Substandard drugs are medicinal products that fail to meet internationally accepted quality and/or bioavailability but have been approved in some countries where they are commercially available.¹ Substandard drugs represent a worldwide public health problem; data from the World Health Organization (WHO) estimates that 1 in 10 medical products in low and middle-income countries are substandard or counterfeit; antibiotics are the most commonly reported substandard medical products.²

Substandard antibiotics are a serious problem since they may lead to antimicrobial resistance development due to several reasons: 1) they may contain the incorrect amount of active ingredients; 2) the active ingredient does not fulfill the pharmaceutical quality minimum requirements, and/or 3) the formulation is poorly designed or manufactured.^3,4 If the active ingredient is not released properly, pharmacokinetic parameters may be altered leading to therapeutic failure. Moreover, insufficient antibiotic concentrations in the body will only kill or inhibit the growth of some bacteria, not all the bacteria producing an infection, leading to antibiotic resistance by natural selection.

Ciprofloxacin is a second-generation fluoroquinolone antibiotic; one of the most successful and widely used compounds of this class. It is active against a broad spectrum of bacteria and is commonly prescribed for the treatment of respiratory and urinary tracts, as well as for sexually transmitted infections.⁵

Due to its efficacy, many non-innovator formulations have been commercialized after patent expiration. Nevertheless, it has been demonstrated that the commercialization and use of substandard oral ciprofloxacin formulations in developing countries have contributed to an increased risk of treatment failure and antimicrobial resistance development.⁶ Thus, an important step to prevent the spreading of substandard drugs is to ensure adequate quality from the drug manufacturer and quality control tests for both branded and generic drugs.⁴ Therefore, quality control and bioequivalence studies of the commercially available ciprofloxacin formulations must be performed.⁶

Several bioequivalence studies for oral ciprofloxacin immediate-release tablets (250 mg^7,8, 500 mg^9-15, 750 mg^16,17), and extended-release ciprofloxacin tablets (1,000 mg)^18,19 have been published. Nevertheless, there are no published reports on in vivo bioequivalence for the 250 mg ciprofloxacin Mexican formulations. Consequently, the aim of this study was to determine the bioequivalence of two immediate-release tablet formulations containing 250 mg of ciprofloxacin manufactured and commercialized in Mexico in healthy volunteers.

Volunteers

Twenty-four young men healthy volunteers participated in the study. They were 21.54 ± 1.61 years old (mean ± SD) and weight of 68.29 ± 9.79 kg. The volunteers were included in the study after a 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 and urinalysis test).

Exclusion criteria included any medical record that may affect the pharmacokinetics or the pharmacodynamic response to the active substance (kidney failure, liver failure, hypertension, co-administration of any other medication), previous history of allergy to fluoroquinolones and participation in clinical trials within two months before the study period. Written informed consent was signed by all volunteers prior to inclusion in the study.

Study design and treatment assignment

The study was performed following a cross-over 2 x 2, randomized design, comparing two oral formulations containing 250 mg of ciprofloxacin: Lemyflox^® by Lemery Laboratories (Mexico City) as the test formulation and Ciproxina^® (Bayer, Mexico City) as reference formulation.

The subjects were randomly assigned to one of the two groups or administration sequences. During the first administration, the subjects of one group received the test formulation while the subjects of the other received the reference formulation. After a seven-day washout, a second administration was delivered. Subjects were switched so that the first group received reference formulation, while the second group received test formulation. Tablets were administered with 250 mL of water after an overnight fast (10 h). Volunteers had breakfast, lunch, and dinner at 4, 8 and 14 h, respectively, after dosing. The total daily calorie intake diet supplied was 1300 kcal. All adverse events were documented.

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 protocols were approved by the Institutional Review Board and the Mexican Regulatory Agency (Comisión Federal para la Protección contra Riesgos Sanitarios). Institutional Ethics Committee approved the protocol and assigned the registry number CEID-CPX-1999-1.

Sample collection

After drug administration, 12 venous blood samples of 10 mL were obtained from each volunteer by venipuncture. The first sample was drawn immediately before drug administration and then at 0.25; 0.5; 0.75; 1.0; 1.5; 2.0; 3.0; 4.0; 6.0; 8.0 and 12.0 h after medication. Blood samples were collected in vacutainer tubes with EDTA as an anticoagulant; plasma was separated by centrifugation and it was transferred in three aliquots in 2 mL cryotubes identified with the project code; volunteer number; sample number and study period. Samples were frozen at -80 °C until analysis.

Analytical procedures

Ciprofloxacin plasma concentrations were determined by High-Pressure Liquid Chromatography (HPLC) procedure described by Vella et al.²⁰, with some modifications. The chromatographic system consisted of a Separation Module Waters No. 2695 and Detector UV-VIS Waters No. 2487, the recording of the data was carried out with an empower data acquisition system (Waters Assoc., Milford, MA, USA).

For analysis, 100 μL of plasma was placed in 2 mL Eppendorf tubes with 10 μL of internal standard (EI) ofloxacin 1 μg/mL and 0.5 mL of dichloromethane, this mix was vortexed for 1 min and centrifuged at 12500 rpm for 5 minutes. 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 and 40 μL aliquots were injected into the chromatographic system.

The mobile phase was 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). Diethylamine 0.01% was added to this mixture. Separations were carried out using a Halo C18 reverse-phase (4.6 x 100 mm, the particle size of 2.7 μm) column. The flow rate was 1.4 mL/min and the detector wavelength was 295 nm. The analytical method was validated according to the Mexican Official Norm²¹, based on selectivity, linearity, precision, accuracy, and stability.

Pharmacokinetic analysis

Pharmacokinetic parameters were obtained using a non-compartmental analysis. Maximum plasma concentration (C_max), time to reach C_max (T_max) were obtained directly from plasma concentrations versus time plots. The area under the curve at the last sampling time (AUC_0-t) was calculated by the trapezoidal rule. The elimination rate constant (Ke) was calculated by log-linear regression of the final phase of elimination, and the elimination half-life (T_½) was estimated as 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 divided by Ke.

Statistical analysis

In order to determine bioequivalence between the two products, logarithmic transformations of the ratios of C_max, AUC_0-t and AUC_0-inf were compared by analysis of variance (ANOVA) for cross-over designs with effects for sequence, period and formulation. Then, 90% confidence intervals were calculated and Schuirmann bilateral test was performed. These calculations were performed in the ANOVA module using the Winnonlin Professional V 8.1 software (2019, Pharsight, Palo Alto CA, USA). Bioequivalence was determined if the ratios of the logarithmic transformation of pharmacokinetic parameters (C_max, AUC_0-t and AUC_0-inf) exhibited 90% confidence intervals values within the interval 80 -125%.

Analytical method

The described analytical method was fully validated complying with the Mexican Official Norm.²¹ Under the described conditions, retention times were 2.73 min and 2.39 min for ciprofloxacin and the internal standard, respectively (Graphic 1, B). No interferences were observed (Graphic 1, D). The relationship between concentration and peak area ratio was found to be linear (R = 0.99) within the range of 0.1 to 6.0 µg/mL of ciprofloxacin plasma concentration.

Pharmacokinetics

Mean plasma ciprofloxacin concentration following administration of the two assayed tablet formulations is shown in Graphic 2. Relevant pharmacokinetic parameters are shown in Table 1. Plasma concentration against time profiles was similar for both formulations. Both ciprofloxacin (reference and test) were well tolerated by all the volunteers. No adverse effects were reported.

Bioequivalence analysis

In order to determine bioequivalence, C_max, AUC_0-t and AUC_0-inf were compared by ANOVA for a crossover design, followed by the 90% confidence intervals and Schuirmann bilateral test. The 90% confidence intervals limits ranged from 82.92 to 100.88% for C_max, from 93.05 to 109.15% in the case of AUC_0-t and from 97.76 to 114.99% for AUC_0-inf. The accepted bioequivalence limits are 80-125%. Shuirmann test showed that the probability that the bioequivalence limits are exceeded is below 0.05 (Table 2). Hence, both formulations are bioequivalent.

In this study, we report the bioequivalence analysis of a test formulation Lemyflox^® (Lemery Laboratories, Mexico) and the reference formulation Ciproxina^® (Bayer, Mexico). The main objective of bioequivalence studies is to assure that the efficacy and safety of a generic or test formulation are similar to that of the reference drug product. A generic drug product is assumed to be bioequivalent if the average bioavailability of the generic formulation is within ± 20% of that of the innovator formulation when both formulations are administered at the same dose in healthy volunteers.

It is important to note that a generic formulation should only be marketed if its quality and bioavailability are equivalent to those of the innovator drug.⁶ Unfortunately, there is evidence of non-innovator substandard drugs exhibiting poor-quality and low bioavailability which are presently being marketed in some countries.^4,22-24 In the case of antibiotics, these substandard products contribute to the extension of antibacterial resistance. Since the information on the bioequivalence of ciprofloxacin formulations manufactured in Mexico is scarce, we decided to examine the bioequivalence of two currently commercialized formulations.

The statistical evaluation of bioequivalence was carried out comparing the C_max, AUC_0-t and AUC_0-inf between the evaluated formulations. Table 2 shows that 90% confidence intervals for the log-transformed T/R ratio of the bioavailability parameters are within the limits set by the regulations 80-125%. In addition, the results of the Schuirmann test confirmed the high probability that the values were within the acceptance criteria (p <0.05).

According to our results, the test formulation is bioequivalent with the reference formulation and no significant differences should be expected regarding its efficacy and/or therapeutic safety.