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Keywords:

  • Copper chelation;
  • Copper toxicosis treatment;
  • Copper-associated hepatitis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background

d-Penicillamine is the most commonly used copper-chelating agent in the treatment of copper-associated hepatitis in dogs. Response to therapy can be variable, and there is a lack of pharmacokinetic information available for dogs. Coadministering the drug with food to alleviate vomiting has been recommended for dogs, which contradicts recommendations for drug administration to humans.

Hypothesis

Coadministration of d-penicillamine with food decreases relative bioavailability and maximum plasma drug concentrations (Cmax) in dogs.

Animals

Nine purpose-bred dogs with a median body weight of 17.0 kg.

Methods

Dogs received d-penicillamine (12.5 mg/kg PO) fasted and with food in a randomized, crossover design. Blood samples were collected before and 0.25, 0.5, 1, 2, 3, 4, 8, 12, and 24 hours after dosing. Total d-penicillamine concentrations were measured using liquid chromatography coupled with tandem quadrupole mass spectrometry. Pharmacokinetic parameters were calculated for each dog.

Results

Two fasted dogs (22%) vomited after receiving d-penicillamine. Mean Cmax ± standard deviation (SD) was 8.7 ± 3.1 μg/mL (fasted) and 1.9 ± 1.6 μg/mL (fed). Mean area under the plasma concentration curve ± SD was 16.9 ± 5.9 μg/mL·h (fasted) and 4.9 ± 3.4 μg/mL·h (fed). There were significant reductions in relative bioavailability and Cmax in fed dogs (P < .001).

Conclusions and Clinical Importance

Coadministration of d-penicillamine with food significantly decreases plasma drug concentrations in dogs. Decreased drug exposure could result in decreased copper chelation efficacy, prolonged therapy, additional cost, and greater disease morbidity. Administration of d-penicillamine with food cannot be categorically recommended without additional studies.

Abbreviations
AUC0-24h

area under the plasma concentration vs. time curve

CAH

copper-associated hepatitis

C max

maximum plasma drug concentration

SD

standard deviation

S-S dimer

symmetric disulfide dimer

t ½

elimination half-life

Copper-associated liver diseases in dogs have gained considerable attention in recent years. The original description of copper toxicosis in the Bedlington Terrier was reported in 1975.[1] Since that time, many additional affected breeds of dogs have been identified.[2-4] Recently, pathologic copper accumulation has been identified in several popular breeds including Doberman Pinschers and Labrador Retrievers.[5-8] Although therapies such as oral administration of zinc salts and low-copper diets are available, these are best suited to treat patients with mild disease or to maintain remission in patients that previously have received more aggressive treatment.[2, 9, 10] Currently, d-penicillamine is the most commonly used copper-chelating drug for treating dogs with copper-associated hepatitis (CAH).[2, 10, 11]

d-Penicillamine, an amino acid with a functional sulfhydryl group, was first identified as a product of penicillin hydrolysis in the 1940s.[12] A decade later, d-penicillamine entered clinical use as a copper-chelating agent after it was discovered in the urine of human patients with liver disease that were receiving penicillin treatment. Urinary d-penicillamine was associated with increased urinary copper excretion.[13] d-Penicillamine also has been used therapeutically in humans to treat rheumatoid arthritis, progressive systemic sclerosis, cystinuria, primary biliary cirrhosis, and other diseases.[14] Its main indication, however, is for treatment of Wilson's disease, a disorder in which a reduction or absence of ATP-7B gene expression results in copper accumulation in the liver, brain, eyes, and kidneys.[15] Since its first description as a therapeutic agent for Wilson's disease in the 1950s, several pharmacokinetic and pharmacodynamic evaluations have been performed.[16-20] Although d-penicillamine is effective in decreasing tissue copper concentrations, adverse effects can occur and include neurologic deterioration, hypersensitivity reactions, proteinuria, autoimmune diseases, and bone marrow suppression.[21-23] The most common adverse effect in humans is gastrointestinal upset which can necessitate a switch to alternative copper-lowering medications.[21, 23] Veterinary use of d-penicillamine has increased with the recent recognition that hepatopathies that occur in several breeds of dogs are copper-associated. However, responses to d-penicillamine treatment are variable and may be less consistent than those observed in Wilson's disease.[2, 24, 15, 25] Little pharmacokinetic information is available for dogs. One report from 1981 describes the pharmacokinetics of d-penicillamine in a single dog,[26] but broad inference cannot be made from a single subject. Furthermore, the analytical methods used in that study predate the accuracy of more modern methods. Although many adverse effects seen in humans have not been reported in dogs, vomiting is common and can preclude treatment in some cases.[2] Administering the drug with food to prevent or decrease vomiting is commonly recommended for dogs treated with d-penicillamine.[2, 24, 11] However, humans are instructed to take the drug on an empty stomach as 25–70% reductions in bioavailability are seen when the drug is administered with a meal.[17, 18] Similar pharmacokinetic studies in dogs have not been reported. The purpose of this study was to describe the pharmacokinetics of PO-administered d-penicillamine in healthy fed and fasted dogs. We hypothesized that administration of d-penicillamine with food would decrease relative bioavailability and maximum plasma drug concentrations (Cmax).

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

Nine purpose-bred dogs, 4 intact males and 5 intact females, were used in this study. Breeds included Beagles (4) and Beagle-Briard crosses (5). The median weight of the dogs was 17.0 kg (range, 11.8–21.6 kg). Dogs were between 12 and 13 months of age and were considered healthy based on the absence of clinical abnormalities and normal hematologic and biochemical evaluations. This study was approved by the Institutional Animal Care and Use Committee at Michigan State University.

Drug Preparation

Commercially available 250 mg d-penicillamine capsules1 were compounded into 12.5 mg/kg doses by the Michigan State University Veterinary Teaching Hospital pharmacy. Analysis of random capsules using liquid chromatography coupled with tandem quadrupole mass spectrometry (LC/MS/MS) established that compounded capsules were prepared to 98.7 ± 4.7% of the intended individual target doses.2 Compounded capsules were stored at room temperature and used within 1 month after compounding.

Study Design and Sample Collection

This study was a randomized crossover design. Dogs were randomly assigned to either the fasted group or the fed group. Study phases were separated by 1 week to allow sufficient time for drug washout and blood volume recovery. All dogs were fasted for 12 hours before beginning the sampling period. In both groups, a capsule containing 12.5 mg/kg of d-penicillamine was administered PO. For the fed group, dogs consumed approximately 25% of their estimated daily resting energy requirements, were given d-penicillamine PO, and then consumed an additional 25% of their estimated resting energy requirements. Food calculations were based on a standard formula for estimating caloric requirements.[27] All dogs were fed the same canned commercial maintenance diet.3 Venous blood samples (3 mL) were collected before and 0.25, 0.5, 1, 2, 3, 4, 8, 12, and 24 hours after d-penicillamine administration. Collected blood sample tubes immediately were placed in wet ice, and within 3 minutes after collection, centrifuged at 4°C for 10 minutes × 1,200 g. Plasma then was collected and stored at −80°C until analysis.

d-Penicillamine Analysis

d-Penicillamine monomer and S-S dimer (symmetric disulfide) concentrations were measured using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), specifically by employment of a Thermo-Finnigan Surveyor HPLC connected to a Thermo-Finnigan TSQ Quantum ESI-MS/MS detector.4 Briefly, 0.5 mL of plasma was protein precipitated and simultaneously extracted by addition of 1 mL acetonitrile. Standard dilutions in water were prepared with concentrations ranging from 0.05 to 100 μg/mL for both monomeric d-penicillamine (>99% purity)5 and its S-S dimer (>99% purity).5 Ionization for LC/MS/MS was accomplished by an electrospray source kept in positive mode. Although noncovalent dimerization is an occasional artifact of electrospray, often with dimers coordinated around a metal ion,[28] the technique employed here could readily distinguish penicillamine monomer from its covalent disulfide dimer. Separate calibration curves were generated for each compound with individual standard compounds. A full report on development of this method is in preparation, but the extraction method is summarized as follows: d-penicillamine monomer was quantitated by positive mode electrospray focusing on the quantitative transition m/z 150 [RIGHTWARDS ARROW] 115 as well as the penicillamine-sodium adduct transition m/z 172 [RIGHTWARDS ARROW] 155, while the S-S dimer was quantitated similarly using m/z 297 [RIGHTWARDS ARROW] 180 and the corresponding dimer sodium adduct from m/z 319 [RIGHTWARDS ARROW] 241. Chromatography on an Alltech Alltima 3 μm 2.1 × 50 mm C18 column6 involved a gradient with 0.1% formic acid in HPLC-grade water (Solvent A) and HPLC-grade acetonitrile (Solvent B) at 300 μL/min throughout with variation as follows: 0–1 minutes, 90%A/10%B; 1-2 min, linear gradient to 10%A/90%B; 2–5 minutes, hold at 10%A/90%B; 5–10 minutes, linear gradient to 90%A/10%B; 10–12 minutes, hold at 90%A/10%B. The linear equation of the line resulting from calibration plots was used to determine sample d-penicillamine monomer and S-S dimer concentrations by interpolating the peak area of samples in the equation of the line. The peak areas of standards and samples were measured using an analytical software package.7 Total d-penicillamine was defined as the sum of d-penicillamine monomer and its S-S dimer.

Assay Validation

The LC/MS/MS method was determined to be appropriate for penicillamine quantitation by examining validation parameters for limits of detection and quantitation (LOD/LOQ), linearity, precision, and accuracy. LOD/LOQ were calculated by examining the SD of blank run responses (minimum of 10) and multiplying by 3.3× for LOD and 10× for LOQ.[29] Linearity was assessed by determination of the coefficient of determination, R2, for linear or quadratic unweighted unforced standard curves. Precision was assessed by refit of standards to their respective standard curves in the ranges 1–1,000 μg/mL for d-penicillamine monomer and 0.5–100 μg/mL for its S-S dimer. Accuracy was assessed for serum samples by assessment of sera spiked at 1.0 and 10.0 μg/mL d-penicillamine S-S dimer.

Validation results are as follows: d-penicillamine monomer LOD = 0.18 μg/ml, LOQ = 0.55 μg/ml; d-penicillamine S-S dimer LOD = 0.10 μg/mL, LOQ = 0.32 μg/mL. Calibrator R2 was 1.000 (SD = 0.0) for d-penicillamine monomer (n = 2) and 0.9982 (SD = 0.0028) for d-penicillamine S-S dimer (n = 7). The average refit values (%RSD) on repeat assessment of calibrators were as follows: (1) d-penicillamine 10.0, 100, and 1,000 μg/mL gave 11.1 (2.1%), 99.9 (0.0%), and 958.2 (3.1%), respectively; (2) d-penicillamine S-S dimer 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg/mL gave 0.60 (17.9%), 1.2 (18.3%), 5.4 (15.0%), 11.4 (12.9%), 49.0 (2.9%), and 100.3 (0.5%), respectively. d-penicillamine S-S dimer spikes returned the following average values: 1.0 μg/mL gave 1.2 μg/mL (%RSD = 17.5%), whereas 10.0 μg/mL gave 10.5 μg/mL (%RSD = 18.1%). The above validation findings are in accord with suggested guidelines of the Food and Drug Administration (FDA) for bioanalytical methods.8

Pharmacokinetic Analysis

Pharmacokinetic analysis was performed for each dog. Both Cmax and time of maximum plasma concentration (Tmax) were obtained directly from experimental data. Area under the plasma concentration versus time curve (AUC0–24h) was determined by the trapezoidal method. The elimination rate constant (λ) was derived from the linear regression of the terminal 3 observations plotted as the log mean concentrations against time. The corresponding terminal-phase elimination half-life (t½) was calculated as: t½ = ln2/λ. Plasma samples with total d-penicillamine concentrations below the LOQ were treated as zero values for all calculations.

Statistical Analysis

Response variables were AUC0–24h, Cmax, and Tmax. Although AUC0–24h and Cmax were normally distributed, Tmax was not. Area under the curve and Cmax were evaluated by means of split plot analysis of variance with the grouping factor of sex and the repeat factors of period and fasting. Comparisons of pharmacokinetic parameters between the fasted and fed dog groups were performed using a Mann–Whitney test (Tmax) or a 2-tailed t-test (AUC0–24h and Cmax). Statistical analyses were performed using commercially available software.9 For all analyses, P ≤ .05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Analysis of the contents of 8 d-penicillamine capsules1 identified a mixture of d-penicillamine monomer (92.3 ± 8.5%) and its S-S dimer (7.7 ± 8.5%).2 Analysis of plasma samples identified detectable concentrations of only the d-penicillamine S-S dimer; the d-penicillamine monomer was not detected in any of the samples. Two fasted dogs (22%) vomited after receiving d-penicillamine. The dogs vomited 46 and 76 minutes after receiving the drug, which roughly corresponded to each dog's Tmax. The study phase was repeated in both dogs, however, both dogs vomited again at approximately the same times. Only data from the original samples were analyzed. In both dogs, fasted AUC0–24h was still approximately 2 times greater than fed AUC0–24h. Maximum plasma drug concentrations from the dogs that vomited were 2nd and 5th highest of the 9 fasted dogs. None of the fed dogs vomited. One dog was excluded from statistical comparisons in the fed group because postfeeding drug concentrations at all time points were below the LOQ.

The mean peak plasma concentration of d-penicillamine (±SD) in fasted dogs (8.7 ± 3.1 μg/mL) was substantially higher than in fed dogs (1.9 ± 1.6 μg/mL) (Table 1). The mean (±SD) AUC0–24h for fasted dogs (16.9 ± 5.9 μg/mL·h) was substantially greater than that for fed dogs (4.9 ± 3.4 μg/mL·h) (Fig 1). The mean percent reductions (±SD) in AUC0–24h and Cmax were 69.7% (±19.4%) and 77.4% (±15.0%), respectively. Overall, there were significant reductions in AUC0–24h and Cmax in fed dogs (P < .001). Coadministration of d-penicillamine with food also significantly delayed Tmax (P = .011) with peak plasma total d-penicillamine concentrations occurring between 1 and 2 hours postdose in fasted dogs and closer to 3 hours in fed dogs. Sex and study period had no effect on Cmax, AUC0–24h, or Tmax. Half-lives were not compared between groups.

Table 1. Pharmacokinetic parameters of d-penicillamine in fasted and fed dogs
Study groupParameterMedianRangeMean ± SD
  1. Values represent the median, range, and mean ± SD, with the exception of t½, which was calculated based on the means of the observed data across time points. SD, standard deviation; Cmax, maximum plasma concentration; Tmax, time to maximum plasma concentration; AUC, area under plasma concentration time curve; t½, elimination half-life.

Fasted (n = 9)Cmax (μg/mL)6.994.92–13.088.65 ± 3.09
Tmax (hours)1.000.5–2.001.17 ± 0.66
AUC0–24h (μg/mL·h)14.0811.67–28.4616.86 ± 5.90
t½ (hours)1.69
Fed (n = 8)Cmax (μg/mL)1.600.51–5.471.88 ± 1.60
Tmax (hours)2.501.00–8.003.00 ± 2.14
AUC0–24h (μg/mL·h)4.210.99–12.224.88 ± 3.40
t½ (hours)2.60
image

Figure 1. Plasma concentrations of total d-penicillamine after a single oral dose (12.5 mg/kg). Results are given as mean (±standard deviation) from 8 (fed) and 9 (fasted) dogs.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Pharmacokinetic parameters of PO-administered d-penicillamine in fasted and fed healthy dogs were investigated in the present study. Significant reductions in Cmax and exposure (AUC0–24h) were observed when the drug was coadministered with food. The pharmacokinetic parameters (AUC0–24h, Cmax, Tmax, t½) in fasted dogs were similar to those reported in pharmacokinetic studies of humans.[14, 20, 30] The 69.7% reduction in relative bioavailability when the drug was administered with food also is consistent with reductions of 25–70% reported in studies of humans.[14, 16, 17] In contrast to reports in humans, we did detect a significant delay in Tmax in fed dogs.[16] Determination of additional drug parameters such as absolute drug bioavailability, clearance, and volume of distribution would require paired intravenous and oral dosing and this warrants additional investigation.

d-Penicillamine is present in multiple forms in biological fluids: free reduced d-penicillamine, protein-bound d-penicillamine, and disulfides.[17] Some reports speculate that the free monomeric form is physiologically active, simply binding copper between the sulfhydryl and amino groups.[31] However, the actual cupruresis induced after drug administration is 1% of the expected effect if this interpretation were true.10 Other reports have suggested that plasma copper speciation and mechanism of drug action are far more complex and incompletely understood.[32, 33] Although attempts were made to detect and quantify both free d-penicillamine monomer and the S-S dimer, we were only able to detect the d-penicillamine S-S dimer. d-penicillamine, like many thiols, is known to readily undergo oxidation reactions under a variety of conditions.[14] Our collection or processing methods may have favored oxidation to the S-S dimer form. Another possibility is that d-penicillamine is primarily present in canine plasma in its S-S dimer form, and that the monomer is either absent, covalently protein bound, or only present in concentrations below the detection limit of our assay. Reports of d-penicillamine pharmacokinetics in humans have produced similar results when attempting to isolate the different forms of d-penicillamine.[17] In many previous studies, total d-penicillamine concentrations are reported after protein precipitation (to release bound d-penicillamine) without further elaboration.[14, 16, 17] Despite varying methodologies and ambiguities in previous reports, the overall pharmacokinetic data in studies of humans generally are in good agreement.[14, 17] Furthermore, investigations have disclosed that the S-S dimer can be spontaneously reduced to free drug in vivo and vice versa.[34] As such, it is appropriate to consider contributions from both sources in pharmacokinetic evaluations.

We observed a longer t½ in fed dogs. Because of profound effects of food on absorption, it is likely that low-level absorption and elimination were occurring simultaneously throughout much of the sampling period. In many fed dogs, drug concentrations postCmax were below the LOQ at later time points. This might have precluded accurate t½ determinations in the fed dogs and, as such, the reported t½ in fed dogs should be considered preliminary, pending further investigations. We acknowledge this limitation and chose not to compare this parameter between fed and fasted dogs. However, pharmacokinetic studies in humans have documented a similar t½ in fed and fasted subjects.[18] Another potential limitation in our study is the determined Cmax relative to sampling times. Most individual fasted dogs had Tmax at 1 or 2 hours, and mean Tmax was 1.2 hours. Our sampling times included a 1 and 2-hour sample. Ideally, a 1.5 hour sample would have been collected to maximize the accuracy of Cmax determinations given the fairly rapid absorption and elimination of d-penicillamine. Some pharmacokinetic parameters also potentially could differ in dogs with CAH.

Although d-penicillamine is not approved by the FDA for use in dogs, it has been used to treat CAH in dogs since the 1970s.[1] d-Penicillamine is FDA approved to treat copper-associated Wilson's disease patients for which explicit guidelines exist. The FDA approved prescribing information specifies “it is important that d-penicillamine be given on an empty stomach, at least 1 hour before meals or 2 hours after meals, and at least 1 hour apart from any other drug, food, or milk.”10 In contrast, d-penicillamine has been administered to dogs 20 minutes[35] or 30 minutes[36] before food and has been recently recommended to be given with food.[2, 24, 11] Published reports in dogs document some hepatic copper-lowering effects when administered 20 or 30 minutes[35, 36] before food. However, other than anecdotal reports, no veterinary studies document efficacy when d-penicillamine is given with a meal.[11]

In addition to differences in drug administration in relationship with food, differences are evident in the dosage per kg of body weight administered to humans compared to dogs. The published recommended dog dosage is 10–15 mg/kg twice daily (20–30 mg/kg/day).[2, 11] The FDA recommended total daily dose in humans is between 750 and 1500 mg per day predicated initially on the degree of cupriuresis, and later on the reduction in nonceruloplasmin plasma copper concentration. 10,[21] On a per kg body weight basis, the usual dosage in humans is approximately one-half of the recommended dosage in dogs.

The findings reported herein establish that d-penicillamine relative bioavailability is decreased by approximately 70% when the drug is administered with food. These findings do not establish that the administration of d-penicillamine with food is without efficacy for copper chelation in dogs, but do suggest that current dosing recommendations lead to substantial waste of a rather expensive drug.

The recommendation to administer d-penicillamine with food recently has been proposed because some dogs vomit the drug.[2, 24] Two fasted dogs in our study vomited after d-penicillamine administration and did so again when the experiment was repeated. Despite vomiting, fasting AUC0–24h in both dogs was still approximately 2 times greater than fed AUC0–24h. Vomiting occurred at approximately the time of peak plasma drug concentration suggesting that the vomiting may have been blood concentration dependent and mediated by way of the chemoreceptor trigger zone.[37] Vomiting of this nature may be dose dependent.

In the aggregate, our results and those of others identify gaps in the knowledge on which to base sound d-penicillamine dosing recommendations for the dog. Although 10–15 mg/kg twice daily d-penicillamine doses administered 20 minutes before feeding has been documented to decrease liver copper concentrations in 1 report, 3 of 43 Labrador Retrievers vomited and were excluded from the study.[35] Individual doses slightly less than 10–15 mg/kg administered on an empty stomach might be less stimulatory of emesis and yet be efficacious. In partial support of this approach, d-penicillamine doses less than 7.0 mg/kg administered twice daily 30 minutes before feeding to 5 Doberman Pinschers resulted in decreased hepatic copper in all dogs in 1 report.[36] Alternatively, a long acting antiemetic could be administered 1 hour before d-penicillamine administration to dogs that have a proclivity to vomit. Regardless of these considerations, efficacy of d-penicillamine administration with food has not been evaluated by controlled studies.

In conclusion, d-penicillamine undergoes rapid absorption and elimination in fasted dogs with peak plasma drug concentrations occurring between 1 and 2 hours. The administration of d-penicillamine with food markedly decreases relative bioavailability and Cmax. The reduction in drug exposure associated with administration with food could result in decreased efficacy and veterinary clinicians should remain mindful of this fact.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors acknowledge Dr Joe Jehl, Kristi Aybar, Sharon Steck, Janice Querubin, and Marlee Richter for their contributions.

Conflict of Interest: Authors disclose no conflict of interest.

Funding: This study was supported by the Michigan State University College of Veterinary Medicine Companion Animal Fund and the Michigan State University College of Veterinary Medicine Trinket Fund.

Footnotes
  1. 1

    Cuprimine, Aton Pharma Inc, Lawrenceville, NJ

  2. 2

    The capsule contents were weighed and, in each case, were greater than the designated values (eg, 250 mg) because of the presence of excipient components. Comparison of such measured values (eg, 360 mg total) to the designated contents enabled adjustments of concentrations made up with the initial assumption of pure compound. Therefore, solutions made up at nominal concentrations of 1.0 mg/mL with pill contents, then diluted 1 : 10 to 100 μg/mL for each formulation could then be adjusted for the purity of the capsule contents. In the example described, the nominal concentration of the diluted standard would be adjusted to 100 μg/mL × (250/360) = 69.4 μg/mL. The percent yield was then based simply on the comparison of the sum of d-penicillamine and d-penicillamine disulfide species with each adjusted target concentration. A finding of 68.9 μg/mL in this example would mean (68.9/69.4) × 100 = 99.3% of the intended individual target dose

  3. 3

    Nestlé Purina Inc, St. Louis, MO

  4. 4

    Thermo-Fisher Scientific Inc, Waltham, MA

  5. 5

    Sigma-Aldrich Co LLC, St. Louis, MO

  6. 6

    Fisher Scientific, www.fishersci.com

  7. 7

    Xcalibur, Thermo-Fisher Scientific Inc, Waltham, MA

  8. 8

    Guidance for Industry, Bioanalytical Method Validation. U.S. Department of Health and Human Services, Food and Drug Administration, 2001. Available from: http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf

  9. 9

    GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA

  10. 10

    Drug Label Section, Current Medication Information. Available from: http://dailymed.nlm.nih.gov/dailymed/druginfo

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References