Protirelin (Thyrel TRH) is a novel, potent and highly conserved neuropeptide that exerts the hormonal control of thyroid-stimulating hormone (TSH) levels as well as neuromodulatory functions. Protirelin is used to treat hypothyroidism by stimulating the release of a hormone called thyroid-stimulating hormone, or TSH, from the anterior pituitary gland.

Physicochemical Properties

Molecular Formula	C16H22N6O4
Molecular Weight	362.3837
Exact Mass	362.17
CAS #	24305-27-9
Related CAS #	Protirelin acetate;120876-23-5;Thyrotropin-Releasing Hormone (TRH), Free Acid;24769-58-2
PubChem CID	638678
Appearance	White to off-white solid powder
Density	1.5±0.1 g/cm3
Boiling Point	942.1±65.0 °C at 760 mmHg
Flash Point	523.6±34.3 °C
Vapour Pressure	0.0±0.3 mmHg at 25°C
Index of Refraction	1.617
LogP	-2.43
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	6
Heavy Atom Count	26
Complexity	597
Defined Atom Stereocenter Count	3
SMILES	C1C[C@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@@H]3CCC(=O)N3)C(=O)N
InChi Key	XNSAINXGIQZQOO-SRVKXCTJSA-N
InChi Code	InChI=1S/C16H22N6O4/c17-14(24)12-2-1-5-22(12)16(26)11(6-9-7-18-8-19-9)21-15(25)10-3-4-13(23)20-10/h7-8,10-12H,1-6H2,(H2,17,24)(H,18,19)(H,20,23)(H,21,25)/t10-,11-,12-/m0/s1
Chemical Name	(S)-N-((S)-1-((S)-2-carbamoylpyrrolidin-1-yl)-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl)-5-oxopyrrolidine-2-carboxamide
Synonyms	Synthetic thyrotropin-releasing hormone; TRF; TRH; TSH-RF; pGlu-His-Pro-NH2; {pGLU}HP-NH2; Protirelin; Antepan; Hirtonin; A-38579; A 38579; A38579; Thyrel TRH
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Protirelin (synthetic pQHP-NH₂) activates the human thyrotropin-releasing hormone receptor (TRHR) and the Caenorhabditis elegans TRHR ortholog TRHR-1. [1]
ln Vitro	Protirelin (pQHP-NH₂) activated the C. elegans TRHR-1 expressed in CHO cells at a concentration of 10 µM in a calcium mobilization assay. [1]
ln Vivo	The effect of thyrotropin-releasing hormone (Protirelin, TRH), one of the hypothalamic-releasing hormones, on body temperature in rats was studied. Protirelin was injected intraperitoneally at doses of 1, 5, 10 and 20 mg/kg into male Wistar rats weighing 200-250 g. Protirelin causes a dose-dependent transient increase in body temperature. Injection of 20 mg/kg Protirelin into thyroidectomized rats caused a significant increase in body temperature in sham-operated animals, but did not show an increase in body temperature. The current results indicate that thyroid hormones may have been involved in the control of postembryonic growth and reproduction in the past [1]. Intraperitoneal (i.p.) injection of Protirelin (TRH tartrate) at doses of 10 and 20 mg/kg induced a dose-dependent, transient hyperthermia in male Wistar rats (200-250 g). The hyperthermia appeared early, with a peak rise in rectal temperature (mean increase of 0.5°C to 1.5°C) observed at 40 minutes post-injection, and lasted for approximately 80 minutes. Doses of 1 and 5 mg/kg did not cause a significant temperature increase. [2] Thyroidectomized rats, which underwent surgery 10 days prior, failed to exhibit the hyperthermic response to a 20 mg/kg i.p. dose of Protirelin. In contrast, sham-operated animals showed a significant temperature rise with the same dose. This result suggests that the hyperthermic action of Protirelin in rats is mediated through the release of thyroid hormone. [2]
Enzyme Assay	A luminescence-based calcium mobilization assay was used to measure GPCR activation. CHO cells expressing the receptor of interest, along with aequorin and a promiscuous human Gα subunit, were loaded with coelenterazine H. After peptide addition, luminescence was measured at 469 nm for 30 seconds. In this assay, Protirelin activated the C. elegans TRHR-1 at 10 µM. [1]
Animal Protocol	Ninety male Wistar rats weighing 200-250 g were used. Protirelin tartrate was dissolved in physiological saline. In the first experiment, different groups of rats received a single intraperitoneal injection of the drug at doses of 1, 5, 10, or 20 mg/kg; a control group received saline only. Rectal temperature was measured using an electronic thermometer inserted 5 cm into the rectum, before and at various time points after injection. The experiment was conducted from 1 p.m. to 4 p.m. in a room maintained at 24 ± 1°C. [2] In the second experiment, rats were subjected to thyroidectomy or sham surgery under thiopental sodium anesthesia. Ten days post-operation, thyroidectomized and sham-operated rats received a single intraperitoneal injection of either Protirelin (20 mg/kg) or saline. Rectal temperature was measured using the same method as in the first experiment. [2]
References	[1]. Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2017 May 16;114(20):E4065-E4074. [2]. Hyperthermia induced by thyrotropin-releasing hormone (TRH, Protirelin) in the rat. Kurume Med J. 1979;26(2):107-12.
Additional Infomation	Protirelin is a tripeptide composed of L-pyroglutamyl, L-histidyl and L-prolinamide residues joined in sequence. It has a role as a human metabolite. It is a peptide hormone and a tripeptide. Protirelin is the pharmaceutically available synthetic analogue of the endogenous peptide thyrotropin-releasing hormone (TRH). It is a tri-peptide tropic hormone, released by the hypothalamus, that stimulates the release of Thyroid Stimulating Hormone (TSH) and prolactin from the anterior pituitary. Although not currently available in any FDA-approved product, protirelin is a component of the TRH Test where it is used to test the response of the anterior pituitary gland in conditions such as secondary hypothyroidism and acromegaly. Protirelin has been reported in Homo sapiens with data available. A tripeptide that stimulates the release of THYROTROPIN and PROLACTIN. It is synthesized by the neurons in the PARAVENTRICULAR NUCLEUS of the HYPOTHALAMUS. After being released into the pituitary portal circulation, TRH (was called TRF) stimulates the release of TSH and PRL from the ANTERIOR PITUITARY GLAND. See also: Transferrin (annotation moved to). Protirelin (pQHP-NH₂) is the synthetic form of thyrotropin-releasing hormone (TRH), a tripeptide highly conserved across vertebrates. It is used as a reference ligand in comparative studies of TRH-like neuropeptide systems across species. In this study, it served as a positive control to demonstrate cross-species activation of TRH receptors. [1]

Solubility Data

Solubility (In Vitro)

DMSO : ~250 mg/mL (~689.88 mM)

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7595 mL	13.7977 mL	27.5953 mL
	5 mM	0.5519 mL	2.7595 mL	5.5191 mL
	10 mM	0.2760 mL	1.3798 mL	2.7595 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.