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Keywords:

  • Topiramate;
  • Pharmacokinetics;
  • Intravenous;
  • Antiepileptic drug

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References

Purpose

Although oral topiramate (TPM) products are widely prescribed for migraines and epilepsy, injectable TPM is not available for human use. We have developed a solubilized TPM formulation using a cyclodextrin matrix, Captisol with the long-term goal of evaluating its safety and efficacy in neonatal seizures. This study in healthy adult volunteers was performed as required by the U.S. Food and Drug Administration (FDA) to demonstrate the pharmacokinetics and safety prior to initiation of studies involving children. This study allowed investigation of absolute bioavailability, absolute clearance, and distribution volume of TPM, information that could not be obtained without using an intravenous TPM formulation.

Methods

This study was an open-label, two-way crossover of oral and intravenous TPM in 12 healthy adult volunteers. Initially two subjects received 50 mg, intravenously and orally. Following evidence of safety in the first two subjects, 10 individuals received 100 mg doses of intravenous and oral TPM randomly sequenced 2 weeks apart. Blood samples were taken just prior to drug administration and at intervals up to 120 h afterwards. TPM was measured using a validated liquid chromatography—mass spectrometry method. Concentration-time data were analyzed using a noncompartmental approach with WinNonlin 5.2.

Key Findings

All subjects completed the study. The mean (±standard deviation) absolute oral bioavailability was 109% (±10.8%). For intravenous and oral TPM the mean distribution volumes were 1.06 L/kg (±0.29) and 0.94 L/kg (±0.24). Clearances were 1.33 L/h (±0.26) and 1.22 L/h (±0.26). The half-life values were 42.3 h (±6.2) and 41.2 h (±7.5). No changes in heart rate, blood pressure, electrocardiography, or infusion site reactions were observed. Mild central nervous system cognitive adverse events and ataxia occurred between dosing and 2 h post dose with both intravenous and oral administration. With intravenous TPM, these adverse effects occurred as early as during the 15-min intravenous infusion.

Significance

In healthy adults, oral TPM is bioequivalent to intravenous TPM, and infusion of 50–100 mg over 15 min is safe. Neurologic effects occurred during the infusion, demonstrating that TPM rapidly diffuses into the brain, which supports its evaluation for neonatal seizures. Results from this pilot study will inform the design of subsequent studies in children and newborns, including controlled clinical trials intended to assess the efficacy and safety of intravenous TPM for neonatal seizures. In addition, our results provide support for the further development of intravenous TPM as bridge therapy for older children and adults in whom oral TPM therapy is interrupted.

Topiramate (TPM), available as tablets and sprinkle capsules, is approved for use in adults for prophylaxis of migraines and for patients ages 2 and older with epilepsy. There is, however, no available intravenous product. We have established a program to develop an injectable TPM formulation to evaluate its treatment in hypoxic-ischemic brain injury in newborns. This report deals with the first study comparing oral and intravenous TPM in volunteers as an early step in this process.

Topiramate has a broad range of pharmacologic effects. TPM blocks voltage-gated sodium channels, augments γ-aminobutyric acid (GABA) at certain subtypes of GABAA receptors, inhibits α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate ionotropic receptors, and is a weak inhibitor of some isoenzymes of carbonic anhydrase (CA-II and CA-IV; Sachdeo, 1998). However, the precise mechanisms by which it exerts its antiseizure effect has not been determined. An important recent finding is that TPM is highly effective in controlling seizures and is neuroprotective in newborn laboratory animal models of status epilepticus and cerebral ischemia (Yang et al., 1998; Niebauer & Gruenthal, 1999; Lee et al., 2000; Cha et al., 2002; Follett et al., 2004; Koh et al., 2004).

Hypoxic-ischemic brain injury in newborns is a significant medical problem with a high mortality rate; grave neurologic sequelae including impaired cognition and neonatal seizures; and serious treatment-related adverse effects. All of these can cause further brain injury leading to significant morbidity in later life.

The drugs of choice to treat neonatal seizures are phenobarbital and phenytoin. However, <50% of newborns respond to therapy with these medications and both are associated with serious adverse effects including further brain injury, acute systemic toxicity, and significant drug interactions (Painter et al., 1999; Bittigau et al., 2003; Glier et al., 2004). A safer, more effective treatment for neonatal seizures combined with the potential for neuroprotection would represent a significant advancement in the treatment of hypoxic-ischemic brain injury. Oral TPM has been used to treat neonatal seizures that fail to respond to first-line therapy, but oral administration in newborns is often unreliable, imprecise, and delays attainment of a therapeutic effect (Silverstein & Ferriero, 2008; Filippi et al., 2009).

We have previously completed a low dose, first-in-human study of intravenous TPM in adult patients taking oral TPM for either epilepsy or migraine headaches (Kriel et al., 2010). The patients received a single 25 mg stable-labeled dose of intravenous TPM in addition to their oral TPM regimen. Both the safety and pharmacokinetics results were used to design the phase I investigation reported herein.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References

Subjects

Twelve healthy volunteers (six female, six male) (11 white, one African American) between 19 and 55 (mean 35) years of age were enrolled. Subjects were recruited by from the Minneapolis/Saint Paul, Minnesota metropolitan area. Inclusion criteria were the following: 18–65 years of age, body mass index (BMI) range 18–32, creatinine clearance (CrCl) >70 ml min−1. Women of childbearing potential were excluded unless they were using an effective method of birth control. Subjects were excluded if they had a history of intolerance to intravenous administration of medication; were taking any medication (over-the-counter medications could not be taken within 7 days of beginning the study); had known hypersensitivity to TPM; had a significant medical history of cardiac, neurologic, psychiatric, oncologic, endocrinologic, metabolic, or hepatic disease; and had used any investigational drug or device in the 30 days prior to screening. Mean weight was 78.2 kg with a range of 58.3–112.3 kg.

Study drug

TPM has limited water solubility (9.8 mg/ml), with poor stability (Doose & Streeter, 2002). Both TPM solubility and stability are improved with the addition of a cyclodextrin, Captisol®. Intravenous TPM was formulated as a 10 mg/ml solution dissolved in a 10% sulfobutylether β-cyclodextrin (Captisol, Ligand Technologies, La Jolla, CA, U.S.A. Lot # CY-04A-05010, 2008). This formulation is concentrated enough TPM to limit the injection volume but does not require amounts of Captisol larger than those in currently approved products. The formulation was manufactured at the University of Iowa, Division of Pharmaceutical Sciences under good manufacturing practices. The University of Minnesota holds a provisional patent on an injectable formulation of topiramate and Captisol®.

Subjects fasted for 2 h prior to and 4 h post dosing. Topamax 50 mg tablets, taken from the same lot (#9AG393; Janssen Pharmaceuticals, Titusville, NJ, U.S.A.), were used for oral dosing as a single dose or as two tablets given with 8 ounces of water. The intravenous TPM doses were administered over 15 min with a syringe pump.

Study design

The study was conducted under an amendment to IND #78993 and approved by the University of Minnesota Institutional Review Board. All subjects provided written informed consent. Subjects were compensated for their participation.

Subjects were admitted into Prism Research, a clinical research facility, the day before the studies at which time they underwent brief physical and neurologic examinations. A electrocardiography (ECG) recording and routine blood tests were obtained. Subjects remained at the research facility for 24 h following dosing for blood draws and safety assessments. They returned at 48, 72, 96, and 120 h after dosing for additional blood draws and safety assessments including vital signs and reporting of adverse events.

Two subjects received 50 mg of intravenous TPM infused over 15 min. After a 2-week washout period, these two subjects received 50 mg of oral TPM. No serious adverse events occurred after the 50 mg intravenous dose; therefore, per protocol, the remaining 10 subjects were randomly assigned to receive either 100 mg of intravenous TPM infused over 15 min or 100 mg of oral TPM. Following a 2-week washout, subjects were given the alternate treatment. This study was not blinded and all subjects received both treatments. The treatment order of intra-venous and oral in the 100 mg dose group was randomly assigned in 1:1 allocation from a random number table. The doses of TPM used in this study were clinically relevant in that they are routinely administered as initial therapy in an acute care setting and fall within the range of maintenance doses used to treat migraines and epilepsy (May et al., 2002).

Blood sampling

Ten milliliter blood samples were collected for determination of plasma TPM concentrations pre-dose, and 5, 15, and 30 min, and 1, 2, 4, 6, 10, 12, 24, 48, 72, 96, and 120 h after administration of the intravenous and oral dose. Samples were immediately centrifuged and the plasma frozen until analysis.

Analytic methods

The analytical method was designed to measure TPM and stable-labeled TPM in plasma using Hewlett-Packard 1100 series (Agilent Technologies, Santa Clara, CA, U.S.A.) with a degasser (Model G1322A), quaternary pump (Model G1311A), autosampler (Model G1313A), and a quadrupole mass spectrometer (model G1946A). ChemStation Software (Agilent Technologies) was used to operate the modules and for data analysis. The analytes were separated using a Zorbax Eclipse XDB C18 column (150 × 3.0 mm, 3.5 micron particle size) (Agilent Technologies) and eluted isocratically with a mobile phase consisting of 20 mm ammonium acetate (Fisher Scientific, Hanover Park, IL, U.S.A.) buffer and methanol (Fisher Scientific) (50:50, v/v). The flow rate was 0.4 ml/min, and the run time was 10 min. The analytes were subsequently detected in the electrospray ionization-mass spectrometry system; the mass spectrometer was run in the positive ion mode. Nitrogen was used as the drying gas and was supplied at a flow of 12 L/min at a temperature of 300°C. The capillary voltage was set at 4,000 V. The high-performance liquid chromatography (HPLC) eluent was introduced in the source via the electrospray interface, generating the negatively charged molecular ion [M-H]-. The samples were analyzed using Selected Ion Monitoring mode (SIM) and quantitation ions were m/z 338, m/z 344, and m/z 350 for TPM, TPMSI, and TPMd12 internal standard, respectively. The samples were run with a seven-concentration standard curve (run in triplicate) and nine quality control samples (low, med, and high run in triplicates). The calibration curve was linear in the concentration range of 0.1–20 μg/ml. The limit of detection was 0.5 ng/ml; the limit of quantification was 0.04 μg/ml. Assay precision for TPM and stable-labeled TPM ranged from 2–5% to 3–5%, respectively, with accuracy values of TPM recovery for TPM in spike plasma ranging from 94–105% to 90–106% for stable-labeled TPM.

Pharmacokinetic analysis

TPM concentration-time data were analyzed using a noncompartmental pharmacokinetic approach with WinNonLin software (version 5.2; Pharsight Corporation, Mountain View, CA, U.S.A.). The elimination rate constant (λz) was determined from the slope of the terminal log-linear portion of the plasma concentration-time curve, and the terminal half-life (t1/2) was calculated as ln 2/(λz). Maximum plasma concentrations (Cmax) and time to maximum concentration (tmax) were obtained by direct observation of the data. The area under the concentration-time curve (AUC) to the last nonzero plasma concentration (Clast) above the lower limit of quantification was calculated as AUClast. The area under the concentration-time curve extrapolated to infinity (AUC0–∞) was calculated as AUClast + (Clast/λz). Clearance (CL) was obtained by dose divided by AUC0–α. Volume of distribution (Vd) was calculated by dose divided by k*AUC0–α. Absolute bioavailability (F) was determined from the following equation:

  • display math

The intravenous formulation was used as the reference product to determine the bioequivalence of oral TPM tablets. Assessment of bioequivalence was determined using a nonreplicate crossover design (see Statistical analysis for details). The parameters were dose normalized because two doses were used (50 and 100 mg). Means and standard deviations for the parameters were also obtained using the descriptive statistics tool in WinNonLin.

Safety monitoring

A neurologist was present to clinically evaluate subjects before, during, and for 2 h following intravenous and oral drug administration. Safety assessments included clinical laboratory tests (sodium, potassium, glucose, serum creatinine, chloride, urea nitrogen, hemoglobin, albumin, carbon dioxide, alanine transaminase, aspartate transaminase, alkaline phosphate, bilirubin, and total plasma protein) and physical examinations with vital signs, continuous 12-lead ECG monitoring, and physical examinations including a brief neurologic examination. An adverse event was defined as any reaction, side effect, or other untoward event, regardless of relationship to study drug, that occurred any time after the subject had signed the consent form and extended until 14 days after the last dose of study drug.

Central nervous system toxicity was assessed by observing tandem gait and evaluating dysmetria by the finger to nose to finger test. The ECG was monitored by a physician prior to, during, and 15 min after the infusion. Blood pressure and pulse were monitored every 2 min during infusion, every 15 min for 1 h after infusion, and then every 8 h until 24 h post dose. A nurse examined the infusion site for irritation and extravagation and asked the patient about infusion site discomfort and other adverse events.

Statistical analysis

The mean and standard deviation were calculated for all the pharmacokinetic parameters. Paired t-test was used to compare the oral to the intravenous pharmacokinetic parameters. A p-value < 0.05 was considered statistically significant. The ratio of least-square means and corresponding 90% confidence intervals were calculated on log transformed data for Cmax/D, AUClast/D, and AUC0-–α/D. Bioequivalence was achieved if the 90% confidence interval bounds for the least-square mean ratio (oral/IV) from log transformed data for Cmax/D, AUClast/D, and AUC0-–α/D fell between 80% and 125%.

The effect of age, height, weight, and sex on t1/2, Vd, CL, AUC0–α, AUC0–α/D was examined using univariate analysis of variance. A p-value < 0.05 was considered significant for all analyses.

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References

Pharmacokinetics results

Once oral TPM reached Cmax, TPM concentrations from the oral and intravenous administrations were similar and declined at the same rate (Fig. 1). Figure 2, which presents concentration-time data in the first 4 h post dose, shows that Cmax following intravenous TPM was slightly higher and occurred earlier than with oral TPM, although the difference was not statistically significant.

image

Figure 1. Mean plasma concentrations versus time after oral and intravenous topiramate (0–120 h). Error bars signify one standard deviation of the mean.

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image

Figure 2. Mean plasma concentrations versus time after oral and intravenous topiramate (0–4 h). Error bars signify one standard deviation of the mean.

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TPM pharmacokinetic parameters determined following oral and intravenous dosing are presented in Table 1. There were no statistically significant differences in any of pharmacokinetic parameters. The mean time to maximum concentration after oral TPM was 1.35 h (range of 0.25–4 h).

Table 1. Mean pharmacokinetic parameters (n = 12)
ParameterIV (mean ± SD)Oral (mean ± SD)p-value
  1. CL, clearance; Vd, volume of distribution; t1/2, half-life; Cmax, maximum plasma concentration; Cmax/D, dose normalized maximum plasma concentration; AUC0-α, area under the plasma concentration time curve from zero to infinity; AUC0-α/D, dose normalized area under the plasma concentration time curve from zero to infinity.

CL (CL/F) (L/h)1.33 ± 0.261.22 ± 0.2600.334
Vd (L/kg)1.06 ± 0.290.94 ± 0.240.279
t1/2 (h)42.3 ± 6.241.18 ± 7.50.693
Cmax (μg/ml)1.99 ± 0.891.801 ± 0.640.542
Cmax/D (μg/ml/mg)0.0212 ± 0.0070.0195 ± 0.0050.512
AUC0-α (h*μg/ml)72.6 ± 21.179.1 ± 26.40.536
AUC0-α/D (h*μg/ml/mg)0.78 ± 0.170.85 ± 0.190.337

The mean absolute bioavailability for orally administered TPM was 109 ± 10.8% with a range of 91.7–129.7% (Fig. 3).

image

Figure 3. Absolute bioavailability by subject. Each diamond represents an individual subject.

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The 90% confidence intervals for the least-square mean ratio (oral/intravenous) from log transformed data are shown in Table 2. The 90% confidence intervals of oral TPM compared to intravenous TPM were within the 80–125% limits for Cmax/D, AUClast/D, and AUC0-α/D. There were no significant sequence or period effects on Cmax/D, AUClast/D, and AUC0-α/D.

Table 2. Bioequivalence of oral as compared to intravenous topiramate (n = 12)
 Ratio (% oral to IV)Lower 90% CIUpper 90% CI
  1. CI, confidence interval; Cmax/D, dose normalized maximum plasma concentration; AUClast/D, area under the plasma concentration time curve from zero to last measurable concentration; AUC0-α/D, dose normalized area under the plasma concentration time curve from zero to infinity.

Ln(Cmax/D)92.2684.17101.13
Ln(AUClast/D)109.24103.20115.62
Ln(AUC0-α/D)108.44102.85114.34

Age, height, weight, and sex had no significant effect on TPM pharmacokinetic parameters.

Safety results

No serious adverse events were reported by subjects following intravenous or oral administration of TPM. All adverse events were classified as either mild or moderate (Table 3). No subjects discontinued the study. There were no changes in heart rate, blood pressure, ECG, or infusion site reactions. No subjects reported local discomfort due to administration of the intravenous formulation.

Table 3. Adverse events after oral and intravenous topiramate (n = 12)
Adverse eventnSeverityRelation to study drugDurationOutcome
Oral administration     
Ataxia1MildDefinitely7 hResolved
Headache2MildPossibly2, 16 hResolved
Cognitive impairment1MildDefinitely5 hResolved
Ecchymosis at venipuncture sites1MildUnlikelyn/aResolved
IV administration     
Abnormal taste1MildPossibly15 minResolved
Hip arthralgia1ModerateUnlikely2 daysResolved
Upper respiratory infection1MildUnlikelyn/aResolved
Headache1MildPossibly1.5 hResolved
Fatigue1MildPossibly2 daysResolved
Rash1MildUnlikely2 daysResolved
Lightheadedness2MildDefinitely1, 2 hResolved
Nystagmus1MildDefinitely2 hResolved
Ataxia3

1-Mild

2-Moderate

Definitely1.5, 2.5, 4 hResolved
Intoxication1MildDefinitely2 hResolved
Cognitive impairment1ModerateDefinitely4 hResolved
Calm sensation1MildDefinitely2 hResolved
Memory impairment1MildDefinitelyn/aResolved
Dizziness1MildDefinitely1 hResolved
IV infiltrate2Mild n/aResolved

Reported side effects occurred between dosing and 2 h following dosing and usually resolved within 4 h regardless of route of medication. The onset of central nervous system cognitive adverse events and ataxia following the intravenous dose usually occurred during infusion or within 15 min postinfusion.

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References

This is the first study investigating the safety and pharmacokinetics of intravenous TPM at clinically relevant doses in subjects not previously exposed to TPM. The results provide new information about TPM. In an earlier, preliminary study in which a subclinical dose (25 mg) of intravenous TPM was given to patients on oral TPM, no serious adverse effects occurred (Kriel et al., 2010). That study provided the basis for the current study in which we investigated the pharmacokinetics and safety of higher doses. Use of healthy volunteers permitted better assessment of the risk and type of adverse effects associated with intravenous TPM that might occur in patients receiving TPM for the first time, as would be the case with neonates.

Intravenous 100-mg TPM doses did not cause any serious adverse effects, but did produce mild central nervous system effects within 15 min following drug administration, indicating rapid diffusion into the central nervous system. This property, which had not previously been characterized, supports the rationale for using intravenous TPM in neonatal seizures, where a rapid onset of action is needed. Furthermore, the intravenous TPM doses, which were given over 15 min, did not cause any changes in vital signs or ECG. Commonly observed adverse reactions known to occur with Topamax, particularly during initiation of therapy include somnolence, dizziness, ataxia, speech disorders and related speech problems, nervousness, psychomotor slowing, abnormal vision, difficulty with memory, paresthesia, diplopia, fatigue, difficulty with concentration or attention, confusion, anorexia, anxiety, and weight decrease (Topamax product label, 2012). As our study was not a full dose ranging trial and only involved a single dose, definitive conclusions about the safety of larger, repeated intravenous TPM doses are not possible. A study involving multiple doses used in clinical practice would permit a more robust investigation of relationship between adverse events and drug concentration.

Absolute bioavailability of oral TPM has been, until now, unknown because no intravenous formulation was available to conduct such as study. In our initial investigation in patients with migraines and epilepsy, we assumed the subjects to be at steady state and found the absolute bioavailability to be 110 ± 16%. [Correction added after online publication 18-March-2013: The absolute bioavailability values have been updated from 97 ± 24%]. The present controlled study in healthy volunteers confirms that observation and permits a more precise determination of TPM bioavailability and bioequivalence. The results from this study further establish that oral TPM is bioequivalent to intravenous TPM with relatively low interpatient variability. Plasma concentrations including peak concentrations attained by intravenous infusion were similar to oral administration, although the Tmax for the oral dose occurred approximately 1.35 h after dose. Should an intravenous TPM formulation be developed for older children and adults needing bridge therapy, the determination that the oral absorption is approximately 100% will simplify the switched from intravenous to oral, or vice versa.

TPM clearances (mean 1.2–1.3 L/kg) after oral and intravenous administration were not statistically different in this study and are similar to previous reports of oral clearance in subjects not taking enzyme-inducing medications (Easterling et al., 1988; Doose et al., 1996; Conway et al., 2003). TPM clearance in healthy volunteers was also similar to the clearance in those not taking inducing co-medication in our previous study of 25 mg doses (1.35 ± 0.37 L/h (Kriel et al., 2010).

As with bioavailability, TPM volume of distribution could not be accurately determined until the development of an intravenous formulation. We found distribution volume to be the same following oral and intravenous administration, and it exhibited relatively low variability (≈25%). Distribution volume is the parameter used to determine loading doses designed quickly to attain targeted drug concentrations. Characterization of TPM's distribution volume, with its low intrasubject variability, permits calculation of intravenous loading doses with good precision. A 1 mg/kg dose would be expected to produce, on average, a Cmax of 1 mg/L with a range of 0.75–1.25 mg/L in two thirds of patients. Future studies investigating the safety of using higher dosing for loading patients are needed.

The mean TPM half-life of 42 h observed in this study was longer than the previously reported value of 21 h (Doose & Streeter, 2002). As confirmation of our result, a similarly long half-life has been reported by Lambrecht et al. (2011). It now appears that TPM has a much longer half-life than previously reported, which may permit less frequent daily dosing than is current practice. In any case, the long half-life suggests that the intravenous TPM may be given once or twice daily while maintaining targeted plasma concentrations.

This study was conducted in healthy volunteers. Future research is needed to determine if intravenous TPM administration can be used for extended periods and at higher doses in adults and children with epilepsy. Intravenous TPM may also be useful in situations where patients on TPM are not able to take medications orally, for example, patients who are undergoing surgery, vomiting, have malabsorption disorders, or who are noncompliant. Safety studies at doses used to load patients are also needed, although patients requiring higher loading doses will likely be in an acute care or hospital setting. Therefore, neurologic and cognitive adverse events, which are more likely to occur with higher doses, may not be as clinically important. Adverse effects would likely be minimal in patients who are switched to intravenous TPM from an oral formulation because drug concentrations after oral and intravenous TPM will be similar when the same dose is given.

The results from this study, combined with substantial evidence of safety in children, set the stage for studies in pediatric populations. Further demonstration of safety and characterization of TPM pharmacokinetics in newborns will inform the design of subsequent controlled clinical trials intended to evaluate the efficacy and safety of intravenous TPM for neonatal seizures.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References

This study was partially funded by the Epilepsy Research Foundation's New Therapy Grants Program.

Study funding: This study was supported by grants from Ligand Pharmaceuticals Incorporated and the Epilepsy Research Foundation New Therapy Grant Program.

Disclosures

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References

Dr. Cloyd receives royalties under a licensing agreement between Ligand and the University of Minnesota. This relationship has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policies. Dr. Cloyd is also a consultant to Upsher Smith Laboratories regarding the development of an extended release topiramate product. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosures
  8. References