Atlantia is a world-class provider of human clinical studies. For over a decade we have delivered clinical results to our many clients around the world, on time and on budget. We strive to offer the highest quality science while maintaining a cost-conscious approach to maximize the value proposition for each of our clients.
Clinical Trial of a Probiotic and Herbal Supplement for Lung Health
Nancy M. Wenger 1 † , Luhua Qiao 1 † , Teodora Nicola 1 , Zoha Nizami 1 , Isaac Martin 1 , Brian A. 1 Halloran 1 , Kosuke Tanaka 1 , Michael Evans 1 , Xin Xu 2,3 , Timothy G. Dinan 4 , Charles Kakilla 4 , 2 Gillian DunnGalvin 4 , Namasivayam Ambalavanan 1 , Kent A. Willis 1 , Amit Gaggar 2,3 , 3 Charitharth Vivek Lal 1* 4 1 Division of Neonatology, Department of Pediatrics, University of Alabama at Birmingham, AL 5 2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Alabama 6 at Birmingham, AL 7 3 Program in Protease and Matrix Biology, University of Alabama at Birmingham, AL 8 4 Atlantia Clinical Trials, Cork, IRE 9
† These authors contributed equally to this work and share first authorship. 10
11 12 13
* Correspondence:
Charitharth Vivek Lal MD, FAAP
clal@uabmc.edu
Keywords: microbiome, gut-lung axis, probiotic, Lactobacillus, short-chain fatty acids, asthma 14
15
Abstract
Introduction: Dysbiosis of the gut microbiome may augment lung disease via the gut-lung axis. 16 Proteobacteria may contribute to tissue proteolysis followed by neutrophil recruitment, lung tissue 17 injury, and perpetuation of chronic inflammation. To study the effects of probiotics across the gut- 18 lung axis, we sought to determine if a Lactobacillus probiotic and herbal blend was safe and well- 19 tolerated in healthy volunteers and asthmatic patients. 20 Methods: We conducted a 1-month randomized, open-label clinical trial in Cork, Ireland with 21 healthy and asthmatic patients who took the blend twice a day. The primary endpoint was safety with 22 exploratory endpoints including quality of life, lung function, gut microbiome ecology, and 23 inflammatory biomarkers. 24 Results: All subjects tolerated the blend without adverse events. Asthmatic subjects who took the 25 blend showed significant improvements in lung function as measured by forced expiratory volume 26 and serum short chain fatty acid levels from baseline to Week 4. The gut microbiome of asthmatic 27 subjects differed significantly from controls, with the most prominent difference in the relative 28 abundance of the proteobacteria Escherichia coli . Administration of the probiotic maintained overall 29 microbial community architecture with the only significant difference being an increase in absolute 30 abundance of the probiotic strains measured by strain-specific PCR. 31 Conclusions: This study supports the safety and efficacy potential of a Lactobacillus probiotic plus 32 herbal blend to act on the gut-lung axis. However, due to the lack of a control group, a longer 33 blinded, placebo-controlled study will be warranted to confirm the efficacy improvements observed 34 in this trial. 35
36
ClinicalTrials.gov Identifier: NCT05173168
37
1
Introduction
The gut-lung axis serves as a powerful means of communication between the microbiome of the gut 38 and the inflammatory and immune microenvironment of the lungs. Patients with respiratory diseases 39 often have gastrointestinal symptoms and show distinct imbalanced gut microbiomes compared to 40 healthy individuals (1-5). Inhaled exposure to environmental toxins such as cigarette smoke and 41 pollution can decrease bacterial diversity and increase inflammation in the gut (6, 7). As such, the 42 state of the intestinal microbiota is deeply connected to lung health and vice versa. 43 Supplementation with probiotic lactic-acid-producing bacteria has been widely studied. Live 44 Lactobacillus strains have been individually clinically studied for supporting gut and lung health in 45 improving and preventing infections (8, 9). Commensal species L. rhamnosus , L. plantarum , and L. 46 acidophilus support maintaining proper uptake of short chain fatty acids (SCFAs) in both healthy and 47 diseased populations (10-12). SCFAs and other metabolites produced by commensal bacteria travel 48 through systemic circulation and mediate inflammatory and immune responses in distal organs (13). 49 In a double-blind, randomized controlled trial of asthmatic patients, a Lactobacillus blend taken once 50 a day for 8 weeks showed immunomodulatory effects via improvement in Th2 cells-associated IL-4 51 and lung function via forced expiratory volume and forced vital capacity (14). Other clinical studies 52 of probiotics, primarily conducted in children, show improvements in asthma severity, allergic 53 response, and immune biomarkers (15-18). Adequate SCFA production is also associated with 54 reduced allergic airway inflammation (19, 20). 55 Bioactive compounds in herbal extracts can further support the anti-inflammatory action of live 56 probiotic strains. Herbal extracts of holy basil leaf, turmeric root, and vasaka leaf can be taken orally 57 to support antioxidant, antitussive, anti-inflammatory, and bronchodilatory effects in the lungs and 58 systemically (21-23). A blend of live Lactobacillus strains with holy basil, turmeric, and vasaka 59 extracts, decreased matrix metalloproteinase 9 (MMP-9) pathway activity, neutrophil recruitment, 60 and pro-inflammatory biomarkers in preclinical in vitro and in vivo models of lung inflammation 61 (24). 62 Our group has focused on attenuating inflammation and supporting lung function through the gut- 63 lung axis using oral probiotic supplementation. In this clinical study, we hypothesized that a 64 preclinically studied (24) Lactobacillus probiotic and herbal blend would demonstrate safety and 65 preliminary biomarker and clinical improvements in healthy and asthmatic populations in a 1-month 66 course of dosing. 67
68
2
Materials and Methods
69
2.1
Trial Design
The study was conducted in accordance with the Declaration of Helsinki and approved by the 70 Clinical Research Ethics Committee of the Cork Teaching Hospitals of University College Cork 71 (ECM 4 (n) 7/9/2021& ECM 5 (7) 10/26/2021 & ECM 3 (lll) 11/16/2021). 72 This study was an open-label, exploratory pilot study to assess the safety of 4 weeks of dosing a 73 probiotic and herbal blend (resB Lung Support, ResBiotic Nutrition) in healthy and asthmatic 74 participants. Forty participants were screened to identify 22 eligible subjects. The study population 75 consisted of n=11 healthy participants and n=11 participants diagnosed with asthma. Within these 76
2
This is a provisional file, not the final typeset article
populations, active smoking status was noted (healthy smokers n=3, asthmatic smokers n=4). 77 Informed consent was obtained from all subjects involved in the study. First patient first visit was 78 November 23, 2021, and last patient last visit was January 20, 2022. 79 The study protocol consisted of four onsite visits over a 6-week period. Participants were pre- 80 screened with an online questionnaire and invited for an onsite screening visit (Visit 1) to confirm 81 their eligibility. A urine drug test was conducted and a urine test for pregnancy was performed for 82 individuals of childbearing potential. No changes were made to the methods after the trial started. 83
84
2.1.1 Participants
Male and female participants ages 18-65 were recruited who were either in general good health at the 85 discretion of the investigator or had asthma and were on stable medication for at least 3 months. 86 Exclusion criteria included pregnancy, acute or chronic illness which by the investigator’s judgment 87 precluded them from participating in the study, 2 hospital admissions in the past 6 months, and use 88 of antibiotics, probiotics, immunosuppressive medications, or oral steroids (>10 mg/day) for >3 days 89 in the previous 12 weeks. Participants were also excluded if they had made any major dietary 90 changes or changed medications or supplements in the 30 days prior to enrollment. 91
Participants were enrolled and conducted site visits at a single site: Atlantia Food Trials, Heron 92 House Office, Blackpool Retail Park, in Cork, Ireland. 93
94
2.1.2 Interventions
All 22 participants took the supplement twice daily for 4 weeks (28 days). One capsule contains 95 8.25x10 9 CFU Lactobacillus plantarum RSB11® , 7.9x10 9 CFU Lactobacillus acidophilus RSB12® , 96 6.4x10 9 CFU Lactobacillus rhamnosus RSB13® , 48.0 mg vasaka ( Adhatoda vasica root) extract, 97 42.0 mg holy basil ( Ocimum sanctum leaf) extract, and 30.0 mg turmeric ( Curcuma longa root) 98 extract. Total CFU count at the time of the last patient out was 2.6x10 10 CFU/capsule. At Visit 1, 99 participants were provided with a 30-day supply plus two days of overage. Study product was stored 100 refrigerated on-site, and participants were instructed to store it refrigerated at home as well. 101 Supplementation started after completion of Visit 2, where participants were instructed to consume 102 one capsule twice daily with food and water at home. At Visits 3 and 4, participants returned all 103 unused investigational product so that compliance could be calculated. Participants had to consume at 104 least 75% (45 capsules) of their supply to be deemed compliant. 105
106
2.1.3 Outcomes
The primary objective of this trial was to measure safety of the blend in healthy and asthmatic study 107 participants. Safety was measured by number of participants experiencing at least one adverse event 108 (AE); number of AEs including causality, severity, and seriousness assessments; number of 109 participants with discontinuations due to AEs; change in systolic blood pressure, diastolic blood 110 pressure, heart rate, and body temperature from baseline to Week 2 and Week 4; change in blood 111 safety parameters via serum chemistry profile and hematology profile from baseline to Week 2 and 112 Week 4. 113 Exploratory objectives for this trial included: change in gut microbiota (16s) sequencing from 114 baseline to Week 4; change in lung function measured by spirometry (Forced Expired Volume in 1 115 second (FEV1) and Forced Vital Capacity (FVC)) from baseline to Week 4; change in oxygen levels 116 (% pulse oxygen levels) from baseline to Week 4; change in SGRQ score from baseline to Week 4. 117
3
At Baseline and Week 4, participants had blood drawn for biomarker analysis, spirometry measured, 118 and completed the Saint George’s Respiratory Questionnaire (SGRQ). Participants supplied a stool 119 sample at Visit 2 and Visit 4. Vitals, safety blood parameters, and adverse events/severe adverse 120 events (AE/SAE) were monitored at each visit. Participants completed an end-of-study product 121 questionnaire at Visit 4. There were no changes to the study protocol after the study initiated. 122
123
2.1.4 Blinding
This was an open label study where study personnel and the participants were aware of the product 124 they were taking; blinding did not apply. 125
126
2.1.5 Statistical Methods
The primary method of analysis was descriptive statistics. Summary statistics for continuous 127 measures were provided for the actual measurements at each visit and change from baseline to each 128 visit. These tables included sample size, minimum and maximum statistics, mean, median, quartiles, 129 and standard deviations. Key variables were categorized into clinical ranges. In the summary tables, 130 counts and percentages were used in the frequency tables. 131 Inferential statistics were run to provide information on the trends within the data. D’Agostino & 132 Pearson test of normality was run to determine if the data was normally distributed. Post-hoc paired 133 t-tests were conducted to assess within group changes from baseline to Week 4 in the healthy and 134 asthmatic populations separately. Participants were further segmented into smokers vs. non-smokers 135 in assessing the changes from baseline to Week 4 in serum SCFA using paired t-tests. All analyses 136 requiring significance testing were two-sided at a 5% significance level. Results were viewed as 137 statistically significant if the p-value was less than or equal to 0.05. 138
139
2.2
Microbiome Amplicon Sequencing and Analysis
Microbial DNA was extracted from human stool samples and 16S amplicon sequencing was 140 performed using the Illumina MiSeq platform at the University of Alabama at Birmingham 141 Microbiome Resource Core Facility under the direction of Dr. Casey Morrow. Sequencing data 142 quality control, alignment and demultiplexing were perform by the using a custom script built using 143 MOTHUR and QIIME 2 with amplicon sequence variants (ASVs) identified using SILVA. 144 Processed ASV tables were imported into MicrobiomeAnalyst for further analysis and data 145 visualization (25, 26). ASVs that appeared in less than two samples, were prevalent in 10% of 146 samples and less than 5% of the inter quartile range were removed, leading to the removal of 504 low 147 abundance features. Cumulative sum scaling was performed but not rarefication or transformation. 148 Alpha diversity was quantified with the Shannon and Chao1 Indices. We visualized beta diversity 149 with principal coordinates analysis of Bray-Curtis dissimilarity matrices and performed significance 150 testing using permutational multivariate analysis of variance (PERMANOVA) and permutational 151 multivariate analysis of dispersion (PERMDISP). Feature selection was performed using 152 MetagenomeSeq and DESeq2. 153
154
2.3
Qualitative PCR
DNA was re-extracted from same fecal material used in MiSeq with Zymobiomics DNA Mini-Prep 155 kit (Zymo Research). PCR (Qiagen Fast Cycling PCR kit) was performed on samples with at least 156 3ng/uL total DNA by pico green quantification (Quant-It dsDNA Assay Kit, ThermoFisher). A 157 common 16S gene-based forward primer was utilized with strain-specific reverse primers at a similar 158 site (0.4uM), in a 25uL reaction with 30ng DNA template, using SYBR Green dye (LTi) to monitor 159
4
This is a provisional file, not the final typeset article
in real time. Analysis of PCR reactions was performed on 2% agarose gels. Densitometry (% and ng 160 values) was calculated from the imaged PCR gels using a GS-900 densitometer and Image Lab 161 software (Version 6.1, Bio-Rad). Bands were gel extracted (Qiagen Qiaquick Gel Extaction Kit) and 162 sequenced to confirm species identity. ZymoBiomics Gut Microbiome Standard (negative control) 163 was used to demonstrate lack of nonspecific amplification. 164
165
2.4
Biomarker Analysis
Serum SCFA levels were measured by GC-MS at Creative Proteomics (Shirley, NY). The following 166 SCFAs were analyzed: acetic acid (C2:0), propionic acid (C3:0), butyric acid (C4:0), isobutyric acid 167 (C4:0i), valeric acid (C5:0); isovaleric acid (C5:0i), hexanoic acid (C6:0). Free short chain fatty acids 168 were derivatized using methyl chloroformate in 1-propanol yielding propyl esters before subsequent 169 liquid-liquid extraction into hexane and analysis on a SLB- 5ms (30x0.25x1.0μm) (Supelco) column 170 and detection using GC-EI-MS in SIM-mode. Instrumental analysis was performed on an Agilent 171 7890 GC coupled to an Agilent 5977 MSD (Agilent Technologies). Quantification was performed 172 against a 5-point calibration curve. 173
174
3
Results
3.1 Oral supplementation with probiotic blend is safe for human consumption 175
Forty total participants were screened to randomize 22 participants with asthma (n=11) or were 176 healthy (n=11) who met study eligibility criteria (Table 1, Figure 1). Smoking status was noted 177 (healthy smokers n=3, asthmatic smokers n=4) because of its well-documented impact on lung health 178 and susceptibility to disease. Three asthmatic participants also reported having a history of allergic 179 rhinitis. Participants who met the eligibility criteria and successfully completed the Screening Visit 180 were enrolled in the trial. Subjects took the probiotic blend twice a day for 4 weeks, and assessments 181 were conducted at Baseline and the end of Week 2 and Week 4. 182 No SAEs or withdrawals due to AEs occurred during this study in the healthy and asthmatic 183 populations, successfully satisfying our primary endpoint (Table 2). There was only one mild 184 gastrointestinal complaint of bloating, which required no action, reported during the trial 185 (Supplemental Table 1). Individual results for vital signs and safety blood parameters were clinically 186 reviewed by the medical doctor and deemed to be safe at all timepoints across all participants 187 (Supplemental Tables 2-3). No participants reported changes in their alcohol habits during the course 188 of the study. 189
190
3.2 Probiotic blend improves lung function in asthmatic subjects
Spirometry measurements were taken at Baseline and Week 4 to assess changes in lung function. 191 Data was normally distributed as measured by D’Agostino & Pearson test of normality: FEV1 192 (K2=1.097, P=0.5778), FVC (K2=0.4601, P=0.7945). In asthmatic participants, average FEV1% 193 increased significantly ( P =0.018) and FVC trended up ( P =0.082) from Baseline to Week 4 (Table 3). 194 The healthy population did not see a change in FEV1% ( P =0.099) or FVC ( P =0.387), and neither 195 group had a significant improvement in FEV/FVC ratio (healthy P =0.113, asthma P =0.284). Three 196 patients showed a decrease in average FEV1 or FVC, but the change was <10% (Supplemental Table 197 4). 198
3.3 Probiotic blend improves quality of life scores as measured by SGRQ 199
5
Participants responded to the Saint George’s Respiratory Questionnaire (SGRQ) at Baseline, Week 2, 200 and Week 4 to evaluate the supplement’s effect on quality of life as measured through symptoms, 201 impact, and total score (Supplemental Table 5). Across total score and the subcategories of 202 symptoms, activity, and impact there was no significant change from baseline to Week 4 in either the 203 healthy or asthmatic participants (Table 4). However, impact scores in asthmatic participants trended 204 towards significance ( P =0.065). Of the participants that smoke, have asthma, or have asthma and 205 smoke, 36% noted an improvement in their overall health, 43% noted less frequent coughing, 43% 206 noted fewer instances of feeling short of breath, and 29% noted fewer cough or breathing-related 207 sleep disturbances. 208
3.4 Majority of participants would recommend probiotic and herbal blend 209
In an end of trial survey, 100% of participants who smoke would recommend the probiotic and 210 herbal blend to friends and family. 90% of asthmatic participants and 82% of healthy participants 211 would recommend the same. 212
3.5 Probiotic blend improves serum short chain fatty acid levels in asthmatic subjects 213
Serum was analyzed for changes in short chain fatty acid (SCFA) levels between baseline and Week 214 4. Participants with asthma showed significant changes in serum SCFA levels. Propionic acid 215 increased across all asthmatics (Figure 2A), propionic acid and isovaleric acid increased significantly 216 in asthmatic non-smokers (Figure 2B), and acetic acid and butyric acid significantly increased in 217 subjects with asthma who smoked (Figure 2C). Across all groups, isobutyric acid and valeric acid did 218 not show a significant change, and hexanoic acid levels were too low for detection. 219
220
3.6
Gut microbiome profile differs in asthmatic subjects
To examine the effects of the probiotic on the intestinal microbiome for potentially adverse signals, 221 we collected stool samples at baseline and Week 4 and analyzed for participants’ gut microbiome 222 signatures. The overall alpha (intra-sample) and beta (inter-sample) diversity was not significantly 223 altered between baseline and after 4 weeks of probiotic administration (Shannon Diversity, P =0.644, 224 T-test; Chao1 Richness Index, P =0.665, T-test, Figure 3A. P=1, R2=0.007, PERMANOVA; 225 P=0.279, PERMDISP, Figure 3B), with only modest differences in colonization at the genus level 226 (Supplemental Table 6). 227 However, the patient’s asthma status did associate with differences in alpha and beta diversity 228 (Chao1, P =0.033 Figure 3C, and P =0.017, R2 = 0.0464, PERMANOVA; P=0.52805, PERMDISP, 229 Figure 3D). The most prominent differences at the genus level were higher relative abundance of E. 230 coli (Log 2 FC 26.6), Bacteroidetes dorei (Log 2 FC 24.2), and B. ovatus (Log 2 FC 21.7) (Supplemental 231 Table 7). 232
233
3.7
Probiotic Lactobacillus strains detected in stool
We performed qPCR using primers validated for strain-specificity. Most samples were not colonized 234 with L. plantarum at baseline (19%), but this signature was augmented in most samples by 235 administration of the probiotic and herbal blend (81%, chi 2 , 12.5, P =0.0004). L. acidophilus 236 colonization was more common at baseline (37%) and was detected in most of the post-exposure 237 samples (62%, chi 2 2, P =0.157). Similarly, L. rhamnosus was detected in most individuals before and 238 after probiotic administration (62% versus 81%, chi 2 , 1.39, P =0.238). 239
6
This is a provisional file, not the final typeset article
In healthy participants, the concentration of L. plantarum increased significantly and L. acidophilus 240 and L. rhamnosus trended upward from baseline to Week 4 after taking the probiotic and herbal 241 blend (Figure 4A). Percent densitometry was calculated from PCR product bands of gels and of L. 242 plantarum and L. acidophilus increased from baseline to Week 4 with L. rhamnosus trending up 243 (Figure 4B). In asthmatic participants, the concentration of all three Lactobacillus strains increased 244 significantly from baseline to Week 4 (Figure 4C). Percent densitometry of L. plantarum and L. 245 acidophilus increased from baseline to Week 4 and L. rhamnosus trended upward (Figure 4D). 246
247
4
Discussion
In our 1-month clinical trial dosing a Lactobacillus probiotic blend to healthy volunteers and 248 asthmatic participants, we found that the supplement was safe and well-tolerated among all subjects, 249 fulfilling our primary endpoint. This was anticipated because prior to this clinical trial, all three 250 probiotic strains have been independently clinically validated and the herbs have a long history of use 251 in preclinical research and traditional medicine (8, 9, 27-30). 252 Although a majority of the participants did show a modest improvement in quality-of-life scores, the 253 changes were not significant in the healthy or asthmatic participants. In the healthy participants, their 254 SGRQ scores were generally good at baseline which meant that any change was incremental. Even 255 the asthmatic population that was more likely to start from a lower baseline (higher score) did not 256 show a significant change. The dosing period of the trial was only four weeks which did not allow for 257 enough time to capture changes in perceived symptoms as a result of taking the blend. 258 There were slight decreases in average FEV1 and/or FVC in 3 asthmatic patients: 2 smokers and 1 259 non-smoker. However, the differences from baseline to Week 4 were small, with increases <10% of 260 the lower Week 4 value. Additionally, it was noted that 10 patients had a >10% difference between 261 their two FEV or FVC readings which could have skewed the average value. Several asthma patients 262 did report increases in SGRQ scores from baseline to Week 4; however, two of the participants with 263 large increases (AN2 and AN4) both contracted lower respiratory tract infections unrelated to taking 264 study product during the course of the trial which may have affected the results of the questionnaire. 265 A third asthma patient with an increase in SGRQ scores has allergic rhinitis, which may have played 266 a role, although they reported no AE or SAEs. Across all participants, there were no related 267 respiratory AEs reported and no SAEs of any kind reported. 268 SCFA levels were not significantly changed in the healthy population from baseline to Visit 4. 269 However, SCFA levels were significantly upregulated in the serum of the asthmatic participants, 270 suggesting a pre-existing deficiency that was addressed through supplementation. SCFAs are known 271 to affect immune cell function and a variety of inflammatory pathways including TNF- , IL-2, IL-6, 272 and IL-10 (31). We observed distinct increases in serum SCFAs acetic acid, propionic acid, and 273 butyric acid in subjects with asthma who smoked, and propionic acid and isovaleric acid in subjects 274 with asthma who did not smoke. Gut dysbiosis diminishes metabolism of anti-inflammatory SCFAs, 275 impairi ng the body’s ability to regulate systemic inflammation and exacerbating allergic lung 276 inflammation (19, 20, 32, 33). 277 An interesting finding was the significant improvement in lung function in the asthmatic group as 278 measured by FEV1%. This change may have been due to the significant uptick in serum SCFAs 279 which traveled through systemic circulation and reduced inflammation in the lungs. Nevertheless, the 280 sample size in this study is too small to determine a direct correlation, and larger studies would be 281 necessary to confirm this correlation. Previous studies have shown an association between 282
7
Lactobacillus administration and immune regulation of Th1/Th2 response, reducing allergic 283 inflammation characteristic of asthma (14, 34). The effect of the herbal extracts can also not be 284 discounted, as turmeric, holy basil, and vasaka have antioxidant and anti-inflammatory properties 285 (22, 35). Although FVC values were not statistically significant, a longer study with more subjects 286 may reveal a larger impact over time. 287 As anticipated, the probiotic induced no significant alterations in the global gut microbiome 288 community composition of the human participants (36). This agrees with prior literature that 289 demonstrates a limited long-term alteration in the gut microbiota from probiotics in healthy adults, 290 that stems from colonization resistance from the microbiota and host factors that perform ecology 291 maintenance (36-40). Alternatively, prior studies of probiotic administration have suggested that the 292 administration of a Lactobacillus probiotic can alter the prevalence of other Lactobacillus strains 293 without altering overall community structure (36). We verified by strain-specific PCR that the three 294 Lactobacillus strains contained in the probiotic blend increased from baseline to Week 4, of note 295 significantly in the asthmatic population. 296 We identified robust differences in the microbiota of asthmatic patients in our cohort, confirming 297 prior observations that community composition was associated with altered microbiota (41, 42). 298 Specifically, an increase in proteobacteria E. coli was particularly notable, since proteobacteria have 299 been linked with increased risk of respiratory disease (43). These differences are interesting and 300 deserving of further validation in a larger cohort of asthmatic individuals. 301 There are several limitations to this study, as its primary focus was safety. The study was unblinded 302 and did not have a placebo group. It is difficult to credit the observed improvements to specific 303 components of the blend. We also did not collect efficacy data at Week 2 (Visit 3), although it is 304 unlikely that an effect would have been observed after two weeks. The magnitude of change in 305 SCFAs required to affect biomarkers is unknown, as we did not assess asthma-specific markers of 306 inflammation in serum such as eosinophils, leukotrienes, or IL-5. Future trials will include a placebo 307 arm and a longer administration period in order to be able to draw stronger conclusions about clinical 308 efficacy endpoints. 309 Information on the type or severity of asthma was not collected. If a participant had intermittent or 310 non-seasonal asthma, their resolution of symptoms or improvement in lung function could have been 311 reflective of the regular course of the disease. In regard to allergic asthma, three of the asthmatic 312 participants reported having allergic rhinitis; however, none reported allergic symptoms during the 313 trial. If a participant did have an allergic asthma attack, it could have skewed the SGRQ or lung 314 function results, providing either a false improvement or false worsening result. However, due to the 315 season in which the trial was conducted, a seasonal attack was less likely. The study was conducted 316 in its entirety between November 23, 2021, and January 20, 2022, a time of the year during which 317 seasonal allergies are rare and do not begin until mid-late January in Ireland. 318 In summary, the Lactobacillus probiotic and herbal blend was found to be safe in healthy and 319 asthmatic subjects, and improvements in lung function were accompanied by potentially beneficial 320 increases in SCFA in asthmatic subjects. Based on the results of this preliminary clinical study, we 321 propose that this blend may improve lung function and inflammation by supplementing microbes in 322 the gut to increase SCFA production in systemic circulation. Clinical relevance of these findings 323 affects both patients with and without existing respiratory conditions who are seeking a means to 324 support their lung health. 325
8
This is a provisional file, not the final typeset article
326
5
Figure Legends
327
Figure 1. CONSORT flow diagram.
Figure 2. Probiotic and herbal blend improves serum SCFA levels in the asthmatic population. 328 Significant increases in serum short chain fatty acid (SCFA) levels as broken down by A) all 329 asthmatic participants, B) asthmatic non-smokers, and C) asthmatic smokers. * P <0.05, ** P <0.01, 330 *** P <0.001. 331 Figure 3. Global microbiome community composition not disrupted by probiotic 332 administration, but asthmatics have a different gut microbiome. ( A ) Alpha diversity is unaltered 333 by probiotic administration. B ) Beta diversity remains similar following probiotic administration. 334 Asthmatic patients have different alpha diversity ( C ) and beta diversity ( D ). 335 Figure 4. Abundance of probiotic Lactobacillus strains increases in stool of participants. In 336 healthy participants, the concentration [ng] of A) L. plantarum increased significantly and L. 337 acidophilus and L. rhamnosus trended upward from baseline to Week 4 after supplementation. B) % 338 densitometry of L. plantarum and L. acidophilus increased significantly and L. rhamnosus trended 339 upward from baseline to Week 4. In asthmatic participants, the concentration [ng] of C) all three 340 Lactobacillus strains increased significantly from baseline to Week 4 after supplementation. D) % 341 densitometry of L. plantarum and L. acidophilus increased significantly and L. rhamnosus trended 342 upward from baseline to Week 4. * P <0.05, ** P <0.01 343
344
9
345
6
Tables
Table 1. Frequency table of participant demographics collected at baseline in the safety population. 346 Mean and SD of baseline measurements for respiratory function and SGRQ scores. 347
Parameter
Healthy N=11 N (%)
Asthma N=11 N (%)
Total N=22 N (%)
Sex
Female Male Total N
8 (72.7%) 3 (27.3%) 11 (100.0%)
7 (63.6%) 4 (36.4%) 11 (100.0%)
15 (68.2%) 7 (31.8%) 22 (100.0%)
Age
Median Range
43 36
43 29
43 41
Race/Ethnicity
White - Irish White – Irish Traveler White ‐ Any Other White
10 (90.9%) - 1 (9.1%) - - - - - - - - 11 (100.0%) 7 (63.6%) 1 (9.1%) 3 (27.3%) 11 (100.0%) 7 (63.6%) 4 (36.4%) 11 (100.0%) Mean / SD - 96.25 / 17.09 91.67 / 22.68 95.00 / 17.66 - 86.63 / 12.42 95.67 / 1.15 89.09 / 11.23
11 (100.0%) - - - - - - - - - - 11 (100.0%) 7 (63.6%) - 4 (36.4%) 11 (100.0%) 10 (90.9%) 1 (9.1%) 11 (100.0%) Mean / SD - 82.29 / 10.56 65.75 / 16.84 76.27 / 14.89 - 77.57 / 16.52 64.00 / 18.78 72.64 / 17.79
21 (95.5%) - 1 (4.5%) - - - - - - - - 22 (100.0%) 14 (63.6%) 1 (4.5%) 7 (31.9%) 22 (100%) 17 (77.3%) 5 (22.7%) 22 (100%) Mean / SD - 89.73 / 15.68 76.86 / 22.48 85.64 / 18.60 - 82.40 / 14.70 77.57 / 21.52 80.86 / 16.78
Background
Black or Black Irish ‐ African Black or Black Irish ‐ Any Other
Black Background
Asian or Asian Irish ‐ Chinese Asian or Asian Irish ‐ Any Other
Asian Background
Other, including Mixed Background Total N
Smoking Status
Non-smoker Past Smoker Current Smoker Total N
Alcohol Consumption Consumes alcohol
Abstains from alcohol Total N
Baseline Respiratory Function FEV1 % Non-smoker Smoker Total FVC % Non-smoker Smoker Total
10
This is a provisional file, not the final typeset article
Baseline SGRQ Scores Symptoms Non-smoker Smoker Total Activity Non-smoker Smoker Total Impact Non-smoker Smoker Total Total Non-smoker Smoker Total
Mean / SD - 4.92 / 8.39 15.35 / 13.38 7.76 / 10.43 - 3.05 / 6.57 24.81 / 21.55 8.99 / 15.04 - 0.60 / 1.71 2.49 / 2.53 1.12 / 2.02 - 2.14 / 3.94 12.25 / 10.65 4.90 / 7.47
Mean / SD - 54.47 / 15.80 53.35 / 18.22 54.06 / 15.80 - 27.52 / 12.51 30.14 / 8.77 28.47 / 10.90 - 23.05 / 15.88 20.94 / 4.70 22.28 / 12.62 - 29.67 / 12.50 29.01 / 6.73 29.43 / 10.37
Mean / SD - 28.04 / 28.23 37.07 / 25.26 30.91 / 27.06 - 14.47 / 15.76 27.85 / 14.19 18.73 / 16.24 - 11.08 / 15.62 13.03 / 10.51 11.70 / 13.97 - 14.99 / 16.64 21.82 / 11.86 17.16 / 15.34
348 349
350
351
352
353
354
355
Table 2. Frequency table for AEs and SAEs by health status and MEDRA SOC in the safety 356 population. 357
Health Status
N
%
Healthy Asthma Healthy
No SAE Reported No SAE Reported No AE Reported Possibly related AE o
11 11
100.0 100.0
6 2
54.5 18.2
Blood pressure increased Blood urea increased
o
Probably related AE o
1
9.1
Abdominal distension
Not related AE
0 2
0.0
Unlikely related AE o
18.2
Hyperphosphatemia
Tachycardia
o
Total
11
100.0
11
Asthma
No AE Reported Possibly related AE Probably related AE
8 0 0 2
72.7
0.0 0.0
Not related AE o
18.2
Lower respiratory tract infection
Unlikely related AE o
1
9.1
Urinary tract infection
Total
11
100.0
358 359
360
361
362
363
364
365
366
367
368
369
Table 3. Paired samples test to assess within group change in lung function parameters from Baseline 370 to Week 4 in the healthy (n=11) and asthma (n=11) populations. 371
Paired Differences
95% Confidence Interval of the Difference
Significance
Mean
SD
SEM
Lower Upper
t
df Two-sided p
Healthy FEV1 Average (%) Week 4 – Baseline Healthy FVC Average (%) Week 4 – Baseline
-5.000
9.110 2.747 -11.120 1.120
-1.820 10 0.099
-2.091
7.661 2.310 -7.238
3.056
-0.905 10 0.387
Healthy FEV1/FVC
-0.036
0.069 0.021 -0.082
0.010
-1.735 10 0.113
12
This is a provisional file, not the final typeset article
Week 4 – Baseline Asthma
8.455
9.974 3.007 1.754
15.155 2.811 10 0.018
FEV1 Average (%) Week 4 – Baseline Asthma FVC Average (%) Week 4 – Baseline
4.364
7.487 2.257 -0.666
9.393
1.933 10 0.082
Asthma FEV1/FVC Week 4 – Baseline
0.033
0.098 0.030 -0.032
0.099
1.132 10 0.284
372 373
374
375
376
377
378
379
380
Table 4. Paired samples test to assess within group change in SGRQ total score and subscales from 381 Baseline to Week 4 in the healthy (n=11) and asthma (n=11) populations. 382
Paired Differences
95% Confidence Interval of the Difference
Significance
Mean
SD
SEM
Lower Upper
t
df Two-sided p
Healthy SGRQ Total Score Week 4 – Baseline
-1.432
2.924 0.882 -3.396
0.532
-1.624 10 0.135
Healthy SGRQ Symptom Score Week 4 – Baseline
-5.829
10.962 3.305 -13.194 1.535
-1.764 10 0.108
13
Healthy SGRQ Activity Score Week 4 – Baseline Healthy SGRQ Impact Score Week 4 – Baseline Asthma SGRQ Total Score Week 4 – Baseline
-0.753
4.789 1.444 -3.970
2.465
-0.521 10 0.614
-0.468
2.623 0.791 -2.230
1.294
-0.592 10 0.567
-3.594
7.967 2.402 -8.946
1.758
-1.496 10 0.165
Asthma SGRQ Symptom Score Week 4 – Baseline
-10.012 20.628 6.220 -23.870 2.846
-1.610 10 0.139
Asthma SGRQ Activity Score Week 4 – Baseline Asthma SGRQ Impact Score Week 4 – Baseline
1.333
10.153 3.061 -5.487
8.154
-.436
10 0.672
-4.451
7.118 2.146 -9.233
0.330
-2.074 10 0.065
383
384
385
7
Conflict of Interest
ResBiotic Nutrition Inc is a university startup out of the University of Alabama at Birmingham of 386 which Dr. Lal is the Founder, Dr. Gaggar is the Chief Medical Officer, and Dr. Ambalavanan and Dr. 387 Willis are Advisors. 388
389
8
Author Contributions
Study conception and strategy: CVL. Study design: NMW, LQ, TN, ZN, XX, AG, CVL. Manuscript 390 preparation and interpretation: NMW, TN, KAW, NA, AG, CVL, BAH. Clinical trial design and 391 execution: NMW, TD, AG, CVL. Clinical data analysis: TN, ZN, LQ, GD, CK, AG, CVL, BAH, 392 KT, ME, IM. All authors reviewed, advised, and approved the final version submitted for publication 393 and agree to be accountable for the work. 394
395
9
Funding
Research reported in this article was supported by the National Heart, Lung and Blood Institute of the 396 National Institutes of Health under award number K08 HL141652 (CVL) and K08 HL151907 397 (KAW). resB Lung Support for clinical trial use was gifted by ResBiotic Nutrition, Inc. 398
14
This is a provisional file, not the final typeset article
399
10
Acknowledgments
400
A preprint of this manuscript has been published online at medRxiv.
401
11
Data Availability Statement
The data that support the findings of this study are openly available in SRA at 402 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA922968, reference number PRJNA922968. 403
404
405
406
407
408
409
410
411
412
413
414
415
References
416
1. Bowerman KL, Rehman SF, Vaughan A, Lachner N, Budden KF, Kim RY, et al. Disease- 417 associated gut microbiome and metabolome changes in patients with chronic obstructive pulmonary 418 disease. Nat Commun. 2020;11(1):5886. 419 2. Yeoh YK, Zuo T, Lui GC-Y, Zhang F, Liu Q, Li AY, et al. Gut microbiota composition 420 reflects disease severity and dysfunctional immune responses in patients with COVID-19. Gut. 421 2021;70(4):698-706. 422 3. Yazar A, Atis S, Konca K, Pata C, Akbay E, Calikoglu M, et al. Respiratory symptoms and 423 pulmonary functional changes in patients with irritable bowel syndrome. Am J Gastroenterol. 424 2001;96(5):1511-6. 425 4. Hilty M, Burke C, Pedro H, Cardenas P, Bush A, Bossley C, et al. Disordered microbial 426 communities in asthmatic airways. PLoS One. 2010;5(1):e8578. 427 5. Marri PR, Stern DA, Wright AL, Billheimer D, Martinez FD. Asthma-associated differences 428 in microbial composition of induced sputum. J Allergy Clin Immunol. 2013;131(2):346-52 e1-3. 429
15
6. Yan S, Ma Z, Jiao M, Wang Y, Li A, Ding S. Effects of Smoking on Inflammatory Markers 430 in a Healthy Population as Analyzed via the Gut Microbiota. Front Cell Infect Microbiol. 431 2021;11:633242. 432 7. Allais L, Kerckhof FM, Verschuere S, Bracke KR, De Smet R, Laukens D, et al. Chronic 433 cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the 434 murine gut. Environ Microbiol. 2016;18(5):1352-63. 435 8. Chong H-X, Yusoff NAA, Hor Y-Y, Lew L-C, Jaafar MH, Choi S-B, et al. Lactobacillus 436 plantarum DR7 improved upper respiratory tract infections via enhancing immune and inflammatory 437 parameters: A randomized, double-blind, placebo-controlled study. Journal of Dairy Science. 438 2019;102(6):4783-97. 439 9. Hojsak I, Abdović S, Szajewska H, Milosević M, Krznarić Z, Kolacek S. Lactobacillus GG in 440 the prevention of nosocomial gastrointestinal and respiratory tract infections. Pediatrics. 441 2010;125(5):e1171-7. 442 10. Kumar A, Alrefai WA, Borthakur A, Dudeja PK. Lactobacillus acidophilus counteracts 443 enteropathogenic E. coli-induced inhibition of butyrate uptake in intestinal epithelial cells. Am J 444 Physiol Gastrointest Liver Physiol. 2015;309(7):G602-7. 445 11. Wullt M, Johansson Hagslatt ML, Odenholt I, Berggren A. Lactobacillus plantarum 299v 446 enhances the concentrations of fecal short-chain fatty acids in patients with recurrent clostridium 447 difficile-associated diarrhea. Dig Dis Sci. 2007;52(9):2082-6. 448 12. Moens F, Duysburgh C, van den Abbeele P, Morera M, Marzorati M. Lactobacillus 449 rhamnosus GG and Saccharomyces cerevisiae boulardii exert synergistic antipathogenic activity in 450 vitro against enterotoxigenic Escherichia coli. Benef Microbes. 2019;10(8):923-35. 451 13. Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, deRoos P, et al. Metabolites 452 produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature. 453 2013;504(7480):451-5. 454 14. Sadrifar S, Abbasi-Dokht T, Forouzandeh S, Malek F, Yousefi B, Salek Farrokhi A, et al. 455 Immunomodulatory effects of probiotic supplementation in patients with asthma: a randomized, 456 double-blind, placebo-controlled trial. Allergy, Asthma & Clinical Immunology. 2023;19(1):1. 457 15. Huang CF, Chie WC, Wang IJ. Efficacy of Lactobacillus Administration in School-Age 458 Children with Asthma: A Randomized, Placebo-Controlled Trial. Nutrients. 2018;10(11). 459 16. Liu A, Ma T, Xu N, Jin H, Zhao F, Kwok LY, et al. Adjunctive Probiotics Alleviates 460 Asthmatic Symptoms via Modulating the Gut Microbiome and Serum Metabolome. Microbiol 461 Spectr. 2021;9(2):e0085921. 462 17. Chen YS, Jan RL, Lin YL, Chen HH, Wang JY. Randomized placebo-controlled trial of 463 lactobacillus on asthmatic children with allergic rhinitis. Pediatr Pulmonol. 2010;45(11):1111-20. 464 18. Wickens K, Barthow C, Mitchell EA, Kang J, van Zyl N, Purdie G, et al. Effects of 465 Lactobacillus rhamnosus HN001 in early life on the cumulative prevalence of allergic disease to 466 11 years. Pediatr Allergy Immunol. 2018;29(8):808-14. 467 19. Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, et al. Gut 468 microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nature 469 Medicine. 2014;20(2):159-66. 470
16
This is a provisional file, not the final typeset article
20. Cait A, Hughes MR, Antignano F, Cait J, Dimitriu PA, Maas KR, et al. Microbiome-driven 471 allergic lung inflammation is ameliorated by short-chain fatty acids. Mucosal Immunology. 472 2018;11(3):785-95. 473 21. Amala R, Sujatha S. Presence of pyrroloquinazoline alkaloid in Adhatoda vasica attenuates 474 inflammatory response through the downregulation of pro-inflammatory mediators in LPS stimulated 475 RAW 264.7 macrophages. Bioimpacts. 2021;11(1):15-22. 476 22. Manarin G, Anderson D, Silva JME, Coppede JDS, Roxo-Junior P, Pereira AMS, et al. 477 Curcuma longa L. ameliorates asthma control in children and adolescents: A randomized, double- 478 blind, controlled trial. J Ethnopharmacol. 2019;238:111882. 479 23. Kelm MA, Nair MG, Strasburg GM, DeWitt DL. Antioxidant and cyclooxygenase inhibitory 480 phenolic compounds from Ocimum sanctum Linn. Phytomedicine. 2000;7(1):7-13. 481 24. Wenger NM, Qiao L, Nicola T, Nizami Z, Xu X, Willis KA, et al. Efficacy of a Probiotic and 482 Herbal Supplement in Models of Lung Inflammation. Microorganisms. 2022;10(11):2136. 483 25. Chong J, Liu P, Zhou G, Xia J. Using MicrobiomeAnalyst for comprehensive statistical, 484 functional, and meta-analysis of microbiome data. Nature Protocols. 2020;15(3):799-821. 485 26. Dhariwal A, Chong J, Habib S, King IL, Agellon LB, Xia J. MicrobiomeAnalyst: a web- 486 based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids 487 Research. 2017;45(W1):W180-W8. 488 27. Dhuley JN. Antitussive effect of Adhatoda vasica extract on mechanical or chemical 489 stimulation-induced coughing in animals. J Ethnopharmacol. 1999;67(3):361-5. 490 28. Maheshwari RK, Singh AK, Gaddipati J, Srimal RC. Multiple biological activities of 491 curcumin: a short review. Life Sci. 2006;78(18):2081-7. 492 29. Mondal S, Varma S, Bamola VD, Naik SN, Mirdha BR, Padhi MM, et al. Double-blinded 493 randomized controlled trial for immunomodulatory effects of Tulsi (Ocimum sanctum Linn.) leaf 494 extract on healthy volunteers. J Ethnopharmacol. 2011;136(3):452-6. 495 30. Lyra A, Hillilä M, Huttunen T, Männikkö S, Taalikka M, Tennilä J, et al. Irritable bowel 496 syndrome symptom severity improves equally with probiotic and placebo. World J Gastroenterol. 497 2016;22(48):10631-42. 498 31. Vinolo MA, Rodrigues HG, Nachbar RT, Curi R. Regulation of inflammation by short chain 499 fatty acids. Nutrients. 2011;3(10):858-76. 500 32. Shin N-R, Whon TW, Bae J-W. Proteobacteria: microbial signature of dysbiosis in gut 501 microbiota. Trends in Biotechnology. 2015;33(9):496-503. 502 33. Saint-Criq V, Lugo-Villarino G, Thomas M. Dysbiosis, malnutrition and enhanced gut-lung 503 axis contribute to age-related respiratory diseases. Ageing Res Rev. 2021;66:101235. 504 34. Torii A, Torii S, Fujiwara S, Tanaka H, Inagaki N, Nagai H. Lactobacillus Acidophilus Strain 505 L-92 Regulates the Production of Th1 Cytokine as well as Th2 Cytokines. Allergology International. 506 2007;56(3):293-301. 507 35. Jamshidi N, Cohen MM. The Clinical Efficacy and Safety of Tulsi in Humans: A Systematic 508 Review of the Literature. Evid Based Complement Alternat Med. 2017;2017:9217567. 509 36. Fuentes S, Egert M, Jiménez-Valera M, Ramos-Cormenzana A, Ruiz-Bravo A, Smidt H, et al. 510 Administration of Lactobacillus casei and Lactobacillus plantarum affects the diversity of murine 511
17
intestinal lactobacilli, but not the overall bacterial community structure. Research in Microbiology. 512 2008;159(4):237-43. 513 37. Heilig HGHJ, Zoetendal EG, Vaughan EE, Marteau P, Akkermans ADL, Vos WMd. 514 Molecular Diversity of <i>Lactobacillus</i> spp. and Other Lactic Acid Bacteria in the Human 515 Intestine as Determined by Specific Amplification of 16S Ribosomal DNA. Applied and 516 Environmental Microbiology. 2002;68(1):114-23. 517 38. Zmora N, Zilberman-Schapira G, Suez J, Mor U, Dori-Bachash M, Bashiardes S, et al. 518 Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique 519 Host and Microbiome Features. Cell. 2018;174(6):1388-405.e21. 520 39. Suez J, Zmora N, Zilberman-Schapira G, Mor U, Dori-Bachash M, Bashiardes S, et al. Post- 521 Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by 522 Autologous FMT. Cell. 2018;174(6):1406-23.e16. 523 40. Liou MJ, Miller BM, Litvak Y, Nguyen H, Natwick DE, Savage HP, et al. Host cells 524 subdivide nutrient niches into discrete biogeographical microhabitats for gut microbes. Cell Host & 525 Microbe. 2022;30(6):836-47.e6. 526 41. Wang Z, Lai Z, Zhang X, Huang P, Xie J, Jiang Q, et al. Altered gut microbiome 527 compositions are associated with the severity of asthma. Journal of Thoracic Disease. 528 2021;13(7):4322-38. 529 42. Frati F, Salvatori C, Incorvaia C, Bellucci A, Di Cara G, Marcucci F, et al. The Role of the 530 Microbiome in Asthma: The Gut – Lung Axis. International Journal of Molecular Sciences. 531 2019;20(1):123. 532 43. Abdel-Aziz MI, Vijverberg SJH, Neerincx AH, Kraneveld AD, Maitland-van der Zee AH. 533 The crosstalk between microbiome and asthma: Exploring associations and challenges. Clinical & 534 Experimental Allergy. 2019;49(8):1067-86. 535
536
18
This is a provisional file, not the final typeset article
Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18Powered by FlippingBook