Pharmacokinetics and Safety of Single and Multiple Doses of Oral N-Acetylcysteine in Healthy Chinese and Caucasian Volunteers: An Open-Label, Phase I Clinical Study

This was a single- and multiple-dose, single-centre, open-label, phase I clinical study. The study was performed at the Phase I Unit of CROSS Research Arzo, Switzerland.

Healthy Chinese and Caucasian men and women aged 18–50 years with a bodyweight of ≥ 45 kg and a body mass index (BMI) of 18–29 kg/m² who provided written informed consent for participating in the study were included. Additional inclusion criteria were: systolic blood pressure (BP) 100–139 mmHg, diastolic BP 50–89 mmHg, heart rate (HR) 50–90 bpm, good comprehension and use of the written and spoken Italian language. In addition, women of childbearing potential were required to use an effective method of contraception, and all women had to have a negative pregnancy test result at screening and Day − 1.

Participants were required to remain at the study site from the evening of Day − 1 until the morning of Day 7.

All participants received NAC 600-mg uncoated tablets (Fluimucil^®; batch number 20039498, expiry December 2021). NAC was administered orally with 150 mL of still mineral water. On Day 1, a single tablet was administered at 08:00 ± 1 h under fasting conditions. After a wash-out period of 48 h, NAC was administered twice daily (at 08:00 ± 1 h before breakfast and at 20:00 ± 1 h before dinner) on Days 3, 4 and 5. On Day 6, a single tablet was administered at 08:00 ± 1 h under fasting conditions. Steady state of NAC was expected to be attained after seven consecutive doses administered up to the morning of Day 6 with a τ of 12 h on the basis of mean half-life values of up to 14 h found in previous studies of the same dosage form.

On Day  1 and Day 5, a standardised light dinner was served. On Day 1 and Day 6, participants were not allowed any food or drinks (except water) for at least 10 h before, and for 5 h after, NAC administration. Water was allowed ad libitum, except for 1 h before and 1 h after NAC administration. During the 4 h following NAC administration, when not involved in study procedures, participants remained seated and were not allowed to lie down.

On Day 1 and Day 6, standardised lunch and dinner were served at approximately 5 h and 12 h after NAC administration, respectively. On Days 2, 3, 4 and 5, standardised breakfast, lunch and dinner were served at approximately 09:00, 13:00 and 20:00, respectively. One cup of tea or coffee was allowed after each meal only. Any other food or drinks containing xanthines (e.g. chocolate) were forbidden for the duration of stay at the study site. Alcohol and grapefruit were forbidden from 24 h before NAC administration on Day 1 until the end of the study. A single cigarette after each meal was allowed during the stay at the study site. Routine daily activities were encouraged, but hazardous, strenuous or athletic activities were not permitted.

In order to measure pharmacokinetic parameters, 6 mL of blood was collected from a vein in the forearm using an indwelling catheter with a switch valve. Blood for pharmacokinetic analyses was collected at 20:00 ± 1 h on Day   1; before NAC administration, and 5, 15, 30 and 45 min and 1, 1.25, 1.5, 2, 3, 4, 6, 8, 12, 16, 24 and 36 h after NAC administration on Day 1 and Day 2; and before NAC administration, and 5, 15, 30 and 45 min and 1, 1.25, 1.5, 2, 3, 4, 6, 8, 12, 16 and 24 h after NAC administration on Day 6 and Day 7. Urine was collected before the end of each of the following intervals: 0–4 h, 4–8 h, 8–12 h, 12–24 h and 24–36 h after NAC administration on Day 1 and Day 2, and 0–4 h, 4–8 h, 8–12 h and 12–24 h on Day 6 and Day 7. The concentrations of NAC in plasma and urine were determined at Ardena Bioanalytical Laboratory, The Netherlands.

The bioanalytical methods used for the quantification of NAC in human Li-heparin plasma and urine were validated for samples stored at ≤  − 70 °C. A full validation of the methods was performed according to the current guidelines. The long-term stability of NAC was tested and the results showed that NAC stored frozen at ≤  − 18 °C or ≤  − 70 °C is stable for up to 65 d ays in plasma and 71 days in urine. Both liquid chromatography with tandem mass spectrometry (LC–MS/MS) methods produced accurate and precise results. Validation studies showed that the absolute biases for the quality control (QC) samples at the low, medium and high levels were − 0.6%, − 5.0% and − 5.6% in plasma, and 4.6%, − 9.0% and − 7.0% in urine, respectively. The precision results [expressed as total coefficients of variation (CV)] were as follows: 5.3%, 2.1% and 1.9% in plasma, and 4.5%, 1.6% and 3.1% in urine, for the low, medium and high QC samples, respectively. Intra-run CVs (repeatability) were 3.0%, 1.0% and 1.0% in plasma, and 4.5%, 2.1% and 2.3% in urine, for the low, medium and high QC samples, respectively. The calibration range covered 20.0–5000 ng/mL in human plasma and 0.100–20.0 µg/mL in human urine.

The pharmacokinetic parameters assessed after single dose administration on Day 1 were: maximum concentration (C_max), time to C_max (t_max), terminal elimination rate constant (λ_z, calculated using log-linear regression from at least three points), area under the concentration–time curve (AUC) until time t (AUC_0–t, calculated using the trapezoidal method), AUC extrapolated to infinity (AUC_0–∞, calculated as AUC_0–t + C_t/λ_z, where C_t is the last measurable drug concentration), proportion of residual AUC extrapolated to infinity (AUC_extra, calculated as 100 × [C_t/λ_z]/AUC_0–∞), AUC from administration to 12 h on Day 1 (AUC_0–12, calculated using the trapezoidal method), t_½ (calculated as ln2/λ_z), volume of distribution [V_z, calculated as dose/(AUC_0–∞ × λ_z)] and total body clearance (CL_t, calculated as dose/AUC_0–∞) of NAC in plasma, and total amount excreted until time t (Ae_0–t), fraction excreted until time t (Fe_0–t) and renal clearance (CL_r, Ae_0–t/AUC_0–∞) of NAC in urine. The pharmacokinetic parameters assessed after multiple doses on Day 6 and Day 7 were: C_max at steady state (C_max,ss), t_max at steady state (t_max,ss), minimum concentration at steady state (C_min,ss), AUC_0–t at steady state (AUC_0–t,ss), AUC during the interval τ at steady state (AUC_τ,ss, calculated using the trapezoidal method), average concentration at steady state (C_ave,ss, calculated as AUC_t,ss/t), accumulation ratio (ARAUC, calculated as AUC_τ,ss/AUC_0–12) and peak–trough fluctuation [PTF, calculated as (C_max,ss − C_min,ss)/C_ave,ss × 100)] of NAC in plasma, and Ae_0–t at steady state (Ae_0–t,ss) of NAC in urine.

The plasma pharmacokinetic parameters were calculated using baseline-corrected concentrations because quantifiable levels of endogenous NAC were detected at both baseline measurements (evening of Day 1 and pre-dose on Day 1). For each participant, an individual mean baseline value was calculated as the arithmetic mean of the two measured baseline concentrations, and this mean value was subtracted from each concentration value measured on Days 1–2 and 6–7. The quality of log-linear regression and, consequently, the reliability of the extrapolated pharmacokinetic parameters (namely λ_z, t_½, AUC_0–∞, AUC_extra, V_z, CL_r and CL_t) was demonstrated by a determination coefficient, R² ≥ 0.8. Individual extrapolated parameters, when considered unreliable, were reported as not calculated. In the pharmacokinetic analysis, the scheduled sampling times were used for calculation, as the deviations observed were considered not relevant for the analysis purposes, except for one sampling time for one participant. As per the study protocol, deviations from the scheduled sampling times were recommended not to exceed predetermined ranges.

Safety assessments included treatment-emergent adverse events (AEs), vital signs (BP and HR), bodyweight, physical examinations and laboratory parameters.

No formal sample size calculation was performed. Instead, we estimated the number of participants that would be sufficient for the descriptive purposes of the study.

Results are presented using descriptive statistics, including the number of observations, geometric mean (for pharmacokinetic parameters only), arithmetic mean, standard deviation (SD), coefficient of variation, minimum, median and maximum for quantitative variables, and frequency for qualitative variables. The pharmacokinetic analysis was performed using Phoenix WinNonlin^® v.6.3 (Pharsight) and SAS^® v.9.3 TS1M1 for Windows^®.

The study protocol was approved by an independent ethics committee (Comitato Etico Cantonale, Canton Ticino, Switzerland). The study was conducted in accordance with the protocol and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Good Clinical Practice guidelines, Declaration of Helsinki and applicable Swiss and European Union regulations.

Mean baseline-corrected plasma concentrations of NAC on Day 1 and Day 6 are shown in Fig. 1a (Chinese participants) and Fig. 1b (Caucasian participants). After a single dose (Day 1), the plasma concentration of NAC increased rapidly in both groups. In Chinese participants, the mean ± SD maximum observed plasma concentration of NAC was 1950.9 ± 1026.1 ng/mL at 45 min after NAC administration, while, in Caucasian participants, the maximum observed plasma concentration was 2743.9 ± 985.2 ng/mL at 1.0 h. After that, plasma concentrations of NAC declined rapidly in both groups. At steady state (Day 6), pre-dose baseline-corrected NAC plasma concentrations were approximately ten times higher than on Day 1 (mean ± SD 36.4 ± 11.5 ng/mL vs. 4.22.5 ± 130.5 ng/mL in Chinese and 39.7 ± 13.9 ng/mL vs. 377.7 ± 92.7 ng/mL in Caucasian participants). Plasma concentration–time curves at steady state were otherwise similar to those after a single dose.

The elimination phase after the single NAC administration could not be defined for two Caucasian participants according to the established condition (quality of extrapolated pharmacokinetic parameters as demonstrated by a determination coefficient, R² < 0.8), and therefore λ_z, t_½, AUC_0–∞, AUC_extra, V_z and CL_t for these individuals were not calculated.

Mean calculated C_max was similar in Chinese and Caucasian participants both after a single dose of NAC and at steady state (Table 2). In Chinese participants, mean calculated C_max was approximately 1.3 times higher at steady state than after a single dose, while, for Caucasians, it was approximately 1.2 times higher. After a single dose, median t_max was 1.25 h in Chinese participants and 1.00 h in Caucasian participants. At steady state, median t_max was 1.00 h in both groups. Mean AUC_0–t and AUC_τ,ss were also similar in Chinese and Caucasian participants. As expected, the extent of exposure increased at steady state relative to single dose administration. However, mean ARAUC was quite low in both groups (1.5 in Chinese and 1.4 in Caucasians). Mean ± SD t_½ was 15.4 ± 3.5 h in Chinese and 18.7 ± 7.2 h in Caucasian participants. Mean V_z was higher than 1000 L in both groups (1250.0 L in Chinese and 1400.8 L in Caucasian participants).

The mean Ae_0–t was small (approximately 2% of the dose) and relatively constant after single and multiple doses of NAC (Table 3). Differences between Chinese and Caucasian participants were minor. The mean Fe_0–t was also relatively low and similar in Chinese and Caucasian participants (Table 3).

Study treatment had no relevant effects on BP, HR, bodyweight or laboratory parameters.