Preclinical Drug Candidates Research Articles

Bridging academic science and clinical research in the search for novel targeted anti-cancer agents.

This review starts with a brief history of drug discovery & development, and the place of Asia in this worldwide effort discussed. The conditions and constraints of a successful translational R&D involving academic basic research and clinical research are discussed and the Singapore model for pursuit of open R&D described. The importance of well-characterized, validated drug targets for the search for novel targeted anti-cancer agents is emphasized, as well as a structured, high quality translational R&D. Furthermore, the characteristics of an attractive preclinical development drug candidate are discussed laying the foundation of a successful preclinical development. The most frequent sources of failures are described and risk management at every stage is highly recommended. Organizational factors are also considered to play an important role. The factors to consider before starting a new drug discovery & development project are described, and an example is given of a successful clinical project that has had its roots in local universities and was carried through preclinical development into phase I clinical trials.

Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9nM, 14.4nM, and 8.1nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates.

A Journey around the Medicinal Chemistry of Hepatitis C Virus Inhibitors Targeting NS4B: From Target to Preclinical Drug Candidates.

Hepatitis C virus (HCV) infection is a global health burden with an estimated 130-170 million chronically infected individuals and is the cause of serious liver diseases such as cirrhosis and hepatocellular carcinoma. HCV NS4B protein represents a validated target for the identification of new drugs to be added to the combination regimen recently approved. During the last years, NS4B has thus been the object of impressive medicinal chemistry efforts, which led to the identification of promising preclinical candidates. In this context, the present review aims to discuss research published on NS4B functional inhibitors focusing the attention on hit identification, hit-to-lead optimization, ADME profile evaluation, and the structure-activity relationship data raised for each compound family taken into account. The information delivered in this review will be a useful and valuable tool for those medicinal chemists dealing with research programs focused on NS4B and aimed at the identification of innovative anti-HCV compounds.

Abstract 3665: Reconstructing the itraconazole scaffold towards improved specificity for hedeghog signaling inhibition

Abstract Inappropriate regulation of the hedgehog (Hh) signaling pathway contributes to the development and progression of a number of human cancers; most notably, basal cell carcinoma (BCC) and medulloblastoma (MB). Clinical validation of small molecule mediated silencing of the Hh pathway was recently demonstrated by the FDA approval of vismodegib for the treatment of locally advanced BCC, representing a first-in-class inhibitor of the Hh signaling pathway. Vismodegib antagonizes Hh signaling by directly binding smoothened (SMO), a critical regulator of the pathway and target of numerous small molecule preclinical and clinical drug candidates. Vismodegib treatments in MB patients revealed novel SMO mutants that demonstrated resistance to vismodegib therapy, which underscores the need for continued development of novel Hh pathway inhibitors. Recently, itraconazole (ITZ) was identified as an inhibitor of Hh signaling in vitro and in vivo. Characterization indicated ITZ, a clinically used antifungal agent, maintains Hh inhibition against vismodegib-resistant SMO mutants. Ongoing structure-activity analysis for the ITZ scaffold is currently being investigated to identify analogues with improved potency and reduced non-specific toxicity. Results from these studies to identify candidates with improved pharmacological profiles and selectivity for Hh signaling modulation will be described herein. Citation Format: Albert M. DeBerardinis, M. Kyle Hadden. Reconstructing the itraconazole scaffold towards improved specificity for hedeghog signaling inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2015-3665

Abstract 3286: A tissue engineered model of Ewing's sarcoma

Abstract More than 90% of the deaths that occur in adolescents and young adults (AYA) suffering from Ewing sarcoma (ES), the second most common bone sarcoma, are a direct result of tumor metastasis from bone and growth within the lung. Yet, little is known about the bone or lung microenvironment and the critical role they play in promoting tumor survival, growth, and differentiation. Current preclinical models poorly reflect how ES behaves in patients and most rely upon 2D cell cultures that place tumor cells on flat plastic containers or within animals. Neither model adequately mimics human tumors and the resulting phenotypic drift from their human counterparts can lead to misleading preclinical results and drug candidates that seem promising in the lab but often disappoint when advanced to the clinic. In an attempt to overcome this notable challenge, our team of cancer biologists, bioengineers, and clinicians pioneered a tissue-engineered ex vivo bone tumor niche that better mimics the native behavior of malignant bone tumors. Briefly, this synthetic osseus niche - fabricated from biologically inert 10 μm electrospun PCL fibers - forms a minature wafer capable of fostering strong cell-matrix adhesion, in vivo-like expression of oncogenic protein targets, and a physiological cell proliferation rate that is better suited for evaluation of experimental cytotoxic drugs. Importantly, this tissue engineered preclinical ES model provides a unique opportunity to assess several of the most promising biologically targeted therapies, including those directed toward the pathognomonic EWS-FLI chromosomal translocation, mTOR, and IGF-1R (a critical drug target responsible for malignant transformation of ES from MSCs and oncogene addiction). Given the nascent convergence between the tissue engineering/regeneration and cancer biology fields, the advantages and potential pitfalls of tissue-engineered bone tumor models will be introduced and recent findings discussed, including identification of cell/ECM interactions that promote ES cell survival, maintain oncogenic proteomic signatures, and preserve an in-vivo like response to conventional and biologically targeted therapies. Unpublished work will be presented that highlights the ability of flow perfusion bioreactors to more uniformly distribute cells and nutrients throughout the ECM-saturated 3D scaffolds while increasing clearance of waste byproducts. In conclusion, a tissue engineered preclinical ES model allows scientists a unique opportunity to modulate and control key interactions between tumor cells, their adjacent microenvironment, and even heterotopic cells while simultaneously monitoring the impact of these interactions on cancer cell behavior. At the expense of reduced biological complexity, the more simplified ex vivo system is expected to shed new light on druggable targets tangent to the cells themselves, and in so doing, ultimately yield innovative therapies able to cure ES patients. Citation Format: Joseph A. Ludwig, Salah Lamhamedi Cherradi, Brian Menegaz, Marco Santoro, Antonios Mikos. A tissue engineered model of Ewing's sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3286. doi:10.1158/1538-7445.AM2015-3286

Tight binding enantiomers of pre-clinical drug candidates

MTDIA is a picomolar transition state analogue inhibitor of human methylthioadenosine phosphorylase and a femtomolar inhibitor of Escherichia coli methylthioadenosine nucleosidase. MTDIA has proven to be a non-toxic, orally available pre-clinical drug candidate with remarkable anti-tumour activity against a variety of human cancers in mouse xenografts. The structurally similar compound MTDIH is a potent inhibitor of human and malarial purine nucleoside phosphorylase (PNP) as well as the newly discovered enzyme, methylthioinosine phosphorylase, isolated from Pseudomonas aeruginosa. Since the enantiomers of some pharmaceuticals have revealed surprising biological activities, the enantiomers of MTDIH and MTDIA, compounds 1 and 2, respectively, were prepared and their enzyme binding properties studied. Despite binding less tightly to their target enzymes than their enantiomers compounds 1 and 2 are nanomolar inhibitors.

Nitroimidazo-oxazole compound DNDI-VL-2098: an orally effective preclinical drug candidate for the treatment of visceral leishmaniasis.

The objective of this study was to identify a nitroimidazo-oxazole lead molecule for the treatment of visceral leishmaniasis (VL). A library of 72 nitroimidazo-oxazoles was evaluated in vitro for their antileishmanial activity against luciferase-transfected DD8 amastigotes of Leishmania donovani. On the basis of their in vitro potency and pharmacokinetic properties, the promising compounds were tested in acute BALB/c mouse and chronic hamster models of VL via oral administration and efficacy was evaluated by microscopic counting of amastigotes after Giemsa staining. The best antileishmanial candidates (racemate DNDI-VL-2001) and its R enantiomer (DNDI-VL-2098) were evaluated in vitro against a range of Leishmania strains. These candidates were further studied in a hamster model using various dose regimens. Cytokine and inducible nitric oxide synthase estimations by real-time PCR and nitric oxide generation by Griess assay were also carried out for DNDI-VL-2098. In vitro screening of nitroimidazo-oxazole compounds identified the racemate DNDI-VL-2001 (6-nitroimidazo-oxazole derivative) and its enantiomers as candidates for further evaluation in in vivo models of VL. DNDI-VL-2098 (IC50 of 0.03 μM for the DD8 strain) showed excellent in vivo activity in both mouse and hamster models, with an ED90 value of 3.7 and <25 mg/kg, respectively, and was also found to be very effective against high-grade infection in the hamster model. Our studies revealed that, along with leishmanicidal activity, DNDI-VL-2098 was also capable of inducing host-protective immune cells to suppress Leishmania parasites in hamsters. These studies led to the identification of compound DNDI-VL-2098 as a preclinical candidate for further drug development as an oral treatment for VL.

Physicochemical Properties, Form, and Formulation Selection Strategy for a Biopharmaceutical Classification System Class II Preclinical Drug Candidate

This work summarizes the pharmaceutical evaluation of a preclinical drug candidate with poor physicochemical properties. Compound 1 is a weakly basic, GPR-119 agonist designated to Biopharmaceutics Classification System Class II because of good permeability in a Caco-2 cell line model and poor solubility. Compound 1 showed good oral bioavailability from a solution formulation at low doses and oral exposure sufficient for toxicological evaluation at high doses from a nanosuspension of Form A-the only known polymorph of 1 during drug discovery. The identification of the thermodynamically stable polymorph, Form B, during early development adversely affected the bioperformance of the nanosuspension. The poor solubility of Form B resulted in a significant reduction in the oral exposure from a nanosuspension to a level that was insufficient for toxicological evaluation of compound 1. Subsequent to the discovery of Form B, multiple form and formulation engineering strategies were evaluated for their ability to enhance the oral exposure of 1. Formulations based on cocrystals and amorphous solid dispersions showed a statistically significant increase in exposure, sixfold and sevenfold, respectively, over the benchmark formulation, a suspension of Form B. The physicochemical characterization of 1, and the solid form and formulation engineering approaches explored to address the insufficient oral exposure of Form B are discussed along with insights on improving the physicochemical properties of the follow-on drug candidates in discovery.

Abstract 1753: GLPG1790: The first Ephrin (EPH) receptor tyrosine kinase inhibitor for the treatment of triple negative breast cancer

Abstract Receptor tyrosine kinases define a clinically relevant class of targets in the field of cancer. Here we report the discovery of a pre-clinical drug candidate directed against the EPH receptor family. Members of this family of receptor tyrosine kinases are over-expressed in diverse cancer types. GLPG1790 is a small molecule, nanomolar inhibitor of various EPH receptors kinases. The compound displayed a remarkable efficacy by once-daily oral administration in a mouse EPHA2 expressing xenograft model (MDA-MB-231). A rapid dose-dependent reduction of tumor growth was achieved, with full inhibition at the oral dose of 30 mg/kg/d. GLPG1790 efficacy at this dose was similar to that of Paclitaxel administered at its maximum tolerated dose. A series of experiments was initiated to confirm the mechanism of action of this compound. GLPG1790 inhibits human EPHA2 kinase activity with an IC50 of 11 nM in a biochemical assay. In the human MDA-MB-231 breast cancer cell line, that expresses a high level of EPHA2 protein, GLPG1790 inhibits receptor phosphorylation with an IC50 of 260 nM, and anchorage-independent growth with similar potency. In addition, in vivo target engagement was demonstrated in the mouse MDA-MB-231 xenograft model. GLPG1790 inhibited EPHA2 receptor phosphorylation after single oral administration of 30 and 100 mg/kg doses. The extent of the effects observed on EPHA2 phosphorylation correlated with intra-tumoral GLPG1790 concentration. Moreover, the MAPK pathway, known to be a major driver of proliferation of this cell line, was inhibited both in in vitro cellular assays and in xenograft target engagement studies. Flow cytometric analyses revealed a cell cycle arrest at the G0/G1 phase for MDA-MB-231 cells treated with GLPG1790. EPHA2 knock-down-based experiments further support the EPH-driven mode of action of GLPG1790. All together these data stimulate the development of GLPG1790 in triple negative breast cancer. This novel mechanism of action is under investigation in other cancer types overexpressing EPH's (melanoma, pancreatic, ovarian, prostatic and colorectal cancers). Citation Format: Philippe Pujuguet, Filip Beirinckx, Carole Delachaume, Jacques Huck, Ellen Van der Aar, Reginald Brys, Luc Van Rompaey, Piet Wigerinck, Laurent Saniere. GLPG1790: The first Ephrin (EPH) receptor tyrosine kinase inhibitor for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2014-1753

Design and synthesis of novel spin-labeled camptothecin derivatives as potent cytotoxic agents

In our continuing search for natural product-based spin-labeled antitumor drugs, 20 novel spin-labeled camptothecin derivatives were synthesized via a Cu-catalyzed one pot reaction and evaluated for cytotoxicity against four human tumor cell lines (A-549, MDA-MB-231, KB, and KBvin). Eighteen of the target compounds (9a, 9b, 9d–9k, 9m–9t) exhibited significant in vitro antiproliferative activity against these four tested tumor cell lines. Compounds 9e and 9j (IC50 0.057 and 0.072μM, respectively) displayed the greatest cytotoxicity against the multidrug-resistant (MDR) KBvin cell line and merit further development into preclinical and clinical drug candidates for treating cancer including MDR phenotype.

Use of microdosing and accelerator mass spectrometry to evaluate the pharmacokinetic linearity of a novel tricyclic GyrB/ParE inhibitor in rats.

Determining the pharmacokinetics (PKs) of drug candidates is essential for understanding their biological fate. The ability to obtain human PK information early in the drug development process can help determine if future development is warranted. Microdosing was developed to assess human PKs, at ultra-low doses, early in the drug development process. Microdosing has also been used in animals to confirm PK linearity across subpharmacological and pharmacological dose ranges. The current study assessed the PKs of a novel antimicrobial preclinical drug candidate (GP-4) in rats as a step toward human microdosing studies. Dose proportionality was determined at 3 proposed therapeutic doses (3, 10, and 30 mg/kg of body weight), and PK linearity between a microdose and a pharmacological dose was assessed in Sprague-Dawley rats. Plasma PKs over the 3 pharmacological doses were proportional. Over the 10-fold dose range, the maximum concentration in plasma and area under the curve (AUC) increased 9.5- and 15.8-fold, respectively. PKs from rats dosed with a (14)C-labeled microdose versus a (14)C-labeled pharmacological dose displayed dose linearity. In the animals receiving a microdose and the therapeutically dosed animals, the AUCs from time zero to infinity were 2.6 ng · h/ml and 1,336 ng · h/ml, respectively, and the terminal half-lives were 5.6 h and 1.4 h, respectively. When the AUC values were normalized to a dose of 1.0 mg/kg, the AUC values were 277.5 ng · h/ml for the microdose and 418.2 ng · h/ml for the pharmacological dose. This 1.5-fold difference in AUC following a 300-fold difference in dose is considered linear across the dose range. On the basis of the results, the PKs from the microdosed animals were considered to be predictive of the PKs from the therapeutically dosed animals.

The Progress Towards the Development of DHQO Derivatives and Related Analogues with Inotropic Effects

During the course of the past two decades, our group has worked towards the development of increasingly potent inotropic agents by making structural modifications to compounds derived from 3,4-dihydro-2(1H)-quinolinone (DHQO) and related analogues. Herein, we describe the design and synthesis of a new series of DHQO derivatives and the subsequent evaluation of their positive inotropic activity towards left atrium stroke volume in isolated rabbit-heart preparations. Some of these derivatives presented favorable in vitro activities compared with the reference drug, milrinone, and compound 10a, in particular is currently being investigated as a preclinical drug candidate. This review also describes our progress towards the development of DHQO derivatives and related analogues with positive inotropic activities from 1999 to 2013.

Increases survival by apo-sGC activation via post-stroke blood brain barrier stabilisation and anti-inflammation

Background Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. In many settings of ischemia, vasodilation to increase perfusion is a suitable approach; yet in stroke it bears the risk of systemic hypotension, shunting of blood from the ischemic to healthy areas, increased infarcts and eventually reduced survival. One potentially innovative and mechanism-based approach are sGC activators, as they represent diseasespecific vasodilators that are potentiated under conditions of oxidative stress and have microvascular selectivity e.g. to unload the acutely failing heart.

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Gaining understanding of common complex diseases and their treatments are the main drivers for life sciences. As we show here, comprehensive protein set analyses offer new opportunities to decipher functional molecular networks of diseases and assess the efficacy and side-effects of treatments in vivo. Using mass spectrometry, we quantitatively detected several thousands of proteins and observed significant changes in protein pathways that were (dys-) regulated in diet-induced obesity mice. Analysis of the expression and post-translational modifications of proteins in various peripheral metabolic target tissues including adipose, heart, and liver tissue generated functional insights in the regulation of cell and tissue homeostasis during high-fat diet feeding and medication with two antidiabetic compounds. Protein set analyses singled out pathways for functional characterization, and indicated, for example, early-on potential cardiovascular complication of the diabetes drug rosiglitazone. In vivo protein set detection can provide new avenues for monitoring complex disease processes, and for evaluating preclinical drug candidates.

The Synthesis and Evaluation of Novel Hydroxyl Substituted Chalcone Analogs with in Vitro Anti-Free Radicals Pharmacological Activity and in Vivo Anti-Oxidation Activity in a Free Radical-Injury Alzheimer’s Model

Alzheimer’s disease (AD) pathogenesis involves an imbalance between free radical formation and destruction. In order to obtain a novel preclinical anti-AD drug candidate, we synthesized a series of novel hydroxyl chalcone analogs which possessed anti-free radical activity, and screened their effects on scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH free radicals in vitro. Compound C7, 4,2'-dihydroxy-3,5-dimethoxychalcone was found to have potent activity in these anti-free radical activity tests. Further research revealed that C7 could elevate glutathione peroxidase (GSH-PX) and super oxide dismutase (SOD) levels and lower malonaldehyde (MDA) level in vivo in the Alzheimer’s model. The indication of C7’s effect on AD needs further study.

Kynurenines in the CNS: recent advances and new questions

Various pathologies of the central nervous system (CNS) are accompanied by alterations in tryptophan metabolism. The main metabolic route of tryptophan degradation is the kynurenine pathway; its metabolites are responsible for a broad spectrum of effects, including the endogenous regulation of neuronal excitability and the initiation of immune tolerance. This Review highlights the involvement of the kynurenine system in the pathology of neurodegenerative disorders, pain syndromes and autoimmune diseases through a detailed discussion of its potential implications in Huntington's disease, migraine and multiple sclerosis. The most effective preclinical drug candidates are discussed and attention is paid to currently under-investigated roles of the kynurenine pathway in the CNS, where modulation of kynurenine metabolism might be of therapeutic value.

A Physico-Biochemical Study on Potential Redox-Cyclers as Antimalarial and Antischistosomal Drugs

The role of redox enzymes in establishing a microenvironment for parasite development is well characterized. Mimicking human glucose-6-phosphate dehydrogenase and glutathione reductase (GR) deficiencies by redox-cycling compounds thus represents a challenge to the design of new preclinical antiparasitic drug candidates. Schistosomes and malarial parasites feed on hemoglobin. Heme, the toxic prosthetic group of the protein, is not digested and represents a challenge to the redox metabolism of the parasites. Here, we report on old and new redox-cycling compounds--whose antiparasitic activities are related to their interference with (met)hemoglobin degradation and hematin crystallization. Three key-assays allowed probing and differentiating the mechanisms of drug actions. Inhibition of β-hematin was first compared to the heme binding as a possible mode of action. All tested ligands interact with the hematin π-π dimer with K(D) similar to those measured for the major antiparasitic drugs. No correlation between a high affinity for hematin and the capacity to prevent β-hematin formation was however deduced. Inhibition of β-hematin formation is consequently not the result of a single process but results from redox processes following electron transfers from the drugs to iron(III)-containing targets. The third experiment highlighted that several redox-active compounds (in their reduced forms) are able to efficiently reduce methemoglobin to hemoglobin in a GR/NADPH-coupled assay. A correlation between methemoglobin reduction and inhibition of β-hematin was shown, demonstrating that both processes are closely related. The ability of our redox-cyclers to trigger methemoglobin reduction therefore constitutes a critical step to understand the mechanism of action of our drug candidates.

Are Meaningful Predictions of Toxicological Outcomes in Clinical Trials Based on In Vitro Diagnostics of Preclinical Drug Candidates Possible?

Are Meaningful Predictions of Toxicological Outcomes in Clinical Trials Based on <i>In Vitro</i> Diagnostics of Preclinical Drug Candidates Possible?

Drug safety is a barrier to the discovery and development of new androgen receptor antagonists

Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required. Non-clinical drug safety studies and safety pharmacology assays were performed on previously approved AR antagonists (bicalutamide, flutamide, nilutamide), next generation antagonists in clinical testing (MDV3100, BMS-641988), and a pre-clinical drug candidate (BMS-501949). In addition, non-clinical studies with AR mutant mice, and EEG recordings in rats were performed. Non-clinical findings are compared to disclosures of clinical trial results. As a drug class, AR antagonists cause seizure in animals by an off-target mechanism and are found in vitro to inhibit GABA-A currents. Clinical trials of candidate next generation AR antagonists identify seizure as a clinical safety risk. Non-clinical drug safety profiles of the AR antagonist drug class create a significant barrier to the identification of next generation AR antagonists. GABA-A inhibition is a common off-target activity of approved and next generation AR antagonists potentially explaining some side effects and safety hazards of this class of drugs.

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Author SummaryStroke is the second leading cause of death worldwide. Today, only one approved therapy exists—a drug that breaks down blood clots—the effectiveness of which is moderate, and it can only be used in about 10% of patients because of contraindications. New therapeutic strategies that are translatable to humans and more rigid thresholds of relevance in pre-clinical stroke models are needed. One candidate mechanism is oxidative stress, which is the damage caused by reactive oxygen species (ROS). Antioxidant approaches that specifically target ROS have thus far failed in clinical trials. For a more effective approach, we focus here on targeting ROS at its source by investigating an enzyme involved in generating ROS, known as NADPH oxidase type 4, or NOX4. We found that NOX4 causes oxidative stress and death of nerve cells after a stroke. Deletion of the NOX4-coding gene in mice, as well as inhibiting the ROS-generating activity of NOX with a pharmacological inhibitor, reduces brain damage and improves neurological function, even when given hours after a stroke. Importantly, neuroprotection was preserved in old male and female Nox4 −/− mice as well as in Nox4 −/− mice subjected to permanent ischemia. NOX4 thus represents a most promising new therapeutic target for reducing oxidative stress in general, and in brain injury due to stroke in particular.