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.     

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.     

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.     

In vitro effects of meloxicam with or without doxorubicin on canine osteosarcoma cells

Cyclooxygenase (COX) inhibitors, already widely used to reduce fever, inflammation and pain, are under increasing consideration as potential agents for the prevention and treatment of neoplasia. As COX-2 was detected in human and canine osteosarcomas, we have evaluated the effect of the preferential COX-2 inhibitor meloxicam on an established D-17 canine osteosarcoma cell line, which expressed, as well as COX-1 and COX-2 also COX-3 (as demonstrated by Western blot). An XTT proliferation kit was used to assess surviving cells after drug treatment. At low concentrations (1, 2, 4 and 10 microm) meloxicam caused an increase in cell numbers while a marked anti-proliferative effect was observed at higher concentrations (100, 200 microm) after 3 days and also 3 weeks of incubation. The chemotherapeutic drug doxorubicin showed a cytotoxic effect at all concentrations (60-1920 nm). Exposure of tumour cells to combinations of meloxicam and doxorubicin revealed synergistic effects (with 240 nm doxorubicin), as well as sub-additive and antagonistic results, especially if combined with concentrations of meloxicam typically found in serum. Care should be taken in concluding, on the basis of one in vitro study, that meloxicam does not have a role in the treatment of canine osteosarcomas given that the results from in vivo studies may differ.     

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.     

Adjuvant therapy with carboplatin and pamidronate for canine appendicular osteosarcoma

Amputation and chemotherapy are the mainstay of treatment for canine appendicular osteosarcoma (OSA). In vitro studies have demonstrated anti‐tumour activity of pamidronate against canine OSA. The purpose of this study was to assess the safety of adding pamidronate to standard post‐operative carboplatin chemotherapy in 17 dogs with appendicular OSA treated with limb amputation. Median disease‐free interval (DFI) and median survival time (MST) were evaluated as secondary endpoints. Incidence of side effects and treatment outcomes were compared to 14 contemporary control patients treated with carboplatin alone. There were no identified side effects to the pamidronate treatment. The median DFI for the study group was 185 days compared to 172 days for the control group (P = 0.90). The MST of the study group was 311 days compared to 294 days for the control group (P = 0.89). Addition of pamidronate to carboplatin chemotherapy for the treatment of canine appendicular OSA is safe and does not impair efficacy of standard carboplatin treatment.     

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.     

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.     

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.     

Double-Blind Placebo-Controlled Trial of Adjuvant Pamidronate with Palliative Radiotherapy and Intravenous Doxorubicin for Canine Appendicular Osteosarcoma Bone Pain

Background: Canine osteosarcoma (OSA) causes focal malignant osteolysis leading to severe pain. Despite the documented efficacy of radiotherapy or IV aminobisphosphonates for managing cancer bone pain, their potential combined therapeutic value has not been reported in OSA-bearing dogs.
Hypothesis: Pamidronate combined with standardized palliative therapy will improve pain control and bone biologic effects in OSA-bearing dogs.
Animals: Fifty dogs with appendicular OSA treated with standardized palliative therapy and either pamidronate or sterile saline.
Methods: Randomized, prospective, double-blinded, placebo-controlled study. Treatment responses for dogs receiving standardized palliative therapy with (n = 26) or without (n = 24) adjuvant pamidronate were serially evaluated for changes in subjective pain scores, urine N-telopeptide (NTx) excretion, primary tumor relative bone mineral density ( r BMD), and computerized pressure platform gait analysis.
Results: Median duration of subjective pain relief for dogs treated with adjuvant pamidronate or placebo was 76 and 75 days, respectively ( P = .39). Forty percent (20/50; pamidronate [11/26] and placebo [9/24]) of dogs experienced durable analgesia, defined by pain alleviation ≥112 days. For patients achieving durable pain control, dogs treated with pamidronate achieved greater reductions in NTx excretion and larger increases in r BMD compared with placebo controls. Changes in peak vertical force assessed by computerized pressure platform gait analysis correlated with pain alleviation in OSA-bearing dogs.
Conclusions and Clinical Importance: Combining pamidronate with standardized palliative therapy is safe, but does not clearly improve pain alleviation. However, in dogs achieving durable pain control, adjuvant pamidronate appears to decrease focal bone resorption in the local tumor microenvironment.     

RADIATION AND CISPLATIN FOR TREATMENT OF CANINE URINARY BLADDER CARCINOMA

Two cases of canine urinary bladder carcinoma were treated with combined radiation and cisplatin. Total radiation dose was 4400 cGy for one dog and 4800 cCy for the other. Cobalt 60 radiation was fractioned using 400 cGy per fraction. Cisplatin was administered intraarterially at a dose of 50 mg/m² divided equally six to seven hours before the first three radiation fractions. Cisplatin was administered before the last three radiation fractions at the same dose and time, but was infused intravenously. Objective evaluation using double contrast cystograms revealed reduction in tumor size in both dogs. The therapy was well-tolerated with minimal side effects.     

The effect of deracoxib and piroxicam on viability of canine osteosarcoma cells in vitro

Introduction:  Cyclooxygenase‐2 (COX‐2) inhibitors are being used increasingly in cancer therapy. Although the effects of COX‐2 inhibitors have been evaluated extensively in carcinomas, less is known about their effects in sarcomas. Since the majority of dogs with appendicular osteosarcoma (OSA) are treated for pain with a non‐steroidal anti‐inflammatory drug (some COX‐2 selective) prior to definitive treatment, it is important to determine the effects that commonly used NSAIDS have on tumor cell growth.
Methods:  Established canine osteosarcoma (POS, HMPOS and COS31) and canine fibroblast cell lines were maintained in culture under standard conditions. Cells were incubated with either deracoxib (1 uM to 500 uM) or piroxicam (1 uM to 1000 uM). Cell viability was assessed at 72 hours by cell counts and the MTT assay. The DNA fragmentation analysis was utilized to assess for apoptosis induction.
Results:  Deracoxib concentrations ≥100 uM and piroxicam concentrations ≥500 uM significantly reduced mean cell viability of all three OSA cell lines (lowest cell viability percentages 20% and 32%, respectively). Deracoxib concentrations ≥250 uM and piroxicam concentrations ≥500 uM also reduced viability of fibroblasts; however, the cell viability percent was reduced to only 54% and 68%, respectively, of the control value. Exposure of OSA cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation.
Conclusions:  Deracoxib and piroxicam demonstrated a cytotoxic effect on canine osteosarcoma cells. There was no evidence of apoptosis induction at the concentrations evaluated. Further investigation will need to be performed to determine whether either drug exhibits anti‐tumor effects in vivo .     

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild‐type p53

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.     

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.     

The Immunotherapy of Canine Osteosarcoma: A Historical and Systematic Review

Osteosarcoma is a malignant mesenchymal neoplasm that accounts for the majority of primary bone tumors in dogs and shares biological and clinical similarities with osteosarcoma in humans. Despite dose intensification with conventional cytotoxic therapies, survival times for dogs and humans diagnosed with high‐grade osteosarcoma have not changed in the past 20 years, with the principal cause of mortality being the development of pulmonary metastases. Given the therapeutic plateau reached for delaying metastatic progression with cytotoxic agents, exploration of alterative adjuvant therapies for improving management of osteosarcoma micrometastases is clinically justified. Evidence suggests that osteosarcoma is an immunogenic tumor, and development of immunotherapies for the treatment of microscopic lung metastases might improve long‐term outcomes. In this review, the history and foundational knowledge of immune interactions to canine osteosarcoma are highlighted. In parallel, immunotherapeutic strategies that have been explored for the treatment of canine osteosarcoma are summarized. With a greater understanding and awareness for how the immune system might be redirected toward combating osteosarcoma metastases, the rational development of diverse immune strategies for managing osteosarcoma holds substantial promise for transforming the therapeutic landscape and improving disease management in both dogs and human beings.     

Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

Analgesic and anti‐inflammatory actions of robenacoxib in acute joint inflammation in dog

Schmid, V. B., Spreng, D. E., Seewald, W., Jung, M., Lees, P., King, J. N. Analgesic and anti‐inflammatory actions of robenacoxib in acute joint inflammation in dog. J. vet. Pharmacol. Therap. 33 , 118–131. The objectives of this study were to establish dose–response and blood concentration–response relationships for robenacoxib, a novel nonsteroidal anti‐inflammatory drug with selectivity for inhibition of the cyclooxygenase (COX)‐2 isoenzyme, in a canine model of synovitis. Acute synovitis of the stifle joint was induced by intra‐articular injection of sodium urate crystals. Robenacoxib (0.25, 0.5, 1.0, 2.0 and 4.0 mg/kg), placebo and meloxicam (0.2 mg/kg) were administered subcutaneously (s.c.) 3 h after the urate crystals. Pharmacodynamic endpoints included data from forceplate analyses, clinical orthopaedic examinations and time course of inhibition of COX‐1 and COX‐2 in ex vivo whole blood assays. Blood was collected for pharmacokinetics. Robenacoxib produced dose‐related improvement in weight‐bearing, pain and swelling as assessed objectively by forceplate analysis (estimated ED₅₀ was 1.23 mg/kg for z peak force) and subjectively by clinical orthopaedic assessments. The analgesic and anti‐inflammatory effects of robenacoxib were significantly superior to placebo (0.25–4 mg/kg robenacoxib) and were non‐inferior to meloxicam (0.5–4 mg/kg robenacoxib). All dosages of robenacoxib produced significant dose‐related inhibition of COX‐2 (estimated ED₅₀ was 0.52 mg/kg) but no inhibition of COX‐1. At a dosage of 1–2 mg/kg administered s.c., robenacoxib should be at least as effective as 0.2 mg/kg of meloxicam in suppressing acute joint pain and inflammation in dogs.     

Single-agent pamidronate for palliative therapy of canine appendicular osteosarcoma bone pain

BACKGROUND: Canine appendicular osteosarcoma (OSA) causes focal bone destruction, leading to chronic pain and reduced quality-of-life scores. Drugs that inhibit pathologic osteolysis might provide additional treatment options for managing cancer-induced bone pain. Aminobisphosphonates induce osteoclast apoptosis, thereby reducing pain associated with malignant osteolysis in human patients with cancer. HYPOTHESIS: Treatment of dogs with pamidronate administered intravenously will alleviate bone pain and reduce pathologic bone turnover associated with appendicular OSA in dogs. ANIMALS: Forty-three dogs with naturally occurring appendicular OSA administered pamidronate intravenously. METHODS: Prospective study. Therapeutic responses in dogs treated with pamidronate administered intravenously and nonsteroidal anti-inflammatory drugs (NSAID) were evaluated by using a numerical cumulative pain index score (CPIS), and by quantifying urine N-telopeptide (NTx) excretion and relative primary tumor bone mineral density (rBMD) assessed with dual energy x-ray absorptiometry. In addition, variables, including pamidronate dose, skeletal mass, baseline and change for CPIS, urine NTx and rBMD during treatment, and baseline tumor volume and radiographic pattern were compared between dogs clinically responsive and nonresponsive to pamidronate therapy. RESULTS: Twelve of 43 dogs (28%) had pain alleviation for >4 months, lasting a median of 231 days. Changes in CPIS and rBMD during treatment were statistically different between responders and nonresponders (P = .046 and .03, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Substantiated by reductions in CPIS and increases in rBMD, single-agent pamidronate administered intravenously with NSAID therapy relieves pain and diminishes pathologic bone turnover associated with appendicular OSA in a subset of dogs.