Preclinical Pharmacology Studies Research Articles

EXPERT EVALUATION OF PRECLINICAL STUDIES OF PRIMARY AND SECONDARY PHARMACODYNAMICS OF MEDICINES

The article dwells upon the results of comparative analysis of the Russian national requirements and EAEU regulations dealing with evaluation of preclinical pharmacological studies. It highlights historical aspects of elaboration of regulatory requirements and scientific and methodological recommendations for conducting and evaluating preclinical pharmacological studies. According to the EAEU Rules of registration and evaluation of medicinal products for human use, the Common Technical Document has to include information on primary pharmacodynamics, secondary pharmacodynamics, safety pharmacology studies, and pharmacodynamic drug interactions, which are subject to evaluation during examination of the preclinical study results. The national guideline on evaluation of medicines does not contain any clear indication of the subject of examination in primary and secondary pharmacodynamic studies, which can make it difficult to prepare a registration dossier in the format of a Common Technical Document. The article formulates basic approaches to expert evaluation of the results of preclinical studies of primary and secondary pharmacodynamics of medicines. It determines the basics of expert evaluation which covers methodological framework of the research, results of the research, characteristics of the safety profile, extrapolation of preclinical data, characteristics of risk factors and of the predictable clinical safety profile for patients.

Abstract 2736: Preclinical pharmacologic and pharmacodynamic studies of a novel and potent IDO1 inhibitor D-0751

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the degradation of the essential amino acid L-tryptophan to kynurenine. It plays an important role in the initial and rate-limiting step in the breakdown of tryptophan. It has been reported that IDO1, an enzyme induced by IFN-gamma, is one of the central regulators of immune responses in various physiologic and pathologic settings and the enzyme is frequently expressed in cancers. Kynurenine produced by IDO1 suppresses T-cell function in the tumor microenvironment, resulting in tumor cell evasion from immune cells. IDO1 inhibitors, such as epacadostat and BMS-986205, have shown preliminary clinical therapeutic response in melanoma and other solid tumors when combined with anti-PD-1 antibody. Here, we report the discovery of a novel and selective IDO1 inhibitor with potent activity in various cell-based assays. Our lead molecule D-0751 inhibits IDO1 activity in HeLa cell and human whole blood assay with IC50 of 0.6 nM and 13 nM, respectively. It activates the human T-cell activity in a HeLa co-culture study with EC50 at 9 nM. D-0751 is optimized to have good PK profile in rodent, dog and monkey as well as slow clearance when incubated with human hepatocyte. Pharmacodynamic (PD) effect has been observed in mouse models (blood and tumor samples) as well as in dog (blood samples) with efficient kynurenine reduction observed at optimal dose level. Taken together, these results support the advancement of D-0751 as a clinical candidate in combination with anti-PD-1/L1 antibody in various solid tumor indications. Citation Format: Yaolin Wang, Zhe Shi, Zixing Han, Zhenwu Wang, Yueheng Jiang, Xing Dai. Preclinical pharmacologic and pharmacodynamic studies of a novel and potent IDO1 inhibitor D-0751 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2736.

Abstract 3890: Interspecies comparison of in vitro endoxifen metabolism

Abstract Endoxifen (N-desmethyl-4-hydroxytamoxifen) is an active metabolite of tamoxifen being developed for use in breast cancer prevention and therapy. To optimize model selection for preclinical toxicology and pharmacology studies, the in vitro metabolism of endoxifen was compared using commercially available pools of hepatocytes from humans, rats, dogs, rabbits, minipigs, and non-human primates. Endoxifen (10 μM) was incubated with hepatocytes for 0, 2, or 4 hours. Cells were harvested, extracted, and analyzed by LC-MS/MS to quantitate levels of parent compound and major metabolites. Although substantial differences in patterns of endoxifen metabolism were seen in different species, endoxifen glucuronide and endoxifen sulfate were the primary metabolites identified in all species. Endoxifen was relatively stable when incubated with human hepatocytes: after 2 and 4 hours of incubation, endoxifen levels were > 20-fold greater than levels of endoxifen glucuronide and > 5-fold greater than levels of endoxifen sulfate. Levels of endoxifen glucuronide and endoxifen sulfate in human hepatocytes were similar at both time points. By contrast, endoxifen was extensively glucuronidated (but not sulfated) in both rabbit and minipig hepatocytes: after 4 hours of incubation, levels of endoxifen glucuronide in rabbit and minipig hepatocytes were > 5 times the level of parent drug. Levels of endoxifen sulfate were very low in both rabbits and minipigs. Rats and dogs demonstrated patterns of endoxifen metabolism that were intermediate to those of humans, rabbits, and minipigs. In rat hepatocytes, levels of parent drug were slightly greater than levels of endoxifen glucronide at 4 hours; little endoxifen sulfate was generated in rat hepatocytes. In dog hepatocytes, levels of endoxifen and endoxifen glucuronide were comparable at 4 hours; sulfation of endoxifen was greater in dogs than in other species, as levels of endoxifen sulfate at 4 hours were approximately 2/3 of the levels of parent drug and endoxifen glucuronide. Although endoxifen was somewhat less stable in hepatocytes isolated from non-human primates than in human hepatocytes, endoxifen metabolism in hepatocytes from non-human primates demonstrated the greatest similarity to metabolism in human hepatocyte pools: at 4 hours, levels of parent drug in monkey hepatocytes were approximately 4 times the levels of endoxifen glucuronide and 8 times the levels of endoxifen sulfate. These data demonstrate that endoxifen is quite stable when incubated with hepatocytes from humans and non-human primates. By contrast, endoxifen is rapidly and extensively glucuronidated in hepatocytes from rabbits and minipigs. Rats and dogs, the two species used most commonly in preclinical safety studies, both demonstrate substantially more rapid endoxifen metabolism than do humans. [Supported by HHSN261201500024I from the National Cancer Institute, DHHS.] Citation Format: Miguel Muzzio, Zhihua Huang, Elizabeth R. Glaze, David L. McCormick. Interspecies comparison of in vitro endoxifen metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3890.

Preclinical Pharmacology and Safety Studies of GalNAc-conjugated Antisense Oligonucleotides Targeting Human PCSK9

Preclinical Pharmacology and Safety Studies of GalNAc-conjugated Antisense Oligonucleotides Targeting Human PCSK9

Abstract B114: Humanized anti-Sialyl-Tn monoclonal antibody-drug conjugates inhibit tumor growth in vitro and in vivo

Abstract Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anticancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely observed in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. Interestingly, we have observed that the level of this carbohydrate is elevated following chemotherapy treatment in both in vitro and in vivo model systems, suggesting a potential role of STn in chemoresistance. We have identified and humanized a panel of mouse monoclonal novel antibodies (Abs) that specifically target with high affinity the STn glycan independent of its carrier protein. We have demonstrated that these Abs are highly selective in binding assays demonstrating robust nanomolar EC50s, possess specificity on cancer tissues as determined with neuraminidase treatment, and exhibit STn-specific glycan binding on Siamab’s proprietary glycan array. Antitumor efficacy was evaluated in vitro and in vivo utilizing humanized anti-STn antibody-drug conjugated (ADC) material using MMAE as the cytotoxic agent. Cell cytotoxicity was demonstrated in a panel of STn-expressing cell lines with low nanomolar IC50s. In vivo, single-dose mouse PK studies were performed with humanized anti-STn ADCs and we determined half-life of approximately 3 days for both SIA01-ADC and SIA02-ADC. We confirmed that the decrease in proliferation in vitro can be translated to a reduction in tumor size in vivo through a series of cell line-derived and patient-derived (PDX) ovarian cancer and pancreatic cancer xenograft models. Inhibition of tumor progression was observed in all models with complete regressions observed in some treatment arms. No significant weight loss occurred in any treatment groups, indicating the therapy was well tolerated by all the groups. A decrease in STn expression was noted in the tumors following treatment, indicating that we were hitting the target against which these Abs were generated. In addition to these preclinical pharmacology studies, we demonstrated that the administration of our anti-STn ADC has an excellent safety profile through the completion of a non-GLP pilot PK/toxicity study in cynomolgus monkeys. No weight loss or deaths occurred in this study and no gross pathology changes were observed in all organs examined. Histopathologic changes were limited to the bone marrow with minimal to mild decreased cellularity and mild decrease in the myeloid to erythroid ratio observed. All clinical chemistry results (liver, kidney function, etc.) were normal throughout study. Changes in hematology parameters–modest neutropenia–were consistent with other MMAE ADCs and therefore, we conclude, are not related to the STn target, which is consistent with our tissue cross-reactivity studies demonstrating that overall normal tissue expression of the STn target is insignificant. Our data demonstrate that high-affinity, STn-selective humanized mAbs show promise as therapies for solid tumors. Citation Format: Daniel Dransfield, Jillian M. Prendergast, David A. Eavarone, Rawan Nazer, Linah Al-Alem, Bo Rueda, Jenna Stein, Jeff Behrens. Humanized anti-Sialyl-Tn monoclonal antibody-drug conjugates inhibit tumor growth in vitro and in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B114.

Latent Warheads for Targeted Cancer Therapy: Design and Synthesis of pro-Pyrrolobenzodiazepines and Conjugates.

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds, and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine moiety. Once the prodrug (pro-PBD) conjugate enters a targeted cell, cleavage of the linker system triggers the generation of a reactive intermediate possessing an aldehyde and aromatic amine. An intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine and, as a result, the diazepine ring. In our pro-PBDs, we mask the aldehyde as a hydrolytically sensitive oxazolidine moiety which in turn is a part of a reductively labile self-immolative linker system. To prove the range of applications for this new class of latent DNA-alkylators, we designed and synthesized several novel latent warheads: pro-PBD dimers and hybrids of pro-PBD with other sequence-selective DNA minor groove binders. Preliminary preclinical pharmacology studies showed excellent biological activity and specificity.

A novel radiolabeled indole derivative as solid tumor imaging agent: in silico and preclinical pharmacological study

Biologically active indole derivative was synthesized and screened for its cytotoxicity against breast cancer cell line (MCF-7) with the IC50 equals 17.5 µg/mL. Docking study showed its high binding affinity to the COX-2 enzyme. It’s radiolabeling with 99mTc and showed high radiochemical yield (92.10 ± 0.57%) and in vitro stability in serum up to 10 h. Biodistribution studies of a novel 99mTc-NSIC complex in tumor-bearing mice showed high T/NT ratio (6.17 ± 0.08 at 30 min. post-injection), suggesting this complex could be used as a solid tumor imaging agent.

Study of specific pharmacological activity of standardized composition of bee product substances for treatment of urogenital system

Introduction. The work presents review of the data literature, which indicate the prospects of creation new highly efficient drugs for the treatment of chronic prostatitis and prostate gland adenoma based bioactive standardized substances of bee products, including powdered honey (PH), propolis phenolic hydrophobic drug (PPHD) and bee pollen (BP). Materials and methods. Results of discussion of preclinical pharmacological studies of standardized substances of bee products composition – PH, PPHD and BP for the treatment of specified pathology is given in the experimental part. Results. It was found that the most pronounced anti-inflammatory effect on the level 40 % the mixture of APIs (PH, PPHD and BP) detects at a dose of 100 mg/kg in relation to the reference drug – trianom capsules in doses of 100 and 130 mg/kg. Conclusions. Usage of bee products is grounded in creation of the new drug on their basis in the form of standardized substances of bee products composition –PH, PPHD and BP for chronic prostatitis and prostate gland adenoma. It was found that the most pronounced anti-inflammatory effect on 40 % the mixture of APIs (PH, PPHD and BP) detects at a dose of 100 mg/kg. It was established that the composition of standard substances of bee products – PH, PPHD and BP at a dose of 100 mg/kg shows a more pronounced specific pharmacological effect in comparison to the reference product – trianom capsules at doses of 100 and 130 mg/kg, which positively affect the course of the pilot prostatitis in male rats caused by dichloroethyl.

Abstract 4606: Preclinical characterization of a novel fully human IgG1 anti-PD-L1 mAb CK-301

Abstract Antibodies targeting Programmed Death-1 (PD-1), or its ligand, PD-L1, have demonstrated remarkable efficacy in subsets of cancer patients, with inhibition of the interaction between PD-1 on T-cells and PD-L1 on tumor cells leading to the recovery of anti-tumor immune response and immune-mediated eradication of tumors. However, not all patients respond to existing PD-1 and PD-L1 targeting agents and relapses to therapy still occur. Therefore, there exists a need to identify additional therapeutics and approaches to engage the immune system to enhance the efficacy of current anticancer therapies. Using phage and yeast display approaches, we have discovered and optimized a novel, fully human, PD-L1 specific IgG1 antibody, CK-301, which exhibits subnanomolar binding affinity for PD-L1. CK-301 blocks binding of PD-L1 to both PD-1 and B7-1 in enzyme-linked immunosorbent assays (ELISA) and cell-based competition assays. Using an assay measuring inhibition of a nuclear factor of activated T-cells (NFAT) reporter caused by PD-L1 binding to PD-1, we demonstrate that CK-301 completely reverses reporter inhibition at concentration of less than 1 µg/ml, IC50 of the dose response curve is 80ng/ml. CK-301 enhances IFN-gamma secretion in allogeneic mixed lymphocyte reaction (MLR) using primary human T-cells and immature dendritic cells. CK-301 can also trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mediated killing of PD-L1+ cell lines, including lymphoma cells. CK-301 has similar subnanomolar affinity for cynomolgus monkey PD-L1 as for human PD-L1, hence we chose Macaca fascicularis for pre-clinical toxicology and safety pharmacology studies. Single-dose administration of CK-301 to monkeys up to the highest tested dose of 100 mg/kg was shown to be safe and demonstrated linear dose-dependent pharmacokinetic (PK) properties over the dose range from 1 to 100 mg/kg with a half-life of 15 days at 100 mg/kg. A first-in-human Phase 1 study of CK-301 is planned to commence in mid-2017. Citation Format: Leonid Gorelik, George Avgerinos, Yune Kunes, Wayne A. Marasco. Preclinical characterization of a novel fully human IgG1 anti-PD-L1 mAb CK-301 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4606. doi:10.1158/1538-7445.AM2017-4606

Traumatic Brain Injury: Preclinical Imaging Diagnostic(s) and Therapeutic Approaches.

Traumatic brain injury (TBI) is the result of an external physical force to the head that harms the brain. TBI is a major public health problem worldwide and mainly results from falls, vehicle accidents and violence. Clinical problem: The management of TBI, causing a wide spectrum of possible health outcomes, has barely changed over the years as encouraging outcomes from many pre-clinical therapeutic and pharmacological studies have only rarely been translated to the clinical situation. New management options: In the last decades management of TBI is rapidly advancing and new innovative imaging modalities with sophisticated treatment options by using nanomedicine based drug delivery systems are under investigation. Nano formulations such as PLGA, exosomes and liposomes have the advantage of a targeted and controlled delivery of their cargo, such as diagnostic probes and/or therapeutic drugs. Here we provide an overview of new promising pre-clinical developments in TBI management that may find their way to the clinic in the near future. Nanotechnology and nanomedicine in TBI intervention may establish new platforms for targeted drug delivery to the traumatized brain to improve the quality of life and survival of TBI patients.

Large-scale identification of adverse drug reaction-related proteins through a random walk model.

Adverse drug reactions (ADRs) are responsible for drug failure in clinical trials and affect life quality of patients. The identification of ADRs during the early phases of drug development is an important task. Therefore, predicting potential protein targets eliciting ADRs is essential for understanding the pathogenesis of ADRs. In this study, we proposed a computational algorithm,Integrated Network for Protein-ADR relations (INPADR), to infer potential protein-ADR relations based on an integrated network. First, the integrated network was constructed by connecting the protein-protein interaction network and the ADR similarity network using known protein-ADR relations. Then, candidate protein-ADR relations were further prioritized by performing a random walk with restart on this integrated network. Leave-one-out cross validation was used to evaluate the ability of the INPADR. An AUC of 0.8486 was obtained, which was a significant improvement compared to previous methods. We also applied the INPADR to two ADRs to evaluate its accuracy. The results suggested that the INPADR is capable of finding novel protein-ADR relations. This study provides new insight to our understanding of ADRs. The predicted ADR-related proteins will provide a reference for preclinical safety pharmacology studies and facilitate the identification of ADRs during the early phases of drug development.

Non-invasive imaging demonstrates clinical features of ankylosing spondylitis in a rat adjuvant model: a case study.

Main features of ankylosing spondylitis like inflammatory erosive osteopenia and bony overgrowth are recapitulated in rats challenged with complete Freund’s adjuvant. In vivo changes induced in the rat spine were followed longitudinally by magnetic resonance imaging (MRI) and assessed terminally by micro-computerized tomography (micro-CT) and histology. Signals reflecting inflammation were detected by MRI at levels L5-L6 throughout the experiment, peaking at day 27 after adjuvant. Bone erosion and formation occurred from this time point onward, as confirmed by micro-CT. Histology confirmed the inflammation and bone remodeling. The present study demonstrates the potential of imaging for longitudinal assessments of spinal changes in this animal model and the excellent correlation between in vivo images and histology underlines its fundamental role in the validation of non-invasive imaging.

Leveraging model-based study designs and serial micro-sampling techniques to understand the oral pharmacokinetics of the potent LTB4 inhibitor, CP-105696, for mouse pharmacology studies

1. Leukotriene B4 (LTB4) is a proinflammatory mediator important in the progression of a number of inflammatory diseases. Preclinical models can explore the role of LTB4 in pathophysiology using tool compounds, such as CP-105696, that modulate its activity. To support preclinical pharmacology studies, micro-sampling techniques and mathematical modeling were used to determine the pharmacokinetics of CP-105696 in mice within the context of systemic inflammation induced by a high-fat diet (HFD).2. Following oral administration of doses > 35 mg/kg, CP-105696 kinetics can be described by a one-compartment model with first order absorption. The compound’s half-life is 44–62 h with an apparent volume of distribution of 0.51–0.72 L/kg. Exposures in animals fed an HFD are within 2-fold of those fed a normal chow diet. Daily dosing at 100 mg/kg was not tolerated and resulted in a >20% weight loss in the mice.3. CP-105696’s long half-life has the potential to support a twice weekly dosing schedule. Given that most chronic inflammatory diseases will require long-term therapies, these results are useful in determining the optimal dosing schedules for preclinical studies using CP-105696.

Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy

Relapsing-remitting multiple sclerosis is commonly associated with motor impairments, neuropathic pain, fatigue, mood disorders, and decreased life expectancy. However, preclinical pharmacological studies predominantly rely on clinical scoring of motor deficit as the sole behavioral endpoint. Thus, the translational potential of these studies is limited. Here, we have assessed the therapeutic potential of a novel anti-inflammatory interleukin-10 (IL-10) non-viral gene therapy formulation (XT-101-R) in a rat relapsing remitting experimental autoimmune encephalomyelitis (EAE) model. EAE induced motor deficits and neuropathic pain as reflected by induction of low-threshold mechanical allodynia, suppressed voluntary wheel running, decreased social exploration, and was associated with markedly enhanced mortality. We also noted that voluntary wheel running was depressed prior to the onset of motor deficit, and may therefore serve as a predictor of clinical symptoms onset. XT-101-R was intrathecally dosed only once at the onset of motor deficits, and attenuated each of the EAE-induced symptoms and improved survival, relative to vehicle control. This is the first pharmacological assessment of such a broad range of EAE symptoms, and provides support for IL-10 gene therapy as a clinical strategy for the treatment of multiple sclerosis.

The impact of early human data on clinical development: there is time to win

Modern accelerator mass spectrometry (AMS) methods enable the routine application of this technology in drug development. By the administration of a (14)C-labelled microdose or microtrace, pharmacokinetic (PK) data, such as mass balance, metabolite profiling, and absolute bioavailability (AB) data, can be generated easier, faster, and at lower costs. Here, we emphasize the advances and impact of this technology for pharmaceutical companies. The availability of accurate intravenous (iv) PK and human absorption, distribution, metabolism, and excretion (ADME) information, even before or during Phase I trials, can improve the clinical development plan. Moreover, applying the microtrace approach during early clinical development might impact the number of clinical pharmacology and preclinical safety pharmacology studies required, and shorten the overall drug discovery program.

The therapeutic potential of natural compounds against Alzheimer's disease: A preclinical pharmacological study in both sexes

Alzheimer's disease (AD), a neurodegenerative neuropsychiatric disorder, is often comorbid with depression and anxiety. Neuropsychiatric disorders are also characterized by sex differences. However, most preclinical pharmacological studies are conducted using only males. Herein, we used male and female twelve-month-old mice (3xTg) expressing mutated forms of human proteins Tau, APP and Presenilin1. These mice are considered a valid animal model of AD. We investigated the effects of the natural compound trans-crocin-4 (TC-4), which is derived from Crocus sativus and the olive compound oleuropein on the cognitive, depressive and anxious profile of 3xTg mice. We found that male and female 3xTg mice exhibited reduced locomotor activity and oleuropeine treatment (100 mg/kg i.p., for 21 days) did not reverse this phenotype. In addition, anxiety- and depressive-like behaviors were not affected by genotype, sex or oleuropeine treatment. Interestingly, oleuropeine exhibited a tendency to enhance cognitive performance in male 3xTg mice. Treatment with TC-4 (50 and 150 mg/kg, i.p., acutely or chronically for 10 days) affected locomotor activity in a sex-differentiated manner. Interestingly, acute TC-4 clearly enhanced cognitive performance in all groups although it reduced center entries in the open field. Additionally, chronic TC-4 treatment enhanced novel object discrimination mainly in male 3xTg mice. Our findings highlight the potential of those natural compounds, which warrant further investigation but also emphasize the benefits of including both males and females in preclinical pharmacological studies.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Discovery of (3-(4-(2-Oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone (AZD1979), a Melanin Concentrating Hormone Receptor 1 (MCHr1) Antagonist with Favorable Physicochemical Properties.

A novel series of melanin concentrating hormone receptor 1 (MCHr1) antagonists were the starting point for a drug discovery program that culminated in the discovery of 103 (AZD1979). The lead optimization program was conducted with a focus on reducing lipophilicity and understanding the physicochemical properties governing CNS exposure and undesired off-target pharmacology such as hERG interactions. An integrated approach was taken where the key assay was ex vivo receptor occupancy in mice. The candidate compound 103 displayed appropriate lipophilicity for a CNS indication and showed excellent permeability with no efflux. Preclinical GLP toxicology and safety pharmacology studies were without major findings and 103 was taken into clinical trials.

Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease.

BackgroundCynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations.Methods and FindingsOur data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications.ConclusionsAltogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.

Orexin Receptor Antagonists: New Therapeutic Agents for the Treatment of Insomnia.

Since its discovery in 1998, the orexin system, composed of two G-protein coupled receptors, orexins 1 and 2, and two neuropeptide agonists, orexins A and B, has captured the attention of the scientific community as a potential therapeutic target for the treatment of obesity, anxiety, and sleep/wake disorders. Genetic evidence in rodents, dogs, and humans was revealed between 1999 and 2000, demonstrating a causal link between dysfunction or deletion of the orexin system and narcolepsy, a disorder characterized by hypersomnolence during normal wakefulness. These findings encouraged efforts to discover agonists to treat narcolepsy and, alternatively, antagonists to treat insomnia. This perspective will focus on the discovery and development of structurally diverse orexin antagonists suitable for preclinical pharmacology studies and human clinical trials. The work described herein culminated in the 2014 FDA approval of suvorexant as a first-in-class dual orexin receptor antagonist for the treatment of insomnia.

Cytotoxic activity of the MK2 inhibitor CMPD1 in glioblastoma cells is independent of MK2.

MAPK-activated protein kinase 2 (MK2) is a checkpoint kinase involved in the DNA damage response. MK2 inhibition enhances the efficacy of chemotherapeutic agents; however, whether MK2 inhibition alone, without concurrent chemotherapy, would attenuate survival of cancer cells has not been investigated. CMPD1 is a widely used non-ATP competitive inhibitor that prevents MK2 phosphorylation. We employed CMPD1 together with MK2 knock-down and ATP-competitive MK2 inhibitor III (MK2i) in a panel of glioblastoma cells to assess whether MK2 inhibition could induce cancer cell death. While CMPD1 was effective at selective killing of cancer cells, MK2i and MK2 knock-down had no effect on viability of glioblastoma cells. CMPD1 treatment induced a significant G2/M arrest but MK2i-treated cells were only minimally arrested at G1 phase. Intriguingly, at doses that were cytotoxic to glioblastoma cells, CMPD1 did not inhibit phosphorylation of MK2 and of its downstream substrate Hsp27. These results suggest that CMPD1 exhibits cytotoxic activity independently of MK2 inhibition. Indeed, we identified tubulin as a primary target of the CMPD1 cytotoxic activity. This study demonstrates how functional and mechanistic studies with appropriate selection of test compounds, combining genetic knock-down and pharmacological inhibition, coordinating timing and dose levels enabled us to uncover the primary target of an MK2 inhibitor commonly used in the research community. Tubulin is emerging as one of the most common non-kinase targets for kinase inhibitors and we propose that potential tubulin-targeting activity should be assessed in preclinical pharmacology studies of all novel kinase inhibitors.