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Beneficial Effects of the Calcium Channel Blocker CTK 01512-2 in a Mouse Model of Multiple Sclerosis

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Abstract

Voltage-gated calcium channels (VGCCs) play a critical role in neuroinflammatory diseases, such as multiple sclerosis (MS). CTK 01512-2 is a recombinant version of the peptide Phα1β derived from the spider Phoneutria nigriventer, which inhibits N-type VGCC/TRPA1-mediated calcium influx. We investigated the effects of this molecule in the mouse model of experimental autoimmune encephalomyelitis (EAE). The effects of CTK 01512-2 were compared to those displayed by ziconotide—a selective N-type VGCC blocker clinically used for chronic pain—and fingolimod—a drug employed for MS treatment. The intrathecal (i.t.) treatment with CTK 01512-2 displayed beneficial effects, by preventing nociception, body weight loss, splenomegaly, MS-like clinical and neurological scores, impaired motor coordination, and memory deficits, with an efficacy comparable to that observed for ziconotide and fingolimod. This molecule displayed a favorable profile on EAE-induced neuroinflammatory changes, including inflammatory infiltrate, demyelination, pro-inflammatory cytokine production, glial activation, and glucose metabolism in the brain and spinal cord. The recovery of spatial memory, besides a reduction of serum leptin levels, allied to central and peripheral elevation of the anti-inflammatory cytokine IL-10, was solely modulated by CTK 01512-2, dosed intrathecally. The intravenous (i.v.) administration of CTK 01512-2 also reduced the EAE-elicited MS-like symptoms, similarly to that seen in animals that received fingolimod orally. Ziconotide lacked any significant effect when dosed by i.v. route. Our results indicate that CTK 01512-2 greatly improved the neuroinflammatory responses in a mouse model of MS, with a higher efficacy when compared to ziconotide, pointing out this molecule as a promising adjuvant for MS management.

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Acknowledgments

We would like to thank Janaína Pasetti Nunes for her valuable technical assistance in histological processing.

Funding

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)-AUX-PE Toxinologia, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), in addition to a Financiadora de Estudos e Projetos (FINEP) research grant “Implantação, Modernização e Qualificação de Estrutura de Pesquisa da PUCRS” (PUCRSINFRA) # 01.11.0014-00, Brazil. Rodrigo B. M. Silva is a pharmacology PhD student receiving grants from CAPES. Maria M. Campos is a researcher career awardee of CNPq (303842-2014-8).

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Authors and Affiliations

  1. Escola de Medicina, Programa de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, 90619-900, Brazil

    Rodrigo B. M. Silva & Maria M. Campos

  2. Escola de Ciências da Saúde, Centro de Toxicologia e Farmacologia, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Avenida Ipiranga, 6681, Porto Alegre, RS, 90619-900, Brazil

    Rodrigo B. M. Silva & Maria M. Campos

  3. Centro de Pesquisa Pré-Clínica, Instituto do Cérebro do Rio Grande do Sul – Brain Institute (BraIns), Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, 90610-000, Brazil

    Samuel Greggio, Gianina T. Venturin & Jaderson C. da Costa

  4. Escola de Ciências da Saúde, Curso de Graduação em Biomedicina, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, 90619-900, Brazil

    Samuel Greggio

  5. Núcleo de Pós-Graduação, Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, Belo Horizonte, 30150-240, Brazil

    Marcus V. Gomez

  6. Escola de Ciências da Saúde, Curso de Graduação em Odontologia, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, 90619-900, Brazil

    Maria M. Campos

  7. Escola de Ciências da Saúde, Programa de Pós-Graduação em Odontologia, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, 90619-900, Brazil

    Maria M. Campos

Authors
  1. Rodrigo B. M. Silva
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  2. Samuel Greggio
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  3. Gianina T. Venturin
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  4. Jaderson C. da Costa
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  5. Marcus V. Gomez
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  6. Maria M. Campos
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Corresponding author

Correspondence to Maria M. Campos.

Ethics declarations

All animal experimental procedures complied with the National Institutes of Health Animal Care Guidelines (NIH publications n° 80-23), and were approved by the Animal Ethics Committee of the Pontifical Catholic University of Rio Grande do Sul (PUCRS, Porto Alegre, Brazil) (protocol number 14/00424).

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic Supplementary Material

Supplementary fig. 1

Experimental design phase 1 (A) and phase 2 (B) for the experimental autoimmune encephalomyelitis (EAE) evoked by MOG35-55 in female C57/BL6 mice. Representative scheme (A and B) shows the EAE induction during 25 days as well as the respective days of treatment with vehicle, CTK 01512-2, ziconotide and fingolimod. In experimental design phase 1 shows the behavioral studies performed as von Frey hairs, hot-plate test, clinical score, neurological severity score and body weight during EAE model. In phase 2, the behavioral tests evaluated were object location test, clinical score, neurological severity score, rotarod and body weight. (GIF 71.6 KB)

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Supplementary fig. 2

Body weight (gain/loss) was assessed during 25 days after subcutaneous administration of MOG35-55. Effects of treatment with CTK 01512-2, ziconotide (Zico) (25, 50, or 100 pmol/site, dosed at days 4, 10, 15, 20 and 24 post-MOG35-55), by intrathecal (i.t.) route, or fingolimod (0.3 mg/kg, dosed once a day, beginning 7 days after the first MOG35-55 injection), by oral route (p.o.), on body weight (gain/loss) (A) at day 5 (B), day 10 (C), day 15 (D), day 20 (E) and day 25 (F) in the model of MOG35-55-evoked experimental autoimmune encephalomyelitis (EAE) in C57/BL6 mice. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Differences in the gain and weight loss was determined by one-way analysis of variance, followed by Newman-Keuls post hoc test. Each column represents the mean and the vertical lines show the standard error mean. The experimental N of each group is provided in the graph A. #p < 0.05, ##p < 0.01 and ###p < 0.001 significantly different from naive values. **p < 0.01 and ***p < 0.001. MOG = myelin oligodendrocyte glycoprotein. (GIF 97.1 KB)

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Supplementary fig. 3

Cytokine production was measured by ELISA in spleen on day 25 day after MOG35-55 elicited multiple sclerosis in mice. Effects of treatment with CTK 01512-2, ziconotide (Zico) (50 pmol/site, dosed at days 4, 10, 15, 20 and 24 days post-MOG35-55), by intrathecal (i.t.) route, or fingolimod (0.3 mg/kg, dosed once a day, beginning 7 days after the first MOG35-55 injection), by oral route (p.o), on production of TNF (A), IL-1β (B), IFN-γ (C), IL-17 (D), IL-23 (E), CCL3 (F) and IL-10 (G) in the model of MOG35-55-evoked experimental autoimmune encephalomyelitis (EAE) in C57/BL6 mice. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Differences in the cytokine formation was determined by one-way analysis of variance, followed by Newman-Keuls post hoc test. Each column represents the mean and the vertical lines show the standard error mean. The experimental N of each group is provided in the graphs (A-G). #p < 0.05, ##p < 0.01 and ###p < 0.001 significantly different from naive values. *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from vehicle values. $p < 0.05 and $$p < 0.01 and significantly different from ziconotide values. CCL3 = chemokine (C-C motif) ligand 3; CFA = complete Freund’s adjuvant; IFN-γ = interferon-gamma; IL = interleukin; MOG = myelin oligodendrocyte glycoprotein; PBS = phosphate-buffered saline; PTX = Pertussis toxin; TNF = tumor necrosis factor; Veh = vehicle. (GIF 85.4 KB)

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Supplementary fig. 4

Cytokine activity was evaluated 25 days after subcutaneous application of MOG35-55 in serum. Effects of treatment with CTK 01512-2, ziconotide (Zico) (50 pmol/site, dosed at days 4, 10, 15, 20 and 24 days post-MOG35-55), by intrathecal (i.t.) route, or fingolimod (0.3 mg/kg, dosed once a day, beginning 7 days after the first MOG35-55 injection), by oral route (p.o), on production of TNF (A), IL-1β (B), IFN-γ (C), IL-17 (D), IL-23 (E), CCL3 (F) and IL-10 (G) in the model of MOG35-55-evoked experimental autoimmune encephalomyelitis (EAE) in C57/BL6 mice. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Differences in the cytokine production was determined by one-way analysis of variance, followed by Newman-Keuls post hoc test. Each column represents the mean and the vertical lines show the standard error mean. The experimental N of each group is provided in the graphs (A-G). #p < 0.05 significantly different from naive values. **p < 0.01 significantly different from vehicle values. CCL3 = chemokine (C-C motif) ligand 3; CFA = complete Freund’s adjuvant; IFN-γ = interferon-gamma; IL = interleukin; MOG = myelin oligodendrocyte glycoprotein; PBS = phosphate-buffered saline; PTX = Pertussis toxin; TNF = tumor necrosis factor; Veh = vehicle. (GIF 54.6 KB)

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Supplementary fig. 5

Effects of treatment with CTK 01512-2, ziconotide (Zico) (50 pmol/site, dosed at days 4, 10, 15, 20 and 24 days post-MOG35-55), by intrathecal (i.t.) route, or fingolimod (0.3 mg/kg, dosed once a day, beginning 7 days after the first MOG35-55 injection), by oral route (p.o), on the representative images of hematoxylin-eosin (HE, inflammation) staining (A), luxol fast blue (LFB, demyelination) staining (B), immunohistochemical for glial fibrillary acidic protein (GFAP, astrocytic marker) (C) and ionized-binding adapter molecule 1 (Iba1, microgila marker) (D) in the model of MOG35-55-evoked EAE in C57/BL6 mice. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Dotted rectangles demonstrate the accumulation of inflammatory infiltrate and red arrows indicate the immunopositivity in brain (cerebral cortex). The images were captured in ×100 magnification. Scale bar = 50 μm. (GIF 867 kb)

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Supplementary fig. 6

Effects of treatment with CTK 01512-2, ziconotide (Zico) (50 pmol/site, dosed at days 4, 10, 15, 20 and 24 days post-MOG35-55), by intrathecal (i.t.) route, or fingolimod (0.3 mg/kg, dosed once a day, beginning 7 days after the first MOG35-55 injection), by oral route (p.o), on [18F]-FDG metabolism of the following brain regions: thalamus (A), hypothalamus (B), superior colliculi (C), inferior colliculi (D), midbrain (E) and cingulate cortex in the model of MOG35-55-evoked experimental autoimmune encephalomyelitis (EAE) in C57/BL6 mice. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Differences in the standardized uptake value (SUV) was determined by two-way analysis of variance. Each column represents the mean and the vertical lines show the standard error mean. The experimental N of each group is provided in the graphs (A-F). #p < 0.05, ##p < 0.01 and ###p < 0.001 significantly different from naive values. *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from vehicle values. $p < 0.05 significantly different from ziconotide values. (GIF 170 kb)

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Supplementary fig. 7

Recombinant peptide CTK 01512-2 treatment prevents MOG35-55-evoked activity loss in mice. Effects of treatment with CTK 01512-2 (CTK), ziconotide (Zico) (0,2 mg/kg, every 3 days, starting on day 7, after the first MOG35-55 injection), by intravenous (i.v.) route, or fingolimod (Fingo, 0.3 mg/kg, once a day, beginning 7 days post-MOG35-55 application) by oral route (p.o.), on the ambulatory movement (A), traveled distance (B) and speed (C) in the model of experimental autoimmune encephalomyelitis (EAE)-affected mice. Representative images of the mouse movements (D) through the arena, 23 days after MOG35-55 application according to the respective treatments. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Differences in the behavioral tests were determined by one-way analysis of variance, followed by Newman-Keuls post hoc test. Each point represents the mean and the vertical lines show the standard error mean. The experimental N of each group is described in the graphs (A-C). #p < 0.05 and ##p < 0.01 significantly different from naive values. *p < 0.05 significantly different from vehicle values. B = baseline; CFA = complete Freund’s adjuvant; MOG = myelin oligodendrocyte glycoprotein; PBS = phosphate-buffered saline; PTX = Pertussis toxin; Veh = vehicle. (GIF 161 kb)

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Supplementary fig. 8

Effects of treatment with CTK 01512-2 (CTK), ziconotide (Zico) (0,2 mg/kg, every 3 days, starting on day 7, after first MOG35-55 injection), given by intravenous (i.v.) route, or fingolimod (Fingo, 0.3 mg/kg, once a day, beginning 7 days post-MOG35-55 application) given by oral route (p.o.), on the thermal nociception (A), body weight (gain/loss) (B) and spleen wet weight (C) in the model of MOG35-55-caused experimental autoimmune encephalomyelitis (EAE) in C57/BL6 mice. Fingolimod was used as a positive control drug for multiple sclerosis treatment. Differences in the hot-plate test, body and spleen weight were determined by one-way analysis of variance, followed by Newman-Keuls or Bonferroni post hoc test. Each point represents the mean and the vertical lines show the standard error mean. The experimental N of each group is described in the graphs (A-C). ##p < 0.01 and ###p < 0.001 significantly different from naive values. **p < 0.01 and ***p < 0.001 significantly different from vehicle values. $$p < 0.01 and $$$p < 0.001 significantly different from ziconotide values. B = baseline; CFA = complete Freund’s adjuvant; MOG = myelin oligodendrocyte glycoprotein; PBS = phosphate-buffered saline; PTX = Pertussis toxin; Veh = vehicle. (GIF 85.2 KB)

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Supplementary Table 1

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Supplementary video 1

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Supplementary video 2

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Supplementary video 3

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Silva, R.B.M., Greggio, S., Venturin, G.T. et al. Beneficial Effects of the Calcium Channel Blocker CTK 01512-2 in a Mouse Model of Multiple Sclerosis. Mol Neurobiol 55, 9307–9327 (2018). https://doi.org/10.1007/s12035-018-1049-1

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