Ciprofloxacin is one of the most successful and widely used broad-spectrum antibacterial compounds of the fluoroquinolones class.¹ It is prescribed for the treatment of bacterial infections, particularly in the urinary and respiratory tracts.^1,2 Oral ciprofloxacin is available as immediate-release tablets (twice-daily administration) and as an extended-release formulation administered once a day.

An extended-release formulation provides the advantage to improve patient compliance maintaining a constant plasma drug concentration over a prolonged period^3,4; thus, decreasing the risk of treatment failure.⁵ Furthermore, modified-release formulations can enhance the therapeutical index of antibiotics.

Ciprofloxacin is a concentration-dependent antibiotic that increases bacteria-killing when increased levels of drugs above the minimum inhibitory concentration (MIC) are achieved. Hence, maximum peak concentration (C_max) and area under the concentration curve (AUC) determine the clinical outcomes of ciprofloxacin. It is demonstrated that the extended-release profile of ciprofloxacin provides higher C_max/MIC and AUC/MIC ratios than the immediate-release formulation⁶, this correlates with a significantly higher antibacterial activity that helps to prevent the development of antimicrobial resistance.⁴

Therefore, extended-release formulations have a crucial primary role in the antibiotic delivery field. However, non-equivalent and substandard ciprofloxacin formulations have an insufficient drug release.^7,8 That leads to subtherapeutic drug levels that increase the risk of treatment failure and antimicrobial resistance development.^2,8,9 Hence, bioequivalence studies of the commercially available oral ciprofloxacin formulations should be monitored.⁹

To the best of our knowledge, there are no published bioequivalence studies for extended-release ciprofloxacin formulations in Mexico. Based on this, the research question proposed is: Are the generic extended-release ciprofloxacin products marketed in Mexico bioequivalent to the reference formulation?

To answer this question, in our study we aimed to determine the bioequivalence of two extended-release ciprofloxacin tablet formulations of 1,000 mg dose manufactured and commercialized in Mexico. We hypothesize that the bioavailability of generic extended-release ciprofloxacin products marketed in Mexico is comparable to that reference drug, and, therefore, it is considered bioequivalent.

Subjects

Seventeen male and nine female healthy Mexican volunteers participated in the study, demographic data of the subjects can be seen in Table 1. The volunteers were included in the study protocol after normal findings from pre-study medical history, physical examination, chest X-ray, ECG, and laboratory tests (including hematological and biochemical parameters, hepatitis, and human immunodeficiency virus serological tests, tests for drug abuse, urinalysis, and pregnancy test).

Exclusion criteria included any medical history that may affect the pharmacokinetics and response of ciprofloxacin (kidney failure, liver failure, hypertension, etc.), co-administration of any other medication, previous history of allergy to fluoroquinolones, participation in clinical trials within two months before the study period, female subjects with sexual activity and risk of pregnancy who are using hormonal contraceptive agents (oral, injectable or implantable), and pregnancy.

Sample Size Calculation

The number of subjects was determined using the intrasubject coefficient of variation (CV%) of previous bioequivalence studies¹⁰, where for C_max, a variation of 25.56% was identified. With this CV% under the regulatory requirements for sample size calculation¹¹ (α = 5% and a power of 80%), the calculated sample size resulted in twenty-four subjects. Two additional subjects were recruited, anticipating some volunteers may retreat; therefore, the final “n” of subjects was twenty-six.

Study Design

The study was randomized, single-blind, 2-period, 2-treatment, crossover, single-dose-per-period, 2-sequence study in healthy subjects under fasting conditions to compare the bioavailability of ciprofloxacin in two extended-release formulations: Cipro XR^® (Bayer Mexico, Mexico) as the reference formulation and Ciproflox DM^® (Senosiain Laboratories, Mexico) as the test formulation.

Subjects were admitted to the study center the day before the administration of the study drug and received a light dinner balanced among protein, carbohydrates, and fat, equivalent to 1,670 Kcal. The following day, volunteers were randomly assigned to one of two groups of administration. The subjects of one group received the first administration with the test formulation; simultaneously, the subjects of the other group received the reference formulation. After a seven-day washout, a second administration was given; volunteers were switched; thus, the first group received the reference formulation, while the second group received the test formulation. After dosing, volunteers had breakfast, lunch, and dinner at 4, 8, and 14 h, respectively. The total daily calorie intake diet supplied was 1,300 Kcal. Safety was assessed by monitoring vital signs during both study periods.

Sample Collection

Seventeen blood samples (6 mL) were obtained by venipuncture from each volunteer. The first sample was collected immediately before drug administration and then during a 24-hour period after medication: 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 8.0, 10.0, 12.0, and 24.0 h. Blood samples were collected in Vacutainer™ tubes with EDTA as an anticoagulant; subsequently, the plasma was separated by centrifugation, and it was transferred in cryotubes. Samples were kept frozen at -80 °C until analysis.

Analytical Procedures

Ciprofloxacin plasma concentrations were determined using a specific High-Pressure Liquid Chromatography (HPLC) method, previously developed and validated.¹⁰ Briefly, 100 μL of plasma was mixed with 10 μL of an ofloxacin solution (1 μg/mL, internal standard) and 0.5 mL of dichloromethane. The mixture was vortexed at high speed for 1 min and centrifuged at 12,500 rpm for 5 min. The aqueous phase was discarded, and the organic phase was evaporated at 30°C under a gentle nitrogen stream. The residue was reconstituted with 200 μL of deionized water. Aliquots of 40 μL were injected into the chromatographic system Waters Alliance 2695 with UV-detector Waters 2487 (Waters Corp., Milford, MA, USA). Analyses were carried out using a C₁₈ column Zorbax Eclipse, 4.6 x 75 mm, particle size 3.5 µm (Agilent, Santa Clara, CA, USA). The column was eluted with a mobile phase composed of a mixture of phosphate buffer (sodium phosphate monobasic 10 mM, adjusted to pH 3 with orthophosphoric acid) and acetonitrile in a ratio of 13:87 (v/v) containing diethylamine 0.01%. The flow rate was 1.4 mL/min, and UV detection was carried out at 295 nm. The analytical method was validated according to Norma Oficial Mexicana.¹¹ Under the described conditions, retention times were 2.73 min and 2.39 min for ciprofloxacin and the internal standard, respectively. The method was linear R²=0.99 within the range of 0.1 to 6 µg/mL of ciprofloxacin plasma concentration.

Pharmacokinetic Analysis

Pharmacokinetic parameters were obtained employing a standard non-compartmental analysis (NCA). This analysis is based on the individual observations of the pharmacokinetic parameters in each subject. Next, the observed values are averaged and analyzed. Therefore, the mean pharmacokinetic parameters in the NCA are built with the averages of the parameters from the individual observations and may differ from the ones observed in each subject. Maximum plasma concentration (C_max) and time to reach C_max (T_max) were obtained from plasma concentrations versus time profiles. The area under the curve at the last sampling time (AUC_0-t) was calculated using the trapezoidal summation method. The elimination rate (Ke) was calculated by log-linear regression of the final phase of elimination; the elimination half-life (T_½) was estimated by dividing 0.693/Ke. The area under the curve extrapolated to infinity (AUC_0-inf) was calculated by adding the quotient of the last measured concentration between Ke.

Bioequivalence Statistical Analysis

An analysis of variance model including sequence, period, and formulation as fixed effects based on linear mixed effect models, considering the type III sum of squares was initially performed. Then, bioequivalence of the test and reference drug products was determined based on the comparison of log-transformed ratios of C_max, AUC_0-t, and AUC_0-inf. According to the requirements set by international regulations^11-13, drug products were bioequivalent if confidence intervals of such variables were within the range of 80-125% with a confidence level of 90% and statistical power p<0.05 of Schuirmann bilateral test. All statistical calculations were performed using the Phoenix/WinNonlin^® software V 8.1 (Certara, LP, Princeton, New Jersey).

Ethics Approval

This study was conducted according to the ethical standards of the Mexican General Health Law and to the ethical principles outlined in the Declaration of Helsinki. The Institutional Review Board and the Mexican Regulatory Agency (Comisión Federal para la Protección contra Riesgos Sanitarios) approved the protocols. Institutional Ethics Committee approved the protocol and assigned the registry number: IICO-CIP-10/DIX-EB-10-022.

Informed Consent

All volunteers signed the written informed consent before inclusion in the study protocol.

Pharmacokinetics

Mean plasma ciprofloxacin concentration determined by HPLC following administration of both formulations is shown in Graphic 1. The summary of pharmacokinetic parameters (C_max, T_max, AUC_0-t, AUC_0-inf and T_½) is in Table 2. All the volunteers tolerated well ciprofloxacin formulations, and no side or adverse effects were observed.

Bioequivalence Statistical Analysis

The 90% confidence intervals limit ranged from 85.69-114.31% for C_max, from 85.16-115.84% in the case of AUC_0-t and 85.39-114.61% for AUC_0-inf. The international accepted bioequivalence limits are 80-125%. Schuirmann test confirmed the high probability that such observation C_max, AUC_0-t, and AUC_0-inf ratios were within 80-125% (p<0.05) (Table 3). These results confirm that both formulations are bioequivalent.

Generic drugs are predominant in current medical practice and as a result of the current economics of modern health care systems.¹⁴ The assessment of bioequivalence of generic drugs is based on the fundamental assumption that two drugs are equivalent when the rate and extent of absorption of the test drug do not show a significant difference from those of the reference drug when they are administered under similar experimental conditions.¹⁵ Thus, bioequivalence studies ensure the efficacy and safety of generic drugs.^7,15

Demonstrating bioequivalence is essential for all drugs, especially for antimicrobial agents. The use of non-equivalent generic antibiotics may lead to clinical failure and, of greater concern, to the selection of antimicrobial resistance, a problem with serious public health, environmental, and economic consequences.^16,17 Therefore, a robust regulatory mandate is required to reduce the entry of such drugs in the market. There is evidence of generic substandard or non-equivalent antibiotics in the market, including ciprofloxacin^8,9,18,19, correlated to a significant development of antimicrobial resistance.

Bioequivalence studies for extended-release ciprofloxacin formulations manufactured in Mexico are scarce; consequently, we decided to analyze the bioequivalence of two currently commercialized extended-release formulations. Pharmacokinetic parameters (C_max, AUC_0-t, and AUC_0-inf) were statistically analyzed for bioequivalence demonstration. The confidence intervals at 90% for the ratio of logarithmic transformations of these pharmacokinetic parameters comply with the requirements set by international regulations of 80-125% (Table 3). Furthermore, the results of the Schuirmann test verified the high probability that these values were within the acceptance criteria (p<0.05). It was concluded that the tested Ciproflox DM^® (Senosiain Laboratories, Mexico) extended-release formulation is bioequivalent to the reference formulation Cipro XR^® (Bayer Mexico, Mexico); thus, its correct use does not lead to therapeutic failure and antimicrobial resistance development.