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Pharmacokinetics of 14C-radioactivity after oral intake of a single dose of 14C-labeled fampridine (4-aminopyridine) in healthy volunteers
Clinical Therapeutics/Volume 31, Number 2,2009
BriefReport
Pharmacokinetics of 14C_Radioactivity After Oral Intake of a Single Dose of 14C-Labeled Fampridine (4-Aminopyridine) in Healthy Volunteers Andrew R. Blight, PhD; and Herbert R. Henney III, PharmD
Acorda Therapeutics, Hawthorne, New York ABSTRACT Background: Fampridine (4-aminopyridine) is a potassium channel blocker that has been evaluated as a treatment for patients with spinal cord injury and multiple sclerosis. Objective: The purpose of this study was to determine the pharmacokinetics of a single dose of an orally administered solution of 14C-Iabeled fampridine in healthy volunteers. Methods: In this open-label, single-dose study conducted in an inpatient setting, healthy adult men were administered an oral solution containing 15 mg of 14C-Iabeled fampridine (100 J..l.Ci) in a fasted state. In addition to blood sampling for analysis of plasma 14C-radioactivity at prescribed intervals over 7 days, all urine and feces were collected for analysis of drug recovery and disposition. Urine samples were also analyzed for metabolic profiling. Plasma pharmacokinetic parameters of the 14C-radiolabeled drug were determined using standard liquid-scintillation techniques. Recovery was calculated to provide the total amount of radioactivity excreted as a proportion of the original dose. Nonhydrolyzed and hydrolyzed urine extracts were analyzed for radioactivity and metabolites using reverse-phase, isocratic high-performance liquid chromatography with spectrophotometric and radioactive detection. Tolerability was assessed through evaluation of vital signs, hematologic and other laboratory parameters, and electrocardiography. Results: The 4 white male subjects had a mean (SO) age of 21 (2) years. No clinically significant abnormalities in vital signs, clinical chemistry, hematology, urinalysis, or electrocardiography were observed either before or during the study. Peak plasma radioactivity was reached at 1 hour after dosing, with a median concentration of 72.9 ng . mL-l. There was complete disappearance of radioactivity by 24 hours 328
(limit of quantitation, 400 disintegrations/min per peak), and the calculated median t 1l2 was 3.14 hours. Total cumulative recovery of 14C-radioactivity was 96.36%, with only 0.51 % of drug recovered in feces. On chromatography, 2 metabolites accounted for a low proportion of total urinary radioactivity (3% and 6% of total radioactivity in the interval from 0 to 4 hours after dosing; 17% and 9% in the interval from 8 to 12 hours after dosing). Three subjects reported mild and transient dizziness occurring 1 halfhour after dosing; this was considered possibly related to the study drug. Conclusion: Fampridine administered as an oral solution was rapidly absorbed and was nearly completely and rapidly eliminated as unchanged drug via urinary excretion, suggesting that it is unlikely to undergo substantial metabolic transformation. (Clin Ther. 2009;31:328-335) © 2009 Excerpta Medica Inc. Key words: fampridine, 4-aminopyridine, pharmacokinetics, drug disposition.
INTRODUCTION Fampridine (4-aminopyridine) is a broad-spectrum blocker of potassium channels at millimolar concentrations. 1,2 However, plasma concentrations obtained in clinical studies are typically <1 J..l.M (94 ng' mL-1); the molecular characteristics of specific potassium channels with sensitivity in this range have not been determined. The clinical neurologic effects of fampridine are consistent with the molecular mechanism of Accepted for publication)anuary 26,2009. Express Track online publication February 18, 2009. doi:l 0.1 016/j.c1inthera.2009.02.004 0149-2918/$ - see front matter © 2009 Excerpta Medica Inc. All rights reserved.
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A.R. Blight and H.R. Henney III
potassium channel blockade. At the cellular level, this action may increase neuronal excitability,3 relieve conduction block in demyelinated axons,4-7 and potentiate synaptic and neuromuscular transmission. 8- 10 However, the relative contribution of these distinct cellular effects to the potential therapeutic benefits and adverse effects of 4-aminopyridine is currently unclear. Over the past 30 years, 4-aminopyridine has been evaluated as a potential treatment for neurologic and neuromuscular conditions such as myasthenia graviS,11 Eaton-Lambert syndrome,12,13 episodic ataxia,14 upbeat and downbeat nystagmus,15,16 and spinal cord injury. 17-21 A number of studies have suggested potential benefits of fampridine as a treatment targeted at overcoming the action-potential conduction deficits associated with demyelination in patients with multiple sclerosis (MS), particularly in terms of improving walking ability and muscle strength.22-29 The pharmacokinetics of fampridine have been investigated in several published studies. 17,18,24,30-32 These studies involved either intravenous or oral delivery of immediate-release (IR) formulations, although the former route is not clinically practical and may be associated with intolerable adverse effects, particularly pain at the injection site. 17,32 Many of these analyses were not systematic pharmacokinetic evaluations but were performed as a part of clinical trials in patients with spinal cord injury or MS. Nonetheless, their findings suggested that fampridine has a narrow therapeutic range, with oral dosing of IR formulations resulting in a high Cmax ' followed by rapid elimination via renal excretion. 30 ,31,33 It has been suggested that this profile, in which Cmax is achieved -1 hour after drug administration, contributes to the occurrence of adverse events, which may occur in a dose-dependent manner. 24 The disposition of fampridine has been evaluated in 2 published studies,30,31 both of which reported high recovery of unchanged drug in the urine (87%91 %), although neither reported on fecal recovery. These studies predominantly involved intravenous administration, which, in the study by Uges et al,31 was used as the standard for determining the bioavailability of oral preparations of enteric-coated and uncoated tablets of 4-aminopyridine. Neither study fully evaluated the potential for metabolic transformation. For therapeutic and drug-registration purposes, it is important to obtain specific information on the dispoFebruary 2009
sition and metabolism of a drug in humans. Therefore, the purpose of the present study was to use the sensitivity associated with radioactive techniques to establish the rate and extent of absorption of a single oral dose of 14C-Iabeled fampridine, as well as to characterize the disposition of this agent with respect to routes of elimination and the balance of total 14C_ radioactivity. In addition, samples were analyzed for the presence of potential metabolites resulting from transformation of the parent compound.
SUBJECTS AND METHODS This study was conducted in accordance with the Declaration of Helsinki (third revision) and was approved by the medical ethics committee of the Stichting Beoordeling Ethiek Bio-Medisch Onderzooek (Assen, The Netherlands). All subjects provided written informed consent before participation. Because of its nature, the study was performed on an inpatient basis at the clinic site (Zuidlaren, The Netherlands) from the afternoon before dosing until the afternoon of day 8 after dosing. The inpatient stay was prolonged if background-corrected radioactivity in the urine was >50 disintegrations per minute (dpm) . mL-1 or was >75 dpm per homogenized fecal sample of -400 mg. The study population consisted of healthy male volunteers aged between 18 and 45 years, with body weight within 15% of normal for body structure and no pathologic findings on the medical history or screening assessments. Screening included a physical examination and medical history, recording of vital signs, laboratory tests, and a 12-lead electrocardiogram (ECG). Grounds for exclusion included, but were not limited to, evidence of clinically relevant pathology, mental handicap, or illness within 5 days before study initiation; a history of food or drug allergies; a history of alcohol or drug abuse or addiction or a positive drug test at screening; treatment with systemic medications during the previous 3 months; smoking of >10 cigarettes/d; exposure to radiation for any reason other than routine radiography within the past year; and an abnormal defecation pattern (ie, less than once every 2 days). 14C-radiolabeled 4-aminopyridine IR was administered as an oral solution of 15 mg (100 J..l.Ci) in 50 mL of water. Administration took place in the morning after a lO-hour fast and was observed by an investigator to ensure compliance. After administration of the solution, the container was washed and equal volumes 329
Clinical Therapeutics
of water were consumed 3 times. An additional 200 mL of water was swallowed 2 hours after dosing, and fasting was continued until lunch. With the exceptions of acetaminophen (paracetamol) and topical agents, no concomitant medications were permitted during the study. Blood sampling was performed either via an indwelling catheter or by direct venipuncture. Samples were collected into heparinized tubes and immediately cooled to O°c. Within 1 hour of blood sampling, plasma was separated by centrifugation for 10 minutes (4°C, 1500g) and stored at -20°C until analyzed. Samples (10 mL) were collected for analysis of 14C_ radioactivity immediately before drug administration and at the following time points after dosing: 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours. All urine and feces were collected during the study. Urine was collected at the following hourly intervals relative to dosing (0 hour): -12 to 0, 0 to 4, 4 to 8, 8 to 12, and 12 to 24 hours, and at 24-hour intervals thereafter until discharge. All samples were processed after determination of their volume; 10-mL samples were analyzed for 14C-radioactivity, and 20-mL samples were used for pharmacokinetic analysis of fampridine and its metabolites when the radioactivity count was >1000 dpm . mL-l. For fecal analysis, pooled samples were used for each 24-hour period and, after determination of their weight, 10 g was obtained from the homogenized samples and analyzed for 14C-radioactivity. Aliquots of plasma, urine, and feces were analyzed for 14C-radioactivity by standard liquid-scintillation techniques using a 1900 TR Liquid Scintillation Counter (Canberra Packard Instruments Co., Downers Grove, Illinois). All analyses were performed in duplicate and included 2 blank matrix samples and 6 quality control samples; values were corrected for background. Descriptive analysis was used for standard plasma pharmacokinetic parameters, including Cmax ' T max' t 1l2 , elimination rate constant (A et ), and AUC o-
fornia) equipped with an SP 8750 organizer and a Waters 717 Autosampler Injector (Waters Corporation, Milford, Massachusetts). Nonhydrolyzed and enzymatically hydrolyzed (~-glucuronidase type HI) urine samples were injected directly into the HPLC system. This system incorporated a Waters pBondapack C18 analytical column (Waters), which was temperatureconditioned (18°C-24°C) using a thermostatic water bath (Julabo Labortechnik GmbH, Seelbach, Germany). Two modes of detection were used: spectrophotometric detection at an ultraviolet wavelength of 263 nm (Perkin Elmer LC-75 spectrophotometric detector, Perkin Elmer Ltd., Beaconsfield, United Kingdom); and radioactivity detection using a Radiomatic 525 TR flow detector (Canberra Packard), with a limit of quantitation determined to be 400 dpm per peak. The chromatograph system was connected to a Foxy 200 fraction collector (Teledyne Isco Inc., Lincoln, Nebraska). The mobile phase, filtered and degassed before use, consisted of 800 mL of buffer (1 M potassium dihydrogen orthophosphate, 1 M phosphoric acid, 1 M I-heptanesulfonic acid, and 0.05 M tetrabutylammonium iodide; pH 3.5) mixed with 35 mL of acetonitrile and 165 mL of water. Mean (percent coefficient of variation) intra-assay reproducibility ranged from 95.93% to 102.43% (1.75%-5.83%), and mean interassay reproducibility ranged from 93.70% to 104.58% (0.58%-3.26%), with a mean recovery of 52.18% to 67.91% (7.03%-18.57%) for sample and 79.62% (12.47%) for internal standard. Tolerability was monitored based on vital signs, hematologic and other laboratory values, and ECGs. Subjects were also asked questions designed to elicit information on the occurrence of adverse events. RESULTS Four white male subjects with a mean (SO) age of 21 (2) years and a mean weight of 70.8 (3.3) kg were enrolled in and completed the study (Table I). No clinically significant changes in vital signs or clinical chemistry, hematology, urinalysis, or ECG findings were observed during the study. Four adverse events of mild intensity were reported, 1 in each subject. Three of these events-dizziness occurring 1 half-hour after dosing-were considered possibly related to the study medication. The fourth event was a sore throat and was not considered related to the study medication. Median plasma concentrations of drug calculated from recovered 14C-radioactivity reached C max Volume 31 Number 2
A.R. Blight and H.R. Henney III
Table I. Characteristics of the 4 male subjects. Characteristic
Value
Age, y Mean (SO) Range
21 (2) 18-22
Weight, kg Mean (SO) Range
70.8 (3.3) 68.1-75.3
Body mass index, kg/m 2 Mean (SO) Range
21.8 (1.4) 20.3-23.6
Oose administered, J..l.Ci Mean (SO) Range
105.5 (1.0) 105-107
-1.0 hour after administration, with subsequent rapid elimination and complete disappearance from the plasma by 24 hours after dosing (Figure 1). The frequency of plasma sampling was too low to provide a more precise determination of T max' The pharmacoki-
netic parameters derived from total 14C-radioactivity are summarized in Table II. The median AUCo-
100
....... I
...J
E
75
t)()
c '-'" c 0
...
.~
I'll
'"'
50
V
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0
U
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E VI
25
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1:[
0
0
12
24
36
48
Time (h) Figure 1. Median plasma concentrations offampridine calculated from 14C-radioactivity recovered in plasma over time after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine in 4 healthy male subjects.
February 2009
331
Clinical Therapeutics
separated from the parent compound at 7.3 minutes (Figure 3). Additionally, 2 minor peaks in metabolites were observed in most chromatograms both before and after hydrolysis. These metabolites had retention times of -4.6 minutes and 8.6 minutes that coincided with those of standard preparations of 2-hydroxy4-aminopyridine (4.40 minutes) and 3-hydroxy4-aminopyridine (8.75 minutes). Quantitatively, the 2 metabolites comprised 3% and 6% of total radioactivity in the interval from 0 to 4 hours after dosing, and 17% and 9% in the interval from 8 to 12 hours after dosing.
Table II. Pharmacokinetic parameters of fampridine derived from plasma 14C-radioactivity after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine (N - 4). Value
Parameter Cmax ' ng . mL-l Median Range
72.9 63.1-86.6
Tmax ' h Median Range Ael , h- 1 Median Range
1.0 1.0-1.0
Median Range
3.14 2.92-4.59
AUCo_co> ng . h/mL-l Median Range -
Although the generalizability of this study is limited by its small, all-male population, administration of a single 15-mg oral dose of 14C-Iabeled 4-aminopyridine was associated with rapid absorption, with peak plasma radioactivity being reached by 1.0 hour after dosing. Elimination from the plasma was also rapid and was complete by 24 hours, with a calculated t 1l2 of 3.14 hours. These data suggest that the drug was well absorbed. No serious adverse events were reported during the study; mild and transient dizzi-
0.220 0.151-0.237
t 1/ 2 , h
Ael
DISCUSSION
341.2 258.0-416.3
elimination rate constant.
.<>
Urine
.t:.. Feces + Total
100
- - - - -ov - - -. - - - - -. - -..... - - -- - - - - -- -...,.- -- - - - - -- --
~ VI
-
------ ... ------
-
0
0 ..... 0
80
~
...~
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u
60
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ill l: :l
0
40
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20
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A
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A
•
A _ • __ •
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_
0 0
24
48
72
96
120
144
168
Time (h) Figure 2. Median cumulative excretion of 14C-radioactivity after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine in 4 healthy male subjects.
332
Volume 31 Number 2
A.R. Blight and H.R. Henney III
ness was reported in 3 subjects shortly after drug administration. The plasma pharmacokinetics of fampridine in this study, measured in terms of radioactivity, agreed with those reported previously for 4-aminopyridine IR, administered as single escalating oral doses of 10, 15, 20, and 25 mg at weekly intervals in healthy volunteers. 33 In that study, the T max and t 1l2 for the 15-mg dose were 1.0 and 3.4 hours, respectively. The Cmax and AUCo-
Table III. Cumulative excretion of 14C-radioactivity after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine (N - 4). Parameter
Value
% of dose Median Range
95.85 93.80-97.50
% of dose Median Range
0.51 0.45-0.53
% of dose Median Range
96.36 94.33-97.95
Aeurine'
A\eces'
Ae total ,
total amount of 14C-radioactivity excreted in urine; Aefeces - total amount of 14C-radioactivity excreted in feces; Aelolal - sum of Aeurine + A\eces'
Aeurine -
856
7.30
642
~
U
6.80
428-
2'44.60
f\\ K. ,",-~.AQ;;;;,3.;.;.70:l0)~~ \...,.~~5~.5~0<::::=J.~:...._ _"-.-I-==-_...:::.t. 3.30 5 6.00 0
o..L...,;:.=o.:;;30::..::....ci1i:..20;;;",...:::::2~.2;;.0,d..
o
0.80
1.70
0~.4_0.....il::,;,I_.40=::::::::::::1:::::2Q-;;.5:::::0:::::12:::,.8::::°::...o::::::::14.:::'ll1o.0""i
1 \9::.4:::.,0""",X C
"0
12.00
13.60
"5
Minutes Figure 3. Characteristic chromatogram (high-performance liquid chromatography with radioflow detection), showing 14C-radioactivity peaks in a hydrolyzed urine sample in the collection interval from 8 to 12 hours after dosing (subject number 1). CPM - counts per minute.
February 2009
333
Clinical Therapeutics
the radioactivity was excreted in urine indicates that fampridine was almost completely absorbed from the oral solution. Findings from several studies have suggested the possibility of enterosystemic recirculation of fampridine, based mainly on observations of fluctuations in plasma concentrations among individual subjects. 17,18,30 The low fecal excretion reported in the present study suggests that absorption is almost complete or that hepatobiliary pathways represent only a minor route of elimination. However, HPLC analysis revealed the presence of a shoulder on the main peak, indicating that whereas most of the compound excreted in the urine was unchanged, excreted compounds may include conjugates of 4-aminopyridine. The occurrence of limited metabolic transformation was also indicated by the 2 minor peaks, consistent with 2-hydroxy-4-aminopyridine and 3-hydroxy-4aminopyridine, although the overall presence of metabolites represented only a small proportion (17% and 9%, respectively) of the total compound. Early work in dogs suggested that 4-aminopyridine metabolites include 3-hydroxy-4-aminopyridine and the sulfate conjugate of 3-hydroxy-4-aminopyridine (data on file, study HWI6379-102 [1992], Acorda Therapeutics, Hawthorne, New York), although a reference standard for identifying the sulfate conjugate was not available for the present study. If conversion of the parent compound to these metabolites via hydroxylation and sulfate conjugation can be confirmed in humans, it would be in contrast to the study by Uges et al,31 in which no evidence of such metabolic transformation was found. In this study, fampridine exhibited almost complete absorption, high urinary excretion, and low metabolic transformation. These properties, along with a narrow therapeutic range, rapid rise to Cmax ' and short t 1l2 , suggest the need for frequent dosing of fampridine to maintain effective plasma levels and improve tolerability. A formulation having improved pharmacokinetic characteristics might enhance the therapeutic potential of fampridine. CONCLUSIONS
The results of this study of 14C-Iabeled fampridine are consistent with previous reports of rapid absorption and nearly complete renal elimination of the drug. The presence of additional chromatographic peaks suggested the occurrence of metabolic transformation, 334
although these peaks accounted for only 17% and 9% of the total compound. ACKNOWLEDGMENTS
This study was supported by Acorda Therapeutics, Hawthorne, New York. Drs. Blight and Henney are employees of Acorda Therapeutics and hold stock in the company. The authors thank E. Jay Bienen, PhD, an independent scientific consultant based in New York City, for editorial support, which was funded by Acorda Therapeutics. REFERENCES 1. Gold MS, Shuster MJ, Levine JD. Characterization of six voltage-gated K+ currents in adult rat sensory neurons.
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1996;75:2629-2646.
2. Coetzee WA, Amarillo Y, Chiu J, et al. Molecular diversity ofK+ channels. Ann N Y Acad Sci. 1999;868:233-285. 3. Bowman WC, Savage AO. Pharmacological actions of aminopyridines and related compounds. Rev Pure Appl Pharmacol Sci. 1981;2:317-371.
4. Sherratt RM, Bostock H, Sears TA. Effects of4-aminopyridine on normal and demyelinated mammalian nerve fibres. Nature. 1980;283:570-572.
5. Targ EF, Kocsis JD. 4-Aminopyridine leads to restoration of conduction in demyelinated rat sciatic nerve. Brain Res. 1985;328:358-361. 6. Blight AR. Effect of 4-aminopyridine on axonal conduction-block in chronic spinal cord injury. Brain Res Bull. 1989;22:47-52.
7. Shi R, Blight AR. Differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cord. Neuroscience. 1997;77:553-562. 8. Lundh H. Effects of 4-aminopyridine on neuromuscular transmission. Brain Res. 1978;153:307-318. 9. Kim YI, Goldner MM, Sanders DB. Facilitatory effects of 4-aminopyridine on normal neuromuscular transmission. Muscle Nerve. 1980;3:105-111.
10. Smith KJ, Felts PA, John GR. Effects of 4-aminopyridine on demyelinated axons, synapses and muscle tension. Brain. 2000;123:171-184. 11. Lundh H, Nilsson 0, Rosen I. Effects of 4-aminopyridine in myasthenia gravis.) Neurol Neurosurg Psychiatry. 1979;42: 171-175. 12. Lundh H, Nilsson 0, Rosen I. 4-Aminopyridine-a new drug tested in the treatment of Eaton-Lambert syndrome.
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13. Agoston S, van Weerden T, Westra P, Broekert A. Effects of 4-aminopyridine in Eaton Lambert Syndrome. Br J Anaesth. 1978;50:383-385.
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sodic ataxia type 2 with the potassium channel blocker 4-aminopyridine. Neurology. 2004; 62: 1623-1625. Glasauer S, Kalla R, Buttner U, et al. 4-Aminopyridine restores visual ocular motor function in upbeat nystagmus. J Neurol Neurosurg Psychiatry. 2005;76:451-453. Kalla R, Glasauer S, Buttner U, et al. 4-Aminopyridine restores vertical and horizontal neural integrator function in downbeat nystagmus. Brain. 2007;130:2441-2451. Hansebout RR, Blight AR, Fawcett S, Reddy K. 4-Aminopyridine in chronic spinal cord injury: A controlled, double-blind, crossover study in eight patients. J Neurotrauma. 1993;10:1-18. Potter Pj, Hayes KC, Hsieh jT, et al. Sustained improvements in neurological function in spinal cord injured patients treated with oral 4-aminopyridine: Three cases. Spinal Cord. 1998;36:147-155. SegaljL, Pathak MS, HernandezjP, et al. Safety and efficacy of4-aminopyridine in humans with spinal cord injury: A long-term, controlled trial. Pharmacotherapy. 1999;19: 713-723. van der Bruggen MA, Huisman HB, Beckerman H, et al. Randomized trial of 4-aminopyridine in patients with chronic incomplete spinal cord injury. J Neurol. 2001 ;248: 665-671. Cardenas DO, Ditunno j, Graziani V, et al. Phase 2 trial of sustainedrelease fampridine in chronic spinal cord injury. Spinal Cord. 2007;45: 158-168. Stefoski 0, Davis FA, Faut M, Schauf CL. 4-Aminopyridine improves clinical signs in multiple sclerosis. Ann Neurol.1987;21:71-77. Davis FA, Stefoski 0, Rush J. Orally administered 4-aminopyridine improves clinical signs in multiple sclerosis. Ann Neurol. 1990;27: 186192. Bever CT jr, Young 0, Anderson PA, et al. The effects of4-aminopyridine
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in multiple sclerosis patients: Results ofa randomized, placebo-controlled, double-blind, concentration-controlled, crossover trial. Neurology. 1994;44: 1054-1059. Polman CH, Bertelsmann FW, van Loenen AC, Koetsier jc. 4-Aminopyridine in the treatment of patients with multiple sclerosis. Long-term efficacy and safety. Arch Neurol. 1994;51 :292-296. Schwid SR, Petrie MD, McDermott MP, et al. Quantitative assessment of sustained-release 4-aminopyridine for symptomatic treatment of multiple sclerosis. Neurology. 1997;48: 817-821. Goodman AD, Cohen jA, Cross A, et al. Fampridine-SR in multiple sclerosis: A randomized, doubleblind, placebo-controlled, doseranging study. Mult Seier. 2007; 13: 357-368. Goodman AD, Brown TR, Cohen jA, et ai, for the Fampridine MSF202 Study Group. Dose comparison trial of sustained-release fampridine in multiple sclerosis. Neurology. 2008;71 :1134-1141. Goodman AD, Brown TR, Krupp L, et al. Sustained release of oral fam-
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pridine in multiple sclerosis: A randomised, double-blind, controlled trial. Lancet. 2009. In press. Evenhuisj, Agoston S, Salt Pj, et al. Pharmacokinetics of 4-aminopyridine in human volunteers. A preliminary study using a new GLC method for its estimation. Br J Anaesth. 1981 ;53:567-570. Uges DR, Sohn Yj, Greijdanus B, et al. 4-Aminopyridine kinetics. Clin Pharmacol Ther. 1982;31 :587-593. Van Diemen HA, Polman CH, Koetsier jC, et al. 4-Aminopyridine in patients with multiple sclerosis: Dosage and serum level related to efficacy and safety. Clin Neuropharmacol.1993;16:195-204. Hayes KC, Katz MA, Devane jG, et al. Pharmacokinetics of an immediaterelease oral formulation of Fampridine (4-aminopyridine) in normal subjects and patients with spinal cord injury. J Clin Pharmacol. 2003; 43:379-385. Ritschel WA. Handbook of Basic Pharmacokinetics-Including Clinical Applications. 4th ed. Hamilton, III: Drug Intelligence Publications; 1992. Glantz SA. Primer ofBiostatistics. New York, NY: McGraw-Hili; 1981.
Address correspondence to: Herbert R. Henney III, PharmD, Acorda Therapeutics, 15 Skyline Drive, Hawthorne, NY 10532. E-mail: hhenney@ acorda.com 335
BriefReport
Pharmacokinetics of 14C_Radioactivity After Oral Intake of a Single Dose of 14C-Labeled Fampridine (4-Aminopyridine) in Healthy Volunteers Andrew R. Blight, PhD; and Herbert R. Henney III, PharmD
Acorda Therapeutics, Hawthorne, New York ABSTRACT Background: Fampridine (4-aminopyridine) is a potassium channel blocker that has been evaluated as a treatment for patients with spinal cord injury and multiple sclerosis. Objective: The purpose of this study was to determine the pharmacokinetics of a single dose of an orally administered solution of 14C-Iabeled fampridine in healthy volunteers. Methods: In this open-label, single-dose study conducted in an inpatient setting, healthy adult men were administered an oral solution containing 15 mg of 14C-Iabeled fampridine (100 J..l.Ci) in a fasted state. In addition to blood sampling for analysis of plasma 14C-radioactivity at prescribed intervals over 7 days, all urine and feces were collected for analysis of drug recovery and disposition. Urine samples were also analyzed for metabolic profiling. Plasma pharmacokinetic parameters of the 14C-radiolabeled drug were determined using standard liquid-scintillation techniques. Recovery was calculated to provide the total amount of radioactivity excreted as a proportion of the original dose. Nonhydrolyzed and hydrolyzed urine extracts were analyzed for radioactivity and metabolites using reverse-phase, isocratic high-performance liquid chromatography with spectrophotometric and radioactive detection. Tolerability was assessed through evaluation of vital signs, hematologic and other laboratory parameters, and electrocardiography. Results: The 4 white male subjects had a mean (SO) age of 21 (2) years. No clinically significant abnormalities in vital signs, clinical chemistry, hematology, urinalysis, or electrocardiography were observed either before or during the study. Peak plasma radioactivity was reached at 1 hour after dosing, with a median concentration of 72.9 ng . mL-l. There was complete disappearance of radioactivity by 24 hours 328
(limit of quantitation, 400 disintegrations/min per peak), and the calculated median t 1l2 was 3.14 hours. Total cumulative recovery of 14C-radioactivity was 96.36%, with only 0.51 % of drug recovered in feces. On chromatography, 2 metabolites accounted for a low proportion of total urinary radioactivity (3% and 6% of total radioactivity in the interval from 0 to 4 hours after dosing; 17% and 9% in the interval from 8 to 12 hours after dosing). Three subjects reported mild and transient dizziness occurring 1 halfhour after dosing; this was considered possibly related to the study drug. Conclusion: Fampridine administered as an oral solution was rapidly absorbed and was nearly completely and rapidly eliminated as unchanged drug via urinary excretion, suggesting that it is unlikely to undergo substantial metabolic transformation. (Clin Ther. 2009;31:328-335) © 2009 Excerpta Medica Inc. Key words: fampridine, 4-aminopyridine, pharmacokinetics, drug disposition.
INTRODUCTION Fampridine (4-aminopyridine) is a broad-spectrum blocker of potassium channels at millimolar concentrations. 1,2 However, plasma concentrations obtained in clinical studies are typically <1 J..l.M (94 ng' mL-1); the molecular characteristics of specific potassium channels with sensitivity in this range have not been determined. The clinical neurologic effects of fampridine are consistent with the molecular mechanism of Accepted for publication)anuary 26,2009. Express Track online publication February 18, 2009. doi:l 0.1 016/j.c1inthera.2009.02.004 0149-2918/$ - see front matter © 2009 Excerpta Medica Inc. All rights reserved.
Volume 31 Number 2
A.R. Blight and H.R. Henney III
potassium channel blockade. At the cellular level, this action may increase neuronal excitability,3 relieve conduction block in demyelinated axons,4-7 and potentiate synaptic and neuromuscular transmission. 8- 10 However, the relative contribution of these distinct cellular effects to the potential therapeutic benefits and adverse effects of 4-aminopyridine is currently unclear. Over the past 30 years, 4-aminopyridine has been evaluated as a potential treatment for neurologic and neuromuscular conditions such as myasthenia graviS,11 Eaton-Lambert syndrome,12,13 episodic ataxia,14 upbeat and downbeat nystagmus,15,16 and spinal cord injury. 17-21 A number of studies have suggested potential benefits of fampridine as a treatment targeted at overcoming the action-potential conduction deficits associated with demyelination in patients with multiple sclerosis (MS), particularly in terms of improving walking ability and muscle strength.22-29 The pharmacokinetics of fampridine have been investigated in several published studies. 17,18,24,30-32 These studies involved either intravenous or oral delivery of immediate-release (IR) formulations, although the former route is not clinically practical and may be associated with intolerable adverse effects, particularly pain at the injection site. 17,32 Many of these analyses were not systematic pharmacokinetic evaluations but were performed as a part of clinical trials in patients with spinal cord injury or MS. Nonetheless, their findings suggested that fampridine has a narrow therapeutic range, with oral dosing of IR formulations resulting in a high Cmax ' followed by rapid elimination via renal excretion. 30 ,31,33 It has been suggested that this profile, in which Cmax is achieved -1 hour after drug administration, contributes to the occurrence of adverse events, which may occur in a dose-dependent manner. 24 The disposition of fampridine has been evaluated in 2 published studies,30,31 both of which reported high recovery of unchanged drug in the urine (87%91 %), although neither reported on fecal recovery. These studies predominantly involved intravenous administration, which, in the study by Uges et al,31 was used as the standard for determining the bioavailability of oral preparations of enteric-coated and uncoated tablets of 4-aminopyridine. Neither study fully evaluated the potential for metabolic transformation. For therapeutic and drug-registration purposes, it is important to obtain specific information on the dispoFebruary 2009
sition and metabolism of a drug in humans. Therefore, the purpose of the present study was to use the sensitivity associated with radioactive techniques to establish the rate and extent of absorption of a single oral dose of 14C-Iabeled fampridine, as well as to characterize the disposition of this agent with respect to routes of elimination and the balance of total 14C_ radioactivity. In addition, samples were analyzed for the presence of potential metabolites resulting from transformation of the parent compound.
SUBJECTS AND METHODS This study was conducted in accordance with the Declaration of Helsinki (third revision) and was approved by the medical ethics committee of the Stichting Beoordeling Ethiek Bio-Medisch Onderzooek (Assen, The Netherlands). All subjects provided written informed consent before participation. Because of its nature, the study was performed on an inpatient basis at the clinic site (Zuidlaren, The Netherlands) from the afternoon before dosing until the afternoon of day 8 after dosing. The inpatient stay was prolonged if background-corrected radioactivity in the urine was >50 disintegrations per minute (dpm) . mL-1 or was >75 dpm per homogenized fecal sample of -400 mg. The study population consisted of healthy male volunteers aged between 18 and 45 years, with body weight within 15% of normal for body structure and no pathologic findings on the medical history or screening assessments. Screening included a physical examination and medical history, recording of vital signs, laboratory tests, and a 12-lead electrocardiogram (ECG). Grounds for exclusion included, but were not limited to, evidence of clinically relevant pathology, mental handicap, or illness within 5 days before study initiation; a history of food or drug allergies; a history of alcohol or drug abuse or addiction or a positive drug test at screening; treatment with systemic medications during the previous 3 months; smoking of >10 cigarettes/d; exposure to radiation for any reason other than routine radiography within the past year; and an abnormal defecation pattern (ie, less than once every 2 days). 14C-radiolabeled 4-aminopyridine IR was administered as an oral solution of 15 mg (100 J..l.Ci) in 50 mL of water. Administration took place in the morning after a lO-hour fast and was observed by an investigator to ensure compliance. After administration of the solution, the container was washed and equal volumes 329
Clinical Therapeutics
of water were consumed 3 times. An additional 200 mL of water was swallowed 2 hours after dosing, and fasting was continued until lunch. With the exceptions of acetaminophen (paracetamol) and topical agents, no concomitant medications were permitted during the study. Blood sampling was performed either via an indwelling catheter or by direct venipuncture. Samples were collected into heparinized tubes and immediately cooled to O°c. Within 1 hour of blood sampling, plasma was separated by centrifugation for 10 minutes (4°C, 1500g) and stored at -20°C until analyzed. Samples (10 mL) were collected for analysis of 14C_ radioactivity immediately before drug administration and at the following time points after dosing: 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours. All urine and feces were collected during the study. Urine was collected at the following hourly intervals relative to dosing (0 hour): -12 to 0, 0 to 4, 4 to 8, 8 to 12, and 12 to 24 hours, and at 24-hour intervals thereafter until discharge. All samples were processed after determination of their volume; 10-mL samples were analyzed for 14C-radioactivity, and 20-mL samples were used for pharmacokinetic analysis of fampridine and its metabolites when the radioactivity count was >1000 dpm . mL-l. For fecal analysis, pooled samples were used for each 24-hour period and, after determination of their weight, 10 g was obtained from the homogenized samples and analyzed for 14C-radioactivity. Aliquots of plasma, urine, and feces were analyzed for 14C-radioactivity by standard liquid-scintillation techniques using a 1900 TR Liquid Scintillation Counter (Canberra Packard Instruments Co., Downers Grove, Illinois). All analyses were performed in duplicate and included 2 blank matrix samples and 6 quality control samples; values were corrected for background. Descriptive analysis was used for standard plasma pharmacokinetic parameters, including Cmax ' T max' t 1l2 , elimination rate constant (A et ), and AUC o-
fornia) equipped with an SP 8750 organizer and a Waters 717 Autosampler Injector (Waters Corporation, Milford, Massachusetts). Nonhydrolyzed and enzymatically hydrolyzed (~-glucuronidase type HI) urine samples were injected directly into the HPLC system. This system incorporated a Waters pBondapack C18 analytical column (Waters), which was temperatureconditioned (18°C-24°C) using a thermostatic water bath (Julabo Labortechnik GmbH, Seelbach, Germany). Two modes of detection were used: spectrophotometric detection at an ultraviolet wavelength of 263 nm (Perkin Elmer LC-75 spectrophotometric detector, Perkin Elmer Ltd., Beaconsfield, United Kingdom); and radioactivity detection using a Radiomatic 525 TR flow detector (Canberra Packard), with a limit of quantitation determined to be 400 dpm per peak. The chromatograph system was connected to a Foxy 200 fraction collector (Teledyne Isco Inc., Lincoln, Nebraska). The mobile phase, filtered and degassed before use, consisted of 800 mL of buffer (1 M potassium dihydrogen orthophosphate, 1 M phosphoric acid, 1 M I-heptanesulfonic acid, and 0.05 M tetrabutylammonium iodide; pH 3.5) mixed with 35 mL of acetonitrile and 165 mL of water. Mean (percent coefficient of variation) intra-assay reproducibility ranged from 95.93% to 102.43% (1.75%-5.83%), and mean interassay reproducibility ranged from 93.70% to 104.58% (0.58%-3.26%), with a mean recovery of 52.18% to 67.91% (7.03%-18.57%) for sample and 79.62% (12.47%) for internal standard. Tolerability was monitored based on vital signs, hematologic and other laboratory values, and ECGs. Subjects were also asked questions designed to elicit information on the occurrence of adverse events. RESULTS Four white male subjects with a mean (SO) age of 21 (2) years and a mean weight of 70.8 (3.3) kg were enrolled in and completed the study (Table I). No clinically significant changes in vital signs or clinical chemistry, hematology, urinalysis, or ECG findings were observed during the study. Four adverse events of mild intensity were reported, 1 in each subject. Three of these events-dizziness occurring 1 half-hour after dosing-were considered possibly related to the study medication. The fourth event was a sore throat and was not considered related to the study medication. Median plasma concentrations of drug calculated from recovered 14C-radioactivity reached C max Volume 31 Number 2
A.R. Blight and H.R. Henney III
Table I. Characteristics of the 4 male subjects. Characteristic
Value
Age, y Mean (SO) Range
21 (2) 18-22
Weight, kg Mean (SO) Range
70.8 (3.3) 68.1-75.3
Body mass index, kg/m 2 Mean (SO) Range
21.8 (1.4) 20.3-23.6
Oose administered, J..l.Ci Mean (SO) Range
105.5 (1.0) 105-107
-1.0 hour after administration, with subsequent rapid elimination and complete disappearance from the plasma by 24 hours after dosing (Figure 1). The frequency of plasma sampling was too low to provide a more precise determination of T max' The pharmacoki-
netic parameters derived from total 14C-radioactivity are summarized in Table II. The median AUCo-
100
....... I
...J
E
75
t)()
c '-'" c 0
...
.~
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'"'
50
V
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0
U
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25
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1:[
0
0
12
24
36
48
Time (h) Figure 1. Median plasma concentrations offampridine calculated from 14C-radioactivity recovered in plasma over time after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine in 4 healthy male subjects.
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331
Clinical Therapeutics
separated from the parent compound at 7.3 minutes (Figure 3). Additionally, 2 minor peaks in metabolites were observed in most chromatograms both before and after hydrolysis. These metabolites had retention times of -4.6 minutes and 8.6 minutes that coincided with those of standard preparations of 2-hydroxy4-aminopyridine (4.40 minutes) and 3-hydroxy4-aminopyridine (8.75 minutes). Quantitatively, the 2 metabolites comprised 3% and 6% of total radioactivity in the interval from 0 to 4 hours after dosing, and 17% and 9% in the interval from 8 to 12 hours after dosing.
Table II. Pharmacokinetic parameters of fampridine derived from plasma 14C-radioactivity after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine (N - 4). Value
Parameter Cmax ' ng . mL-l Median Range
72.9 63.1-86.6
Tmax ' h Median Range Ael , h- 1 Median Range
1.0 1.0-1.0
Median Range
3.14 2.92-4.59
AUCo_co> ng . h/mL-l Median Range -
Although the generalizability of this study is limited by its small, all-male population, administration of a single 15-mg oral dose of 14C-Iabeled 4-aminopyridine was associated with rapid absorption, with peak plasma radioactivity being reached by 1.0 hour after dosing. Elimination from the plasma was also rapid and was complete by 24 hours, with a calculated t 1l2 of 3.14 hours. These data suggest that the drug was well absorbed. No serious adverse events were reported during the study; mild and transient dizzi-
0.220 0.151-0.237
t 1/ 2 , h
Ael
DISCUSSION
341.2 258.0-416.3
elimination rate constant.
.<>
Urine
.t:.. Feces + Total
100
- - - - -ov - - -. - - - - -. - -..... - - -- - - - - -- -...,.- -- - - - - -- --
~ VI
-
------ ... ------
-
0
0 ..... 0
80
~
...~
""tl
u
60
...
ill l: :l
0
40
E
«
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20
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_
0 0
24
48
72
96
120
144
168
Time (h) Figure 2. Median cumulative excretion of 14C-radioactivity after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine in 4 healthy male subjects.
332
Volume 31 Number 2
A.R. Blight and H.R. Henney III
ness was reported in 3 subjects shortly after drug administration. The plasma pharmacokinetics of fampridine in this study, measured in terms of radioactivity, agreed with those reported previously for 4-aminopyridine IR, administered as single escalating oral doses of 10, 15, 20, and 25 mg at weekly intervals in healthy volunteers. 33 In that study, the T max and t 1l2 for the 15-mg dose were 1.0 and 3.4 hours, respectively. The Cmax and AUCo-
Table III. Cumulative excretion of 14C-radioactivity after oral administration of a single dose of 15 mg 14C-labeled 4-aminopyridine (N - 4). Parameter
Value
% of dose Median Range
95.85 93.80-97.50
% of dose Median Range
0.51 0.45-0.53
% of dose Median Range
96.36 94.33-97.95
Aeurine'
A\eces'
Ae total ,
total amount of 14C-radioactivity excreted in urine; Aefeces - total amount of 14C-radioactivity excreted in feces; Aelolal - sum of Aeurine + A\eces'
Aeurine -
856
7.30
642
~
U
6.80
428-
2'44.60
f\\ K. ,",-~.AQ;;;;,3.;.;.70:l0)~~ \...,.~~5~.5~0<::::=J.~:...._ _"-.-I-==-_...:::.t. 3.30 5 6.00 0
o..L...,;:.=o.:;;30::..::....ci1i:..20;;;",...:::::2~.2;;.0,d..
o
0.80
1.70
0~.4_0.....il::,;,I_.40=::::::::::::1:::::2Q-;;.5:::::0:::::12:::,.8::::°::...o::::::::14.:::'ll1o.0""i
1 \9::.4:::.,0""",X C
"0
12.00
13.60
"5
Minutes Figure 3. Characteristic chromatogram (high-performance liquid chromatography with radioflow detection), showing 14C-radioactivity peaks in a hydrolyzed urine sample in the collection interval from 8 to 12 hours after dosing (subject number 1). CPM - counts per minute.
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333
Clinical Therapeutics
the radioactivity was excreted in urine indicates that fampridine was almost completely absorbed from the oral solution. Findings from several studies have suggested the possibility of enterosystemic recirculation of fampridine, based mainly on observations of fluctuations in plasma concentrations among individual subjects. 17,18,30 The low fecal excretion reported in the present study suggests that absorption is almost complete or that hepatobiliary pathways represent only a minor route of elimination. However, HPLC analysis revealed the presence of a shoulder on the main peak, indicating that whereas most of the compound excreted in the urine was unchanged, excreted compounds may include conjugates of 4-aminopyridine. The occurrence of limited metabolic transformation was also indicated by the 2 minor peaks, consistent with 2-hydroxy-4-aminopyridine and 3-hydroxy-4aminopyridine, although the overall presence of metabolites represented only a small proportion (17% and 9%, respectively) of the total compound. Early work in dogs suggested that 4-aminopyridine metabolites include 3-hydroxy-4-aminopyridine and the sulfate conjugate of 3-hydroxy-4-aminopyridine (data on file, study HWI6379-102 [1992], Acorda Therapeutics, Hawthorne, New York), although a reference standard for identifying the sulfate conjugate was not available for the present study. If conversion of the parent compound to these metabolites via hydroxylation and sulfate conjugation can be confirmed in humans, it would be in contrast to the study by Uges et al,31 in which no evidence of such metabolic transformation was found. In this study, fampridine exhibited almost complete absorption, high urinary excretion, and low metabolic transformation. These properties, along with a narrow therapeutic range, rapid rise to Cmax ' and short t 1l2 , suggest the need for frequent dosing of fampridine to maintain effective plasma levels and improve tolerability. A formulation having improved pharmacokinetic characteristics might enhance the therapeutic potential of fampridine. CONCLUSIONS
The results of this study of 14C-Iabeled fampridine are consistent with previous reports of rapid absorption and nearly complete renal elimination of the drug. The presence of additional chromatographic peaks suggested the occurrence of metabolic transformation, 334
although these peaks accounted for only 17% and 9% of the total compound. ACKNOWLEDGMENTS
This study was supported by Acorda Therapeutics, Hawthorne, New York. Drs. Blight and Henney are employees of Acorda Therapeutics and hold stock in the company. The authors thank E. Jay Bienen, PhD, an independent scientific consultant based in New York City, for editorial support, which was funded by Acorda Therapeutics. REFERENCES 1. Gold MS, Shuster MJ, Levine JD. Characterization of six voltage-gated K+ currents in adult rat sensory neurons.
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Address correspondence to: Herbert R. Henney III, PharmD, Acorda Therapeutics, 15 Skyline Drive, Hawthorne, NY 10532. E-mail: hhenney@ acorda.com 335