A composition and photodynamic therapy method that shorten the infectious period in infected individuals and induce a sustained humoral response and cellular T-cell response to a target antigen.

Abstract

The present invention provides a photodynamic therapy method for shortening the infectious period of an infection caused by a target microorganism, comprising applying a photosensitive substance to a target treatment area and applying light of a wavelength absorbed by the photosensitive substance to the target treatment area. The present invention also provides a method for inducing a sustained humoral response and a cellular T cell response to an antigen of a target microorganism, comprising applying a photosensitive substance to a target treatment area and applying light of a wavelength absorbed by the photosensitive substance to the target treatment area.

Description

[Technical Field]

[0001] The present invention provides compositions and photodynamic therapy methods that shorten the infectious period in infected individuals and induce sustained humoral and cellular T-cell responses to target antigens. [Background technology]

[0002] The 2019 COVID-19 pandemic resulted in more than 590 million infections and at least 6.44 million deaths worldwide. This pandemic has affected people of all ages and occupations around the world, with a clearly defined upper respiratory tract viral etiology, and in some cases, depending on the strain and target group, severe lower respiratory tract infections, often leading to acute respiratory failure and death.

[0003] COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 ("SARS-CoV-2") and its variants (e.g., UK, South Africa, California, Alpha, Delta, Omicron, etc.). Rapid vaccine development has resulted in widespread herd immunity against at least lethal variants in most countries. Nevertheless, despite the significant reductions in morbidity and mortality seen with the most effective mRNA vaccines, none of the current interventions have been able to prevent new pandemic waves from the Omicron variant (BA.1, BA.1.1, BA.2, etc.), and even if protection against future viral genotypes is achieved, the extent of that protection remains uncertain. Therefore, public health authorities advise health authorities and the public not to consider the fight against the worst pandemic of our time to be over. [Overview of the project]

[0004] The present invention provides a composition and a photodynamic therapy method for shortening the infectious period of individuals infected with disease-causing microorganisms, such as SARS-CoV-2 or other viruses.

[0005] The present invention also provides compositions and photodynamic therapeutic methods that induce sustained humoral and cellular T-cell responses to antigens of microorganisms causing such diseases.

[0006] The features and embodiments of this invention will become clearer from the following detailed description, claims, and drawings. The drawings are briefly described below. [Brief explanation of the drawing]

[0007] [Figure 1] This is a diagram illustrating part of the application process of the photosensitive chlorhexidine composition of the present invention. [Figure 2] This is a diagram illustrating the forward-illuminated light application process of the present invention using an optical applicator. [Figure 3] Figure 2 illustrates the back-illuminated light application process of the present invention using the optical applicator shown. [Figure 4] This is an illustration of the forward-illuminated light application process of the present invention using a different optical applicator. [Figure 5] Figure 4 illustrates the back-illuminated light application process of the present invention using the optical applicator shown. [Figure 6] This is a flowchart of the patient registration process for the clinical trial detailed in Example I. [Figure 7] This is a table of baseline characteristics of patients in the clinical trial detailed in Example I. [Figure 8A] This figure shows the results of the infectivity assay and RT-PCR performed on nasopharyngeal swabs in the clinical trial detailed in Example I. [Figure 8B] This figure shows the results of the infectivity assay and RT-PCR performed on nasopharyngeal swabs in the clinical trial detailed in Example I. [Figure 8C] This figure shows the results of the infectivity assay and RT-PCR performed on nasopharyngeal swabs in the clinical trial detailed in Example I. [Figure 9] This table shows the risk of infectivity according to the PCR test delta-Ct at 3 and 7 days after the start of treatment for the clinical trial detailed in Example I. [Figure 10] This table shows the risk of transmissibility at day 7, corresponding to different Ct cutoff points in antigen and PCR testing procedures for the clinical trial detailed in Example I. [Figure 11A] This graph shows the results of humoral immunoassays performed in the clinical trials detailed in Example I. [Figure 11B] This graph shows the results of humoral immunoassays performed in the clinical trials detailed in Example I. [Figure 11C] This graph shows the results of humoral immunoassays performed in the clinical trials detailed in Example I. [Figure 11D] This graph shows the results of humoral immunoassays performed in the clinical trials detailed in Example I. [Figure 12] This is a table of antibody assays for the clinical trials detailed in Example I. [Figure 13] This is a table of T-cell immunoassays for the clinical trials detailed in Example I. [Figure 14A] This table shows the results of cellular immunoassays performed in the clinical trials detailed in Example I. [Figure 14B] This table shows the results of cellular immunoassays performed in the clinical trials detailed in Example I. [Figure 14C] This table shows the results of cellular immunoassays performed in the clinical trials detailed in Example I. [Figure 14D]A table showing the results of a cellular immune assay conducted for the clinical trial detailed in Example I. [Figure 14E] A table showing the results of a cellular immune assay conducted for the clinical trial detailed in Example I. [Figure 15] A table of COVID-19 symptoms detected during the clinical trial detailed in Example I.

Mode for Carrying Out the Invention

[0008] Photodynamic therapy basically involves using light energy to activate one or more photosensitive substances of a photosensitive composition, such that as a result, those photosensitive substances can then directly transfer energy to a substrate / target (type I reaction), or interact with molecular oxygen to generate reactive oxygen species (type II reaction). These reactions have been shown to kill disease-causing microorganisms, probably mainly through lipid peroxidation, membrane damage, and damage to intracellular components. Thus, photodynamic therapy has been used to treat patients suffering from various infectious diseases caused by viruses, bacteria, and fungi (hereinafter collectively referred to as "target microorganisms").

[0009] It has previously been shown that combining a low concentration of chlorhexidine with a photosensitive substance increases the effect of photodynamic therapy in the elimination of target microorganisms. See U.S. Patent Nos. 8,247,406 and 8,618,091.

[0010] In the early stages of the COVID-19 pandemic, in single-cell RNA sequencing tests in healthy human subjects, particularly high expression of viral entry factors was shown, and the ACE2 receptor located in goblet cells and multiciliated cells of the nasal epithelium or olfactory epithelium was the most affected target. However, these viral entry factor proteins were also expressed at lower but still significant levels in other tissues, particularly the multiciliated airway epithelium. These data are useful for understanding both the rapid spread of severe COVID-19 and its predominantly pneumonic phenotype, while also highlighting the importance of the nose as an entry site and potential initial replication reservoir in the first few days after SARS-CoV-2 inoculation into the host.

[0011] In the photodynamic therapy method (hereinafter referred to as "PDT") of the present invention, which activates a photosensitive substance (e.g., methylene blue) in a target treatment area (e.g., the anterior nasal cavity) by light, it has been shown that (i) the infectious period of an infectious disease (e.g., COVID-19) caused by a target microorganism (e.g., SARS-CoV-2) is shortened, and (ii) a persistent humoral response and cellular T cell response against an antigen of such a target microorganism (e.g., SARS-CoV-2) are induced.

[0012] The PDT of the present invention is composed of the following process. First, an applicator (e.g., an impregnated swab) containing a photosensitizing composition (hereinafter referred to as "PC") is provided. The PC contains a photosensitive substance (e.g., phenothiazinium) and optionally a low concentration of chlorhexidine. The photosensitive substance of the PC used in the clinical trial considered in Example I was methylene blue of the phenothiazinium system at a concentration of 0.01% (weight %) of the total weight.

[0013] Other photosensitive substances that affect both Type I and Type II photochemical reactions are also suitable for use in PC. Here, Type I reactions are electron abstraction redox reactions when light is applied, and Type II reactions are those that produce singlet oxygen (via molecular oxygen) when light is applied. Preferred phenothiaziniums for PC include not only methylene blue, but also toluidine blue and those discussed in U.S. Patent Application Publication 2004-0147508. Another preferred photosensitive substance for PC is indocyanine green. The present invention also intends to use two or more photosensitive substances, such as methylene blue and toluidine blue. The above photosensitive substances are illustrative and are not intended to limit the scope of the present invention in any way.

[0014] In certain embodiments, the photosensitive substance may be tetrapyrrole or its derivatives, such as porphyrin, chlorin, bacteriochlorin, phthalocyanine, naphthalocyanine, texaphylline, verdin, purpurin, or pheophorbid, phenothiazine, etc., as described in U.S. Patents No. 6,211,335; No. 6,583,117; and No. 6,607,522 and U.S. Patent Application Publication No. 2003-0180224. For example, suitable classes of compounds that can be used as photosensitive substances include macrocyclic compounds derived from pyrrole, porphyrin, chlorin, bacteriochlorin, isobacteriochlorin, phthalocyanine, naphthalocyanine, porphycene, porphycyanine, pentaphylin, saffrin, benzochlorin, chlorophyll, azaporphyrin, 5-aminolevulinic acid (a porphyrin metabolic precursor), synthetic diporphyrin and dichlorin, phenyl-substituted tetraphenylporphyrin, indium methylpyropheophorbide chloride, 3,1-Mesotetrakis(opropionamidophenyl)porphyrin, Bardine, Purpurin, Zinc Naphthalocyanine, Anthracendione, Anthrapyrazole, Aminoanthraquinone, Phenoxazine dye, Chlorin, Benzoporphyrin derivatives, Sulfonated Aluminum Phthalocyanine, Tetrasulfonated derivatives, Sulfonated Aluminum Naphthalocyanine, Chloroaluminum Sulfonated Phthalocyanine, Phenothiazine derivatives, Chalcogenapyryllium dye, Cationic Serena and Telrapyryllium derivatives, Ring-substituted Cationic Phthalocyanine, Pheophorbid Alpha, Hydroporphyrin, Phthalocyanine, Hematoporphyrin, Protoporphyrin, Uroporphyrin III, Coproporphyrin III, Protoporphyrin IX, 5-Aminolevulinic Acid, Pyrromethane Boron Fluoride Difluorides), indocyanine green, zinc phthalocyanine, dihematoporphyrin, benzoporphyrin derivatives, carotenoporphyrin, hematoporphyrin and porphyrin derivatives, rose bengal, bacteriochlorin A, epigallocatechin, epicatechin derivatives, hypocrelin B, urocanic acid, indoleacrylic acid, rhodium complexes, etiobenzochlorin, octaethylbenzochlorin, sulfonated Pc-naphthalocyanine, silicon naphthalocyanine, chloroaluminum sulfonated phthalocyanine, phthalocyanine derivatives, iminium salt complexes of benzochlorin, and other iminium salt complexes, DNA-binding fluorescent dyes, psoralen, acridine compounds, suprofen, tiaprofenic acid, nonsteroidal anti-inflammatory drugs, methylpheophorbide-α-(hexyl-ether), and other pheophorbides, furanocoumarin hydroperoxide, Victoria blue Examples include BO, methylene blue, toluidine blue, porphycene compounds, and combinations thereof.

[0015] Photosensitive substances may be present in PC in any appropriate amount. For example, they may be about 0.005% to about 1% by weight, about 0.01% to about 0.1% by weight, about 0.01% to about 0.05% by weight, and less than or equal to about 1% by weight. Percentages relative to total weight (wt%) can also be converted to percentages of weight relative to total volume (weight-volume%) or percentages of volume relative to total volume (volume%). For the purposes of this specification, the concentration of photosensitive substances may be expressed in weight % (% wt), weight-volume % (% w / v), or volume % (% v / v), and such expressions of concentration shall include their equivalent expressions (for example, if expressed as weight %, then equivalent concentrations measured in weight-volume % and volume %) shall also be included). When used herein, the term "approximately" means + / - 20% of the expressed value.

[0016] The concentration of chlorhexidine used in the PC for this test was 0.25% by weight as chlorhexidine gluconate. Other forms of chlorhexidine, such as chlorhexidine digluconate, chlorhexidine dihydrochloride, and chlorhexidine diacetate, are also suitable for use in PC. Exemplary suitable concentrations are about 1% by weight; about 0.5% by weight; about 0.25% by weight; about 0.125% by weight; about 0.125% by weight to about 1% by weight; about 0.125% by weight to about 0.5% by weight; about 0.2% by weight to about 0.3% by weight; about 0.125% by weight to about 0.3% by weight; less than about 1% by weight but greater than about 0.1% by weight; less than about 0.8% by weight but greater than about 0.1% by weight. It is preferable that chlorhexidine be provided at a concentration that reduces and / or eliminates potential irritation and hypersensitivity to host tissue at the treatment site. This reduction and / or elimination of potential irritation and hypersensitivity is particularly useful when the host tissue in the treatment area is a sensitive tissue such as the nasal mucosa.

[0017] PC optionally further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is a diluent, auxiliary agent, excipient, or vehicle, and the other components of PC (e.g., photosensitive substances and chlorhexidine, etc.) are administered together with the carrier. The pharmaceutically acceptable carrier is preferably approved by federal or state regulatory authorities for use in animals, and more specifically in humans, or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias. The pharmaceutically acceptable carrier is preferably a sterile liquid. Examples of pharmaceutically acceptable carriers, but not limited to, include water, physiological saline, dextrose solution, glycerol solution, and phosphate-buffered saline.

[0018] A pharmaceutically acceptable carrier, when combined with a photosensitive substance and chlorhexidine, is more preferably one that allows the PC to have a viscosity low enough to flow into the target treatment site, while also having a viscosity high enough to be retained within the target treatment site. Further compositions that become liquid after application to the target treatment site are intended, such as compositions that melt or become a solution at the target treatment site. Alternatively, as a non-limiting example, by using an inverted-phase polymer substrate, the PC can be gelled after being applied to the treatment site as a liquid. This allows the PC to effectively cover the treatment site while simultaneously retaining the composition within the treatment site.

[0019] The PDT may further include any cleaning procedure of the user's nostrils, particularly the nasal passages within the upper part of the nostrils. During the test, each non-infected user blew their nose with a tissue. This nostril cleaning procedure can also be performed by wiping each nostril with a clean cotton swab. Furthermore, such clean cotton swabs may be pre-soaked in a cleaning solution, such as water or saline solution, before being used in the nostril cleaning procedure.

[0020] After the nasal cavity has been cleansed, PDT further involves inserting an applicator 10 containing PC12 into one of the user's nostrils 14, as shown in Figure 1, and applying PC to the intranasal treatment site 16 inside the nostril 14, ensuring that a sufficient amount of PC12 adheres evenly to the intranasal treatment site 16. The applicator 10 is disposable and preferably used only once. Depending on the size of the user's nostrils (there may be multiple nostrils), it is preferable to insert the applicator 10 into each nostril 14 to a depth of at least approximately 2 cm to 3 cm. The intranasal treatment site 16 is defined as the inner surface of the nostril 14, including all areas located below the nasal turbinates, including the inferior nasal turbinate region, but is not limited to this. Particular care should be taken to ensure a good coverage area in the anterior intranasal pocket-like region of the intranasal treatment site 16 (i.e., the tip of the nose). The PC application process is repeated for the user's other nostril, so that the PC 12 is uniformly and sufficiently deposited on the intranasal treatment site 16 within the second nostril (not shown in Figure 1).

[0021] After the PC application process is completed for both nostrils, PDT is further performed with a photoapplication process. The photoapplication process consists of applying light of a wavelength that can be absorbed by the photosensitive substance(s) contained in the PC to each of the intranasal treatment sites. This photoapplication process can be implemented using any suitable method and light source disclosed in the art, such as laser diodes, light-emitting diodes, infrared and enhanced pulsed light beams, or a combination thereof.

[0022] Before applying light to the intranasal treatment site, optionally, the PC may be placed in contact with the intranasal treatment site for a short period ranging from less than 15 seconds to more than 120 seconds; this time serves as a pre-incubation period.

[0023] In one embodiment of PDT, the photoapplication process is performed twice. One of these is performed to irradiate the posterior aspect of the intranasal treatment site, as shown in Figures 3 and 5, and the other is performed to irradiate the anterior aspect of the intranasal treatment site 16, as shown in Figures 2 and 4. For posterior irradiation of the intranasal treatment site, the PDT inserts the photodiffuser tip 18 straight into the user's nostril 14 by orienting the photodiffuser tip 18 toward the back of the user's head, as shown in Figures 3 and 5, to perform a 2-minute irradiation cycle at a wavelength matched to the primary absorption wavelength of the photosensitizer selected for use. After this posterior irradiation is complete, the PDT may optionally remove the photodiffuser tip 18 from the user's nostril 14 and repeat the photoapplication process for both nostrils 14 in preparation for anterior irradiation of the intranasal treatment site 16. In order to irradiate the intranasal treatment site 16 forward, in PDT, as shown in Figures 2 and 4, the light diffuser tip 18 is inserted into the user's nostril 14 by pointing it forward (away from the face) and towards the tip and inside of the user's nose for a 2-minute irradiation cycle at a wavelength matched to the primary absorption wavelength of the photosensitive substance selected for use. Once the forward irradiation is complete, the light diffuser tip 18 is removed from the user's nostril 14. In the second embodiment, in PDT, as shown in Figures 3 and 5, the light diffuser tip 18 is inserted straight into the user's nostril 14 by pointing it towards the back of the user's head for a 2-minute irradiation cycle at a wavelength matched to the primary absorption wavelength of the photosensitive substance selected for use, the light diffuser tip 18 is removed, PC is reapplied to the nostril, and the light diffuser tip 18 is reinserted in the same manner as in the second treatment cycle. For this test, the optical diffuser tip 18 was driven by a light source capable of producing sufficient optical output at wavelengths of 630 nm to 690 nm for irradiation cycles. An example of a suitable light source is a Class 1 laser device consisting of two 700 mW channels. As mentioned above, PDT can also be achieved using other suitable light sources.

[0024] Depending on the concentration of the photosensitive material and the output of the light-emitting device(s), the time required to apply light to the treatment site is short, for example, about 15 seconds to less than 5 minutes, preferably about 1 minute to 3 minutes and about 2 minutes to 4 minutes. The amount of light energy supplied per area during each cycle of light application is about 2 J / cm². 2 ~About 45J / cm 2 A range of approximately 18 J / cm², preferably about 18 J / cm². 2 ~About 36J / cm 2 It is preferable that multiple phototherapy treatments (for example, about 2 to 10 times, about 3 to 5 times, etc.) are applied to each treatment site, thereby applying a total accumulated light energy to the treatment site that may be substantially higher than the light energy that can be supplied during each cycle.

[0025] If desired, PDT can be repeated multiple times (for example, approximately 2 to 5 times). It is preferable that the application of light to the treatment site does not cause physiological damage to the treatment site and / or surrounding host tissue. [Examples]

[0026] Example I A single-center, randomized, placebo-controlled, single-blind clinical trial was conducted at a university hospital in northern Spain. Participants were enrolled from the University of Navarra COVID19 Safe Campus Program and the Clinica Universidad de Navarra COVID19 Surveillance program. Both programs were established during the 2020 pandemic, after the lifting of the initial population lockdown in Spain, to conduct active surveillance and limit the spread of new viruses within university campuses and hospitals. These programs were conducted using either rapid antigen tests or PCR tests, based on both randomized testing and individualized testing through contact tracing.

[0027] The inclusion criteria for this study required patients to be 18 years of age or older, to present within 48 hours of a positive SARS-CoV-2 test result based on real-time PCR (less than 27 cycles) or rapid antigen testing, and to have a disease level limited to asymptomatic or mild cases. All concomitant medications except angiotensin receptor blockers or immunosuppressants were permitted. All patients consented to receive either a non-painful intranasal photoirradiation process or an equivalent placebo.

[0028] Patients with severe co-existing conditions were excluded. Individuals who reported being unable to tolerate the insertion of a photocurator due to the size, shape, or anatomical variations of their mouth or nose, individuals known to have allergic reactions to components of intranasal decongestant treatment, including methylene blue or chlorhexidine gluconate, individuals with moderate or severe COVID-19 disease, and individuals unable to receive necessary follow-up examinations were excluded from the study.

[0029] Participants were randomly assigned in a 1:1 ratio to either the control group or the treatment group in each block using a computer program after inclusion. Personnel performing the randomization were also assigned other tasks, such as rating selection / exclusion criteria, obtaining informed consent from patients, and completing baseline and follow-up questionnaires. These personnel did not administer any treatment to either group, nor did they collect any biological samples.

[0030] The control treatment was performed using saline solution and a switched-off laser irradiation device, and the intervention was blinded to patients using polarized glasses. The treatment was not blinded to the investigators. Participants receiving the placebo treatment followed the same timing and cycle protocol as the intervention group.

[0031] The PDT of the present invention was performed using a CE-marked Steriwave® Nasal Photodisinfection System (NPS, SW4000, Ondine Biomedical Inc., Vancouver, BC, Canada). Briefly, the NPS is a Class II medical device comprising a power source ("Light Source"), a Nasal Light Illuminator (NLI) consisting of a single-use bidirectional laser-conducting injection-molded nasal applicator, and a methylene blue preparation at a concentration of 0.01% (wt%) of total weight and chlorhexidine gluconate at a concentration of 0.25% (PC) of total weight. Chlorhexidine gluconate is approved in Canada and Europe for the elimination of potentially pathogenic microorganisms from the anterior nasal meatus. Topically applied PC binds to microbial cell wall components, and red light (having a wavelength of 664 nm to 670 nm) is absorbed by photosensitive molecules, generating reactive oxygen species (ROS) responsible for lethal microbial cell wall disruption. It should be noted that the wavelength of red light may be 630nm-690nm, 650nm-680nm, and 660nm-670nm. Furthermore, the present invention can also be carried out using light having a wavelength of 500nm-800nm.

[0032] Nasopharyngeal (NP) swabs were taken pre-treatment at baseline, 3 days post-treatment, and 7 days after the start of treatment. NP samples were collected and placed in Universal Transport Media (UTM, Copan) for immediate RNA extraction (TANBead® Nucleic Acid Extraction Kit) and RT-PCR (Cepheid Xpert Xpress SARS-CoV-2, genes E and N), and retained in UTM for in vitro infectivity assays in a biosafety level 3 facility. Aliquots of total nucleic acids extracted from baseline NP swabs were used for SARS-CoV-2 sequencing.

[0033] The NPS was performed by pre-trained medical professionals. The procedure was initiated by applying PC to the inside of the anterior nostrils, including the patient's nostrils and nasal passages. The operator then connected the NLI to a power source and inserted the two-pronged nasal end of the NLI into the patient's nostril. In this protocol, the Light Source was switched on, and three 4-minute irradiation cycles were applied, with two new additional PCs used after each irradiation cycle to ensure equivalent performance. This 4-minute x 3-cycle scheme was repeated on days 2 and 3. In total, each patient received nine 4-minute cycles, for a total of 36 minutes of treatment. To avoid self-contamination, the NLI cables were labeled "L" for the left nostril and "R" for the right nostril. A new set of NLI was used daily to avoid self-contamination. Subjects were asked to blow their noses before treatment, but local treatment of the nose was not permitted. The placebo-controlled group (arm) followed the same protocol as the intervention group for three consecutive days, including the application of saline solution and the introduction of a switched-off PDT device.

[0034] The primary outcome of this study was reduced infectivity after 3 days of treatment. Prior to the start of enrolment, considering the evidence that persistent PCR positive results were due to nucleic acid residues rather than viable virus, in vitro infectivity assays using Vero-E6 cells were preferred over RT-PCR as the endpoint, but both techniques were performed for each sample. Secondary outcomes were reduced safety and infectivity at other time points (days 7 and 14 after the start of treatment). Biological correlations in this study included analysis of immunogenicity of nucleocapsid, spike, and the entire SARS-CoV-2 genome in patients at weeks 10 and 20 post-treatment, as well as genomic sequencing of samples, as described below.

[0035] Patients were questioned about symptoms present 24 hours prior to baseline visit, and on days 3 and 7. The symptoms collected included pharyngitis, chills, newly occurring or worsening cough, dyspnea, chest tightness, body temperature above 38°C, fatigue, myalgia, loss of smell, loss of taste, headache, gastrointestinal symptoms, dyssomnia, general malaise, and nasal congestion. These symptoms were assessed using a four-category scale (none, mild, moderate, or severe).

[0036] To assess safety, all immediate local effects at the treatment site, as well as all delayed local effects, were determined. The severity and likelihood of these effects being associated with the procedure, as subjectively measured by the investigating physician, were also recorded. These questions were asked during interviews immediately after the procedure and at each subsequent follow-up visit.

[0037] Other variables, such as sex, age, completion of initial COVID-19 vaccination, COVID-19 booster vaccination, previous COVID-19 infection, weight, height, and vital signs including body temperature, heart rate, systolic blood pressure, diastolic blood pressure, and blood oxygen saturation, were also collected.

[0038] Sample size was calculated based on the difference in delta-Ct before and after intervention, following the application of PDT to infected samples for 4 and 8 minutes in a preliminary in vitro study. At 99% power, a very small sample size (6 patients per group) was sufficient to demonstrate microbiological efficacy. Given the wide range of ages and co-existing conditions among the subjects, it was difficult to make assumptions regarding their clinical course and viral load reduction. Furthermore, it was known that viral load spontaneously decreased in this study population (i.e., mostly vaccinated and otherwise healthy individuals). A dropout rate of 10% was also taken into consideration. Based on these factors, a sample size of 100 patients (50 patients per group) was proposed. However, due to the rapid containment of the sixth wave of the COVID-19 pandemic in Spain (December 2021–February 2022), it was not possible to reach the complete sample size.

[0039] To compare various quantitative variables between the control and intervention groups, the Mann-Whitney U test was performed for variables that did not satisfy normality, and the Student's t-test was performed for variables that did satisfy normality. The median, interquartile range, mean, and standard deviation were calculated for each test. The chi-squared test was performed for qualitative variables.

[0040] In the infectivity test analysis, a multiple linear regression model was performed to calculate the beta coefficient and their respective 95% confidence intervals (95% CI) to validate the effectiveness of the treatment. To assess the ability to reduce infectivity in diagnostic tests, a multivariate adjusted logistic regression model was performed to estimate the odds ratio (OR) and its 95% CI. Both models were adjusted for sex, age, number of initial symptoms, COVID vaccine, COVID booster medication, and prior SARS-CoV-2 infection.

[0041] All p-values ​​shown are two-tailed p-values. Statistical analysis was performed using Prism software (GraphPad Software, San Diego, California) and STATA 13.0.

[0042] In vitro infectivity assay: Confluent monolayers of Vero-E6 cells were subcultured in 96-well plates until re-confluent. These cells were then infected with all patient-derived samples pre-diluted 1:2 in infection medium (Minimum Essential Medium (MEM) with 0.2% BSA, 2 mM glutamine, and 20 mM Hepes), and incubated at 37°C for 4 hours. After removing the inoculum, Eagle MEM with 10% fetal bovine serum and antibiotics was added to each well of infected cells. Uninfected cells were used as negative controls. After 72 hours, cells were collected, lysed using Dynabeads® MyOne® Silane beads, and the amount of SARS-CoV-2 genome was analyzed by RT-PCR (Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2, BGI, ORF1ab gene and human β-actin gene). These values ​​were expressed as 2^(-ΔCt)*1000.

[0043] Specific anti-SARS-CoV-2 humoral and cellular responses: To analyze participants' serological responses, blood samples were taken 10 and 20 weeks after enrollment in the clinical trial.

[0044] Anti-SARS-CoV-2 antibodies were detected using four different commercially available chemiluminescence tests. First, total antibodies (IgG+IgM) against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein were quantified using the Elecsys® anti-SARS-CoV-2 S test (Roche Diagnostics, Germany) on the cobas e601 platform. Second, total antibodies (IgG+IgM) (anti-N) against the viral nucleocapsid were qualitatively detected using the Elecsys® anti-SARS-CoV-2 test (Roche Diagnostics, Germany). Third, anti-SARS-CoV-2 specific IgG against the nucleocapsid and spike protein was detected using the COVID-19 VIRCLIA® IgG Monotest (Vircell SL, Spain). Fourth, anti-SARS-CoV-2 IgM+IgA against nucleocapsid and spike protein was detected using COVID-19 VIRCLIA® IgM+IgA Monotest (Vircell SL, Spain). Serum samples were pre-inactivated at 56°C for 30 minutes. Interpretation of various immunoassays was performed as recommended by each manufacturer.

[0045] Cell-mediated immune responses to SARS-CoV-2 were measured using QuantiFERON® SARS-CoV-2 Starter and Extended Sets (QIAGEN, USA). The Starter Set contained specific peptides (S1 subdomain, S2 subdomain, RBD subdomain) derived from the spike antigen to evaluate CD4 T cell immune responses (Ag1 tube) and CD4+CD8 T cell immune responses (Ag2 tube). The Extended Set contained additional specific peptides (S domain, N domain, and M domain) derived from the entire SARS-CoV-2 genome to test complete and specific CD4 T cell-mediated immune responses (Ag3 tube). After inoculation, the tubes were incubated at 37°C for 20–24 hours, and then plasma IFN-γ concentrations were measured using QuantiFERON® ELISA (QIAGEN, USA). Samples were considered reactive if IFN-γ production greater than 0.15 IU / mL was observed in any tube after stimulation.

[0046] Sequencing analysis of COVID-19 variants: Libraries were prepared from swab samples using a commercially available kit (COVIDSeq Assay, Illumina), and sequencing was performed using NextSeq2000 (Illumina). Analysis was performed using Kraken (Illumina). Phylogenetic tree analysis was performed using Nextclade.org.

[0047] In vitro preliminary test to evaluate the photodynamic effect against SARS-CoV2: Before this test, the effectiveness of PDT against SARS-CoV2 survival ex vivo was unknown. For this reason, an in vitro preliminary test was carried out in the biosafety level 3 (BSL3) facility of the Gene Therapy Division of the Center for Applied Medical Research (CIMA) at the University of Navarra. For this purpose, nasopharyngeal swab samples with a high SARS-CoV-2 load from severe COVID-19 patients were cultured in confluent Vero-E6 cells, and the supernatant was collected 72 hours after inoculation. Then, the virus was filtered, and titration was performed using a lysis plate assay with a Vero-E6 cell monolayer, and as a result, a titer of 4.3×10 7 plaque-forming units (PFU / ml) was obtained. 20 μL of a virus solution containing 1×10 4 PFU and 1×10 5 PFU of SARS-CoV-2 was prepared using phosphate-buffered saline (PBS). Then, each sample was mixed with 180 μl of a photosensitive substance preparation (PF) and added to a small plastic reservoir (nasal tip reservoir) that simulates the human nostril. As a control, the virus sample was mixed with 180 μl of PBS without using PF. Then, NLI was applied to each sample for 0 minutes, 4 minutes, or 8 minutes, and the sample was gently shaken every 2 minutes to supply oxygen to the sample again. Then, the NLI was removed, each sample was collected, and analyzed by RT-PCR to quantify the amount of the SARS-CoV-2 genome. Viral RNA was extracted from each sample using Dynabeads™ MyOne™ Silane beads (ThermoFisher) and quantified by RT-qPCR using oligonucleotides specific to the SARS-CoV-2 nucleocapsid gene. From the results obtained, 10 4 plaque-forming units or 10 5When plaque-forming units (PFUs) were treated with PDT exposure for 4 to 8 minutes, increased destruction of SARS-CoV-2 viral nucleic acids was observed. The inventors retained this data not as evidence of in vivo efficacy, but simply as proof that PDT has a detrimental effect on viable viral particles.

[0048] Results: 79 patients were screened in Spain between December 21, 2021 and February 15, 2022, during the sixth wave of the COVID-19 pandemic. One patient was excluded for not meeting the criteria. Of the 78 patients randomly assigned in a 1:1 ratio, two patients in the treatment group discontinued treatment (one due to local irritation and the other for personal reasons), and one patient in the placebo group discontinued treatment (for personal reasons). The 75 patients who completed the trial were included in the analysis. Therefore, the retention rate was 96.1%. See Figure 6.

[0049] The population consisted mainly of young adults with mild symptoms, no history of natural SARS-CoV-2 infection (79%), who had completed a course of vaccination (i.e., received two doses of mRNA-based vaccine) (93%), and who were expected to have high viral loads. See Figure 7. No significant differences were observed between the intervention group and the placebo group, except for measured blood pressure, which was not considered clinically significant in terms of its effect or absolute value.

[0050] Patients who received treatment showed a significant reduction in infectivity from baseline to 3 days after treatment (p<0.0001), while placebo patients did not show a significant reduction in infectivity (p=0.24). See Figure 8A. In both the placebo group and treated patients, infectivity was significantly reduced from day 3 to day 7, but this reduction was more pronounced in the treated group (p=0.003) than in the placebo group (p=0.0089) (Figure 8A).

[0051] Figures 8A and 8C show the results of infectivity assays and RT-PCR for nasopharyngeal swabs. Regarding Figure 8A, subjects enrolled in this clinical trial received nasopharyngeal swabs at baseline (day 0), day 2, and day 7. Samples were collected, placed in a viral transport medium, and then used for infectivity analysis in VeroE6 cells. 72 hours after infection, the supernatant was tested for the presence and quantity of SARS-CoV-2 using RT-PCR for the Orf1b gene and human beta-actin. Referring to Figure 8A, the data are expressed as the common logarithm of the delta of the fluorescence threshold cycle number for beta-actin and the Orf1b gene. Wilcoxon's test was used for paired data to compare baseline and D2, and D2 and D7. Regarding Figure 8B, the RT-PCR results for the E and N genes of SARS-CoV-2 from nasopharyngeal swab samples immediately after collection are shown. Referring to Figure 8B, the data are expressed as the fluorescence threshold cycle number. Wilcoxon's test was used for paired data to compare baseline with D2, and D2 with D7. Figure 8C shows the percentages of "positive" and "negative" individuals in each of the two groups at baseline, D2, and D7, with cycle thresholds selected accordingly.

[0052] In a multivariate adjusted linear regression model, the protective effect of the treatment was demonstrated on day 3, with a mean β coefficient of -812 (95% CI -478660 to -1.3, p < 0.05). A trend was also observed on day 7, with a mean β coefficient of -9.3 (95% CI -269.1 to 3.1, p NS). Please refer to Figure 9, which shows the risk of infectivity according to PCR test delta-Ct 3 and 7 days after the start of treatment.

[0053] Throughout the study period, the mean PCR cycles increased in both the treatment and placebo groups. However, in treated patients, a significant difference was observed in RT-PCR cycles for both the E and N genes 7 days after the start of treatment (Figure 8B). Analysis of the probability of PCR negativity on day 7 showed protection by intranasal PDT compared to PCR positivity on day 7, with odds ratios of 0.16 (95% CI: 0.05~0.52) and 0.15 (95% CI: 0.04~0.58), respectively, using thresholds of 32 cycles and 34 cycles. See Figures 10 and 8C.

[0054] Figures 11A to 11D show the results of humoral immunoassays. Patients were followed for 20 weeks after intranasal PDT treatment. Plasma and peripheral CD4 T lymphocyte and CD8 T lymphocyte samples were collected at weeks 10 and 20. The detection of total anti-spike antibody in plasma is shown in Figure 11A. The detection of total anti-nucleocapsid antibody in plasma is shown in Figure 11B. The change in median levels at week 20 compared to week 10 is shown in Figure 11C. The percentage change in median antibody levels at week 20 compared to week 10 is shown in Figure 11D. The results of antibody assays and cellular immunoassays are shown in Figures 12 and 13, respectively. Figures 14A to 14E show the results of cellular immunoassays. Quantiferon assays were used to analyze specific T cell responses to specific purified peptides (S1 subdomain, S2 subdomain, RBD subdomain) derived from the spike antigen. This analysis used either isolated CD4 (shown in Figure 14A) or a CD4 / CD8 combination (shown in Figure 14B). Both CD4 and CD8 responses to additional specific peptides (S-domain, N-domain, and M-domain) derived from the whole genome of SARS-CoV-2 were also tested in T cells from both placebo-receiving and PDT-treated subjects (shown in Figure 14C). The median change in IFN units at 20 weeks compared to 10 weeks for the three assays is shown in Figure 14D. The percentage change in median IFN units at 20 weeks compared to 10 weeks is shown in Figure 14E.

[0055] Antibody quantification at 10 and 20 weeks post-treatment showed no difference in the production of anti-spike antibodies (Figure 11A) and anti-nucleocapsid antibodies (Figure 11B) between the control group and the PDT group (Figure 12). A significant decrease in anti-spike antibodies was observed in the placebo group at 20 weeks (p<0.001), but not in the PDT group (p=0.1336) (Figure 11A). Measurement of interferon (IFN) synthesis by CD4 T cells after exposure to SARSCoV2 spike antigen revealed that the median IFN units in the placebo group were lower than those in the PDT group at both 10 and 20 weeks (Figures 13, 14A). The combined CD4 T cell response and CD8 T cell response to the spike protein also showed a higher trend in individuals treated with PDT, with the median at 20 weeks being almost double that of the placebo group (p=0.0971) (Figures 13, 14B). When CD4 and CD8 cells were exposed to the entire SARS-CoV-2 genome product, a T cell response was observed in the PDT group at both 10 and 20 weeks (p=0.0293) (Figure 13, Figure 14C). At these time points, specific T cell immunity to SARS-CoV-2 was significantly reduced in the control group from 10 to 20 weeks, whereas the level of SARS-CoV-2-induced IFN production was similar in individuals treated with PDT from 10 to 20 weeks (Figure 14C).

[0056] COVID-19 RNA sequencing was successful for 72 patients (96% of the population). The results showed that 97.2% of patients had the Omicron 21 K variant (B.1.1.529 or BA.1), with only two exceptions: one patient had Omicron 21 L (BA.2), and one patient had colonized the Delta 21 J variant.

[0057] Analysis of symptom progression on days 3 and 7 after the start of treatment revealed a significant decrease in the prevalence of chest tightness and headache in the PDT treatment group compared to the placebo group on day 3. Please refer to Figure 15, which presents a list of COVID-19 symptoms detected during the clinical trial.

[0058] Regarding the safety of the intervention, a total of 53 mild adverse events were reported in 32 patients (5 in the control group and 27 in the intervention group). One patient in the intervention group (2.7%) withdrew from the study due to severe itching in the nasal cavity on the first day after treatment.

[0059] The COVID-19 pandemic has presented serious challenges to the health and economies of individuals, societies, and nations worldwide. Since the end of 2019, several variants of SARS-CoV-2 have triggered successive waves of regional outbreaks of the virus with varying clinical and biological profiles, and clinical outcomes range from severe COVID-19 pneumonia and death to completely asymptomatic cases.

[0060] In the early stages of SARS-CoV-2 infection, particularly in carriers exhibiting mild symptoms of less lethal variants such as Omicron, local eradication of the nasal passages—the most likely site of viral entry and initial replication—may be associated with inhibiting viral spread in various clinical and nonclinical situations. Our study is considered to be the first prospective randomized trial to explore the tolerance and relevance of intranasal eradication in individuals with mild SARS-CoV-2 infection.

[0061] The primary endpoint of this study (i.e., reduction in infectivity as measured by an in vitro infectivity assay using nasopharyngeal swab samples) was achieved. The efficacy of intranasal PDT was demonstrated in individuals with high viral load (Ct < 26 and / or positive antigen test at enrollment), mild symptoms, and otherwise healthy. The treatment consisted of 12-minute treatments with methylene blue PDT for three consecutive days.

[0062] Interestingly, even though the PDT treatment was applied only to the nasal passages, the treatment reduced SARS-CoV-2 colonization throughout the upper respiratory tract compared to placebo. This may be due to the nasal mucosa being a major site of viral replication in the case of omicron variants and / or vaccination status. This result strengthens the hypothesis regarding the relevance of intranasal eradication in a disease that can progress to a systemic stage within days. The effect of PDT was observed as early as 3 days post-treatment using an in vitro infectivity assay with NP swabs. Although considered to be unreliable readout information, RT-PCR cycles for NP samples were also higher in the treatment group on day 7, further demonstrating the long-lasting effect of intranasal eradication. Consequently, PDT treatment had a significant protective effect in avoiding PCR positivity on day 7. No serious adverse effects were observed during treatment.

[0063] A rapid decrease in viral load was observed in the entire study population (placebo and PDT) on days 3 and 7, which may obscure the magnitude of the PDT effect. A potential explanation is that the majority of the study population was vaccinated (93%). Indeed, vaccination has been associated with a more rapid decrease in viral RNA in SARS-CoV-2 infected patients. On the other hand, almost all patients were primarily infected with the omicron variant (21 K, BA.1), which has better biological properties compared to other variants (97.2%). Less pathogenic variants may have less persistence in the upper respiratory tract compared to alpha or delta variants. These two facts—the high percentage of vaccinated individuals and the rapid decrease in viral load—highlight the effectiveness of PDT in eliminating infectious viral particles, with the viral reduction in the treatment group being significantly higher than in the placebo group as early as day 3.

[0064] The inventors have shown that treatment with PDT has a significant and sustained effect on SARS-CoV-2 specific T lymphocyte-mediated immunity, which is significant and unexpected. The inventors showed that in individuals treated with intranasal PDT, the median IFN unit production by CD4 T lymphocytes and CD8 T lymphocytes was nearly doubled. Most importantly, while there was a significant decrease between 10 and 20 weeks post-treatment in the placebo-treated individuals, all T cell responses assessed after PDT treatment persisted. The inventors believe this is the first evidence of PDT-mediated immune enhancement against SARS-CoV-2.

[0065] The sustained immune effects induced by PDT application lead to novel immune strategies. For example, photosensitive substances and antigens can be used to induce photochemical internal transfer of antigens to antigen-presenting cells (APCs), thereby facilitating adjuvant-free cross-priming of CD8 T lymphocytes.

[0066] The present invention further includes topical treatment with PDT to reduce the viral load in patients with an early viral infection or a mild viral infection not caused by SARS-CoV-2 (i.e., caused by another virus). The present invention also includes topical treatment with PDT to reduce the bacterial load in patients with an early bacterial infection or a mild bacterial infection. Finally, the present invention includes topical treatment with PDT to reduce the fungal load in patients with an early fungal infection or a mild fungal infection.

[0067] The topical treatment using PDT discussed in this application can be applied to anyone.

[0068] The descriptions and illustrations presented herein are intended to inform those skilled in the art of the present invention, its principles, and its practical applications. Those skilled in the art can adapt and apply the invention in various forms to best suit the requirements of their particular use. Accordingly, the specific embodiments of the invention described herein are not intended to exhaust or limit the invention. Accordingly, the scope of the invention should not be determined by reference to the above description, but rather by reference to the appended claims, and the entire scope of equivalents of such claims is encompassed. All disclosures of papers and references, including patent applications and publications, constitute part of this specification by reference in all respects.

Claims

1. The use of a photosensitive molecule in the manufacture of a topical drug for photodynamic therapeutic treatment to shorten the infectious period of an infection caused by a target microorganism, wherein the topical drug is applied to a target region and light is applied to the target region.

2. The use of the photosensitive molecule according to claim 1, wherein the target region is the anterior nostril.

3. The use of the photosensitive molecule according to claim 1 or 2, wherein the target microorganism is a coronavirus including SARS-CoV-2 or a variant of SARS-CoV-2.

4. The use of a photosensitive molecule according to any one of claims 1 to 3, wherein the photosensitive molecule is a phenothiazine.

5. The use of the photosensitive molecule according to any one of claims 1 to 4, wherein the photosensitive molecule is methylene blue.

6. The use of a photosensitive molecule according to any one of claims 1 to 5, wherein the concentration of the photosensitive substance is about 1% by weight or less of the topical drug.

7. The use of a photosensitive molecule according to any one of claims 1 to 6, wherein the topical drug further comprises chlorhexidine at a concentration of about 1% by weight or less.

8. The use of photosensitive molecules in topical drug compositions that induce sustained humoral and cellular T-cell responses to antigens of microorganisms causing such diseases.

9. The use of the photosensitive molecule according to claim 8, wherein the target region is the anterior nostril.

10. The use of the photosensitive molecule according to claim 8 or 9, wherein the target microorganism is a coronavirus including SARS-CoV-2 or a variant of SARS-CoV-2.

11. The use of a photosensitive molecule according to any one of claims 8 to 10, wherein the photosensitive molecule is a phenothiazine.

12. The use of the photosensitive molecule according to any one of claims 8 to 11, wherein the photosensitive molecule is methylene blue.

13. The use of a photosensitive molecule according to any one of claims 8 to 12, wherein the concentration of the photosensitive substance is about 1% by weight or less of the topical drug.

14. The use of a photosensitive molecule according to any one of claims 8 to 13, wherein the topical drug further comprises chlorhexidine at a concentration of about 1% by weight or less.

15. The use of a photosensitive molecule according to any one of claims 8 to 14, wherein the photosensitive substance is methylene blue at a concentration of 0.01% by weight of the photosensitive composition, and the concentration of chlorhexidine is 0.25% by weight of the photosensitive composition.

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