Treatment of epilepsy in dogs continues to evolve as newer-generation anticonvulsants become available to supplement or, in some cases, replace traditional first-line medications. In approximately 20% to 30% of dogs treated with traditional antiseizure medications (ie, phenobarbital or potassium bromide), seizures are not adequately controlled or adverse effects develop.1 Medications approved for seizure management in humans are now added as supplementary treatment for some patients with refractory epilepsy, and some drugs are increasingly used as single or primary treatments.2–4 Levetiracetam has a unique mechanism of action and has been approved for treatment of seizures in humans. Studies5,6 have been conducted to evaluate the efficacy and tolerability of levetiracetam in dogs with some positive findings.
Despite potential benefit of levetiracetam for treating seizures in dogs, requirements for frequent administration as well as the high financial cost have prohibited its use for some patients. Long-acting levetiracetam products have been used in human medicine to address the concern about administration frequency, allowing once-daily administration in some patients.7 For humans, FDA-approved, bioequivalent generic formulations are available, and if such products could be used in dogs, financial concerns could also be addressed. However, reports regarding the pharmacokinetics or bioequivalence of generic ER products in dogs are lacking. The purpose of the study reported here was to evaluate the pharmacokinetic profile and potential bioequivalence of generic and brand-name ER formulations of levetiracetam in healthy dogs. Our hypothesis was that generic formulations would have similar bioequivalence and pharmacokinetic profiles in dogs, compared with those of brand-name formulations, thereby providing a more cost-efficient, convenient treatment option for epilepsy in this species.
Materials and Methods
Animals
Six client-owned dogs (3 males and 3 females) between 4 and 8 years of age and weighing between 22.0 and 28.0 kg were enrolled in the study. Dogs were determined to be healthy on the basis of unremarkable results of physical and neurologic examinations and clinicopathologic tests (CBC, serum biochemical analysis, and urinalysis). All owners provided informed consent, and the study protocol was approved by the University of Georgia College of Veterinary Medicine Clinical Research Committee.
Levetiracetam administration
Food was withheld from dogs for at least 12 hours before the experiment began. Dogs were allotted to 2 groups of 3 dogs each to facilitate blood sample collection for formulation assessment. One intact 500-mg tablet of each of 4 formulations of levetiracetam was administered PO to each dog on 4 occasions that were separated by a minimum washout period of 7 days. Formulations included 2 generic products (generic-ER1a and generic-ER2b), 1 brand-name ER productc (brand-ER), and 1 brand-name IR productd (brand-IR). Order of product administration was randomized for each dog by use of a predetermined schedule.e
Experimental protocol
For each administration, a 20-gauge, 1.25-inch intravenous catheterf was inserted into a cephalic vein to facilitate sample collection. Three milliliters of blood was collected from each dog at the time of catheter placement (0 hours) as well as 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, and 36 hours after drug administration. Blood samples were allowed to coagulate for 30 minutes. All samples were then centrifugedg for 10 minutes at 1,489 × g. Serum was harvested by use of pipettes, transferred immediately to 2.0-mL microtubes, and stored at −80°C until analysis. Values of physiologic variables, including heart rate (0 and 0.5 hours), respiration rate (0, 0.25, and 0.5 hours), and arterial blood pressure as measured with the ultrasonic Doppler method (0, 0.25, and 0.5 hours), were recorded. Adverse effects in dogs, including changes in mentation, behavior, gait, vital signs (eg, heart rate, respiratory rate, and arterial blood pressure), and other overt signs of illness (eg, vomiting or diarrhea), were recorded when identified.
Pharmacokinetic analysis
Serum from clinically normal, untreated dogs was used for preparation of standard and quality control solutions. The levetiracetam standard (calibration) solutionh was serially diluted to 96, 48, 24, 12, 6, 3, 1.5, 0.75, 0.375, and 0.1875 μg/mL. Quality control samples were prepared separately at concentrations of 48, 6, and 0.75 μg/mL. For each sample analysis (serum, standard, quality control, and blank), 25 μL of sample was added to 25 μL of internal standard solution (clonazepamh [7.12 μg/mL]). Protein was removed from samples by addition of 450 μL of ice-cold acetonitrile, tube contents were mixed by use of a vortex device for 30 seconds, and tubes were centrifuged for 20 minutes at 14,000 × g. A 100-μL aliquot of supernatant was extracted, and 400 μL of mobile phase was added. Contents were then mixed and transferred to an autosampler vial for analysis by means of ultra-high-performance liquid chromatography with tandem mass spectrometry.i Samples (2 μL) were separated on a C18 stationary phase columnj with isocratic mobile phase consisting of 0.1% formic acid and acetonitrile (20:80 [vol:vol]). Measurement of levetiracetam and internal standard (clonazepam) concentrations was performed at a flow rate of 0.4 mL/min by use of electrospray ionization.k Results for the method were linear from 1.875 to 480 ng/mL, with a limit of quantification of 1.8 ng/mL; accuracy was > 90%, and variation was ≤ 15%. Nitrogen gas was used as the drying nebulizer and collision gas (10 L/min at 325°C and 2460.7 cm H2O). Capillary voltage was set at 3,500 V. Mass spectral data were acquired in positive-ion mode, and mass transitions were monitored by means of multiple-reaction monitoring (Appendix).
Pharmacokinetic analysis
Pharmacokinetic values were estimated from a noncompartmental analysis of serum concentrations of levetiracetam after oral administration of brand-IR, brand-ER, generic-ER1, and generic-ER2 by use of statistical modeling software.l Parameters included Cmax, Tmax, terminal rate constant (β), AUC, half-life, Vd, and Cl. The AUC was estimated by use of the logarithmic trapezoidal method, with extrapolation of the degree of drug exposure from the time of last measured concentration to infinity. Time to maximum serum concentration of levetiracetam was estimated on the basis of data collected at the predetermined sample collection points for generation of drug concentration versus time profiles. The Cmax and AUC were normalized to dose, and Cl and Vd were normalized to F. Published data6 for dogs and humans suggested that F is equal to 1.0; therefore, Cl and Vd represent good estimates of the fraction of drug absorbed (Cl/F and Vd/F, respectively).
Statistical analysis
Statistical analysis of data from all dogs that received each formulation of levetiracetam (n = 5 or 6) was performed with the aid of statistical software.m Results are reported as mean ± SD. Estimates of pharmacokinetic variables were compared among formulations by use of 1-way ANOVA. Differences were considered significant at P ≤ 0.05.
Results
Animals
All 6 dogs were used in pharmacokinetic analysis of levetiracetam for brand-IR, brand-ER, and generic-ER1. Only 5 dogs were used in the testing of generic-ER2 because of dog availability. Pharmacokinetic profiles of the 4 formulations were summarized (Table 1). Extended-release formulations had a significantly longer mean Tmax than did the IR formulation. Mean ± SD Cmax for levetiracetam was greatest for brand-IR (34.9 ± 6.4 μg/mL), compared with values for all ER formulations (brand-ER, 29.4 ± 4.1 μg/mL; generic-ER1, 21.9 ± 3.4 μg/mL; and generic-ER2, 27.8 ± 4.0 μg/mL; Figure 1). Some differences were evident for Tmax and Cmax within the group of ER formulations, with generic-ER1 having a significantly lower Cmax, compared with the value for brand-ER.
Mean ± SD linear (A) and logarithmic (B) serum concentrations of levetiracetam in healthy dogs at various points after oral administration of one 500-mg tablet of brand-IR (white inverted triangles [n = 6]), brand-ER (black inverted triangles [6]), generic-ER1 (white circles [6]), or generic-ER2 (black circles [5]). Time of administration was designated as time 0. Formulations were evaluated individually in a randomized crossover design, and each administration was separated by a minimum washout period of 7 days. Logarithmic transformation of data was performed for the curves in panel B to facilitate interpretation at the 24-hour measurement point.
Citation: American Journal of Veterinary Research 76, 8; 10.2460/ajvr.76.8.719
Mean ± SD linear (A) and logarithmic (B) serum concentrations of levetiracetam in healthy dogs at various points after oral administration of one 500-mg tablet of brand-IR (white inverted triangles [n = 6]), brand-ER (black inverted triangles [6]), generic-ER1 (white circles [6]), or generic-ER2 (black circles [5]). Time of administration was designated as time 0. Formulations were evaluated individually in a randomized crossover design, and each administration was separated by a minimum washout period of 7 days. Logarithmic transformation of data was performed for the curves in panel B to facilitate interpretation at the 24-hour measurement point.
Citation: American Journal of Veterinary Research 76, 8; 10.2460/ajvr.76.8.719
Mean ± SD linear (A) and logarithmic (B) serum concentrations of levetiracetam in healthy dogs at various points after oral administration of one 500-mg tablet of brand-IR (white inverted triangles [n = 6]), brand-ER (black inverted triangles [6]), generic-ER1 (white circles [6]), or generic-ER2 (black circles [5]). Time of administration was designated as time 0. Formulations were evaluated individually in a randomized crossover design, and each administration was separated by a minimum washout period of 7 days. Logarithmic transformation of data was performed for the curves in panel B to facilitate interpretation at the 24-hour measurement point.
Citation: American Journal of Veterinary Research 76, 8; 10.2460/ajvr.76.8.719
Mean ± SD values of pharmacokinetic variables for levetiracetam in healthy dogs after oral administration of one 500-mg tablet of brand-IR, brand-ER, generic-ER1, or generic-ER2.*
| Variable | Brand-IR | Brand-ER | Generic-ER1 | Generic-ER2 |
|---|---|---|---|---|
| Tmax (h) | 2.29 ± 1.42 | 6.00 ± 2.19† | 5.33 ± 2.07 | 8.00 ± 2.83† |
| Cmax (μg/mL) | 34.9 ± 6.40 | 29.4 ± 4.1‡ | 21.9 ± 3.4† | 27.8 ± 4.0† |
| β (1/h) | 0.173 ± 0.037 | 0.157 ± 0.017 | 0.159 ± 0.021 | 0.161 ± 0.030 |
| t1/2 (h) | 4.17 ± 0.92 | 4.47 ± 0.50 | 4.44 ± 0.68 | 4.42 ± 0.81 |
| AUC (h•μg/mL) | 313 ± 80 | 361 ± 71‡ | 280 ± 62 | 380 ± 30‡ |
| Vd (mL) | 9,780 ± 2000 | 9,020 ± 1,110‡ | 11,800 ± 32,60 | 8,440 ± 2,110‡ |
| Cl (mL/h) | 1,680 ± 435 | 1,420 ± 270‡ | 1,850 ± 443 | 1,310 ± 94.7‡ |
Formulations of levetiracetam were evaluated individually in a randomized crossover design, and each evaluation was separated by a minimum washout period of 7 days. Data from 6 dogs are represented for brand-IR, brand-ER, and generic-ER1; data from 5 dogs are represented for generic-ER2.
Value differs significantly (P ≤ 0.05) from that for brand-IR.
Value differs significantly (P ≤ 0.05) from that for generic-ER1.
β = Terminal rate constant. t1/2 = Half-life.
The AUCs did not differ significantly between ER formulations and the IR formulation; however, generic-ER1 had a significantly lower AUC (280 ± 62 h•μg/mL) than did the other ER formulations (generic-ER2, 380 ± 30 h•μg/mL; brand-ER, 361 ± 71 h•μg/mL). Overall, calculated half-lives as well as elimination rate constants were similar among all formulations. Volume of distribution did not differ significantly between ER and IR formulations. On the other hand, Vd and Cl were significantly lower for brand-ER and generic-ER2, compared with respective values for generic-ER1 (Table 1). The relative F of generic-ER1 and generic-ER2 was 0.77 and 1.05, respectively, compared with F for the IR formulation.
All formulations appeared to be tolerated well by all dogs at the administered dose. Adverse effects were limited to mild sedation in 1 dog after it received brand-ER.
Discussion
Of the many anticonvulsants that are commercially available, levetiracetam appears to have a unique mechanism of action, with minimal adverse effects.3,8 Development of ER and generic formulations of the drug could facilitate treatment of seizures in dogs. However, comparison of pharmacokinetic data for various formulations is important for ensuring effective use of these and other drugs in a growing spectrum of medications available for this purpose.
In the present study, pharmacokinetic profiles of 4 formulations of levetiracetam were evaluated after oral administration of one 500-mg tablet of each formulation to 6 healthy dogs. Intraindividual comparisons of the pharmacokinetics of the 4 formulations were considered when the present study was designed; however, given the variability in pharmacokinetics that can be detected within individual dogs, we chose to report summary data for each formulation of levetiracetam to provide findings that may be generalized to other dogs. Values of Cmax in the study were similar among all ER formulations, and all values were lower than the Cmax for the IR formulation. All serum concentrations of levetiracetam in the study dogs were within the therapeutic range for humans (5 to 45 μg/mL) by 12 hours after administration; the therapeutic range has not been established for dogs. This finding suggested that the Cmax obtained with a single dose of an ER formulation, albeit lower than with the more commonly used brand-IR, may be sufficient to achieve a drug concentration sufficient to control clinical signs in dogs with seizures. A subtherapeutic serum concentration of levetiracetam was evident for all formulations by 24 hours after administration, suggesting a need for twice-daily administration to maintain a therapeutic concentration. The Tmax was greater for the ER formulations as well, compared with the Tmax for the IR formulation. This finding was not unexpected given that ER formulations generally have slower rates of dissolution or release from tablets than do IR formulations, and such rates would affect the rate and extent of drug absorption. These differences between ER and IR formulations can result in ER formulations providing more gradual changes in serum drug concentration (lower Cmax), which could minimize the degree of fluctuation in serum concentrations of active drug and reduce the potential for adverse effects with long-term administration.
The finding that AUCs were similar between brand-ER and generic-ER2 suggested that the total degree of drug exposure and F did not differ significantly between these formulations of levetiracetam and brand-IR. This lack of a difference would be clinically important when considering duration of effect when a dog is originally given brand-IR, followed by a transition to brand-ER, because the amount of drug exposure would be similar between the 2 formulations. However, it is important to consider that the AUC of generic-ER1 was significantly different from that of the other ER formulations of levetiracetam. Given existing data for humans and dogs that suggest levetiracetam is rapidly and completely absorbed after oral administration (F = 1.0),6 administration of generic-ER1 to the dogs of the present study resulted in a lower F or an apparent increase in Cl/F relative to values for the other formulations. Administration of various formulations may consequently result in differences in the degree of drug exposure that could contribute to ineffective treatment. In addition, although clinical effects of levetiracetam administration were not evaluated in the present study, fluctuations in serum drug concentrations could lead to an increase in the potential for adverse effects, depending on a patient's response to the fluctuations.
Some differences were identified among ER formulations administered to dogs of the present study. One generic formulation (generic-ER1) had a lower relative F than did brand-ER; findings were similar for the other generic formulation (generic-ER2). The AUC, Vd, and Cl differed significantly between generic-ER1 and generic-ER2. It is unclear whether these differences would be clinically important, but the findings emphasized that differences can exist between 2 generic ER products from different manufacturers. Breakthrough seizures have reportedly occurred in humans in which a transition was made from a name brand to a generic formulation, but this concern has been challenged.9 In a study10 involving dogs, the peak plasma concentration, elimination half-life, and F were similar between 1 brand and 1 generic formulation of phenobarbital. In that study, seizure frequency was no different when various formulations of phenobarbital were administered, and adverse effects were similar for the various formulations of phenobarbital used.10 Although studies involving larger sample sizes are needed, a difference in the incidence of adverse effects may not be clinically important when comparing the clinical effects of ER and IR levetiracetam.
Limitations of the present study included small sample size, administration of single versus multiple doses, and lack of evaluation of the safety and efficacy of the various formulations of levetiracetam in dogs with seizures. Although the sample size was sufficient for detection of significant differences in pharmacokinetic properties among the formulations, the fact that only 6 dogs were included and that those dogs were healthy limited application of the findings to the general population of dogs with seizures. In our experience, levetiracetam has been used in dogs with minimal adverse effects; however, some dogs might have difficulty tolerating the drug. In addition, anticonvulsant medications are often administered with food, which could affect bioequivalence and F by altering release and systemic absorption of the drugs. Studies involving dogs with seizures and dogs from which food has not been withheld are needed before conclusions can be made about the clinical impact of various levetiracetam formulations. Because a therapeutic serum concentration of levetiracetam has not been fully established for dogs, it is also unclear whether the serum drug concentrations achieved with oral administration of one 500-mg tablet of levetiracetam in our study would be therapeutic. Furthermore, dogs with seizures can be treated with multiple medications, and the coadministration of other mediations might affect the pharmacokinetic profile of levetiracetam.11 Ideally, interactions among anticonvulsant medications and ER formulations of levetiracetam would be assessed before conclusions are made regarding the benefits of ER formulations in the treatment of dogs with seizures.
In the present study, ER formulations of levetiracetam had promising pharmacokinetic profiles in healthy dogs and achieved serum drug concentrations similar to those used to guide treatment of humans with seizures. Serum concentrations of levetiracetam achieved with administration of the ER formulations were maintained for a period that would allow a longer interval between doses than is needed for IR administration. A longer interval would facilitate dog-owner compliance with treatment regimens and reduce the potential for adverse effects by providing more constant serum drug concentrations than might be achieved with IR formulations.
The specific effective dose should theoretically be comparable to that chosen when IR formulations are used. In a previous study,5 administration of an IR formulation of levetiracetam (10 to 20 mg/kg, PO, q 8 h) was identified as effective in 9 of 14 dogs with seizures. A similar degree of efficacy might therefore be achieved with ER formulations of levetiracetam, which would also permit less frequent dose administration. In addition, the generic ER formulations evaluated in the present study had pharmacokinetic profiles similar to those of existing brand-name ER formulations, suggesting generic ER products would be similarly effective in the treatment of dogs with seizures, although some differences were identified between generic ER formulations that may or may not be clinically important. Although other studies are needed to confirm the effectiveness of ER formulations of levetiracetam in dogs, the present findings suggested that consistent administration of 1 formulation rather than transitioning between formulations should be practiced when treating dogs with seizures.
Acknowledgments
Supported in part by Virbac Animal Health; the Department of Medicine and Surgery, College of Veterinary Medicine, University of Georgia; and the Auburn University Internal Grant Program for the Specialized Pharmaceutical and Experimental Center for Translational Research and Analysis. Dr. Arnold and Mr. Nie were supported by the National Institutes of Health (R01 EB016100).
Presented as a poster at the 2014 American College of Veterinary Internal Medicine Forum, Nashville, Tenn, June 2014.
The authors thank Lisa Reno, Cody Mannino, and Mallory Gammage for technical assistance.
ABBREVIATIONS
| AUC | Area under the drug concentration-versus-time curve |
| Cl | Systemic clearance |
| Cmax | Maximum serum drug concentration |
| ER | Extended release |
| F | Bioavailability |
| IR | Immediate release |
| Tmax | Time to maximum serum drug concentration |
| Vd | Volume of distribution |
Footnotes
Lupin Pharmaceuticals Inc, Baltimore, Md.
Actavis, Parsippany, NJ.
Keppra ER, UCB Inc, Smyrna, Ga.
Keppra IR, UCB Inc, Smyrna, Ga.
Random.org. Randomness and Integrity Services Ltd, Dublin, Ireland. Available at: www.random.org. Accessed Oct 15, 2012.
Surflo intravenous catheter, Terumo Medical Corp, Elkton, Md.
Model CR 412, Jouan Inc, Winchester, Va.
Sigma Chemical Co, St Louis, Mo.
Agilent 1290 Infinity binary LC system coupled with Agilent 6460 triple quadrapole mass spectrometer, Agilent Technologies, Santa Clara, Calif.
ZORBAX SB-C18 column (2.1 × 50 mm, 1.8 μm), Agilent Technologies, Santa Clara, Calif.
Agilent jet stream electrospray ionization source, Agilent Technologies, Santa Clara, Calif.
WinNonlin Professional, version 5.3, Pharsight Corp, Mountain View, Calif.
SPSS Statistics, version 16.0.0, IBM Corp, Armonk, NY.
References
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Appendix
Precursor ion-to-product ion transitions used for quantification and qualification of levetiracetam in canine serum samples.
| Variable | Precursor ion (m/z) | Product ion (m/z) |
|---|---|---|
| Levetiracetam quantification | 171.1 | 125.9 |
| Levetiracetam qualification | 171.1 | 154.0 |
| Clonazepam quantification | 316.1 | 269.9 |
| Clonazepam qualification | 316.1 | 240.9 |
