LH-releasing activity of potent LH-RH analogs in vitro

The LH-releasing activity of eight superactive analogs of LH-RH was measured in pituitary cells in primary culture. Introduction of the C-terminal ethylamide modification into [D-Ala⁶]- and [D-Leu⁶]-LH-RH (two peptides already 3 times more active than LH-RH) increases their activities 10-fold. [D-Phe⁶]- and [D-Trp⁶]-LH-RH are 90 and 100 times more active than LH-RH, respectively. The ethylamide derivatives of these two compounds are however approximately six times less active than the parent peptides.     

The role of N-acyl groups in the inhibitory activity of LH-RH analogues

Inhibitory analogues of luteinizing hormone-releasing hormone (LH-RH) were prepared with formyl-D-Trp¹, acetyl-D-Trp¹, valeryl-D-Trp¹, tartaryl-D-Trp¹, diacetyl-tartaryl-D-Trp¹, acetyl-Gly¹, and acetyl-Sar¹ successively replacing the position one in the analogue [D-Trp¹, D-p-Cl-Phe², D-Trp³, D-Phe⁶, D-Ala¹⁰]-LH-RH. The formyl-D-Trp¹ and acetyl-D-Trp¹ analogues yielded 100% blockade of ovulation at the 10 μg dose; the others were less potent and inhibited ovulation at the 50 μg dose. The inhibitory potency seems to correlate with the polarity of the acyl group.     

Luteinizing hormone-releasing hormone analogs with increased anti-ovulatory activity

A series of LH-RH antagonist analogs has been developed in which inhibitory activities have been increased to a potentially clinically useful level. The new peptides, which are typified by [N-acetyl-D-p-Cl-Phe^1,2, D-Trp³, D-Phe⁶,D-Ala¹⁰]-LH-RH and [N-acetyl-D-Trp^1,3,D-p-Cl-Phe²,D-Phe⁶, D-Ala¹⁰]-LH-RH, most importantly contain new modification to positions 1, 2 and 10, and induce full blockade of ovulation at single doses as low as 10 μg per rat (50 μg/kg). Various ring substituents on D-Trp or D-Phe in position 1 or other D-amino acid replacements in position 10 did not significantly improve anti-ovulatory activity. Incorporation of N-Me-Leu in position 7 was slightly detrimental to activity.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

Synthesis and biological properties of (D-Ala-6, des-Gly-NH2-10)-LH-RH ethylamide, a peptide with greatly enhanced LH- and FSH- releasing activity

A nonapeptide analog of luteinizing hormone-releasing hormone (LH-RH), [D-Ala⁶, des-Gly-NH₂¹⁰]-LH-RH ethylamide, was prepared by solid-phase methodology. The peptide was assayed against LH-RH in two $\text{in vivo}$ systems and was found to be many times more potent than the naturally occurring hormone. In one of the tests, based on elevation of LH and FSH levels after infusion into immature male rats, the analog showed LH-releasing activity of 1600% and FSH-releasing activity of 1200% compared to LH-RH.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown

Analogs of luteinizing hormone- releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct ³H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe⁶,desGly¹⁰]-LHRH ethylamide and [D-Ser(Bu^t)⁶,desGly¹⁰]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al₂O₃ catalyst to high specific radioactives. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe⁶,desGly¹⁰]- and [D-Ser(Bu^t)⁶, desGly¹⁰]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of tha analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.     

Synthesis and biological activities of analogs of luteinizing hormone-releasing hormone (LH-RH) substituted in position 1 or 2

Syntheses are described of [Pro¹]-LH-RH, [Orotic acid¹]-LH-RH, [Glu¹]-LH-RH, [Ser²]-LH-RH, [Leu²]-LH-RH, [Gln²]-LH-RH and [Phe²]-LH-RH. The LH-releasing hormone (LH-RH) activity of each of these peptides was compared with that of natural LH-RH $\text{in vivo}$. [Glu¹]-LH-RH and [Phe²]-LH-RH had significant LH-RH activity, while all the other analogs possessed extremely low activities. These findings are briefly discussed in the light of the structure-activity relationship for LH-RH.     

Cytotoxic Peptides: Naphthoquinonyl Derivatives of Luteinizing Hormone-Releasing Hormone

Summary In an attempt to produce efficient cytotoxic derivatives of luteinizing hormone-releasing hormone (LH-RH), two novel 1,4-naphthoquinone derivatives of [d-Lys⁶]-LH-RH were synthesized primarily by solid-phase peptide synthesis, in good yield and high purity. The ability of each analog to produce reactive oxygen species using enzymatic reduction, i.e. NADPH-cytochrome P-450 reductase, was evaluated employing electron spin resonance (ESR) spectroscopy and spin-trapping techniques. The ESR results suggest that the novel cytotoxic analogs are extremely effective in generating oxygen radicals.     

Synthesis and biological properties of (Leu-6)-LH-RH and (D-Leu-6,desGly-NH210)-LH-RH ethylamide

[Leu⁶,desGly-NH₂¹⁰]-LH-RH ethylamide and [Leu⁶]-LH-RH, two analogs of LH-RH, were prepared by the solid phase method. LH- and FSH-releasing activity of these peptides was assayed against LH-RH by subcutaneous administration in immature male rats. When the integrated levels of LH and FSH over a 6 hr period after the injection were used as parameter of the LH- and FSH-releasing activities, the [Leu⁶]-LH-RH showed LH-releasing activity of 9 times and FSH-releasing activity of 5 times greater than that of LH-RH. [Leu⁶,desGly-NH₂¹⁰]-LH-RH ethylamide showed LH- and FSH-releasing activities of 53.6 and 14.5 times greater, respectively, than that of LH-RH.     

Syntheses and biological evaluation of analogs of luteinizing hormone-releasing hormone (LH-RH) modified in position 2, 3, 4 or 5

Syntheses by the conventional methods as well as the chemical, physical and biological properties are described of the following analogs of the LH-releasing hormone (LH-RH): [Leu³]-LH-RH, [Phe³]-LH-RH, [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH, Des-His²-[Phe⁵]-LH-RH, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH. $\text{In vivo}$ assays showed that [Leu³]-LH-RH did not release LH in doses as high as 5 – 25 μg, having less than 0.0008% of LH-RH activity, while [Phe³]-LH-RH had 0.43% of the LH-RH activity of natural LH-RH. The LH-RH activities of [Trp²] [His³]-LH-RH, Des-Trp³-LH-RH and Des-His²-[Phe⁵]-LH-RH were extremely low. On the other hand, [Ala⁴]-LH-RH, [Phe⁵]-LH-RH and [Ala⁴] [Phe⁵]-LH-RH had significant LH-RH activity. The structure-activity relationship of LH-RH is discussed on the basis of these findings.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

On the importance of position one of ovulation inhibitors, as based on studies on [D-Phe2, Pro3, D-Phe6]-LHRH.

Substitution of cyclopentylcarbonyl-(Cpc) for 1 in the effective and potent antiovulatory inhibitor, [D-Phe², Pro³, D-Phe⁶]-LHRH (I) retained the $\text{in vitro}$ potency. We know of no other inhibitor of the luteinizing hormone releasing hormone (LHRH) with a modification at position 1, which is as potent $\text{in vitro}$. This result agrees with the concept of the role of 1, D-Phe², Pro³, D-Phe⁶]-LHRH did not inhibit ovulation in rats at the same dosage as did I; this result is under study to circumvent. Des-Gly¹⁰-[D-Phe², Pro³, D-Phe⁶]-LHRH ethylamide and [Glu¹, D-Phe², Pro³, D-Phe⁶]-LHRH were significantly less active $\text{in vitro}$ than I.     

Analgesic effect of antagonists of substance P

[D-Pro²,D-Phe⁷,D-Trp⁹]-SP and [D-Pro²,D-Trp^7,9]-SP havebeen shown to be antagonists of substance P. The hindlimb scratching syndrome of mice, known to be caused by substance P was absent when these peptides were injected into substance P-treated mice. Substance P shortens “tail withdrawal time” from hot water; the two peptides greatly prolonged tail withdrawal time. Antidromic stimulation of the saphenous nerve (rat), known to release substance P and to induce vasodilatation plasma extravasation, was also greatly inhibited by [D-Pro²,D-Phe⁷,D-Trp⁹]-SP. These peptides presumably cause anti-nociceptor effects (analgesia) by inhibition of substance P at receptors and favor the concept that substance P is a sensory neurotransmitter of nociceptive messages.     

D-pGlu1,D-Phe2,D-Trp3,6]-LRF. A potent luteinizing hormone releasing factor antagonist in vitro and inhibitor of ovulation in the rat.

The decapeptide D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂ was synthesized in gram quantities by solid phase techniques on a methyl benzhydrylamine resin. This peptide was purified by ion exchange and partition chromatographies. It was fully characterized by amino acid analysis, tlc in four systems, optical rotation and high pressure liquid chromatography (reversed phase) using a triethyl ammonium phosphate buffer. This peptide was found to inhibit ovulation by 100% in the rat at a subcutaneous dose of 250μg in corn oil when administered at noon on the day of proestrus. Furthermore, it was found to be long acting since 1.5mg blocked ovulation in 9 out of 10 rats when administered as early as 19 hrs before the afternoon of proestrus. [D-pGlu¹, D-Phe², D-Trp^3,6]-LRF reduces the amount of LH normally secreted in response to LRF by 50% at a molar ratio (IDR₅₀) of 3/1 ([antagonist])/[LRF]). Three other analogs, equally well-characterized and with the D-pGlu¹ modification, i.e. [D-pGlu¹, D-Phe², Pro³, D-Trp⁶]-LRF, [D-pGlu¹, D-Phe², Phe³, D-Trp⁶]-LRF and [D-pGlu¹, D-Phe^2,6, D-Trp³]-LRF were found to be somewhat less potent ($\text{IDR}{}_{50}\text{=}\text{150}\text{1}\text{,}\text{60}\text{1}\text{,}\text{5}\text{1}$) respectively.     

High affinity binding sites for a somatostatin-28 analog in rat brain

Using an iodinated analog of a large (28 residues) and biologically active form of somatostatin, ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵]SS-28, it was possible to demonstrate saturable and high affinity binding sites (dissociation constant = 0.46 ± 0.04 nM) in rat cortical membranes. Somatostatin, somatostatin-28, as well as two potent analogs, [D-Trp⁸] somatostatin and [D-Trp²²] somatostatin-28, could completely displace the radiogland in the nanomolar range whereas the inactive analog Des-Trp⁸-somatostatin and the unrelated peptide GnRH showed no affinity for these binding sites; octa- and nona-peptide analogs of somatostatin were inactive. High binding was found in hippocampus, amygdala, tuberculum olfactorium, caudate-putamen and cortex; moderate binding in midbrain and hypothalamus, and no binding in the cerebellum. These results suggest that specific somatostatin receptors can be measured within the brain with ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵] SS-28 as radioligand.     

Synthesis and biological activities of pseudopeptide analogues of LH-RH: agonists and antagonists

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH₂S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the $\text{in}\text{vitro}$ activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro¹, D-Phe², D-Trp^3,6]LH-RH antagonist was completely inhibitory at 30 ng $\text{in}\text{vitro}$ and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed $\text{in}\text{vivo}$ antiovulatory activity at the 250 μg level.     

Cytotoxic Peptides: Naphthoquinonyl Derivatives of Luteinizing Hormone-Releasing Hormone

In an attempt to produce efficient cytotoxic derivatives of luteinizing hormone-releasing hormone (LH-RH), two novel 1,4-naphthoquinone derivatives of [D-Lys⁶]-LH-RH were synthesized primarily by solid-phase peptide synthesis, in good yield and high purity. The ability of each analog to produce reactive oxygen species using enzymatic reduction, i.e. NADPH-cytochrome P-450 reductase, was evaluated employing electron spin resonance (ESR) spectroscopy and spin-trapping techniques. The ESR results suggest that the novel cytotoxic analogs are extremely effective in generating oxygen radicals.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Inactivation of two hyperactive LH-RH analogues by rat hypothalamic peptidases.

Hyperactive analogues of luteinizing hormone-releasing hormone (LH-RH) are beleived to derive their properties from either increased binding affinity to anterior pituitary receptor sites or through decreased susceptibility to enzymic degradation. To investigate the latter suggestion and to examine the possible sites of hypothalamic peptidases inactivating LH-RH, D-Ser(TBU)6-EA10-LH-RH and D-Leu6-EA10-LH-RH, which are known to have considerably greater activity than LH-RH, were incubated with a hypothalamic supernatant fraction containing active peptidases degrading LH-RH, and their gonadotrophin-releasing ability after incubation with the enzymes was tested in normal, adult male rats; LH-RH was also tested in the same way. From a comparison of the relative losses of biological activity, both the LH-RH analogues treated proved to be more resistant to the hypothalamic peptidases than LH-RH itself; the D-Leu6-EA10-LH-RH retained its gonadotrophin-releasing activity longer than the D-Ser(TUB)6-EA10-LH-RH. These findings indicate that increased activity of the analogues may, in part be due to increased resistance to enzymic inactivation and suggest initial sites of cleavage at the Gly-leu and Pro-Gly NH2 bonds in the LH-RH decapeptide by the hypothalamic enzymes. Studies on the action of peptidases on LH-RH and its analogues may yield useful information in the design of peptidase with increased biological activity.