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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).)

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.

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.