Behavioral effects of intracerebral administration of luteinizing hormone releasing hormone (LHRH) in rats

The effects of LHRH intracerebrally infused on acquisition of conditioned avoidance responses (CARs) and spontaneous motility were studied in adult male rats. The results were the following: 1) LHRH (1 and 2.5 micrograms/rat) administered through a cannula stereotaxically implanted into the lateral ventricle induced an impairment in the acquisition of CARs along with an increase in global motility, rearing, head shaking and grooming behavior; 2) LHRH 1 microgram/rat injected into the hippocampus or nucleus accumbens induced also an impairment in acquisition which is evident 15 min after treatment. In contrast, intrastriatal injection induced an immediate disruption of this behavior; and 3) there is a good dose-response relationship for intrastriatal LHRH between 7.8 and 62.5 ng/rat. The results suggest that the estriatum could be the locus of the LHRH-induced inhibition of CARs. Then the possibility of an involvement of the dopamine nigrostriatal system is discussed.     

Characterization of the degradation products of luteinizing hormone releasing hormone

The degradation products of luteinizing hormone releasing hormone [LH/RH; 1; gonadorelin releasing hormone (GnRH); less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2] were determined in aqueous solution (pH 6.5) at 25, 37, 50, and 80 degrees C. The predominant route of degradation involved the cleavage of the less than Glu-His and Trp-Ser peptide bonds to give peptides 5-9 and hydrolysis of the terminal Gly-NH2 to the free acid form in peptides 4 and 10. Racemization of the serine and histidine residues to give peptides 2 and 3 was a second route of degradation.     

Pharmacological evidence of catecholaminergic involvement in the behavioral effects of luteinizing hormone releasing hormone in rats

The influence of L-DOPA on the behavioral effects of LHRH was studied in male rats. Subcutaneous administration of LHRH (100 micrograms/kg) caused a significant disruption in the acquisition of a conditioned avoidance response (CAR) and a significant increase in head shaking behavior (HSB). Pretreatment with this hormone antagonized the stimulatory action of amphetamine (1 mg/kg, IP) in acquisition of CARs, spontaneous motor activity (SMA) and rearing behavior (RB). L-DOPA (100 mg/kg, IP), administered after LHRH, stimulated SMA, RB and HSB. In addition L-DOPA antagonized the effect of LHRH on acquisition of CARs and counteracted the antagonism between LHRH and amphetamine in acquisition of CARs and SMA. These findings indicate that LHRH could exert its behavioral effects through an inhibitory action upon brain catecholamine synthesis. The suppression of CARs may be the response to DA antagonism and the interaction with amphetamine could be mediated by an inhibition of both DA and NE activities. The possibility of an interaction between LHRH and central serotonin mechanisms is also discussed.     

Influence of luteinizing hormone releasing hormone (LHRH) on the behavioral effects of amphetamine in rats

The influence of luteinizing hormone releasing hormone (LHRH) on the behavioral effects induced by several doses of D-amphetamine (0.25, 0.5, 1.0 and 2.0 mg/kg IP) was studied. A dose response relation was previously established for the effects of LHRH (50, 100 and 200 micrograms/kg SC) on acquisition and retention of conditioned avoidance responses (CARs). The neuropeptide impaired acquisition and improved retention of CARs, without modifying spontaneous motor activity. Pretreatment with 100 micrograms/kg of LHRH antagonizes the enhancement in acquisition of CARs due to D-amphetamine 0.5, 1.0 and 2.0 mg/kg, the impairment in retention induced by amphetamine 1.0 and 2.0 mg/kg, and the hypermotility and the increased rearing behavior induced by amphetamine 1.0 and 2.0 mg/kg. These results suggest that brain catecholamines, particularly dopamine, could play a role in the behavioral effects of LHRH. Interactions between LHRH and central dopaminergic mechanisms are discussed.     

Stability of carthamin fromCarthamus tinctorius in aqueous solution: pH and temperature effects

Thermal stability of a red pigment, carthamin, fromCarthamus tinctorius was investigated to explore possible applications as natural color additives for foods, cosmetics, and nutraceuticals. Degree of degradation reactions of carthamin at acidic, neutral and alkaline conditions were determined with UV/Vis spectral measurements. Decomposition half lives of carthamin at 25°C were 4.0 h, 5.1 h, and 12.5 h at pH 5.0, pH 7.0, and pH 12.0, respectively, indicating that carthamin is much more stable at alkaline pH than acidic or neutral conditions. The activation energies of carthamin at pH 5.0, pH 7.0, and pH 12.0 were 15.6, 15.7 and 16.8 kcal/mol, respectively.     

Oral absorption studies of lipid-polylysine conjugates of thyrotropin releasing hormone (TRH1) and luteinizing hormone releasing hormone (LHRH1)

The lipoamino acids and their oligomers provide an excellent means of enhancing peptide lipophilicity and also helping to increase the stability of the peptide and protect it from enzymatic degradation. Thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) were extended on the N-terminal with one and two lipoamino acids and labelled with the ³H-acetyl group. TRH and LHRH conjugates were also prepared where the compounds were extended with two lipoamino acids, a polylysine unit and the N-terminal labelled with the ³H-acetyl group. The higher lipophilicity resulted in a higher Caco-2 cell association and also a higher rate of oral uptake. The addition of the polylysine system increased the water solubility, as well as the oral uptake of the conjugates. The conjugates developed have been absorbed and detected after oral administration and appear to be stable for a considerable time in vivo.     

Oral absorption studies of lipid-polylysine conjugates of thyrotropin releasing hormone (TRH1) and luteinizing hormone releasing hormone (LHRH1)

The lipoamino acids and their oligomers provide an excellent means of enhancing peptide lipophilicity and also helping to increase the stability of the peptide and protect it from enzymatic degradation. Thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) were extended on the N-terminal with one and two lipoamino acids and labelled with the ³H-acetyl group. TRH and LHRH conjugates were also prepared where the compounds were extended with two lipoamino acids, a polylysine unit and the N-terminal labelled with the ³H-acetyl group. The higher lipophilicity resulted in a higher Caco-2 cell association and also a higher rate of oral uptake. The addition of the polylysine system increased the water solubility, as well as the oral uptake of the conjugates. The conjugates developed have been absorbed and detected after oral administration and appear to be stable for a considerable time in vivo.     

Facilitation of copulatory performance in male rats by naloxone: Effects of hypophysectomy, 17 α-estradiol,and luteinizing hormone releasing hormone

Three experiments were performed to explore the mechanism whereby systemic administration of the opiate receptor antagonist, naloxone hydrochloride (20 mg/kg) causes reductions in the frequency of intromissions preceding ejaculation and latency to ejaculation in sexually experienced male rats. Administration of naloxone to male rats which were hypophysectomized in addition to being castrated and implanted SC with 30 mm silastic capsules containing testosterone caused such behavioral changes, suggesting that these behavioral effects of naloxone do not result from interference with the binding of endorphin of pituitary origin. Surprisingly, a significant facilitatory effect of naloxone on sexual performance was absent in castrated controls bearing 30 mm testosterone implants. Recent evidence suggests that 17α-hydroxylated estrogens, which may be produced in gonadally intact males, possess appreciable affinity for opioid receptors. However, daily administration of 17α-estradiol (50 μg) to castrated, testosterone-implanted males failed to make them as behaviorally responsive to naloxone as gonadally intact animals. Administration of LHRH (1 μg given SC 1.5 hr prior to testing) caused a significant reduction in ejaculation latency in gonadally intact males but not in castrated males bearing 30 mm testosterone implants. It is suggested that the facilitatory effect of naloxone on masculine sexual performance results, in part, from a drug-induced release of LHRH.     

Antagonists of luteinizing hormone releasing hormone bind to rat mast cells and induce histamine release

It was reported previously that administration of certain synthetic antagonists of LHRH to rats produced allergy-like symptoms that were attributed to their histamine releasing action. In the present study the interaction of LHRH analogs with rat peritoneal mast cells was investigated in vitro. Potent antagonists of LHRH showed strong in vitro histamine releasing activity from rat peritoneal mast cells. Membrane preparations of rat pituitary glands showed specific binding of radioiodinated LHRH antagonist as well as LHRH agonist. However, rat peritoneal mast cells and membrane preparations from those cells bound antagonist but not the agonist. Furthermore, the LHRH antagonist did not bind to membranes prepared from tissues such as prostate, liver, kidney, and brain. Competitive displacement curves of the [125I]-antagonist with different LHRH analogs showed that the ability of the analogs to compete for binding sites on mast cells was related to their histamine releasing activity. We conclude that histamine release from rat mast cells induced by LHRH analogs is mediated by specific binding of the active peptides to cell membranes. Furthermore, using rat mast cells, the binding assay in conjunction with histamine releasing assay may be utilized to predict the in vivo histamine releasing potential of new LHRH peptides which are of clinical importance.     

Antagonists of luteinizing hormone releasing hormone with novel unnatural amino acids at position six

Five new antagonists of luteinizing hormone releasing hormone (LHRH) containing novel unnatural amino acids at position six are reported. They are very effective in the rat antiovulatory assay. Using saline as vehicle, antagonist-[N- Ac-d -2-Nal¹, d -4-Cl-Ph², d -3-Pal³, Arg⁵, d -A2⁶,d -Ala¹⁰-LHRH inhibited ovulation completely at 1 μg/rat and three of the other antagonists showed some antiovulatory activity at μpg/rat.     

Muscarine and luteinizing hormone releasing hormone attenuate adrenaline induced hyperpolarization in amphibian sympathetic ganglia.

The adrenaline-induced hyperpolarization (AdH) and the responses evoked by muscarine and luteinizing hormone releasing hormone (LHRH) were recorded from neurones in amphibian sympathetic ganglia by means of the sucrose gap technique. The amplitude of the AdH was reduced when 'M-channel' closure was promoted by superfusion of LHRH or muscarine. 4-Aminopyridine (4-AP, 1 mM) antagonized the AdH, but not the depolarization evoked by muscarinic agonists. This implies that the channels involved in the electrogenesis of the AdH have different pharmacological properties from 'M-channels' and that the AdH is not generated by the opening of 'M-channels' outside their normal voltage range. Possible explanations for the attenuation of the AdH by muscarine and LHRH might be that (i) intracellular biochemical changes produced by these substances somehow interfere with the generation of the AdH or that (ii) muscarine and LHRH have allosteric interactions with the adrenoceptor mediating the AdH.     

Treatment of pre-menopausal advanced breast cancer with goserelin--a long-acting luteinizing hormone releasing hormone agonist.

Twenty-two pre-menopausal evaluable patients with advanced breast cancer (median age 39 years; ER positive 19, unknown three; prior adjuvant chemotherapy 16) were treated with the LHRH agonist goserelin depot (Zoladex). Serum levels of 17 beta-estradiol and progesterone were suppressed by goserelin within 3-4 weeks of therapy, while serum leuteinizing hormone and follicle stimulating hormone titers remained in the low level of the normal range. Complete or partial response was documented in seven of 22 cases (32%) and occurred in all major sites of disease. Tumor response was documented in women regularly menstruating at the start of therapy. Median time to disease progression was 23 weeks; median duration of response was 64 weeks; overall survival was 141 weeks. Zoladex was well tolerated: only hot flushes in 82% and reversible cutaneous pigmentation in the site of injection in 45% of the patients were observed. In our experience the activity of Zoladex was comparable to that of oophorectomy, without the psychological trauma and the morbidity related to surgical castration.     

Effect of luteinizing hormone releasing hormone (LHRH) agonists on body weight in rats

As the potential clinical applications of LHRH agonists increase, the issue of side effects on nonreproductive organ systems assumes greater significance. LHRH agonists administered continuously or in twice-daily injections accelerated body weight gain in female rats. Two different LHRH agonists produced similar effects on body weight in female rats. When Leuprolide (LHRHa) was given to female rats in twice-daily injections, the adrenal weight increased after 21 days of treatment, whereas administration by osmotic minipumps produced no significant effect on adrenal weight. When another agonist, Zoladex (LHRHz), was given continuously for 52 and 229 days to female rats, the adrenal weight decreased. No change was noted in adrenal weight or body-weight gain of male rats given LHRHz continuously for 51 and 62 days. Reproductive-organ weights were significantly reduced in all LHRH-agonist-treated animals. Serum progesterone in female rats and testosterone in male rats were significantly decreased following treatment with LHRHz. No significant differences were noted in serum prolactin, growth hormone, or corticosterone. The mechanisms and significance of these findings are discussed.     

Potency of synthetic luteinizing hormone releasing hormone preparations in rat anterior pituitary cell cultures.

Selected synthetic luteinizing hormone releasing hormone preparations were assayed, and their potencies were determined relative to one sample utilizing primary cultures of enzymatically dispersed rat anterior pituitary cells. Preliminary cell culture experiments indicated that luteinizing hormone releasing hormone had to be in constant contact with cells for continued luteinizing hormone secretion. Luteinizing hormone levels in media reached a maximum concentration after 4 hr of continuous luteinizing hormone releasing hormone exposure. Cell culture bioassay was selected over the bioassay employing chronically ovariectomized steroid-blocked rats due to greater sensitivity and economy. The assay of each luteinizing hormone releasing hormone preparation was replicated four to seven times. Preparations from several companies were less potent (p less than 0.05) than the reference product. Contaminants were disclosed by TLC in preparations with potencies lower than the reference product.     

Development of luteinizing hormone releasing hormone (LHRH) delivery systems for vaginal mucosal route

The objective of this study was to find a rational dosage form for vaginal mucosal delivery of LHRH. Vaginal absorption of LHRH was estimated by measuring its ovulation inducing effect in rat and in vitro vaginal membrane permeation study in rabbit. The effectsof different hydrogel bases, such as Polycarbophil and Pemulen compared with solutions on vaginal membrane permeation of LHRH were investigated. Sodium laurate, disodium ethylenediamine tetraacetate (EDTA) and sodium tauro-24,25-dihydrofusidate (STDHF), which are effective peptidase inhibitors were chosen as additives to a LHRH hydrogel delivery system and LHRH solutions. A Polycarbophil hydrogel formulation showed 3.4 times increase in LHRH vaginal membrane permeability compared with a solution formulation. Vaginal membrane permeability from the Polycarbophil was greater than that from Pemulen hydrogels. This may be due to the larger bioadhesive values. LHRH solution with EDTA(2%), STDHF(1%) and sod. laurate(0.5%) showed 4.1 times, 4.8 times and 6.0 times of ovulation inducing activity compared with control. These results suggest that enzyme inhibition effect of EDTA, STDHF and sod. laurate may result in substantial enhancement of vaginal absorption. By administration of Polycarbophil hydrogels containing LHRH the ovulation inducing activity was 3.3 times greater than the solutions. This result indicates the bioadhesive hydrogels as well as peptidase inhibition significantly improved absorption of LHRH. By coadministration with these inhibitors the ovulation inducing activity of Polycarbophil hydrogel containing LHRH was comparable with subcutaneous administration in ovulation inducing activity.     

Alcohol effects on naloxone-stimulated luteinizing hormone, prolactin and estradiol in women

Plasma luteinizing hormone (LH), estradiol, prolactin and progesterone levels were measured in nine normal adult women prior to and following administration of naloxone and oral ingestion of ethanol or placebo-control solution. Each subject served as her own control in a double-blind study carried out during the midluteal phase of the menstrual cycle. The mean (+/- SD) progesterone level was 13.9 +/- 1.3 during control conditions and 13.9 +/- 1.7 during alcohol conditions. The mean peak blood alcohol level was 100 +/- 13 mg/dl within 45-60 min after initiation of drinking. Under placebo-control conditions, naloxone stimulated a significant increase in plasma LH and prolactin but did not increase estradiol or progesterone. Alcohol did not attenuate the significant naloxone stimulation of LH, and progesterone levels were equivalent under alcohol and control conditions. Alcohol significantly enhanced naloxone stimulation of prolactin and estradiol. Alcohol administration significantly augmented the naloxone-induced increase in plasma prolactin levels. After alcohol administration, naloxone also induced a significant increase in plasma estradiol levels, which was sustained throughout the 180-min sampling period. The mechanisms underlying alcohol's enhancement of naloxone-stimulated prolactin and estradiol remain to be determined. The alcohol-related increase in naloxone-stimulated prolactin secretion may reflect increased hypothalamic and/or pituitary sensitivity to alcohol following endogenous opioid blockade by naloxone or an effect of increased estrogen levels. The significant increase in plasma estradiol levels following concurrent naloxone and alcohol administration may occur as a consequence of alterations in steroid biotransformation associated with intrahepatic ethanol catabolism.