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Integrating clinical pathology data with anatomic pathology data is a common practice when reporting findings in the context of nonclinical toxicity studies and aids in understanding and communicating the nonclinical safety profile of test articles in development. Appropriate pathology data integration requires knowledge of analyte and tissue biology, species differences, methods of specimen acquisition and analysis, study procedures, and an understanding of the potential causes and effects of a variety of pathophysiologic processes. Neglecting these factors can lead to inappropriate data integration or a missed opportunity to enhance understanding and communication of observed changes. In such cases, nonclinical safety information relevant to human safety risk assessment may be misrepresented or misunderstood. This “Points to Consider” manuscript presents general concepts regarding pathology data integration in nonclinical studies, considerations for avoiding potential oversights and errors in data integration, and focused discussion on topics relevant to data integration for several key organ systems including liver, kidney, and cardiovascular system.

RO7297089, an anti-B-cell maturation antigen (BCMA)/CD16A bispecific tetravalent antibody, is being developed as a multiple myeloma (MM) therapeutic. This study characterized nonclinical pharmacokinetics (PK), pharmacodynamics (PD), soluble BCMA (sBCMA), and soluble CD16 (sCD16) changes following administration of RO7297089 to support clinical trials. Unbound and total RO7297089 concentrations were measured in cynomolgus monkeys. RO7297089 exhibited a bi-phasic systemic concentration-time profile, similar to a typical human immunoglobulin 1 antibody. Target engagement by RO7297089 led to a robust increase (~100-fold) in total systemic sBCMA levels and relatively mild increase (~2-fold) in total sCD16 levels. To describe the relationship of nonclinical PK/PD data, we developed a target-mediated drug disposition (TMDD) model that includes the systemic target engagement of membrane BCMA (mBCMA), sBCMA, membrane CD16 (mCD16), and sCD16. We then used this model to simulate the PK/PD relationship of RO7297089 in MM patients by translating relevant PK parameters and target levels, based on the literature and newly generated data such as baseline sCD16A levels. Our model suggested that the impact of TMDD on RO7297089 exposure may be more significant in MM patients due to significantly higher expression levels of both mBCMA and sBCMA compared to healthy cynomolgus monkeys. Based on model simulations, we propose more frequent dosing of RO7297089 compared to regular monthly frequency in the clinic at the beginning of treatment to ensure sustained target engagement. This study demonstrates a translational research strategy for collecting relevant nonclinical data, establishing a TMDD model, and using simulations from this model to inform clinical dose regimens. Graphical Abstract

Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there are few reports on the utility of these renal biomarkers in mice, another important and widely used preclinical animal species for drug development studies. The purpose of this study was to assess the value of these recently qualified biomarkers for the early detection of drug-induced kidney injury (DIKI) in different strains of mice using multiple assay panels. To this end, we evaluated biomarker response to kidney injury induced by several nephrotoxic agents including amphotericin B, compound X, and compound Y. Several of the biomarkers were shown to be sensitive to DIKI in mice. When measured, urinary albumin and neutrophil gelatinase-associated lipocalin were highly sensitive to renal tubular injury, regardless of the assay platforms, mouse strain, and nephrotoxic agents. Depending on the type of renal tubular injury, kidney injury molecule-1 was also highly sensitive, regardless of the assay platforms and mouse strain. Osteopontin and cystatin C were modestly to highly sensitive to renal tubular injury, but the assay type and/or the mouse strain should be considered before using these biomarkers. Calbindin D28 was highly sensitive to injury to the distal nephron in mice. To our knowledge, this is the first report that demonstrates the utility of novel urinary biomarkers evaluated across multiple assay platforms and nephrotoxicants in different mice strains with DIKI. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in mice for toxicology studies.

Transforming growth factor β (TGFβ) signaling has been recently shown to reduce antitumor response to PD-L1 blockade, leading to a renewed enthusiasm in developing anti-TGFβ therapies for potential combination with cancer immunotherapy agents. Inhibition of TGFβ signaling in nonclinical toxicology species is associated with serious adverse toxicities including cardiac valvulopathies and anemia. Previously, cardiovascular toxicities have been thought to be limited to small molecule inhibitors of TGFβ receptor and not considered to be a liability associated with pan-TGFβ neutralizing monoclonal antibodies (mAbs). Here, we report the toxicity findings associated with a potent pan-TGFβ neutralizing mAb (pan-TGFβ mAb; neutralizes TGFβ1, 2, and 3) after 5 weekly intravenous doses of 10, 30, and 100 mg/kg, followed by a 4-week recovery period, in mice and cynomolgus monkeys. Mortality was observed due to acute bleeding and cardiovascular toxicity in mice at ≥ 30 mg/kg and prolonged menstruation in female monkeys at 100 mg/kg. Additional findings considered to be on-target exaggerated pharmacology included generalized bleeding and cardiovascular toxicity in mice and monkeys; histopathologic changes in the teeth, tongue, and skin in mice; and abnormal wound healing and microscopic pathology in the bone in monkeys. Importantly, our data indicate that the cardiovascular toxicities associated with the inhibition of TGFβ signaling are not limited to small molecule inhibitors but are also observed following administration of a potent pan-TGFβ inhibiting mAb.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE

Clinical pathology reporting practices are diverse among individuals and organizations involved in nonclinical toxicology studies. Clear, informative, and consistent reporting of clinical pathology results increases their value and avoids misinterpretation, resulting in decreased drug development costs. In recent years, certain common practices in clinical pathology reporting have been embraced by industry leaders and more consistently utilized across the pharmaceutical industry. The purpose of this manuscript is to review current clinical pathology reporting practices and to provide nonbinding suggestions to improve consistency, quality, and value of clinical pathology reports generated in support of nonclinical toxicology studies.

Coronaviruses are positive stranded RNA viruses that cause respiratory, enteric and central nervous system diseases in many species, including humans. Until recently, the relatively low burden of disease in humans caused by few of these viruses impeded the development of coronavirus specific therapeutics. However, the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV), and more recently, Middle East respiratory syndrome coronavirus (MERS-CoV), has impelled the development of such drugs. This review focuses on some newly identified SARS-CoV inhibitors, with known mechanisms of action and their potential to inhibit the novel MERS-CoV. The clinical development of optimized versions of such compounds could be beneficial for the treatment and control of SARS-CoV, the current MERS-CoV and other future SARS-like epidemics.

We have previously shown that SSYA10-001 blocks severe acute respiratory syndrome coronavirus (SARS-CoV) replication by inhibiting SARS-CoV helicase (nsp13). Here, we show that SSYA10-001 also inhibits replication of two other coronaviruses, mouse hepatitis virus (MHV) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). A putative binding pocket for SSYA10-001 was identified and shown to be similar in SARS-CoV, MERS-CoV, and MHV helicases. These studies show that it is possible to target multiple coronaviruses through broad-spectrum inhibitors.

More than a year ago, a novel coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was first identified as the causative agent of a severe pneumonia in several patients in the Middle East [1]. As of August 5, 2013, a total of 94 cases have been identified in the Middle East (Saudi Arabia, Jordan, Qatar, and the United Arab Emirates), Europe (United Kingdom, France, Italy, and Germany), and North Africa (Tunisia), with a case fatality rate of 56%. Since this coronavirus is closely related to severe acute respiratory syndrome coronavirus (SARS-CoV), an epidemic that was short-lived but alarming in 2002–2003 that resulted in approximately 8000 cases and 800 deaths, several health agencies that include governmental, public health, clinical, and laboratory authorities have all moved rapidly to respond to this new virus outbreak. Following identification of MERS-CoV, details about its genomic sequence and organization, species tropism, and host-cell receptor (dipeptidyl peptidase 4) have been reported [2,3]. Although there have been several reports of inhibitors of SARS-CoV, to date, there are no approved drugs or vaccines for the treatment of SARS-CoV. Studies have shown that ribavirin has a very minimal effect in controlling adverse outcomes from SARS-CoV infection. Its use has attracted a lot of criticism due to its unproven and low efficacy and unwarranted side effects [4]. At non-toxic concentrations, ribavirin has no direct in vitro activity against SARS-CoV [5]. Among some of the promising agents for the control of SARS-CoV infection are interferons. A large number of recombinant interferons that belong to the three classes (α, β and γ) have been tested for their antiviral activities against SARS-CoV in vitro. Interferon-α and -β were shown to be active against SARS-CoV, with interferon-β being the most active of the three [6]. More recently, Falzarano et al [7] proposed a potential therapeutic regimen for the treatment of MERS-CoV based on cell-based experiments. In this report, the authors compared the use of both interferon-α2b and ribavirin individually or in combination for the inhibition of MERS-CoV in two different cell lines, Vero and LLC-MK2. When used individually, to completely eliminate the cytopathic effect (CPE) of SARS-CoV infection, each drug required concentrations as high as 1000 U/ml interferon-α2b (IFN- α2b) and 200 µg/ml ribavirin. However, when the two drugs were combined they acted synergistically, and only 125 U/ml IFN-α2b and 25 µg/ml ribavirin were required to completely eliminate the CPE. This represented an 8- and 16-fold decrease in the amount of IFN-α2b and ribavirin respectively, which is required to attain the same reduction when used individually. While the combination therapy portends to be a promising strategy, these concentrations are still very much higher than the current doses at which these two drugs are being used for Hepatitis C virus (HCV) infections in humans [8]. High doses of ribavirin have been associated with hemolytic anemia, elevated transaminase levels and bradycardia [9]. Despite the lower doses of these drugs for the treatment of HCV, many side effects accompany this combination therapy, which include depression, suicide, relapse of drug abuse/overdose, bacterial infections and many others (http://www.hepatitis.va.gov/provider/reviews/treatment-side-effects.asp#S1X). Nonetheless, given the severity of this disease and the lack of any available treatments, this is a promising finding that should be further explored. Given the similarity of MERS-CoV with SARS-CoV, what lessons can we learn from past work on SARS-CoV, or other positive strand RNA viruses? MERS-CoV was recently shown to use dipeptidyl peptidase 4 as its functional receptor to gain entry into cells [3]. While this virus is utilizing a receptor different than the one used by SARS and other respiratory coronaviruses, there appears to be some similarities in their mechanisms of entry. MERS-CoV has been shown to require cathepsins B and L for entry, similar to SARS-CoV [10]. Cathepsin L inhibitors have been shown to prevent SARS-CoV entry in SARS-CoV-pseudotyped assays (SARS-CoV env / HIV core) [11]. Published cathepsin L inhibitors include oxocarbazate, MDL28170 and SSAA09E1. MDL28170 and oxocarbazate inhibit SARS-CoV-pseudotyped virus with EC50s of ~100 nM and ~300 nM respectively [12,13]. These two compounds appear to be peptidomimetic in nature, thereby limiting their use only to parenteral administration if they were to be considered for therapeutic purposes. SSAA09E1, a non-peptidomimetic small molecule that inhibited SARS-CoV-pseudotyped virus with an EC50 of ~7 µM can also inhibit live SARS-CoV in Vero cells (unpublished data). Therefore, any of these cathepsin L inhibitors would be worth testing for inhibition of MERS-CoV as well as of other coronaviruses that require cathepsin L for entry. In thinking about treatments of viral infections, one has to also consider the challenge of drug resistance, which is the result of the ability of viruses to rapidly evolve and escape treatments through multiple and diverse mechanisms of resistance. There is an extensive body of clinical data on development of resistance against antivirals that are used in the clinic for the treatment of Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) infections. For HIV, remarkable progress has been made with highly active anti-retroviral therapies (HAART), which are based on combinations of drugs that almost exclusively target directly the virus. HAART regimens significantly suppress the viral load in the blood plasma of HIV patients, resulting in significant decreases in HIV-related deaths. However, prolonged treatment inevitably results in resistance to all of these available drugs. [14–16]. Similar benefits have been reported in the treatment of HBV-infected patients with antiretrovirals. For many years, HCV has been treated with a combination therapy of pegylated interferon and ribavirin. Clinical trials have shown sustained virological response rates (SVRs) of ~50% (efficacy), which does not even provide equivalent results in clinical practice [17]. HCV has also evolved various mechanisms to develop resistance to these therapies. For example, HCV circumvents the IFN response by blocking downstream signaling actions via STAT1, STAT2, IRF9 and JAK-STAT pathways [18,19]. Hence, MERS coronavirus can also develop resistance to IFN treatment. Ribavirin is a nucleoside analog that after activation by host kinases to a nucleotide, it can be used by viral polymerases and incorporated into the nascent viral genomes, causing mutations by allowing base mismatches. During the amplification of hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. Hence, ribavirin's antiviral activity is likely to result from error catastrophe caused by increased mutagenesis of viral genomes thereby reducing the fitness of the virus. Another mechanism through which ribavirin may exhibit its antiviral activity is by depletion of intracellular GTP pools (by inhibition of the cellular Inosine monophosphate dehydrogenase [IMPDH] by the 5′-monophosphate metabolite of ribavirin) [20]. This mechanism may be useful in explaining the drug's general cytotoxic and anti-DNA replication effect as well as some effect on DNA viral replication. While reports of viral resistance to ribavirin are rare, its reliance on host factors to be able to convert to its mono- and tri-phosphate active forms stands as a potential problem. Darryl Falzarano et al [7] showed the difference in the efficacies of ribavirin depending on the cell types, as ribavirin was more effective in LLC-MK2 cells than the Vero cells. Since, MERS-CoV naturally infects respiratory epithelial cell, it will be more expedient to monitor the effect of ribavirin in respiratory epithelial cells to see if comparable viral inhibition will be observed. With limited efficacy of this therapy, coupled with multiple side effects and resistance development, many scientists have been working on IFN-free therapy that involves development and the use of small molecule compounds that directly inhibit the viral replication proteins. The efficacies of these compounds are being tested alone or in combination with ribavirin or pegylated IFNs. These compounds are referred to as “Direct Acting Antivirals”. Examples include the recently approved boceprevir and telaprevir NS3/4A protease inhibitors, as well as NS5A inhibitors and NS5B polymerase inhibitors [21] that are in clinical trials. For coronaviruses, several reports of SARS-CoV small molecule compounds that inhibit the viral replication proteins, or prevent entry of the virus into cells, are well documented [11,12,22,23]. All these compounds, with known mechanisms of action, introduce potential leads for the development of MERS and other coronaviruses therapeutics. In conclusion, the recent report that interferon-α2b and ribavirin have some synergistic activity in cell-based assays is indeed encouraging, as these drugs are currently used to treat hepatitis C and are therefore widely available even though they are imperfect but with known pharmacological properties. Moreover, the study highlights how past research in related viruses can provide useful insights in the treatment of emerging diseases.

Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS coronavirus (SARS-CoV) and for which there are currently no approved treatments. We report the discovery and characterization of small-molecule inhibitors of SARS-CoV replication that block viral entry by three different mechanisms. The compounds were discovered by screening a chemical library of compounds for blocking of entry of HIV-1 pseudotyped with SARS-CoV surface glycoprotein S (SARS-S) but not that of HIV-1 pseudotyped with vesicular stomatitis virus surface glycoprotein G (VSV-G). Studies on their mechanisms of action revealed that the compounds act by three distinct mechanisms: (i) SSAA09E2 { N -[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide} acts through a novel mechanism of action, by blocking early interactions of SARS-S with the receptor for SARS-CoV, angiotensin converting enzyme 2 (ACE2); (ii) SSAA09E1 {[( Z )-1-thiophen-2-ylethylideneamino]thiourea} acts later, by blocking cathepsin L, a host protease required for processing of SARS-S during viral entry; and (iii) SSAA09E3 [ N -(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide] also acts later and does not affect interactions of SARS-S with ACE2 or the enzymatic functions of cathepsin L but prevents fusion of the viral membrane with the host cellular membrane. Our work demonstrates that there are at least three independent strategies for blocking SARS-CoV entry, validates these mechanisms of inhibition, and introduces promising leads for the development of SARS therapeutics.

Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC 50s) of 5.70 and 5.30 μM, respectively. This compound also has inhibitory activity (50% effective concentration [EC 50]=8.95 μM) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC 50]= >250 μM), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target. Copyright © 2012, American Society for Microbiology. All Rights Reserved., This work was supported in part by NIH grants AI076119, AI094715, AI079801, and AI074389. We also acknowledge support by a grant from the Ministry of Knowledge and Economy, Bilateral International Collaborative R&D Program, Republic of Korea.