Composition and photodynamic therapy method for shortening infection period and inducing persistent body fluid and T cell response of infected person to target antigen

Abstract

The invention provides a photodynamic therapy method for shortening the infection period of infection caused by targeted microorganisms. The photodynamic therapy method comprises the following steps: applying a photosensitizer to a targeted body treatment area; and applying light to the target body treatment region at the wavelength absorbed by the photosensitizer. The invention also provides a method of inducing a sustained humoral and cellular T cell response against a target microbial antigen, comprising: administering a photosensitizer to a targeted body treatment area; and applying light to the target body treatment region at the wavelength absorbed by the photosensitizer.

Description

Technical Field

[0001] This invention provides a composition and photodynamic therapy that shortens the infection period and induces sustained humoral and cellular T-cell responses against target antigens in infected individuals. Background Technology

[0002] COVID-19 infection is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (“SARS-CoV-2”) and its variants (such as those in the UK, South Africa, California, Alpha, Delta, Omeprung, etc.). Due to the rapid development of vaccines, most countries have achieved widespread herd immunity, at least against lethal variants. However, as the most effective mRNA vaccines have shown, despite significant reductions in morbidity and mortality, all current interventions have failed to prevent a new wave of the Omeprung variants (BA.1, BA.1.1, BA.2, etc.), and the effectiveness of protection against future viral genotypes (if any) remains uncertain. Therefore, public health authorities advise health authorities and the public not to assume that the fight against the most severe pandemic of our time is over.

[0003] The present invention provides a composition and photodynamic therapy for shortening the infection period of individuals infected with pathogenic microorganisms such as SARS-CoV-2 or other viruses.

[0004] The present invention also provides a composition and a photodynamic therapy for inducing sustained humoral and cellular T-cell responses against such pathogenic microbial antigens. Attached Figure Description

[0005] The features and inventive aspects of the invention will become more apparent after reading the following detailed description, claims, and drawings, in which a brief description is provided below:

[0006] Figure 1 This is a diagram illustrating a portion of the application process of the photosensitized chlorhexidine composition of the present invention;

[0007] Figure 2 This is a graphical description of the front illumination light application process of the present invention using an applicator head;

[0008] Figure 3 Is using Figure 2 A graphical description of the post-illumination light application process of the application head of the present invention;

[0009] Figure 4 This is a graphical description of the front illumination light application process of the present invention using another applicator head;

[0010] Figure 5 Is using Figure 4 A graphical description of the post-illumination light application process of the application head of the present invention;

[0011] Figure 6 This is a flowchart of the patient recruitment process for the clinical study described in detail in Example I;

[0012] Figure 7 This is the patient baseline characteristics table of the clinical study described in detail in Example I;

[0013] Figure 8A -C shows the results of nasopharyngeal swab infectivity assays and RT-PCR performed for the clinical studies detailed in Example I;

[0014] Figure 9 The clinical study described in Example I showed the risk of infection based on PCR detection of ΔCt at 3 and 7 days after the start of treatment;

[0015] Figure 10 This is a risk table of transmissibility within 7 days based on antigen testing and different Ct cutoff points in PCR testing treatment during clinical studies described in detail in Example I;

[0016] Figure 11A -D shows the results of humoral immunity assays performed for the clinical studies detailed in Example I;

[0017] Figure 12 This is the antibody assay table for the clinical study described in detail in Example I;

[0018] Figure 13 This is the T-cell immunoassay table from the clinical study described in detail in Example I;

[0019] Figure 14A -E shows the results of a cellular immunoassay performed for the clinical study detailed in Example I;

[0020] Figure 15 This is a table of symptoms of COVID-19 infection detected during the clinical study described in detail in Example I. Summary of the Invention

[0021] Photodynamic therapy primarily involves using light energy to activate one or more photosensitizers in a photosensitizing composition. These photosensitizers can either directly transfer energy to the 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 pathogenic microorganisms, likely primarily through lipid peroxidation, membrane damage, and damage to intracellular components. Therefore, photodynamic therapy has been used to treat patients with various infectious diseases caused by viruses, bacteria, and fungi (collectively referred to as "targeted microorganisms").

[0022] The combination of low-concentration chlorhexidine with photosensitizers has previously been shown to enhance the efficacy of photodynamic therapy in eliminating targeted microorganisms. See U.S. Patent Nos. 8,247,406B2 and 8,618,091B2.

[0023] In the early stages of the COVID-19 pandemic, single-cell RNA sequencing studies in healthy human subjects revealed particularly high expression of viral entry factors, with the most severely affected target being the ACE2 receptor located in goblet and multimarginal hair cells of the nasal epithelium. Although the expression levels of these proteins were also low in other tissues, they remained significant, especially in the multimarginal hair airway epithelium. While these data contribute to understanding the rapid spread and dominant pneumonia phenotype of severe COVID-19 infection, they also highlight the importance of the nose as an entry site and potential major replication reservoir in the first few days after SARS-CoV-2 inoculation.

[0024] The photodynamic therapy method of the present invention (hereinafter referred to as "PDT"), which involves photoactivating a photosensitizer (e.g., methylene blue) on a treatment area of ​​the body (e.g., the anterior nasal cavity), has been shown to (i) shorten the duration of infection (e.g., COVID-19 infection) caused by a targeted microorganism (e.g., SARS-CoV-2); and (ii) induce sustained humoral and cellular T-cell responses against antigens of such targeted microorganisms (e.g., SARS-CoV-2).

[0025] The PDT of the present invention comprises the following process. First, an application head (e.g., a pre-dipped swab) containing a photosensitizer (hereinafter “PC”) is provided. The PC includes a photosensitizer (e.g., phenothiazine) and optionally a low concentration of chlorhexidine. The photosensitizer used in the clinical studies discussed in Example I was phenothiazine-onium methylene blue at a concentration of 0.01% (%wt) of the total weight.

[0026] Other photosensitizers affecting type I and type II photoreactions are also suitable for PC, where type I reaction produces an electron-extracting redox reaction upon light application, and type II reaction produces singlet oxygen (via molecular oxygen) upon light application. Preferred phenothiazine-onium photosensitizers for PC include not only methylene blue, but also toluidine blue, and those discussed in U.S. Patent Publication No. 2004-0147508. Another preferred photosensitizer for PC is indocyanine green. The invention also contemplates the use of two or more photosensitizers, such as methylene blue and toluidine blue. The above photosensitizers are merely examples and are not intended to limit the scope of the invention in any way.

[0027] In some embodiments, the photosensitizer may be tetrapyrrole or its derivatives, such as porphyrin, chlorins, bacteriochlorins, phthalocyanine, naphthylphthalocyanine, texaphyrins, verdins, violetin, or pheophytic acid, phenothiazine, etc., as described in U.S. Patent Nos. 6,211,335, 6,583,117, 6,607,522, and U.S. Patent Publication No. 2003-0180224. For example, suitable classes of compounds that can be used as photosensitizers include pyrrole-derived macrocyclic compounds, porphyrins, dihydroporphyrins, chlorophyll, isochlorophyll, phthalocyanines, naphthylphthalocyanines, porphyrinenes, porphycyanines, pentaporphyrins, blue porphyrins, benzodihydroporphyrins, chlorophyll, azaporphyrins, the metabolic porphyrin precursor 5-aminolevulinic acid, synthetic bisporphyrins and bisdihydroporphyrins, phenyl-substituted tetraphenylporphyrins, and methyl chloropyrophosphates. Indium pheophylate, 3,1-meso-tetra(propionylaminophenyl)porphyrin, chlorophyll, violet pigment, zinc naphthylphthalocyanine, anthraquinone, anthraphenazole, aminoanthraquinone, phenoxazine dyes, dihydroporphyrin, benzo[a]porphyrin derivatives, aluminum sulfonate phthalocyanine, tetrasulfonated derivatives, aluminum sulfonate phthalocyanine, aluminum chlorosulfonate phthalocyanine, phenothiazine derivatives, thiopyranium dyes, cationic selenium / tellurium pyranium derivatives, cyclically substituted cationic phthalocyanine, α-pheophylate, hydride porphyrin Phthalocyanine, hematoporphyrin, protoporphyrin, uroporphyrin III, coprophyrin III, protoporphyrin IX, 5-aminolevulinic acid, pyrrolimethane difluoride, indocyanine green, zinc phthalocyanine, dihematoporphyrin, benzoporphyrin derivatives, carotenoid porphyrin, hematoporphyrin and porphyrin derivatives, rose red, chlorophyll A, epigallocatechin, epicatechin derivatives, bamboo red phyllogen B, uric acid, indoleacrylic acid, rhodium complex, primordial benzodihydroporphyrin, octaethyl Benzodihydroporphyrin, sulfonated phthalocyanine, naphthalocyanine silicon, chlorosulfonated phthalocyanine aluminum, phthalocyanine derivatives, imine salt benzodihydroporphyrin and other imine salt complexes, DNA-binding fluorescent dyes, psoralen, acridine compounds, sulprofen, xeroflavone, nonsteroidal anti-inflammatory drugs, methyl pheophytic acid-A-(hexyl ether) and other pheophytic acid, furanocoumarin hydroperoxide, Victoria Blue BO, methylene blue, toluidine blue, porphyrinene compounds and combinations thereof.

[0028] Photosensitizers may be present in PC in any suitable amount. Examples are between about 0.005% wt and about 1% wt, between about 0.01% wt and about 0.1% wt, between about 0.01% and about 0.05% wt, and not exceeding about 1% wt. Total weight percentage (%wt) can also be converted to total weight volume percentage (%w / v) or total volume volume percentage (%v / v). For the purposes of this specification, the concentration of the photosensitizer may be expressed in %wt, %w / v, or %v / v, and such expression of concentration is intended to include its equivalent value (e.g., if expressed in %wt, it is intended to include the equivalent concentration measured in %w / v and %v / v). The term "about" as used in this specification shall refer to + / - 20%.

[0029] The concentration of chlorhexidine used in the PC used in the study was 0.25% by weight chlorhexidine gluconate. Other forms of chlorhexidine, such as chlorhexidine digluconate, chlorhexidine dihydrochloride, chlorhexidine diacetate, etc., are also suitable for PC. Exemplary suitable concentrations are approximately 1% wt; approximately 0.5% v / v; approximately 0.25% wt; approximately 0.125% wt; between approximately 0.125% wt and approximately 1% wt; between approximately 0.125% wt and approximately 0.5% wt; between approximately 0.2% wt and approximately 0.3% wt; between approximately 0.125% wt and approximately 0.3% wt; less than approximately 1% wt but greater than approximately 0.1% wt; less than approximately 0.8% wt but greater than approximately 0.1% wt. Chlorhexidine is preferably provided at a concentration that reduces and / or eliminates potential irritation and sensitivity of the treatment site to the host tissue. When the host tissue in the treatment area is sensitive, such as the nasal mucosa, reducing and / or eliminating this potential irritation and sensitivity is particularly helpful.

[0030] PC may optionally include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is a diluent, adjuvant, excipient, or carrier used to administer other components of the PC (e.g., photosensitizers and chlorhexidine). Pharmaceutically acceptable carriers are preferably approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more particularly in humans. Pharmaceutically acceptable carriers are preferably sterile liquids. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, glucose solutions, glycerol solutions, phosphate-buffered saline solutions, etc.

[0031] Further preferably, when a pharmaceutically acceptable carrier is combined with a photosensitizer and chlorhexidine, it allows the PC to have a sufficiently low viscosity to flow into the targeted therapeutic site, while having a sufficiently high viscosity to retain the PC within the targeted therapeutic site. Other compositions that are expected to become liquid upon application to the targeted therapeutic site, such as compositions that melt or dissolve at the targeted therapeutic site, are also possible. Alternatively, the PC may gel after being applied as a liquid to the therapeutic site, as a non-limiting example, by using an inverse polymer substrate; this would allow the PC to effectively cover the therapeutic site while also retaining the components of the therapeutic site.

[0032] PDT can also include optional cleaning of the user's nostrils, particularly the nasal cavity contained within the upper nostrils. During the study, each uninfected user blew their nose with a tissue. This nostril cleaning process can also be achieved by wiping each nostril with a clean swab. Furthermore, this clean swab can be soaked in a cleaning solution, such as water, saline solution, or the like, before being used for the nostril cleaning process.

[0033] Once the nostrils are cleaned, PDT also includes applying PC by inserting an applicator 10 containing PC 12 into one of the user's nostrils 14 and wiping the nasal treatment area 16 inside the nostril 14, so that PC 12 is evenly and generously deposited on the nasal treatment area 6, such as Figure 1 As shown. Preferably, the applicator 10 is for single use. Depending on the size of the user's nostrils, the applicator 10 is preferably inserted into each nostril 14 at least about 2 to 3 cm deep. The nasal treatment area 16 is defined as the inner surface of the nostril 14, including but not limited to all areas below the nasal turbinate, including the inferior nasal turbinate region. Preferably, particular attention is paid to achieving good coverage in the foremost nasal pouch area (i.e., the tip of the nose) of the nasal treatment area 16. In the user's other nostril ( Figure 1 The PCC application process is repeated (not shown) to deposit PC 12 evenly and in large quantities on the nasal treatment site 16 inside the second nostril.

[0034] After completing the PC application process for both nostrils, PDT also includes a light application process. The light application process involves applying light to each nasal treatment site at a wavelength that can be absorbed by the photosensitizer contained in the PC. This light application process can be achieved using any publicly available suitable method and light source, such as laser diodes, light-emitting diodes, infrared and enhanced pulsed beams, or combinations thereof.

[0035] Before applying light to the nasal treatment site, the PC is optionally in contact with the nasal treatment site for a short period of time, ranging from less than 15 seconds to more than 120 seconds, as a pre-incubation period.

[0036] In one embodiment of PDT, the light application process is performed twice: once for example... Figure 3 and Figure 5The posterior lighting of the nasal treatment area shown is used once. Figure 2 and Figure 4 The anterior illumination of the nasal treatment site 16 is shown. For posterior illumination of the nasal treatment site, PDM involves inserting the tip 18 of a light diffuser directly into the user's nostril 14, as shown. Figure 3 and Figure 5 As shown, a 2-minute illumination cycle is performed by orienting the light diffuser tip 18 toward the back of the user's head to match the primary absorption wavelength of the selected sensitizer. Once this rearward illumination is complete, PDT may optionally include removing the light diffuser tip 18 from the user's nostrils 14 and repeating the PC application process for both nostrils 14 to prepare for forward illumination of the nasal treatment site 16. For forward illumination of the nasal treatment site 16, PDT involves inserting the light diffuser tip 18 into the user's nostrils 14, as... Figure 2 and Figure 4 As shown, a 2-minute illumination cycle is performed by orienting the distal tip of the light diffuser tip 18 forward (away from the face) and inward toward the user's nose to a wavelength matching the dominant absorption wavelength of the selected sensitizer. After completing the anterior illumination, the light diffuser tip 18 is removed from the user's nostril 14. In a second embodiment, PDM involves inserting the light diffuser tip 18 directly into the user's nostril 14, as... Figure 3 and Figure 5 As shown, the method involves orienting the light diffuser tip 18 towards the back of the user's head and irradiating for 2 minutes with a wavelength matching the primary absorption wavelength of the selected sensitizer. The light diffuser tip 18 is then removed, PC is reapplied to the nostrils, and the light diffuser tip 18 is reinserted in the same manner for a second treatment cycle. For this study, the light diffuser tip 18 was powered by a light source capable of producing sufficient light power at wavelengths between 630 nm and 690 nm for the illumination cycle. An example of a suitable light source is a Class 1 laser device consisting of two 700 mW channels. As mentioned above, other suitable light sources can also be used to implement PDT.

[0037] Depending on the photosensitizer concentration and the power of the light-emitting device, applying light to the treatment site may only require a short time, for example, from about 15 seconds to less than about 5 minutes, preferably from about 1 minute to about 3 minutes, and from about 2 minutes to about 4 minutes. The light energy provided per unit area during each light irradiation cycle is preferably about 2 J / cm². 2 Approximately 45 J / cm 2 Within the range, preferably around 18 J / cm 2 Up to approximately 36 J / cm 2 It is preferable to perform multiple light treatments on each treatment site (e.g., about 2 to about 10 times, about 3 to about 5 times, etc.) so that the total cumulative light energy applied to the treatment site is much higher than the light energy provided in each cycle.

[0038] If necessary, PDT can be repeated multiple times (e.g., approximately 2 to approximately 5 times). Preferably, applying light to the treatment site does not cause physiological damage to the treatment site and / or surrounding host tissue.

[0039] Example I

[0040] A single-center, randomized, placebo-controlled, single-blind clinical trial was conducted at a university hospital in northern Spain. Participants were recruited from the University of Navarra's COVID19 Safe Campus project and the Clinica Universidad de Navarra COVID19 Surveillance project. Both projects were established during the 2020 pandemic to actively monitor and limit the spread of the novel virus within university campuses and hospitals following the initial implementation of population lockdowns in Spain. These procedures were based on randomized and contact-tracing individual testing using rapid antigen or PCR tests.

[0041] Inclusion criteria for this study required patients to be 18 years of age or older, to have tested positive for SARS-CoV-2 within 48 hours of diagnosis based on real-time PCR (less than 27 cycles) or rapid antigen testing, and to be asymptomatic or have only mild illness. All concomitant medications were permitted except for angiotensin receptor blockers or immunosuppressants. All patients consented to a painless nasal light therapy procedure or a placebo equivalent.

[0042] Patients with severe comorbidities were excluded. Individuals who reported intolerance to light head insertion due to variations in the size, shape, or anatomy of the mouth and nose, individuals with known allergies to non-colonizing nasal treatment ingredients (including methylene blue or chlorhexidine gluconate), individuals with moderate or severe COVID-19 infection, and individuals unable to attend necessary follow-up were excluded from the study.

[0043] Following inclusion in the trial, participants were randomly assigned by block to either the control or treatment group (1:1) using a computer program. The person performing randomization was also assigned additional tasks such as assessing inclusion / exclusion criteria, obtaining informed consent from patients, and completing baseline and follow-up questionnaires. This person did not receive treatment in either group, nor were any biological samples collected.

[0044] The control group received saline solution, while the sham treatment used a switched-off laser illumination device. Polarized glasses were used to keep patients unaware of the intervention. No blinding was applied to the investigators. Participants in the placebo group followed the same time and periodicity protocol as the intervention group.

[0045] The PDT of this invention uses Steriwave with the CE marking. TMThe nasal light disinfection system (NPS, SW4000, Ondine Biomedical Inc., Vancouver, BC, Canada) is used. In short, the NPS is a Class II medical device comprising a power source (“light source”), a nasal illuminator (NLI) consisting of a disposable, bidirectional, laser-conductive, injection-molded nasal applicator, 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, approved in Canada and Europe for the removal of potentially pathogenic microorganisms from the anterior nasal passages. The locally applied PC binds to components of the microbial cell wall, and red light (wavelengths between 664 nm and 670 nm) is absorbed by the photosensitizer molecules, generating reactive oxygen species (ROS) that cause the destruction of lethal microbial cell walls. Note that the wavelengths of the red light can also be between 630 nm and 690 nm, 650 nm and 680 nm, and 660 nm and 670 nm. Furthermore, light with wavelengths between 500 nm and 800 nm can also be used to implement this invention.

[0046] Nasopharyngeal swabs were performed as baseline examinations before treatment, 3 days after treatment, and 7 days after treatment initiation. NP samples were collected in universal transport medium (UTM, Copan) for immediate RNA extraction. Nucleic acid extraction kits and RT-PCR (Cepheid Xpert Xpress SARS-CoV-2, genes E and N) were used and stored in UTMs for in vitro infectiousness detection in a biosafety level 3 facility. Total nucleic acids extracted from baseline NP swabs were aliquoted for SARS-CoV-2 sequencing.

[0047] The NPS was used by trained healthcare professionals. The procedure was initiated by wiping the patient's anterior nasal cavity, including the nostrils and nasal passages, with a PC. The operator then connected the NLI to a power source and inserted the nasal ends of the NLI into the patient's nostrils. In this protocol, the light source was turned on, and two new additional PCs were used for a 4-minute illumination cycle after each illumination cycle, for a total of 3 cycles to ensure consistent performance. This 4-minute x 3 protocol was repeated on Day 2 and Day 3. Each patient received a total of 36 minutes of treatment, consisting of 9 four-minute cycles. To avoid self-contamination, the NLI cables were labeled "L" for the left nostril and "R" for the right nostril. A new set of NLIs was used daily to prevent self-contamination. Subjects were asked to blow their nose before treatment but were not allowed to receive any local treatment. The placebo control group received saline solution and a closed PDT device for three consecutive days, following the intervention group's protocol.

[0048] The primary outcome of this study was reduced infectivity 3 days after treatment. Prior to recruitment, given evidence of persistence of PCR-positive results due to residual nucleic acid rather than live virus, both techniques were performed on each sample, but in vitro infectivity testing using Vero-E6 cells was preferred over RT-PCR as the endpoint. Secondary outcomes were safety and reduced infectivity at other time points (7 and 14 days after treatment initiation). The biological relevance of this study included analysis of immunogenicity of patients at 10 and 20 weeks post-treatment for nucleocapsid proteins, spike proteins, and total SARS-CoV-2 genome, as well as genomic sequencing of specimens, as described below.

[0049] Patients were asked about symptoms they experienced 24 hours prior to the baseline visit, on day 3, and on day 7. Collected symptoms included sore throat, chills, new or worsening cough, difficulty breathing, chest tightness, fever exceeding 38 degrees Celsius, fatigue, muscle pain, loss of smell, loss of taste, headache, gastrointestinal symptoms, difficulty sleeping, malaise, and nasal congestion. Symptoms were collected using a four-category scale (none, mild, moderate, or severe).

[0050] To assess safety, any immediate and delayed local treatment effects were identified. The severity of these effects, as well as investigator-subjective measurements of their treatment-related likelihood, were also documented. These questions were raised immediately after treatment and in interviews at each subsequent follow-up visit.

[0051] Other variables were also collected, such as gender, age, completion of initial COVID-19 vaccination, booster dose of COVID-19 vaccine, previous COVID-19 infection, weight, height and body temperature, heart rate, systolic and diastolic blood pressure, and vital signs such as blood oxygen saturation.

[0052] Sample size was calculated based on the difference in ΔCt before and after the intervention in preliminary in vitro studies, 4 and 8 minutes after PDT administration to infected samples. A small sample size (6 per group) is required to demonstrate microbial efficacy at 99% power. Given the wide range of ages and comorbidities among participants, it was difficult to hypothesize about their clinical course and viral load decline. We also knew that viral load would spontaneously decline in our study population (i.e., the majority of vaccinated and other healthy individuals). Furthermore, a 10% dropout rate was considered. Taking all this into account, a sample size of 100 patients was recommended, with 50 in each group. Due to the rapid decline of the sixth wave of COVID-19 infections in Spain (December 2021 to February 2022), we were unable to reach the full sample size.

[0053] To compare different quantitative variables between the control and intervention groups, the Mann-Whitney U test was performed on patients who did not conform to normality, and the Student's t-test was performed on patients who did conform to normality. The median, interquartile range, mean, and standard deviation were calculated. The Chi-square test was performed on the qualitative variables. 2 test.

[0054] In the analysis of infectious disease trials, multiple linear regression models were performed, and beta coefficients and their respective 95% confidence intervals (95% CI) were calculated to validate treatment efficacy. To assess the ability of diagnostic tests to reduce transmissibility, multivariate adjusted logistic regression models were performed, and odds ratios (ORs) and their 95% confidence intervals were estimated. Both models were adjusted for factors including: sex, age, number of initial symptoms, COVID-19 vaccine, COVID-19 booster dose, and prior SARS-CoV-2 infection.

[0055] All presented p-values ​​are two-tailed. Prism software (GraphPad, San Diego, CA) and STATA 13.0 were used for statistical analysis.

[0056] In vitro infectivity assay: Confluent Vero-E6 cell monolayers were passaged to confluence in 96-well plates and infected with samples from all patients previously diluted 1:2 with infection medium (minimum essential medium - MEM containing 0.2% BSA, 0.2%, 2mM glutamine, and 20mM Hepes) and incubated at 37°C for 4 hours. After inoculum removal, Eagle's MEM containing 10% fetal bovine serum and antibiotics was added to each well of the infected cells. Uninfected cells served as a negative control. After 72 hours, cells were collected and cultured using Dynabeads... TM MyOne TM Silane beads were lysed, and the number of Severe Acute Respiratory Syndrome Coronavirus-2 genomes was analyzed by RT-PCR (a real-time fluorescent RT-PCR kit for detecting SARS-CoV-2, BGI, ORF1ab genes, and human β-actin). These values ​​are expressed as 2^(-ΔCt)*1000.

[0057] Specific anti-SARS-CoV2 humoral and cellular responses: Blood samples were taken at 10 and 20 weeks after entering the clinical trial to analyze the serological responses of participants.

[0058] Anti-SARS-CoV-2 antibody detection was performed using four different commercial chemiluminescence assays. First, it was performed on the cobase601 platform. The Anti-SARS-CoV-2 S (Roche Diagnostics, Germany) test quantifies total antibodies (IgG + IgM) against the SARS-CoV-2 spike protein receptor-binding domain (RBD). Secondly, it uses... The Anti-SARS-CoV-2 test (Roche Diagnostics, Germany) qualitatively detects total antibodies (IgG+IgM) in the viral nucleocapsid (anti-N). Third, it uses data from COVID-19 infection... The IgG monomer assay (Vircell SL, Spain) detects SARS-CoV-2-specific IgG against the nucleocapsid and spike protein. Fourth, it uses SARS-CoV-2 infection... The IgM+IgA monomer assay (Vircell SL, Spain) detects anti-SARS-CoV-2 IgM+IgG against the nucleocapsid and spike protein. Serum samples were previously inactivated at 56°C for 30 minutes. Interpretation of different immunoassays is as per each manufacturer's recommendations.

[0059] use The SARS-CoV-2 Starter and Extended Sets (QIAGEN, USA) measure cell-mediated SARS-CoV-2 immune responses. The starter kit includes specific peptides from the spike antigen (S1, S2, RBD subdomains) to assess immune responses from CD4 (Ag1 tube) and CD4+CD8 (Ag2 tube) T cells. The extended kit contains additional specific peptides from the entire SARS-CoV-2 genome (S, N, and M domains) to investigate complete, specific CD4 and CD8 T cell-mediated immune responses (Ag3 tube). After inoculation, the tubes are incubated at 37°C for 20–24 hours, followed by... ELISA (QIAGEN, USA) measures the concentration of IFN-γ in plasma. A sample is considered reactive when any tube shows an IFN-γ production higher than 0.15 IU / mL after stimulation.

[0060] Sequencing analysis of SARS-CoV-2 infection variants: Libraries were prepared from swab samples using a commercial kit (COVIDSeq assay, Illumina) and sequenced using NextSeq2000 (Illumina). Analysis was performed using Kraken (Illumina). Phylogenetic analysis was conducted using Nextclade.org.

[0061] Preliminary in vitro study evaluating the effects of photodynamic therapy on SARS-CoV-2: Prior to this study, the efficacy of photodynamic therapy on SARS-CoV-2 survival in vitro was unclear. Therefore, a preliminary in vitro study was conducted at the Biosafety Level 3 (BSL3) facility of the Gene Therapy Department at the Centre for Applied Medicine Research (CIMA) at the University of Navarre. For this purpose, nasopharyngeal swab samples with high viral loads of SARS-CoV-2 from severely ill SARS-CoV-2-infected patients were cultured in fusion Vero-E6 cells, and the supernatant was collected 72 hours post-inoculation. The virus was then filtered and titrated using a Vero-E6 cell monolayer lysis plate assay at a titer of 4.3 x 10⁻⁶ / mL. 7 Plaque-forming units (PFU / ml) were prepared in 20 μL solutions using phosphate-buffered saline (PBS) solution. 4 and 1x10 5 PFU of SARS-CoV-2 viral solution was then mixed with 180 μl of photosensitizer preparation (PF) and added to a small plastic reservoir (nasal tip reservoir) mimicking a human nostril. As a control, the viral sample was mixed with 180 μl of PBS without PF. NLI was then applied to each sample for 0, 4, or 8 minutes, gently shaking the NLI every two minutes to reoxygenate the sample. The NLI was then removed, and each sample was collected and analyzed by RT-PCR to quantify the amount of SARS-CoV-2 genome. Dynabeads was used. TM MyOne TM Viral RNA was extracted from each sample using silane beads (ThermoFisher) and quantified by RT-qPCR using oligonucleotides specific to the SARS-CoV-2 nucleocapsid gene. The results confirmed increased destruction of SARS-CoV-2 viral nucleic acid when treated with 4 to 8 minutes of PDT exposure to 10⁴ or 10⁵ plaque-forming units (PFU). We did not retain these data suggesting in vivo efficacy, but only demonstrate the detrimental effects of PDT on live viral particles.

[0062] Results: During the sixth wave of the COVID-19 pandemic in Spain, 79 patients were screened between December 21, 2021, and February 15, 2022. One patient was excluded due to not meeting the criteria. Of the 78 patients randomly assigned 1:1, two withdrew from the treatment group (one due to local irritation, the other for personal reasons), and one patient in the placebo group (for personal reasons). 75 patients who completed the study were included in the analysis. Therefore, the retention rate was 96.1%. See also Figure 6 .

[0063] The population primarily consisted of young people with mild symptoms, who had not been naturally infected with SARS-CoV-2 (79%), had completed the full vaccination course (i.e., two doses of mRNA-based vaccine) (93%), and were expected to have high viral loads. See Figure 7 Apart from measured blood pressure, no significant differences were observed between the intervention and placebo groups, although the effects and absolute values ​​were considered clinically irrelevant.

[0064] Infectivity was significantly reduced in treated patients from baseline to 3 days post-treatment (p<0.0001), while it was not reduced in placebo patients (p=0.24). See [link to relevant documentation]. Figure 8A From day 3 to day 7, the infectivity of both the placebo group and the treated patients was significantly reduced, although the reduction was greater in the treatment group (p = 0.003) than in the placebo group (p = 0.0089). Figure 8A ).

[0065] Figure 8A -C shows the results of nasopharyngeal swab infectivity assays and RT-PCR. For Figure 8A In the clinical study, recruited participants underwent nasopharyngeal swab examinations at baseline (day 0), day 2, and day 0. Samples were collected in viral transport medium and subsequently used to analyze the infectivity of VeroE6 cells. At 72 hours post-infection, the presence and quantity of SARS-CoV-2 in the supernatant were detected using RT-PCR with the Orf1b gene and human β-actin. (See reference...) Figure 8A Data are expressed as log10 of the δ between the fluorescence threshold cycles of β-actin and Orf1b genes. Wilcoxon tests were used to compare baseline with D2 and D2 with D7 for paired data. Figure 8B Immediately after collection, RT-PCR was performed on the E and N genes of SARS-CoV-2 from nasopharyngeal swab samples. (Refer to...) Figure 8B Data are presented as fluorescence threshold cycles. Wilcoxon tests were used to compare baseline with D2 and D2 with D7. C. At baseline D2 and D7, the percentages of “positive” and “negative” individuals in both groups depended on the chosen cycle threshold.

[0066] In the multiple adjusted linear regression model, a protective effect was observed after 3 days of treatment, with a mean β coefficient of -812 (95% CI -478660–-1.3, p<0.05). A trend was observed after 7 days, with a mean β coefficient of -9.3 (95% CI -269.1–-3.1, pNS). See also Figure 9 The table shows the risk of infection based on PCR detection of ΔCt at 3 and 7 days after the start of treatment.

[0067] During the study, the mean PCR cycle increased in both the treatment and placebo groups, although the treatment group showed a significant difference in RT-PCR cycles for the E and N genes 7 days after treatment initiation. Figure 8B In analyzing the probability of a negative PCR result at 7 days, nasal PDT showed a protective effect against a positive PCR result at 7 days, with odds ratios of 0.16 (95% CI: 0.05–0.52) and 0.15 (95% CI: 0.04–0.58), respectively, at thresholds of 32 or 34 cycles. See also Figure 10 ; Figure 8C .

[0068] Reference Figure 11A -D indicates the results of humoral immunity assays. Patients were followed up for 20 weeks after nasal PDT treatment. Plasma and peripheral CD4 and CD8 T lymphocyte samples were collected at weeks 10 and 20. Figure 11A As shown, the total anti-spike antibody in plasma is detected. The detection method for total anti-nucleocapsid antibody in plasma is as follows. Figure 11B As shown. Figure 11C This shows the change in the median level at 20 weeks compared to 10 weeks. (For example...) Figure 11D The figure shows the percentage change in median antibody levels at 20 weeks relative to 10 weeks. Results of antibody assays and cellular immunoassays are shown in [the figures]. Figure 12 and Figure 13 (See also) Figure 14A -E indicates the results of a cell immunoassay. The Quantiferon assay is used with isolated CD4 (e.g.) Figure 14A (as shown) or a combination of CD4 / CD8 (such as) Figure 14B (As shown) Analyzed specific T cell responses to specific purified peptides targeting the spike antigen (S1, S2, RBD subdomains). T cells from placebo and PDT-treated subjects, or additional specific peptides from the SARS-CoV-2 whole genome (S, N, and M domains), were used to test CD4 and CD8 responses (e.g. Figure 14C (As shown). Figure 14D The change in median IFN in units at 20 weeks is shown compared to the 10-week values ​​for the three assays. The percentage change in median IFN in units at 20 weeks compared to 10 weeks is shown below. Figure 14E As shown.

[0069] Antibody quantification at 10 and 20 weeks post-treatment showed a difference in anti-spike antibody levels between the control group and the PDT group. Figure 11A ) and antinucleocapsid antibodies ( Figure 11B There is no difference in the generation of () Figure 12 The placebo group showed a significant decrease in anti-spike antibody levels at week 20 (p<0.001), but the PDT group did not (p=0.1336). Figure 11AMeasurements of interferon (IFN) synthesis by CD4 T cells after exposure to the SARS-CoV-2 spike antigen showed that, compared with PDT, the placebo group had a lower median IFN unit at 10 and 20 weeks. Figure 13 ; Figure 14A In individuals treated with PDT, the combined response of CD4 and CD8 T cells to the spike protein also trended upward, with a median nearly double that of placebo at 20 weeks (p = 0.0971). Figure 13 ; Figure 14B When CD4 and CD8 cells were exposed to the SARS-CoV-2 whole genome product, increased T cell responses were observed in the PDT group at both 10 and 20 weeks (p = 0.0293) (Table 5). Figure 4 C). At both time points, the control group showed a significant decrease in SARS-CoV-2-specific T-cell immunity over 10 to 20 weeks, while in individuals treated with PDT, SARS-CoV-2-induced interferon production levels were similar over 10 to 20 weeks. Figure 14C ).

[0070] RNA sequencing of SARS-CoV-2 infection was successfully performed in 72 patients (96% of the total population). The results showed that 97.2% of the patients had the Omeprone 21K variant (B.1.1.529 or BA.1), with only two exceptions: one patient had Omeprone 21L (BA.2) and one patient was colonized by the Delta 21J variant.

[0071] When analyzing symptom progression on days 3 and 7 after treatment initiation, the proportion of patients experiencing chest tightness and headache on day 3 was significantly lower in the PDT group compared to the placebo group. See also Figure 15 It provides a list of symptoms of COVID-19 infection detected during clinical trials.

[0072] Regarding the safety of the intervention, 53 mild adverse events were reported among the 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 after the first day of treatment due to severe nasal itching.

[0073] The COVID-19 pandemic has presented enormous challenges to the health and economies of individuals, societies, and nations around the world. Since the end of 2019, several variants of SARS-CoV-2 have caused successive waves of viral outbreaks with different clinical and biological characteristics, ranging from severe COVID-19 infection and death to completely asymptomatic cases.

[0074] In the early stages of SARS-CoV-2 infection, particularly in mildly symptomatic carriers of low-lethal variants such as Omeprón, localized nasal decolonization—the site of viral entry and initial replication—may be associated with suppressing viral transmission in various clinical and non-clinical settings. We believe our study is the first prospective randomized trial to explore the tolerance and associations of nasal decolonization in mildly symptomatic SARS-CoV-2 infected individuals.

[0075] The primary endpoint of the trial (i.e., a reduction in infectivity as measured in vitro from nasopharyngeal swab samples) was achieved. We demonstrate the effect of nasal PDT on other healthy individuals with high viral loads and mild symptoms (Ct < 26 and / or positive antigen detection at recruitment). Treatment consisted of three consecutive days of 12-minute methylene blue PDT.

[0076] Interestingly, although PDT treatment was only applied to the nasal cavity, it reduced SARS-CoV-2 colonization throughout the upper respiratory tract compared to placebo. This is likely due to the fact that the nasal mucosa is a primary site of viral replication in the Omeprone variant and / or under vaccination status. This result reinforces the hypothesis that nasal decolonization is associated with disease that can progress to a systemic stage within days. The effect of PDT was observed in vitro using NP swabs for infectivity assays 3 days after treatment. Although considered a less reliable reading, the RT-PCR cycling in NP samples from the treatment group was higher at 7 days, further indicating the durable effect of nasal decolonization. Therefore, PDT treatment plays an important protective role in avoiding PCR positivity within 7 days. No serious adverse events occurred during treatment.

[0077] We observed a sharp decline in viral load across our entire population (placebo and PDT) on days 3 and 7, which may have suppressed the extent of the effect of PDT. One possible explanation is that the majority of our population was vaccinated (93%): in fact, vaccination is associated with a faster decline in viral RNA in patients infected with SARS-CoV-2. On the other hand, almost all patients were primarily infected with the Omeprone variant (21K, BA.1) (97.2%), which exhibits favorable biological characteristics compared to other variants. The low-invasive variants may have lower persistence in the upper respiratory tract compared to α or δ variants. These two facts—the high proportion of vaccinated individuals and the rapid decline in viral load—highlight the efficacy of PDT in eliminating infectious viral particles, with the treatment group showing a significantly higher attenuation after three days of treatment compared to the placebo group.

[0078] Importantly and unexpectedly, we found that PDT treatment had a profound and durable effect on SARS-CoV-2-specific T lymphocyte-mediated immunity. We found that in individuals receiving nasal PDT, CD4 and CD8 T lymphocytes produced nearly twice the median IFN units. Most importantly, while the placebo group showed significant attenuation between weeks 10 and 20 post-treatment, all assessed T-cell responses persisted after PDT treatment. We consider this the first evidence that PDT enhances immunity against SARS-CoV-2.

[0079] The sustained effects of PDT on immunity offer us novel immunization strategies. For example, we can use combinations of photosensitizers and antigens to induce photochemical internalization of antigens in antigen-presenting cells (APCs), which facilitates their adjuvant-free crosspriming to CD8 T lymphocytes.

[0080] This invention also includes topical PDT to reduce viral load in patients with early or mildly symptomatic viral infections caused by non-SARS-CoV-2 (i.e., those caused by other viruses). This invention also includes topical PDT to reduce bacterial load in patients with early or mildly symptomatic bacterial infections. Finally, this invention includes topical PDT to reduce fungal load in patients with early or mildly symptomatic fungal infections.

[0081] The local PDT treatment discussed in this application can be applied to anyone.

[0082] The explanations and illustrations provided herein are intended to familiarize those skilled in the art with the invention, its principles, and its practical applications. Those skilled in the art can adapt and apply the invention in various forms, which may best suit the requirements of a particular application. Therefore, the specific embodiments of the invention set forth are not intended to be exhaustive or limiting. Consequently, the scope of the invention should not be determined by reference to the foregoing description, but rather by reference to the appended claims and the full scope of their equivalents. All disclosures in articles and references, including patent applications and publications, are incorporated herein by reference for all purposes.

Claims

1. Use of a photosensitive molecule in the preparation of a topical medicament for photodynamic therapy to shorten the infection period of an infection caused by a targeted microorganism, wherein the topical medicament is applied to a targeted body region and light is applied to the targeted body site.

2. Use of the photosensitive molecule according to claim 1, wherein the targeted body region is the anterior nasal cavity.

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

4. Use of the photosensitive molecule according to any one of the preceding claims, wherein the photosensitive molecule is a thiophene.

5. Use of the photosensitive molecule according to any one of the preceding claims, wherein the photosensitive molecule is methylene blue.

6. Use of the photosensitizer according to any one of the preceding claims, wherein the concentration of the photosensitizer does not exceed about 1% wt of the topical drug.

7. Use of the photosensitive molecule according to any one of the preceding claims, wherein the topical drug further comprises chlorhexidine at a concentration not exceeding about 1% wt.

8. Use of photosensitizing molecules in topical pharmaceutical compositions that induce sustained humoral and cellular T-cell responses against such pathogenic microbial antigens.

9. Use of the photosensitive molecule according to claim 8, wherein the targeted body region is the anterior nasal cavity.

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

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

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

13. Use of the photosensitizer according to any one of claims 8 to 12, wherein the concentration of the photosensitizer does not exceed about 1% wt of the topical drug.

14. Use of the photosensitive molecule according to any one of claims 8 to 13, wherein the topical drug further comprises chlorhexidine at a concentration not exceeding about 1% wt.

15. Use of the photosensitive molecule according to any one of claims 8 to 14, wherein the photosensitizer is methylene blue at a concentration of 0.01% wt of the photosensitive composition, and the concentration of chlorhexidine is 0.25% wt of the photosensitive composition.

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