Prevention of infection by highly pathogenic viruses using topical application of povidone-iodine on mucous membranes
11246887 · 2022-02-15
Assignee
Inventors
Cpc classification
A61P1/00
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K47/36
HUMAN NECESSITIES
A61K9/127
HUMAN NECESSITIES
International classification
A61K9/127
HUMAN NECESSITIES
A61K47/32
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
This invention is directed to methods for prevention of infections by highly pathogenic viruses by applying to the nasal mucous membranes topical preparations comprising the broad-spectrum antimicrobial agent povidone-iodine.
Claims
1. A method of reducing SARS-CoV-2 viral load in a human subject infected with SARS-CoV-2, the method comprising application to the nasal passages of the human subject 1 to 12 times daily commencing after or around the time of exposure to the SARS-CoV-2 and/or thereafter for preferably up to 21 days, an effective amount of a pharmaceutical preparation comprising povidone-iodine (PVP-I) at a concentration of greater than 0.10% w/v and less than about 1.25% w/v, wherein the application causes the reduction of the SARS-CoV-2 viral load in the human.
2. The method as claimed in claim 1 wherein the pharmaceutical preparation is applied to the human subject prior to exposure of the subject to SARS-Co-V-2 or a person infected with SARS-CoV-2.
3. The method as claimed in claim 2 wherein the human subject is a healthcare worker who is, or is likely to be, exposed to the virus or infected patients.
4. The method as claimed in claim 1, wherein the PVP-I concentration in the pharmaceutical preparation applied to the nasal passages is about 0.1% to about 1.0% w/v.
5. The method as claimed in claim 1, wherein the PVP-I concentration in the pharmaceutical preparation applied to the nasal passages is about 0.2% to about 0.5% w/v.
6. The method as claimed in claim 1, wherein the PVP-I concentration in the pharmaceutical preparation applied to the nasal passages is about 0.2% to about 0.45% w/v.
7. The method as claimed in claim 1, wherein the pharmaceutical preparation is in a dosage form selected from the group consisting of intranasal solutions, liposomal preparations, drops, sprays, gels, aerosols, and inhalants.
8. The method as claimed in claim 1 wherein the pharmaceutical preparation is an aqueous based intranasal preparation where water makes up from about 80% w/v to about 96% w/v of the total preparation.
9. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of potassium iodide of from about 0.005 to about 0.05% w/v of the total preparation.
10. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of potassium iodate of from about 0.001 to about 0.03% w/v of the total preparation.
11. The method as claimed in claim 1 wherein the pharmaceutical preparation is an aqueous based intranasal preparation where water makes up from about 80% w/v to about 96% w/v of the total preparation and has a pH of from about 3-6.
12. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of humectant from about 1 to about 10% w/v of the total preparation.
13. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of a polar solvent from about 0.2 to about 2% w/v of the total preparation.
14. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of a counter-irritant or local anaesthetic from about 0.001 to about 1% w/v of the total preparation.
15. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of a preservative from about 0.01 to about 0.5% w/v of the total preparation.
16. The method as claimed in claim 1 wherein the pharmaceutical preparation additionally includes an amount of a buffer from about 0.05 to about 0.5% w/v of the total preparation.
Description
BRIEF DESCRIPTION OF FIGURES
(1)
(2)
DETAILED DESCRIPTION OF THE INVENTION
(3) To date, PVP-I has never been proposed as a preventative for infections caused by HP viruses by application to the nasal passages. Its use as a topical antiseptic on hands and skin to prevent infection by viruses, including HP viruses, is known and widely applied as part of barrier regimens for preventing access of the virus to the interior of the human body.
(4) Australian patent application 2014206143 to Molloy and Goodall, discloses that application of PVP-I to the nasal passages can be used to treat and prevent infections caused by common cold viruses, but there is no suggestion that a similar application to the nasal passages could prevent infection and/or shedding by/of HP viruses or diseases associated with secondary infections, nor is it obvious because SARS, MERS and AIV, for example, are considered lower respiratory tract infections (LRI) with very different clinical presentation and pathology to an infection limited to the upper respiratory tract (URI) such as the common cold. Furthermore Ebola is considered to be a consequence of systemic infection.
(5) The present invention discloses for the first time that application of PVP-I to the nasal passages has utility in the reduction of risk of infection and transmission of infection, the prevention of illness as well as the decrease risk of secondary infections caused by and associated with HP viruses such as Ebolaviruses, such as EBOV, pathogenic coronaviruses, such as SARS-CoV and MERS-CoV, and pandemic influenza viruses, such as AIV, and other emergent HP viruses.
EBOV
(6) In the case of EBOV and other filoviruses, whether the exposure occurs by direct contact with infected body fluids or through airborne virus, at some point the virus needs to gain access to the internal human body in order to initiate and propagate the infection. A likely portal for such access is the mucous membranes of the face, the respiratory tract and in particular the nasal passages.
(7) As is the case with the common cold, the eyes may be one portal, as could occur when someone rubs their eyes with fingers contaminated with virus. However due to the physiology of the human body it is most likely that any virus entering through the eyes would find its way to the nasal passages and replicate there as part of the process of establishing a productive infection.
(8) The oral cavity might also be a useful portal for the virus but the constant secretion of saliva and excretory mechanisms such as swallowing would argue against this as an important access point for the virus. In contrast, the nasal passages represent a highly receptive area for an infection and in the absence of a cold or other nasal infection, secretions are limited and as taught by Australian patent application 2014206143 to Molloy et al, previously referenced herein, normal mucociliary clearance takes around 15 minutes. The nasal passages offer another important advantage for filoviruses such as EBOV in that the nasal passages contain monocytes that act as sentinel cells for infection, also as taught in Australian Patent 2014206143 to Molloy et al, previously referenced herein. Such immune cells are known to become infected by EBOV and to act as a ‘Trojan horse’ for the EBOV infection that spreads to organs throughout the body. These immune cells are not commonly found in the oral cavity.
(9) Further the presence of EBOV or EBOV RNA in the nasal passages would cause the monocytes to release cytokines thereby attracting neutrophils to the nasal passages, which would also be susceptible to infection by EBOV and in fact, rather than only fight the infection, which is their normal role, it is postulated that their infection would accelerate and propagate the infection along with the infected monocytes.
(10) Therefore, the present invention discloses for the first time that through the unexpected combination of these factors, the nasal passages surprisingly offer a uniquely attractive portal for an initial entry of EBOV into the human host as a prelude to systemic replication and disease. However, it is this point of entry that also makes EBOV especially susceptible to attack and destruction by PVP-I through application to the nasal passages.
(11) It is known that PVP-I rapidly destroys EBOV and likely all other filoviruses at low PVP-I concentrations. Also, as taught in Australian Patent 2014206143 to Molloy et al previously referenced herein, it is known that PVP-I is toxic to immune cells such as monocytes at low PVP-I concentrations. This knowledge combined with the disclosure above of the mechanism of ingress and infection by EBOV has led the current inventors to a new discovery and that is that the repeated intranasal application of PVP-I according to the inventive method would not only eliminate any EBOV present in the nasal passages before an infection can be initiated but would destroy any infected monocytes or neutrophils and eliminate non-infected monocytes or neutrophils present so that could not become subsequently infected by EBOV. In essence, PVP-I application to the nasal passages would transform the nasal passages from a convenient portal of access for EBOV to a secondary barrier of protection from EBOV infection, greatly reducing the overall risk of EBOV infection.
(12) Australian Patent 2014206143 to Molloy et al previously referenced herein teaches that for use in the nasal passages, a suitable concentration of PVP-I is between 0.10% w/v and 2.5% w/v, with the lower value representing the level below which there is little or no effective antimicrobial capacity and the upper value representing the level above which PVP-I is known to cause ciliotoxicity in the nose. For use in the treatment of the common cold the patent teaches against the use of liposomal preparations of PVP-I, as defined in the patent, because of their slower performance and reduced antiviral activity in the face of mucin inactivation, secretory clearance and a most resilient virus, human rhinovirus. In the case of Ebolaviruses, however, which are enveloped viruses and far more sensitive to PVP-I, and with the absence of the rhinorrhoea present in colds, these factors are less applicable. Indeed the slower release and longer residence time associated with liposomal PVP-I formulations, or other slow release/longer residence time forms, for example gels may be an advantage for preventative applications such as EBOV infection. Therefore, the current invention does not exclude liposomal formulations of PVP-I.
(13) As to frequency and timing of application, this will depend on the circumstances of the exposure to EBOV or similar viruses. In the case of healthcare workers who are using PPE and associated barrier techniques while they are exposed to infected individuals, it is not practical to apply a preparation to the nose while the PPE are in place and to do so may increase exposure to viruses. The first opportunity to apply a PVP-I intranasal preparation could be before or while applying PPE (i.e., as part of a PPE “suiting up” regime) or could be at the time of removing the PPE after completing work activities, where the purpose of the application of intranasal PVP-I is to eliminate any viruses that might somehow have circumvented the PPE and other barriers, for example due to failure of a respirator or by the worker inadvertently touching the exterior of a face mask or gown during removal and subsequently touching their nose or eyes allowing the virus to potentially find its way to the nasal passages. In those circumstances, the worker should use the product immediately after removing the PPE in accordance with recommended safe work practices and thereafter at a frequency of up to 12 times daily for the period equivalent to the incubation period for the virus, which in the case of EBOV is up to 21 days after the most recent exposure to the virus.
(14) In the case of people who do not have PPE and may be exposed to virus, such as family members, the PVP-I intranasal preparation would need to be used continuously during the exposure period at a frequency of up to 12 times daily and after the exposure at a similar frequency for the period equivalent to the incubation period for the virus, which in the case of EBOV is up to 21 days after the most recent exposure to the virus.
(15) In every case, the volume of the PVP-I intranasal preparation should be sufficient to reach all parts of the nasal passages, which in the case of a liquid PVP-I intranasal preparation as taught in Australian Patent 2014206143 to Molloy et al previously referenced herein may represent a volume of up to 1 mL applied to each nostril of the exposed person.
(16) The PVP-I intranasal preparation may be in the form a solution, drops, spray, gel, cream, aerosol, or inhalant.
AIV
(17) In the case of AIV and as disclosed by Shinya et al (“Influenza virus receptors in the human airway.” Nature, 440: 435-6) H5N1 only productively replicates in the cells of the lower respiratory tract, not the nasal passages. Therefore, the application of PVP-I to the nasal passages would not be regarded by one skilled in the art as a productive means of treating or preventing AIV infection.
(18) However, the present inventors have discovered that where human-human transmission occurs, it would normally occur as the result of the sneezing or coughing of an infected person, with the virus carried by droplets. Such droplets are large enough to deposit in the nasal passages and while the virus may not replicate in the nasal cells, the nasal passages are expected to act as an important initial staging point for establishing infection and/or prior to further ingress to the lower respiratory tract. For example, the virus could be carried by mucociliary clearance to the throat from where it can readily migrate to the bronchi.
(19) Further, in the case of animal-human transmission of AIV, as might occur with people tending, handling or otherwise exposed to animals, especially poultry, infected with AIV, the virus would be carried on dust and other particles and also very likely find its way to the nasal passages and lodge there prior to further migration into the lungs.
(20) In either animal-human or human-human transmission, the virus could also be carried on hands and the person may self-inoculate by touching their eyes or nose with contaminated fingers. Again, the nasal passages could be important in ferrying the virus ultimately to the lungs.
(21) Therefore, the present invention discloses for the first time that whether a person is exposed to infected animals or humans, the application of PVP-I to the nasal passages provides an important protective effect against infection and/or replication/shedding of virus immediately post infection.
(22) It is known that PVP-I rapidly destroys H5N1 and all other influenza viruses, pandemic or otherwise, at low PVP-I concentrations. This knowledge combined with the disclosure above of the mechanism of ingress and infection by AIV has led the current inventors to a new discovery and that is that the repeated intranasal application of PVP-I according to the inventive method would eliminate any AIV present in the nasal passages before the virus can migrate to the lower respiratory tract and establish a productive infection. In essence, the nasal application of PVP-I would transform the nasal passages from a convenient portal of access for AIV to a secondary barrier of protection from AIV infection, greatly reducing the overall risk of AIV infection.
(23) For reasons already discussed, a suitable concentration of PVP-I is between 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range within such concentrations.
(24) As to frequency and timing of application, similar considerations apply to those already discussed for EBOV. In the case of animal or healthcare workers who are using PPE and associated barrier techniques while they are exposed to AIV infected animals or people, the worker should use the product before or while applying PPE or immediately after removing the PPE and thereafter at a frequency of up to 12 times daily for the period equivalent to the incubation period for the virus, which in the case of AIV may be as long as 17 days.
(25) In the case of people who do not have PPE and may be exposed to virus, such as family members, the PVP-I intranasal preparation would need to be used continuously during the exposure period at a frequency of up to 12 times daily and after the exposure at a similar frequency for the period equivalent to the incubation period for the virus, which in the case of AIV is up to 17 days after the most recent exposure to the virus.
(26) In every case, the volume of the PVP-I intranasal preparation should be sufficient to reach all parts of the nasal passages, which in the case of a liquid PVP-I intranasal preparation may represent a volume of up to 1 mL applied to each nostril of the exposed person.
(27) The PVP-I intranasal preparation may be in the form a solution, drops, spray, gel, cream, aerosol, or inhalant.
HP Coronaviruses (e.g., SARS and MERS)
(28) The MERS-CoV causes a LRI, which can result in severe pneumonia with acute respiratory distress and multiple-organ failure leading to a high mortality rate. Like AIV, transmission is likely through airborne droplets or direct or indirect contact with the virus.
(29) Adney et al (“Replication and Shedding of MERS-CoV in Upper Respiratory Tract of Inoculated Dromedary Camels.” Emerging Infectious Diseases, 20, 12 (2014): 1999-2005) showed that MERS-CoV actively replicated in the upper respiratory tract of camels.
(30) Memish et al (“Middle East respiratory syndrome coronavirus infections in health care workers.” N Engl J Med 369 (2013):884-886) reported asymptomatic carriers of MERS CoV often had mild URI symptoms as well suggesting that the virus might infect the nasal passages of humans, but in a subsequent study Memish et al (“Prevalence of MERS-CoV Nasal Carriage and Compliance With the Saudi Health Recommendations Among Pilgrims Attending the 2013 Hajj.” JID 210 (2014): 1067-1072) found no evidence of nasal carriage of MERS-CoV, at least in asymptomatic subjects.
(31) For these reasons and the fact that SARS and MERS are considered lower respiratory tract infections, the application of PVP-I to the nasal passages would not be regarded by one skilled in the art as a productive means of treating or preventing SARS or MERS infections.
(32) However, where human-human transmission occurs, it would normally occur as the result of the sneezing or coughing of an infected person, with the virus carried by droplets. Such droplets are large enough to deposit in the nasal passages and while the virus may not replicate in the nasal cells, the nasal passages may act as an important initial staging point for further ingress to the lower respiratory tract. For example, the virus could be carried by mucociliary clearance to the throat from where it can readily migrate to the bronchi.
(33) Further, in the case of animal-human transmission of SARS or MERS, as might occur with people tending, handling or otherwise exposed to infected animals, the virus would be carried on dust and other particles and also likely find its way to the nasal passages and lodge there prior to further migration into the lungs.
(34) In either animal-human or human-human transmission, the virus could also be carried on hands and the person may self-inoculate by touching their eyes or nose with contaminated fingers. Again, the nasal passages could be important in ferrying the virus ultimately to the lungs. Furthermore, Sungnak and colleagues have recently reported evidence that cells of the nasal passages may be particularly susceptible to infection and have postulated a potential role in initial viral infection, spread and clearance of SARS-CoV-2 (Sungnak, et al., 2020. Nature Comms. 26: 681-687. “SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes”).
(35) Therefore, the present invention discloses for the first time that whether a person is exposed to infected animals or humans, the application of PVP-I to the nasal passages may provide an important protective effect against ingress to the lower respiratory tract and productive infection. This would be especially important for health care workers and other susceptible people who could be exposed to infected individuals which have tested positive, for instance, for SARS-CoV-2, display the symptoms of COVID-19 disease, or for people who are testing individuals to determine if they are positive for the virus
(36) The examples provided herein show that PVP-I also inactivates SARS-CoV-2 (
(37) This knowledge combined with the disclosure above of the mechanism of ingress and infection by SARS or MERS coronaviruses has led the current inventors to a new discovery and that is that the repeated intranasal application of PVP-I according to the inventive method would eliminate any SARS-CoV or MERS-CoV present in the nasal passages before the virus can migrate to the lower respiratory tract and establish a productive infection.
(38) This would include “suppressing’ viral infection in an individual infected, for instance with SAR-CoV-2 or other viruses, in order to reduce the risk of transmission.
(39) The concept of “suppressing” viral infection indicates any aspect of viral infection, such as viral replication, time course of infection, amount (titer) of virus, lesions, and/or one or more symptoms is curtailed, inhibited, or reduced (in terms of severity and/or duration) in an individual or population of individuals treated with a PVP-I composition in accordance with the invention as compared to an aspect of viral infection in an individual or population of individuals not treated in accordance with the invention. Reduction in viral titer includes, but is not limited to, elimination of the virus from an infected site or individual. Viral infection can be assessed by any means known in the art, including, but not limited to, measurement of virus particles, viral nucleic acid or viral antigens, detection of symptoms and detection and/or measurement of anti-virus antibodies. Anti-virus antibodies are widely used to detect and monitor viral infection and generally are commercially available. In addition, viral infection can be assessed by other means known in the art including, but not limited to, PCR, in situ hybridization with virus specific probes, TCID.sub.50 assays, infectious center assays, plaque assays, etc.
(40) For reasons already discussed, a suitable concentration of PVP-I is between 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range within such concentrations.
(41) As to frequency and timing of application, similar considerations apply to those already discussed for EBOV and AIV. In the case of animal or healthcare workers who are using PPE and associated barrier techniques while they are exposed to AIV infected animals or people, the worker should use the product prior to or at the same time as applying PPE, or immediately after removing the PPE and thereafter at a frequency of up to 12 times daily for the period equivalent to the incubation period for the virus, which in the case of SARS is up to 10 days and for MERS is up to 14 days.
(42) In the case of people who do not have PPE and may be exposed to virus, such as family members, the PVP-I intranasal preparation would need to be used continuously during the exposure period at a frequency of up to 12 times daily and after the exposure at a similar frequency for the period equivalent to the incubation period for the virus, or up to 10 days for SARS and 14 days for MERS after the most recent exposure to the virus.
(43) In every case, the volume of the PVP-I intranasal preparation should be sufficient to reach all parts of the nasal passages, which in the case of a liquid PVP-I intranasal preparation may represent a volume of up to 1 mL applied to each nostril of the exposed person.
(44) The PVP-I intranasal preparation may be in the form a solution, drops, spray, gel, cream, aerosol, or inhalant.
(45) In relation to the HP viruses discussed above in certain embodiments the nasal application provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 5-20 mins which persists for about 8 hrs after being exposed to said virus after one application within the nasal passage. In an embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 5-20 mins which persists for about 6 hrs after being exposed to said virus after one application within the nasal passage. In an embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 5-20 mins which persists for about 4 hrs after being exposed to said virus after one application within the nasal passage. In another embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 5-20 mins which persists for 2 hrs after being exposed to said virus after one application within the nasal passage. In another embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 5-20 mins which persists for 1 hr after being exposed to said virus after one application within the nasal passage.
(46) In relation to the HP viruses discussed above in certain embodiments the nasal application provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 10 mins which persists for about 8 hrs after being exposed to said virus after one application within the nasal passage. In an embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 10 mins which persists for about 6 hrs after being exposed to said virus after one application within the nasal passage. In an embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 10 mins which persists for about 4 hrs after being exposed to said virus after one application within the nasal passage. In another embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 10 mins which persists for 2 hrs after being exposed to said virus after one application within the nasal passage. In another embodiment the composition provides a clinically significant Log reduction of at least several log units (e.g. 2-4 log units) within about 10 mins which persists for 1 hr after being exposed to said virus after one application within the nasal passage.
Secondary Infections
(47) It is well known that so called “secondary infections” can be a common complication of a primary viral infection, particularly in the case of a primary respiratory viral disease. These secondary infections are often bacterial infections. Secondary infections occurs during or after an infection from another pathogen, commonly viruses. These secondary diseases are thought to be facilitated by a number of factors, including viral mediated enhanced attachment and colonization, viral mediated enhanced pathogenesis and viral mediated immune-modulation. Secondary infections are responsible for increased morbidity and in some cases mortality and have been shown to increase the societal and individual impact of HP viruses. For example, during the H1N1 2009 influenza pandemic secondary infection by a variety of micro-organisms including Mycoplasma pneumoniae, S. aureus, K. pneumoniae, S. pneumoniae, M. catarrhalis, P. aeruginosa, S. pyogenes, and Streptococcus agalactiae was shown to be present in approximately a third to a half of all fatalities (Morris et al., Front. Microbiol. 2017; 8: 1041 “Secondary Bacterial Infections Associated with Influenza Pandemics” and references therein). Similarly secondary infections have been commonly reported following autopsy of fatal COVID-19 disease there are concerns that these may contribute to mortality and morbidity associated with the recent pandemic (Cox et al., Lancet, Microbe 2020; 1: E11 “Co-infections: potentially lethal and unexplored in COVID-19”, and references therein). These secondary infections present a complex diagnostic challenge since they may comprise a variety of species and may prove difficult to resolve from micro-organisms colonizing the patient benignly or in some cases previously benign organisms may become pathogenic following the primary infection. PVP-I is reported to have broad spectrum anti-microbial activity and rapidly inactivates a wide range of micro-organisms including antibiotic resistant strains (Kanagalingam, et al., Int J Clin Practice. 2015 69; 11: 1247-1256, “Practical use of povidone-iodine antiseptic in the maintenance of oral health and in the prevention and treatment of common oropharyngeal infections” and references therein). The use of PVP-I has been proposed as a universal “disinfectant” for the purposes maintaining, for example, oral health associated with microbial infections. Importantly, due to its mode of action and broad spectrum activity it is not expected that knowledge of the identity or antibiotic susceptibility of the micro-organism responsible for the secondary infection will be required nor the lack of this knowledge limit the utility of PVP-I.
(48) Secondary infections, often associated with bacteria and fungi, are a known cause of serious morbidity and mortality in viral diseases, particularly respiratory viruses. Secondary infections are also thought to have played a role in recent pandemics caused by HP viruses. They are often difficult to diagnose and, for example in the case of antibiotic resistant organisms, can pose treatment challenges. The primary virus infection is thought to predispose a patient to secondary diseases through, for example, promoting colonization, immune-modulation and other factors such as facilitating increased virulence. Diseases such as bacterial pneumonia caused by secondary infections are a result of bacterial infection of the lower respiratory tract.
(49) For these reasons the application of PVP-I to the nasal passages would not be regarded by one skilled in the art as a productive means of treating or preventing secondary infections.
(50) Therefore, the present invention discloses for the first time that whether a person is exposed to infection with a HP virus, the application of PVP-I to the nasal passages may provide an important protective effect against secondary infections and the diseases associated with secondary infections. This would be especially important for health care workers and other susceptible people who may be at increased risk.
(51) It is known that PVP-I has a broad spectrum antimicrobial effect (2006), (previously referenced herein), and is unaffected by antibiotic resistance. In some cases, the organisms involved in secondary diseases may colonise the nasal passages in a benign fashion prior to the primary infection.
(52) This knowledge combined with the disclosure above of the mechanism of ingress and infection by HP viruses has led the current inventors to a new discovery and that is that the repeated intranasal application of PVP-I according to the inventive method would eliminate micro-organisms present in the nasal passages, thereby preventing the promotion of colonization and exacerbated disease, and limit their spread before they can migrate to the lower respiratory tract (or elsewhere) and establish a productive secondary infection.
(53) For reasons already discussed, a suitable concentration of PVP-I is between 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range within such concentrations.
(54) As to frequency and timing of application, similar considerations apply to those already discussed for primary infection with HP viruses. In the case of animal or healthcare workers who are using PPE and associated barrier techniques while they are exposed to HP virus infected animals or people, the worker should use the product prior to or at the same time as applying PPE, or immediately after removing the PPE and thereafter at a frequency of up to 12 times daily for the period equivalent to the incubation period for the virus.
(55) In the case of people who do not have PPE and may be exposed to virus, such as family members, the PVP-I intranasal preparation would need to be used continuously during the exposure period at a frequency of up to 12 times daily and after the exposure at a similar frequency for the period equivalent to the incubation period for the virus.
(56) In every case, the volume of the PVP-I intranasal preparation should be sufficient to reach all parts of the nasal passages, which in the case of a liquid PVP-I intranasal preparation may represent a volume of up to 1 mL applied to each nostril of the exposed person.
(57) It is intended that the preparations be applied at “ambient temperature” which refers to the temperature in the environment at which the method of the current invention is conducted. Typically ambient temperature will be about 10° C. to about 30° C. Importantly the term “ambient temperature” means that neither the formulation nor the nasal passages of the subject to be treated are exposed to external heating in carrying out the method of the present invention.
Intranasal Preparations
(58) As disclosed herein the PVP-I intranasal preparations may be in the form a solution, drops, spray, gel, cream, aerosol, or inhalant.
(59) Suitable concentration of PVP-I is between 0.10% w/v and 1.25% w/v, such as about 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, or about 1.2% or any range within such concentrations.
(60) In certain embodiments the PVP-I intranasal preparations are aqueous based intranasal preparations where water makes up from about 80% w/v to about 96% w/v of the total preparation.
(61) In certain embodiments the PVP-I preparations disclosed herein additionally includes an amount of iodide and/or iodate salts of from about 0.003 to about 0.05% w/v of the total preparation.
(62) In certain embodiments the PVP-I preparations disclosed herein may also include one or more of the following: solubilising agents, polar solvents, dry acidulant, sequestrants, alkaline agents, counter-irritant agents, local anaesthetic and preservatives.
(63) In certain embodiments the PVP-I intranasal preparations are aqueous based intranasal preparations where water makes up from about 80% w/v to about 96% w/v of the total preparation and has a pH of from about 2-7 such as a pH range of about 3-6.
(64) In certain embodiments the PVP-I preparations disclosed herein additionally includes an amount of humectant such as hyaluronic acid, polyethylene glycol or glycerol (glycerine) from about 1 to about 10% w/v of the total preparation.
(65) In certain embodiments the PVP-I preparations disclosed herein additionally includes an amount of a polar solvent, such as ethanol, from about 0.2 to about 2% w/v of the total preparation.
(66) In certain embodiments the PVP-I preparations disclosed herein additionally includes an amount of a counter-irritant or local anaesthetic, such as menthol, from about 0.001 to about 1% w/v of the total preparation.
(67) In certain embodiments the PVP-I preparations disclosed herein additionally includes an amount of a preservative, such as a quaternary ammonium salt preservative, from about 0.01 to about 0.5% w/v of the total preparation.
(68) In certain embodiments the PVP-I preparations disclosed herein additionally includes an amount of buffer such as sodium phosphate, citrate or acetate from about 0.05 to about 0.5% w/v of the total preparation.
(69) The pharmaceutical preparation may further comprise at least one pharmaceutically acceptable diluent, excipient or carrier.
(70) The diluent, excipient or carrier may be a flavor agent, sweetener, colouring agent, solvent, buffer, alcohol, polymer, surfactant or other diluent, excipient or carrier designed to optimize the nasal delivery, intranasal distribution, stability, effectiveness, acceptability, tolerability of the preparation.
EXAMPLES
Aqueous-Intranasal Preparation Examples
Example 1
(71) TABLE-US-00001 Per 100 mL g % w/v PVP-I 0.50 0.50% Potassium Iodide 0.010 0.010% Hyaluronic Acid 1.00 1.00% Sodium Citrate 0.15 0.15% Eucalyptus 0.01 0.010% Ethanol 0.49 0.490% Benzalkonium Chloride 0.01 0.01% Water 97.83 97.83% 100.00 100.0%
Example 2
(72) TABLE-US-00002 Per 100 mL g % w/v PVP-I 0.50 0.50% Potassium Iodide 0.010 0.010% Potassium Iodate 0.005 0.005% Glycerol 5.00 5.00% Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Menthol 0.01 0.010% Water 93.84 93.84% 100.00 100.0%
Example 3
(73) TABLE-US-00003 Per 100 mL g % w/v PVP-I 0.50 0.50% Potassium Iodide 0.010 0.010% Potassium Iodate 0.005 0.005% Glycerol 5.00 5.00% Sodium Hydrogen Phosphate 0.15 0.15% Sodium Hydroxide 0.75 0.75% Ethanol 0.49 0.49% Menthol 0.01 0.010% Water 93.08 93.08% 100.00 100.0%
Example 4
(74) TABLE-US-00004 Per 100 mL g % w/v PVP-I 0.50 0.50% Povidone 0.300 0.30% Glycerol 3.00 3.00% Sodium Citrate 0.15 0.15% Citral 0.49 0.49% Water 95.56 95.56% 100.00 100.0%
Example 5
(75) TABLE-US-00005 Per 100 mL g % w/v PVP-I 0.50 0.50% Povidone 0.030 0.030% Potassium Iodide 0.010 0.010% Polyethylene Glycol 4000 2.00 2.00% Tween 20 0.05 0.05% Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Peppermint Oil 0.01 0.010% Water 96.76 96.76% 100.00 100.0%
Example 6
(76) TABLE-US-00006 Per 100 mL g % w/v PVP-I 0.50 0.50% Sodium Iodide 0.010 0.010% Sodium Acetate 0.15 0.15% Sorbitol 0.01 0.010% Lutrol 0.50 0.500% Water 98.83 98.83% 100.00 100.0%
Example 7
(77) TABLE-US-00007 Per 100 mL g % w/v PVP-I 0.50 0.50% Lutrol 2.00 2.00% Sodium Citrate 0.15 0.15% Saccharine 0.01 0.010% Water 97.34 97.34% 100.00 100.0%
Example 8
(78) TABLE-US-00008 Per 100 mL g % w/v PVP-I 0.50 0.50% Polyethylene Glycol 4000 3.00 3.00% Sodium Hydrogen Phosphate 0.15 0.15% Xylitol 1.00 1.00% Water 95.35 95.35% 100.00 100.0%
Example 9
(79) TABLE-US-00009 Per 100 mL g % w/v PVP-I 0.50 0.50% Triacetin 2.00 2.00% Sodium Acetate 0.15 0.15% Tween 20 0.05 0.05% Ethanol 0.49 0.49% Eucalyptus Oil 0.01 0.010% Water 96.80 96.80% 100.00 100.0%
Example 10
(80) TABLE-US-00010 Per 100 mL g % w/v PVP-I 1.00 1.00% Potassium Iodide 0.02 0.02% Hyaluronic Acid 0.50 0.50% Sodium Citrate 0.15 0.15% Eucalyptus 0.01 0.01% Ethanol 0.49 0.49% Benzalkonium Chloride 0.01 0.01% Water 97.82 97.82% 100.00 100.00%
Example 11
(81) TABLE-US-00011 Per 100 mL g % w/v PVP-I 1.00 1.00% Potassium Iodide 0.02 0.02% Potassium Iodate 0.01 0.01% Glycerol 5.00 5.00% Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Menthol 0.01 0.01% Water 93.32 93.32% 100.00 100.00%
Example 12
(82) TABLE-US-00012 Per 100 mL g % w/v PVP-I 1.00 1.00% Povidone 0.60 0.60% Glycerol 3.00 3.00% Sodium Citrate 0.15 0.15% Citral 0.49 0.49% Water 94.76 94.76% 100.00 100.00%
Example 13
(83) TABLE-US-00013 Per 100 mL g % w/v PVP-I 1.00 1.00% Povidone 0.06 0.06% Potassium Iodide 0.02 0.02% Polyethylene Glycol 4000 2.00 2.00% Tween 20 0.05 0.05% Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Peppermint Oil 0.01 0.01% Water 96.22 96.22% 100.00 100.00%
Example 14
(84) TABLE-US-00014 Per 100 mL g % w/v PVP-I 1.00 1.00% Sodium Iodide 0.02 0.02% Sodium Acetate 0.15 0.15% Sorbitol 0.01 0.01% Lutrol 0.50 0.50% Water 98.32 98.32% 100.00 100.00%
Example 15
(85) TABLE-US-00015 Per 100 mL g % w/v PVP-I 1.00 0.50% Lutrol 2.00 2.00% Sodium Citrate 0.15 0.15% Saccharine 0.01 0.01% Water 96.84 96.84% 100.00 100.00%
Example 16
(86) TABLE-US-00016 Per 100 mL g % w/v PVP-I 1.00 0.50% Polyethylene Glycol 4000 3.00 3.00% Sodium Hydrogen Phosphate 0.15 0.15% Xylitol 1.00 1.00% Water 94.85 94.85% 100.00 100.00%
Example 17
(87) TABLE-US-00017 Per 100 mL g % w/v PVP-I 1.00 0.50% Triacetin 2.00 2.00% Sodium Acetate 0.15 0.15% Tween 20 0.05 0.05% Ethanol 0.49 0.49% Eucalyptus Oil 0.01 0.01% Water 96.30 96.30% 100.00 100.00%
Example 18
(88) TABLE-US-00018 Per 100 mL g % w/v PVP-I 0.20 0.20% Potassium Iodide 0.02 0.02% Hyaluronic Acid 0.50 0.50% Sodium Citrate 0.15 0.15% Eucalyptus 0.01 0.01% Ethanol 0.49 0.49% Benzalkonium Chloride 0.01 0.01% Water 98.62 98.62% 100.00 100.00%
Example 19
(89) TABLE-US-00019 Per 100 mL g % w/v PVP-I 0.20 0.20% Potassium Iodide 0.02 0.02% Potassium Iodate 0.01 0.01% Glycerol 5.00 5.00% Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Menthol 0.01 0.01% Water 93.32 93.32% 100.00 100.00%
Example 20
(90) TABLE-US-00020 Per 100 mL g % w/v PVP-I 0.20 0.20% Povidone 0.60 0.60% Glycerol 3.00 3.00% Sodium Citrate 0.15 0.15% Citral 0.49 0.49% Water 95.56 95.56% 100.00 100.00%
Example 21
(91) TABLE-US-00021 Per 100 mL g % w/v PVP-I 0.20 1.00% Povidone 0.06 0.06% Potassium Iodide 0.02 0.02% Polyethylene Glycol 4000 2.00 2.00% Tween 20 0.05 0.05% Sodium Hydrogen Phosphate 0.15 0.15% Ethanol 0.49 0.49% Peppermint Oil 0.01 0.01% Water 97.02 97.02% 100.00 100.0%
Example 22
(92) TABLE-US-00022 Per 100 mL g % w/v PVP-I 0.20 1.00% Sodium Iodide 0.02 0.02% Sodium Acetate 0.15 0.15% Sorbitol 0.01 0.01% Tween 20 Lutrol 0.50 0.50% Water 99.12 99.12% 100.00 100.0%
Example 23
(93) TABLE-US-00023 Per 100 mL g % w/v PVP-I 0.20 0.50% Lutrol 2.00 2.00% Sodium Citrate 0.15 0.15% Saccharine 0.01 0.01% Water 97.64 97.64% 100.00 100.00%
Example 24
(94) TABLE-US-00024 Per 100 mL g % w/v PVP-I 0.20 0.50% Polyethylene Glycol 4000 3.00 3.00% Sodium Hydrogen Phosphate 0.15 0.15% Xylitol 1.00 1.00% Water 95.65 95.65% 100.00 100.00%
Example 25
(95) TABLE-US-00025 Per 100 mL g % w/v PVP-I 0.20 0.50% Triacetin 2.00 2.00% Sodium Acetate 0.15 0.15% Tween 20 0.05 0.05% Ethanol 0.49 0.49% Eucalyptus Oil 0.01 0.01% Water 96.30 96.30% 100.00 100.00%
Biological Data
a) SARS-CoV-2
(96) PVP-I nasal intranasal preparation of the examples inactivates SARS-CoV-2 in a cell culture system
Introduction
(97) This study assessed the ability of PVP-I to inactivate SARS-CoV-2, shown to replicate efficiently within the upper respiratory tract. PVP-I was assessed against saline and media only controls in parallel to assess the presence/absence of virucidal activity.
Study Objective
(98) The objective of this study is to compare and assess the virucidal activity of PVP-I against the SARS-CoV-2 strain (BetaCoV/Australia/VIC01/2020) in the African Green Monkey Kidney (Vero) human cell line.
(99) The impact on virus replication will be qualitatively assessed for SARS-CoV-2 RNA using a SARS specific TaqMan Real-time PCR assay targeting the E-gene.
Materials
Virus
(100) SARS-CoV-2 (BetaCoV/Australia/VIC01/2020) was isolated and grown by the Victorian Infectious Diseases Reference Laboratory from a positive patient specimen in January, 2020. Whole-genome sequencing confirmed the presence of SARS-CoV-2 (GenBank ID: MT007544).
Placebo and Povidone Iodine Solutions
(101) PVP-I examples according to the description were prepared at a concentration of PVP-I between 0.1% w/v and 1.25% w/v (designated as “PVP-I”—this examples section)
Cells
(102) Vero cells were grown in Eagle's Minimum Essential Medium (Sigma-Aldrich, North Ryde, Australia) catalogue number (M2279), supplemented with 1× Non-essential amino acids (NEAA) (Gibco, Mount Waverley, Australia; catalogue number 11140050) and 10% heat-inactivated foetal bovine serum (FBS: Bovogen, Melbourne, Australia; catalogue number SFBS, lot# 1502B).
Methods
Plating 96-Well Plates
(103) Vero cells were trypsinised, counted and seeded into 96-well plates at a cell density of 1×104 cells/well in 200 ul media and incubated at 37° C., 5% CO.sub.2. Outer perimeter wells of the plate were not used, to minimise potential edge effects.
Incubation of Virus with Treatment Solutions
(104) 95 ul of each test solution (saline or PVP-I) was incubated with 5 ul of each virus at 37° C. for 1 minute. At the end of incubation, the test solution/virus mix was diluted 1:10 with ice cold EMEM 2% FBS media. Immediately after adding ice cold media, 100 ul of test mixture for the samples was added to the plates in triplicate (column 2 below). Note: PVP-I was tested in duplicate (PVP-I #1, PVP-I #2). Samples were thoroughly mixed and 100 ul transferred to column 3 with fresh pipette tips. 100 ul was transferred from column 3 to column 4 and so on until column 10. 100 ul from column 10 was discarded, leaving a final volume of 200 ul in the wells. Column 11 contained cells alone with fresh EMEM 2% which served as a no virus control. Plates were incubated at 37° C., 5% CO2 atmosphere for 48 hours before harvesting.
(105) TABLE-US-00026 Uninfected dilution factor: 3 9 27 81 243 729 2187 6561 19683 log(10) dilution 0.5 1 1.4 1.9 2.4 2.9 3.3 3.8 4.3 CONTROL 2 3 4 5 6 7 8 9 10 A Sample 1 B C D Sample 2 E F G H
Purification of SARS-CoV RNA
(106) SARS-CoV-2 RNA was purified from 200 ul supernatant/media from each well of the 96-well plates using the QIAamp 96 virus QlAcube HT Kit and processed on the QIAcube robotic extraction platform (QIAgen, Hilden, Germany) to confirm the presence/absence of replicating virus.
Taqman RT-PCR of SARS-CoV RNA
(107) Purified SARS-CoV-2 RNA was reverse transcribed to cDNA using the Bioline Sensifast cDNA kit (Catalogue number CSA-01148; London, UK). Real-time assays were performed using the published SARS E-gene assay[1] with primers and probes (IDT, Singapore) and ABI TaqMan Fast Universal PCR Master Mix (2×) (catalogue number 4352042; Thermofisher, Vilnius, Lithuania). Assays were performed on a Thermofisher ABI 7500 Fast Real Time PCR machine.
Results
Taqman RT-PCR Assessment of SARS-CoV-2 RNA in 96-Well Plate Supernatants
(108) To confirm that the presence or absence of replicating SARS-CoV-2, 200 ul supernatant samples from all triplicate wells were analysed for the presence of SARS-CoV-2 RNA by Taqman RT-PCR.
(109) To establish a baseline of RT-PCR cycle threshold (Ct) values for non-replicating, non-viable virus, initial inoculum samples (0 h post infection) were titrated 1:3 (˜0.5 log10) to 1:19683 (4.3 log10) and subjected to the E-gene specific Taqman RT-PCR. SARS-CoV-2 alone, in the presence of saline, and Nasodine, show a linear relationship between Ct-value and dilution factor (see
48 Hr Study
(110) Following 48 h of incubation on Vero cells, SARS-CoV-2 displayed robust replication both within and without the presence of saline (
Conclusion of 48 H Study
(111) The objective of this study was to compare and assess the virucidal activity of PVP-I solution against the newly emerged SARS-CoV-2, the causative agent of COVID-19 disease.
(112) Using real-time TaqMan RT-PCR methods it was determined that PVP-I possesses clear virucidal activity against SARS-CoV-2 (−2.4 Log10 reduction, or >99% kill) compared to saline alone.
96 Hr Study
(113) Following 96 h of incubation on Vero cells, SARS-CoV-2 in media alone displayed robust replication (
Conclusion
(114) The objective of this study was to compare and assess the virucidal activity of PVP-I solution against the newly emerged SARS-CoV-2, the causative agent of COVID-19 disease. Using real-time TaqMan RT-PCR methods it was determined that PVP-I possesses clear virucidal activity against SARS-CoV-2 and based on the RT-PCR data obtained effectively eliminated the replication competent SARS-CoV-2 present in the sample compared to media alone as evidenced by the lack of viral replication in the dilutions cultured from PVP-I treated samples compared to the equivalent untreated samples. A comparison of the viral RNA copy number associated with cultures derived from the least diluted untreated control sample with the equivalent PVP-I sample indicated a −3.6 Log10 reduction in RNA copies or a >99.97% PVP-I mediated kill. The extent of this quantifiable “kill” is limited by the titre of measurable viable virus in the control sample.