Pharmaceutical composition for treating and preventing HPV

Abstract

The present invention relates to a pharmaceutical composition for use in the treatment and prevention of HPV infection and / or related cancers, wherein said composition is capable of combating the implantation of HPV transformed cells and its subsequent proliferation and tissue invasion.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing and treating HPV infection and related lesions, wherein the composition can prevent the implantation of HPV-transformed cells and inhibit their subsequent proliferation and clonal expansion. Background Art

[0002] HPV infection is a common viral infection that causes benign proliferative lesions of the skin or mucosal epithelium (condyloma acuminatum or warts). There are more than 200 types of infection that are transmitted through direct skin-to-skin contact or direct mucosal-to-mucosal contact.

[0003] Most HPV infections are completely clinically inapparent. However, in some cases, small hyperplastic lesions may develop that persist for several months and eventually clear up on their own. However, it can have a significant impact on the patient's health, depending on its localization. The main clinical manifestations are as follows:

[0004] I. Genital warts. This is the most widespread sexually transmitted disease (STD) worldwide. It is mainly caused by HPV types 6 and 11 and appears as flat lesions, small cauliflower-like bumps, or tiny stalk-like projections. In women, genital warts mostly appear on the vulva, but may also appear near the anus, on the cervix, or in the vagina. In men, genital warts appear on the penis and scrotum or around the anus. Genital warts rarely cause discomfort or pain, although they may be itchy or painful. They last for 6-18 months and then clear up on their own. Despite this, they are often a source of deep anxiety, discomfort, and shame, and have a serious impact on personal and intimate life.

[0005] II. Common warts. Common warts, mainly caused by HPV-1, HPV-2, HPV-5, etc., appear as rough, raised cauliflower-like bumps, usually on the hands and fingers. In most cases, common warts are just unsightly, but they can also cause pain or cause easy injury or bleeding.

[0006] III. Plantar warts. Plantar warts are usually caused by HPV types 1, -2, -3, -4, -27, -29, -57, with HPV-1 considered the most prevalent. They are hard, granular growths that usually appear on the heel or sole of the foot. These warts may cause discomfort.

[0007] IV. Flat warts. Flat warts are slightly raised lesions on the top of the head. They may appear anywhere, but children usually get them on the face, and men tend to get them in the beard area. Women tend to get them on their legs.

[0008] V. Laryngeal papilloma. Children born to mothers infected with anogenital condyloma acuminatum (either clinically apparent or not) may develop juvenile laryngeal papilloma during early childhood. This is a biologically benign pediatric productive laryngeal infection, primarily caused by HPV-11, and is characterized by the growth of vocal cord condyloma acuminatum. These lesions may be large enough to compromise voice, feeding, and cause progressive respiratory distress with the potential risk of suffocation. Laryngeal condyloma acuminatum must be surgically removed, but the recurrence rate is high (nearly 40%), so it is not uncommon for children to undergo multiple surgeries each year.

[0009] VI. Dysplastic cervicovaginal lesions. HPV infection of the cervicovaginal epithelium is a very common phenomenon, estimated to occur at least once in any woman's lifetime. When these lesions are infected by several types of the AlphaPapillomavirus genus, the so-called high-risk HPV (HR-HPV), they produce moderate to severe dysplastic lesions. These lesions tend to persist and may eventually lead to invasive cervical cancer. Although cancer is indeed a rare complication of viral infection, the very high infection rates make it a fairly common phenomenon. In fact, cervical cancer is the third most common cause of neoplastic death in women worldwide and the eighth most common cause of neoplastic death in Italy and Southern Europe. In addition to the obvious burden of personal anxiety, pain and death associated with any type of neoplastic disease, cervical cancer imposes a very large burden on society, families and communities, as it affects middle-aged women at the most productive and quality stage of their lives, as it occurs most frequently between the ages of 30 and 50.

[0010] VII. Oropharyngeal cancer. It is now generally recognized that HR-HPV infection also plays a driving role in a subset of oropharyngeal and tonsillar cancers, the incidence of which is now significantly increasing.

[0011] VIII. Anorectal dysplasia and anorectal cancer. In part related to the mechanisms that occur in cervicovaginal lesions, HR-HPV is also a cause of anorectal cancer, a disease that is widespread and extremely underexplored and underdiagnosed among middle-aged homosexual men and male and female HIV patients.

[0012] HPV-specific vaccines have become available since the beginning of the 21st century. These vaccines have an excellent safety and efficacy profile and have the potential to completely eradicate any type of infectious and neoplastic HR-HPV-related conditions. However, despite this favorable background, vaccination campaigns have suffered a notable failure. In fact, due to economic, social and cultural reasons, coverage is not satisfactory in any region and the figures are steadily worsening (Gabutti G et al., 2021). Sadly, low vaccine coverage occurs mainly in low- and middle-income countries, as well as in social groups and geographical areas where HPV infections and related cancers are more prevalent. Therefore, in the following decades, it is expected that more than 50% of the world's population will remain completely susceptible to many potentially preventable neoplastic diseases.

[0013] Early diagnosis of preneoplastic lesions provides a second-line, non-vaccine-based tool for preventing HR-HPV-related conditions. Indeed, the development of a full neoplastic phenotype requires a long and complex series of events since HR-HPV infection is established. This process, known as neoplastic progression, usually takes several years to complete and, at least in the case of cervical cancer, is accompanied by a distinctive pattern of progressive severe cytological histological signs. Based on these signs, early detection of preneoplastic lesions can be easily accomplished before invasive growth begins.

[0014] However, once a dysplastic cervical lesion is detected, it must be surgically removed. For this purpose, there are several different technical methods, mainly including laser excision, electrosurgery (called loop electrosurgical excision procedure) (LEEP) or cold knife conization. Each of them is quite conservative, less invasive and suitable for day surgery. On the contrary, they all need to be performed by high-quality and therefore limited surgical equipment, are expensive and technically demanding, and what is worse, all of these methods are burdened with high recurrence rates, thus requiring close follow-up and often repeated surgical operations. Such a high recurrence rate is due to incomplete excision of the primary lesion and secondary implanted cells due to the sampling / excision process. These facts are also the reasons for the high recurrence rate of juvenile laryngeal papilloma mentioned above.

[0015] Sadly, the same sociological and psychological negative attitudes towards HPV vaccination also apply to cervical screening campaigns, which in reality are strictly adhered to by only a minority of the general population. Furthermore, it must be considered that the option of early detection of preneoplastic lesions is not feasible for anorectal, oropharyngeal and laryngeal cancers, since no specific cytohistological markers for preneoplastic lesions have been identified in these areas to date.

[0016] Therefore, the burden of HPV-related conditions is expected to continue worldwide for many years.Therefore, there is a great need for treatments that can reduce / antagonize the implantation of HPV-transformed cells, inhibit or limit the spread of already established lesions and prevent their secondary spread / invasion.

[0017] Based on the above considerations, new strategies for preventing and treating HPV-related cancers are urgently needed. These new strategies must be able to overcome the shortcomings of existing methods.

[0018] As we all know, polyphenols are a large family of molecules that are mainly distributed in food, health products and medicinal plants. They are very popular and are considered to have a wide range of pharmacological properties, including antioxidant, antimicrobial, antidiabetic, cytoprotective and antitumor activities. (Leri M et al., 2020)(Sur S et al., 2017)(Zhou Y et al., 2016)(Cardona F et al., 2013).

[0019] Flavonoids are a subclass of polyphenols in plant secondary metabolites that are widely found in fruits and vegetables and have been reported to have a variety of immunomodulatory, anti-inflammatory and antibacterial functions in mammals ( A et al., 2014)(Kopustinskiene DM et al., 2020).

[0020] The number of scientific reports on the potential antitumor effects of polyphenols and flavonoids is steadily increasing (SurS et al., 2017)(Zhou Y et al., 2016), however, the available data present a rather heterogeneous and sometimes contradictory picture, making it impossible to draw consistent conclusions. In addition, there are currently few reports dealing with the relevant field of metastatic processes or secondary implantation of transformed pre-neoplastic cells. During metastasis, cancer cells migrate from the primary site of clonal growth, invade the local extracellular matrix (ECM), enter the blood / lymph flow, migrate through the stroma of topologically distant and histologically different tissues, and establish secondary tumor growth. This requires the coordinated and sequential activation and inactivation of many specific cellular functions, including but not limited to adhesion, cytoskeletal remodeling, matrix digestion and deposition, and polarization / depolarization. In fact, the polyphenol prototype gallic acid was shown to reduce the migration of gastric cancer cells (Ho HH et al., 2010) and glioma cells (Lu J et al., 2010) in wound healing and Boyden chamber assays. Caffeic acid was found to reduce the invasiveness of PC3 prostate cancer cells by 50% (Lansky EP et al., 2005). Abel et al., (2018a; 2018b) reported that some purified polyphenolic fractions could partially reduce cell adhesion of PC3 and DU145 human prostate cancer cells on collagen-I (but not on fibronectin-I) coated plastic and reduce invasion of PC3 cells but not DU145 cells into Matrigel. However, all these effects were limited in extent, elicited only at very high concentrations, and were always accompanied by severe toxic effects. Summary of the invention

[0021] According to the present invention, it has now been surprisingly found that some polyphenolic compounds are effective against HPV.

[0022] Specifically, according to the present invention, the 16 polyphenols / flavonoids (hereinafter also referred to as RNPs) listed in Table 1 were tested as potential components of new pharmaceutical formulations for treating HPV-related conditions.

[0023] Table 1

[0024]

[0025]

[0026] Interestingly, among these 16 candidates, 6 showed significant anti-clonal effects. That is, kaempferol (9), galangin (10) and luteolin (12) were shown to have significant anti-clonal and anti-proliferative effects on HPV-transformed cells. According to the present invention, these three molecules can provide a core composition for innovative treatment to prevent the implantation of HPV-transformed cells and their subsequent proliferation and tissue invasion. Specifically, as described in Example 1, the combination of kaempferol (9), galangin (10) and luteolin (12) showed a synergistic effect against HPV infection and related diseases.

[0027] Furthermore, according to the invention, a second group of molecules consisting of chrysin (14), quercetin (15) and apigenin (16) also showed a good anti-clonal effect, albeit milder than one of the first three compounds, and could represent a group of complementary substances to improve the pharmacological profile of the core composition. In fact, their association could induce a clear synergistic effect, i.e. their combination induced an inhibitory effect of the same order or higher than that induced by the individual compounds. These data are summarized in Table 2.

[0028] Table 2

[0029]

[0030]

[0031] Table 2 shows the anti-clonal effects of the isolated polyphenols and their combinations. The inhibition relative rate (IRR) (column 4) provides the efficacy ranking of the molecules. Assuming quercetin, the less active molecule, as the efficacy unit, the IRR was calculated as the CID of the probe molecule 50 Reference CID of quercetin 50 Inversely proportional to.

[0032] Finally, according to the present invention, other molecules were found, namely vanillic acid (1) and caffeic acid (2), which, although without any direct activity on their own, are able to induce a certain degree of clonal inhibition when combined with galangin or quercetin, and the same is true for coumaric acid (3), ferulic acid (4), benzoic acid (5), chlorogenic acid (6) and protocatechuic acid (7), when they are combined with galangin, chrysin or apigenin ( Figure 9-11 ). This third group of compounds may have potential value as synergists.

[0033] Therefore, the present invention advantageously provides a pharmaceutical composition of low molecular weight polyphenols / flavonoids that can prevent the implantation, clonal expansion and secondary migration of HPV-transformed neoplastic cells. The purpose of the composition according to the present invention is to prevent the initiation of HPV transformation; to provide local drug treatment for established HPV-related lesions; to prevent recurrence after surgical excision.

[0034] More specifically, the pharmaceutical composition according to the present invention can be advantageously used for:

[0035] -Prevention of secondary implantation of HPV lesions after colposcopy and surgical excision of CIN II / CIN III cervical lesions.

[0036] -Prevention of secondary implantation of HPV lesions following other endoscopic / diagnostic or surgical excision of dysplastic / neoplastic lesions in the oropharynx and laryngeal cavity.

[0037] -Prevention of primary implantation and secondary spread of HPV lesions following oral-dental procedures in patients with a history of oropharyngeal and laryngeal dysplastic / neoplastic lesions.

[0038] -Prevention of HR-HPV infection in the female genital tract after unprotected / unprotected sexual intercourse.

[0039] - Painless, self-administered treatment for CIN-I or insignificant HR-HPV infection of the female genital tract.

[0040] This is a fairly common condition for which only a “watch and wait option” is currently available.

[0041] - Medical treatment of recurrent laryngeal papilloma of the juvenile type, a biologically benign, potentially fatal disease.

[0042] -Prevention of anal HPV infection and its secondary dysplastic / neoplastic lesions in HIV-positive patients and homosexual men, a condition that is difficult to suspect and diagnose and is increasing rapidly.

[0043] -Preventing nosocomial transmission of HR-HPV infection during endoscopic diagnosis

[0044] -Prevent recurrence of genital condyloma acuminatum after surgical removal.

[0045] Therefore, a specific object of the present invention is a pharmaceutical composition comprising or consisting of luteolin, galangin and kaempferol and one or more pharmaceutically acceptable excipients and / or adjuvants.

[0046] The luteolin, galangin and kaempferol compounds according to the present invention may be synthetic compounds or extracted from natural sources such as plants.

[0047] In the pharmaceutical composition according to the present invention,

[0048] Luteolin may be present in an amount of 1 to 20 μM, such as 1 to 10 μM, preferably 2 to 12 μM, such as 2 to 8 μM, more preferably 4 to 7 μM,

[0049] Galangin may be present in an amount of 10 to 40 μM, preferably 20 to 40 μM, more preferably 20 to 30 μM, even more preferably 25 μM,

[0050] Kaempferol may be present in an amount of 10 to 40 μM, preferably 20 to 40 μM, such as 20 to 30 μM, more preferably 22 to 30 μM, even more preferably 12 to 30 μM, such as 25 μM or 15 μM.

[0051] According to the present invention, the pharmaceutical composition may further comprise one, more than one or all compounds selected from chrysin, quercetin and apigenin.

[0052] The chrysin, quercetin and apigenin compounds according to the present invention may be synthetic compounds, or they may be isolated from natural sources such as plants.

[0053] In the pharmaceutical composition according to the present invention,

[0054] Chrysin may be present in an amount of 10 to 50 μM, preferably 15 to 30 μM, more preferably 20 μM or 25 μM,

[0055] Quercetin may be present in an amount of 40 to 80 μM, preferably 50 to 60 μM, more preferably 50 μM,

[0056] Apigenin may be present in an amount of 10 to 50 μM, preferably 10 to 40 μM, more preferably 15 to 30 μM, more preferably 25 μM.

[0057] According to an embodiment of the present invention, the pharmaceutical composition may also include one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevicornulin, preferably one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, more preferably caffeic acid and / or ferulic acid.

[0058] The vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevifolia compounds according to the present invention may be synthetic compounds, or they may be isolated from plants or from other natural sources.

[0059] According to an embodiment of the present invention, the pharmaceutical composition is not honey.

[0060] According to an embodiment of the present invention, the pharmaceutical composition does not contain other flavonoids or polyphenols other than luteolin, galangin and kaempferol.

[0061] According to another embodiment of the present invention, the pharmaceutical composition does not contain other flavonoids or polyphenols other than luteolin, galangin, kaempferol, chrysin, quercetin and apigenin.

[0062] According to another embodiment of the present invention, the pharmaceutical composition does not contain other flavonoids or polyphenols other than luteolin, galangin, kaempferol, chrysin, quercetin, apigenin, vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, and protocatechuic acid.

[0063] According to another embodiment of the present invention, the pharmaceutical composition does not contain other flavonoids or polyphenols other than luteolin, galangin, kaempferol, chrysin, quercetin, apigenin, vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevifolia.

[0064] According to the present invention, each of the vanillic acid, coumaric acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevicornulin compounds may be present in an amount of <0.100 mM, such as <0.050, while each of the caffeic acid and ferulic acid compounds are present in an amount of 20 to 100 μM.

[0065] The invention also relates to a pharmaceutical composition as defined above for medical use.

[0066] Furthermore, the present invention relates to a pharmaceutical composition as defined above for use in the prevention and treatment of viral infections and / or related diseases.

[0067] According to the present invention, the viral infection can be an HPV infection, such as human αHPV infection, and the related disease can be selected from the group consisting of: HPV-related cancers, HPV dysplastic lesions, HPV laryngeal papilloma, HPV genital warts, α papillomavirus subclinical anogenital and cervicovaginal infection, and α papillomavirus latent anogenital and cervicovaginal infection (i.e., simple molecular positivity).

[0068] According to an embodiment of the present invention, the pharmaceutical composition for the above use is not honey.

[0069] According to the present invention, the pharmaceutical composition can be administered by a route selected from the group consisting of: local route, intravenous route, intramuscular route, organ selective extracorporeal perfusion, intracavitary perfusion, intraperitoneal perfusion, intracerebroventricular or subarachnoid continuous perfusion.

[0070] Furthermore, according to the present invention, the pharmaceutical composition can be delivered via liposomes, nanoparticles; radiofrequency, electroporation, ion electrophoresis or shock wave delivery systems.

[0071] Another object of the present invention is a combination of luteolin, galangin and kaempferol for separate and sequential use in the prevention and treatment of viral infections and / or related diseases.

[0072] For the above-mentioned single or sequential use, the combination according to the present invention may also comprise one, more than one or all compounds selected from chrysin, quercetin and apigenin.

[0073] In addition, for the above-mentioned single or sequential use, the combination according to the present invention may also contain one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and pine pinus; preferably one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, more preferably caffeic acid and / or ferulic acid.

[0074] According to the invention, "single use" is understood to mean the simultaneous administration of the compounds of the combination according to the invention, wherein each compound is in a different pharmaceutical form.

[0075] According to the invention, "sequential use" is understood to mean the consecutive administration of the compounds of the combination according to the invention, wherein each compound is in a different pharmaceutical form.

[0076] Single and sequential use is in the order of hours, such as in the same day, same morning, same afternoon. In other words, all compounds in the combination must be administered within a period of time as part of a single drug prescription so that their therapeutic effects can combine with each other and achieve a synergistic effect.

[0077] According to the combination of the present invention, the viral infection can be an HPV infection, such as a human αHPV infection, and the associated disease can be selected from the group consisting of: HPV-related cancers, HPV dysplastic lesions, HPV laryngeal papilloma, HPV genital warts, α papillomavirus subclinical anogenital and cervicovaginal infection, and α papillomavirus latent anogenital and cervicovaginal infection (i.e., simple molecular positive). According to the present invention, the compound of the combination can be administered by a route selected from the group consisting of: a topical route, an intravenous route, an intramuscular route, an organ selective in vitro perfusion, an intracavitary perfusion, an intraperitoneal perfusion, an intraventricular or subarachnoid continuous perfusion.

[0078] According to the present invention, the compound of the combination can be delivered via liposomes, nanoparticles; radiofrequency, electroporation, ion electrophoresis or shock wave delivery systems.

[0079] The present invention also relates to one or more compounds selected from luteolin, galangin, kaempferol, chrysin, quercetin and apigenin for use in preventing and treating viral infections and / or related diseases.

[0080] According to the present invention, luteolin, galangin, kaempferol, chrysin, quercetin and apigenin may be synthetic compounds, or they may be isolated from other natural sources such as plants.

[0081] According to the present invention, the viral infection can be an HPV infection, such as a human αHPV infection, and the related disease can be selected from the group consisting of: HPV-related cancers, HPV dysplastic lesions, HPV laryngeal papilloma, HPV genital warts, α papillomavirus subclinical anogenital and cervicovaginal infection, and α papillomavirus occult anogenital and cervicovaginal infection (i.e., simple molecular positivity).

[0082] According to the present invention, the more compounds may be selected from the group consisting of: kaempferol and galangin; kaempferol and luteolin; kaempferol and chrysin; kaempferol and quercetin; kaempferol and apigenin; galangin and luteolin; galangin and chrysin; galangin and quercetin; galangin and apigenin; luteolin and chrysin; luteolin and quercetin; luteolin and apigenin; chrysin and quercetin; quercetin and apigenin; kaempferol, galangin and luteolin; kaempferol, galangin and chrysin; kaempferol, galangin and quercetin; kaempferol, galangin and apigenin; kaempferol, luteolin and chrysin; Naphtha, luteolin, and quercetin; kaempferol, luteolin, and apigenin; kaempferol, chrysin, and quercetin; kaempferol, chrysin, and apigenin; kaempferol, quercetin, and apigenin; galangin, luteolin, and chrysin; galangin, luteolin, and quercetin; galangin, luteolin, and apigenin; galangin, chrysin, and quercetin; galangin, chrysin, and quercetin; galangin, chrysin, and apigenin; galangin, quercetin, and apigenin; luteolin, chrysin, and quercetin; luteolin, chrysin, and apigenin; luteolin, quercetin, and apigenin; chrysin, quercetin, and apigenin; kaempferol, galangin, luteolin, and chrysin; kaempferol, galangin, luteolin, and chrysin; kaempferol, galangin, luteolin, and apigenin; kaempferol, galangin, chrysin, and quercetin; kaempferol, galangin, chrysin, and apigenin; kaempferol, galangin, quercetin, and apigenin; kaempferol, luteolin, chrysin, and quercetin; kaempferol, luteolin, chrysin, and quercetin; kaempferol, luteolin, chrysin, and apigenin; kaempferol, luteolin, chrysin, and apigenin; kaempferol, luteolin, quercetin, and apigenin; kaempferol, chrysin, quercetin, and apigenin; galangin, luteolin, chrysin, and quercetin; galangin, luteolin, quercetin, and apigenin; galangin, luteolin, quercetin, and apigenin; galangin, chrysin, quercetin, and apigenin; luteolin, chrysin, quercetin, and apigenin; Kaempferol, galangin, luteolin, chrysin and quercetin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, quercetin and apigenin; kaempferol, galangin, chrysin, quercetin and apigenin; kaempferol, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; preferably kaempferol, galangin and luteolin; chrysin and quercetin; kaempferol and apigenin; galangin and luteolin; chrysin and luteolin; or luteolin and apigenin.

[0083] The present invention also relates to a pharmaceutical composition for preventing and treating viral infection and / or related diseases, comprising one or more compounds selected from luteolin, galangin, kaempferol, chrysin, quercetin and apigenin, and one or more pharmaceutically acceptable excipients and / or adjuvants. According to the present invention, the viral infection may be HPV, such as human αHPV infection, and the related disease may be selected from the group consisting of: HPV-related cancer, HPV dysplastic lesions, HPV laryngeal papilloma, HPV genital warts, α-papillomavirus subclinical anogenital and cervicovaginal infection, and α-papillomavirus latent anogenital and cervicovaginal infection (i.e., simple molecular positive).

[0084] According to an embodiment of the present invention, the pharmaceutical composition for the above use is not honey.

[0085] According to the present invention, the more compounds in the pharmaceutical composition may be selected from the group consisting of: kaempferol and galangin; kaempferol and luteolin; kaempferol and chrysin; kaempferol and quercetin; kaempferol and apigenin; galangin and luteolin; galangin and chrysin; galangin and quercetin; galangin and apigenin; luteolin and chrysin; luteolin and quercetin; luteolin and apigenin; chrysin and quercetin; quercetin and apigenin; kaempferol, galangin and luteolin; kaempferol, galangin and chrysin; kaempferol, galangin and quercetin; kaempferol, galangin and apigenin; kaempferol, luteolin and quercetin; and chrysin; kaempferol, luteolin, and quercetin; kaempferol, luteolin, and apigenin; kaempferol, chrysin, and quercetin; kaempferol, chrysin, and apigenin; kaempferol, quercetin, and apigenin; galangin, luteolin, and chrysin; galangin, luteolin, and quercetin; galangin, luteolin, and apigenin; galangin, chrysin, and quercetin; galangin, chrysin, and apigenin; galangin, quercetin, and apigenin; luteolin, chrysin, and quercetin; luteolin, chrysin, and apigenin; luteolin, quercetin, and apigenin; chrysin, quercetin, and apigenin; kaempferol, galangin, luteolin, and chrysin; kaempferol, galangin, luteolin, and chrysin; , luteolin and quercetin; kaempferol, galangin, luteolin and apigenin; kaempferol, galangin, chrysin and quercetin; kaempferol, galangin, chrysin and apigenin; kaempferol, galangin, quercetin and apigenin; kaempferol, luteolin, chrysin and quercetin; kaempferol, luteolin, chrysin and apigenin; kaempferol, luteolin, chrysin and apigenin; kaempferol, luteolin, quercetin and apigenin; kaempferol, chrysin, quercetin and apigenin; galangin, luteolin, chrysin and quercetin; galangin, luteolin, chrysin and apigenin; galangin, luteolin, quercetin and apigenin; galangin, chrysin, quercetin and apigenin; luteolin, chrysin , quercetin and apigenin; kaempferol, galangin, luteolin, chrysin and quercetin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, quercetin and apigenin; kaempferol, galangin, chrysin, quercetin and apigenin; kaempferol, luteolin, chrysin, quercetin and apigenin; galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; preferably kaempferol, galangin and luteolin; chrysin and quercetin; kaempferol and apigenin; galangin and luteolin; chrysin and luteolin; or luteolin and apigenin.

[0086] In the pharmaceutical composition according to the invention as defined above

[0087] Luteolin may be present in an amount of 1 to 20 μM, such as 1 to 10 μM, preferably 2 to 12 μM, such as 2 to 8 μM, more preferably 4 to 7 μM,

[0088] Galangin may be present in an amount of 10 to 40 μM, preferably 20 to 40 μM, more preferably 20 to 30 μM, even more preferably 25 μM,

[0089] Kaempferol may be present in an amount of 10 to 40 μM, preferably 20 to 40 μM, such as 20 to 30 μM, more preferably 22 to 30 μM, even more preferably 12 to 30 μM, such as 25 μM or 15 μM,

[0090] Chrysin may be present in an amount of 10 to 50 μM, preferably 15 to 30 μM, more preferably 20 μM or 25 μM.

[0091] Quercetin may be present in an amount of 40 to 80 μM, preferably 50 to 60 μM, more preferably 50 μM.

[0092] Apigenin may be present in an amount of 10 to 50 μM, preferably 10 to 40 μM, more preferably 15 to 30 μM, more preferably 25 μM.

[0093] The pharmaceutical composition according to the present invention may also contain one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevicornulin; preferably caffeic acid, ferulic acid, vanillic acid, coumaric acid, benzoic acid, chlorogenic acid, protocatechuic acid, more preferably caffeic acid and / or ferulic acid.

[0094] For example, the pharmaceutical composition can include vanillic acid, galangin, and apigenin; caffeic acid, galangin, and apigenin; coumaric acid, galangin, chrysin, and apigenin; ferulic acid, galangin, chrysin, and apigenin; benzoic acid, galangin, chrysin, and apigenin; chlorogenic acid, galangin, chrysin, and apigenin; or protocatechuic acid, galangin, chrysin, and apigenin.

[0095] As mentioned above, the vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevifolia compounds according to the present invention may be synthetic compounds, or they may be isolated from natural sources such as plants.

[0096] According to the present invention, each of the vanillic acid, coumaric acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevicornulin compounds may be present in an amount of <0.100 mM, such as <0.050 mM, while each of the caffeic acid and ferulic acid compounds are present in an amount of 20 to 100 μM.

[0097] According to the present invention, the pharmaceutical composition can be administered by a route selected from the group consisting of: local route, intravenous route, intramuscular route, organ selective extracorporeal perfusion, intracavitary perfusion, intraperitoneal perfusion, intracerebroventricular or subarachnoid continuous perfusion.

[0098] According to the present invention, the pharmaceutical composition can be delivered via liposomes, nanoparticles; radiofrequency, electroporation, ion electrophoresis or shock wave delivery system.

[0099] The present invention also relates to a combination of one or more compounds selected from luteolin, galangin, kaempferol, chrysin, quercetin and apigenin for use alone or sequentially to prevent and treat viral infections and / or related diseases. According to the present invention, the viral infection may be HPV, such as human αHPV infection, and the related disease may be selected from the group consisting of: HPV-related cancers, HPV dysplastic lesions, HPV laryngeal papilloma, HPV genital warts, α-papillomavirus subclinical anogenital and cervicovaginal infection, and α-papillomavirus latent anogenital and cervicovaginal infection (i.e., simple molecular positive).

[0100] The combination for the above-mentioned single or sequential use according to the present invention may comprise one, more than one or all compounds selected from luteolin, galangin, kaempferol, chrysin, quercetin and apigenin.

[0101] In addition, the combination for use alone or sequentially according to the present invention may also contain one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevifolia, preferably caffeic acid, ferulic acid, vanillic acid, coumaric acid, benzoic acid, chlorogenic acid, protocatechuic acid, more preferably caffeic acid and / or ferulic acid.

[0102] According to the invention, "single use" is understood to mean the simultaneous administration of the compounds of the combination according to the invention, wherein each compound is in a different pharmaceutical form.

[0103] According to the invention, "sequential use" is understood to mean the consecutive administration of the compounds of the combination according to the invention, wherein each compound is in a different pharmaceutical form.

[0104] In particular, the combination according to the invention may comprise one or more compounds selected from the group consisting of: kaempferol and galangin; kaempferol and luteolin; kaempferol and chrysin; kaempferol and quercetin; kaempferol and apigenin; galangin and luteolin; galangin and chrysin; galangin and quercetin; galangin and apigenin; luteolin and chrysin; luteolin and quercetin; luteolin and apigenin; chrysin and quercetin; chrysin and apigenin; quercetin and apigenin; kaempferol, galangin and luteolin; kaempferol, galangin and chrysin; kaempferol, galangin and quercetin; kaempferol, galangin and apigenin; Kaempferol, luteolin, and chrysin; Kaempferol, luteolin, and quercetin; Kaempferol, luteolin, and apigenin; Kaempferol, chrysin, and quercetin; Kaempferol, chrysin, and apigenin; Kaempferol, quercetin, and apigenin; Kaempferol, quercetin, and apigenin; Galangin, luteolin, and chrysin; Galangin, luteolin, and quercetin; Galangin, luteolin, and apigenin; Galangin, chrysin, and quercetin; Galangin, chrysin, and apigenin; Galangin, quercetin, and apigenin; Luteolin, chrysin, and quercetin; Luteolin, chrysin, and apigenin; Luteolin, quercetin, and apigenin; Chrysin, quercetin, and apigenin; Kaempferol, galangin, luteolin, and quercetin; Luteolin, chrysin, and apigenin; Luteolin, quercetin, and apigenin; Chrysin, quercetin, and apigenin; Kaempferol, galangin, luteolin, and quercetin kaempferol and chrysin; kaempferol, galangin, luteolin and quercetin; kaempferol, galangin, luteolin and apigenin; kaempferol, galangin, chrysin and quercetin; kaempferol, galangin, chrysin and apigenin; kaempferol, galangin, quercetin and apigenin; kaempferol, luteolin, chrysin and quercetin; kaempferol, luteolin, chrysin and apigenin; kaempferol, luteolin, chrysin and apigenin; kaempferol, luteolin, quercetin and apigenin; kaempferol, chrysin, quercetin and apigenin; kaempferol, luteolin, chrysin and apigenin; galangin, luteolin, chrysin and quercetin; galangin, luteolin, chrysin and apigenin; galangin, luteolin, quercetin and apigenin; , chrysin, quercetin and apigenin; luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin and quercetin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, chrysin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; kaempferol, galangin, luteolin, chrysin, quercetin and apigenin; preferably kaempferol, galangin and luteolin; chrysin and quercetin; kaempferol and apigenin.

[0105] According to the present invention, the compounds of the combination may be administered by a route selected from the group consisting of topical route, intravenous route, intramuscular route, organ selective extracorporeal perfusion, intracavitary perfusion, intraperitoneal perfusion, intracerebroventricular or subarachnoid continuous perfusion.

[0106] According to the present invention, the compounds of the combination can be delivered via liposomes, nanoparticles; radiofrequency, electroporation, ion electrophoresis or shock wave delivery systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The present invention will now be described in an illustrative and non-limiting manner according to preferred embodiments of the invention, with particular reference to the Examples and the accompanying drawings, in which:

[0108] Figure 1 :The anti-cloning effect of RNP1-16 on HeLa cells.

[0109] Panel A): 50 μM of RNP 1-8 was applied to cells in rows AH, columns 1-6 (6 replicates per condition). None of the compounds induced any significant effect compared to control untreated cells (rows BH, columns 7-12). The positive control (NP) of the experiment is shown in row A, wells 7-12.

[0110] Panel B): Anti-cloning effect of RNP 9-16. In columns 1-6 of rows A, B, D, F, G and H, kaempferol (9), galangin (10), luteolin (12), chrysin (14), quercetin (15) and apigenin (16) clearly inhibited the cloning activity almost completely. No effect was observed for RNP11 brevifolia and RNP13 rutin. Panels C and D: Schematic topology of the treatments and growth conditions on panels A and B, respectively. Comparable results were obtained for both SiHa and ME-180 cells.

[0111] Figure 2 : Live cell analysis of the antiproliferative effect of RNPs. Siha cells were seeded in 96-well plates at 1000 cells / well and allowed to adhere overnight. The plates were then washed twice with PBS and supplemented with 100 microliters / well of fresh medium plus 50 μM of each compound. Images were taken every 12 hours; growth curves were drawn and the percentage of confluence was plotted against time. Panel A: For RNPs 1-8, 11, and 13-15, no antiproliferative effect was detected. In fact, their growth curves outlined a single spindle, completely covering and obscuring the growth curve of control untreated cells. In contrast, a clear antiproliferative effect was shown in Panel B, where growth curves for RNP 9 kaempferol (solid squares), RNP 12 luteolin (solid diamonds), RNP 16 apigenin (solid circles), and control untreated cells (solid stars) are depicted. Luteolin induced an almost complete proliferation block, while apigenin and kaempferol achieved partial blockade of approximately 20% and 40%, respectively. Each point represents the mean of six independent replicates. The bar width represents the standard deviation (SD). Consistent results were also obtained for HeLa and ME-180 cells.

[0112] Figure 3 : Titration of the anti-clonal effects of galangin and luteolin. Panel A. Hela cells were challenged with serial two-fold dilutions of 50 μM luteolin (columns 1-6) and 100 μM galangin (columns 7-10). Hela control untreated cells are in columns 11-12. The clonal activity of luteolin (solid box) and galangin (dashed box) was significantly reduced. The percentage of clonal inhibition was best assessed by the percentage of confluence assessed by live cell image analysis shown in panels B and C, which shows the CID of luteolin 50 The CID of galangin was 6.25 μM. 50 is 12.5 μM (solid line and dashed line boxes in Figure A, respectively).

[0113] Figure 4 : Kaempferol / Apigenin synergistic activity. In this experiment, kaempferol and apigenin were combined or applied to Siha cells in decreasing concentrations, as shown in the topology (Figure C). Cells treated with 12.5 μM kaempferol (well F: 6, 7, solid line boxes in Figure A) or 12.5 μM apigenin (well A: 1, 2 line boxes) were not different from control untreated cells (well G: 6, 7 dotted boxes). Interestingly, they were each applied at 12.5 μM simultaneously, inducing a sharp decrease in cloning efficiency (well C: 6, 7 solid line ellipses). The histogram in Figure B describes the number of colonies in each well evaluated by visual inspection and counting. Similar results were obtained for HeLa and ME-180 cells.

[0114] Figure 5 : Synergistic effect of combined administration of chrysin and quercetin on HeLa cells. Panel A: Endpoint anti-clonal effect of chrysin / quercetin at 12.5 / 25 μM induced stronger clonal inhibition (dashed ellipse) than chrysin (dashed box) or quercetin (segmented box) alone. Panel B: Topological schematic of treatment and growth conditions on the plate. Panels C and D: Live cell analysis of the conditions highlighted in panel A. The growth curve of the chrysin / quercetin combination at 12.5 / 25 μM (triangular line in panel D) was always lower than the curves of chrysin and quercetin alone (circular line in panel C and inverted triangle line in panel D, respectively). Almost overlapping results were also obtained in Ca-Ski cells.

[0115] Figure 6: Synergistic effect of galangin / luteolin combination. Figure A: End-point anti-clonal effect of galangin and luteolin applied in decreasing concentrations on SiHa cells. Cells treated with 6.25μM luteolin (wells D: 11, 12, solid squares in Figure a) or 25.0μM galangin (wells F: 3, 4, line squares) were no different from control untreated cells (wells G: 3, 9 line ellipses). Interestingly, their simultaneous application at 6.25 / 25.0μM, respectively, induced a significant decrease in cloning efficiency (wells D: 3, 4, solid ellipses). The histogram in Figure B describes the number of colonies in each well evaluated by visual inspection and counting.

[0116] Panel C is a topography of the plate treatment. Each condition was analyzed in duplicate. Similar results were obtained in HeLa cells.

[0117] Figure 7 : Synergistic effect of chrysin / luteolin. Figure A: End-point anti-clonal effect of chrysin and luteolin applied in decreasing concentrations on Siha cells. Cells treated with 6.25μM luteolin (wells D: 11, 12, solid squares in Figure a) or 25.0μM galangin (wells F: 3, 4, line squares) were no different from control untreated cells (wells G: 3, 11 line ellipses). Interestingly, their simultaneous application at 6.25 / 25.0μM, respectively, induced a significant decrease in cloning efficiency (wells D: 3, 4, solid ellipses). The number of colonies in each well evaluated by visual inspection and counting is depicted in a histogram in Figure B. Figure C is a topological diagram of the plate treatment. Each condition was analyzed in duplicate.

[0118] Figure 8 : Synergistic effect of luteolin / apigenin combination. Figure A: Endpoint anti-clonal effect of luteolin and apigenin applied to Siha cells in decreasing concentrations. Cells treated with 6.25μM luteolin (well D: 11, 12, solid line boxes in Figure a) or 25.0μM apigenin (well F: 3, 4, line segment boxes) were no different from control untreated cells (well G: 1, 12 line segment ellipses). Interestingly, their simultaneous application at 6.25 / 25.0μM, respectively, induced a significant decrease in cloning efficiency (well D: 3, 4, solid line ellipses). In Figure B, the histogram describes the number of colonies in each well evaluated by visual inspection and counting. Figure C is a topological schematic diagram of the plate treatment. Each condition was analyzed in duplicate.

[0119] Fig. 9: Shows that both vanillic acid and caffeic acid have a synergistic effect when applied together with galangin and apigenin. Hela cells were treated with 50 μM vanillic acid or caffeic acid (on the left and right sides of the plate, respectively) in combination with other compounds (25 μM for each compound except galangin applied at 12.5 μM). Panel A shows the endpoint clonal analysis. In Panel B, a topological sketch of the treatment is shown. It can be seen that both vanillic acid and caffeic acid induced almost complete clonal inhibition when combined with galangin (solid ellipse in Panel A) or apigenin (dashed ellipse in Panel A), while neither of them induced any effect when compared to control untreated cells. Vanillic acid is in the solid box, caffeic acid is in the line segment box, and control untreated cells are in the line-dot-line box. For the individual effects of galangin 12.5 μM and apigenin 25.0 μM, see respectively. Figure 3 , Hole D: 7-10 and Figure 8 , Wells F: 3, 4. The extent of clonal inhibition is best assessed by percent confluence as assessed by live cell image analysis shown in panels B and C. Panel D is a schematic topology of plate processing. Each condition was analyzed in triplicate.

[0120] Fig.10 : Coumaric acid and ferulic acid have a synergistic effect with galangin, chrysin and apigenin. HeLa cells were treated with 50 μM of coumaric acid or ferulic acid (left and right sides of the plate, respectively) and 25 μM of the other compounds (except galangin applied at 12.5 μM). Panel A shows the endpoint clonal analysis. As can be seen, coumaric acid and ferulic acid induced an almost complete clonal inhibition of HeLa cells when combined with galangin (solid ellipse) or chrysin (segmented ellipse) or apigenin (dashed ellipse). When applied alone, none of them induced any effect: coumaric acid is in the solid box, ferulic acid in the segmented box, and control untreated cells in the dashed box. For the individual effects of galangin 12.5 μM, chrysin 25.0 μM and apigenin 25.0 μM, see respectively. Figure 3 , hole D: 7-10, Figure 7 , hole F: 3, 4 and Figure 8 , wells F: 3, 4. The extent of clonal inhibition is best assessed by percent confluence as assessed by live cell image analysis as shown in panels B and C. Panel D is a graphic topography of plate treatments. Each condition was analyzed in triplicate. Overlapping results were also obtained with Ca-Ski or ME-180 cells.

[0121] Fig.11: Synergistic effects of benzoic acid, chlorogenic acid and protocatechuic acid with galangin, chrysin and apigenin. HeLa cells were treated with 50 μM of benzoic acid or chlorogenic acid or protocatechuic acid and 25 μM of the other compounds as shown in Figures E, F and G. It can be seen that their combination with 25 μM of galangin (solid ellipse) or chrysin (segmented ellipse) or apigenin (dashed ellipse) induced an almost complete clonal inhibition of HeLa cells. In contrast, when applied alone, none of them induced any effect: benzoic acid is in the solid box, chlorogenic acid in the segmented box, and control untreated cells in the dashed box. For the individual effects of galangin 12.5 μM, chrysin 25.0 μM and apigenin 25.0 μM, respectively, see Figure 3 , hole D: 7-10, Figure 7 , hole F: 3, 4 and Figure 8 , Well F: 3, 4. The histograms in panels B, C, and D depict the number of colonies per well assessed by visual inspection and counting under the highlighted conditions. Similar results were obtained using SiHa cells.

[0122] Fig.12 : Synergistic effect of simultaneous administration of kaempferol, galangin, and luteolin to Siha cells. Kaempferol, galangin, luteolin, and their combination were applied to SiHa cells, wherein kaempferol, galangin, luteolin were each at a concentration of 25 μM and the combined concentrations were 12.5 / 12.5 / 6.25 μM. The treatments were allowed to stay on the cells for 2 or 4 / 8 / 16 / 24 hours. Thereafter, the treatments were washed off, the cells were washed twice with PBS, fresh medium was replenished, and the anti-clonal effect was then monitored by live cell image analysis.

[0123] As can be seen, RNP9, RNP10 or RNP12 require almost 24 hours to induce approximately 50% clonal inhibition. In contrast, for the triple combination, clonal activity is easily inhibited with only an 8-hour treatment, while the extent is significantly greater than 50% with a 16-hour long treatment. Finally, the clonal inhibition achieved by the triple combination after 24 hours is much stronger than that of the single components. Therefore, the triple combination is superior to its single components in terms of shorter induction time and extent of action. Similar results were obtained with HeLa cells. DETAILED DESCRIPTION

[0124] Embodiment 1: Study on the anti-HPV effect of the polyphenolic compounds according to the present invention.

[0125] Materials and methods

[0126] The following molecules (hereinafter also referred to as RNPs) have been analyzed for their antitumor activity.

[0127] 1. Vanillic Acid

[0128] 2. Caffeic Acid

[0129] 3. Coumaric acid

[0130] 4. Ferulic acid

[0131] 5. Benzoic acid

[0132] 6. Chlorogenic Acid

[0133] 7. Protocatechuic acid

[0134] 8. Pinosin

[0135] 9. Kaempferol

[0136] 10. Galangin

[0137] 11. Brassinolide

[0138] 12. Luteolin

[0139] 13. Rutin

[0140] 14. Chrysin

[0141] 15. Quercetin

[0142] 16. Apigenin

[0143] Chemicals

[0144] Polyphenols were obtained as 99% purified material from Sigma-Aldrich Co (Merk Life Science Srl. Via Monte Rosa, 93. Milano - Italy).

[0145] cell

[0146] A total of 6 cell lines listed in Table 3 were used for this study. The cell lines were obtained from cell biology research centers in Italy, the United Kingdom and Germany. This study was based entirely on in vitro experiments and no information or materials derived from patients were used, so no ethical issues were raised. Informed consent was not required according to national and international ethical rules.

[0147] Table 3

[0148]

[0149]

[0150]

[0151] Note: Cell line characteristics are fully described at www.cellosaurus.org. Available accession numbers are reported in column 5. NA: Not available.

[0152] HeLa cells are the oldest continuous cell line, originally derived from HPV18-positive metastatic cervical cancer (Scherer WF et al., 1953). Siha cells are an epithelial cell line derived from HPV16-positive invasive cervical squamous cell carcinoma (Friedel F et al., 1970). Ca-Ski cells are cell lines containing multiple copies of transcriptionally active HPV16 genomes (Baker CC et al., 1984). Both Hela and Siha were gifts from Professor MS Campo of the Beatson Cancer Research Institute in Glasgow, Scotland. Due to the versatility and ease of use of these two cell lines, they have been used in most reported experiments. Ca-Ski cells were kindly provided by Professor Matthias Dürst of the German Cancer Research Center in Heidelberg, Federal Republic of Germany. These cells used with Siha provide a model of genetic instability and variability of cervical cancer. HK-168 is an immortalized, non-tumorigenic, continuous cell line derived from human skin keratinocytes transformed in vitro with a complete HPV16 genome (DeMarco F et al., 2007). This cell line is thought to recapitulate the biological features of transformed dysplastic cells and was used in preliminary experiments to set experimental doses and conditions. ME-180 cells are a cell line containing a small number of transcriptionally active copies of HPV68 and provide an in vitro model for HPV-induced dysplastic / neoplastic lesions with an intermediate carcinogenic risk (YeeCC et al., 1985). HaCaT cells were derived from solar skin lesions by Petra Boukamp et al. (1988) and were used herein as an in vitro equivalent of UV-induced pre-neoplastic epithelial cells to exclude possible direct toxic effects of polyphenols. Both HaCaT and ME-180 were generously donated by Professor Matthias Dürst. HeLa, Siha, Ca-Ski, ME-180 and HaCaT were all grown in high glucose DMEM supplemented with 10% fetal calf serum (FCS). In high Ca 2+ HK-168 cells that retain terminal differentiation capacity at 40 and 100% FCS concentrations were grown in K-SFM, a chemically defined medium supplemented with human EGF and bovine pituitary extract (ThermoFisher Scientific, Life Technologies, Monza–MI). Each cell line was subcultured twice weekly at an appropriate split ratio according to the specific proliferation index. In no case was antibiotics or antimycotics added to the culture medium. For this study, all cell lines were obtained from the cell archival facility of our institution.

[0153] Anti-clonogenic activity

[0154] Anti-clonal activity was assessed using a current standard qualitative plating efficiency assay and quantitative live cell imaging analysis (INCUCYTE S3 Live Cell Analysis Imaging System – Sartorius Welwyn Garden City, Hertfordshire, UK). For the qualitative plating efficiency assay, cells were plated at a “clonal” density (i.e., 0.5 cells / mm 2 ) were inoculated in medium containing the test molecule or in normal medium and incubated without any further manipulation. After 9-14 days, the culture was decanted and stained with 0.2% CV / methanol for 5 minutes at room temperature, depending on the cell line-specific proliferation index. All conditions were tested in triplicate in 6-well culture plates. The anti-clone formation activity of the treatments was evaluated using a comparison of the colony forming units (CFU) of the treated cultures with those of the control cultures. The lowest concentration that was able to reduce the colony forming units (CFU) of the treated group by 50% compared to the control group was defined as the colony inhibition concentration (CID 50 ). For quantitative assessment, cells were seeded in 96-well microplates at the above density and conditions and grown in an INCUCYTE S3 imaging system during the subsequent 8-day incubation. Images were acquired every 12 hours and then monitored by automated image analysis for colony formation and percentage of growth area covered (confluence). Analysis parameters were as follows: Segment Adjustment = 0.4; Well Fill < 5,000 μm 2 ; Adjusted pixels = -2. When the target consists of at least 24 cells, i.e., with a 24,000 μm 2 If the area is above 0.05 and the eccentricity is <0.750, it is evaluated as a colony.

[0155] For antiproliferative effects, cells were plated at 1000 cells / well (i.e., 30 cells / mm 2 ) were seeded in 96-well microplates at a density that allowed exponential growth rates for subsequent 8-day incubation. After overnight adhesion, the cell monolayers were washed and supplemented with fresh medium containing the different test molecules, and normal medium was used as a cell growth control.

[0156] Cell growth was then monitored using an automated INCUCTE S3 imaging system. Images were acquired every 12 hours. Analysis parameters were as follows: segment adjustment = 0.9; pore filling < 7,000 μm 2 ; Adjust pixels = 0.

[0157] Statistical analysis

[0158] The data of growth curve, anti-clone and anti-proliferation assays were obtained from at least three independent experiments. Each data point of quantitative anti-proliferation or anti-clone experiments represents the mean value (± SD) of eight independent replicates. Data were analyzed and plotted using Prism 6.0 software (GraphPad Software, San Diego, CA).

[0159] result

[0160] Anti-cloning effect of polyphenols on Hela cells Figure 1 As shown. It can be seen (panel a) that components 1) vanillic acid, 2) caffeic acid, 3) coumaric acid, 4) ferulic acid, 5) benzoic acid, 6) chlorogenic acid, 7) protocatechuic acid and 8) pinus acid, used alone, do not have any significant anti-clonal effect. In contrast (in panel b), kaempferol (9), galangin (10) and luteolin (12), chrysin (14), quercetin (15) and apigenin (16), each induced almost complete clonal inhibition when applied at 50 μM, a concentration far lower than that of any of the multiple polyphenols in most over-the-counter nutritional preparations. To confirm and extend this observation, parallel experiments were performed using HPV-16 transformed Siha cells and the data obtained were almost superimposable (data not shown). Interestingly, in addition to the reported anti-clonal effect, compound 12) luteolin showed a strong anti-proliferative effect on established tissue cultures. Compounds 9) and 16) also showed significant but mild effects. Figure 2 , Figure B).

[0161] To quantify these clonal inhibitory effects, limiting dilution experiments were performed to determine their 50% clonal inhibitory dose (CID 50 ). Luteolin (12) and galangin (10) anti-clonal titration as Figure 3 As shown. The experimental concentrations are shown on the left side of the plate lid. Luteolin is in columns 1-6 (left side of the plate); galangin is in columns 7-10 on the right side of the plate. Untreated control cells are in columns 11-12. Almost complete inhibition can be seen in wells C: 1-6, while approximately 50% inhibition is clearly seen in wells D: 1-6. The percentage of inhibition is best assessed by live cell image analysis as plotted in panels (b) and (c). As can be seen, the CID of luteolin 50 was 6.25 μM, while the CID of galangin 50 The higher concentrations of both compounds were completely inhibitory.

[0162] Based on this evidence, it is reasonable to assume that a core formulation containing the two most potent anticlonal compounds, luteolin and galangin, and the two most potent antiproliferative agents, luteolin and kaempferol, at equal or different molar concentrations, may have potent anticlonal and antiproliferative effects and therefore represent a new pharmacological agent that could be used to prevent the implantation of HPV-transformed cells and their subsequent proliferation and tissue invasion. Fig.12 The clear synergistic effect of their triple combination shown in was confirmed.

[0163] In addition to their direct anti-clonal effects, compounds 14 chrysin, 15 quercetin, and 16 apigenin may have further synergistic effects when administered in combination with each other or with kaempferol, galangin, and luteolin. In fact, the synergistic effect of chrysin / quercetin, such as Figure 5 As shown in Figure 2, the synergistic effect of kaempferol / apigenin is Figure 4 As shown, the synergistic effects of luteolin / chrysin and luteolin / apigenin are Figure 7 and 8 shown.

[0164] Based on this further evidence, it is reasonable to assume that a second group of compounds consisting of 14) chrysin, 15) quercetin, and 16) apigenin (either alone or in combination, and in proportions yet to be determined) may provide a significant synergistic enhancement of the pharmacodynamic activity of the core formulation when combined with the core formulation.

[0165] The third group, consisting of 1-7 molecules, namely vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid and protocatechuic acid, although they do not possess any direct anti-clonogenic activity on their own, are able to induce a certain degree of inhibition when combined with the core compounds and / or compounds of the second group: in fact, vanillic acid and caffeic acid have a synergistic effect with galangin and apigenin ( Fig. 9 ), and coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, and protocatechuic acid have synergistic effects with galangin, apigenin, and chrysin ( Figure 10-11 ).from Fig.12 As can be seen in the Figure 1, almost 24 hours are required for RNP9, RNP10 or RNP12 to induce approximately 50% clonal inhibition. In contrast, for the triple combination, clonal activity is easily inhibited with only an 8-hour treatment, while the extent is significantly greater than 50% with a 16-hour long treatment. Finally, the clonal inhibition achieved by the triple combination after 24 hours is much stronger than that of the single components. Therefore, the triple combination is superior to its single components in terms of shorter induction time and extent of action. Similar results were obtained with HeLa cells. Therefore, the third group of compounds is expected to achieve a convenient in vivo enhancement effect.

[0166] As mentioned above, the experiments were conducted on different HPV cell lines belonging to different HPV types, such as HPV16, HPV18 and HPV68. These experiments convince those skilled in the art that the above-mentioned favorable results can be obtained for any type of αHPV infection and related diseases.

[0167] Indeed, viral strains are often distinguished and classified by differences in their natural host range, cell and tissue selection tropism, pathogenicity and cytopathology, structural and chemico-physical properties, antigenic characteristics, and relatedness of protein and genomic sequences.

[0168] However, papillomaviruses (PV) have presented exceptions to the classical taxonomic rules since their initial description (Gissmann and zur Hausen, 1976; Favre et al., 1975; Orth et al., 1978). As knowledge continued to grow, these differences became increasingly greater and ultimately led to the development of a set of specific and dedicated classification criteria that are indeed very different from the standard classification criteria (de Villiers EM, 2013)(Munoz N. et al., 2003)(Castle PE, 2009), https: / / pave.niaid.nih.gov / analyze / l1_taxonomy_tool. The core reason for this special situation is that none of the more than 200 known HPVs (only a few of the hundreds of animal PVs) can actually be cultured using conventional techniques like most other viral strains and biological entities. Therefore, the structural and functional data required for standard classification are simply not available, and the entire PV classification has to be based entirely on genomic data alone. Therefore, the usual classification of variants, types, species, genera and families cannot be based on the observation of truly autonomous biological entities, but on arbitrary numerical criteria of homology. For example, two different PV isolates are assumed to belong to different species as long as they differ from each other by more than 30% of the bases in their L1 region, or, for example, once they differ by less than 2%, they are designated as two variants of the same type. Therefore, HPV virus types do not refer to clearly different biological entities like human herpesvirus type 1 (HSV-1) or type 2 (HSV-2). Instead, HPV types are referred to as "arbitrarily" distinguishable theoretical entities. To reflect this situation, pathologists and clinicians usually refer to HPV by the colloquial term "type" instead of the more correct "species". However, although based entirely on statistical considerations, this classification system still has clinical relevance because all HPV types associated with human pathology are actually included in the genus alpha papillomavirus. The seven HPV types 16, 31, 33, 35, 52, 57 and 58 belonging to species α-9, and the six types 18, 39, 45, 59, 68 and 70 belonging to species α-7, are actually members of a single pathological class, share common anatomical targets and induce a fairly uniform range of pathological changes (i.e., CIN-I, CIN-II, CIN-III), and as a group, cause almost all human cervical cancers (CastlePE 2009). Their affinity is further reflected in the fact that the nine-valent HPV vaccine containing seven of them can provide cross-reactive protection against an extended group of related oncogenic types (Huh WK et al., 2017).Furthermore, although the two HPV types 6 and 11 belonging to the related species α-10 cause genital condyloma acuminatum (i.e., benign epithelial hyperplasia rather than malignant neoplastic lesions), they share the same genomic organization and the same target tissues of species α-9 and α-7, and the biochemical function profiles of their early proteins are closely related and partially overlapping. Accordingly, these types, namely types 6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 57, 58, 59, 68, and 70, are currently considered to be a fairly homogeneous group of parasites and are represented by the anogenital PV.

[0169] Finally, it must be noted that the vast majority of biochemical and molecular biological data on αPV proteins are derived almost exclusively from studies based on the expression of subgenomic fragments from the HPV16 or HPV18 genomes, which were analyzed by extensive engineering techniques, such as transient or stable transfection of cells and lentiviral-related expression systems. In contrast, only a small part of the data comes from experiments on a few spontaneously immortalized cell lines. And these few cell lines always contain HPV16 genomes (SiHa; Ca-Ski; etc.) or HPV18 genomes (HeLa; KB; etc.). The only notable exception to this rule is ME-180 cells, a spontaneously immortalized cell line that contains the α-10 HPV68 type genome. No other continuous cell lines containing any other HPV genome are available for cell biology experiments. Therefore, according to the best current technical tools and scientific knowledge, the data reported in this article are based on the most extensive experimental cell models available, and the comments and speculations presented in this article are based on the latest advances in the current scientific knowledge of HPV biology. Therefore, for those skilled in the art, the above experimental results are reasonable for the human alpha papillomavirus genus as a whole.

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Claims

1. A pharmaceutical composition, comprising luteolin, galangin and kaempferol, and one or more excipients and / or adjuvants; or, the pharmaceutical composition consists of luteolin, galangin and kaempferol, and one or more excipients and / or adjuvants.

2. The pharmaceutical composition according to claim 1, wherein Luteolin is present in an amount of 1 to 20 μM, preferably 2 to 12 μM, more preferably 4 to 7 μM, galangin is present in an amount of 10 to 40 μM, preferably 20 to 40 μM, more preferably 20 to 30 μM, even more preferably 25 μM, Kaempferol is present in an amount of 10 to 40 μM, preferably 20 to 40 μM, more preferably 22 to 30 μM, even more preferably 12 to 30 μM, such as 25 μM or 15 μM.

3. The pharmaceutical composition according to any one of claims 1-2, wherein the pharmaceutical composition further comprises one, more than one or all compounds selected from chrysin, quercetin and apigenin.

4. The pharmaceutical composition according to claim 3, wherein Chrysin is present in an amount of 10 to 50 μM, preferably 15 to 30 μM, more preferably 20 μM or 25 μM, quercetin is present in an amount of 40 to 80 μM, preferably 50 to 60 μM, more preferably 50 μM, Apigenin is present in an amount of 10 to 50 μM, preferably 10 to 40 μM, more preferably 15 to 30 μM, more preferably 25 μM.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition further comprises one, more than one or all compounds selected from vanillic acid, caffeic acid, coumaric acid, ferulic acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus serrata, rutin and brevicornulin, preferably caffeic acid and / or ferulic acid.

6. The pharmaceutical composition of claim 5, wherein each of the vanillic acid, coumaric acid, benzoic acid, chlorogenic acid, protocatechuic acid, pinus, rutin and brevifolia compounds are present in an amount of <0.100 mM, and each of the caffeic acid and ferulic acid compounds are present in an amount of 20 to 100 μM.

7. A pharmaceutical composition as defined in any one of claims 1 to 6 for medical use.

8. A pharmaceutical composition as defined in any one of claims 1 to 6 for use in the prevention and treatment of viral infections and / or related diseases.

9. The pharmaceutical composition according to claim 8, for use according to claim 8, wherein the viral infection is HPV infection, such as human αHPV infection, and the associated disease is selected from the group consisting of: HPV-related cancers, HPV dysplastic lesions, HPV laryngeal papillomas, HPV genital warts, α papillomavirus subclinical anogenital and cervicovaginal infections, and α papillomavirus occult anogenital and cervicovaginal infections.

10. The pharmaceutical composition according to any one of claims 7-9, for use according to any one of claims 7-9, wherein the pharmaceutical composition is administered by a route selected from the group consisting of: local route, intravenous route, intramuscular route, organ selective extracorporeal perfusion, intracavitary perfusion, intraperitoneal perfusion, intraventricular or subarachnoid continuous perfusion.

11. The pharmaceutical composition according to any one of claims 7-10, for use according to any one of claims 7-10, wherein the pharmaceutical composition is delivered by liposomes, nanoparticles; radiofrequency, electroporation, ion electrophoresis or shock wave delivery system.

12. A combination of luteolin, galangin and kaempferol for separate and sequential use in the prevention and treatment of viral infections and / or related diseases.

13. The combination according to claim 12 for use according to claim 12, wherein the viral infection is an HPV infection, such as a human alpha HPV infection, and the associated disease is selected from the group consisting of: HPV-associated cancers, HPV dysplastic lesions, HPV laryngeal papillomas, HPV genital warts, alpha papillomavirus subclinical anogenital and cervicovaginal infections and alpha papillomavirus occult anogenital and cervicovaginal infections.

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