Zuluaga et al. BMC Res Notes (2015) 8:546 DOI 10.1186/s13104-015-1507-z Pharmacodynamics of nine generic products of amikacin compared with the innovator in the neutropenic mouse thigh infection model Zuluaga et al. Zuluaga et al. BMC Res Notes (2015) 8:546 DOI 10.1186/s13104-015-1507-z RESEARCH ARTICLE Pharmacodynamics of nine generic products of amikacin compared with the innovator in the neutropenic mouse thigh infection model Andres F. Zuluaga1,2, Carlos A. Rodriguez1,2, Maria Agudelo1,2,4 and Omar Vesga1,2,3,4* Abstract  Background:  Previously, we validated the mouse thigh infection model to test the therapeutic equivalence of generic antibiotic products. Here, our aim was to compare the in vivo efficacy of amikacin products in clinical use in Colombia using this animal model. Results:  All except one generic product had the same in vitro potency, judging by the lack of differences on MIC and MBC compared with the innovator. However, eight of nine generic products failed in the neutropenic mouse thigh infection model to achieve the innovator’s maximum effect (Emax ≤ 5.65 for the generics vs. 6.58 log10 CFU/g for the innovator) against Escherichia coli SIG-1, after subcutaneous treatment every 6 h with doses ranging from 1.5 to 3072 mg/kg per day. Conclusion:  As we demonstrated previously with other antibiotics such as vancomycin, gentamicin and oxacillin, the generic products of amikacin failed the in vivo efficacy testing. The therapeutic equivalence should be assessed in vivo before clinical approval of generic products. Keywords:  Amikacin, Animal models, Generics, Therapeutic equivalence, Antimicrobial resistance © 2015 Zuluaga et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background Generic substitution of medications is a common prac- tice [1–3]. Worldwide, there is an abbreviated approval pathway for generic drugs of small molecules in which the comparative clinical trials are waived [4, 5], after demon- strating good manufacturing practices and bioequivalence in healthy volunteers [6, 7]. Furthermore, bioequivalence is waived for intravenous generics under the assump- tion that pharmaceutical equivalence predicts therapeu- tic equivalence accurately [8]. This approach has certainly rendered the desired economic results [9], but at the price of neglecting solid evidence documenting the clinical fail- ure of intravenous generics of vancomycin and cefuroxime [10]. Besides, an animal infection model was validated by our group to determine the therapeutic equivalence of antimicrobials [11, 12], in which many generic products of vancomycin [13], oxacillin [14, 15], gentamicin [16], mero- penem [17], lincomycin [18], ampicillin [19], and penicillin G [20] failed to kill the same number of microorganisms as the innovators. Of great concern, those generics of van- comycin that failed therapeutic equivalence selected the resistant subpopulation of Staphylococcus aureus [21], whilst therapeutically equivalent generics of ciprofloxa- cin were indistinguishable from the innovator in terms of selection of resistant Pseudomonas aeruginosa [22]. Amikacin is derived from kanamycin and its structure confers stability towards many enzymes, mainly from Gram negative bacteria, that hydrolyze other aminoglycosides [23]. This quality makes it the preferred aminoglycoside to prescribe along with a β-lactam to treat diverse nosocomial infections. During the execution of this study, the sudden discontinuation of the innovator product (Amikin®, Bristol Open Access *Correspondence: omar.vesga@udea.edu.co 3 Department of Internal Medicine, Universidad de Antioquia, Calle 70 No. 52‑21, Medellín, Colombia Full list of author information is available at the end of the article Page 3 of 8Zuluaga et al. BMC Res Notes (2015) 8:546 Myers-Squibb) forced us to stop the in  vivo comparative experiments. In view of the impossibility of obtaining addi- tional data, we decided to publish the available evidence. Results Antibiotics Table  1 lists the products tested with their pharmaceutical form, lot numbers, manufacturers and distributors. The dem- onstration of pharmaceutical equivalence for Carlon, Gencol, Pisa, Scalpi and Sigma generic products was published previ- ously by our group [8]. Seven of nine generic products (78 %) were produced in Colombia while the other two (Genven and Pisa) were made in Venezuela and Mexico, respectively. The Farmionni-Lubelca consortium manufactured three (Scalpi, Serpharma, Zokumey) of the nine generics tested (33 %), but they were analyzed as independent products. Susceptibility testing Table 2 shows the MIC and MBC of all products against E. coli SIG-1 or P. aeruginosa ATCC 27853. All but one generic amikacin product exhibited the same in  vitro efficacy of the innovator; the exception was Serpharma, which MIC and MBC were 10- and 16-fold higher against both strains (P < 0.05 by Dunn’s multiple comparison test). These results were reproducible in assays performed in different days. Reliability of the animal model to test therapeutic equivalence The repeatability of the PD parameters was assessed in two different days with the same batch of the innovator (batch 99A106). Figure 1 shows that there was no differ- ence in the non-linear regression (NLR) from two inde- pendent experiments (P = 0.39 by CFA) with innovator amikacin. Therapeutic equivalence testing Untreated animals had 7.04–7.34 log10 CFU/g when treatment started (0  h) and 9.07–9.78 log10 CFU/g 24  h later when therapy was finished (net growth = 2.24 ± 0.29 log10 CFU/g). All products tested yielded valid non-linear regressions describing the dose–response relationships obtained by Hill’s Equa- tion (Fig. 2). The PD parameters for the innovator were Emax = 6.58 ± 0.40 log10 CFU/g, ED50 = 272 ± 44.6 mg/ kg per day, and N = 1.02 ± 0.12, while the magnitudes of primary (Emax, ED50, N) and secondary (BD, 1LKD, and 2LKD) parameters of the other nine products are sum- marized in Table 3. Except for Carlon product (Fig. 2, panel a), the remain- ing eight generics failed to reach the innovator’s Emax, which ranged from 5.10 to 5.65 log10 CFU/g; in the best case, it was one order of magnitude lower than the inno- vator. It means that the innovator killed ~3.80 million microorganisms per gram of tissue at the maximal total dose used, whilst the most effective generic killed only 0.45 million. Although two generics (Gencol and Pisa) had greater potency than the innovator comparing their bacteriostatic dose (≤110  ±  8.30 vs. 144  ±  12.7  mg/ kg per day), both also had significantly lower Emax (P = 0.0003). Table 1  General description of the amikacin products studied a  The pharmaceutical equivalence (same potency and concentration of the active ingredient) tested by microbiological assay was published elsewhere [8] Amikacin product Form Demonstrated pharmaceutical equivalencea Batch Manufacturer Distributor BMS (innovator) 1 g in 4 ml Not applicable 99A106 Grove, Ecuador The manufacturer 0.5 g in 2 ml 00G030 05H091A Bristol-Myers Squibb, Ecuador Carlon 0.5 g in 2 ml Yes 111V0203 Carlon, Colombia The manufacturer FormasG 0.5 g in 2 ml No 02525 Vitropharma, Colombia Formas genericas farmaceuticas, Colombia Gencol 0.25 g in 2 ml Yes 0100 0200 Chalver, Colombia The manufacturer Genven 0.5 g in 2 ml No 904037 Leti for Genven, Venezuela The manufacturer Pisa 0.5 g in 2 ml Yes 060865 011306 PiSa, Mexico ECAR, Colombia Quimicol 0.5 g in 2 ml No 3780199 Quimicol, Colombia The manufacturer Scalpi 0.5 g in 2 ml Yes AK030072 Farmionni-Lubelca, Colombia Farmionni scalpi, Colombia Serpharma 0.25 g in 2 ml No AK020086 Farmionni-Lubelca, Colombia Serpharma, Colombia 0.1 g in 2 ml AK010172 Sigma (reference) 1 g powder Yes 120K1643 Sigma Chemical Co, USA The manufacturer Zokumey 0.25 g in 2 ml No AK020035 Farmionni-Lubelca, Colombia Zokumey pharma, Colombia Page 4 of 8Zuluaga et al. BMC Res Notes (2015) 8:546 Discussion Here, our results with amikacin indicate that almost all generics (eight of nine products) failed therapeutic equivalence in a head-to-head in  vivo comparison with the innovator, independently of their pharmaceutical equivalence. These data are similar to previous results with other antibiotics [13, 15, 17], reinforcing the idea that therapeutic equivalence of generic antimicrobials cannot be predicted from pharmaceutical equivalence or in vitro testing and therefore requires in vivo studies [11, 12]. The reliability of the thigh infection model to test the efficacy of antibiotics was assessed in two independent experiments with the innovator, exhibiting the same PD profile (Fig. 1). Besides, the similar in vivo lower efficacy of three generic products from the same manufacturer (all produced by Farmionni-Lubelca) confirmed the con- sistency of the model’s findings. Generic drugs are necessary to regulate drug price. But the scant information provided in the abbreviated way used by generic manufacturers have arisen some theoretical concerns [10, 24, 25] that are experimentally supported by our results. In this context, the “contamina- tion” of bioequivalent generic heparin with oversulfated chondroitin sulfate killed approximately 1000 patients around the world [26], but generic antibiotics may entail an even worse problem: antimicrobial resistance [27, 28]. We already demonstrated that so called “bioequivalent” generics of vancomycin devoid of therapeutic equiva- lence do enrich resistant subpopulations of S. aureus after exposure for only 12 days in the neutropenic murine thigh infection model [21]. In contrast, fully equivalent generic products of ciprofloxacin do not exhibit difference on resistance profile [22]. Here, similar to the vancomycin case, the therapeutically nonequivalent generics of ami- kacin do not sterilize the thighs even at the highest dose (3072 mg/kg per day), leaving at least 3 million bacterial cells per gram of tissue exposed to the antibiotic, but alive. Then, the risk of resistance is not a minor point [29]. The study by Miller et  al. supports our hypoth- esis about the impact on resistance of the massive use of generic products of amikacin failing therapeutic Table 2  Comparison of the in vitro biological potency of the amikacin products studied Concentrations are expressed as geometric mean and range (Min. and Max) in mg/L MIC minimal inhibitory concentration, MBC minimal bactericidal concentration ** p value <0.05 by Dunn’s multiple comparison test Product Escherichia coli SIG-1 Pseudomonas aeruginosa ATCC 27853 MIC Min Max MBC Min Max MIC Min Max MBC Min Max BMS 1.59 1.00 2.00 1.78 1.00 2.00 4.00 4.00 4.00 10.08 8.00 16.00 Carlon 4.00 4.00 4.00 8.00 8.00 8.00 5.66 4.00 8.00 5.66 4.00 8.00 FormasG 4.00 4.00 4.00 4.00 4.00 4.00 5.66 4.00 8.00 8.00 8.00 8.00 Gencol 2.00 2.00 2.00 2.00 2.00 2.00 4.00 4.00 4.00 8.00 4.00 16.00 Genven 2.00 2.00 2.00 2.00 2.00 2.00 2.83 2.00 4.00 5.66 4.00 8.00 Pisa 2.00 2.00 2.00 2.83 2.00 4.00 4.00 4.00 4.00 11.31 8.00 16.00 Quimicol 2.83 1.00 8.00 4.00 2.00 8.00 3.36 2.00 4.00 6.73 4.00 16.00 Scalpi 2.83 2.00 4.00 2.83 2.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 Serpharma** 19.03 16.00 32.00 22.63 16.00 32.00 38.05 32.00 64.00 64.00 64.00 64.00 Sigma 1.41 1.00 2.00 2.83 2.00 4.00 4.00 4.00 4.00 8.00 8.00 8.00 Zokumey 2.00 2.00 2.00 4.00 2.00 8.00 4.00 4.00 4.00 11.31 8.00 16.00 Fig. 1  Reliability of the neutropenic murine thigh infection model with the innovator of amikacin (BMS) in two independent experi- ments. The non-significant P value (0.393) from global curve fitting analysis (CFA) indicates that the underlying populations are better described by a single curve, confirming the model’s reliability for test- ing therapeutic equivalence Page 5 of 8Zuluaga et al. BMC Res Notes (2015) 8:546 equivalence [30]. They demonstrated that the amino- glycoside resistance mechanisms changed with the time (comparing studies before and after 1990’s decade) and geographical region, according to the increased usage of these drugs. According to Miller et  al., the baseline resistance level of Citrobacter spp., Enterobacter spp. or Klebsiella spp. to amikacin was lower than 20  % before 1987 (when generic consumption was limited), moment at which a continuous rise was observed reaching 60  % in countries like Chile, Uruguay, Mexico and Venezuela. They also found that it was precisely during this period of massive use of generic antimicrobials (after 1987), that new enzymes capable to degrade amikacin appeared, such as AAC(6′)-I alone or combined with other enzyme like AAC(6′)-I +  AAC(3)-II. Although only speculative with the available data, the possibility that therapeutic nonequivalent generics could enhance enzymatic resist- ance deserves scientific testing [29, 31]. There are at least two hypotheses to explain the find- ings. First, Bau et al. described the X-ray crystal structure of amikacin [32], establishing that the spatial relationship depends on two bifurcated hydrogen (H) bonds that are Fig. 2  In panel a, the in vivo activity of the Carlon generic product of amikacin compared with the innovator (BMS). The non-significant P value of the curve-fitting analysis (0.055) indicates that the generic is therapeutically equivalent to the innovator, however the higher data dispersion reduced the power of the test to detect significant differences from 87 % to 63 %. In panel b, the in vivo activity of eight generic products of amika- cin compared with the innovator (BMS). The global curve-fitting analysis (P < 0.05) demonstrates that the generics are described by independent curves, characterized by reduced Emax compared with the innovator (see Table 3), indicating that they lack therapeutic equivalence, despite similar in vitro activity Table 3  In vivo pharmacodynamic parameters of nine generics and the innovator product of amikacin AdjR2 adjusted coefficient of determination, Sy|x standard error of the estimate, CFA curve fitting analysis, Emax maximum effect, SE standard error, ED50 effective dose to kill 50 % of Emax, N slope, BD bacteriostatic dose, 1LKD and 2LKD 1- and 2-log kill dose, respectively Amikacin AdjR2 Sy|x Emax SE ED50 SE N SE BD SE 1LKD SE 2LKD SE P value (CFA) BMS (innovator) 0.97 0.37 6.58 0.40 272 44.6 1.02 0.12 144 12.7 266 19.9 490 40.8 NA  Carlon 0.93 0.65 5.58 0.33 160 27.0 2.01 0.49 132 17.3 190 28.6 288 55.8 0.055 FormasG 0.98 0.36 5.65 0.19 216 19.6 2.01 0.27 169 16.7 242 17.7 357 23.9 0.013 Gencol 0.99 0.24 5.10 0.14 122 11.0 2.26 0.43 110 8.30 157 15.5 248 37.1 0.001 Genven 1.00 0.17 5.36 0.07 209 13.2 3.35 0.31 185 11.9 232 13.6 301 17.8 <0.0001 Pisa 0.98 0.38 5.45 0.22 96 14.1 1.36 0.22 77 9.70 133 16.5 257 40.2 0.001 Quimicol 0.98 0.41 5.53 0.22 241 30.4 2.61 0.56 208 27.9 275 30.1 380 43.2 0.004 Scalpi 0.98 0.38 5.65 0.36 324 48.8 1.35 0.24 228 26.4 384 36.6 684 78.3 0.004 Serpharma 0.99 0.22 5.58 0.17 343 23.6 1.76 0.20 261 17.4 395 21.0 624 39.0 <0.0001 Zokumey 0.95 0.51 5.25 0.24 145 25.7 1.57 0.35 117 18.7 191 30.1 347 68.5 0.044 Page 6 of 8Zuluaga et al. BMC Res Notes (2015) 8:546 necessary for the internal conformation of the amikacin molecule. The first H-bond is common with the molecule of kanamycin to control the A/B ring orientation but the second H-bond is required to define the conformational angles around the B/C ring junction. Any subtle change in the position of the second H-bond or its lack could reduce significantly the in  vivo efficacy of amikacin. To test this hypothesis, one could compare simultaneously the chemical structure of innovator and generic by X-ray crystallography or NMR studies, however, the innovator is no longer available. Second, that impurities or different excipients might explain the failure of amikacin gener- ics [33], but it is less likely because the process for semi- synthesis of amikacin from acylation of kanamycin A is a well-known process [34]. Conclusions In vitro susceptibility tests do not predict the in  vivo efficacy of generic products of amikacin. Considering the potential impact on antimicrobial resistance of non- therapeutically equivalent generics, more studies com- paring the molecular and chemical identity, as well as head-to-head studies in validated animal models of infec- tion should be required before approval of generic ami- kacin products, although therapeutic equivalence will be difficult to establish without a gold standard (innovator product). Methods Antibiotics All amikacin products were bought from reputable drug- stores and handled following the instructions of each manufacturer. The innovator drug was included in all experiments as the gold standard [35]. Additionally, the reference powder (Sigma Aldrich, USA), a product not designed for clinical use, was used. Bacteria and media E. coli SIG-1 (an ampicillin-resistant clinical isolate) was selected for in  vitro and in  vivo experiments. For susceptibility testing, Pseudomonas aeruginosa ATCC 27853 was used as control [36]. Culture media included trypticase soy broth and agar for in vivo studies and cat- ion-adjusted Mueller–Hinton broth and agar for suscep- tibility testing (Becton–Dickinson, USA). Susceptibility testing Minimal inhibitory (MIC) and bactericidal (MBC) con- centrations of nine generic products, the reference pow- der and the innovator of amikacin were determined twice by broth microdilution following the Clinical Laboratory Standard Institute method [36]. To compare the in vitro potency, the differences between geometric means were assessed by Kruskal–Wallis (KW) test fol- lowed by Dunn’s multiple comparison test (GraphPad Prism 6.05) [37]. Animal model The University of Antioquia Animal Experimentation Ethics Committee approved the protocol. Six-week- old, 23–27  g, female murine-pathogen free mice of the strain Udea:ICR(CD-1) were used [38]. Mice were ren- dered neutropenic by injecting two intraperitoneal doses of cyclophosphamide (Cytoxan®, Bristol-Myers Squibb, Puerto Rico) given 4 days (150 mg/kg) and 1 day (100  mg/kg) before infection [39]. An intramuscular injection (0.1 mL) per thigh of a log-phase culture with ~7 log10 CFU of E. coli SIG-1 per mL was used. Two hours later (0  h), infected animals began a 24  h-treat- ment with each amikacin product (N ≥  10 mice/prod- uct), allocating two animals per dose and using at least five total doses that ranged from no effect (1.5 mg/kg per day) to maximal effect (3072 mg/kg per day). Each dose was administered by the subcutaneous route (0.2  mL) every 6  h to optimize fCmax/MIC and fAUC/MIC, the pharmacodynamic (PD) indices related to the efficacy of amikacin in mice and humans with normal renal func- tion [40, 41]. Untreated infected control mice were sacri- ficed just after inoculation (−2 h), at the onset (0 h), and at the end of experiment (24 h), while treated mice were euthanized at 24 h. To determine antibacterial efficacy, both thighs of each mouse were dissected under aseptic technique and homogenized independently in sterile saline (1:10). After serial dilutions and manual plating, the cultures were incubated for 18 h at 37 °C under air atmosphere before colony counting and data registration in a database (Microsoft Excel®, USA). In this model, one thigh weighs 1 g and the limit of detection is 100 CFU/thigh. Statistical analysis For each total dose (independent variable), the net anti- bacterial effect (E, dependent variable) was calculated by subtracting the CFU/g obtained in thighs of infected mice from the 24 h untreated controls. Nonlinear regres- sion of the dose–effect data from each product fitted to Hill’s model provided the primary PD parameters maximum effect (Emax), effective dose killing 50  % of the Emax (ED50), and slope (N), as well as the secondary PD parameters bacteriostatic dose (BD) and the doses required to kill the first (1LKD) and second (2LKD) log of bacteria (SigmaPlot 12.3). To test the therapeutic equivalence, the magnitudes of these parameters were compared (each generic vs. the innovator) by curve fit- ting analysis (GraphPad Prism 6.05) as was described thoroughly elsewhere [13]. The quality of the nonlinear Page 7 of 8Zuluaga et al. BMC Res Notes (2015) 8:546 regressions was assessed by the adjusted coefficient of determination (adj.R2), the standard error of estimate (Sy|x), the fulfillment of the assumptions of normality and homoscedasticity (constant variance), and the absence of multicollinearity (variance inflation factor). Accepting a 5 % chance for a type I error (α-error) and expecting residuals’ standard deviations ≤0.5 log, the treatment of 10 animals per product to compare nine generic prod- ucts with the innovator confers 87 % power to reject the null hypothesis (H0: generics = innovator product) if the magnitude of the difference on antibacterial efficacy is >1 log10 CFU/g. Abbreviations AAC(6′)-I: aminoglycoside 6′-N-acetyltransferase type I; AAC(3)-II: aminoglyco- side 3-N-acetyltransferase type II; Adj. R2: adjusted coefficient of determination; ATCC: American Type Culture Collection; AUC: area under the curve concentra- tion–time; BD: bacteriostatic dose; Cmax: the maximum concentration; CFA: curve fitting analysis; CFU: colony forming unit; CLSI: Clinical and Laboratory Standard Institute; Emax: maximum effect; ED50: effective dose to kill 50 % of Emax; MIC: minimal inhibitory concentration; MBC: minimal bactericidal con- centration; N: Slope; NLR: non-linear regression; Sy|x: standard error of the esti- mate; SE: standard error; 1LKD and 2LKD: 1- and 2-log kill dose, respectively. Authors’ contributions AFZ contributed during the experimental process, took care of the animals, performed the analysis and interpretation of data and wrote the first manu- script. CAR took care of the animals, helped during in vitro experimentation, and reviewed the first manuscript. MA performed the experiments and took care of the animals. OV conceived, directed, and designed the study, obtained funding, edited all drafts and rewrote the final version of the paper. All authors read and approved the final manuscript. Author details 1 GRIPE [Grupo Investigador de Problemas en Enfermedades infecciosas], Uni- versidad de Antioquia, Calle 70 No. 52‑21, Medellín, Colombia. 2 Department of Pharmacology and Toxicology at Medical School, Universidad de Antioquia, Calle 70 No. 52‑21, Medellín, Colombia. 3 Department of Internal Medicine, Universidad de Antioquia, Calle 70 No. 52‑21, Medellín, Colombia. 4 Infec- tious Diseases Unit, Hospital Universitario San Vicente Fundación, Medellín, Colombia. Acknowledgements This work was supported by the University of Antioquia UdeA and Sistema General de Regalías of Colombia (BPIN: 2013000100183)—Ruta N—Centro de Informacion y Estudio de Medicamentos y Tóxicos (CIEMTO). The authors wish to express their gratitude to Beatriz E. Salazar and Wilson Galvis for their help during the experimental execution. Competing interests Zuluaga has received honoraries for unrelated lectures from Allergan, Amgen, Lilly, Mundipharma, Novonordisk, Pfizer, Roche and Sanofi. Rodriguez has received honoraries for unrelated lectures from Roche and Amgen. Agudelo and Vesga have no conflicts of interest to declare. None of these companies or any other pharmaceutical company were involved in the design, execution, or publication of this study. Availability of supporting data The data supporting the results of this article are included within the article. There are no additional files. Received: 2 June 2015 Accepted: 21 September 2015 References 1. Dettelbach HR. A time to speak out on bioequivalence and therapeutic equivalence. J Clin Pharmacol. 1986;26(5):307–8. 2. Rheinstein PH. Therapeutic inequivalence. Drug Saf. 1990;5(Suppl 1):114–9. 3. Frank RG. The ongoing regulation of generic drugs. N Engl J Med. 2007;357(20):1993–6. doi:10.1056/NEJMp078193. 4. Zarowitz BJ. The generic imperative. Geriatr Nurs. 2008;29(4):223–6. doi:10.1016/j.gerinurse.2008.06.003. 5. Howland RH. Evaluating the bioavailability and bioequivalence of generic medications. J Psychosoc Nurs Ment Health Serv. 2010;48(1):13–6. doi:10.3928/02793695-20091204-07. 6. Arnold ME. Implications of differences in bioanalytical regulations between Canada, USA and South America. Bioanalysis. 2011;3(3):253–8. doi:10.4155/bio.10.187. 7. Carpenter D, Tobbell DA. Bioequivalence: the regulatory career of a pharmaceutical concept. Bull Hist Med. 2011;85(1):93–131. doi:10.1353/ bhm.2011.0024. 8. Zuluaga AF, Agudelo M, Rodriguez CA, Vesga O. Application of micro- biological assay to determine pharmaceutical equivalence of generic intravenous antibiotics. BMC Clin Pharmacol. 2009;9:1. 9. Glantz LH, Annas GJ. Impossible? Outlawing state safety laws for generic drugs. N Engl J Med. 2011;365(8):681–3. doi:10.1056/NEJMp1107832. 10. Mastoraki E, Michalopoulos A, Kriaras I, Mouchtouri E, Falagas M, Karatza D, et al. Incidence of postoperative infections in patients undergoing coronary artery bypass grafting surgery receiving antimicrobial prophy- laxis with original and generic cefuroxime. J Infect. 2008;56(1):35–9. 11. Zuluaga AF, Rodriguez CA, Agudelo M, Vesga O. About the validation of animal models to study the pharmacodynamics of generic antimi- crobials. Clin Infect Dis Off Publ Infect Dis Soc Am. 2014;59(3):459–61. doi:10.1093/cid/ciu306. 12. Agudelo M, Rodriguez CA, Zuluaga AF, Vesga O. Relevance of various animal models of human infections to establish therapeutic equivalence of a generic product of piperacillin/tazobactam. Int J Antimicrob Agents. 2015;45(2):161–7. doi:10.1016/j.ijantimicag.2014.10.014. 13. Vesga O, Agudelo M, Salazar BE, Rodriguez CA, Zuluaga AF. Generic van- comycin products fail in vivo despite being pharmaceutical equivalents of the innovator. Antimicrob Agents Chemother. 2010;54(8):3271–9. doi:10.1128/AAC.01044-09. 14. Rodriguez CA, Zuluaga AF, Salazar BE, Agudelo M, Vesga O. Experimental comparison of 11 generic products (GP) of oxacillin (OXA) with the origi- nal compound (OC) in terms of concentration of active principle (CAP), in vitro activity, and in vivo efficacy, using the neutropenic murine thigh infection model (NMTIM), Abstract A-1305. 54th interscience conference on antimicrobial agents and chemotherapy (ICAAC); Washington, DC: American Society for Microbiology; 2004. 15. Rodriguez CA, Agudelo M, Zuluaga AF, Vesga O. In vitro and in vivo comparison of the anti-staphylococcal efficacy of generic products and the innovator of oxacillin. BMC Infect Dis. 2010;10:153. 16. Zuluaga AF, Agudelo M, Cardeno JJ, Rodriguez CA, Vesga O. Determina- tion of therapeutic equivalence of generic products of gentamicin in the neutropenic mouse thigh infection model. PLoS One. 2010;5(5):e10744. 17. Agudelo M, Rodriguez CA, Pelaez CA, Vesga O. Even apparently insignifi- cant chemical deviations among bioequivalent generic antibiotics can lead to therapeutic nonequivalence: the case of meropenem. Antimicrob Agents Chemother. 2014;58(2):1005–18. doi:10.1128/AAC.00350-13. 18. Salazar BE, Zuluaga AF, Rodriguez CA, Agudelo M, Vesga O. Experimen- tal comparison of 7 generic products of lincomycin with the original compound in terms of concentration of active principle, in vitro activity, and in vivo efficacy, using the neutropenic murine thigh infection model. Abstract A-1879. 44th interscience conference on antimicrobial agents and chemotherapy; Washington, DC: American Society for Microbiology; 2004. 19. Zuluaga AF, Salazar BE, Loaiza S, Agudelo M, Vesga O. Therapeutic equiva- lence with the original compound of 8 generic products of ampicillin determined in the neutropenic murine thigh infection model. Abstract E-2033. 44th interscience conference on antimicrobial agents and chemo- therapy; Washington, DC: American Society for Microbiology; 2004. Page 8 of 8Zuluaga et al. BMC Res Notes (2015) 8:546 20. Agudelo M, Zuluaga AF, Rodriguez CA, Salazar BE, Vesga O. Determination of therapeutic equivalence for 5 generic products of penicillin G using the neutropenic murine thigh infection model. Abstract A-1877. 44th interscience conference on antimicrobial agents and chemotherapy. Washington, DC: American Society for Microbiology; 2004. 21. Rodriguez CA, Agudelo M, Zuluaga AF, Vesga O. Generic vancomycin enriches resistant subpopulations of Staphylococcus aureus after expo- sure in the neutropenic mouse thigh infection model. Antimicrob Agents Chemother. 2011. doi:10.1128/AAC.05129-11. 22. Rodriguez CA, Agudelo M, Zuluaga AF, Vesga O. Impact on resistance of the use of therapeutically equivalent generics: the case of cipro- floxacin. Antimicrob Agents Chemother. 2015;59(1):53–8. doi:10.1128/ AAC.03633-14. 23. Gonzalez LS III, Spencer JP. Aminoglycosides: a practical review. Am Fam Physician. 1998;58(8):1811–20. 24. Jones RN, Fritsche TR, Moet GJ. In vitro potency evaluations of various piperacillin/tazobactam generic products compared with the contem- porary branded (Zosyn, Wyeth) formulation. Diagn Microbiol Infect Dis. 2008;61(1):76–9. 25. Meredith P. Bioequivalence and other unresolved issues in generic drug substitution. Clin Ther. 2003;25(11):2875–90. 26. Blossom DB, Kallen AJ, Patel PR, Elward A, Robinson L, Gao G, et al. Out- break of adverse reactions associated with contaminated heparin. N Engl J Med. 2008;359(25):2674–84. 27. Norrby SR, Nord CE, Finch R. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect Dis. 2005;5(2):115–9. doi:10.1016/S1473-3099(05)01283-1. 28. Alanis AJ. Resistance to antibiotics: are we in the post-antibiotic era? Arch Med Res. 2005;36(6):697–705. doi:10.1016/j.arcmed.2005.06.009. 29. Toutain PL, Bousquet-Melou A. Rebuttal to the reaction of the EGGVP to the review article ‘the consequences of generic marketing on antibi- otic consumption and the spread of microbial resistance: the need for new antibiotics’. J Vet Pharmacol Ther. 2014;37(6):618–23. doi:10.1111/ jvp.12166. 30. Miller GH, Sabatelli FJ, Hare RS, Glupczynski Y, Mackey P, Shlaes D, et al. The most frequent aminoglycoside resistance mechanisms—changes with time and geographic area: a reflection of aminoglycoside usage pat- terns? Aminoglycoside Resistance Study Groups. Clin Infect Dis Off Publ Infect Dis Soc Am. 1997;24(Suppl 1):S46–62. 31. Rodriguez CA, Agudelo M, Gonzalez M, Zuluaga AF, Vesga O, editors. Generic piperacillin-tazobactam (TZP) enriches resistant subpopulations of Escherichia coli after exposure in the neutropenic mouse thigh infec- tion model (NMTIM). 53 interscience conference on antimicrobial agents and chemotherapy. American Society for Microbiology; 2013. 32. Bau R, Tsyba I. Crystal structure of amikacin. Tetrahedron. 1999;55(52):14839–46. doi:10.1016/s0040-4020(99)00983-7. 33. Amikacin Sulfate. 2010. http://www.drugs.com/monograph/amikacin- sulfate.html. Accessed 12 Dec 2010. 34. Mangia A. Acylation of kanamycin a. Google Patents; 1998. 35. Schiffman DO. Evaluation of amikacin sulfate (Amikin). A new aminogly- coside antibiotic. JAMA. 1977;238(14):1547–50. 36. Clinical, Laboratory Standards I. Performance standards for antimicrobial susceptibility testing, approved standard M100-S19. Wayne: Clinical and Laboratory Standards Institute (CLSI); 2009. 37. Glantz SA. Primer of biostatistics. 7th ed. New York: McGraw-Hill; 2012. p. 489. 38. Zuluaga A, Salazar B, Galvis W, Loaiza S, Agudelo M, Vesga O. Foundation of the first functional MPF animal facility in Colombia. Iatreia [Spanish]. 2003;16:115–31. 39. Zuluaga AF, Salazar BE, Rodriguez CA, Zapata AX, Agudelo M, Vesga O. Neutropenia induced in outbred mice by a simplified low-dose cyclophosphamide regimen: characterization and applicability to diverse experimental models of infectious diseases. BMC Infect Dis. 2006;6:55. 40. Craig WA. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clin Infect Dis. 1998;26(1):1–10. 41. Craig WA, Redington J, Ebert SC. Pharmacodynamics of amikacin in vitro and in mouse thigh and lung infections. J Antimicrob Chemother. 1991;27(Suppl C):29–40. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit