Taiji Asami

Chronic Administration of the Metastin/Kisspeptin Analog KISS1-305 or the Investigational Agent TAK-448 Suppresses Hypothalamic Pituitary Gonadal Function and Depletes Plasma Testosterone in Adult Male Rats

Metastin/kisspeptin, a hypothalamic peptide, plays a pivotal role in controlling GnRH neurons. Here we studied the effect of chronic sc administration of two kisspeptin analogs, KISS1-305 and TAK-448, on hypothalamic-pituitary-gonadal function in male rats in comparison with a GnRH analogue leuprolide or bilateral orchiectomy (ORX). The prototype polypeptide, KISS1-305 (1-4 nmol/h), caused substantial elevations of plasma LH and testosterone, followed by abrupt reductions of both hormone levels. Notably, testosterone levels were reduced to castrate levels within 3 d and remained depleted throughout the 4-wk dosing period, an effect that was faster and more pronounced than leuprolide (1 nmol/h) dosing. KISS1-305 also reduced genital organ weight more profoundly than leuprolide. In mechanistic studies, chronic KISS1-305 administration only transiently induced c-Fos expression in GnRH neurons, suggesting that GnRH-neural response was attenuated over time. Hypothalamic GnRH content was reduced to 10-20% of control at 3 wk without any changes in Gnrh mRNA expression. Dosing with the investigational peptide TAK-448 was also studied to extend our understanding of hypothalamic-pituitary functions. Similar to ORX, TAK-448 (0.1 nmol/h) depleted testosterone and decreased GnRH content by 4 wk. However, in contrast to ORX, TAK-448 decreased gonadotropin levels in pituitary and plasma samples, implying the suppression of GnRH pulses. These results suggest that chronic administration of kisspeptin analogs disrupts endogenous kisspeptin signals to suppress intrinsic GnRH pulses, perhaps by attenuating GnRH-neural response and inducing continuous GnRH leakage from the hypothalamus. The potential utility of kisspeptin analogs as novel agents to treat hormone-related diseases, including prostate cancer, is discussed.

Design, Synthesis, and Biological Evaluation of Novel Investigational Nonapeptide KISS1R Agonists with Testosterone-Suppressive Activity

Metastin/kisspeptin is a 54 amino acid peptide ligand of the KISS1R receptor and is a critical regulator of GnRH secretion. The N-terminally truncated peptide, metastin(45-54), possesses a 10-fold higher receptor-binding affinity than full-length metastin and agonistic KISS1R activity but is rapidly inactivated in rodent plasma. We have developed a decapeptide analog [D-Tyr(45),D-Trp(47),azaGly(51),Arg(Me)(53)]metastin(45-54) with improved serum stability compared with metastin(45-54) but with decreased KISS1R agonistic activity. Amino acid replacements at positions 45-47 led to an enhancement of KISS1R agonistic activity and metabolic stability. N-terminal truncation resulted in a stable nonapeptide, [D-Tyr(46),D-Pya(4)(47),azaGly(51),Arg(Me)(53)]metastin(46-54), compound 26, which displayed KISS1R binding affinities comparable to metastin(45-54) and had improved serum stability. Compound 26 reduced plasma testosterone in male rats and is the first short-length metastin analog to possess testosterone suppressive activities. Compound 26 has led to the elucidation of investigational analogs TAK-683 and TAK-448, both of which have undergone clinical evaluation for hormone-dependent diseases such as prostate cancer.

Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45-54), has 3-10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe(50)-Gly(51) and Gly(51)-Leu(52) as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and D-tryptophan (D-Trp), produced analogs that were highly stable in mouse serum. D-Trp(47) analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution.

Trypsin resistance of a decapeptide KISS1R agonist containing an Nω-methylarginine substitution

Metastin/kisspeptin is an amidated peptide with 54 amino acid residues isolated from human placental tissues as a ligand of the orphan G-protein-coupled receptor KISS1R that is expressed throughout the central nervous system and in a variety of endocrine and gonadal tissues. Compared to the full-length metastin protein, the N-terminal truncated peptide metastin(45-54) has 3-10 times higher receptor affinity and enhanced ability to increase intracellular calcium concentration which is essential for activation of protein kinases involved in intracellular signaling in a number of pathways that affect reproduction and cell migration. However, metastin(45-54) is rapidly inactivated in serum. In this study, we designed and synthesized a number of metastin(45-54) analogs and evaluated their agonistic activity and trypsin resistance. Among analogs with substitutions of arginine at position 53, N(ω)(-)methylarginine analog 8 showed 3-fold more potent agonistic activity compared with metastin(45-54). Furthermore, analog 8 was shown to resist trypsin cleavage between positions 53 and 54. This substitution may be useful in the development of other Arg-containing peptides for which the avoidance of cleavage is desired.

Design and synthesis of ETA receptor antagonists and study of ETA receptor distribution

Many oligopeptides were designed to find ETA receptor antagonists on the hypothesis that an ETA receptor can recognize two hydrophobic parts of ET-1, i.e., Val-Tyr-Phe and Ile-Ile-Trp, over a short distance. They were synthesized from the benzyl ester of the C-terminal amino acid by stepwise chain elongation using the solution method. The binding affinity of the synthetic peptides to the endothelin receptors was examined in porcine cardiac ventricular muscle membrane for ETA receptor and in bovine whole brain membrane for ETB receptor. Hexamethyleneiminocarbonyl-Leu-trp-ala-beta ala-tyr-phe (TTA-386) was selected as an ETA receptor-selective competitive antagonist to ET-1. It competed against ET-1 at ETA receptor sites and showed one-third the binding affinity of ET-1 for ETA receptor and < 1/10,000 the affinity for ETB receptor. It inhibited the ET-1-induced increase of cytosolic free calcium concentration in A-10 cells. The 125I-labeled hexapeptide (125I-TTA-386) was prepared to distinguish the distribution of ETA receptor from ETB receptor. Scatchard plot analysis of saturation binding of 125I-TTA-386 to porcine cardiac membranes showed the same Bmax value as that of 125I-ET-1. Autoradiographic studies showed that ETA receptors are most abundant in the cardiac muscle, intestine, large bowel, spleen, and testis.

Effects and Therapeutic Potentials of Kisspeptin Analogs: Regulation of the Hypothalamic-Pituitary-Gonadal Axis

The hypothalamic peptide kisspeptin (metastin), the endogenous ligand of the G protein-coupled receptor KISS1R, plays a critical role in controlling GnRH release from hypothalamic GnRH neurons and thereby regulates hypothalamic-pituitary-gonadal functions. Although the therapeutic potential of kisspeptin is attractive, its susceptibility to proteolytic degradation limits its utility. To overcome this, KISS1R agonists or antagonists as peptide analogs or small molecules have been investigated. Kisspeptin analogs have been most extensively studied by reducing the length of the peptide from the original 54 amino acids to 10 amino acids or less and by substituting key amino acid residues. Also, 2 investigational kisspeptin agonist analogs have been evaluated in clinical studies in men; in agreement with animal studies, abrupt elevations in gonadotropin and testosterone levels were observed as an acute effect, followed by rapid reductions in these hormones as a chronic effect. Some studies of small-molecule KISS1R antagonists have also been published. In this review, we present a brief overview on kisspeptin/KISS1R physiology in reproductive functions and summarize the available knowledge of both agonists and antagonists. We also focus on the kisspeptin agonist analogs by summarizing key pharmacological findings from both clinical and preclinical studies, and discuss their potential therapeutic utility.

Development and validation of sensitive sandwich ELISAs for two investigational nonapeptide metastin receptor agonists, TAK-448 and TAK-683

TAK-448 and TAK-683, investigational agents with potential utility in the treatment of prostate cancer, are potent low molecular weight metastin receptor agonists consisting of nine amino acids. Monoclonal antibodies (mAbs) against these agents were developed to facilitate their evaluation in preclinical studies. Six mAbs were obtained from four immunogens. Three mAbs recognized the C-terminal of TAK-683 and TAK-448, two recognized the N-terminal of TAK-683, and one recognized the N-terminal of TAK-448. Using various combinations of these six mAbs, sandwich ELISAs for TAK-448 and TAK-683 were developed. These assays were highly sensitive, specific, and accurate. The detection limit for TAK-448 and TAK-683 was 3 and 5 pg/mL, respectively, and there was no interference from rat plasma, rat metastin, or analogs of TAK-448/TAK-683. Recovery achieved ≤±10% with intra-/inter-day assay precision coefficient of variation <10%. The assay demonstrated high stability and sample pre-treatment was not required. Each assay detected the dose-dependent concentration of TAK-448 and TAK-683 in blood 24h after a single intravenous administration of 0.1 and 1mg/kg doses. In conclusion, sensitive sandwich ELISAs were developed to detect the small peptides TAK-448 and TAK-683. The novel assays reliably quantified these nonapeptides in rat plasma, and thus will be useful for preclinical studies of these agents. This methodology may be applicable to the development of similar assays for other short peptides.

Physicochemically and pharmacokinetically stable nonapeptide KISS1 receptor agonists with highly potent testosterone-suppressive activity.

Modifications of metastin(45-54) produced peptide analogues with higher metabolic stability than metastin(45-54). N-terminally truncated nonapeptide 4 ([D-Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46-54)) is a representative compound with both potent agonistic activity and metabolic stability. Although 4 had more potent testosterone-suppressant activity than metastin, it possessed physicochemical instability at pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn48 and Ser49; substitution of Ser49 with Thr49 reduced this instability and maintained KISS1 receptor agonistic activity. Furthermore, [D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54) (14) showed 2-fold greater [Ca2+]i-mobilizing activity than metastin(45-54) and an apparent increase in physicochemical stability. N-terminal acetylation of 14 resulted in the most potent analogue, 22 (Ac-[D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54)). With continuous administration, 22 possessed 10-50-fold more potent testosterone-suppressive activity in rats than 4. These results suggested that a controlled release of short-length KISS1 receptor agonists can suppress the hypothalamic-pituitary-gonadal axis and reduce testosterone levels. Compound 22 was selected for further preclinical evaluation for hormone-dependent diseases.

A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects

Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.

A Short-Length Peptide YY Analogue with Anorectic Effect in Mice

Peripheral administration of PYY3–36, a fragment of peptide YY (PYY), has been reported to reduce food intake by activating the neuropeptide Y2 receptor (Y2R). An N-terminally truncated PYY analogue, benzoyl-[Ala26,Ile28,31]PYY(25–36) (1), showed a relatively potent agonist activity for Y2R but a weak anorectic activity by intraperitoneal administration (2000 nmol/kg) in lean mice because of its markedly poor biological stability in the mouse serum. Notably, two cyclohexylalanine (Cha) substitutions for Tyr residues at positions 27 and 36 (4) improved the stability in the mouse serum concomitant with enhanced anorectic activity. Further optimization at positions 27, 28, 30, and 31 revealed that 21, containing Cha28 and Aib31 residues, showed a more potent anorectic activity than PYY3–36 at a low dose of 300 nmol/kg. The minimum effective dose by intraperitoneal administration of 21 was 30 nmol/kg (ca. 52 μg/kg) in mice, suggesting the biologic potential of short-length PYY3–36 analogues with a potent anorectic effect.