Design and Synthesis of an Investigational Nonapeptide KISS1 Receptor (KISS1R) Agonist, Ac- d -Tyr-Hydroxyproline (Hyp)-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2 (TAK-448), with Highly Potent Testosterone-Suppressive Activity and Excellent Water Solubility

Article abstract of DOI:10.1021/acs.jmedchem.6b00379

Metastin/kisspeptin is an endogenous ligand of KISS1 Receptor (KISS1R). Metastin and KISS1R are suggested to play crucial roles in regulating the secretion of gonadotropin-releasing hormone (GnRH), and continuous administration of metastin derivatives attenuated the plasma testosterone levels in male rats. Our optimization studies of metastin derivatives led to the discovery of 1 (Ac-d-Tyr-d-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH₂, TAK-683), which suppressed plasma testosterone in rats at lower doses than those of leuprolide. Although 1 possessed extremely potent pharmacological activity, 20 mg/mL aqueous solution of 1 has a gel formation property. In order to improve this physicochemical property, we substituted d-Trp at position 47 with a variety of amino acids; we identified that substitution with cyclic amino acids, which could change peptide conformation, retained its potency. Especially, analogue 24 (TAK-448) with trans-4-hydroxyproline (Hyp) at position 47 showed not only superior pharmacological activity to 1 but also excellent water solubility. Furthermore, 20 mg/mL aqueous solution of 24 did not show gel formation up to 5 days.

Full text of DOI:10.1021/acs.jmedchem.6b00379

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Article
Design and Synthesis of an Investigational Nonapeptide
KISS1 Receptor (KISS1R) Agonist Ac-D-Tyr-Hyp-Asn-Thr-
2
Phe-azaGly-Leu-Arg(Me)-Trp-NH (TAK-448) with Highly Potent
Testosterone-Suppressive Activity and Excellent Water Solubility
Naoki Nishizawa, Yoshihiro Takatsu, Satoshi Kumano, Atsushi Kiba, Junko Ban,
Shunichirou Tsutsumi, Hisanori Matsui, Shin-ichi Matsumoto, Masashi Yamaguchi,
Yukihiro Ikeda, Masami Kusaka, Tetsuya Ohtaki, Fumio Itoh, and Taiji Asami
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00379 • Publication Date (Web): 02 Sep 2016
Downloaded from http://pubs.acs.org on September 3, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society.
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Design and Synthesis of an Investigational Nonapeptide KISS1 Receptor
(KISS1R) Agonist Ac-D-Tyr-Hyp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2
(TAK-448) with Highly Potent Testosterone-Suppressive Activity and Excellent
Water Solubility
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Naoki Nishizawa*,†, Yoshihiro Takatsu†, Satoshi Kumano†, Atsushi Kiba†, Junko Ban‡,
Shunichirou Tsutsumi‡, Hisanori Matsui†, Shin-ichi Matsumoto†, Masashi Yamaguchi†,
Yukihiro Ikeda‡, Masami Kusaka‡, Tetsuya Ohtaki†, Fumio Itoh†, Taiji Asami*,†
†Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, Fujisawa,
Japan
‡CMC Center, Takeda Pharmaceutical Company Ltd, Osaka, Japan
ABSTRACT
Metastin/kisspeptin is an endogenous ligand of KISS1R. Metastin and KISS1R are
suggested to play crucial roles in regulating the secretion of GnRH and continuous
administration of metastin derivatives attenuated the plasma testosterone levels in
male rats. Our optimization studies of metastin derivatives led to the discovery of 1
(Ac-D-Tyr-D-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2, TAK-683), which
suppressed plasma testosterone in rats at lower doses than those of leuprolide.
Although 1 possessed an extremely potent pharmacological activity, 20-mg/mL
aqueous solution of 1 has a gel formation property. In order to improve this
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physicochemical property, we substituted D-Trp at position 47 with a variety of amino
acids; we identified that substitution with cyclic amino acids, which could change
peptide conformation, retained its potency. Especially, Hyp47 analogue 24 (TAK-448)
showed not only superior pharmacological activity to 1, but also excellent water
solubility. Furthermore, 20-mg/mL aqueous solution of 24 did not show a gel
formation up to five days.
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INTRODUCTION
Metastin/kisspeptin is a peptide with 54 amino acids isolated from human placental
tissues, as an endogenous ligand of an orphan G protein-coupled receptor GPR54, also
known as hOT7T175, and AXOR12, currently termed KISS1 receptor (KISS1R).1-6
The mRNA of the KISS1 gene, which encodes metastin, is expressed in the rodent and
human placenta and brain. The expression of metastin and KISS1R mRNA shows
similar pattern in various regions of the rat and human nervous systems. KISS1R is
expressed mainly in hypothalamic tissues; the expression levels in other brain areas
are low.4−11
At first, metastin and KISS1R were thought to correlate with cancer metastasis;1,2
however, subsequent studies clarified that metastin-KISS1R signaling plays a key role
in reproduction and regulation of the hypothalamic-pituitary-gonadal (HPG) axis. The
relationship between inactivating mutations of KISS1R and idiopathic
hypogonadotropic hypogonadism in humans and mice was reported by two
independent groups in 2003.9,10 Following studies suggested that metastin significantly
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induced gonadotropin-releasing hormone (GnRH) release in mammals and other
species.4,12 Single administration of metastin markedly elevated the plasma levels of
the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in adult male
Wistar rats.13 In contrast, chronic administration of metastin analogues was found to
increase both plasma LH and testosterone levels transiently, followed by a profound
decrease below the detectable level in male rats.14-16 Our immunohistochemical
analysis of rat brain tissue suggested the suppression of intrinsic GnRH pulses and
downstream pituitary-gonadal functions by the chronic administration of metastin
analogues. This may be due to the attenuation of the responsiveness of GnRH neurons
to endogenous metastin stimulation and the stimulation of GnRH neurons to
continuously release low levels of GnRH.14 The central roles of kisspeptin/KISS1R
signaling suggest that activation or suppression of the HPG axis by appropriate dosing
of KISS1R agonists should potentially prevent or treat a variety of
sex-hormone-dependent diseases.
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Native metastin is the 54-amino-acid peptide, but metastin (45-54), which is an
N-terminally truncated analogue, shows full in vitro activities. Furthermore, the
short-length peptide displays higher potency (3-10 times) than native metastin,1 which
indicates that the C-terminal region of metastin contains the active core of biological
activity. Generally, peptides, which are thought to be poor drug candidates, are fragile
molecules rapidly degraded by various exogenous proteases in blood circulation.
Several groups and we have reported that rational modification of metastin (45-54) led
to increased biological stability and enhanced the therapeutic potential; however, few
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reports demonstrated in vivo activity.15-24 Our previous optimization studies of
N-terminal truncated metastin derivatives led to the discovery of a nonapeptide 1
(Ac-D-Tyr-D-Trp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2, TAK-683) as a novel
class of anti-prostate cancer drug.16,25 Compound 1 suppressed plasma testosterone in
rats at lower doses than a GnRH super agonist, leuprolide, with four-week sustained
administration. However, the aqueous solubility of 1 was much lower than that of
leuprolide, and 20-mg/mL aqueous solution of 1 has a gel formation property.
Therefore, we performed synthetic studies to improve these physicochemical
properties of 1.
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RESULTS
Chemistry
We synthesized all peptides by use of the standard Fmoc-based solid phase synthetic
method. Following purification of the obtained crude peptides using preparative HPLC
led to homogeneity. The purity of each peptide was verified by analytical
reversed-phase and ion-exchange HPLC, and the structure was assigned using
matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry (MS).
Biological Activities
All synthesized peptides were evaluated for their agonistic activities as first
screening by fluorometric imaging plate reader (FLIPR) assay, which determined the
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ligand-induced elevation of Ca2+ in Chinese hamster ovary (CHO) cells expressing
human KISS1R. We observed the maximum response in the presence of peptide and
expressed the response as a percent of the corrected one to a maximal concentration of
the control peptide, metastin (45-54). Nonlinear regression of smoothed data
determined the potency (EC50) of peptides. Peptides with good agonistic activities
were further evaluated for their testosterone suppressive activities in rats with a
six-day sustained subcutaneous administration study (0.1 nmol/h, i.e., 50
nmol/kg/week) as second screening. In addition, low-dose administrations of the
selected compounds, which showed good activities at the second screening, were
carried out to evaluate their minimum effective doses. Results of in vivo screening are
shown in Table 1. The minimum effective doses were defined as the minimum dosing
rate to suppress the testosterone level below the measurement limit in all individual
rats (testosterone < 0.04 ng/mL of plasma level, n=5).
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Design and syntheses of comprehensive nonapeptide metastin analogues
In order to improve the physicochemical properties of 1, some of the component
amino acids should be replaced with appropriate amino acids. First, we performed
sequential replacement studies at each residue of 1 with a variety of amino acids;
small side chain (Ala), branched (Leu), aromatic (Phe), acidic (Asp and Glu) and basic
(Lys) amino acids, then determined no possibility to improve physicochemical
properties by substitution between positions 48 and 54 (data not shown). On the other
hand, substitutions at the N-terminus and position 47 retained capabilities to improve
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the physicochemical characteristics. Especially, amino acid replacements of D-Trp at
position 47 with small side chain amino acid D-Ala 2, aliphatic D-Leu 3, basic D-Lys 4,
acidic D-Glu 5 and stereoisomer Trp 6 retained in vitro/in vivo activities (Table 1).
These results suggested that the side chain at position 47 was not important for the
interaction with KISS1R, and might be useful to improve physicochemical
characteristics of 1. Thus, we focused on analogues substituted at position 47.
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Design and syntheses of nonapeptide metastin analogues substituted at position
47
There were two different ways to increase peptide solubility. One possible way was
to replace D-Trp47 with a hydrophilic amino acid such as an acidic (e.g., Asp and Glu),
basic (e.g., Lys and Arg) or neutral amino acid (e.g., Ser and Thr). The second
approach was to replace D-Trp47 with L-amino acids, N-substituted amino acids (e.g.,
N-methylalanine (MeAla)), β-amino acids (e.g., β-alanine (β-Ala)), cyclic amino acids
(e.g., Pro) and achiral amino acids (i.e. Gly and 2-aminoisobutyric acid (Aib)), which
could change the peptide conformation.
As described above, a broad spectrum of amino acids were tolerable in position 47,
and especially L-Trp substitution 6 was superior to D-Trp 1 in terms of testosterone
suppressive activity (Table 1). Generally, D-amino acid substitutions enhance
metabolic stability of the scissile peptide bond. D-Amino acids at position 47 had been
thought to contribute to the increased stability of metastin analogues in circulating
blood. However, Trp47 analogue 6 was found to show enhanced in vivo activity, which
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led us to synthesize L-amino acid substitutions as well as D-amino acid analogues in
order to obtain more potent and water soluble analogues than those of 1.
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On the basis of these results, we prepared Lys47 7 and Glu47 8 analogues, which
enhanced not only agonistic activities but also in vivo activities. The activities of 7 and
8 were more potent than those of their corresponding D-amino-acid analogues 4 and 5,
respectively. Compounds 7 and 8 showed comparable or more potent activity with
respect to that of 1 in a six-day sustained subcutaneous administration study in rats.
It was expected that the polar substitution of Glu or Lys at position 47 would enhance
the water-solubility of each compound.
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Substitutions at position 47 with small side chain amino acids, N-substituted amino
acids and achiral amino acids were also expected to improve physicochemical
properties by changing the peptide conformation. Similar to the results for Trp and
D-Trp analogues, Ala analogue 10 demonstrated more potent activity than that of
D-Ala analogue 2. On the other hand, replacement with N-substituted amino acids (i.e.,
MeAla 12, D-MeAla 13 and sarcosine (Sar) 14) reduced in vitro/in vivo activities.
Interestingly, replacement with achiral amino acids (i.e., Gly 9 and Aib 11) retained
the pharmacological activities, which were comparable or more potent than those of 1.
Gly 9 and Aib 11 analogues were predicted to have better water solubility because of
the removal of hydrophobic indole moiety of 1.
Replacements of Trp47 6 with β-amino acids, β-Ala 15, 2-aminobenzoic acid
(Abz(2)) 16 azetidine-3-carboxylic acid (Aze(3)) 21 and piperidine-3-carboxylic acid
(Pic(3)) 22 did not provide good results for biological activities. [Ca2+]i–mobilizing
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activities of 15, 16, 21 and 22 for human KISS1R decreased more than three-fold
compared to 6. Testosterone suppressive activities of 15, 16 and 22 were not observed
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with
a
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rats.
Compound
23
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1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), which is a constrained Phe
analogue, had good in vitro activities; however, significant testosterone suppressive
activity was not demonstrated at 0.01 nmol/h. On the other hand, 17, which has
L-form cyclic α-amino acid Pro at position 47, showed higher pharmacological
activities in rats than those of 1, although in vitro activities of 17 were lower than
those of 1. These results led us to consider that the analogues replaced with cyclic
amino acids at position 47 might induce an increase in both testosterone suppressive
activity and water solubility. Therefore, we carried out further studies on replacements
with other cyclic α-amino acids.
As a result, we discovered the following:
(1) Four-membered cyclic α-amino acid (azetidine-2-carboxylic acid (Aze(2)) 19 and
cyclic β-amino acid (azetidine-3-carboxylic acid (Aze(3)) 21 showed comparable
agonistic activities; on the other hand, as for six-membered cyclic amino acids, cyclic
α-amino acid (piperidine-2-carboxylic acid (Pic(2)) 20) was more favorable than
cyclic β-amino acid (Pic(3) 22).
(2) When comparing cyclic α-amino acids, pyrrolidine ring 17 was preferable to other
rings (19 and 20).
(3) Insertion of a sulfur atom to the ring (thiazolidine-2-carboxylic acid (Thz) 25)
proved ineffective for in vivo activity. Consequently, trans-4-hydroxyproline (Hyp) 24
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was the most suitable amino acid at this position among the cyclic amino acids
examined in this study. Hyp47 analogue 24 completely suppressed the blood
testosterone level in a sustained administration study of rats at a dose rate of 0.01
nmol/h. Moreover, plasma testosterone in four-fifths of individuals was suppressed at
a dose rate of 0.003 nmol/h. The most striking aspect of the present results is that the
effect of 24 was approximately three-fold higher than that of 1.
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Design and syntheses of nonapeptide metastin analogues with combined
replacements at positions 47 and 50
It has been reported that halogenation of peptides may change the physicochemical
properties and enhance tissue distribution and pharmacokinetic stability.26-30 We
thought introduction of a halogen atom into the benzene ring of Phe at position 50
might cause an increase in pharmacological activity, thus we examined further
modifications of the position 47 substituted analogues.
Firstly, Phe at position 50 of compound 1 was replaced by 2-fluorophenylalanine
(Phe(2F)), 3-fluorophenylalanine (Phe(3F)), 4-fluorophenylalanine (Phe(4F)) and
4-chlorophenylalanine (Phe(4Cl)). Among these peptides, Phe(3F)50 analogue 27 had
the highest potency in rat pharmacological studies (Table 1). Moreover, we performed
similar modifications on the analogues with Trp47, Lys47, Glu47, Gly47, Aib47 and Hyp47,
respectively. As a result, Trp47 analogue 30 represented decreased testosterone
suppressive activities in rats. On the other hand, a combination of Lys47 and Phe(3F)50
31 resulted in equivalent activities to those of the corresponding parent (Phe(3F)50)
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compound 27; and 32 (Glu47 and Phe(3F)50) demonstrated a slight increase in activity.
Among all halogenated analogues, Glu analogue 32 and Hyp analogue 35 suppressed
plasma testosterone in all rats at a dose of 0.01 nmol/h; however, none of the peptides
including Gly 33 and Aib 34 analogues had significant testosterone suppressive
activity at the lower dose, 0.003 nmol/h.
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Although in vitro agonistic activity against rat KISS1R of 32 was lower than those
of 1 and 27, 32 showed good pharmacological activities with in vivo studies in rats,
and such data suggests that the pharmacokinetic parameters of 32 were improved from
those of 1 and 27. Since the agonistic activity of 32 on human KISS1R was higher
than that on rat KISS1R, testosterone suppressive activity of 32 in humans might be
more potent than that of 1.
Continuous administration of compounds 1, 24 and 32 in male rats
In vivo efficacy of the two selected derivatives were evaluated in terms of
steady-state concentration of plasma testosterone after one week and four weeks of
continuous administration of peptides in intact male rats. Approximately steady-state
testosterone concentrations by the infusion of all peptides were produced up to one
week. We treated the value below the detection limit (0.04 ng/mL of plasma level) in
radioimmunoassay as 0.04.
As is the case with 1,16 continuous administration of 24 or 32 at a dose of 30 pmol/h
for one week inhibited plasma testosterone in all rats (Figure 1, Table 2). In a
one-week study, 10 pmol/h dosing of 1 did not attenuate testosterone concentrations
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completely in all rats. On the other hand, the plasma testosterone levels in all rats
decreased below the limit of measurement in radioimmunoassay when 24 or 32 was
dosed at 10 pmol/h. Furthermore, plasma testosterone for all rats administered at 10
pmol/h of 24 was suppressed for one week. As for the four-week studies, testosterone
suppressive activities of 24 and 32 were more potent than those of 1; approximately
one-third dose of 24 showed similar efficacy to that of 1.
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Pharmacokinetic parameters of compounds 24 and 32 in rats
The pharmacokinetic parameters of 1 and highly potent analogues, 24 and 32, were
examined by use of IGS/SD rats. All peptides were injected intravenously and
subcutaneously at 1 mg/kg. Mean pharmacokinetic variable estimates of 1 were as
follows: C5min, 2509 ng/mL; VdSS, 1225 mL/kg; and CLtotal, 819 mL/h/kg (Table 3).
Area under the plasma concentration-time curve (AUC0-24h) values after intravenous
injections improved slightly with the replacement of D-Trp47 in 1 with Hyp 24 or Glu
32. The VdSS values for 24 and 32 were 0.3-fold lower than that of 1, probably because
of the reduced hydrophobicity. Reduced metastin analogues are supposed to be
excreted by the kidneys and urine; therefore, the decrease in the VdSS values attenuated
the CLtotal values of 24 and 32 after administration. The bioavailability (BA) values of
24 and 32 increased dramatically from 9.4 for 1 to 43.9 and 94.5, respectively. The
hydrophilic substitution at position 47 is thought to cause the improvement in the
absorption rates across subcutaneous tissues. These replacements are extremely
effective in terms of improving the AUC values in subcutaneous dosing.
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Water solubility of nonapeptide metastin analogues substituted at position 47
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On the basis of the above-mentioned results, we selected seven compounds,
including 24 and 32, which had different amino acids at position 47, and determined
their solubility in water at a concentration of 20 mg/mL (Table 4). Gel-forming ability
of these compounds was also evaluated visually up to five days after dissolution.
Initially 1 was dissolved completely; however, the clear solution turned into gel within
three hours at room temperature. Among the six compounds synthesized in this study,
Aib47 11, Hyp47 24 and Lys47 31 analogues showed excellent water solubility at the
same concentrations, and gelation was not observed up to five days. Furthermore, 24
was completely dissolved in water up to the concentration of 500 mg/mL. Meanwhile,
the Trp47 6 and Glu47 32 analogues exhibited poor solubility and the solution turned
into gel as with 1. The remarkable difference in water solubility between 1 and 24
with only one amino acid substitution was assumed to be due to the difference in total
hydrophobicity and global conformation of these peptides. Preliminary circular
dichroism (CD) analysis of 1 and 24 in water showed a slight decrease in β-sheets
content (1: 34.8%, 24: 21.9%) (Figure S1). Further investigation of the
physicochemical analysis of 2 and 24 would clarify the effect of the amino acid
substitution to improve water solubility and avoid gelation.
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CONCLUSION
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In this study, we performed synthetic studies to improve the physicochemical
properties of an aqueous solution of 1. As a result, we determined that replacement at
position 47 of 1 can modify the physicochemical characteristics without any loss in
pharmacological activity. By replacing D-Trp47 with Lys, Aib and Hyp, 20-mg/mL
aqueous solution of the compounds acquired no gelation properties. Among these
peptides, the Hyp47-substituted analogue 24 was completely soluble in water at a
concentration of 500 mg/mL. The water solubility of 24 was dramatically improved
compared with that of 1. Testosterone suppressive activities of 24 in rats were
approximately three times more potent than of 1. The minimum effective doses of 1
and 24 for decreasing plasma testosterone to levels similar to surgical castration were
100 and 30 pmol/h, respectively. The extremely high pharmacological activity and the
excellent solubility of 24 are expected to promote development of sustained-release
devices for a novel class of drugs for sex hormone-dependent diseases, e.g., prostate
cancer. These results of in vitro and in vivo studies strongly suggested that a
controlled release of KISS1R agonists suppress the HPG axis to achieve suppression
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of
testosterone
concentrations
and
prompted
us
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24
(Ac-D-Tyr-Hyp-Asn-Thr-Phe-azaGly-Leu-Arg(Me)-Trp-NH2, TAK-448) as a clinical
candidate of metastin analogue.31-35
EXPERIMENTAL SECTION
Instruments and Materials
Solid phase syntheses were carried out by manual Fmoc SPPS consisting of
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Fmoc cleavage with 20% piperidine/DMF (20 min) and Fmoc amino acid
condensation reaction using DIPCDI/HOAt (4 eq.), or by automated peptide
synthesizer ABI 433A (as per the Fmoc/DCC/HOBt 0.25-mmol protocol). All final
compounds were purified to homogeneity of ≥95% in RP-HPLC analysis with UV
detection at 210 and 254 nm using the following two methods: MERCK Chromolith®
FastGradient RP-18 end-capped column (2.0 × 50 mm), linear density gradient elution
with eluents A/B = 95/5−25/75 (14 min), using 0.1% TFA in water as eluent A and
0.1% TFA-containing acetonitrile as eluent B, flow rate: 0.5 mL/min; and
Phenomenex Luna® 5-µm SCX 100 Å column (4.6 × 100 mm), linear density gradient
elution with eluents A/B = 100/0−0/100 (20 min) using 20-mM sodium phosphate
buffer (pH 2.1)/acetonitrile (75/25) as eluent A and 0.5-M NaCl containing a 20-mM
sodium phosphate buffer (pH 2.1)/acetonitrile (75/25) as eluent B, flow rate: 1.0
mL/min. The identity of the peptides was confirmed by MALDI-TOF-MS analysis.
Commercially available amino acid derivatives and resins were purchased from
Novabiochem, Watanabe Chemical Industries, Peptide Institute, Bachem, AnaSpec,
Chem-Impex International and American Peptide Company, while other reagents, such
as coupling and de-protection reagents, were purchased from Wako Pure Chemical
Industries, Novabiochem, Watanabe Chemical Industries and Nacalai Tesque.
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General procedure for synthesis of metastin analogues
All peptides were synthesized in the similar manner as the previous compounds.16
Detailed
synthesis
procedure
for
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Ac-[D-Tyr46,Hyp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54) 24 is described as
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a representative compound (Supporting Information).
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Calcium Mobilization Assay
As per the method described in the previous article, the increased activity of the
intracellular Ca ion level was measured using FLIPR.16 A change in the intracellular
calcium ion level was measured against the passage of time. The ratio of EC50 values
of intracellular Ca ion-increasing activity between samples and metastin (45-54) are
shown in Table 1. See Supporting Information for the detailed assay protocol.
Evaluation of blood testosterone level decreasing effect of metastin peptide
derivatives using mature male rat
A metastin peptide derivative (hereinafter referred to as peptide) was dissolved in
50% DMSO aqueous solution (Otsuka Pharmaceutical Co., Ltd) to prepare a peptide
solution with a concentration of 0.1, 0.03, 0.01 or 0.003 mM. Five ALZET osmotic
pumps (Model 2001, 0.2 mL in volume, release rate: 0.001 mL/h, DURECT
Corporation) were filled with this peptide solution. The ALZET pumps filled with the
peptide solution were implanted subcutaneously in five nine-week-old CD (SD) IGS
male rats (Charles River Japan, Inc.) on their backs under ether anesthesia by one
pump/animal. For negative control, five ALZET osmotic pumps were filled with 50%
DMSO aqueous solution, which were similarly implanted in five males CD (SD) IGS
rats, respectively. These pump-implanted rats were fed for six days under normal
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feeding conditions. After weighing, the animal was decapitated to collect blood. After
0.03 mL/mL blood of aprotinin solution (Trasylol, Bayer) containing 0.1 g/mL EDTA
2Na was added to the blood, the plasma was separated and recovered by centrifugation
at 1,800 × g for 25 min. From the plasma obtained, 0.05 mL was used for
radioimmunoassay (DPC Total Testosterone Kit, Diagnostic Products Corporation) to
measure the plasma testosterone level in each rat.
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Pharmacokinetics of metastin analogues in rats
Ten groups of SD rats (three per group) were used to evaluate the pharmacokinetics
of metastin analogues as follows. 1, 24 or 32 was given intravenously or
subcutaneously to rats at a dose of 1 mg/kg. Blood samples of 100 μL to determine the
concentration of 1, 24 or 32 were collected at predetermined time intervals, namely 5,
10, 15, and 30 minutes, and 1, 2, 4, 8, and 24 hours after injections. The plasma was
collected by removing plasma proteins using 0.2% formic acid/methanol (1:4, v/v) and
acetonitrile, followed by centrifugation at 15,000 rpm for five minutes. The
supernatant obtained was treated with 0.4% aqueous formic acid and centrifuged at
15,000 rpm for five minutes. The plasma concentration of 1, 24 or 32 was determined
by liquid chromatography-mass spectrometry. The pharmacokinetic parameters
associated with each group were assessed using noncompartmental analysis. The
plasma concentration five minutes after injection (C5min), area under the plasma
concentration-time curve up to the last time (AUC0-24h), mean residence time (MRT),
volume of distribution at steady state (Vdss), and total body clearance (CLtotal) for each
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rat after intravenous administration, were obtained. The maximum plasma
concentration (Cmax), time to maximum plasma concentration (Tmax), area under the
plasma concentration-time curve up to last time (AUC0-24h), mean residence time
(MRT), and bioavailability (BA) for each rat after subcutaneous administration, were
also obtained.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
1. General procedure for synthesis of metastin analogues (PDF)
2. Table S1. Chemical data for nonapeptide metastin analogues (PDF)
3. Calcium mobilization assay (PDF)
4. Molecular formula strings and the associated biochemical and biological data
(CSV)
5. Figure S1. Far-UV circular dichroism spectra of metastin analogues (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: taiji.asami@takeda.com. Phone: +81-466-32-1186. Fax: +81-466-29-4453.
*E-mail: naoki.nishizawa@takeda.com. Phone: +81-466-32-1187. Fax:
+81-466-29-4453.
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Notes
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The authors declare no competing financial interest.
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ABBREVIATIONS USED36
Abz(2), 2-aminobenzoic acid; Ac2O, acetic anhydride; Aib, 2-aminoisobutylic acid;
Nω-methylarginine;
Arg(Boc2,Me),
β-Ala,
β-alanine;
Arg(Me),
Nω,ω'-bis-tert-butoxycarbonyl-Nω-methylarginine; Aze(2), azetidine-2-carboxylic acid;
Aze(3), azetidine-3-carboxylic acid; Boc, tert-butyloxycarbonyl; azaGly, azaglycine;
tBu, tert-butyl; CDI, N,N'-carbonyldiimidazole; DIEA, N,N'-diisopropylethylamine;
DIPCDI, N,N'-diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide;
Fmoc,
9-fluorenylmethoxycarbonyl;
HOBt, N-hydroxybenzotriazole;
HOAt,
Hyp,
N-1-hydroxy-7-azabenzotriazole;
trans-4-hydroxyproline; MeAla, N-methylalanine; MeGly, N-methylglycine; Pbf,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;
Phe(4Cl),
Phe(3F),
Pic(2),
4-chlorophenylalanine
3-fluorophenylalanine
;
;
Phe(2F),
Phe(4F),
2-fluorophenylalanine
4-fluorophenylalanine
;
;
piperidine-2-carboxylic acid; Pic(3), piperidine-3-carboxylic acid; RP-HPLC,
reversed-phase high performance liquid chromatography; Sar, sarcosine,
N-methylglycine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Thz,
thiazolidine-4-carboxylic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid;
TIS: triisopropylsilane; Trt, triphenylmethyl (trityl). Amino acid symbols denote
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L-configuration unless indicated otherwise.
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of [3H][D-Pen2, D-Pen5]enkephalin, and two halogenated analogs after
intravenous administration. J. Pharmacol. Exp. Ther. 1991, 259, 1109-1117.
(30) Liu, H-M; Liu, X. F.; Yao, J. L.; Wang, C. L.; Yu, Y.; Wang, R. Utilization of
combined chemical modifications to enhance the blood–brain barrier
permeability and pharmacological activity of endomorphin-1. J.Pharmacol. Exp.
Ther. 2006, 319, 308-316.
(31) Asami, T.; Nishizawa, N. Metastin derivatives and use thereof. WO2007072997.
(32) Kuze, Y.; Jinno, F.; Kondo, T.; Asahi, S. Highly sensitive liquid
chromatography-tandem mass spectrometry method for quantification of
TAK-448 in human plasma. J. Pharm. Biomed. Anal. 2013, 83, 75-81.
(33) Yoshida, N.; Nishizawa, N.; Matsui, H.; Moriya, Y.; Kitada, C.; Asami, T.;
Matsumoto, H. Development and validation of sensitive sandwich ELISAs for
two investigational nonapeptide metastin receptor agonists, TAK-448 and
TAK-683. J. Pharm. Biomed. Anal. 2012, 70, 369-377.
(34) Matsui, H.; Masaki, T.; Akinaga, Y.; Kiba, A.; Takatsu, Y.; Nakata, D.; Tanaka,
A.; Ban, J.; Matsumoto, S. I.; Kumano, S.; Suzuki, A.; Ikeda, Y.; Yamaguchi,
M.; Watanabe, T.; Ohtaki, T.; Kusaka, M. Pharmacologic profiles of
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7
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9
10
11
investigational kisspeptin/metastin analogues, TAK-448 and TAK-683, in adult
male rats in comparison to the GnRH analogue leuprolide. Eur. J. Pharmacol.
2014, 735, 77-85.
(35) MacLean, DB.; Matsui, H.; Suri, A.; Neuwirth, R.; Colombel, M. Sustained
exposure to the investigational Kisspeptin analog, TAK-448, down-regulates
testosterone into the castration range in healthy males and in patients with
prostate cancer: results from two Phase 1 studies. J. Clin. Endocrinol. Metab.
2014, 99, E1445-1153.
12
13
14
15
16
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18
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20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(36) Abbreviations used for amino acids and designation of peptides follow the rules
of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem.
1972, 247, 977−983. Amino acid symbols denote the L-configuration unless
indicated otherwise.
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Table 1. Biological activities of metastin (45-54) and nonapeptide metastin
analogues substituted at positions 47 and 50
5
6
Ac-D-Tyr-AA47-Asn-Thr-AA50-azaGly-Leu-Arg(Me)-Trp-NH2
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
testosterone suppressive activityb
number of rats (T < 0.04 ng/mL, n = 5)
agonist activities EC50 nM (95% CI)a
human
compound
AA47
AA50
rat
4.2 (3.0−6.1)
1.3 (1.1−1.6)
16 (7.9−33)
12 (5.7−26)
18 (6.5−50)
40 (21−79)
4.8 (2.5−9.3)
6.8 (3.3−14)
11 (5.9−21)
46 (10−200)
6.0 (3.2−11)
17 (6.4−47)
43 (15−130)
7.3 (5.9−9.0)
20 (15−30)
11 (6.4−18)
47 (11−200)
9.3 (7.6−11)
110 (96−130)
16 (9.7−26)
17 (11−28)
15 (10−21)
45 (23−85)
7.0 (4.0−12)
36 (15−81)
23 (10−54)
1.8 (0.91−3.6)
4.8 (3.3−7.1)
4.7 (1.8−13)
2.8 (1.7−4.8)
1.2 (0.80−1.9)
7.1 (5.8−8.6)
20 (14−28)
7.4 (7.0−8.0)
7.0 (4.7−11)
8.5 (6.5−11)
0.1 nmol/h
0.03 nmol/h
0.01 nmol/h
0.003 nmol/h
NDc
5
NDc
4
NDc
0
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
0
metastin(45-54)
0.96 (0.55−1.7)
0.33 (0.25−0.44)
2.7 (0.92−8.1)
2.0 (0.71−5.9)
3.6 (1.1−11)
1
D-Trp
D-Ala
D-Leu
D-Lys
D-Glu
Phe
NDc
5
NDc
NDc
NDc
NDc
5
2
Phe
3
NDc
5
3
Phe
NDc
2
4
Phe
5
Phe
22 (9.7−52)
5
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
0
NDc
5
6
Trp
Phe
0.61 (0.25−1.5)
1.6 (1.1−2.3)
2.8 (2.1−3.6)
5.2 (2.3−12)
7
Lys
Phe
4
8
Glu
Phe
5
4
NDc
3
9
Gly
Phe
5
NDc
5
10
Ala
Phe
1.2 (0.76−2.0)
2.4 (1.6−3.6)
4.4 (1.5−13)
11
Aib
Phe
3
NDc
NDc
NDc
NDc
NDc
5
12
MeAla
Phe
0
13
D-MeAla
Phe
1.8 (1.4−2.4)
4.9 (3.0−8.1)
3.3 (2.0−5.4)
5.2 (2.5−11)
3
NDc
0
14
Sar
Phe
15
β-Ala
Abz(2)
Pro
Phe
16
Phe
0
NDc
NDc
5
17
Phe
0.48 (0.20−1.2)
5.8 (3.0−11)
NDc
1
NDc
NDc
NDc
NDc
NDc
NDc
4
18
D-Pro
Phe
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
NDc
19
Aze(2)
Pic(2)
Aze(3)
Pic(3)
Tic
Phe
1.9 (1.1−3.4)
2.8 (1.5−5.3)
2.3 (1.3−4.3)
5.5 (4.3−7.0)
0.49 (0.20−1.2)
5.2 (2.3−12)
NDc
NDc
NDc
0
20
Phe
3
NDc
0
21
Phe
22
Phe
23
Phe
4
NDc
1
24
Hyp
Phe
5
NDc
0
NDc
NDc
0
25
Thz
Phe
2.3 (1.3−4.3)
0.65 (0.31−1.4)
1.4 (1.1−1.8)
1.9 (0.62−6.0)
0.56 (0.27−1.1)
0.073 (0.043−0.12)
0.43 (0.16−1.1)
0.47 (0.27−0.84)
1.1 (0.81−1.6)
1.2 (0.81−1.7)
0.75 (0.36−1.6)
NDc
NDc
5
26
D-Trp
D-Trp
D-Trp
D-Trp
Phe(2F)
Phe(3F)
Phe(4F)
Phe(4Cl)
Phe(3F)
Phe(3F)
Phe(3F)
Phe(3F)
Phe(3F)
Phe(3F)
27
4
NDc
NDc
NDc
NDc
0
28
0
50
51
52
53
54
55
56
57
NDc
NDc
NDc
NDc
NDc
NDc
NDc
29
0
30
Trp
Lys
Glu
Gly
Aib
Hyp
1
31
4
32
5
NDc
NDc
5
33
0
34
0
58
35
0
59
EC50 values of agonist activities were determined as concentrations of peptide analogs that gave half-maximum [Ca2+] mobilizing activities. b Plasma testosterone
suppressive activities of peptide analogs at 0.1, 0.03, 0.01 or 0.003 nmol/h. Peptides were dissolved in 50% DMSO to prepare 0.1, 0.03, 0.01 or 0.003 mM solutions used in
ALZET® osmotic pumps implanted subcutaneously in five CD(SD)IGS male rats. After six days, plasma testosterone levels were measured using radioimmunoassay.
Testosterone suppression was expressed as the number of rats with plasma testosterone levels below the limit of detection (0.04 ng/mL). c Not determined.
a
60
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Table 2.
Plasma testosterone suppressive activities by continuous
4
5
6
administration of 1, 24 and 32
7
8
9
Ac-D-Tyr-AA47-Asn-Thr-AA50-azaGly-Leu-Arg(Me)-Trp-NH2
testosterone suppressive activity (number of rats, T < 0.04 ng/mL, n = 5)a
dose (pmol/h)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
10
30
100
compound
AA47
-Trp
AA50
Phe
1 wk
4 wk
1 wk
4 wk
1 wk
4 wk
1 wk
5
4 wk
5
1
D
0
5
0
0
1
0
3
5
5
2
3
4
5
5
5
4
5
3
NDb
NDb
NDb
NDb
24
Hyp
Phe
32
Glu
Phe(3F)
aPlasma testosterone suppressive activities by continuous administration of compound 1, 24 or 32 in male rats at 3, 10, 30, or 100
pmol/h. Compound 1, 24 or 32 dissolved in 50% DMSO to prepare 3, 10, 30, or 100 μM peptide solutions was added to ALZET®
osmotic pumps which were implanted subcutaneously in 5 Crl:CD(SD) male rats. After 1 or 4 weeks, the plasma testosterone level of
each rat was measured using RIA. The value below the limit of detection (0.04 ng/mL) in RIA was treated as 0.04. bNot determined.
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Table 3. Pharmacokinetic parameters of metastin analogues after intravenous
and subcutaneous administration in rats
6
7
8
9
1
24
32
compound
iv
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C5min (ng/mL)
2509 ± 287
1225 ± 79
0.69 ± 0.02
562 ± 37
2850 ± 121
1547 ± 105
0.62 ± 0.04
2633 ± 165
1331 ± 275
0.53 ± 0.03
406 ± 58
AUC0-24h (ng･h/mL)
MRT (h)
Vdss(mL/kg)
CLtotal (mL/h/kg)
sc
403 ±
5
819 ± 52
648 ± 45
771 ± 143
Cmax (ng/mL)
Tmax (h)
100 ± 44
0.19 ± 0.10
115 ± 44
0.86 ± 0.15
9.4 ± 3.6
342 ± 40
703 ± 97
0.39 ± 0.19
1258 ± 71
1.09 ± 0.13
94.5 ± 20.2
0.33 ± 0.14
679 ± 196
1.21 ± 0.34
43.9 ± 13.0
AUC0-24h (ng･h/mL)
MRT (h)
B.A. (%)
Peptides were administered intravenously or subcutaneously to SD rats at a dose of 1 mg/kg. Blood
samples were collected at 5, 10, 15, 30 min, 1, 2, 4, 8, 24 h after injection. Plasma was collected by
removing plasma proteins using 0.2% formic acid/methanol (1:4, v/v) and acetonitrile, followed by
centrifugation at 15,000 rpm for 5 min. The obtained supernatant was treated with 0.4% aqueous formic acid
and centrifuged at 15,000 rpm for 5 min. The plasma concentration of peptides was determined by LC/MS.
The C5min, AUC0–24h, MRT, Vdss, and CLtotal were obtained for each rat after intravenous administration and
expressed as mean ± standard deviation (n=3). The Cmax, Tmax, AUC0–24h, MRT, and B.A. were obtained for
each rat after subcutaneous administration and expressed as mean ± standard deviation (n=3).
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Table 4. Physicochemical profiles of nonapeptide metastin analogues substituted at positions 47 and 50
4
5
Ac-D
-Tyr-AA47-Asn-Thr-AA50-azaGly-Leu-Arg(Me)-Trp-NH2
6
7
8
9
physicochemical propertiesa
aqueous solution (20 mg/mL)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
compound
AA47
-Trp
AA50
Phe
initial appearance
soluble
gel-forming ability
gelation within 3 hr
1
6
D
Trp
Gly
Aib
Hyp
Lys
Glu
Phe
poorly soluble
soluble
gelation instantly
9
Phe
apperance of suspended solid after 40 hr
no gelation for 5 days
11
24
31
32
Phe
soluble
Phe
soluble
no gelation for 5 days
Phe(3F)
soluble
no gelation for 5 days
Phe(3F)
poorly soluble
gelation in the presence of liquid phase after 48 hr
aPhysicochemical properties of metastin analogues were evaluated in terms of water solubility at the concentration of 20 mg/mL and gel-forming
ability up to 5 days after dissolution.
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