• View in gallery

    Mean ± SEM cyclosporine concentrations measured in 8 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg) alone (black circles with dashed line) or oral administration of that same dose of cyclosporine 1 hour after oral administration of metoclopramide (0.3 to 0.5 mg/kg; white squares with dashed-and-dotted line), 2 g of PWG (black diamonds with solid line), or both metoclopramide (0.3 to 0.5 mg/kg) and 2 g of PWG (black squares with dotted line). A Latin square design was used with a 14-day washout period between treatments.

  • View in gallery

    Values for CL/F of cyclosporine determined in 8 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg, PO) alone or oral administration of that same dose of cyclosporine 1 hour after oral administration of metoclopramide (0.3 to 0.5 mg/kg), 2 g of PWG, or both metoclopramide (0.3 to 0.5 mg/kg) and 2 g of PWG. A Latin square design was used with a 14-day washout period between treatments. Each symbol and line combination represents results for 1 of the 8 dogs in the study.

  • View in gallery

    Mean ± SEM cyclosporine concentrations measured in 6 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg) alone (black circles with dashed line) or oral administration of that same dose of cyclosporine 1 hour after oral administration of 10 g of PWG (white squares with solid line). The 6 dogs are a subgroup of the 8 dogs included in part 1 of the study and whose results are reported in Figures 1 and 2; concentrations for cyclosporine alone reported here were extracted from the data for the same 6 dogs in part 1 of the study.

  • View in gallery

    Values for CL/F of cyclosporine determined in 6 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg, PO) alone or after oral administration of 10 g of PWG. The 6 dogs are a subgroup of the 8 dogs included in part 1 of the study and whose results are reported in Figures 1 and 2; concentrations for cyclosporine alone reported here were extracted from the data for the same 6 dogs in part 1 of the study. Each symbol and line combination represents results for 1 of the 6 dogs in the study.

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Effects of powdered whole grapefruit and metoclopramide on the pharmacokinetics of cyclosporine in dogs

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  • 1 Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
  • | 2 Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
  • | 3 Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
  • | 4 Department of Pharmacology and Experimental Therapeutics, School of Medicine, Tufts University, Boston, MA 02111.
  • | 5 Department of Pharmacology and Experimental Therapeutics, School of Medicine, Tufts University, Boston, MA 02111.

Abstract

Objective—To determine whether oral administration of metoclopramide or a commercially available powdered whole grapefruit (PWG) nutraceutical in combination with cyclosporine enhances systemic availability of cyclosporine in dogs.

Sample—8 healthy mixed-breed dogs in part 1 and 6 of these 8 dogs in part 2.

Procedures—Cyclosporine pharmacokinetics were determined over the course of 24 hours after oral administration of cyclosporine (5 mg/kg) alone, cyclosporine with metoclopramide (0.3 to 0.5 mg/kg), cyclosporine with 2 g of PWG, or cyclosporine combined with both metoclopramide and 2 g of PWG by use of a Latin square crossover study with a 14-day washout period between treatments. Sixty days later, 6 of the 8 dogs were given 10 g of PWG followed by cyclosporine, and pharmacokinetic parameters were compared with those previously obtained after administration of cyclosporine alone.

Results—Although metoclopramide or coadministration of metoclopramide and 2 g of PWG had no effect on the pharmacokinetic parameters of cyclosporine, compared with results for cyclosporine alone, the higher (10-g) dose of PWG resulted in 29% faster mean time to maximal plasma cyclosporine concentration, 54% larger area under the curve, and 38% lower apparent oral clearance.

Conclusions and Clinical Relevance—Adjustment of the cyclosporine dose may not be needed when metoclopramide is coadministered orally to prevent common adverse effects of cyclosporine. Powdered whole grapefruit has the potential to reduce the required orally administered dose of cyclosporine but only when PWG is used in an amount (at least 10 g) that is currently not cost-effective.

Abstract

Objective—To determine whether oral administration of metoclopramide or a commercially available powdered whole grapefruit (PWG) nutraceutical in combination with cyclosporine enhances systemic availability of cyclosporine in dogs.

Sample—8 healthy mixed-breed dogs in part 1 and 6 of these 8 dogs in part 2.

Procedures—Cyclosporine pharmacokinetics were determined over the course of 24 hours after oral administration of cyclosporine (5 mg/kg) alone, cyclosporine with metoclopramide (0.3 to 0.5 mg/kg), cyclosporine with 2 g of PWG, or cyclosporine combined with both metoclopramide and 2 g of PWG by use of a Latin square crossover study with a 14-day washout period between treatments. Sixty days later, 6 of the 8 dogs were given 10 g of PWG followed by cyclosporine, and pharmacokinetic parameters were compared with those previously obtained after administration of cyclosporine alone.

Results—Although metoclopramide or coadministration of metoclopramide and 2 g of PWG had no effect on the pharmacokinetic parameters of cyclosporine, compared with results for cyclosporine alone, the higher (10-g) dose of PWG resulted in 29% faster mean time to maximal plasma cyclosporine concentration, 54% larger area under the curve, and 38% lower apparent oral clearance.

Conclusions and Clinical Relevance—Adjustment of the cyclosporine dose may not be needed when metoclopramide is coadministered orally to prevent common adverse effects of cyclosporine. Powdered whole grapefruit has the potential to reduce the required orally administered dose of cyclosporine but only when PWG is used in an amount (at least 10 g) that is currently not cost-effective.

Cyclosporine is a potent immunosuppressive and immune modulatory drug used in humans1,2 and other animals.3,4 Although cyclosporine administration has obvious benefits, its commonly associated adverse effects, such as vomiting, weight loss, anorexia, and elevation in liver enzyme activities,4,5 are of considerable concern in veterinary medicine. Additionally, the expense of treatment with cyclosporine often precludes its routine use in veterinary patients. Thus, there is growing interest in veterinary medicine to reduce the overall daily dose of cyclosporine while maintaining therapeutic concentrations in the blood without inducing additional adverse effects.6

Despite development of optimized oral dosage forms of cyclosporine (eg, the currently available micro-emulsified formulation), the absolute bioavailability of cyclosporine in dogs remains relatively low (approx 35%).4,7 This low bioavailability is thought to result in part from extensive metabolism of cyclosporine by CYP3A enzymes located in the small intestinal mucosa and liver.3–5,8–10 Consequently, various attempts have been made to increase cyclosporine bioavailability by administering it in combination with CYP3A inhibitors that should attenuate the metabolism of cyclosporine and increase amounts of unmetabolized cyclosporine in the blood of both humans5,11–13 and dogs.5,6,14,15 Such combinations are examples of intentional therapeutic (as opposed to unintentional adverse) drug-drug interactions used to reduce the cost of expensive treatments.

In clinical veterinary medicine, cyclosporine frequently is administered with ketoconazole, a CYP3A inhibitor. Depending on the dose of ketoconazole administered, the total dose of cyclosporine may be decreased by 75% to 90% in dogs.14,15 Although ketoconazole may enhance the bioavailability of cyclosporine, ketoconazole often causes unwanted adverse effects, such as anorexia, nausea, vomiting, and elevated hepatocyte-specific enzyme activities in blood.16 Therefore, other drug combinations that increase cyclosporine bioavailability without introducing additional adverse effects need to be identified.

Studies in human17–21 and veterinary22 medicine have revealed that the coadministration of grapefruit juice with various drugs, including cyclosporine, can markedly improve drug bioavailability. In human studies,17,19 grapefruit juice administered prior to oral administration of cyclosporine enhanced the blood concentration of cyclosporine by 45% to 62%. Grapefruit juice contains substantial amounts of furanocoumarins, which are compounds that cause the distinctive bitter taste of this fruit juice. Furanocoumarins are also potent inhibitors of intestinal CYP3A enzymes,18 and it is believed that inhibition of CYP3A-mediated metabolism of cyclosporine is the main mechanism by which grapefruit juice increases cyclosporine bioavailability.18,20,23 In addition, grapefruit juice appears to have minimal effects on hepatic CYP3A.23 Consequently, furanocoumarins in grapefruit juice are unlikely to induce the adverse hepatic effects seen with ketoconazole administration.

Although oral administration of grapefruit juice is easily achieved in most human subjects, it is impractical for veterinary patients in part because of the juice's bitterness and acidity, which would likely limit voluntary ingestion by veterinary patients, and because of the relatively large volume of juice needed to achieve an inhibitory effect (as much as 100 mL). However, a PWG preparation in a convenient capsular form has been marketed as a nutraceutical for weight loss in humans. Consequently, we hypothesize that this preparation might provide a more convenient alternative to liquid grapefruit juice, be a safer alternative to ketoconazole, and reduce the required dose and related expense of cyclosporine treatment in veterinary patients.

Metoclopramide, an antiemetic and gastrointestinal prokinetic drug,24,25 frequently is coadministered with cyclosporine by veterinary dermatologists when initiating cyclosporine treatment in dogs to reduce several adverse effects of cyclosporine, including nausea and vomiting. There is also evidence in humans that metoclopramide may enhance the bioavailability of cyclosporine in that coadministration of metoclopramide with cyclosporine results in a small but significant increase (29%) in the AUC.26 The mechanism underlying this effect is currently unclear, although on the basis of results of an in vitro study,27 metoclopramide does not appear to inhibit CYP3A enzymes. To the authors' knowledge, there have been no studies conducted to evaluate the effect of metoclopramide on cyclosporine pharmacokinetics in dogs.

The primary purpose of the study reported here was to determine whether coadministration of a commercially available PWG nutraceutical could enhance the systemic availability of cyclosporine in a group of healthy dogs. We also evaluated the effects of metoclopramide on the pharmacokinetics of cyclosporine given alone or in combination with PWG in the same dogs.

Materials and Methods

Animals—Eight adult crossbred dogs from the University of Pennsylvania School of Veterinary Medicine research colony were included in part 1 of the study. These dogs comprised 2 sexually intact females and 6 sexually intact males with a mean age of 1.5 years (range, 1 to 2 years) and mean body weight of 17.5 kg (range, 13.9 to 21.2 kg). In part 2 of the study, only 6 of the original 8 dogs were used because 2 male dogs were not available for participation in the study at that time. Consequently, part 2 included 2 sexually intact females and 4 sexually intact males with a mean age of 1.5 years (range, 1 to 2 years) and mean body weight of 20 kg (range, 14.5 to 23.8 kg). Dogs were housed as pairs in temperature-controlled cages at 20° to 22°C and 50% to 60% humidity with a 12-hour light cycle. Dogs were assessed as healthy on the basis of results of physical examination, CBC, biochemical profile, and urinalysis performed 7 days prior to initiation of the study. The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Drugs and reagents—A commercially available PWGa (capsules that each contained 500 mg of PWG) was obtained. The recommended dose on the label of this product as a dietary supplement for humans was 1.5 to 3 g/d (ie, 3 to 6 capsules/d); thus, a dose of 2 g/dog (4 capsules/dog) was chosen for part 1 of the study. For part 2 of the study, a higher dose of 10 g/dog (20 capsules/dog) was evaluated on the basis of the results of an in vitro bioactivity study28 in which investigators used the same PWG product and suggested that a dose of up to 21 g might be required to achieve relevant CYP3A inhibition. Twenty capsules (10 g) were considered the maximum number that could be administered to dogs of this body size while maintaining gastric acceptability.

To ensure the consistency of the inhibitory effect between doses of the PWG product, the contents of representative capsules (n = 8) from the same lot used in this study were each assayed for their ability to inhibit activity of CYP3A (triazolam hydroxylation) in vitro by the use of dog liver microsomes. At a PWG concentration of 1.25 mg/mL, there was a mean decrease of 41% in triazolam 1′-hydroxylation activity with minimal variation among capsules (coefficient of variation, 4%). Complete details of the bioassay method and results have been reported elsewhere.28

Microemulsified cyclosporineb was used at a dose (5 mg/kg) recommended for the treatment of atopic dermatitis.7 Metoclopramidec was used at the antiemetic dose of 0.3 to 0.5 mg/kg.24

All drugs were administered orally via voluntary consumption when offered (most dogs readily consumed the 4-capsule dose of PWG) or, when necessary, by gently placing the drugs in the pharyngeal region and closing the dog's mouth until swallowing was observed. Voluntary consumption of the 20-capsule dose of PWG in part 2 of the study was aided by coating the capsules with a small amount of a maintenance diet as a flavor enhancer.

Effect of 2 g of PWG, metoclopramide, and their combination—Food was withheld from all dogs for 12 hours prior to all treatments. Water was available throughout the study. To account for possible carryover effects, the dogs were assigned to receive the following 4 treatments in a Latin square design: cyclosporine (5 mg/kg, PO, once), metoclopramide (0.3 to 0.5 mg/kg, PO; mean dose, 0.4 mg/kg) followed 1 hour later by cyclosporine (5 mg/kg, PO, once), PWG (2 g [ie, 4 capsules], PO; mean dose, 114 mg/kg) followed 1 hour later by cyclosporine (5 mg/kg, PO, once), and PWG (2 g, PO) with metoclopramide (0.3 to 0.5 mg/kg, PO) followed 1 hour later by cyclosporine (5 mg/kg, PO, once). Dogs were fed a standard maintenance diet 2 hours after administration of cyclosporine. Treatments were administered 14 days apart to minimize prior treatment effects, such as induction of drug-metabolizing enzyme genes.7,24,29 Treatments were repeated until all dogs in the study had received all 4 treatments.

Effect of 10 g of PWG—After completion of part 1 of the study and a washout period of > 60 days, 6 of the original 8 dogs received a higher dose of PWG (10 g [20 capsules], PO, which corresponded to a mean dose of 500 mg/kg) followed 1 hour later by cyclosporine (5 mg/kg, PO, once). Dogs were fed a standard maintenance diet 2 hours after administration of cyclosporine. Pharmacokinetic results derived for each dog in part 2 were compared with results determined previously for administration of cyclosporine alone for the same dog in part 1.

Collection of blood samples—Blood samples were collected immediately before (time 0) and 5, 30, 60, and 90 minutes and 2, 4, 6, 8, 10, and 24 hours after oral administration of cyclosporine. All blood samples were collected into tubes that contained EDTA. Blood samples were immediately refrigerated at 4°C for the first 24 hours and then frozen at −20°C pending analysis.

Cyclosporine analysis via HPLC-MS-MS—Whole blood cyclosporine analysis was performed at the Toxicology Laboratory of the University of Pennsylvania Hospital. Cyclosporine was measured via HPLC-MS-MS on the basis of a method described in another study.30 Briefly, 200 μL of EDTA-anticoagulated whole blood was spiked with cyclosporine D as an internal standard and mixed with 800 μL of a protein-precipitating solution that consisted of 70% methanol and 30% zinc sulfate. The sample was centrifuged, and 100 μL of supernatant was injected into an HPLC systemd for online solid-phase extractione and analytic separation.f A mixture of 80% methanol and 20% 30mM ammonium acetate buffer (pH, 5.1) was used as a wash solution, and the eluting solution consisted of 97% methanol and 3% 30mM ammonium acetate buffer (pH, 5.1). An MS systemg with a switching valve was used for the specific detection of cyclosporine and the internal standard. Ions were generated by atmospheric pressure chemical ionization operating in positive ionization mode. Ions were detected by use of multiple reaction monitoring with the peak area ratio for cyclosporine (mass-to-charge ratio, 1,219.95 → 1,203.15) relative to the internal standard (mass-to-charge ratio, 1,233.94 → 1,217.25) used for quantification. The percentage recovery of cyclosporine from blood was > 90%, and the limit of quantitation was 35 ng/mL. The assay was linear from 35 to 550 ng/mL, with an interassay precision of < 10% within this range. For concentrations > 550 ng/mL, serial dilutions were made by use of a negative control matrix and reanalyzed. The resultant concentration value then was multiplied by the appropriate dilution factor to derive the undiluted concentration. Dilutions of up to 1:10 were validated for assay accuracy and precision.

Pharmacokinetic analysis—Pharmacokinetic parameters were derived from whole blood concentrations of cyclosporine and time after drug administration data via noncompartmental analysis conducted by use of commercially available software.h Derived parameters included Cmax, Tmax, T1/2, AUC, and CL/F. Weight-normalized CL/F values were derived by dividing the cyclosporine dose by the total AUC and the body weight of the specific dog. Total AUC values were derived through extrapolation of the terminal elimination phase beyond the last measured time point, with the extrapolated area representing < 10% of the total AUC.

Statistical analysis—To detect a difference as small as 25% between treatment groups with 80% power and α set at 0.05, it was determined that 8 dogs would need to be included in the study. Dogs were assigned by use of a Latin square design into 4 treatments groups to adjust for carryover effects. In part 1 of the study, a repeated-measures ANOVA that adjusted for order and period effects was used to determine differences among treatment groups. A Dunnett t test was used for post hoc analysis to make pairwise comparisons between cyclosporine alone and each of the other 3 treatments. For part 2 of the study, a paired t test was used to compare cyclosporine pharmacokinetic parameters obtained after administration of 10 g of PWG, relative to the results obtained for the same 6 dogs after administration of cyclosporine alone in part 1 of the study. Because all dogs received the 10-g PWG treatment following the part 1 treatments, it was not possible to control for a treatment order effect in the data analysis of part 2. Significance was set a priori at values of P < 0.05. All analyses were performed by use of statistical software.i

Results

Animals—No adverse effects (including vomiting, diarrhea, or decreased appetite) potentially associated with the study treatments were observed in any of the dogs.

Effect of 2 g of PWG, metoclopramide, and their combination on cyclosporine pharmacokinetics—Cyclosporine concentrations did not differ between cyclosporine alone and any of the other treatments over time (Figure 1). Additionally, there were no significant differences detected between cyclosporine alone and any of the other treatments for any of the pharmacokinetic parameters evaluated (Table 1). Because it was possible that the extent of any effect could have differed among dogs, a trend plot of CL/F data for each dog for each treatment was constructed (Figure 2). However, there were still no obvious trends in CL/F values for each dog with respect to treatment.

Table 1—

Values for cyclosporine pharmacokinetics determined for 8 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg) alone or oral administration of that same dose of cyclosporine 1 hour after oral administration of metoclopramide (0.3 to 0.5 mg/kg), 2 g of PWG, or both metoclopramide (0.3 to 0.5 mg/kg) and 2 g of PWG.

VariableCyclosporine aloneCyclosporine and metoclopramideCyclosporine and 2 g of PWGCyclosporine and metoclopramide and 2 g of PWG
MeanMeanMean difference from cyclosporine95% CIMeanMean difference from cyclosporine95% CIMeanMean difference from cyclosporine95% CI
AUC (μg/mL•min)399341−58−257 to 142374−25−224 to 17549496−104 to 295
Cmax(ng/mL)1,1281,015−113−417 to 191977−152−456 to 1531,22192−212 to 397
Tmax(min)9486−8−30 to 15984−18 to 2675−19−41 to 3
T1/2(min)697682−15−268 to 237667−30−283 to 22272225−228 to 278
CL/F (mL/min/kg)14.316.92.6−0.05 to 515.10.74−2 to 314.2−0.2−3 to 2

Values did not differ significantly (P ≥ 0.05) between cyclosporine alone and all other treatments.

CI = Confidence interval.

Figure 1—
Figure 1—

Mean ± SEM cyclosporine concentrations measured in 8 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg) alone (black circles with dashed line) or oral administration of that same dose of cyclosporine 1 hour after oral administration of metoclopramide (0.3 to 0.5 mg/kg; white squares with dashed-and-dotted line), 2 g of PWG (black diamonds with solid line), or both metoclopramide (0.3 to 0.5 mg/kg) and 2 g of PWG (black squares with dotted line). A Latin square design was used with a 14-day washout period between treatments.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.687

Figure 2—
Figure 2—

Values for CL/F of cyclosporine determined in 8 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg, PO) alone or oral administration of that same dose of cyclosporine 1 hour after oral administration of metoclopramide (0.3 to 0.5 mg/kg), 2 g of PWG, or both metoclopramide (0.3 to 0.5 mg/kg) and 2 g of PWG. A Latin square design was used with a 14-day washout period between treatments. Each symbol and line combination represents results for 1 of the 8 dogs in the study.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.687

Effect of 10 g of PWG on cyclosporine pharmacokinetics—The treatment consisting of 10 g of PWG administered with cyclosporine resulted in higher cyclosporine concentrations over time, compared with results for administration of cyclosporine alone (Figure 3). Compared with results for administration of cyclosporine alone, mean cyclosporine CL/F values were significantly (P = 0.004) lower (approx 38% lower) after administration with 10 g of PWG (Figure 4; Table 2). This was also reflected by a significantly (P = 0.03) higher (54% higher) mean AUC. In addition, mean Tmax values were significantly (P = 0.01) reduced (by 29%); however, mean Cmax values were not significantly (P = 0.16) affected by administration of 10 g of PWG. In contrast to lower CL/F values, mean T1/2 values were essentially identical and did not differ significantly (P = 0.95) regardless of PWG administration.

Table 2—

Mean ± SD cyclosporine pharmacokinetics determined for 6 healthy dogs* after oral administration of a single dose of cyclosporine (5 mg/kg) alone or after oral administration of cyclosporine (5 mg/kg) and 10 g of PWG.

VariableCyclosporine aloneCyclosporine and 10 g of PWGP value
AUC (μg/mL•min)402 ± 29621 ± 930.03
Cmax (ng/mL)1,075 ± 1051,331 ± 1490.16
Tmax (min)105 ± 775 ± 100.01
T1/2 (min)734 ± 81742 ± 770.95
CL/F (mL/min/kg)14.2 ± 1.18.8 ± 1.10.004

The 6 dogs are a subgroup of the 8 dogs used in part 1 of the study and whose results are reported in Table 1.

Values reported here were extracted from the data for the same 6 dogs obtained in part 1 of the study.

Values of P < 0.05 were considered significant.

Figure 3—
Figure 3—

Mean ± SEM cyclosporine concentrations measured in 6 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg) alone (black circles with dashed line) or oral administration of that same dose of cyclosporine 1 hour after oral administration of 10 g of PWG (white squares with solid line). The 6 dogs are a subgroup of the 8 dogs included in part 1 of the study and whose results are reported in Figures 1 and 2; concentrations for cyclosporine alone reported here were extracted from the data for the same 6 dogs in part 1 of the study.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.687

Figure 4—
Figure 4—

Values for CL/F of cyclosporine determined in 6 healthy dogs after oral administration of a single dose of cyclosporine (5 mg/kg, PO) alone or after oral administration of 10 g of PWG. The 6 dogs are a subgroup of the 8 dogs included in part 1 of the study and whose results are reported in Figures 1 and 2; concentrations for cyclosporine alone reported here were extracted from the data for the same 6 dogs in part 1 of the study. Each symbol and line combination represents results for 1 of the 6 dogs in the study.

Citation: American Journal of Veterinary Research 72, 5; 10.2460/ajvr.72.5.687

Discussion

In another study28 conducted by our research group, we determined that the PWG preparation used in the study reported here can inhibit metabolism of the CYP3A probe (triazolam) in vitro as determined by use of dog liver microsomes, with an inhibitory potency comparable to that of liquid grapefruit juice. Consequently, in the present study, it was hypothesized that PWG should inhibit the metabolism of cyclosporine (also a CYP3A substrate) in vivo in dogs. In support of this hypothesis, results of the present study confirmed that administration of PWG was associated with an increase in blood concentrations of cyclosporine, increased AUC, and decreased CL/F, presumably through inhibition of intestinal or hepatic CYP3A-mediated metabolism of cyclosporine. However, the effect was a dose-dependent phenomenon, with no effect observed at a dose of 2 g of PWG and a mild effect (a 38% decrease in CL/F) observed at a dose of 10 g of PWG.

The extent of the inhibitory effect of 10 g of PWG is similar to, but slightly smaller than, the effect that has been reported in dogs for 100 mL of liquid grapefruit juice and 10 g of lyophilized grapefruit juice; administration of those doses resulted in a decrease in praziquantel clearance of approximately 50%.22 It is possible that there could be greater inhibition for a higher dose of PWG. Analysis of results for our research group's previous study28 also suggested that a dose of 21 g of PWG would be equivalent to 100 mL of liquid grapefruit juice. However, this would have required administration of 42 capsules of PWG in vivo, which was thought to be impractical. More substantial inhibitory effects have been observed with ketoconazole, which reduces cyclosporine clearance and decreases the required dose of cyclosporine by as much as 90%.15 This difference in inhibitory efficacy of ketoconazole versus grapefruit products on the clearance of cyclosporine and other CYP3A substrates may relate to the tissues that are affected. In the case of grapefruit juice products (at least at the commonly used doses), the inhibition is primarily restricted to the intestinal wall and does not affect the liver, most likely because there is limited penetration of the inhibitory constituents (furanocoumarins) into the enterohepatic circulation.23 However, ketoconazole has good systemic penetration and inhibits both intestinal wall and hepatic CYP3A. In support of this contention, analysis of data obtained in the study reported here revealed a decrease in the CL/F of cyclosporine with administration of 10 g of PWG, which is consistent with intestinal wall inhibition and associated enhanced systemic absorption of cyclosporine; however, there also was no effect on T1/2 of cyclosporine, which is consistent with minimal inhibition of CYP3A-mediated hepatic metabolism.

We believe this to be the first study conducted to investigate the potential for CYP3A inhibition in vivo by this formulation of PWG in any species. In addition, we believe this to be the first study conducted to investigate the effects of metoclopramide on cyclosporine pharmacokinetics in dogs. However, analysis of results of the present study revealed no effect of metoclopramide (0.3 to 0.5 mg/kg administered 60 minutes before cyclosporine) on any of the cyclosporine pharmacokinetic parameters evaluated in the dogs administered cyclosporine alone or cyclosporine in combination with 2 g of PWG. In humans, metoclopramide coadministration is reported31,32 to increase the rate and extent of absorption of various drugs, including, but not limited to, cyclosporine. Metoclopramide administration has been reported26 to cause a 29% increase in cyclosporine bioavailability in humans. However, it should be pointed out that the dose of metoclopramide used in that study26 in humans was divided and given at 3 time points (30 minutes prior to cyclosporine administration, in conjunction with cyclosporine administration, and 30 minutes after cyclosporine administration). This frequency of dosing may not be practical in veterinary medicine. Furthermore, the present study was designed to detect a 25% decrease in cyclosporine CL/F, and it is possible that the effect was smaller than this. A decrease of < 25% is not likely to have much clinical relevance (eg, dose reduction of cyclosporine as a cost-savings measure). Consequently, on the basis of our findings, we would conclude that adjustment of the cyclosporine dose is not needed when cyclosporine is coadministered with metoclopramide in canine patients.

Although no obvious adverse effects were observed with a single administration of 2 or 10 g of PWG, it is possible that such effects might be observed with repeated administration. The commercial product used in this study was specifically marketed for weight loss in humans at a dose of 1.5 to 3 g/d (3 to 6 capsules/d). Although unsubstantiated, it is claimed by the manufacturers that the large amount of pectin in the product isolated from the grapefruit seeds, pulp, and rind may make people feel full, thereby causing weight loss through appetite suppression. The effect on appetite for long-term administration of PWG in dogs is not known.

One of the purposes of evaluating the potential dose reduction for cyclosporine as a result of administration of PWG was to determine if there would be a reduced cost of overall treatment. Therefore, a preliminary cost-benefit analysis was performed. For a 20-kg dog receiving the formulation of microemulsified cyclosporineb used in the present study at a dosage of 5 mg/kg, the cost per 100-mg dose was $7.33 as of October 2009.33 For the PWG capsulesa used in the present study, the cost per dose (20 capsules at $0.24/capsule) was $4.80 as of October 200934 (ie, approx 65% of the cost of a cyclosporine dose). Consequently, the predicted 38% reduction in cyclosporine dose provided through PWG coadministration would provide no cost savings and would in fact be more expensive than for cyclosporine alone. In this scenario, cost savings would only be achieved with PWG doses < 12 capsules/treatment. Because the present study only evaluated single-dose regimens, it is possible that a dose of < 10 g of PWG within a multiple-dose regimen may be sufficient to achieve the same inhibitory effect as would a single 10-g dose of PWG. Such a possibility could be evaluated in future studies.

The present study had several limitations that should be considered when interpreting the data. Although we attempted to control for possible treatment-order effects in part 1 by use of a Latin square design, in part 2 of the study, treatment with the 10-g dose of PWG consistently followed all other treatments, including administration of cyclosporine alone from part 1 that was used for comparison. Although it is possible that there may have been an influence of the treatments in part 1 on the 10-g PWG treatment, this was unlikely because > 60 days were allowed to elapse between the initial treatments (part 1) and the 10-g PWG treatment (part 2). As mentioned previously, we only evaluated administration of single doses of cyclosporine, and it is possible that the effect may differ when repeated administration of cyclosporine is used, as is likely to be the case for the clinical use of cyclosporine in dogs.

Finally, in contrast to traditional pharmaceuticals, nutraceutical products, including dietary supplement-type products such as PWG, are only loosely regulated and generally do not have standardized content for active ingredients. In turn, our results may have been affected by variation in inhibitory effects among capsules. However, we examined the ability of PWG to inhibit CYP3A activity in human and dog liver microsomes in vitro and found only minimal variation (coefficient of variation, < 6%) in inhibitory effect28 among 8 capsules randomly selected from the same product lot used in the study reported here. Nevertheless, it is possible that more variability may be observed among different lots of this product over time, which may need to be taken into consideration if this product were to be prescribed to veterinary patients.

We concluded on the basis of analysis of results of the present study that metoclopramide can be administered together with cyclosporine to help alleviate common adverse effects (including nausea and vomiting) of cyclosporine without the need for adjustment of the cyclosporine dose. Grapefruit-containing products may provide a safe and effective method to enhance cyclosporine bioavailability in dogs. A commercially available PWG nutraceutical product was found to increase blood concentrations of cyclosporine, thereby reducing the required dose of cyclosporine, although only at relatively large doses (at least 10 g) of PWG, which may limit any cost advantage of combined PWG and cyclosporine treatment over monotherapy with cyclosporine. Therefore, additional studies are needed to identify safe and cost-effective methods to reduce the required dose of cyclosporine in dogs.

ABBREVIATIONS

AUC

Area under the cyclosporine concentration-time curve

CL/F

Apparent oral clearance

Cmax

Maximum detected cyclosporine concentration

CYP3A

Cytochrome P450 3A

HPLC

High-performance liquid chromatography

MS

Mass spectrometry

PWG

Powdered whole grapefruit

T1/2

Elimination half-life

Tmax

Time to maximum concentration

a.

Grapefruit Solution capsules, Solution Health Systems, Hollywood, Fla.

b.

Atopica, Novartis Animal Health, Greensboro, NC.

c.

Barr Laboratories Inc, Pomona, NY.

d.

Agilent 1100 series HPLC system, Agilent Technologies Inc, Santa Clara, Calif.

e.

Eclipse XBD C8 column, 4.6 × 12.5 mm, 5 μm, Agilent Technologies Inc, Santa Clara, Calif.

f.

Nova-Pak C18 column, 2.1 × 150 mm, 4 μm, Waters Corp, Milford, Mass.

g.

API 2000 triple quadrupole MS system, PE Sciex Instruments, Foster City, Calif.

h.

WinNonLin software, Pharsight Corp, Mountain View, Calif.

i.

SAS software, version 9.2, SAS Institute Inc, Cary, NC.

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Contributor Notes

Dr. Cerundolo's present address is Dick White Referrals, Veterinary Specialist Centre, Station Farm, London Rd, Six Mile Bottom, Suffolk, CB8 0UH, England.

Dr. Shofer's present address is Department of Emergency Medicine, School of Medicine, University of North Carolina, Chapel Hill, NC 27599.

Dr. Radwanski was supported by an American College of Veterinary Dermatology Resident Research grant provided by Novartis. Dr. Court was supported by grant GM-061834 from the National Institute of General Medical Sciences, National Institutes of Health.

Presented in abstract form at the 24th North American Veterinary Dermatology Forum, Savannah, Ga, April 2009.

The authors thank Dr. Leslie M. Shaw, Dr. Michael C. Milone, JoAnn Gardiner, and Sam Miller for technical assistance.

Address correspondence to Dr. Radwanski (nradwans@yahoo.com).