RU2843868C1 - METHOD OF USING ALLOSTERIC THYROTROPHIC HORMONE RECEPTOR AGONIST, ETHYL-2-(4-(4-(5-AMINO-6-(TERT-BUTYLCARBAMOYL)-2-(METHYLTHIO)THIENO[2,3-d]-PYRIMIDIN-4-YL)PHENYL)-1H-1,2,3-TRIAZOL-1-YL) ACETATE, FOR COMPENSATION OF THYROID DEFICIENCY CAUSED BY DIABETES MELLITUS TYPE 2 - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, namely to a method for thyroid hormone recovery in rats with type 2 diabetes mellitus. Method for restoring thyroid hormone levels in rats with type 2 diabetes mellitus using a low-molecular allosteric direct thyrotrophic hormone receptor agonist – compound TPY3m, ethyl-2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, with its single or five-day intraperitoneal or oral administration, characterized in that said compound in type 2 diabetes mellitus is used to restore deficiency of thyroid hormones, causing an increase in the level of free thyroxine (FT4) in the blood with single intraperitoneal and oral administration on average by not less than 25% and 20%, respectively and during five-day intraperitoneal and oral administration is not less than 25% and 20%, respectively, increase in the level of total thyroxine (TT4) in the blood with a single intraperitoneal and oral administration on average of not less than 40% and 35%, respectively and during five-day intraperitoneal and oral administration is not less than 50% and 60%, respectively, increase in the level of free triiodothyronine (FT3) in the blood with a single intraperitoneal and oral administration on average of not less than 35% and 30%, respectively and during five-day intraperitoneal and oral administration is not less than 50% and 50%, respectively, increase in the level of total blood triiodothyronine (TT3) after a single intraperitoneal and oral administration on average of not less than 35% and 20%, respectively and during five-day intraperitoneal and oral administration is not less than 15% and 25%, respectively, and does not have an undesirable effect on thyroliberin-stimulated levels of thyroid hormones, the absence of significant changes, as well as on levels of thyrotrophic hormone (TSH) and testosterone in blood, absence of significant changes when using the compound TPY3m in a daily dose of 15 mg/kg for intraperitoneal administration and in a daily dose of 40 mg/kg for oral single or five-day administration.

EFFECT: invention enables normalizing the thyroid hormone levels decreased in type 2 diabetes mellitus.

1 cl, 7 dwg, 1 ex

Description

Field of technology

The invention relates to medicine, namely to pharmaceuticals, and in particular is intended to compensate for the deficiency of thyroid hormones in type 2 diabetes mellitus (T2DM).

State of the art

There is an article by the authors of the patent (Bakhtyukov AA, Derkach KV, Fokina EA, Sorokoumov VN, Zakharova IO, Bayunova LV, Shpakov AO Development of low-molecular-weight allosteric agonist of the thyroid-stimulating hormone receptor with thyroidogenic activity // Reports of the Russian Academy of Sciences. Life Sciences. 2022. Vol. 503. No. 1. Pp. 161-165. doi: 10.31857/S2686738922020032; English version - Bakhtyukov AA, Derkach KV, Fokina EA, Sorokoumov VN, Zakharova IO, Bayunova LV, Shpakov AO Development of low-molecular-weight allosteric agonist of thyroid-stimulating hormone receptor with thyroidogenic activity // Doklady Biochemistry and Biophysics. 2022. V. 503. No. 1. P. 67-70. doi: 10.1134/S1607672922020016. PMID: 35538280. WOS:000793157400004), which describes the synthesis and physicochemical characteristics of a new allosteric agonist of the thyroid stimulating hormone (TSH) receptor - compound TPY3m, ethyl 2-(4-(4-(5-amino-6-( tert- butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)- 1H -1,2,3-triazol-1-yl) acetate. It was found that TPY3m stimulates thyroxine production when administered intraperitoneally to healthy Wistar rats, and also stimulates the expression of thyroidogenesis genes in the FRTL-5 rat thyrocyte cell culture and in the rat thyroid gland (TG). It was also shown that the TPY3m compound does not reduce the expression of the TSH receptor gene, and when administered together with TSH (in FRTL-5 cells) or thyroliberin (in the rat TG), it even restores the expression of this gene, reduced under conditions of increased TSH concentration in the culture fluid (FRTL-5 cells, in vitro experiments) or in the blood (rats, in vivo experiments).

A disadvantage of the article is that TPY3m was used to stimulate thyroid hormone synthesis in healthy rats without signs of thyroid deficiency, and only with a single intraperitoneal injection. Also, the effect of TPY3m on the stimulating effect of thyroliberin on the production of thyroid hormones in rats with thyroid hormone deficiency was not studied. It should be noted that in hypothyroid rats with thyroxine and triiodothyronine deficiency, both the activity of the thyroid axis and the functioning of negative feedback in it are altered, which inevitably and significantly affects the pharmacological profile of the studied drug and its effect on thyroliberin-dependent functions of the thyroid axis. In addition, the article does not contain data on the possible effect of TPY3m on the level of testosterone in the blood of animals, since, due to the similar structural and functional organization of the TSH and luteinizing hormone (LH) receptors, there is a risk of activation of the LH receptor and steroidogenesis dependent on it by this compound.

There is an article by the authors of the patent (Derkach K.V., Sorokoumov V.N., Morina I.Yu., Kuznetsova V.S., Romanova I.V., Shpakov A.O. Regulatory effects of thienopyrimidine derivative TPY3m on thyroid status after its five-day oral and intraperitoneal administration to rats // Cell technologies in biology and medicine. 2024. No. 2. P. 116-120. doi: 10.47056/1814-3490-2024-2-116-120; English version Derkach K.V., Sorokoumov V.N., Morina I.Y., Kuznetsova V.S., Romanova I.V., Shpakov A.O. Regulatory Effects of 5-Day Oral and Intraperitoneal Administration of a Thienopyrimidine Derivative on the Thyroid Status in Rats // Bull Exp Biol Med. 2024. Sep 12. doi: 10.1007/s10517-024-06223-8. PMID: 39266923). The referenced article is taken as a prototype.

In this article, the activity of the TPY3m compound was studied after oral and intraperitoneal administration to rats for five days and it was shown that with both routes of administration, this compound causes an increase in thyroid hormone levels, but has a weak effect on the TSH level in the blood. At the same time, TPY3m does not have an inhibitory effect on the stimulation of thyroliberin production of thyroid hormones and does not have a damaging effect on thyroid tissue, which indicates the prospects of its use as an activator of the thyroid axis with long-term use. The disadvantage of this article, chosen as a prototype, is that TPY3m was used to stimulate the synthesis of thyroid hormones only in healthy rats without signs of thyroid deficiency, including that caused by type 2 diabetes, and the effect of TPY3m on the stimulating effect of thyroliberin was also studied only in healthy rats without signs of thyroid deficiency. As is known, the effects of thyroid system regulators, including those administered sequentially, differ significantly at normal and reduced levels of thyroid hormones. It is important that the lack of data on the effect of a drug with TSH receptor agonist activity on rats with thyroid hormone deficiency does not allow an adequate assessment of its therapeutic potential in hypothyroidism. The article lacks data on the possible effect of TPY3m on testosterone levels, which may change in the case of TPY3m exposure to the LH receptor, related to the TSH receptor.

In 2009, American scientists Susan Neumann and her colleagues (Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA) developed new compounds NCGC00168126-01, N-(4-(5-(3-(furan-2-ylmethyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide, and NCGC00165237-01, N-(4-(5-(3-benzyl-5-hydroxy-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyloxy)phenyl)acetamide, with direct TSH receptor agonist activity (Neumann S, Huang W, Titus S, Krause G, Kleinau G, Alberobello AT, Zheng W, Southall NT, Inglese J, Austin CP, Celi FS, Gavrilova O, Thomas CJ, Raaka BM, Gershengorn MC. Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice. Proc Natl Acad Sci U S A. 2009 Jul 28;106(30):12471-6. doi: 10.1073/pnas.0904506106. Epub 2009 Jul 10. PMID: 19592511; PMCID: PMC2708975). A detailed analysis of the biological activity of these compounds was given later in a review by these authors (Neumann S, Gershengorn MC. Small molecule TSHR agonists and antagonists. Ann Endocrinol (Paris). 2011 Apr;72(2):74-6. doi: 10.1016/j.ando.2011.03.002. Epub 2011 Apr 20. PMID: 21511239; PMCID: PMC3467701). When acting on cells with the expressed TSH receptor, NCGC00168126-01 dose-dependently increased the cAMP level with an EC50 value of 660 nM. Its effectiveness was comparable to TSH. NCGC00165237-01 had even higher in vitro activity and stimulated adenylate cyclase activity with an EC50 of 40 nM. NCGC00168126-01 and NCGC00165237-01 were selective for the TSH receptor and had little effect on the stimulation of adenylate cyclase by gonadotropins in cells expressing the LH receptor, which is structurally related to the TSH receptor. Incubation of human thyrocytes (24 h) with 30 μM NCGC00165237-01 increased thyroglobulin expression in them to the same extent as treatment with 18 nM TSH. When administered intraperitoneally and orally to mice, NCGC00165237-01 significantly increased total thyroxine levels and enhanced radioactive iodine uptake by rat thyroid thyrocytes.

The disadvantage of the article is that compounds NCGC00168126-01 and NCGC00165237-01 were not used to treat animals with thyroid hormone deficiency, which does not allow us to judge their prospects as pharmacological drugs for the treatment of hypothyroidism. The authors studied only a single administration of these compounds to rodents and did not study their effect on thyroliberin-stimulated production of thyroid hormones. At the same time, from the point of view of clinical application, a study of the course of drug administration is extremely important, since this allows us to evaluate the stability of the effect of the studied drugs over time and identify possible side effects of such therapy. Another disadvantage is the limited range of the study of the thyroid hormone pattern, as well as the lack of an assessment of the effect of compounds NCGC00168126-01 and NCGC00165237-01 on the concentration of endogenous TSH in the blood. Moreover, both compounds NCGC00168126-01 and NCGC00165237-01 are not derivatives of thieno[2,3-d]-pyrimidine, but belong to a different class of heterocyclic compounds, which fundamentally differs from the compound TPY3m we developed.

In 2020, American scientists Raul Latif and co-authors synthesized the compound MSq1 (2-(4-propylphenyl)-N-(4-pyridinylmethyl)-4-quinolinecarboxamide), which demonstrated the activity of a direct TSH receptor agonist, selective for G _q/11 -protein-specific signaling cascades through which TSH activates the phospholipase pathway and calcium signaling in target cells (Latif R, Morshed SA, Ma R, Tokat B, Mezei M, Davies TF. A Gq Biased Small Molecule Active at the TSH Receptor. Front Endocrinol (Lausanne). 2020 Jun 26;11:372. doi: 10.3389/fendo.2020.00372. PMID: 32676053; PMCID: PMC7333667). The authors showed that the compound MSq1, being an activator of the TSH receptor-coupled G _q/11 protein, demonstrates pronounced antiproliferative activity, including influencing the proliferative signals generated by TSH and stimulating autoantibodies to the TSH receptor, which can help achieve a significant therapeutic effect in modulating thyroid growth.

The disadvantage of the article is that the compound developed by the authors does not affect the production of thyroid hormones and, accordingly, is not able to affect the thyroid status, including hypothyroidism of any etiology. This is due to the lack of effect of compound MSq1 to influence the adenylate cyclase signaling pathway in target cells. Thus, based on the pharmacological profile, compound MSq1 cannot be used to correct hypothyroid conditions, including those caused by type 2 diabetes. Along with this, the authors did not study the use of compound MSq1 in vivo, since all their studies are limited to in vitro experiments with various thyrocyte cultures, which does not indicate in favor of the clinical relevance of the compound they developed. It should be noted that compound MSq1 is not a derivative of thieno[2,3-d]-pyrimidine, belonging to a different class of heterocyclic compounds, which differs from the compound TPY3m we developed.

In the article by Michael Allen and co-authors (Allen MD, Neumann S, Gershengorn MC. Small-molecule thyrotropin receptor agonist activates naturally occurring thyrotropin-insensitive mutants and reveals their distinct cyclic adenosine monophosphate signal persistence. Thyroid. 2011 Aug;21(8):907-12. doi: 10.1089/thy.2011.0025. Epub 2011 Jul 11. PMID: 21745101; PMCID: PMC3148121) it was established that the previously developed and described compound NCGC00161870, (S)-N-(4-((5-(3-benzyl-5-hydroxy-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyl)oxy)phenyl)acetamide hydrochloride (also designated as C2), currently manufactured by MedKoo Biosciences, Inc. (CAS# ML109 HCl), as a small molecule agonist of the TSH receptor, in the culture of HEK-EM293 cells expressing mutant forms of the TSH receptor (Cye ⁴¹ Ser or Leu ²⁵² Pro substitutions) increased the level of cAMP, while TSH was inactive.

The disadvantage of this article is that the authors did not study the effect of the compound NCGC00161870 on thyroid hormone levels in vivo, either after a single or long-term administration, and did not investigate its effect on thyroliberin-stimulated activity. They write about its potential effectiveness in subclinical hypothyroidism caused by inactivating mutations in the TSH receptor, which does not apply to hypothyroidism caused by type 2 diabetes and other metabolic disorders, but this assumption is not substantiated by the authors. In addition, the compound NCGC00161870 is not a thieno[2,3-d]-pyrimidine derivative, but belongs to a different class of heterocyclic compounds, which fundamentally differs from the compound TPY3m developed by us.

In 2016, it was shown that the enantiomers of compound NCGC00161870 (C2), (S)-N-(4-((5-(3-benzyl-5-hydroxy-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)-2-methoxybenzyl)oxy)phenyl)acetamide, are characterized by different abilities to stimulate the production of thyroid hormones, and the most active among them is the E2 enantiomer (Neumann S, Padia U, Cullen MJ, Eliseeva E, Nir EA, Place RF, Morgan SJ, Gershengorn MC. An Enantiomer of an Oral Small-Molecule TSH Receptor Agonist Exhibits Improved Pharmacologic Properties. Front Endocrinol (Lausanne). 2016 Jul 27;7:105. doi: 10.3389/fendo.2016.00105. PMID: 27512388; PMCID: PMC4961696). It was shown that five-day administration of E2 to mice resulted in a more pronounced increase in thyroxine levels compared to that caused by recombinant TSH, indicating the high potential of E2 as a thyroidogenic drug in vivo.

A shortcoming of this article is that the studies were performed on healthy rodents and the effect of compound E2, the enantiomeric form of NCGC00161870 (C2), on animals with thyroid hormone deficiency (hypothyroidism), including that caused by T2DM, was not studied. All experiments were limited to assessing the effect of E2 on basal thyroxine levels in healthy mice; its effect on the entire pattern of thyroid hormones and on the TSH level in the blood was not studied; the effect of the drug on thyroliberin-stimulated production of thyroid hormones was not assessed, which is important for assessing its effect on endogenous TSH-stimulated thyroidogenesis. In addition, compound E2, one of the enantiomers of NCGC00161870, is not a thieno[2,3-d]-pyrimidine derivative, belonging to a different class of heterocyclic compounds, which differs from the compound TPY3m developed by us.

In 2018, German and American scientists led by Mervin Gershengorn developed the compound NCGC00379308, N-(1-phenylethyl)-2-(1-piperazinyl)-4-quinazolinamine, also designated D3-βArr, with the activity of a positive allosteric modulator of the TSH receptor, which selectively increased the translocation of β1-arrestin to the TSH receptor, but did not affect the activity of G _s -protein- and G _q/11 -protein-mediated cascades, demonstrating specificity for β-arrestin signaling (Neumann S, Eliseeva E, Boutin A, Barnaeva E, Ferrer M, Southall N, Kim D, Hu X, Morgan SJ, Marugan JJ, Gershengorn MC. Discovery of a Positive Allosteric Modulator of the Thyrotropin Receptor: Potentiation of Thyrotropin-Mediated Preosteoblast Differentiation In Vitro. J Pharmacol Exp Ther. 2018 Jan;364(1):38-45. doi: 10.1124/jpet.117.244095. Epub 2017 Oct 31. PMID: 29089368; PMCID: PMC5729612). Compound NCGC00379308 potentiated the effects of TSH on osteoblast precursor growth and differentiation via β-arrestin mechanisms.

A disadvantage of this article is that the compound NCGC00379308 did not affect the levels of thyroid hormones, which is due to the lack of its effect on G _s proteins and the enzyme adenylate cyclase. The authors did not study the activity of the compound in vivo, which does not allow us to assess the clinical relevance of the created allosteric modulator of the TSH receptor. The data obtained by the authors indicate that the compound NCGC00379308 is not intended for the correction of thyroid hormone deficiency (hypothyroidism), including that caused by T2DM. Along with this, the compound NCGC00379308 is not a derivative of thieno[2,3-d]-pyrimidine, which differs from the compound TPY3m developed by us.

A paper by Marvin Gershengorn's group presents data on the ability of the LH receptor agonist compound Org41841, 5-amino-N-tert-butyl-4-(3-methoxyphenyl)-2-methylsulfanylthieno[2,3-d]pyrimidine-6-carboxamide, now manufactured by MedKoo Biosciences, Inc. (CAS# 301847-37-0), influence TSH receptor activity (Moore S, Jaeschke H, Kleinau G, Neumann S, Costanzi S, Jiang JK, Childress J, Raaka BM, Colson A, Paschke R, Krause G, Thomas CJ, Gershengorn MC. Evaluation of small-molecule modulators of the luteinizing hormone/choriogonadotropin and thyroid stimulating hormone receptors: structure-activity relationships and selective binding patterns. J Med Chem. 2006 Jun 29;49(13):3888-96. doi: 10.1021/jm060247s. Using cell cultures, a relatively weak agonist activity of this compound towards the TSH receptor was demonstrated (defined as a partial agonist).

The disadvantage of the article is that the compound Org41841 has not been studied in vivo, and that it activates the TSH receptor with low efficiency, but stimulates the LH receptor and its dependent steroidogenic cascades with high efficiency. This indicates the low specificity of Org41841 in relation to TSH-dependent cascades and the impossibility of its use for the correction of thyroid pathology, including hypothyroidism caused or associated with T2DM.

There are a significant number of articles, including those by the authors of the patent, which describe the development and biological activity of low-molecular compounds that are ligands of the allosteric sites of the TSH receptor, with the activity of inverse allosteric agonists, negative allosteric modulators or neutral allosteric antagonists of the TSH receptor:

1) Neumann S, Kleinau G, Costanzi S, Moore S, Jiang JK, Raaka BM, Thomas CJ, Krause G, Gershengorn MC. A low-molecular-weight antagonist for the human thyrotropin receptor with therapeutic potential for hyperthyroidism. Endocrinology. 2008 Dec;149(12):5945-50. doi: 10.1210/en.2008-0836. Epub 2008 Jul 31. PMID: 18669595; PMCID: PMC2613050.

2) Neumann S, Huang W, Eliseeva E, Titus S, Thomas CJ, Gershengorn MC. A small molecule inverse agonist for the human thyroid-stimulating hormone receptor. Endocrinology. 2010 Jul;151(7):3454-9. doi: 10.1210/en.2010-0199. Epub 2010 Apr 28. PMID: 20427476; PMCID: PMC2903937.

3) Neumann S, Eliseeva E, McCoy JG, Napolitano G, Giuliani C, Monaco F, Huang W, Gershengorn MC. A new small-molecule antagonist inhibits Graves' disease antibody activation of the TSH receptor. J Clin Endocrinol Metab. 2011 Feb;96(2):548-54. doi: 10.1210/jc.2010-1935. Epub 2010 Dec 1. PMID: 21123444; PMCID: PMC3048317.

4) Neumann S, Pope A, Geras-Raaka E, Raaka BM, Bahn RS, Gershengorn MC. A drug-like antagonist inhibits thyrotropin receptor-mediated stimulation of cAMP production in Graves' orbital fibroblasts. Thyroid. 2012 Aug;22(8):839-43. doi: 10.1089/thy.2011.0520. Epub 2012 Jul 11. PMID: 22784331; PMCID: PMC3407388.

5) Turcu AF, Kumar S, Neumann S, Coenen M, Iyer S, Chiriboga P, Gershengorn MC, Bahn RS. A small molecule antagonist inhibits thyrotropin receptor antibody-induced orbital fibroblast functions involved in the pathogenesis of Graves ophthalmopathy. J Clin Endocrinol Metab. 2013 May;98(5):2153-9. doi: 10.1210/jc.2013-1149. Epub 2013 Mar 12. PMID: 23482611; PMCID: PMC3644605.

6) Neumann S, Nir EA, Eliseeva E, Huang W, Marugan J, Xiao J, Dulcey AE, Gershengorn MC. A selective TSH receptor antagonist inhibits stimulation of thyroid function in female mice. Endocrinology. 2014 Jan;155(1):310-4. doi: 10.1210/en.2013-1835. Epub 2013 Dec 4. PMID: 24169564; PMCID: PMC3868809.

7) Marcinkowski P, Hoyer I, Specker E, Furkert J, Rutz C, Neuenschwander M, Sobottka S, Sun H, Nazare M, Berchner-Pfannschmidt U, von Kries JP, Eckstein A, Schülein R, Krause G. A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy. Thyroid. 2019 Jan;29(1):111-123. doi: 10.1089/thy.2018.0349. Epub 2018 Dec 15. PMID: 30351237.

8) Sarkar R, Bolel P, Kapoor A, Eliseeva E, Dulcey AE, Templin JS, Wang AQ, Xu X, Southall N, Klubo-Gwiezdzinska J, Neumann S, Marugan JJ, Gershengorn MC. An Orally Efficacious Thyrotropin Receptor Ligand Inhibits Growth and Metastatic Activity of Thyroid Cancers. J Clin Endocrinol Metab. 2024 Aug 13;109(9):2306-2316. doi: 10.1210/clinem/dgae114. PMID: 38421044; PMCID: PMC11318999

Articles by the authors of the patent

9) Derkach K.V., Bakhtyukov A.A., Sorokoumov V.N., Shpakov A.O. New functional antagonist of the thyroid-stimulating hormone receptor based on thieno[2,3-d]pyrimidine // Reports of the Russian Academy of Sciences. Life Sciences. 2020. Vol. 491. No. 1. P. 141-145. doi: 10.31857/S2686738920020080 (Derkach K.V., Bakhtyukov A.A., Sorokoumov V.N., Shpakov A.O. New Thieno-[2,3-d]-pyrimidine-based Functional Antagonist for the Receptor of Thyroid Stimulating Hormone // Doklady Biochemistry and Biophysics. 2020. V. 491. P. 77-80. doi: 10.1134/S1607672920020064.

10) Derkach K.V., Fokina E.A., Bakhtyukov A.A., Sorokoumov V.N., Stepochkina A.M., Zakharova I.O., Shpakov A.O. Study of biological activity of a new neutral thyroid-stimulating hormone receptor antagonist based on thieno[2,3-d]-pyrimidine // Bulletin of Exp. Biology and Medicine. 2021. Vol. 172. No. 12. P. 711-715. doi: 10.47056/0365-9615-2021-172-12-711-715 (Derkach K.V., Fokina E.A., Bakhtyukov A.A., Sorokoumov V.N., Stepochkina A.M., Zakharova I.O., Shpakov A.O. The Study of Biological Activity of a New Thieno[2,3-D]-Based Neutral Antagonist of Thyrotropin Receptor // Bull. Exp. Biol. Med. 2022. No. 6. P. 713-716. WOS:000789759100013).

9) Derkach K.V., Bakhtyukov A.A., Sorokoumov V.N., Lebedev I.A., Didenko E.A., Shpakov A.O. Low-molecular-weight inverse agonist of the thyrotropin receptor, active both upon intraperitoneal and oral administration // I.M. Sechenov Russian Physiological Journal. 2024. Vol. 110. No. 1. P. 108-121. https://doi.org/10.31857/S0869813924010078 (Derkach K.V., Bakhtyukov A.A., Sorokoumov V.N., Lebedev I.A., Didenko E.A., Shpakov A.O. Low Molecular Weight Thyrotropin Receptor Inverse Agonist is Active upon both Intraperitoneal and Oral Administration // J. Evol. Biochem. 2024. V. 60. No. 1. P. 295-305.

The disadvantage of the above studies is that, according to their pharmacological profile, all the compounds studied and described in the above articles are not direct (full or partial) agonists of the TSH receptor and belong to the pharmacological classes of inverse allosteric agonists, negative allosteric modulators or neutral allosteric antagonists of the TSH receptor. The drugs developed and described in these articles do not have the activity of stimulating the production of thyroid hormones (on the contrary, they reduce it), which makes it impossible and even unacceptable to use them to correct thyroid deficiency and treat various forms of hypothyroidism, including those caused by or associated with type 2 diabetes.

The objective of the claimed technical solution is to develop a method for treating hypothyroidism caused by type 2 diabetes mellitus using a low-molecular allosteric direct agonist of the thyroid-stimulating hormone receptor - the compound TPY3m, ethyl-2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, capable of normalizing the levels of thyroid hormones reduced in type 2 diabetes mellitus.

Disclosure of the essence of the technical solution

The technical solution to the above problem is solved by developing a method for using a low-molecular allosteric direct agonist of the thyroid-stimulating hormone receptor - the TPY3m compound, ethyl 2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, with its single or five-day intraperitoneal or oral administration to normalize the levels of thyroid hormones in rats with a deficiency of these hormones caused by long-term type 2 diabetes mellitus, and characterized in that the said compound in type 2 diabetes mellitus is used to restore the deficiency of thyroid hormones, causing an increase in the level of free thyroxine (FT4) in the blood with a single intraperitoneal and oral administration by an average of no less than by 25% and 20%, respectively, and with five-day intraperitoneal and oral administration by at least 25% and 20%, respectively, an increase in the level of total thyroxine (TT4) in the blood with a single intraperitoneal and oral administration on average by at least 40% and 35%, respectively, and with five-day intraperitoneal and oral administration by at least 50% and 60%, respectively, an increase in the level of free triiodothyronine (FT3) in the blood with a single intraperitoneal and oral administration on average by at least 35% and 30%, respectively, and with five-day intraperitoneal and oral administration by at least 50% and 50%, respectively, an increase in the level of total triiodothyronine (TT3) in the blood with a single intraperitoneal and oral administration on average by at least 35% and 20%, respectively, and with five-day intraperitoneal and oral administration by at least 15% and 25%, respectively, and does not have an undesirable effect on thyroliberin-stimulated levels of thyroid hormones (no significant changes), as well as on the levels of thyroid stimulating hormone (TSH) and testosterone in the blood (no significant changes), when using the TPY3m compound at a daily dose of 15 mg / kg for intraperitoneal and at a daily dose of 40 mg / kg for oral administration.

Brief list of drawings

Fig. 1 - shows the effect of a single treatment of male rats with type 2 diabetes and thyroid hormone deficiency (hypothyroidism) with TPY3m (i.p., 15 mg/kg; orally 40 mg/kg), thyroliberin (intranasally, 100 IU/rat) and sequentially TPY3m and thyroliberin at the same doses on the level of free thyroxine in the blood.

K - control rats; SD - rats with T2DM without treatment with drugs; SD+TRH - rats with T2DM that were treated once with thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(i/p) - rats with T2DM that were treated once with intraperitoneally administered TPY3m (15 mg/kg); SD+TPY3m(i/p)+TRH - rats with T2DM that were treated sequentially with TPY3m (i/p, 15 mg/kg) and thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(per) - rats with T2DM that were treated once with orally administered TPY3m (40 mg/kg); DM+TPY3m(per)+TRH - rats with DM2, which were treated sequentially with TPY3m (orally, 40 mg/kg) and thyroliberin (intranasally, 100 IU/rat). Differences with the control ( ^a ) and diabetic ( ^b ) groups are statistically significant at p <0.05. Data are presented as M±SEM , in all groups n = 8. The level of free thyroxine was determined using the ELISA kit from Immunotech (Russia). Blood was taken from the tail vein of rats under local anesthesia 3.5 hours after the administration of TPY3m.

Fig. 2 - shows the effect of a single treatment of male rats with type 2 diabetes and thyroid hormone deficiency (hypothyroidism) with TPY3m (i.p., 15 mg/kg; orally 40 mg/kg), thyroliberin (intranasally, 100 IU/rat) and sequentially TPY3m and thyroliberin at the same doses on the level of total thyroxine in the blood.

K - control rats; SD - rats with T2DM without treatment with drugs; SD+TRH - rats with T2DM that were treated once with thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(i/p) - rats with T2DM that were treated once with intraperitoneally administered TPY3m (15 mg/kg); SD+TPY3m(i/p)+TRH - rats with T2DM that were treated sequentially with TPY3m (i/p, 15 mg/kg) and thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(per) - rats with T2DM that were treated once with orally administered TPY3m (40 mg/kg); DM+TPY3m(per)+TRH - rats with DM2, which were treated sequentially with TPY3m (orally, 40 mg/kg) and thyroliberin (intranasally, 100 IU/rat). Differences with the control ( ^a ) and diabetic ( ^b ) groups, as well as between the DM+TRH and DM+TPY3m(per)+TRH ( ^c ) groups are statistically significant at p < 0.05. Data are presented as M ± SEM , in all groups n = 8. The level of total thyroxine was determined using the ELISA kit from Immunotech (Russia). Blood was taken from the tail vein of rats under local anesthesia 3.5 hours after the administration of TPY3m.

Fig. 3 - shows the effect of a single treatment of male rats with type 2 diabetes and thyroid hormone deficiency (hypothyroidism) with TPY3m (i.p., 15 mg/kg; orally 40 mg/kg), thyroliberin (intranasally, 100 IU/rat) and sequentially TPY3m and thyroliberin at the same doses on the level of free triiodothyronine in the blood.

K - control rats; SD - rats with T2DM without treatment with drugs; SD+TRH - rats with T2DM that were treated once with thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(i/p) - rats with T2DM that were treated once with intraperitoneally administered TPY3m (15 mg/kg); SD+TPY3m(i/p)+TRH - rats with T2DM that were treated sequentially with TPY3m (i/p, 15 mg/kg) and thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(per) - rats with T2DM that were treated once with orally administered TPY3m (40 mg/kg); DM+TPY3m(per)+TRH - rats with DM2, which were treated sequentially with TPY3m (orally, 40 mg/kg) and thyroliberin (intranasally, 100 IU/rat). Differences with the control ( ^a ) and diabetic ( ^b ) groups are statistically significant at p <0.05. Data are presented as M±SEM , in all groups n = 8. The level of free triiodothyronine was determined using the ELISA kit from Immunotech (Russia). Blood was taken from the tail vein of rats under local anesthesia 3.5 hours after the administration of TPY3m.

Fig. 4 - shows the effect of a single treatment of male rats with type 2 diabetes and thyroid hormone deficiency (hypothyroidism) with TPY3m (i.p., 15 mg/kg; orally 40 mg/kg), thyroliberin (intranasally, 100 IU/rat) and sequentially TPY3m and thyroliberin at the same doses on the level of total triiodothyronine in the blood.

K - control rats; SD - rats with T2DM without treatment with drugs; SD+TRH - rats with T2DM that were treated once with thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(i/p) - rats with T2DM that were treated once with intraperitoneally administered TPY3m (15 mg/kg); SD+TPY3m(i/p)+TRH - rats with T2DM that were treated sequentially with TPY3m (i/p, 15 mg/kg) and thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(per) - rats with T2DM that were treated once with orally administered TPY3m (40 mg/kg); DM+TPY3m(per)+TRH - rats with DM2, which were treated sequentially with TPY3m (orally, 40 mg/kg) and thyroliberin (intranasally, 100 IU/rat). Differences with the control ( ^a ) and diabetic ( ^b ) groups, as well as between the DM+TRH and DM+TPY3m(per)+TRH ( ^c ) groups are statistically significant at p < 0.05. Data are presented as M ± SEM , in all groups n = 8. The level of total triiodothyronine was determined using the ELISA kit from Immunotech (Russia). Blood was taken from the tail vein of rats under local anesthesia 3.5 hours after the administration of TPY3m.

Fig. 5 - shows the effect of a single treatment of male rats with type 2 diabetes and thyroid hormone deficiency (hypothyroidism) with TPY3m (i.p., 15 mg/kg; orally 40 mg/kg), thyroliberin (intranasally, 100 IU/rat) and sequentially TPY3m and thyroliberin at the same doses on the level of thyroid stimulating hormone (TSH) in the blood.

K - control rats; SD - rats with T2DM without treatment with drugs; SD+TRH - rats with T2DM that were treated once with thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(i/p) - rats with T2DM that were treated once with intraperitoneally administered TPY3m (15 mg/kg); SD+TPY3m(i/p)+TRH - rats with T2DM that were treated sequentially with TPY3m (i/p, 15 mg/kg) and thyroliberin (intranasally, 100 IU/rat); SD+TPY3m(per) - rats with T2DM that were treated once with orally administered TPY3m (40 mg/kg); DM+TPY3m(per)+TRH - rats with DM2, which were treated sequentially with TPY3m (orally, 40 mg/kg) and thyroliberin (intranasally, 100 IU/rat). Differences with the control ( ^a ) and diabetic ( ^b ) groups are statistically significant at p <0.05. Data are presented as M±SEM , in all groups n =8. TSH level was determined using the Rat Thyroid Stimulating Hormone ELISA kit (Cusabio Biotech Co., Ltd., China). Blood was collected from the tail vein of rats under local anesthesia 3.5 hours after TPY3m administration.

Fig. 6 - shows the effect of a single treatment of male rats with type 2 diabetes and thyroid hormone deficiency (hypothyroidism) with TPY3m (i.p., 15 mg/kg; orally 40 mg/kg), thyroliberin (intranasally, 100 IU/rat) and sequentially TPY3m and thyroliberin at the same doses on the blood testosterone level.

K - control rats; SD - rats with T2DM without treatment with drugs; SD+TPY3m(i/p) - rats with T2DM that were treated once with intraperitoneal TPY3m (15 mg/kg); SD+TPY3m(per) - rats with T2DM that were treated once with orally administered TPY3m (40 mg/kg). Data are presented as M±SEM , in all groups n = 8. There were no statistically significant differences between testosterone levels in the study groups. Testosterone levels in the blood were determined using an ELISA kit from Alkor-Bio (Russia). Blood was taken from the tail vein of rats under local anesthesia 3.5 hours after TPY3m administration.

Fig. 7 - shows the effect of a five-day treatment of male rats with T2DM and thyroid hormone deficiency (hypothyroidism) with TPY3m TPY3m (i.p., 15 mg/kg/day; orally 40 mg/kg/day) on the levels of thyroid hormones (FT4, TT4, FT3, TT3), TSH and testosterone in the blood.

K(5) - control rats, SD(5) - rats with T2DM without treatment with drugs, SD+TPY3m(i/p)(5) - rats with T2DM treated for 5 days with intraperitoneal TPY3m (15 mg/kg); SD+TPY3m(per)(5) - rats with T2DM treated for 5 days with orally administered TPY3m (40 mg/kg). Differences with the control ( ^a ) and diabetic ( ^b ) groups are statistically significant at p <0.05. Data are presented as M±SEM , in all groups n = 6. The level of thyroid hormones in the blood was determined using ELISA kits from Immunotech (Russia), the concentration of TSH in the blood was determined using the ELISA kit “Rat Thyroid Stimulating Hormone ELISA kit” (“Cusabio Biotech Co., Ltd.”, China), the level of testosterone in the blood was determined using the ELISA kit from Alkor-Bio (Russia). Blood was taken from the tail vein of rats under local anesthesia 3.5 hours after the last (on the 5th day) administration of TPY3m.

Implementation of a technical solution

In the technical solution, the following concepts are understood under the terms used:

TPY3m - ethyl 2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, a derivative of the heterocyclic compound thieno[2,3-d]-pyrimidine, a low-molecular allosteric agonist of the thyroid-stimulating hormone (TSH) receptor developed by us, which stimulates the activity of the TSH receptor and signaling cascades dependent on it, including those carried out through the G _s protein and the enzyme adenylate cyclase. It is effective as a stimulator of thyroid hormone production, including in case of deficiency (hypothyroidism).

Type 2 diabetes mellitus (T2DM) (insulin-resistant or non-insulin-dependent diabetes) is a chronic endocrine disease in which the body cannot effectively use insulin produced in sufficient quantities by the beta cells of the pancreas, which is caused by the development of insulin resistance; the main symptom of T2DM is hyperglycemia, it is often associated with obesity, dyslipidemia and arterial hypertension.

An agonist is a chemical compound (ligand) that, upon specific interaction with a receptor molecule, changes its functional state, transferring it to an active conformation and, thereby, causing a biological response of the target cell to the action of the agonist.

An allosteric agonist is a chemical compound (allosteric ligand) that interacts with the allosteric site of a hormone receptor, causing its activation in the absence of an endogenous ligand (hormone) interacting with the high-affinity orthosteric site.

An allosteric site of a receptor is a site of the receptor that does not coincide with the high-affinity orthosteric site, the target of an endogenous ligand (hormone), the binding of which by the ligand (allosteric regulator) affects the stability and equilibrium of the active conformations of the receptor, both bound to the orthosteric agonist and free of it.

The hypothalamic-pituitary-thyroid axis (GGT axis) is an endocrine complex that includes the hypothalamus, pituitary gland, and thyroid gland. It regulates metabolic processes, response to stress, thermogenesis (including sudden temperature fluctuations), and is in close interaction with other components of the endocrine system. As an effector hormone, it includes triiodothyronine, which is formed from thyroxine by its conversion by deiodinases.

Hypothyroidism is a clinical syndrome that develops due to long-term, persistent deficiency of thyroid hormones in the body or the development of resistance to TSH at the tissue level.

Insulin resistance (IR) is a decrease in the sensitivity of target tissues to insulin, which is a characteristic feature of type 2 diabetes, and is assessed using the IR index, calculated as the product of insulin and glucose concentrations in the blood with the appropriate conversion factor.

Intraperitoneal glucose tolerance test (IGTT) is a laboratory test for the diagnosis of impaired glucose tolerance (prediabetes, metabolic syndrome) and type 2 diabetes, which includes a glucose load (intraperitoneal administration of a glucose solution) followed by monitoring of blood glucose levels (in rats, usually for 120 min).

Levothyroxine is a drug (sodium salt of L-thyroxine) used in the treatment of hypothyroid conditions as a replacement therapy; after absorption and partial metabolism in the liver and kidneys, it has a regulatory effect on the development and growth of tissues and metabolism.

Luteinizing hormone (LH) is a glycoprotein hormone produced by the gonadotrophs of the adenohypophysis after their stimulation by gonadotropin-releasing hormone; when acting on reproductive tissues, it stimulates the synthesis and secretion of sex steroid hormones (androgens, estrogens, and progesterone). In men, LH acts on the Leydig cells of the testes, activating the synthesis of testosterone in them and controlling spermatogenesis; in women, it acts on the follicular cells of the ovaries and the corpus luteum, stimulating ovulation and activating the synthesis of estrogens and progesterone in the follicle cells.

Impaired glucose tolerance is a condition characterized by normal fasting glucose levels, its absence in urine, but elevated blood glucose levels during a glucose load or consumption of high-carbohydrate foods (assessed in an oral glucose tolerance test or IGTT).

Testosterone is the main male sex steroid hormone (androgen class), synthesized from cholesterol in the Leydig cells of the testes under the influence of LH produced by the adenohypophysis; it is responsible for the development of primary and secondary sexual characteristics in men, regulates spermatogenesis, and is involved in the control of metabolic and anabolic processes.

Thyroid hormones are iodinated derivatives of the amino acid tyrosine - thyroxine (tetraiodothyronine) and triiodothyronine, which have similar physiological properties and are produced by thyrocytes (follicular cells) of the thyroid gland from thyroglobulin under the influence of TSH produced by the adenohypophysis. In the blood, thyroid hormones exist in free and plasma protein-bound forms, according to which free (FT4) and total (TT4) thyroxine and free (FT3) and total (TT3) triiodothyronine are distinguished.

Thyroid-stimulating hormone (TSH) is a glycoprotein hormone produced by the thyrotrophs of the adenohypophysis after their stimulation by thyroliberin, it acts on specific receptors located on the surface of the epithelial cells of the thyroid gland, stimulating their production of thyroxine and its conversion to triiodothyronine.

Thyroliberin is a hypothalamic tripeptide, a TSH-releasing hormone, which causes increased secretion of TSH (and, to a lesser extent, prolactin) by the thyrotrophs of the anterior pituitary gland and is involved in the control of some mental functions.

It is known that when studying the effect of the intraperitoneally and orally administered compound TPY3m, ethyl 2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, a low molecular weight allosteric agonist of the thyroid stimulating hormone (TSH) receptor, on the levels of thyroid hormones - thyroxine and triiodothyronine, including those stimulated by thyroliberin, as well as on the levels of TSH and testosterone in the blood of male rats with T2DM induced by a high-fat diet (duration of 16 weeks) and streptozotocin injection (15 mg / kg body weight, intraperitoneally, 10 weeks after the start of a high-fat diet), the single and 5-day administration of the drug, as well as the following daily doses: for intraperitoneal administration of TPY3m - 15 mg/kg, for oral administration of TPY3m - 40 mg/kg.

The TPY3m compound was synthesized by the authors of the patent using their own original method, as described previously (Bakhtyukov AA, Derkach KV, Fokina EA, Sorokoumov VN, Zakharova IO, Bayunova LV, Shpakov AO Development of a low-molecular allosteric agonist of the thyroid-stimulating hormone receptor with thyroidogenic activity // Reports of the Russian Academy of Sciences. Life Sciences. 2022. Vol. 503. No. 1. Pp. 161-165. doi: 10.31857/S2686738922020032; English version - Bakhtyukov AA, Derkach KV, Fokina EA, Sorokoumov VN, Zakharova IO, Bayunova LV, Shpakov AO Development of low-molecular-weight allosteric agonist of thyroid-stimulating hormone receptor with thyroidogenic activity // Doklady Biochemistry and Biophysics. 2022. V. 503. No. 1. P. 67-70. doi: 10.1134/S1607672922020016. PMID: 35538280. WOS:000793157400004), and characterized by physicochemical parameters confirming its chemical structure. Spectrum ¹ H-NMR (400 MHz, chloroform -d ): δ 8.09 - 8.03 (m, 3H Ar phenyl + Ar triazol), 7.76 (d, J = 7.9 Hz, 2H Ar phenyl), 5.32 (s, 2H CArC H ₂ C(O)), 5.26 (m, 3H NH ₂ + NH), 4.34 (q, J = 7.1 Hz, 2H CH ₂ C(O)O Et ), 2.69 (s, 3H MeS), 1.48 (s, 9H tBu), 1.36 (t, J = 7.2 Hz, 3H CH ₃ C(O)O Et ). Mass spectrum (ESI+, 100 V, CH ₃ OH): found - 526.1695 [M+H]; calculated for C ₂₄ H ₂₈ N ₇ O ₃ S ₂ ⁺ - 526.1690.

Thyroliberin (Sigma, USA) was administered intranasally, in saline, at a dose of 100 μg/rat, as described by us previously (Derkach KV, Bogush IV, Berstein LM, Shpakov AO. The Influence of Intranasal Insulin on Hypothalamic-Pituitary-Thyroid Axis in Normal and Diabetic Rats. Horm Metab Res. 2015 Nov;47(12):916-24. doi: 10.1055/s-0035-1547236. Epub 2015 Mar 6. PMID: 25750079). TPY3m was dissolved in DMSO and administered intraperitoneally in a volume of 200 μl DMSO and orally (through a gastric tube) in the same volume; control and intact diabetic rats were given their solvents instead of TPY3m and thyroliberin at the same times and in the same volume. In single-dose experiments, 8 rats were used per group (male Wistar rats), in five-day experiments - 6 rats per group. TPY3m (or its solvent DMSO) was administered at 10:00 and blood was collected under local anesthesia 3.5 hours after drug administration. In the thyroliberin-treated groups, TSH-releasing factor was administered 30 min after TPY3m (or DMSO) to ensure preliminary binding of TPY3m to the allosteric site of the TSH receptor in thyrocytes, even before the onset of thyroliberin-stimulated TSH secretion. The development of T2DM was assessed by metabolic and hormonal markers of this disease, such as increased body weight, basal and postprandial hyperglycemia, hyperinsulinemia, increased insulin resistance index, dyslipidemia (hypertriglyceridemia), selecting animals with developed disease for experiments.

A single intraperitoneal (15 mg/kg) and oral (40 mg/kg) administration of TPY3m resulted in restoration of decreased levels of thyroid hormones - free (FT4) and total (TT4) thyroxine and free (FT3) and total (TT3) triiodothyronine - in the blood of male rats with type 2 diabetes, indicating normalization of the thyroid status in diabetic animals with hypothyroidism. At the same time, a single intraperitoneal or oral administration of TPY3m did not have an undesirable effect on thyroliberin-stimulated levels of thyroid hormones and on the basal TSH level in the blood of animals, which indicates the absence of its inhibitory effect on the hypothalamic and pituitary links of the thyroid axis and demonstrates the preservation of TSH receptor activation by the endogenous hormone under the stimulating effect of TPY3m. Single intraperitoneal or oral administration of TPY3m also had no adverse effect on blood testosterone levels, indicating the absence of a significant effect of TPY3m on the LH receptor and the selectivity of its stimulating effect on the TSH receptor.

Five-day administration of TPY3m, both intraperitoneally (15 mg/kg) and orally (40 mg/kg), resulted in restoration of the decreased levels of thyroid hormones in the blood of male rats with type 2 diabetes - FT4, TT4, FT3 and TT3, which indicates normalization of the thyroid status in rats with type 2 diabetes and hypothyroidism. At the same time, with long-term (five-day) administration of TPY3m also did not have any undesirable effects on the levels of TSH and testosterone in the blood of animals.

Conclusion. Single and five-day administration of TPY3m, ethyl 2-(4-(4-(5-amino-6-( tert -butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)- 1H -1,2,3-triazol-1-yl) acetate, a low molecular weight allosteric agonist of the TSH receptor, both intraperitoneally (15 mg/kg) and orally (40 mg/kg), resulted in a clearly expressed restoration of significantly reduced levels of thyroid hormones - free (FT4) and total (TT4) thyroxine and free (FT3) and total (TT3) triiodothyronine - in the blood of male rats with T2DM, indicating normalization of the thyroid status in diabetic animals with hypothyroidism, without having an undesirable effect on thyroliberin-stimulated production of thyroid hormones, TSH and testosterone levels.

As an example of a technical solution, data are presented on the effect of TPY3m upon single and five-day administration, intraperitoneally at a dose of 15 mg/kg and orally at a daily dose of 40 mg/kg, to male rats with hypothyroidism (thyroid hormone deficiency) caused by T2DM induced by a long-term high-fat diet and a low dose of streptozotocin, on the basal and thyroliberin-stimulated levels of thyroid hormones, as well as on the level of TSH and testosterone in the blood of animals.

To induce T2DM in rats, a 16-week high-fat diet was used, which included daily consumption by animals of 5-7 g of a mixture enriched with saturated fats and their single treatment with streptozotocin at a dose of 15 mg/kg (intraperitoneally, 10 weeks after the start of the diet). The development of T2DM was assessed by metabolic and hormonal markers of this disease, including an increase in body weight, the development of basal and postprandial hyperglycemia, hyperinsulinemia, an increased insulin resistance index, and hypertriglyceridemia.

In case of single administration of TPY3m, control and six diabetic groups of male Wistar rats (8 animals each) were randomly formed: (1) control rats (group C); (2) rats with T2DM without drug treatment (group DM); (3) rats with T2DM, which were treated once with thyroliberin (intranasally, 100 IU/rat) (group DM+TRH); (4) rats with T2DM, which were treated once with intraperitoneal TPY3m (15 mg/kg) (group DM+TPY3m(ip)); (5) rats with T2DM, which were treated sequentially with TPY3m (ip, 15 mg/kg) and thyroliberin (intranasally, 100 IU/rat) (group DM+TPY3m(ip)+TRH). (6) T2DM rats treated with a single oral dose of TPY3m (40 mg/kg) (T2DM+TPY3m(per) group); (7) T2DM rats treated sequentially with TPY3m (orally, 40 mg/kg) and thyroliberin (intranasally, 100 IU/rat) (T2DM+TPY3m(per)+TRH group).

In the case of a five-day treatment of male Wistar rats with hypothyroidism induced by T2DM, four groups (6 animals in each) were formed: (1) control rats, which received TPY3m in its vehicle DMSO instead of TPY3m (group K(5)); (2) rats with T2DM, which received TPY3m in its vehicle DMSO instead of TPY3m (group DM(5)); (3) rats with T2DM, which were treated for five days with intraperitoneally administered TPY3m (15 mg/kg) (group DM+TPY3m(ip)(5)); (4) rats with T2DM, which were treated for five days with orally administered TPY3m (40 mg/kg) (group DM+TPY3m(per)(5)).

In diabetic rats, all forms of thyroid hormones - FT4, TT4, FT3 and TT3 - were decreased in the blood without changing the TSH level, which indicates the development of hypothyroidism in them. A single treatment of animals with the compound TPY3m, ethyl 2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, a low molecular weight allosteric agonist of the TSH receptor, both intraperitoneally (15 mg/kg) and orally (40 mg/kg), led to the restoration of thyroid hormone levels (Figs. 1-4) and did not have an undesirable effect on the TSH level (Fig. 5). In this case, the levels of triiodothyronine, FT3 and TT3, the main effector hormone of the thyroid system, were restored to the greatest extent, and did not differ from those in the control group (Figs. 3, 4). This indicates the effectiveness of TPY3m with both routes of administration for restoring the thyroid status in hypothyroidism caused by type 2 diabetes. When administered together with TPY3m, the stimulating effect of thyroliberin on the production of thyroid hormones was preserved (Figs. 1-4), and the thyroliberin-stimulated TSH level did not change significantly (Fig. 5), which indicates the absence of competition between TPY3m and TSH for binding to the TSH receptor and the absence of an inhibitory effect of TPY3m on the hypothalamic and pituitary links of the thyroid axis. Interestingly, oral administration of TPY3m slightly but significantly increased thyrotropin-stimulated TT4 and TT3 production (Figs. 2, 4). This provides additional evidence that it does not negatively affect the ability of endogenous TSH, the secretion of which is stimulated by thyrotropin, to activate thyroidogenesis in the thyroid gland of diabetic rats with hypothyroidism. Importantly, the effect of TPY3m was specific for the TSH receptor, since it did not affect testosterone levels regulated through the LH receptor related to the TSH receptor (Fig. 6).

Five-day administration of TPY3m to diabetic rats with hypothyroidism by both routes of delivery, intraperitoneal and oral, also resulted in restoration of the levels of thyroid hormones - FT4, TT4, FT3 and TT3 without significant changes in the TSH level (Fig. 7). The only exception was the TT3 level in the DM+TPY3m(i.p.)(5) group, which did not differ significantly from that in the DM(5) group, but did not differ from that in the control group either, indicating its restoration under conditions of long-term TPY3m therapy (Fig. 7). As with a single administration, five-day administration of TPY3m, both intraperitoneal and oral, did not cause changes in the testosterone level compared to the DM(5) group (Fig. 7), indicating the selectivity of TSH receptor activation by its allosteric agonist TPY3m.

The claimed method for restoring thyroid hormone levels in rats with type 2 diabetes mellitus using a low molecular weight allosteric direct agonist of the thyroid stimulating hormone receptor - the TPY3m compound, ethyl 2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, with its single or five-day intraperitoneal or oral administration, where the said compound in type 2 diabetes mellitus is used to restore thyroid hormone deficiency, using the TPY3m compound at a daily dose of 15 mg/kg for intraperitoneal and at a daily dose of 40 mg/kg for oral single or five-day administration, causing

an increase in the level of free thyroxine (FT4) in the blood with a single intraperitoneal and oral administration on average by at least 25% and 20%, respectively, and with five-day intraperitoneal and oral administration by at least 25% and 20%, which is confirmed by the data presented in Fig. 1,

an increase in the level of total thyroxine (TT4) in the blood with a single intraperitoneal and oral administration on average by at least 40% and 35%, respectively, and with five-day intraperitoneal and oral administration by at least 50% and 60%, respectively, which is confirmed by the data presented in Fig. 2,

an increase in the level of free triiodothyronine (FT3) in the blood with a single intraperitoneal and oral administration on average by at least 35% and 30%, respectively, and with five-day intraperitoneal and oral administration by at least 50% and 50%, respectively, which is confirmed by the data presented in Fig. 3,

an increase in the level of total triiodothyronine (TT3) in the blood with a single intraperitoneal and oral administration by an average of at least 35% and 20%, respectively, and with a five-day intraperitoneal and oral administration by at least 15% and 25%, respectively, which is confirmed by the data presented in Fig. 4.

Claims (7)

A method for restoring thyroid hormone levels in rats with type 2 diabetes mellitus using a low molecular weight allosteric direct agonist of the thyroid stimulating hormone receptor, the TPY3m compound, ethyl 2-(4-(4-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]-pyrimidin-4-yl)phenyl)-1H-1,2,3-triazol-1-yl) acetate, by single or five-day intraperitoneal or oral administration, characterized in that said compound is used in type 2 diabetes mellitus to restore thyroid hormone deficiency, causing an increase in the level of free thyroxine (FT4) in the blood with a single intraperitoneal and oral administration by an average of at least 25% and 20%, respectively, and with five-day intraperitoneal and oral administration by at least 25% and 20%, respectively, an increase in the level of total thyroxine (TT4) in the blood with a single intraperitoneal and oral administration by an average of at least 40% and 35%, respectively, and with five-day intraperitoneal and oral administration by at least 50% and 60%, respectively, an increase in the level of free triiodothyronine (FT3) in the blood with a single intraperitoneal and oral administration by an average of at least 35% and 30%, respectively, and with five-day intraperitoneal and oral administration by at least 50% and 50%, respectively, an increase in the level of total triiodothyronine (TT3) in the blood with a single intraperitoneal and oral administration by an average of at least 35% and 20%, respectively, and with five-day intraperitoneal and oral administration by at least 15% and 25%, respectively, and does not have an adverse effect on thyroliberin-stimulated thyroid hormone levels, no significant changes, as well as on the levels of thyroid stimulating hormone (TSH) and testosterone in the blood, no significant changes, when using the TPY3m compound at a daily dose of 15 mg/kg for intraperitoneal administration and at a daily dose of 40 mg/kg for oral single or five-day administration.