key: cord-0307331-h9ygbsvc
authors: Raghavan, K.; Dedeepiya, V. D.; Suryaprakash, V.; Rao, K.-S.; Ikewaki, N.; Sonoda, T.; Levy, G. A.; Iwasaki, M.; Senthilkumar, R.; Preethy, S.; Abraham, S. J.
title: Beneficial Effects of novel Aureobasidium Pullulans strains produced Beta-1,3-1,6 Glucans on Interleukin-6 and D-Dimer levels in COVID-19 patients; results of a randomized multiple-arm pilot clinical study
date: 2021-08-14
journal: nan
DOI: 10.1101/2021.08.09.21261738
sha: d27f705e6ff591422d6e5aca177b0215d34f76e0
doc_id: 307331
cord_uid: h9ygbsvc

Objective: Cytokine storm and Coagulopathy have been implicated as major causes of morbidity and mortality in COVID-19 patients. A black yeast Aureobasidium pullulans AFO 202 strain produced beta 1,3 1,6 glucan has been reported to offer potential immune enhancement and metabolism balancing, as well as mitigation of coagulopathy risks. The N 163 strain produced beta glucan is an efficient anti-inflammatory immune modulator. In this pilot clinical study, we report the beneficial effects of these two beta glucans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. Methods: A total of 24 RT PCR positive COVID 19 patients were recruited (Age range: 18~62; 17 males and 7 females). Patients were randomly divided into three groups (Gr): Gr. 1 control (n=8); Gr. 2: AFO-202 beta glucan (n=8); and Gr. 3, a combination of AFO-202 and N-163 beta glucans (n=8). All three groups received the standard care while groups 2 and 3 received additional supplementation of beta glucans for 30 days. In addition to basic clinical parameters, we periodically evaluated D Dimer, IL6, erythrocyte sedimentation rate (ESR), C reactive protein (CRP), the neutrophil to lymphocyte ratio (NLR), the lymphocyte to CRP ratio (LCR) and the leukocyte CRP ratio (LeCR). Results: The duration of hospital stay for all three groups was nearly equivalent. There was no mortality of the subjects in any of the groups. Intermittent oxygen was administered from day of admission for up to four to five days with mask (two to four Lpm) to two subjects in Gr. 2 and one subject in Gr. 3. None of the subjects required ventilation. The D Dimer values in Gr. 1, which was on average 751 ng/ml at baseline, decreased to 143.89 ng/ml on day 15, but increased to 202.5 ng/ml on day 30, which in groups 2 and 3 decreased on day 15 and continued to remain at normal levels until day 30. IL6 levels decreased on day 15 from an average of 7.395 pg/ml to 3.16 pg/ml in the control, 26.18 pg/ml to 6.94 pg/ml in Gr. 2 and 6.25 pg/ml to 5.22 pg/ml in Gr. 3. However, when measured on day 30, in Gr. 1, the IL-6 increased to 55.37 pg/ml while there was only slight marginal increase in Gr. 2 but within normal range, and the levels further decreased to less than 0.5 pg/ml in Gr. 3. The same trend was observed with ESR. LCR and LeCR increased significantly in Gr. 3. NLR decreased significantly in groups 2 and 3. There was no difference in CRP within the groups. Conclusion: In this exploratory study, consumption of Aureobasidium pullulans produced beta glucans for thirty days, results in a significant control of IL6, D Dimer and NLR, a significant increase in LCR, LeCR and marginal control of ESR in COVID 19 patients. As these beta glucans are well known food supplements with decades of a track record for safety, based on these results, we recommend larger multi-centric clinical studies to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome.

perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted  https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint 6 Abstract:

Objective: Cytokine storm and Coagulopathy have been implicated as major causes of morbidity and mortality in COVID-19 patients. A black yeast Aureobasidium pullulans AFO-202 strain produced beta 1,3-1,6 glucan has been reported to offer potential immune enhancement and metabolism balancing, as well as mitigation of coagulopathy risks. The N-163 strain produced beta glucan is an efficient anti-inflammatory immune modulator.

In this pilot clinical study, we report the beneficial effects of these two beta glucans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients.

Methods: A total of 24 RT-PCR positive COVID-19 patients were recruited (Age range:

18~62; 17 males and 7 females). Patients were randomly divided into three groups (Gr):

Gr. 1 control (n=8); Gr. 2: AFO-202 beta glucan (n=8); and Gr. 3, a combination of AFO-202 and N-163 beta glucans (n=8). All three groups received the standard care while groups 2 and 3 received additional supplementation of beta glucans for 30 days. In addition to basic clinical parameters, we periodically evaluated D-Dimer, IL-6, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), the neutrophil to lymphocyte ratio (NLR), the lymphocyte to CRP ratio (LCR) and the leukocyte-CRP ratio (LeCR).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted  https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint Results: The duration of hospital stay for all three groups was nearly equivalent. There was no mortality of the subjects in any of the groups. Intermittent oxygen was administered from day of admission for up to four to five days with mask (two to four Lpm) to two subjects in Gr. 2 and one subject in Gr. 3 . None of the subjects required ventilation. The D-Dimer values in Gr. 1, which was on average 751 ng/ml at baseline, decreased to 143.89 ng/ml on day 15, but increased to 202.5 ng/ml on day 30, which in groups 2 and 3 decreased on day 15 and continued to remain at normal levels until day 30. IL-6 levels decreased on day 15 from an average of 7.395 pg/ml to 3.16 pg/ml in the control, 26.18 pg/ml to 6.94 pg/ml in Gr. 2 and 6.25 pg/ml to 5.22 pg/ml in Gr. 3. However, when measured on day 30, in Gr. 1, the IL-6 increased to 55.37 pg/ml while there was only slight marginal increase in Gr. 2 but within normal range, and the levels further decreased to less than 0.5 pg/ml in Gr. 3. The same trend was observed with ESR. LCR and LeCR increased significantly in Gr. 3. NLR decreased significantly in groups 2 and 3. There was no difference in CRP within the groups.

In this exploratory study, consumption of Aureobasidium pullulans produced beta glucans for thirty days, results in a significant control of IL6, D-Dimer and NLR, a significant increase in LCR, LeCR and marginal control of ESR in COVID-19

patients. As these beta glucans are well known food supplements with decades of a track All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The COVID-19 is an ongoing pandemic with nearly 200 million people affected worldwide and more than four million deaths to date [2] . Many patients are hospitalized 10 to 12 days after a positive RT-PCR test [3] . Recovery occurs within 14 days in most of the affected patients, but it may take more than 25 days in some patients [4] . Even patients with mild symptoms may progress to requiring oxygen therapy and mechanical ventilation and/or leading to death. The progression to severe disease is attributed to the hyperactive inflammatory response leading to a cytokine storm, thereby causing organ damage [5] . A dysfunctional immune-coagulation response activating the coagulation cascade leading to a hypercoagulable state may cause adverse clinical outcomes including death. Patients with co-morbidities such as diabetes, dyslipidemia and risk of coagulation are prone to a rapid progression of the disease and an immune-inflammatory-coagulation dysregulation-related adverse outcome [5] . Several interventions are part of the standard treatment regimen, including pharmacological agents such as anti-virals, IL6 inhibitors, All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint anti-coagulants, steroids and supplements such as vitamins or zinc [6] . The solidarity trial, an international clinical trial to help find an effective treatment for COVID-19 continues to generate evidence for the use of these interventions, and the search for effective COVID-19 therapeutics continues [6] . Many of the pharmacological interventions have associated side effects, and in regard to the supplements, zinc or ascorbic acid have not been able to significantly decrease the duration of symptoms compared with standard care [3] .

Given this background, the biological-response modifier glucans (BRMG) such as the Aureobasidium pullulans produced β-glucans have been suggested as an alternative adjunct treatment based on their potential to activate both the innate and adaptive arms of the host immune response against COVID-19 [7, 8] . This BRMG has been reported to reduce the levels of IL-1β, IL-2, IL-4, IL-6, IL-12, TNF-α, IFN-γ, and sFasL while increasing IL8 and sFAS, thereby exerting an effective optimal defence against viral infection without hyperinflammation [9] . The metabolic effects of the A. pullulans beta glucan in normalizing blood glucose and lipid levels add to their benefits for use in COVID-19 as fasting blood glucose is an independent outcome of COVID-19 severity and mortality [10] . The potential of this BRMG in acting as a prophylactic supplement to help combat coagulopathy associated with COVID-19 has also been described [11] . All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted  In an animal study using healthy SD rats, AFO-202 beta glucan was beneficial in decreasing neutrophil to lymphocyte ratio (NLR) while increasing lymphocyte to CRP ratio (LCR) and the leukocyte-CRP ratio (LeCR) in 15 days [12] , whereas in a nonalcoholic steatohepatitis (NASH) disease animal model, it has been demonstrated that N-163 beta glucan has the potential for anti-inflammatory, anti-fibrotic immune modulation [13] . Similarly, in healthy human volunteers, the reported decrease in pro-inflammatory markers and increase in anti-inflammatory markers in an advantageous manner with the same beta glucans [14] , thus supporting their ability to aid recovery in COVID-19 was considered worth studying. In this report, we hereby present the evaluation of the beneficial outcome of β-glucans produced by A. pullulans (AFO-202 and N-163 strains) in patients with COVID-19.

The present study was an open label, prospective, randomized, comparative, multiple arm pilot clinical study to evaluate the immune enhancement and immunomodulatory efficacy of supplementation with A. pullulans' novel strains produced beta glucans compared to those who undergo a conventional therapeutic regimen alone in adult subjects with COVID-19 caused by SARS-CoV2(B-CoV).

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint Adult subjects between 18 and 65 years (both ages and sexes inclusive) who were confirmed to be positive for SARS-CoV2 by way of RT-PCR with or without comorbidities, and with mild to moderate COVID-19 symptoms [15] but who required hospitalization were included in the study. Severe COVID-19-affected patients requiring intensive care, children and pregnant women were excluded.

There were three groups (Gr.) comprised of eight subjects each (Total n =24).

The control group receiving the standard treatment, which consisted of Inj.

Remdesivir 200 mg (Day 1), Inj. Remdesivir 100 mg (Day 2 to Day 5) Inj. Solumedrol 80mg IV BD Inj. Clexane 40mg OD, broad spectrum antibiotic bronchodilators and supportive measures.

The intervention was standard treatment along with AFO-202 beta glucan supplement at 3 gm per day (1.0 gm granule in a sachet containing 42mg active ingredient of ß-1,3-1,6 Glucan, with each meal).

The intervention was AFO-202 beta glucan at 3 gm per day (1.0 gm granule with each meal) in combination with N-163 beta glucan at 10 gm per day (10 gm gel in a sachet with 90mg of ß-1,3-1,6 Glucan, with one of the meals every day).

The primary outcome was improvement in the clinical symptoms of COVID-19: time taken for improvement, complete recovery, recurrence in typical symptoms from baseline. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint

The secondary outcome was evaluation for required hospitalization, mortality, progression to critical care admission, oxygen/life-support and tests for biochemical parameters such as D-Dimer, IL6, ESR, CRP, NLR, LCR and LeCR.

Data were analysed using Microsoft Excel and Origin 2021b statistical software. Results are presented as mean or mean ± standard deviation for the continuous normal variables.

Independent sample T-test and Kruskal-Wallis Test was used for comparison between the groups. A p-value < 0.05 was considered significant.

The baseline characteristics of the patients are presented in Table 1 The oxygen saturation increased in all the groups in the 15-and 30-day follow-up but the difference was not significant. The temperature and heart rate also showed no significant difference among the groups (figure 2).

In the control group, the mean fasting blood glucose (FBG) levels on day 0 were 210.14 mg/dl which decreased to 121.5 mg/dl on day 30. In Gr. 2, the mean FBG decreased from 154.14 mg/dl to 103 mg/dl in 30 days. In Gr. 3, it decreased from 238 mg/dl to 140 mg/dl in 30 days (figure 3). The difference among the groups was not significant. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint

The immunological profile from pathogenesis to progression to a severe disease in COVID-19 leading to a cytokine storm assessed by elevation of biomarkers such as IL6 and coagulopathy assessed by the elevation of markers such as D-Dimer has been reported to be unique [16] . Though many recover after one or two weeks of illness, the second to fourth week after initial onset has been reported to be stormy as many develop immune hyper-activation leading to cytokine storm marked by elevation of markers such as NLR and CRP, decrease of LCR and LeCR [17] , or coagulopathy disruption evident by elevation of markers such as D-Dimer and ferritin. Standard care of management that has been recommended by professional bodies includes steroid based immune suppression or anti-coagulants [6] . Even under appropriate treatment, morbidity, mortality and related correlations to the pathogenesis remain uncertain [6]. There have been reports of bleeding diathesis after anti-coagulants [18], and the appropriate timing for administration of these pharmacological agents are still being debated [19] . At this hour, a safer and more efficient interventional strategy to modulate the immune system to prevent or tackle the cytokine storm and to manage the coagulopathy cascade is needed.

A balanced immune enhancement with metabolism regulation potential, of AFO-202 beta glucan has been reported earlier, while an efficient immune-modulation including anti-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint fibrotic effects has been reported using N-163 beta glucan [7] [8] [9] [10] [11] [12] [13] [14] . Specially, in healthy volunteers' alleviation of glucotoxicity by balancing of blood glucose with AFO-202 and alleviation of lipotoxicity by balancing of lipid profile when AFO-202 beta glucan is combined with N-163 beta glucan has been reported [14] . Such combinations yielding beneficial effects in animal studies [13] of NASH models have proven the anti-fibrotic and anti-inflammatory immunomodulatory capabilities. The above studies in healthy animal models, human volunteers and diseased animal models have demonstrated the potentials of these beta glucans offering a balanced immunomodulation along with the necessary immune enhancement. Having used them alone or in combination, we report results herein in COVID-19 patients yielding both benefits, such as balanced modulation of inflammatory associated parameters including IL-6, NLR, LCR and LeCR. However, in addition to the beneficial effects on the immune system, the added benefits in regulating the coagulopathy evident by decrease in D-Dimer is a unique dual advantage which has never been reported with a single standalone agent or a molecule for COVID-19 in the literature, to our knowledge. D-dimer has been reported to have the highest C-index to predict in-hospital mortality in COVID-19 patients [5] . A D-dimer level greater than 0.5 μg/ml is associated with severe infection, and a value greater than 1 μg/ml is associated with increasing odds of in-All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in [20] . In the current study, the combination of AFO-202 and N-163 beta glucans were able to bring down the D-Dimer values which was greater than 1.6 μg/ml at baseline to normal values in 15 days (figure 5) that continued to be maintained even until day 30 against the control group in whom it increased. Though all the participants received anti-coagulants, the normalization of D-Dimer was significant when supplemented with beta glucans as in Gr.2 and 3.

Likewise, IL-6 levels have been reported to be significantly elevated and associated with adverse clinical outcomes in COVID-19. Inhibition of IL-6 has been suggested as a novel target for managing dysregulated host responses in patients with COVID-19 [21] . In the current study, the IL-6 values were decreased from values predictive of higher mortality due to COVID-19 > 24 pg/ml [22] to normal values which were maintained in the beta glucan groups only while it increased to values predictive of higher mortality in the control groups at day 30 (figure 4). The significant and steady decrease in IL6 and D-Dimer (figure 4 and 5) substantiate the anti-inflammatory and anti-coagulation benefits of beta glucans. The observation that patients in groups 2 and 3 had normal levels of IL-6, D-Dimer and ESR in contrast to increased levels on day 30 in the control group strongly suggests their relevance to potential management of post-COVID or long-COVID All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in reported in the earlier clinical study in terms of immunomodulation in healthy volunteers [14] and anti-inflammatory, anti-fibrotic effects in NASH animal models [13] , warrant a larger study to confirm the same. In such larger multi-centric clinical trials, evaluations on other biochemical and immunological parameters along with radiological diagnostics can help decipher the various underlying mechanisms to further validate the advantageous effects of these safety-proven beta-glucans so that they could be recommended for All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 14, 2021. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint 34 baseline (day 0), day 15 and day 30 wherein the decrease was statistically significant in Gr.2 and 3 (p < 0.05). perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 14, 2021. ; https://doi.org/10.1101/2021.08.09.21261738 doi: medRxiv preprint

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2: (Standard treatment + AFO-202 beta glucan supplementation)

3: (Standard treatment + AFO-202 and N-163 beta glucan supplementation)

Leukocyte to c-reactive protein (LeCR) values in subjects with COVID-19

Standard treatment + AFO-202 beta glucan supplementation); and F. Gr. 3: (Standard treatment + AFO-202 and N-163 beta glucan supplementation) at baseline (day 0), day 15 and day 30

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