FormalPara Key Points

The bioequivalence of two formulations of azithromycin tablets was demonstrated under both fasted and fed conditions in healthy Chinese volunteers. The test and the reference azithromycin tablets were bioequivalent and well tolerated.

1 Introduction

Azithromycin is the first macrolide antibiotic. It works by inhibiting bacterial growth. It binds to the 50S ribosomal subunit of susceptible organisms, thereby interfering with protein synthesis [1, 2]. The current clinical forms of azithromycin commonly used include injections, tablets, granules, capsules, dispersible tablets, sustained-release tablets and so on. Due to its acid stability, great tissue penetration, and a significantly longer half-life (t½), it is widely used in clinical practice [3]. Azithromycin exhibits antibacterial activity against a number of sensitive bacteria such as Haemophilus influenzae, Streptococcus pneumoniae, Chlamydia pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Streptococcus agalactis. It is mainly used in respiratory infections caused by sensitive bacteria, skin and soft tissue infections, as well as simple genital infections caused by Chlamydia trachomatis and non resistant gonococci [4, 5].

Azithromycin is rapidly absorbed and reached peak plasma concentration at around 2–3 h [6]. The absolute oral bioavailability of azithromycin is reported to be approximately 37% after oral administration [7, 8]. The antimicrobial activity of azithromycin is reduced at low pH. It is distributed throughout the body widely, which may be related to the clinical activity. After a single oral dose of azithromycin, the final elimination half-life is approximately 68 h. Biliary excretion is the main pathway for the clearance of azithromycin, and it is basically excreted in prototype form [9].

Bioequivalence study in China is initiated by the National Medical Products Administration (NMPA), which supports a generic consistency evaluation program for ensuring that generic products manufactured in China meet the required standards and provide equivalent therapeutic effects as their reference products. This trial was designed to evaluate the bioequivalence of two azithromycin tablets. Azithromycin has a wide first pass effect, resulting in high within-subject variation after oral administration. We conducted a single-center, open-label, single-dose, randomized, 3-way crossover study to investigate the pharmacokinetic (PK) characteristics and bioequivalence of two formulations of azithromycin. The study also aimed to assess the safety of the two formulations under fasted and fed conditions in healthy Chinese subjects.

2 Materials and Methods

2.1 Study Design

This was a single-center, open-label, single-dose, randomized, 3-way crossover bioequivalence study with two independent groups (fasting group and fed group) conducted at Huzhou Central Hospital, China. Thirty-six healthy Chinese subjects were enrolled in each group (fasting and fed), and randomly assigned to three treatment sequences by a randomization schema (Table S1, see electronic supplementary material [ESM]). Each treatment sequence consisted of three periods, with a 14-day washout period between each period. The test formulation was 250 mg of azithromycin tablets (batch no. AT20031701, Shijiazhuang No.4 Pharmaceutical Co., Ltd, Shijiazhuang, China). The reference formulation was also 250 mg of azithromycin tablets (Zithromax®, batch no. R80439, Pfizer Laboratories Div Pfizer Inc, New York, USA).

Subjects were administered 250 mg of azithromycin tablets (test/reference, T/R) on days 1, 15 and 29. In the fasting state, the subjects took either a T or R azithromycin tablet along with 240 mL of water in the morning, after fasting for a minimum of 10 h overnight. In the fed state, the subjects were also fasted for at least 10 h, and were orally administered one T or R formulation of azithromycin with 240 mL of water within 30 min of eating a high-fat diet. This high-fat diet consisted of approximately 150 kcal from protein, 250 kcal from carbohydrates, and 500–600 kcal from fat.

The trial design was approved by the Ethics Committee at Huzhou Central Hospital (Ethics approval No: 202112015-01). The trial was conducted following the principles outlined in the Declaration of Helsinki, Good Clinical Practice (GCP), and the guidelines set by the National Medical Products Administration (NMPA). Prior to their involvement, all participants provided their informed consent by signing the appropriate forms.

2.2 Subjects

Chinese subjects were required to be aged 18–65 years (including 18 and 65 years) with a body mass index range of 19–26 kg/m2. The average body weight of males was ≥ 50 kg, and that of females was ≥ 45 kg. All participants demonstrated a comprehensive understanding of the objectives, methodology, and potential adverse effects prior to the commencement of the trial. Additionally, they willingly signed an informed consent form before any research procedure. Based on clinical history, vital sign examinations, physical examinations, clinical laboratory tests, and 12-lead electrocardiograms (ECGs), it was established that all subjects were healthy.

The exclusion criteria were as follows: (i) a history of two or more drug or food allergies, lactose intolerance, or known to be allergic to drug ingredients; (ii) history of arthralgia, hives, angioneurotic edema and other diseases with clinical significance, or had any disease that increases the risk of bleeding (intracranial hemorrhage, gastrointestinal bleeding or perforation, thrombocytopenia with clinical significance, coagulation dysfunction, etc.); (iii) had used any medication (including prescription drugs, over-the-counter drugs, herbal medicines, etc.) or health products within 2 weeks, or any drugs that inhibit or induce drug metabolism within 30 days prior to screening; (iv) history of drug abuse within 6 months before screening or positive drug screen; (v) smoking more than five cigarettes/day, drinking too much tea or caffeinated beverages (> 8 cups/day, 250 mL/cup) or alcohol abuse within 3 months before screening; (vi) had consumed foods that inhibit or induce drug metabolism (including grapefruit and lime) within 7 days before administration; (vii) blood donation or significant blood loss (> 400 mL) within 3 months; (viii) an abnormal diet (including high potassium, low fat, dieting, low sodium, etc.) within 30 days; (ix) participated in clinical trials of other drugs within 3 months; (x) had pregnancy plans and declined to use effective contraception for the following 6 months or female subjects who were lactating.

2.3 Estimation of Sample Size

The main PK parameters of azithromycin included maximum plasma concentration (Cmax) and area under the concentration–time curve (AUC) in this study. According to previous research results [12,13,14,15], the within-subject coefficient of variation (CVw) of both Cmax and AUC under fasting conditions exceeded 30%. The CVw of Cmax was set to 35%. The desired test power was set at 80% with a significance level of 0.05. The expected geometric mean ratio (GMR) was 90%. The estimated sample size was 30, using up to a 74–134% widened interval to calculate sample size. Due to the lengthy duration required for cleaning, a total of 36 subjects were allocated to both fasting and fed conditions, taking into account the shedding rate.

2.4 Pharmacokinetic Assessment

Blood samples (4 mL) were collected at 0 h (within 1 h before administration), 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 12, 24, 48, 72, 96, and 120 h after oral administration of azithromycin tablets in the fasting study. Blood samples (4 mL) were collected at 0 h (within 1 h before oral administration), 0.5, 1, 1.5, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 8, 12, 24, 48, 72, 96, and 120 h after oral administration of azithromycin tablets in the fed study. The samples were centrifuged 1700×g for 10 min at 4 ℃ within 1.5 h after blood collection. The plasma was separated into two aliquots and stored at − 80 ℃ until PK analysis.

2.5 Plasma Drug Concentration Analysis

A validated high-performance liquid chromatography–tandem mass spectrometry (HPLC‒MS/MS) bioanalytical method was used to quantify the concentration of azithromycin in plasma. The chromatographic separation was carried out using a Shimadzu LC20ADXR liquid chromatograph system and performed on an Ultimate XB-C18 (50 × 2.1 mm, 5 μm, Welch Materials) column maintained at 30 °C. Aqueous formic acid 0.1% and ammonium acetate and acetonitrile 10.0 mM were used in mobile phase with a flow rate of 0.9 mL/min. Each injection took 5.2 min to analyze. The analytes were detected by a Triple Quad 4500 tandem triple quadrupole mass spectrometer (Applied Biosystems/Sciex) with an electrospray source. The mass spectrometer was operated in the positive ionization mode, and quantification was performed using multiple reaction monitoring chromatograms. The transitions monitored for azithromycin and the internal standard azithromycin-13C-d6 were m/z 749.5/591.5 and 753.5/595.5, respectively. The assay exhibited linearity within the concentration range of 2–1000 ng/mL, and the lower limit of quantitation (LLOQ) was 2 ng/mL. In plasma samples, the intra- and inter-day precision for the LLOQ of azithromycin was found to be <  6.4% and 5.8%, respectively. Further, the intra-day accuracy for LLOQ ranged from 96.6% to 103.9%, while the inter-day accuracy was 99.2%. The intra- and inter-day precision for the rest of the quality control concentrations was found to be <  5.0% and 4.5%, respectively. Further, the intra-day accuracy ranged from 100.2 to 107.2%, while the inter-day accuracy was 102.4% to 104.7%.

2.6 Safety Assessment

During the study, the doctor assessed the safety and tolerability of the treatment. Clinical symptoms, physical examination, ECG, vital signs, and clinical laboratory assessments (such as blood biochemistry, urinalysis) were assessed at the screening and end of each study. Clinical research physicians promptly documented all adverse events (AEs) and assessed the severity of each event associated with the drug being studied. The Common Terminology Criteria for Adverse Events version 5.0 was used to grade AEs.

2.7 Pharmacokinetic and Statistical Analysis

Noncompartmental analysis of the PK parameters was conducted by Phoenix WinNonLin 8.2 (Certara, Princeton, New Jersey, USA). The estimation of sample size was performed using PASS 11.0.7 software (NCSS, Kaysville, Utah, USA). SAS 9.4 Statistical Package (SAS, Cary, North Carolina, USA) was used for randomization. The within-subject standard deviations (SWR) of the main PK parameters (Cmax, area under the concentration–time curve from time 0 to the time of the last measurable plasma concentration [AUC0–t] and area under the concentration–time curve from time 0 extrapolated to infinity [AUC0–∞]) were calculated using the reference-scaled average bioequivalence method (RSABE) of the reference formulation. The average bioequivalence method (ABE) was applied to evaluate the bioequivalence when SWR of the reference formulation was < 0.294 (CVW% <  30%). The average bioequivalence method (RSABE) was applied to evaluate the bioequivalence when SWR of the reference formulation was ≥0.294 (CVW% ≥ 30%).

3 Results

3.1 Pharmacokinetics and Bioequivalence

A total of 164 subjects were screened and 72 healthy Chinese subjects were enrolled in the trial and were randomized to treatment (Fig. 1). There were 36 subjects in the fasting state and 36 subjects in the fed state. Baseline demography and characteristics of the healthy Chinese subjects are listed in Table 1.

Fig. 1
figure 1

Subject disposition

Full size image
Table 1 Baseline demographic characteristics of subjects
Full size table

In the fasting state, one subject (K007, T-R-R) was withdrawn after the 24-h blood sample collection in the third period because of personal reasons. As the concentration of the first blood sample (0.5 h) reached Cmax in the first period, one subject (K020, T-R-R) was excluded from the bioequivalence set (BES) in the first period. The AUC%Extrap of two subjects (K008, R-T-R; K016, R-T-R) in the second period and two subjects (K007, T-R-R; K015, T-R-R) in the third period were >20%, so the AUC0–∞ of these subjects in the relating periods were not included in the bioequivalence analysis. Therefore, a total of 36 subjects were included in the PK parameter sets (PKPS) and the BES in the fasting condition.

In the fed state, one subject (C024, R-T-R) was withdrawn because of personal reasons and one subject (C027, R-T-T) was withdrawn because of receiving a COVID-19 vaccine injection in the washout stage after the 120-h blood sample collection in the second period. The AUC%Extrap of two subjects (C025, T-R-R; C031, R-R-T) in the first period, three subjects (C003, R-R-T; C025, T-R-R; C031, R-R-T) in the second period and two subjects (C011, T-R-R; C025, T-R-R) in the third period were > 20%, so the AUC0–∞ of these subjects were excluded from the BES in the relating periods. A total of 36 subjects were included in the PKPS and the BES in the fed condition.

The plasma concentration–time curves of azithromycin, following a single-dose oral administration in both fasting and fed conditions are presented in Fig. 2. The PK parameters are summarized in Table 2. After a single oral dose of a 250-mg test or reference azithromycin tablet, the mean values of time to maximum concentration (Tmax), AUC0–t, AUC0–∞, t½, apparent volume of distribution (Vd) and total body clearance (CL) were similar for both treatments in fasting and fed states. Median Tmax was about 2.5 h and the mean value was about 50 h. In the fasting state, the within-subject variability values of Cmax were 52.5% for test and 38.8% for reference. The within-subject variability value of AUC0–t was 32.4% for test and 31.0% for reference. In the fed state, the within-subject variability values of Cmax were 49.0% for test and 42.2% for reference. The within-subject variability values of AUC0–t were 30.9% for test and 23.5% for reference.

Fig. 2
figure 2

A Mean plasma concentration–time curves of azithromycin after administration of the test (T) and reference (R) formulations to the healthy subjects in a fasting state. B Mean plasma concentration–time curves of azithromycin after administration of T and R formulations to the healthy subjects in a fed state. The error bars represent the standard error

Full size image
Table 2 Main PK parameters of azithromycin after administration of the test or reference tablets to healthy subjects
Full size table

Bioequivalence evaluations of the T/R formulations are summarized in Table 3. In the fasting state, the bioequivalence of Cmax was evaluated by the RSABE approach (SWR > 0.294) and the bioequivalence of AUC0–t and AUC0–∞ were evaluated by the ABE approach (SWR <  0.294). The geometric mean ratio (GMR) of T/R for Cmax was 106.49%, and the 95% upper confidence bound was < 0. GMRs of AUC0–t and AUC0–∞ were 103.34% and 101.28%, and the 90% confidence intervals (CIs) of the T/R were 95.90–111.35% and 94.85–108.15%, respectively. In fed state, the bioequivalence of Cmax was evaluated by the RSABE approach (SWR >0.294), while the bioequivalence of AUC0–t and AUC0–∞ were evaluated by the ABE method (SWR < 0.294). The GMR for Cmax was 99.80%, while the 95% upper confidence bound value was < 0. The GMRs of AUC0–t and AUC0–∞ were 97.07% and 98.15%, and the 90% CIs of T/R were 90.02–104.68% and 90.66–106.25%, respectively.

Table 3 Bioequivalence evaluation of major pharmacokinetic parameters
Full size table

3.2 Safety Analysis

In both the fasting and fed states, azithromycin had good tolerance. Twenty-seven AEs occurred in 17 (47.22%) healthy subjects, of which 13 (48.15%) were drug related in the fasting state. After taking the T formulation, eight AEs occurred in six healthy subjects (16.7%, 6/36). After taking the R formulation, 19 AEs occurred in 12 healthy subjects (33.3%, 12/36). Twenty-seven AEs occurred in 17 (47.22%) healthy subjects, of which 12 (44.44%) were drug related in the fed state. After taking the T formulation, 12 AEs occurred in 10 healthy subjects (28.6%, 10/35). After taking the R formulation, 15 AEs occurred in 10 healthy subjects (27.8%, 10/36). No serious adverse events (SAEs) or AEs leading to withdrawal occurred in either the fasting or fed state.

In this trial, several drug-related AEs occurred, including decreased potassium levels (4 cases), increased presence of red blood cells in urine (4 cases), and diarrhea (3 cases), which were consistent with the adverse reactions reported in the manual. Prompt and appropriate actions were taken to address all AEs.

4 Discussion

This was a single-dose, reference-replicated, 3-period crossover trial comparing the bioequivalence of a test azithromycin tablet with a reference tablet in healthy Chinese subjects under fasting and fed conditions. The azithromycin tablet was well tolerated, and there were no deaths or SAEs in any groups.

There have been studies on the pharmacokinetics and bioequivalence of generic azithromycin formulations, such as tablets [10,11,12,13], granules [14], capsules [15, 16] and eye drops [17]. The median Tmax of the test drug was 2.4960 (0.496–4.996) h and 2.4960 (0.996–4.996) h for the reference drug in the fasting state, which was similar in the fed state. These values were within the Tmax value range of azithromycin found in the previous literature [10, 11, 13] of 1.00–3.00 h. The t½ values for the two formulations were consistent with those reported in Indonesian [13] and Jordanian subjects [16] (about 50 h). However, t½ values of Indian subjects [11] were shorter (about 35 h) compared with Chinese subjects, influenced by race differences. The Cmax and AUC values could not be directly compared with those reported in previous literature due to variations in the dosage, with 500 mg used in those studies.

This study was conducted under both fasted and fed conditions after a single oral dose of azithromycin tablets. It appears that, for both test and reference tablets, the PK parameters, such as Tmax, AUC0–t, AUC0–∞, t½, Vd and CL, were similar in both fasting and fed states. For Cmax, the level was lower in the fasting state compared with the fed state, with no statistically significant differences. But in the previous literature [10], for both test and reference tablets, administration in the fed state caused a 21–29% decrease in azithromycin AUC0-72 in healthy Chinese subjects. The reason for the decrease could be that the high-fat meal might have delayed gastric emptying, resulting in extended gastric residence and degradation of azithromycin. A future study is warranted to confirm the food effect in azithromycin tablets, which may also be helpful for understanding and improving the clinical implications of azithromycin tablet dosing.

In this study, the within-subject variability values of Cmax and AUC were > 30% (as shown in Table 2), and azithromycin is regarded as a highly variable drug after oral administration according to international consensus. Our findings achieved comparable results with those reported in previous bioequivalence studies of a single-dose azithromycin tablet [13] or capsule [15, 16] in Asian populations, such as Indonesian, Jordanian and Indian subjects, with the within-subject variability values of Cmax and AUC ranging from 31% to 49%. The current study included only Chinese subjects, which might affect the generalizability of the study to other populations. This study employed the RSABE approach, using a partial replication design to evaluate the bioequivalence of two different formulations of azithromycin tablets. The RSABE approach is specifically designed for highly variable drugs and adjusts the bioequivalence limits by scaling them to the within-subject variability of the reference product used in the study. Additionally, it imposes a limit of 0.8–1.25 on the geometric mean ratio between the test and reference formulations [18]. In this study, SWR for Cmax in both fasting and fed conditions was > 0.294, while SWR for AUC0–t and AUC0–∞ in both fasting and fed conditions was < 0.294. The bioequivalence of Cmax was evaluated by the RSABE approach, and the bioequivalence of AUC0–t and AUC0–∞ was evaluated by the ABE approach.

The AUC%Extrap of four cases in the fasting condition and seven cases in the fed condition were > 20%, so the AUC0–∞ of these subjects in the relating periods were not included in the bioequivalence analysis. We used sensitivity analysis to evaluate the bioequivalence of the two formulations by including the above data of the AUC%Extrap. In the fasting state, SWR for AUC0–∞ was 0.1801, GMR was 103.18%, and the 90% CI of T/R was 96.22–110.64%. In the fed state, SWR for AUC0–∞ was 0.1439, GMR was 96.66%, and the 90% CI of T/R was 89.91–103.91%. As a result, the AUC0–∞ of these subjects in the relating periods were not included in the bioequivalence analysis.

The safety assessment indicated that all AEs were mild and were promptly and appropriately addressed, and followed up until the event returned to stable or normal. The AEs in our study were increased triglycerides, uric acid, urinary RBC, decreased potassium, and diarrhea, which had already been reported previously [10]. There was no significant difference in the incidence of AEs between the test drug and the reference drug when administered under both fasting and fed conditions. In short, in healthy Chinese subjects, the two formulations of azithromycin were well tolerated regardless of fast or fed conditions.

5 Conclusion

The test azithromycin tablets produced by Shijiazhuang No.4 Pharmaceutical Co., Ltd were equivalent to Zithromax® produced by Pfizer Laboratories Div Pfizer Inc (USA). The results proved the pharmacokinetics of the generic azithromycin tablet to be similar to those of Zithromax® after oral administration of a single 250-mg dose in healthy Chinese subjects. The trial demonstrated that the generic azithromycin tablets and Zithromax® were bioequivalent under both fasting and fed conditions.