Gonadotropins, including human chorionic gonadotropin (hCG), are used to induce ovulation, but they have a number of side effects, including ovarian hyperstimulation syndrome (OHSS). A possible alternative is allosteric luteinizing hormone (LH)/hCG receptor agonists, including the compound TP4/2 we developed, which remains active when administered orally. The aim was to study the effectiveness of TP4/2 (orally, 40 mg/kg) as an ovulation inducer in FSH-stimulated immature female rats, compared with hCG (s.c., 15 IU/rat). TP4/2 stimulated progesterone production and corpus luteum formation; time-dependently increased the ovarian expression of steroidogenic genes (Star, Cyp11a1, Cyp17a1) and genes involved in ovulation regulation (Adamts-1, Cox-2, Egr-1, Mt-1); and increased the content of metalloproteinase ADAMTS-1 in the ovaries. These effects were similar to those of hCG, although in some cases they were less pronounced. TP4/2, in contrast to hCG, maintained normal LH levels and increased the ovarian expression of the LH/hCG receptor gene, indicating preservation of ovarian sensitivity to LH, and did not cause a sustained increase in expression of vascular endothelial growth factor-A involved in OHSS. Thus, TP4/2 is an effective ovulation inducer that, unlike hCG, has a lower risk of OHSS and ovarian LH resistance due to its moderate stimulating effect on steroidogenesis.

The approaches to correct thyroid deficiency include replacement therapy with thyroid hormones (THs), but such therapy causes a number of side effects. A possible alternative is thyroid-stimulating hormone (TSH) receptor activators, including allosteric agonists. The aim of this work was to study the effect of 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 (TPY3m), a TSH receptor allosteric agonist developed by us, on basal and thyroliberin (TRH)-stimulated TH levels and the hypothalamic-pituitary-thyroid (HPT) axis in male rats with high-fat diet/low-dose streptozotocin-induced type 2 diabetes mellitus (T2DM). Single and three-day administration of TPY3m (i.p., 20 mg/kg) was studied, and the effect of TPY3m on the HPT axis was compared with that of levothyroxine. TPY3m increased TH levels when administered to both healthy and diabetic rats, normalizing thyroxine and triiodothyronine levels in T2DM and, unlike levothyroxine, without negatively affecting TSH levels or the expression of hypothalamic and pituitary genes responsible for TSH production. TPY3m pretreatment preserved the stimulatory effects of TRH on TH levels and thyroid gene expression. This indicates the absence of competition between TPY3m and endogenous TSH for TSH receptor activation and is supported by our in vitro results on TPY3m- and TSH-stimulated adenylate cyclase activity in rat thyroid membranes. Morphological analysis of thyroid glands in diabetic rats after three-day TPY3m administration shows an increase in its functional activity without destructive changes. To summarize, TPY3m, with the activity of a partial allosteric agonist of the TSH receptor, was created as a prototype of drugs to correct thyroid insufficiency in T2DM.

Polycystic ovary syndrome (PCOS) is associated with impaired ovarian steroidogenesis and ovulation, which necessitates the development of effective ovulation inducers for PCOS. The aim of the study was to evaluate the effects of allosteric luteinizing hormone receptor agonist TP03 and human chorionic gonadotropin (hCG) on ovarian steroidogenesis, as well as ovulation in prepubertal female rats with dehydroepiandrosterone(DHEA)-induced PCOS. Taking into account differences in progesterone levels, cohorts with high (PCOS(H)) and low (PCOS(L)) progesterone were formed and treated with Follimag and Cetrotide. After 48 h, TP03 (25 mg/kg) or hCG (25 IU/rat) were injected, and hormone levels, gene expression, and ovarian morphology were assessed. The PCOS(H)-cohort exhibited irregular estrous cycles, ovarian cysts, and increased ovarian mass and estradiol levels, but the number of corpora lutea (CL) was maintained. In the PCOS(L)-cohort, ovarian weight was increased, and Star, Cyp11a1, and Adamts1 gene expression as well as the CL number were decreased. In both cohorts, TP03 and hCG increased progesterone levels and the expression of steroidogenesis (Star, Cyp11a1) and ovulation (Cox2, Adamts1, Egr1) genes, as well as inducing CL formation. Thus, TP03, like hCG, stimulates steroidogenesis and ovulation in PCOS-rats with different progesterone levels, which provides the first evidence of the effectiveness of allosteric LHR agonists as ovulation triggers in PCOS.

BACKGROUND: Rodent models of polycystic ovary syndrome, including those induced by dehydroepiandrosterone, are widely used to study the pathogenesis of this disorder; however, ovarian steroidogenesis and ovulation markers in this model remain insufficiently characterized. There are no data on the ability of luteinizing hormone receptor agonists to stimulate ovulation in rats with this model of polycystic ovary syndrome, which is important for assisted reproductive technologies. AIM: This study aimed to investigate ovarian steroidogenesis, ovulation markers, and ovarian morphology in infantile female rats with dehydroepiandrosterone-induced polycystic ovary syndrome, and to assess the effects of the allosteric agonist TP03, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide, on these parameters. METHODS: Female Wistar rats, starting at 23–26 days of age, were treated with dehydroepiandrosterone for 6 weeks (6 mg/100 g/day, subcutaneously). The following parameters were assessed: body and ovarian weight, serum levels of testosterone, progesterone, and estradiol (enzyme-linked immunosorbent assay), ovarian gene expression (reverse-transcription polymerase chain reaction), and ovarian morphology (number of tertiary and preovulatory follicles, corpora lutea, and follicular cysts) by microscopy. Rats with polycystic ovary syndrome were stimulated with an ovulation-inducing drug at a dose of 15 IU/rat, followed by TP03 at a dose of 25 mg/kg 48 h later; the studied parameters were evaluated 16 h later. RESULTS: In dehydroepiandrosterone-induced rats, ovarian weight, the number of tertiary follicles and corpora lutea of previous cycles, progesterone levels, and the expression of genes responsible for steroidogenesis (Star, Cyp11a1, Cyp17a1) and ovulation (Adamts-1, Vegf-a) were decreased, whereas testosterone levels were increased and follicular cysts were detected in the ovaries, consistent with the pathogenesis of polycystic ovary syndrome. Treatment with ovulation-inducing drugs increased ovarian weight, progesterone levels, and Cyp17a1 gene expression, and stimulated ovulation, leading to the formation of corpora lutea. Luteinized unruptured follicles were also detected in the ovaries; this finding was associated with decreased expression of the ADAMTS-1 metalloproteinase and VEGF-A genes involved in follicular rupture. CONCLUSION: Treatment of infantile rats with dehydroepiandrosterone-induced polycystic ovary syndrome using TP03 induces ovulation; however, along with normal corpora lutea, luteinized unruptured follicles are formed as a result of decreased expression of genes involved in follicular rupture.

Gonadotropins are widely used clinically for controlled ovulation induction, but they have a number of serious side effects, including ovarian hyperstimulation. Low-molecular-weight allosteric agonists of the luteinizing hormone (LH) receptor, including our newly developed compound 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido) phenyl)thieno[2,3-d]-pyrimidine-6-carboxamide (TP03), may be an alternative. However, their pharmacokinetics and tissue distribution remain largely unstudied.    Aim of the study was to investigate the dynamics of changes in TP03 blood levels during its use to induce ovulation in immature female rats stimulated with follimag, as well as to evaluate TP03 distribution in the ovaries and liver of the animals.    Material and methods.  Immature female rats stimulated with follimag were administered with TP03 (15 mg/kg, intraperitoneally). Blood estradiol and progesterone levels, as well as the number of preovulatory follicles and corpora lutea in the ovaries, were then assessed over 24 hours. TP03 levels in plasma, ovaries, and liver were measured using reversed-phase HPLC with tandem mass spectrometry.    Results.  TP03 increased blood progesterone content (peaking at 8 hours) and resulted in corpora lutea formation after 16–24 hours. TP03 was detectable in plasma for 24 hours, reaching its maximum concentration (Cmax, 3530 ng/ml) 0.25 hours (Tmax) after administration, indicating rapid absorption of the compound. The half-life (t1/2) of TP03 was 3.11 hours, and the mean residence time (MRT) in the body was 4.29 hours. TP03 accumulation in the ovaries and liver was demonstrated as early as 1 hour after administration.    Conclusions.  TP03 stimulates the LH receptor, increases progesterone production, and induces ovulation in immature female rats. Its high effectiveness as an ovulation inducer is largely due to its rapid entry into the bloodstream and effective absorption in the ovaries.