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Pediatric Endocrinology Branch (A.R.A., G.P.C., K.P.), Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development; The Warren Magnuson Clinical Center (K.A.C.); Laboratory of Medicinal Chemistry (K.C.R.), National Institute of Diabetes and Digestive and Kidney Diseases; Clinical Neuroendocrinology Branch (G.C., D.R., P.W.G.), National Institute of Mental Health; and Laboratory of Clinical Studies (J.D.H., J.P., M.G., S.L.), Primate Unit, Intramural Research Program, National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Alejandro Ayala, M.D., Building 10, Room 9D-42, 10 Center Drive, Bethesda, Maryland 20892. E-mail: ayalaa{at}nih.gov.
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Introduction |
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Numerous studies in rats have suggested that CRHR-1 antagonists may attenuate stress-related behaviors without significantly compromising adrenal function. Antalarmin [N-butyl-N-ethyl-{2,5,6-trimethyl-7-(2,4,6)trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl] amine] is a pyrrolopyrimidine of lipophilic nature with high selectivity for the CRHR-1. The nonpeptidic nature of antalarmin makes it suitable for oral administration. Using forced immobilization as a model of acute stress, Wong et al. (7) reported that antalarmin administered for 8 wk to rats did not blunt stress-stimulated ACTH and corticosterone release. Furthermore, antalarmin administered for 11 d to adult male rats had no effect on body weight, plasma leptin levels, blood glucose, or fat cell leptin messenger RNA levels (8), yet the adrenal cortices of these animals were 30% smaller due to an increase in the apoptotic rate of adrenocortical cells. Short-term administration of antalarmin in rhesus macaques significantly influenced the behavioral and physiologic responses to intense psychosocial stress, the intruder paradigm. Specifically, antalarmin decreased the stress-induced anxiety-like behaviors, plasma ACTH, cortisol, norepinephrine, and epinephrine; furthermore, no toxicity was observed in these studies (9). Other studies involving a myriad of nonpeptide CRHR-1 antagonists have been shown to possess anxiolytic and antidepressant effects. The compound DMP695 was found to abolish CRH-mediated activation of noradrenergic neurons in the locus coeruleus, a key mediator of the stress response (10). The antagonist CRA1000 decreased the number of escape failures in rats subjected to a learned helplessness test (11), whereas Griebel et al. (12) reported similar anxiolytic effects with the compound SSR125543A.
In situ hybridization studies indicate a heterogeneous anatomic distribution of the CRHR subtypes in the brain. More importantly, it is now recognized that these receptors not only differ in anatomic distribution among species but also in structure, binding characteristics, and function (13, 14). Indeed, there is a significant interspecies difference in the distribution of these receptors within the CNS (15, 16), which may translate into different behavioral and endocrine outcomes according to the species studied (rats, nonhuman primates, or humans). Because of the above-mentioned differences, animal experiments would not necessarily translate into similar findings in humans. Social separation is a potent stressor for rhesus monkeys of all ages and results in substantial disruption of behavior and physiologic arousal, particularly during the initial hour of separation (10, 17). There is scant information regarding the effects of chronic administration of a CRH antagonist in higher mammals. Because nonhuman primates are phylogenetically related to humans, we sought to investigate the behavioral and hormonal responses to long-term (chronic) administration of antalarmin in rhesus macaques. In doing so, we used a previously validated stress paradigm involving social separation.
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Materials and Methods |
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Eight preadolescent (4–6 kg) male rhesus monkeys from the Laboratory of Comparative Ethology (National Institute of Child Health and Human Development) at the NIH Animal Center (Poolesville, MD) were used. Half of these monkeys had been raised by their mothers (mother-reared) and half had been raised with their peers (peer-reared) during the first 6 months of their lives. The monkeys studied belonged to the same social group and were housed in two social caging systems for the duration of the study. All animals were healthy, as documented by veterinary and routine laboratory evaluation. All animals were on a regular day/light cycle (12-h cycles) and were fed with standard monkey chow daily. The NIH Animal Care and Use Committee approved all procedures employed in this study.
Study design
A placebo-controlled, double-blind, cross-over design was used. The study lasted 114 d and comprised the following consecutive phases (Fig. 1
): adaptation (14 d duration; d 0–14); baseline 1 (14 d duration; d 15–28), social separation 1 (14 d duration; d 29–42); recovery (30 d duration, d 43–72) and, after the cross-over, baseline 2 (14 d duration; d 74–86), social separation 2 (14 d duration; d 87–100), and recovery 2 (14 d duration; d 101–114).
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Baseline. During a 2-wk baseline period, before the onset of social separation, focal animal behavioral data were obtained daily. Each of the individual behavioral scoring sessions lasted 5 min. Once a week during this period, the monkeys were anesthetized and cerebrospinal fluid (CSF) and plasma samples were obtained.
Social separation. A 2-wk social separation phase followed the baseline phase. Social separation was used as model of stress, as previously reported (18). During these 2 wk, at noon on each Monday, solid panels separated the individuals in the quad cages so that each monkey occupied one separate quadrant of the caging system. Behavior was recorded daily for two 5-min periods from each monkey (Monday through Thursday). On Thursday, all of the monkeys were anesthetized to obtain CSF and blood samples. On Friday mornings, the panels dividing the monkeys in each quad were removed, and the monkeys were reunited. Immediately after the reunion of the monkeys, behavioral data were obtained from each monkey for 10 min. An additional 5-min reunion score was collected on Mondays before reinsertion of the panels for separations.
Washout. The social separations were followed by a 1-month recovery period. As in the baseline phase, during the recovery period, the monkeys were socially housed in their respective quad caging systems, they received placebo treats once daily as previously described, and CSF and blood samples were obtained once a week. The monkeys receiving drug and those receiving placebo were crossed over so that the monkeys originally receiving antalarmin received placebo and monkeys previously receiving placebo received antalarmin.
Antalarmin administration
Antalarmin (20 mg/kg) or placebo was administered daily in the morning as a 5-g flavored primate treat (Bioserve, Frenchtown, NJ). Active drug and placebo had similar appearance and were found in a pilot study to be well accepted, tolerated, and palatable to a similar extent by monkeys (data not shown). To ensure proper compliance, after the treat containing either study drug or matching placebo was handed to each animal in the cage, the animals were watched consuming the medicated treat; when a monkey did not consume it, the treat was recovered, combined with another flavorful vehicle (e.g. frosting, jam, etc.), and offered again to the monkey.
Serum and CSF hormonal assays
Cisternal CSF and femoral venous blood samples were obtained once a week on the fourth day of separation under ketamine anesthesia (15 mg/kg im). Additional serum samples were obtained 1 and 2 h after separation after the animals were hand caught. The blood and CSF samples were immediately placed on wet ice and centrifuged at 4 C for 20 min. The samples were then placed on liquid nitrogen and stored at –70 C until assayed. CSF samples were assayed for CRH, antalarmin, norepinephrine, and epinephrine.
End points: both endocrine and behavioral end points were the main foci of this study
Behavioral end points. Behavioral data were recorded daily for 10 min from each monkey using a computerized scoring template. The behavioral data obtained from each individual included the frequency and duration of the following behaviors: locomotion (e.g. the movement from one location to another), passive (e.g. no motion, the monkey sits and scans its environment), vocalization, stereotypic locomotion (e.g. repetitive movement), self-grooming or scratching, oral manipulation of a body part, self-clasping (e.g. manual grasping of a body part), and environmental exploration (e.g. tactile or oral manipulation of their environment).
Endocrine end points
Femoral venous blood (10 cc) samples and cisternal CSF (3 cc) were obtained between 1330 and 1400 h during the following phases of the study adaptation: baseline, 1 h (acute stress 1), 2 h (acute stress 2), and 4 d (chronic stress) after social separation, and recovery. Samples were obtained under ketamine anesthesia (15 mg/kg im) as follows: the animals were caught individually and subsequently anesthetized. All the samples were drawn within 15 min of anesthesia. The following hormones were measured: plasma (ACTH, cortisol), and the catecholamines (norepinephrine and epinephrine). In addition, CSF CRH, 3, 4-dihydroxyphenylglycol, dihydroxyphenylalanine, dopamine, and 3,4-dihydroxyphenyl acetic acid were measured. Antalarmin levels were determined in concurrent plasma and CSF samples.
Analytical methods
As previously reported, plasma ACTH and cortisol were assayed by immunoradiometric assay. CRH in CSF was measured by RIA, and catecholamines in plasma and CSF were assayed by HPLC coupled with electrochemical detection (10).
Antalarmin in plasma and CSF was measured by HPLC as follows: hexane extracts were analyzed using a gas chromatograph (model 6890, Hewlett Packard, Portland, OR) equipped with a mass selective detector (model 5973, Hewlett Packard). The analytical column used was a 15 M [;times]; 250 µm DB-1 capillary (J and W Scientific, Folsom, CA), with antalarmin eluting at 15.7 min under the operating conditions. Quantitation of antalarmin was accomplished using electron impact ionization with single ion monitoring of 349 m/z, representing the loss of an ethyl group from the amine. The area of the peak corresponding to antalarmin was compared with a calibration curve generated using six standards fith a correlation of 0.99993. The detection limit of this assay was 0.1 ng/ml.
Statistical methods
Statistical analyses were designed to answer the following questions: 1) did the separation paradigm have the intended effects, i.e. did separation result in changes in hormone levels and/or behavior; 2) did antalarmin attenuate the behavioral response(s) to stress; 3) did antalarmin blunt the hormonal response to stress; and 4) did mother-reared and peer-reared monkeys have different behavioral and/or hormonal baselines, did they react differently to the stressor, and did they have different responses to antalarmin?
Because preliminary analyses showed similar hormonal and behavioral patterns in the two rearing groups (data not shown), the peer-reared and mother-reared animals were combined for analysis to increase statistical power. Thus, a two-way repeated-measures ANOVA was used to assess the effect of antalarmin and the separation paradigm on hormones and behavior. The design included within animal effects of drug (antalarmin vs. placebo) and study phase. For plasma cortisol and ACTH, the phases included adaptation, baseline with drug, acute stress (end of first hour of d 1 separation), and chronic stress (end of d 4 separation). After initially analyzing the wk 1 and 2 separation data separately, data from wk 1 and 2 were combined because the results of the two analyses were similar. For CSF concentrations, phases included only adaptation, baseline, and chronic stress. Analysis of behavioral data included, additionally, a reunion phase. Planned contrasts included the comparison of baseline with each other phase. Data were transformed logarithmically before analysis, when necessary, to bring the distributions closer to a normal distribution.
To ascertain that the separation paradigm was an adequate stressor, we tested for phase differences, comparing separation phases with the baseline phase. The two-way interaction of drug, and phase was the test of greatest interest. A significant result would indicate that hormonal patterns over the phases of the experiment were different, depending on whether the monkeys were taking the drug or placebo. Conservative Greenhouse-Geisser corrections were used when the Greenhouse- Geisser 
was small (an indication of nonconstant covariance) to minimize type II error. All tests were assessed at the 95% probability level. SAS statistical software (SAS Institute, Cary, NC) was used for all analyses.
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Results |
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Indices of hypothalamus-pituitary-adrenal (HPA) function.
Figure 2 depicts plasma ACTH (upper panel) and cortisol (lower panel) concentrations in animals treated with antalarmin or placebo during adaptation, baseline, acute stress, and chronic stress. Overall, plasma ACTH showed the following changes over the phases of the study: 1) ACTH decreased between adaptation and baseline [F(1,7) = 18.7, P < 0.005], indicating that the animals had adjusted to the novel environment; 2) social separation induced a robust rise in ACTH levels [F(1,7) = 156.0, P < 0.0001], indicating that social separation was an adequate model for acute stress (baseline vs. acute); and 3) after chronic stress, by d 4 of separation, ACTH levels were no longer significantly different from baseline levels [F (1, 7) = 0.00]. There was no significant effect of antalarmin and no antalarmin by phase interaction.
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Levels of CRH in the CSF were detectable across all phases of the study and ranged between approximately 50 and 400 ng/ml (19). Analysis of CSF CRH showed no significant main effect or interaction involving the drug. Only the phase effect was significant [F(2, 14) = 13.3, P < 0.001], with lower levels during adaptation [mean = 75.7, F(1,7) = 17.4, P < 0.005] or chronic stress [mean = 73.0, F (1, 7) = 18.7, P < 0.005], compared with baseline (mean = 90.4).
Serum catecholamines.
Figure 2
shows plasma norepinephrine (upper panel) and epinephrine (lower panel) in animals treated with antalarmin or placebo during adaptation, baseline, acute stress, and chronic stress. Similar to the ACTH and cortisol, social separation significantly increased catecholamine levels; norepinephrine rose approximately 3-fold over baseline [F(1,7) = 28.4, P < 0.005], whereas epinephrine increased to a much greater extent of approximately 30-fold, compared with baseline [F(1,7) = 41.64, P < 0.001]. Moreover, no differences in rearing effects (mother vs. peer reared) on the plasma levels of these hormones were observed at the onset of the experiment (adaptation); norepinephrine and epinephrine increased significantly during acute stress in both the antalarmin and placebo groups (no significant drug by phase interactions); and norepinephrine and epinephrine remained slightly elevated after chronic stress when compared with baseline [F(1,7) = 5.54, P < 0.05, NE; F (1, 7) = 12.29, P < 0.01, epinephrine].
CSF catecholamines
No significant changes in CSF norepinephrine or epinephrine levels were found due to drug or phase (data not shown).
Behavioral observations
The comparisons were of two types: primary, a posteriori planned comparisons that are directly related to our hypothesis, and secondary, comparisons that were unrelated to the primary hypothesis that antalarmin would reduce stress. The primary comparisons were few in number (two), and the secondary comparisons were not statistically significant. Five behaviors were selected as having adequate frequency for analysis: self-directed, environmental exploration, passivity, vocalization, and locomotion (Fig. 3). Each showed significant behavioral effects due to the experimental paradigm. Three of these behaviors showed significant changes due to separation. Self-directed behavior and environmental exploration each decreased significantly during acute stress, compared with baseline [F(1,7) = 55.9, P < 0.0001; F(1,7) = 902, P < 0.05]. Self-directed behavior continued to be reduced after chronic stress [F(1,7) = 6.36, P < 0.05, base vs. chronic]. In contrast, passive behavior increased dramatically during acute stress [F(1,7) = 517.9, P < 0.0001, base vs. acute] and remained highly elevated after chronic stress [F(1,7) = 78.4, P < 0.0001, base vs. chronic].
Both self-directed behavior and locomotion were significantly greater at baseline than during adaptation [F(1,7) = 7.54, P < 0.05; F(1,7) = 5.98, P < 0.05]. Self-directed behavior was significantly reduced during reunions [F(1,7) = 24.9, P < 0.005], but both locomotion and vocalization were increased during reunions, compared with baseline [F(1,7) = 12.4, P < 0.01; F(1,7) = 17.4, P < 0.005].
Only one behavior, environmental exploration, showed any effect due to the drug: environmental exploration occurred more frequently when animals were taking antalarmin than when taking placebo [F(1,7) = 9.2, P < 0.05] (Fig. 4).
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Antalarmin was detected in the plasma of each animal while they were taking the active drug, confirming that each monkey had indeed swallowed the tablet and that antalarmin had been absorbed by the gastrointestinal tract of each animal (Fig. 1). In contrast, antalarmin was undetectable in plasma during placebo administration, confirming that the washout period had been of sufficient duration to clear the drug from the circulation. Antalarmin levels in plasma showed substantial interindividual variability, suggesting differences in gastrointestinal absorption. Levels of antalarmin in the CSF were either very low or undetectable, suggesting poor permeability of the blood-brain barrier (BBB) to this dose of antalarmin in this primate species.
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Discussion |
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Stress-induced CSF CRH increases have been documented in various primate stress models and humans (21, 22, 23). Contrary to previously published experiments, an unexpected finding was that CSF CRH, norepinephrine, and epinephrine levels were not increased during stress (data not shown). Moreover, CSF levels of CRH, norepinephrine, and epinephrine did not change with antalarmin administration. The pathophysiologic mechanisms of chronic stress may, however, differ from those observed during acute stress (24). The lower serum levels of plasma ACTH, cortisol, and catecholamines during chronic stress, compared with acute stress, suggest that time-dependent habituation to the stressor and a concomitant dampening of the behavioral stress response are likely to take place. The mechanism by which the HPA axis response to chronic stress is sustained despite the blockade of the CRHR-1 remains to be elucidated. One explanation may lie in the interplay between CRHR-1 and CRHR-2, (25, 26). Humans and monkeys have higher densities of CRHR-2 in the anterior pituitary than rodents (27), and it is therefore plausible that in the event of CRHR-1 blockade, CRHR-2-mediated HPA axis activation may ensue. The finding that mice lacking both the CRHR-1 and CRHR-2 exhibit lower ACTH and cortisol levels than those lacking either the CRHR-1 or CRHR-2 supports this idea (28). However, other studies (29) using different mutant lines of the CRHR-2 knock-out suggest a minor role of the CHRR-2 in HPA axis regulation. Recently Broadbear et al. (30) reported that self-injection (iv) of antalarmin failed to decrease ACTH and cortisol serum levels in adult rhesus monkeys. In another study, Wong et al. (7) found that antalarmin-treated rats had intact ACTH and corticosterone responses to immobilization stress after 8 wk of daily ip antalarmin administration. However, antalarmin blocked the ACTH (31) observed in rats treated with the CRHR-1 antagonist CP-154,526 (32). Alternative explanations for such variation in HPA axis reactivity include the activation of the HPA axis by other ACTH secretagogues, differences in duration of treatment and the nature of the stressor, and inherent pharmacologic differences among CRH antagonists (24).
The ACTH secretagogue action of CRH is exerted via the CRHR-1. Vasopressin, a known ACTH secretagogue, may play a major role in maintaining the integrity of the HPA axis during CRH receptor blockade (33). Studies involving CRHR-1–/– mutant mice suggest that a selective compensatory activation of the hypothalamic vasopressinergic, but not the oxytocinergic system, maintains basal ACTH secretion and HPA system activity (34). Moreover, conditional mutagenesis studies involving genetically engineered mice (Crhr1loxP/loxPCaMKIICre) in which gene-targeting technologies convey neuroanatomical specificity of the knockout suggest that that limbic CRHR-1 plays a major role in the central control of HPA system feedback and hormonal adaptation to stress (31, 35). Urocortin, a mammalian CRH-related peptide as well as other peptides that show close structural homology with CRH may also regulate ACTH secretion. Rat corticotrophs express both CRH and urocortin mRNA, suggesting that these peptides may regulate ACTH release through an auto/paracrine mechanism. It is therefore possible that urocortin, a CRHR-2 ligand that also activates CRHR-1, acts as a compensatory factor in the release of ACTH in the event of CRHR-1 blockade. The possibility of an existing CRH inhibitory factor has also been suggested (36). The down-regulation of this putative HPA axis inhibitor could also explain the normal peripheral stress response in CRHR-1 antagonist-treated animals.
Environmental exploration involves examination or manipulation of environmental stimuli. These behaviors include foraging through wood shavings on the cage floor; tactile or oral exploration of primate toys, cage walls, and components; and eating and drinking. Antalarmin facilitated environmental exploration, a behavior usually suppressed in stressful conditions (18), which was not observed while the same animals were taking placebo. Using rat and primate models, several groups reported similar anxiolytic effects of CRHR-1 antagonists. In models of learned helplessness and conditioned fear involving rats exposed to repetitive electric shock, antalarmin decreased the expression and development of conditioned fear (37). Moreover, mice lacking the CRHR-1 exhibited less anxiety-like behavior, and transgenic mice that overexpressed CRH were more prone to stress-related behavior (28). Other nonpeptide CRH antagonists with close structural similarity to antalarmin also exerted anxiolytic effects in rats (32, 38, 39). In a previous study, single-dose antalarmin increased exploratory and sexual behaviors otherwise suppressed during stressful events in adult rhesus monkeys (10). Using a similar study design, He et al. (40) demonstrated that an analog of the compound DMP696 reduced stereotypical mouth movements in rhesus monkeys by 50%. In the present study, we observed a less robust effect of chronic administration antalarmin on anxiety-like behavior, compared with a previous acute, single-dose study. This divergence may be partially explained by methodological differences among the studies (i.e. nature and duration of the stressor, the animals tested), drug pharmacokinetics (variable drug absorption and bioavailability), induction of drug-neutralizing enzymes in the brain, variable (interindividual) HPA axis responsiveness to stressors (41), or possibly drug tolerance. Moreover, studies performed with the CRH type 1 antagonist, R121919, suggest that the anxiolytic effects of CRHR-1 antagonists may depend on the innate ability to mount a response to stress (42). Because age per se is thought to modulate the activity of the HPA axis in response to social separations (43), it is possible that we did not observe other behavioral changes because our study involved young (preadolescent) monkeys, as opposed to the adult monkeys used in an earlier study.
Antalarmin and the closely related compound CP-126,154 penetrate the BBB (9, 27). However, the optimal daily nontoxic dose of antalarmin needed to achieve CNS penetration, and the desired anxiolytic effect is not known. Although plasma antalarmin was detected in all the monkeys receiving the drug, we found lower CSF levels of antalarmin in preadolescent monkeys, compared with our previously published data in adult male rhesus monkeys. Hence, suboptimal CNS penetration or increased CSF clearance of antalarmin in younger monkeys may partially explain the decreased behavioral effects observed in this study. Also, alterations of BBB permeability may be precipitated by chronic repeated stress (44). Therefore, the difference in the nature and degree of the stressor may account for the lower antalarmin CSF levels observed in our study.
Certain limitations of our study need to be taken into consideration. The small number of animals in this experiment could explain why we did not find changes in other behavioral and endocrine parameters. Serum antalarmin levels showed significant variability, probably reflecting variable uptake, absorption rate, and metabolism of the compound. Although our dosing regimen was based on previous findings, the optimal dose of antalarmin remains to be determined.
To the best of our knowledge, our study is the first masked placebo-controlled trial involving long-term oral administration of the CRH antagonist antalarmin to nonhuman primates. CRHR-1 antagonists are potential therapeutic agents for psychiatric and somatic disorders associated with a maladaptive stress response. Marked differences are observed in the distribution of CRH neurons and CRHR-1 in humans, primates, and rodents. Therefore, comparing paradigms and outcomes in animal and human studies is a major concern to investigators who are interested in studying the safety and effectiveness of CRH antagonists. The phylogenetic similarity between nonhuman primates and humans narrows such a gap, and we can infer that similar behavioral, autonomic, and neuroendocrine responses will occur in humans. Indeed, a dose-escalating (phase 1), open-label study involving depressed subjects who received the CRHR-1 receptor blocker R121919 found that plasma cortisol and ACTH secretion rates and their respective circadian rhythms were not altered (45). The authors, however, reported a significant reduction in depression and anxiety scores. Furthermore, relapse was observed in a substantial number of patients after the drug was discontinued. These results lend further credence to the hope that CRH antagonists may be a novel class of anxiolytics and antidepressants. Nonetheless, because of the small number of patients involved in this study, the lack of a control group, and the possibility of a placebo effect, caution should be exercised.
In summary, we report the first masked placebocontrolled study involving chronic oral administration of a nonpeptide CRHR-1 antagonist in nonhuman primates. Our study suggests that chronic CRHR-1 receptor blockade does not blunt the HPA axis, sympathetic, and adrenomedullary hormonal responses to stress but increases environmental exploration, a behavior that is normally suppressed during stressful events. Taken together, these finding suggest that CRHR-1 antagonists may be a valid treatment of stress- related disorders, including anxiety and depression. Nonetheless, many uncertainties remain. Because antalarmin did not affect other measures of anxiety, further studies involving a larger number of subjects and different dosing regimens are needed to better elucidate the putative role and safety of orally administered CRHR-1 antagonists in clinical practice.
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Acknowledgments |
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Footnotes |
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Received December 18, 2003.
Accepted July 27, 2004.
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