The study was performed as a randomized, open-label, two-formulation, two-sequence, two-period, single-dose crossover bioequivalence study with a wash-out period of 7 days, and conducted at the Guangdong Province Traditional Chinese Medical Hospital, Guangzhou, China. The study was approved by the independent Ethics Committee of the hospital (registration number: A2017-002-1-02) on the basis of the principles of some international guidelines including the CIOMS (Council for International Organizations of Medical Sciences) International Ethical Guidelines for Biomedical Research Involving Human Subjects, the Good Clinical Practice, the World Medical Association’s Declaration of Helsinki, and WHO Operational Guidelines for Ethics Committees for Biomedical Research Review. The signed informed consent was acquired from every enrolled volunteer before formal screening.
2.2 Test and Reference Products
The ciprofloxacin hydrochloride tablet (Kuinuoxian®, drug specification: 250 mg; purity: 98.1%; batch number: 01170801; expiry date: July, 2020) used as the test product w manufactured by Baiyunshan Pharmaceutical General Factory (Guangzhou, China), which attached to Baiyunshan Pharmaceutical Holdings Limited (GPHL), Guangzhou, China.
We chose the ciprofloxacin hydrochloride tablet (Ciprobay®, drug specification: 250 mg; purity: 98.4%; batch number: BXN14RC; expiry date: August, 2020) manufactured by Bayer Vital GmbH, Leverkusen, Germany, as the reference product, in accordance with the Procedures for Selection and Determination of Reference Product for Generic Chemicals, which was published by the National Medical Products Administration (NMPA), Beijing, China. The reference product was in the recommended list of NMPA.
2.3 Inclusion and Exclusion Criteria
Subjects who met all the following requirements could be included:
- Subjects aged at least 18 years old, at most 45 years old, with appropriate sex ratio
- Male subjects weighed ≥ 50 kilogram and female subjects weighed ≥ 45 kilogram, with a body mass index (BMI) between 19-26 kg/m2
- All medical examination results showed no abnormality, or abnormality without clinical significance
- Female subjects with negative pregnancy tests and male subjects had no fertility planning from two weeks before administration to the next six months, they and their partners used effective contraception and had no egg or sperm donation programs
- Subjects were fully aware of the purpose, nature, methods, and possible adverse events of the study, volunteered to serve as subjects, and signed informed consent before the study began
- Subjects were able to communicate well with the researchers, and understood and complied with the strict requirements of the study.
Subjects who met any of the following criteria would be excluded:
- Allergic physique, or allergy to the food and drug, especially for penicillin, clarithromycin, cephalosporin
- Surgical or medical history, illness with clinical significance, which would endanger the safety of subjects or have an impact on study results
- Difficulty for swallowing, or gastrointestinal disease, which affected drug absorption
- Positive test results for hepatitis B surface antigen (HBsAg), anti-HIV antibodies, hepatitis C antibody, or treponema pallidum antibody
- Gestation or lactation for the female subjects
- Diarrhea after drinking milk in fed study
- Positive urinary drug screen for morphine, methamphetamine, ketamine, dimethylene dioxyamphetamine, tetrahydrocannabinolic acid, cocaine
- Habitual use of Chinese herbs or functional vitamins
- Participation in another clinical trials within three months prior to the screening
- Blood donation or blood loss exceeded 400 mL within three months prior to the screening, or intention to donate blood during the clinical trial within three months after the end of the clinical trial
- Use of any medication or health products within three months prior to the screening
- Smoking > 5 cigarettes per day within three months prior to the screening, or disagreement to avoid using any tobacco products during the trial
- Alcohol drinking averaged > 14 units per week or > 2 units per day within 3 months prior to the screening, 1 unit was equivalent to 17.7 mL ethanol, or disagreement to avoid using any ethanol products during the trial
- Values for alcohol breath test results were > 0.0mg /100 mL
- A long history of consuming excess caffeinated beverages, which were more than eight drinks a day, 1 drink was 250 mL, or intake of food or drinks rich in methyl xanthine purines or caffeine (coffee, strong tea, cola, chocolate, etc) within 48 hours prior to the administration, and failure to stop the intake during the clinical trial
- Intake of special diet, such as pitaya, mango, pomelo, the food and drinks prepared from these fruits, or strenuous exercise within 48 hours prior to the administration, or other factors that could affect the absorption, distribution, metabolism, or excretion of ciprofloxacin
- Intolerableness to venipuncture blood collection or history of dizziness
- Special requirements for diet, and failure to follow the uniform diet
- Occurrence of acute illness during the screening period, or before the administration
- Any other factors the researchers considered inappropriate for volunteers to participate in the study.
After signing the informed consent forms, all enrolled volunteers would undergo a series of inquiries and examinations, such as demographic data, medical history, allergy, the history of smoking and drinking, physical examination, weight and height measurement, vital signs, 12-lead electrocardiogram (ECG), chest radiography, laboratory analysis of blood routine, urine routine and blood biochemistry, serum pregnancy test (female only) and disease markers for syphilis, HIV (human immunodeficiency virus), and hepatitis B and C viruses. The screening tests were performed within 7 days prior to the first period of administration.
The volunteers who met all the inclusion criteria and did not meet any of the exclusion criteria during screening period were notified to enroll in the Phase Ⅰ Clinical Research Center of the hospital for a uniform lunch and dinner one day before the trial. In the fasting study, a total of 107 volunteers were recruited, 93 of whom were excluded. For the fed study, 26 of 90 volunteers were eligible, among whom 24 volunteers were assigned to receive a randomized order and the remaining 2 volunteers were excluded because the number of subjects had exceeded the predetermined number (24).
The following assessments were performed again before administration: health survey (recent medical history, medication history, smoking and drinking), vital sign measurement, female pregnancy test, alcohol breath test, drug screening in urine. In accordance with these assessments, the researchers checked the inclusion/exclusion criteria again and made the final decision, and all enrolled subjects must guarantee to fast for at least 10 hours before administration.
Under the terms of the informed consent, the subject had the rights to withdraw from the study and revoke the informed consent without any reason at any time, also without any impact on their rights, interests, and entries into other clinical studies. The researchers would let the subjects withdraw from the study if the subjects were unfit to continue the study in the following cases:
- Necessity to stop the study from the point of view of medical ethics
- Occurrence of serious adverse events (SAEs)
- Poor compliance involved failure to accept the examination and administration according to regulations, use of drug or food affecting safety assessment and pharmacokinetic analyses
- Other behaviors that could affect the test results
2.5 Study Drug Administration
Subjects were randomly assigned to receive a single oral dose of the test (T) or reference (R) product of ciprofloxacin hydrochloride 250 mg, in light of a balanced randomization schedule generated by SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and the study drug was administrated in the order of T-R or R-T during each period.
In the fed study, 24 subjects were randomly assigned to two groups at a 1:1 ratio. After an overnight fast of ≥10 hours. Ciprofloxacin hydrochloride tablets were administered with 240 mL of water after standardized high-fat and high-calorie (800-1000 kilocalorie) meals, the meals were eaten up within 30 minutes, and chewing drugs was forbidden during administration. Subjects were granted a free access to water until 1 hour before study drug dosing and 1 hour after administration, and fasted until 4 hours after administration. Standard light meals were provided 4 and 10 hours after administration of ciprofloxacin hydrochloride tablets. Two dosing periods were separated by a 7-day washout period, and two groups were cross-administered on the seventh day. For the fasting study, the only difference from the fed study was that subjects fasted until 4 hours after administration.
Vital signs (pulse, blood pressure, body temperature) were assessed prior to dosing and at 2 (± 0.5) h, 4 (± 0.5) h, 12 (± 0.5) h, 24 (± 0.5) h after administration. The assessments should delay until sampling was completed, if there was a scheduling conflict between the assessments and sampling. Adverse events (AEs), concomitant medication usage and non-drug treatments were tracked through the whole study. Subjects were permitted to leave the Phase Ⅰ Clinical Research Center of the hospital until all blood sample collections were completed. Snacks, areca nuts, alcoholic drinks and smoking were forbidden from the enrollment to the final follow-up visit.
2.6 Sample Collection
Subjects were admitted to the Phase Ⅰ Clinical Research Center of the hospital one day prior to each period. After an overnight fast (≥10 hours), the baseline blood samples were collected within one hour before study drugs dosing. Collection of postdose blood samples were within two minutes of the specified times. Five hours after administration of study drug, the time deviation could be extended to three minutes. Venous blood samples of upper extremities were collected into labeled ethylene diamine tetraacetic acid (EDTA) tubes for pharmacokinetic analyses before administration and at 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12.0, 24.0 and 36.0 h after administration in each period, and there were 16 time points in total.
Within 1 hour after collection, blood samples of 4 mL were centrifuged at 3000 rpm for 10 minutes at 4 °C to separate plasma in a precooled centrifuge. Each plasma sample was divided into two aliquots, then transferred to corresponding labeled tubes, and one of which was in reserve. The plasma samples were transferred to -60 °C within 1 hour after centrifugation. After packaging with dry ice by professional cold chain logistics company (Shengsheng Supply Chain Management s.a, Shanghai, China), the plasma samples were transferred to the analytical facility (MicroConstants., Inc, Beijing, China) for assay, and temperature monitoring and adequate dry ice were supplied during transportation.
2.7 Analytical Method and Validation
Ciprofloxacin plasma concentration was determined by a means of liquid chromatography-tandem mass spectrometry method. A XSelect HSS T3, 5.0 µm, 100 x 2.1 (Waters corporation. Milford, Massachusetts, U.S.A) mm column was used for separation at a flow-rate of 0.5 mL/min with column temperature of 30°C. The mobile phase consisted of water with 20 mM ammonium formate (Acros Organics, Belgium) and 0.1% formic acid (ROE, U.S.A): methanol (Honeywell, U.S.A) with 0.1% formic acid (65:35, vol/vol), all chemicals and reagents were HPLC (High Performance Liquid Chromatography) grade. Ciprofloxacin (batch number: 130451-201203, purity: 84.2%, expiry date: March 7, 2018) used as a reference substance was obtained from the National Institutes for Food and Drug Control, Beijing, China. Ciprofloxacin hydrochloride-D8 (batch number: 1519-039A3, purity: 99.5%, expiry date: October 11, 2019), as an internal standard (IS), was purchased from TLC Pharmaceutical standards Ltd, Shanghai, China. HPLC instrumentation (model number: 1100) was purchased from Agilent Technologies Inc (California, U.S.A). Mass spectrometric detection was performed in a multiple reaction monitoring (MRM) mode using electrospray ionization source, and the mass spectrometer was interfaced to a computer workstation running MassLynx v.4.1 (Waters, Milford, Massachusetts, U.S.A). The standard curve was found to be linear over the concentration range of 1.87 - 935 ng/mL, with lower limit of quantification (LLOQ) of 1.87 ng/mL, and the LLOQ of quality control (QC) was also 1.87 ng/mL. The inspection contents of analytical methodology validation included between-run precision and accuracy, within-run precision and accuracy, recovery rate, selectivity (specificity), matrix effects, dilution reliability, stepwise dilution reliability, residual effect of HPLC injection, analytical batch length, reproducibility for reinjection of 72 hours, placement stability of processed samples, freeze-thaw stability, stability of samples, matrix effects, hemolysis effects, recovery rate, accuracy and precision, reproducibility for reinjection of 72 hours.
After the end of the two period of sampling, the subjects underwent a series of safety assessments before hospital discharge, which included physical examination, vital signs, 12-lead ECG, chest radiography, laboratory analysis of blood routine, urine routine and blood biochemistry, serum pregnancy test. The subjects would be permitted to leave the Phase Ⅰ Clinical Research Center of the hospital, if their assessments were satisfactory.
The subjects were followed up 3-7 days after hospital discharge, and inquired whether there were any subsequent AEs, occurrence of which would be recorded, the follow-up would be continued until the disappearance of the AEs. If the subjects experienced AEs during the study, they would be followed up until the AEs stabilized, disappeared, or lost to follow-up.
2.9 Safety Assessments
Safety Set (SS) consisted of all subjects who had received the study drug at least once after enrollment and undergone safety assessments, the role of SS was to evaluate the safety of the study drug. The incidence, time, severity, and relationship to the study drug of AEs were recorded by nursing and observations of the medical staff, and results of laboratory and pathology throughout the study. Clinical parameters for the safety assessments were obtained from physical examination, vital signs, 12-lead ECG, and laboratory analysis of blood routine, urine routine and blood biochemistry. The researchers calculated the amount and frequency of abnormal clinical parameters in the study, then detailly listed the abnormal clinical parameters and corresponding clinical explanations, and concretely described the changes in vital signs.
2.10 Pharmacokinetic and Statistical Analyses
Chromatogram collection and chromatographic peak integral of ciprofloxacin and IS was performed by MassLynx v.4.1 (Waters, Milford, Massachusetts, U.S.A). Linear fitting of the peak area ratio of ciprofloxacin to IS and the concentration of the reference substance of ciprofloxacin was to obtain a standard curve equation (weight = 1/x2). The peak areas and concentrations obtained from MassLynx v.4.1 were imported into the Microsoft Office Excel (Microsoft Corporation, Washington, U.S.A) to be used to calculate the mean, deviation, standard deviation, and variable coefficient (CV). The data were stored on a computer hard drive interfaced to the mass spectrometer for subsequent pharmacokinetic analyses.
Phoenix WinNonlin version 6.4 (Pharsight Corporation, Mountain View, California) were used for calculation of pharmacokinetic parameters, which included Cmax, AUC0-t, AUC0-∞, Tmax, t1/2, and λz.
- AUC0-t = the area under the plasma concentration–time curve from time 0 to time t, it was calculated by linear trapezoidal method, where time t was the last timepoint to collect blood sample
- AUC0-∞ = the area under the plasma concentration-time curve from time 0 to infinity, it was calculated as AUC0-t + Ct/λz, where Ct was the last measurable drug concentration, and λz was apparent elimination rate constant derived from linear regression analysis of ln(concentration-time) curve during the elimination phase
- Tmax = the time to maximum concentration (Cmax), both Tmax and Cmax were actual measured values
- t1/2 = ln2/λz, terminal elimination half-life
2.11 Bioequivalence Analyses
Bioequivalence of the two ciprofloxacin hydrochloride tablets was determined by analysis of variance (ANOVA) after ln-transformation of the Cmax, AUC0-t, and AUC0-∞, which was performed using Statistical Analysis Software (SAS) version (SAS Institute Inc., USA) 9.4 with a non-compartment model. A test product was considered bioequivalent to a reference product if 90% CIs of the test/reference GMRs for ln-transformed pharmacokinetic parameters (Cmax, AUC0-t, and AUC0-∞) were within the acceptance range of 80.00% – 125.00%.