Gemcitabine is a synthetic analogue of cytosine ara-binoside that exhibits antitumor activity through inhibition of DNA replication and cell growth as a result of incorporation of gemcitabine into replicating DNA and through inhibition of repair mechanisms by masked DNAChain termination.1 Gemcitabine has biological activity against a variety of human cancers and has been approved for use as a single agent in the treatment of human patients with pancreatic adenocar-cinoma and in combination protocols for the treatment of patients with non-small cell lung cancer, metastatic breast cancer, or ovarian cancer.2–7 In addition, clinical trials of gemcitabine, administered either as a single agent or as part of a multiagent protocol, in people with various solid tumors, including transitional cell carcinoma, advanced biliary carcinoma, and soft tissue sarcoma, have demonstrated stabilization of disease.8–11
The potential activity of gemcitabine against OSA cells has been investigated in vitro and in vivo. In 1 study,12 gemcitabine inhibited viability and growth of human OSA cell lines in vitro and, when administered by means of IP injection, inhibited lung metastasis in mice inoculated SC with a murine OSA cell line. Additionally, aerosolized gemcitabine inhibited growth of OSA xenografts and lung metastasis in mice.13 Although gemcitabine has not been evaluated extensively in human patients with OSA, 1 study11 demonstrated stabilization of metastatic disease in 5 of 7 patients with progressive localized or metastatic chemoresistant OSA for 13 to 96 weeks.
The ability of gemcitabine to act synergistically with platinum compounds has been investigated both in vitro and in human clinical trials. For example, gemcitabine and carboplatin were found to induce synergistic killing of several human non-small cell lung cancer cell lines.14,15 Administration of a combination of gemcitabine and cisplatin resulted in synergistic cytotoxicosis in human squamous cell carcinoma and platinum-resistant ovarian carcinoma cell lines in vitro.16 Finally, cytotoxicosis was significantly improved, compared with effects obtained with any single agent alone, when cisplatin or carboplatin was added to gemcitabine in the treatment of human endometrial carcinoma cell lines.17 With respect to clinical patients, the combination of gemcitabine and carboplatin has been approved for use in women with platinum-sensitive recurrent ovarian cancer.7 In patients with advanced non-small cell lung cancer, the combination of gemcitabine with carboplatin or cisplatin has significantly improved response rates and survival times.4
Bisphosphonates have also been used to treat a variety of diseases, including primary bone tumors and bone metastases, and the bisphosphonate pamidronate disodium has been shown to decrease the viability of canine OSA cell lines in vitro.18 Furthermore, a recent study19 demonstrated that bisphosphonates enhance the cytotoxic effects of gemcitabine in vitro.
Gemcitabine has not been used extensively in veterinary oncology A phase I study20 determined that administration of gemcitabine to dogs biweekly at a dosage of 675 mg/m2, IV, resulted in minimal toxicosis. Clinically, administration of gemcitabine (2 mg/kg, IV, over 20 to 30 minutes) and carboplatin (10 mg/kg, IV, as an bolus) to dogs with various cancers did not result in severe toxicoses but was associated with low response rates.21 However, little is known about the efficacy of gemcitabine in dogs with OSA.
The present study was designed as a first step in determining whether gemcitabine could be useful in the treatment of dogs with OSA. Specifically, the purpose of the study reported here was to evaluate in vitro biological activity of gemcitabine against canine OSA cell lines. In addition, we wanted to determine whether gemcitabine acted synergistically with carboplatin or pamidronate.
Materials and Methods
Four canine OSA cell linesa (OSA8, OSA16, OSA32, and OSA36) maintained in RPMI-1640 mediumb supplemented with 10% fetal bovine serum,c nonessential amino acids, sodium pyruvate, HEPES, penicillin, streptomycin, and L-glutamine were used in the study
Effect of gemcitabine on cell viability—The WST-1 assay was used to assess the effect of gemcitabine on cell viability. In brief, OSA cells (2,000 cells/well) were seeded in 96-well plates in 150 μϵ of RPMI-1640 medium with 1% fetal bovine serum and incubated overnight at 37°C and 5% CO2. Gemcitabined (0.01, 0.1, 1, 10, 100, or 1,000μM) was added to the wells, and plates were incubated for an additional 72 hours. Cell viability was then assessed with the WST-1 assay,e performed in accordance with the manufacturer's specifications. Because gemcitabine has a short elimination half-life, viability was also assessed after gemcitabine was incubated with the cells for only 2 hours to mimic expected in vivo drug exposure time. For all assays, cells treated with doxorubicin (0.5μM) were used as the positive control,22 and untreated cells were used as the negative control. Absorbance was quantified with an ELISA plate readerf at a wavelength of 440 nm, and cell viability was calculated as a percentage of viability for the negative control wells (ie, 100 X absorbance of treated wells/absorbance of untreated wells). All samples were analyzed in triplicate, and each experiment was repeated 3 times with each of the cell lines. For each of the OSA cell lines, a logarithmic regression curve constructed with the cell viability data was used to calculate the IC50 for gemcitabine.
Effect of gemcitabine on cell cycle distribution—The propidium iodide staining method was used to assess the effect of gemcitabine on cell cycle distribution. In brief, OSA cells (5 × 105 cells/well) were seeded in 6-well plates in 3 mE of RPMI-1640 medium with 1% fetal bovine serum and incubated overnight at 37°C and 5% CO2. Gemcitabine (1, 10, or 100μM) was added to the wells, and plates were incubated for an additional 48 hours. Cells were then collected, fixed in 70% ethanol, incubated with 0.5 mE of propidium iodide staining solution consisting of propidium iodide (50 μg/mL) and RNAse (10 μg/mL) in PBS solution containing 0.1% glucose, and analyzed by means of flow cytometry. Data were analyzed by means of standard software.g Cells treated with doxorubicin (0.5μM) were used as the positive control, and untreated cells were used as the negative control. All samples were analyzed in duplicate, and each experiment was repeated 3 times with each cell line.
Effect of gemcitabine on apoptosis—Apoptosis was assessed by measuring caspase-3/7 activity. In brief, OSA cells (5,000 cells/well) were seeded in 96-well plates in 150 μϵ of RPMI-1640 medium with 1% fetal bovine serum and incubated overnight at 37°C and 5% CO2. Gemcitabine (1, 10, or 100μM) was added to the wells, and plates were incubated for an additional 24 hours. Caspase-3/7 activity was then measured with a commercial assayh performed in accordance with the manufacturer's specifications. Fluorescence was quantified with an ELISA plate reader at an excitation wavelength of 354 nm and emission wavelength of 442 nm. Cells treated with doxorubicin (0.5μM) were used as the positive control, and untreated cells were used as the negative control. Samples were analyzed in triplicate, and each experiment was repeated 3 times with each of the cell lines.
Effects of gemcitabine in combination with pamidronate—To determine the effects of a combination of gemcitabine and pamidronate, cell viability was assessed after incubation of OSA cells for 72 hours with gemcitabine (0, 10, or 100μM) and pamidronatei (0, 1, 10, or 100μM).
Effects of gemcitabine in combination with carboplatin—To determine the effects of a combination of gemcitabine and carboplatin, cell viability was assessed after incubation of OSA cells for 72 hours with gemcitabine (0, 1, 10, or 100μM) and carboplatin1‘ (0 or 250μM). A carboplatin concentration of 250μM was chosen because this is the reported peak plasma concentration in dogs following bolus IV administration of a standard dose (300 mg/m2) of carboplatin.23
To determine the effect of a combination of gemcitabine and carboplatin on apoptosis, caspase-3/7 activity was measured after a 24-hour incubation of OSA cell lines with gemcitabine (0, 1, 10, or 100μM) and carboplatin (250μM).
To identify a synergistic interaction between gemcitabine and carboplatin, cell viability was assessed after incubation of OSA cells for 72 hours with each drug alone and with a combination of gemcitabine and carboplatin. For the drug combination assays, cells were incubated with solutions containing both drugs at a fixed ratio (gemcitabine-to-carboplatin ratio, 1:100), with concentrations of gemcitabine ranging from 0.0625 to 8μM and concentrations of carboplatin ranging from 6.25 to 800μM. Synergy was analyzed by use of the CI method, as described,24 with standard software.k The amount of each drug, either alone or in combination, needed to achieve a given effect level (ie, reduction in cell viability) was used to calculate CI values, which were then used to define the nature of the drug interaction, with a CI < 1 indicating synergism, a CI > 1 indicating antagonism, and a CI = 1 indicating additivity. The dose-reduction index was determined by comparing the ratio of the concentrations required to obtain a given degree of growth inhibition for gemcitabine and carboplatin individually and in combination.
Finally, to determine whether sequence of administration of gemcitabine and carboplatin had an effect on cell viability, OSA cells were exposed sequentially to gemcitabine (1, 10, or 100μM) then carboplatin (250μM) or to carboplatin (250μM) then gemcitabine (1, 10, or 100μM). Cells were incubated with the first drug for 2 hours. Medium was then removed with suction, cells were rinsed 3 times with PBS solution, the second drug was added, and cells were incubated an additional 2 hours. Medium was again removed, and cells were rinsed. Cell viability was then assessed after a final 72-hour incubation period.
Statistical analysis—Data were summarized as mean and SD. All data were transformed to natural logarithms prior to statistical analysis to equalize variances across treatment groups and achieve approximate normality. For the cell viability and apoptosis assays, the Dunnett multiple comparisons method was used to compare results for treated cells with results for positive and negative control cells. An overall α value of 0.05 was maintained. For cell viability experiments involving pamidronate and gemcitabine, a value of P < 0.025 was considered significant to adjust for the 2 concentrations of gemcitabine (10 and 100μM) that were used. To determine whether sequence of administration of gemcitabine and carboplatin (gemcitabine then carboplatin vs carboplatin then gemcitabine) had an effect on cell viability, results were analyzed by use of 2-way ANOVA incorporating a term for the interaction between order and concentration of gemcitabine. If the interaction was significant (P < 0.05), pairwise comparisons of the 2 administration sequences were performed for each concentration of gemcitabine; a Bonferroni correction was used with a P value cutoff of 0.017. If the interaction was not significant, mean effect of order across concentration of gemcitabine was examined. The Student t test was used to compare results of incubation with gemcitabine for 2 versus 72 hours. All statistical analyses were performed with commercially available software^ Unless otherwise indicated, values of P < 0.05 were considered significant.
Results
Effect of gemcitabine on cell viability—For all 4 canine OSA cell lines, percentage cell viability after incubation with gemcitabine for 72 hours decreased as concentration of gemcitabine increased (Figure 1). For the OSA8, OSA16, and OSA36 cell lines, IC50 concentrations were 5.7, 10.3, and 15.3 μM, respectively. An IC50 concentration could not be calculated for the OSA32 cell line because a 50% decrease in cell viability was not observed with any of the gemcitabine concentrations that were evaluated.
Mean ± SD cell viability, determined by use of a WST-1 assay, for 4 canine OSA cell lines incubated with gemcitabine (0, 0.01, 0.1, 1, 10, 100, or 1,000μM) for 72 hours. Cell viability was calculated as mean absorbance of treated cells divided by mean absorbance of untreated cells.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
No consistent significant differences in cell viability were observed when cells were incubated with gemcitabine for 2 versus 72 hours (Figure 2). Most often, when a significant difference was observed, percentage cell viability was lower after incubation with gemcitabine for 2 hours than after incubation with gemcitabine for 72 hours.
Mean ± SD cell viability for canine OSA cell lines OSA8 (A), OSA16 (B), OSA32 (C), and OSA36 (D) following incubation with gemcitabine for 2 hours (open circles) or 72 hours (closed triangles). *,†Values were significantly (*P < 0.05; †P < 0.01) different following incubation for 2 hours versus incubation for 72 hours.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
Effect of gemcitabine on cell cycle distribution— For all 4 OSA cell lines, incubation with gemcitabine was associated with a substantial increase in the percentage of dead cells (sub-G0/G1 phase; Figure 3).
Mean ± SD percentage of cells in the sub-G0/G1 phase (ie, dead cells), determined by use of the propidium iodide staining method, following incubation of 4 canine OSA cell lines with gemcitabine (0, 1, 10, or 100μM) for 48 hours (A) and representative histograms demonstrating cell cycle distribution following incubation of each cell line with gemcitabine (10μM) for 48 hours (B; thin lines indicate untreated [control] cells and bold lines indicate cells treated with gemcitabine).
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
Effect of gemcitabine on apoptosis—Significant increases in caspase-3/7 activity were detected following incubation of cell lines OSA8 and OSA36 with gemcitabine at concentrations > 1μM (Figure 4). However, for all concentrations of gemcitabine evaluated, caspase-3/7 activity was not significantly increased when cell lines OSA 16 and OSA32 were incubated with gemcitabine.
Mean ± SD caspase-3/7 activity, measured in relative fluorescence units (RFU) with a commercial assay, in canine OSA cell lines OSA8 (A), OSA16 (B), OSA32 (C), and OSA36 (D) following incubation with gemcitabine (0, 1, 10, or 100μM) alone (closed squares) or in conjunction with carboplatin (250μM; open squares) for 24 hours. *Significantly (P < 0.05) different from value obtained when cells were incubated with gemcitabine alone. †Significantly (P < 0.05) different from value for untreated (ie, 0μM gemcitabine) control cells.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
Effects of gemcitabine in combination with pamidronate—For cells lines OSA8, OSA16, and OSA36, incubation with pamidronate alone at a concentration of 100μM resulted in a significant decrease in cell viability, compared with viability of untreated cells (Figure 5). In contrast, incubation of the OSA32 cell line with pamidronate alone did not result in a significant decrease in cell viability. For the OSA16 and OSA32 cell lines, but not the OSA8 and OSA36 cell lines, a significant decrease in cell viability was observed when cells were incubated with a combination of gemcitabine (10μM) and pamidronate (100μM), compared with viability when cells were incubated with gemcitabine alone (10μM). However, significant differences were not observed with any of the other gemcitabine-pamidronate combinations that were tested.
Mean ± SD cell viability for canine OSA cell lines OSA8 (A), OSA16 (B), OSA32 (C), and OSA36 (D) following incubation with gemcitabine at concentrations of 0μM (open triangles), 10μM (open circles), or 100μM (closed squares) in conjunction with various concentrations of pamidronate (0, 1, 10, or 100μM) for 72 hours. *Significantly (P < 0.05) different from viability for untreated control cells.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
Effects of gemcitabine in combination with carboplatin—For all 4 canine OSA cell lines, cell viability was significantly decreased when cells were incubated with a combination of gemcitabine and carboplatin, compared with viability when cells were incubated with carboplatin alone (Figure 6). Evaluation of CI values (Figure 7) indicated that for all 4 cell lines, gemcitabine and carboplatin interacted synergistically, in that most CI values were < 1 over a range of drug effect levels. Strong synergism (CI < 0.3) occurred at drug effect levels corresponding to drug concentrations < for gemcitabine and 100μM for carboplatin in all cell lines, except that for the OSA8 and OSA16 cell lines, the drug interaction was not synergistic at drug effect levels < 0.2 (corresponding to 0.0625μM gemcitabine and 6.25μM carboplatin), and for the OSA36 cell line, the drug interaction was only moderately synergistic at drug effect levels < 0.2. For all cell lines, moderate to slight synergism was observed at drug effect levels corresponding to concentrations > for gemcitabine and 100μM for carboplatin. Calculation of the dose-reduction index indicated that the concentration of gemcitabine necessary to inhibit growth of 50% of OSA cells could be reduced by 15.5-fold (OSA8), 18.8-fold (OSA16), 46.6-fold (OSA32), and 21.4-fold (OSA36) with the addition of carboplatin.
Mean ± SD cell viability for canine OSA cell lines OSA8 (A), OSA16 (B), OSA32 (C), and OSA36 (D) following incubation with gemcitabine (0, 1, 10, and 100μM) alone (closed squares) or in combination with carboplatin (250μM; open squares). ^Significantly (P < 0.05) different from value obtained when cells were incubated with gemcitabine alone.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
Combination index values for canine OSA cell lines OSA8 (A), OSA16 (B), OSA32 (C), and OSA36 (D) following incubation with gemcitabine and carboplatin alone and in combination at a fixed gemcitabine-to-carboplatin concentration ratio of 1:100 for 72 hours. Fraction affected equates to percentage reduction in cell viability. A CI value > 1 indicates synergy.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
For the OSA8, OSA16, and OSA36 cell lines, caspase-3/7 activity was significantly higher when cells were incubated with gemcitabine in combination with carboplatin, compared with activity when cells were incubated with gemcitabine alone (Figure 4). For the OSA32 cell line, there was a significant increase in caspase-3/7 activity when cells were incubated with gemcitabine at concentrations of 1 or 100μM in combination with carboplatin, but not when incubated with gemcitabine at a concentration of 10μM in combination with carboplatin.
For the OSA8 and OSA36 cell lines, cell viability was significantly lower when cells were incubated with carboplatin first, then gemcitabine, compared with viability when cells were incubated with gemcitabine followed by carboplatin (Figure 8). For the OSA8 cell line, a significant interaction between order of drug administration and concentration of gemcitabine was not detected, indicating that the magnitude of the difference did not differ with concentration of gemcitabine. For the OSA36 cell line, a significant interaction was identified; however, cell viability was consistently lower when carboplatin was administered first. For the OSA16 cell line, significant effects of drug administration order were detected only with gemcitabine concentrations of 1 and 100μM. For the OSA32 cell line, the sequence of drug administration did not have a significant effect on cell viability.
Mean ± SD cell viability for canine OSA cell lines OSA8 (A), OSA16 (B), OSA32 (C), and OSA36 (D) following incubation with gemcitabine (1, 10, or 100μM) for 2 hours followed by incubation with carboplatin (250μM) for 2 hours (open circles) and following incubation with carboplatin (250μM) for 2 hours followed by incubation with gemcitabine (1, 10, or 100μM) for 2 hours (closed squares). *Values were significantly (P < 0.017) different at the indicated gemcitabine concentration. †Values were significantly (P < 0.001) different at the indicated gemcitabine concentration.
Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.799
Discussion
In the present study, we evaluated the in vitro activity of gemcitabine against canine OSA cell lines alone and in combination with compounds known to have activity against OSA. For all 4 cell lines, dose-dependent growth inhibition was observed when the cell lines were incubated with gemcitabine, and for 3 of the 4 cell lines, IC50 concentrations ranged from 5.7 to 15.3μM (for the remaining cell line, an IC50 concentration could not be calculated). These results are similar to those reported for human OSA cells treated with gemcitabine in vitro.12 A previous study25 of the pharmacokinetics of gemcitabine in dogs found that administration of gemcitabine at the maximum tolerated dose (22 mg/kg or 675 mg/m2) resulted in plasma concentrations of 20 to 30 μg/mL, equivalent to 67 to 100μM. However, continuous bathing of OSA cells in gemcitabine for 72 hours does not reflect the in vivo pharmacokinetics. Therefore, to account for the short elimination half-life of gemcitabine, OSA cell lines in the present study were also incubated with gemcitabine for only 2 hours. Although differences in cell viability were observed when results of incubation for 2 versus 72 hours were compared, a significant difference was not consistently observed across all concentrations, and the magnitude of the decrease in cell viability was similar for the 2 exposure durations at each gemcitabine concentration. Interestingly, when a significant difference was observed, most often, cell viability was lower after 2 hours of incubation with gemcitabine than after 72 hours of incubation.
We also found in the present study a dose-dependent increase in the proportion of cells in the sub-G0/G1 phase following incubation of all 4 cell lines with gemcitabine. For cell lines OSA8 and OSA36, a corresponding increase in caspase-3/7 activity was identified, suggesting that apoptosis was the mechanism of cell death. For cell lines OSA16 and OSA32, the increase in the proportion of cells in the sub-G0/G1 phase following incubation with gemcitabine was modest and a significant increase in caspase-3/7 activity was not identified, indicating that the OSA16 and OSA32 cell lines were more resistant to the effects of gemcitabine. These data suggest that gemcitabine may act, in part, by inhibiting cell viability in resistant OSA cell populations, rather than directly inducing cell death. Moreover, it may explain the fact that in clinical trials involving human patients with metastatic OSA, gemcitabine primarily caused disease stabilization.
Given the potential resistance of OSA cells to single-agent chemotherapy, we evaluated gemcitabine in combination with compounds known to have activity against OSA to determine whether additive or synergistic effects could be identified. Pamidronate has been evaluated as a potentially active compound against canine OSA cell lines in vitro, with concentrations ranging from 100 to 1,000μM resulting in decreased cell viability of OSA cell lines after 48 and 72 hours of drug exposure.18 Our results were consistent with these published results. However, recent clinical trials did not find a significant survival benefit when pamidronate was added to chemotherapy treatment following amputationm or during palliative radiation therapy in combination with chemotherapy.26 Published data regarding peak tissue concentrations achieved following pamidronate administration are not available; however, peak tissue concentrations following administration of zoledronate have been evaluated, with concentrations approaching 1μM in soft tissue, 5μM in healthy bone, and > 5μM in areas of pathological bone resorption.11 Pamidronate is a second-generation bisphosphonate and is not as potent as the third-generation bisphosphonate zoledronate. Given findings for zoledronate, it is unlikely that concentrations of pamidronate > 100μM can be consistently achieved in vivo. Therefore, our data indicate that at biologically relevant drug concentrations, pamidronate does not inhibit viability of OSA cell lines when used as a single agent or in combination with gemcitabine. As such, it is unlikely that combinations of pamidronate and gemcitabine would exhibit biological activity in vivo.
Carboplatin and gemcitabine are ideal candidates for use in combination, as they have different but complementary mechanisms of action and acceptable toxicity profiles. Indeed, these chemotherapeutics have demonstrated synergistic activity in the treatment of a variety of malignancies in human patients. In the present study, a significant decrease in cell viability was identified for all 4 OSA cell lines when carboplatin and gemcitabine were used in combination, compared with cell viability when carboplatin was used alone. Interestingly, for cell lines OSA16 and OSA32, the gemcitabine-carboplatin combination decreased cell viability by > 50% when gemcitabine concentrations > 10μM were used. For all 4 cell lines, the decrease in cell viability obtained when either drug was used alone was not as great as that obtained when the drugs were combined. Importantly, the combination of gemcitabine and carboplatin was synergistic for all 4 OSA cell lines. These data are consistent with findings for a variety of human cancer cell lines.
Results of a clinical study21 in which carboplatin and gemcitabine were administered on the same day to dogs suggest that the recommended doses of these drugs are 10 mg of carboplatin/kg and 2 mg of gemcitabine/kg, given 4 hours apart. These doses correspond to plasma concentrations of gemcitabine of approximately 10μM and carboplatin of approximately 250μM, which are similar to the drug concentrations at which synergistic drug activity was observed in the present study.
It has been hypothesized that when carboplatin and gemcitabine are given together, gemcitabine inhibits DNA repair of platinum-induced damage through its inhibition of ribonucleotide reductase. Gemcitabine is also incorporated into DNA during the repair process, thereby contributing to inhibition of further DNA replication and repair. Thus, it has been suggested that when gemcitabine is administered in conjunction with a platinum compound, the gemcitabine be administered before the platinum compound to ensure that a sufficient intracellular concentration of gemcitabine is present prior to initiation of DNA damage by the platinum compound. In the present study, a significant and consistent decrease in cell viability was achieved when OSA cell lines OSA8 and OSA36 were treated with carboplatin prior to gemcitabine, compared with cell viability when the treatment schedule was reversed. This was also true for the OSA16 cell line for most of the gemcitabine concentrations used. Conversely, for the OSA32 cell line, the sequence of drug administration did not have a significant effect on cell viability. We hypothesize that the increase in activity observed in 3 of the 4 OSA cell lines when carboplatin was administered prior to gemcitabine was due to inhibition of ribonucleotide reductase by the platinum drug, leading to a decrease in deoxycytidine triphosphate.27,28 We further hypothesize that the decrease in deoxycytidine triphosphate in turn released the inhibition of deoxycytidine kinase by deoxycytidine triphosphate, the rate-limiting enzyme in the activation of gemcitabine. If this were the case, gemcitabine activity would have increased substantially as a result of the increase in deoxycytidine kinase activity and increased incorporation of gemcitabine into DNA. Gemcitabine is also an inhibitor of ribonucleotide reductase thus contributing to further inhibition of DNA replication and repair.
The clinical effects of drug sequence have been evaluated extensively, and results have been inconsistent. In 1 study,15 administration of carboplatin in conjunction with gemcitabine resulted in synergistic activity against a lung carcinoma cell line only when carboplatin was administered prior to gemcitabine. It was further demonstrated that administration of carboplatin 4 hours prior to gemcitabine was associated with higher response rates and longer survival times in human patients with non-small cell lung cancer, compared with results for patients treated with these drugs in the reverse order.15 In contrast, another study29 demonstrated that the sequence of administration of carboplatin and gemcitabine did not affect toxicity, pharmacodynamics, the maximum tolerated dose, or response rates in patients with non-small cell lung cancer. These conflicting data suggest that the actual sequence of gemcitabine and platinum compound administration may not be the determining factor regarding response to therapy.
The present study offers new data regarding the biological activity of gemcitabine with or without carboplatin against canine OSA cell lines. However, there were several limitations that should be addressed regarding the in vitro nature of the study. First, despite the activity of gemcitabine as a single agent and in combination with carboplatin against OSA cell lines, our results may not accurately reflect the true activity of these drugs in vivo in dogs with OSA owing to their complicated pharmacokinetics. Second, although we attempted to mimic in vivo drug exposure by conducting some experiments with only a 2-hour incubation period, we did not conduct all experiments in this manner. For example, experiments in which carboplatin was combined with gemcitabine (with the exception of the sequence of administration experiments) were conducted with 72hour drug exposure durations. Because we know that carboplatin has activity in dogs with OSA,30,31 our goal in the combination experiments was to demonstrate that gemcitabine in combination with carboplatin had more activity against canine OSA cell lines, compared with carboplatin alone, and that the combination was synergistic. It is possible that by decreasing the duration of drug exposure from 72 to 2 hours, results would have been different. However, we believe that the drug combination would continue to have been more effective, compared with carboplatin alone, given the results of our sequence of administration studies.
In summary, our data demonstrate that as is the case with human OSA cell lines, gemcitabine has biological activity against canine OSA cell lines and that this activity is synergistic when gemcitabine is combined with carboplatin at biologically relevant drug concentrations. These results lay the groundwork for future evaluations of gemcitabine administered alone or in combination with carboplatin for dogs with OSA.
ABBREVIATIONS
| CI | Combination index |
| IC50 | Concentration required for 50% inhibition of cell viability |
| OSA | Osteosarcoma |
| WST-1 | Water soluble tetrazolium-1 |
Provided by Dr. Jaime Modiano, College of Veterinary Medicine and Cancer Center, University of Minnesota, Minneapolis, Minn.
Gibco/Invitrogen, Grand Island, NY.
Gemini, Sacramento, Calif.
Eli Olly, Indianapolis, Ind.
Roche, Indianapolis, Ind.
Molecular Devices, Sunnyvale, Calif.
Cell Quest Pro software, Becton-Dickinson Biosciences, San Jose, Calif.
SensoEyte Homogenous AMC Caspase-3/7 Assay Kit, AnaSpec, San Jose, Calif.
Teva Pharmaceuticals, North Wales, Pa. j. Hospira Inc, Eake Forest, Ill.
Calcusyn, version 1.2, Biosoft, Cambridge, England.
SAS, version 9.1, SAS Institute Inc, Cary, NC.
Kozicki A, Chun R, Kurzman I, et al, School of Veterinary Medicine, University of Wisconsin, Madison, Wis: Personal communication, 2008.
Fan T, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Ill: Personal communication, 2008.
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