Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro.

Antagonistic analogs of growth hormone-releasing hormone (GHRH) suppress growth of various tumors in vivo. This effect is exerted in part through inhibition of the GHRH-GH-insulin-like growth factor (IGF)-I axis. Nevertheless, because autocrine/paracrine control of proliferation by IGF-II also is a major factor in many tumors, the interference with this growth-stimulating pathway would offer another approach to tumor control. We thus investigated whether GHRH antagonists MZ-4-71 and MZ-5-156 also act on the tumor cells directly by blocking the production of IGF-II. An increase in the IGF-II concentration in the media during culture was found in 13 of 26 human cancer cell lines tested. Reverse transcription-PCR studies on 8 of these cell lines showed that they also expressed IGF-II mRNA. Antagonists of GHRH significantly inhibited the rate of proliferation of mammary (MDA-MB-468 and ZR-75-1), prostatic (PC-3 and DU-145), and pancreatic (MiaPaCa-2, SW-1990, and Capan-2) cancer cell lines as shown by colorimetric and [3H]thymidine incorporation tests and reduced the expression of IGF-II mRNA in the cells and the concentration of IGF-II secreted into the culture medium. Growth and IGF-II production of lung (H-23 and H-69) and ovarian (OV-1063) cancer cells that express mRNA for IGF-II and excrete large quantities of IGF-II also was marginally suppressed by the antagonists. These findings suggest that antagonistic analogs of GHRH can inhibit growth of certain tumors not only by inhibiting the GHRH-GH-IGF-I axis, but also by reducing the IGF-II production and by interfering with the autocrine regulatory pathway.

Expression of growth hormone-releasing hormone (GHRH) messenger ribonucleic acid and the presence of biologically active GHRH in human breast, endometrial, and ovarian cancers.

GHRH is produced in a variety of extrahypothalamic tissues, including some neoplasms. We have previously reported that GHRH antagonists can inhibit the growth of various human cancers xenografted into nude mice. These observations suggest that locally produced GHRH might directly affect tumor cell proliferation. To investigate this possibility, we have examined the local production of GHRH in human endometrial, ovarian, and breast cancers obtained after surgery or grown in nude mice as xenografts. We have also examined whether the GHRH produced in these tumors is biologically active. RT-PCR and Southern blotting showed expression of messenger ribonucleic acid for GHRH in 17 of 22 endometrial and 17 of 22 ovarian cancer specimens and in all of the human endometrial, ovarian, and breast cancer xenografts studied. Acid extracts of endometrial cancer specimens and breast cancer xenografts that expressed the GHRH gene contained immunoreactive GHRH peptide, as assessed by RIA for GHRH. The level of immunoreactive GHRH detected was equivalent to 2.7-6.4 ng GHRH-(1-29)/g tissue. Purified extract from one of these tumor samples induced a powerful stimulation of GH release from rat pituitary cells. The presence of biologically and immunologically active GHRH and messenger ribonucleic acid for GHRH in human breast, endometrial, and ovarian cancers supports the hypothesis that locally produced GHRH may play a role in the proliferation of these tumors.

Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted in vivo activities.

Some antagonists of human growth hormone-releasing hormone (hGH-RH) synthesized previously were shown to inhibit in vivo proliferation of various human cancers in nude mice. However, the activity of these analogs requires an increase to assure clinical efficacy. In an attempt to prepare hGH-RH antagonists with a high and protracted activity, we synthesized and biologically tested 22 antagonistic analogs of hGH-RH(1-29)NH2. The ability of the antagonists to inhibit hGH-RH-induced GH release was evaluated in vitro in a superfused rat pituitary system, as well as in vivo after i.v. injection into rats. The binding affinity of the peptides to GH-RH receptors also was determined. All antagonistic analogs had the common core sequence [PhAc-Tyr1,D-Arg2, Phe(4-Cl)6 (para-chlorophenylalanine), Abu15 (alpha-aminobutyric acid), Nle27]hGH-RH(1-29)NH2 and contained Arg, D-Arg, homoarginine (Har), norleucine (Nle), and other substitutions. The following analogs were determined to have a high and/or protracted antagonistic activity: [PhAc-Tyr1,D-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27, D-Arg29]hGH-RH(1-29)NH2 (JV-1-10), [PhAc-Tyr1,D-Arg2,Phe(4-Cl)6, Abu15,Nle27,D-Arg28,Har29]hGH-RH(1-29)NH2 (MZ-6-55), [PhAc-Tyr1, D-Arg2,Phe(4-Cl)6,Arg9,Abu15,Nle27,D-Arg28,Har29 ]hGH-RH(1-29)NH2 (JV-1-36), and [PhAc-Tyr1,D-Arg2,Phe(4-Cl)6,Har9,Tyr(Me)10,Abu15, Nle27,D-Arg28,Har29]hGH-RH(1-29)NH2 (JV-1-38). Among the peptides tested, analog JV-1-36 showed the highest GH-RH antagonistic activity in vitro and also induced a strong and prolonged inhibition of GH release in vivo for at least 30 min. The antagonist JV-1-38 was slightly less potent than JV-1-36 both in vitro and in vivo but proved to be very long-acting in vivo, suppressing the GH-RH-induced GH release even after 60 min. High and protracted in vivo activities of these antagonists indicate an improvement over earlier GH-RH analogs. Some of these hGH-RH antagonists could find clinical applications in the treatment of cancers dependent on insulin-like growth factors I and II.

Dynamic insulin secretion from perifused rat pancreatic islets.

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 microM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 microM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K+ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED50 = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED50 = 89 nM) and DA (ED50 = 2.2 microM). gamma-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances.

Differences in hormone release patterns from the anterior pituitary and the pineal gland.

Patterns of hormone release from peptide- and monoamine-hormone secreting cells were compared. LH, GH and PRL secretion from rat anterior pituitary cells and melatonin secretion from the rat pineal gland fragments were studied in a dynamic, in vitro system. The following fundamental differences were found: 1. Adenohypophysial cells respond to a specific stimulus (releasing hormones) with hormone secretion within seconds. There is a 90-minute lag, however, between the beginning of specific stimulation (norepinephrine, 1 microM for 30 min) and the onset of melatonin (MT) release from the pineal cells. 2. Hormone secretion from the pituitary cells returns to the basal value five to 10 minutes after the stimulus has been stopped. Once initiated, MT release from the pineal lasts for five to seven hours even if the NE stimulus was stopped one hour before the beginning of the MT response. 3. A transitory increase in potassium concentration (by 50-100 mM) induces a sharp, distinct rise of hormone secretion from pituitary cells, similar in shape and size experienced as response to releasing hormone stimulation. Pineal cells do not respond to elevation of potassium concentration at all. 4. High concentration of tropic hormones can be extracted from pituitary cells at the end of the superfusion experiment. Their hormone content is equivalent to the amount released from non-stimulated cells in a 50 to 80 hours period (LH and GH cells). No significant quantity of MT can be extracted from non-stimulated, disintegrated pineal cells. This diversity in the control of hormone secretion from anterior pituitary and the pineal gland can be considered as a model for peptide and monoamine-hormone producing glands.

Development of a radioimmunoassay for some agonists of growth hormone-releasing hormone.

A radioimmunoassay (RIA) method was developed for determination of superactive GH-RH agonist Dat1,Ala15,Nle27 GH-RH(1-28)Agm29 (MZ-2-51) and some of the related analogs in biological fluids. The analogs were radioiodinated using the Bolton-Hunter method. For the generation of antibodies, rabbits were immunized with MZ-2-51 and its C-terminal derivative Nle27 GH-RH(17-28)Agm29, conjugated to bovine serum albumin with glutaraldehyde. The resulting antibodies exhibited high affinity and very low cross-reactivity with related, naturally occurring peptides, enabling us to set up a sensitive and specific RIA. High cross-reactions with some of the MZ-2-51 derivatives like MZ-3-149 (40%) and related compounds made it possible for us to also assay these analogs with the same antibody. At B/Bo of 23-37%, the final dilutions of the antibodies ranged from 1:35000 to 1:120000. The minimal detectable concentration of MZ-2-51 was 1.4 fmol (4.6 pg)/tube. The intra- and inter-assay variations were 2.2-4.1% and 9.3-13.9%, respectively. The antibody permitted direct determination of the analogs, without extraction, from biological fluids and tissue extracts. The analogs proved to be stable in serum, and no special treatment of sample was required. Pharmacodynamic studies were performed in rats. Serum levels of GH-RH(1-29) and two of its analogs were monitored following subcutaneous injection. Serum concentration of the analogs and GH-RH(1-29) reached a peak 15 min after injection and returned to basal levels within 90-120 min. Serum GH levels also reached a peak in 15 min, but declined more slowly in the case of analogs than GH-RH(1-29). The biological half-life of both analogs was significantly longer than that of GH-RH(1-29), probably due to their reduced enzymatic degradation.

Evaluation of luteinizing hormone-releasing hormone antagonistic activity in vitro.

Antagonistic analogues of luteinizing hormone-releasing hormone (LHRH) belong to a class of compounds that can be utilized for treatment of some hormone-dependent cancers and gynecologic disorders. Recently, we synthesized and tested a large number of LHRH analogues for LHRH antagonistic activity in the dispersed pituitary cell superfusion system. This fast, reliable, and dynamic system made it possible for us not only to evaluate the relative amounts of an analogue required for suppression of the LH-releasing activity of exogenous LHRH but also provided quantitative data on dynamic interactions between the LHRH analogue, LHRH receptors, and LH secretion. Three experimental paradigms were used: (i) LHRH responses after preincubation with the antagonist, (ii) pulsatile, simultaneous infusion of LHRH and the antagonistic analogue, and (iii) effects of the analogues on ongoing, continuous LH secretion induced by prolonged stimulation with LHRH. From the data obtained, we conclude that (i) the suppression of the LHRH-induced LH release was more effective and longer lasting when the cells were preincubated with the antagonistic analogues before the LHRH stimulation than in the case of simultaneous exposure; (ii) not only the potency but also the time of onset and the duration of the LH release-suppressing activity varied according to the different peptides used, resulting in different shapes of response curves; and (iii) from the accurate data obtained in this dynamic system, quantitative parameters of the in vivo interactions between the antagonists and LHRH on the LHRH receptor can be calculated.

Permeability of the murine blood-brain barrier to some octapeptide analogs of somatostatin.

Analogs of somatostatin are being investigated clinically for the treatment of various malignancies, including brain tumors. We studied the ability of three therapeutically promising radioactively labeled somatostatin octapeptide analogs, RC-160, RC-121, and RC-161, to cross the blood-brain barrier (BBB) after peripheral or central injection. After i.v. injection, intact RC-160 was recovered from the blood and the brain. The entry rates were different from each compound but were generally low. By contrast, entry across the intact BBB increased 220 times when RC-160 was given in a serum-free perfusate. This suggests that some serum-related factor, probably the previously described protein binding or an aggregation-promoting factor, is the main determinant in limiting the blood-to-brain passage of somatostatin analogs. Entry into the brain was not inhibited by the addition of unlabeled analog to the perfusate, showing that passage was probably by diffusion across the membranes that comprise the BBB rather than by saturable transport. By contrast, a saturable system was found to transport peptide out of the central nervous system (CNS). The clearance from the CNS of RC-160 and RC-121, but not RC-161, was faster than could be accounted for by reabsorption of cerebrospinal fluid. Transport of radioactively labeled RC-160 out of the CNS was inhibited by unlabeled RC-160 or somatostatin but was not affected by some other peptide known to cross the BBB by their own transport systems. More than 80% of the radioactivity recovered from the blood after intracerebroventricular injection of RC-160 was eluted by HPLC at the position of the labeled analog, showing that the peptide had crossed the BBB in intact form. Our results indicate the presence of a saturable transport system in one direction across the BBB for some superactive analogs of somatostatin.

Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo. A histological and immunohistochemical study.

Sustained delivery systems (microcapsules, microparticles, or implants) developed for once a month administration of peptides are efficacious and convenient. Long acting formulations of several bioactive peptides are based on microcapasules of a biodegradable polymer poly(DL-lactide-co-glycolide) (PLG), but a better understanding is required of the mechanism of the peptide release from the microcapsules, which is assumed to be primarily by diffusion through pores. In order to clarify this mechanism, microcapsules and microparticles of the agonist [D-Trp6]-LHRH and microcapsules of the LHRH antagonist SB-75 were given i.m. to rats 2 h and 1, 2, 4, 7, 14 and 21 days before histological and immunohistochemical investigation. Signs of biodegradation of the PLG matrix could be seen the first day after the injection, in a form of vacuole development in the interior of the particles and connected with the presence of macrophages within the matrix. The microcapsules showed excellent tissue-compatibility, and no significant foreign body reaction was detected. Immunohistochemical study on the microcapsules revealed no visible decrease in peptide concentration in the remnants of the matrix even 2 weeks after the injection. Evaluation of serum [D-Trp6]-LHRH showed that after an initial burst, both microcapsules and microparticles maintained elevated serum [D-Trp6]-LHRH levels for more than 3 weeks. Our results suggest that the previously proposed mechanisms do not reflect the experimental findings, particularly for the insoluble peptides. The peptide release from the PLG microcapsules or microparticles appears to be controlled mostly by the speed of the biodegradation of the polymer matrix and the diffusion of the peptides from the PGL is negligible.

Development of radioimmunoassay for a potent luteinizing hormone-releasing hormone antagonist. Evaluation of serum levels after injection of [Ac-3-(2-naphthyl)-D-Ala1, D-Phe(pCl)2, 3-(3-pyridyl)-D-Ala3, D-Cit6, D-Ala10] LHRH.

Highly potent antagonistic analogs of luteinizing hormone-releasing hormone (LHRH), free of edematogenic effects have been developed. These analogs proved to be potent inhibitors of LH, follicle stimulating hormone (FSH) and sex steroid levels in animals and human beings. The clinical utility of these compounds would be greatly enhanced by a sustained delivery system, capable of maintaining therapeutic peptide levels in blood over an extended period of time. Consequently, long acting formulations of microcapsules were prepared from one of the most potent antagonists, [Ac-3-(2-naphthyl)-D-Ala1, D-Phe (pCl)2, 3-(3-pyridyl)-D-Ala3, D-Cit6, D-Ala10] LHRH (SB-75). The microcapsules consisted of 2% w/w SB-75 in poly-DL-lactide-co-glycolide (PLGA), a biocompatible, biodegradable polymer. To facilitate pharmacokinetic studies necessary for experimental and clinical investigation of the microencapsulated analog, a highly sensitive and specific radioimmunoassay was developed. The antibody against SB-75 was generated in rabbits. No significant cross-reaction could be detected with several natural peptides and analogs tested. The sensitivity of the assay is 0.6 pg/tube. The RIA is suitable for direct determination of SB-75 level in 20 microliters serum. The two lots of SB-75 microcapsules exhibited different pharmacokinetic release patterns. Single intramuscular injection of 20 mg SB-75 microcapsules, PLGA batch No. 001, into female rats maintained elevated serum SB-75 levels for three weeks. The suppression of LH secretion during this period was indicated by histological findings. The ovaries in the treated group were polyfollicular and no corpora lutea were present, indicating a prolonged ovarian inactivity due to LH deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly potent metallopeptide analogues of luteinizing hormone-releasing hormone.

Metal complexes related to the cytotoxic complexes cisplatin [cis-diamminedichloroplatinum(II)] and transbis(salicylaldoximato)copper(II) were incorporated into suitably modified luteinizing hormone-releasing hormone (LH-RH) analogues containing D-lysine at position 6. Some of the metallopeptides thus obtained proved to be highly active LH-RH agonists or antagonists. For instance, SB-40, a PtCl2-containing metallopeptide in which platinum is coordinated to an N epsilon-(DL-2,3-diaminopropionyl)-D-lysine residue [D-Lys(DL-A2pr] at position 6, showed 50 times higher LH-releasing potency than the native hormone. SB-95, [Ac-D-Nal(2)1,D-Phe(pCl)2, D-Pal(3)2, Arg5,D-Lys[DL-A2pr(Sal2Cu)]6,D-Ala10]LH-RH, where Nal(2) is 3-(2-naphthyl)alanine, Pal(3) is 3-(3-pyridyl)alanine, and copper(II) is coordinated to the salicylideneimino moieties resulting from condensation of salicylaldehyde with D-Lys(DL-A2pr)6, caused 100% inhibition of ovulation at a dose of 3 micrograms in rats. Most metallopeptide analogues of LH-RH showed high affinities for the membrane receptors of rat pituitary and human breast cancer cells. Some of these metallopeptides had cytotoxic activity against human breast cancer and prostate cancer cell lines in vitro (this will be the subject of a separate paper on cytotoxicity evaluation). Such cytostatic metallopeptides could be envisioned as targeted chemotherapeutic agents in cancers that contain receptors for LH-RH-like peptides.

Highly potent analogues of luteinizing hormone-releasing hormone containing D-phenylalanine nitrogen mustard in position 6.

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by linking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. To obtain highly potent alkylating analogues of LH-RH, the D enantiomer of Mel was incorporated into position 6 of the native hormone and some of its antagonistic analogues. Of the peptides prepared, [D-Mel6]LH-RH (SB-05) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,Arg5,D-Mel6,D-Ala10++ +]LH-RH [SB-86, where Nal(2) is 3-(2-naphthyl)alanine and Pal(3) is 3-(3-pyridyl)alanine] possessed the expected high agonistic and antagonistic activities, respectively, and also showed high affinities for the membrane receptors of rat pituitary cells, human breast cancer cells, human prostate cancer cells, and rat Dunning R-3327 prostate tumor cells. These two analogues exerted cytotoxic effects on human and rat mammary cancer cells in vitro. Thus these two D-Mel6 analogues seem to be particularly suitable for the study of how alkylating analogues of LH-RH could interfere with intracellular events in certain cancer cells.

Comparison of different agonists and antagonists of luteinizing hormone-releasing hormone for receptor-binding ability to rat pituitary and human breast cancer membranes.

A sensitive multipoint assay capable of measuring receptors for LHRH and its analogs using 5-10 micrograms membrane protein/incubation tube was used to determine binding characteristics of different agonists and antagonists of LHRH in membranes of male rat pituitary and human breast cancer specimens. This method also permitted Scatchard analysis of the receptor binding in pellet fractions of human breast cancer biopsies remaining from estrogen and progesterone receptor assays. The potent agonist [D-Trp6]LHRH bound to at least two classes of receptor sites, one with high affinity and one with low affinity in both rat pituitary and human breast cancer samples. The analysis of displacement curves of LHRH by agonists and antagonists showed that LHRH also bound to two classes of receptor sites in pituitary and one receptor site with lower affinity in human breast cancer membranes. Among the antagonists synthesized in our laboratory, SB-030, SB-077, SB-088, and SB-090 appeared to be the most potent in displacing labeled [D-Trp6]LHRH and showed the highest binding affinity to the pituitary and breast cancer membranes. Labeled antagonists showed somewhat less affinity to membranes of pituitaries and human cancers than the agonists and bound to only a single class of receptor population. Both agonists and antagonists were able to bind to membranes of human breast cancer samples, and some antagonists were very potent in this respect. Certain LHRH agonists or antagonists could be capable of exerting direct inhibitory effects on breast cancers depending upon the presence and characteristics of LHRH receptors.

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues.

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.

Highly potent antagonists of luteinizing hormone-releasing hormone free of edematogenic effects.

To eliminate the undesirable edematogenic effect of the luteinizing hormone-releasing hormone (LH-RH) antagonists containing basic D amino acids at position 6, exemplified by [Ac-D-Phe(pCl)1,2,D-Trp3,D-Arg6,D-Ala10]LH-RH [Phe(pCl) indicates 4-chlorophenylalanine], analogs with D-ureidoalkyl amino acids such as D-citrulline (D-Cit) or D-homocitrulline (D-Hci) at position 6 were synthesized and tested in several systems in vitro and in vivo. HPLC analysis revealed that the overall hydrophobicity of the D-Cit/D-Hci6 analogs was similar to that of the basic D-Arg6 antagonists. In vitro, most of the analogs completely inhibited LH-RH-mediated luteinizing hormone release in perfused rat pituitary cell systems at an antagonist to LH-RH molar ratio of 5:1. In vivo, the most active peptides, [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Trp3,D-Cit6,D-Ala10]LH-RH [Nal(2) indicates 3-(2-naphthyl)alanine] and its D-Hci6 analog, caused 100% inhibition of ovulation in cycling rats in doses of 3 micrograms and suppressed the luteinizing hormone level in ovariectomized female rats for 47 hr when administered at doses of 25 micrograms. Characteristically, these peptides did not exert any edematogenic effects even at 1.5 mg/kg. These properties of the D-Cit/D-Hci6 antagonists may make them useful clinically.

The influence of gonadectomy, androgen exposure, or a gonadal graft in the neonatal rat on the volume of the sexually dimorphic nucleus of the preoptic area.

Although the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the adult rat has been shown to be modified by the hormone environment early in postnatal life, the present study was performed to clarify several fundamental questions related to this process. This study was designed to evaluate the ability of exogenous testosterone propionate (TP), or a gonadal graft, to influence SDN-POA volume in rats which were gonadectomized as neonates. Orchidectomy on day 1 resulted in an approximately 50% decrease in adult SDN-POA volume; however, the influence of the testes on their resulting SDN-POA volume was replaced affectively by the administration of 100 micrograms or 1 mg of TP on postnatal day 2 or by a testicular (but not ovarian) graft on the day of castration. In the female, ovariectomy on postnatal day 1 failed to alter SDN-POA volume relative to that of sham-operated females. Exogenous TP, but neither testicular nor ovarian grafts, resulted in a larger SDN-POA volume when observed in the adult female. Thus, the development of the SDN-POA of the neonatal male is significantly influenced by the hormonal activity of the testes at this time period. While the SDN-POA of the neonatal female is potentially responsive to androgen, the role played by the ovaries in the development of the SDN-POA remains unclear. In addition, the different response of the developing male and female SDN-POA to a testicular graft suggests that the hormonal sensitivity of this nucleus may differ in the two sexes.