PET Imaging of CRF1 with [11C]R121920 and [11C]DMP696: is the target of sufficient density?
Introduction
Corticotropin-releasing factor (CRF) is a 41-amino acid peptide, first isolated from hypothalamus, that was shown to activate the hypothalamic-pituitary-adrenal (HPA) axis by stimulating the release of adrenocorticotropic hormone by the pituitary [1]. CRF is present in widespread brain regions and is a central mediator of the behavioral, autonomic, endocrine and immune responses to stress [2]. The physiological effects of CRF are mediated by at least two G-protein-coupled receptors, CRF1 and CRF2 [3]. CRF also binds to CRF-binding protein (CRF-BP), a membrane-associated protein that regulates local availability of CRF at its receptors [4].
Rodent studies indicate that the CRF1 and CRF2 receptors are pharmacologically distinct and have different anatomical distributions [5]. In the rodent, CRF1 mediates effects on the HPA axis and anxiety-related behaviors [6], and CRF2 affects feeding behaviors [7]. There are important species differences in the distributions of mRNA and protein of these two receptors between nonhuman primate and rodent [8], [9]. In pituitary, CRF1 protein level depends on post-transcriptional regulatory mechanisms [10], suggesting dissociation of protein density from mRNA level for this receptor.
CRF hypersecretion is implicated in the pathogenesis of mood and anxiety disorders. Higher CRF in cerebrospinal fluid has been reported in major depression [11], [12], [13], as well as PTSD [14], Tourette's syndrome [15], anorexia nervosa [16] and suicide [17], [18], [19], [20]. Postmortem studies of suicides report higher pontine [21] and frontal cortical [22] CRF peptide, and lower frontal cortical CRF-binding sites [23] and CRF1 mRNA [22]. CRF1 antagonists are in development as potential therapeutic agents in mood and anxiety disorders [24], [25]. The CRF1 antagonist R121919 reduces anxiety-related behaviors in rodent [6], [26], and anxiolytic and antidepressant effects are suggested in man [27].
A positron emission tomography (PET) radioligand for CRF1 would permit in vivo quantification of regional brain binding in psychiatric disorders and suicidal behaviors as well as receptor occupancy relationships for therapeutic drug studies. There have been only a few reports on the development of CRF1 radioligands for PET or single photon emission tomography (SPECT) imaging. With the use of high affinity, selective, nonpeptide CRF1 antagonist antalarmin (N-butyl-N-ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo-[2,3]pyrimidin-4-yl]-amine; Ki=2.5 nM) as a template, several fluoro-substituted analogues were synthesized [28]. One with subnanomolar affinity for CRF1 (Ki=0.91 nM) proved to have poor penetration of the blood–brain barrier (BBB), prompting the design of more hydrophilic analogues with calculated log P values (ClogP) less than that of antalarmin (ClogP=7.0) [29]. An iodinated analogue for SPECT imaging was successfully converted to a targeted I-125 pure product, but, unfortunately, it also had reduced affinity (Ki=14 nM) for CRF1 [30]. Fluorinated and iodinated derivatives based on the high affinity CRF1 antagonist CP-154,526 (Ki=0.48 nM) have also been synthesized. Two with subnanomolar binding affinities (Ki=0.52 nM, 0.94 nM) were radiolabeled with F-18 and I-123, respectively [31]. Yet, in rodent both showed low accumulation in brain and very poor uptake and retention in regions of high CRF1 density. More recently, a high affinity (Ki=1.9 nM), lower lipophilicity (ClogP=3.05) brominated derivative of CP-154,526 was synthesized and labeled with Br-76 [32]. Biodistribution studies in rat indicated [76Br]MJL-1-109-2 crosses the BBB, and autoradiography demonstrated a pattern of CRF1-specific binding comparable with the known distribution of CRF1. Yet, to date, testing in PET has not been reported.
We have previously reported on a potential CRF1 radioligand [11C]SN003 [33]. We now report on two new small molecule CRF1 antagonist ligands that are candidates for PET imaging, R121920 and DMP969, that we have labeled with carbon-11 and tested for in vivo binding in baboon. Since one of the essential criteria for the success of a PET radioligand is sufficient density of the target protein [34], we also performed membrane-enriched tissue homogenate and autoradiographic binding studies of CRF1 in baboon and human brain to determine whether CRF1 protein density is sufficient for PET.
Section snippets
Synthesis of [11C]R121920 and [11C]DMP696
The methodologies for the preparation of precursors and the radiosyntheses for [11C]R121919 and [11C]DMP696 have been described previously [35], [36]. Table 1 shows the molecular weights, melting points, partition coefficients (logPo/w) and Ki for these ligands along with synthesis time, radiochemical yield, purity and specific activity (SA). Our previously reported CRF1 radioligand, [11C]SN003 [33], is included in the table for comparison purposes. Fig. 1 shows the chemical structures of all
Metabolism of [11C]R121920 and [11C]DMP696 in baboon
Both [11C]R121920 and [11C]DMP696 were rapidly metabolized, with mean times to 50% of parent ligand remaining in plasma of 12.9 min for [11C]R121920 and 9.2 min for [11C]DMP696. Fig. 2A and B shows the remaining percentage of unmetabolized parent compound for the two radioligands as a function of time.
PET imaging in baboon
Both [11C]R121920 and [11C]DMP696 demonstrated a pattern of distribution consistent with BBB penetration and early accumulation of the parent compound in the brain. Representative time–activity
Discussion
We report on the development of two novel C-11 CRF1 radioligands, [11C]R121920 and [11C]DMP696, for PET imaging. However, similar to our prior report of the CRF1 radioligand [11C]SN003 [33], in vivo testing of these ligands failed to demonstrate detectable specific binding in baboon brain using PET. The imaging analyses included several brain regions known to contain significant levels of the CRF1 receptor in primate such as cerebellum and multiple cortical regions [9], [48], [49]. The
Conclusion
In summary, we report on two new C-11 radioligands for CRF1 and PET, [11C]DMP696 and [11C]R121920. Like our prior finding with [11C]SN003, we found no evidence of detectable specific binding for CRF1 in vivo in baboon using PET. Our in vitro survey of human brain regions assessing the binding density or Bmax of CRF1 indicates the amount of target protein in brain areas assessable by PET is quite low and near the limit of detectability. Moreover, we found a major species difference in binding
Acknowledgments
This study was supported by NIMH grant MH066620 (P.I.: J.S. Dileep Kumar). The authors are grateful for the work of the imaging team in the Division of Neuroscience and Virginia Johnson for her assistance with the autoradiography images.
References (53)
Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders
Psychoneuroendocrinology
(1995)- et al.
Corticotropin releasing factor (CRF) binding protein: a novel regulator of CRF and related peptides
Front Neuroendocrinol
(1995) - et al.
Brain penetrance, receptor occupancy and antistress in vivo efficacy of a small molecule corticotropin releasing factor type I receptor selective antagonist
Neuropsychopharmacology
(2002) - et al.
Corticotrophin-releasing factor receptors: from molecular biology to drug design
Trends Pharmacol Sci
(1996) - et al.
Corticotropin releasing hormone receptors: two decades later
Peptides
(2004) - et al.
Low cerebrospinal fluid corticotropin-releasing hormone concentrations in eucortisolemic depression
Biol Psychiatry
(1997) - et al.
Elevated cerebrospinal fluid corticotropin-releasing factor in Tourette's syndrome: comparison to obsessive compulsive disorder and normal controls
Biol Psychiatry
(1996) - et al.
Elevated CSF CRF in suicide victims
Biol Psychiatry
(1989) - et al.
HPA-related CSF neuropeptides in suicide attempters
Eur Neuropsychopharmacol
(1992) - et al.
Decreased corticotropin-releasing hormone (CRH) concentrations in the cerebrospinal fluid of eucortisolemic suicide attempters
J Psychiatr Res
(2001)
A follow up study of suicide attempters: increase of CSF-somatostatin but no change in CSF-CRH
Eur Neuropsychopharmacol
The rationale for corticotropin-releasing hormone receptor (CRH-R) antagonists to treat depression and anxiety
J Psychiatr Res
Effects of the high-affinity corticotropin-releasing hormone receptor 1 antagonist R121919 in major depression: the first 20 patients treated
J Psychiatr Res
Synthesis and biological activity of fluoro-substituted pyrrolo[2,3-d]pyrimidines: the development of potential positron emission tomography imaging agents for the corticotropin-releasing hormone type 1 receptor
Bioorg Med Chem Lett
CRHR1 Receptor binding and lipophilicity of pyrrolopyrimidines, potential nonpeptide corticotropin-releasing hormone type 1 receptor antagonists
Bioorg Med Chem
The development of a potential single photon emission computed tomography (SPECT) imaging agent for the corticotropin-releasing hormone receptor type
Bioorg Med Chem Lett
Synthesis and characterization of fluorinated and iodinated pyrrolopyrimidines as PET/SPECT ligands for the CRF1 receptor
Nucl Med Biol
Synthesis and in vivo evaluation of [11C]SN003: a potential PET ligand for CRF1 receptors
Bioorg Med Chem
Predicting the success of a radiopharmaceutical for in vivo imaging of central nervous system neuroreceptor systems
Mol Imaging Biol
Autoradiographic localization of CRF1 and CRF2 binding sites in adult rat brain
Neuropsychopharmacology
The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites
Nucl Med Biol
Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin
Science
Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders
Mol Psychiatry
Characterization of corticotropin-releasing factor receptor subtypes
Ann N Y Acad Sci
Expression of corticotropin releasing hormone receptors type I and type II mRNA in suicide victims and controls
Mol Psychiatry
Autoradiographic and in situ hybridization localization of corticotropin-releasing factor 1 and 2 receptors in nonhuman primate brain
J Comp Neurol
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