Microbiome Metabolic Therapies Reduce Microbiota-Associated Ammonia in Ex Vivo Fecal Samples From Healthy Subjects and Patients With Minimal Hepatic Encephalopathy and Demonstrate Improved Tolerability Over Lactulose in a Clinical Study

Jasmohan S. Bajaj,1 Kelsey Miller,2 Jie Tan,2 Jonathan Lawrence,2 Robert S. Mittleman,3 Brian Meehan2

1Virginia Commonwealth University School of Medicine and Hunter Holmes McGuire VA Medical Center, Richmond, Virginia2Kaleido Biosciences, Inc, Lexington, Massachusetts; 3Former employees of Kaleido Biosciences, Inc.

INTRODUCTION

OBJECTIVES

RESULTS

Steve Fukuda, Michael Mahowald, and Ruth Thieroff-Ekerdt contributed to the design and conduct of the clinical food study. PRECISIONscientia provided editorial support.

TABLE 2. CLINICAL STUDY SAFETY AND TOLERABILITY SUMMARY

• There were no serious adverse events, and TEAEs were primarily limited to the GI tract (ie, diarrhea, abdominal distension, and flatulence). Three subjects from the lactulose cohort had study product interruptions that did not lead to discontinuation of study product. All study product-related TEAEs had resolved by the end of the study

• KB174 gave rise to a greater ex vivo responder frequency than lactulose in fecal communities derived from both healthy subjects (75% vs 21%, respectively) and patients with MHE (32% vs 20%, respectively) (Figure 3). A fecal community is deemed an ex vivo responder when incubation with a glycan reduces ammonia levels equal to or greater than 2-fold, relative to a no-added carbon control. This threshold was chosen as an indicator of glycan efficacy after considering the broad range of glycan-mediated ammonia responses in initial ex vivo screens

• Fecal bacterial communities derived from healthy subjects and patients with MHE displayed differences in composition (Figure 4 and Table 1). Despite these differences, KB174 mediated ammonia reduction in samples derived from both cohorts

RESULTS (CONT)

ACKNOWLEDGEMENTS

FIGURE 4. EX VIVO COMPARISON OF HEALTHY AND MHE MICROBIOMES

• Safety was reported as treatment-emergent adverse events (TEAEs). Gastrointestinal tolerability was assessed with the GI tolerability questionnaire, the Bristol Stool Scale, and assessment of bowel frequency and urgency daily for the duration of the study. The GI tolerability questionnaire assessed the severity and frequency of GI symptoms, including nausea, vomiting, abdominal cramping, gas/flatulence, and reflux, on 4-point scales (range, 0-64). The Bristol Stool Scale assessed stool consistency (range, 0-7; scores of 6 or 7 were scored as diarrhea); subjects scored all bowel movements according to the scale for the duration of the study

• To assess the ability of a MMT (KB174) to reduce microbiota-associated ammonia in fecal microbiome samples from healthy subjects and patients with minimal hepatic encephalopathy (MHE) compared with lactulose, a non-absorbed disaccharide and the current first-line treatment for HE, in an ex vivo assay

• To assess the safety and tolerability of two MMTs (KB174 and MMT2) compared with lactulose in a clinical study with healthy subjects

METHODSEx vivo assays• KB174 was tested in ex vivo assays with fecal microbiome samples from 25 patients with MHE and 28 healthy

subjectsClinical study• This was a randomized open-label clinical study in healthy subjects • Subjects were between 18 and 65 years of age, body mass index of 20 to 40, with no history of gastrointestinal (GI)

conditions (inflammatory bowel disease, irritable bowel syndrome, or GI malignancy), and not taking GI-related medications or supplements (including probiotics, prebiotic dietary supplements, and laxatives)

• Subjects were randomized to one of two MMTs, KB174 (n=12) or MMT2 (n=12) titrated up to 90 g/d (9-45 g twice a day [BID]), or lactulose (10g/15mL, n=12) titrated up to 80 g/d (120 mL/d, three times a day [TID]; Figure 1)

• KB174 reduced ammonia values in fecal samples from healthy subjects and those from patients with MHE (Figure 2). Additionally, KB174 was significantly more effective in reducing ammonia levels than lactulose in both cohorts (healthy: 55% vs 41% reduction compared with control group; MHE: 37% vs 25% reduction; **P<0.001)

KB174 (n=12)

MMT2 (n=12)

Lactulose (n=12)

Number of subjects reporting ≥1 TEAEs 7 7 12

Treatment interruption due to GI intolerance, n 0 0 3

Number of subject de-escalations, n 1 1 7

Average change in daily stool frequency +0.3 -0.3 +1.2

REFERENCES1. Ferenci P. Hepatic encephalopathy. Gastroenterol Rep (Oxf). 2017;5(2):138-147.2. Miller K, et al. Identification of novel glycans that target gut microbiota-associated ammonia production. Poster presented at: The

International Liver Congress™ 2019; April 10-14, 2019; Vienna, Austria.3. Miller K, et al. Developing a drug discovery platform to target gut microbiota-associated ammonia production. Poster presented at:

Microbiome: Chemical Mechanisms and Biological Consequences; March 10-14, 2019; Montreal, Québec, Canada.

EASL | The Digital International Liver Congress. August 27-29, 2020. Poster 4015.

MDS2

MDS1

HealthyMHE

• The ex vivo screening platform enabled assessment of glycan-mediated effects on ammonia values in fecal samples derived from both healthy and cirrhotic patient populations

• Screening efforts resulted in the identification of KB174, an MMT that effectively lowered ammonia values in samples from healthy subjects as well as patients with MHE

• Additionally, KB174 reduced ammonia levels more effectively than lactulose in both magnitude as well as ex vivo responder frequency in both cohorts

• In a clinical study, KB174 was better tolerated than lactulose in healthy subjects

CONCLUSIONS

• The ability of KB174 to reduce ammonia more effectively than lactulose in ex vivo studies as well as its better tolerability in healthy subjects supports further development to potentially treat patients with hepatic encephalopathy

• A clinical food study to assess the effect of KB174 on gut microbial nitrogen metabolism in patients with well-compensated cirrhosis has been completed (Bhandari BR, et al, EASL 2020, NCT03855956)

SUMMARY

TABLE 1. DIFFERENCES IN TAXONOMY BETWEEN HEALTHY AND MHE FECAL COMMUNITIES

Community structure ****P=0.001 (Adonis test)

Shannon diversity P=0.492 (Wilcoxon rank sum)

Richness ****P=0.0028 (Wilcoxon rank sum)

Taxa MHE communities showed significant enrichment for Lactobacillaceae and Enterobacteriaceae and significant depletion of Lachnospiraceae and Eubacteriaceaerelative to healthy communities

FIGURE 3. EX VIVO AMMONIA RESPONDER FREQUENCY

Healthy MHE

KB174LactuloseKB174Lactulose0

10

20

30

40

50

60

80

%Ex

viv

oResponder 70

***

0.17

***P<0.0001

• Hyperammonemia plays a key role in the pathogenesis of hepatic encephalopathy, and a significant amount of ammonia derives from the action of gut microbiota1

• Microbiome Metabolic Therapies (MMT™) are proprietary ensembles of novel synthetic glycans that have the potential to:– Reduce the gut contribution to blood ammonia levels and lead to improved clinical outcomes for patients with cirrhosis2– Support the diversity of the gut microbiome in the management of hepatic encephalopathy

• An ex vivo screening platform was established to collect data on the effect of glycans, including lactulose and MMTs3. Via this platform, fecal samples from a healthy subject were incubated with more than 200 glycans, and levels of microbiota-derived ammonia were measured– One of the top ammonia-reducing glycans from the screen was KB174

• KB174 may reduce ammonia by enriching for commensal strains that consume ammonia at the expense of those that produce ammonia via ureases and deaminases

****Indicates statistical significance (p< 0.05)

FIGURE 1. CLINICAL STUDY DESIGN

*MMT2 was another compound identified during screening and utilized in the clinical study

Washout(~2 wk)

“Run-in”(~2 wk)

Study product intake escalation period (21 days)

18 g (9 g BID)

36 g(18 g BID)

54 g (27 g BID)

72 g (36 g BID)

90 g (45 g BID)

18 g (9 g BID)

36 g(18 g BID)

54 g (27 g BID)

72 g (36 g BID)

90 g(45 g BID)

10 g (3.3 g TID)

20 g (6.7 g TID)

40 g (13.3 g TID)

60 g (20 g TID)

80 g(27.7 g TID)

Period 1(5 days)

Period 2(4 days)

Period 3(4 days)

Period 4(4 days)

Period 5(4 days)

15 mL(5 mL TID)

30 mL(10 mL TID)

60 mL(20 mL TID)

90 mL(30 mL TID)

120 mL(40 mL TID)

KB174(n=12)MMT2*(n=12)

Lactulose(n=12)

STUDY PRODUCT

For lactulose only

FIGURE 2. MAGNITUDE OF EX VIVO AMMONIA REDUCTION

KB174LactuloseWater KB174LactuloseWater

15

10

5

Compound

Am

mon

ia (m

M)

** **

** **** **

Healthy MHE