The literature is still controversial in relation to therapeutic differences between innovative, generic, and similar anti-seizures medications (ASM). Topiramate (TPM) is an ASM used in the treatment of various seizure types and in different epileptic syndromes, as well as in other groups of morbidities, and it is available in many generic and similar forms, besides the innovator. The aim of this translational work was to compare different brands of TPM by using animal models of seizures induced by pentylenetetrazole (PTZ).
Five brands of TPM (one reference, two similar and two generics) were tested in mice. Animals were previously treated with TPM (n=6/brand) and latencies from PTZ injection to onset of manifestations, first seizure and death were measured and compared between groups. Experiment was conducted in two settings: acute seizure model (PTZ 80 mg/kg) and kindling model (PTZ 20, 30, and 40 mg/kg in 8 alternate days).
The experiment did not demonstrate significant differences between the TPM brands regarding the protective effect in the acute seizure and kindling models.
In conclusion, results can be explained by true therapeutic equivalence or insufficiency of the PTZ model to reveal differences among brands.
Epilepsy is a brain disease according to the International League Against Epilepsy (ILAE), defined by at least two 24-hour unprovoked or reflex epileptic seizures, one unprovoked or reflex epileptic seizure, and a probability of new seizures within 10 years similar to the risk of recurrence after two seizures and the diagnosis of an epileptic syndrome.
Along the years, the use of generic drugs in the treatment of epilepsies has been debated due to the risk of unpredictable consequences such as the recurrence of crises or the appearance of adverse effects.
Five brands of TPM were purchased from the conventional trade (drugstore), all in the presentation of packages with 60 coated tablets of 25 mg each. Besides reference (innovative), brands were chosen as the two market sales leaders, both for generic and similar. In the experiments, the drugs were designated as: reference-R (Topamax®; Jansen-Cilag, São Paulo, Brazil), generic 1-G1 (Biossintética, São Paulo, Brazil), generic 2-G2 (Eurofarma, São Paulo, Brazil), similar 1-S1 (Amato®; Eurofarma) and similar 2-S2 (Égide®; Libbs, São Paulo, Brazil).
Swiss albino mice (20–30 g), aged 45 to 60 days, obtained from the Núcleo de Biologia Experimental of the University of Fortaleza were used. The specific-pathogen-free animals were housed in appropriate cages (individually ventilated cages, Tecniplast [Buguggiate, Varese, Italy]) and kept at room temperature of 22–24°C on a 12:12 hours light:dark cycle. They received standard feed (Purina, São Paulo, Brazil) and water ad libitum. All protocols were in strict compliance with the standards established by Brazil’s National Council on Animal Experimentation Control and received approval from the Committee on Animal Research and Ethics of UNIFOR (#6879070220).
The TPM-coated tablets were crushed and homogenized, then diluted in 0.9% NaCl in the proportion of 1 tablet of 25 mg to 2.5 mL of 0.9% sodium chloride, producing a concentration of 10 mg/mL. The animals (n=6/group) were treated orally (gavage;
Sixty minutes after the administration of TPM or vehicle, all groups received an intraperitoneal injection of PTZ (80 mg/kg; 0.1 mL/10 g of weight) to induce seizures. During the 60 minutes immediately following the administration of PTZ, the animals remained in individual cages and had their behavior recorded on video, using a smartphone device (iPhone®; Apple Inc., Los Altos, CA, USA) for reviewing and time measuring. Primary outcomes were latencies (in seconds) to the onset of manifestations, to first seizure and to death. The manifestations considered in this analysis were those listed on the Racine scale.
Along 16 consecutive days, animals were daily treated in the regimen described above (2.3). Starting on the second day and every other day thereafter, 60 minutes after the administration of TPM or vehicle, all groups received an intraperitoneal injection of PTZ in incremental doses (20 mg/kg on days 2, 4, 6, and 8; 30 mg/kg on days 10 and 12; 40 mg/kg on days 14 and 16). During the 30 minutes immediately following the administration of PTZ, the animals remained in individual cages and had their behavior recorded on video, using a smartphone device (iPhone®; Apple Inc.) for reviewing and time measuring. Primary outcomes were latencies (in seconds) to the onset of manifestations, to first seizure and to death. The manifestations considered in this analysis were the also those considered in the acute seizure model (2.4). Events not observed during 30-minute observation periods were assigned latencies of 1,800 seconds.
All quantitative variables were tested for normal distribution using the Shapiro-Wilk test and the results are presented as median (interquartile interval) for non-normal distribution. The medians of each group (n=6) in seconds to first manifestations, first seizure and death were compared. Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s post-test. For comparisons of three or more paired groups, the Friedman test was used. In addition, the Kaplan-Meier survival analysis was performed to investigate which group presented a relationship with the fastest onset of the studied events. The median time until the event was estimated within the groups, with respective 95% confidence intervals. The log-rank test, a non-parametric test, was used to compare survival curves between two or more groups. Data were analyzed using SPSS software for Macintosh, version 23 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 5.01 for Windows (GraphPad Software, La Jolla, CA, USA;
In the acute PTZ-induced seizure model, 97.2% of the animals presented manifestations, 94.4% had at least one seizure and 75% died within the 60-minute observation period (
As an additional finding, when analyzed as categorical variable, there was a statistically significant difference in the frequency of deaths (but not of manifestations or seizures) among the groups (
Brands of generics and similar were chosen using a commercial performance criterion to enhance the relevance of the findings for the community. The acquisition of the drugs in the conventional trade intended to mimic the exposure of real patients to the variety of brands. Furthermore, manufacturers were not made aware of this experiment to exempt the study form conflicts of interest with any of the tested brands.
In general, the results were compatible with true therapeutic equivalence among the five brands. However, statistical significance in a minority of variables and comparisons suggests that real differences concerning therapeutical properties still exist. A few aspects inherent to the model and the experiment design might also have contributed to the findings. Although the PTZ model in mice has been used in the developmental stages of TPM,
Another possible factor was the reduced group size and consequent limited statistical power. Nevertheless, the animal model proved to be a suitable alternative for comparative studies between brands since it offers some advantages: same morbid condition, genetic homogeneity, matched distribution of age and sex, besides objective, reliable and reproducible outcomes. Despite the limitations, the experiment met the expectation of overcoming the previously mentioned methodological aspects inherent to studies in humans. The findings support the hypothesis of therapeutic equivalence between the reference, generics, and similar forms of TPM. Other outlines of the PTZ model or even other experimental models may offer more accuracy for the purpose of comparing different brands of ASMs in an alternative and translational setting.
The authors declare that they have no conflicts of interest.
George Linard Silva Malveira: Investigation, Conceptualization, Methodology, Writing-original draft, review&editing. Sacha Aubrey Alves Rodrigues Santos: Methodology, Writing-original draft, review& editing. Gerlânia de Oliveira Leite: Methodology, Writing-original draft. Adriana Rolim Campos: Conceptualization, Methodology, Writing-original draft, review, editing, Supervision, Project administration, Funding acquisition.
Groups and events evaluated in animals
Total group (n=36) | |
---|---|
Was there a first manifestation? | |
No | 1.0 (2.8) |
Yes | 35.0 (97.2) |
First manifestation ΔT1 (seconds) | 53.5 (48.0–73.0) |
Convulsion | |
No | 2.0 (5.6) |
Yes | 34.0 (94.4) |
Convulsion ΔT2 (seconds) | 95.0 (57.5–327.5) |
Death | |
No | 9.0 (25.0) |
Yes | 27.0 (75.0) |
Death ΔT3 (seconds) | 433.5 (215.5–2540.5) |
Total group quantitative data expressed as median and interquartile range in parentheses.
Comparison between times until events (first manifestation, seizure and death) according to the studied groups
Acute PTZ-induced seizure model | ||||||
---|---|---|---|---|---|---|
| ||||||
First manifestation ΔT1 (seconds) | First seizure ΔT2 (seconds) | Death ΔT3 (seconds) | ||||
Drugs | 0.307 | 0.307 | 0.129 | |||
Control | 51.0 (37–58) | 53.5 (38–59) | 145.0 (105–441) | |||
Reference | 68.0 (62–74) | 75.5 (69–154) | 2,088.5 (426–3,600) | |||
Generic 1 | 50.5 (46–65) | 126.5 (53–210) | 319.0 (204–1,481) | |||
Generic 2 | 46.0 (43–55) | 151.5 (46–335) | 537.0 (172–629) | |||
Similar 1 | 60.5 (48–78) | 201.0 (66–517) | 1,976.0 (235–3,600) | |||
Similar 2 | 53.5 (51–84) | 193.0 (108–522) | 741.5 (256–3,600) |
Values are presented as median and interquartile range in parentheses.
PTZ, pentylenetetrazole.
Kruskal-Wallis test was used.
Kaplan-Meier analysis with comparison of times until the first manifestation, first seizure and death according to the studied groups
First manifestation | First seizure | Death | ||||
---|---|---|---|---|---|---|
Drugs | 0.393 | 0.329 | 0.184 | |||
Control | 55 (48.210–61.790) | 57 (51.908–62.092) | 441 (98.100–783.800) | |||
Reference | 66 (59.305–72.695) | 72 (58.421–85.579) | 963 (0.000–5926.226) | |||
Generic 1 | 53 (41.118–64.882) | 72 (51.631–92.369) | 414 (329.130–498.870) | |||
Generic 2 | 61 (44.026–77.974) | 87 (59.841–114.159) | 840 (227.200–1,452.700) | |||
Similar 1 | 49 (38.816–59.184) | 100 (11.730–188.260) | 783 (0.000–2,146.000) | |||
Similar 2 | 53 (47.342–58.658) | 141 (42.550–239.450) | 705 (414.700–995.200) |
Values are presented as median and interquartile range in parentheses.
The log-rank test was used.
Acute pentylenetetrazole-induced seizure model
Group | |||||||
---|---|---|---|---|---|---|---|
| |||||||
Control | Reference | Generic 1 | Generic 2 | Similar 1 | Similar 2 | ||
Any manifestation | 6 (100.0) | 5 (83.3) | 6 (100.0) | 6 (100.0) | 6 (100.0) | 6 (100.0) | 0.399 |
At least 1 seizure | 6 (100.0) | 5 (83.3) | 6 (100.0) | 6 (100.0) | 6 (100.0) | 5 (83.3.0) | 0.516 |
Death | 6 (100.0) | 3 (50.0) | 5 (83.3) | 6 (100.0) | 3 (50.0) | 4 (66.7.0) | 0.133 |
Values are presented as number (%), chi-square test or likelihood ratio.
Kindling model. Latencies to first manifestation and first seizure
Group | |||||||
---|---|---|---|---|---|---|---|
| |||||||
Control | Reference | Generic 1 | Generic 2 | Similar 1 | Similar 2 | ||
First | |||||||
manifestation | |||||||
Day 1 | 116 (110–1,800) | 1,055 (30–1,800) | 1,044 (251–1,800) | 235 (116–1,800) | 164.5 (97–1,800) | 184 (120–223) | 0.629 |
Day 2 | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 0.820 |
Day 3 | 80 (52–132) | 389 (219–1,800) | 51.5 (22–106) | 296.5 (197–1800) | 245 (131–370) | 107 (82–259) | 0.005 |
Day 4 | 115 (49–147) | 118 (87–1,800) | 1,108.5 (200–1800) | 77 (52–98) | 164.5 (102–237) | 135.5 (77–273) | 0.019 |
Day 5 | 30.5 (10–73) | 103 (88–132) | 86.5 (78–161) | 108 (33–131) | 86.5 (21–140) | 75 (51–110) | 0.285 |
Day 6 | 74 (25–84) | 80 (56–97) | 95 (77–131) | 105.5 (90–153) | 127 (105–147) | 177 (102–1,800) | 0.065 |
First seizure | |||||||
Day 1 | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1.000 |
Day 2 | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1.000 |
Day 3 | 975 (128–1,800) | 1,800 (473–1,800) | 1,800 (567–1,800) | 1,800 (1,800–1,800) | 1,800 (1,800–1,800) | 1,099.5 (347–1,800) | 0.499 |
Day 4 | 214 (173–1,800) | 220 (100–1,800) | 1,800 (1,800–1,800) | 960 (100–1,800) | 319 (102–1,800) | 1,800 (1,800–1,800) | 0.234 |
Day 5 | 1,800 (1,800–1,800) | 972.5 (125–1,800) | 223.5 (181–1,800) | 1,800 (150–1,800) | 190.5 (151–1,800) | 956.5 (98–1,800) | 0.507 |
Day 6 | 1,108.5 (238–1,800) | 119 (66–424) | 164 (118–1,800) | 1,800 (160–1,800) | 131 (107–592) | 1,018.5 (108–1,800) | 0.188 |
Values are presented as median and interquartile range in parentheses.
Using the Kruskal-Wallis test with Dunn's post-test there was
Generic 1 vs. reference.
Generic 1 vs. generic 2.