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Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Parkinson's disease (PD) is a basal ganglia movement disorder characterized by progressive degeneration of the nigrostriatal dopaminergic system. Immunohistochemical methods have been widely used for characterization of dopaminergic neuronal injury in animal models of PD, including the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model. However, conventional immunohistochemical techniques applied to tissue sections have inherent limitations with respect to loss of 3D resolution, yielding insufficient information on the architecture of the dopaminergic system. To provide a more comprehensive and non-biased map of MPTP-induced changes in central dopaminergic pathways, we used iDISCO immunolabeling, light-sheet fluorescence microscopy (LSFM) and deep-learning computational methods for whole-brain three-dimensional visualization and automated quantitation of tyrosine hydroxylase (TH)-positive neurons in the adult mouse brain. Mice terminated 7 days after acute MPTP administration demonstrated widespread alterations in TH expression. Compared to vehicle controls, MPTP-dosed mice showed a significant loss of TH-positive neurons in the substantia nigra pars compacta and ventral tegmental area. Also, MPTP dosing reduced overall TH signal intensity in basal ganglia nuclei, i.e. the substantia nigra, caudate-putamen, globus pallidus and subthalamic nucleus. In contrast, increased TH signal intensity was predominantly observed in limbic regions, including several subdivisions of the amygdala and hypothalamus. In conclusion, mouse whole-brain 3D imaging is ideal for unbiased automated counting and densitometric analysis of TH-positive cells. The LSFM–deep learning pipeline tracked brain-wide changes in catecholaminergic pathways in the MPTP mouse model of PD, and may be applied for preclinical characterization of compounds targeting dopaminergic neurotransmission.

In development of peptide therapeutics, rodents are commonly-used preclinical models when screening compounds for efficacy endpoints in the early stages of discovery projects. During the screening process, some peptides administered subcutaneously to rodents caused injection site reactions manifesting as localized swelling. Screening by postmortem evaluations of injection site swelling as a marker for local subcutaneous histamine release, were conducted in rats to select drug candidates without this adverse effect. Histological analysis of skin samples revealed that the injection site reactions were concurrent with mast cell degranulation, resulting in histamine release. Mast cell activation can be mediated by MRGPRX2, a GPCR that induces a pseudo-allergenic immune response. The present study demonstrates that a commercially-available cell-based MRGPRX2 assay reliably identifies compounds that induce histamine release or localized edema in ex vivo human and rodent skin samples. In vitro screening was subsequently implemented using the MRGPRX2 assay as a substitute for postmortem injection site evaluation, thus achieving a significant reduction in animal use. Thus, in cases where injection site reactions are encountered during in vivo screening, to enable faster screening during the early drug discovery process, an MRGPRX2 in vitro assay can be used as an efficient, more ethical tool with human translational value for the development of safer pharmacotherapies for patients.

Weight management pharmacotherapies seldom induce body weight loss that is comparable to that produced by bariatric surgery. In this regard, combination therapies targeting multiple signaling pathways that regulate energy balance may provide a means to achieve greater weight loss efficacy while also allowing the use of lower doses of each drug, and thus mitigating potential gastrointestinal tolerability issues commonly observed with current therapeutics for weight management. Amylin and GLP-1 are peptide hormones that regulate appetite. Upon ingestion of a meal, amylin is co-secreted with insulin from pancreatic beta-cells, while GLP-1 is secreted from enteroendocrine cells in the intestine. Both native peptides have a short half-life and reduce food intake, delay gastric emptying and decrease glucagon levels. Amylin and GLP-1 analogues have been developed for the treatment of diabetes, as well as weight management. The long-acting once-weekly GLP-1 analogue, semaglutide is approved for the treatment of type 2 diabetes and is in clinical development for weight management. AM833 (NNC0174-0833) is a long-acting, once-weekly human amylin analogue that is also in clinical development for weight management. Here, we present the combined effect of AM833 and semaglutide on weight loss in rodent models of obesity. Diet-induced obese (DIO) rats and mice on a high energy diet were dosed subcutaneously once-daily for 24 or 28 days with sub-maximal doses of AM833, semaglutide or two modes of combination treatments. The first combination mode consisted of concurrent co-dosing of both drugs, while the second entailed add-on of AM833 after one week of treatment with semaglutide. Body weight and food intake were measured daily. Body composition was assessed by magnetic resonance scan pre- and post-treatment. In the DIO rat, both concurrent (-13.1% ± 0.7%) and add-on (-12.8% ± 1.2%) treatment modes induced equivalent weight loss that was greater than each monotherapy (-6.3% ± 0.7%, 2 nmol/kg semaglutide and -5.8% ± 0.9%, 2 nmol/kg AM833) relative to initial body weights. Both combination groups achieved normalization of body weight to that of lean age-matched control rats. Reductions in cumulative food intake corresponded with the extent of weight loss. In the DIO mouse, weight loss in the combination groups was not significantly different (-9.6% ± 1.5%, concurrent vs. -11.5% ± 1.2%, add-on) but was greater than that observed in each monotherapy group (-1.9% ±1.2%, 1 nmol/kg semaglutide and +1.5% ± 2.2%, 10 nmol/kg AM833). In the DIO mouse, body weight did not normalize to match that of lean controls with combination treatment. In both rodent models, combination therapy at submaximal doses of AM833 and semaglutide achieved significantly greater weight loss compared to the monotherapy groups highlighting the potential of this combination for further clinical development.

Amylin is a peptide hormone that is co-secreted with insulin from pancreatic β-cells. It is member of the calcitonin family and activates amylin receptors (AMYRs) in the calcitonin receptor (CTR) family. During recent years, several AMYR agonists have been in clinical development for treatment of diabetes and obesity. In contrast to pramlintide, which is a short acting amylin analogue approved for use in combination with meal-time insulin in patients with diabetes, the more recent development candidates have been dual acting analogues activating both the CTR and the AMYRs. In rodents the CTR plays a significant role in calcium homeostasis, while in humans this has been associated with little long-term effect on the plasma calcium levels (Ganong, 1993; Longo et al., 2011); Rodents are usually chosen as first choice pre-clinical models for in vivo testing and introducing acute and profound hypocalcaemia can lead to general neurological and neuromuscular disturbances, which can be challenging in clinical development programs. AM833 (NNC0174-0833) is a long acting acylated dual acting amylin analogue activating both CTR and AMYRs. When combined with the long acting GLP-1 analogue semaglutide for weight management, both preclinical studies in rat models of obesity and clinical trials in humans with obesity have demonstrated significant weight loss potential. In young rats aged 7–9 weeks, single sc dose of 3–300 nmol/kg AM833 induced a reduction of total and ionized calcium of 40–50% from baseline with lowest level around 12 hours after dosing. A similar response was seen after administration of salmon calcitonin which also activates both CTR and AMYRs. Tachyphylaxis was introduced, with no effect on calcium found after sub chronic daily treatment for 2 weeks. Age played a key role in the sensitivity to calcium lowering with almost no effect found in 11 months old rats. When administering AM1213 (NNC0174-1213), a selective AMYR agonist with little activation of the CTR, no or little calcium lowering of around 10% was seen. These data indicated that the calcium lowering properties are CTR mediated. The induction of hypercalcemia was a rodent specific phenomenon as no calcium lowering outside of normal range was seen in young dogs and rabbits. Clinical data confirmed that CTR induced calcium lowering was not human relevant, but these data showed that understanding physiology and pharmacology in the animal models of investigation as well as human translational relevance is of outmost importance in the progress of an early development program. References: GaNong WF. Calcitonin. In: Review of medical physiology. 16th ed. Appleton & Lange. 1993. Longo DL et al. Calcitonin. In: Harrison’s principles of internal medicine. 18th ed. McGraw-Hill; 2011.

Many hormone-receptor signalling pathways trigger Ca2+ oscillations subsequent to inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Because of the Ca2+ dependency of the IP3 receptor, processes that affect Ca2+ removal from the vicinity of the IP3R Ca2+ release channel can be important modulators of hormonal signalling. This dissertation examines (1) the role of the ER luminal Ca2+ -binding chaperone calreticulin in the modulation of Ca2+ uptake by two SERCA isoforms, (2) cytosolic Ca2+ buffering by the EF-hand CaBP, parvalbumin and (3) the influence of thyroid hormone and its receptor on mitochondrial Ca2+ buffering. These goals are achieved by applying molecular biology techniques in conjunction with confocal imaging of IP3-induced Ca2+ wave activity in Xenopus laevis oocytes. Differential modulation of SERCA2 isoforms by calreticulin. In Xenopus oocytes, overexpression of SERCA isoforms increases IP3-induced Ca2+ wave frequency. Overexpression of the ER resident molecular chaperone, calreticulin (CRT), results in a sustained elevation of intracellular Ca2+ with a concomitant inhibition of repetitive Ca2+ waves following IP3R activation. This sustained elevation is also observed when CRT was co-expressed with SERCA 2b. CRT is a member of a novel class of ER chaperones with lectin activity. These chaperones bind to N-linked glycosylation residues to modulate protein folding. SERCA 2b has a luminal facing N-linked glycosylation site (N1036) that is not present in SERCA 2a. If the inhibition of Ca2+ wave activity is due to CRT interacting with the glycosylation site on SERCA 2b, then coexpression of CRT with SERCA 2a should not produce the inhibitory effect. Effects on IP3-induced Ca 2+ signalling are examined by coexpression of a CRT mutant with SERCA 2b, 2a and the mutant SERCA2b N1036A to determine whether CRT differentially modulates Ca2+ uptake by these ATPases. Spontaneous Ca2+ sparks evoked by parvalbumin. The EF hand CaBP, parvalbumin, buffers cytosolic Ca 2+ concentrations. Parvalbumin is reported to facilitate skeletal muscle relaxation and protects neuronal cells from high Ca2+ loads. Both Mg2+ and Ca2+ can bind to parvalbumin. At resting cytosolic concentrations, parvalbumin is bound to Mg2+. The kinetics of Ca2+ buffering by parvalbumin are determined by its slow Mg2+ dissociation rate. By injecting purified protein or by overexpressing parvalbumin in oocytes, the effects of Ca2+ buffering by parvalbumin on Ca2+ release events is examined.

Background MCL-1 is a prosurvival B-cell lymphoma 2 family protein that plays a critical role in tumor maintenance and survival and can act as a resistance factor to multiple anticancer therapies. Herein, we describe the generation and characterization of the highly potent and selective MCL-1 inhibitor ABBV-467 and present findings from a first-in-human trial that included patients with relapsed/refractory multiple myeloma (NCT04178902). Methods Binding of ABBV-467 to human MCL-1 was assessed in multiple cell lines. The ability of ABBV-467 to induce tumor growth inhibition was investigated in xenograft models of human multiple myeloma and acute myelogenous leukemia. The first-in-human study was a multicenter, open-label, dose-escalation study assessing safety, pharmacokinetics, and efficacy of ABBV-467 monotherapy. Results Here we show that administration of ABBV-467 to MCL-1-dependent tumor cell lines triggers rapid and mechanism-based apoptosis. In vivo, intermittent dosing of ABBV-467 as monotherapy or in combination with venetoclax inhibits the growth of xenografts from human hematologic cancers. Results from a clinical trial evaluating ABBV-467 in patients with multiple myeloma based on these preclinical data indicate that treatment with ABBV-467 can result in disease control (seen in 1 patient), but may also cause increases in cardiac troponin levels in the plasma in some patients (seen in 4 of 8 patients), without other corresponding cardiac findings. Conclusions The selectivity of ABBV-467 suggests that treatment-induced troponin release is a consequence of MCL-1 inhibition and therefore may represent a class effect of MCL-1 inhibitors in human patients. Plain language summary Apoptosis is a type of cell death that removes abnormal cells from the body. Cancer cells can have increased levels of MCL-1, a protein that helps cells survive and prevents apoptosis. ABBV-467 is a new drug that blocks the action of MCL-1 (an MCL-1 inhibitor) and could promote apoptosis. In animal models, ABBV-467 led to cancer cell death and delayed tumor growth. ABBV-467 was also studied in a clinical trial in 8 patients with multiple myeloma, a blood cancer. In 1 patient, ABBV-467 treatment prevented the cancer from getting any worse for 8 months. However, in 4 out of 8 patients ABBV-467 increased the levels of troponin, a protein associated with damage to the heart. This concerning side effect may impact the future development of MCL-1 inhibitors as anticancer drugs. Yuda et al. report the development and evaluation of a novel selective MCL-1 inhibitor, ABBV-467. The compound is efficacious in preclinical models but increases in cardiac troponin are seen in a first-in-human study, suggesting potential cardiac toxicity of the drug.