Suppression and recovery of LH secretion by a potent and selective GnRH-receptor antagonist peptide in healthy early follicular-phase women are mediated via selective control of LH secretory burst mass.

AIM: GnRH antagonists are competitive inhibitors of GnRH receptors. Their administration induces prompt suppression of the gonadal axis. In animals, GnRH antagonists upregulate the activity of GnRH-secreting neurones, which could cause gonadotrophin rebound following inhibition. The aim of this study was to evaluate the effects of a potent GnRH antagonist, Teverelix (TEV), on the gonadal axis in healthy young women. SUBJECTS AND MEASUREMENTS: In nine women [20-35 years old, body mass index (BMI) 19-25 kg/m2] in the early follicular phase, serum LH and FSH levels were evaluated every 10 min from 08.00 to 12.00 h before, and 24 h and 96 h after TEV injection (2.5 mg in 1 ml subcutaneously on day 0). Serum gonadotrophin and oestradiol levels were also evaluated at baseline and at 6, 8, 12, 48, 72 h after TEV. RESULTS: The antagonist reduced both serum LH and FSH concentrations; LH levels were significantly and promptly reduced at +6 h (nadir at +8 h) until +48 h and recovered at +72 h, while FSH levels were reduced (P<0.05) 24 h after the antagonist and normalized at +48 h. LH (but not FSH) concentrations at +96 h exceeded baseline (P<0.05). TEV suppressed oestradiol concentrations (P<0.05) with a nadir at +24 h, comparable reduction at +48 h and recovery to baseline at +72 h. Deconvolution analysis showed that the antagonist peptide suppressed (P<0.02) the pulsatile production rate, burst mass and amplitude of LH on day 1. Pulsatile FSH secretion also fell at this time (P<0.05). LH and FSH pulse frequency were not modified by TEV. At +96 h, LH pulsatility did not significantly differ from that at baseline. Suppression of mean LH or FSH concentrations did not affect the relative pattern regularity (approximate entropy) of LH and FSH secretion. CONCLUSIONS: This study demonstrates that the acute administration of a potent GnRH antagonist induces prompt inhibition of the gonadal axis lasting for 2 days in women due to mechanistically specific suppression of LH secretory burst mass and the mean FSH secretion rate. The trend toward rebound release of LH following the end of the pharmacological effect of the antagonist could reflect a rise in endogenous GnRH activity.

EP1572: a novel peptido-mimetic GH secretagogue with potent and selective GH-releasing activity in man.

EP1572 UMV1843 [Aib-DTrp-DgTrp-CHO]) is a new peptido-mimetic GH secretagogue (GHS) showing binding potency to the GHS-receptor in animal and human tissues similar to that of ghrelin and peptidyl GHS. EP1572 induces marked GH increase after s.c. administration in neonatal rats. Preliminary data in 2 normal young men show that: 1) acute i.v. EP1572 administration (1.0 microg/kg) induces strong and selective increase of GH levels; 2) single oral EP1572 administration strongly and reproducibly increases GH levels even after a dose as low as 0.06 mg/kg. Thus, EP1572 is a new peptido-mimetic GHS with potent and selective GH-releasing activity.

Radioimmunoassay of meterelin and pharmacokinetics after single injection and implant administration in dogs.

A sensitive and specific radioimmunoassay for a novel luteinizing-hormone-releasing-hormone (LHRH) agonist, [2-Me-D-Trp6, DesGly10]LHRH ethylamide (meterelin), was developed for documenting the pharmacokinetic parameters of this peptide following its intravenous (iv) and subcutaneous (sc) administration in dogs. The assay was also used for monitoring meterelin in plasma following its release from d,l-lactide-glycolide implants in the same species. Rabbit antisera generated against [DespyroGlu1] meterelin and conjugated to bovine serum albumin with glutaraldehyde showed high specificity, whereas crossreactivity to LHRH and its fragments and to analogs with substitutions at residues 6 and 10 was found insignificant. The assay was validated in terms of accuracy (recovery range, 94.0-105.4%), in terms of precision (intra- and interassay variations of 10.0-12.4% and 8.6-11.3%, respectively), and in terms of sensitivity (minimum detectable dose of 2.7 pg/assay). Following iv acute administration, a biexponential decline of plasma meterelin levels was observed, with distribution and elimination half-lives of 5.9 +/- 2.5 and 106 +/- 22 min, respectively. After sc acute administration, the elimination half-life was in the range of 103 to 173 min. The systemic clearance (CLT) ranged from 1.6 to 2.6 mL/min/kg, and the volume of distribution at steady state (Vdss) varied from 285 to 438 mL/kg. The elimination half-life (T1/2 beta), Vdss, and ClT were not significantly different after both routes of administration over the 1-100-microgram/kg dose range of peptide studied. Castrate levels of testosterone were attained 10 days after sc administration of the implant, lasted for up to 247 days, and were well correlated with plasma levels of meterelin.

Radioimmunoassay of Antarelix, a luteinizing hormone releasing-hormone antagonist, in plasma and its application for pharmacokinetic study in dogs.

A procedure for the radioimmunoassay (RIA) of Antarelix (teverelix) in plasma has been developed for the pharmacokinetic study of this potent LHRH antagonist in dogs. Antiserum was produced by coupling the deamidated Antarelix analog to bovine serum albumin by a carbodiimide reaction and immunizing rabbits with the conjugate. The crossreactivity of the antiserum with LHRH, LHRH agonist Metereline and LHRH antagonists tested was negligible, except for Antide which displayed a crossreactivity of 33%. No crossreactivity with Antarelix metabolites could be detected. The RIA is suitable for the direct determination of Antarelix in plasma, with a minimum detectable level of 1.12 fmol/assay. The accuracy and precision of the method were assessed with plasma samples spiked with Antarelix at concentrations ranging from 0.4 to 6.4 pmol/ml. The recovery was 104.8% with intra- and interassay CV between 1 and 3.7%. Pharmacokinetic profiles of Antarelix in dogs were established following an i.v. or a s.c. dose of 10 micrograms/kg.

Small peptides as potent releasers of growth hormone.

Newly synthesized peptides are described which release growth hormone in the infant rat and their biological activity is compared with known GHRPs. Some of these peptides are the most potent GHRPs reported to date.

Radioimmunoassay for hexarelin, a peptidic growth hormone secretagogue, and its pharmacokinetic studies.

A radioimmunoassay (RIA) method for hexarelin, a peptidic growth hormone secretagogue, has been developed and applied to pharmacokinetic studies in dogs following an IV dose of 1 microgram/kg, and three SC doses of 1, 10, and 100 micrograms/kg. The sensitivity of the assay was determined to be 1.34 fmol/assay. Cross-reactivity of the antiserum with nine hexarelin analogues was less than 1% upon modification of positions 3, 4, or 5 of the peptide. No apparent cross-reaction with endogenous hexarelin metabolites were observed. Intra- and interassay coefficients of variation were less than 3% and 4%, respectively. Intravenous bolus pharmacokinetics of hexarelin displayed a high terminal half-life of 120 min, a fractional plasma clearance of 4.28 ml/min/kg, and a volume of distribution at steady state of 387.7 ml/kg. Following SC administration of hexarelin, despite the increase in dose administered, both clearance (3.93-5.17 ml/min/kg) and volume of distribution (316-544 ml/kg) parameters remained constant over the dose range studied.

Growth hormone releasing activity by intranasal administration of a synthetic hexapeptide (hexarelin).

OBJECTIVE: Hexarelin is a new synthetic growth hormone releasing peptide. We have tested the efficacy of intranasal (i.n.) administration of hexarelin to stimulate plasma GH and have compared this to the intravenous (i.v.) administration of the peptide. PATIENTS: Ten children with familial short stature (FSS) aged 5.5-15.5 years and two known GH deficient patients aged 24 and 28 years without GH treatment. METHODS: All 12 subjects were submitted to i.v. (1 microgram/kg) and i.n. (20 micrograms/kg) hexarelin tests with a one-week interval between tests. Blood samples for GH, TSH, fT4 and T3 were obtained at 0, 15, 30, 60, 90 and 120 minutes. The hormone determinations were made by standard radio-immunoassays (RIA). RESULTS: Both the i.n. and i.v. administration of hexarelin induced a large GH response, the mean (+/- SD) being 72.2 +/- 35.5 mU/l for the i.n. test and 79.6 +/- 53.0 mU/l for the i.v. test. The peak GH in the i.v. test occurred at 15-30 minutes and in the i.n. test between 30 and 60 minutes. The GH deficient patients showed no GH response in either test. Plasma TSH decreased in the FSS children from a mean (+/- SD) of 1.0 +/- 0.26 to 0.64 +/- 0.2 mU/l (P < 0.005) during the i.n. test and from 1.0 +/- 0.3 to 0.7 +/- 0.3 mU/l (P < 0.05) during the i.v. test. In the isolated GH deficient patient, plasma TSH decreased from 1.06 +/- 0.38 mU/l to 0.86 +/- 0.17 during the i.v. test and from 1.60 +/- 0.01 to 1.11 +/- 0.06 mU/l during the i.n. test. There were no significant changes in plasma fT4 or T3 in any of the tests. CONCLUSIONS: The synthetic hexapeptide hexarelin is a potent pituitary GH stimulator when administered intranasally. The GH response was similar to that observed after intravenous hexarelin. Simultaneously, there was a significant decrease in plasma TSH but the concentrations remained in the normal range. These findings appear to be of theoretical and practical relevance to the investigation and management of short children.

Suppression of allogeneic reactivity in vitro by the syncytiotrophoblast membrane glycocalyx of the human term placenta is carbohydrate dependent.

Immunosuppressive factors isolated from trophoblast are known to block both innate and major histocompatibility complex (MHC)-dependent cell-mediated immune responses in vitro and, in some cases, in vivo. We investigated the biochemical nature of these factors, which is presently unknown. Immunosuppressive activity, assessed by inhibition of two-way MLR, was extracted from term syncytiotrophoblast microvilli using 3 M KCl. The activity resisted both extensive pronase digestion and heating to 90 degrees C for 1 h, demonstrating that intact membrane proteins were not required. Although purified protein-linked oligosaccharides released by hydrazinolysis from the syncytiotrophoblast membrane were themselves inactive, they blocked the immunosuppressive activity of the KCl extract. After pronase digestion, the activity could be fractionated by TSK 55S gel filtration, followed by C18 reverse-phase chromatography. Sequential exoglycosidase digestion of hydrazine-released sugars of the active fraction demonstrated that it contained neutral N-linked oligomannose and hybrid oligosaccharides, which normally make up < 3% of the total syncytiotrophoblast-derived protein glycan library. These glycopeptides of the active fraction were associated with membrane phospholipid micelles. The possible mechanism by which incompletely processed N-linked oligosaccharides expressed by a variety of syncytiotrophoblast membrane glycoproteins may block allogeneic reactivity when presented as polyvalent sugar groups is discussed.

Growth hormone-releasing activity of hexarelin in humans. A dose-response study.

Hexarelin is a new hexapeptide (His-D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2) that stimulates the release of growth hormone both in vitro and in vivo. In this double-blind, placebo-controlled, rising-dose study we evaluated the growth hormone releasing activity of hexarelin in healthy human subjects. Twelve adult male volunteers received single intravenous boluses of 0.5, 1 and 2.micrograms.kg-1 hexarelin as well as placebo. For safety, drug doses were given in a rising-dose fashion with placebo randomly inserted into the sequence. Plasma growth hormone concentrations increased dose-dependently after the injection of the peptide, peaking at about 30 min and then decreasing to baseline values within 240 min with a half-life of about 55 min. The mean peak plasma growth hormone concentrations (Cmax) were 3.9, 26.9, 52.3, 55.0 ng.ml-1 after 0, 0.5, 1 and 2 micrograms.kg-1, respectively. The corresponding areas under the curve of growth hormone plasma levels from drug injection to 180 min (AUC0-180) were 0.135, 1.412, 2.918 and 3.695 micrograms.min.ml-1. The theoretical maximum response (Emax) and the dose that produces half of the maximum response (ED50) were estimated using logistic regression. The calculated ED50 values were 0.50 and 0.64 microgram.kg-1 for Cmax and AUC0-180, respectively. The corresponding Emaxs were 55.1 ng.ml-1 and 3936 ng.min.ml-1, thus indicating that the effect after the 2 micrograms.kg-1 dose is very close to the maximal response.(ABSTRACT TRUNCATED AT 250 WORDS)

Antarelix (EP 24332) a novel water soluble LHRH antagonist.

A novel water soluble LHRH antagonist (EP 24332-Antarelix) is described. Its activity in vitro and in vivo in several animal models is given. Antarelix (Ac-D-Nal, D-Cpa, D-Pal, Ser, Tyr, D-Hci, Leu, Lys-(iPr), Pro, D-Ala-NH2) in view of its potency, modest histamine-liberating activity and high water solubility has been selected for further development.

Serum lipid and lipoprotein abnormalities in human African trypanosomiasis.

We have studied the serum lipoprotein system in human African trypanosomiasis (Trypanosoma brucei gambiense infection). The study was carried out on 74 Congolense patients suffering from sleeping sickness and 34 Congolense control subjects living in the endemic region of Boko Songho. We have determined the serum concentrations of lipids (triglycerides, cholesterol, phospholipids) and apolipoproteins (apolipoprotein A-I and B), and the separation of serum lipoproteins by electrophoresis. For the patients infected with T. b. gambiense, in comparison with control subjects, the results have shown (i) a significant increase in triglyceride concentration and a decrease in cholesterol concentration; (ii) a significant rise in apolipoprotein B concentration and a significant reduction in apolipoprotein A-I concentration; and (iii) an increase in low density lipoproteins and a decrease in high density lipoproteins. We conclude, therefore, that human African trypanosomiasis is associated with marked alterations in the composition and levels of host lipoproteins.

Study of proteolytic activities released by incubation of trypanosomes (Trypanosoma brucei brucei) in pH 5.5 and pH 7.0 phosphate/glucose buffers.

Proteolytic activities released by overnight incubation of Antwerpeen Trypanozoon antigenic type (AnTat) 1.1 trypanosomes at 4 degrees C in pH 5.5 and pH 7.0 phosphate/glucose buffers were analyzed in the supernatants obtained after centrifugation of the parasite suspensions. The assays used the fluorogenic substrates N-alpha-benzyloxycarbonyl-L-phenyl-alanyl-L-arginine-7-amido-4- methylcoumarin (Z-Phe-Arg-NMec) and N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine-7- amido-4-methylcoumarin (Z-Arg-Arg-NMec) at two different pHs (6.0 and 8.3). Z-Phe-Arg-NMec hydrolysis was inhibited by 2 microM L-trans-epoxysuccinyllencylamido(4-guanidino)butane (E-64) to a greater extent in the pH 7.0 supernatant than in the pH 5.5 supernatant. Z-Arg-Arg-NMec hydrolysis by the two supernatants was not significantly inhibited by 2 microM E-64. At pH 8.3 this activity was increased more than 2-fold by the addition of dithiothreitol. The hydrolysis activities were analyzed in collected eluates after fractionation of the supernatants by gel permeation high-performance liquid chromatography. Z-Phe-Arg-NMec hydrolytic activity inhibited by 2 microM E-64 was maximal at a retention time of 33 min (approx. Mr 30,000). In addition, a hydrolytic activity against the substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec gave a peak showing a maximum at a retention time of 29 min (approx. Mr 70,000).

Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat.

Hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levels were stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations.

Variant surface glycoprotein of Trypanosoma brucei brucei AnTat 1.1: influence of the isolation conditions upon the disulfide linked dimer/monomer ratio.

1. Using the variant surface glycoprotein (VSG) isolation procedure described by Baltz et al. ([1976] Ann. Immunol. (Inst. Pasteur) 127 C, 761-774) which involves suspension of the trypanosomes in a pH 5.5 buffer, the Antwerpen trypanozoon antigenic type (AnTat) 1.1 VSG is mainly obtained as a disulfide linked dimeric form with a trace amount of a monomeric form. 2. The use of a parasite suspension buffer at pH 7.0 results in a slight decrease of the VSG dimer/monomer ratio. 3. pH 5.5 and 7.0 supernatants of centrifuged parasite suspensions were submitted to kinetic incubations at different temperatures and pH, and we found conditions involving transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer (shifting the pH 5.5 supernatant to pH 7.0 and incubation at room temperature). 4. This transformation of the AnTat 1.1 VSG dimer into the AnTat 1.1 VSG monomer is activated by the addition of 1 mM reduced glutathione, and is inhibited by the addition of 1 mM oxidized glutathione or 0.1 mM N-ethylmaleimide or cadmium acetate.

Gonadotropic axis and Trypanosoma brucei gambiense infection.

A gonad endocrine survey on 46 Congolese patients (15 women and 31 men) with parasitologically confirmed trypanosomiasis found amenorrhoea in 60% of the women and impotence in 70% of the men. The basic gonad endocrine examination showed a decrease in oestradiol levels in about 65% of the women. Both amenorrhoea and low oestrogen levels were observed in the second phase (P2) of the disease, but low oestrogen levels were sometimes noted in the first phase of the disease (P1). In the men, about 50% of the cases (P2) showed a decrease in testosterone. However, as in the women, the variation of testosterone was also observed in the first phase (P1). A static and dynamic examination of the hypothalamic-pituitary-gonadal axis was undertaken in order to investigate the origin of these hypogonadisms. A supra - or extra-hypophyseal origin is discussed.

Evidence of myristylated disulfide-linked dimer of variant surface glycoprotein of Trypanosoma brucei-brucei.

1. Variant surface glycoprotein (VSGs) of Trypanosoma brucei-brucei may exist as a disulfide-linked dimer in both forms: myristylated (mfVSG) and non-myristylated (sVSG), as judge by fluorography and immunoblotting of SDS-PAGE under non-reducing conditions. 2. The dimeric VSG form is labeled with [3H]-myristic acid in our incorporation conditions. 3. AnTat 1.1 trypanosomes preincubated with tunicamycin and incubated with [3H]-myristic acid synthesized a labeled molecule that has an apparent molecular weight slightly smaller than the native form, and that also corresponds to a disulfide-linked dimer.

Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat 1.1 of Trypanosoma brucei brucei.

Variant surface glycoprotein (VSG) of Trypanosoma brucei brucei AnTat 1.1 was released by means of the procedure described by Baltz et al. ([1976], Ann. Immunol. [Inst. Pasteur] 127C, 761-774). The concanavalin-A chromatography yielded 3 VSG fractions according to the addition, in the elution buffer, of alpha-methyl-D-mannopyranoside, beta-mercaptoethanol, and sodium dodecyl sulfate. These VSG fractions showed heterogeneous behaviour on reverse-phase high performance liquid chromatography. The 3 VSG fractions as well as the myristylated VSG of AnTat 1.1 essentially consist of dimer VSG forms linked through a disulfide bridge, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and nonreducing conditions.