Journal of Pharmaceutical and Biomedical Analysis 70 (2012) 369–377 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al h om epage: www.elsevier.com/locate/jpba Development and validation of sensitive sandwich ELISAs for two investigational nonapeptide metastin receptor agonists, TAK-448 and TAK-683 Nobuyo Yoshida a,∗ , Naoki Nishizawa b , Hisanori Matsui c , Yuu Moriya d , Chieko Kitada b , Taiji Asami b , Hirokazu Matsumoto a a Advanced Science Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan b Medicinal Chemistry Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan c Oncology Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan d Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan a r t i c l e i n f o Article history: Received 17 February 2012 Received in revised form 18 May 2012 Accepted 22 May 2012 Available online 1 June 2012 Keywords: TAK-448 TAK-683 Metastin Sandwich ELISA Monoclonal antibodies a b s t r a c t 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 24 h after a single intravenous administration of 0.1 and 1 mg/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. © 2012 Published by Elsevier B.V. 1. Introduction Metastin (also termed kisspeptin) is a novel endogenous reg- ulatory peptide that acts as the ligand of GPR54, an orphan G protein-coupled receptor [1–3]. Metastin is a 54 amino acid polypeptide that functions as a key regulator of hypothala- mic gonadotropin-releasing hormone neurons in several species, including humans [4–8]. Recently, it was reported that chronic or Abbreviations: ANA, analog; cEIA, competitive enzyme immunoassay; CV, coef- ficient of variation; HRP, horseradish peroxidase; IC50, half maximal inhibitory concentration; KLH, keyhole-limpet hemocyanin; p-6, position 6; PK, pharmacoki- netics; RE, relative error. ∗ Corresponding author. Tel.: +81 466 32 1344; fax: +81 466 29 4417. E-mail addresses: nobuyo.yoshida@takeda.com (N. Yoshida), naoki.nishizawa@takeda.com (N. Nishizawa), matsui.hisanori@takeda.com (H. Matsui), yuu.moriya@takeda.com (Y. Moriya), taiji.asami@takeda.com (T. Asami), hirokazu.matsumoto@takeda.com (H. Matsumoto). repeated administration of metastin led to inhibition of pituitary or gonadal function in rats, monkeys, and humans [9–12]. These findings suggest that the metastin/GPR54 system is a potential therapeutic target for sex hormone-related diseases, but the bio- logical activity and stability of metastin need to be improved to be useful for clinical development. Therefore, two investigational metastin analogs (TAK-448 and TAK-683) have been developed that show enhanced in vitro activity and improved in vivo stability [13,14]. Both TAK-448 and TAK-683 are synthetic peptides consisting of nine amino acids and both are analogs of the C-terminal decapep- tide of metastin, which binds to GPR54 with similar affinity to that of the full-length peptide. The only difference between TAK-448 and TAK-683 is the second amino acid of the N-terminal, which is hydroxyproline (Hyp) and d-tryptophan (d-Trp) for each peptide, respectively. In our previous investigation, chronic administration of either TAK-448 or TAK-683 at a dose of <30 pmol/kg/h sup- pressed the plasma testosterone concentration to the castration 0731-7085/$ – see front matter © 2012 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jpba.2012.05.033