Improved nonclinical safety profile of a novel, highly selective inhibitor of the immunoproteasome subunit LMP7 (M3258).

M3258 is the first selective inhibitor of the immunoproteasome subunit LMP7 (Large multifunctional protease 7) in early clinical development with the potential to improve therapeutic utility in patients of multiple myeloma (MM) or other hematological malignancies. Safety pharmacology studies with M3258 did not reveal any functional impairments of the cardiovascular system in several in vitro tests employing human cardiomyocytes and cardiac ion channels (including hERG), guinea pig heart refractory period and force contraction, and rat aortic contraction as well as in cardiovascular function tests in dogs. Following single dose M3258 administration to rats, no changes were observed on respiratory function by using whole body plethysmography, nor did it change (neuro)behavioral parameters in a battery of tests. Based on pivotal 4-week toxicity studies with daily oral dosing of M3258, the identified key target organs of toxicity were limited to the lympho-hematopoietic system in rats and dogs, and to the intestine with its local lymphoid tissues in dogs only. Importantly, the stomach, nervous system, heart, lungs, and kidneys, that may be part of clinically relevant toxicities as reported for pan-proteasome inhibitors, were spared with M3258. Therefore, it is anticipated that by targeting highly selective and potent inhibition of LMP7, the resulting favorable safety profile of M3258 together with the maintained potent anti-tumor activity as previously reported in mouse MM xenograft models, may translate into an improved benefit-risk profile in MM patients.

Comparison of the Sysmex XT-2000iV with microscopic differential counts of canine bone marrow.

Canine bone marrow is frequently assessed in the advanced preclinical research environment. Automated analysis provides time savings and objectivity over the gold standard of microscopic (cytologic) evaluation. We compared the analysis of 90 canine bone marrow samples by the Sysmex XT-2000iV hematology analyzer (Sysmex Corp., Kobe, Japan) with cytologic evaluation. Gates for cell populations were created in the system's WBC/BASO channel. Variables "total nucleated red blood cells" (total_NRBC), "poly- and orthochromatic nucleated red blood cells" (poly_orth_NRBC), "total neutrophils" (total_NEUT), "mature neutrophils" (mature_NEUT), and myeloid-to-erythroid (M:E) ratio were compared with cytologic evaluation. Intra-assay repeatability and total error (TE) were calculated for both methods. Intra-assay repeatability was 0.95-2.48% for the XT-2000iV and 8.32-23.23% for cytology. Observed TE for the automated measurement was 5.16-46.8% and for cytology 22.70-76.74%. Spearman rank correlation was excellent for M:E ratio (0.91) and fair for the other populations (0.65-0.71). Absolute bias for M:E ratio was low (-0.114). A negative absolute bias of -7.71% for the XT-2000iV was found for poly_orth_NRBC, whereas the bias was positive for total_NEUT (7.10%) and mature_NEUT (14.67%). M:E ratio of canine bone marrow samples can be precisely determined using the Sysmex XT-2000iV WBC/BASO channel. Total_NRBC, poly_orth_NRBC, total_NEUT, and mature_NEUT can be estimated rapidly. With distinctly lower coefficient of variation and observed TE compared with cytology, automated measurement provides advantages in terms of standardization, and it is suited to the advanced preclinical research environment where large numbers of samples are investigated.

Optimized gating and reference ranges of reticulated platelets in dogs for the Sysmex XT-2000iV.

BACKGROUND: Canine reticulated platelets (r-PLTs) i.e., juvenile PLTs reflecting thrombopoiesis can be measured automatically with the hematology analyzer Sysmex XT-2000iV using manual gating options. However, the impact of interferences on r-PLT measurements performed with the gates published previously (Pankraz et al., Vet Clin Path 38:30-38, 2009; Gelain et al., High fluorescent platelets fraction in macrothrombocytopenic Norfolk terrier, 2010) is largely unknown. The aim was to compare different published gates for measurement of r-PLTs with the Sysmex XT-2000iV with an own, optimized gate ("Oellers-gate") and to establish reference intervals (RIs) in > 120 dogs. Data of 362 measurements of diseased and healthy dogs were analyzed retrospectively. Several gates were applied and RIs for r-PLTs and platelet indices were established for pet dogs and a group of 153 healthy Beagles kept under defined housing conditions. Intra-assay precision (CV) was also assessed. RESULTS: In 30/362 samples, interferences consistent with small erythrocytes/reticulocytes were seen in the previously published gates but not in the "Oellers-gate". Good correlation was found between the different gates (rs: 0.88-1.00). RIs for the "Pankraz-gate", the "Gelain-gate", and the "Oellers-gate" were 0.0-1.2, 0.2-3.7 and 0.2-3.9 % respectively. CVs were ranging between 22 and 41 %. CONCLUSIONS: Optimization of previously published gates minimized interferences of small erythrocytes with r-PLT measurements.

Differences in hematologic variables in rats of the same strain but different origin.

BACKGROUND: Wistar rats are frequently used in toxicologic studies; however, the impact of animal source on hematologic reference intervals (RI) is not known. OBJECTIVE: The purpose of this study was to compare hematologic data of Wistar rats obtained from 2 breeders and to calculate preliminary RI for the Sysmex XT-2000iV hematology analyzer. METHODS: Blood collected in EDTA from 75 10-13-week-old rats (male, n = 38; female, n = 37) from breeder 1 and 60 rats (male, n = 30; female, n = 30) from breeder 2 was analyzed using a Sysmex XT-2000iV hematology analyzer. Results were analyzed for differences using a t-test or Mann-Whitney U-test. Sex-specific 95% RI were calculated for rats from both breeders. RESULTS: Significant breeder-specific differences were found for WBC, RBC, platelet, lymphocyte, monocyte, eosinophil, and reticulocyte counts and MCV, MCH, MPV, and plateletcrit for both sexes, and for HCT and hemoglobin concentration for female rats. The greatest differences (P < .0001) between rats from breeders 1 and 2, respectively, were found for WBC (female: 2.2-5.9 × 10(9) /L vs 4.2-9.4 × 10(9) /L 38; male: 3.4-9.5 vs 6.4-14.4 × 10(9) /L), lymphocyte (female: 1.7-4.8 × 10(9) /L vs 3.4-8.2 × 10(9) /L; male: 2.6-7.8 vs 5.2-12.4 × 10(9) /L), and platelet (female: 591-836 × 10(9) /L vs 804-1282 × 10(9) /L; male: 573-998 × 10(9) /L vs 861-1247 × 10(9) /L) counts. CONCLUSION: Differences in hematologic RI between Wistar rats obtained from different breeders underscore the need to evaluate untreated control animals for comparison, rather than using historical RI, to detect compound-related effects in preclinical safety studies. Valid RI for control animals are needed and should be verified when using a new supplier to avoid misinterpretation of data. Low reference limits for some variables, such as WBC counts, in rats from one source may preclude their use in studies in which detection of leukopenia is required.

Phenotyping of congenic dipeptidyl peptidase 4 (DP4) deficient Dark Agouti (DA) rats suggests involvement of DP4 in neuro-, endocrine, and immune functions.

BACKGROUND: Treatment of diabetes type 2 using chronic pharmacological inhibition of dipeptidyl peptidase 4 (DP4) still requires an in-depth analysis of models for chronic DP4 deficiency, because adverse reactions induced by some DP4 inhibitors have been described. METHODS: In the present study, a novel congenic rat model of DP4 deficiency on a "DP4-high" DA rat genetic background was generated (DA.F344-Dpp4(m)/ SvH rats) and comprehensively phenotyped. RESULTS: Similar to chronic pharmacological inhibition of DP4, DP4 deficient rats exhibited a phenotype involving reduced diet-induced body weight gain and improved glucose tolerance associated with increased levels of glucagon-like peptide-1 (GLP-1) and bound leptin as well as decreased aminotransferases and triglycerides. Additionally, DA.F344-Dpp4(m)/SvH rats showed anxiolytic-like and reduced stress-like responses, a phenomenon presently not targeted by DP4 inhibitors. However, several immune alterations, such as differential leukocyte subset composition at baseline, blunted natural killer cell and T-cell functions, and altered cytokine levels were observed. CONCLUSIONS: While this animal model confirms a critical role of DP4 in GLP-1-dependent glucose regulation, genetically induced chronic DP4 deficiency apparently also affects stress-regulatory and immuneregulatory systems, indicating that the use of chronic DP4 inhibitors might have the potential to interfere with central nervous system and immune functions in vivo.

Humane endpoints in the efficacy testing of swine erysipelas vaccines.

For licensing the efficacy of vaccines for veterinary use has to be demonstrated by well-controlled laboratory experiments in which vaccinated and untreated animals of the target species are challenged. Erysipelas challenge tests cause extreme suffering of the unprotected animals with high fever, apathy, large skin lesions, and even death. This paper describes a standardised procedure for the vaccination challenge test and gives due consideration to the welfare of the animals. By monitoring and using clinical signs observed during the test it is possible to minimise animal pain and distress, thus preventing unnecessary animal suffering.