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Nonclinical safety and pharmacokinetic data for monomethyl auristatin E (MMAE) and 14 vedotin antibody-drug conjugates (ADC) were evaluated to determine patterns of toxicity, consistency of pharmacokinetic results, and species differences between rats and monkeys. Most nonclinical toxicities were antigen-independent, common across ADCs, and included hematologic, lymphoid, and reproductive toxicity related to MMAE pharmacology. Hematologic toxicity was the dose-limiting toxicity (DLT) or predominant toxicity for the majority of vedotin ADCs in both species. Tissue expression of the targeted antigen of an ADC rarely correlated with DLT; only two ADCs had antigen-dependent skin DLTs. For two additional ADCs, antigen-dependent delivery of MMAE in the bone marrow may have exacerbated the antigen-independent hematologic DLT. The highest tolerated doses and pharmacokinetics were similar within a given species, with rats tolerating higher doses than monkeys. Studies longer than 1 month in duration detected the same or fewer toxicities than 1-month studies and had no additional findings that affected the human risk assessment. These data support opportunities to streamline ADC toxicity assessments without compromising human starting dose selection or target organ identification., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Doxorubicin-related heart failure has been recognized as a serious complication of cancer chemotherapy. This paper describes a cardiovascular safety pharmacology study with chronic dosing of doxorubicin in a non-human primate model designed to characterize the onset and magnitude of left ventricular dysfunction (LVD) using invasive and non-invasive methods. Methods: Cynomolgus monkeys ( N = 12) were given repeated intravenous injections of doxorubicin over 135 days (19 weeks) with dosing holidays when there was evidence of significantly decreased hematopoiesis; a separate group ( N = 12) received vehicle. Arterial and left ventricular pressure telemetry and cardiac imaging by echocardiography allowed regular hemodynamic assessments and determination of LVD. Blood samples were collected for hematology, clinical chemistry, and assessment of cardiac troponin (cTnI) and N-terminal pro b-type natriuretic peptide (NT-proBNP). Myocardial histopathology was a terminal endpoint. Results: There was variable sensitivity to the onset of treatment effects, for example 25% of doxorubicin-treated animals exhibited LVD (e.g., decreases in ejection fraction) following 50-63 days (cumulative dose: 8-9 mg/kg) on study. All animals deteriorated into heart failure with additional dosing 135 days (total cumulative dose: 11-17 mg/kg). Reductions in arterial pressure and cardiac contractility, as well as QTc interval prolongation, was evident following doxorubicin-treatment. Both cTnI and NT-proBNP were inconsistently higher at the end of the study in animals with LVD. Measurements collected from control animals were consistent and stable over the same time frame. Minimal to mild, multifocal, vacuolar degeneration of cardiomyocytes was observed in 7 of 12 animals receiving doxorubicin and 0 of 12 animals receiving vehicle. Conclusions: This repeat-dose study of doxorubicin treatment in the cynomolgus monkey demonstrated a clinically relevant pattern of progressive heart failure. Importantly, the study revealed how both telemetry and non-invasive echocardiography measurements could track the gradual onset of LVD., Competing Interests: All authors were employed by Amgen, Inc. NE is employed by Seattle Genetics. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Engwall, Everds, Turk and Vargas.)

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20 + human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Access and use of historical control data was identified as a top stakeholder concern across organizations according to results of a survey of needs and challenges related to nonclinical data conducted by the FDA/PhUSE Nonclinical Working Group in 2011. There is a perception there may be additional ways to capitalize on historical control data to enhance studies or submissions across industry, academia, and government. During the working sessions of the FDA/PhUSE Computational Sciences Symposium in March 2012, a Historical Control subgroup of the FDA/PhUSE Nonclinical Working Group was formed to investigate how the industry might more effectively harness the vast amount of data from untreated/vehicle control animals. The subgroup includes broad representation of stakeholders with interest in nonclinical data., Methods: This paper describes progress to date and includes results of a second survey to determine how organizations use and would like to use historical control data., Results: Respondents to the survey strongly support that historical control data are useful and should be in an accessible format. Four potential project options were posed in the survey, with an overall positive response; also, several write-in options were suggested by respondents., Conclusions: Community-supported projects to increase the availability of well-annotated and scientifically curated collections of historical control data appear to be of most interest.

In nonsmall cell lung cancer (NSCLC), the threonine(790)-methionine(790) (T790M) point mutation of EGFR kinase is one of the leading causes of acquired resistance to the first generation tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib. Herein, we describe the optimization of a series of 7-oxopyrido[2,3-d]pyrimidinyl-derived irreversible inhibitors of EGFR kinase. This led to the discovery of compound 24 which potently inhibits gefitinib-resistant EGFR(L858R,T790M) with 100-fold selectivity over wild-type EGFR. Compound 24 displays strong antiproliferative activity against the H1975 nonsmall cell lung cancer cell line, the first line mutant HCC827 cell line, and promising antitumor activity in an EGFR(L858R,T790M) driven H1975 xenograft model sparing the side effects associated with the inhibition of wild-type EGFR.

The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1: in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1: was shown in vitro and in a bile-duct cannulated rat (14)C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3: , was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1: to rats significantly reduced the formation of M3: and prevented liver toxicity, whereas direct administration of M3: reproduced the toxicity. Structural changes were introduced to 1: to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2: ), and addition of a methyl group to the benzylic carbon (compound 3: ) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3: (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Most published reviews of preclinical toxicological clinical pathology focus on the fundamental aspects of hematology, clinical chemistry, coagulation, and urinalysis in routine toxicology animal species, for example, rats, mice, dogs, and nonhuman primates. The objective of this continuing education course was to present and discuss contemporary examples of nonroutine applications of clinical pathology endpoints used in the drug development setting. Area experts discussed bone turnover markers of laboratory animal species, clinical pathology of pregnant and growing laboratory animals, clinical pathology of nonroutine laboratory animal species, and unique applications of the Siemens Advia(®) hematology analyzer. This article is a summary based on a presentation given at the 31st Annual Symposium of the Society of Toxicologic Pathology, during the Continuing Education Course titled "Nontraditional Applications of Clinical Pathology in Drug Discovery and Preclinical Toxicology.", (© 2014 by The Author(s).)

Gene expression profiling is a tool to gain mechanistic understanding of adverse effects in response to compound exposure. However, little is known about how the common handling procedures of experimental animals during a preclinical study alter baseline gene expression. We report gene expression changes in the livers of female Sprague-Dawley rats following common handling procedures. Baseline gene expression changes identified in this study provide insight on how these changes may affect interpretation of gene expression profiles following compound exposure. Rats were divided into three groups. One group was not subjected to handling procedures and served as controls for both handled groups. Animals in the other two groups were weighed, subjected to restraint in Broome restrainers, and administered water via oral gavage daily for 1 or 4 days with tail vein blood collections at 1, 2, 4, and 8 hours postdose on days 1 and 4. Significantly altered genes were identified in livers of animals following 1 or 4 days of handling when compared to the unhandled animals. Gene changes in animals handled for 4 days were similar to those handled for 1 day, suggesting a lack of habituation. The altered genes were primarily immune function related genes. These findings, along with a correlating increase in corticosterone levels suggest that common handling procedures may cause a minor immune system perturbance.

Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥ 50 mg/kg had unexpected acute thrombocytopenia (nadir ~3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab')2 portion of mAbY.1 nor the F(ab')2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.

AMG X, a human neutralizing monoclonal antibody (mAb) against a soluble human protein, caused thrombocytopenia, platelet activation, reduced mean arterial pressure, and transient loss of consciousness in cynomolgus monkeys after first intravenous administration. In vitro, AMG X induced activation in platelets from macaque species but not from humans or baboons. Other similar mAbs against the same pharmacological target failed to induce these in vivo and in vitro effects. In addition, the target protein was known to not be expressed on platelets, suggesting that platelet activation occurred through an off-target mechanism. AMG X bound directly to cynomolgus platelets and required both the Fab and Fc portion of the mAb for platelet activation. Binding to platelets was inhibited by preincubation of AMG X with its pharmacological target or with anti-human Fc antibodies or by preincubation of platelets with AMG X F(ab')(2) or human immunoglobulin (IVIG). AMG X F(ab')(2) did not activate platelets. Thus, platelet activation required both recognition/binding of a platelet ligand with the Fab domain and interaction of platelet Fc receptors (i.e., FcγRIIa) with the Fc domain. These findings reflect the complexity of the mechanism of action of mAbs and the increasing awareness of potential for unintended effects in preclinical species.

Genome-wide gene expression profiling has become standard for assessing potential liabilities as well as for elucidating mechanisms of toxicity of drug candidates under development. Analysis of microarray data is often challenging due to the lack of a statistical model that is amenable to biological variation in a small number of samples. Here we present a novel non-parametric algorithm that requires minimal assumptions about the data distribution. Our method for determining differential expression consists of two steps: 1) We apply a nominal threshold on fold change and platform p-value to designate whether a gene is differentially expressed in each treated and control sample relative to the averaged control pool, and 2) We compared the number of samples satisfying criteria in step 1 between the treated and control groups to estimate the statistical significance based on a null distribution established by sample permutations. The method captures group effect without being too sensitive to anomalies as it allows tolerance for potential non-responders in the treatment group and outliers in the control group. Performance and results of this method were compared with the Significant Analysis of Microarrays (SAM) method. These two methods were applied to investigate hepatic transcriptional responses of wild-type (PXR(+/+)) and pregnane X receptor-knockout (PXR(-/-)) mice after 96 h exposure to CMP013, an inhibitor of β-secretase (β-site of amyloid precursor protein cleaving enzyme 1 or BACE1). Our results showed that CMP013 led to transcriptional changes in hallmark PXR-regulated genes and induced a cascade of gene expression changes that explained the hepatomegaly observed only in PXR(+/+) animals. Comparison of concordant expression changes between PXR(+/+) and PXR(-/-) mice also suggested a PXR-independent association between CMP013 and perturbations to cellular stress, lipid metabolism, and biliary transport.

8-2 fluorotelomer alcohol is a fluorinated chemical intermediate used to manufacture specialty polymers and surfactants. The potential subchronic toxicity and the reversibility of the effects of this chemical were evaluated following approximately 90 days of oral gavage dosing to Crl:CD(SD)IGS BR rats. A complete toxicological profile, including neurobehavioral assessments and hepatic beta-oxidation, were conducted at selected intervals and a group of rats was included for a 90-day postdosing recovery period. Dose levels tested were 0 (control), 1, 5, 25, and 125 mg/kg. No test-substance-related mortality occurred at any dose level. Rats at 125 mg/kg developed striated teeth, such that these animals were switched to ground chow at 77 days. No treatment-related alterations in body weight, food consumption, neurobehavioral parameters, or hematology/clinical chemistry were found. Hepatic beta-oxidation was increased in males at 125 mg/kg and in females at 25 and 125 mg/kg. In both males and females, plasma fluorine levels were increased at 125 mg/kg and urinary fluorine was elevated at > or =5 mg/kg. Degeneration/disorganization of enamel organ ameloblast cells was observed at 125 mg/kg in males, but not females. Liver weight increases accompanied by focal hepatic necrosis were observed at both 25 and 125 mg/kg, and chronic progressive nephrotoxicity occurred in female rats at 125 mg/kg. With the exception of hepatocellular necrosis in males at 125 mg/kg and the increased incidence and severity of chronic progressive nephropathy in females at 125 mg/kg, all other changes showed evidence of reversibility. The no-observed-adverse-effect level was 5 mg/kg.

Inhalation studies were conducted to determine the potential subchronic toxicity of a mixture of trans-1,2-dichloroethylene (70%), cis-1,2-dichloroethylene (5%), and perfluorobutylethylene (25%). Groups of rats were exposed to 0, 400, 2000, or 8000 ppm concentrations of the mixture vapor 6 h/day, 5 days/wk, for a total of 20 exposures. Subgroups of rats were further observed during a 1-mo recovery period. Functional observational battery (FOB) and motor activity (MA) behavioral tests were conducted prior to initiation of the exposures, during exposure wk 4, and after a 1-mo postexposure recovery period. Clinical pathology evaluations were conducted at the end of the exposure period and after a 1-mo recovery period. At the end of the 4-wk exposure period, tissues from rats were collected, histologically processed, and evaluated by light microscopy. Test substance-related, biologically significant decreased body weights and body weight gains occurred in male and female rats exposed to 8000 ppm. In addition, test substance-related, statistically significant decreases in food consumption and/or food efficiency were observed in male rats exposed to 8000 ppm. During exposures to 8000 ppm, some rats exhibited tremors and ataxia. Usually tremors and ataxia were observed within 1 h after initiation of the daily exposure period and were observed during each exposure day. Tremors were also observed during 1 exposure day in the 2000 ppm animals. In addition to the tremors and ataxia, rats exposed to 2000 ppm or 8000 ppm had a diminished and/or no alerting response to a sharp, sound stimulus during each of the daily exposure periods. These effects were transient since no clinical observations of compromised neurological function were detected when the rats were evaluated upon return to the animal room following exposure. Daily reoccurrence of this apparently acute effect in the 8000 ppm group did not produce enduring neurological changes since there were no test substance-related effects on FOB parameters or on MA conducted the day following the last exposure or during the recovery period. In addition, there were no toxicologically significant changes in hematology, clinical chemistry, or urinalysis parameters in either males or females for any exposure concentration; and there were no test substance-related gross or microscopic morphological changes in males or females administered any exposure concentration. Under the conditions of the study, the no-observed-effect level (NOEL) was 400 ppm in males and females based on clinical signs of toxicity during exposure to 2000 or 8000 ppm.

A semiautomated quantitative assay for rat serum alkaline phosphatase (ALP) isoenzyme determination was developed, incorporating selective precipitation of bone alkaline phosphatase (BALP) with wheat germ lectin and differential inhibition with levamisole for determination of intestinal alkaline phosphatase (IALP). The assays for each isoenzyme were linear over a broad range of activities. The within-run and between-run coefficients of variation were less than 11% for all 3 isoenzymes. Dilution of serum with saline results in an artifactual overestimation of BALP activity. Comparison of ALP and ALP isoenzyme activity in rats of various ages showed that BALP activity drops dramatically with increasing age of rats. IALP activity is greater in immature rats compared to that in mature rats. While there was no difference between male and female rats at 4 wk of age with regard to total ALP activity and activity of any of the isoenzymes, total ALP activity and activity of the individual isoenzymes were higher in males than in females at most ages over 4 wk. Gavage with corn oil resulted in increased serum IALP activity, and bile duct ligation resulted in increased liver alkaline phosphatase activity. This combined assay for the 3 ALP isoenzymes in rat serum is an efficient means of analysis of large numbers of samples and should increase markedly the specificity of serum ALP activity in identifying the target organ in toxicologic studies when serum ALP activity is increased.