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Corrigendum
Effects of the synthesized growth hormone releasing peptide,
KP-102, on growth hormone release in sodium glutamate
monohydrate-treated low growth rats
Terutake Nakagawaa, Kiyoharu Ukaia,T, Tadashi Ohyamaa,
Masao Koidab, Hitoshi Okamurac
aCentral Research Institute, Kaken Pharmaceutical Co. Ltd., 14 Shinomiya,
Minamikawara-cho, Yamashina-ku, Kyoto 607, JapanbDepartment of Pharmacology, Faculty of Pharmaceutical Sciences, Setunan University, Osaka, Japan
cDepartment of Anatomy, Kobe University School of Medicine, Kobe, Japan
Abstract
KP-102 (D-Ala-D-b-Nal-Ala-Trp-D-Phe-Lys-NH2), a new second generation hexapeptide, has a potent growth
hormone (GH)-releasing action in vivo and in vitro. Here, we evaluated the GH-releasing action of KP-102 under
pentobarbital (PB) anesthesia in neonatally sodium-glutamate-monohydrate-treated low growth (NMSG- LG) rats.
The plasma GH level in NMSG-LG rats after i.v. administration of KP- 102 at 100 Ag/kg was 1/6.7 (95% CL. 1/
14.7–1/3.0) of that in normal rats given the same dose (p b 0.01). However, the increase was significant compared
with that in normal rats after saline administration (p b 0.0l). The plasma GH releasing action of KP-102 at 100 Ag/kg i.v. in rats with lesions in the bilateral hypothalamic arcuate nuclei (ARC), was about 1/6.3 (95% C.L. 1/12.4–1/
3.2) of that in normal rats under PB anesthesia (p b 0.01). When KP-102 was injected into the ARC at doses of
0.0002, 0.02 and 2 Ag/rat, GH release was dose-related (p b 0.01) under PB anesthesia. KP-102 at 2 Ag i.c.v. also
increased the plasma GH levels (p b 0.01) to about 1/8.3 (95% C.L. 1/22.7–1/3.1) of that by systematic
administration, at the same potency as the ARC injection (1/13.7 and 95% C.L. 1/37.2–1/5.0). These findings
suggest that KP-102 potently stimulates the GH release by a direct or indirect antagonism of somatostatin (SRIF)
and growth hormone releasing hormone (GHRH) release in the hypothalamus and by a direct action on the
pituitary. Furthermore, the GH-releasing action of KP-102 was similar and additive upon both regions in vivo at
the maximum effective dose. Moreover, since the GH-release in response to KP-102 administration differed
Life Sciences 76 (2005) 2753–2761
www.elsevier.com/locate/lifescie
0024-3205/$ -
doi:10.1016/j.l
DOI of orig
T Correspond
E-mail add
see front matter D 2005 Elsevier Inc. All rights reserved.
fs.2005.02.002
inal article: 10.1016/S0024-3205(96)00356-6.
ing author. Tel.: +81 75 594 0787; fax: +81 75 594 0790.
ress: [email protected] (K. Ukai).
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–27612754
between NMSG-LG and normal rats, and since KP-102 increased the GH release even in NMSG-LG rats, it should
be evaluated in the hypophysial GH secretion tests, and may be used to treat the hypophysial GH secretion
insufficiency.
D 2005 Elsevier Inc. All rights reserved.
Keywords: KP-102; Growth hormone releasing peptide; Arcuate nuclei lesion; i.c.v.
A family of synthetic hexapeptides (GHRPs) exhibits growth hormone (GH) releasing actions
developed from Met-enkephalin analogs, and the actions of the representative hexapeptide, GHRP-6
have been extensively studied [1,2]. KP-102 (D-Ala-D-b-Nal- Ala-Trp-D-Phe-LysNH2), a recently
synthesized member of this GHRP family, has a stronger growth hormone (GH)-releasing action than
GHRP-6 [3,4]. These GHRPs act on both the hypothalamus and pituitary, but their relative actions at
the two sites remain to be clarified. Marked decreases in the GH releasing action of hexapeptide
GHRPs by impairment of the hypothalamohypophysial system have been confirmed by using GHRP-6
in the goat and rat models of hypothalamohypophysial separation, and in patients with hypothalamo
hypophysial impairments [5–7]. Clinically, many patients with a GH deficiency reportedly have
hypothalamic dysfunction rather than low hypophysial GH release [8]. Thus, the action of KP-102
should be studied in a hypothalamohypophysial impairment model. Large doses of sodium glutamate
monohydrate (MSG) administered to neonatal rats cause low thalamic weight as well as decreased
body weight and length as compared with normal rats [9]. In this study, the effects of KP-102 on GH
release under pentobarbital (PB) anesthesia were evaluated in neonatally MSG-treated low growth
(NMSG-LG) rats. Also, neurons in the hypothalamic arcuate nuclei (ARC) are considered to be
selectively damaged in this model [9,10]. Therefore, the roles of the hypothalamus and the pituitary in
the mechanism of action of KP-102 were evaluated after its systematic administration in PB
anesthetized rats with ARC lesions, and after an injection into the ARC and intra-third cerebral
ventricle (i.c.v.) in normal rats anesthetized with PB.
Materials and methods
Test compound and reagents
KP-102 was synthesized at our institute. Sodium glutamate monohydrate (Wako Pure Chemical
Industries, Ltd., Tokyo, Japan), and sodium pentobarbital (RNembutal; Dainippon Pharmaceutical Co.,
Ltd., Osaka, Japan) were purchased. KP-102 was dissolved in physiological saline at 1 mg/ml and
diluted immediately before use. Since KP-102 is absorbed by glass, the polyethylene test tubes were
coated with silicone.
Animals
Male and female SD strain rats were purchased from SLC Inc. and used after acclimation in an animal
room at 248C under 50–70% humidity for at least 1 week.
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–2761 2755
GH releasing action in NMSG-LG rats
NMSG-LG rats were generated by intraperitoneal administration (i.p.) of MSG to male neonates (born
to female rats purchased on Day 16 of gestation) at 4 mg/kg body weight 5 times, namely, 1, 3, 5, 7, and
9 days after birth. The control group was administered i.p. physiological saline (NS-NG). The animals
were raised normally thereafter and studied at the age of 10 weeks. The animals were fixed in the supine
position under anesthesia with sodium pentobarbital (40 mg/kg, i.p.). A polyethylene tube was inserted
into the right jugular vein and the left femoral artery for drug administration and blood sampling, and the
animals were left in this state for 30 minutes to stabilize the postoperative conditions. Blood (150 Al) wascollected every 5 minutes for over 30 minutes. Saline and KP-102 100 Ag/kg were administered
intravenously (i.v.) to NS-NG rats, and saline and KP-102 100 Ag/kg were also administered to NMSG-
LG rats, each consisting of 6 animals.
GH releasing action in ARC-lesioned rats
ARC lesions were produced in 10-week-old male rats. The animals were fixed on a stereotaxic
apparatus under halothane anesthesia, then the probes of a lesion generator (Radionics, INC.; Model
RFG-4A, Burlington, USA) were inserted into the bilateral ARC (A: 3.3, L: 0.2, D: 10) according to the
brain atlas of Paxinos and Watson. A high frequency electric current was applied for 2 minutes at 708C.After 60 minutes, the operation for drug administration and blood sampling was performed and the
animals were allowed to stabilize for 30 minutes. Blood (150 Al) was collected every 5 minutes for over
30 minutes after drug administration. KP-102 was administered intravenously at a dose of 100 Ag/kg to
normal, sham-operated and ARC-lesioned groups, each consisting of 12–17 animals. After the end of the
experiment, the site of the ARC lesion was histologically examined and the 17 of 20 rats destroyed
bilaterally were used.
Effect of injection of KP-102 into the ARC and the i.c.v. on GH-releasine action in normal rats
To evaluate the direct effects of the drug on the ARC, a polyethylene tube for blood sampling was
inserted into the left femoral artery of 10-week-old male rats under sodium pentobarbital anesthesia (40
mg/kg, i.p.). A 30-gauge stainless steel cannula for drug injection was then inserted into the right ARC
or the third cerebral ventricle (A: 3.3, L: 0.0, D: 10.2) as described above, and the drug was infused at a
rate of 1 Al/min for over 2 minutes. Blood (150 Al) was collected every 5 minutes for 30 minutes after the
drug infusion. In the first experiment, saline and KP-102 at doses of 0.0002,0.02, or 2 Ag/rat wereadministered into the ARC of 6 animals each. In the second experiment, saline and KP-102 were
administered at a dose of 2 Ag/rat into the third ventricle or ARC to 4 animals each.
After the experiment, 2% Evans blue was infused via cannula to confirm the histological examination
and 28 of the 32 rats and all 8 rats were used in the ARC injection and i.c.v. groups respectively.
GH assay
The blood samples were immediately centrifuged at 48C at 1,500 � g for 15 minutes, and the plasma
was stored at �208C or below until the GH assay. Plasma GH was measured by means of a
radioimmunoassay (RIA) using materials supplied by NIDDK (National Hormone Distribution Program,
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–27612756
NIH, Bethesda, USA: The rGH and anti-rGH serum were kindly provided by Dr. C. Y. Bowers). The
assay used iodinated purified rGH and anti-rGH serum. Values are expressed in terms of NIDDK-rat-
GH-RP-2 standard (potency 2 IU/mg), as ng/ml of plasma.
Statistical analysis
The experiments proceeded according to a multifactorial design using time as the secondary factor.
The raw data were converted to logarithms to obtain a nearly homogeneous and normal distribution, and
the analysis of variance (ANOVA) was performed. When replication was uneven, harmonic means were
used [11]. From the time-concentration curve of GH, which corresponded to the secondary factor of
time, the area under the curve (AUC) was determined by trapezoidal approximation, and the ANOVA
was performed after logarithmic conversion. The significance of the differences on multiple comparisons
was examined by the LSD method. Since the detection limit of the GH by RIA is 0.8 ng/ml, values of
0.8 ng/ml or less were regarded as 0.8 ng/ml and the degree of freedom of the error variance was
adjusted as missing data in the statistical analysis. To examine the reproducibility of the means obtained
in each experiment under the same conditions, the 95% confidence limits (95% C.L.) were calculated
using the following formula,
jy1� y2jNffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiFVeldVeld 1=n1ð Þ þ FVe2d Ve2d 1=n2ð Þ
pN N ð1Þ
where Fvel and Fve2 are F table (p=0.05), vel and ve2 are the degree of freedom of Ve, Vel and Ve2 are
the error variance of ANOVA, nl is the number of repetitions used to calculate the mean value yl, and n2
is the number of repetitions used to calculate the mean value y2 [11].
Results
GH-releasing action in NMSG-LG rats
The means and the standard deviations of the wet weight of the pituitary and the body weight in the
NMSG-LG rats were 6.2 F 0.5 mg (p b 0.01) and 276 F 30 g (p b 0.0l), respectively, being below the
10.6 F 1.0 mg and 366 F 24 g, respectively, of the neonatally saline treated, normal growth (NS-NG)
rats, corresponding with the reported data [9,10]. Similarly, the means and standard deviations of the
body length (from the nose tip to the origin of the tail) and the tail length in the NMSG-LG rats were
21.3F 0.7 cm (p b 0.01) and 16.4F 1.1 cm (p b 0.01), respectively, being below the 24.0F 1.2 cm and
19.8 F 0.9 cm, respectively, in the NS-NG rats.
At the 0 min point, the difference in the basal GH release between the saline and KP-102 administration
NS-NG rats and between the saline and KP- 102 administration NMSG-LG rats was not significant, but
that between the NS-NG rats and NMSG-LG rats administered either saline or KP-102 was significant.
After drug administration, the differences in GH release between the saline and KP-102 administration
NS-NG rats, between the saline and KP-102 administration NMSG-LG rats, and between the NS-NG rats
and NMSG-LG rats administered saline or KP-102, respectively, were significant. The AUC (0–30 min)
means and standard deviations in the saline-administered control NMSG-LG and NS-NG rats were 1.767
F 0.299 and 2.588 F 2.32 ng/ml in logarithmic coordinates, respectively, those in the NMSG-LG and
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–2761 2757
NS-NG rats administered KP -102 at 100 Ag/kg i.v. were 3.371 F 0.185 and 4.194 F 0.281 ng/ml in
logarithmic coordinates, respectively. The effect of KP-102 was significantly lower in the NMSG-LG rats
and was about 1/6.7 (95% C.L. 1/14.7–1/3.0), being the reverse logarithm of the difference between the
mean values (p b 0.01) and variance of error in ANOVA, of that in the NS-NG rats (Fig. 1).
GH releasing action in ARC-lesioned rats
At the 0 min point before KP-102 administration, the basal GH release was not significantly different
between the control, the sham, and the ARC-lesioned groups. No significant difference in AUC (0–30
min) was evident between the control and sham groups, but a significant decrease of GH release was
observed in the ARC-lesioned group as compared with the control (p b 0.01) and sham (p b 0.05) groups.
The means and standard deviations of the AUC (0–30 min) in the control, sham, and ARC-lesioned groups
in logarithmic coordinates were 4.239 F 0.143, 4.056 F 0.152, and 3.441 F 0.63 ng/ml, respectively.
The AUC (0–30 min) of GH release in the ARC-lesioned group was about 1/6.3 (95% C.L. 1/12.4 –
1/3.2) of that in the control group (Fig. 2).
Effect of injection of KP-102 into the ARC and the i.c.v. on GH release in normal rats
At the 0 min point before KP-102 administration, the basal GH release was not significantly different
between the control, 0.0002,0.02, and 2 Ag KP-102 groups. No significant difference in AUC (0–30
min) was found between the 0.0002 Ag KP-102 and saline (control) groups , but a significant increase of
GH release was noted in the 0.02 (p b 0.1) and 2 Ag (p b 0.01) groups as compared with the control
group, and the test of linearity based on the orthogonal coefficient indicated a dose-related (p b 0.01)
stimulation of plasma GH secretion by KP-102. The means and standard deviations of AUC (0–30 min)
Fig. 1. GH releasing action of KP-102 in NS-treated normal and NMSG-treated low growth rats. Each data points and vertical
bars represent the means F S.D. (n = 6). o, Saline i.v. and D, KP-102 100 Ag/kg i.v. in NS-treated rats. !, Saline i.v. and E,
KP-102 100 Ag/kg i.v. in NMSG-treated rats.
Fig. 2. GH releasing action of KP-102 100 Ag/kg i.v. in ARC-lesioned rats. Each columns and vertical bars show the means FS.D. of the AUC (0–30 min). NS: not significantly different, *: p b 0.05, **: p b 0.0l.
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–27612758
in logarithmic coordinates in the control, 0.0002,0.02, and 2 Ag KP-102 groups were 2.761 F 0.40 ng/
ml), 2.701 F 0.217, 3.103 F 0.365, and 3.717 F 0.161 ng/ml, respectively (Fig. 3).
At the 0 min point before KP-102 administration, the difference in the basal GH release between the
saline ARC, KP-102 ARC, saline i.c.v., and KP-102 i.c.v. groups was not significant. A significant
increase of GH release (p b 0.01) was noted in rats given 2 Ag KP-102 i.c.v. as compared with the saline
(i.c.v.) group, but no significant difference in the AUC was evident between the KP-102 groups injected
2 Ag into the ARC and i.c.v.. The means and standard deviations for the saline i.c.v., KP-102 2 Ag i.c.v.,
Fig. 3. GH-releasing action after an injection of KP-102 into the ARC. Columns and vertical bars show the means F S.D. (n =
5) of the AUC (0–30 min). y: p b 0.1, **: p b 0.01.
Fig. 4. GH releasing action on injection of KP-102 into the ARC and i.c.v.. Columns and vertical bars show the means FS.D.(n = 4) of the AUC (0–30 min). **: p b 0.01.
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–2761 2759
saline ARC, and KP-102 2 Ag ARC groups were 2.279 F 0.198 (the mean value in the NS-NG rats
administered saline in the initial experiment was 2.588, which was in the 95% C.L.), 3.058 F 0.459,
2.609F 0.236, and 3.273F 0.429 ng/ml, respectively (Fig. 4). Although this experiment did not include
the systemic administration of KP-102 at 100 Ag/kg i.v., the mean values of the saline i.c.v. and ARC
control groups were not significantly different from that of the NS-NG group in the initial experiment.
This suggests the high reproducibility of the experiments and the compensation for the KP-102 i.v.
group. The GH-releasing actions of KP-102 i.c.v. and ARC were 1/8.3 (95% C.L. 1/22.7-1/3.1) and 1/
13.7 (95% C.L. 1/37.2–1/5.0) as compared with the initial systemic i.v. administration of KP-102 at 100
Ag/kg, which was 90% or more of the effective dose in S-shape dose response curve [3]. From modified
Eq. (1), orthogonal comparison of [(saline i.v. in the NS-NG rats group) + (KP-102 i.v. in NS-NG rats
group)-(KP-102 i.v. in ARC lesioned rats group)-(KP-102 i.c.v. group)] did not reveal any significant
difference (p b 0.05). This result means that the action of KP-102 is not synergistic but additive on the
hypothalamus and pituitary in stimulating GH release, although statistically not significant, because
when drugs show S-shape concentration-response curves, strict statistical distinction of whether the
concurrent actions of two drugs are synergistic or additive is difficult [12].
Discussion
GH is synthesized and stored in the pituitary. Its continuous release into the peripheral circulation is
regulated by the hypothalamic nervous system. The ARC in the hypothalamus releases growth hormone
releasing hormone (GHRH) into the hypophysial portal blood and stimulates the release of GH from the
pituitary. The periventricular hypothalamic nucleus (Pe) of the hypothalamus releases somatostatin
(SRIF) into the hypophysial portal blood and inhibits GH release from the pituitary [8]. KP-102 exerts
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–27612760
direct actions on the pituitary in vitro. A study on cultured pituitary cells has shown that about 10 times
more GH is released than GHRP-6, which is a hexapeptide GHRP of an earlier generation, and that the
action site of GHRPs differs from that of GHRH [3,4]. However, GHRPs are more effective in vivo than
in vitro [3].
We injected KP-102 into the unilateral ARC to examine the direct actions of the drug on hypothalamic
ARC. The plasma GH concentration increased in a dose-related manner. The central action of KP-102
was also confirmed by i.c.v. administration. Similar results have been obtained using GHRP-6 i.c.v. in
guinea pigs, and the action of GHRP-6 is antagonized by the i.c.v. administration of the long-acting
SRIF agonist, sandostatin [13]. Since GHRH i.c.v. does not induce GH release, that release upon GHRP-
6 i.c.v. administration may not be the result of centrally administered peptide leaking into the blood
stream and reaching the pituitary to directly release GH [13].
Moreover, the direct action of KP-102 on hypothalamic ARC is supported by the expression of the fos
gene specifically in the hypothalamic ARC detected by fos immunohistology after GHRP-6 and KP-102
were systematically administered [14,15]. The c-fos gene expression was induced by KP-102 only in the
source of the GHRH nerves of the ARC, and its distribution did not completely overlap with that of the
GHRH nerves [15]. How these c-fos gene expressing nerves are related to the SRIF antagonizing action
of KP-102 is an interest for future studies along with the U factor [16]. Furthermore, when KP-102 was
injected directly into the ARC or i.c.v. at a dose of 2 Ag, the AUC of GH release was about 1/10 of that
observed when the drug was systematically administered to the control group in two experiments and
this difference is thought to be due to the lack of direct actions of KP-102 on the pituitary.
The impairment of GH release in the NMSG-LG rats is considered to be due to selective lesion of
ARC neurons without damage to Pe neurons, which release SRIF [8,10]. In ARC-lesioned rats, part of
the median eminence and Pe were damaged according to a histological examination and basal SRIF
release is considered to have decreased. No difference was found in either model, although GH release of
KP-102 was extremely decreased to about 1/6.5 of that in the normal rats. Both experiments were
performed under PB anesthesia, which inhibits SRIF release into the hypophysial portal blood [17].
The basal GH release of the control group in the NMSG-LG rats was significantly lower than that in
the NS-NG rats throughout the observation period. This decrease is considered to be a result of a
decrease in the release of GHRH due to lesion of the hypothalamic ARC, as SRIF release is already
inhibited by PB anesthesia [17]. The GHRP analogue, hexarelin induces GHRH release into the
hypophysial portal blood in nonanesthetized sheep [18].
The GH-releasing action of GHRH with PB anesthesia was about 30 times that without anesthesia,
but the action of KP-102 was nearly the same with and without PB anesthesia [3]. GH release is slightly
increased by PB alone [3]. Thus, the combination of GHRH and SRIF inhibitor has a synergistic action
[12].
These findings suggest that KP-102 has not only a PB-like inhibitory or antagonistic action against
SRIF release [19] but also GHRH releasing action in the hypothalamus. Moreover, the analysis of
orthogonal comparison suggested that the effect on the hypothalamus and the pituitary is additive rather
than due to a primary action on either of the two regions.
The GH release after i.v. administration of KP-102 at 100 Ag/kg to the NMSG-LG rats was 1/6.7 of
that in the NS-NG rats, but it was nearly equivalent to that obtained by administration of GHRH at 10-
100 g/kg i.v. to normal, non-anesthetized rats [3]. Therefore, if the pituitary retains the ability to release
GH, KP-102 should increase GH release to a therapeutically sufficient level despite some attenuation of
its efficacy. Pharmacological tests are necessary to diagnose GH insufficiency, but several problems are
T. Nakagawa et al. / Life Sciences 76 (2005) 2753–2761 2761
associated with the conventional diagnostic procedure [20]. The GH-release function of the NMSG-LG
rats was distinguishable from that of normal rats by the response to KP-102 and the KP-102-induced
sufficient GH releasing action even in the NMSG-LG rats. Therefore, the effect of KP-102 upon
hypophysial GH secretion should be evaluated, and the drug may be used to diagnose and treat the
hypophysial GH secretion insufficiency.
Acknowledgments
The authors would like to thank Dr. E. Osada and Dr. H. Moritoki for their suggestions, and Mr. H.
Ueo for technical assistance.
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