Differentiating effect of pamidronate on canine osteo‐sarcoma cell lines in vitro

Introduction: Pamidronate has been traditionally used to manage pathologic osteoclastic disorders. In addition to its effects on osteoclasts, pamidronate has also been demonstrated to promote phenotypic maturation and inhibition of proliferation in osteoblasts. Canine osteosarcoma (OSA) consists of malignant, undifferentiated osteoblasts. The objective of this study was to determine if micromolar concentrations of pamidronate could induce malignant osteoblastic differentiation as evaluated by an increase in alkaline phosphatase (ALP) activity and/or osteocalcin (OC) production, two specific markers of normal osteoblastic activity. Methods: Two canine OSA cell lines (HMPOS and COS 31) were used for all experiments. Cells were incubated for 48 or 72 hours with various pamidronate concentrations (0, 0.1, 1, 5, 10, and 20 μM). After incubation, the supernatants were sampled and the relative amounts of viable cells were determined with a cell proliferation assay (Cell Titer 96^® AQ_uous, Promega). An ALP detection kit (Starbright^®, Sigma^®) was used to measure the ALP activity and an ELISA (Osteocalcin EIA kit, Biomedical Technologies) was used to determine the concentration of osteocalcin in the supernatants. The ALP and osteocalcin values were corrected for the amount of viable cells. Results: Pamidronate induced a dose‐dependent reduction in the number of viable COS 31 and HMPOS cells at both 48 and 72 hours. A dose‐dependent elevation in ALP activity from baseline was observed. At 20 μM, a 2.3‐fold increase was observed for HMPOS at 72 hours, while a 1.43‐fold increase was observed for COS 31 at 72 hours. Very low level (less than 2 ng/ml) of osteocalcin pre‐ and post‐pamidronate treatment was detected for both COS 31 and HMPOS. Conclusion: The data suggests that pamidronate increases alkaline phosphatase activity in canine OSA cells in a dose‐dependent manner. However, cytotoxic assays are needed in order to accurately characterize any concurrent decrease in the number of viable cells. The potential differentiating effect of pamidronate on malignant osteoblasts provides an additional argument for its use in the palliative treatment of OSA.     

Investigating the in vitro interactive effects of ionizing radiation and pamidronate in a canine osteosarcoma cell line

Introduction: Dogs with osteosarcoma suffer intense bone pain. Conventional palliative treatment options include ionizing radiation with or without systemic chemotherapy. Intravenous pamidronate is a first‐line therapeutic agent in people with painful skeletal metastases and is currently being evaluated for the management of osteolytic pain associated with canine osteosarcoma. The theoretical combination of radiation and pamidronate for managing bone pain appears to be a rational treatment option, however, in vitro experiments demonstrating additive or supraadditve cytotoxic effects of these two modalities would further support the clinical institution of this novel combination therapy. The purpose of this study was to evaluate the in vitro cytotoxicity of ionizing radiation and pamidronate in a canine OSA cell line and to characterize the interaction between these two therapeutic modalities when used in combination. Methods: The canine osteosarcoma cell line, HMPOS, was subjected independently to varying doses of ionizing radiation (100 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy, or 1000 cGy) or different concentrations of pamidronate (10, 25, 50, 66, 75, 100, 150, 200, and 250 μM). Cells were harvested following 48 hours of incubation and cell viability was assessed by flow cytometric analysis. Dose‐response curves were generated, and a theoretical ED₅₀ for each treatment modality calculated. An ED₅₀ isobologram was created to determine if different combinations of ionizing radiation and pamidronate (isoboles) would demonstrate subadditive, additive, or supraadditve cytotoxic effects. Results: The ED₅₀ of ionizing radiation and pamidronate were 614 cGy and 65.3 μM, respectively. Of the six different isobole combinations evaluated, three produced supraadditve effects, one produced a subadditive effect, and two produced additive effects. Conclusions: Both ionizing radiation and pamidronate independently exert in vitro cytotoxic effects. Positive interactive effects can be generated, but only with specific isobole combinations. The results from this in vitro study support the use of specific combinations of ionizing radiation and pamidronate in order to maximize the cytotoxic properties of both treatment modalities.     

The effects of cyclooxygenase‐2 inhibition on apoptosis and cell cycle distribution using two in vitro models of nasal squamous cell carcinoma

Objective: Despite numerous attempts using a variety of therapeutic modalities, response rates and survival times for canine nasal squamous cell carcinoma remain poor. The goals of this study are to determine if a COX‐2 selective inhibitor induces apoptosis and alters cell cycle distribution in two in vitro models of nasal squamous cell carcinoma, and to establish a basis for future clinical trials using COX‐2 inhibitors as radiosensitizing agents. Methods: The nasal squamous cell carcinoma cell lines FAT‐7 (rat) and RPMI 2650 (human) were purchased from ATCC (Manassas, VA). Cell pellets were stained for COX‐2 expression using standard immunohistochemical techniques. Following the determination of growth kinetics for each cell line, cells were plated in triplicate using 25 cm² tissue culture flasks and incubated with different concentrations of a COX‐2 selective inhibitor (0, 1, 10, 50 and 100 μM) for 72 hours. Cells were stained with Annexin‐V and propidium iodide (Oncogene Research Products) and analyzed with flow cytometry to assess cell viability. Cell cycle distribution was determined using a propidium iodide methodology and flow cytometric analysis. Results: Preliminary results show that the addition of a COX‐2 selective inhibitor caused a dose‐dependent cytotoxicity on the FAT‐7 cells. Viability at 72 hours ranged from 95.4% for control samples to 7.46% for cells incubated at 100 μM. Changes in cell cycle distribution were also detected. Conclusions: Future study is warranted to determine if the addition of a COX‐2 selective inhibitor will increase response rates and overall survival in a population of spontaneously arising canine nasal squamous cell carcinomas.     

Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei ., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.
Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.
Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC₅₀ for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.
Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro . This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.     

In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2‐carboxyethyl)phosphine (Pt‐TCEP), has been evaluated in canine (D‐17) and human osteosarcoma (U2‐OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 μM) of Pt‐TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer‐term effect of Pt‐TCEP was evaluated in the colony‐forming unit assay after 24 hours exposure to the Pt‐TCEP (2 and 3 μM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt‐TCEP (3 μM). Its pro‐apoptotic activity was examined after 24 hours treatment with Pt‐TCEP (1.25, 2.50, 5, 10 and 20 μM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 μM Pt‐TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC₅₀ of Pt‐TCEP was 5.93 ± 0.12 in D‐17 and 3.45 ± 0.14 in U2‐OS cell lines after 24 hours, and 1.77 ± 0.14 in D‐17 and 1.53 ± 0.11 in U2‐OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt‐TCEP induced caspase‐dependent apoptosis. The expression of the anti‐apoptotic Bcl‐X_L protein was decreased after Pt‐TCEP treatment in both cell lines. The results confirmed anti‐cancer activity of Pt‐TCEP against canine and human osteosarcoma cell lines.     

The radiosensitizing effect of the aurora kinase inhibitors,ENMD‐2076, on canine mast cell tumours in vitro

ENMD‐2076 is an aurora kinase inhibitor that also has multi‐target tyrosine kinase inhibitor properties. In this study, the mRNA and the protein expression of aurora‐A and aurora‐B were evaluated in three canine mast cell tumour cell lines. Dose‐dependent cytotoxicity was seen in the cells treated, and it affected the cell cycle with cells in the G2/M phase being selectively killed. The cells were also evaluated for radiosensitivity with/without ENMD‐2076, and radiosensitization was seen after 3 Gy and 6 Gy exposures with ENMD‐2076 for 48 h. Protein expression of caspase‐3 was gradually increased, and the expression intensity was highest at 24 h post irradiation in cells without ENMD‐2076 treatment, which indicates that radiation exposure with ENMD‐2076‐induced cell death faster than radiation treatment alone. Our study results suggest the potential usefulness of treating canine mast cell tumours with aurora kinase inhibitors alone or in conjunction with radiation therapy.     

Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE‐dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI‐1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC₅₀ values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI‐1 cells. In C2 cells, the combination “ibrutinib + midostaurin” produced synergistic growth‐inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE‐dependent histamine release in primary MCT cells, with IC₅₀ values ranging from 0.05 to 0.1 μM in NI‐1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti‐proliferative effects in canine neoplastic MCs and counteracts IgE‐dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.     

The effects of sulforaphane on canine osteosarcoma proliferation and invasion

Recent evidence in in vitro and in vivo models suggests that sulforaphane (SFN), found in raw cruciferous vegetables, may have utility in chemoprevention, as an antineoplastic agent and as a free radical scavenger. The effects of SFN alone or with doxorubicin on cell viability were examined, as well as cell cycle kinetics, invasion capabilities and apoptosis in three canine osteosarcoma cell line (D17, OS 2.4 and HMPOS). Results showed that SFN could not induce cell death at potentially physiological concentrations (<50 μM), but significantly diminished cell invasion and downregulation of focal adhesion kinase (FAK) signaling. Modest cell cycle changes were observed in each cell line. When doxorubicin was used in conjunction with SFN, there was a protective effect to doxorubicin‐induced cytotoxicity in D17 and OS 2.4 cells. Further studies examining SFN as a supplement are warranted, particularly in light of pro‐proliferative and cytoprotective properties in canine osteosarcoma.     

In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells

Benzimidazole anthelmintics have reported anti‐neoplastic effects both in vitro and in vivo. The purpose of this study was to evaluate the in vitro chemosensitivity of three canine glioma cell lines to mebendazole and fenbendazole. The mean inhibitory concentration (IC₅₀) (±SD) obtained from performing the MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay after treating J3T, G06‐A, and SDT‐3G cells for 72 h with mebendazole were 0.030 ± 0.003, 0.080 ± 0.015 and 0.030 ± 0.006 μM respectively, while those for fenbendazole were 0.550 ± 0.015, 1.530 ± 0.159 and 0.690 ± 0.095 μM; treatment of primary canine fibroblasts for 72 h at IC₅₀ showed no significant effect. Immunofluorescence studies showed disruption of tubulin after treatment. Mebendazole and fenbendazole are cytotoxic in canine glioma cell lines in vitro and may be good candidates for treatment of canine gliomas. Further in vivo studies are required.     

Investigation of the effect of pamidronate disodium on the in vitro viability of osteosarcoma cells from dogs

OBJECTIVE: To determine the effect of pamidronate disodium on the in vitro viability of osteosarcoma cells and non-neoplastic cells from dogs. SAMPLE POPULATION: 3 osteosarcoma and 1 fibroblast cell lines derived from dogs. PROCEDURE: Cell counts and cell viability assays were performed in cultures of osteosarcoma cells (POS, HMPOS, and COS31 cell lines) and fibroblasts after 24, 48, and 72 hours of incubation with pamidronate at concentrations of 0.001 to 1000 microM or with no drug (control treatment). Percentage viability was determined in cell samples for each concentration of pamidronate and each incubation time. A DNA fragmentation analysis was performed to assess bisphosphonate-induced apoptosis. RESULTS: Osteosarcoma cell viability decreased significantly in a concentration- and time-dependent manner at pamidronate concentrations ranging from 100 to 1000 microM, most consistently after 48 and 72 hours' exposure. In treated osteosarcoma cells, the lowest percentage cell viability was 34% (detected after 72 hours' exposure to 1000 microM pamidronate). Conversely, 72 hours' exposure to 1000 microM pamidronate did not significantly reduce fibroblast viability (the lowest percentage viability was 76%). After 72 hours of exposure, pamidronate did not cause DNA fragmentation in POS or HMPOS cells. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that pamidronate may have the potential to inhibit osteosarcoma growth in dogs, possibly through a nonapoptotic mechanism. The clinical relevance of these in vitro findings remains to be determined, but administration of pamidronate may potentially be indicated as an adjuvant treatment in chemotherapeutic protocols used in dogs.     

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.     

Biological activity of dihydroartemisinin in canine osteosarcoma cell lines

OBJECTIVE: To evaluate the biological activity of dihydroartemisinin on canine osteosarcoma cell lines in vitro. SAMPLE POPULATION: 4 canine osteosarcoma cell lines. PROCEDURES: Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100 microM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2',7'-dihydrofluorescein diacetate and flow cytometry. RESULTS: The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6 microM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.     

In vitro effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine hemangiosarcoma cell lines

This study evaluated the in vitro activity of masitinib mesylate against canine hemangiosarcoma (HSA) cell lines after treatment with increasing concentrations of masitinib mesylate (0.01-100?μM) for 24, 48 and 72 h. Results indicated that masitinib mesylate caused a dose- and time-dependent decrease in HSA cell proliferation. The 50% inhibitory concentration (IC(50) ) at 72 h for three HSA cell lines (DEN, Fitz and SB) was found to be 8.56, 9.41 and 10.65?μM, respectively. Further investigation demonstrated that masitinib mesylate induced apoptosis in all HSA cell lines, including activation of caspase-3/7. Measurement of VEGF levels in cell supernatant found a statistically significant increased VEGF in close proximity to the IC(50) of each cell line followed by a decline back towards baseline. These findings indicate that masitinib mesylate causes dose-dependent HSA cell death in vitro and supports future clinical trials of masitinib for canine HSA.     

Effect of simvastatin on cell proliferation and Ras activation in canine tumour cells

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP‐binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration‐ and time‐dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase‐3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP‐bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin‐induced cytotoxic effects in a variety of canine tumour cells.     

Effects of vitamin D and retinoids on the differentiation and growth in vitro of canine osteosarcoma and its clonal cell lines

Although canine osteosarcoma is one of the most malignant, aggressive and lethal neoplasms originating from undifferentiated bone cells, it may retain some capacity for normal differentiation. The purpose of this study was to ascertain if the residual capacity for differentiation could be used to suppress its malignant properties. We tested the efficacy of vitamin D and retinoids in inducing differentiation and inhibiting growth of the POS canine osteosarcoma and four of its clonal cell lines, POS 14A (fibroblast type), POS 53B (chondroblast type), POS 53C (undifferentiated type) and POS 53D (osteoblastic type). Treatment with 10(-10)to 10(-8)M concentrations of calcitriol, OCT, cholecalciferol, all-trans retinoic acid (ATRA) and 9-cis retinoic acid for 48-120 hours changed the morphology of POS, POS 53B, POS 53C and POS 53D cells to cells that were elongated and spindle-shaped. Increased number of cytoplasmic organelles and pronounced nuclear activities were induced by concentrations of 10(-8)M and 10(-7)M for 120 hours. All drugs at concentrations of 10(-10)to 10(-8)M for 72 hours inhibited POS growth dose-dependently. OCT significantly reduced the cell number in all cell lines when used at concentrations between 10(-9)and 10(-8)M for 72 hours and exerted significant anti-proliferative effects for eight days culture. This study demonstrated that changed morphology and inhibition of growth was induced by treatment of the cells with these vitamins, that the loss of control of differentiation in the neoplasia was not irreversible and that these drugs may be useful in the clinic.     

Investigation of the effects of deracoxib and piroxicam on the in vitro viability of osteosarcoma cells from dogs

OBJECTIVE: To determine whether exposure of canine osteosarcoma cells to deracoxib or piroxicam results in decreased viability, whether the cytotoxic effects of deracoxib and piroxicam involve induction of apoptosis, and whether deracoxib is a more potent inhibitor of osteosarcoma cell growth than piroxicam. SAMPLE POPULATION: 1 fibroblast and 3 osteosarcoma cell lines. PROCEDURE: Cell counts and viability assays were performed using osteosarcoma cells (POS, highly metastatic POS, and canine osteosarcoma cell 31) and fibroblasts after 72 hours of incubation with deracoxib at concentrations of 0.5 microM to 500 microM or piroxicam at concentrations of 1 microM to 1,000 microM. Percentage viability was determined for each concentration. A DNA fragmentation analysis was performed to assess drug-induced apoptosis. RESULTS: Concentration of deracoxib required for 50% inhibition of cell viability (IC50) was reached in all 3 osteosarcoma cell lines and ranged from 70 to 150 microM, whereas the IC50 for piroxicam was only reached in the POS cell line at 500 microM. Neither deracoxib nor piroxicam induced sufficient toxicity in fibroblasts to reach an IC50. Exposure of osteosarcoma cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation. CONCLUSIONS AND CLINICAL RELEVANCE: Intermediate and high concentrations of deracoxib and high concentrations of piroxicam were cytotoxic to osteosarcoma cells; neither drug inhibited cell viability at typical plasma concentrations in dogs. Deracoxib inhibited viability of cells at concentrations that did not affect fibroblast viability. There was no evidence of apoptosis induction for either drug; however, only 1 cell line was evaluated for apoptosis induction and only for a limited selection of drug concentrations.     

Pharmacokinetics of single-dose intravenous levetiracetam administration in normal dogs

Objective: To determine plasma pharmacokinetics of levetiracetam after a single intravenous dose (60 mg/kg) in normal dogs using a high‐performance liquid chromatography assay validated for canine plasma. Design: Pharmacokinetic study. Setting: A university‐based canine research facility. Animals: Six healthy adult dogs. Interventions: Intravenous drug administration, multiple blood sample procurement. Measurements and main results: There were no obvious adverse effects associated with the intravenous (IV) bolus administration of levetiracetam in any of the dogs. Plasma levetiracetam concentrations remained above or within the reported therapeutic range for humans (5–45 μg/mL) for all dogs, for all time periods evaluated. Mean and median (in parentheses) values for pharmacokinetic parameters included the following: maximum plasma concentration, 254 μg/mL (254 μg/mL); half‐life, 4.0 hours (4.0 hours); volume of distribution at steady state, 0.48 L/kg (0.48 L/kg); clearance, 1.4 mL/kg/min (1.5 mL/kg/min); and median residence time, 6.0 hours (6.0 hours). Conclusions: In normal dogs, a 60 mg/kg IV bolus dose of levetiracetam is well tolerated and achieves plasma drug concentrations within or above the therapeutic range reported for humans for at least 8 hours after administration. Based on the favorable pharmacokinetics and tolerability demonstrated for IV levetiracetam in this study, in addition to previously demonstrated efficacy of oral levetiracetam, IV levetiracetam may be a useful treatment option for emergency management of canine seizure activity.     

Evaluation of the effects of histone deacetylase inhibitors on cells from canine cancer cell lines

OBJECTIVE: To determine whether exposure of canine cancer cells to histone deacetylase (HDAC) inhibitors S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide (OSU-HDAC42) or suberoylanilide hydroxamic acid (SAHA) results in increased histone acetylation and decreased cell viability and whether any changes in viability involve induction of apoptosis or alterations in progression of the cell cycle. Sample POPULATION: 9 canine cancer cell lines. PROCEDURES: Cells from 9 canine cancer cell lines were treated with dimethyl sulfoxide vehicle, OSU-HDAC42, or SAHA, then assays of cell viability were performed. Histone acetylation was assessed by use of western blot analysis. Apoptosis was assessed via ELISA to detect fragmentation of cytoplasmic nucleosomal DNA and western blot analysis to detect cleavage of caspase 3. Cell cycle analysis was performed by use of propidium iodide staining and flow cytometry. RESULTS-Concentrations of OSU-HDAC42 and SAHA required to achieve 50% inhibition of cell viability (IC(50)) were reached in cells of 6 and 4 canine cancer cell lines, respectively, and ranged from approximately 0.4 to 1.3 microM for OSU-HDAC42 and 0.6 to 4.8 microM for SAHA. Cells from T-cell lymphoma, mast cell tumor, osteosarcoma, and histiocytic sarcoma lines were most sensitive to HDAC inhibition, with IC(50)s of < 1 microM for OSU-HDAC42 and < 5 microM for SAHA. Induction of apoptosis was indicated via cleavage of caspase 3 and increases in cytoplasmic nucleosomes and the subG(1) cell population. CONCLUSIONS AND CLINICAL RELEVANCE: Micromolar concentrations of HDAC inhibitors OSU-HDAC42 and SAHA induced histone acetylation, cytotoxicity, and apoptosis in canine cancer cells. In general, OSU-HDAC42 was more potent than SAHA.