Non-peptide Antagonist Research Articles

Targeting the Urotensin II/UT G Protein-Coupled Receptor to Counteract Angiogenesis and Mesenchymal Hypoxia/Necrosis in Glioblastoma.

Glioblastomas (GBMs) are the most common primary brain tumors characterized by strong invasiveness and angiogenesis. GBM cells and microenvironment secrete angiogenic factors and also express chemoattractant G protein-coupled receptors (GPCRs) to their advantage. We investigated the role of the vasoactive peptide urotensin II (UII) and its receptor UT on GBM angiogenesis and tested potential ligand/therapeutic options based on this system. On glioma patient samples, the expression of UII and UT increased with the grade with marked expression in the vascular and peri-necrotic mesenchymal hypoxic areas being correlated with vascular density. In vitro human UII stimulated human endothelial HUV-EC-C and hCMEC/D3 cell motility and tubulogenesis. In mouse-transplanted Matrigel sponges, mouse (mUII) and human UII markedly stimulated invasion by macrophages, endothelial, and smooth muscle cells. In U87 GBM xenografts expressing UII and UT in the glial and vascular compartments, UII accelerated tumor development, favored hypoxia and necrosis associated with increased proliferation (Ki67), and induced metalloproteinase (MMP)-2 and -9 expression in Nude mice. UII also promoted a “tortuous” vascular collagen-IV expressing network and integrin expression mainly in the vascular compartment. GBM angiogenesis and integrin αvβ3 were confirmed by in vivo 99mTc-RGD tracer imaging and tumoral capture in the non-necrotic area of U87 xenografts in Nude mice. Peptide analogs of UII and UT antagonist were also tested as potential tumor repressor. Urotensin II-related peptide URP inhibited angiogenesis in vitro and failed to attract vascular and inflammatory components in Matrigel in vivo. Interestingly, the UT antagonist/biased ligand urantide and the non-peptide UT antagonist palosuran prevented UII-induced tubulogenesis in vitro and significantly delayed tumor growth in vivo. Urantide drastically prevented endogenous and UII-induced GBM angiogenesis, MMP, and integrin activations, associated with GBM tumoral growth. These findings show that UII induces GBM aggressiveness with necrosis and angiogenesis through integrin activation, a mesenchymal behavior that can be targeted by UT biased ligands/antagonists.

Possible Protective Effect of Endothelin-1 Receptor Antagonist on Renal Impairment in Type 2 Diabetic Rats

Background: Diabetic nephropathy (DN) is characterized by persistent albuminuria and progressive decline in glomerular filtration rate (GFR). Bosentan is a non-peptide mixed antagonist of ET-1. Aim: To investigate the possible protective role of bosentan on renal impairment in T2DM rats and the mechanisms concerned. Materials: Fifty rats were randomly divided into five equal groups; Non-Diabetic (ND), Diabetic non-treated (DN), Diabetic Bosentan-treated (D-Bos.), Diabetic Metformin-treated (D-Met.), Diabetic combined Bosentan and Metformin-treated groups (D-Bos& Met.). T2DM was induced by Streptozotocin (STZ) (35mg/kg) and high fat diet (HFD) for 3 weeks. Both Bosentan and metformin were given orally for 4 weeks. At the end of the experiment, 24hrs urine samples were collected to measure urine (volume, albumin, NAG and creatinine), renal blood flow velocity (RBFV) and peripheral renal resistance (RVR) were measured using Doppler technique. Blood was collected directly from abdominal aorta to estimate fasting serum glucose, glycosylated hemoglobin (HBA1c), serum insulin, complete lipid profile, renal function tests and parameters of oxidative stress: malondialdehyde (MDA), total antioxidant capacity (TAC) and tumor necrosis factor-α (TNFα). Insulin resistance, serum LDL-cholesterol, urinary albumin creatinine ratio (UACR) and creatinine clearance (CC) were calculated. Results: Serum cysteine c, BUN, urinary albumin and UACR were significantly lowered in both D-Bos. & D-Met when compared to the corresponding values in DN while urinary volume, urinary creatinine, CC and RBFV were significantly elevated but serum creatinine, NAG & RVR were significantly lowered in D-Bos. only. Conclusion: Bosentan and metformin treatment both have a renoprotective effect. Combined treatment showed better results.

Vasopressin V1B Receptor Antagonists as Potential Antidepressants.

Accumulating evidence shows that certain populations of depressed patients have impaired hypothalamus-pituitary-adrenal (HPA) axis function. Arginine-vasopressin (AVP) is one of the primary factors in HPA axis regulation under stress situations, and AVP and its receptor subtype (V1B receptor) play a pivotal role in HPA axis abnormalities observed in depression. Based on this hypothesis, several non-peptide V1B receptor antagonists have been synthesized, and the efficacies of some V1B receptor antagonists have been investigated in both animals and humans. V1B receptor antagonists exert antidepressant-like effects in several animal models at doses that attenuate the hyperactivity of the HPA axis, and some of their detailed mechanisms have been delineated. These results obtained in animal models were, at least partly, reproduced in clinical trials. At least 2 V1B receptor antagonists (TS-121 and ABT-436) showed tendencies to reduce the depression scores of patients with major depressive disorder at doses that attenuate HPA axis hyperactivity or block the pituitary V1B receptor. Importantly, TS-121 showed a clearer efficacy for patients with higher basal cortisol levels than for those with lower basal cortisol levels, which was consistent with the hypothesis that V1B receptor antagonists may be more effective for patients with HPA axis hyperactivity. Therefore, V1B receptor antagonists are promising approaches for the treatment of depression involving HPA axis impairment such as depression.

A Robust Bioassay of the Human Bradykinin B2 Receptor That Extends Molecular and Cellular Studies: The Isolated Umbilical Vein

Bradykinin (BK) has various physiological and pathological roles. Medicinal chemistry efforts targeted toward the widely expressed BK B2 receptor (B2R), a G-protein-coupled receptor, were primarily aimed at developing antagonists. The only B2R antagonist in clinical use is the peptide icatibant, approved to abort attacks of hereditary angioedema. However, the anti-inflammatory applications of B2R antagonists are potentially wider. Furthermore, the B2R antagonists notoriously exhibit species-specific pharmacological profiles. Classical smooth muscle contractility assays are exploited over a time scale of several hours and support determining potency, competitiveness, residual agonist activity, specificity, and reversibility of pharmacological agents. The contractility assay based on the isolated human umbilical vein, expressing B2R at physiological density, was introduced when investigating the first non-peptide B2R antagonist (WIN 64338). Small ligand molecules characterized using the assay include the exquisitely potent competitive antagonist, Pharvaris Compound 3 or the partial agonist Fujisawa Compound 47a. The umbilical vein assay is also useful to verify pharmacologic properties of special peptide B2R ligands, such as the carboxypeptidase-activated latent agonists and fluorescent probes. Furthermore, the proposed agonist effect of tissue kallikrein on the B2R has been disproved using the vein. This assay stands in between cellular and molecular pharmacology and in vivo studies.

Targeting GRP receptor: Design, synthesis and preliminary biological characterization of new non-peptide antagonists of bombesin

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration.GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP.We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues’ side chains in a suitable manner for binding to GRP-R.Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.

Neonatal Suckling, Oxytocin, and Early Infant Attachment to the Mother.

The neuropeptide oxytocin (OT) promotes maternal care and social affiliation in adults but its importance in infant attachment still remains unknown. True animal models of infant attachment are extremely rare, and the sheep (in complement to non-human primates) is one of the few that provides the opportunity to investigate its neuroendocrinological basis. In the lamb, access to the udder has strong rewarding properties for the establishment of a preferential relationship with the mother. Therefore, the present study explored the possible involvement of OT through its release during close social contacts with the mother. The first experiment revealed that lambs having free access to the udder from birth onward developed, by 12 h of age, a clear preference for their mothers over another maternal ewe. Delaying access to the udder for six, four or even only 2 h starting at birth, by covering the ewe’s udder, resulted in the lack of such a preference without affecting general activity. These effects persisted in most cases at 24 h but by 72 h of age a bond with the mother was clearly expressed. Experiment two showed that social interactions with the mother were followed by a release of OT in the plasma when lambs had the possibility to suckle. Non-nutritive interactions were without effects. Preliminary data on two subjects suggested that OT might also increase in the cerebrospinal fluid after suckling. Finally, in the third experiment, oral administration of a non-peptide OT receptor antagonist (L-368-899, Merck) over the first 4 h after birth led to decreased exploration of the mother’s body compared to lambs receiving saline, and impaired the expression of a preference for the mother at 24 h. The effects were no longer observed at 48 h. Our findings demonstrate that both delayed access to the mother’s udder and OT receptor antagonist alter the onset of mother preference in newborn lambs. This suggests that central OT facilitates the development of filial attachment through its release during suckling.

Relugolix: A novel gonadotropin-releasing hormone antagonist for prostate cancer

Androgen deprivation therapy (ADT) is currently the mainstay of treatment for advanced prostate cancer. The peptide formulations of gonadotropin-releasing hormone (GnRH) antagonists need to be given subcutaneously every month. This led to the development of an oral, nonpeptide GnRH antagonist formulation relugolix which promptly lowers the levels of testosterone, luteinizing hormone, and follicular-stimulating hormone. On December 18, 2020, the US Food and Drug Administration approved relugolix for the treatment of adult advanced prostate cancer. The recommended loading dose of 360 mg on the 1st day of treatment, followed by 120 mg once daily orally, approximately the same time each day. The maximum plasma concentration (Tmax) is obtained within 2.25 h and is metabolized to a major extent by CYP3A mediated mechanism. Hot flushes, musculoskeletal pain, and fatigue are some of its common adverse effects. High rates of testosterone suppression with a limited adverse event profile make it an ideal therapy for the treatment of advanced prostate cancer.

The development of proteinase-activated receptor-2 modulators and the challenges involved.

Protease-activated receptor-2 (PAR2) has been extensively studied since its discovery in the mid-1990. Despite the advances in understanding PAR2 pharmacology, it has taken almost 25 years for the first inhibitor to reach clinical trials, and so far, no PAR2 antagonist has been approved for human use. Research has employed classical approaches to develop a wide array of PAR2 agonists and antagonists, consisting of peptides, peptoids and antibodies to name a few, with a surge in patent applications over this period. Recent breakthroughs in PAR2 structure determination has provided a unique insight into proposed PAR2 ligand binding sites. Publication of the first crystal structures of PAR2 resolved in complex with two novel non-peptide small molecule antagonists (AZ8838 and AZ3451) revealed two distinct binding pockets, originally presumed to be allosteric sites, with a PAR2 antibody (Fab3949) used to block tethered ligand engagement with the peptide-binding domain of the receptor. Further studies have proposed orthosteric site occupancy for AZ8838 as a competitive antagonist. One company has taken the first PAR2 antibody (MEDI0618) into phase I clinical trial (NCT04198558). While this first-in-human trial is at the early stages of the assessment of safety, other research into the structural characterisation of PAR2 is still ongoing in an attempt to identify new ways to target receptor activity. This review will focus on the development of novel PAR2 modulators developed to date, with an emphasis placed upon the advances made in the pharmacological targeting of PAR2 activity as a strategy to limit chronic inflammatory disease.

Procalcitonin Exerts a Mediator Role in Septic Shock Through the Calcitonin Gene-Related Peptide Receptor.

Clinically, procalcitonin represents the most widely used biomarker of sepsis worldwide with unclear pathophysiologic significance to date. Pharmacologically, procalcitonin was shown to signal through both calcitonin receptor and calcitonin gene-related peptide receptor in vitro, yet the identity of its biologically relevant receptor remains unknown. Prospective randomized animal investigations and in vitro human blood studies. Research laboratory of a university hospital. C57BL/6J mice and patients with post-traumatic sepsis. Procalcitonin-deficient mice were used to decipher a potential mediator role in experimental septic shock and identify the relevant receptor for procalcitonin. Cecal ligation and puncture and endotoxemia models were employed to investigate septic shock. Disease progression was evaluated through survival analysis, histology, proteome profiling, gene expression, and flow cytometry. Mechanistic studies were performed with cultured macrophages, dendritic cells, and gamma delta T cells. Main findings were confirmed in serum samples of patients with post-traumatic sepsis. Procalcitonin-deficient mice are protected from septic shock and show decreased pulmonary inflammation. Mechanistically, procalcitonin potentiates proinflammatory cytokine expression in innate immune cells, required for interleukin-17A expression in gamma delta T cells. In patients with post-traumatic sepsis, procalcitonin positively correlates with systemic interleukin-17A levels. In mice with endotoxemia, immunoneutralization of interleukin-17A inhibits the deleterious effect of procalcitonin on disease outcome. Although calcitonin receptor expression is irrelevant for disease progression, the nonpeptide calcitonin gene-related peptide receptor antagonist olcegepant, a prototype of currently introduced antimigraine drugs, inhibits procalcitonin signaling and increases survival time in septic shock. Our experimental data suggest that procalcitonin exerts a moderate but harmful effect on disease progression in experimental septic shock. In addition, the study points towards the calcitonin gene-related peptide receptor as relevant for procalcitonin signaling and suggests a potential therapeutic application for calcitonin gene-related peptide receptor inhibitors in sepsis, which warrants further clinical investigation.

Naltrexone a potential therapeutic candidate for COVID-19

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease (COVID-19) that has resulted in a global pandemic. At the time of writing, approximately 16.06 million cases have been reported worldwide. Like other coronaviruses, SARS-CoV-2 relies on the surface Spike glycoprotein to access the host cells, mainly through the interaction of its Receptor Binding Domain (RBD) with the host receptor Angiotensin-Converting Enzyme2 (ACE2). SARS-CoV-2 infection induces a profound downstream pro-inflammatory cytokine storm. This release of the pro-inflammatory cytokines is underpinning lung tissue damage, respiratory failure, and eventually multiple organ failure in COVID-19 patients. The phosphorylation status of ERK1/2 is positively correlated with virus load and ERK1/2 inhibition suppressed viral replication and viral infectivity. Therefore, molecular entities able to interfere with binding of the SARS-CoV-2 Spike protein to ACE2, or damping hyperinflammatory cytokines storm, blocking ERK1/2 phosphorylation have a great potential to inhibit viral entry along with viral infectivity. Herein, we report that the FDA-approved non-peptide opioid antagonist drug, naltrexone suppresses high fat/LPS induced pro-inflammatory cytokine release both from macrophage cells and Adipose Tissue Macrophage. Moreover, Low Dose Naltrexone (LDN) also showed its activity as an ERK1/2 inhibitor. Notably, virtual docking and simulation data also suggest LDN may disrupt the interaction of ACE2 with RBD. LDN may be considered as a target as the treatment and (or) adjuvant therapy for coronavirus infection. Clinical toxicity measurements may not be required for LDN since naltrexone was previously tested and is an approved drug by the FDA. Communicated by Ramaswamy H. Sarma

Optimization of Reaction Parameters for the Synthesis of 177Lu-DOTAELA

The article provides a comparison of the theoretically calculated and experimentally determined yield of the reaction 176Lu (n, �A)177Lu. Also, it provides the results of the studies on lutetium-177 labeling of a non-peptide antagonist of gonadotropin-releasing hormone (GnRH) elagolix (ELA) associated with a chelating DOTA (DOTAELA). The synthesized DOTAELA complex was labeled with the 177Lu isotope.177Lu was produced by the reaction (n, �A) using the enriched LuCl3 target at the reactor WWR�CK. Production of 177Lu by the (n, �A) reaction from the enriched 176Lu target achieved by irradiation for 17 days. All stages of the complex preparation were evaluated by paper chromatography. The optimal technological parameters for the synthesis of the complex 177Lu-DOTAELA are: pH - 4.5, 90-100 �aC and 40 min. The obtained optimal parameters made it possible to produce a labeled complex of 177Lu�CDOTAELA with a radiochemical yield of �Y 95%.

Identification of novel Urotensin-II receptor antagonists with potent inhibition of U-II induced pressor response in mice

Urotensin II (U-II) has been found to be one of the most potent vasoconstrictor (Ames et al., 1999; Bohm et al., 2002) reported till date. U-II exerts its response via activation of a G-protein coupled receptor, Urotensin II receptor(UT). Binding of U-II to UT leads to an instant increase in the inositol phosphate turnover and intracellular Ca2+. Such an instant Ca2+ release and potent vasoconstriction exerted by U-II is expected to have an important role in the progression of cardiac diseases.We have previously shown that UT antagonist DS37001789 prevents U-II induced blood pressure elevation in mice (Nishi et al., 2019) in a dose dependent manner, with potent efficacy at 30 and 100 mg/kg. Further to this, we have also shown that DS37001789 ameliorates mortality in pressure-overload mice with heart failure (Nishi et al., 2020). We therefore conducted an extensive structure-activity relationship studies to identify molecules with superior efficacy. In the present manuscript, we report the identification of two potent, non-peptide small molecule antagonists of Urotensin II receptor (UT), RCI-0879 and RCI-0298 which blocked the action of U-II, both in vitro and in vivo. These molecules were found to be very potent in in vitro Ca2+ and radioligand binding assays using human and mouse UT over-expressing CHO cells. RCI-0879 and RCI-0298 also exhibited superior efficacy in in vivo mouse pressor response model using C57BL/6 mice, compared to our initial molecules (Nishi et al., 2019) and demonstrated ED50 values of 3.2 mg/kg and 6.8 mg/kg respectively. Our findings reported herewith, further strengthen our concept and belief in UT antagonization as a potential therapeutic approach for the management of chronic heart failure.

Crystal structure of the human oxytocin receptor.

The peptide hormone oxytocin modulates socioemotional behavior and sexual reproduction via the centrally expressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a nonpeptidic antagonist developed as an oral drug for the prevention of preterm labor. Our structure reveals insights into the detailed interactions between the G protein-coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helices IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Furthermore, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg2+ that acts as potent, positive allosteric modulator for agonist binding. Together, these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics.

99mTc]Tc-DGA1, a Promising CCK2R-Antagonist-Based Tracer for Tumor Diagnosis with Single-Photon Emission Computed Tomography.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK2R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK2R antagonist Z-360 carrying an acyclic tetraamine, for [99mTc]Tc labeling. Preclinical comparison of [99mTc]Tc-DGA1 with [99mTc]Tc-DG2 (CCK2R-agonist reference) was conducted in HEK293-CCK2R/CCK2i4svR cells and mice models, qualifying [99mTc]Tc-DGA1 for further study in patients with CCK2R-positive tumors and single-photon emission computed tomography/CT.

Substance P enhances the local activation of NK1R-expressing c-kit+ cardiac progenitor cells in right atrium of ischemia/reperfusion-injured heart

BackgroundLocalization of neurokinin 1 receptor (NK1R), the endogenous receptor for neuropeptide substance P (SP), has already been described for the right atrium (RA) of the heart. However, the biological role of SP/NK1R signal pathways in the RA remains unclear.Sprague-Dawley rats were randomly divided into 4 groups (n = 22 each); subjected to sham, ischemia/reperfusion-injury (I/R), I/R with 5 nmole/kg SP injection (SP + I/R), and SP + I/R with 1 mg/kg RP67580 injection (RP, a selective non-peptide tachykinin NK1R antagonist) (RP/SP + I/R). The left anterior descending coronary artery was occluded for 40 min followed by 1 day reperfusion with SP or SP + RP or without either. After 1 day, both atria and ventricles as well as the heart apexes were collected.ResultsSP promoted the expression of c-Kit, GATA4, Oct4, Nanog, and Sox2 in only the RA of the SP + I/R rats via NK1R activation. In agreement with these observations, NK1R-expressing c-Kit+ Nkx2.5+GATA4+ cardiac progenitor cells (CPCs) in the ex vivo RA explant outgrowth assay markedly migrated out from RA1 day SP + I/R approximately 2-fold increase more than RA1 day I/R. Treatment of SP promoted proliferation, migration, cardiosphere formation, and potential to differentiate into cardiomyocytes. Using RP inhibitor, NK1R antagonist not only inhibited cell proliferation and migration but also reduced the formation of cardiosphere and differentiation of c-Kit+ CPCs.ConclusionSP/NK1R might play a role as a key mediator involved in the cellular response to c-Kit+ CPC expansion in RA of the heart within 24 h after I/R.

MON-176 Discovery and Identification of Late Stage Selective Nonpeptide ACTH Antagonists for the Treatment of Cushing’s Disease, Ectopic ACTH Secreting Tumors, and Congenital Adrenal Hyperplasia

Adrenocorticotropic hormone (ACTH) is an important modulator of steroidal hormone synthesis and secretion from the adrenal gland and its selective activity at the melanocortin type 2 receptor (MC2) dictates the synthesis and secretion of cortisol (corticosterone in rats). Excess ACTH action contribute to the pathophysiology of Cushing’s disease (CD), ectopic ACTH secreting tumors (EAS), and Congenital Adrenal Hyperplasia (CAH). Cushing’s disease results from a microadenoma derived from pituitary corticotrophic cells that secretes excess ACTH, whereas EAS arises from nonpituitary ACTH secreting tumors. Excess ACTH action at the adrenal gland and resulting hypercortisolemia presents in a myriad of symptoms that result in high morbidity. CAH results from inactivating mutations in steroid synthesis pathways, resulting in lack of cortisol and aldosterone production. Lack of negative feedback by cortisol at the level of the pituitary causes the over-secretion of ACTH, and overproduction of adrenal androgens, causing significant virilization and reduction in quality of life. We hypothesize that blocking ACTH action directly via a selective MC2 receptor antagonist may provide an important new therapeutic mechanism for these patients.To test this hypothesis, Crinetics launched an iterative medicinal chemistry program to identify potent and selective nonpeptide ACTH antagonists with pharmaceutical and safety characteristics suitable for evaluation in human clinical trials. Unlike most other G protein coupled receptors, MC2 requires the presence of an accessory protein (MRAP) for cell surface expression and recognition of ACTH. Using CHO-K cells stably expressing this MC2-MRAP complex, iterative optimization led to the discovery of multiple chemical classes of highly potent, nonpeptide MC2 receptor selective antagonist leads, which were then further optimized for drug-like characteristics. We identified multiple compounds that exhibit high potency for human and rat MC2 receptors (hMC2 Kb <1 nM), while having no activity at the MC1, MC3, MC4, or MC5 receptors. Leading ACTH antagonists were also evaluated for drug like characteristics, including good stability in liver microsomes, lack of inhibition of cytochromes P450 and the hERG ion channel, and were shown to exhibit good exposure upon oral dosing in both rats and dogs. These ACTH antagonists acutely suppress corticosterone secretion in an ACTH-challenge model in rats. In a 7-day hypercortisolemia model in which rats receive an implanted minipump that continually secretes ACTH, corticosterone levels were decreased, and body weight loss and adrenal hypertrophy were prevented with ACTH antagonist treatment. The culmination of these studies has led to a subset of candidate molecules that are being evaluated in genotoxicity, safety pharmacology, and general toxicology studies to enable evaluation in human clinical trials.

OR19-03 Effects of Nonpeptide Orally Bioavailable ACTH Antagonists on Adrenal Gland Size and Function in Rats

Cushing’s disease (CD) and Ectopic ACTH syndrome (EAS) stem from excess circulating adrenocorticotropic hormone (ACTH) and resulting hypercortisolemia. In CD, excess ACTH is secreted from pituitary tumors, whereas excess ACTH in EAS arises from nonpituitary tumors. ACTH acts on the adrenal melanocortin type 2 (MC2) receptor to control the synthesis and secretion of adrenal hormones, including the stress hormone cortisol (corticosterone in rats) which accounts for the comorbidities of CD and EAS. Availability of a potent ACTH antagonist that can normalize cortisol in patients with diseases of excess ACTH will be a major advance in endocrinology. Additionally, an ACTH antagonist will have utility in congenital adrenal hyperplasia (CAH) because of its ability to block production of excess adrenal androgens.Crinetics is evaluating and developing ACTH antagonists for the treatment of diseases of excess ACTH. To our knowledge, these compounds represent the first potent nonpeptide ACTH antagonists to demonstrate in vitro potency and in vivo efficacy. As a result, the direct effects of sustained MC2 receptor blockade on the structure and function of the adrenal gland have never been able to be assessed. We examined the effects of several orally bioavailable ACTH antagonists across a range of doses on Sprague-Dawley rat adrenal gland weight, histology, and hormone levels in repeat dosing (7-14 days) studies. Sustained MC2 receptor antagonism dose dependently blocked activity of ACTH at the level of the adrenal gland and reduced plasma corticosterone levels. In the normal rat, this resulted in dose-dependent atrophy of the adrenal gland as assessed by organ weights and microscopically. The atrophy was primarily observed in the cortisol producing zona fasciculata, as well as in the zona reticularis, with smaller reductions noted in the aldosterone producing zona glomerulosa. Additionally, hypertrophy of the adrenal glands caused by continuous subcutaneous administration of exogenous ACTH was reversed by treatment with an ACTH antagonist. The adrenal effects were accompanied by expected changes in corticosterone levels. These preclinical findings demonstrate the therapeutic potential of ACTH antagonism and provide a strong rationale for development of an orally bioavailable drug that can be used to combat CD, EAS, and CAH.

NK1R antagonist decreases inflammation and metastasis of breast carcinoma cells metastasized to liver but not to brain; phenotype-dependent therapeutic and toxic consequences.

Substance P a neuro-immune mediator acts on Neurokinin-1 and -2 receptors (NK1R and NK2R). Inhibitors of NK1R are considered to be safe and effective approaches for cancer treatment since Aprepitant, a non-peptide antagonist of NK1R is widely used for chemotherapy-induced emesis and has cytotoxic and antitumor effects in various models for cancer. On the other hand, our previous findings demonstrated that systemic inhibition of NK1R may decrease cytotoxic anti-tumoral immune response. Hence, actual consequences of inhibition of neurokinin receptors under in vivo conditions in a syngeneic model of carcinoma should be determined. The effects of highly potent and selective non-peptide mouse NK1R and NK2R antagonists RP 67580 and GR 159897, respectively, on metastatic breast carcinoma were evaluated. Specifically, 4T1 breast cancer cells metastasized to brain (denoted as 4TBM) and liver (denoted as 4TLM) were used to induce tumors in Balb-c mice. Changes in tumor growth, metastasis and immune response to cancer cells were determined. We here observed differential effects of NK1R antagonist depended on the subset of metastatic cells. Specifically, inhibition of NK1R markedly increased liver metastasis of tumors formed by 4TBM but not 4TLM cells. On the contrary, NK1R antagonist decreased inflammatory response and liver metastasis in 4TLM-injected mice. 4TLM tumors act more aggressively inducing more inflammatory response compared to 4TBM tumors. Hence, differential effects of NK1R antagonist are at least partly due to extend and type of the inflammatory response evoked by specific subset metastatic cells. These findings demonstrate the necessity for understanding the immunological consequences of tumor-microenvironment interactions.

Glucagon and Glucagon-like Peptide-1 Receptors: Promising Therapeutic Targets for an Effective Management of Diabetes Mellitus

G-protein-coupled receptors (GPCRs) are membrane-bound proteins, which are responsible for the detection of extracellular stimuli and the origination of intracellular responses. Both glucagon and glucagon-like peptide-1 (GLP-1) receptors belong to G protein-coupled receptor (GPCR) superfamily. Along with insulin, glucagon and GLP-1 are critical hormones for maintaining normal serum glucose within the human body. Glucagon generally plays its role in the liver through cyclic adenosine monophosphate (cAMP), where it compensates for the action of insulin. GLP-1 is secreted by the L-cells of the small intestine to stimulate insulin secretion and inhibit glucagon action. Despite extensive research efforts and the multiple approaches adopted, the glycemic control in the case of type-2 diabetes mellitus remains a major challenge. Therefore, a deep understanding of the structure-function relationship of these receptors will have great implications for future therapies in order to maintain a normal glucose level for an extended period of time. The antagonists of glucagon receptors that can effectively block the hepatic glucose production, as a result of glucagon action, are highly desirable for the tuning of the hyperglycemic state in type 2 diabetes mellitus. In the same manner, GLP-1R agonists act as important treatment modalities, thanks to their multiple anti-diabetic actions to attain normal glucose levels. In this review article, the structural diversity of glucagon and GLP-1 receptors along with their signaling pathways, site-directed mutations and significance in drug discovery against type-2 diabetes are illustrated. Moreover, the promising non-peptide antagonists of glucagon receptor and agonists of GLP-1 receptor, for the management of diabetes are presented with elaboration on the structure-activity relationship (SAR).

Novel and ecofriendly UV-Spectrophotometry methods for estimation of Tolvaptan using hydrotropic agent

The aim of present investigation is to establish simple and economical UV- Spectrophotometric methods for estimation of Tolvaptan using Zero order UV-Spectrophotometric absorbance method and Zero Order- Area under curve (AUC) method with the application of hydrotropic solubilization phenomenon. Tolvaptan is non-peptide vasopressin V2 receptor antagonist. It is completely insoluble in water. Hydrotropic agent, sodium lauryl Sulfate (SLS) 5% w/v was used to enhance the solubility of Tolvaptan. Maximum absorption for Tolvaptan was found to be at 269 nm. The methods are based upon measurement of absorbance at 269 nm and integration of area under curve for analysis of Tolvaptan in the wavelength range of 263.2-282.2 nm. The drug followed linearity in the concentration range of 3 - 18 µg/mL with correlation coefficient value r2> 0.99 for both methods. The proposed methods were validated for accuracy, precision, repeatability and ruggedness, as per ICH guidelines. The proposed methods were applied for qualitative and quantitative estimation of Tolvaptan in pharmaceutical formulation and results were found in good agreement with the label claimed. Developed methods can be used for routine analysis of Tolvaptan in bulk and tablets. Keywords: Sodium Lauryl Sulphate, Tolvaptan, UV- Spectrophotometry - Area under Curve, UV-Spectrophotometry, Hydrotropy .