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.     

The second-generation curcumin analogue RL71 elicits G2/M cell cycle arrest and apoptosis in canine osteosarcoma cells

Canine osteosarcoma is an aggressive cancer, comprising 85% of canine bone neoplasms. Current treatment practices of surgery and chemotherapy increase 1-year survival by only 45%. The curcumin analogue RL71, has demonstrated potent in vitro and in vivo efficacy in several models of human breast cancer through increased apoptosis and cell cycle arrest. Thus, the present study aimed to investigate efficacy of curcumin analogues in two canine osteosarcoma cell lines. Osteosarcoma cell viability was assessed using the sulforhodamine B assay and mechanisms of action were determined by analysing the levels of cell cycle and apoptotic regulatory proteins via Western blotting. Further evidence was obtained using flow cytometry to detect cell cycle distribution and the number of apoptotic cells. RL71 was the most potent curcumin analogue with EC₅₀ values of 0.64 ± 0.04 and 0.38 ± 0.009 μM (n = 3) in D-17 (commercial) and Gracie canine osteosarcoma cells, respectively. RL71 significantly increased the ratio of cleaved-caspase 3 to pro-caspase 3 and the level of apoptotic cells at the 2× and 5× EC₅₀ concentration (p < 0.001, n = 3). Furthermore, at the same concentration, RL71 significantly increased the number of cells in the G2/M phase. In conclusion, RL71 has potent cytotoxic activity in canine osteosarcoma cells triggering G2/M arrest and apoptosis at concentrations achievable in vivo. Future research should further investigate molecular mechanisms for these changes in other canine osteosarcoma cell lines prior to in vivo investigation.     

The effects of baicalein on canine osteosarcoma cell proliferation and death

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti‐inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage‐independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response.     

Evaluation of satraplatin in dogs with spontaneously occurring malignant tumors

Background: Satraplatin is the 1st orally bioavailable platinum anticancer drug. Objective: Our objectives were to evaluate efficacy in vitro against a canine cancer cell line, to determine the maximally tolerated dose (MTD) of satraplatin in tumor‐bearing dogs, to identify the dose‐limiting and other toxicities in dogs, and to record pharmacokinetics (PK). Animals: Dogs with macro‐ or microscopic malignant neoplasia. Methods: D17 canine osteosarcoma cells first were evaluated in a clonogenic survival assay. Then, dogs with a diagnosis of malignant neoplasia were prospectively entered in standard 3 + 3 cohorts. Additional patients were entered at the MTD to assess efficacy. Total and free platinum (by ultrafiltrate) concentrations were determined with inductively coupled plasma mass spectroscopy. Results: Satraplatin inhibited clonogenic survival in vitro at clinically relevant and achievable concentrations. Twenty‐three dogs were treated, 14 with PK evaluation. The MTD was 35 mg/m²/d for 5 days, repeated every 3–4 weeks. Bioavailability was 41%. PK variables (mean ± SD) at the MTD included T_max 1.8 (± 0.7) hours, C_max 72 (± 26) ng/mL, area under concentration (AUC)_{0–24 h} 316 (± 63) h × ng/mL, and MRT 7 (± 1.3) hours. Higher AUC after the 5th versus the 1st dose suggested drug accumulation. Interestingly, platelets consistently reached nadir sooner than did neutrophils (day 14 versus 19). Myelosuppression was dose‐limiting and gastrointestinal toxicity was mild. Conclusions and Clinical Importance: Satraplatin was well tolerated in tumor‐bearing dogs, thus warranting further investigation in a phase II trial.     

Effects of clopidogrel and aspirin on platelet aggregation, thromboxane production, and serotonin secretion in horses

Background: Critically ill horses are susceptible to thrombotic disease, which might be related to increased platelet reactivity and activation. Objectives: To compare the effect of oral clopidogrel and aspirin (ASA) on equine platelet function. Animals: Six healthy adult horses. Methods: Horses received clopidogrel (2 mg/kg PO q24h) or ASA (5 mg/kg PO q24h) for 5 days in a prospective randomized cross‐over design. Platelet aggregation responses to adenosine diphosphate (ADP) and collagen via optical aggregometry, and platelet secretion of serotonin (5HT) and production of thromboxane B₂ (TXB₂) by ELISA were evaluated. In horses receiving clopidogrel, high‐performance liquid chromatography analysis for clopidogrel and its carboxylic‐acid metabolite SR 26334 was performed. Results: SR 26334 was identified in all clopidogrel‐treated horses, although the parent compound was not detected. Clopidogrel resulted in decreases in ADP‐induced platelet aggregation persisting for 120 hours after the final dose. ADP‐induced platelet aggregation decreased from a baseline of 70.2 ± 14.7% to a minimum of 15.9 ± 7.7% 24 hours after the final dose (P < .001). Collagen‐induced aggregation decreased from a baseline of 93 ± 9.5% to a minimum of 70.8 ± 16.9% 48 hours after the final dose (P < .001). ASA did not decrease platelet aggregation with either agonist. ASA decreased serum TXB₂ from a baseline value of 1310 ± 1045 to 128 ± 64 pg/mL within 24 hours (P < .01). Conclusions and Clinical Importance: Clopidogrel effectively decreases ADP‐induced platelet aggregation in horses, and could have therapeutic applications for equine diseases associated with platelet activation.     

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.     

Pharmacokinetics and bone tissue concentrations of lincomycin following intravenous and intramuscular administrations to cats

Albarellos, G. A., Montoya, L., Denamiel, G. A. A., Velo, M. C., Landoni, M. F. Pharmacokinetics and bone tissue concentrations of lincomycin following intravenous and intramuscular administrations to cats. J. vet. Pharmacol. Therap.  35 , 534–540. The pharmacokinetic properties and bone concentrations of lincomycin in cats after single intravenous and intramuscular administrations at a dosage rate of 10 mg/kg were investigated. Lincomycin minimum inhibitory concentration (MIC) for some gram‐positive strains isolated from clinical cases was determined. Serum lincomycin disposition was best‐fitted to a bicompartmental and a monocompartmental open models with first‐order elimination after intravenous and intramuscular dosing, respectively. After intravenous administration, distribution was rapid (T_1/2(d) = 0.22 ± 0.09 h) and wide as reflected by the volume of distribution (V_(d(ss))) of 1.24 ± 0.08 L/kg. Plasma clearance was 0.28 ± 0.09 L/h·kg and elimination half‐life (T_1/2) 3.56 ± 0.62 h. Peak serum concentration (C_max), T_max, and bioavailability for the intramuscular administration were 7.97 ± 2.31 μg/mL, 0.12 ± 0.05 h, and 82.55 ± 23.64%, respectively. Thirty to 45 min after intravenous administration, lincomycin bone concentrations were 9.31 ± 1.75 μg/mL. At the same time after intramuscular administration, bone concentrations were 3.53 ± 0.28 μg/mL. The corresponding bone/serum ratios were 0.77 ± 0.04 (intravenous) and 0.69 ± 0.18 (intramuscular). Lincomycin MIC for Staphylococcus spp. ranged from 0.25 to 16 μg/mL and for Streptococcus spp. from 0.25 to 8 μg/mL.     

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.     

Chilled and post‐thaw storage of sperm in different goldfish types

The effective storage time of sperm after stripping (for 48 hr in 6‐hr intervals) and after thawing (for 6 hr in 2‐hr intervals) in Black moor, Oranda and Calico goldfish types was investigated. Variations in sperm density were also measured in all lines. The efficiency of a sperm cryopreservation method formerly developed for common carp was recorded in all three goldfish lines. Motility parameters ((pMOT, %), curvilinear velocity (VCL, μm/s) and straightness (STR, %)) of Black moor sperm did not decrease significantly during 48 hr of storage. A significant reduction in the Oranda type compared to the fresh control was observed in pMOT after 42 (23 ± 2%) and VCL after 36 (94 ± 12 μm/s) hours (pMOT 84 ± 5%, VCL 150 ± 11 μm/s). In the Calico type, pMOT decreased significantly already after 18 (42 ± 26%) and VCL after 6 (105 ± 8 μm/s) hours (fresh: pMOT 92 ± 5%, VCL 151 ± 6 μm/s). A high pMOT immediately following thawing was measured in Oranda (46 ± 12%) and Calico (55 ± 15%) types, whereas a reduced pMOT was recorded in Black moor (24 ± 19%). In Calico, pMOT showed a significant reduction after 6 hr (19 ± 11%) in comparison with the initial value, with no changes observed in VCL and STR. None of the parameters changed in the Black moor and Oranda types. Evidence was found that different goldfish lines have different sperm quality and characteristics. Further studies can investigate the possible effects of chilled and post‐thaw storage on the fertilizing capacity of sperm in the Black moor, Oranda and Calico goldfish types.     

The use of pulsed electromagnetic field (PEMF) therapy to provide post-operative analgesia in the dog

Buprenorphine is an effective analgesic when administered epidurally to humans. The purpose of this study was to compare epidural buprenorphine (B; n = 10) with epidural morphine (M; n = 10) for post‐operative analgesia in dogs undergoing cranial cruciate ligament repair. All dogs were premedicated with acepromazine (0.1 mg kg^?1 IM), induced with propofol (4–6 mg kg^?1 IV) and maintained with halothane in oxygen. Dogs were randomly assigned to receive B (0.004 mg kg^?1) or M (0.1 mg kg^?1) in the lumbosacral epidural space in a total volume of 0.2 mL kg^?1. End‐tidal halothane and CO₂ and temperature were recorded every 15 minutes until extubation (t = 0). A numerical rating pain score (SPS) was recorded at t = 0, 1, 2, 4, 6, 10 and 24 hours by a blinded observer. Dogs received rescue morphine (1.0 mg kg^?1 IM) if indicated by SPS and the time of rescue analgesic administration was recorded. Observable side‐effects such as urinary retention, sedation or pruritus were recorded. Data were analyzed with repeated measures anova . Mean ± SD body weight (kg) and age (yrs) for B dogs was 34.2 ± 10.8 and 5.5 ± 2.8; for M dogs these values were 36.6 ± 13.5 and 5.9 ± 3.3. Mean ± SD SPS for B dogs at t = 0, 1, 2, 4, 6, 10 and 24 hours were 1.2 ± 0.75, 3.2 ± 2.0, 4.5 ± 4.3, 4.6 ± 3.4, 4.7 ± 3.0, 5.0 ± 4.9 and 5.1 ± 3.5. For M dogs these values were 1.7 ± 0.5, 2.6 ± 2.0, 3.7 ± 0.75, 4.2 ± 2.2, 4.1 ± 3.0, 3.1 ± 2.1 and 3.9 ± 1.9. There were no significant differences between B and M with respect to SPS, times or frequency of rescue morphine administration, end‐tidal halothane and CO₂, or esophageal temperature. Fifty per cent of dogs in both groups required rescue morphine. Buprenorphine is as effective as morphine for epidural analgesia in healthy dogs undergoing hindlimb orthopedic surgery.     

Intrinsic radiosensitivity and repair of sublethal radiation-induced damage in canine osteosarcoma cell lines

OBJECTIVE: To characterize the radiosensitivity and capacity for sublethal damage repair (SLDR) of radiation-induced injury in 4 canine osteosarcoma cell lines. SAMPLE POPULATION: 4 canine osteosarcoma cell lines (HMPOS, POS, COS 31, and D17). PROCEDURES: A clonogenic colony-forming assay was used to evaluate the cell lines' intrinsic radiosensitivities and SLDR capacities. Dose-response curves for the cell lines were generated by fitting the surviving fractions after radiation doses of 0 (control cells), 1, 2, 3, 6, and 9 Gy to a linear quadratic model. To evaluate SLDR, cell lines were exposed to 2 doses of 3 Gy (split-dose experiments) at an interval of 0 (single 6-Gy dose), 2, 4, 6, or 24 hours, after which the surviving fractions were assessed. RESULTS: Mean surviving fraction did not differ significantly among the 4 cell lines at the radiation doses tested. Mean surviving fraction at 2 Gy was high (0.62), and the alpha/beta ratios (predictor of tissue sensitivity to radiation therapy) for the cell lines were low (mean ratio, 3.47). The split-dose experiments revealed a 2.8- to 3.9-fold increase in cell survival when the radiation doses were applied at an interval of 24 hours, compared with cell survival after radiation doses were applied consecutively (0-hour interval). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that these canine osteosarcoma cell lines are fairly radioresistant; alpha/beta ratios were similar to those of nonneoplastic, late-responding tissues. Future clinical investigations should involve increasing the fraction size in a manner that maximizes tumor killing without adverse effects on the nonneoplastic surrounding tissues.     

Pharmacokinetics of azithromycin in lactating dairy cows with subclinical mastitis caused by Staphylococcus aureus

Lucas, M. F., Errecalde, J. O., Mestorino, N. Pharmacokinetics of azithromycin in lactating dairy cows with subclinical mastitis caused by Staphylococcus aureus. J. vet. Pharmacol. Therap. 33 , 132–140. Azithromycin is a time‐dependent antimicrobial with long persistence. The main characteristics of azithromycin suggest that it could be useful for treating bovine mastitis caused by Staphylococcus aureus. To investigate this possibility, its pharmacokinetic (PK) behavior was studied. Six Holstein lactating cows with subclinical mastitis were administered two 10 mg/kg intramuscular (i.m.) doses of azithromycin, with a 48‐h interval. Milk and plasma concentrations were measured by microbiological assay. The MIC₉₀ was determined in 51 S. aureus isolations to calculate pharmacokinetic/pharmacodynamic (PK/PD) parameters. Milk maximal concentration (C_max) was 7.76 ± 1.76 μg/mL (16.67 h post‐first administration) and 7.82 ± 2.18 μg/mL (14 h post‐2^nd administration). In plasma C_max was 0.18 ± 0.03 μg/mL (2 h post‐1^rst administration) and 0.11 ± 0.03 μg/mL (14 h post‐2^nd administration). Azithromycin was eliminated from the milk with a half‐life (T½λ) of 158.26 ± 137.7 h after 2^nd administration, meanwhile plasma T½λ resulted shorter(13.97 ± 11.1 h). The mean area under the concentration vs. time curve from 0 to 24 h (AUC_0‐24h) was 153.82 ± 34.66 μg·h/mL in milk secretion and 2.61 ± 0.59 μg·h/mL in plasma. Infection presence in the quarters had a significant effect (P < 0.05) on the area under the concentration vs. time curve from 0 to infinity (AUC_0‐∞) and clearance from the mammary gland (Cl_mam/F). Moreover, it had influence on milk bioavailability (F_milk), T½λ, AUC_0‐∞ and mean residence time (MRT) in milk, which values resulted increased in mastitic quarters. In this study, it was determined that the production level and the mammary health status have an influence on PK parameters of azithromycin treatments in bovine mastitis.     

The effects of L‐659,066, a peripheral α2‐adrenoceptor antagonist,and verapamil on the cardiovascular influences of dexmedetomidine in conscious sheep

Raekallio M. R., Honkavaara J. M., Vainio O. M. The effects of L‐659,066, a peripheral α₂‐adrenoceptor antagonist, and verapamil on the cardiovascular influences of dexmedetomidine in conscious sheep. J. vet. Pharmacol. Therap. 33 , 434–438. We investigated whether administration of L‐659,066, a peripheral α₂‐adrenoceptor antagonist, or verapamil, a calcium‐channel antagonist, would prevent the cardiovascular effects of dexmedetomidine. Eleven sheep received three intravenous treatments with a randomized, cross‐over design: dexmedetomidine (5 μg/kg, DEX); DEX with L‐659,066 (250 μg/kg, DEX + L); and verapamil (0.05 mg/kg) 10 min prior to DEX (Ver + DEX). Haemodynamics were recorded at intervals upto 40 min. Acute increases in mean arterial pressure (MAP) (106 ± 10.7 to 120.8 ± 11.7 mmHg), central venous pressure (CVP) (3.3 ± 3.2 to 14.7 ± 5.0 mmHg) and systemic vascular resistance (SVR) (1579 ± 338 to 2301 ± 523 dyne s/cm⁵), and decreases in cardiac output (CO) (5.36 ± 0.87 to 3.93 ± 1.30 L/min) and heart rate (HR) (88.6 ± 15.3 to 49.7 ± 5.5/min) were detected with DEX. The peak SVR remained lower after Ver + DEX (1835 ± 226 dyne s/cm⁵) than DEX alone, but the other parameters did not significantly differ between these treatments. 2 min after drug delivery, differences between DEX and DEX + L were statistically significant for all measured haemodynamic parameters. With DEX + L, an early decrease in MAP (99.9 ± 6.8 to 89.3 ± 6.6 mmHg) was detected, and DEX + L induced a slight but significant increase in CVP and a decrease in HR at the end of the observation period, while SVR and CO did not significantly change. All animals were assessed as deeply sedated from 2–20 min with no differences between treatments. L‐659,066 has great potential for clinical use to prevent the cardiovascular effects of dexmedetomidine mediated by peripheral α₂‐adrenoceptors, whereas the effects of verapamil were marginal.     

Disposition kinetics of albendazole and metabolites in laying hens

Bistoletti, M., Alvarez, L., Lanusse, C., Moreno, L. Disposition kinetics of albendazole and metabolites in laying hens. J. vet. Pharmacol. Therap.  36 , 161–168. An increasing prevalence of roundworm parasites in poultry, particularly in litter‐based housing systems, has been reported. However, few anthelmintic drugs are commercially available for use in avian production systems. The anthelmintic efficacy of albendazole (ABZ) in poultry has been demonstrated well. The goal of this work was to characterize the ABZ and metabolites plasma disposition kinetics after treatment with different administration routes in laying hens. Twenty‐four laying hens Plymouth Rock Barrada were distributed into three groups and treated with ABZ as follows: intravenously at 10 mg/kg (ABZ i.v.); orally at the same dose (ABZ oral); and in medicated feed at 10 mg/kg·day for 7 days (ABZ feed). Blood samples were taken up to 48 h posttreatment (ABZ i.v. and ABZ oral) and up to 10 days poststart feed medication (ABZ feed). The collected plasma samples were analyzed using high‐performance liquid chromatography. ABZ and its albendazole sulphoxide (ABZSO) and ABZSO₂ metabolites were recovered in plasma after ABZ i.v. administration. ABZ parent compound showed an initial concentration of 16.4 ± 2.0 μg/mL, being rapidly metabolized into the ABZSO and ABZSO₂ metabolites. The ABZSO maximum concentration (C_max) (3.10 ± 0.78 μg/mL) was higher than that of ABZSO₂C_max (0.34 ± 0.05 μg/mL). The area under the concentration vs time curve (AUC) for ABZSO (21.9 ± 3.6 μg·h/mL) was higher than that observed for ABZSO₂ and ABZ (7.80 ± 1.02 and 12.0 ± 1.6 μg·h/mL, respectively). The ABZ body clearance (Cl) was 0.88 ± 0.11 L·h/kg with an elimination half‐life (T_1/2el) of 3.47 ± 0.73 h. The T_1/2el for ABZSO and ABZSO₂ were 6.36 ± 1.50 and 5.40 ± 1.90 h, respectively. After ABZ oral administration, low ABZ plasma concentrations were measured between 0.5 and 3 h posttreatment. ABZ was rapidly metabolized to ABZSO (C_max, 1.71 ± 0.62 μg/mL) and ABZSO₂ (C_max, 0.43 ± 0.04 μg/mL). The metabolite systemic exposure (AUC) values were 18.6 ± 2.0 and 10.6 ± 0.9 μg·h/mL for ABZSO and ABZSO₂, respectively. The half‐life values after ABZ oral were similar (5.91 ± 0.60 and 5.57 ± 1.19 h for ABZSO and ABZSO₂, respectively) to those obtained after ABZ i.v. administration. ABZ was not recovered from the bloodstream after ABZ feed administration. AUC values of ABZSO and ABZSO₂ were 61.9 and 92.4 μg·h/mL, respectively. The work reported here provides useful information on the pharmacokinetic behavior of ABZ after both i.v. and oral administrations in hens, which is a useful first step to evaluate its potential as an anthelmintic tool for use in poultry.     

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.     

Comparison of Plasma Fentanyl Concentrations by Using Three Transdermal Fentanyl Patch Sizes in Dogs

Objective—To compare plasma fentanyl concentrations attained after the application of three transdermal fentanyl patch sizes (50, 75, and 100 μg/hour) in dogs. Design—Repeated Latin square controlled study. Animals—Six intact, mixed-breed adult dogs (2 males, 4 females) weighing 19.9 ± 3.4 kg. Methods—Each dog was randomly assigned to receive each of three treatments: 50 (P50), 75 (P75), or 100 (P100) μg/hour transdermal patches. Patches were left in place for 72 hours. Jugular venous blood was collected at 1,2, 4, 8, 12, 24, 36, 48, 60, and 72 hours after patch application and for 1, 2, 4, 8, and 12 hours after patch removal. Plasma fentanyl concentrations were measured using a radioimmunoassay technique. After a 96-hour washout period, each dog was moved to another treatment group and received a different patch size. Results—The following results were obtained (mean ± SD): average plasma fentanyl concentration from 24 to 72 hours, 0.7 ± 0.2 ng/mL (P50), 1.4 ± 0.5 ng/mL (P75), 1.2 ± 0.5 ng/mL (P100); the total area under the concentration versus time curve (0 hours to infinity), 46 ± 12.2 ng/h/mL (P50), 101.2 ± 41.4 ng/h/mL (P75), 80.4 ± 38.3 ng/h/mL (P100); and the apparent elimination half-life, 3.6 ± 1.2 hours (P50), 3.4 ± 2.7 hours (P75), and 2.5 ± 2.0 hours (P100). There was a high degree of variability in plasma fentanyl concentrations achieved. Plasma fentanyl concentrations declined rapidly after patch removal. Conclusions—The attainment of steady-state plasma concentrations takes up to 24 hours, and there is a great deal of variability in the final concentrations reached in different individuals. In this study, the 100 μg/hour patches did not provide statistically increased plasma concentrations when compared with the 50 μg/hour patches. Clinical Relevance—Because of the interindividual and intraindividual variation in plasma fentanyl concentrations, patches should be applied 24 hours before the anticipated time that analgesia will be required. Adequacy of analgesia and potentially deleterious side effects, such as sedation and respiratory depression, should be monitored while the patches are in place. Skin reactions may occur, and the patches should be removed if such skin irritation is seen. After the patch is removed, it is expected that analgesia will wane rapidly because of the brief elimination half-life.     

Effects of transplantation sites on tumour growth,pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice

To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT‐xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC‐xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin–radixin–moesin (p‐ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p‐ERM expression at later time points. The expression of ezrin and p‐ERM in the primary tumours of IT‐xenografted mice was higher than those in SC‐xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells.     

The bisphosphonates alendronate and zoledronate are inhibitors of canine and human osteosarcoma cell growth in vitro

Bisphosphonates (BPs) are a class of non‐hydrolysable analogues of pyrophosphate that have high affinity for bone mineral and are inhibitors of bone resorption. The in vitro effects of two nitrogen‐containing BPs, alendronate (ALE) and zoledronate (ZOL), on growth, induction of apoptosis and effects on cell‐cycle distribution in two canine and two human osteosarcoma (OSA) cell lines are investigated here. Both significantly (P < 0.001) reduced cell growth in all cell lines, as assessed by a colorimetric assay with IC₅₀ values in the range of 7.3–61.4 µM and 7.9–36.3 µM for ALE and ZOL, respectively. Both BPs caused a significant (P < 0.001) dose‐dependent increase in the proportion of cells undergoing apoptosis, as assessed both by cell‐cycle analysis and by annexin‐V binding. Both ALE and ZOL altered the proportion of cells in each phase of the cell cycle, but the extent and proportion was both drug and cell line dependent. These data indicate that the nitrogen‐containing BPs have direct anti‐tumour activity against canine and human OSA cells.     

Investigating the molecular mechanism of pamidronate‐induced in vitro cytotoxicity in canine osteosarcoma cells

Introduction: Pamidronate is used to treat metastatic bone lesions in human cancer patients. By directly inhibiting the mevalonate pathway, pamidronate disrupts GTPase‐binding protein prenylation, resulting in osteoclast apoptosis. Pamidronate has been demonstrated to exert dose‐ and time‐dependent cytotoxic effects in several canine malignant osteoblastic cell lines. However, the exact cytotoxic mechanism remains speculative. The purpose of this study was to investigate and characterize the molecular mechanism of pamidronate‐induced cytotoxicity in canine malignant osteoblasts. Methods: The involvement of farnesyl or geranylgeranyl pyrophosphate synthase inhibition was evaluated by performing rescue experiments with cells incubated for 48 hours with cytotoxic concentrations of pamidronate (50 and 100 μM), with or without farnesol (FOH) and geranylgeraniol (GGOH). Cell lysates were fluorometrically‐assessed for caspase‐3‐like activity (R&D Systems) following incubation with varying concentrations of pamidronate (control, 50 and 100 μM) for 48 hours. The expression of a prenylated GTPase protein, Rap1 A, was qualitatively evaluated with immunoblotting using a polyclonal goat antibody (Santa Cruz Biotechnology) in cells incubated for 48 hours with varying concentrations of pamidronate (0, 1, 10, 50, and 100 μM). Results: In cells treated with lower cytotoxic concentrations of pamidronate (50 μM) for 48 hours, the addition of GGOH, but not FOH, was able to diminish the degree cytotoxicity, p ≤ 0.05. However, cells incubated with higher cytotoxic concentrations of pamidronate (100 μM), neither GGOH nor FOH were able to reduce the level of pamidronate‐induced cytotoxicity. Caspase‐3‐like activity directly correlated with the degree of pamidronate‐induced cytotoxicity. Cells treated with pamidronate at 50 μM and 100 μM increased caspase‐3‐like activity by 5.3 and 7.1‐fold, respectively over untreated controls. The detection of Rap1A by immunoblotting requires further optimization. Conclusions: Pamidronate‐induced cytotoxicity in canine osteosarcoma cells may share a similar mechanism as has been demonstrated for osteoclasts. Inhibition of the mevalonate pathway appears to contribute to the observed cytotoxicity at lower doses of pamidronate. In addition, caspase‐3‐like activity contributes to the apoptotic process induced by pamidronate in malignant canine osteoblasts.