Numerous lines of investigation indicate that different types of stress from diverse causes elevates corticotropin releasing hormone (CRH) in the brain and periphery and that interaction of CRH with its receptors launches multiple coping responses in the organism via the HPA axis and direct action on CRH neurons. Many human disorders of the central nervous and periphery are linked to elevation or depletion of the CRH receptor system tone. These include gastrointestinal disorders, eating disorders, depression, anxiety-related disorders, obsessive compulsive disorder and others. CRH also plays a role in the immune response to various stressors, and in premature delivery in pregnancy and in insulin resistance. Chronic insulin resistance has many detrimental effects, which can lead to permanent disorders such as hypertension, obesity and type II diabetes mellitus. Substance abuse and its consequences are also major stressors and contribute to many current societal and medical problems including a major role in the spread of acquired immune deficiency syndrome, drug-resistant tuberculosis and hepatitis. In order to gain further insight into the pathogenesis of stress-related disorders, to probe sites for possible intervention, and to develop potential treatments for these disorders, we have designed, synthesized and evaluated novel nonpeptide ligands which act on CRH receptors, cocaine receptors [DA transporter proteins (DAT)] and the opioid receptors. Significance: Systemically active, nonpeptide agonists and antagonists will be invaluable research tools for definitive preclinical and clinical studies of the variety of disorders already known to be modulated by the CHR system and for the discovery of new roles of this system in normal and pathological states. Further elucidation of the underlying mechanisms of these disorders will enable the development of targeted therapeutic and diagnostic interventions. The development of PET and SPECT imaging agents for in vivo quantitation of CRH receptor subtypes in animals and conscious humans has vast potential for understanding the function of the CRH system in normal, drug-altered and disease states. Such imaging studies may provide (a) insight in the fundamental nature of behavior, drug action and drug abuse on the CNS, (b) methodology for the development of clinical correlates with drug receptor dysfunction as adjuncts for diagnoses and (c) the ability to further evaluate the effects of treatment disorders that are linked to the CRH system. In addition, CRH receptor imaging methodology should be of value in the discovery and development of novel medications for the routine diagnosis and treatment CRH related disorders. With the current global threat aimed at civilian populations, the development of posttraumatic stress disorder (PTSD) on a national scale becomes a security as well as a health care priority. The linkage of abnormal stress responsivity and HPA dysfunction looms as a long term problem from our current societal disruptions. Chemical Synthesis: We previously described antalarmin, our lead compound in this work. We have now designed and synthesized potential PET and SPECT ligands that showed high affinity for CHR receptors. One of these (LWH-154) was a fluorine labeled candidate based on antalarmin that showed subnanomolar affinity and was labeled with tritium to high specific activity (69 Ci/mmol). A closely related compound with a terminal hydroxyl group in the side chain also showed subnanomolar affinity. One of the potential SPECT ligands, a high affinity iodo analog of NBI-27914, was labeled to high specific activity with 125I via a tributyltin intermediate. We also synthesized a high affinity bromo derivative as in this series as that could be labeled with 76Br to provide a potential PET ligand from the same tributyltin intermediate. We have also addressed synthesized a number of potential PET ligands with lower lipophilicity than antalarmin related compounds to enable passage into the brain and for clearance of nonspecific binding within the constraints of the half-life of the positron emitting isotope. One example is a bromo-[1,5-a]pyrazolo-1,3,5-triazine analog of DMP696 that showed Clog P 3.05 and Ki = 1.3 nM suggesting that the 76Br-labeled material as a potential PET ligand. Our chemical studies also includes the synthesis of two 13C antalarmin isotopes with double 13C labeling in different positions required for pharmacokinetic studies. We also developed a practical synthesis of multigram amounts of the pure crystalline serotonin 5HT2a antagonist MDL 100907, its enantiomer and their 3-phenolic derivatives and N-nor derivatives. MDL 100907 is the most selective antagonist known for the serotonin 5HT2a receptor. The 3-phenolic derivative was required as precursor of the 13C labeled drug for our primate PET studies of the action of antalarmin on 5HT2a receptor. Biological Results: Our initial characterization of antalarmin showed that antalarmin displaced 125I ovine CRH from rat pituitary, cerebellum and frontal cortex homogenates with respective Ki values of 1.9, 1.3 and 1.4 nM. No displacement was observed in heart homogenates consistent with CRH1 receptor selectivity. In vivo, antalarmin (20 mg/kg ip) suppressed secretion of CRH-induced ACTH and corticosterone. It also suppressed carrageenin-induced subcutaneous inflammation indicating that this and other such compounds may be valuable tools for the study of disorders involving hypersecretion of CRH. Our later studies showed that antalarmin has many diverse effects including: 1. Attenuation of immobilization-stress-induced stomach ulcer, colonic hypermotility, mucin depletion and struggling behavior in the rat. Antalarmin was more effective that the anxiolytics diazepam and buspirone and the antidepressants buspirone and fluoxetine in these tests. 2. Extension of gestation time in sheep by virtue of its antagonism of fetal CHR. 3. Attenuation of behavioral, neuroendocrine and autonomic responses in primates after oral administration in the intruder paradigm, an intense social stressor. These findings underscore the importance of CRH in the central nervous system and periphery.
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