Periodic Reporting for period 4 - NOVIRUSES2BRAIN (''One size fits all'' unique drug to eradicate multiple viral species simultaneously from the central nervous system of co-infected individuals)
Periodo di rendicontazione: 2023-06-01 al 2024-08-31
Viruses that infect the brain and the rest of the central nervous system pose a serious global health threat as they may lead to severe neurological impairments. Viruses such as Zika (ZIKV), Dengue (DENV), HIV, and SARS-CoV-2 have been responsible for significant public health crises. Notably, the Zika virus outbreak in South America underscored the devastating impact of neurotropic viral infections, particularly on pregnant women, where translocation across the blood-placenta barrier (BPB) led to congenital neurological disorders, including microcephaly.
Despite the high risk of co-infections with Aedes-borne viruses like Zika, Dengue, and Chikungunya, as well as other neurotropic viruses such as HIV and SARS-CoV-2, current drug development strategies largely ignore the reality of multiple infections. There remains an urgent need for antiviral treatments capable of crossing biological barriers, particularly the blood-brain barrier (BBB), to effectively target these viruses where they reside in the brain. Also, the recent COVID-19 pandemic raised awareness for the need to develop broad spectrum antivirals.
The NOVIRUSES2BRAIN project aims to develop innovative peptide-based drug candidates capable of crossing the BBB and BPB to combat brain-residing viruses. By designing antiviral peptide-porphyrin conjugates (PPCs) with enhanced brain targeting and efficacy, the project seeks to establish a new paradigm in antiviral drug development, ensuring that critical infections can be treated even when they affect the brain. The final phase of the project has successfully demonstrated the ability of some lead compounds to cross the BBB and reduce viral loads in vivo in preclinical models, this proof of concept being a significant step towards the preparation of future clinical trials.
Despite the high risk of co-infections with Aedes-borne viruses like Zika, Dengue, and Chikungunya, as well as other neurotropic viruses such as HIV and SARS-CoV-2, current drug development strategies largely ignore the reality of multiple infections. There remains an urgent need for antiviral treatments capable of crossing biological barriers, particularly the blood-brain barrier (BBB), to effectively target these viruses where they reside in the brain. Also, the recent COVID-19 pandemic raised awareness for the need to develop broad spectrum antivirals.
The NOVIRUSES2BRAIN project aims to develop innovative peptide-based drug candidates capable of crossing the BBB and BPB to combat brain-residing viruses. By designing antiviral peptide-porphyrin conjugates (PPCs) with enhanced brain targeting and efficacy, the project seeks to establish a new paradigm in antiviral drug development, ensuring that critical infections can be treated even when they affect the brain. The final phase of the project has successfully demonstrated the ability of some lead compounds to cross the BBB and reduce viral loads in vivo in preclinical models, this proof of concept being a significant step towards the preparation of future clinical trials.
We designed and synthesized a pool of antiviral peptide-porphyrin conjugates (PPCs) capable of crossing the blood-brain barrier (BBB) and blood-placenta barrier (BPB) while exhibiting antiviral activity. To evaluate their ability to traverse the BBB and BPB, as well as their impact on the integrity and functionality of both barriers, we tested the PPCs in in vitro models using mouse and human brain cell lines. Some PPCs efficiently crossed the BBB and BPB models without compromising barrier integrity. Further investigations revealed how these PPCs are transported inside cells, shedding light on their drug delivery mechanisms to the brain. To assess their effectiveness, we tested the antiviral activity of the PPCs against ZIKV, DENV, HIV, and SARS-CoV-2 in vitro. Four PPCs demonstrated potent anti-infective efficacy against most of these viruses. The most promising PPCs, identified through in vitro studies, continued to be synthesized for subsequent in vivo studies. ZIKV infection during pregnancy can lead to microcephaly, a birth defect characterized by an abnormally small head. Therefore, we assessed the therapeutic potential of PPCs using a neonatal mouse model of ZIKV infection. We evaluated viral load in the brain and spleen, clinical signs, mortality rates, brain damage, and brain inflammation. We successfully ranked drug leads for in vivo efficacy based on their effectiveness and cytotoxic safety profiles. P-H3 and P-H1, at 7 mg/kg, demonstrated the highest efficacy. An adult disease mouse model was also used, in which PPCs were initially administered at a low concentration. Although preliminary data showed that PPCs did not significantly reduce viral load in the brains of infected adult mice, some PPC-treated animals exhibited a lower viral load compared to untreated infected mice, suggesting that a higher dose of PPCs may lead to significant viral load reduction. Currently, we are conducting assays using a higher concentration of PPCs in adult mouse models.
To extend toxicity evaluations to non-rodent species, we characterized toxic effects in two well-established model organisms: Galleria mellonella (wax moth larvae) and Danio rerio (zebrafish). Our previous in vivo toxicology studies showed that PPCs administration in non-infected newborn mice did not impact survival. Building on this, we assessed the toxicity of P-H1 and P-H3 in Galleria mellonella larvae. Larvae at the last instar stage were injected with PPCs at different doses. Both P-H1 and P-H3 were generally well tolerated, except for the highest dose of P-H1 (140 mg/Kg), which resulted in a 10% reduction in larval survival.
We conducted in vivo ADME-tox assays, starting with the biodistribution of selected conjugates and their physiological clearance from the blood using adult female animal models. Pharmacokinetic profiles were generated for different candidates, with steady-state determinations performed at different doses. P-H3 stood out due to its extended half-life and robust pharmacokinetic parameters, making it highly suitable for further therapeutic development. Following P-H3, P-H1 demonstrated high brain penetration, positioning it as a promising candidate for targeting central nervous system (CNS) disorders.
Beyond laboratory research, the project team is engaging with regulators, including the European Medicines Agency (EMA), to explore the potential clinical pathway for these antiviral therapies. Contact with clinical trial sponsors such as SwissPTH and industrial stakeholders, such as Dompe, have also been carried out. Dissemination efforts included presentations at national and international scientific conferences, publications of research papers, patent filings, and outreach via social media, TV, and public engagement events.The next steps will involve completing toxicology studies to confirm the safety of these drug candidates in animals, paving the way for future human trials. The project’s promising findings could contribute to new treatments for infection by brain-targeting viruses and provide crucial insights for antiviral drug development.
To extend toxicity evaluations to non-rodent species, we characterized toxic effects in two well-established model organisms: Galleria mellonella (wax moth larvae) and Danio rerio (zebrafish). Our previous in vivo toxicology studies showed that PPCs administration in non-infected newborn mice did not impact survival. Building on this, we assessed the toxicity of P-H1 and P-H3 in Galleria mellonella larvae. Larvae at the last instar stage were injected with PPCs at different doses. Both P-H1 and P-H3 were generally well tolerated, except for the highest dose of P-H1 (140 mg/Kg), which resulted in a 10% reduction in larval survival.
We conducted in vivo ADME-tox assays, starting with the biodistribution of selected conjugates and their physiological clearance from the blood using adult female animal models. Pharmacokinetic profiles were generated for different candidates, with steady-state determinations performed at different doses. P-H3 stood out due to its extended half-life and robust pharmacokinetic parameters, making it highly suitable for further therapeutic development. Following P-H3, P-H1 demonstrated high brain penetration, positioning it as a promising candidate for targeting central nervous system (CNS) disorders.
Beyond laboratory research, the project team is engaging with regulators, including the European Medicines Agency (EMA), to explore the potential clinical pathway for these antiviral therapies. Contact with clinical trial sponsors such as SwissPTH and industrial stakeholders, such as Dompe, have also been carried out. Dissemination efforts included presentations at national and international scientific conferences, publications of research papers, patent filings, and outreach via social media, TV, and public engagement events.The next steps will involve completing toxicology studies to confirm the safety of these drug candidates in animals, paving the way for future human trials. The project’s promising findings could contribute to new treatments for infection by brain-targeting viruses and provide crucial insights for antiviral drug development.
NOVIRUSES2BRAIN employs an interdisciplinary approach integrating medicinal chemistry, biochemistry, biophysics, and in vivo virology studies to address the pressing need for antiviral drugs that can penetrate the brain. Currently, there are no approved drugs specifically targeting Zika or other Aedes-borne viruses, highlighting the urgency of developing novel broad-spectrum antivirals. Building on previous discoveries of molecules with antiviral activity and others with the ability to cross biological barriers, the project has successfully combined these properties into peptide-drug conjugates, namely a small library of peptide-porphyrin conjugates (PPCs). The project’s impact extends beyond scientific achievements. Engagement with regulatory bodies such as the European Medicines Agency (EMA) will facilitate discussions on potential clinical pathways for these antivirals. Dissemination efforts have included presentations at national and international conferences, publication of research findings, patent applications, and outreach activities through social media, television, and public engagement events.Looking ahead, the final phase of the project will focus on completing physiological toxicology studies to confirm the safety of these drug candidates in animal models, paving the way for human trials. The outcomes of NOVIRUSES2BRAIN could revolutionize antiviral treatment strategies by providing effective therapeutics for brain-targeting viruses, addressing an unmet medical need, and strengthening Europe’s capacity to develop antiviral drugs.