WO2021167609A1

WO2021167609A1 - Method for treating and preventing coronavirus (covid-19) infection by direct pulmonary administration of a selected microorganism

Info

Publication number: WO2021167609A1
Application number: PCT/US2020/018951
Authority: WO
Inventors: Xin Rui XIA
Original assignee: Xia Xin Rui
Priority date: 2020-02-18
Filing date: 2020-02-20
Publication date: 2021-08-26

Abstract

A new use of a selected microorganism is disclosed. The invention relates to a method for treating and preventing Coronavirus (COVID-19) infection in humans by direct pulmonary administration of an effective amount of a selected microorganism to the humans. The microorganism will stimulate the localized lung immune system, and produce antimicrobial and antivirus substances against COVID-19 infection, which is a new strategy for treating and preventing current world-wide epidemic COVID-19 infection. This method is simple, cost effective and using a commercially available strain that can be used to treat and prevent COVID- 19 infection for large populations.

Description

SPECIFICATIONS

FIELD OF THE INVENTION

The present invention relates to a means of treating and preventing respiratory infections in humans, and, more particularly, to a method for treating and preventing Coronavirus (COVID-19) infection in humans by direct pulmonary administration of an effective amount of probiotic Bacillus subtilis spores to the humans.

BACKGROUND OF THE INVENTION

The outbreak of Coronavirus (COVID-19) is spreading, claimed thousands of lives. The ever-spreading epidemic disaster, particularly in China, cause world-wide panic and enormous economic loss. There is no effective treatment method except supportive medications and depending on the immune system of the patients.

This invention presents a method for treating and preventing Coronavirus (COVID-19) infection in humans by direct pulmonary administration of an effective amount of probiotic Bacillus subtilis spores to the humans.

Bacillus subtilis is a Gram-positive, rod-shaped bacterium (4-10 micrometers (pm) long and 0.25-1.0 pm in diameter). It can form an endospore (1.4pm in diameter), to survive extreme chemical, physical and mechanical conditions [1, 2]. Bacillus subtilis spores have been widely used as probiotics for gut health of animals including humans. Probiotics are live microorganisms that confer health benefits to the host when administered in adequate amounts [1,3]. After survival of Bacillus subtilis spores from stomach acid, the spores rapidly germinate and become active bacteria in the warm, moist and nutritious small intestine and gut environment. The exponential proliferation of the vegetative cells of Bacillus subtilis will produce its probiotic health benefits.

Several health benefits of oral probiotic supplement of bacillus subtilis spores have been reported [4-7]: (i) Competitive exclusion to expel pathogenic microbials out of the Gl track; (ii) Immunostimulation to activate the innate immune system of the host to produce immunoglobulin G (IgG), macrophage and development of a gut-associated lymphoid system (GALT); (iii) Antiviral and antimicrobial activity, during proliferation of Bacillus subtilis, several active substances such as subtilisin, polymyxin, bacteriocin, gramicidin, etc. are produced, which are active against pathogenic bacteria or endogenous Infected conditional pathogens.

Huang et al [7] studied the immunostimulatory activity of Bacillus spores. Bacillus species, typically Bacillus subtilis, are being used as probiotics and mounting evidence indicates that Bacillus species are important for development of a robust gut-associated lymphoid system (GALT). A number of gut isolates of Bacillus incorporating three species, B. subtilis, Bacillus licheniformis and Bacillus flexus were used to evaluate the nature of interaction between spores and the GALT. In mice orally administered with spores, evidence of cell proliferation was determined in the germinal centers of Peyer's patches. Stimulation of antigen presenting cells and T lymphocytes was also markedly enhanced. Cytokines were shown to be induced in spleens and mesenteric lymph nodes of mice including the proinflammatory cytokines, tumour necrosis factor-a and IL-6.

Lefevre et al [8] reported that Bacillus subtilis probiotic oral consumption reduced the incidence of upper respiratory infection in elderly subjects in a newly published clinical trial. One hundred subjects aged 60-74 were included in this randomized, double-blind, placebo-controlled, parallel-arms study. Subjects consumed either the placebo or the probiotic (2xl0⁹ B. subtilis CU1 spores daily) by short periodical courses of 10 days intermittently, alternating 18-day course of break. This scheme was repeated 4 times during the study. Symptoms of gastrointestinal and upper/lower respiratory tract infections were recorded daily by the subjects throughout the study (4 months). Blood, saliva and stool samples were collected in a predefined subset of the first forty-four subjects enrolled in the study. Consumption of B. subtilis CU1 significantly increased fecal and salivary secretory IgA concentrations compared to the placebo. A post-hoc analysis on this subset showed a decreased frequency of respiratory infections in the probiotic group compared to the placebo group. This study provides evidence that B. subtilis CU1 supplementation during the winter period may be a safe effective way to stimulate immune responses in elderly subjects.

Marseglia et al [9] reported the efficacy of Bacillus clausii spores in the prevention of recurrent respiratory infections in children in a clinical pilot study. This pilot study was aimed to assess the efficacy and the safety of B month treatment with Bacillus clausii in the prevention of recurrent respiratory infections (RRI) in children. Eighty children with RRI were studied where 40 of them were randomly treated with Bacillus clausii for 3 months, and followed up for further 3 months; 40 were included in the control group during the same period. Children treated with B. clausii had shorter duration of Rl in comparison with the control group both during the treatment phase (mean 11.7 days vs 14.37; p=0.037) and the follow-up period (mean 6.6 days vs 10.92; p=0.049). This effect was evident also in allergic children during the follow-up. In conclusion, this pilot study provides the first preliminary evidence that B. clausii may exert a significant and persistent impact on Rl in children and is safe and well tolerated.

There are very limited reports on use of Bucillus subtilis spores in respiratory system. Yang et al [10] studied the effects of intranasal administration with Bacillus subtilis on immune cells in the nasal mucosa and tonsils of swine, which has similar respiratory immune system with humans. The study demonstrated that B. subtilis was able to reach the lamina propria of the nasal mucosa, nasopharyngeal tonsils and soft palate tonsils 2 hours after intranasal administration for Bacillus subtilis spore aqueous suspension. The number of dendritic cells, immunoglobulin A+ B cells and T cells in the nasal mucosa and tonsils significantly increased (P<0.05) following administration with B. subtilis. No obvious differences were observed in the morphological structure following B. subtilis administration. It suggested a further study into the intranasal administration of B. subtilis in humans to enhance the immunity of human nasal mucosa to respiratory diseases.

Hong et al [11] reported that Alveolar macrophages treated with Bacillus subtilis spore protect mice infected with Respiratory Syncytial Virus (RSV), which is a major pathogen that infects lower respiratory tract and causes a common respiratory disease. Despite serious pathological consequences with this virus, effective treatments for controlling RSV infection remain unsolved, along with poor innate immune responses induced at the initial stage of RSV infection (Figure 1). Alveolar macrophage (AM) is one of the primary innate immune cell types in the respiratory tract and may contribute to protective responses against RSV infection. As an effective strategy for enhancing anti-viral function of AM, the study suggests the intranasal administration of Bacillus subtilis spore which induces expansion of AM in the lung with activation and enhanced production of inflammatory cytokines along with several genes associated with Ml macrophage differentiation. Such effect by spore on AM was largely dependent on TLR-MyD88 signaling and, most importantly, resulted in a profound reduction of viral titers and pathological lung injury upon RSV infection. The protective role of AM in RSV infection and its functional modulation by B. subtilis spore may be a useful and potential therapeutic approach against RSV.

The present inventor believe that The health benefits from oral probiotics of Bacillus subtills spores to senor and children against respiratory virus infection, could be applied to alleviate the current Coronavirus infection, particularly to those with gut disorder symptoms. However, the health benefits from oral probiotics is not enough to against the severity of localized lung infection from current Coronavirus. Therefore, this inventor proposes to directly administrate the probiotic Bacillus subtilis spores to the respiratory system. To date, there is no clinical study report to intentionally, direct administrate probiotics to the lung yet. Even though recently Noci et al (2018) has demonstrated an antibiotic or probiotic aerosol therapy in modulation of the pulmonary microbiota and promote immunosurveillance against lung metastases [12]. The present inventor has developed a method to treat mouth and sinus candidiasis for himself using an aerosol of probiotic Bacillus subtills spores, the health benefit of which is the base for this invention conceived.

It is known that the lung and Gl tract have distinct anatomic and biological features and functions, but these two organs share the same embryological origin and both contain mucosa-lined luminal surfaces, it is where microbial community inhabits. Until very recently, it was believed that the lungs were sterile or free from bacteria [13,14] After the first report of lung microbiome in healthy subjects in 2010 by Hilty [15], extensive studies have been made to realize that a complex variety of microorganisms inhabit on the mucous layer and the epithelial surfaces in the lung, including bacteria, fungi, viruses and bacteriophages [12-19]

The new knowledge of lung microbiome justifies the therapeutic strategy of present invention. A new concept of "Lung-probiotics" is introduced in present invention. The knowledge of health benefits from oral probiotics could be conferred to the respiratory system with pulmonary administration of Lung-probiotics because the lung microbial microbiome lives on the lung mucosa-lined luminal surfaces similar to that harboring gut microbiome in microscopic views (Figure 2). The localized health benefits of Lung-probiotics (immune- stimaulation, antiviral and antimicrobial and competitive exclusion) could be a new strategy to treat or prevent the current epidemic COVID19 infection.

Bacillus subtilis spores are selected as the first Lung-probiotics because it is safe and non-pathogenic, particularly, it has good physical and mechanical properties that allow its pulmonary administration as a micro-mist or aerosol without jeopardize it integrity [1,4]. Moreover, Bacillus probiotics are the only aerobic bacteria could proliferate at lung mucosa-layers with a pH value around 6.6 [1,18,19] Most other commonly used oral probiotics, such as Lactobacillus and Bifidobacteria species are lactic acid producing, anaerobic can only be used for gut health [1,21]. In fact, not every Bacillus spp. can be used as probiotics, some species are well known pathogens, for example, Bacillus anthracis causing cutaneous anthrax and inhalation anthrax diseases; Bacillus cereus are causing food poisoning [1,22,23] Therefore, the selection of a correct strain for Lung-probiotic is of critical importance to clearly understand the phenotypic and genotypic characteristics. It is better to choose a strain having a history of safe use for human and having GRAS status (Generally Recognized as Safe by USFDA). Bacillus Subtilis Spores is one of Bacillus probiotics obtained GRAS status [1,24,25]

To the inventor's knowledge, this is the first invention to intentionally, direct pulmonary administration of probiotic Bacillus subtilis spores to the humans. The direct Lung-probiotic administration could be an effective method to treat and prevent world-wide epidemic of COVID-19 infection by localized immunostimulation, antimicrobial and antiviral activity of the probiotic strain.

DETAIL DESCRIPTIONS

Embedment 1. Micro-mist pulmonary administration

(1) Weigh 2.32 gram of probiotic Bacillus subtilis spores containing about (30 xlO⁹ cfu) into a beaker (Figure 3A).

(2) Add 300mL drinking water at ambient temperature (sterilized by autoclave or boiling for lOmin).

(3) Stir spore powder to suspend the spores uniformly in the water (Figure 3B). This prepared aqueous suspension has a spore concentration of 1 xlO⁸ cfu/mL.

(4) Transfer the spore suspension to a sprayer bottle. (5) Administrate the micro-mist of the spore suspension by using a micro-mist nebulizer (Figure 4) by deep breathing the micro-mist into the lung through mouth and nose alternatively (so the entire respiratory system is treated).

(6) For a target delivery amount of lxlO⁹ cfu, 10 mL of the 1 x 10^s cfu/mL spore suspension is needed.

(7) Multiple administrations can be made daily depending on the health conditions and tolerance response of the humans. A preferred embedment is once a day.

Note: The pulmonary administration using micro-mist nebulizer is not quantitative. In fact, the depth of delivery in the respiratory track is governed by the particle size of the spore micro-mist. The smaller the particle size, the deeper will be delivered into the respiratory track as shown in Figure 4. Thus, the performance of the micro-mist nebulizer is critical for pulmonary administration. Fortunately, our lung is bidirectional that we breath in and out, in which micro spore particles can be fairly delivered to the lung. Moreover, it will multiply once every BOmin after rapid germination in the warm, moist, and nutritious environment of our lung.

Figure 4 and 5 shows two types of micro-mist nebulizers, in fact, any micro-mist nebulizer for liquid suspension pulmonary drug delivery can be used.

Embedment 2. Micro-aerosol pulmonary administration

Pulmonary administration of the probiotic bacillus subtilis spores can also be achieved by using micro aerosol inhalers in dry powder of the spores as shown in Figure 6.

Embedment 3. Mass pulmonary administration

The micro-mist of aqueous spore suspension and aerosol of spore micro-powder can be generated by using commercial equipment as shown in Figure 7, which can be used for pulmonary administration of probiotic Bacillus subtilis spores to large number of peoples for treatment and prevention from COVID-19 infection. A therapeutic dose of Bacillus subtilis spore could be achieved by deep breathing in the spore mist for a given amount of time, for example 10 min. The feasibility of this embedment is because the Bacillus subtilis spores can be cost effective produced in large quantity; it is safe non-pathogenic to humans or animal. It is also environmental-friendly. In fact, there are plenty of the bacteria in soil, on grass or plants.

Figure 7 is only an illustration of a powerful vibration mesh nebulizer having multiple nebulizer heads connected by plastic tubes. In fact, each of the nebulizer heads should be placed in an isolated compartment to prevent cross infection between patients during treatment.

Precautions and warnings for application of this invention

Warning 1: Application of present invention of Lung-probiotics must follow current well-developed medical protocols. Unknow outcomes could be encountered, particular, the correct strain of probiotics and the product purity and quality and many unseen factors could jeopardize the outcome. Even though gut probiotics have become a routine world-wide use, Lung-probiotic is a new application. Doctors, medical experts and microbiome scientists must be involved for effective application of present invention. Warning 2: After treating with this invention, the infectious ability will not be eliminated immediately. More important, the infectious ability still exist even though the innate immune system is activated and the individual feel cured. Well-developed quarantine and protection protocols must be followed.

Warning 3: The applicability of this invention to life threatening patients must be closely monitored because Bacillus subtilis bacteria could enter the circulation system through damaged epithelial layer of the respiratory track. Antibiotics to systemic infection of Bacillus subtilis bacteria must be ready; although this has never been reported in oral probiotic use for centuries.

Warning 4: The effectiveness and side effects must be tested using pigs for any new administration protocols and new batch production of Lung-probiotic Bacillus subtilis spores. Pigs must be pathogen free in order to obtain clinical outcomes for human references since regular pig may already have Bacillus subtilis in their respiratory system from their close contact to the soil.

REFERENCES

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Claims

What is claimed is:

1. A method for treating and preventing COVID-19 Coronavirus infection in humans by direct pulmonary administration of an effective amount of probiotic Bacillus subtilis spores to the humans.

2. The method of claim 1, in which the probiotic Bacillus subtilis spores can be suspended in water and administrated pulmonary via nasal or mouth in a form of micro-mist generated from the Bacillus subtilis spores suspension by using commercial available micro-mist nebulizers.

3. The method of claim 1, in which the probiotic Bacillus subtilis spores can also be pulmonary administrated via nasal or mouth in an aerosol form generated by using a commercially available micro particular powder nebulizer.

4. The method of claim 1, in which a powerful commercial aerosol nebulizer with multiple spray nozzles can be used for pulmonary administration for multiple patients simultaneously.

5. The method of claim 1, in which the effect amount of probiotic spores is in a range of 1 - lxl015cfu. A preferred embedment is 1x109 cfu at a spore concentration of 1x108 cfu/mL.

6. Bacillus subtilis spores is selected as the first Lung-probiotics for treating and preventing current epidemic Coronavirus (COVID-19) infection. Other aerobic Bacillus strains of well- known oral probiotics, such as, Bacillus clausii, Bacillus licheniformis, Bacillus coagulans etc. can also be used as Lung-probiotics to provide health benefits for a broad-spectrum of respiratory diseases, such as common flu, RSV, RRL, Pneumonia, Asthma, Respiratory Tract Candidiasis, Tuberculosis etc., therefore claimed in this invention.