Eltrombopag is a first-in-class, orally bioavailable, small-molecule, nonpeptide agonist of the thrombopoietin receptor (TpoR), which is being developed as a treatment for thrombocytopenia of various etiologies. In vitro studies have demonstrated that the activity of eltrombopag is dependent on expression of TpoR, which activates the signaling transducers and activators of transcription (STAT) and mitogen-activated protein kinase signal transduction pathways. The objective of this preclinical study is to determine if eltrombopag interacts selectively with the TpoR to facilitate megakaryocyte differentiation in platelets. Functional thrombopoietic activity was demonstrated by the proliferation and differentiation of primary human CD34+ bone marrow cells into CD41+ megakaryocytes. Measurements in platelets in several species indicated that eltrombopag specifically activates only the human and chimpanzee STAT pathways. The in vivo activity of eltrombopag was demonstrated by an increase of up to 100% in platelet numbers when administered orally (10 mg/kg per day for 5 days) to chimpanzees. In conclusion, eltrombopag interacts selectively with the TpoR without competing with Tpo, leading to the increased proliferation and differentiation of human bone marrow progenitor cells into megakaryocytes and increased platelet production. These results suggest that eltrombopag and Tpo may be able to act additively to increase platelet production.

Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic-pituitary-adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article-related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias.This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies:

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Cephalosporin treatment in man has been associated with a low incidence of hemolytic anemia, thrombocytopenia, and neutropenia; some cases have been shown to be immune-mediated. This triad of blood dyscrasias was also demonstrated in our laboratory in a series of toxicity studies in dogs of two cephalosporin compounds, cefonicid and cefazedone; these studies provided evidence for drug-associated immune hemolytic anemia, based on conventional laboratory tests. To further investigate possible immune mechanisms of the cephalosporin-induced cytopenias, we measured erythrocyte-associated, platelet-associated (PAIgG), and serum antineutrophil IgG over the course of cephalosporin treatment, using highly sensitive 125I-staphylococcal protein A (SPA) assays, as well as the direct antiglobulin test; we compared these findings with the hematologic changes. Intravenous treatment with high doses of cefazedone (540 mg/kg/day, increased to a maximum of 840 mg/kg/day for 4 months or until hematologic effects were evident) resulted in a high incidence of anemia (7/14), thrombocytopenia (11/14), and neutropenia (7/14). Of the affected dogs examined, 6/7 with anemia, 9/9 with thrombocytopenia, and 7/7 with neutropenia showed increased levels of the respective cell-associated antibody, compared with untreated controls. Unaffected dosed animals generally did not show these changes. In 3/3 dogs examined following remission of thrombocytopenia, PAIgG returned to levels comparable with controls; as one of these dogs suffered a relapse, increased PAIgG was again observed. Animals sacrificed during cytopenic episodes showed cytologic and histologic evidence of increased hemophagocytosis. We conclude that antibody-mediated blood cell destruction contributes to all three cephalosporin-induced cytopenias in the dog.

An inappropriate blood-to-anticoagulant ratio can cause an artifactual prolongation of the activated partial thromboplastin time (APTT) and prothrombin time (PT). In a drug safety study in dogs, w e observed a 4-to 5-second increase in the APTT from baseline coincident with increased hematocrit values (56% to 65%) secondary to drug-induced vomiting and diarrhea. The PT and platelet counts were unchanged, and there was no clinical evidence of bleeding associated with venipuncture. Although we were unable to sample the same dogs to investigate the possible effect of hemoconcentration on the prolonged APTT, the question was addressed by an in vitro study. The hematocrit value for citrated blood samples collected from t is well documented in human patients that a marked I increase in the hematocrit can artifactually prolong the APTT and PT because of an inappropriate blood-to-anticoagulant ratio.' When the hematocrit value is above 55%, there is a relative decrease in plasma volume. As a result, when the blood is centrifuged, the plasma fraction will contain an increased concentration of sodium citrate, causing excessive anticoagulation. The ideal concentration in the standard blood/anticoagulant (9: 1) mixture must be maintained between 0.109 mol/L (3.2%) and 0.129 mol/L (3.8%) of sodium citrate for accurate interpretation of the APTT and PT results.2In a drug toxicity study conducted in beagle dogs, we observed a 4-to 5-second prolongation for the APTT in dogs with high hematocrits (ie, 57% to 65%) ( Table I). The hemoconcentration was attributed to dehydration associated with drug-induced vomiting and diarrhea. The observation prompted an in vitro assessment of the effects of increased hematocrit on the APTT values in dogs.A 4.5-mL whole blood specimen anticoagulated with 0.5 mL of 0.129 mol/L (3.8%) buffered citrate solution (9: 1) was obtained from 3 healthy beagle dogs. A spun hematocrit (Drummond Microhematocrit, Broomall, PA) was determined for each specimen. The blood tubes were centrifuged at 2200 rpm for 15 minutes at 4°C and aliquots of plasma and red blood cell layer were retained to prepare blood specimens with high hematocnts.The 3 dogs were bled again as described above. Appropriately measured aliquots of this whole blood were dispensed into each of two 2.7 mL tubes each containing 0.3 mL of 0.129 mol/L buffered citrate. One tube was filled completely with whole blood, the other tube was filled with whole blood and an aliquot of hemoconcentrated citrated blood. The proper fill level was maintained in all tubes. A spun microhematocrit was determined on all tubes before centrifugation at 2200 rpm for 15 minutes at 4°C to harvest test plasma for determination of the PT and APTT by a photometric clot detection method using the Electra 700 (Medical Laboratory Automation Inc, Mt. Vernon, NY). Dade Actin Activated Cephaloplastin Reagent (liquid rabbit brain) and Dade Calcium Chloride, 0.02 mol/L (Baxter Diagnostics, Deerfield, IL) were used for APTT determinations and Dade Thromboplastin C (dried rabbit br...