Abstract
Background/Aim
Growth hormone (GH) secretion patterns differ across species. Humans exhibit a nocturnal surge, while rodents exhibit ultradian pulses. In cynomolgus monkeys, diurnal and daily variations and responsiveness to exogenous GH-releasing hormone (GHRH) remain insufficiently defined in non-clinical studies. This study aimed to characterize GH secretion patterns and evaluate responsiveness to exogenous GHRH in adult male cynomolgus monkeys for pituitary toxicity studies.
Materials and Methods
Serum from 10 animals was collected between 10:00 and 22:30 and again at 10:30 on the following day to evaluate diurnal variation. Serum from 10 additional animals was collected once daily between 9:00 and 10:00 across five days to evaluate daily variation. In the GH stimulation test, four animals received intravenous pralmorelin hydrochloride (as GHRH) and four received physiological saline between 11:00 and 11:30. Serum was collected before and at 0.5, 1, and 2 h after administration. GH concentration was measured with enzyme-linked immunosorbent assay.
Results
Diurnal variation was observed, with concentrations increasing from 10:00 to 11:30, transiently dropping to their lowest at 12:30 and peaking at 22:30, similar to the pattern in humans. Daily variation was also observed inter- and intra-individually across five days. In the stimulation test, compared to the control group, the GHRH group showed higher GH concentrations at 0.5 h (p<0.05), as in humans, and a greater area under the curve (p<0.05).
Conclusion
In adult male cynomolgus monkeys, diurnal and daily GH variations and responsiveness to exogenous GHRH were confirmed. Morning GHRH administration in the stimulation test, when basal GH is low and diurnal influence is minimal, and multi-timepoint sampling are recommended for reliable GH assessment. These findings suggest that cynomolgus monkeys are a suitable model for pituitary toxicity studies.
Keywords: Growth hormone, diurnal and daily variations, growth hormone-releasing hormone, growth hormone stimulation, cynomolgus monkey
Introduction
Non-human primates are phylogenetically close to humans and exhibit comparable endocrine rhythms. The cynomolgus monkey (Macaca fascicularis) is a well-known non-human primate that is widely used in non-clinical toxicological research (1). Cynomolgus monkeys are a more suitable model for evaluation of growth hormone (GH) dynamics and pituitary endocrine function (hypothalamic/pituitary/somatotropic axis) than rodents, as their pituitary anatomy and regulatory pathways are similar to those of humans.
In the clinic, GH stimulation tests are preferable to random GH measurements, as random measurements are often uninformative in evaluation of GH secretion because of pulsatility. These stimulation tests (e.g., insulin-induced hypoglycemia, arginine loading, clonidine loading) induce GH release to assess pituitary reserve. In humans, the growth hormone-releasing hormone (GHRH) + arginine test is recognized as a reliable alternative to the more common insulin tolerance test for diagnosing GH deficiency (2). GHRH administration allows direct assessment of the ability of the pituitary to secrete GH by bypassing hypothalamic influence. If pituitary somatotrophs are intact, exogenous GHRH elicits a marked increase in GH, whereas a blunted response may indicate reduced pituitary function. Thus, the GH stimulation test using GHRH is important in distinguishing pituitary from hypothalamic causes of GH deficiency and is a useful tool in endocrine evaluation (3).
In non-clinical studies, measurement of hormone levels such as GH in laboratory animals can provide early indication of endocrine disruption. An abnormal GH secretion pattern or response to stimulation in toxicology studies may indicate drug-induced pituitary dysfunction. The presence of individual, diurnal, and daily variations in blood hormone levels are well known, and obtaining data on these variations may prove valuable for evaluating effects on the endocrine system. While reference values for hematological and biochemical parameters in routine clinical pathology in cynomolgus monkeys are available (4), to the best of our knowledge, no data exists on diurnal and daily variations in GH secretion in cynomolgus monkeys. Therefore, the present study aimed to evaluate the diurnal and daily variations of serum GH concentrations in male cynomolgus monkeys. In addition, we evaluated an endogenous GH response to exogenous GHRH in a GH stimulation test to confirm that the response can be evaluated in cynomolgus monkeys similarly to in humans.
Materials and Methods
Animals. Male cynomolgus monkeys were used for characterization of the GH secretion profile and response to exogenous GHRH. GH secretion is affected by sex hormones, and menstrual cycle-associated fluctuations have been reported (5). Endogenous estrogen variations may affect the GH/IGF-1 axis, and menstrual cycle phase has been suggested to affect GH sensitivity. Therefore, males were selected over females to ensure consistency of assessment. Twenty-eight male cynomolgus monkeys (Macaca fascicularis, 3 to 7 years old, China origin) were used in this study. All animals were housed in individual cages (cage size: 680 mm [D] × 620 mm [W] × 770 mm [H]) in a temperature- and humidity-controlled animal room. Environmental controls were set to maintain a temperature range of 23˚C to 29˚C, a relative humidity range of 30% to 70%, 15 air changes per hour, and 12 h per day of artificial light (7:00 to 19:00). Solid food (approximately 12 g × 9 pieces, HF Primate J 12G 5K9J, Purina Mills, LLC., Arden Hills, MN, USA) was provided to each animal between 14:00 and 16:00 once daily. On the day before blood sampling, all remaining food was removed at approximately 17:00 or later for overnight fasting. Water was available ad libitum from an automatic water supply system. The study was approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety Research Laboratories, which is fully certified by AAALAC International.
Examination of diurnal and daily variation of GH. Ten animals were used to evaluate diurnal variation in GH secretion. Blood was drawn at the following times: 10:00, 11:00, 11:30, 12:30, 14:30, 18:30, 22:30, and 10:30 on the following day. The blood sampling times were determined in consideration of the clinical observation schedule and toxicokinetic sampling points in non-clinical toxicity studies. A separate group of 10 animals was used to evaluate daily variation in GH secretion. Blood was collected at a single time point between 9:00 and 10:00 for five consecutive days.
Assessment of GH stimulation test. Eight animals were used. The control group and the pralmorelin hydrochloride (GHRH) group each consisted of four animals. Pralmorelin hydrochloride (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan) was administered intravenously once at a dose of 10 μg/kg body weight (BW) between 11:00 and 11:30. In the control group, physiological saline (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan.) was administered in a similar manner as pralmorelin hydrochloride to the GHRH group. Administration was conducted in the morning to minimize diurnal variation. Blood was drawn before administration and at 0.5, 1, and 2 h after administration.
Blood sampling. Blood was drawn from the femoral vein under restraint to obtain serum. The collected serum was stored in a deep freezer at −70˚C or below until analysis.
GH analysis. Serum GH concentration was measured with enzyme-linked immunosorbent assay (ELISA) using a Quantikine Human Growth Hormone ELISA kit (DGH00, R&D Systems, Inc., Minneapolis, MN, USA).
Statistical analysis. For GH concentration, the mean values and standard deviation (SD) were calculated. For diurnal variation, the coefficient of variation (CV) was calculated at each sampling time point to evaluate the variation in concentration among animals. For daily variation, the CV was calculated to evaluate the variation in concentration among animals on each sampling day as well as within the same animal over five consecutive days. For diurnal variation, statistical significance was determined using one-way analysis of variance (ANOVA) for comparison between 10:00 and each sampling point. For daily variation, statistical significance was determined using one-way ANOVA for comparison between Day 1 and each sampling day. For the GH stimulation test, the area under the curve (AUC) was calculated using the trapezoidal method. Statistical significance was determined using Student’s t-test for comparison between the control and GHRH groups, as well as pre- and post-administration values. The MiTOX System (Mitsui E&S Systems Research Inc., formerly Mitsui Zosen Systems Research Inc., Chiba, Japan) was used for these statistical analyses with p<0.05 considered statistically significant.
Results
Diurnal variation of GH. No statistically significant differences were observed in GH concentrations at any time point (Figure 1, Table I). The lowest GH concentration (mean±SD: 2.747±2.510 ng/ml) was observed at 12:30, and the SD at this time point was lower than at other time points. The GH concentrations at 18:30 (10.556±7.217 ng/ml) and 22:30 (10.934±8.825 ng/ml) were higher than at other time points. GH concentrations increased once at 11:30 (8.289±7.899 ng/ml); transiently decreased to their lowest at 12:30 (2.747±2.510 ng/ml); and then increased again in the evening (18:30; 10.556±7.217 ng/ml), peaking at 22:30 (10.934±8.825 ng/ml). The SD was low at 12:30, and the GH concentration was low from 11:00 to 12:30. The GH concentration at 10:30 on the following day was comparable to that at 10:00 on the previous day. High inter-individual variation in GH concentration was observed at all time points with a wide CV range, while the overall pattern of GH secretion was consistent.
Figure 1.
Serum growth hormone (GH) concentrations at different time points on the same day in adult male cynomolgus monkeys. GH concentrations were measured using the samples obtained at 10:00, 11:00, 11:30, 12:30, 18:30, 22:30, and 10:30 on the following day (n=10 per time point). Data are expressed as the mean+standard deviation (SD). There are no statistically significant differences between 10:00 and the other sampling points.
Table I. Serum growth hormone (GH) concentrations at different time points on the same day in adult male cynomolgus monkeys.
SD: Standard deviation; CV: coefficient of variation; Min: minimum; Max: maximum.
Daily variation of GH. No statistically significant differences were observed in GH concentrations at a single time point between 9:00 and 10:00 over five consecutive days (Figure 2, Table II). The CV of GH concentration among animals on each sampling day ranged from 100.1% to 123.3%. The CV of GH concentration for each animal over five consecutive days ranged from 18.1% to 183.1%. High inter- and intra-individual variation was noted in GH concentrations during the five-day period.
Figure 2.
Serum growth hormone (GH) concentrations at a single time point between 9:00 and 10:00 on five consecutive days in adult male cynomolgus monkeys. GH concentrations were measured using samples obtained at a single time point between 9:00 and 10:00 on Days 1 through 5 (n=10 per day). Data are expressed as the mean+standard deviation (SD). There are no statistically significant differences between Day 1 and the other sampling days.
Table II. Serum growth hormone (GH) concentrations at a single time point between 9:00 and 10:00 on five consecutive days in adult male cynomolgus monkeys.
Blood sampling time: a single time point between 9:00 and 10:00. SD: Standard deviation; CV: coefficient of variation; Min: minimum; Max: maximum.
Assessment of GH stimulation test. GH concentrations at 0.5 h after administration in the control and GHRH groups were significantly higher than those before administration (Figure 3). Compared to the control group, GH concentrations in the GHRH group were significantly increased at 0.5 h after administration. GH concentrations in the GHRH group peaked (35.213±14.633 ng/ml) at 0.5 h after administration, were decreased at 1 h, and had returned to pre-administration values at 2 h. The AUC in the GHRH group was significantly greater than that in the control group, indicating that GH secretion was enhanced by GHRH administration (Figure 4).
Figure 3.
Serum growth hormone (GH) concentrations after growth hormone-releasing hormone (GHRH) (pralmorelin hydrochloride) administration in adult male cynomolgus monkeys. GH concentrations were measured using samples obtained before administration and at 0.5, 1, and 2 h after administration of GHRH (pralmorelin hydrochloride) in adult male cynomolgus monkeys (n=4 per group). Data are expressed as the mean+standard deviation (SD). *p<0.05, **p<0.01 vs. pre-administration value. #p<0.05 vs. physiological saline group.
Figure 4.
Area under the curve (AUC) for growth hormone (GH) from preadministration to 2 h after growth hormone-releasing hormone (GHRH) (pralmorelin hydrochloride) administration in adult male cynomolgus monkeys (n=4 per group). Data are expressed as the mean+standard deviation (SD). *p<0.05 vs. physiological saline group. BW: Body weight.
Discussion
In this study, we examined the diurnal and daily variation of GH and GH response to GHRH stimulation in adult male cynomolgus monkeys, which are widely used in non-clinical drug development studies. In examination of diurnal variation in the present study, the lowest concentration was observed during the daytime (12:30); while the highest concentrations were observed at night (22:30), when concentrations were approximately 3.7 times higher than during the daytime. In humans, GH concentrations have been reported to be low during the daytime (<5 ng/ml from 6:00 to 12:00) and high at night (approximately 15 ng/ml from 22:00 to 2:00) (6), suggesting that the diurnal rhythm in cynomolgus monkeys is similar to that in humans (6-8). In contrast, while humans and cynomolgus monkeys show a similar nighttime increase in GH secretion, GH concentrations in rats have been reported to be high during the day (200 to 300 ng/ml) and low at night, as rats are nocturnal (9-11).
In the present study, although there were no significant differences in GH concentrations among the five days investigated, large inter- and intra-individual fluctuations were observed, suggesting daily variations in GH concentration. Our results are similar to those seen in humans (12).
In the GH stimulation test in this study, GHRH induced a distinct GH peak at 0.5 h post-administration, which is consistent with previous studies in humans and rhesus monkeys (13-15). This result suggests that cynomolgus monkeys exhibit GH responsiveness to exogenous GHRH similar to that seen in humans. Due to the diurnal and daily variations in GH secretion in male cynomolgus monkeys (which are similar to those observed in humans) (12,16), a single time-point measurement is insufficient for accurately evaluating GH secretion disorders. Implementing dynamic stimulation tests may be of value in compensating for these fluctuations. These tests standardize conditions by introducing an external stimulus, making them highly effective for evaluating pituitary endocrine function (hypothalamic/pituitary/somatotropic axis). In the control group, a small increase in GH concentration was noted, suggesting stress associated with administration.
In diurnal assessment of GH concentration in this study, SD values were low at the 12:30 time point, and GH concentrations were low from 11:00 to 12:30. The GH stimulation test was conducted in the morning, resulting in a GH peak similar to that seen in humans. These results suggest that the GH stimulation test in cynomolgus monkeys is best conducted in the morning, as it is in humans and rhesus monkeys (13-15).
Conducting the stimulation test at a time of low basal GH secretion can show a clearer GH increase after stimulation. In the present study, GH concentrations reached their minimum around midday. GHRH stimulation during the low-baseline period minimizes interference from endogenous GH fluctuations, thereby allowing a more discernible and reliable increase in GH levels.
Although rats are commonly used in hormone-related animal studies, GH secretion in rats is known to occur in a more frequent and irregular pulselike pattern (9,17), unlike the secretion pattern in humans and non-human primates. In addition to being technically challenging, continuous blood sampling in rats has limitations in quantitatively capturing temporal variations (18-20). In contrast, cynomolgus monkeys allow for continuous blood sampling and have a secretion pattern closer to that of humans, making them a more suitable model for temporal assessment of GH secretion. In non-clinical studies in cynomolgus monkeys, routine blood sampling is generally performed via restraint rather than catheterization. In the present study, the variations of GH secretion were evaluable in restrained cynomolgus monkeys. Therefore, these data provide valuable reference values for GH secretion under the same blood sampling conditions commonly used in non-clinical studies.
In the present study, we established that there are diurnal and daily variations in GH secretion in adult male cynomolgus monkeys and that the GH stimulation test can evaluate responsivity of the pituitary in cynomolgus monkeys. Morning administration of GHRH in the GH stimulation test, timed to correspond to the lowest basal GH secretion and minimal diurnal rhythmic influence, is suitable for accurate assessment. In particular, the GH stimulation test can be useful for evaluating toxicological studies targeting the pituitary gland. Considering the diurnal variation in GH secretion in cynomolgus monkeys, multi-point assessments and the GH stimulation test are suitable for accurate GH evaluation in cynomolgus monkeys, similar to how GH is evaluated in humans.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Authors’ Contributions
Y.T. planned the study. Y.T. performed the data analysis. Y.T., T. Yoshikawa, T. Yamada, and H.K. drafted the manuscript. All Authors read and approved the final manuscript.
Acknowledgements
The Authors thank the research members of the Clinical Pathology Department and Laboratory Animal Management Department at Drug Safety Research Laboratories, Shin Nippon Biomedical Laboratories, Ltd. for providing expertise that greatly assisted the research.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
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