4-Aminopyridine

IC50: 170 and 230 μM at KV1.1 and KV1.2, respectively

K+ channels function to conduct K+ ions down their electrochemical gradient, doing so both rapidly and selectively. Biologically, these channels act to set or reset the resting potential in a number of cells. In excitable cells, such as neurons, the delayed counterflow of potassium ions shapes the action potential. 4-Aminopyridine (4-AP or fampridine) is a potassium channel-blocking agent.

In vitro: 4-AP acts by selectively blocking fast, voltage-gated K+ channels in excitable tissues. In axons, K+ channel blockade increases the safety factor1 across demyelinated internodes and 4-AP can, therefore, restore conduction in focally demyelinated axons. 4-AP also increases calcium (Ca2+) influx at presynaptic terminals thereby enabling an enhancement of neuroneuronal or neuromuscular transmission in normally myelinated neurons [1].

In vivo: Investigations of the effects of 4-AP on neurologic deficits in animal in vivo models of demyelinating disease or SCI have yielded inconsistent results. Some trials have shown indications of potential neurological benefit, such as enhanced motor evoked potentials or reflex activity, while others have yielded no evident gains in function [1].

Clinical trial: The randomized clinical trials which have been completed to date indicate that K+ channel blockade by 4-Aminopyridine may prove to be a useful strategy for ameliorating central conduction deficits. Diverse neurological gains have been reported for both motor and sensory domains [1].

Reference:
[1] Hayes KC.  The use of 4-aminopyridine (fampridine) in demyelinating disorders. CNS Drug Rev. 2004 Winter;10(4):295-316.

Antiseizure Effects of Ketogenic Diet on Seizures Induced with Pentylenetetrazole, 4-Aminopyridine and Strychnine in Wistar Rats.[Pubmed:28262846]

Niger J Physiol Sci. 2017 Mar 6;31(2):115-119.

The ketogenic diet (KD) is a cheap and effective alternative therapy for most epilepsy. There are paucity of experimental data in Nigeria on the usefulness of KD in epilepsy models. This is likely to be responsible for the poor clinical acceptability of the diet in the country. This study therefore aimed at providing experimental data on usefulness of KD on seizure models. The study used 64 Wistar rats that were divided into two dietary groups [normal diet (ND) and ketogenic diet (KD)]. Animal in each group were fed for 35days. Medium chain triglyceride ketogenic diet (MCT-KD) was used and it consisted of 15% carbohydrate in normal rat chow long with 5ml sunflower oil (25% (v/w). The normal diet was the usual rat chow. Seizures were induced with one of Pentelyntetrazole (PTZ), 4-Aminopyridine (AP) and Strychnine (STR). Fasting glucose, ketosis level and serum chemistry were determined and seizure parameters recorded. Serum ketosis was significantly higher in MCT-KD-fed rats (12.7 +/-2.6) than ND-fed (5.17+/-0.86) rats. Fasting blood glucose was higher in ND-fed rats (5.3+/-0.9mMol/l) than in MCT-KD fed rats (5.1+/-0.5mMol/l) with p=0.9. Seizure latency was significantly prolonged in ND-fed compared with MCT-KD fed rats after PTZ-induced seizures (61+/-9sec vs 570+/-34sec) and AP-induced seizures (49+/-11sec vs 483+/-41sec). The difference after Str-induced seizure (51+/-7 vs 62+/-8 sec) was not significan. The differences in seizure duration between ND-fed and MCT-KD fed rats with PTZ (4296+/-77sec vs 366+/-46sec) and with AP (5238+/-102sec vs 480+/-67sec) were significant (p<0.05), but not with STR (3841+/-94sec vs 3510+/-89sec) respectively. The mean serum Na+ was significantly higher in MCT-KD fed (141.7+/-2.1mMol/l) than ND-fed rats (137+/-2.3mMol/l). There was no significant difference in mean values of other serum electrolytes between the MCT-KD fed and ND-fed animals. MCT-KD caused increase resistance to PTZ-and AP-induced seizures, but has no effect on STR-induced seizures. This antiseizure property is probably mediated through GABAergic receptors (PTZ effect) and blockade of membrane bound KATP channels (AP effect) with some enhancement by serum ketosis.

Subacute Fluoxetine Reduces Signs of Hippocampal Damage Induced by a Single Convulsant Dose of 4-Aminopyridine in Rats.[Pubmed:27989232]

CNS Neurol Disord Drug Targets. 2017;16(6):694-704.

BACKGROUND: Epilepsy is a central disorder associated with neuronal damage and brain hypometabolism. It has been reported that antidepressant drugs show anticonvulsant and neuroprotective effects in different animal models of seizures and epilepsy. AIMS: The purpose of this study was to investigate the eventual short-term brain impairment induced by a single low convulsant dose of the potassium channel blocker 4-Aminopyridine (4-AP) and the eventual neuroprotective effects exerted by fluoxetine, a prototypical selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitor (SSRI). METHOD: In vivo 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) and several histological assessments were carried out in adult male rats after i.p. administration of 3 mg/kg 4-AP for evaluating eventual brain metabolism impairment and signs of hippocampal damage. We also evaluated the effects of a short-term fluoxetine treatment (10 mg/kg, i.p. for 7 days) in this seizure model. RESULTS: [18F]FDG PET analysis revealed no changes in the regional brain metabolism on day 3 after 4-AP injection. The histological assessments revealed signs of damage in the hippocampus, a brain area usually affected by seizures. Thus, reactive gliosis and a significant increase in the expression of caspase-9 were found in the aforementioned brain area. By contrast, we observed no signs of neurodegeneration or neuronal death. Regarding the effects of fluoxetine, this SSRI showed beneficial neurologic effects, since it significantly increased the seizure latency time and reduced the abovementioned 4-AP-induced hippocampal damage markers. CONCLUSION: Overall, our results point to SSRIs and eventually endogenous 5-HT as neuroprotective agents against convulsant-induced hippocampal damage.

4-Aminopyridine ameliorates experimental autoimmune neuritis in Lewis rats.[Pubmed:28284349]

J Neuroimmunol. 2017 Apr 15;305:72-74.

We investigated the effect of 4-Aminopyridine (4-AP) on experimental autoimmune neuritis (EAN) using a 4-AP-treated group in which 4-AP was administered in the diet, and a control group (n=10 per group). Electrophysiological and pathological assessment was performed in the sciatic nerve. The EAN clinical scores were significantly lower in the 4-AP-treated group than in the control group (p<0.05). The motor conductance velocity two weeks post-immunization was significantly higher in the 4-AP-treated group (p<0.05). Finally, 4-AP did not lead to pathological changes. Thus, 4-AP might be a potential therapeutic agent in demyelinating neuropathy.

Clinically Relevant Levels of 4-Aminopyridine Strengthen Physiological Responses in Intact Motor Circuits in Rats, Especially After Pyramidal Tract Injury.[Pubmed:28107804]

Neurorehabil Neural Repair. 2017 Apr;31(4):387-396.

BACKGROUND: 4-Aminopyridine (4-AP) is a Food and Drug Administration-approved drug to improve motor function in people with multiple sclerosis. Preliminary results suggest the drug may act on intact neural circuits and not just on demyelinated ones. OBJECTIVE: To determine if 4-AP at clinically relevant levels alters the excitability of intact motor circuits. METHODS: In anesthetized rats, electrodes were placed over motor cortex and the dorsal cervical spinal cord for electrical stimulation, and electromyogram electrodes were inserted into biceps muscle to measure responses. The motor responses to brain and spinal cord stimulation were measured before and for 5 hours after 4-AP administration both in uninjured rats and rats with a cut lesion of the pyramidal tract. Blood was collected at the same time as electrophysiology to determine drug plasma concentration with a goal of 20 to 100 ng/mL. RESULTS: We first determined that a bolus infusion of 0.32 mg/kg 4-AP was optimal: it produced on average 61.5 +/- 1.8 ng/mL over the 5 hours after infusion. This dose of 4-AP increased responses to spinal cord stimulation by 1.3-fold in uninjured rats and 3-fold in rats with pyramidal tract lesion. Responses to cortical stimulation also increased by 2-fold in uninjured rats and up to 4-fold in the injured. CONCLUSION: Clinically relevant levels of 4-AP strongly augment physiological responses in intact circuits, an effect that was more robust after partial injury, demonstrating its broad potential in treating central nervous system injuries.

In Vitro electrophysiological activity of nerispirdine, a novel 4-aminopyridine derivative.[Pubmed:19413590]

Clin Exp Pharmacol Physiol. 2009 Nov;36(11):1104-9.

Summary 1. The non-selective K(+) channel blocker 4-Aminopyridine (4-AP) has shown clinical efficacy in the treatment of neurological disorders such as multiple sclerosis. The clinical usefulness of 4-AP is hampered by its ability to produce seizures. Nerispirdine, an analogue of 4-AP, is currently under clinical investigation for the treatment of multiple sclerosis. In contrast with 4-AP, nerispirdine is not proconvulsant, suggesting mechanistic differences between the two drugs. 2. Using whole-cell patch-clamp electrophysiology, we compared the effects of 4-AP and nerispirdine on the cloned human K(+) channels K(v)1.1 and K(v)1.2, expressed in Chinese hamster ovary cells, and on voltage-dependent Na(+) channels recorded from human SH-SY5Y cells. 3. Nerispirdine inhibited K(v)1.1 and K(v)1.2 with IC(50) values of 3.6 and 3.7 micromol/L, respectively. 4-Aminopyridine was approximately 50-fold less potent at blocking these channels. Nerispirdine also inhibited voltage-dependent Na(+) channel currents recorded from human SH-SY5Y cells with an IC(50) of 11.9 micromol/L when measured from a -70 mV holding potential. In contrast, 4-AP had no effect on Na(+) channel currents. 4. The results demonstrate that nerispirdine, like 4-AP, can inhibit axonal K(+) channels and that this mechanism may underlie the ability of the drug to enhance neuronal conduction. Unlike 4-AP, nerispirdine can also inhibit neuronal Na(+) channels, a mechanism that may explain why nerispirdine lacks proconvulsant activity.

Different state dependencies of 4-aminopyridine binding to rKv1.4 and rKv4.2: role of the cytoplasmic halves of the fifth and sixth transmembrane segments.[Pubmed:10411564]

J Pharmacol Exp Ther. 1999 Aug;290(2):569-77.

4-Aminopyridine (4AP) binding to rKv1.4 occurs preferentially in the activated state, whereas binding to rKv4.2 occurs in the rested state. To explore structural basis for the different state dependencies of 4AP binding, regions of rKv1.4 that are likely to form the 4AP-binding site and/or the activation gate were replaced by the corresponding rKv4.2 sequences one at a time, and the resulting effects on channel gating and 4AP binding were examined. Replacing the amino acid sequence of rKv1.4 in the intracellular loop between the fourth and fifth transmembrane segments (S4 and S5) with that of rKv4.2 did not alter channels' gating properties or the state dependence of 4AP binding. On the other hand, replacing the rKv1.4 sequence in the cytoplasmic half of S5 (N-S5) or S6 (C-S6) with that of rKv4.2 markedly altered the voltage dependence and kinetics of activation gate function. Importantly, these mutations transferred the rested-state 4AP-binding preference from the donor to the host channel. These data can be explained by a scheme in which the function of the activation gate determines the state dependence of 4AP binding, although the relationship between the binding site and the gate may be similar between rKv1.4 and rKv4.2. The amino acid sequences in the N-S5 and C-S6 domains contribute to this activation gate function.

Reverse use dependence of Kv4.2 blockade by 4-aminopyridine.[Pubmed:8930194]

J Pharmacol Exp Ther. 1996 Nov;279(2):865-76.

4-Aminopyridine (4AP) can block various K channels with different state dependences; block occurs in the activated state or in the closed state. The use of K channel clones to study the mechanism and structural determinants responsible for the state dependence of 4AP actions has been hampered by the fact that, for all the K channel clones examined so far, 4AP binding and unbinding occur mainly in the activated state. We report here that 4AP binding to a fast inactivating K channel encoded by Kv4.2 in Xenopus oocytes occurred exclusively in the closed state. The binding rate was slow and independent of membrane voltage in the range from -80 to -120 mV. The binding rate was linearly related to 4AP concentration, yielding apparent binding and unbinding rate constants of 0.012 mM-1 s-1 and 0.062 s-1, respectively. 4AP dissociation from Kv4.2 occurred in two processes, a slow process in the closed state (in a voltage range from -70 to -40 mV) and a fast process in the activated state, which suggested that the closure of the activation gate of Kv4.2 did not prevent the entry or exit of 4AP molecules but slowed these processes. 4AP slowed the rate of Kv4.2 decay during depolarization, consistent with the notion that channel inactivation occurred only after 4AP dissociation. Inactivating Kv4.2 channels prevented 4AP binding. Therefore, 4AP binding and Kv4.2 inactivation were mutually exclusive. This, in conjunction with the observation that 4AP blocked Kv4.2 channels from the intracellular side of the cell membrane, suggests that the 4AP binding site is on the cytoplasmic surface of the Kv4.2 channel at, or adjacent to, the domains involved in channel inactivation. The distinct features of 4AP actions on the time course of transient outward current in human ventricular myocytes suggest that Kv4.2-like subunits are important in the formation of these channels in human heart.

Closed- and open-state binding of 4-aminopyridine to the cloned human potassium channel Kv1.5.[Pubmed:7473178]

J Pharmacol Exp Ther. 1995 Nov;275(2):864-76.

The effect of 4-Aminopyridine (4-AP) on membrane and gating currents of Kv1.5 channels was studied in a human cell line. The rank order of block was cell-attached > whole cell >> outside-out macropatches, which suggested that 4-AP blocked channels from the cytosolic face. Stimulation after exposure to 4-AP during rest resulted in a "supernormal" current immediately after the onset of depolarization, followed by open channel block during maintained depolarization. 4-AP remained trapped in closed channels, and unblock after drug washout required depolarization. At higher concentrations, 4-AP induced channel block by binding to closing or nonconducting channels. This effect could be reduced by hyperpolarization and higher pulsing rates. Block of Kv1.5 channels under steady-state conditions was correlated with a dose-dependent reduction in gating charge movement. The normalized voltage dependence of gating was shifted to more negative potentials by 4-AP. A model incorporating both open and closed channel binding of 4-AP reproduced data, including the "supernormal" current, by shifting the Po - V curve slightly to the left. The results show that 4-AP can both enhance and block Kv1.5 current and suggest that 4-AP can bind with different affinities to more than one site on Kv1.5 channels.