TnP is a family of patented synthetic peptides which is in a preclinical development stage with valuable potential therapeutic indication for multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system (CNS). The use of a preclinical animal model, such as experimental autoimmune encephalomyelitis (EAE) has deepened our knowledge of the immunomodulatory functions of TnP as a drug. We have shown that TnP possesses a disease suppressive function in EAE, ameliorating disease severity by 40% and suppressing the accumulation of T helper (Th)1- and Th17-producing lymphocytes (by 55% and 60%, respectively) in CNS along with activated microglia/macrophages populations (by 33% and 50%, respectively), and also conferred a protective effect anticipating the remyelination process to day 66 compared to day 83 of untreated cuprizone-mice. Here we expanded our knowledge about its effects compared with current first-line disease-modifying therapies (DMT). We demonstrated that prophylactic treatment with TnP generated similar protection to betaseron (30%) or was more effective than glatiramer (44% versus 6%) or fingolimod (50% versus 19%) against the development of clinical symptoms. Although TnP controlled the leukocyte infiltration (87% versus 82%) into demyelinated areas of the spinal cord in the same way as betaseron and fingolimod, it was more effective (72% to 78% decrease) in the long-term control of neuronal degeneration compared to them. Also, when compared to glatiramer, TnP was more efficient in reversing leukocytes infiltration into the spinal cord (55% versus 24%), as well as induced a higher percentage of regulatory cells in spleen (2.9-fold versus 2.3-fold increase over vehicle-treated EAE mice) an in the spinal cord (8-fold versus 6-fold increase over vehicle-treated EAE mice). This specialized TnP profile for inducing immune tolerance and neuronal regeneration has significant therapeutic potential for the treatment of MS and other autoimmune diseases.
Peptides as drugs have advanced and continue to grow with scientific innovation, expanding into new indications and molecular targets. Peptides represent a small portion (2%) of the world drug market, but together successful market peptides such as Copaxone (glatiramer acetate), Lupron, Zoladex, Sandostatin, and Velcade totaled about $25 billion in sales in 2018.
There are currently ∼105 approved peptide drugs on the market, ∼200 in clinical development and ∼600 in the preclinical discovery stage. 86% of peptide drugs with therapeutic purposes or as approved molecular probes and diagnostic tools are of natural origin or synthetic derivatives from a natural source and the vast majority of them have a molar mass <2000 g/mol. The peptide market is growing twice as fast as the rest of the drug market, suggesting that peptides may soon occupy an ever larger niche in the therapeutic arsenal for metabolic, central nervous system (CNS), oncology, cardiovascular and chronic inflammatory diseases.
The oceans are an exceptionally rich source of bioactive metabolites, including the TnP peptide extracted from the venom of the niquim, the Brazilian fish Thalassophryne nattereri. Our group identified and patented the family of analog peptides called the TnP family which invention relates to synthetic peptides containing a sequence of 13 L-amino acids and with a disulfide bond between Cys4 and Cys13 in its structure, and is in a preclinical development stage with valuable potential therapeutic indicated for chronic inflammatory diseases such as asthma and multiple sclerosis (MS). Measures aimed to improve the metabolic stability and half-life of the cyclic peptide TnP were performed, including N-terminal amidation. Our data in the patent show that cyclic TnP has remarkable resistance to the action of proteolytic enzymes such as trypsin and pepsin, and its high solubility allows it to be rapidly absorbed. In mice, we observed that immunization with TnP at different doses, in the presence or absence of adjuvant, does not generate specific antibodies and, therefore, the molecule is not immunogenic.
The use of experimental systems, such as experimental autoimmune encephalomyelitis - EAE, has deepened our knowledge of the immunomodulatory functions of TnP as a drug. In the extensive work carried out by our group, we found that subcutaneous treatment with TnP successfully improves the severity of clinical signs of myelin oligodendrocyte glycoprotein (MOG)-induced EAE, delaying the onset of maximal symptoms (4 days) and decreasing the severity of symptoms by 40% compared with control EAE mice treated with vehicle alone. TnP beneficially interferes with the immune circuit at various stages by partially IL-10-dependent mechanisms, including suppressing activation of conventional dendritic cells (DC) and providing the emergence of plasmacytoid DC and regulatory cells during the EAE induction phase; blocking the transit and infiltration of leukocytes into the CNS by suppressing matrix metalloproteinase (MMP)-9 activity and CD18 expression; blocking the reactivation and permanence of Th1 and Th17 lymphocytes in the CNS; the prevention of microglial expansion and macrophage infiltration into the CNS; favoring the localized increase in regulatory T cells; and finally, suppressing demyelination in the spinal cord of EAE mice and leading to accelerated remyelination in a cuprizone model.
In view of the advance in the preclinical development chain, the safety and biodistribution of TnP were recently investigated using zebrafish as a model for preclinical toxicology studies. We reveal a broad therapeutic index with non-lethal doses ranging from 1 nM to 10 μM, without neurotoxicity or cardiotoxic effects. The low frequency of TnP-induced abnormalities in embryos was associated with the high safety of the molecule and the ability of the developing embryo to process and eliminate it. TnP crossed the blood-brain barrier without disturbing the development of the normal architecture of the forebrain, midbrain, and hindbrain.
Currently, the TnP family invention is patented in Brazil (PI0602885-3A2, original #PI0703175-0, 09/04/2019); Europe – WO/2008/009085 (2008); United States – US20100144607 (2012); Canada - CA2657338 (2013); China - CN101511861 (2013); Hong Kong - HK1135406 (19/09/2013); India – IN94/MUMNP/2009 (2013); South Korea - KR1020090037900 (2014), and Japan – JP2010503613 (2014), and thanks to its low toxicity and pharmacological characteristics, it is presented as a candidate for the development of new pharmacological strategies to reduce the effects of multiple sclerosis. Here the aim of our work is to expand our knowledge about its effects compared with current disease-modifying therapies (DMTs).
The first large scale human clinical trial in patients with relapsing-remitting MS (RRMS) using interferon beta (IFN-β) was published in 1993 and showed that relapse rates were reduced by 34% in high dose IFN-β1b and by 8% in lower dose compared to placebo group, and severity of relapses was also reduced.
To study the manifestations of EAE as well as TnP compared to betaseron subcutaneous (s.c.) prophylactic treatment in the CNS, we used the MOG 35–55 peptide-induced EAE model in the susceptible strain. Female C57BL/6J (BL6) mice induced with EAE and treated with vehicle exhibited chronic disease progression with onset around day 11 post-induction (p.i.), exhibiting mean maximal clinical score of 2.3 ± 0.09 at day 17 p.i. that was maintained in chronic phase, grade 2–2.1.
TnP (3 mg/Kg) or betaseron (10,000 UI/mice) treatments started with s.c. daily application by 10 days during induction phase. EAE symptoms appeared on day 12 p.i. for both treated-groups (0.1 ± 0.01 and 0.2 ± 0.01, respectively). TnP as well as betaseron both ameliorated the clinical manifestations of EAE decreasing by 30% the mean maximal clinical score from 2.3 ± 0.09 to 1.6 ± 0.02. TnP delayed the onset of maximal symptoms by 2 days (19) and betaseron delayed the onset of symptoms by 1 day (18). The beneficial effect was stable over time and sustained until day 30.
Because immune cell infiltration of the CNS is considered a hallmark of EAE, we investigated the effect of TnP compared to betaseron on leukocyte infiltration in the CNS by the analysis of hematoxylin and eosin (H&E) or with Luxol fast blue (LFB) paraffin sections at day 17. While vehicle-treated EAE mice presented extensive infiltration of inflammatory cells per mm 2 (254 ± 4.3) and areas of demyelination (score 3), tissue from TnP- (34 ± 2.3 and score 1.5) or betaseron-treated mice (45 ± 1.9 and score 1.2) lacked severe signs of inflammation with preserved myelinated areas.
IFNs do not directly exert a neuroprotective effect; however, through their direct effect on encephalitogenic CD4+Th1 cells, altering their activation state trigger a decrease in neuronal demyelination, which prevents further neuronal damage. Next, Fluoro-Jade C was used to evaluate the degree of neurodegeneration in damage areas of the spinal cord of vehicle- or treated-EAE mice and the corrected total cell fluorescence (CTCF) was obtained. Vehicle-treated EAE mice exhibited a significantly increased number of degenerating neurons on day 17 (1.12 ± 0.01). Degeneration progressively intensified up to day 30 p.i. in this group, from central to the peripheral areas (2.29 ± 0.01). The TnP-treated group of mice showed a mild degeneration (0.54 ± 0.01) in the central region that remained similar until day 30 (0.65 ± 0.01). In contrast, the intense neuronal degeneration seen on day 17 (3.38 ± 0.01) in betaseron-treated group was observed to increase at day 30 (3.99 ± 0.01). In summary, these data show that subcutaneous treatment with TnP was more effective in the long-term control of neuronal degeneration.
Like IFN-β, glatiramer exerts broad immunomodulatory effects that are incompletely understood. In patients, glatiramer significantly reduced disease symptoms and development of new lesions by up to 30% in RRMS, although it showed no improvement in long-term efficacy on progression of disability. Next we examined the role of TnP in the control of EAE symptoms compared with glatiramer in a prophylactic regimen of subcutaneous application.
Mice that received TnP showed substantially fewer neurological deficits by the measurement of the mean maximal clinical score of 0.9 ± 0.03 than mice that received glatiramer (1.5 ± 0.03) or vehicle-treated EAE mice (1.6 ± 0.01). The decrease in mean maximal clinical score induced by glatiramer was 6% in the first 5 days compared to 44% of decrease caused by TnP treatment which lasted for 30 days. In addition, we observed that TnP delayed the peak of symptoms by 4 days, while glatiramer accelerated it by 1 day.
At the peak of disease (day 17), microglia and macrophage infiltrations were examined in the CNS of vehicle- or treated-groups. The percentage of CD45 high CD11b high macrophages infiltrating the brain or spinal cord decreased by 74% and 55%, respectively, after TnP treatment compared to a 26% and 24% of decrease caused by glatiramer. In addition, TnP caused a 37% of decrease in CD45 low CD11b low microglia in the brain and 79% in the spinal cord compared to percentages of 12% and 60% induced by glatiramer.
Glatiramer treatment results in the skewing of auto-reactive lymphocytes away from the pathogenic effector cell responses towards regulatory functions. The next step was to compare the ability of prophylactic treatment with TnP to induce regulatory T and B cells in the presence of EAE. As showed in an increase in the percentage of activated CD19+CD1d+CD5+B regulatory (Breg) during the priming phase was also observed in the spleen of TnP- (2.9-fold) as well as glatiramer- (2.3-fold) compared to vehicle-treated EAE mice, consistent with a systemic effect of both treatments.
We also examined the percentage of conventional CD4+CD25+FOXP3+T regulatory (Treg) cells in the CNS. A higher percentage of CD4+ Treg cells was detected in the CNS mainly in the spinal cord of TnP-treated mice during the peak phase of the disease (2.3-fold and 8-fold increase), compared to a percentage (2-fold and 6-fold increase) of these cells in the CNS of glatiramer-treated mice.
