Validation of Sysmex XT-2000iV analyzer-generated quantitative bone marrow differential counts in cynomolgus monkeys, Beagle dogs, and CD-1 mice.

BACKGROUND: In a previous study, the validation of rat bone marrow (BM) collection, processing, and analysis using the Sysmex XT-2000iV (Sysmex Corporation, Kobe, Japan) hematology analyzer showed that the Sysmex hematology analyzer produced BM differential counts that were comparable to those obtained with microscopic differential counts. OBJECTIVE: This study was conducted to expand the validation of the Sysmex TNCC (total nucleated cell count) and 5-part BM differential in cynomolgus monkeys, Beagle dogs, and CD-1 mice, which are alternate species that are also frequently used in preclinical safety studies. METHODS: The Sysmex 5-part BM differential counts were generated with a two-step process, whereby proliferating and maturing erythroid and myeloid cells were determined by preset gating and lymphocytes were determined using species-specific B- and T-lymphocyte antibodies and a magnetic cell-sorting method (MACS). Agreement with microscopic myelograms with 500-cell differential counts was determined from BM suspensions of 62 cynomolgus monkeys, 47 Beagle dogs, and 44 CD-1 mice. RESULTS: The correlation coefficients between methods for myeloid to erythroid (M:E) ratios in all three species was > 0.928. The Bland-Altman differences between methods were approximately ± 0.3 units for the M:E ratio in dogs and mice, and +0.6 and -0.4 in monkeys. The upper limits of agreement for all three species were ≤7% for maturing myeloid cells, ≤6% for maturing erythroid cells, and ≤4% for proliferating myeloid cells, proliferating erythroid cells, and lymphocytes. CONCLUSIONS: The Sysmex XT-2000iV produces an automated M:E ratio and a 5-part differential count equivalent to microscopic differential counts in cynomolgus monkeys, Beagle dogs, and CD-1 mice.

From the Cover: Fenretinide, Troglitazone, and Elmiron Add to Weight of Evidence Support for Hemangiosarcoma Mode-of-Action From Studies in Mice.

Pharmaceuticals and chemicals produce hemangiosarcomas (HS) in mice, often by nongenotoxic, proliferative mechanisms. A mode-of-action (MOA) for hemangiosarcoma was proposed based on information presented at an international workshop (Cohen et al., Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18.). Five key elements of the MOA were articulated and included hypoxia, macrophage activation, increased angiogenic growth factors, dysregulated angiogenesis/erythropoiesis, and endothial cell proliferation. The goal of the current study was to add to the weight-of-evidence for the proposed MOA by assessing these key elements with 3 different compounds of varying potency for HS induction: fenretinide (high), troglitazone (intermediate), and elmiron (low). Multiple endpoints, including hypoxia (hyproxyprobe, transcriptomics), endothelial cell (EC) proliferation, and clinical and anatomic pathology, were assessed after 2, 4, and 13-weeks of treatment in B6C3F1 mice. All 3 compounds demonstrated strong evidence for dysregulated erythropoiesis (decrease in RBC and a failure to increase reticulocytes) and macrophage activation (4- to 11-fold increases); this pattern of hematological changes in mice might serve as an early biomarker to evaluate EC proliferation in suspected target organs for potential HS formation. Fenretinide demonstrated all 5 key elements, while troglitazone demonstrated 4 and elmiron demonstrated 3. Transcriptomics provided support for the 5 elements of the MOA, but was not any more sensitive than hypoxyprobe immunohistochemistry for detecting hypoxia. The overall transcriptional evidence for the key elements of the proposed MOA was also consistent with the potency of HS induction. These data, coupled with the previous work with 2-butoxyethanol and pregablin, increase the weight-of-evidence for the proposed MOA for HS formation.

Comparison of the Sysmex XT-2000iV and microscopic bone marrow differential counts in Wistar rats treated with cyclophosphamide, erythropoietin, or serial phlebotomy.

BACKGROUND: In a previous study, it was demonstrated that bone marrow analysis using the Sysmex XT-2000iV hematology analyzer produced differential counts in untreated rats that were comparable to microscopic differential counts. OBJECTIVE: The aim of this study was to modulate hematopoiesis in rats in vivo either through pharmacologic treatment or serial phlebotomy, and to determine whether the Sysmex XT-2000iV could accurately analyze bone marrow quantitative changes when compared with results obtained by microscopy. METHODS: Rats were treated once with 0, 5, 20, and 40 mg/kg cyclophosphamide (CP), 0, 50, 100 IU/kg erythropoietin (EPO) on 4 consecutive days, or serial phlebotomy of 1-2 mL of blood for 4 days. Modulation of hematopoietic populations in bone marrow was evaluated using the Sysmex XT-2000iV hematology analyzer, and compared with microscopic differential counts. RESULTS: Correlation coefficients between M:E ratios determined by Sysmex and the microscopic method were 0.94, 0.96, and 0.98 for CP, EPO, or serial phlebotomy treatments, respectively. Mean concordance correlation coefficients for M:E demonstrated method agreement of 0.63, 0.92, and 0.85 for the 3 treatments. Quantitative automated and microscopic bone marrow differential counts were within the expected 95% confidence intervals for CP, EPO or serial phlebotomy. CONCLUSIONS: The Sysmex XT-2000iV provides quantitative bone marrow differential counts of bone marrow cell series in rats with treatment-induced changes which are comparable to microscopic differential counts. Reliable automatic bone marrow differential counting allows increased throughput, sensitivity, reproducibility, and enhanced interpretation of bone marrow evaluation in rodent preclinical studies.

Validation of Sysmex XT-2000iV generated quantitative bone marrow differential counts in untreated Wistar rats.

BACKGROUND: Preclinical drug trials frequently require assessment of bone marrow toxicity in animals to evaluate hematopoietic safety. Since the gold standard, cytologic evaluation, is time consuming and requires highly trained individuals, automated methods remain intriguing. OBJECTIVE: The Sysmex XT-2000iV hematology analyzer allows user-developed customizable gating. This study was conducted to validate the gating of bone marrow cell populations in Sysmex cytograms from untreated rats. METHODS: B- and T-lymphocytes and myeloid cells were experimentally depleted from Charles River Wistar Han IGS (CRL: WI [Han]) rat whole bone marrow suspension using a magnetic cell sorting (MACS) method. The positively and negatively selected populations were used to verify select gates within the Sysmex cytogram. Intra- and inter-animal precision, comparability between right and left femur, as well as agreement with microscopic myelograms based on 500 counted cells, were determined. RESULTS: Intra-sample precision and right-to-left femur comparability confirmed that gating was reproducible and stable. In 50 tested rats, myeloid to erythroid ratios (M:E) were 1.32 ± 0.33 in males and 1.38 ± 0.29 in females by Sysmex compared to 1.36 ± 0.32 in males and 1.42 ± 0.32 in females by microscopic evaluations. Bland-Altman differences between methods was ≤ ± 0.35 units for M:E, ≤ 5.4% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, ≤ 6.0% for maturing myeloid cells, ≤ 3.4% for proliferating myeloid cells, and ≤ 4.1% for lymphocytes. CONCLUSIONS: In untreated control Charles River Wistar Han IGS (CRL: WI [Han]) rats, the Sysmex XT-2000iV produced an automated M:E and 5-part differential count equivalent to microscopic differential counts.

Pregabalin induces hepatic hypoxia and increases endothelial cell proliferation in mice, a process inhibited by dietary vitamin E supplementation.

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Hemangiosarcoma in mice administered pregabalin: analysis of genotoxicity, tumor incidence, and tumor genetics.

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.

Mode of action associated with development of hemangiosarcoma in mice given pregabalin and assessment of human relevance.

Pregabalin increased the incidence of hemangiosarcomas in carcinogenicity studies of 2-year mice but was not tumorigenic in rats. Serum bicarbonate increased within 24 h of pregabalin administration in mice and rats. Rats compensated appropriately, but mice developed metabolic alkalosis and increased blood pH. Local tissue hypoxia and increased endothelial cell proliferation were also confirmed in mice alone. The combination of hypoxia and sustained increases in endothelial cell proliferation, angiogenic growth factors, dysregulated erythropoiesis, and macrophage activation is proposed as the key event in the mode of action (MOA) for hemangiosarcoma formation. Hemangiosarcomas occur spontaneously in untreated control mice but occur only rarely in humans. The International Programme on Chemical Safety and International Life Sciences Institute developed a Human Relevance Framework (HRF) analysis whereby presence or absence of key events can be used to assess human relevance. The HRF combines the MOA with an assessment of biologic plausibility in humans to assess human relevance. This manuscript compares the proposed MOA with Hill criteria, a component of the HRF, for strength, consistency, specificity, temporality, and dose response, with an assessment of key biomarkers in humans, species differences in response to disease conditions, and spontaneous incidence of hemangiosarcoma to evaluate human relevance. Lack of key biomarker events in the MOA in rats, monkeys, and humans supports a species-specific process and demonstrates that the tumor findings in mice are not relevant to humans at the clinical dose of pregabalin. Based on this collective dataset, clinical use of pregabalin would not pose an increased risk for hemangiosarcoma to humans.

Key components of the mode of action for hemangiosarcoma induction in pregabalin-treated mice: evidence of increased bicarbonate, dysregulated erythropoiesis, macrophage activation, and increased angiogenic growth factors in mice but not in rats.

In carcinogenicity studies, pregabalin increased hemangiosarcoma incidence in mice but not in rats. Investigative studies, ranging in length from 24 h to 12 months, were conducted in mice (1000 or 5000 mg/kg) and rats (900 mg/kg) to evaluate a potential mode-of-action scheme for tumor formation. Three areas were evaluated: (1) hematopoiesis (because endothelial and hematopoietic cells arise from the same precursor and hemangiosarcomas are primarily located in mouse hematopoietic tissues), (2) angiogenic growth factors (because increased angiogenic growth factors may stimulate vascular tumors), and (3) pulmonary/blood gas parameters (because hypoxia is a known driver for endothelial cell proliferation). In mice, pregabalin rapidly increased platelet and megakaryocyte counts, activated platelets and bone marrow erythrophages, decreased the myeloid-to-erythroid (M:E) ratio (49%), and produced bone marrow and splenic congestion and extramedullary hematopoiesis (EMH). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor immunohistochemical staining were also increased in mouse bone marrow and spleen and vascular endothelial growth factor receptor 2 immunolabeling was increased in liver. Serum bicarbonate was increased within 24 h of pregabalin administration, persisted over time, and was accompanied by decreased respiratory rate (up to 34%) and increased partial pressure of carbon dioxide (pCO(2)), resulting in sustained metabolic alkalosis and elevated blood pH in mice. In contrast, in rats, pregabalin decreased overall bone marrow cellularity, including decreased number of megakaryocytes (24%) with no evidence of erythrophages, no change in M:E ratio, no EMH, and no increase in angiogenic growth factors or blood pH. Persistent alterations in serum bicarbonate, respiratory function, and blood gas parameters in mice, without adequate compensatory mechanisms, has the potential to create chronic tissue hypoxia, an accepted driver of endothelial cell proliferation.

Hemangiosarcoma in rodents: mode-of-action evaluation and human relevance.

Although rarely occurring in humans, hemangiosarcomas (HS) have become important in evaluating the potential human risk of several chemicals, including industrial, agricultural, and pharmaceutical agents. Spontaneous HS arise frequently in mice, less commonly in rats, and frequently in numerous breeds of dogs. This review explores knowledge gaps and uncertainties related to the mode of action (MOA) for the induction of HS in rodents, and evaluates the potential relevance for human risk. For genotoxic chemicals (vinyl chloride and thorotrast), significant information is available concerning the MOA. In contrast, numerous chemicals produce HS in rodents by nongenotoxic, proliferative mechanisms. An overall framework is presented, including direct and indirect actions on endothelial cells, paracrine effects in local tissues, activation of bone marrow endothelial precursor cells, and tissue hypoxia. Numerous obstacles are identified in investigations into the MOA for mouse HS and the relevance of the mouse tumors to humans, including lack of identifiable precursor lesions, usually late occurrence of the tumors, and complexities of endothelial biology. This review proposes a working MOA for HS induced by nongenotoxic compounds that can guide future research in this area. Importantly, a common MOA appears to exist for the nongenotoxic induction of HS, where there appears to be a convergence of multiple initiating events (e.g., hemolysis, decreased respiration, adipocyte growth) leading to either dysregulated angiogenesis and/or erythropoiesis that results from hypoxia and macrophage activation. These later events lead to the release of angiogenic growth factors and cytokines that stimulate endothelial cell proliferation, which, if sustained, provide the milieu that can lead to HS formation.

Toxicity and toxicokinetics of the cyclin-dependent kinase inhibitor AG-024322 in cynomolgus monkeys following intravenous infusion.

PURPOSE: Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects. METHODS: Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m(2), respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred at > or =3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose. RESULTS: No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen at > or =6 mg/kg. AG-024322 at > or =6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes at > or =6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent. CONCLUSION: The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0-24.5) of 2.11 microg h/mL.

Cutaneous lesions in the rat following administration of an irreversible inhibitor of erbB receptors, including the epidermal growth factor receptor.

CI-1033 (canertinib) is an irreversible inhibitor of the erbB family of transmembrane tyrosine kinase receptors, including the epidermal growth factor (EGF) receptor. Various inhibitors of the EGF receptor, including CI-1033, have resulted in cutaneous toxicity in humans as a common adverse event. In a chronic toxicity study in rats, CI-1033 produced cutaneous lesions with morphologic characteristics similar to that reported in man. Here the authors describe in detail the dermal changes observed, along with other noteworthy findings of that study. Male and female Wistar rats (15/sex/group) were administered CI-1033 for 27 weeks at 2.5, 5, or 10 mg/kg (15, 30, or 60 mg/m(2), respectively) by gavage. Control animals (15/sex) received vehicle alone (aqueous 0.5% methylcellulose) in a dose volume of 5 mL/kg. Six animals/sex/dose were included for toxicokinetic evaluations. Skin lesions were the primary drug-related toxicity and occurred at > or = 2.5 mg/kg in a dose-dependent fashion. The major gross lesions were papules that evolved into crusts and scales that were first observed in weeks 1 and 3, respectively. Alopecia developed in conjunction with the papular eruptions. Skin changes were most pronounced in females, possibly due to higher drug levels. In week 13, CI-1033 plasma AUC(0-24) values were 527 to 1980 ng.h/mL in males and 844 to 2920 ng x h/mL in females at 2.5 to 10 mg/kg. Microscopic changes could be described as 3 patterns that affected the tail and body (haired skin). Pattern 1 consisted of epidermal changes that started as a superficial, perivascular spongiotic dermatitis with evolving epidermal hyperplasia, scale-crusts, and areas of ulceration. Areas of hyperplasia on the tail were often associated with the development of new hair follicles. Pattern 2 was characterized by a suppurative to pyogranulomatous infundibular folliculitis. Pattern 3 consisted of abnormally oriented hair follicles with malformed hair shafts that were associated with a deeper (isthmic) folliculitis; this correlated with alopecia. Elevations in bone marrow myeloid counts correlated with a peripheral leukocytosis, consistent with inflammatory changes in the dermis. In addition, hepatic cholestasis and epithelial atrophy in the gastrointestinal tract and vagina occurred at > or = 2.5 mg/kg. In conclusion, CI-1033 produced cutaneous lesions involving the epidermis and hair follicle, and the morphologic characteristics were similar to that reported in clinical studies with various inhibitors of the EGF receptor. These changes are consistent with pharmacologic inhibition of the EGF receptor in these tissues and demonstrate that the rat can serve as an animal model for investigating the mechanisms for this toxicity.

Roles of exercise and pharmacokinetics in cerivastatin-induced skeletal muscle toxicity.

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are associated with adverse skeletal muscle effects, but the underlying mechanisms remain unclear. To determine whether toxicity involves the level of drug exposure in muscle tissue and to test the effect of exercise on cerivastatin (CVA)-induced skeletal muscle damage, female rats were administered vehicle or CVA at 0.1, 0.5, and 1.0 mg/kg/day by gavage for two weeks and exercised or not on treadmills for 20 min/day. Clinical chemistry and plasma and tissue pharmacokinetics were evaluated; light and transmission electron microscopy (TEM) of Type I and Type II fiber-predominant skeletal muscles were performed. Serum levels of AST, ALT, CK, and plasma lactic acid were significantly elevated dose-dependently. CVA treatment decreased psoas and quadriceps weights. At 1 mg/kg all muscles except soleus demonstrated degeneration. Exercise-exacerbated severity of CVA-induced degeneration was evident in all muscles sampled except soleus and quadriceps. Early mitochondrial involvement in toxicity is suggested by the numerous membranous whorls and degenerate mitochondria observed in muscles at 0.5 mg/kg. No significant differences in CVA concentrations between either EDL and soleus or plasma and muscle were found. We found that CVA had no effect on cleaved caspase 3. In summary, we found that treadmill exercise exacerbated the incidence and severity of CVA-induced damage in Type II fiber-predominant muscles. Tissue exposure is likely not the key factor mediating CVA-induced skeletal muscle toxicity.

Sustained inhibition of rat myometrial gap junctions and contractions by lindane.

BACKGROUND: Gap junctions increase in size and abundance coincident with parturition, forming an intercellular communication network that permits the uterus to develop the forceful, coordinated contractions necessary for delivery of the fetus. Lindane, a pesticide used in the human and veterinary treatment of scabies and lice as well as in agricultural applications, inhibits uterine contractions in vitro, inhibits myometrial gap junctions, and has been associated with prolonged gestation length in rats. The aim of the present study was to investigate whether brief exposures to lindane would elicit sustained inhibition of rat uterine contractile activity and myometrial gap junction intercellular communication. METHODS: To examine effects on uterine contraction, longitudinal uterine strips isolated from late gestation (day 20) rats were exposed to lindane in muscle baths and monitored for changes in spontaneous phasic contractions during and after exposure to lindane. Lucifer yellow dye transfer between myometrial cells in culture was used to monitor gap junction intercellular communication. RESULTS: During a 1-h exposure, 10 micro M and 100 micro M lindane decreased peak force and frequency of uterine contraction but 1 micro M lindane did not. After removal of the exposure buffer, contraction force remained significantly depressed in uterine strips exposed to 100 micro M lindane, returning to less than 50% basal levels 5 h after cessation of lindane exposure. In cultured myometrial myocytes, significant sustained inhibition of Lucifer yellow dye transfer was observed 24 h after lindane exposures as brief as 10 min and as low as 0.1 micro M lindane. CONCLUSION: Brief in vitro exposures to lindane have long-term effects on myometrial functions that are necessary for parturition, inhibiting spontaneous phasic contractions in late gestation rat uterus and gap junction intercellular communication in myometrial cell cultures.