Β-subunit Of Chorionic Gonadotropin Research Articles

Multiple mechanisms of allosteric regulation of the luteninizing hormone receptor

The regulatory effects of luteinizing hormone (LH) and chorionic gonadotropin (CG) are realized through the activation of the G-protein coupled LH/CG receptor (LH/CG-R). The result of this is the activation of various types of G proteins, which leads to stimulation (Gs) or inhibition (Gi) of the cAMP-dependent pathway and stimulation of calcium signaling (Gq/11, Gi), and the recruitment of β-arrestins, which prevent G protein signaling through receptor internalization and downregulation, but can also activate the mitogen-activated protein kinase cascade. Despite a certain similarity in the effects of LH and CG, there are differences between them both in efficiency and in the pattern of regulation of LH/CG-R. This is a consequence of differences in the affinity of LH and CG to the orthosteric site of the receptor, as well as differences at the level of allosteric regulation of the receptor, which is due to the presence of a C-terminal extension in the β-subunit of CG, including sites for O-glycosylation, and the variability of N-glycosylation of α- and β-subunits of gonadotropins. Moreover, the number of N-glycans, the degree of their branching and charge differ, which leads to different efficiency of activation of intracellular cascades, affecting the physiological response of the reproductive system to gonadotropins. Of great importance is the formation of homodi(oligo)meric complexes of LH/CG-R and its heterocomplexes with the follicle-stimulating hormone receptor, where protomers allosterically influence the efficiency of LH/CG-R activation and the bias of signal transduction. Taking into account the large number of allosteric sites in LH/CG-R, the development of low-molecular allosteric regulators is underway, including agonists based on thieno[2,3-d]-pyrimidine and peptides derived from the cytoplasmic loops of LH/CG-R. These regulators can become prototypes of drugs for correcting the functions of the reproductive system. This review is devoted to the analysis of data on the similarities and differences in the signaling and physiological effects of gonadotropins with LH activity, the role of allosteric mechanisms in this, and the prospects for creating allosteric regulators of LH/CG-R.

Biomarkers of placental dysfunction. Clinical experience

The objective: to determine the diagnostic value of biomarkers of placental dysfunction at different stages of gestation.Materials and methods. The research was carried out in 2015–2022 on the basis of the municipal non-commercial enterprise “Maternity House No.5” of the Odesa City Council.We analyzed the course and clinical outcomes of pregnancies in 118 women (the main group) who developed and established placental dysfunction (PD) during pregnancy at different stages of gestation. The control group included 78 healthy women whose pregnancies occurred without PD.In women of both groups the frequency of detection of risk factors for PD was assessed by analyzing the content of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) biomarkers in blood serum, Pregnancy-Associated Plasma Protein A (PAPP-A) and β-subunit of chorionic gonadotropin (hCG).Fetal growth retardation was determined by deviations of fetal metric indicators (abdominal circumference, head circumference, biparietal size, thigh length, estimated fetal weight) below the 10th percentile.Statistical processing of the obtained results was carried out using Statistica 14.0 software (TIBCO, USA).Results. Among the examined women of both groups, cases where the pregnancy occurred at the age of 30 years or more and was the first pregnancy that did not end in abortion prevailed. The average age of women in the main group was 33.4±2.3 years, the control group – 29.3±1.4 years.The main group had more women with a history of multiple abortions (odds ratio (OR)=5.6, 95% confidence interval (CI): 1.9–18.8). Disorders of the menstrual cycle in history were found in 39 (33.1%) women of the main group and in 18 (23%) women of the control group (OR=1.6, 95% CI: 0.9–3.2). Concomitant gynecological diseases were represented by uterine fibroids – in 16 (13.6%) pregnant women of the main group and in 8 (10.2%) women of the control group (OR=1.4, 95% CI: 0.6–3.4), benign ovarian tumors – in 13 (11.0%); OR=20.1, 95% CI: 1.2–343.1), mastopathy – in 19 (16.1%; OR=30.8, 95% CI: 1.8–517.6) women of the main group.A significant number of women in the main group had a hypertrophic nutritional status – the body mass index (BMI) at the time of pregnancy was on average 26.7±0.9 kg/m2, women in the control group were represented by the normotrophic type (BMI – 22.2±0.3 kg/m2), in 4 women the indicator was more 30 kg/m2.In the vast majority of pregnant women (68 persons – 57.6%) of the main group the signs of placental malperfusion were registered up to 34 weeks. In 12 (10.2%) cases a primary PD was established.The content of PIGF in women of the main group at the 20th week of pregnancy decreased to 83±4 pg/ml, which is significantly less than the reference values (≥100 pg/ml). On the other hand, in the control group the PIGF concentration was 147±8 pg/ml (p<0.05). The sFlt-1 amount in the main group corresponded to the level of 3395±62 pg/ml. Therefore, the ratio of sFlt-1/PlGF was equal to 40.8±0.4, which is prognostically unfavorable.The PAPP-A indicator, according to monitoring data at the 20th week of pregnancy, was 0.77±0.08 mU/l. In pregnant women with lower values in the future PD occurred earlier. As for the indicators of the β-subunit of hCG, the levels of this hormone in most cases corresponded to the normative values, amounting to an average of 4.7±0.1 IU/l in a pregnancy with a male fetus and 8133±21 IU/l in a pregnancy with a female fetus.Signs of preeclampsia were determined in 22 (18.6%) pregnant women of the main group. The value of the sFlt-1/PlGF ratio in these pregnant women exceeded 50.0.Conclusions. In the vast majority of pregnant women of the main group (68 – 57.6%) the signs of placental malperfusion were found up to 34 weeks. In 22 (18.6%) pregnant women with placental dysfunction (PD) the signs of preeclampsia were detected, the development of which increases when the sFlt-1/PlGF ratio exceeds 50, starting from the II trimester of pregnancy, which is an early biomarker of PD.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG β-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115-149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture.

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) β-subunit, was attached between the β- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-wt was 32-34 and 34-36 kDa, respectively, and that of LH-M and FSH-M was 40-43 and 42-45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8-10 kDa. The EC50 value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.

A Two-Step Hysteroscopic Management for Cesarean Scar Pregnancy: A Proposal Method

Background: Cesarean Scar Pregnancy (CSP) is a cause of severe maternal morbidity. Currently, no guideline for its management is shared. We assessed safety and effectiveness of Methotrexate (MTX) administration within the sub-chorionic space under hysteroscopic guidance, followed by resectoscopic placental removal. Methods: Five patients suffering from type 2 CSP underwent a sequential treatment based on hysteroscopic techniques. Pregnancy termination was firstly obtained by injection of 80 mg of MTX within the intervillous spaces of placental site. The intervention was performed in an office setting using a 16Fr hysteroscope. MTX was administered by a 17-gauge needle suitable for the operative channel of hysteroscope. Subsequently, based on the decline of Human Chorionic Gonadotropin β-subunit (β-HCG), we timed a placental removal using a 27-Fr resectoscope, under conscious sedation. Results: In all women a diagnosis of CSP was achieved between 6 and 8 gestational age weeks. Hysteroscopic MTX administration resulted easily, quickly, painlessly and uneventfully in all patients. A substantial decrease of β-HCG was obtained in all patients within 15 days from the MTX administration. After a mean time of 27 days from MTX a resectoscopic removal of CSP was carried-out without any recorded adverse outcome. After 30 days from surgery β-HCG returned to non-pregnant level and normal physical findings were found in all patients. Conclusions: Hysteroscopy-guided MTX sub-chorionic administration resulted safe and effective for CSP termination. It was followed by successful and uneventful resectoscopic placenta removal in all patients. When hysteroscopy facilities are available, this combined therapy can be an option to treat CSP.

Biliary hCGβ Is a Potential Novel Marker for Prediction of Biliary Neoplasia in Primary Sclerosing Cholangitis Patients

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with an increased risk of cholangiocarcinoma (CCA). Novel markers, to complement or replace CA19-9, are urgently needed for the screening of PSC-associated biliary neoplasia. Previous studies have suggested that serum trypsinogen-2 and human chorionic gonadotropin β-subunit (hCGβ) may serve as such markers. Using highly specific in-house immunoassays, we studied trypsin(ogen)-2 and -3, SPINK1 and hCGβ in bile samples of 214 patients, referred for endoscopic retrograde cholangiography. We found that biliary trypsinogen-2 was decreased (p = 0.027) and hCGβ was elevated (p < 0.001) in PSC patients who were diagnosed 1.6 years (median, range 0.1–8.8 years) later with CCA or in whom biliary dysplasia was observed at least twice in brush cytology (n = 11) as compared to PSC patients without CCA or repeated dysplasia (n = 171). The other studied markers did not show significant differences between these groups. Our results warrant further evaluation of hCGβ as a predictive marker for PSC-associated biliary neoplasia.

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

ABSTRACT To identify the specific region of eCG involved in FSH-like activity, the following mutant expression vectors were constructed targeting the amino acid residues 102–104 of the eCG β-subunit: single mutants, eCGβV102G/α, eCGβF103P/α, and eCGβR104K/α; double mutants, eCGβV102G;F103P/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α; triple mutant, eCGβV102G;F103P;R104K/α. The LH-like and FSH-like activities of eCG mutants were examined in CHO-K1 cells expressing rat LH/CG receptor and rat FSH receptor. The levels of eCGβV102G/α, eCGβR104K/α, and eCGβV102G;R104K/α in the culture supernatant were markedly lower than those of eCGβ/α-wt. The other mutants and rec-eCGβ/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α were approximately 61%, 52%, and 54%, respectively, of those of eCG-wt. The Rmax values of the mutants were 58.9%–78.8% those of eCG-wt with eCGβR104K/α exhibiting the lowest value. The FSH-like activities of single mutants were only 16%–20% of those of eCG-wt. Additionally, the FSH-like activity of double mutants was less than 10% of that of eCG-wt. In particular, the FSH-like activities of βV102G;R104K/α and βF103P;R104K/α were 2.5–2.9% of that of eCG-wt. These results suggest that the amino acid residues 102–104 of the eCG β-subunit are dispensable and that the residue 104 of the eCG β-subunit plays a pivotal role in signal transduction through the rat FSH receptor. Thus, these mutants may aid future studies on eCG interactions with mammalian FSH receptors in vitro and in vivo.

Activin-inhibitory action on lactotrophs is decreased in lactotroph hyperplasia.

Among all the hormone-secreting pituitary tumours, prolactinomas are the most frequently found in the clinic. Since dopamine is the primary inhibitor of lactotroph function, dopamine agonists represent the first-line therapy. However, a subset of patients exhibits resistance to these drugs, and therefore, alternative treatments are desired. As activins inhibit prolactin gene expression through the inhibition of Pit-1 involving the p38MAPK pathway, in the present work, we studied the local activin system as an alternative inhibitory system for lactotroph hyperplasia treatment. We used two different mouse models of prolactinoma: transgenic mice with overexpression of the human chorionic gonadotropin β-subunit (hCGβ) and mice lacking dopamine receptor type 2. In both models, females, but not males, develop lactotroph hyperplasia from the fourth month of life. We found reduced expression of pituitary activin subunits and activin receptors in hyperplastic pituitaries from both models compared with wild-type counterparts. Consequently, hyperplastic pituitaries presented a reduced activin-inhibitory action on prolactin secretion. Additionally, while female wild-type lactotrophs presented high levels of phospho-p38MAPK, it was lost in prolactinomas, concomitant with decreased activin expression, increased Pit-1 expression and tumour development. In contrast, male pituitaries express higher mRNA levels of activin subunits βA and βB, which would suggest a stronger activin inhibitory function on lactotrophs, protecting this sex from tumour development, despite genotype. The present results highlight the importance of the activin inhibitory action on lactotroph function and place the local activin system as a new target for the treatment of dopamine agonist-resistant prolactinomas.

ИЗМЕНЕНИЕ ГОРМОНАЛЬНОГО СТАТУСА У ЖЕНЩИН С ГРИППОМ А(Н3N2) В ПЕРВОМ ТРИМЕСТРЕ БЕРЕМЕННОСТИ

The contents of β-subunit of chorionic gonadotropin (β-CG), estriol, progesterone, dehydroepiandrosterone (DEAS), placental lactogen and cortisol were studied in 164 women of child-bearing age at the 7-9th weeks of pregnancy. The first group (control) consisted of 30 women with physiologic course of pregnancy. The second group (comparison) had 32 patients with the threat of premature delivery of non-infectious etiology. The third group included 36 patients with influenza virus А(Н3N2) (anti-virus antibody titers were 1:4-1:16); the fourth group had 34 women with influenza virus А(Н3N2) (antibody titers were 1:8-1:32); in the fifth group there were 32 patients with influenza virus А(Н3N2) (antibody titers were 1:32-1:128) and the threat of premature delivery in the period of reconvalescence. The patients of the fifth group had the decrease of the level of β-CG till 40.5±2.0 mМЕ/ml, of estriol till 3.4±0.21 nmole/l, of progesterone till 35.1±3.13 nmole/l, of DEAS till 7.3±0.68 nmole/l, of placental lactogen till 12.1±1.0 nmole/l, as well as the increase of cortisol till 738.7±22.08 nmole/l in comparison with 73.0±2.0 mМЕ/ml (р<0.001), 5.9±0.33 nmole/l (р<0.001), 70.6±4.14 nmole/l (р<0.001); 7.3±0.68 nmole/l (р<0.001), 12.1±1.0 nmole/l (р<0.001) and 567.8±24.02 nmole/l (р<0.001) in the first group, respectively. The differences of hormonal status in the fifth and third group consisted of the drop of concentration of β-CG by 22.8% (р<0.001), of estriol by 26.1% (р<0.01), of progesterone by 32.1% (р<0.001) and placental lactogen by 32.4% (р<0.001). At the same time there were no significant changes in the concentration of DEAS and there were registered higher indicators of cortisol, i.e. the increase by 11.5% (р<0.05), which suggested the significance of the growth of antivirus antibody titers in the suppression of synthesis of hormones in ovaries and in syncytiotrophoblast as a result of direct and indirect negative influence of influenza infection.

Hyperprolactinemia induced by hCG leads to metabolic disturbances in female mice.

The metabolic syndrome is a growing epidemic; it increases the risk for diabetes, cardiovascular disease, fatty liver, and several cancers. Several reports have indicated a link between hormonal imbalances and insulin resistance or obesity. Transgenic (TG) female mice overexpressing the human chorionic gonadotropin β-subunit (hCGβ+ mice) exhibit constitutively elevated levels of hCG, increased production of testosterone, progesterone and prolactin, and obesity. The objective of this study was to investigate the influence of hCG hypersecretion on possible alterations in the glucose and lipid metabolism of adult TG females. We evaluated fasting serum insulin, glucose, and triglyceride levels in adult hCGβ+ females and conducted intraperitoneal glucose and insulin tolerance tests at different ages. TG female mice showed hyperinsulinemia, hypertriglyceridemia, and dyslipidemia, as well as glucose intolerance and insulin resistance at 6 months of age. A 1-week treatment with the dopamine agonist cabergoline applied on 5-week-old hCGβ+ mice, which corrected hyperprolactinemia, hyperandrogenism, and hyperprogesteronemia, effectively prevented the metabolic alterations. These data indicate a key role of the hyperprolactinemia-induced gonadal dysfunction in the metabolic disturbances of hCGβ+ female mice. The findings prompt further studies on the involvement of gonadotropins and prolactin on metabolic disorders and might pave the way for the development of new therapeutic strategies.

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in Pichia pastoris.

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris. Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC50 of 7μgml(-1), and 30% of cells were annexin V-positive after treatment with 20μg endo-CTP ml(-1) for 48h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague-Dawley rats was much longer than that of its commercial counterpart (Endostar). A long-acting endostatin can be produced using CTP technology.

Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG β-subunit

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) β-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity.

Stormy Encounter with Partial Hydatidiform Mole

We report a case of a 40-year-old multiparous woman who underwent total abdominal hysterectomy due to massive vaginal bleeding from partial molar pregnancy. Post-operatively, she developed high-grade fever, profuse sweating and shortness of breath. Examination revealed tachycardia, hypertension, elevated jugular venous pressure, crackles on both lower lung fields, with no palpable thyroid mass. Free thyroxine (FT4) and human chorionic gonadotropin β-subunit (β-hCG) were markedly elevated, while thyroid stimulating hormone (TSH) was significantly suppressed. With a Burch and Wartofsky score of 55, thyroid storm from the molar pregnancy was considered. She was given propylthiouracil (PTU), propranolol and hydrocortisone. Resolution of her signs and symptoms were noted 2 to 3 days following treatment.

Short-Term Pharmacological Suppression of the Hyperprolactinemia of Infertile hCG-Overproducing Female Mice Persistently Restores Their Fertility

Female infertility is often associated with deregulation of hormonal networks, and hyperprolactinemia is one of the most common endocrine disorders of the hypothalamic-pituitary axis affecting the reproductive functions. We have shown previously that transgenic female mice overexpressing human chorionic gonadotropin β-subunit (hCGβ+ mice), and producing elevated levels of bioactive LH/hCG, exhibit increased production of testosterone and progesterone, are overweight and infertile, and develop hyperprolactinemia associated with pituitary lactotrope adenomas in adult age. In the present study, we analyzed the influence of the hyperprolactinemia of hCGβ+ females on their reproductive phenotype by treating them with the dopamine agonists, bromocriptine and cabergoline. Long-term bromocriptine treatment of adult mice was effective in the control of obesity, pituitary growth, and disturbances in the hormone profile, demonstrating that hyperprolactinemia was the main cause of the hCGβ+ female phenotype. Interestingly, short-term treatment (1 wk) with cabergoline applied on 5-wk-old mice corrected hyperprolactinemia, hyperandrogenism, and hyperprogesteronemia, prevented pituitary overgrowth, normalized gonadal function, and recovered fertility of adult hCGβ+ females after hormone-induced and natural ovulation. The same cabergoline treatment in the short term applied on 3-month-old hCGβ+ females failed to recover their reproductive function. Hence, we demonstrated that the short-term cabergoline treatment applied at a critical early stage of the phenotype progression effectively prevented the hyperprolactinemia-associated reproductive dysfunction of hCG-overproducing females.

Conversion of TSH Heterodimer to a Single Polypeptide Chain Increases Bioactivity and Longevity

TSH is a dimeric glycoprotein hormone composed of a common α-subunit noncovalently linked to a hormone-specific β-subunit. Previously, the TSH heterodimer was successfully converted to an active single-chain hormone by genetically fusing α and β genes with [TSHβ- carboxyl-terminal peptide (CTP)-α] or without (TSHβ-α) the CTP of human chorionic gonadotropin β-subunit as a linker. In the present study, TSH variants were expressed in Chinese hamster ovarian cells. The results indicated that TSHβ-α single chain has the highest binding affinity to TSH receptor and the highest in vitro bioactivity. With regard to the in vivo bioactivity, all TSH variants increased the levels of T(4) in circulation after 2 and 4 h of treatment. However, the level of T(4) after treatment with TSH-wild type was significantly decreased after 6 and 8 h, compared with the levels after treatment with the other TSH variants. TSHβ-α and TSHβ-CTP-α single chains exhibited almost the same bioactivity after 8 h of treatment. Evaluating the half-life of TSH variants, TSHβ-CTP-α single chain revealed the longest half-life in circulation, whereas TSH-wild type exhibited the shortest serum half-life. These findings indicate that TSH single-chain variants with or without CTP as a linker may display conformational structures that increase binding affinity and serum half-life, thereby, suggesting novel attitudes for engineering and constructing superagonists of TSH, which may be used for treating different conditions of defected thyroid gland activity. Other prominent potential clinical use of these variants is in a diagnostic test for metastasis and recurrence of thyroid cancer.

Corifollitropin alfa for female infertility

Introduction: Follicle-stimulating hormone (FSH) is essential for the development of ovarian follicles. Urinary-derived and recombinant FSH (rFSH) preparations are widely used in infertility treatment but have to be administered daily to achieve steady-state serum levels. Corifollitropin alfa is a hybrid molecule with a prolonged half-life.Areas covered: The development and clinical testing of corifollitropin alfa, including the pharmacodynamics and kinetics, efficacy and drug safety. Searches were performed using the Medline database.Expert opinion: Corifollitropin alfa is composed of the FSH α-subunit and a hybrid of the FSH β-subunit and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β-subunit. The rationale of developing such a molecule was to reduce patient burden, by reducing the number of injections required to sustain multifollicular growth. Two strengths of corifollitropin are available (for patients ≤ 60 kg and > 60 kg). Compared with a daily dose of 200 IU of rFSH, 150 mcg of corifollitropin is equivalent in safety and pregnancy outcomes in women > 60 and < 90 kg using an antagonist protocol. Another RCT in women ≤ 60 kg also confirmed safety and efficacy of follicular stimulation (100 mcg of corifollitropin versus 150 IU of rFSH), but it was not powered to demonstrate equivalence in terms of pregnancy rates.

Designing a Long-Acting Human Growth Hormone (hGH) by Fusing the Carboxyl-Terminal Peptide of Human Chorionic Gonadotropin β-Subunit to the Coding Sequence of hGH

Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-beta-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.

Absorption, Distribution, Metabolism and Excretion of Corifollitropin Alfa, a Recombinant Hormone with a Sustained Follicle-Stimulating Activity

The follicle-stimulating hormone (FSH) analog corifollitropin alfa (Org 36286) is a potent FSH receptor agonist with an extended plasma half-life due to the fusion of the carboxy-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit and the human FSH β-subunit. The absorption, tissue distribution, metabolism and excretion of corifollitropin alfa were studied in rats following a single subcutaneous administration of [¹²⁵I]corifollitropin alfa. The biological activity of [¹²⁵I]corifollitropin alfa was confirmed by an in vitro FSH receptor transactivation assay. Radioactivity in blood, serum, tissues and excreta was determined by radiometry up to 168 h post dosage. A drug-specific distribution occurred mainly to ovaries and the renal system. The distribution was similar in albino and pigmented rats, ruling out effects of melanin binding on distribution. Metabolites were studied in urine and serum by SDS-PAGE. The maximum concentration of 12 h indicated a slow absorption and excretion. Radioactivity was mainly (86%) excreted via urine. 90% of the radioactivity in serum was identified as [¹²⁵I]corifollitropin alfa, whereas only 7–15% of the radioactivity in urine was identified as [¹²⁵I]corifollitropin alfa and its dissociation products, the α- and β-subunits (including its CTP part). The remainder of the radioactivity in either matrix represented low-molecular-weight compounds resulting from catabolism and deiodination. In conclusion, the metabolic fate of corifollitropin alfa strongly resembles that of endogenous glycoprotein hormones, which predominantly consists of kidney clearance and the urinary excretion of the intact protein in parallel to kidney catabolism.

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

BackgroundMucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.ResultsThe present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in N. benthamiana L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated in vitro by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed E. coli enterotoxin B subunit:H. sapiens mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing Y. enterocolitica UDP-GlcNAc 4-epimerase gene and C. elegans UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.ConclusionPlant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.