Potent Prolactin Research Articles

Spectrofluorimetric and stability-indicating thin layer chromatographic methods for determination of cabergoline, a prolactin inhibitor in pharmaceuticals

Simple, Economic, and selective spectrofluorimetric and stability-indicating thin layer chromatographic (TLC) with fluorescence detection methods were developed for the determination of Cabergoline, a potent prolactin inhibitor, and long-acting dopamine receptor agonist, in bulk drug and pharmaceutical dosage forms based on its native fluorescence. Method A was based on measuring the fluorescence intensity at 338 nm after excitation at 280 nm. The measured fluorescence was directly proportional to the concentration of the drug over the range of 50.0–450.0 ng/mL with a limit of detection of 14.4 and a limit of quantification of 43.7 ng/mL. The TLC method (method B) was employed on TLC silica gel 60 F254 aluminum sheets previously exposed to concentrated (30–34 %) hydrochloric acid vapor. Ethyl acetate: n-hexane: diethylamine system with a ratio of (10: 3: 1, v/v/v) developing system was used. The retention factor (Rf) of Cabergoline was 0.58 ± 0.03. Linearity was found to be in the range of 100.0–1500.0 ng/band. The LOD and LOQ were 25.4 and 76.9 ng/band, respectively. The methods were validated successfully according to ICH guidelines.

Abstract 3720: Blockade of the short-form of prolactin receptor induces FOXO3a/EIF-4EBP1-mediated cell death in uterine cancer

Abstract Purpose: Extra-pituitary prolactin (PRL) is involved in development and progression of uterine carcinoma. However, the underlying mechanisms of PRL/PRLR axis in uterine cancer are not well understood, and current means of targeting this axis are limited. Here, we used a potent PRL antagonist (G129R) to interfere with oncogenic activities of uterine cancer cells and further inhibit tumor growth. Experimental Design: We performed integrated analyses by comparing expression of PRLR isoforms in a broad panel of tumors and normal countparts using TCGA and GTex databases. Therapeutic effects of G129R treatment in uterine cancers were assessed in both orthotopic PTENwt and PTENmut models. Additionally, we carried out reverse phase protein assay (RPPA) at in vivo condition from the resulting tumors and at in vitro conditions to interrogate downstream targets and mechanism of action for G129R. Further functional analyses including cell viability, cell cycle and nascent protein synthesis were accessed in two types of uterine cancer cells under serum-depleted condition. Results: We found that PRLR_S1a, a short form of PRLR that is generated from alternative splicing during transcription, is expressed in human uterine cancers to a greater extent than other isoforms. Blocking activities of the PRL/PRLR_S1a axis with the antagonist G129R significantly reduced growth and progression of both orthotopic PTENwt and PTENmut uterine tumors. Data from reverse-phase protein arrays (RPPA) by in the resulting tumors showed that pathways involved in epithelial-mesenchymal transition such as PI3K/mTOR activities were significantly downregulated by G129R treatment. In addition, based on the RPPA data from the in vitro uterine cancer cells treated with G129R as monotherapy or in combination with PRL, we discovered a novel function of G129R in initiating cell death mediated by nuclear FOXO3a and inducing the G0/G1 arrest in both PTENwt and PTENmut uterine cancer cells. Furthermore, G129R decreased the nascent protein synthesis in these uterine cancer cells mediated by transcriptional suppressor EIF-4EBP1. Collectively, our findings reveal a new role of the PRLR_S1a isoform in uterine cancer and identify G129R as a potential therapeutic in uterine cancer. Citation Format: Yun-Fei Wen, Ying Wang, Shaolin Ma, Anca Chelari Raicu, Keith A. Baggerly, Anil K. Sood. Blockade of the short-form of prolactin receptor induces FOXO3a/EIF-4EBP1-mediated cell death in uterine cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3720.

Effects of the dopamine agonist cabergoline on the pulsatile and TRH-induced secretion of prolactin, LH, and testosterone in male beagle dogs

In the present study, the pulsatile serum profiles of prolactin, LH and testosterone were investigated in eight clinically healthy fertile male beagles of one to six years of age. Serum hormone concentrations were determined in blood samples collected at 15 min intervals over a period of 6 h before (control) and six days before the end of a four weeks treatment with the dopamine agonist cabergoline (5 μg kg −1 bodyweight/day). In addition, the effect of cabergoline administration was investigated on thyrotropin-releasing hormone (TRH)-induced changes in the serum concentrations of these hormones. In all eight dogs, the serum prolactin concentrations (mean 3.0 ± 0.3 ng ml −1) were on a relatively constant level not showing any pulsatility, while the secretion patterns of LH and testosterone were characterised by several hormone pulses. Cabergoline administration caused a minor but significant reduction of the mean prolactin concentration (2.9 ± 0.2 ng ml −1, p < 0.05) and did not affect the secretion of LH (mean 4.6 ± 1.3 ng ml −1 versus 4.4 ± 1.7 ng ml −1) or testosterone (2.5 ± 0.9 ng ml −1 versus 2.4 ± 1.2 ng ml −1). Under control conditions, a significant prolactin release was induced by intravenous TRH administration (before TRH: 3.8 ± 0.9 ng ml −1, 20 min after TRH: 9.1 ± 5.9 ng ml −1) demonstrating the role of TRH as potent prolactin releasing factor. This prolactin increase was almost completely suppressed under cabergoline medication (before TRH: 3.0 ± 0.2 ng ml −1, 20 min after TRH: 3.3 ± 0.5 ng ml −1). The concentrations of LH and testosterone were not affected by TRH administration. The results of these studies suggest that dopamine agonists mainly affect suprabasal secretion of prolactin in the dog.

Intra-arterial injection of prolactin-releasing peptide elevates prolactin gene expression and plasma prolactin levels in rainbow trout.

Prolactin-releasing peptide (PrRP), recently isolated from the brain of mammals and teleosts, is a strong candidate for being a stimulatory hormone of pituitary prolactin secretion. The present study examined whether or not PrRP is capable of inducing prolactin gene expression and elevating plasma prolactin levels in vivo in cannulated rainbow trout. Following a single intra-arterial injection of chum salmon PrRP (40 nmol kg(-1)) through a dorsal aorta catheter, plasma prolactin levels increased (P<0.05) rapidly (2 min and 30 min), and prolactin mRNA levels were elevated (P<0.05) in pituitaries sampled 8 h after the injection. In contrast, plasma levels of somatolactin were decreased (P<0.05) and growth hormone and somatolactin mRNA levels were not significantly affected by PrRP. Thus, PrRP appears to be a potent prolactin secretagogue as well as prolactin transcription inducer in vivo in the rainbow trout.

A Practical Synthesis of Cabergoline.

Cabergoline is an N-acylurea derived from 9,10-dihydrolysergic acid, which is a potent prolactin inhibitor. It is marketed by Pharmacia as Dostinex for the treatment of hyperprolactinemia and is currently under active development for the treatment of a variety of CNS disorders. In the existing process, the N-acylurea is formed by the reaction of an amide with a large excess of ethyl isocyanate at elevated temperatures. An improved process was developed that eliminates this hazardous reaction. The amide is reacted with phenyl chloroformate and then with ethylamine, which provides a mild and efficient means of forming the unsymmetrical N-acylurea.

A practical synthesis of cabergoline.

Cabergoline is an N-acylurea derived from 9,10-dihydrolysergic acid, which is a potent prolactin inhibitor. It is marketed by Pharmacia as Dostinex for the treatment of hyperprolactinemia and is currently under active development for the treatment of a variety of CNS disorders. In the existing process, the N-acylurea is formed by the reaction of an amide with a large excess of ethyl isocyanate at elevated temperatures. An improved process was developed that eliminates this hazardous reaction. The amide is reacted with phenyl chloroformate and then with ethylamine, which provides a mild and efficient means of forming the unsymmetrical N-acylurea.

Prolactin receptor antagonists.

Most prolactin (PRL) in the circulation is produced by the pituitary. However, a wide variety of traditional target tissues of the hormone have also been shown to produce their own prolactin. The amount produced per cell is low, but may well be sufficient for autocrine/paracrine activity. Although dopamine agonists allow one to study the target tissue effects of pituitary PRL, other agents, such as PRL receptor (PRLR) antagonists, are needed to analyze autocrine/paracrine loops. With PRLR antagonists, it should be possible to dissect out the role of extrapituitary prolactin in both the normal physiology, and the pathophysiology of various tissues. In tissues where the locally produced PRL may promote disease, such antagonists have the potential to be important therapeutics. This article briefly, but critically, reviews current understanding of PRL-receptor interactions and initial signaling, and describes the development of both growth hormone (GH) and PRL antagonists within that context. In the final section, results with a very potent PRL antagonist further one theme of the article, which is whether the simple receptor dimerization model explains all signal transduction following PRLR binding.

Periodic use of cabergoline, a prolactin inhibitor, for control of reproductive success in rats: Work in progress

Cabergoline is a potent prolactin inhibitor which stimulates dopaminergic receptors, acting as a prolactin-inhibiting-factor (PIF) agonist. Pairs of mature and fertile Wistar rats were fed 3 doses of cabergoline in an oily vehicle (treatment, n = 5 each), or the vehicle only (control, n = 10) as a top dressing on feed, given once or twice weekly for 9 wk, followed by a 6-wk recovery period. Reproductive success was evaluated by counting pregnancies resulting in delivery and pups born alive or dead over the 15-wk period. Groups of 5 pairs each responded to treatment with 100 or 150 μg cabergoline fed once weekly, or 50 μg twice weekly, with reduction or prevention of reproductive success. Pregnancies either did not go to term, or the pups were stillborn or died within 12 h of birth (P < 0.001). Treatment with 50 μg cabergoline once a week was only partially effective (P > 0.05). Rats in the control group experienced unimpaired reproductive success. No carry-over effects on pup survival into the recovery period were observed; reproductive success was restored within 2 to 3 wk. Treatment with cabergoline did not affect body weight, the estrus cycles or the mating frequency. No aversion to cabergoline was observed over time.

Underfeeding-induced suppression of mammary tumors: counteraction by estrogen and haloperidol.

The purpose of this study was to investigate the mechanism by which underfeeding induces regression of carcinogen-induced mammary tumors in the rat and to determine if tumor regression in underfed rats could be prevented on a chronic basis by maintaining elevated circulating levels of estrogen and/or prolactin (PRL) by treatment with estradiol benzoate (EB) and a dopamine receptor blocker, haloperidol (HAL). Female rats with 7,12-dimethylbenzanthracene-induced mammary tumors were fed ad libitum (full-fed), half-fed (HF), or half-fed and treated wtih EB (HF+EB), HAL (HF+HAL), or both (HF+EB+HAL) for 15 weeks. Tumor diameter, tumor number, and body weight were determined each week. At the end of the experiment, hypothalamic concentrations of catecholamines, indoleamines, and their metabolites were determined by high performance liquid chromatography. Tumor diameter, tumor number, and body weight increased progressively in the full-fed rats, but decreased significantly in the HF rats. Treatment of HF rats with EB, HAL, or both prevented tumor regression, but had no effect on body weight, which declined continuously. In the HF rats, there was an increase in the concentration of dopamine and a decrease in the concentration of serotonin in the hypothalamus, whereas treatment with HAL reversed these effects. EB had no effect on neurotransmitter concentrations in the HF rats, but treatment of HF+EB animals with HAL decreased the dopamine concentration. The changes in dopamine and serotonin observed in HF rats are known to inhibit PRL secretion, whereas HAL, which blocked these changes, is a well established stimulator of PRL secretion. Since the mammary tumors are dependent on PRL for development and growth, it is probable that the regression of these tumors in the HF rats was ultimately due to a decrease in PRL secretion, and the prevention of this regression in HF+HAL rats was ultimately due to an increase in PRL secretion. EB, a potent PRL stimulator, probably blocked tumor regression in HF+EB rats by increasing PRL secretion by a direct effect on the pituitary.

Differential Effects of Pituitary Stalk-Section on Posterior Pituitary and Hypothalamic Contents of Prolactin-Releasing Factor, Oxytocin, Dopamine and Beta-Endorphin

We have recently shown that the posterior pituitary (neurointermediate lobe) contains a potent prolactin (PRL)-releasing factor (PRF) which is distinct from known PRL secretagogues. Posterior pituitary PRF appears to be a small peptide of an unknown cellular origin. Using pituitary stalk-sectioned (SS) male rats, the objectives of this study were: (1) to determine if PRF is transported from the hypothalamus or is synthesized within the pituitary gland, and (2) to compare changes in PRF activity with alterations in the posterior pituitary content of beta-endorphin (beta-END), oxytocin (OXY), and dopamine (DA). One or two weeks following pituitary SS or sham surgery (SHAM), acid extracts of the posterior pituitary and medial basal hypothalamus (MBH) were analyzed for their hormone content. PRF activity was assessed by determining the stimulation of PRL secretion from perifused anterior pituitary cells, DA was measured by HPLC, and OXY and beta-END levels were determined by RIAs. OXY and DA concentrations in the posterior pituitary were reduced more than 95% at both 1 and 2 weeks after SS. PRF activity in the posterior pituitary was significantly reduced by 75 and 90%, 1 and 2 weeks after SS, respectively. In contrast, beta-END levels in the posterior pituitary at these times increased 20 and 60%, as compared to SHAM rats. Unlike the posterior pituitary, OXY levels in the MBH increased 123% 1 week following SS, and 1,260% at 2 weeks. These increases may reflect the accumulation of OXY-containing secretory vesicles in the severed nerves. DA concentrations in the MBH showed a biphasic pattern. DA levels were initially decreased by 70%, and then increased, but remained 30% below SHAM levels. The reason for these alterations in DA levels is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)

Potent in vivo and in vitro prolactin inhibiting activity of sauvagine, a frog skin peptide.

The effect of sauvagine (SAU), a frog skin peptide, on prolactin (PRL) levels was studied in vivo and in vitro. Subcutaneous administration of SAU (20 micrograms/kg) reduced plasma PRL levels in normal adult male rats and suppressed the suckling-induced rise of PRL in lactating rats even at doses of 1 and 5 micrograms/kg. Perfusion of isolated and dispersed rat pituitary cells in vitro with increasing doses of SAU (from 5 x 10(-10) to 1.7 x 10(-8)M) induced a significant dose-related decrease of PRL secretion in the eluate. These results indicate that SAU is a potent PRL inhibiting factor and that its action is exerted at the pituitary level. If SAU or a SAU-related peptide is present in the mammalian brain, it can be tentatively hypothesized that this peptide plays an important role in the control of PRL secretion.

Prolactin and contact sensitivity.

Hypophysectomized (Hypo-X) rats did not develop contact dermatitis in response to dinitrochlorobenzene (DNCB). Syngeneic pituitary grafts placed under the kidney capsule or daily treatment with prolactin restored the DNCB-reactivity of Hypo-X animals. Combined treatment with other pituitary hormones was ineffective. Treatment of normal rats with a potent prolactin antagonist drug, bromocriptine, was as effective in inhibiting contact sensitivity as was hypophysectomy. These results indicate the contact sensitivity is a prolactin dependent reaction.

Role of glucocorticoids and progesterone in the development of rough endoplasmic reticulum involved in casein biosynthesis

Hydrocortisone acetate injected into pseudopregnant rabbits induced casein synthesis and a parallel accumulation of casein mRNA. These effects were not accompanied by any enrichment of total RNA in the mammary cell. Hydrocortisone acetate did not favour the attachment of polysomes to endoplasmic reticulum. Casein mRNA concentration was enhanced in free and membrane-bound polysomes. After long treatments, the concentration of casein mRNA reached a plateau in membrane bound polysomes whereas it continued to be accumulated in free polysomes, suggesting that a substantial part of casein synthesis is then carried out by free polysomes. Progesterone injected with high doses of prolactin was unable to prevent the stimulatory action of prolactin on the synthesis of casein, the accumulation of casein mRNA and mammary gland growth, as judged by DNA content. By contrast, the increase in the total RNA content of mammary gland was still significantly reduced by progesterone. In addition, progesterone inhibited almost completely the formation of membrane-bound polysomes and the anchorage of casein mRNA to endoplasmic reticulum. From these data, it was concluded that the formation of the endoplasmic reticulum is not a prerequisite for the initiation of casein synthesis. Glucocorticoids do not play a major role in the formation of the endoplasmic reticulum and the Golai apparatus and in the binding of casein synthesizing polysomes to membranes. Progesteronne is capable of inhibiting preferentially and gradually the stimulation of cellular functions requiring the most potent prolactin stimulation.

Effect of CB154 on serum hormone level and lactogenesis in dairy cows.

During two weeks prepartum, one hundred mg of 2-Br-α-ergocryptine (CB154), a potent prolactin inhibitor, was subcutaneously administered to dairy cows 8 to 10 times. Simultaneous injections of an antiserum to bovine prolactin around parturition were also given to a CB154-administered cow. The serum prolactin (PRL) level decreased to less than 2ng/ml immediately after the commencement of CB154 injection, and the low level continued until calving. PRL surge near parturition which usually emerged in normal calving, completely disappeared. The low serum PRL level continued till at least two weeks after calving. No reduction of serum growth hormone and triiodothyronine levels was detected. Lactogenesis was more or less suppressed by CB154 administration. The mean milk production in three cows during a week postpartum was 58.5% of that of the previous lactation. It took 30 to 50 days to attain the milk yield of the previous lactation. In the cow administered with CB154 and an antiserum to bovine PRL simultaneously, the milk production reduced to 43.7% of that of the previous lactation. The milk production was not achieved to that of the previous lactation even on the 50th day after calving. The concentrations of total milk protein, IgG, and β-lactoglobulin in colostrum and milk from the cows treated with CB154 were significantly higher than those from the control cows. On the contrary, the concentration of a-lactalbumin in colostrum from the cows given CB154 was 40% of that the control cows (p<0.001). The concentrations of lactose and a-lactalbumin on the 6th day postpartum were also significantly lower than those of the control cows. These results indicate that the suppression of PRL secretion around parturition after CB154 treatment induces the reduction of syntheses of a-lactalbumin, lactose and the depression of milk production during lactogenesis.

Ergot alkaloids. Synthesis of 6-alkyl-8-ergolenes and 6-methyl-8-aminoergolines as potential prolactin inhibitors.

The synthesis of several N-6 derivatives of elymoclavine (3) and potential alkylating derivatives of 6-methyl-8-aminoergolines (12) is described. These compounds were screened for prolactin-inhibiting ability and 6-propyl-8-hydroxymethyl-8-ergolene (9) was found to be as active as the most potent prolactin inhibitors reported to date. The total synthesis of racemic methyl dihydrolysergate I (23), having a trans C, D ring fusion, from the tricyclic ketone 18 is also described.

Potent prolactin and growth hormone releasing activity of more analogues of Met-enkephalin.

MORPHINE stimulates prolactin and growth hormone secretion1,2, so the identification of brain peptides with opiate-like activity (methionine-enkephalin and leucine-enkephalin)3 raised the possibility that these peptides, beside their role as modulators of pain4, could also be involved in the control of neuro-endocrine functions. We did in fact find that Met-enkephalin administered intraventricularly led to a stimulation of prolactin and growth hormone release5–7. β-Endorphin (β-LPH61–91) was, however, found to be 500 to 2,000 times more potent than Met-enkephalin (β-LPH61–65) in stimulating the release of the pituitary hormones. Since both peptides show similar affinity for the opiate receptor when binding studies are performed at 0 °C in conditions of minimal enzymatic degradation8,9, the markedly different biological activities of the two peptides after intracerebral injection are likely to be due to a more rapid inactivation of Met-enkephalin. We show here that the two analogues resistant to enzymatic degradation, [D-Ala2]Met-enkephalin and [D-Ala2]Met-enkephalinamide can stimulate prolactin and growth hormone release after intraventricular injection at a level of potency which is 500 to 5,000 times higher than that of the natural Met-enkephalin molecule.

EFFECT OF PROLACTIN INHIBITORY AGENTS ON THE ECTOPIC ANTERIOR PITUITARY AND THE MAMMARY GLAND IN RATS

The purpose of the present study was to investigate the biological effectiveness of two highly potent prolactin (PRL) inhibitors, lisuride hydrogen maleate (LMH) and 2-Br-alpha-ergocryptine (CB-154), in the absence of hypothalamic factors acting directly at the level of the anterior pituitary. Hypophysectomized female rats bearing 4 transplanted pituitaries beneath the kidney capsules were treated with oestradiol benzoate (OeB) and progesterone (P) with or without simultaneous administration of LHM or CB-154 for 22 days in order stimulate or inhibit lobula-alveolar growth of the mammary glands. In addition to the investigation of the mammary glands by DNA determination and assessment of the histological pictures, the aim of this study was directed towards the influence of the substances tested at the level of the anterior pituitary remote from the hypothalmus. In this connection the changes in the different cells within the ectopic pituitaries as revealed by immunoenzyme-cytochemical studies were investigated. The results obtained support the classical view of a neuroendocrine regulation of mammary gland growth and the importance of oestrogens, P and PRL within this system. Both ergot derivatives LHM and CB-154 were able to antagonize the stimulatory effect of OeB combined with P on the mammary gland. With regard to the mechanism of action of LHM and CB-154 it is concluded that both substances act via a direct action on dopaminergic receptors within the ectopic anterior pituitary.

Prolactin response to N3im-methyl-thyrotropin releasing hormone in euthyroid subjects.

The N3im-methyl analogue of thyrotropin releasing hormone (methyl-TRH) was compared with ordinary TRH as a prolactin (PRL) releaser in 32 euthyroid volunteers (20 male and 12 female, ages 17-66 years). The mean PRL response to 100 mug of methyl-TRH was greater (P less than 0.025) than the PRL response to 500 mug of TRH at 10 min and at all sampling times from 30 to 240 min after administration of the releasing factors. The mean peak PRL (at 10 min), maximum deltaPRL, and integrated PRL response area were greater (P less than 0.025) after administration of methyl-TRH than after TRH. The PRL response to methyl-TRH was greater (P less than 0.005) for the 12 women than for the 20 men in this study. The mean baseline PRL was correlated with the peak PRL (r=0.74, P less than 0.01) and the maximum deltaPRL (r=0.58, P less than 0.01) after methyl-TRH. Following administration of methyl-TRH, the peak PRL was correlated with the peak TSH (r=0.43, P less than 0.05), the maximum deltaPRL was correlated with the maximum deltaTSH (r=0.43, P less than 0.05), and integrated PRL response area was correlated with the integrated TSH response area (r=0.44, P less than 0.05). Similar correlations between PRL and TSH responses were seen after giving TRH. Age was inversely correlated with baseline PRL (r=-0.55, P less than 0.01), with maximum deltaPRL (r=-0.64, P less than 0.01), and with the PRL response area (r=0.48, P less than 0.01) after administration of methyl-TRH or TRH. Methyl-TRH did not significantly alter serum levels of growth hormone (16 subjects) and luteinizing hormone and follicle stimulating hormone (14 subjects). The results of this study indicate that methyl-TRH is a more potent prolactin releaser than TRH. Like TRH, methyl-TRH has specificity in its effects on the pituitary, releasing only TSH and PRL in normal man.