WO2007097624A1

WO2007097624A1 - Pharmaceutical compositions for the treatment of influenza infections

Info

Publication number: WO2007097624A1
Application number: PCT/NL2007/050072
Authority: WO
Inventors: Nisar Ahmed Khan; Robbert Benner; Gert Wensvoort
Original assignee: Biotempt B.V.
Priority date: 2006-02-21
Filing date: 2007-02-20
Publication date: 2007-08-30

Abstract

The invention relates to the field of the production of a pharmaceutical composition for the treatment of a subject suffering or believed to be suffering from an acute influenza virus infection. Provided is the use of a peptide consisting of 3 to 6 amino acids for the production of a pharmaceutical composition for the treatment of a subject, preferably a human subject, suffering or believed to be suffering from an influenza infection. Also provided is a pharmaceutical composition comprising at least one antiviral peptide consisting of 3 to 6 amino acids in combination with an additional antiviral agent.

Description

P80341PC00

Title: PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF INFLUENZA INFECTIONS

FIELD OF THE INVENTION

The invention relates to the field of the production of a pharmaceutical composition for the treatment of a subject suffering or believed to be suffering from an acute influenza virus infection, such as influenza caused by an avian influenza virus.

BACKGROUND OF THE INVENTION Influenza virus is an enveloped RNA virus of the orthomyxovirus family with a genome that is composed of eight (influenza A and B) or seven (influenza C) segments. Influenza A and B are responsible for the annual outbreaks of epidemic influenza in humans. The virus capsid contains two major antigenic proteins, hemagglutinin (HA) and neuraminidase (NA). There are 16 different HA subtypes (Hi— H 16) and nine different NA subtypes (Nl- N9), all of which have been found among influenza A viruses. Wild birds are the primary natural reservoir for all subtypes of influenza A viruses and are thought to be the source of influenza A viruses in all other animals. Although most influenza viruses cause asymptomatic or mild infection in birds, infection with certain strains of H5 and H7 viruses can cause widespread disease and death among wild and domestic birds such as chickens and turkeys. Pigs are susceptible to avian, human and swine influenza viruses and thus have a potential to be infected with influenza viruses from different species (e.g. ducks and humans) at the same time. In this circumstance, active replication of both virus subtypes within the same host may result in reassortment of the genome RNA segments, creating a new virus containing a novel combination of HA and/or NA capsid proteins, a process known as antigenic shift. Antigenic shift may result in the emergence of a new influenza A subtype which can cross the species barrier and infect humans, who may have little or no immunity to the new virus. If the virus can be transmitted easily from person to person, an influenza pandemic could occur. Several instances of human infections and outbreaks following interspecies transmission of avian influenza have been reported since 1997. Most cases have resulted from contact with infected animals or contaminated surfaces. In 1997, the infection of 18 individuals with an unchanged avian influenza A virus (H5N1), six of whom died, was another sign that the threat of an influenza pandemic still exists, although serological investigations showed that human-to- human transmission was very rare. In 2003, H5N1 and H9N2 infections were confirmed in Hong Kong with several reported cases of mortality. Since January 2004, outbreaks of avian H5N1 influenza have been reported in several countries in Asia. These outbreaks were believed to have been contained earlier in the year, but beginning in June 2004, new deaths were reported in Vietnam caused by a highly pathogenic H5N1 avian influenza. This second H5 strain has some immunological differences to the 1997 strain suggesting that vaccines derived from the 1997 seed strains may be ineffective against it. In 1999, H9N2 avian influenza was confirmed in two children in Hong Kong and several additional cases from mainland China. In 2003, another outbreak of H9N2 infection was confirmed in Hong Kong. The largest avian influenza outbreak occurred in the Netherlands in 2003 and involved an H7N7 virus. Recent evidence has emerged suggesting that approximately 1000 people, mainly farmers and poultry workers, were infected by the virus as a result of handling poultry. Of these, approximately 500 complained of symptoms consisting mainly of conjunctivitis and influenza-like illness. In another unexpected finding, those who developed symptoms after being infected passed the virus on to approximately 59% of their household contacts.

Currently, authorities in many countries are alarmed by reports of a rapidly spreading viral pneumonia caused by the so-called avian influenza virus H5N1 with sometimes lethal consequences for man. However, the threat is by no means coming from H5N1 alone, H7N7virus was lethal to man in 2003, and other isolates of avian influenza virus (AIV) may proof to be lethal to man in the future. Symptoms of avian influenza infection, which is believed to be spread through droplets by sneezing and coughing, include high fever, chills, coughing, nasal congestion, and breathing difficulty in chickens and other fowl, leading to sometimes high mortality. In human cases the illness causes high fever, muscle aches and shaking chills, followed by coughing and labored breathing. A second phase in a smaller group of patients is followed by coughing, labored breathing, and hypoxia; sometimes a patient's condition is severely aggravated leading to death. Many nations are now proposing radical action. Since 1997, highly pathogenic avian H5N1 influenza viruses have caused concern about their human pandemic potential. Although the mass slaughter of poultry Hong Kong stopped the H5N1 outbreak in 1997, the precursors of the virus continue to circulate in southern China. Reassortment of these precursor viruses with other avian influenza viruses has generated multiple genotypes of H5N1 viruses in recent years. The continued cocirculation of these viruses in wild aquatic birds and poultry in China has created the potential for avian-to-human and human- to-human transmission. Although only 2 cases of human H5N1 infection were reported in early 2003, a new H5N1 antigenic variant has spread widely across Asia since December 2003, causing deaths in 50% of confirmed cases, including probable human-to-human transmission in a family cluster of the disease in Thailand.

Vaccination and antiviral treatment are currently seen as the 2 options for the control of influenza. Although vaccine is the preferred method of prophylaxis, at least 6 months are required to produce vaccine against currently circulating influenza viruses, including new H5N1 antigenic variants. The application of antiviral drugs during the early stages of a pandemic may help to control it. Two classes of drugs are currently available for prophylaxis and treatment of influenza virus infection: M2 ion channel blockers (amantadine and rimantadine) and neuraminidase inhibitors (NAIs; oseltamivir and zanamivir). Prophylactic oseltamivir was used in 2003 during an H7N7 avian influenza virus outbreak in the Netherlands; however, its efficacy is unclear because the initiation of treatment was delayed. Lack of data about widespread, prolonged prophylactic use of oseltamivir was mentioned as an obstacle faced by health planners during the H7N7 outbreak.

AI is caused by a virus herein also called the AI virus (AIV). Incubation period after infection with the AIV is usually short: 1 to 3 (but is seen up to 16) days followed by headache, sore throat, muscle aches, chills, and fullness in the nose. Then there is often a profuse watery discharge from the nose which gradually thickens and becomes mucopurulent and decreases in volume. In some cases the infection resolves in about a week, however, in other cases the illness generally continues with a fever greater than 100 degrees Fahrenheit. The fever is sometimes associated with further chills, headache, malaise, and body aches. Some also experience mild respiratory symptoms at the outset. While most of the patients in a first phase start out with the usual flu-like symptoms, and then seemingly go on to recover, in a second phase some patients take, even after apparent recovery, a sudden turn for the worse. The clinical signs and laboratory abnormalities that indicate that serious problems are developing are as follows: LDH becomes elevated, creatine kinase becomes elevated, neutrophilia develops, platelets are often suppressed, prothrombin time goes up and systemic clotting accelerates. Other indicative clinical signs include migratory pneumonias and many patients become hypoxic. Some of the initial AI cases problems grow so serious that patients will require mechanical ventilation. These problems are usually caused by large-scale damage produced by proinflammatory cytokines and other inflammatory mediators.

Where diagnostic techniques aimed at detecting AIV and AI infections seem to be rapidly evolving, currently no specific treatment of the AI infection other than the known anti-virals and only symptomatic or perfunctory treatments directed against the second phase consequences of AI are available. Complications that are further aggravating the second phase condition are manifold and for example comprise bacterial infections or chlamydial infections which may require antibiotic treatment. The other complications include acute respiratory disorder syndrome (ARDS), multiple organ disorder syndrome (MODS) or multiple organ failure (MOF), systemic inflammatory response syndrome (SIRS) which may well be a direct secondary effect of viral immune activation in the host and thus immune dysregulation, and sepsis, septic shock and death which may be partly from the primary infection or from a combined infection. Doctors are trying to figure out what works best.

SUMMARY OF THE INVENTION

The invention provides use of a short (3 to 6 amino acid long) peptide or functional analogue thereof for the production of a pharmaceutical composition for the treatment of a subject, preferably a human, suffering or believed to be suffering from an influenza infection, in particular a so-called avian influenza infection. The invention in particular provides use of at least one such peptide or functional analogue thereof for the production of a pharmaceutical composition for the treatment of a subject suffering or believed to be suffering from AI, in particular when a severe acute respiratory infection is caused by an infection with a AI virus, and especially when said subject is at risk to be suffering or believed to be suffering from a late phase and possibly lethal course of AI infection. Also provided herein is method for treatment of a subject suffering or believed to be suffering from an influenza infection, comprising administering a pharmaceutical composition comprising a therapeutically effective amount ϋf an antiviral peptide consisting of 3 to 6 amino acids. A combination therapy comprising the administration of one or more antiviral peptides of the invention and at least one additional antiviral agent was found to be particularly advantageous. Remarkably, in the second phase of AI the AI viruses often are not found active in the pulmonary system, and it is the prominent inflammatory response that is the main course of the worsening of the patient's condition against which treatment with a pharmaceutical composition according to the invention is most preferred. The use according to the invention is particularly useful in those cases wherein said subject is at risk to be suffering or believed to be suffering from such a secondary or late phase and possibly lethal course of AI characterized by one or more clinical signs or laboratory findings that indicate such an immunological reaction, for example characterized by clinical signs or laboratory findings selected from the group of elevated lactic dehydrogenase levels, elevated creatine kinase levels, neutrophilia, elevated prothrombine time, depressed platelets, elevated d-dimers, migratory pneumonia and progressive hypoxia with evolution to ARDS with and without DIC, SIRS, or MODS. In the present application the use of a peptide consisting of 3 to 6 amino acids is provided for the production of a pharmaceutical composition for the treatment of a subject, preferably a human subject, suffering or believed to be suffering from an influenza infection. Said influenza infection may be an avian influenza infection, for example avian influenza caused by avian influenza virus H5N1. More specifically, the use of the small antiviral peptides LQG, AQG, LQGV, AQGV, MTR, MTRV, WC, QWC, VLPALP, and VLPALPQ, and functional analogue peptides, or mixtures of peptide (analogues), is described.

Amino acids are given here in the one-letter-code. These peptides (in their active state only at about 3 to 7 amino acids long) have unsurpassed immunological activity that they exert by regulating expression of genes encoding inflammatory mediators such as cytokines. In one embodiment, the invention provides use of at least one of the aforementioned antiviral peptides or functional analogues thereof for the production of a pharmaceutical composition for the treatment of a subject suffering or believed to be suffering from influenza infection, particularly AI. In onepreferred embodiment, peptide LQGV or AQGV is individually selected for treatment. In another embodiment, peptide LQGV, MTRV or WC is selected individually or in any combination with each other. For example, LGQV is used alone or in combination with MTRV and WC. As another example, MTRV is used alone or in combination with LQGV or WC. Likewise, provided is the use of WC alone or in combination with MTRV or LQGV. In a specific aspect, the invention provides a pharmaceutical composition comprising a mixture of LQGV, MTRV and WC. The relative amounts of individual antiviral peptides in a mixture of peptides can vary. They can be the same or different.

As is exemplified herein below, it was found that the antiviral peptides of the invention are advantageously used in combination with an additional antiviral agent, which may be of peptidic or non-peptidic nature. In one aspect the invention therefore relates to the use of one or more antiviral peptides as described above for the manufacture of a pharmaceutical composition, wherein said composition comprises an additional antiviral agent. Exemplary additional antiviral agents include those known in the art and those still to be discovered. Preferably, it is an M2 ion channel blocker or a neuramidase inhibitor. A suitable additional antiviral agent for use in combination with a peptide or peptide mixture according to the invention is oseltamivir.

Furthermore, the invention provides the treatment of a subject suffering or believed to be suffering from disease caused by an influenza virus, such as AIV, by administration of a therapeutically effective amount of a short (3 to 6 amino acid long) antiviral peptide or functional analogue, or mixture of such short peptides, together with a pharmaceutically acceptable diluent to said subject. A particular useful pharmaceutically acceptable diluent is sterile water or an isotonic salt solution such as 0.9% saline or phosphate buffered salt solution (PBS). In a preferred embodiment, the invention provides the treatment of a subject suffering or believed to be suffering from disease caused by an infection with the AI virus by administration of a pharmaceutical composition comprising a pharmacologically effective amount of any of the short antiviral peptides LQG, AQG, LQGV, AQGV, MTR, MTRV, WC, QWC, VLPALP, and VLPALPQ, or functional analogue peptides, or mixture of said antiviral peptides, together with a pharmaceutically acceptable diluent to said subject. In one embodiment, peptide LQGV or AQGV is individually selected for treatment. In another embodiment, treatment comprises administering peptide LQGV, MTRV or WC individually or in any combination with each other. For example, LGQV is used alone or in combination with MTRV and WC. As another example,

MTRV is used alone or in combination with LQGV or WC. Likewise, provided is the use of WC alone or in combination with MTRV or LQGV. In a specific aspect, the invention provides a method for treatment of a subject suffering or believed to be suffering from an influenza infection, for instance avian influenza infection such as avian influenza infection caused by H5N1, comprising administering a pharmaceutical composition comprising a mixture of LQGV, MTRV and WC.

The invention provides the treatment of a subject suffering or believed to be suffering from inflammatory disease caused by an infection with Al-virus by administration of a pharmaceutical composition comprising a pharmacologically effective amount of a short, aforementioned peptide capable of regulating expression of genes encoding inflammatory mediators such as cytokines. Variants and modifications of these peptides that have functionally equivalent or analogue activity can be synthesized and tested for their activity easily by the person skilled in the art, using for example animal experiments, such as experiments with mice as explained herein. In another embodiment the invention provides a pharmaceutical composition for the treatment of AI-related disease comprising such a peptide or functional analogue thereof, and use of a gene-regulatory peptide or functional analogue thereof for the production of a pharmaceutical composition for the treatment of AI-related disease. The term "pharmaceutical composition" as used herein is intended to cover both the active functional compound or compounds alone or a composition containing the compound or compounds together with a pharmaceutically acceptable carrier, diluent or excipient. Acceptable diluents of a pharmaceutical composition as described herein are for example physiological salt solutions or phosphate buffered salt solutions. In one embodiment of the present invention, said pharmaceutical composition is administered in an effective concentration to an animal or human systemically, e.g. by intravenous, intra-muscular or intraperitoneal administration. A pharmaceutical composition according to the invention may of course also be formulated for oral administration. Preferred dosage regimes of trimeric peptide (i.e. LQG, AQG and WC) range from 0.1 mg/kg to 10 mg/kg, with optimum dosing to be found at 0.5 mg/kg to 5 mg/kg. Preferred dosage regimes of tetrameric, pentameric, hexameric or heptameric peptide (i.e. LQGV, AQGV, QWC, VLPALP and VLPALPQ) range from 0.3 mg/kg to 30 mg/kg, with optimum dosing to be found at 1 mg/kg to 10 mg/kg. In a specific aspect, a dosage regimen of 1-10 mg/kg is used, for example a mixture comprising two or three antiviral peptides is administered at around 5 mg/kg for each peptide. Peptide compositions can be given once, or daily, at regular intervals, preferably every 8 hours, with a bolus injection or per infusionem. Oral treatment is possible but likely not first choice in intensive care patients.

Also, the invention provides a method for the treatment of a subject, preferably a human, suffering or believed to be suffering from a so-called avian influenza infection, comprising a combination treatment with a short (3 to 6 amino acid long) antiviral peptide or functional analogue thereof, said subject also being treated or having been treated with an additional anti-viral compound such as one or more M2 ion channel blockers and/or one or more neuraminidase inhibitors. The combination therapy may comprise administration of antiviral peptides simultaneously with the at least one additional antiviral compound, for example in the form of a pharmaceutical composition comprising an admixture of antiviral peptide(s) of the invention and at least one additional antiviral agent. It may also comprise the parallel treatment in the form of administering separate formulations. In yet another embodiment, the therapy with peptides and additional antiviral agent overlaps only partially or not at all. The invention in particular provides use of at least one such peptide or functional analogue thereof for the production of a pharmaceutical composition for the treatment of a subject suffering or believed to be suffering from AI, in particular when a severe acute respiratory infection is caused by an infection with a AI virus, and especially when said subject is at risk to be suffering or believed to be suffering from a late phase and possibly lethal course of AI infection, and has already been treated with such an anti-viral compound. This use according to the invention is particularly useful in those cases wherein said subject, already having been treated with an antiviral, is still at risk to be suffering or believed to be suffering from such a secondary or late phase and possibly lethal course of AI. In particular, the use of the small peptides LQG, AQG, LQGV, AQGV, MTR, MTRV, WC, QWC, VLPALP, and VLPALPQ, or mixtures thereof, in combination therapy with aforementioned antivirals is provided. In a preferred embodiment, peptide treatment is combined with oseltamivir treatment. These peptides (in their active state often only at about 3 to 6 amino acids long) were shown to have unsurpassed immunological activity that they exert by regulating expression of genes encoding inflammatory mediators such as cytokines and work synergistically with anti-viral compounds. In one embodiment, the invention provides use of at least one of the aforementioned peptides or functional analogues thereof for the production of a pharmaceutical composition for the treatment of a subject suffering or believed to be suffering from AL In one embodiment, peptide LQGV or AQGV is individually selected for treatment. In a preferred embodiment, treatment with LQGV, MTRV and/or WC is provided, more preferably treatment with a mixture of at least two, or all three of these peptides is provided. The invention also provides a pharmaceutical composition comprising a mixture of the peptides LQGV, MTRV and WC.

In an even more preferred embodiment, LQGV, MTRV and/or WC treatment is combined with, preferably oral, oseltamivir treatment, or with treatment with a functionally analogous neuraminidase inhibitor. Oseltamivir is orally active in a variety of species. Interspecies differences in esterase activity require that different doses are administered to ensure that plasma concentrations of the carboxylate achieve levels associated with antiviral activity. To achieve the same area under the curve (AUC) as an oral dose of 75 mg twice daily in humans, mice for example require an oseltamivir dose of 10 mg/kg, ferrets 5 mg/kg and chickens 120 mg/kg.

Furthermore, the invention provides the treatment of a subject suffering or believed to be suffering from disease caused by an infection with the AI virus by administration of a short (3 to 6 amino acid long) peptide or functional analogue together with a pharmaceutically acceptable diluent to said subject. A particular useful pharmaceutically acceptable diluent is sterile water or an isotonic salt solution such as 0.9% saline or phosphate buffered salt solution (PBS). In a preferred embodiment, the invention provides the treatment of a subject suffering or believed to be suffering from disease caused by an infection with the AI virus by administration of a pharmaceutical composition comprising a pharmacologically effective amount of any of the small peptides LQG, AQG, LQGV, AQGV, MTR, MTRV, WC₁ QWC, VLPALP, and VLPALPQ, or functional analogue peptides, together with a pharmaceutically acceptable diluent to said subject. In a preferred embodiment, peptide LQGV or AQGV is individually selected for treatment.

Patients who are in the onset or amidst the late immune dysfunctional complications of AI are preferably treated with a pharmaceutical composition for the treatment of AI as provided herein. Such patients often present with evidence of progressive hypoxia as seen by lowered oxygen and its saturation on arterial blood gases as well as evidence through oximetry. These patients typically have presented with the compilation of signs and symptoms described earlier which typify at AI like illness and this is associated with laboratory findings including some of the following: lymphopenia, thrombocytopenia, positive d-dimers, elevated partial prothrombin times, elevated LDH, and elevated CK. These patients may well need supplementary oxygen or even artificial ventilatory support. These patients may as well be treated with antivirals such as ribavirin at 400 mg three times per day orally or through an IV preparation, with antibiotics, such as cefotaxime and clarithromycin (or levofloxacin) to target common pathogens causing community-acquired pneumonia according to established treatment protocols, with corticosteroids (prednisolone at a dose of up to 1 mg per kilogram of body weight per day), or with other anti-inflammatory drugs or drugs (such as vaccines) specifically aimed at fighting the AI virus. Suitable patients to be treated often have progressive pneumonia in the form of ARDS on sequential X-rays or CTs. They are treated with either a bolus of pharmaceutical composition every eight hour or by continuous drip administration of a total therapeutic dose until treatment is no longer deemed needed. Outcome is measured by survival, time of resolution of hypoxia, and time and course of resolution of pneumonia and other immune dysregulation processes which include normalization of coagulation, CK, LDH, and the cascade of THl and TH2 cytokines/chemokines.

Preparing a pharmaceutical composition for the treatment of AI from chemically synthesized peptides. Examples of useful peptides to be included in such a pharmaceutical composition are LQG, AQG, WC, MTR, LQGV, AQGV, QWC, MTRV, VLPALP and VLPALPQ.

Peptide synthesis: The peptides were prepared by solid-phase synthesis (Merrifield, 1963) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology (Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos, 1991) as the solid support. The side-chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: (i) removal of the alpha-amino Fmoc-protection by piperidine in dimethylformamide (DMP), (ii) coupling of the Fmoc amino acid (3 eq.) with diisopropylcarbodiimide (DIC)/1- hydrozybenzotriazole (HOBt) in DMF/N- methylformamide (NMP) and (in) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H2O/trϋsopropylsilane (TIS) 95:2.5:2.5. After 30 minutes TIS was added until decoloarization. The solution was evaporated in vacuo and the peptide precipitated with diethylether. The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). HPLC conditions were as follows: column: Vydac TP21810C18 (10 x 250 mm) (column selection is crucial since peptides have shorter retention time); elution system: gradient system of 0.1% TFA in water v/v (A) and 0.08-1% TFA in acetonitrile (ACN) v/v (B); flow rate 6 ml/min.; absorbance was detected from 190-370 nm. There were different gradient systems used. For example for peptide LQGV: 10 minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For peptide VLPALP: 5 minutes 5% A followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40⁰C. The remaining TFA was exchanged against acetate by eluting two times over a column with anion exchange resin (Merck II) in acetate form. The elute was concentrated and lyophilized in 28 hours.

Treatment protocol

Treatment with a pharmaceutical composition according to the invention may for example comprise infusions with Ringer's lactate for the first 24 hours, said Ringer's lactate provided with a pharmaceutical composition as provided herein, preferably, 1 -1000 mg/1 peptide such as VLPALPQ, LGQV, WC, AQGV or MTRV, or mixtures of two or three of such peptides. During treatment of severely affected patients, for example under mechanical ventilation, it is often important to keep the volume up, and, if needed, provide the pharmaceutical composition (e.g. peptide or functional analogue thereof) in even further hypotonic solutions, such as in 0.3 to 0.6% saline.

Peptide compositions can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/1, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example every eight to twelve hours in a bolus injection or per infusionem until the patient stabilizes, are recommended.

The administration of peptide may be done as a single dose, as a discontinuous sequence of various doses, or continuously for a period of time sufficient to permit substantial modulation of gene expression. In the case of a continuous administration, the duration of the administration may vary depending upon a number of factors which would readily be appreciated by those skilled in the art. The administration dose of the peptide may be varied over a fairly broad range. The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end. The optimal dose or doses for a particular patient should and can be determined by taking into consideration relevant factors such as the condition, weight and age of the patient, and other considerations of the physician or medical specialist involved.

In another example wherein a peptide as provided herein is useful in treatment of AI disease, the invention provides a pharmaceutical composition for systemic application, in particular selected from the group pharmaceutical compositions for intravenous, intraperitoneal, intrathoracal, or intramuscular administration, comprising a peptide or functional analogue thereof, and use of a peptide or functional analogue thereof for the production of a pharmaceutical composition for systemic application. Such a composition is most useful to treat the body as a whole and in a preferred embodiment thereby affects essentially not or only little the area to which it is applied.

In another example wherein a peptide as provided herein is useful in treatment of AI, it is herein provided to modulate AI disease in a subject comprising providing said subject with one or more of the aforementioned short peptides or functional analogues thereof wherein said subject is also provided with an agent directed against disseminated intravascular coagulation, in particular wherein said agent comprises Activated Protein C activity. Such an agent to modulate disseminated intravascular coagulation (DIC) comprises preferably (recombinant) human Activated Protein C. It is preferably given to the patient per infusionem, whereby peptide can be given in the same infusion, the peptide (or analogue) concentration preferably being from about 1 to about 1000 mg/1, but the peptide can also been given in a bolus injection. Doses of 1 to 5 mg/kg bodyweight, for example every eight hours in a bolus injection or per infusionem until the patient stabilizes, are recommended. In another example wherein a gene-regulatory peptide as provided herein is useful as a modulator of NF-kappaB to be used in treatment of AI disease the invention provides combination therapy that include the concomitant treatment of the patient with a (monoclonal) antibody directed against a cytokine, such as TNF-alpha, IL-6 or IL- 12, with a (monoclonal) antibody or directed against AIV.

Synthetic peptides can be obtained using various procedures known in the art. These include solid phase peptide synthesis (SPPS) and solution phase organic synthesis (SPOS) technologies. SPPS is a quick and easy approach to synthesize peptides and small proteins. The C-terminal amino acid is typically attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The peptide, or its functional analogue, modification or derivative, can be administered as the entity as such or as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines). A selected peptide and any of the derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in-situ as a conjugate or be released locally after reaching a targeted tissue or organ. The term "pharmaceutical composition" as used herein is intended to cover both the active peptide alone or a composition containing the peptide together with a pharmaceutically acceptable carrier, diluent or excipient. Acceptable diluents of a peptide as described herein are for example physiological salt solutions or phosphate buffered salt solutions.

Furthermore, aforementioned peptides, or a modifications or analogues thereof, can be chemically synthesized using D- and/or L-stereoisomers. For example, a short peptide that is a retro-inverso of any of the aforementioned short peptides is produced. The concept of polypeptide retro-inversion (assembly of a natural L-amino acid-containing parent sequence in reverse order using D- amino acids) has been applied successfully to synthetic peptides. Retro-inverso modification of peptide bonds has evolved into a widely used peptidomimetic approach for the design of novel bioactive molecules which has been applied to many families of biologically active peptides. The sequence, amino acid composition and length of a peptide will influence whether correct assembly and purification are feasible. These factors also determine the solubility of the final product. The purity of a crude peptide typically decreases as the length increases.

The yield of peptide for sequences less than 15 residues is usually satisfactory, and such peptides can typically be made without difficulty. The overall amino acid composition of a peptide is an important design variable. A peptide's solubility is strongly influenced by composition. Peptides with a high content of hydrophobic residues, such as Leu, VaI, He, Met, Phe and Trp, will either have limited solubility in aqueous solution or be completely insoluble. Under these conditions, it can be difficult to use the peptide in experiments and treatments, and it may be difficult to purify the peptide if necessary. To achieve a good solubility, it is advisable to keep the hydrophobic amino acid content below 50% and to make sure that there is at least one charged residue for every five amino acids. At physiological pH Asp, GIu, Lys, and Arg all have charged side chains. A single conservative replacement, such as replacing Ala with GIy, or adding a set of polar residues to the N- or C-terminus, may also improve solubility. Peptides containing multiple Cys, Met, or Trp residues can also be difficult to obtain in high purity partly because these residues are susceptible to oxidation and/or side reactions. If possible, one should choose sequences to minimize these residues. Alternatively, conservative replacements can be made for some residues. For instance, Norleucine can be used as a replacement for Met, and Ser is sometimes used as a less reactive replacement for Cys. If a number of sequential or overlapping peptides from a protein sequence are to be made, making a change in the starting point of each peptide may create a better balance between hydrophilic and hydrophobic residues. A change in the number of Cys, Met, and Trp residues contained in individual peptides may produce a similar effect. A peptide modification may include phosphorylation (e.g on a Tyr, Ser or Thr residue), N-terminal acetylation, C-terminal amidation, C-terminal hydrazide, C-terminal methyl ester, fatty acid attachment, sulfonation (tyrosine), N-terminal dansylation, N-terminal succinylation, tripalmitoyl-S-Glyceryl

Cysteine (PAM3 Cys-OH) as well as farnesylation of a Cys residue. Systematic chemical modification of a gene-regulatory peptide can for example be performed in the process of peptide optimization.

Synthetic peptides can be obtained using various procedures known in the art. These include solid phase peptide synthesis (SPPS) and solution phase organic synthesis (SPOS) technologies. SPPS is a quick and easy approach to synthesize peptides and small proteins. The C-terminal amino acid is typically attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products.

LEGEND TO THE FIGURES

Fig. 1. Mortality rate for each treatment group of influenza virus-infected mice

Groups of 10 Balb/c mice were inoculated i.n. on DO with 20 LD50 of H5N1 virus or PBS, and treated with peptide mixture BP-AI 100 in combination with the additional antiviral agent oseltamivir (Tamiflu) from DO until D 13, with BP-AI 100 in combination with Tamiflu from day 4 (D4) until day 13 (D 13) or with Tamiflu alone from DO until D 13. Number of surviving mice on each study day is indicated for each treatment group. Arrow indicates time of virus administration (DO). Note that one mouse died soon after oral administration of Tamiflu (indicated by the asterisk) infected in the group receiving BP-AI 100/T treatment from DO to 14 (group 1). Example 1: The effect of anti-viral peptides in influenza virus infected mice.

This Example exemplifies the in vivo anti-viral efficacy of the small peptides of the invention in mice following intranasal administration of a lethal dose of influenza virus. The study was conducted by permission of the Dutch Ministry of Agriculture.

Animals The Dutch study plan, describing the animal experiment performed in this study registered under number 2006043c, was approved by the Ethical Review Committee (ERC) of the Animal Sciences Group prior to study start.

Species/breed: Mouse, Balb/c BYJ (Specific Pathogen Free, SPF). Initial age: 7 weeks old on Study Day 0. Number and Sex: Maximum 50 female animals. Origin/Source: Charles River Laboratories, Maastricht. Group identification: Cages were labelled with Study No., Group No., Date

Commenced.

Animal details were recorded on Form DB 155.001.2.

Management

Cage space per animal/ Mice were kept in filtertop cages providing space per animal Stocking rate: in accordance with animal welfare regulations.

General maintenance: Cleaning before and after the animal phase was performed according to ASG's Standard Operating Procedures. Daily care of the animals such as cleaning of cages, feeding, refreshing and changing litter, was recorded on form DB 155.015

Husbandry precautions: Upon entry to room, the hygiene regimen was performed according to SOP 2.6.0116.

Feeding and watering Feed and potable water was available ad libitum. Water was method: refreshed once a day.

Feeding and watering was performed according to ASG's current Standard Operating Procedure (SOP) DB-SB-03-001. Feed: Product code 2110 provided by Arie Blok Animal Feed.

Room identification: The room was labelled with Study No., Unit No./ Room No., Name Investigator.

Environmental control: Mice were maintained between a minimum and maximum temperature of 20°C and 24°C.

Air temperature and relative humidity was continuously monitored and recorded on form DB 155.003.

Study design

A total number of 50 SPF Balb/c mice, 6 weeks of age at the time of arrival, were used in the study and were randomly allocated to one of the groups as outlined in the table below.

Table 1

All animals were acclimatized and maintained for a period of 7 days prior to the intranasal administration of virus or placebo on study day 0 as indicated above. Animals were treated daily with a peptide mixture herein referred to as "BP-AI 100" and/or Tamiflu up to day 14 (D 14) as indicated in Table 1, either starting on the day of inoculation (DO) or on day 4 (D4).

During the animal phase of the study, pathogenicity was evaluated by daily clinical assessments and recording of weight as an additional indicator of disease. Surviving animals were euthanized and bled on study day 18. For further details see Section 5. Procedures

Virus and Test Item Administration

Method of Inoculum Preparation The virus material was stored at -75°C ± 10⁰C and thawed prior to administration in a 37°C water bath until the ice has just melted. Once thawed, the material was diluted in cold PBS until a final dose of 20 LD50 per 50 μl and kept on ice until administration to the mice.

Method of Test Item Preparation Peptide mixture BP-AI 100 comprising the peptides LQGC, MTRV and WC was provided by Biotempt BV and stored at -20⁰C. On study day 0 the material was thawed and stored at 4⁰C during the course of the study. Prior to administration, the material was diluted in phosphate buffered saline (Gibco) until the recommended dose (using 6 mL for the red vials and 10 mL for the grey vials). If the content of one vial was not sufficient to inject all animals scheduled for treatment, a second vial was opened. Diluted material was stored at 4°C and used on the next day.

Tamiflu® (oseltamivir) was diluted in distilled water according to the instruction of the manufacturer (Hoffmann-La Roche) until a final concentration of 2 mg/mL, aliquoted, and also stored at 4⁰C until administration.

Dose level and Method of Administration

The animals were anaesthetized with ketamin/ xylazin intraperitoneally and inoculated with approximately 50 μl of diluted virus or PBS (placebo group). Animals, which received also an intraperitoneal injection with peptide on DO (group 1 and 4), were anaesthetized first and treated last.

The virus and placebo control was administered by intranasal inoculation using a sterile pipette tip for each mouse as indicated in SOP 2.6 0068 and recorded on Form DB 155.002.4. Unused material was returned to the lab for titration. Dose level and Method of Test Item Administration

The animals received BP-AI 100 by intraperitoneal injection of a 200 μl volume according to standard procedure. This corresponded to a dose of 5 mg/kg body weight for each of the anti- viral peptides. The animals received 100 μl of Tamiflu by the oral route using a knob-headed needle. Administrations were recorded on Form DB 155.002.4.

Safety Precautions

Precautions were taken when handling the inoculum as indicated in SOP 2.6.0116.

Methods and Observations

General Health Observations

Only animals assessed as being suitable for the study following the health assessment on the day of arrival were placed on acclimatization. General health observations were performed on each animal once daily (during normal servicing procedures in the morning) from Day -1 to 15 (except DO) or for as long as they remained in the study. Observations of general health were assessed on the basis of the categories specified in the Appendix.

Weighing of Animals

All animals were weighed daily starting on Day -1 until D 15 (except DO) and on D 18. Animals were also weighed in case of death prior to the end of the study. Body weight in gram was recorded according to SOP DB-IV-45-011 on form DB 155.006.2. Calibration of the weighing scale used for the study was carried out according to SOP 2.1.1.087 in place at ASG.

Production ofAntisera To obtain post-infection sera, blood was collected from surviving animals of group 1 (n=9), 2 (n=3), 3 (n=2), 4 (n=10) and 5 (n=9) on study day 18. Blood samples were pooled per group. However, blood sampled from the mouse in group 1, which had escaped from virus inoculation, was kept apart. Serum was prepared following standard procedures. Sera were stored at -2O⁰C until assayed for the presence of virus neutralizing antibodies. Study Completion / Withdrawal

The animal phase of the study was terminated on study day 18. Animals were killed by administration of barbiturate followed by exsanguination.

Results

Table 1. Total clinical score

Groups of 10 Balb/c mice were inoculated i.n. on DO with 20 LD50 of H5N1 virus or PBS, and treated with BP-AI 100 in combination with Tamiflu from DO until D 13, with BP-AI 100 in combination with Tamiflu from D4 until D 13, or with Tamiflu alone from DO until D 13. Total clinical scores are summarized for each dose group on each study day. Total clinical scores were determined by multiplying the number of mice with score 3 (upper table) or multiplying the number of mice with score 3 added to the multiplicity of death cases receiving score 4 (below). Note that only 9 mice of group 1 (BP-AI 100/T 0-14) were infected.

Total clinical score per day *

Study Day

Treatment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

¹ one mouse was not infected

Total clinical score per day *

Study Day

Treatment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

¹ one mouse was not infected Table 2. Onset of respiratory signs following infection

Groups of 10 Balb/c mice were inoculated i.n. on DO with 20 LD50 of H5N1 virus or PBS, and treated with BP-AI 100 in combination with Tamiflu from DO until D 13, with BP-AI 100 in combination with Tamiflu from D4 until D 13, or with Tamiflu alone from DO until D13. Mice were examined daily for clinical signs for 14 days p.i. Number of mice out of 10 showing respiratory distress (dyspnoe) are summarized for each dose group on each study day. In groups where mice died before completion of the study, the number of the remaining mice are indicated. Note that only 9 mice were infected in group 1 (BP-AI 100/T0-14).

Number of mice showing respiratory distress '

Study Day

Treatment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

PBS/PB AI-100/T(0-14) 0 0 0 0 0 0 0 0 0 0 0 0 0 T (0-14) 0 0 0 0 0 0 0 0/9 0/9

* a score of 3 or more ^a out of 10 unless indicated otherwise

¹ by accident one mouse was not infected

Table 3 shows the number of surviving mice on each study day is indicated for each treatment group. Figure 1 shows the corresponding mortality curves.

Table 3. Mortality

Number of living mice on each study day

Study Day

Treatment -1 5 6 7 8 9 10 11 12 13 14 15 18 Total deaths

BP-AI 100/T(0 -14) 9 9 9 9 9 9 9 9 9 9 9 9 9 9 8 8 8 8 1 BP-AM00/T(4-14) 10 10 10 10 10 10 10 10 10 6 4 4 3 3 3 3 3 3 7 H5N1 10 10 10 10 10 10 10 10 9 6 5 4 4 3 2 2 2 2 8

PBS/BP-AI100/T(0-14) 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 0 T (0-14) 10 10 10 10 10 10 10 10 10 9* 9 9 9 9 9 9 9 9 1

¹ mouse died soon after oral administration of Tamiflu Conclusions

• Challenge with 20 LD50 of H5N1 influenza virus caused a mortality rate of 80% indicating that a lethal dose was used in this study.

• The BP-AI 100/Tamifiu combination therapy itself does not negatively affect mouse health.

• Treatment of mice with Tamiflu delayed onset of clinical signs, decreased severity of disease and reduced mortality.

• Tamiflu treatment combined with peptides is superior to treatment with Tamiflu alone: mice stayed healthy for one day longer and clinical scores were a lower over the entire period.

• The moderate effect observed with the combination therapy starting at 4 days p.i. is probably due to the less effective acting of Tamiflu if treatment is started later in the infection period.

Claims

1. Use of a peptide consisting of 3 to 6 amino acids for the production of a pharmaceutical composition for the treatment of a subject, preferably a human subject, suffering or believed to be suffering from an influenza infection.

2. Use according to claim 1, wherein said influenza infection is an avian influenza infection, preferably avian influenza infection caused by avian influenza virus H5N1.

3. Use according to claim 1 or 2, wherein said peptide is selected from the group of peptides LQG, AQG, LQGV, AQGV, MTR, MTRV, WC, QWC, VLPALP, and VLPALPQ, and mixtures thereof.

4. Use according to any one of claims 1-3, wherein said composition comprises a mixture of the peptides LQGV, MTRV and WC.

5. Use according to any one of claims 1-4, wherein said composition comprises an additional antiviral agent, preferably an M2 ion channel blocker or a neuramidase inhibitor, more preferably oseltamivir.

6. A method for the treatment of a subject, preferably a human, suffering or believed to be suffering from an influenza infection, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of an antiviral peptide consisting of 3 to 6 amino acids.

7. Method according to claim 6, wherein said subject is suffering or believed to be suffering from avian influenza infection, preferably avian influenza infection caused by avian influenza virus H5N1.

8. A method according to claim 6 or 7, wherein said antiviral peptide is selected from the group of peptides LQG₁ AQG, LQGV, AQGV, MTR, MTRV, WC, QWC, VLPALP, and VLPALPQ, and mixtures thereof.

9. Method according to any one of claims 6 to 8, comprising a combination treatment with an additional antiviral agent, preferably an M2 ion channel blocker or a neuramidase inhibitor, more preferably oseltamivir.

10. Method according to any one of claims 6 to 9, to wherein said pharmaceutical composition comprises a mixture of the peptides LQGV, MTRV and WC.

11. Method according to any one of claims 6 to 10, wherein said antiviral peptide or mixture thereof is administered in a dosage regime ranging from 0.1 mg/kg to 30 mg/kg of body weight, preferably from 1 mg/kg to 10 mg/kg.

12. A pharmaceutical composition comprising at least one antiviral peptide consisting of 3 to 6 amino acids in combination with an additional antiviral agent.

13. Pharmaceutical composition of claim 12, wherein said additional antiviral agent is an M2 ion channel blocker or a neuramidase inhibitor, preferably oseltamivir.

14. Pharmaceutical composition according to claim 12 or 13, wherein said antiviral peptide is selected from the group consisting of the peptides LQG, AQG, LQGV, AQGV, MTR, MTRV, WC, QWC, VLPALP, and VLPALPQ, and mixtures thereof.

15. Pharmaceutical composition according to claim 14, comprising a mixture of the peptides LQGV, MTRV and WC.

16. A pharmaceutical composition comprising a mixture of the peptides LQGV, MTRV and WC.

17. Pharmaceutical composition according to any one of claims 12 to 16, wherein said composition is formulated for oral administration.