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Oral Pharmacokinetic Drug-drug Interactions between Amifampridine and Acetaminophen in Rats
Yakhak Hoeji 2024;68(2):98-104
Published online April 30, 2024
© 2024 The Pharmaceutical Society of Korea.

Jin-Woo Cheong*,†, Yeo-Dim Park*,†, Dang-Khoa Vo*, and Han-Joo Maeng*,#

*College of Pharmacy, Gachon University
Correspondence to: #Han-Joo Maeng, Ph.D., College of Pharmacy, Gachon University, 191 Hambakmoe-ro, Yeonsu-gu, Incheon 21936, Republic of Korea
Tel: +82-32-820-4935, Fax: +82-32-820-4829
E-mail: hjmaeng@gachon.ac.kr
These authors contributed equally to this work.
Received February 15, 2024; Revised March 6, 2024; Accepted March 31, 2024.
Abstract
Amifampridine, the first-line medication for Lambert-Eaton myasthenic syndrome (LEMS), is extensively metabolized by N-acetyltransferase 2 (NAT2). Drug-drug interactions (DDIs) can occur when co-administered with a NAT2 inhibitor and amifampridine. Acetaminophen is a widely used analgesic for mild to moderate pain, which is also known as a NAT2 inhibitor. In this work, we studied the effects of acetaminophen on the amifampridine pharmacokinetics in rats. Both acetaminophen (300 mg/kg) and amifampridine (2 mg/kg) were administered orally. In acetaminophen-treated rats, the systemic exposure to amifampridine significantly increased, and the ratio of the area under the plasma concentration-time curve for 3-N-acetylamifampridine to amifampridine (AUCm/AUCp) decreased markedly, which is likely due to the inhibition of NAT2 by acetaminophen. Also, the urinary amount excreted was increased in the acetaminophen-treated group, but the renal clearance remained unchanged. This oral pharmacokinetic drug-drug interaction study showed that orally administered acetaminophen significantly inhibits the NAT2-based metabolism of amifampridine and may cause meaningful DDIs.
Keywords : Amifampridine, N-acetyltransferase 2 (NAT2), Acetaminophen, 3-N-acetylamifampridine, Drug-drug interactions (DDIs)
Introduction

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disabling neuromuscular disease, caused by autoantibodies that target presynaptic voltage-gated calcium channels. It decreases the quantal release of acetylcholine, which causes dysautonomia, diminished or absent reflexes, and muscle weakness.1) These symptoms develop after 40 years of age, with peak incidence between 50 and 70 years of age.2) LEMS is also linked to small cell lung cancer, where 50% of LEMS patients are concurrently diagnosed with cancer or receive a diagnosis of cancer within two years of the first diagnosis.3) Amifampridine (3,4-diaminopyridine; 3,4-DAP) (Fig. 1A) has been clinically used to manage the symptoms of LEMS. The mechanism involves inhibiting fast voltage-gated potassium channels in the presynaptic neuron, preventing the outflow of potassium ions. Acetylcholinecontaining vesicles are exocytosed as a result of this prolonging presynaptic depolarization and increasing calcium influx.4) Even though amifampridine is not recommended for use in those who have a history of seizures, it has shown to be effective and tolerable in multiple clinical trials with no significant side effects recorded.1,5) In November 2018, amifampridine received approval from the United States Food and Drug Administration (US FDA) under the brand name, Firdapse® for the treatment of LEMS in adults.3) Currently, it is recommended as the first-line medication therapy for managing the symptoms of LEMS.



Fig. 1. Chemical structures of amifampridine (A) and its formed metabolite, 3-N-acetylamifampridine (B), and acetaminophen (C).

Following oral administration, amifampridine is quickly absorbed and has an elimination half-life from 20 minutes to 2 hours, with considerable inter-individual variability. The maximal pharmacological effect is achieved after 1.5 hours and maintained for 3~8 hours.6) It undergoes 3-N-acetylation to form a single major inactive metabolite, 3-N-acetylamifampridine (Fig. 1B), that subsequently undergoes renal elimination. In particular, around 80% of a dose of amifampridine that is taken orally undergoes metabolism and is eliminated from the body. N-acetylation is carried out by two polymorphic enzymes, N-acetyltransferase types 1 (NAT1) and 2 (NAT2). NAT2 polymorphism has a major impact on the rate of acetylation; in humans, “slow” acetylator phenotypes accumulate amifampridine to higher levels than “fast” acetylator phenotypes.7) This polymorphism in NAT2 can affect their effectiveness and increase the likelihood of adverse effects and toxicity. The NAT2 slow acetylator phenotype has been found to be more prevalent in African and Caucasian groups (40-70%) compared to Pacific Asian populations (10-30%).8) According to recent research, amifampridine is primarily eliminated by N-acetylation metabolism in both acetylator phenotypes, independent of renal function.9) This result suggests that rather than renal function level, metabolic acetylator phenotype has a greater impact on the pharmacokinetic profile of amifampridine.

NATs are drug-metabolizing enzymes that catalyze the conjugation of an acetyl group from acetyl coenzyme A (acetyl-CoA) to arylamines, hydrazines, and N-hydroxyarylamines.10) Their roles include detoxification and xenobiotic metabolic inactivation, and both polymorphic enzymes have different substrate profiles. NAT1 preferentially acetylates the arylamines 4-aminobenzoic acid and 4-aminosalicylate, whereas NAT2 has higher activity towards the hydrazines isoniazid and hydralazine, and the arylamine sulfamethazine. Different tissue distributions and patterns are another characteristic of human NATs; specifically, NAT2 is primarily expressed in the liver and gastrointestinal system.11) Several NAT2 inhibitors have been reported such as acetaminophen (Fig. 1C). A common over-the-counter medication used as an antipyretic and painkiller is acetaminophen, which is structurally identical to the acetylated product of NAT substrates. When combined with acetaminophen, it significantly reduced the acetylating capability in both in vitro and in vivo experiments.12) The mechanism by which acetaminophen inhibits N-acetylation by NAT2 is unknown, but it is suggested that acetaminophen may bind to the active site of the enzyme and act as an end-product feedback inhibitor. These findings suggest that inhibition of acetylation by acetaminophen is likely to lead to important drug-drug interactions (DDIs), which can increase the action of a drug and cause unanticipated side effects. Peripheral and oral paresthesia are the most commonly reported drug-related side effects associated with amifampridine.3) Additionally, some severe adverse effects such as seizures have been associated with excessive doses of amifampridine. Consequently, an in-depth DDI study between amifampridine and acetaminophen is required.

In our recent study, the effect of acetaminophen, a NAT2 inhibitor, on the pharmacokinetics of amifampridine, a NAT2 substrate, was investigated, both in vitro and in vivo.13) The inhibitory effects of NAT2 inhibitors, including acetaminophen, were evaluated in vitro. The study confirmed that using the rat liver S9 fraction, amifampridine is extensively metabolized through the acetylation pathway, and inhibitory effects of acetaminophen, and other well-known NAT2 inhibitors such as apocynin and cimetidine were also indicated. Furthermore, we found that acetaminophen inhibits N-acetylation in a mixed inhibition manner, by binding to both unbound and bound NAT2 with amifampridine. It also showed a time-dependent inhibition, with a Kinact of 0.0031 min-1. These in vitro findings suggest that acetaminophen significantly suppresses amifampridine’s NAT2-based metabolism. For an in vivo pharmacokinetic study, the pharmacokinetic interaction between amifampridine and acetaminophen was examined in rats. In our previous study, rats were given an intraperitoneal injection of acetaminophen and then amifampridine orally. There was a significant increase in systemic exposure to amifampridine and a decrease in total clearance (CL/F) when acetaminophen was co-administered intraperitoneally. The AUC ratio of 3-N-acetylamifampridine to amifampridine (AUCm/AUCp) significantly decreased by 65% in the acetaminophen-treated group. The study demonstrated acetaminophen inhibits acetylation of amifampridine and changes pharmacokinetics in rats, indicating that NAT2-mediated DDIs require further investigation.

However, considering that acetaminophen is mainly administered orally, further in vivo study should be needed. Because NAT2 is expressed not only in the liver but also in the intestine, oral administration of acetaminophen may influence a greater alteration of amifampridine pharmacokinetics than intraperitoneal administration of acetaminophen. Therefore, the purpose of this study was to investigate the pharmacokinetics of amifampridine when co-administered with acetaminophen orally in rats.

Methods

Materials

Acetaminophen, amifampridine, cremophor® EL, and tolbutamide were acquired from Sigma Aldrich (St Louis, MO, USA). Phosphate-buffered saline (PBS) was acquired from Welgene (Gyeongsangbuk-do, Korea). The source of ondansetron was Cadila, located in Ahmedabad, India. The supplier from Huons (Seongnam, Korea) supplied the heparin. The supplier of PEG400 was Samchun, located in Seoul, Korea. Zoletil 20 and Rompun 2% injection, respectively, were acquired from Bayer Pharmaceuticals (Leverkusen, Germany) and Vibrac SA (Carros, France). This investigation utilized methanol and acetonitrile of high-performance liquid chromatography (HPLC) grade, both of which were procured from Honeywell Burdick and Jackson Co. (Ulsan, Korea). An AquaMAX ultrapure water purification system (YL Instruments, Anyang, Korea) was utilized to purify the water. All additional solvents and compounds were of HPLC or reagent grade and were utilized without additional purification.

In vivo pharmacokinetic interaction study in rats

Male Sprague Dawley (SD) rats were acquired from Nara Bio-Tech., Korea. The rats were acclimated to the laboratory settings for a minimum of one week. Throughout this period, they were granted unrestricted availability to nourishment and hydration and were subjected to a light and dark cycle alternating every 12 hours. The animal investigations were conducted under the approval of the Institutional Animal Care and Use Committee at Gachon University (GIACUC-R2019020, July 1st, 2019).

The rats were divided into two groups, the control group, and the acetaminophen-treated group, using random assignment. A combination of Rompun and Zoletil was injected intraperitoneally (IP) to the rats to induce anesthesia. Subsequently, the femoral artery was cannulated using polyethylene sterile tubing PE#50 (outside diameter: 0.965 mm) to collect blood samples. For the drug-drug interaction study, a dose of 2 mg/kg of amifampridine, dissolved in saline at a concentration of 2 mg/mL, was given orally to both the control group (n=5) and the acetaminophentreatment group (n=7) using the rat oral Zonde. The acetaminophen drug solution was dissolved in a vehicle consisting of Cremophor® EL, PEG, and DDW (in a ratio of 5:55:40, v/v/v) at a concentration of 150 mg/mL. This solution was administered orally for the acetaminophen treatment group only, 1 hour before the amifampridine administration whereas the vehicle was administered orally for the control group. A volume of approximately 0.25 mL of blood was subsequently obtained from the femoral artery into polystyrene PE Beckman® type micro-centrifuge tubes at 0, 5, 15, 30, 60, 90, 120, 240, 360, and 480 min after amifampridine administration. The blood samples were centrifugated at 14,000 rpm for 15 min at 4°C. Plasma samples were then separated and stored at -20°C until analysis.

To analyze amifampridine and 3-N-acetylamifampridine, 50 μL of every plasma sample was mixed with 100 μL of a working solution containing ondansetron at a concentration of 5 ng/mL in acetonitrile. To quantify acetaminophen levels, the plasma samples were diluted by a factor of 10 using blank plasma. The diluted plasma sample (100 μL) was combined with 200 μL of IS solution (tolbutamide 200 ng/mL in methanol). After the mixture was vortexed and centrifuged, the supernatant was extracted and used for LC-MS/MS analysis.

Simultaneously, urine samples were collected in 50 mL conical tubes during a period of 24 h, divided into three intervals (0-4, 4-8, and 8-24 h). Urine samples were 500-fold diluted using PBS. Consequently, 200 μL of the IS was spiked into an Eppendorf tube containing 100 μL of diluted urine. The process of urine sample preparation was similar to plasma. The supernatants were transferred into vials to be used for injection in the LC-MS/MS analysis.

Quantification of plasma and urine samples using LC-MS/MS

The quantitative analysis of amifampridine, 3-N-acetylamifampridine, and acetaminophen was conducted using the tandem mass detection system (6495 Triple Quadrupole, Agilent Technologies, Santa Clara, CA, USA) coupled with the HPLC system (1290 infinity) and electrospray ionization (ESI+) Jet Stream ion source (Agilent Technologies, Santa Clara, CA, USA). The SynergiTM Polar-RP 80Å Column (150×2.0mm, 4 μm; Phenomenex, Torrance, CA) was used in conjunction with the Security Guard Column (4.0×3.0 mm; Phenomenex) to separate and analyze the samples. The HPLC system employed a mobile phase composed of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The elution process employed a gradient elution with a flow rate of 0.2 mL/min. The MRM transitions of m/z 110.1→93.0 for amifampridine, 152.1→110.1 for 3-N-acetylamifampridine, and 294.1→170.0 for the IS (ondansetron) were simultaneously utilized.

Also, MRM transitions of m/z 152.1→110.0 and 271.1→155.0 were applied for acetaminophen and tolbutamide (IS), respectively. The column and autosampler were maintained at temperatures of 25 and 4°C, respectively, and the injection volume was 2 μL.

The calibration curves were created by graphing the ratios of peak areas acquired from the analyte and the internal standard (Y-axis) against the corresponding concentrations of the analyte (X-axis). A weighted least-squares linear regression analysis was performed, using a weight of 1/x.

Data Analysis

Using non-compartmental analysis using WinNonlin® 8.3 software (Pharsight Co., Mountain View, CA, USA), the pharmacokinetic parameters were obtained such as the area under the plasma concentration-time curve from time zero to last sampling point (AUClast), the area under the plasma concentration-time curve from time zero to infinity (AUCinf), elimination half-life (t1/2), mean residence time (MRT), oral clearance (CL/F), and oral volume of distribution (Vd/F). The peak plasma concentration (Cmax) and the time to reach Cmax (Tmax) were directly read from the plasma concentration data. A statistical analysis was conducted utilizing the Student’s t-test (two-tailed) to compare the means of the control and experimental groups. Any change that was considered statistically significant had a p-value of less than 0.05. The data is displayed as means with their corresponding standard deviations (SDs).

Results

At the dose of 300 mg/kg, the rats were administered with acetaminophen orally. The plasma concentration levels were observed, where the Cmax was 92.2 μg/mL and the level was maintained to be 52.57 μg/mL at 480 min (Table 1 and Fig. 2). In both control rats and acetaminophen-treated rats, the plasma concentrations of amifampridine and 3-N-acetylamifampridine were determined to investigate the DDIs between acetaminophen and amifampridine. In the acetaminophen-treated group, Cmax and AUCinf of amifampridine significantly increased by 177% and 349%, respectively, compared to the control group (Table 2 and Fig. 3A). Cmax of 3-N-acetylamifampridine was significantly reduced by about 33% in the acetaminophen-treated group (Table 2 and Fig. 3B). However, AUCinf of 3-N-acetylamifampridine was unchanged. Notably, the ratio of AUCm to AUCp reduced considerably from 21.99±2.33 to 5.30±1.93 (p<0.001) following the administration of acetaminophen. This observation shows that orally administered acetaminophen does inhibit the NAT2-mediated metabolism of amifampridine to its metabolite, 3-N-acetylamifampridine, in rats.



Fig. 2. The plasma concentration-time profile of acetaminophen after 300 mg/kg PO administration of acetaminophen in rats (n=7).
Data are expressed as means±SDs.



Fig. 3. Plasma concentration-time profiles of amifampridine (A) and 3-N-acetylamifampridine (B) after PO administration of 2 mg/kg amifampridine in control (n=5) and acetaminophen-treated (n=7) rats.
Open and closed circles represent the control and acetaminophen-treated rats, respectively. Data are expressed as means±SDs.

Pharmacokinetic parameters of acetaminophen after oral administration of 300 mg/kg acetaminophen in acetaminophen-treated rats (n=7).

Data are expressed as means±SDs.

Parameters Acetaminophen-treated rats
Cmax (μg/mL) 92.23±21.22
Tmax (min) 076.43±111.83
AUClast (μg×min/mL) 30020±10400
AUCinf (μg×min/mL) 110800±89000
t1/2 (min) 774.3±641.2
MRT (min) 1150±913
CL/F (mL/min/kg) 3.718±1.681
Vd/F (mL/kg) 2930±707

AUCinf, area under the plasma concentration-time curve from time 0 to infinity; AUClast, area under the plasma concentration-time curve from time 0 to the last quantifiable point; CL/F, clearance divided by bioavailability (F); Cmax, maximum plasma concentration; Tmax, time to reach Cmax; MRT, mean residence time; t1/2, half-life; Vd/F, volume of distribution divided by F.



Pharmacokinetic parameters of amifampridine and 3-N-acetylamifampridine after oral administration of amifampridine (2 mg/kg) in control (n=5) and acetaminophen-treated rats (n=7).

Data are expressed as means±SDs.

Pharmacokinetic parameters Amifampridine 3-N-acetylamifampridine
Control Acetaminophen-treated Control Acetaminophen-treated
Cmax (μg/mL) 0.07333±0.03190 000.2028±0.0683** 0.7812±0.1647 000.5270±0.1026**
Tmax (min) 29.00±35.60 38.57±27.19 72.00±40.25 0248.6±171.2*
AUClast (μg×min/mL) 9.661±1.124 0039.57±9.02*** 199.3±25.9 172.4±33.5
AUCinf (μg×min/mL) 10.16±1.44 0045.66±9.51*** 224.6±50.7 231.5±51.6
t1/2 (min) 96.59±25.20 126.0±16.6 123.2±42.2 220.5±98.1
MRT (min) 158.5±54.4 191.8±25.3 231.2±70.4 0384.8±110.1*
CL/F (mL/min/kg) 200.1±28.9 0045.36±9.38*** - -
Vd/F (mL/kg) 27290±4360 0008218±2001*** - -
CLr (mL/min) 3.135±0.889 2.441±0.913 4.886±0.490 4.573±1.484
AUCm/AUCp - - 21.99±2.33 0005.297±1.934***

AUCm/AUCp, AUCinf ratio of 3-N-acetylamifampridine to amifampridine; CLr, renal clearance.

*p<0.05, **p<0.01 and ***p<0.001 compared with control rats



Urine samples were collected for up to 24 hours in order to study the effects of acetaminophen on amifampridine and 3-N-acetylamifampridine excretion in the urine. The cumulative urinary amount of amifampridine was significantly increased in the acetaminophen-treated group. The control rats exhibited a total urine recovery of amifampridine of 5.93%, while the acetaminophen-treated rats showed a recovery of 19.87% (Fig. 4A). In contrast, the urinary excretion of 3-N-acetylamifampridine seemed to decrease at several time points in the presence of acetaminophen, but the difference was not significant (Fig. 4B). The total amount of the metabolite excreted was not significantly different between the two groups. However, the renal clearance of both amifampridine and 3-N-acetylamifampridine was unaltered, suggesting that the urinary excretion of amifampridine and 3-N-acetylamifampridine was unaffected by acetaminophen pretreatment. (Table 2).



Fig. 4. Cumulative urinary amount of amifampridine (A) and 3-N-acetylamifampridine (B) after PO administration of 2 mg/kg amifampridine in control (n=5) and acetaminophen-treated (n=7) rats.
Open and closed circles represent the control and acetaminophen-treated rats, respectively. Data are expressed as means±SDs.

In Table 3, the AUCinf of amifampridine and 3-N-acetylamifampridine of the control and acetaminophen-treated group were compared with the AUCinf value we obtained in our previous study, where acetaminophen was treated to rats via intraperitoneal route.13) AUCinf of amifampridine in acetaminophentreated rats increased dramatically (by 349%) in comparison to the control group, as indicated above (Table 3). This increase in amifampridine’s AUCinf is more than the increased extent (by 186%) that was obtained with intraperitoneal acetaminophen administration.13)

Comparative analysis for dosing routes (IP or PO) of acetaminophen on the pharmacokinetics of amifampridine and its metabolite, 3-N-acetylamifampridine, in drug-drug interaction studies.

Data are expressed as means±SDs.

Parameters Amifampridine 3-N-acetylamifampridine
Acetaminophen-IP Acetaminophen-PO Acetaminophen-IP Acetaminophen-PO
AUCcontrol (μg×min/mL) 9.112±1.616 10.16±1.44 221.4±40.7a 224.6±50.7
AUC+acetaminophen (μg×min/mL) 26.03±6.99a 45.66±9.51 221.3±23.5a 231.5±51.6
AUCR 2.857 4.494 1.000 1.030

aData obtained from Ref. 13.

IP, intraperitoneal; PO: per oral; AUCcontrol, AUCinf of control group; AUC+acetaminophen, AUCinf of acetaminophen-treated group; AUCR, ratio of AUC+acetaminophen to AUCcontrol.


Discussion

This work aimed to examine the effects of acetaminophen on amifampridine oral pharmacokinetics, taking into account NAT2-mediated metabolism. The metabolic process that converts amifampridine into 3-N-acetylamifampridine, which expresses genetic polymorphism among various people or populations, involves the enzyme NAT2. One common and well-studied genetic variation in human populations is NAT2 polymorphism.14) Individuals can be classified into rapid, intermediate, or slow acetylator phenotypes based on the NAT2 alleles they possess. Diverse states of acetylation can modify the drug metabolism or detoxification, which can lead to side effects, enhancing susceptibility to numerous cancers, and inducing genetic damage such as DNA adduct formation.8) Thus, NAT2 activity is essential for maximizing medication therapy while minimizing side effects.

Amifampridine is a first-line therapy for the treatment of LEMS. While it will remain the drug of the first choice for LEMS patients in the coming years, little is known about the pharmacokinetics of amifampridine or DDIs. It is commonly known that NAT2 mainly metabolizes amifampridine. In addition, some drugs are known to inhibit the activity of NAT2, such as acetaminophen. Co-administration of acetaminophen with amifampridine, a NAT2 substrate, has great potential for relevant DDIs. In our previous study, the DDIs between acetaminophen and amifampridine in rats were first investigated.13) An in vitro metabolic study revealed that acetaminophen had an inhibitory effect on the NAT2-mediated metabolism of amifampridine, including its inhibition mechanism. Furthermore, the injection of acetaminophen intraperitoneally in rats influenced the in vivo pharmacokinetics of amifampridine. Namely, the systemic exposure to amifampridine was significantly increased and the total clearance (CL/F) of amifampridine significantly decreased in the acetaminophen-treated rats via the intraperitoneal route, compared to control rats. But the goal of the current investigation was to look at the interactions between oral acetaminophen and amifampridine in vivo. Acetaminophen is normally recognized as a mild analgesic that is available without a prescription. Although it was recently reported that acetaminophen inhibits the metabolism of amifampridine in vivo, further studies with diverse processes are needed to observe and predict the accurate interactions between two drugs. For safety, we should be fully aware of the DDIs associated with commonly used medications.

In this study, we focused on the effects of orally administered acetaminophen on the pharmacokinetics of amifampridine and its metabolite, 3-N-acetylamifampridine. Similar to the previous study,13) t1/2 almost stayed the same after acetaminophen treatment. This is probably caused by both a lower CL/F and a lower volume of distribution (Vd/F) (Table 2). The AUC ratio of 3-N-acetylamifampridine to amifampridine (AUCm/AUCp) significantly decreased by 76% after acetaminophen administration (Table 2). It is unclear what exactly causes a lower volume of distribution (Vd/F). However, it is hypothesized that oral acetaminophen might hinder amifampridine from being broken down as soon as it enters the intestines and liver, increasing the drug’s bioavailability (F). Additionally, the decreased distribution of amifampridine in body tissues caused by acetaminophen could be considered as a potential mechanism. The intraperitoneal injection resulted in an approximately 186% increase in AUCinf and a 64% decrease in CL/F. However, oral administration of acetaminophen to rats resulted in a 349% increase in AUCinf and a 77% drop in CL/F, which exhibits a more significant alteration compared to the intraperitoneal route (Table 3).

The cumulative urinary recovery of amifampridine showed a significant increase of 235% in acetaminophen-treated rats (Fig. 4A). However, the renal clearance remained unchanged between the groups, likely due to the similar increase in cumulative urinary amount to the increase in plasma AUCinf of acetaminophen-treated rats. Acetaminophen did not have a marked effect on the total amount of 3-N-acetylamifampridine excreted in the urine, and there was no difference in the renal clearance of the metabolite between the control and acetaminophen-treated rats.

The route of administration is a critical determinant of pharmacokinetic studies. For example, one of the most widely method used in animal studies is the intraperitoneal route, where the substance is injected into the peritoneum (body cavity). It is quick, easy to master, and minimally stressful for animals. In addition, rodents can safely receive significant amounts of solution (up to 10 mL/kg) through the intraperitoneal route, which is particularly beneficial for substances that have low solubility.15) When given intraperitoneally, the drug is quickly absorbed and it is commonly used to avoid the GI tract and potential degradation/modification of drugs. However, acetaminophen can inhibit the first-pass metabolism of amifampridine not only in the liver but also in the intestine, since acetaminophen is distributed into the tissues during systemic circulation. To mitigate this concern, we studied the in vivo pharmacokinetic DDIs between amifampridine and acetaminophen in rats, both administered via oral route. By oral administration, we could fully evaluate the inhibitory effects of acetaminophen on the NAT2-mediated metabolism of amifampridine in the intestine and liver.

Collectively, these overall results effectively show that acetaminophen changed the pharmacokinetic profiles of amifampridine and 3-N-acetylamifampridine in vivo. Moreover, it showed that the metabolic inhibition of amifampridine by acetaminophen is stronger when it is administered orally. This is probably due to the DDIs between amifampridine and acetaminophen in both the liver and intestine. The metabolism of amifampridine with acetaminophen administered via the intraperitoneal route is also affected in the intestine during systemic circulation, but we can expect that the inhibitory effect of acetaminophen was much stronger when it was administered orally.

Conclusion

The study confirmed that acetaminophen inhibits the acetylation metabolism of amifampridine in rats. In this study, we focused on the inhibitory effects of orally administered acetaminophen on amifampridine pharmacokinetics. The ratio of oral plasma exposure of 3-N-acetylamifampridine to amifampridine was reduced in the presence of oral acetaminophen treatment. Compared to the intraperitoneal dose of acetaminophen, the inhibitory effects were greater when acetaminophen was orally administered, which shows that orally administered acetaminophen extensively inhibits the NAT2-mediated metabolism of amifampridine. Consequently, as DDIs between acetaminophen and amifampridine probably result in higher toxicity, they should be carefully assessed.

Acknowledgment

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2021R1F1A1060378).

Conflict of Interest

All authors declare that they have no conflict of interest.

Authors’ Positions

Jin-Woo Cheong : Undergraduate student

Yeo-Dim Park : Graduate student

Dang-Khoa Vo : Graduate student

Han-Joo Maeng : Professor

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