Characterization of Ebola Virus Disease (EVD) in Rhesus Monkeys for Development of EVD Therapeutics.

Recent Ebola virus (EBOV) outbreaks in West Africa and the Democratic Republic of the Congo have highlighted the urgent need for approval of medical countermeasures for treatment and prevention of EBOV disease (EVD). Until recently, when successes were achieved in characterizing the efficacy of multiple experimental EVD therapeutics in humans, the only feasible way to obtain data regarding potential clinical benefits of candidate therapeutics was by conducting well-controlled animal studies. Nonclinical studies are likely to continue to be important tools for screening and development of new candidates with improved pharmacological properties. Here, we describe a natural history study to characterize the time course and order of progression of the disease manifestations of EVD in rhesus monkeys. In 12 rhesus monkeys exposed by the intramuscular route to 1000 plaque-forming units of EBOV, multiple endpoints were monitored for 28 days following exposure. The disease progressed rapidly with mortality events occurring 7-10 days after exposure. Key disease manifestations observed consistently across the infected animals included, but were not limited to, viremia, fever, systemic inflammation, coagulopathy, lymphocytolysis, renal tubular necrosis with mineralization, and hepatocellular degeneration and necrosis.

Filgotinib, a JAK1 Inhibitor, Modulates Disease-Related Biomarkers in Rheumatoid Arthritis: Results from Two Randomized, Controlled Phase 2b Trials.

INTRODUCTION: The Janus kinase (JAK) inhibitor therapeutic class has shown significant clinical benefit in the treatment of rheumatoid arthritis (RA). We sought to gain insight into the mode of action and immunological effects of filgotinib, a JAK1 selective inhibitor, in active RA by analyzing secreted and cell-based biomarkers key to RA pathophysiology in two phase 2b trials of filgotinib in active RA. METHODS: Immune cell subsets and 34 serum biomarkers were analyzed longitudinally over 12 weeks using blood samples collected from patients with active RA receiving filgotinib (100 or 200 mg once daily) or placebo (PBO) in the two phase 2b trials (DARWIN 1, on a background of methotrexate, and DARWIN 2, as monotherapy). RESULTS: Consistently across both studies, filgotinib treatment decreased multiple immune response biomarkers that have key roles in RA for immune response, and decreased markers that promote matrix degradation, angiogenesis, leukocyte adhesion, and recruitment. Filgotinib did not significantly modulate T and natural killer (NK) lymphoid subsets, but slightly increased B cell numbers after 12 weeks. Multiple correlations were observed for changes in biomarkers with disease activity score 28-CRP. MIP1ß showed modest predictivity at baseline for ACR50 response at 12 weeks in the 100 mg filgotinib dose across both studies (AUROC, 0.65 and 0.67, p < 0.05). CONCLUSIONS: Filgotinib regulates biomarkers from multiple pathways, indicative of direct and indirect network effects on the immune system and the stromal response. These effects were not associated with reductions of major circulating lymphoid populations. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01888874, NCT01894516.

GS-0976 Reduces Hepatic Steatosis and Fibrosis Markers in Patients With Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: De novo lipogenesis is increased in livers of patients with nonalcoholic steatohepatitis (NASH). Acetyl-coenzyme carboxylase catalyzes the rate-limiting step in this process. We evaluated the safety and efficacy of GS-0976, an inhibitor of acetyl-coenzyme A carboxylase in liver, in a phase 2 randomized placebo-controlled trial of patients with NASH. METHODS: We analyzed data from 126 patients with hepatic steatosis of at least 8%, based on the magnetic resonance imaging-estimated proton density fat fraction (MRI-PDFF), and liver stiffness of at least 2.5 kPa, based on magnetic resonance elastography measurement or historical biopsy result consistent with NASH and F1-F3 fibrosis. Patients were randomly assigned (2:2:1) to groups given GS-0976 20 mg, GS-0976 5 mg, or placebo daily for 12 weeks, from August 8, 2016 through July 18, 2017. Measures of hepatic steatosis, stiffness, serum markers of fibrosis, and plasma metabolomics were evaluated. The primary aims were to confirm previous findings and evaluate the relation between dose and efficacy. RESULTS: A relative decrease of at least 30% from baseline in MRI-PDFF (PDFF response) occurred in 48% of patients given GS-0976 20 mg (P = .004 vs placebo), 23% given GS-0976 5 mg (P = .43 vs placebo), and 15% given placebo. Median relative decreases in MRI-PDFF were greater in patients given GS-0976 20 mg (decrease of 29%) than those given placebo (decrease of 8%; P = .002). Changes in magnetic resonance elastography-measured stiffness did not differ among groups, but a dose-dependent decrease in the fibrosis marker tissue inhibitor of metalloproteinase 1 was observed in patients given GS-0976 20 mg. Plasma levels of acylcarnitine species also decreased in patients with a PDFF response given GS-0976 20 mg. GS-0976 was safe, but median relative increases of 11% and 13% in serum levels of triglycerides were observed in patients given GS-0976. CONCLUSIONS: In a randomized placebo-controlled trial of patients with NASH, we found 12-week administration of GS-0976 20 mg decreased hepatic steatosis, selected markers of fibrosis, and liver biochemistry. ClinicalTrials.gov ID NCT02856555.

Interrogation of transcriptomic changes associated with drug-induced hepatic sinusoidal dilatation in colorectal cancer.

Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.

Acetyl-CoA Carboxylase Inhibitor GS-0976 for 12 Weeks Reduces Hepatic De Novo Lipogenesis and Steatosis in Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Increased de novo lipogenesis (DNL) contributes to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase catalyzes the rate-limiting step in DNL. We evaluated the safety and efficacy of GS-0976, a small molecule inhibitor of acetyl-CoA carboxylase, in patients with NASH. METHODS: In an open-label prospective study, patients with NASH (n = 10) received GS-0976 20 mg orally once daily for 12 weeks. NASH was diagnosed based on a proton density fat fraction estimated by magnetic resonance imaging (MRI-PDFF) ≥10% and liver stiffness by magnetic resonance elastography (MRE) ≥2.88 kPa. The contribution from hepatic DNL to plasma palmitate was measured by 14 days of heavy water labeling before and at the end of treatment. We performed the same labelling protocol in an analysis of healthy volunteers who were not given DNL (controls, n = 10). MRI-PDFF and MRE at baseline, and at weeks 4 and 12 of GS-0976 administration, were measured. We analyzed markers of liver injury and serum markers of fibrosis. RESULTS: The contribution of hepatic DNL to plasma palmitate was significantly greater in patients with NASH compared with controls (43% vs 18%) (P = .003). After 12 weeks administration of GS-0976, the median hepatic DNL was reduced 22% from baseline in patients with NASH (P = .004). Compared with baseline, reductions in MRI-PDFF at week 12 (15.7% vs 9.1% at baseline; P = .006), liver stiffness by MRE (3.4 kPa vs 3.1 kPa at baseline; P = .049), TIMP metallopeptidase inhibitor 1 (275 ng/mL vs 244 ng/mL at baseline; P = .049), and serum level of alanine aminotransferase (101 U/L vs 57 U/L at baseline; P = .23) were consistent with decreased hepatic lipid content and liver injury. At week 12, 7 patients (70%) had a ≥30% decrease in MRI-PDFF. CONCLUSION: In an open-label study, patients with NASH given GS-0976 for 12 weeks had reduced hepatic DNL, steatosis, and markers of liver injury. ClinicalTrials.gov no: NCT02856555.

Proof of Concept for an Automated Image Analysis Method to Quantify Rat Bone Marrow Hematopoietic Lineages on H&E Sections.

The bone marrow is an important site for assessment of the hematopoietic toxicity of new drug candidates. Here, we extended our previous work, where we developed a computer algorithm to automatically quantitate overall bone marrow cell density by analyzing digitized images of standard hematoxylin and eosin (H&E) slides of rat bone marrow and further evaluated the capability to quantify myeloid: erythroid + lymphoid (M:EL) ratio and megakaryocyte cell density. We tested the algorithm in a toxicity study, where rats were dosed with two molecules known to affect bone marrow composition, monomethyl auristatin E, and a Bcl-xL inhibitor. The image analysis method detected significant changes in M:EL and megakaryocyte number that were either not found or semiquantitatively described by manual microscopic observation of the same slides. The image analysis results were consistent with other more established but time-consuming methods that measure changes in bone marrow cell composition: smear cytology, flow cytometry, and microscopic assessment. Our work demonstrates the feasibility of a rapid and more quantitative assessment of changes in bone marrow cell lineage composition using a computer algorithm compared to microscopic examination of H&E-stained bone marrow sections.

Individual serum bile acid profiling in rats aids in human risk assessment of drug-induced liver injury due to BSEP inhibition.

Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.

MEK and ERK Kinase Inhibitors Increase Circulating Ceruloplasmin and Cause Green Serum in Rats.

Inhibition of the mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is an attractive therapeutic approach for human cancer therapy. In the course of evaluating structurally distinct small molecule inhibitors that target mitogen-activated protein kinase kinase (MEK) and ERK kinases in this pathway, we observed an unusual, dose-related increase in the incidence of green serum in preclinical safety studies in rats. Having ruled out changes in bilirubin metabolism, we demonstrated a 2- to 3-fold increase in serum ceruloplasmin levels, likely accounting for the observed green color. This was not associated with an increase in α-2-macroglobulin, the major acute phase protein in rats, indicating that ceruloplasmin levels increased independently of an inflammatory response. Elevated serum ceruloplasmin was also not correlated with changes in total hepatic copper, adverse clinical signs, or pathology findings indicative of copper toxicity, therefore discounting copper overload as the etiology. Both ERK and MEK inhibitors led to increased ceruloplasmin secretion in rat primary hepatocyte cultures in vitro, and this increase was associated with activation of the Forkhead box, class O1 (FOXO1) transcription factor. In conclusion, increased serum ceruloplasmin induced by MEK and ERK inhibition is due to increased synthesis by hepatocytes from FOXO1 activation and results in the nonadverse development of green serum in rats.

Bruton's Tyrosine Kinase Small Molecule Inhibitors Induce a Distinct Pancreatic Toxicity in Rats.

Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinases involved in B-cell and myeloid cell signaling. Small molecule inhibitors of BTK are being investigated for treatment of several hematologic cancers and autoimmune diseases. GDC-0853 ((S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selective and reversible oral small-molecule BTK inhibitor in development for the treatment of rheumatoid arthritis and systemic lupus erythematosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days or longer caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. Similar findings were not observed in mice or dogs at much higher exposures. Hemorrhage in the peri-islet vasculature emerged between four and seven daily doses of GDC-0853 and was histologically similar to spontaneously occurring changes in aging SD rats. This suggests that GDC-0853 could exacerbate a background finding in younger animals. Glucose homeostasis was dysregulated following a glucose challenge; however, this occurred only after 28 days of administration and was not directly associated with onset or severity of pancreatic lesions. There were no changes in other common serum biomarkers assessing endocrine and exocrine pancreatic function. Additionally, these lesions were not readily detectable via Doppler ultrasound, computed tomography, or magnetic resonance imaging. Our results indicate that pancreatic lesions in rats are likely a class effect of BTK inhibitors, which may exacerbate an islet-centered pathology that is unlikely to be relevant to humans.

Therapeutic Antibody-Induced Vascular Toxicity Due to Off-Target Activation of Nitric Oxide in Cynomolgus Monkeys.

PRO304186, a humanized monoclonal antibody targeting soluble interleukin-17 A and F, was developed for autoimmune and inflammatory disease indications. When administered to cynomolgus monkeys PRO304186 induced unexpected adverse effects characterized by clinical signs of hematemesis, hematochezia, and moribundity. Pathology findings included hemorrhage throughout the gastrointestinal tract without any evidence of vascular wall damage or inflammatory cellular infiltration. Mechanistic investigation of these effects revealed mild elevations of serum MCP-1 and IL-12/23 but without a classical proinflammatory profile in PRO304186-treated animals. In vitro studies demonstrated off-target effects on vascular endothelial cells including activation of nitric oxide synthase leading to production of nitric oxide (NO) accompanied by increased mitochondrial membrane depolarization, glutathione depletion, and increased paracellular permeability. Additionally, endothelial cell-PRO304186-conditioned medium reduced myosin light chain phosphorylation in vascular smooth muscle cells. Furthermore, an ex vivo study utilizing segments from cynomolgus aorta and femoral artery confirmed PRO304186-induced endothelium-dependent smooth muscle relaxation and vasodilation mediated via NO. Finally, a single dose of PRO304186 in cynomolgus monkeys induced a rapid and pronounced increase in NO in the portal circulation that preceded a milder elevation of NO in the systemic circulation and corresponded temporally with systemic hypotension; findings consistent with NO-mediated vasodilation leading to hypotension. These changes were associated with non-inflammatory, localized hemorrhage in the gastrointestinal tract consistent with hemodynamic vascular injury associated with intense local vasodilation. Together, these data demonstrate that PRO304186-associated toxicity in monkeys was due to an off-target effect on endothelium that involved regional NO release resulting in severe systemic vasodilation, hypotension, and hemorrhage.

Balancing Blood Sample Volume with 3Rs: Implementation and Best Practices for Small Molecule Toxicokinetic Assessments in Rats.

Improved small molecule bioanalytical sensitivity and concomitant decreased sample volume requirements provide an opportunity to reconsider how toxicokinetic (TK) data are collected in rat toxicity studies. Often, satellite groups of rats are designated to separate procedural effects of TK blood collection from the primary toxicity evaluation. Blood microsampling (i.e., ≤50 µL) decreases the blood volume collected such that TK samples can be collected from toxicity groups without impacting toxicity assessment. Small plasma sampling uses slightly higher blood volumes (i.e., 200 µL) with comparable technical feasibility and, importantly, allows multiple analyses with no negative impact on study interpretation. Our "base case" study designs utilize sparse TK sampling from sample toxicity group rats (1-2 samples/rat). Alternate designs with satellite animals may still be warranted based on study objectives (e.g., biomarkers), intolerability, or smaller rat strains; however, we propose these as exceptions rather than standard practice and with a focus to use the fewest animals possible. We review the state of knowledge in bioanalytical and blood sampling techniques and support the paradigm whereby TK sampling of main study animals significantly decreases the overall number of rats required for toxicity assessments and refines study interpretation with additional data options. These efforts maintain a commitment to the 3Rs (replacement, reduction, and refinement) while maintaining high-quality TK evaluations on toxicity studies.

Balancing Efficacy and Safety of an Anti-DLL4 Antibody through Pharmacokinetic Modulation.

PURPOSE: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. EXPERIMENTAL DESIGN: The F(ab')2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab')2) was generated and assessed in efficacy and toxicity studies. RESULTS: Anti-DLL4 F(ab')2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab')2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. CONCLUSIONS: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab')2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans.

Preclinical models of nicotinamide phosphoribosyltransferase inhibitor-mediated hematotoxicity and mitigation by co-treatment with nicotinic acid.

Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.

Effect of selective LRRK2 kinase inhibition on nonhuman primate lung.

Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

Development of a safety biomarker signature to detect hepatic sinusoidal dilation associated with an anti-DLL4 biotherapeutic.

CONTEXT: Biomarkers of lesion-specific drug induced liver toxicity are currently lacking. OBJECTIVE: To develop a biomarker signature using routine clinical pathology parameters that predict hepatic sinusoidal dilation related to anti-DLL4 biotherapeutics. METHODS: Random forest and factor analysis was used to construct a signature of routine laboratory tests to detect microscopically confirmed sinusoidal dilation of the liver. RESULTS: A biomarker signature was developed comprising two scores (S1 and S2) with area under the curve (AUC) for sinusoidal dilation prediction of 0.81, 0.85 and 0.96 in three rat studies and 0.48 and 0.81 in two monkey studies. CONCLUSION: A unique, two-dimensional signature of liver parameters and red blood cell parameters could detect sinusoidal dilation in multiple preclinical species.

Use of optimized aminotransferase methods in regulated preclinical studies.

BACKGROUND: The serum activities of ALT and AST are key indicators of liver toxicity in the drug safety evaluation of laboratory animals and patients. To ensure that the full aminotransferase activity is measured, exogenous Pyridoxyl-5-Phosphate (P5P) cofactor is included in the assay reagent. Clinical pathology laboratories make a choice to use aminotransferase assays with or without the added P5P cofactor, and the impact of assay selection on safety assessment is not well understood. OBJECTIVES: The objective of this report was to investigate the effect of aminotransferase assay selection on the detection of liver toxicity based on a literature review. METHODS: Literature in public databases was searched using combinations of the search terms alanine aminotransferase, aspartate aminotransferase, pyridoxyl-5-phosphate, holoenzyme, apoenzyme, enzyme inhibition, artifact, clinical pathology, toxicology, and safety assessment. Regulations or guidance documents published by health authorities specifying clinical pathology evaluation in nonclinical and clinical safety studies of biopharmaceuticals, chemicals, and devices were also reviewed. RESULTS: Aminotransferase testing is not standardized in safety assessment studies and consequently, laboratories use aminotransferase assays with or without P5P cofactor. Individual studies have demonstrated mean differences of approximately 10-20% in serum ALT activity in animal and human populations. The impact of aminotransferase testing without P5P on detection of toxicity and decision-making in drug development has not been systematically evaluated. CONCLUSIONS: The use of different assays for measuring aminotransferase activity contributes to the variability in data between laboratories and studies. Standardizing aminotransferase assays is an avenue for improving the diagnostic performance in drug safety evaluation.

Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier.

Unexpected hematologic effects of biotherapeutics in nonclinical species and in humans.

Biotherapeutics are expanding the arsenal of therapeutics available for treating and preventing disease. Although initially thought to have limited side effects due to the specificity of their binding, these drugs have now been shown to have potential for adverse drug reactions including effects on peripheral blood cell counts or function. Hematotoxicity caused by a biotherapeutic can be directly related to the activity of the biotherapeutic or can be indirect and due to autoimmunity, biological cascades, antidrug antibodies, or other immune system responses. Biotherapeutics can cause hematotoxicity primarily as a result of cellular activation, cytotoxicity, drug-dependent and independent immune responses, and sequelae from initiating cytokine and complement cascades.  The underlying pathogenesis of biotherapeutic-induced hematotoxicity often is poorly understood. Nonclinical studies have generally predicted clinical hematotoxicity for recombinant cytokines and growth factors.  However, most hematologic liabilities of biotherapeutics are not based on drug class but are species specific, immune-mediated, and of low incidence. Despite the potential for unexpected hematologic toxicity, the risk-benefit profile of most biotherapeutics is favorable; hematologic effects are readily monitorable and managed by dose modification, drug withdrawal, and/or therapeutic intervention.  This article reviews examples of biotherapeutics that have unexpected hematotoxicity in nonclinical or clinical studies.