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].

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].

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The 3,4-DAP release profile from Lycopodium clavatum exine microcapsules (LEMs) co-encapsulated with shellac was studied in simulated biological fluids. When incubated in simulated gastric fluid (SGF, pH 1.5) for up to 8 hours, the LEMs retained 72.2 ± 4.0% of the loaded 3,4-DAP, demonstrating limited release in an acidic environment. Conversely, substantial 3,4-DAP release was observed upon incubation in phosphate-buffered saline (PBS, pH 7.4), confirming pH-dependent release suitable for enteric delivery. [2]
The release was also shown to be time-dependent and proportional to the loading percentage (w/w) of 3,4-DAP (tested at 2.3%, 4.9%, 7.4%, and 10.1%) when measured in PBS (pH 7.4). [2]

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].

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Four randomised controlled trials involving a total of 54 patients with LEMS showed that 3,4-diaminopyridine treatment resulted in significant improvement in muscle strength score, myometric limb measurement, or compound muscle action potential (CMAP) amplitude. [1]

Whole-cell patch-clamp electrophysiology in HEK293T cells: HEK293T cells were transfected with plasmids encoding Kv3.3, Kv3.4, Cav2.1, or Cav1.2 channel subunits along with a GFP marker. Recordings were performed at room temperature using an amphotericin-B-based perforated patch configuration. The pipette solution for potassium current recordings contained potassium methane sulfonate, KCl, Hepes, and MgCl2. The bath solution contained NaCl, Hepes, glucose, CaCl2, and MgCl2. For calcium current recordings, cesium-based solutions were used. Cells were voltage-clamped, and currents were activated by depolarizing steps from -100 mV to +20 or +40 mV. Currents were recorded before and after application of 3,4-DAP dissolved in extracellular saline. The percent inhibition was calculated by comparing peak current amplitudes. [3]

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

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Pharmacokinetic studies were conducted in CD-1 mice. For intravenous (IV) pharmacokinetics, 3,4-DAP was administered intravenously, and plasma concentrations were measured over time. [2]
For oral pharmacokinetics and efficacy studies, several formulations were tested: 1) 3,4-DAP phosphate (the clinical salt form) administered orally. 2) LEM formulations with varying compositions: a) LEMs loaded with 7.4% 3,4-DAP and 40% shellac, b) LEMs loaded with 10.1% 3,4-DAP and 40% shellac, c) LEMs loaded with 10.1% 3,4-DAP, 50% LEM, and 40% shellac (optimized ratio). 3) Control formulations: 3,4-DAP mixed with shellac alone (without LEMs), and 3,4-DAP loaded into LEMs without shellac. All oral doses were administered at 25 mg/kg (based on 3,4-DAP) unless specified otherwise for the free drug control (10 mg/kg). [2]
For the BoNT/A lethality assay, female CD-1 mice were intoxicated with 5 LD50 of BoNT/A. Treatment groups received either empty LEMs (25 mg/kg LEMs), free 3,4-DAP (10 mg/kg), or the optimized LEM formulation (10.1% 3,4-DAP / 50% LEM / 40% shellac, delivering 25 mg/kg of LEMs per mouse). The primary endpoint was time to death. [2]

Absorption, Distribution and Excretion
Orally administered amifapyridine is rapidly absorbed in the human body, reaching peak plasma concentrations within 0.6 to 1.3 hours. In fasting subjects, a single oral dose of 20 mg amifapyridine resulted in a mean peak plasma concentration (Cmax) of 16 to 137 ng/mL. Based on the recovery of unmetabolized amifapyridine and its major metabolite, 3-N-acetylated amifapyridine, from urine, its bioavailability is approximately 93-100%. Food intake reduces amifapyridine absorption and exposure, and shortens the time to peak concentration (Tmax). Based on geometric mean ratios, food intake, on average, reduces Cmax by approximately 44% and AUC by approximately 20%. Systemic exposure to amifapyridine is influenced by the overall metabolic acetylation activity of NAT enzymes and the NAT2 genotype. NAT enzymes are highly polymorphic, resulting in different slow acetylation (SA) and rapid acetylation (RA) phenotypes. Slow-acetylated amifapiridine is more likely to cause increased systemic exposure, potentially requiring higher doses to achieve therapeutic effects. Following oral administration, over 93% of amifapiridine is excreted by the kidneys within 24 hours. Of the total renal excretion, approximately 19% is the parent drug and approximately 74–81.7% is metabolites. In healthy volunteers, plasma volume of distribution for amifapiridine indicates that RUZURGI is a drug with a moderate to high volume of distribution. In rats, the steady-state volume of distribution after infusion at a dose of 2 mg/kg was 2.8 ± 0.7 L/kg. Drug concentrations are highest in excretory organs (including the liver, kidneys, and gastrointestinal tract) and some glandular tissues (such as the lacrimal glands, salivary glands, mucous membranes, pituitary gland, and thyroid gland). Tissue concentrations are generally similar to or higher than plasma concentrations. The overall clearance pathway for amifapiridine includes metabolism and renal clearance; it is primarily cleared from plasma via N-acetylation metabolism. Following a single oral administration of 20 or 30 mg RUZURGI to healthy volunteers, the apparent oral clearance (CL/F) of amifapyridine ranged from 149 to 214 L/h.

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Metabolism/Metabolite
Amifapyridine is primarily metabolized by N-acetyltransferase 2 (NAT2) to 3-N-acetylamifapyridine, which is considered an inactive metabolite.

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Biological Half-Life
The mean elimination half-life of amifapyridine is 3.6 to 4.2 hours, and the mean elimination half-life of its metabolite 3-N-acetylamifapyridine is 4.1 to 4.8 hours.

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The plasma half-life of 3,4-diaminopyridine after intravenous injection in mice is approximately 1 hour. [2] The bioavailability (F) of oral administration of 3,4-diaminopyridine phosphate (immediate-release clinical formulation) in mice is approximately 57%. This formulation can rapidly increase plasma concentrations. [2] The initial LEM formulation (with a 3,4-diaminopyridine loading of 7.4%) had much lower oral bioavailability, approximately 17%, but its plasma concentration peak was broader and shifted to the right compared to immediate-release phosphate. [2] Increasing the 3,4-diaminopyridine loading in the LEM formulation to 10.1% (containing 40% shellac) improved bioavailability. Further optimization by adjusting the LEM to shellac ratio to 50%:40% (3,4-diaminopyridine loading of 10.1%) significantly improved the maximum plasma concentration (Cmax) and area under the curve (AUC). The absolute bioavailability of this optimized formulation could not be precisely calculated, but it was significantly higher. [2] The 3,4-diaminopyridine formulation made with shellac alone (without LEM) failed to produce any measurable plasma concentrations. [2]
After using the optimized LEM formulation, plasma concentrations of 3,4-diaminopyridine remained above the therapeutic threshold of 10–15 µM for more than 3 hours following oral administration. [2]

Hepatotoxicity
The clinical application of ampicillin is limited, but adverse reactions are mainly neurological and gastrointestinal. No elevated serum ALT was reported in pre-marketing studies of ampicillin, but a safety review report from the U.S. Food and Drug Administration (FDA) showed elevated serum ALT in a small number of patients. Nevertheless, there have been no reports of clinically significant liver injury associated with ampicillin use. Therefore, even if ampicillin-induced liver injury occurs, it is certainly very rare. Probability Score: E (Unlikely a cause of clinically significant liver injury, but its use is limited). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no clinical information regarding the use of ampicillin during lactation, nor is there information regarding the presence of ampicillin or its metabolite 3-N-acetaminopyridine in breast milk. If a mother needs to take ampicillin, this is not a reason to stop breastfeeding, but the infant should be closely monitored for excessive crying or irritability, adequate weight gain, and developmental milestones.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
The in vitro human plasma protein binding rates of 3,4-diaminopyridine and 3-N-acetaminopyridine were 25.3% and 43.3%, respectively.

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The most common side effects of 3,4-diaminopyridine are perioral tingling and paresthesia of the fingers; some patients have reported gastrointestinal symptoms. [1]
The most common serious adverse event is seizures; this risk appears to be dose-related, and has been reported at daily doses of approximately 100 mg. [1] There have been reports of iatrogenic toxicity following administration of 360 mg of 3,4-diaminopyridine (3,4-DAP), resulting in supraventricular tachycardia. [1] One patient died of myocardial infarction a few weeks after starting the drug, but the causal relationship is unclear. [1] QT interval prolongation is often mentioned as a possible side effect, but it was not observed in any of the 27 patients studied. [1]

[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
In clinical trials, treatment with ampicillin in patients with hypothalamic-Lemus syndrome (LEMS) showed improvements in compound muscle action potential (CMAP), muscle function, and quantitative myasthenia gravis (QMG) scores. A case report of a male patient with LEMS and euthyroid Hashimoto's thyroiditis who experienced mild QTc interval prolongation after treatment with 90 mg ampicillin combined with 100 mg azathioprine was previously described. In vitro studies have shown that ampicillin can modulate cardiac conduction and induce rhythmic contractions in various arteries across multiple animals. Furthermore, it stimulates potassium-induced dopamine and norepinephrine release in rat hippocampal slices and upregulates acetylcholine release in the brain. It may also enhance adrenergic and cholinergic neuromuscular transmission in the gastrointestinal tract. In a pharmacokinetic study, no effect of ampicillin phosphate on cardiac repolarization as assessed by QTc interval was observed. Heart rate, atrioventricular conduction, or cardiac depolarization (measured by heart rate, PR interval, and QRS interval) remained unchanged. 3,4-Diaminopyridine is the first-line symptomatic treatment for Lambert-Eaton myasthenia gravis (LEMS). [1] It is usually taken at 10-20 mg daily, 2-4 times. [1] Its mechanism of action is to block potassium channels, prolonging depolarization of motor nerve endings, thereby keeping pathological calcium channels open for a longer period, increasing calcium influx and acetylcholine release. [1] A recent Cochrane systematic review summarized its efficacy based on four randomized controlled trials. [1] Most side effects are dose-related, and peak dose limits its therapeutic window. [1] Some potential improvements are mentioned, such as extended-release tablets or combination therapy with pyridostigmine, but these require further investigation. [1]

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.)