hormone analogs: a building block approach to the construction of tetracyclic peptidomimetics.

A building block based approach was used to synthesize a pair of tetracyclic peptidomimetics that constrain all but one of the rotational degrees of freedom of the hypothalamic tripeptide hormone thyroliberin. One of the analogs bound to the thyroliberin endocrine receptor (TRH-R) with an affinity greater than that of an analog without constraints. The tetracyclic peptidomimetics were found to be partial agonists for the TRH-R receptor.