In vitro evaluation of the effects of 4-aminopyridine on cytochrome P450 enzymes

Background Dalfampridine extended release tablets (dalfampridine-ER, known as prolonged-, modified, or sustained-release fampridine tablets in some countries) are approved for the improvement of walking in patients with multiple sclerosis (MS). Dalfampridine-ER is an extended release formulation of 4-aminopyridine (4-AP). Dalfampridine-ER is incorporated into MS management strategies that may include disease-modifying and symptomatic therapies. Since several symptomatic therapies are partially or fully metabolized by enzymes of the hepatic cytochrome P450 system (CYP450) it is important to evaluate drug–drug interactions through potential effects of dalfampridine-ER on CYP450. Methods The ability of 4-AP to inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 in a direct and time-dependent manner was evaluated using pooled human liver microsomes. 4-AP concentrations were 0.03, 0.1, 0.3, 1, 3, 10, and 30 μM, representing 0.1–100-times the average plasma 4-AP concentration (30 ng/mL; 0.32 μM) at therapeutic dosing; the concentration inhibiting 50% of each enzyme activity (IC50) was determined. The ability of 4-AP (0.025, 0.25, 2.5, and 25 μM) to induce the expression of CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4/5 enzymes was evaluated using primary cultures of freshly isolated human hepatocytes from non-transplantable livers. The enzyme-inducing effects of 4-AP were compared with the prototypical inducers. Metabolites were assayed using high-performance liquid chromatography-tandem mass spectrometry techniques. All inhibition and induction assays included positive controls. Results 4-AP did not directly inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4/5, but at a concentration of 30 μM, CYP2E1 was inhibited by 12%, resulting in an estimated IC50 value of 125 μM. None of the enzymes demonstrated time-dependent inhibition by 4-AP. There was little or no effect by 4-AP on enzyme induction, with enzyme activities approximately equivalent to vehicle control. A main limitation was the inability to estimate effectiveness of 4-AP relative to prototypical CYP450 inducers. Conclusion The likelihood of drug–drug interactions is remote in patients with MS who may be taking dalfampridine-ER concomitantly with medications that are metabolized by CYP450 pathways.


Background
Dalfampridine extended release tablets (dalfampridine-ER, known as prolonged-, modified, or sustained-release fampridine tablets in some countries), are approved for the improvement of walking in patients with multiple sclerosis (MS) 1 . This approval was based on two Phase 3 clinical trials that demonstrated a significant improvement in walking speed relative to placebo among patients who responded to treatment 2,3 .
Dalfampridine-ER is an extended release formulation of dalfampridine or 4-aminopyridine (4-AP), a potassium channel blocker that has been extensively used as a tool for characterizing potassium channels. Its putative mechanism of action in MS is relief of the conduction block in demyelinated axons 4 , although it may also enhance neurotransmission at synapses 5,6 . Since this drug is incorporated into MS management strategies that may include a number of other drugs, including disease-modifying therapies (DMTs), it is important to consider its potential for drug-drug interactions.
Many drug-drug interactions are mediated by the hepatic cytochrome P450 system (CYP450). Interferons have been suggested to impact the CYP450 system, thereby potentially altering the pharmacokinetics and pharmacodynamic effects of concomitant medications 7 . However, the CYP450 system itself is not involved in the metabolism of traditional, injectable DMTs for MS, such as the interferons, glatiramer acetate, and natalizumab, and 4-AP itself is largely excreted unchanged in the urine (discussed below), suggesting low potential risk for drug interactions with concomitant use of dalfampridine-ER. Indeed, in a sub-group analysis of the clinical trials, the tolerability and efficacy of dalfampridine-ER appeared to be similar in patients using and not using these therapies 8 . Those results suggested that dalfampridine-ER can be effective regardless of DMT use, and is likely to have a safety profile in patients using DMTs that is similar to those not using them. However, in contrast to DMTs, several pharmacologic therapies used for the symptomatic treatment of MS are partially or fully metabolized by enzymes of the CYP450 system, including baclofen and tizanidine for spasticity, tolterodine for neurogenic bladder, dextromethorphan/quinidine for pseudobulbar affect, and a variety of antidepressants that may be used for depression or neuropathic pain. In addition to these treatments related to MS, there may be a variety of other drugs prescribed for concurrent conditions in individual patients.
The pharmacokinetics of 4-AP have been evaluated in several studies [9][10][11][12][13] , which have included the demonstration that it is rapidly and completely excreted by the urinary route, predominantly as an unchanged compound 11 . Excretion of unchanged drug suggests that 4-AP does not undergo extensive hepatic metabolism and is therefore not an important substrate of the CYP450 system. However, drug-drug interactions might nevertheless be possible if 4-AP were to inhibit or induce CYP450 enzymes involved in the metabolism of other drugs. Therefore, the purpose of this in vitro study was to determine whether 4-AP has an effect, either by induction or inhibition, on components of the CYP450 system that could potentially result in drug interactions. The study was performed in accordance with US Food and Drug Administration (FDA) guidance for performing drug interaction studies 14 , and includes   evaluation of the clinically relevant enzymes CYP1A2,  2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4/5.

Chemicals and reagents
The 4-AP was produced by Regis Technologies (Morton Grove, IL). Stock and working solutions of 4-AP were prepared fresh daily in high-purity water (for inhibition studies) or dimethyl sulfoxide (DMSO).

In vitro CYP450 inhibition
The ability of 4-AP to inhibit the drug-metabolizing enzymes CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 in a direct and time-dependent manner was evaluated, as previously established 15,16 , using human liver microsomes pooled from 16 samples that were prepared and characterized at XenoTech, LLC (Lenexa, KS). In brief, duplicate incubations were conducted at 37 AE 1 C in 400 mL incubation mixtures containing potassium phosphate buffer (50 mM, pH 7.4), MgCl 2 (3 mM), EDTA (1 mM, pH 7.4), and P450 marker substrates at concentrations approximately equal to their Km (Table 1). For CYP3A4/5 inhibition, two substrates were used (testosterone and midazolam) as recommended by the FDA 14 . Reactions were initiated by addition of an NADPH-generating system containing NADP (1 mM), glucose-6-phosphate (5 mM), and glucose-6-phosphate dehydrogenase (1 Unit/mL). After 5 min, the reactions were terminated by addition of an equal volume of acetonitrile (v/v) containing an appropriate internal standard (Table 1). Precipitated protein was removed by centrifugation (920 g, 10 min, 10 C). Calibration and quality control metabolite standards were prepared in zero time incubations.
The concentrations of 4-AP that were evaluated (0.03, 0.1, 0.3, 1, 3, 10, and 30 mM) are $0.1-100-times the average plasma 4-AP concentration (30 ng/mL; 0.32 mM) that was measured in a clinical trial of the therapeutic dose of dalfampridine-ER (10 mg administered twice daily) 2 . Because of the possibility that 4-AP may bind to microsomal protein or lipids, an attempt was made to use standard conditions of 0.1 mg/mL microsomal protein, 5 min incubation time, and 50 mM phosphate buffer concentration, for all reactions with the exceptions of coumarin 7-hydroxylation and midazolam 1 0 -hydroxylation assays. For these reactions, slightly different protein concentrations were used (Table 1) to allow the rate of reaction to be measured under initial rate conditions.
To evaluate 4-AP as a direct-acting inhibitor, the pooled microsomes were incubated with the marker substrates in the presence and absence of the range of 4-AP concentrations to determine the concentration inhibiting 50% of enzyme activity (IC 50 ) value. For time-dependent inhibition, the same concentrations of 4-AP were preincubated with the microsomes and an NADPH-generating system for 30 min. After pre-incubation, the marker substrate was added, and the incubation was continued for 5 min to measure residual P450 activity. All reactions were terminated as described above. Known direct-acting and metabolism-dependent inhibitors were included as positive controls ( Table 2) 14 . Samples were analyzed as described in the Analytical methods section.

In vitro CYP450 induction
Evaluation of 4-AP as an inducer of the expression of CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4/5 enzymes was performed using primary cultures of freshly isolated human hepatocytes with a Matrigel overlay. Of note, CYP2D6 was not examined because this enzyme is recognized by the FDA as being non-inducible 14 . Hepatocytes were obtained from non-transplantable livers from three individual donors according to previously described methods 17 . Culture and treatment procedures were performed as described by Madan et al. 18 . The viability of each preparation was analyzed by trypan blue exclusion, and cultures were allowed to adapt for 3 days prior to treatment.
All working solutions were prepared fresh daily prior to hepatocyte treatment. Approximately 24 h following the final treatment, cultures were visualized to confirm morphological integrity prior to isolation of microsomes, which was carried out according to the methods described by Madan et al. 18 .
Metabolites were quantified by back-calculation of a weighted (1/x), linear, least-squares regression. The regression fit was based on analyte/internal standard peak-area ratios calculated from calibration standard samples, which were prepared from authentic metabolite standards. Peak areas were integrated with Applied Biosystems/MDS SCIEX (Foster City, CA).

Statistical analyses
For inhibition studies, the IC 50 data were processed with a validated, custom software program (DI IC50 LCMS Template version 2.0.3) for Microsoft Excel (Office 2000 version 9.0, Microsoft Inc., Redmond, WA). When inhibition of enzyme activity was observed, the data were processed for determination of IC 50 values by non-linear regression with XLfit3 (Version 3.0.5, ID Business Solutions Ltd., Guildford, Surrey, UK). This software uses the Levenberg-Marquardt algorithm for non-linear regression fitting of the data to the following 4-parameter sigmoidal-logistic IC 50 equation: Background was set to 0 with a range up to 100 to express data as a percentage of control. This software has been verified for its ability to calculate IC 50 values that lie within the concentration range of 4-AP. When less than 50% inhibition is observed, the data are not extrapolated; hence, IC 50 values are reported as being greater than the highest concentration of 4-AP tested.
For the induction studies, data were processed using the validated, custom software program (EI Interim Data Engine, version 1.2.1) for Microsoft Office Excel 2003 (version 11.0, Microsoft Corporation, Redmond, WA). The individual rates of reaction from replicate samples were averaged, and for those groups with n ! 3, standard deviations were determined. Statistically significant differences between group means were calculated by equal variance and normality tests to determine if the data were parametrically distributed. For parametrically distributed data sets, with the exception of isoniazid and saline treatment groups, a one-way repeated measures analysis of variance (ANOVA) was carried out to test for significant differences between group means. For isoniazid and saline, a Student's t-test was used to test for differences. For non-parametrically distributed data sets, with the exception of isoniazid and saline treatment groups, a Kruskal-Wallis ANOVA was performed. The ANOVA was followed by a Dunnett's post-hoc test to identify the group means that were significantly different from the controls. Statistical  Guidance by the FDA on drug interaction studies suggests that a drug with a change in enzyme activity that results in effectiveness !40% relative to the positive control can be considered an enzyme inducer 14 .

In vitro CYP450 inhibition
Under the experimental conditions used, there was no direct inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4/5 at 4-AP concentrations up to 30 mM (Figure 1 and Table  4). However, at a 4-AP concentration of 30 mM, there was $12% inhibition of CYP2E1, resulting in an estimated IC 50 value of 125 mM and a K i for the 4-AP-mediated inhibition of CYP2E1 of 62.5 mM; both of which exceed the highest concentration tested.
When 4-AP was pre-incubated for 30 min in the presence of NADPH-fortified human liver microsomes, there was little or no evidence of time-dependent inhibition for any of the enzymes (Figure 1 and Table 4). Relative to direct inhibition, small increases in inhibition were observed with pre-incubation for CYP1A2, 2A6, 2B6, and C19, although none of these changes was substantial CYP2E1, which had the greatest time-dependent inhibition, and was only inhibited by 19% at 30 mM of 4-AP.

In vitro CYP450 induction
Viability of the hepatocyte preparations was between 83.6% and 91.1%, and these values were considered acceptable for the assays. During and after adaptation to culture, the hepatocytes were judged to be morphologically normal with adequate confluency for treatment. Evaluation of the morphological integrity after final treatment showed that the hepatocytes did not demonstrate any overt signs of toxicity resulting from the test articles.
The mean microsomal CYP enzyme activities after hepatocyte treatment with test articles are summarized in Table 5. As shown in Figure 2, which expresses these activities as the relative increase compared with the vehicle control, hepatocytes responded as expected to treatment with prototypical CYP450 inducers. Treatment with the known inducers resulted in a 35.6-fold increase in activity of CYP1A2 with omeprazole; a 10.7-fold increase in CYP2B6 with phenobarbital; a significant (p50.05) 2.6-fold increase in CYP2E1 with isoniazid; significant (p50.05) increases in CYP2C9 (1.9-fold) and CYP3A4/5 (6.6-fold) with rifampin; and a 4.2-fold increase in CYP2C19 with rifampin. For CYP1A2 and CYP2B6, although significance was determined among all the treatment groups as a result of Kruskal-Wallis One-way ANOVA on ranks (p50.05), Dunnett's test was unable to specify which individual groups were significantly different.
In contrast to the known inducers, 4-AP demonstrated little or no effect on enzymes. Enzyme activities in the presence of 4-AP were approximately equivalent to the DMSO vehicle control, with a very narrow range of increases, 0.89-1.2-fold, across concentrations and enzymes ( Figure 2).
Relative effectiveness could not be calculated for 4-AP for CYP2B6 and 2C9, and for three of the four 4-AP concentrations for 2C19 because individual values included negative numbers that precluded determination of an average value (Table 6). However, for CYP1A2 and 3A4/5, as well as 2C19 (2.5 mM concentration only), none of the effectiveness values exceeded the FDA's cutoff of 40% (Table 6); the highest effectiveness value was 3.29 AE 5.70% for the 2.5 mM concentration for CYP3A4/5. It should also be noted that, since 4-AP was dissolved in DMSO and the prototypical CYP2E1 inducer (isoniazid) was dissolved in saline, the potential of 4-AP to induce CYP2E1 activity could not be evaluated based on effectiveness.

Discussion
Although 4-AP is primarily excreted in the urine as an unchanged drug and is thus not significantly metabolized by CYP enzymes 11,20,21 , it is important to determine if 4-AP may induce or inhibit CYP activity to further predict if drug-drug interactions may occur with the other drugs regularly used by MS patients. The current analyses confirm that, at therapeutic plasma concentrations of 4-AP, there is little potential of drug-drug interactions through this mechanism. In particular, there were no effects on CYP3A4, which is the most common CYP450 isozyme involved in drug metabolism, and little or no evidence of effects on other evaluated isozymes at 4-AP concentrations that were up to 100-times the mean therapeutic plasma concentration.
While 4-AP did not demonstrate direct inhibitory activity for most enzymes evaluated, there was evidence of low direct inhibition, $12%, of CYP2E1 at 30 mM. However, this inhibition was only observed at the highest 4-AP concentration, 30 mM, which is $100-times higher than the average plasma 4-AP concentration of 0.32 mM at the therapeutic dose of dalfampridine-ER 2 , and resulted in an estimated IC 50 for CYP2E1 of 125 mM. Furthermore, based upon FDA draft guidance 14 , a ratio of the [I]/K i of at least 0.1, where [I] is the concentration of inhibitor exposed to the active site of the enzyme, warrants clinical drug interaction studies. Since [I] would operationally translate to the C max (peak plasma concentration), the resulting ratio for 4-AP would be 0.01, suggesting additional drug interaction studies are not warranted based on the estimated K i of 62.5 mM for CYP2E1, and a maximum individual C max of 67 ng/mL (0.71 mM) that has been reported with therapeutic doses of dalfampridine-ER in a clinical trial 2 . It should be noted that, although CYP2E1 has been shown to be the predominant enzyme responsible for 3-hydroxylation of 4-AP, the parent compound undergoes very limited metabolic processing 22 , with clearance primarily as unchanged parent compound in the urine 11 .
For both CYP2E1 and several other cytochromes, preincubation appeared to have a slight impact on inhibition, although there was no substantial increase in CYP2E1 or other cytochrome inhibition relative to the direct assay. However, it is possible that, with longer incubation, a greater inhibitory effect might be observed.
In the induction assays, treatment of three preparations of cultured human hepatocytes with 4-AP at concentrations up to 80-times the therapeutic plasma concentration had little or no effect on CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 activity. FDA guidance suggests that a drug that produces a change !40% of the positive control can be considered as an enzyme inducer and warrants in vivo evaluation; 4-AP had almost no effect on CYP activity for those values that could be calculated. While 4-AP effectiveness values could not adequately be estimated for CYP2B6, 2C9, 2E1, and 2C19, it may nevertheless be considered unlikely that 4-AP would have a Phenacetin O-deethylation 430    Table 3, and are for three determinations (one from each microsomal preparation) unless otherwise indicated. clinically significant impact on the induction of these enzymes since there was no increase in activity with 4-AP relative to the vehicle control, whereas hepatocytes responded as expected to treatment with positive controls.

Conclusions
This in vitro study demonstrated that 4-AP, at concentrations up to 100-fold higher than mean therapeutic plasma concentrations, neither inhibits CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4/5 nor induces 1A2, 2B6, 2C9, 2C19, 2E1, and 3A4/5 to a clinically relevant extent. These data suggest that the likelihood of drug-drug interactions is remote in patients with MS who may be taking dalfampridine-ER concomitantly with medications that are metabolized by these pathways.

Transparency
Declaration of funding This study was funded by Acorda Therapeutics, Inc., Ardsley, New York.
Declaration of financial/other relationship AC and AB are employees and stockholders of Acorda Therapeutics, Inc., Ardsley, New York.