WO2009059388A1

WO2009059388A1 - Use of antagonists of platelet receptor factor for the treatment of infections caused by the influenza virus

Info

Publication number: WO2009059388A1
Application number: PCT/BR2008/000349
Authority: WO
Inventors: Ricardo Tostes Gazzinelli; Mauro Martins Teixeira; Cristina Couto Garcia; Alexandre de Magalhães VIEIRA MACHADO; Remo De Castro Russo
Original assignee: Universidad Federal De Minas Gerais- Ufmg
Priority date: 2007-11-08
Filing date: 2008-11-10
Publication date: 2009-05-14
Also published as: BRPI0705586A2; BRPI0705586B8; BRPI0705586B1

Abstract

The present invention refers to the receptor antagonists of the platelet activation factor (PAF), their analogues and/or derivatives for treating infections caused by the influenza virus.

Description

"USE OF ANTAGONISTS OF PLATELET RECEPTOR FACTOR FOR THE TREATMENT OF INFECTIONS CAUSED BY THE INFLUENZA VIRUS"

The flu virus belongs to the Orthomyxoviridae (from Greek, orthos - correct and myxa - mucus) family, and is an enveloped, spherical and filamentous virus with a single stranded negative-sense RNA. The Orthomyxoviridae family is made up of four different genuses: influenza A, B, C and thogotovirus. Distinction between genuses is made mainly through differences in coding sequences of nucleocapsid and matrix proteins. Another distinction between genuses is perceived through their material genetic composition: while genuses A and B have eight different RNA segments, genus C has 7 such segments. Hemagglutinin (HA) and neuraminidase (NA) - two glycoproteins present on the surface of viral particles of A and B influenza virus - are their main antigenic determinants. The genus C of influenza virus has, in turn, only one surface glycoprotein. (Knipe, et al, 2001).

The Influenza A viruses are classified in different subtypes, which are based on serologic differences and genetic composition of their surface glycoproteins. So far, sixteen types of hemagglutinin, H1-H16, and nine types of neuraminidase, N1-N9 (Cox e Subbarao, 1999) have been identified. Clinically speaking, these A-type viruses are more important as they are highly contagious, causing infections in the upper respiratory tract of humans of all ages, swines and equines and also in several species of birds (Julkunen, et al, 2001 ; Knipe, et al, 2001).

Wild aquatic birds are the reservoir of all influenza A subtypes, whose infection is asymptomatic in these animals. However, aquatic birds may release high concentrations of influenza viruses through their feces for a long period, thus allowing other birds to be infected as well as other species sometimes. In the latter case, infections can be very severe, as in human cases, caused by the avian influenza virus subtype H5N1. (Widjaja, et al, 2004). A peculiar characteristic of the Influenza virus is its ability to cause recurrent annual epidemics and occasional endemics in human population, by spreading swiftly and affecting people of all ages. Epidemics are caused by viruses which have undergone mutations in proteins HA and/or NA, in such a way that the antibodies generated during previous infections or vaccinations are not anymore able to recognize and neutralize new influenza variants. (Cox & Subbarao, 1999). New hemagglutinin genes appear in wild fowl and can adapt in mammals, causing such pandemic surges. A pandemic surge occurs after a new modified virus subtype has appeared which causes more severe infections than those caused by isolated viruses from seasonal flu, in individuals with higher immunological susceptibility. (Reid, et al, 1999). The first report on a pandemic caused by the influenza virus dates back to

1580. It started in Asia and spread all over Europe, Africa and Americas (Garcia- Garcia and Ramos, 2006). The greatest ever-reported influenza pandemic occurred in 1918, known as the Spanish flu, which killed from 20 million to 40 million people around the world. A recently isolated virus from tissues of victimized people by this pandemic revealed that it belonged to the H1 N1 subtype. New pandemics surged in 1957, 1968 and 1977 again caused by other subtype viruses, respectively H2N2, H3N2 and H1 N1 (Webby e Webster, 2003). From 1997, the highly pathogenic subtype H5N1 , spread by fowl, were able to infect humans in Hong Kong and other subtypes also of high pathogenicity - H9N2, H7N7, H7N3, H7N2 e H7N1 - have been isolated from humans or birds in different parts of the world and since then another pandemic is expected to occur (Garcia-Garcia and Ramos, 2006; Webby e Webster, 2003).

In 2001 , the World Health Organization - WHO - created a global agenda for monitoring and controlling the influenza virus so as to improve diagnosis techniques, vaccine production and knowledge on risks of new pandemic surge (Webby e Webster, 2003).

The first human virus isolated by Wilson Smith was adapted in order to infect the central nervous system of mice, by creating the virus strain A/WSN/33 (A from Influenza A; WS, from Wilson Smith; N, from neuroadapted and 33, as it was isolated in 1933). Such a virus property is due to a mutation in the gene called neuraminidase, which in position 130, does not have a glycosylation site found in other strains (Li, et al, 1993). Besides being used in neurotropic model, the WSN/H1 N1 strain is also basic in study models of lung infection in mice (Bot, et al, 1998) and for constructing recombinant viruses (Kash, et al, 2004).

The flu is an acute respiratory disease with higher prevalence and high transmission potential. The virus replicates in column cells of the respiratory tract epithelium, where it accesses respiratory secretions and is transmitted through coughing, sneezing and speaking. A single person is able to transmit the virus to a good number of susceptible people. Subtypes H1 N1 and H2N3 can cause the most common annual endemic, the symptoms of which are fatigue, headache, sore throat, fever, cough, body ache and nasal congestion that appear one or two days after infection (Cox & Subbarao, 1999).

Viruses that were multiplied in the respiratory epithelium of the affected organism infect alveolar macrophages that eventually die by apoptosis while epithelium cells die by necrosis. These signs activate production of cytokines- TNF-α and IL-1 and chemokines - MCP-1/CCL2, RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, IP- 10/CXCL10 and IL-8/CXCL8 (Julkunen, et al, 2001). The release of such inflammatory mediators is responsible for the recruiting of neutrophiles, macrophages and lymphocytes T to the lung tissue after infection. This initial inflammatory response is associated with the most severe forms of the illness (La Gruta, et al, 2007). Study models in animals and humans show that influenza A virus activates neutrophiles, in this way stimulating the production of oxygen reactive species. Such neutrophil activation is associated with bacterial infections that follow common flu (Hartshorn, et al, 1990). In addition to the contribution of neutrophiles in this lung inflammatory process, cells T CD8 account for the lysis of infected cells and production of proinflammatory cytokines, such as IFN-γ and TNF-α, but also contribute to the infection resolution. On the other hand, these cells also trigger an exacerbated lung damage, typical in this pathology, just in the same way as macrophages do, which release large quantities of oxygen and nitrogen reactive species in the pro-inflammatory response, causing tissue damage (La Gruta, et al, 2007). It is possible to conclude that an immune response to Influenza virus involves a great quantity of cells and inflammatory mediators. Such inflammatory response is crucial for eliminating the pathogen, but exacerbation of such processes implies a greater tissue damage which results in the observed morbidity in certain surges of flu. The flu is a significant risk to human population, especially to children, aged and immune-compromised people, even in its most "mild" annual epidemic form. In the 1990s, 36,000 deaths and 200,000 hospitalizations were recorded resulting from the flu, which is a disease that also leads to economic loses as it debilitates the affected individual for a week or more in addition to provoking high expenditure for the public the health system (Molinari, et al, 2007). Furthermore, viral infection eases the way to adhesion to and co-infection of other bacteria, such as Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae, by the damaged epithelium, triggering pneumonia (Julkunen, et al, 2001). Therefore, the search of new more efficacious vaccines and palliative treatments aims at reducing such indices, in addition to preventing or diminishing the reach of an imminent pandemic. Today, the viral neuraminidase surface glycoprotein, involved in releasing visions of infected cells through cleavage of sialic acid residues, has been a target inhibiting therapies of viral activity. Although it shows to have several regions of antigenic variation, the site of this glycoprotein is highly preserved in the subtypes of Influenza A and B. Therefore, Neuraminidase inhibitors, such as Zanamivir and Oseltamivir, have proved to be promising antiviral strategies (Colman, 1999; Kacergius, et al, 2006; Sidwell, et al, 1998). Another viral target in the development of pharmaceuticals is M2 protein, responsible for the loss of viral envelope within the infected cell, which has led to the development of Amantadine and Rimantadine antiviral medications. These two antiviral strategies are used for both prophylaxis and treatment of the flu (Regoes and Bonhoeffer, 2006).

Neuraminidase inhibitors are drugs that are both expensive and difficult to produce and thus, as it happens with the use of M2 inhibitor drugs, whose side effects are more intense, resistant virus strains or subtypes may appear. During an epidemic, when selective pressure becomes greater, resistant strains might survive and undoubtedly would kill thousands of people (Regoes e Bonhoeffer, 2006). Clinical studies also show that Oseltamivir and Zanamivir have their use restrained to individuals of 12 and 5 years of age, respectively (Langley and Faughnan, 2004).

The presence of specific antibodies for the hemagglutinin viral glycoprotein - liable to antigenic variation in sites of systemic or mucous infection - ensures immediate protection against viral infection. Therefore, producing vaccines that trigger the emergence of specific antibodies against the most frequent virus subtypes is now under way. On the other hand, combating virus within infected cells is accomplished by cell-mediated immunity against inner and more preserved virus proteins, such as nucleoprotein - NP, or proteins RNA polymerase PB2 and PA. Therefore, the presence of memory T lymphocytes containing specific epitopes for such preserved proteins may be crucial for patient survival after an avian flu pandemic (Subbarao & Joseph, 2007). At present, vaccination against subtypes causing annual epidemics is designed for high-risk population groups presenting the highest mortality rates caused by cases of flu and is delivered annually (Fleming, 2001). The World Health Organization recommends that vaccine production should be annual and based on variants of subtypes H1 N1 , H3N2 and influenza B. The positive result of subtypes use was of 88% in 10 years, which may affect the vaccine efficacy (Langley & Faughnan, 2004). There are now two major types of vaccine, the Trivalent Inactivated Influenza Vaccine - TIV and Live Attenuated Influenza Vaccine - LAIV. TIV is applied via intramuscular injection and contains purified hemagglutinin and neuraminidase, which triggers high IgG antibody response; it is indicated for individuals of 6 months of age with a reduction of 65% of flu cases by laboratory diagnosis. The attenuated virus vaccine applied via intranasal route has a better response by IgA in primary routes of virus entrance - the mucous membranes; it is indicated for sound individuals raging from 5 to 49 years of age and it can trigger respiratory symptoms, such as nasal secretions for two days with a 79% to 80% protection. As respiratory mucosa is the influenza virus entrance way, an efficacious inducement of immune response by IgA is crucial for impeding infection and intensive viral replication in the respiratory tract (Nichol & Treanor, 2006)

A pandemic scenario represents a great challenge for vaccine production as millions of people may be affected in the world (20% of the population) in a swift spread. Vaccines against influenza for humans are produced in embryonated chicken eggs and the whole production process requires 6 to 10 months to be completed. A pandemic would take 6 to 10 months to reach the whole planet after its first upsurge. Therefore, technical and clinical hindrances for vaccine production against the kind of virus causing a possible pandemic should be overwhelmed so that a larger number of people could be timely immunized. Several laboratories are working already all around the world in the development of an efficacious vaccine against H5N1 subtype, but its production is mainly concentrated in European, American and Asian countries. What is to be known is whether vaccine distribution would be evenly distributed among countries as a whole or whether it would benefit only those producing it. The longer the time is required for the pandemic to spread, the greater the chance for gathering sufficient vaccine doses for the population. (Stόhr & Esveld, 2004; Subbarao & Joseph, 2007)

As for avian influenza viruses of H5N1 subtype, many devastating effects of the infection in humans were shown to be derived from an exacerbated and uncontrolled production of some cytokines. Therefore, pharmacological approaches designed to face the so-called "cytokine storm", resulting from an infection outbreak, could be a simple and inexpensive alternative for treating the influenza virus with a subsequent reduced morbidity. Excessive immune reaction to the virus leads to lung inflammation and lung damage which may lead to multiple organ failure and eventually to death. Regulating this response may represent an alternative strategy to prevent damages provoked by the illness. The use of statines and other pharmaceuticals reducing blood lipid levels has proved to be efficient as to the lethality associated to influenza (Butler, 2007; Rainsford, 2006). A combination of antiviral and anti-inflammatory strategies may potentiate the protection effect of each strategy applied separately, in this way reducing lung damage (Ottolini, et al, 2003)

The platelet activation factor - PAF is a phospholipid with direct effects on inflammatory and immune responses (Weijer, et al, 2003). Its receptor contains 7 transmembrane domains and it is coupled with the heterotrimeric G protein, able to activate several intracellular signaling routes (Honda, et al, 2002). In addition to the classical effect of platelet aggregation, PAF mediates leukocyte transmigration, superoxide production and VEGF expression directly related to the angiogenesis in tissues with inflammatory response (Ishii & Shimizu, 2000). Neutrophil transmigration is mediated by PAF, since the phospholipid mediator activates the cell by modulating the adhesion molecule expression which facilitates its passage through endothelium and stimulating the production of reactive oxygen species (Condliffe, et al, 1996).

Several studies relate PAF to respiratory illnesses as it acts by increasing vascular permeability and mediating the loss of vascular matrix and hydroxyproline (Uhlig, et al, 2005). Animal models of asthma show that - in addition to the fact that PAF is produced by cells like alveolar macrophages, platelets, neutrophiles, mastocytes and eosinophils and be able to activate them all - the mediator provokes bronchoconstriction, airway hyperresponsivity, plasma exudation and increased mucous secretion that are crucial responses to the development of lung inflammation (Ishii & Shimizu, 2000). The first study showing an increased PAF production after a viral infection was accomplished in vitro, by means of a mononuclear phagocyte infection by respiratory syncytial virus, the main cause of lung inflammation in children (Villani, et al, 1991). PAF is known to play a crucial role in neuropathogenesis associated with infection by the human immunodeficiency virus through increased inflammatory response and viral replication (Martin, et al, 2000). Another recent study has shown increased PAF levels and reduced PAF acetyl hydrolysis - its main negative modulator - in patients bearing chronic type-C hepatitis (Caini, et al, 2007).

Respiratory tract infections are the third cause of death in the world (Murray and Lopez, 1997). Such high morbidity is related to T lymphocyte recruiting and activation for virus elimination, which occurs excessively in the lungs leading to tissual damages, airway occlusion and systemic production of inflammatory mediators. Among these mediators, especially TNF-α and IL-6 mediators lead to most respiratory infection symptoms, such as cachexia, fever and lack of appetite (Hussel, et al, 2004). Therefore, strategies that block excessive activation of immune system in response to viral infection are potential targets for pharmacological intervention aiming at treating symptoms and reducing morbidity. A reduced recruiting of inflammatory lung cells may convey a significant therapeutic potential when reducing clinic manifestations without changing necessarily the course of pathogen elimination (Hussel, et al, 2004).

As PAF receptor favors bacterial adhesion and invasion and as infections by influenza virus also favor bacterial infections leading to pneumonia, two works have investigated the receptor blocking in pneumonia cases by Streptococcus pneumoniae.

The first study used a competitive antagonist of PAF receptor before post-influenza bacterial infection which delayed lethality, although treated animals have presented higher bacteremia suggesting that bacteremia would contain mechanisms independent from PAF receptor (McCullers & Rehg, 2002). The other study assessed the role of

PAF receptor by using knockout animals for this receptor and a reduced lethality was evidenced in knockout mice, which was caused by post-influenza pneumonia.

Therefore, the receptor blocking in influenza infection may result in smaller susceptibility to a subsequent infection by Streptococcus pneumoniae (van der Sluijs, et al, 2005).

The researchers of the present invention have discovered that using PAF receptor antagonists, their analogues and/or derivatives are useful for treating infections provoked by the influenza virus.

Delivery of antagonists of the platelet receptor factor (PAF) to mammals - in an adequate therapeutic concentration - is efficacious in treating infections caused by the influenza virus.

The antagonists of the platelet receptor factor (PAF) can be delivered via endovenous, intramuscular, oral, subcutaneous, transdermal or intraperitoneal route.

Additionally, antagonists of the platelet receptor factor (PAF) can be administered in association with other pharmaceuticals for treating infections caused by the influenza virus in such a way as to optimize the prescribed treatment.

The patent document US5559109 describes a treatment method of a pathologic condition mediated by PAF through delivery of a PAF antagonist called N-acryl- piperazine or its derivatives. The patent document US6099836 describes sequences of polynucleotides encoding PAF plasma acetylhydrolase enzime. Methods for producing recombinant of this enzyme as well as other methods for treating pathologic conditions, such as injury by ischemia-reperfusion, and products derived from recombinant acetylhydrolase enzyme.

The patent document US20050032713A1 describes the use of PAF antagonists as an analgesic agent limiting the release of inflammatory mediators. The use of such antagonists in pharmaceutical or nutritional is said to be beneficial for treating acute and chronic pains, excessive uterine contraction, and septic shock as well as for angiogenesis inhibition and tumoral cell proliferation.

The patent document EP0459432A1 describes the use of PAF-acetic antagonists for treating pathologic conditions caused by lipoproteins (normal or modified). The PAF-acetic antagonist is selected from a group of hydrophilic or non- hydrophilic, triazolate-diazepine molecules or analogues. Ginkgolides components, or mixtures of them as well as their synthetic derivatives are also included as PAF-acetic antagonists.

The patent document US20020127287A1 describes treatment and prevention of mediated PAF disturbances by delivering an effective amount of at least one PAF antagonist. PAF antagonists are gingkolides which are topically delivered or added to food.

The patent document US5631246 describes the use of PAF in order to increase von Willebrand blood factor levels and/or factor VIII. This is particularly applied to treating von Willebrand disease and hemophilia.

The patent document WO9001927A2 describes the use of PAF antagonists for treating autoimmune disturbances as idiopathic purple thrombocytopenia. PAF antagonists may be used alone or combined with another immunosuppressive substance, as cyclosporine A.

The patent document US6277846 describes the use of PAF-acetic antagonists as antipruritic agents. This invention also reveals a method for treating itching by delivering a therapeutically effective amount of a PAF antagonist. For example, PAF antagonists can be selected among PAF analogue molecules, isolate natural PAF products with PAF antagonist activity and triazolbenzodiazepines. PAF antagonists are preferably topically delivered on the inflicted spot; systemic routes are also reported to be possible. The following examples are presented so as to give more details concerning the present invention. However, the examples presented do not limit the invention in any way.

EXAMPLE 1 : Standardization of DL-50:

In order to determine DL-50, a lethal dose for 50% of the group, C57/BL6 male mice, aged 8 to 10 weeks, were infected with the WSN strain of H1 N1 serotype Influenza virus, with inocula of 10³, 10⁴, 10⁵ e 10⁶ plaque forming units - PFU. A control group, called Mock, was inoculated with sterile saline solution (FIGURE 01). Based on this curve, the DL-50 of virus WSN in mice C57 was between 1x10⁴ and IxIO⁵ PFU.

EXAMPLE 2: Delivering PAF receptor antagonist, PCA 4248, in mice infected by influenza Wild mice C57, aged from 8 to 10 weeks, were infected with 10⁶ PFU of WSN viruses and divided into two groups. For their "treatment", the first group received, subcutaneously twice a day, the vehicle used for dilution of PCA 4248, 200 μl of a 5% solution of 98% ethylic alcohol diluted in sterile PBS. The second group received 200 μl of the antagonist, a subcutaneous dose equivalent to 5 mg/kg of the animal, every 12 hours since the 3^rd day after infection up to the 10^th day. The animals survived for 21 days.

While the animals treated with the vehicle showed a100% lethality after 8 days of infection, 37.5% of animals treated with PCA 4248 were alive and were sacrificed for serum collection (FIGURE 02).

EXAMPLE 3: Delivering PAF receptor antagonist, PCA 4248, in mice infected by influenza in combination with Oseltamivir phosphate neuraminidase.

Wild mice C57, aged from 8 to 10 weeks, were infected with 10⁶ PFU of WSN viruses and divided into 4 groups, in addition to one uninfected group. The first group, called vehicle, received, subcutaneously once a day (morning), the vehicle used for dilution of PCA 4248, 200 μl of a 5% solution of 98% ethylic alcohol diluted in sterile PBS and also 100 μl of a NaCI 0,9% sterile solution - the vehicle used for diluting Tamiflu, which was orally delivered once a day (night). The second group received 200 μl of PAF antagonist receptor, a subcutaneous dose of 5 mg/kg per animal, every 12 hours. The third group received 100 μl Oseltamivir phosphate neuraminidase by oral route, whose commercial brand is named Tamiflu, in a dose equivalent to 1mg/kg per animal delivered at every 12 hours. The fourth group received 100 μl Tamiflu by oral route, a dose equivalent to 1mg/kg per animal, every 12 hours and then 200 μl of PAF antagonist receptor, a subcutaneous dose equivalent to 5 mg/kg per animal at every 12 hours. The treatment was started 3 days after infection and, on the fifth day, 5 to 6 animals per group were sacrificed for bronchoalveolar wash, viewing to assess leukocyte recruitment for the alveolar space.

Nine days after infection by 10⁶ PFU of WSN viruses, the group treated only with a 1 mg/kg Tamiflu dose had no survivors; the group treated with the vehicle had 13% of its representatives; the group treated with 5 mg/kg of PAF antagonist receptor still had 22.2% of infected animals, while the group treated with a combination of Tamiflu and PAF antagonist receptor could count 62.8% survivors. Such results show the protection potentiation conferred by the PAF receptor antagonist when combined with the antiviral Tamiflu. All groups presented a significant increase in total and differential counts of leukocytes in relation the uninfected group. The group treated with PCA 4248 and the group treated with PCA 4248 combined with antiviral Tamiflu presented smaller leukocyte and neutrophil total recruitment in relation to animals treated with vehicle and Tamiflu alone, which evidenced the antagonist action in recruiting leukocytes to the infection site.

BRIEF DESCRIPTION OF FIGURES:

FIGURE 1 : Lethality: Standardization of DL-50 of WSN virus in C57/BL6 mice: the lethal dose value for 50% of animals lies between 1x10⁴ and 1x10⁵ PFU.

FIGURE 2: Treatment with PCA: treating with PAF receptor antagonist, PCA 4248, has delayed and reduced lethality of infection by WSN lethal inoculum.

FIGURE 3: total count and neutrophiles five days after: cellular recruiting at bronchoalveolar wash after infection by WSN 10⁶ PFU inoculum; groups treated with PCA 4248 and PCA 4248 together with antiviral Tamiflu presented smaller total recruitment of leukocytes and neutrophiles, as compared to animals treated with vehicle or Tamiflu alone.

Claims

1. Use of receptor antagonists of platelet activation factor (PAF), their analogues and/or derivatives, which is characterized by being used in a pharmaceutical preparation for treating infections caused by the influenza virus.

2. Use of receptor antagonists of platelet activation factor (PAF), their analogues arid/or derivatives, in accordance with claim 1 , characterized by comprise the A-, B- and C-type viruses.

3. Use of receptor antagonists of platelet activation factor (PAF)₁ their analogues and/or derivatives, in accordance with claim 2, characterized by comprise an adequate therapeutic concentration for treating infections caused by influenza virus.

4. Use of receptor antagonists of platelet activation factor (PAF), their analogues and/or derivatives, in accordance with claim 3, characterized by being delivered to mammals.

5. Use of receptor antagonists of platelet activation factor (PAF), their analogues and/or derivatives, in accordance with claim 4, characterized by the possibility of being delivered via endovenous, intramuscular, oral, subcutaneous, transdermical or intraperitoneal routes.

6. Use of receptor antagonists of platelet activation factor (PAF), their analogues and/or derivatives, in accordance with claim 5, characterized by the possibility of being delivered in association with other pharmaceuticals reputedly useful for treating infections caused by the influenza virus.