Cav2.1 and Cav1.2 currents in HEK293T cells are unaffected by amifampridine (1.5 μM), whereas Kv3.3 and Kv3.4 currents are severely reduced by about 10% [3]. In frogs and humans, amifampridine (0-100 μM) dose-dependently lengthens the presynaptic AP (action potential) waveforms at the NMJ [3].

After being intoxicated with BONT/A, amifampridine (10 mg/kg; once) can counteract muscular paralysis [2]. Amifampridine has an hour-long plasma half-life and a roughly 57% bioavailability (F) in mice when administered once at doses of 2.5 mg/kg (IV) and 10 mg/kg (PO) [2]. Amifampridine has a relatively short plasma half-life and, after crossing the blood-brain barrier, can cause epileptic seizures at high concentrations [2].

Animal/Disease Models: CD-1 mice (female, 25 g, 6 weeks old) [2]
Doses: 10 mg/kg
Route of Administration: BoNT/A administration (IP) followed by po (oral gavage) once (IP)
Experimental Results: demonstrated that either LEM alone (182 ± 43 minutes) or the maximum safe oral dose of 3,4-DAP alone (225 ± 24 minutes) Dramatically increased the time to death after toxin administration (216 ± 29 minutes). However, when the 10/50/40 3,4-DAP/LEM/shellac formulation was administered at 25 mg/kg, the time to death was 302 ± 26 minutes, a 40% increase compared to toxin alone.

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Animal/Disease Models: CD-1 mice (30-35 g, 8 weeks old) [2]
Doses: 2.5 mg/kg (IV); 10 mg/kg (PO)
Route of Administration: intravenous (iv) (iv)injection, oral administration, once (drug pharmacokinetic/PK/PK analysis)
Experimental Results: pharmacokinetic/PK/PK parameters of Amifampridine in CD-1 mice [1]. IV (2.5 mg/kg) PO (10 mg/kg) t1/2 (h) 1.04 1.28 AUC0-24 (μM·h) 4.29 9.72 F (%) 100 56.7

Absorption, Distribution and Excretion
Orally-administered amifampridine is rapidly absorbed in humans to reach the peak plasma concentrations within by 0.6 to 1.3 hours. A single oral dose of 20 mg amifampridine in fasted individuals resulted in mean peak plasma concentrations (Cmax) ranging from 16 to 137 ng/mL. Bioavailability is approximately 93-100% based on recoveries of unmetabolised amifampridine and a major 3-N-acetylated amifampridine metabolite in urine. Food consumption decreases amifampridine absorption and exposure with a decrease in the time to reach maximum concentrations (Tmax). It is approximated that food consumption lowers the Cmax on average by ~44% and lowers AUC by ~20%. based on geometric mean ratios. Systemic exposure to amifampridine is affected by the overall metabolic acetylation activity of NAT enzymes and NAT2 genotype. The NAT enzymes are highly polymorphic that results in variable slow acetylator (SA) and rapid acetylator (RA) phenotypes. Slow acetylators are more prone to increased systemic exposure to amifampridine, and may require higher doses for therapeutic efficacy.
Following oral administration, more than 93% of total amifampridine is renally eliminated within 24 hours. About 19% of the total renally-excreted dose is in the parent drug form, and about 74-81.7% of the dose is in its metabolite form.
In healthy volunteers, the volume of distribution for plasma amifampridine indicated that RUZURGI is a drug with a moderate to a high volume of distribution. After a 2 mg/kg infusion in rats, the volume of distribution at steady-state was 2.8 ± 0.7 L/kg. Drug concentrations were highest in organs of excretion, including the liver, kidney, and the gastrointestinal tract, and some tissues of glandular function, such as lacrimal, salivary, mucous, pituitary, and thyroid glands. Concentrations in tissues are generally similar to or greater than concentrations in plasma.
Overall clearance of amifampridine is both metabolic and renal; it is primarily cleared from the plasma via metabolism by N-acetylation. Following oral administration of a single 20 or 30 mg dose of RUZURGI to healthy volunteers, amifampridine apparent oral clearance (CL/F) was 149 to 214 L/h.

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Metabolism / Metabolites
Amifampridine is extensively metabolized by N-acetyltransferase 2 (NAT2) to 3-N-acetyl-amifampridine, which is considered an inactive metabolite.

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Biological Half-Life
The average elimination half-life of amifampridine was 3.6 to 4.2 hours and 4.1 to 4.8 hours for the 3-N-acetyl amifampridine metabolite.

Hepatotoxicity
Amifampridine has had limited clinical use, but adverse events have been largely neurologic and gastrointestinal. Serum ALT elevations were not reported in the prelicensure studies of amifampridine but were reported as occurring in a small proportion of patients in safety reviews by the Food and Drug Administration. Nevertheless, there have been no reports of clinically apparent liver injury associated with its use. Thus, liver injury from amifampridine must be rare if it occurs at all.
Likelihood score: E (unlikely cause of clinically apparent liver injury, but experience with its use is limited).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of amifampridine during breastfeeding on the presence of amifampridine or the 3-N-acetyl-amifampridine metabolite in breastmilk. If amifampridine is required by the mother, it is not a reason to discontinue breastfeeding, but the infant should be carefully monitored for excessive crying or fussiness, adequate weight gain, and developmental milestones.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
In vitro human plasma protein binding of amifampridine and 3-N-acetyl amifampridine was 25.3% and 43.3%, respectively.

[1]. Lambert-Eaton myasthenic syndrome: from clinical characteristics to therapeutic strategies. Lancet Neurol. 2011 Dec;10(12):1098-107.

[2]. Lycopodium clavatum exine microcapsules enable safe oral delivery of 3,4-diaminopyridine for treatment of botulinum neurotoxin A intoxication. Chem Commun (Camb). 2016 Mar 18;52(22):4187-90.

[3]. A high-affinity, partial antagonist effect of 3,4-diaminopyridine mediates action potential broadening and enhancement of transmitter release at NMJs. J Biol Chem. 2021 Jan-Jun;296:100302.

Pharmacodynamics
Administration of amifampridine to patients with LES in clinical trials resulted in improvement of the compound muscle action potential (CMAP), muscle function, and quantitative myasthenia gravis (QMG) score. One case of a slight prolongation of the QTc interval in male patient with LEMS and euthyroid Hashimoto’s disease treated with 90 mg of amifampridine in combination with 100 mg azathioprine was reported. _In vitro_, amifampridine was shown to modulate cardiac conduction and induce phasic contractions in different arteries from several species. In addition, it stimulated potassium-evoked dopamine and noradrenaline release in rat hippocampal slices and upregulate acetylcholine release in the brain. It may also potentiate adrenergic and cholinergic neuromuscular transmission in the gatrointestinal tract. In a single pharmacokinetic study, no effect was observed of amifampridine phosphate on cardiac repolarization as assessed using the QTc interval. There were no changes in heart rate, atrioventricular conduction or cardiac depolarization as measured by the heart rate, PR and QRS interval durations.

C5H7N3

109.13

109.063

54-96-6

Amifampridine phosphate;446254-47-3

5918

Off-white to gray solid powder

1.3±0.1 g/cm3

369.3±22.0 °C at 760 mmHg

216-218 °C(lit.)

204.9±9.5 °C

0.0±0.8 mmHg at 25°C

1.676

-0.09

2

3

0

8

74.1

0

OYTKINVCDFNREN-UHFFFAOYSA-N

InChI=1S/C5H7N3/c6-4-1-2-8-3-5(4)7/h1-3H,7H2,(H2,6,8)

3,4-Diaminopyridine

3,6-DAP; 3,4-Diaminopyridine; BRN-0110232; BRN 0110232; BRN0110232; NSC 521760; NSC-521760; NSC521760; SC10; Trade name: Firdapse.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (22.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (22.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (22.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)