RNA VIRAL CHALLENGES

Besides HIV and HCV, First Health Pharmaceuticals extends its commitment to further develop RNA Helicase inhibiting lead compounds to other RNA viruses such as Ebola virus, Dengue virus, West Nile virus, Yellow Fever virus, Chikungunya virus.

Aedes Mosquito viral vector

It should be noted that several of these viruses such as WNV, YFV, DENV and ZIKV are ARthropod-Born (ARBO) viruses, meaning that their viral vector is a mosquito and more specifically, a tropical Tiger mosquito of the Aedes genre. Due to several factors such as increasing travel, global warming and urbanisation, Tiger mosquitoes of different species are expanding their territory worldwide, invading also regions of Europe and North America that were previously spared from Arbovirus epidemics.

Aedes Aegypti is a known vector of several viruses including Yellow Fever virus, Dengue virus and Chikungunya virus. Hundreds of imported cases are reported in Europe every year, including fatal cases. Therefore, the establishment of this mosquito raises in Europe concerns about autochthonous arbovirus transmission, particularly in southern Europe. where climatic conditions are more suitable for the re-establishment of this species. In 2012, a large outbreak of dengue fever occurred in the Portuguese Autonomous Region of Madeira associated with Ae. Aegypti. The epidemic started in October 2012 and by early January 2013 more than 2 000 cases of dengue fever had been reported, with an additional 78 cases reported among European travellers returning from the island.

The success of this invasive species has largely been due to globalisation. It thrives in densely populated areas that lack reliable water supplies, waste management and sanitation. Historically, Aedes Aegypti has moved from continent to continent via ships, and this method of dispersal is thought to present the highest risk of introducing this mosquito into continental Europe from Madeira. It is suggested that Aedes Aegypti evolved its domestic behaviour in West Africa and its widespread distribution and colonisation in the tropics led to the highly efficient inter-human transmission of viruses such as dengue. This domestic behaviour can provide protection against environmental conditions (as it rests indoors) and numerous suitable habitats as oviposition sites, but can also result in increased sensitivity to control measures used to eliminate them.

The European Tiger mosquito Aedes albopictus was the primary vector in the important 2007 outbreak of Zika virus in Gabon. The virus currently spreads in the Americas by means of the Aedes Aegypti vector, but there is clear evidence to assume that Aedes albopictus is a viable vector of Zika virus as well. Laboratory experiments have shown that south east Asian populations of Aedes albopictus secrete the virus in their saliva. There is little doubt that the European and North American albopictus populations will compete against Zika vectors as well. It should, as such, be taken into serious consideration that vector born Zika could become endemic in Europe and North America as well.

Dengue

Dengue fever is a mosquito-born viral infection that has rapidly spread in all regions of WHO in recent years. Dengue virus is transmitted by female mosquitoes mainly of the species Aedes Aegypti (tiger mosquito). Severe Dengue affects most Asian and Latin American countries and has become a leading cause of hospitalization and death among children in these regions. The incidence of Dengue has grown dramatically around the world in recent decades. The actual numbers of Dengue cases are underreported and many cases are misclassified. One recent estimate indicates 390 million Dengue infections per year, of which 96 million have clinically manifested (with any severity of disease). Another study, of the prevalence of Dengue, estimates that 3,9 billion people, in 128 countries, are at risk of infection with Dengue viruses. Not only is the number of cases increasing as the disease spreads to new areas, but explosive outbreaks are occurring. The threat of a possible outbreak of Dengue fever now exists in Europe and local transmission of Dengue was reported for the first time in France and Croatia in 2010 and imported cases were detected in 3 other European countries. In 2012 an outbreak of Dengue on the Madeira islands of Portugal resulted in over 2000 cases and imported cases were detected in mainland Portugal and 10 other countries in Europe.

Severe haemorrhagic Dengue is a potentially deadly complication due to plasma leaking, fluid accumulation, respiratory distress, severe bleeding, or organ impairment. Warning signs occur 3–7 days after the first symptoms in conjunction with a decrease in temperature (below 38°C/100°F) and include: severe abdominal pain, persistent vomiting, rapid breathing, bleeding gums, fatigue, restlessness and blood in vomit. The next 24–48 hours of the critical stage can be lethal; proper medical care is needed to avoid complications and risk of death. Shock (Dengue shock syndrome) and hemorrhage (Dengue hemorrhagic fever) occur in less than 5% of all cases of Dengue, however those who have previously been infected with other serotypes of Dengue virus (“secondary infection”) are at an increased risk. This critical phase, while rare, occurs relatively more commonly in children and young adults.

Developing a vaccine against the disease is challenging. With five different serotypes of the Dengue virus that can cause the disease, the vaccine must immunize against all five types to be effective. Vaccination against only one serotype could possibly lead to severe Dengue hemorrhagic shock (DHS), when infected with another serotype because of an antibody-dependent enhancement. A common problem faced in Dengue-endemic regions is when mothers become infected with Dengue after giving birth, offspring carry the immunity from their mother and are susceptible to hemorrhagic fever if infected with any of the other three serotypes.

There is no specific treatment for Dengue fever. For severe Dengue medical care by physicians and nurses, experienced with the effects and progression of the disease, can save lives – decreasing mortality rates from more than 20% to less than 1%. Maintenance of the patient’s body fluid volume is critical to severe Dengue care.

One of our compounds successfully inhibits both West Nile Virus (WNV) and Dengue Virus (DENV) with sub-Micromolar effective concentrations. The compound also has excellent low toxicity in several CC50 assays. As such, this Translation Inhibitor compound should be considered as a highly promising drug, candidate against both WNV and DENV and improvements are currently being made on the molecular structure in order to further enhance the antiviral efficacy.

Zika Virus

Zika virus is a member of the Flaviviridae family and is transmitted to humans by mosquitoes. It is related to other pathogenic vector born flaviviruses including dengue, West-Nile and Japanese encephalitis viruses, but it produces a comparatively mild disease in humans.

Since 2007 Zika virus has caused several outbreaks in the Pacific, and since 2015 it further spread in the Americas. These were the first documented transmissions outside of its traditional endemic areas, in Africa and Asia. Zika virus is considered an emerging infectious disease with the potential to spread to new areas where the Aedes mosquito vector is present. There is no evidence of transmission of Zika virus in Europe to date.

An evolving outbreak of Zika virus infections is currently spreading in the Americas and the Pacific region, coinciding with an increase in cases of microcephaly and other adverse outcomes during pregnancy and of Guillain–Barré syndrome (GBS) in adults. On 1 February 2016 WHO declared a Public Health Emergency of International Concern (PHEIC) regarding clusters of microcephaly cases and neurological disorders in some areas affected by Zika virus.

The Zika virus is known to pass the maternal-foetal barrier of the placenta in infected mothers, potentially causing harm to the Central Nervous System of unborn babies, with several cases of microcephaly, being reported during the 2015/16 South American Zika fever outbreak.

Our Translation Inhibitor class antiviral compounds have proved to be active against Zika virus and the development of two types of specific Zika antivirals is being pursued.

Our first approach starts from the point of view that it would be better for unborn babies if they were never infected with the Zika virus in the first place, while at the same time it would be desirable to protect the foetus from exposure to any unnecessary chemical agents during its development. As such, we are currently focusing our attention on the development of a Zika antivirus specifically intended to protect the mother in a prophylactic way, without the compound surpassing the maternal-foetal placenta barrier. Such an antivirus could subsequently be subscribed to mothers during the first months of their pregnancy, whenever and wherever the risk of Zika contagion increases..

A pre-selection of compounds, susceptible to be blocked by the placenta barrier has taken place among existing portfolios of active Translation Inhibitors, which are known to posses the desired anti Zika Virus activity;  selection criteria were a reduced lipid solubility and a high molecular weight. These compounds are subject to a placenta-focused ADME optimization model and subsequently reviewed in in-vitro distribution assays.

A secondary development approach focuses on a Zika antiviral to use in already infected mothers and foetuses. This compound will have to surpass both the maternal-foetal barriers as well as the blood brain barrier of the unborn. It should be clear that the first mentioned approach will have to be preferred, considering the still unknown effects of Translation Inhibitors on foetal development. However, considering the seriousness and rapid expansion of the Zika / microcephaly threat, it might very well be that even this approach will prove to outweigh the involved risks.

Ebola Virus

The 2014 Ebola outbreak in western Africa with subsequent confirmed cases in both the US and Europe has made it clear that outbreaks of Ebola hemorrhagic fever are hard to control and may spread beyond the boundaries of their host territory. Ebola virus belongs to the class of Filoviridae Group V negative sense ssRNA viruses and like all RNA viruses it has a very high mutation rate compared to DNA viruses, because viral RNA polymerases lack the proof-reading ability of DNA polymerases. This is why it is difficult to make effective vaccines to prevent diseases caused by RNA viruses. No approved, effective, antiviral treatment is currently available and as such the Ebola case fatality rate is very high. A handful of experimental drugs are available, and some seem to be able to offer treatment benefit, however none of the current options has, thus far, been very successful in clinical trials.

Deadly as Ebola hemorrhagic fever may seem, from an epidemiological point of view, the overall Ebola death rate (even during the 2014 epidemic) is low, when compared, for instance, to the far more common Malaria or Dengue Fever. This is largely due to the fact that overall relatively few people are infected during an Ebola outbreak. Ebola is less contagious than other more common viruses such as Influenza viruses. Direct contact with body fluids is necessary and infection usually only occurs after the manifestation of the first disease symptoms after which the unfortunate patient quickly becomes incapacitated, limiting his further movements and thus the geographical expansion of the viral vector.

11.000 Ebola fatalities during 2014 outbreak represented, of course, a high death toll, but almost insignificant when compared to the over 660.000 deaths caused each year by malaria. The significance of Ebola hemorrhagic fever is strongly connected to the fear of the virus spreading to the western world and the significant resources involved in containing the regular annual outbreaks from their Central African host territory. Considering the scale of 2014 Ebola epidemic, the high costs involved in the containment and management of the outbreak and the difficulties involved in vaccine development, the development of an effective hemorrhagic fever antivirus has become a priority on the global political agenda.

Our RNA Translation Inhibitor compounds were tested in vitro against the Ebola virus and proved effective at clinically relevant micro molar concentrations. Further testing and development are currently taking place in collaboration with the United States Army Medical Research Institute for infectious diseases (USAMRIID), which owns qualified facilities for researching Biohazard Level 4 viruses.

Therapeutic Index

The in-vitro toxicity of the compounds is expressed by the Half Maximal Cytotoxic Concentration (CC50). This value expresses the concentration of the compound that will lead to death of 50% of the cells in a treated population,  respect to an untreated one; thus, “the higher the concentration is, the less toxic are the compounds”. In our case we have data from different in-vitro models used to test the efficacy against virus infections of our molecules (EC50 value), which provide to us  the possibility to make both direct comparisons between the Cytotoxic concentrations in the different cell types (CC50) and the comparison with the effective concentration (EC50). Just to give an example, we tested on LucUbiNeo-ET hepatic cells, peripheral blood mononuclear cells PBMC, baby hamster kidney cells BHK-21, HUH7 hepatocarcinoma cell lines and more.

From the obtained results it immediately becomes clear that both the PBMC, the BHK-21 and the HUH7 assays show no cytotoxic problems whatsoever, with very high (off the scale) concentration levels along the line; which is, of course, a very promising result. Assays based on hepatic LucUbiNeo-ET cells do provide more responsive data on potentially cytotoxic concentrations of some of the compounds in parallel to their efficacy. Such a data is taken in serious consideration when evaluating the potential toxicity of the compounds. On the basis of these considerations, we may conclude that all our drug candidate compounds provide high (very satisfying) Cytotoxic Concentrations in the hepatic LucUbiNeo-ET cell assay as well as in the MTT assay in other cell types, with CC50 values, ranging from 70µM to 200µM.

High cytotoxic (CC50) concentrations in combination with low, sub-micro molar (more effective) EC50 concentrations provide insight in the possible toxic side effects of prolonged use of pharmaceuticals. The multiplier between the effective and the cytotoxic concentrations is known as the Therapeutic Index and a high Index value is considered  a very reassuring factor for every drug candidate. For instance, a multiplier of fifteen would mean that a patient can take fifteen times the dosage to cure his disease before it becomes toxic. In our case, sub-micro molar efficacy concentrations combined with ≥200µM Cytotoxic Concentrations make therapeutic indexes exceeding 500x in several of our antiviral drug candidates. This is, of course, highly promising when it comes to scenarios of long term drug exposure, frequently associated with challenging viral infections like HCV and HIV.