• View in gallery

    Representative scan event chromatograms of canine blank plasma spiked with the internal standard, pioglitazone (500 ng/mL [negative control]). The upper panel represents the chromatogram for the protonated pioglitazone ion [M + H]+ (m/z, 3571 [monitored ions, 135, 199, and 106, with a collision energy of 27, 43, and 44 V, respectively]). The lower panel represents the chromatogram for the protonated rosiglitazone [M + H]+ (m/z, 358.1 [monitored ions, 135, 119, and 107, with a collision energy of 37 53, and 47 V, respectively]). The peak in the lower panel represents cross talk because the pioglitazone and rosiglitazone differ in size by only 1 Da.

  • View in gallery

    Representative scan event chromatograms of canine blank plasma spiked with 500 ng of pioglitazone/mL and 300 ng of rosiglitazone/mL. The upper and lower panels represent the chromatograms for the protonated pioglitazone and rosiglitazone ions, respectively, as in Figure 1. The unintegrated peak seen at 6.69 minutes represents the carbon 13 isotope of pioglitazone.

  • View in gallery

    Representative scan event chromatograms of canine plasma obtained 3 hours after oral administration of 4 mg of rosigliatzone/m2 and spiked with 500 ng of pioglitazone/mL. The upper and lower panels represent the chromatograms for the protonated pioglitazone and rosiglitazone ions, respectively, as in Figure 1. The unintegrated peak seen at 6.69 minutes represents the carbon 13 isotope of pioglitazone.

  • View in gallery

    Calibration curve of rosiglitazone over a range of 1 to 1,000 ng/mL in canine plasma.

  • View in gallery

    Plasma rosiglitazone concentration-time profiles of 3 cancer-bearing dogs.

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Evaluation of an extractionless high-performance liquid chromatography-tandem mass spectrometry method for detection and quantitation of rosiglitazone in canine plasma

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  • 1 Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.
  • | 2 Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.
  • | 3 Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.
  • | 4 Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.
  • | 5 Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.
  • | 6 Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.
  • | 7 Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

Abstract

Objective—To develop a simple extractionless method for detection of rosiglitazone in canine plasma and test the method in a pharmacokinetic study after oral administration of rosiglitazone in dogs.

Animals—3 client-owned dogs with cancer.

Procedures—High-performance liquid chromatography-tandem mass spectrometry was performed on canine plasma. The 3 dogs with cancer in the pharmacokinetic study were assessed via physical examination and clinicopathologic evaluation and considered otherwise healthy. Food was withheld for 12 hours, and dogs were administered a single dose (4 mg/m2) of rosiglitazone. Plasma was collected at various times, processed, and analyzed for rosiglitazone.

Results—The developed method was robust and detected a minimum of 0.3 ng of rosiglitazone/mL. Mean ± SD maximum plasma concentration was 205.2 ± 79.1 ng/mL, which occurred at 3 ± 1 hours, and mean ± SD elimination half-life was 1.4 ± 0.4 hours. The area under the plasma rosiglitazone concentration-versus-time curve varied widely among the 3 dogs (mean ± SD, 652.2 ± 351.3 ng/h/mL).

Conclusions and Clinical Relevance—A simple extractionless method for detection of rosiglitazone in canine plasma was developed and was validated with excellent sensitivity, accuracy, precision, and recovery. The method enabled unambiguous evaluation and quantitation of rosiglitazone in canine plasma. This method will be useful for pharmacokinetic, bioavailability, or drug-drug interaction studies. Oral rosiglitazone administration was well tolerated in the dogs.

Abstract

Objective—To develop a simple extractionless method for detection of rosiglitazone in canine plasma and test the method in a pharmacokinetic study after oral administration of rosiglitazone in dogs.

Animals—3 client-owned dogs with cancer.

Procedures—High-performance liquid chromatography-tandem mass spectrometry was performed on canine plasma. The 3 dogs with cancer in the pharmacokinetic study were assessed via physical examination and clinicopathologic evaluation and considered otherwise healthy. Food was withheld for 12 hours, and dogs were administered a single dose (4 mg/m2) of rosiglitazone. Plasma was collected at various times, processed, and analyzed for rosiglitazone.

Results—The developed method was robust and detected a minimum of 0.3 ng of rosiglitazone/mL. Mean ± SD maximum plasma concentration was 205.2 ± 79.1 ng/mL, which occurred at 3 ± 1 hours, and mean ± SD elimination half-life was 1.4 ± 0.4 hours. The area under the plasma rosiglitazone concentration-versus-time curve varied widely among the 3 dogs (mean ± SD, 652.2 ± 351.3 ng/h/mL).

Conclusions and Clinical Relevance—A simple extractionless method for detection of rosiglitazone in canine plasma was developed and was validated with excellent sensitivity, accuracy, precision, and recovery. The method enabled unambiguous evaluation and quantitation of rosiglitazone in canine plasma. This method will be useful for pharmacokinetic, bioavailability, or drug-drug interaction studies. Oral rosiglitazone administration was well tolerated in the dogs.

Rosiglitazone ((RS)-5-[4-(2-[methyl(pyridin-2-yl) amino]ethoxy)benzyl]thiazolidine-2,4-dione) is a synthetic PPAR7 agonist and an effective FDA-approved antidiabetic agent in humans.1 Peroxisome proliferator-activated receptor 7 is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors that plays a critical role in multiple cellular processes including energy metabolism and differentiation.2,3 Results of multiple studies indicate that PPARγ can function as a tumor suppressor.3–20 Consequently, ligands that behave as PPARγ agonists have been investigated as inhibitors of angiogenesis, for their ability to reduce tumor cell growth, and for their ability to regulate growth, differentiation, and gene regulation in a number of cancer cells and in murine preclinical models.3,7–19,21 Interestingly, synergistic antineoplastic activity between rosiglitazone and carboplatin, a platinum-based chemotherapeutic agent used extensively in both human and veterinary cancer clinics, has been detected via in vitro and in vivo murine studies, indicating that this combination may enhance tumor control.10,21 Although several pharmacokinetic studies22–27 have been described for rosiglitazone in humans, no pharmacokinetic studies have been described in dogs. Furthermore, no methods for determination of the concentration of rosiglitazone in canine plasma have been reported. Therefore, the purpose of the study reported here was to develop a simple extractionless method for detection of rosiglitazone in canine plasma and test the method in a pharmacokinetic study in dogs after oral administration of rosiglitazone.

Materials and Methods

Dogs—This study was approved by the Animal Care and Use Committee at the University of California-Davis Veterinary Medical Teaching Hospital. After the analytic method was validated, it was applied to a pharmacokinetic study of rosiglitazone in 3 client-owned, otherwise healthy, cancer-bearing dogs with informed consent. To be included in the study, dogs were required to have serum creatinine, alanine aminotranfersase, alkakine phosphatase, and total bilirubin concentrations or activities within reference ranges and be free of evidence of cardiac disease. A CBC, serum biochemical panel, and urinalysis were performed prior to administration of rosiglitazone. Dogs ranged from 7 to 10.4 years in age and from 21.1 to 27.7 kg in weight and included a spayed female Border Collie (dog 1), a castrated male terrier (dog 2), and a spayed female German Shepherd Dog (dog 3).

Rosiglitazone (4 mg/m2) was administered orally to dogs after food was withheld for 12 hours. Dogs readily swallowed the tablets. Dogs were hospitalized for 28 hours for observation and venous blood samples were collected into heparinized evacuated tubes from a jugular vein catheter prior to drug administration (time 0) and then at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, and 24 hours following administration. The blood samples were centrifuged at 4,000 × g for 15 minutes at 22 ± 2°C, and the plasma was removed and stored in a cryogenic vial at −80°C until analysis. Plasma rosiglitazone concentrations were inspected on a semilogarithmic plot of concentration versus time for each dog. Pharmacokinetic parameters were evaluated from the plasma concentration-time profile.

All dogs were administered carboplatin IV 24 hours after initiation of the pharmacokinetic study. Dogs were discharged with instructions for owners to administer 4 mg of rosiglitazone/m2, PO once daily and a questionnaire for owners to monitor drug administration and variables such as water intake, appetite, urination, vomiting, energy level, attitude, exercise tolerance, fecal consistency, and frequency of defecation. Toxicoses were graded by the authors according to the Veterinary Cooperative Oncology Group criteria for adverse events.28 Dogs 1 and 3 received carboplatin at 300 mg/m2, IV. Dog 2 was administered carboplatin at 280 mg/m2 because of a grade III thrombocytopenia28 that occurred at a dose of 300 mg/m2 administered prior to enrollment in the study. A CBC was performed 7 to 14 days after carboplatin administration, and a CBC and a complete serum biochemical panel were performed 3 to 6 weeks after initiation of rosiglitazone administration. Dogs were followed for a minimum of 6 weeks with daily administration of rosiglitazone at 4 mg/m2 along with carboplatin administered IV every 3 weeks.

Apparatus, chemicals, and reagents—The HPLC systema was interfaced to a mass spectrometerb by use of an electrospray interface. Instrument control and data acquisition were performed by use of software.c A 10-μL aliquot of the sample was injected into the system by use of an autosampler.a The LC separation was performed on a carbon 18 columnd (2 μm; 100 × 2.1 mm) with a precolumn containing the same resin. Column temperature was maintained at 27 ± 2°C.

Rosiglitazonee and the internal standard, pioglitazone,f were of > 99% purity. All solvents were of HPLC-tandem mass spectrometry grade and included acetonitrile,g purified water,g methanol,h glacial acetic acid,h and 98% pure formic acid.i Blank canine plasma samples were obtained from healthy canine blood donors from the University of California-Davis blood bank.

Acetonitrile and 0.2% formic acid were selected as mobile-phase solvents. A gradient program was used, and the percentage of organic solvent changed linearly as follows: 0 minutes, 10%; 0.4 minutes, 10%; 7.5 minutes, 50%; 8.5 minutes, 90%; 9 minutes, 90%; 9.01 minutes, 10%; and 14 minutes, 10%. Flow rate was 0.4 mL/min.

Nitrogen was used as the sheath gas and the auxiliary gas, with the sheath gas flow rate set at 45 lb/square inch and the auxillary gas at 10 (arbitrary units). A spray voltage of 4,000 V was used in positive ionization mode. The capillary temperature was set to 355°C and the source collision-induced dissociation setting to 2 units. Argonj was used as the collision gas with a gas pressure of 2.0 mm Hg. The tube lens voltage was 126 V The peak width was 0.50. Selected reaction monitoring was used with 3 transitions monitored for rosiglitazone and the internal standard. For rosiglitazone, the protonated ion [M+H]+ 358.1 was isolated and fragmented, with the monitored ions being 135, 119, and 107 with a collision energy of 37, 53, and 47 V, respectively. For pioglitazone, the protonated ion [M+H]+ 357.1 was isolated and fragmented, with the monitored ions being 135, 199, and 106 with collision energies of 27, 43, and 44 V, respectively. For all transitions, the scan width was 0.01 m/z, scan time was 0.25 seconds, and quadrupole 1 and quadrupole 3 (Q1 and Q3) had a peak width of 0.5 Da. The sum of all 3 selected reaction monitoring transitions was used for quantitation.

Preparation of stock and standard solutions—Stock solutions of rosiglitazone and pioglitazone were prepared separately. The stock solution of rosiglitazone was prepared in methanol to yield the primary standard solution with a concentration of 1 mg/mL. The working standard solutions of rosiglitazone were prepared in volumetric flasks by diluting the primary standard solution with methanol, giving final concentrations of 10 and 1 ng/μL. The quality control samples (10, 50, 300, and 500 ng/mL) were prepared by dilution of the working standard solution with drug-free canine plasma. The stock solution of pioglitazone was prepared in dimethyl sulfoxide to yield the primary standard solution with a concentration of 1 mg/mL. The working standard solution of pioglitazone was prepared in a volumetric flask by diluting the primary standard solution with 9 parts acetonitrile to 1 part 1M acetic acid (vol:vol), giving a final concentration of 500 ng/mL. Calibration samples ranged from 1 to 1,000 ng/mL. The desired concentration of rosiglitazone was placed in chromocol vials and evaporated to dryness under nitrogen stream. The samples were reconstituted with drug-free canine plasma.

Preparation of samples—All quality controls, calibration curve, plasma, and patient samples were prepared as follows. The samples were mixed with equal volumes of internal standard with 9 parts acetonitrile to 1 part 1M acetic acid (vol:vol) to precipitate proteins. The samples were capped, shaken for 60 seconds at 22 ± 2°C, refrigerated at 4°C for 20 minutes, shaken at 22 ± 2°C for 20 seconds, and centrifugedk at 3,830 × g for 10 minutes at 4°C; then a 10-μL aliquot of the supernatant was injected into the system for analysis.

Calibration curve—The linearity of the method was evaluated by use of a calibration curve in the range of 1 to 1,000 ng/mL of rosiglitazone, including the LLOQ. Blank canine plasma was spiked with rosiglitazone at concentrations of 1, 10, 20, 50, 100, 200, 300, 400, 500, and 1,000 ng/mL, and the internal standard was spiked at a concentration of 500 ng/mL. The calibration was obtained by plotting the area ratios of rosiglitazone and internal standard versus the concentration of rosiglitazone via least squares linear regression analysis.

Accuracy and precision—For intraday accuracy and precision, samples were spiked with rosiglitazone at concentrations of 1, 50, 500, and 3,000 ng/mL (n = 6 each). For interday accuracy and precision, samples were spiked with rosiglitazone at concentrations of 1, 50, 500, and 3,000 ng/mL (n = 18 each). The LLOQ of the method was defined as the lowest concentration at which acceptable reproducibility could be guaranteed.

Stability—Stability of rosiglitazone in canine plasma was tested by use of quality control samples for 3 freeze-thaw cycles (−80°C; 22°C), at room temperature (22°C), and at refrigeration temperature (4°C). The stability of rosiglitazone in canine plasma was determined over 3 freeze-thaw cycles (−80° to 22°C) within 3 days. In each freeze-thaw cycle, the spiked canine plasma samples were frozen (−80°C) for approximately 24 hours, thawed at 22°C for 15 to 30 minutes, and returned to the freezer or processed and analyzed. The stability of the 10 ng/mL quality control sample was tested after keeping the samples at 22°C for 4, 8, 24, 48, and 72 hours. The stability of the quality control sample was also tested after keeping the samples at 4°C for 4, 8, 24, 28, and 72 hours. Results were compared with those obtained from freshly prepared samples. The long-term stability of 9 randomly selected patient samples was assessed after 6 months of storage at −80°C.

Results

Accuracy, precision, and sensitivity—Patient-matched blank plasma obtained prior to treatment with rosiglitazone or from canine blood donors served as blank samples. Representative chromatograms were prepared from canine blank plasma spiked with the internal standard (500 ng/mL [Figure 1]), canine blank plasma spiked with rosiglitazone (300 ng/mL) and internal standard (500 ng/mL [Figure 2]), and canine plasma obtained 2 to 4 hours after oral administration of rosiglitazone (4 mg/m2) and spiked with the internal standard (500 mg/mL [Figure 3]). Because the peaks of rosiglitazone and the internal standard pioglitazone were well resolved temporally, with retention times of 6.10 and 6.67 minutes, respectively, identification of both compounds was assured. The unintegrated peak at 6.69 minutes represents the carbon 13 isotope of pioglitazone. No endogenous peak from plasma was found to interfere with the elution of either rosiglitazone or the internal standard. Analysis could be achieved within the 14-minute duration of the total chromatography run.

Figure 1—
Figure 1—

Representative scan event chromatograms of canine blank plasma spiked with the internal standard, pioglitazone (500 ng/mL [negative control]). The upper panel represents the chromatogram for the protonated pioglitazone ion [M + H]+ (m/z, 3571 [monitored ions, 135, 199, and 106, with a collision energy of 27, 43, and 44 V, respectively]). The lower panel represents the chromatogram for the protonated rosiglitazone [M + H]+ (m/z, 358.1 [monitored ions, 135, 119, and 107, with a collision energy of 37 53, and 47 V, respectively]). The peak in the lower panel represents cross talk because the pioglitazone and rosiglitazone differ in size by only 1 Da.

Citation: American Journal of Veterinary Research 72, 2; 10.2460/ajvr.72.2.263

Figure 2—
Figure 2—

Representative scan event chromatograms of canine blank plasma spiked with 500 ng of pioglitazone/mL and 300 ng of rosiglitazone/mL. The upper and lower panels represent the chromatograms for the protonated pioglitazone and rosiglitazone ions, respectively, as in Figure 1. The unintegrated peak seen at 6.69 minutes represents the carbon 13 isotope of pioglitazone.

Citation: American Journal of Veterinary Research 72, 2; 10.2460/ajvr.72.2.263

Figure 3—
Figure 3—

Representative scan event chromatograms of canine plasma obtained 3 hours after oral administration of 4 mg of rosigliatzone/m2 and spiked with 500 ng of pioglitazone/mL. The upper and lower panels represent the chromatograms for the protonated pioglitazone and rosiglitazone ions, respectively, as in Figure 1. The unintegrated peak seen at 6.69 minutes represents the carbon 13 isotope of pioglitazone.

Citation: American Journal of Veterinary Research 72, 2; 10.2460/ajvr.72.2.263

Three rosiglitazone analytic series in duplicate were used to verify the calibration curve for the range of rosiglitazone of 1 to 1,000 ng/mL in canine plasma. The calibration curve was quadratic (r2 = 0.999; Figure 4) with the best fit equation as follows:
article image
2
where area ratio is the peak area ratio of the analyte to internal standard and concentration is the concentration of the analyte.
Figure 4—
Figure 4—

Calibration curve of rosiglitazone over a range of 1 to 1,000 ng/mL in canine plasma.

Citation: American Journal of Veterinary Research 72, 2; 10.2460/ajvr.72.2.263

The accuracy and precision of the calibration curve standards were determined (Table 1). The LLOQ of rosiglitazone in canine plasma was verified as 1 ng/mL. This concentration represented accuracy from 80% to 120% with a precision within 20%. Accuracy and precision of the LLOQ were 112% and 5%, respectively. The LLOQ was 0.3 ng/mL at a signal-to-noise ratio of 3.

Table 1—

Accuracy and precision of calibration standards of a method for determining the concentration (ng/mL) of rosiglitazone in 6 canine plasma samples.

Known concentrationDetermined concentration (mean ± SD)Accuracy (%)Precision (CV [%])
1.01.1 ± 0.12110.412.3
10.09.5 ± 0.8494.88.4
20.017.9 ± 1.5489.57.7
50.048.3 ± 3.4696.56.9
100.0105.0 ± 5.56105.05.6
200.0208.2 ± 12.35104.16.2
300.0302.9 ± 18.67101.06.2
400.0402.8 ± 12.70100.73.2
500.0493.7 ± 25.1598.75.0
1,000.01,058.0 ± 7.74105.80.8

CV = Coefficient of variation.

Results of intraday and interday accuracy and precision for rosiglitazone at concentrations of 1 to 3,000 ng/mL of canine plasma were summarized (Table 2). Interday accuracy and precision ranged from 76.3% to 111.6% and 2.1% to 12.6%, respectively. Intraday accuracy and precision ranged from 74.2% to 126.7% and 1% to 8.2%, respectively.

Table 2—

Intraday and interday accuracy and precision of a method for determining the concentration (ng/mL) of rosiglitazone in canine plasma samples.

Known concentrationDetermined concentration (mean ± SD)Accuracy (%)Precision (CV [%])
Intraday (n = 6)
11.1 ± 0.1107.05.9
5039.5 ± 0.879.01.5
500488.0 ± 5.197.61
3,0003007.7 ± 38.0100.31.3
Interday (n = 18)
11.1 ± 0.1111.612.6
5038.1 ± 1.476.32.8
500480.8 ± 10.696.22.1
3,0002649.8 ± 304.688.310.2

CV = Coefficient of variation.

Stability—Rosiglitazone (10 ng/mL) stability in canine plasma was determined (Table 3). There was only a +0.36% deviation from the original concentration of 10 ng/mL, which confirmed the stability through 3 freezethaw cycles. Rosiglitazone in canine plasma was stable at 22° and 4°C for up to 72 hours. Deviation from 10 ng/mL ranged from 2% to 10.6% and 2.8% to 13.6% in the refrigerator. Rosiglitazone stability in canine plasma after oral administration and storage for 6 months at −80°C was determined (Table 4). Rosiglitazone was not found to be stable in canine plasma stored at −80°C for 6 months. Deviation of the 9 long-term samples was 65.3% (range, 52.6% to 73.4%).

Table 3—

Concentrations of rosiglitazone (10 ng/mL) in canine plasma at 20° and 4°C after various storage periods. Deviation is the percentage by which the concentration differs from the original concentration of 10 ng/mL.

Temperature and timeMean concentrationDeviation (%)
20°C
4 h10.52
8 h9.610.6
24 h9.710.3
48 h9.98.3
72 h107.4
4°C
4 h10.24.8
8 h9.313.6
24 h9.412.9
48 h10.42.8
72 h10.24.7

Initial control sample concentration was 10.7 ng/mL.

Table 4—

Initial concentrations (ng/mL) of rosiglitazone and concentrations after storage at −80°C for 6 months.

InitialAfter 6 monthsDeviation (%)
150.3104.269.3
295.9163.055.1
50.332.564.6
29.422.677.1
63.850.078.4
230.6121.452.6
146.095.465.3
170.1106.562.6
142.294.266.2
113.474.866.0
239.7135.456.5

Pharmacokinetic application—To determine whether this method would be useful in a clinical veterinary setting, the method was applied to analyze plasma samples obtained from cancer-bearing dogs that were free of other diseases, after administration of a single dose of rosiglitazone (4 mg/m2). No adverse effects were detected for any of the dogs at the time the drug was given or during the 24-hour postadministration observation period.

There was marked interpatient variability in the plasma rosiglitazone concentration-time profiles for the 3 dogs (Figure 5). Mean ± SD maximum plasma concentration was 205.2 ± 79.1 ng/mL. The elimination half-life was 0.99 hours in dog 1, 1.37 hours in dog 2, and 1.80 hours in dog 3 (mean ± SD, 1.4 ± 0.4 hours). The area under the plasma rosiglitazone concentration-versus-time curve varied widely at 350.7 ng/h/mL for dog 1; 1,038 ng/h/mL for dog 2; and 567.9 ng/h/mL for dog 3 (mean ± SD, 652.2 ± 351.4 ng/h/mL).

Figure 5—
Figure 5—

Plasma rosiglitazone concentration-time profiles of 3 cancer-bearing dogs.

Citation: American Journal of Veterinary Research 72, 2; 10.2460/ajvr.72.2.263

Dog 1 had a transient grade 1 decrease in appetite28 after the first dose of rosiglitazone, then recovered with a normal appetite that remained through the duration of daily administration of rosiglitazone during a 6-week period. Dog 2 had grade 2 thrombocytopenia28 (90,000 platelets/μL) without clinical signs 14 days after administration of carboplatin at 280 mg/m2 and while receiving rosiglitazone daily at 4 mg/m2, PO. The platelet count had returned to the reference range 9 days later (23 days after beginning administration of rosiglitazone daily and 24 days after administration of carboplatin). After the second concurrent carboplatin dose, this dog also developed grade 2 diarrhea,28 which resolved with administration of metronidazole (10 mg/kg, PO, q 12 h) for 5 days. The diarrhea did not recur during sub-sequent treatments with rosiglitazone and carboplatin. Dog 3 had a grade 1 increase in blood creatinine concentration28 (1.3 mg/dL [reference range, 0.3 to 1.2 mg/dL) that resolved without intervention as well as a transient grade 1 anorexia28 that resolved without intervention. None of the dogs required hospitalization for adverse effects. All adverse effects resolved without long-term consequences.

Discussion

Rosiglitazone is an orally administered PPARγ agonist that may be of use as an antineoplastic agent in dogs. On the basis of preliminary data of in vitro and in vivo studies in mice, rosiglitazone may enhance the antitumor activity of carboplatin.

In the present study, rosiglitazone was absorbed in dogs after oral administration, although the interdog variability was high. Rosiglitazone in canine plasma had good stability through 1 to 3 freeze-thaw cycles and delayed processing (at 22° or 4°C), but not with delayed analysis (6 months at −80°C). A previous study27 of un-extracted rosiglitazone in humans revealed stability in human plasma stored at −20°C for 7 months. Possible explanations for the difference are that rosiglitazone is stable in plasma stored at −20°C but not in plasma stored at −80°C, that there are inherent differences between canine and human plasma, or that rosiglitazone is not stable after it has been metabolized (the present study used patient samples obtained after oral administration of rosiglitazone, whereas the previous study used spiked plasma samples). The HPLC method described here can be used for pharmacokinetic, bioavailability, or drug-drug interaction studies of rosiglitazone in dogs.

All 3 dogs in this study readily accepted and tolerated the orally administered rosiglitazone. Emesis of the tablets did not occur, and no adverse reactions were observed during the 24-hour observation period after the first dose. The combination of carboplatin and rosiglitazone was administered for 6 weeks (daily administration of rosiglitazone and IV administration of carboplatin every 3 weeks) with only mild adverse effects, and all dogs recovered from those without complications. Adverse events did not require hospitalization in any dog, and all dogs recovered without permanent long-term effects. These adverse events were similar to adverse events observed in patients that receive carbo-platin alone without rosiglitazone.29–33 Rosiglitazone appeared to be well tolerated after oral administration at 4 mg/m2 once daily for a minimum of 6 weeks in dogs.

ABBREVIATIONS

HPLC

High-performance liquid chromatography

LLOQ

Lower limit of quantitation

[M+H]+

Protonated molecular ion

m/z

Mass-to-charge ratio

PPARγ

Peroxisome proliferator—activated receptor γ

a.

Agilent 1100 HPLC System with Leap Autosampler, Thermo-Electron Corp, Woldbronn, Germany.

b.

Finnigan TSQ Quantum Ultra mass spectrometer, Thermo Finnigan LLC, San Jose, Calif.

c.

ThermoFisher Xcaliber software, version 2.0, ThermoFisher Corp, Waltham, Mass.

d.

ACE carbon 18 column, Advanced Chromatography Technologies, Aberdeen, Scotland.

e.

Fisher Scientific, Houston, Tex.

f.

Alexis Biochemicals, San Diego, Calif.

g.

Burdick and Jackson, Muskegon, Mich.

h.

Fischer Scientific, Fair Lawn, NJ.

i.

Acros Organics, Fair Lawn, NJ.

j.

AirGas, Radnor, Pa.

k.

Sorval Super T21 centrifuge, Sorvall Products LP, Newtown, Conn.

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Contributor Notes

Supported by the University of California-Davis Center for Companion Animal Health Grant #2007-45-R. Ms. Guerrero was supported by the Toni Wiebe Memorial Research Fund.

Address correspondence to Dr. Rodriguez (dvmrodriguez@ucdavis.edu).