Evaluation of the agonist PET radioligand [¹¹C]GR103545 to image kappa opioid receptor in humans: kinetic model selection, test-retest reproducibility and receptor occupancy by the antagonist PF-04455242.

INTRODUCTION: Kappa opioid receptors (KOR) are implicated in several brain disorders. In this report, a first-in-human positron emission tomography (PET) study was conducted with the potent and selective KOR agonist tracer, [(11)C]GR103545, to determine an appropriate kinetic model for analysis of PET imaging data and assess the test-retest reproducibility of model-derived binding parameters. The non-displaceable distribution volume (V(ND)) was estimated from a blocking study with naltrexone. In addition, KOR occupancy of PF-04455242, a selective KOR antagonist that is active in preclinical models of depression, was also investigated. METHODS: For determination of a kinetic model and evaluation of test-retest reproducibility, 11 subjects were scanned twice with [(11)C]GR103545. Seven subjects were scanned before and 75 min after oral administration of naltrexone (150 mg). For the KOR occupancy study, six subjects were scanned at baseline and 1.5 h and 8 h after an oral dose of PF-04455242 (15 mg, n=1 and 30 mg, n=5). Metabolite-corrected arterial input functions were measured and all scans were 150 min in duration. Regional time-activity curves (TACs) were analyzed with 1- and 2-tissue compartment models (1TC and 2TC) and the multilinear analysis (MA1) method to derive regional volume of distribution (V(T)). Relative test-retest variability (TRV), absolute test-retest variability (aTRV) and intra-class coefficient (ICC) were calculated to assess test-retest reproducibility of regional VT. Occupancy plots were computed for blocking studies to estimate occupancy and V(ND). The half maximal inhibitory concentration (IC50) of PF-04455242 was determined from occupancies and drug concentrations in plasma. [(11)C]GR103545 in vivo K(D) was also estimated. RESULTS: Regional TACs were well described by the 2TC model and MA1. However, 2TC VT was sometimes estimated with high standard error. Thus MA1 was the model of choice. Test-retest variability was ~15%, depending on the outcome measure. The blocking studies with naltrexone and PF-04455242 showed that V(T) was reduced in all regions; thus no suitable reference region is available for the radiotracer. V(ND) was estimated reliably from the occupancy plot of naltrexone blocking (V(ND)=3.4±0.9 mL/cm(3)). The IC50 of PF-04455242 was calculated as 55 ng/mL. [(11)C]GR103545 in vivo K(D) value was estimated as 0.069 nmol/L. CONCLUSIONS: [(11)C]GR103545 PET can be used to image and quantify KOR in humans, although it has slow kinetics and variability of model-derived kinetic parameters is higher than desirable. This tracer should be suitable for use in receptor occupancy studies, particularly those that target high occupancy.

Comparison of actigraphy and polysomnography to assess effects of zolpidem in a clinical research unit.

OBJECTIVE: This study sought to compare devices that use actigraphy for measuring sleep endpoints in the clinical research unit (CRU) and home environment. The abilities of polysomnography (PSG) and actigraphy monitors to detect drug effects in a CRU were also investigated. METHODS: Eleven healthy subjects were recruited and monitored with PSG for four consecutive nights in a CRU after receiving no treatment (night 1, N1), and then placebo or 5 mg day(-1) or 10 mg day(-1) zolpidem in a randomised, cross-over design. Subjects wore two devices that use actigraphy (a Respironics® Actiwatch® on the wrist and a BodyMedia® Sensewear® Armband on the upper-arm) on the non-dominant arm for five nights at home and four nights in the CRU during PSG. RESULTS: Wake after sleep onset (WASO) and total sleep time (TST) measured by PSG and estimates of WASO by the Actiwatch decreased significantly with 5mg but not 10mg of zolpidem versus placebo. Direct activity (counts/min) with the Actiwatch decreased in response to zolpidem (both 5 and 10 mg day(-1)) versus placebo. Armband recordings of direct activity were similar to the Actiwatch but not significantly different versus placebo. Both actigraphy device estimates of TST were approximately 1h longer in CRU versus home. Agreement between actigraphy estimates of TST and WASO and PSG values of TST and WASO were closer during nights with zolpidem treatment. CONCLUSIONS: PSG can detect the effects of zolpidem on sleep in a CRU setting. Actigraphy can provide useful assessment of sleep, but direct activity endpoints may be more effective than estimates of TST and WASO.

Quantitative PK-PD model-based translational pharmacology of a novel kappa opioid receptor antagonist between rats and humans.

Pharmacokinetic-pharmacodynamic (PK-PD) modeling greatly enables quantitative implementation of the "learn and confirm" paradigm across different stages of drug discovery and development. This work describes the successful prospective application of this concept in the discovery and early development of a novel κ-opioid receptor (KOR) antagonist, PF-04455242, where PK-PD understanding from preclinical biomarker responses enabled successful prediction of the clinical response in a proof of mechanism study. Preclinical data obtained in rats included time course measures of the KOR antagonist (PF-04455242), a KOR agonist (spiradoline), and a KOR-mediated biomarker response (prolactin secretion) in plasma. Clinical data included time course measures of PF-04455242 and prolactin in 24 healthy volunteers following a spiradoline challenge and single oral doses of PF-04455242 (18 and 30 mg). In both species, PF-04455242 successfully reversed spiradoline-induced prolactin response. A competitive antagonism model was developed and implemented within NONMEM to describe the effect of PF-04455242 on spiradoline-induced prolactin elevation in rats and humans. The PK-PD model-based estimate of K(i) for PF-04455242 in rats was 414 ng/mL. Accounting for species differences in unbound fraction, in vitro K(i) and brain penetration provided a predicted human K(i) of 44.4 ng/mL. This prediction was in good agreement with that estimated via the application of the proposed PK-PD model to the clinical data (i.e., 39.2 ng/mL). These results illustrate the utility of the proposed PK-PD model in supporting the quantitative translation of preclinical studies into an accurate clinical expectation. As such, the proposed PK-PD model is useful for supporting the design, selection, and early development of novel KOR antagonists.

Comparison of the stress response in rats to repeated isoflurane or CO2:O2 anesthesia used for restraint during serial blood collection via the jugular vein.

The primary objective of this study was to compare the effect of 2 commonly used anesthetics, isoflurane and CO2, on the physiologic stress hormone, corticosterone, in rats during serial blood collections. Circulating corticosterone concentrations were monitored during serial jugular blood sampling in rats exposed to either isoflurane or CO2 anesthesia. Blood was drawn under anesthesia at 6 time points (initial sampling and 0.5, 1, 2, 3, and 24 h after initial sampling) across a 24-h period. The results indicated that corticosterone levels in both anesthesia groups showed a similar pattern of stimulation, indicated by a sharp increase in circulating concentrations by the 0.5-h time point, with further elevation at 1 h, and a decline at subsequent time points. Isoflurane-treated animals showed higher baseline levels of corticosterone at the initial sampling. However, the increase in corticosterone at subsequent time points was significantly higher for animals exposed to CO2, suggesting that the cumulative effects of repeated exposure to stressful stimuli was more evident for this type of anesthesia. The apparent reduction in stress effects of isoflurane as opposed to CO2, coupled with other published adverse effects of CO2, suggests that isoflurane is the better choice when anesthesia is needed for serial blood collection. However, availability of equipment for delivery of anesthetic, scavenging of waste gasses, familiarity of personnel with the anesthetic, and the potential effect of the anesthetic on research endpoints should all be considered in choosing between the 2 anesthetics.

Noradrenergic regulation of prolactin secretion at the level of the paraventricular nucleus of the hypothalamus: functional significance of the alpha-1b and beta-adrenergic receptor subtypes.

Previous research has demonstrated that in the Siberian hamster, both photoperiod and estrous cyclicity alter the profile of noradrenergic activity with the paraventricular nucleus of the hypothalamus (PVN), and that noradrenergic activity is correlated with changes in circulating levels of prolactin. Work from our laboratory has demonstrated an inhibitory role for norepinephrine (NE) acting at the alpha-2 receptor subtype within the PVN on serum prolactin levels; however, the functional significance of other adrenergic receptor subtypes on this system is unknown. The purpose of this study was to investigate the functional significance of the alpha-1b and beta-adrenergic receptor subtypes at the level of the PVN on circulating levels of prolactin. These experiments were performed in male Siberian hamsters using reverse microdialysis coupled with serial blood sampling. In Experiment 1, infusion of l-phenylephrine hydrochloride (alpha-1b agonist) initiated a dose-dependent increase in circulating prolactin, whereas infusion of chloroethylclonidine (alpha-1b antagonist) induced a significant dose-dependent decline in prolactin. In Experiment 2, intraparaventricular administration of propranolol (beta antagonist) initiated a significant increase in prolactin levels in a dose-dependent manner, whereas isoproterenol (beta agonist) induced a dose-dependent decline in prolactin. The results of this study indicate that both the alpha-1b and beta-adrenergic receptor subtypes have a significant role in regulating circulating levels of prolactin at the level of the PVN in the Siberian hamster.

Male-induced estrus synchronization in the female Siberian hamster (Phodopus sungorus sungorus).

Olfactory cues play an integral role in the organization of events that mediate reproductive success. In a variety of species, priming pheromones, in particular, are important for ensuring reproductive fitness. To date, very little research has focused on how male-emitted priming pheromones, such as those that regulate the onset of puberty and estrus synchronization in females, affect the reproductive physiology of the female Siberian hamster (Phodopus sungorus sungorus). This lack of research may be due to the physiology of the Phodopus genus; vaginal cytology cannot be used as a reliable indicator of estrus or ovulation. Using a jugular cannulation technique to determine estrous stage by blood analysis of prolactin and luteinizing hormone, we sought to determine if male priming pheromones affect estrous cyclicity in the female Siberian hamster and, if so, whether the production of these priming pheromones is androgen dependent. Our results showed that females exposed to bedding from mature, intact males showed a significantly higher incidence of proestrus 3 days later than did females exposed to the bedding of mature, gonadectomized males. Therefore, we found that not only do male Siberian hamsters emit chemical signals that induce estrus synchronization, but also that this ability is likely to be androgen dependent.

5HT and 5HIAA dialysate levels within the arcuate nucleus of the hypothalamus: relationship with photoperiod-driven differences in serum prolactin and luteinizing hormone in the Siberian hamster.

This study examined the relationship between dialysate levels of serotonin (5HT), and its major metabolite 5HIIAA within the arcuate nucleus of the hypothalamus (ARC) and serum gonadotropin levels under two different in vivo paradigms. Experiment 1 evaluated the relationship between dialysate levels of 5HT and 5HIAA within the ARC and circulating prolactin (PRL) and lutenizing hormone (LH) levels under long- and short-day photoperiod conditions. In experiment 2, the profile of 5HT and 5HIAA dialysate levels within the ARC on the afternoon of proestrous was investigated to determine if changes in serotonergic neurotransmission are correlated with preovulatory surges in LH and PRL. Adult male and female Siberian hamsters were housed either in long-day (16L:8D) or short-day (10L:14D) photoperiods for 8 weeks. Dialysis samples were collected every hour for 5 h (12.00-17.00 h) and blood samples were collected via a jugular cannula every hour for analysis of LH and PRL levels. ARC 5HT and 5HIAA dialysate levels were significantly higher in short-day exposed female hamsters, correlating with suppressed basal LH and PRL secretion when compared to their long-day counterparts. Short-day housed male hamsters displayed significantly higher dialysate levels of 5HIAA than males exposed to a long-day photoperiod-5HT was below the lower limit of detection regardless of photoperiod exposure. Long-day females in proestrus showed no change in dialysate levels of 5HT or 5HIAA within the ARC just prior to the onset of the afternoon surge of LH and PRL. Our results indicate that elevated 5HT and 5HIAA dialysate levels within the ARC may regulate photoperiod effects upon LH and PRL secretion, but not the preovulatory surges of LH and PRL.

Infusion of alpha-2-adrenergic agents into the paraventricular and arcuate nuclei of the hypothalamus in the Siberian hamster: opposing effects on basal prolactin.

Although the alpha(2)-adrenergic receptor subtype has been shown to have a significant influence on circulating levels of prolactin (PRL), its exact role remains unclear. A multitude of studies have demonstrated that blockade of the alpha(2)-receptor can either elevate or decrease circulating levels of PRL. Alpha(2)-receptor-mediated control of both stimulatory and inhibitory arms of the PRL regulatory system may explain this discrepancy. Activation of the alpha(2)-receptor has been shown to inhibit the activity of its target cell, and therefore antagonism of the alpha(2)-receptor within a stimulatory component (e.g., paraventricular nucleus (PVN) of the hypothalamus) would theoretically have the opposite effect that it would have within an inhibitory component (arcuate nucleus of the hypothalamus). Thus, the purpose of this study was to investigate the functional role of the alpha(2)-adrenergic receptor in modulating circulating levels of PRL both at the level of the PVN and arcuate using reverse microdialysis of alpha(2)-adrenergic agents coupled with serial blood sampling in the male Siberian hamster. Male hamsters were fitted with a jugular cannula for serial blood sampling, and an indwelling microdialysis probe for intrahypothalamic drug administration between 08:00 and 10:00 h. Blood samples were collected every hour for 5 h (12:00-17:00 h). During the third sampling period, atipamezole (alpha(2)-antagonist) or medetomidine (alpha(2)-agonist) at one of three doses were infused into the PVN or the arcuate to assess effects on basal PRL. At the level of the PVN, infusion of atipamezole initiated an increase in basal PRL in a dose-dependent fashion, whereas infusion of medetomidine induced a significant decline in basal PRL in a dose-dependent fashion. In the arcuate, only the highest dose of atipamezole had an effect on PRL, and this was in the opposite direction from that seen in the PVN. Infusion of medetomidine did not have a significant effect on basal PRL levels; however, a trend toward a significant elevation was observed for the highest dose. These results suggest that the alpha(2)-receptor subtype may have opposite effects on circulating levels of PRL within the PVN and arcuate regions, and may explain why antagonism of the alpha(2)-receptor has been shown to initiate both surges and declines in basal levels of PRL.