WO2021198962A1 - Method for treating viral diseases - Google Patents

Abstract

A pharmaceutical composition comprising methionine enkephalin (MENK) alone or in combination with one or more antiviral agents for use in treatment or prevention of viral infections in a mammal comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition.

Description

METHOD FOR TREATING VIRAL DISEASES

FIELD OF THE INVENTION

The present invention relates to compositions for use and methods of preventing or treating in patients suffering from viral diseases such as Influenza A and novel coronavirus 2019 (COVID-19) comprising administering to a patient in need thereof a therapeutically effective amount of methionine enkephalin (MENK) alone or in combination with naltrexone or a pharmaceutically acceptable salt thereof.

BACKGROUND

The 2019-20 Wuhan coronavirus outbreak, formally the outbreak of novel coronavirus (COVTD- 19 or 2019-nCoV), is an ongoing viral epidemic primarily affecting Mainland China, along with isolated cases in 27 other countries and territories.

In early December 2019 a new coronavirus, designated 2019-nCoV, was identified in Wuhan, the capital of China's Hubei province, after 41 people developed pneumonia without a clear cause (2019-nCoV acute respiratory disease). The virus is capable of spreading from person to person and how has shown to spread from mother to child in the womb. The incubation period (time from exposure to onset of symptoms) ranges from 2 to 14 days, but it may be contagious during this period and after recovery. Symptoms include fever, coughing and breathing difficulties, and the virus can be fatal. Wuhan and Hubei Province have borne the brunt of the epidemic as the sudden shutdown of transportation links into and around the area slowed the shipping of vital medical supplies. The fatality rate in Wuhan is 4.1 percent and 2.8 percent in Hubei, compared to 0.17 percent elsewhere in mainland China.

A larger number of people may have been infected, but not detected (especially mild cases). According to official figures, As of 6 February 2020, there are 28,368 people infected of which 3,863 (14%) are in critical condition and 565 deaths have been attributed to the virus since the first confirmed death on 9 January, with 1,387 recoveries.

Ultimately, the outbreak could be controlled with a protective vaccine to prevent COVID-19 infection. While vaccine research should be pursued intensely, there exists today no therapy to treat COVID-19 upon infection, despite an urgent need to find options to help these patients and preclude potential death. Against that background the present invention seeks to alleviate the problems in the prior art in fighting infections. SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising methionine enkephalin (MENK) alone or in combination with one or more antiviral agents for use in treatment or prevention of viral infections in a mammal comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition. According to the invention the potential options to treat COVID-19 in patients, with an emphasis on the necessity for speed and timeliness. The present invention uses MENK alone or in combination with naltrexone to prevent and treat viral disorders specifically Influenza A and COVID-19 while also helping to recruit the immune system to build lasting immunity thus improving known treatment regimens. Without the wish to be bound by theory, the present invention thus involves the use of naltrexone and methionine enkephalin (“MENK”) MENK, the endogenous neuropeptide, is suggested to be involved in the regulatory loop between the immune and neuroendocrine systems, with modulation of various functions of cells related to both the innate and adaptive immune systems. The experiments showed that MENK serves as an immune modulator to the pathway between DCs and CD4+T cells. We studied changes of DCs in key surface molecules, the activity of acid phosphatases (ACPs), the production of IL-12, and the effects on murine CD4+T cell expansion and their cytokine production by MENK alone, and in combination with interkeukin-2 (IL-2) or interferon- (IFN- ). Further it was found that MENK could markedly induce the maturation of DCs through the addition of surface molecules such as MHC class II, CD86, and CD40 on murine DCs, the production of IL-12, and the down-regulation of ACP inside DCs, (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). It was also found that MENK alone or in combination with IL-2 or IFN- , could markedly up-regulate both CD4+T cell expansion and the CD4 molecule expression in vivo and in vitro and that MENK alone, or MENK+ IL-2, could enhance the production of interferon- from CD4+T cells. Moreover, MENK alone, or MENK+ IFN-, could enhance the production of IL-2 from CD4+T cells. It is therefore concluded that MENK can exert positive modulation to the pathway between dendtritic cells and CD4+T cells.

In another embodiment the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal further comprising sequential or simultaneously administration of a therapeutically effective amount of a pharmaceutical composition comprising either (+)- naltrexone or (-)-naltrexone or the mixture of naltrexone stereoisomers or a pharmaceutically acceptable salt thereof.

Further, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal, wherein the antiviral agent is an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal). In another embodiment of the invention the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal, wherein said viral infection is selected from the group consisting of influenza A, novel coronavirus 2019 (COVED 19) , 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV, SARS-CoV, molluscum contagiosum infection, HTLV infection, HTLV-1 infection, hepatitis-A, HCV, HBV, HIV/AIDS infection, human papilloma virus infection, herpes virus infection, genital herpes infection, viral dysentery, flu, measles, rubella, chickenpox, mumps, polio, rabies, mononucleosis, ebola, respiratory syncytial virus, dengue fever, yellow fever, lassa fever, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, viral meningitis, west Nile fever, arbovirus, parainfluenza, smallpox, Epstein-Barr virus, dengue hemorrhagic fever, cytomegalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, a hepatitis, cold sores, ocular herpes, meningitis, encephalitis, shingles, encephalitis, California serogroup viral, St. Louis encephalitis, rift valley fever, hand, foot, & mouth disease, hendra virus, enteroviruses, astrovirus, adenoviruses, Japanese encephalitis, lymphocytic choriomeningitis, roseola infantum, sandfly fever, S ARS, warts, cat scratch disease, slap-cheek syndrome, orf, pityriasis rosea and lyssavirus.

In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to claim 4 wherein the viral infection is influenza A infection. In a preferred embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to claim 4 wherein the viral infection is novel coronavirus 2019 (COVID 19) .

In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to claims any of the previous claims wherein the anti- viral agent is selected from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine and etravirine.

In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to claims any of the previous claims wherein said combination of anti-viral agents is selected from the group consisting of rilpivine abacavir and lamivudine; abacavir, dolutegravir and lamivudine; abacavir lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumerate; emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; and lopinavir and ritonavir.

Further according to the invention, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein the anti-viral agent is selected from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof.

I another embodiment the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to claims any of the previous claims further comprising one or more pharmaceutically acceptable excipients.

In yet another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein the excipients can be charged aqueous species that has a net negative charge or a net positive charge. Further according to the invention, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal further comprising administration of a therapeutically effective amount of a compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin and riboflavin or combinations thereof. In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal further comprising administration of a therapeutically effective amount of a compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins or combinations thereof.

Further according to the invention, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal further comprising administration of a therapeutically effective amount of an immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE or combinations thereof.

In another embodiment, the pharmaceutical composition for use in treatment or prevention of viral infections in a mammal further comprising the administration of an antibacterial agent. In yet another embodiment, the pharmaceutical composition for use in treatment or prevention of viral infections in a mammal according to claim 10 wherein the antibacterial agent is Azithromycin.

In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the anti-viral agent is remdesivir.

Further according to the invention, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the anti-viral agent is favipiravir.

In yet another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein the naltrexone or its pharmaceutically acceptable salt is in immediate release form. In yet another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein the amount of naltrexone is between about 1.0 mg and about 8.0 mg.

In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein the amount of naltrexone is between about 0.5 mg and about 6.0 mg.

In another embodiment, the pharmaceutical composition for use in treatment or prevention of viral infections in a mammal wherein the amount of naltrexone is between about 0.05 mg and about 4.5 mg.

In yet another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein said immediate release pharmaceutical composition is for administration once in a 24-hour period.

In yet another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein said mammal is a human.

In yet another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein said pharmaceutically acceptable salt of naltrexone is a hydrochloride salt.

Further according to the invention, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein said immediate release composition releases the pharmaceutically acceptable salt of naltrexone completely within about 60 minutes. In another embodiment, the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal wherein the pharmaceutical composition is for an administration route selected from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration.

In another embodiment the pharmaceutical composition may be for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the pharmaceutical composition is in the form of a capsule or tablet.

In another embodiment, the pharmaceutical composition for use in treatment or prevention of viral infections in a mammal wherein the composition is in the form of a rapidly dissolving film.

Further, according to the invention the composition for use and method further comprises administration of a therapeutically effective amount of a pharmaceutical composition comprising either (+)-naltrexone or (-)-naltrexone or the mixture of naltrexone stereoisomers or a pharmaceutically acceptable salt thereof.

In another embodiment of the invention the antiviral agent is an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal).

According to the invention the viral infection is chosen from the group consisting of influenza A, novel coronavirus 2019 (COVID19) , 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV, SARS-CoV, molluscum contagiosum infection, HTLV infection, HTLV-1 infection, hepatitis-A, HCV, HBV, HIV/AIDS infection, human papilloma virus infection, herpes virus infection, genital herpes infection, viral dysentery, flu, measles, rubella, chickenpox, mumps, polio, rabies, mononucleosis, ebola, respiratory syncytial virus, dengue fever, yellow fever, lassa fever, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, viral meningitis, west Nile fever, arbovirus, parainfluenza, smallpox, Epstein-Barr virus, dengue hemorrhagic fever, cytomegalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, a hepatitis, cold sores, ocular herpes, meningitis, encephalitis, shingles, encephalitis, California serogroup viral, St. Louis encephalitis, rift valley fever, hand, foot, & mouth disease, hendra virus, enteroviruses, astrovirus, adenoviruses, Japanese encephalitis, lymphocytic choriomeningitis, roseola infantum, sandfly fever, SARS, warts, cat scratch disease, slap-cheek syndrome, orf, pityriasis rosea and lyssavirus.

In a preferred embodiment the viral infection is influenza A infection.

In another preferred embodiment the viral infection is novel coronavirus 2019 (COVID19). In another embodiment of invention the anti-viral agent is chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine and etravirine. In yet another embodiment of the invention the combination of anti-viral agents is chosen from the group consisting of rilpivine abacavir and lamivudine; abacavir, dolutegravir and lamivudine; abacavir lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumerate; emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; and lopinavir and ritonavir.

In yet another embodiment of the invention the pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients. In another embodiment of the invention the excipients can be charged aqueous species that has a net negative charge of a net positive charge.

In another embodiment of the invention the method further comprises administration of a therapeutically effective amount of a compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin and riboflavin or combinations thereof. In another embodiment of the invention the method further comprisies administration of a therapeutically effective amount of a compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins or combinations thereof.

In another embodiment of the invention the method further comprises administration of a therapeutically effective amount of an immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE or combinations thereof.

In another embodiment of the invention the anti-viral agent is chosen from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof, alone or in combination with the administration of an antibacterial agent preferably azithromycin. In another embodiment of the invention the anti-viral agent is remdesivir and/or favipiravir.

In another object of the invention the naltrexone or its pharmaceutically acceptable salt, preferably the hydrochloride salt is in immediate release form.

In another embodiment of the invention the amount of naltrexone is between about 1.0 mg and about 8.0 mg. In another embodiment of the invention the amount of naltrexone is between about 0.5 mg and about 6.0 mg.

In another embodiment of the invention the amount of naltrexone is between about 0.05 mg and about 4.5 mg.

In another embodiment of the invention the immediate release pharmaceutical composition is for administration once in a 24-hour period.

In another embodiment of the invention the mammal is a human.

In another embodiment of the invention the immediate release composition releases the pharmaceutically acceptable salt of naltrexone completely within about 60 minutes.

In another embodiment of the invention the pharmaceutical compositions comprising either MENK or naltrexone or a pharmaceutically acceptable salt thereof or both is for an administration route chosen from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration, preferably in a capsule or tablet or a rapidly dissolving film.

Further embodiments:

1. A method for treating or preventing viral infections in a mammal comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising methionine enkephalin (MENK) alone or in combination with one or more antiviral agents.

2. The method according to embodiment 1 further comprising administration of a therapeutically effective amount of a pharmaceutical composition comprising either (+)-naltrexone or (-)- naltrexone or the mixture of naltrexone stereoisomers or a pharmaceutically acceptable salt thereof.

3. The method according to embodiments 1 or 2 wherein the antiviral agent is an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal).

4. The method according to any of the previous embodiments 1 -3 wherein said viral infection is chosen from the group consisting of influenza A, novel coronavirus 2019 (COVTD19) , 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKIJI (beta coronavirus), MERS-CoV, SARS-CoV, molluscum contagiosum infection, HTLV infection, HTLV-1 infection, hepatitis-A, HCV, HBV, HIV/AIDS infection, human papilloma virus infection, herpes virus infection, genital herpes infection, viral dysentery, flu, measles, rubella, chickenpox, mumps, polio, rabies, mononucleosis, ebola, respiratory syncytial virus, dengue fever, yellow fever, lassa fever, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, viral meningitis, west Nile fever, arbovirus, parainfluenza, smallpox, Epstein-Barr virus, dengue hemorrhagic fever, cytomegalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, a hepatitis, cold sores, ocular herpes, meningitis, encephalitis, shingles, encephalitis, California serogroup viral, St. Louis encephalitis, rift valley fever, hand, foot, & mouth disease, hendra virus, enteroviruses, astrovirus, adenoviruses, Japanese encephalitis, lymphocytic choriomeningitis, roseola infantum, sandfly fever, SARS, warts, cat scratch disease, slap-cheek syndrome, orf, pityriasis rosea and lyssavirus.

5. The method according to embodiment 4 wherein the viral infection is influenza A infection.

6. The method according to embodiment 4 wherein the viral infection is novel coronavirus 2019 (COVTD19).

7. The method according to any of the previous embodiments wherein the anti-viral agent is chosen from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine and etravirine.

8. The method according to any of the previous embodiments wherein said combination of anti viral agents is chosen from the group consisting of rilpivine abacavir and lamivudine; abacavir, dolutegravir and lamivudine; abacavir lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumerate; emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; and lopinavir and ritonavir.

9. The method according any of the previous embodiments wherein the anti-viral agent is chosen from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof.

10. The method to according any of the previous embodiments further comprising one or more pharmaceutically acceptable excipients.

11. The method according to embodiment 10 wherein the excipients can be charged aqueous species that has a net negative charge of a net positive charge.

12. The method according to any of the previous embodiments further comprising administration of a therapeutically effective amount of a compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin and riboflavin or combinations thereof.

13. The method according to any of the previous embodiments further comprising administration of a therapeutically effective amount of a compound selected from the group consisting of L- arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins or combinations thereof.

14. The method according to any of the previous embodiments further comprising administration of a therapeutically effective amount of an immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE or combinations thereof.

15. The method according to embodiment 9 further comprising the administration of an antibacterial agent.

16. The method according to embodiment 15 wherein the antibacterial agent is Azithromycin.

17. The method according to any of the previous embodiments wherein the anti -viral agent is remdesivir.

18. The method according to any of the previous embodiments wherein the anti -viral agent is favipiravir.

19. The method according to any of the previous embodiments wherein the naltrexone or its pharmaceutically acceptable salt is in immediate release form.

20. The method according to any of the previous embodiments wherein the amount of naltrexone is between about 1.0 mg and about 8.0 mg.

21. The method according to any of the previous embodiments wherein the amount of naltrexone is between about 0.5 mg and about 6.0 mg.

Brief Description of Drawings

Fig. 1 shows the experimental design of the inventive concepts.

Fig. 2 shows Influenza virus titer and TCID50.

Fig. 3 shows the optimal concentration of MENK on RAW264.7 anti-influenza virus.

Fig. 4 shows the morphology change and apoptosis rate of RAW264.7 cells infected with H1N1 treated with MENK.

Fig. 5 shows MENK inhibition of the replication of influenza virus in RAW264.7 cells. Fig. 6 shows MENK enhanced pro-inflammatory cytokine production by RAW264.7 cells infected with HINT

Fig 7 shows MENK upregulated MOR expression on RAW264.7 cells infected with HI Nl. Fig 8 shows MENK inhibited RAW264.7 cells infected with H1N1 through up-regulating TLR4 and NF-KB.

Fig 9 shows fluorescence microscopy images of the subcellular localization of TLR4 in MENK inhibited RAW264.7 cells infected with H1N1 under different treatment conditions, expressed on cell membrane.

Fig 10 shows the fluorescence microscopy images of the subcellular localization of NF-KB p65 in MENK inhibited RAW264.7 cells infected with HI N1 under different treatment conditions, expressed on cell nucleus.

DETAILED DESCRIPTION OF THE INVENTION

Terms and Definitions Used

Abbreviations: MENK, methionine enkephalin; IAV, influenza A virus; MOR, m-opioid receptor; hpi, hour post infection; TLRs, Toll-like receptors; PRRs, pathogen- recognition receptors; NF- KB, nuclear factor KB; NP, nucleoprotein. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. For example, such salts include salts from ammonia, L-arginine, betaine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine (2,2'- iminobis(ethanol)), diethylamine, 2-(diethylamino)-ethanol, 2-aminoethanol, ethylenediamine, N- ethyl-glucamine, hydrabamine, 1H- imidazole, lysine, magnesium hydroxide, 4-(2-hydroxyethyl)- morpholine, piperazine, potassium hydroxide, 1 -(2-hydroxy ethylj-pyrrolidine, sodium hydroxide, triethanolamine (2,2',2"-nitrilotris(ethanol)), tromethamine, zinc hydroxide, acetic acid, 2,2- dichloro-acetic acid, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 2,5-dihydroxybenzoic acid, 4-acetamido-benzoic acid, (+)-camphoric acid, (+)- camphor- 10-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, decanoic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, ethylenediaminetetraacetic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, D- glucoheptonic acid, D-gluconic acid, D-glucuronic acid, glutamic acid, glutaric acid, 2-oxo- glutaric acid, glycerophosphoric acid, glycine, glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, DL-lactic acid, lactobionic acid, lauric acid, lysine, maleic acid, (-)-L-malic acid, malonic acid, DL-mandelic acid, methanesulfonic acid, galactaric acid, naphthalene- 1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1 -hydroxyl- naphthoic acid, nicotinic acid, nitric acid, octanoic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid (embonic acid), phosphoric acid, propionic acid, (-)-L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (-+-)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid and undecylenic acid. Further pharmaceutically acceptable salts can be formed with cations from metals such as aluminium, calcium, lithium, magnesium, potassium, sodium, zinc and the like (see Pharmaceutical salts, Berge, S. M. etal., J. Pharm. Sci., (1977), Vol.66, pp.1-19).

Pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or a mixture thereof.

Salts of other acids than those mentioned above which for example are useful for purifying or isolating the naltrexone ( e.g . trifluoro acetate salts), also comprise a part of the invention. Typically, a pharmaceutically acceptable salt of a compound of naltrexone may be readily prepared by using a desired acid or base as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as hydrochloric acid may be added to an aqueous suspension of a compound of naltrexone and the resulting mixture evaporated to dryness (lyophilized) to obtain the acid addition salt as a solid. Alternatively, a compound of naltrexone may be dissolved in a suitable solvent, for example an alcohol such as isopropanol, and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration. The acid addition salts of the compounds of naltrexone may be prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the invention.

Also included are both total and partial salts, that is to say salts with 1, 2 or 3, preferably 2, equivalents of base per mole of acid of formula I or salts with 1, 2 or 3 equivalents, preferably 1 equivalent, of acid per mole of base of formula I.

Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates of the compound of the invention are within the scope of the invention. The salts of naltrexone may form solvates ( e.g ., hydrates) and the invention also includes all such solvates. The meaning of the word “solvates” is well known to those skilled in the art as a compound formed by interaction of a solvent and a solute (i.e., solvation). Techniques for the preparation of solvates are well established in the art (see, for example, Brittain. Polymorphism in Pharmaceutical Solids. Marcel Decker, New York, 1999.).

The invention also encompasses prodrugs of the compounds of formula I, i.e., compounds which release an active parent drug (naltrexone) in vivo when administered to a mammalian subject. A prodrug is a pharmacologically active or more typically an inactive compound that is converted into a pharmacologically active agent by a metabolic transformation. Prodrugs of naltrexone are prepared by modifying functional groups present in naltrexone in such a way that the modifications may be cleaved in vivo to release the parent compound. In vivo, a prodrug readily undergoes chemical changes under physiological conditions (e.g., are acted on by naturally occurring enzyme(s)) resulting in liberation of the pharmacologically active agent. Prodrugs of naltrexone wherein a hydroxyl or amino, of naltrexone is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino or carboxy group, respectively. Examples of prodrugs include esters (e.g., acetate, formate, and benzoate derivatives) of compounds of formula I or any other derivative, which upon being brought to the physiological pH or through enzyme action is converted to the active parent drug. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in the art (see, for example, Bundgaard. Design of Prodrugs. Elsevier, 1985).

Prodrugs may be administered in the same manner as the active ingredient to which they convert or they may be delivered in a reservoir form, e.g, a transdermal patch or other reservoir which is adapted to permit (by provision of an enzyme or other appropriate reagent) conversion of a prodrug to the active ingredient slowly over time, and delivery of the active ingredient to the patient.

The term “carrier” refers to a diluent, excipient, and/or vehicle with which an active compound is administered. The pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin, 18th Edition.

A “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the present application includes both one and more than one such excipient.

Naltrexone may be formulated for administration in any convenient way for use in human or veterinary medicine and the invention therefore includes within its scope pharmaceutical compositions comprising a compound of the invention adapted for use in human or veterinary medicine. Such compositions may be presented for use in a conventional manner with the aid of one or more suitable carriers. Acceptable carriers for therapeutic use are well-known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, in addition to, the carrier any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s). Unless otherwise noted the weight of the active ingredient refers to the weight of the free form of the active ingredient. If it is in a different form the skilled person knows how to adjust the weight accordingly.

While it is possible that a naltrexone or MENK may be administered as the bulk substance, it is preferable to present the active ingredient in a pharmaceutical formulation, e.g., wherein the agent is in admixture with a pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

Accordingly, the invention further provides a pharmaceutical composition comprising naltrexone or pharmaceutically acceptable salt thereof or MENK in admixture with a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, excipient, and/or vehicle with which an active compound is administered.

Naltrexone or MENK may be used in combination with other therapies and/or active agents. Accordingly, the invention provides, in a further aspect, a pharmaceutical composition comprising MENK or naltrexone or a solvate, hydrate, enantiomer, diastereomer, N-oxide or pharmaceutically acceptable salt thereof, a second active agent, and a pharmaceutically acceptable carrier.

The pharmaceutical compositions may comprise as, in addition to, the carrier any suitable binder, lubricant, suspending agent, coating agent and/or solubilizing agent.

Preservatives, stabilizers, dyes and flavoring agents also may be provided in the pharmaceutical composition. Antioxidants and suspending agents may be also used.

The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention may be prepared by processes known in the art, for example see W002/00196.

The term "Immediate release" is defined as a release of compound from a dosage form in a relatively brief period of time, generally up to about 60 minutes.

Routes of Administration and Unit Dosage Forms

The routes for administration include oral ( e.g ., as a tablet, capsule, or as an ingestible solution), topical, mucosal (e.g., as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g., by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracere- broventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, epidural and sublingual. The compositions of the invention may be especially formulated for any of those administration routes. In preferred embodiments, the pharmaceutical compositions of the invention are formulated in a form that is suitable for oral delivery.

There may be different composition/formulation requirements depending on the different delivery systems. It is to be understood that not all of the compounds need to be administered by the same route. Likewise, if the composition comprises more than one active component, then those components may be administered by different routes. By way of example, the pharmaceutical composition of the invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by multiple routes.

Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile. For example, naltrexone may be coated with an enteric coating layer. The enteric coating layer material may be dispersed or dissolved in either water or in a suitable organic solvent. As enteric coating layer polymers, one or more, separately or in combination, of the following can be used; e.g., solutions or dispersions of methacrylic acid copolymers, cellulose acetate phthalate, cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethylethylcellulose, shellac or other suitable enteric coating layer polymer(s). For environmental reasons, an aqueous coating process may be preferred. In such aqueous processes methacrylic acid copolymers are most preferred.

When appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges, which can be formulated in a conventional manner.

When the composition of the invention is to be administered parenterally, such administration includes one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.

Pharmaceutical compositions of the invention can be administered parenterally, e.g., by infusion or injection. Pharmaceutical compositions suitable for injection or infusion may be in the form of a sterile aqueous solution, a dispersion or a sterile powder that contains the active ingredient, adjusted, if necessary, for preparation of such a sterile solution or dispersion suitable for infusion or injection. This preparation may optionally be encapsulated into liposomes. In all cases, the final preparation must be sterile, liquid, and stable under production and storage conditions. To improve storage stability, such preparations may also contain a preservative to prevent the growth of microorganisms. Prevention of the action of micro-organisms can be achieved by the addition of various antibacterial and antifungal agents, e.g, paraben, chlorobutanol, or ascorbic acid. In many cases isotonic substances are recommended, e.g, sugars, buffers and sodium chloride to assure osmotic pressure similar to those of body fluids, particularly blood. Prolonged absorption of such injectable mixtures can be achieved by introduction of absorption-delaying agents, such as aluminium monostearate or gelatin.

Dispersions can be prepared in a liquid carrier or intermediate, such as glycerin, liquid polyethylene glycols, triacetin oils, and mixtures thereof. The liquid carrier or intermediate can be a solvent or liquid dispersive medium that contains, for example, water, ethanol, a polyol (e.g, glycerol, propylene glycol or the like), vegetable oils, non-toxic glycerine esters and suitable mixtures thereof. Suitable flowability may be maintained, by generation of liposomes, administration of a suitable particle size in the case of dispersions, or by the addition of surfactants. For parenteral administration, the compound is best used in the form of a sterile aqueous solution, which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Sterile injectable solutions can be prepared by mixing a compound of formula I with an appropriate solvent and one or more of the aforementioned carriers, followed by sterile filtering. In the case of sterile powders suitable for use in the preparation of sterile injectable solutions, preferable preparation methods include drying in vacuum and lyophilization, which provide powdery mixtures of the aldosterone receptor antagonists and desired excipients for subsequent preparation of sterile solutions.

The compound according to the invention may be formulated for use in human or veterinary medicine by injection ( e.g ., by intravenous bolus injection or infusion or via intramuscular, subcutaneous or intrathecal routes) and may be presented in unit dose form, in ampoules, or other unit-dose containers, or in multi-dose containers, if necessary with an added preservative. The compositions for injection may be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, solubilizing and/or dispersing agents. Alternatively, the active ingredient may be in sterile powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

Naltrexone or MENK can be administered (e.g., orally or topically) in the form of tablets, rapidly dissolving films, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate -, delayed-, modified-, sustained-, pulsed-or controlled-release applications.

Naltrexone or MENK may also be presented for human or veterinary use in a form suitable for oral or buccal administration, for example in the form of solutions, gels, syrups, mouth washes or suspensions, or a dry powder for constitution with water or other suitable vehicle before use, optionally with flavoring and coloring agents. Solid compositions such as tablets, rapidly dissolving films, capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules, bullets or premix preparations may also be used. Solid and liquid compositions for oral use may be prepared according to methods well-known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients which may be in solid or liquid form.

The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.

Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

The compositions may be administered orally, in the form of rapid or controlled release tablets, microparticles, mini tablets, capsules, sachets, and oral solutions or suspensions, or powders for the preparation thereof. In addition to the new solid-state forms of pantoprazole of the invention as the active substance, oral preparations may optionally include various standard pharmaceutical carriers and excipients, such as binders, fillers, buffers, lubricants, glidants, dyes, disintegrants, odourants, sweeteners, surfactants, mold release agents, antiadhesive agents and coatings. Some excipients may have multiple roles in the compositions, e.g., act as both binders and disintegrants. Examples of pharmaceutically acceptable disintegrants for oral compositions include starch, pre gelatinized starch, sodium starch glycolate, sodium carboxymethylcellulose, croscarmellose sodium, microcrystalline cellulose, alginates, resins, surfactants, effervescent compositions, aqueous aluminum silicates and cross-linked polyvinylpyrrolidone.

Examples of pharmaceutically acceptable binders for oral compositions include acacia; cellulose derivatives, such as methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose or hydroxyethylcellulose; gelatin, glucose, dextrose, xylitol, polymethacrylates, polyvinylpyrrolidone, sorbitol, starch, pre-gelatinized starch, tragacanth, xanthane resin, alginates, magnesium-aluminum silicate, polyethylene glycol or bentonite. Examples of pharmaceutically acceptable fillers for oral compositions include lactose, anhydrolactose, lactose monohydrate, sucrose, dextrose, mannitol, sorbitol, starch, cellulose (particularly microcrystalline cellulose), dihydro- or anhydro-calcium phosphate, calcium carbonate and calcium sulphate.

Examples of pharmaceutically acceptable lubricants useful in the compositions of the invention include magnesium stearate, talc, polyethylene glycol, polymers of ethylene oxide, sodium lauryl sulphate, magnesium lauryl sulphate, sodium oleate, sodium stearyl fumarate, and colloidal silicon dioxide. Examples of suitable pharmaceutically acceptable odourants for the oral compositions include synthetic aromas and natural aromatic oils such as extracts of oils, flowers, fruits ( e.g ., banana, apple, sour cherry, peach) and combinations thereof, and similar aromas. Their use depends on many factors, the most important being the organoleptic acceptability for the population that will be taking the pharmaceutical compositions.

Examples of suitable pharmaceutically acceptable dyes for the oral compositions include synthetic and natural dyes such as titanium dioxide, beta-carotene and extracts of grapefruit peel.

Examples of useful pharmaceutically acceptable coatings for the oral compositions, typically used to facilitate swallowing, modify the release properties, improve the appearance, and/or mask the taste of the compositions include hydroxypropylmethylcellulose, hydroxypropylcellulose and acrylate-methacrylate copolymers.

Examples of pharmaceutically acceptable sweeteners for the oral compositions include aspartame, saccharin, saccharin sodium, sodium cyclamate, xylitol, mannitol, sorbitol, lactose and sucrose. Examples of pharmaceutically acceptable buffers include citric acid, sodium citrate, sodium bicarbonate, dibasic sodium phosphate, magnesium oxide, calcium carbonate and magnesium hydroxide.

Examples of pharmaceutically acceptable surfactants include sodium lauryl sulphate and polysorbates.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Naltrexone or MENK may also, for example, be formulated as suppositories e.g., containing conventional suppository bases for use in human or veterinary medicine or as pessaries e.g., containing conventional pessary bases.

Naltrexone or MENK may be formulated for topical administration, for use in human and veterinary medicine, in the form of ointments, creams, gels, hydrogels, lotions, solutions, shampoos, powders (including spray or dusting powders), pessaries, tampons, sprays, dips, aerosols, drops (e.g., eye ear or nose drops) or pour-ons. For application topically to the skin, the agent of the invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol, and water. Such compositions may also contain other pharmaceutically acceptable excipients, such as polymers, oils, liquid carriers, surfactants, buffers, preservatives, stabilizers, antioxidants, moisturizers, emollients, colourants, and odourants. Examples of pharmaceutically acceptable polymers suitable for such topical compositions include acrylic polymers; cellulose derivatives, such as carboxymethylcellulose sodium, methylcellulose or hydroxypropylcellulose; natural polymers, such as alginates, tragacanth, pectin, xanthan and cytosan.

Examples of suitable pharmaceutically acceptable oils which are so useful include mineral oils, silicone oils, fatty acids, alcohols, and glycols.

Examples of suitable pharmaceutically acceptable liquid carriers include water, alcohols or glycols such as ethanol, isopropanol, propylene glycol, hexylene glycol, glycerol and polyethylene glycol, or mixtures thereof in which the pseudopolymorph is dissolved or dispersed, optionally with the addition of non-toxic anionic, cationic or non-ionic surfactants, and inorganic or organic buffers. Examples of pharmaceutically acceptable preservatives include sodium benzoate, ascorbic acid, esters of p-hydroxybenzoic acid and various antibacterial and antifungal agents such as solvents, for example ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methyl paraben, ethyl paraben and propyl paraben).

Examples of pharmaceutically acceptable stabilizers and antioxidants include ethylenediaminetetraacetic acid (EDTA), thiourea, tocopherol and butyl hydroxyanisole. Examples of pharmaceutically acceptable moisturizers include glycerine, sorbitol, urea and polyethylene glycol.

Examples of pharmaceutically acceptable emollients include mineral oils, isopropyl myristate, and isopropyl palmitate.

The compounds may also be dermally or transdermally administered, for example, by use of a skin patch. For ophthalmic use, the compounds can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.

As indicated, naltrexone or MENK can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134AT) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, e.g, using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g, sorbitan trioleate.

Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.

For topical administration by inhalation the compounds according to the invention may be delivered for use in human or veterinary medicine via a nebulizer.

The pharmaceutical compositions of the invention may contain from 0.01 to 99% weight per volume of the active material. For topical administration, for example, the composition will generally contain from 0.01-10%, more preferably 0.01-1% of the active material.

Naltrexone or MENK can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

The pharmaceutical composition or unit dosage form of the invention may be administered according to a dosage and administration regimen defined by routine testing in the light of the guidelines given above in order to obtain optimal activity while minimizing toxicity or side effects for a particular patient. However, such fine tuning of the therapeutic regimen is routine in the light of the guidelines given herein. The dosage of the active agents of the invention may vary according to a variety of factors such as underlying disease conditions, the individual’s condition, weight, gender and age, and the mode of administration. An effective amount for treating a disorder can easily be determined by empirical methods known to those of ordinary skill in the art, for example by establishing a matrix of dosages and frequencies of administration and comparing a group of experimental units or subjects at each point in the matrix. The exact amount to be administered to a patient will vary depending on the state and severity of the disorder and the physical condition of the patient. A measurable amelioration of any symptom or parameter can be determined by a person skilled in the art or reported by the patient to the physician.

The pharmaceutical composition or unit dosage form may be administered in a single daily dose, or the total daily dosage may be administered in divided doses. In addition, co-administration or sequential administration of a further compound for the treatment of the disorder may be desirable. To this purpose, the combined active principles are formulated into a simple dosage unit.

For combination treatment where the compounds are in separate dosage formulations, the compounds can be administered concurrently, or each can be administered at staggered intervals. For example, the compound of the invention may be administered in the morning and the additional antiviral compound may be administered in the evening, or vice versa. Additional compounds may be administered at specific intervals too. The order of administration will depend upon a variety of factors including age, weight, gender and medical condition of the patient; the severity and aetiology of the disorders to be treated, the route of administration, the renal and hepatic function of the patient, the treatment history of the patient, and the responsiveness of the patient. Determination of the order of administration may be fine-tuned and such fine-tuning is routine in the light of the guidelines given herein.

MENK, as an immune adjuvant, has potential immune-regulatory activity on innate and adaptive immune cells. The aim of this work was to investigate the antiviral elfect of MENK on influenza virus-infected murine macrophage cells (RAW264.7) and its underlying mechanisms. The results showed that MENK markedly inhibited influenza A virus (H1N1) replication in pre- and post- MENK treatment, especially in pre-MENK treatment. The mechanisms exploration revealed that MENK (10 mg/mL) significantly inhibited the nucleoprotein (NP) of influenza virus and up- regulated levels of IL-6, TNF-a and IFN-b compared with those in H1N1 control group. Further experiments confirmed that antiviral effects of MENK was associated with promotion of opioid receptor (MOR) as well as activation of NF-KB p65 inducing cellular antiviral status. The data suggest that MENK should be potential candidate for prophylactic or therapeutic treatment against H1N1 influenza virus.

Influenza A virus is the most serious influenza type with high morbidity and mortality widespread, resulting in a mild to moderately severe respiratory disease, and even systemic complications, and be- come the first infectious disease with global disease surveillance [1-3] In addition, mutant and re-emerging influenza strain might increase pathogenicity of the virus through altering viral receptor-binding specificity [4] and increasing viral polymerase activity [5], escape from immunity induced by prior infection and vaccination, and potentially develop into a rising global threat [6,7] Vaccines represent the most effective methods to prevent and control influenza virus infection, targeting antigenic drift in influenza virus HA protein [8] However, vaccines have drawbacks, including inadequate protection, high cost, difficulty in predicting representative strains, and time requirements for design and production [9-12] Therefore, novel anti-influenza strategies with high efficacy and low side effects are urgently in demand to prevent and control influenza epidemics.

Macrophages are antigen-presenting cells (APCs) and are known as innate immunity factor of defense against and eliminate infectious viruses. In the early stages of infection, rapid innate immune cells are effective in controlling respiratory epithelial cells infected with influenza virus and viral replication [13,14] Macrophages, as important specialized phagocytic cells of the innate immune system, express pathogen-recognition receptors (PRRs), with the capacity of lysosomal degradation, presenting antigens and secreting antiviral cytokines and chemokines. Toll like receptors (TLRs) as a kind of PRRs, activate cellular signaling pathways that induce nuclear factor KB (NF-KB) transduction, with the function of regulating inflammation involving macrophage capacity [17,18] During influenza virus infection, macrophages produce proinflammatory cytokines and antiviral agent, in- duce acute-phase inflammation, enhance recruitment, activate other immune cells, and finally control early virus replication through establishing antiviral immunity [19,20] MENK, an endogenous opioid peptide, composed of Tyr-Gly-Gly- Phe-Met, is potential to regulate both endocrine and immune systems via binding to opioid receptors (m,d,k) [21], MENK could trigger the second messengers Ca2+ and cAMP to modulate the phagocytic and boost pathogen elimination [22], Previous studies of our laboratory showed that MENK enhanced the production of cytokines, such as IL-1, IL-6, and exerted bidirectional modulation of cytotoxic activities by macrophage [23,24], Recently, published results indicated that MENK increased the level of macrophage surface marker CD64 and the ex- pression of inflammatory cytokines TNF-a to exert an antitumor activity [25], Our team demonstrated that MENK had anti-influenza virus activity by inhibiting inflammatory responses via binding to opioid receptors in vivo [26], However, the function of MENK in controlling influenza virus replication and related immunological regulation in macrophages remains obscured. Therefore, the following work was conducted with intention to find related clues.

Examples

Materials and methods Virus and cell culture

The influenza strain A/PR/8/34 (H1N1; PR8) was kindly provided by China Center for Disease Control and Prevention (Beijing, China). The virus was amplified in the allantoic cavities of lOd- old embryo chicken eggs [27], According to Reed and Muench methods, 50% tissue culture infective dose (TCID50) as a standardized indicator for viral titers was calculated by Hemagglutination test (HA). Murine macrophage cell line (RAW264.7) was purchased from the Cell Resource of Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 pg/mL streptomycin under a humidified atmosphere containing 5% C02 at 37 °C. When RAW264.7 cells were infected with influenza virus, the maintenance medium was changed to serum-free DMEM supplemented with 2 pg/mL TPCK-trypsin (Sigma, USA).

Reagents

MENK (>99% purity) was provided by America peptide Inc. RNeasy mini kit (74104) was purchased from Qiagen. One Step SYBR® Prime Script™ RT-PCR Kit (RR066A) was purchased from TaKaRa. The mAbs of Influenza A Virus Nucleoprotein (ab20343), NF- KB (abl6502), TLR4 (ab22048) and MOR (ab 10275) were purchased from Abeam. DyLight®488 IgG (H + L) was purchased from Earthox. Cell infection and treatment with MENK

The experiment was assigned to four groups: normal control group (Normal-C, cells were not infected and not treated with MENK), influenza virus infected control group (H1N1-C, cells were infected with H1N1 only), pre-treatment of MENK group (pre-MENK, cells were treated with MENK 24 h prior to H1N1 infection), and post-treatment of MENK group (post-MENK, cells were treated with MENK 1 h post (infection) as shown in Fig. 1.

Fig. 1. Experimental design. (A) RAW 264.7 cells were treated with MENK 24 h priorto influenza A/PR/8/34 H1N1 virus infection. (B) RAW 264.7 cells were treated with MENK 1 h after virus infection.

Table 1

PCR Primer Sequences.

Japan) using DP Manager software. Cell viability assay The effect of MENK on RAW264.7 cells proliferation was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS). RAW264.7 cells (1 x 104 cells/ well) were seeded in 96- well plates and treated with MENK (20, 10, 1, 10-1 ~ 10- 12 mg/mL). After 24, 48 and 72 h cultivated, MTS reagent was added to each well, and incubated for additional 4 h. The absorbance value (OD) at 490 nm was measured using a microplate reader (Bio- Rad, USA), and data were analyzed by GraphPad Prism software.

Antiviral effects of MENK

Pre-MENK treatment: RAW264.7 cells (2 x 104 cells/well) were seeded in 96 well plates for 24 h at 37 °C with 5% C02 and treated with MENK (10, 1, 10-1 and 10-2 mg/mL). After 24 h, MENK were removed and the cells were washed twice with 1 x PBS, inoculated 100 TCID50 H1N1 onto cells for 1 h, washed away unattached viruses, then added 100 μL DMEM to each well.

Post-MENK treatment: Virus inoculation was the same way as pre-MENK treatment. The virus- infected cells were treated with MENK (10, 1, 10-1 and 10-2 mg/mL) for 24 h. At 24 , 48 and 72 h pi, OD values were measured by MTS method. Data were analyzed to find out the optimal concentration of MENK. Observation of cell morphology

The RAW264.7 cells were infected with 100 TCID50 H1N1 and treated with optimal concentration of MENK (10 mg/mL) at pre-treatment and post-treatment model for 24 h. After 24, 48 and 72 h pi, morphology changes were observed under conventional light microscopy. The nuclei morphology of virus-infected cells was stained by Hoechst 33258. Images were captured using a fluorescence microscope (Olympus, Japan).

Membranal and intracellular molecules analysis For membranal molecules staining, the cells were washed and incubated with anti-TLR4 (1:100, 22048, Abeam) antibody for 1 h at room temperature, added secondary antibody DyLight®488 IgG (H + L) for 30 min. The cells were washed in cold PBS (1 x ) and resuspended in 300 pL cold PBS (1 x ) containing 2% fetal calf serum (FCS). For intracellular molecules staining, the cells were washed and added 100 pL Fixation/ Permeabilization solution (554714, BD Biosciences), incubated at 4°C for 20 min, washed in BD Perm/WashTM Bulfer (1 x ), added anti-influenza NP (1:100, 20343, Abeam), anti-NF-kB p65(l:100,

16502, Abeam) antibody, then washed and added DyLight®488 IgG (H + L) for 30 min. The cells were washed in BD Perm/WashTM Bulfer (1 x ), resuspended in 300 pL BD Perm/WashTM Bulfer (1 x ) and acquired by FACS Calibur (BD Biosciences).

RNA extraction and qPCR analysis

RNA was extracted from RAW264.7 cells according to the specification of RNeasy (74104, Qiagen). qPCR was performed using One Step SYBR Prime Script RT-PCR Kit (RR066A, TaKaRa) with QuantStudio 6 Flex Real-time PCR system (ABI, USA). qPCR reactions completed as follow: 5 min at 42 °C and 10 s at 95 °C, followed by 40 cycles-3 s at 95 °C, 30 s at 60 °C- and a melting curve step. Primer sequences are details in Table 1. Gene expression was calculated using the 2-AACT method.

ELISA assay

The cell culture supernatants were harvested at 24, 48, 72 h pi and stored at -20 °C until analysis. The concentrations of IL-6, TNF-a and IFN-b were determined using BD ELISA kit (BD Biosciences) according to instructions included in ELISA kit.

Immunofluorescence

The steps of RAW264.7 cells infected with 100TCID50 were as above. The cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 (except MOR dection) and blocked with 2% BSA for 30 min at room temperature, incubated with anti-influenza NP (1:100, 20343,

Abeam), anti-TLR4 (1:100, 22048, Abeam), anti-NF-kB p65 (1:100, 16502, Abeam), anti- MOR (1:100, abl0275) antibody at 4 °C overnight, followed by incubated for 1 h at room temperature with secondary antibody DyLight®488 IgG (H + L). Finally, added 25 pL DAPI- containing anti-fluorescence quencher to stain the cell nuclei. Images were acquired with a fluorescence microscope (Olympus, Japan)

Statistical analyses

All data were present as mean ± SEM and evaluated by one-way analysis of variance (ANOVA) or Student’s t-test. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software, USA). Values of ★p<0.05 or ★★p<0.01 were considered statistically significant.

Results

Influenza virus titer and TCID50 The virus titer increased from 1:32 to 1:512 by chicken embryo culture (Fig. 2A). To determine the appropriate infection dose for ap- plication in cell experiment, the TCID50 was determined. The results showed that 100TCID50 was 1 : 28.2 (Fig. 2B), equivalent to diluting the influenza A virus (H1N1) stock by 28.2 times to infect RAW264.7 cells.

The effect of MENK on cell viability and antiviral ability in RAW264.7 cells To find an optimal concentration for subsequent experiment, we detected the effects of MENK on cell viability and antiviral ability in RAW264.7 cells infected with influenza A virus (H1N1). The concentrations of MENK ranged form 20~10-7 mg/mL promoted cell proliferation, and 10, 1, 10-1 and 10-2 mg/mL were significant obviously in a dose-dependent manner (p < 0.01 or p < 0.05) (Fig. 3A). Based on the results, 10, 1, 10-1 and 10-2 mg/mL MENK were taken to investigate the optimal concentration of MENK against HINT As shown in Fig. 3B, pre-MENK and post- MENK treatment (10 and 1 mg/mL) statistically increased the proliferation rates of RAW264.7 cells at 24 h, 48 h and 72 h pi compared with that in the H1N1-C group (p < 0.01 or p < 0.05), and 10 mg/mL MENK upregulated the higher proportion of cell proliferation than other concentration. Based on these results, the experiments were performed with MENK at optimal concentration of 10 mg/mL.

MENK affected the morphological changes of RAW264.7 cells

The microscope images showed that RAW264.7 cells in the Normal- C group exhibited an initial state, mostly round and translucent. With lasting infection, RAW 264.7 cells displayed remarkable changes in cell morphology, typically produced longer protruding pseudopodia, changed to irregular polymorphism, and appeared to state of aging and death with vacuoles and granular substances in the cytoplasm. The cells in the pre-MENK group and post-MENK group had a long fusiform shape, and with many pseudopods, and occasional vacuoles and particulate substances appeared (Fig. 4A).

Hoechst 33,258 staining showed that the nuclei of the Normal-C group were dark blue, while the H1N1-C group had some apoptotic cells with densely or fragmented stained nucleus. In contrast, the nucleus in pre-MENK group and post-MENK group showed sporadic hyperchromatism, and small amount of fragmented dense in Fig. 4B and C. These results indicated that MENK inhibited the apoptosis of macrophages caused by influenza virus.

MENK inhibited influenza virus replication

The nucleoprotein (NP) of influenza virus encapsulated the negative strand of the viral RNA and was essential for replicative transcription. The relative viral amplification was markedly higher (112,760-fold) at 48 h pi in H1N1-C group, while 38,972-fold in pre-MENK group, and 60,534- fold was observed in post-MENK group compared with that in Normal-C group (Fig. 5A).

The flow cytometry results showed that the expression of Influenza NP significantly increased in RAW264.7 cells infected with influenza virus, and the mean fluorescence intensity (MFI) decreased with last- ingness of infection. The difference between H1N1-C group and Normal-C group was significant at 24, 48, 72 h pi (p < 0.01). In comparison to the H1N1-C group, the level of Influenza NP decreased at each time point in pre-MENK group and post-MENK group (p < 0.01 or p < 0.05) as shown in Fig. 5B and C.

The expression of influenza NP protein was localized by immuno- fluorescence staining and the result showed that cells did not present NP protein in Normal-C group, but at a high level in RAW264.7 cells infected virus at 48 h. MENK treatment down-regulated the expression of influenza NP in pre-MENK group and post-MENK group (Fig. 5D). These findings illustrated that both pre- and post-administration of MENK could reduce influenza virus replication on RAW264.7 cells effectively, and pre-treatment was more effective. MENK promoted productions of inflammatory cytokines

As shown in Fig. 6A, the levels of inflammatory cytokines at mRNA levels increased on 24, 48 and 72 h post-infection with HlNl(IL-6, 4.67-fold/3.42-fold/ 2.71-fold; TNF-a, 6.02-fold/ 4.60- fold/ 3.25-fold; and IFN-b, 6.58-fold/ 4.65-fold/ 2.51-fold) (p < 0.01). In comparison to H1N1-C group, the pre-treatment of MENK increased transcription of the above cytokines (IL-6, 8.02-fold/ 10.79-fold/ 5.43-fold; TNF-a, 12.45-fold/ 17.23-fold/ 9.98-fold; and IFN-b, 11.37- fold/ 15.54- fold/ 7.08-fold) (p < 0.01). Similarly, the post-treatment of MENK upregulated cytokines production (IL-6, 5.88-fold/ 7.79-fold/ 5.16-fold; TNF-a, 10.90-fold/ 14.07-fold/ 7.83-fold; and IFN-b, 9.67-fold/ 11.05- fold/ 5.64-fold) (p < 0.01 or p < 0.05). To further confirm changes of cytokines, the levels of IL-6, TNF-a and IFN-b in the cell supernatant were detected by ELISA. As shown in Fig. 6B, the secretions of IL-6, TNF-a and IFN- b in H1N1-C group were significantly higher than that in the Normal-C group on 24, 48 and 72 h pi (p < 0.01). Compared with H1N1-C group, pre-treatment of MENK significantly enhanced the level of IL-6, TNF-a and IFN-b in the cell supernatant at 24, 48 and 72 h pi (p < 0.01). Those cytokines in post- MENK group to some extent had the same tendency as pre-MENK group (p < 0.01 or p < 0.05). All data suggested that MENK mediated the antiviral effects in RAW264.7 cells by regulating the pro-inflammatory cytokines (IL-6, TNF-a) and Type I IFN (IFN-b).

MENK upregulated MOR expression

To correlate the observation above and assess the mechanisms basis for the effect of MENK, the mRNA expression of MOR in RAW264.7 cells was measured. There was no significant change of MOR between the Normal-C group and H1N1-C group. However, the expression of MOR obviously increased in pre-MENK group (4.10-fold) and post- MENK group (3.65-fold) as shown in Fig. 7A.

Immunofluorescence staining showed opioid receptors were located on cell membrane. There were no significant changes of the fluorescence intensity in cells infected with H1N1, while the intensity re- markly enhanced in pre-MENK group and post-MENK group (Fig. 7B). These results further indicated that MENK exerted antiviral function by upregulating receptors as part of the mechanism of H1N1 therapy.

MENK upregulated the level of TEL4 and NF-kB p65 There were no significant changes in the expression of TLR7 mRNA in four groups of RAW264.7 cells (p>0.05), but TLR4 mRNA changed to varying degrees. Thus, we detected the expressions of PRRs-related factors (TLR4 and NF-KB p65) by qPCR, FCM and immunofluorescence staining. The qPCR results showed that the mRNA levels of TLR4 were up-regulated in pre-MENK group (3.68-fold) and post-MENK group (3.31-fold) (p < 0.01). However, there was no significant difference in TLR4 mRNA between Normal-C group and H1N1-C group (p>0.05). Compare with H1N1-C group, the expressions of NF-KB p65 mRNA in- creased in pre-MENK group (5.73-fold) (p < 0.01) and post-MENK group(4.69-fold) (p < 0.05) (Fig. 8A).

FCM analysis further confirmed changes of TLR4/NF-KB p65 in RAW264.7 cells infected with influenza virus and treated with MENK. As shown in Fig. 8B and C, pre-MENK and post-MENK treatment significantly increased TLR4 and NF-KB p65 expressions at 24, 48, 72 h pi compared to those on untreated cells (p < 0.05 or p < 0.01).

Further description of the figures Fig. 2. Influenza virus titer and TCID50. (A) Influenza virus titer of chicken embryo culture were detected by Hemagglutination test (HA). (B) Dilution ratio of the virus TCID50 using Reed- Muench method.

Fig. 3. The optimal concentration of MENK on RAW264.7 anti-influenza virus. (A) Effect of MENK on proliferation of RAW264.7 cells. (B) The effect of significant concentration of MENK on RAW264.7 anti-influenza virus. Data represent the mean ± SEM of three independent experiments ★p < 0.05, ★★p < 0.01 versus the H1N1-C.

Fig. 4. Morphology change of RAW264.7 cells infected with H1N1 treated with MENK. (A) Light microscope morphology of RAW264.7 cells at 24, 48, 72 hpi. Morphology of infected cells produced longer processus pseudopodia, changed to irregular polymorphism, and appeared vacuoles and granular substances in the cytoplasm. Pre-MENK treatment had a long fusiform shape, and with many pseudopods. Some cells occasionally appeared vacuoles and particulate substances in Post-MENK group. (B) Hoechst staining of RAW264.7 cells at 24, 48, 72 hpi. Infected cells had a few apoptotic cells with nucleus stained by dense or fragmented at 48 and 72 hpi, sporadic hyperpigmentation of nucleus in Pre-MENK group and Post-MENK group. (C) Panel represents apoptosis cells. Data represent the mean ± SEM of three independent experiments, ★p <0.05, ★★p<0.01 versus H1N1-C group.

Fig. 5. MENK inhibited the replication of influenza virus in RAW264.7 cells. (A) Gene expression of virus was quantified at 48 hpi by quantitative PCR in RAW264.7 cells. (B) FCM analyzed the expression of influenza NP in RAW264.7 cells infected with H1N1 at 24, 48,72 hpi. (C) Panel represents the mean fluorescence intensity (MFI). (D) The localization and expression of influenza NP protein in RAW264.7 cells at 48 hpi. Influenza NP expressed on the cell nucleus was shown in green (DyLight 488), and in blue (DAPI) in nuclus. Data represent the mean ± SEM of three independent experiments. ★p<0.05, ★★p<0.01 versus H1N1-C group.

Fig. 6. MENK enhanced pro-inflammatory cytokine production by RAW264.7 cells infected with H1N1. (A) qPCR result to determine gene expressions of IL-6, TNF-a and IL-Ib on RAW264.7 cells at 24, 48,72 hpi. (B) ELISA result to determine expressions of IL-6, TNF-a and IL-Ib in the cell supernatant. Results of qPCR presented as fold increase over the Normal-C group. Data represent the mean ± SEM of three independent experiments, ★p < 0.05, ★★p < 0.01 versus H1N1-C group.

Fig. 7. MENK upregulated MOR expression on RAW264.7 cells infected with HINT (A) Gene expressions of MOR were quantified at 48 hpi by quantitative PCR in RAW264.7 cells. (B) The localization and expression of MOR in RAW264.7 cells at 48 hpi. MOR expressed on the cell membrane was shown in green (DyLight 488), and nucleus in blue (DAPI). Results were presented as fold increase over the Normal-C group. Data represent the mean ± SEM of three independent ex- periments. ★p<0.05, ★★p<0.01 versus H1N1-C group.

Fig. 8. MENK inhibited RAW264.7 cells infected with H1N1 through up-regulating TLR4 and NF-KB. (A) Gene expressions of TLR7, TLR4 and NF-KB p65 were quantified at 48 hpi by quantitative PCR in RAW264.7 cells. (B) FCM analysed the expression of TLR4 and NF-KB p65 in RAW264.7 cells infected with H1N1 at 24, 48,72 hpi. (C) Panel represented mean fluorescence intensity (MFI). Figures 9 and 10 The localization and expression of TLR4 and NF-KB p65 protein in RAW264.7 cells at 48 hpi. TLR4/ NF-KB p65 expressed on the cell membrane/nucleus was shown in green (DyLight 488), and nucleus in blue (DAPI). Data represent the mean ± SEM of three independent experiments. ★p<0.05, ★★p<0.01 versus H1N1-C group. However, there was no significant difference of levels of TLR4 between Normal-C group and H1N1-C group (p>0.05) As shown in Fig. 8D, the fluorescence microscopy images showed the subcellular localization of TLR4/NF-KB p65 in macrophages under different treatment conditions, expressed on cell membrane and nucleus respectively. The fluorescence intensity of TLR4 showed no significant change after cell infection, while pre- and post-MENK administration significantly enhanced the TLR4 expression. A small amount of NF-KB p65 expressed in Normal-C group, and significantly increased after cells infected. The expression of NF-KB p65 in pre-MENK group and post-MENK group were higher than that in H1N1-C group (p < 0.05 or p < 0.01). Our results indicated that MENK could mediate the antiviral effects in RAW264.7 cells by modulating the key effectors of TLR4 and NF- KB p65 expressions, related to PRRs signaling pathway.

General conclusions

Macrophages are an efficient phagocytic component of innate immune response to infection, and play a critical role in the clearance of pathogenic molecules and apoptotic cells, then activate adaptive immune response [28-30] Several studies have shown that clodronate liposome- mediated depletion of macrophages led to virus multiply re- plication and systemic spread of virus, exacerbated the progress of disease [31,32] MENK, as an immune regulating factor, can regulate NK cells, DC, macrophages, CD4+T cells, and CD8+T cells via binding to opioid receptor expression on immune cells [33-38]

The current data demonstrated that the optimal concentration of MENK treatment increased the proliferation rate of RAW264.7 cells infected with virus. Moreover, pre- and post-treatment of MENK de- creased influenza virus replication in RAW264.7 cells, and pre-treatment was more effective. After influenza virus infection, the macro- phages rapidly produced typellFN, cytokines and chemokines, which would regulate inflammatory response and immune response. TNF-a, a major pro-inflammatory cytokine, is produced by macrophages infected with influenza, could amplify production of other pro-inflammatory cytokines and chemokines, accelerate the recruitment of neutrophils and monocytes to the site of infection [39] IL-6 seems to play protective role in the model of IAV infection, accelerate viral clearance and limit inflammatory response [40], and blocking IL-6 might induce in- adequate for inflammation control. Type I IFN (IFN-a/b) expressed by virtually all cells, known as important mediator of virus elimination and activated antiviral state for protecting against acute influenza virus infection [41] Our results showed that MENK administration significantly enhanced the level of IL-6, TNF-a and IFN-b, suggested that MENK upregulated the non-specific immune response and enhanced level of inflammatory cytokines to accelerate viral clearance in RAW264.7 cells infected with H1N1, which may be one aspect of its antiviral efficacy through reverse the immunophenotype of Macro- phages. Our team has reported that MENK exerted an antitumor activity by inducing tumor-associated macrophages (TAMs) polarization from M2 to Ml type [25,42]

Previous experiments confirmed that MENK showed no direct killing effect on influenza virus (published in Chinese). When MENK was co-cultured with virus for 24 h, 48 h and 72 h, there was no change in virus titer. So, the consideration was what factors make MENK regulate RAW264.7 cells against influenza virus infection. Our data demonstrated that the MOR expressed on cell membrane was upregulated by MENK, while it did not change in H1N1-C group. This indicated that MENK played antiviral effects via binding to MOR. It has been reported that knockdown opioid receptor significantly cancelled anti- tumor function of MENK [25] Therefore, data from this study furthered the understanding that MENK’s antiviral effects were achieved by binding to opioid receptors. It was also found that the degree of upregulation of receptors and inflammatory cytokines in pre-MENK administration was higher than post-MENK. This suggested that MENK in prophylactic administration, up-regulated the status of macrophages before infection. Once the virus invaded, macrophages rapidly exerted immunobiological effects to recognize, phagocytose and eliminate virus, which may be the main reason for better preventive effect than that of therapeutic administration. Previous experiments of MENK inhibiting influenza virus infection in vivo also supported this view [26] Furthermore, to explore the underlying antiviral mechanisms of MENK, we followed the search in pattern-recognition receptors (PRRs)- related factors of TLR and NF-KB p65. TLRs are important receptors of PRRs, emerged as key sensors of innate immunity to viruses and highly expressed on immune cells. TLR7 is the major PRRs for the recognition of ssRNA viruses [43,44], but the findings here showed that there was no regulatory effects on TLR7 by MENK, and an interesting finding was that MENK could significantly upregulate the expression of TLR4 in RAW264.7 cells. However, as previously reported activation of TLR7 pathway by influenza virus infection was inhibited by MENK treatment in mice. The reason may be that MENK perform different functions in vivo and in vitro. In vivo, MENK bound to opioid receptors on the surface of various immune cells and stromal cells, such as DC, macrophages, NK, T cells, epithelial cells, endothelial cells, etc. These various cells exerted multiple effects to eliminate infection. Therefore, TLR7 pathway and downstream cytokines induced by influenza virus were not excessively activated. In vitro, MENK acted as an immunomodulator and had a positive immunoregulatory function, which promoted the conversion of macrophage types into classical Ml type with proinflammatory activity, upregulated the TLR4 pathway and induced the release of inflammatory cytokines. The previous findings further confirmed this point. MENK triggered activation of BMDCs via upregulating TLR4 through MyD88/NF-KB signaling pathway in vitro [45] According to documented reports, activation of TLR4-signaling during influenza infection induced an exaggerated inflammatory response in vivo [46-48] Our results indicated that MENK treatment significantly increased TLR4 and NF-KB p65 expression in RAW264.7 cells infected with influenza virus, while there was no significant difference in level of TLR4 between Normal-C group and H1N1-C group. Therefore, our data indicated that MENK maybe activate TLR4-NF-KB p65 signaling, increased inflammatory cytokines and typelIFNs, induced celluar antiviral state. Our team recently found MENK regulated cytokines production by downregulating TLR-MyD88-TRAF6-NF-KB p65 signaling pathway as a treatment for type 2 diabetes mellitus (T2DM) [49] Through comprehensive analysis, MENK tended to inhibit the TLR pathway in vivo, to reduce the expression of inflammatory factors and to upregulate TLR pathway and its downstream cytokines, then stimulated innate immune cells to exert anti-effects in vitro. Taken together, these studies indicate that MENK up-regulates macrophage opioid receptor, activates macrophages and positively regulates macrophage function to augment immune inflammatory response inducing celluar antiviral state, resulted in inhibition of influenza virus invasion and intracellular replication. Thus, the innate cellular immune regulation of MENK can provide new routes for the prevention and treatment of influenza virus. Moreover, MENK acts on innate immune cells by binding to opioid receptors, rather than the virus itself, supporting its use as an adjuvant of influenza vaccine. Therefore, our results illustrate that MENK also has the potential to be an effective nonspecific agent or vaccine adjuvant in preparation of pro-phylactic or therapeutic influenza vaccines. In addition to the work above MENK, as an immune adjuvant, has potential immune-regulatory activity on innate and adaptive immune cells. The aim of this work was to investigate the antiviral effect of MENK on influenza virus-infected murine macrophage cells (RAW264.7) and its underlying mechanisms. The results showed that MENK markedly inhibited influenza A virus (H1N1) replication in pre- and post-MENK treatment, especially in pre-MENK treatment.

The mechanisms exploration revealed that MENK (10 mg/mL) significantly inhibited the nucleoprotein (NP) of influenza virus and up-regulated levels of IL-6, TNF-a and IFN-b compared with those in H1N1 control group. Further experiments confirmed that antiviral effects of MENK was associated with promotion of opioid receptor (MOR) as well as activation of NF-KB p65 inducing cellular antiviral status. The data suggest that MENK should be potential candidate for prophylactic or therapeutic treatment against H1N1 influenza virus.

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Claims

1. A pharmaceutical composition comprising methionine enkephalin (MENK) alone or in combination with one or more antiviral agents for use in treatment or prevention of viral infections in a mammal comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1 for use in treatment or prevention of viral infections in a mammal according to claim 1 further comprising sequential or simultaneously administration of a therapeutically effective amount of a pharmaceutical composition comprising either (+)-naltrexone or (-)-naltrexone or the mixture of naltrexone stereoisomers or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claims 1-2, wherein the antiviral agent is an interferon, an immunomodulator, a viral replication inhibitor, an antisense agent, a therapeutic vaccine, a viral polymerase inhibitor, a nucleoside inhibitor, a viral protease inhibitor, a viral helicase inhibitor, a virion production inhibitor, a viral entry inhibitor, a viral assembly inhibitor, and an antibody therapy (monoclonal or polyclonal).

4. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims, wherein said viral infection is selected from the group consisting of influenza A, novel coronavirus 2019 (COVID19) , 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV, SARS-CoV, molluscum contagiosum infection, HTLV infection, HTLV-1 infection, hepatitis-A, HCV, HBV, HIV/AIDS infection, human papilloma virus infection, herpes virus infection, genital herpes infection, viral dysentery, flu, measles, rubella, chickenpox, mumps, polio, rabies, mononucleosis, ebola, respiratory syncytial virus, dengue fever, yellow fever, lassa fever, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, viral meningitis, west Nile fever, arbovirus, parainfluenza, smallpox, Epstein-Barr virus, dengue hemorrhagic fever, cytomegalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, a hepatitis, cold sores, ocular herpes, meningitis, encephalitis, shingles, encephalitis, California serogroup viral, St. Louis encephalitis, rift valley fever, hand, foot, & mouth disease, hendra virus, enteroviruses, astrovirus, adenoviruses, Japanese encephalitis, lymphocytic choriomeningitis, roseola infantum, sandfly fever, SARS, warts, cat scratch disease, slap-cheek syndrome, orf, pityriasis rosea and lyssavirus.

5. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 4 wherein the viral infection is influenza A infection.

6. The pharmaceutical composition for use in treatment or prevention of viral infections in a mammal according to claim 4 wherein the viral infection is novel coronavirus 2019 (COVID19).

7. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the anti-viral agent is selected from the group consisting of abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate, zidovudine (AZT), atazanivir, darunavir, fosamprenavir, indinavir, nelfinavir, ritonavir, saquinavir, tipranavir, enfuviritide, maraviroc, dolutegravir, elvitegravir, raltegravir, cobicistat, efavirenz, nevirapine and etravirine.

8. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein said combination of anti-viral agents is selected from the group consisting of rilpivine abacavir and lamivudine; abacavir, dolutegravir and lamivudine; abacavir lamivudine and zidovudine; atazanavir and cobicistat; darunavir and cobicistat; efavirenz, emtricitabine and tenofovir disoproxil fumerate; elvitegravir, cobicistat, emtricitabine, tenofovir alafenamide fumerate; elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumerate; emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine and tenofovir disoproxil fumerate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumerate; lamivudine and zidovudine; and lopinavir and ritonavir.

9. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the anti-viral agent is selected from the group consisting of hydroxychloroquine and chloroquine and pharmaceutically acceptable salts thereof.

10. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims further comprising one or more pharmaceutically acceptable excipients.

11. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 10 wherein the excipients can be charged aqueous species that has a net negative charge or a net positive charge.

12. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims further comprising administration of a therapeutically effective amount of a compound selected from the group consisting of ascorbic acid, cyanocobalamin, magnesium sulfate, pantothenate, nicotinic acid, pyridoxin, calcium D pantothenate, thiamin and riboflavin or combinations thereof.

13. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims further comprising administration of a therapeutically effective amount of a compound selected from the group consisting of L-arginine, L-homoarginine, homocysteine, L-glutamine and immunoglobins or combinations thereof.

14. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims further comprising administration of a therapeutically effective amount of an immunoglobin selected from the group consisting of IgG, IgA, IgM, IgD and IgE or combinations thereof.

15. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 9 further comprising the administration of an antibacterial agent.

16. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 15 wherein the antibacterial agent is Azithromycin.

17. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the anti-viral agent is remdesivir.

18. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the anti-viral agent is favipiravir.

19. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claims 2 to 18, wherein the naltrexone or its pharmaceutically acceptable salt is in immediate release form.

20. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 2 to 19 wherein the amount of naltrexone is between about 1.0 mg and about 8.0 mg.

21. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 2 to 19 wherein the amount of naltrexone is between about 0.5 mg and about 6.0 mg.

22. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 2 to 19 wherein the amount of naltrexone is between about 0.05 mg and about 4.5 mg.

23. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claims 19 to 22 wherein said immediate release pharmaceutical composition is for administration once in a 24-hour period.

24. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any one of the previous claims wherein said mammal is a human.

25. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 2 to 19 wherein said pharmaceutically acceptable salt of naltrexone is a hydrochloride salt.

26. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to claim 19 to 25 wherein said immediate release composition releases the pharmaceutically acceptable salt of naltrexone completely within about 60 minutes.

27. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the pharmaceutical composition is for an administration route selected from the group consisting of oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal and rectal administration.

28. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the pharmaceutical composition is in the form of a capsule or tablet.

29. The pharmaceutical composition according to any of the previous claims for use in treatment or prevention of viral infections in a mammal according to any of the previous claims wherein the composition is in the form of a rapidly dissolving film.