EP4731232A1

EP4731232A1 - Porous carbon particles for use in therapy

Info

Publication number: EP4731232A1
Application number: EP24736512.5A
Authority: EP
Inventors: Rajiv Jalan
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2023-06-20
Filing date: 2024-06-20
Publication date: 2026-04-29
Also published as: WO2024261472A1

Abstract

The invention relates to the treatment or prevention of disease by modulating the microbiome of the gut using porous carbon particles comprising micropores of diameter 2 nm or less and meso pores/small macropores of diameter 30 nm to 500 nm, but substantially no mesopores of diameter greater than 2 nm and less than 30nm, and substantially no large macropores of diameter greater than 500 nm.

Description

POROUS CARBON PARTICLES FOR USE IN THERAPY

Field of the invention

The invention relates to the treatment or prevention of disease by modulating the microbiome of the gut using porous carbon particles comprising micropores of diameter 2 nm or less and meso pores/small macropores of diameter 30 nm to 500 nm, but substantially no mesopores of diameter greater than 2 nm and less than 30nm, and substantially no large macropores of diameter greater than 500 nm. The invention also relates to methods for the treatment or prevention of disease, using such porous carbon particles to modulate the microbiome of the gut.

Background to the invention

Recent studies have suggested that the gut microbiome plays a central role in the regulation of inflammation. The gut microbiome is made up of a varied community of microorganisms that inhabit the intestinal tract. The microorganisms are responsible for performing a number of functions, including metabolising nutrients, regulating the immune system and providing a natural defence against infection, that all combine to affect the health of the host. Within the gut microbiome, particular microorganisms are associated with inflammatory markers.

Various multisystem conditions are connected to inflammation, including atherosclerosis, diabetes mellitus, arthritis and Alzheimer’s disease. Inflammation may be stimulated by particular bacteria which can promote a leaky gut and allow bacterial components to reach the bloodstream, triggering a cascade of interleukin and other cytokine pathways, promoting inflammation on a systemic scale.

When dysbiosis and translocation of bacterial components including lipopolysaccharide (LPS) stimulate a systemic inflammation, multiple organ dysfunction may result. For example, gut-derived endotoxaemia and bacterial translocation have been shown to play a central role in the acceleration of atherosclerosis. However therapeutic options to target these factors are currently limited to long-term antibiotics with the attendant problem of infection with resistant organisms.

Orally administered adsorbent porous carbon particles have been used for centuries for the treatment or prevention of various disorders without any major side effects. Activated carbons are widely used to treat poisoning. A microporous carbon, AST-120 (available under the trade name KREMEZIN® from Kureha Corp., Japan) is used to treat patients with renal failure. However clinical trials evaluating the efficacy of AST-120 in the management of hepatic encephalopathy have proven negative.

WO 2013/136094 discloses the use of carbon particles with a bimodal pore size distribution in the treatment of liver disease.

Summary of the invention

The invention relates to the treatment or prevention of disease by modulating the microbiome of the gut.

Accordingly the invention provides porous carbon particles for use in a method of preventing or treating disease, wherein said porous carbon particles comprise micropores of diameter 2 nm or less and mesopores/small macropores of diameter 30 nm to 500 nm, but substantially no mesopores of diameter greater than 2 nm and less than 30nm, and substantially no large macropores of diameter greater than 500 nm, and said method comprises administering the porous carbon particles to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease.

The invention also provides a method of treating or preventing disease, the method comprising administering an effective amount of such porous carbon particles to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease.

The invention also provides use of such porous carbon particles in the manufacture of a medicament for modulating the microbiome of the gut, thereby treating or preventing disease.

Brief description of the drawings

Figure 1: Pore distribution for phenolic resin derived carbons as measured by nitrogen adsorption and determined using BJH method for TE9 carbons unactivated to activated to 29 and 47% bum off. Activation primarily increases the pores in the micropore (<2nm diameter). The small macropores (50-500nm) are largely unaltered with no introduction of pores in the 2-50nm range (mesopores).

Figure 2: Representative pore size distribution of porous carbon particles of the invention, measured by Mercury porosimetry (A: TE7 test carbon; B: TE8 test carbon). The larger peak at above 30,000 is due to the voids between the carbon particles and not to porosity.

Figure 3: Increase in the macropore volume (50-500nm) with the increase in the pore former concentration from TE3 to TE7 for carbon activated to approximately 50% bum off along. No significant introduction of pores in the mesopore range (2-50nm) or change in the pores in the micropore domain.

Figure 4: A Effect of degree of activation on the pore structure as determined by mercury porosimetry, evolution of pore volume on a cm3/gm basis and reduction in bulk density caused by activation; B Effect of degree of activation on pore structure as determined by mercury Porosimetry, change in the mercury pore volume in cm3/cm3 as a function of activation extent.

Figure 5: Physical characteristics of Yaq-001 by scanning electron microscopy demonstrating meso-macroporous domains (Scale bars: from left to right: 200 pm, 40 pm, 400 nm and 400 nm) (n=3).

Figure 6: Mercury porosimetry analysis of Yaq-001. Yaq-001 1002, Yaq-001 1003, and Yaq-001 1004 indicated different batches of Yaq-001. The different batches of Yaq-001 (1002, 1003 & 1004) produced a consistent pore size distribution plot in the meso-macroporous range from 30-200nm.

Figure 7: Adsorption kinetics of albumin, myoglobin, caffeine and endotoxin by Yaq- 001 reflective of adsorption characteristics of macro-, micro- and mesoporous domains respectively (n=3).

Figure 8: Adsorption of a range of bile acids including sodium glycochenodeoxy cholate hydrate, sodium glycocholate hydrate, lithocholic acid, chendeoxycholic acid, cholic acid and deoxycholic acid by Yaq-001(n=3).

Figure 9: Growth curves of E. coli (n=3) and Staph, aureus (n=3) in the presence of Yaq-001, amoxicillin and vehicle. Yaq-001 was not observed to influence bacterial growth kinetics of representative gram-positive and gram-negative bacteria.

Figure 10: Rats underwent bile duct ligation for 4 weeks as a model of cirrhosis (n=23- 37/group) in Experiment 1 and the treatment groups received Yaq-001 for 2 weeks before sacrifice.

Figure 11: 4-week body weight in four groups: Sham (n=36), Sham+Yaq-001 (n=30), BDL (n=37) and BDL+Yaq-001 (n=44) in Experiment 1 and 2. Significantly lower final body weights were observed in BDL compared to Sham controls (p<0.001). Yaq-001- treated BDL rats had a significantly higher body weights compared to untreated-BDL rats (p<0.05).

Figure 12: Plasma alanine transaminase (ALT) concentrations measurements in Sham (n=17), Sham+Yaq-001 (n=14), BDL (n=17) and BDL+Yaq-001 (n=26) groups. Portal pressure (PP) measurements in Sham (n=17), Sham+Yaq-001 (n=19), BDL (n=14) and BDL+Yaq-001 (n=26) groups. Significantly higher ALT and PP were observed in BDL compared to Sham controls (p<0.0001). Yaq-001 -treated BDL rats had a significantly lower ALT and PP compared to untreated-BDL rats (p<0.01, p<0.05).

Figure 12A: TUNEL assay of liver tissue with quantification of staining by digital image analysis. Significantly higher TUNEL assay was observed in BDL compared to Sham controls (p<0.0001). Yaq-001 -treated BDL rats had a significantly lower TUNEL assay compared to untreated-BDL rats (p<0.05) indicative of a reduction in liver cell death with Yaq-001 treatment.

Figure 13: Biochemical profiles of Yaq-001 treatment in cirrhotic rats. (A) Plasma alkaline phosphatase (ALP) concentrations in Sham (n=16), Sham+Yaq-001 (n=l l), BDL (n=18) and BDL+Yaq-001 (n=26) groups. Significantly higher ALP concentrations were observed in BDL compared to Sham controls (p=0.0002). (B) Plasma total bilirubin (TBIL) concentrations in Sham (n=17), Sham+Yaq-001 (n=13), BDL (n=18) and BDL+Yaq-001 (n=26) groups. Significantly higher bilirubin concentrations were observed in BDL compared to Sham controls (p<0.0001). (C) Plasma albumin levels in Sham (n=24), Sham+Yaq-001 (n=22), BDL (n=18) and BDL+Yaq-001 (n=26) groups. Significantly lower albumin levels were observed in BDL compared to Sham controls (p<0.0001).

Figure 14: (E) Plasma total bile acids concentrations in Sham (n=4), Sham+Yaq-001 (n=7), BDL (n=6) and BDL+Yaq-001 (n=6) groups. No significant difference was observed in BDL compared to Sham controls. Yaq-001 treated Sham rats had a significantly lower bile acids concentration compared to untreated Sham rats (p<0.05). (F) Mean Arterial Pressure (MAP) measurements in Sham (n=13), Sham+Yaq-001 (n=l 1), BDL (n=l 1), BDL+Yaq-001 (n=14). Significantly lower MAPs were observed in BDL compared to Sham controls (p<0.001). Yaq-001 treatment had no effect on MAP.

Figure 15: Haematoxylin & Eosin and PicoSirius Red staining of liver tissue with Collagen Proportionate area (CPA) in cirrhotic rats. BDL was associated with a significant increase in CPA compared to Sham controls (p<0.0001). Yaq-001 had no effect on CPA in either groups suggestive that the observed reduction in PP is independent of fixed intrahepatic resistance due to fibrosis.

Figure 16: Arterial ammonia concentrations in Sham (n=7), Sham+Yaq-001(n=5), BDL (n=19), BDL+Yaq-001(n=21) groups. Portal venous ammonia concentrations in Sham (n=6), Sham+Yaq-001(n=5), BDL (n=13), BDL+Yaq-001(n=18) groups. Significantly increased arterial ammonia concentrations and portal venous ammonia concentrations were observed in BDL compared to Sham controls (p<0.0001, p=0.0002). Yaq-001 significantly decreased arterial and portal venous ammonia concentrations in BDL rats (p=0.003, p=0.001).

Figure 17: Serum creatinine in Sham (n=19), Sham+Yaq-001 (n=17), BDL (n=20), BDL+Yaq-001 (n=17) and urea in Sham (n=28), Sham+Yaq-001 (n=23), BDL (n=30), BDL+Yaq-001 (n=34) groups. Yaq-001 markedly decreased serum creatinine and urea levels in BDL rats (p=0.047, p=0.042).

Figure 18: (G) Plasma D-lactate in Sham (n=7), Sham+Yaq-001 (n=8), BDL (n=6), BDL+Yaq-001 (n=7). D-lactate was significantly increased in the BDL group compared with Sham animals (p=0.024). Yaq-001 therapy resulted in a significant reduction in plasma D-lactate in BDL rats (p=0.02). (H) Portal venous [Sham (n=6), Sham+Yaq-001 (n=5), BDL (n=12) and BDL+Yaq-001 (n=7)] and arterial endotoxin concentrations [Sham (n=6), Sham+Yaq-001 (n=5), BDL (n=12) and BDL+Yaq-001 (n=7)]. Portal venous [Sham (n=6), Sham+Yaq-001 (n=5), BDL (n=12) and BDL+Yaq-001 (n=13)] and arterial plasma bacterial DNA positivity [Sham (n=6), Sham+Yaq-001 (n=6), BDL (n=12) and BDL+Yaq-001 (n=7)]. Significantly higher portal venous endotoxin and arterial endotoxin were observed in BDL rats compared to Sham rats (p<0.001). Significantly higher portal venous plasma bacterial DNA positivity was observed in BDL rats compared to Sham rats (p=0.026). Yaq-001 administration was associated with a significant reduction of portal venous endotoxin and arterial endotoxin compared to untreated-BDL rats (p<0.0001, p=0.004). Yaq-001 administration reduced the positive rate of DNA, which was not statistically different (p=0.072).

Figure 19: Effect of Yaq-001 on multiorgan function in ACLF. (A) Rats underwent sham biliary surgery or bile duct ligation (BDL) for 4 weeks. The treated group received Yaq-001 for two weeks prior to LPS injection. Animals were sacrificed either at coma stages or 6 hours after LPS injection (n=9-16/group). (B) Kaplan-Meier analysis of BDL+LPS rats with (n=16) or without (n=12) Yaq-001 treatment. Yaq-001 treatment significantly improved the survival of BDL-LPS rats compared to untreated-BDL+LPS rats (log rank test, p=0.003). (C) Plasma ALT concentrations in Sham+LPS (n=7), Sham+LPS+Yaq-001 (n=5), BDL+LPS (n=10) and BDL+LPS+Yaq-001 (n=9) groups. PP measurements in Sham+LPS (n=8), Sham+LPS+Yaq-001 (n=10), BDL+LPS (n=9) and BDL+LPS+Yaq-001 (n=9) groups. Yaq-001 -treated BDL+LPS rats had a significantly lower ALT and PP compared to untreated-BDL+LPS rats (p<0.005). Figure 20: Biochemical profiles of Yaq-001 treatment in cirrhotic rats. (A) Plasma alkaline phosphatase (ALP) concentrations in Sham (n=16), Sham+Yaq-001 (n=l l), BDL (n=18) and BDL+Yaq-001 (n=26) groups. Significantly higher ALP concentrations were observed in BDL compared to Sham controls (p=0.0002). (B) Plasma total bilirubin (TBIL) concentrations in Sham (n=17), Sham+Yaq-001 (n=13), BDL (n=18) and BDL+Yaq-001 (n=26) groups. Significantly higher bilirubin concentrations were observed in BDL compared to Sham controls (p<0.0001). (C) Plasma albumin levels in Sham (n=24), Sham+Yaq-001 (n=22), BDL (n=18) and BDL+Yaq-001 (n=26) groups. Significantly lower albumin levels were observed in BDL compared to Sham controls (p<0.0001).

Figure 21: (D) Haematoxylin & Eosin and PicoSirius Red staining of liver tissue with Collagen Proportionate area (CPA) in cirrhotic rats. BDL was associated with a significant increase in CPA compared to Sham controls (p<0.0001). Yaq-001 had no effect on CPA in either groups suggestive that the observed reduction in PP is independent of fixed intrahepatic resistance due to fibrosis. (E) 4 week body weights in Sham+LPS (n=9), Sham+LPS+Yaq-001 (n=10), BDL+LPS (n=16) and BDL+LPS+Yaq-001 (n=12) groups. Yaq-001 -treated Sham+LPS and BDL+LPS rats had a slightly higher body weights compared to untreated rats. (F) Mean Arterial Pressure (MAP) measurements in Sham (n=13), Sham+Yaq-001 (n=l l), BDL (n=l l), BDL+Yaq-001 (n=14). Significantly lower MAPs were observed in BDL compared to Sham controls (p<0.001). Yaq-001 treatment had no effect on MAP.

Figure 22: Brain water percentage in Sham+LPS (n=4), Sham+LPS+Yaq-001 (n=4), BDL+LPS (n=7), BDL+LPS+Yaq-001 (n=13) groups. Arterial ammonia concentrations in Sham+LPS (n=5), Sham+LPS+Yaq-001 (n=5), BDL+LPS (n=7), BDL+LPS+Yaq- 001 (n=7) groups. Portal venous ammonia concentrations in Sham+LPS (n=5), Sham+LPS+Yaq-001 (n=5), BDL+LPS (n=6), BDL+LPS+Yaq-001 (n=5) groups. Yaq- 001 decreased brain water percentage and arterial/portal venous ammonia concentrations in BDL+LPS rats compared to untreated rats (p=0.008, p=0.007, p=0.032). Figure 23: Serum creatinine in Sham+LPS (n=4), Sham+LPS+Yaq-001 (n=3), BDL+LPS (n=12) and BDL+LPS+Yaq-001 (n=6) groups. Serum urea in Sham+LPS (n=8), Sham+LPS+Yaq-001 (n=4), BDL+LPS (n=12) and BDL+LPS+Yaq-001 (n=8) groups. Yaq-001 significantly decreased creatinine levels in BDL+LPS rats (p<0.05). Figure 24: Absolute portal venous total leukocyte, neutrophil and monocyte populations in Sham (n=5), Sham+Yaq-001 (n=5), BDL (n=4) and BDL+Yaq-001 (n=4) groups. Absolute arterial total leukocyte, neutrophil and monocyte populations in Sham (n=5), Sham+Yaq-001 (n=5), BDL (n=5) and BDL+Yaq-001 (n=4) groups. BDL was associated with a significant increase in leukocyte, neutrophil and monocyte levels in portal vein and artery compared to Sham controls (p=0.0001, p=0.0002, p=0.018), (p=0.0009, p=0.0004, p=0.016) respectively. Yaq-001 significantly decreased total leukocyte and neutrophil level in portal vein and artery (p<0.05 for both).

Figure 25: Constitutive and LPS-induced ROS production in CD163— gated liver non- parenchymal cell fraction (n=3-5/group), portal venous monocytes (n=3-4/group) and portal venous neutrophil populations (n=3-4/group) (expressed as a percentage of the parent population) in Sham, Sham+Yaq-001, BDL and BDL+Yaq-001. BDL resulted in a significant increase in LPS-induced monocyte and neutrophil ROS production (p<0.05). Yaq-001 significantly attenuated LPS-ROS production both in PV monocytes and Kupffer cell populations (p<0.05).

Figure 26A-C: Effect of Yaq-001 on gene expression profiles in the liver and gut in BDL rats. Rats underwent bile duct ligation for 4 weeks as a model of cirrhosis (n=3- 4/group) and the treatment groups received Yaq-001 for 2 weeks before sacrifice. Liver and colon were collected for transcriptomic analysis. Functional enrichment analysis of liver in pairwise three groups based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The significant changed pathways were shown in panels including inflammation, TLR signaling, cell death, cell senescence and intracellular signaling. Figure 27A-C: Functional enrichment analysis of colon in pairwise three groups based on KEGG database. The significantly changed pathways are shown in panels including inflammation, TLR signaling, cell death, cell senescence and intracellular signaling. Figure 28A-C: Effect of Yaq-001 on gene expression profiles in the brain and kidneys in BDL rats. Rats underwent bile duct ligation for 4 weeks as a model of cirrhosis (n=3-4/group) and the treatment groups received Yaq-001 for 2 weeks before sacrifice. Brain and kidneys were collected for transcriptomic analysis. Functional enrichment analysis of brain is shown pairwise for three groups based on the KEGG database. The significantly changed pathways were shown in panels including inflammation, TLR signaling, cell senescence and intracellular signaling.

Figure 29A-C: Functional enrichment analysis of kidney shown pairwise for three groups based on the KEGG database. The significantly changed pathways are shown in panels including inflammation and TLR signaling.

Figure 30: Yaq-001 treatment in cirrhotic rats is associated with a distinct microbiome. Heatmap of gut microbiome associated with the effect of Yaq-001 as determined by 16S PCR at the family level. The Family Porphyromonadaveae with asterisk was statistically differently abundant between BDL (n=7) vs Sham (n=6), and between BDL+Yaq-001 (n=7) vs BDL groups (n=7) (Wilcoxon rank sum test, p<0.05). The abundance of this family was statistically higher in BDL group than in Sham group, and its abundance statistically decreased in the BDL+Yaq-001 group than in the BDL group. The other six families in the heatmap were with significant fold change value between BDL vs Sham, and between BDL+Yaq-001 vs BDL groups (|log2FC|>2). Of these, five were more abundant in the BDL group than in the Sham group. The abundance largely decreased in the Yaq-001 -treated group than in the BDL group. In addition, of these, one family was less abundant in the BDL group than in the Sham group. The abundance largely increased in the Yaq-001 -treated group than in the BDL group.

Figure 31: Heatmap of gut microbiome at the Genus level. The Genus Barnesiella with asterisk was statistically differently abundant between BDL vs Sham, and between BDL+Yaq-001 vs BDL groups (Wilcoxon rank sum test, p<0.05). The abundance of this genus was statistically higher in BDL group than in Sham group, and its abundance statistically decreased in the BDL+Yaq-001 group than in the BDL group. The other 19 genera in the heatmap were with significant fold change value between BDL vs Sham, and between BDL+Yaq-001 vs BDL groups (|log2FC|>2). Of these, 14 were more abundant in the BDL group than in the Sham group. The abundance largely decreased in the Yaq-001 -reated BDL group. In addition, of these, 5 genera were less abundant in the BDL group than in the Sham group. The abundance largely increased in the Yaq-001- treated group than in the BDL group.

Figure 31A: The correlations between interested DEGs and gut microbiome at family/genus. DEGs were Top 20 and covariant with the effection of Yaq-001 treatment. Nodes represent either genes (lower semi-circular part) or bacteria (upper semi-circular part) at the family and genus level. The nodes are colored based on the log-fold change for the differential gene expression and bacteria abundance differences. The red nodes indicate an increase and blue nodes indicate a decrease. Edges represent the correlation coefficient calculated between genes and microbial genus or family with red indicating a positive correlation and blue a negative correlation. Correlation coefficients greater or equal to 0.4 were plotted in plot C (Spearman’s coefficient >= 0.4), and D shows all correlations.

Figure 32: Yaq-001 treatment in cirrhotic rats is associated with a distinct microbiome signature. (C) The Venn plot of the bacteria changed with the effectiveness of the Yaq- 001 at Family level. Left: The left circle indicates 21 families with significant fold change values between BDL and Sham (|log2FC|>2). The lower circle indicates 15 families with significant fold change values between BDL+ Yaq-001 and BDL (|log2FC|>2). The right circle indicates 22 families without significant fold change values between BDL+Yaq-001 and Sham (|log2FC|<=2). The overlapped part indicates 6 families with abundance changed with the treatment outcomes. Right: The left circle indicates 7 families with abundance being significantly different between BDL and Sham (Wilcoxon rank sum test, p<0.05). The lower circle indicates 1 family with abundance being significantly different between BDL+Yaq-001 and BDL (Wilcoxon rank sum test, p<0.05). The right circle indicates 32 families without abundance being significantly different between BDL+Yaq-001 and Sham (Wilcoxon rank sum test, p>0.05). The overlapped part indicates 1 family with abundance differed with the treatment outcomes. (D) The Venn plot of the bacteria changed with the effectiveness of the Yaq-001 at Genus level. Left: The left circle indicates 68 genera with significant fold change values between BDL and Sham (|log2FC|>2). The lower circle indicates 56 genera with significant fold change values between BDL+Yaq-001 and BDL (|log2FC|>2). The right circle indicates 59 genera without significant fold change value between BDL+Yaq-001 and Sham (|log2FC|<=2). The overlapped part indicates 19 genera with abundance changed with the treatment outcomes. Right: The left circle indicates 17 genera with abundance being significantly different between BDL and Sham (Wilcoxon rank sum test, p<0.05). The lower circle indicates 8 genera with abundance being significantly different between BDL+Yaq-001 and BDL (Wilcoxon rank sum test, p<0.05). The right circle indicates 114 genera without abundance being significantly different between BDL+Yaq-001 and Sham (Wilcoxon rank sum test, p>0.05). The overlapped part indicates 1 genus with abundance differed with the treatment outcomes. Figure 33: Percentage changes in inflammatory markers at weeks 4 and 12 from baseline in placebo (light blue) and actively treated (dark blue) groups: (A) Whole blood reactive oxygen species; (B) Leucocyte Count; (C) C-reactive protein; (D) Interleukin-6; (E) C-X-C motif chemokine ligand 10.

Figure 34: Faecal concentrations of cytokines and D-lactate in placebo-treated (light blue), actively treated (dark blue) at baseline, weeks 4 and 12. Comparative values for healthy controls (green) and decompensated cirrhosis (red) derived from previous studies (PMID: 32838247) have been used for reference.

Figure 35: Lactulose: Rhamnose ratios as a marker of intestinal permeability in placebo and actively treated groups at baseline, week 4 and week 12. Normal value <0.05.

Figure 36: Endotoxin Activity Assay values at baseline, weeks 4 and week 12 in placebo treated (light blue) and actively treated (dark blue) patients. Reference ranges for low, intermediate and high endotoxin activity values are highlighted in red. Percentage differences in serological markers of bacterial translocation markers Bactericidal Permeability Increasing protein (BPI), Lipopolysaccharide Binding Protein (LBP) and endotoxin as measured by the LAL assay; Intestinal Fatty Acid Binding Protein (IFABP) at weeks 4 and 12 compared to baseline in placebo (light blue) and active (dark blue) treated groups.

Figure 37: Reduction in stool and serum markers of gut inflammation and permeability are associated with changes in gut microbiome composition. Differences in markers of gut disease after treatment by Yaq-001 are associated with the gut microbiome. A) Heatmap showing the mean Log2(FC) difference from the baseline of faecal abundance and concentration of markers of cirrhosis. B) Circos plot of significant spearman correlations between genus abundance and gut disease markers (negative and positive correlation chords are coloured blue and red respectively). Segment bar coloured blue to red by the mean Log2(FC) from the baseline at week four in the active arm as coloured by A.

Figure 38: Heatmap showing the mean Log2(FC) difference from baseline of faecal abundance of selected bacterial species known to be over-represented in cirrhosis. Figure 39A and B: Differences in serum and faecal bile acid concentrations after treatment with Yaq-001 are associated with changes to the gut microbiome. Heatmaps showing the mean Log2(FC) difference from the baseline faecal abundance and concentration of markers of cirrhosis, as well as graphed plots showing the mean Log2(FC) difference of total bile acids.

Figure 40: Differences in serum and faecal bile acid concentrations after treatment with Yaq-001 are associated with changes to the gut microbiome. Circos plot of significant spearman correlations between genus abundance and gut disease markers (negative and positive correlation chords are coloured blue and red respectively). Segment bar coloured blue to red by the mean Log2(FC) from the baseline at week four in the active arm as coloured by A.

Figure 41: Differences in serum short chain fatty acid concentrations after treatment with Yaq-001 are associated with changes to the gut microbiome. A) Heatmap showing the mean Log2(FC) difference from the baseline faecal abundance and concentration of markers of cirrhosis. B) Circos plot of significant spearman correlations between genus abundance and gut disease markers (negative and positive correlation chords are coloured blue and red respectively). Segment bar coloured blue to red by the mean Log2(FC) from the baseline at week four in the active arm as coloured by A.

Figure 42: Differences in faecal short chain fatty acid concentrations after treatment with Yaq-001 are associated with changes to the gut microbiome. Heatmap showing the mean Log2(FC) difference from the baseline faecal abundance and concentration of markers of cirrhosis. Circos plot of significant spearman correlations between genus abundance and gut disease markers (negative and positive correlation chords are coloured blue and red respectively). Segment bar coloured blue to red by the mean Log2(FC) from the baseline at week four in the active arm as coloured by A.

Detailed description of the invention

Throughout this specification, the word “comprise”, or variations such as “comprised” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein the term “carbon particle of controlled porosity” is equivalent to “porous carbon particle”.

As used herein, the term “micropore” refers to pores with diameter of 2 nm or less, as measured by nitrogen adsorption and mercury porosimetry methods and as defined by IUPAC. As used herein, the term “mesopore” refers to pores with diameter of greater than 2 nm and less than 50 nm, as measured by nitrogen adsorption and mercury porosimetry methods and as defined by IUPAC.

As used herein, the term “macropore” refers to pores with diameter of 50 nm or more, as measured by nitrogen adsorption and mercury porosimetry methods and as defined by IUPAC.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the general knowledge.

Gut-derived bacterial ligands, in particular endotoxin, drive a dysregulated inflammatory response which has been implicated in numerous conditions related to inflammation. This dysregulated inflammatory response can be central in the progression of disease. Gut bacteria and their ligands migrate from the intestine to the systemic circulation by a process termed bacterial translocation. Bacterial translocation rates are driven by increased bacterial overgrowth, increased intestinal permeability and diminished integrity of innate immune surveillance mechanisms which increase with progressive disease. Markers of bacterial translocation such as endotoxin and bacterial DNA have been shown to be associated with acceleration of disease, including atherosclerosis, and diminished survival highlighting their pathogenic importance.

The microbiome of a subject suffering from a disease may exhibit many pathogenic features characterized by reduced diversity and a reduction of autochthonous bacteria with a reciprocal expansion in proteobacteria, gram negative organisms, which produce with highly immunogenic lipopolysaccharide. The mechanisms of why this change occurs is unknown but disease-specific selection pressures are likely to contribute. Whilst antibiotics have been shown to reduce mortality in certain complications, their use is associated with bacterial superinfection and antibiotic resistance. Furthermore, antibiotics reduce bacterial diversity rendering the microbiome less resilient to a subsequent insult.

One of the consequences of bacterial translocation in different disease states is that the endotoxin-sensing pathways (such as toll-like receptor 4) in different organ systems are known to be primed resulting in heightened susceptibility to organ injury. The present inventors therefore investigated the adsorption of free endotoxin without exerting direct effects on bacterial growth kinetics with the aim of attenuating susceptibility to organ injury without producing the deleterious effects on the microbiome. The inventors developed a synthetic non-antibiotic, endotoxin sequestrant and investigated its use as a novel therapeutic strategy to restore the microbiome, reduce bacterial translocation and systemic inflammation and treat inflammation-based complications in disease states.

In one aspect, therefore, the invention relates to porous carbon particles for use in a method of preventing or treating disease, wherein said porous carbon particles comprise micropores of diameter 2 nm or less and mesopores/small macropores of diameter 30 nm to 500 nm, but substantially no mesopores of diameter greater than 2 nm and less than 30nm, and substantially no large macropores of diameter greater than 500 nm, and said method comprises administering the porous carbon particles to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease.

The present inventors have found that such non absorbable porous carbon particles having a controlled porosity to provide a relatively high proportion of pores in the meso-to-macroporous range, or in the micro- and small macro ranges, confer a high adsorptive capacity for larger biologically relevant molecules such as bacterial toxins in addition to smaller intraluminal targets. The porous carbon particless are nonabsorbable and therefore mediate their effect locally at the gut-barrier interface. However unlike conventional non-absorbable antibiotics, the porous carbon particles of the present invention have been shown not to affect detrimentally the growth of bacteria which are important to maintain gut ecology.

Conventionally produced activated carbon (e.g. granular activated carbon) is normally microporous, having pores of diameter less than 2 nm (TUPAC definition), with little or no pore volume in the mesopore (2-50 nm) or macropore (greater than 50 nm) range.

The porous carbon particles for use in the present invention may have 20% to 90% of the total pore volume made up of pores having a mean diameter of 2 nm or less (micropores), and 75% or more of the remainder of the total pore volume (i.e. the pore volume made up of pores having a mean diameter greater than 2 nm) is made up of pores having a mean diameter of from 30 nm to 500 nm (mesopores/small macropores).

Thus, the porous carbon particles may have a bimodal distribution of pore sizes whereby the total pore volume is distributed between the micropore and large mesopore/small macropore ranges, with substantially no mesopores of diameter greater than 2 nm and less than 30nm or large macropores of diameter greater than 500 nm. The presence of large macropores is preferably minimised as pores of mean diameter above 500 nm will reduce the physical strength of the particles and provide little or no improvement in adsorption.

Typically, mesopores of diameter greater than 2 nm and less than 30 nm make up 20% of the total pore volume or less, more preferably 15% or less, still more preferably 10% or less. Typically, large macropores of diameter greater than 500 nm make up 20% of the total pore volume or less, more preferably 15% or less, still more preferably 10% or less. Typically, mesopores of diameter greater than 2 nm and less than 30 nm, and large macropores of diameter greater than 500 nm together make up 20% of the total pore volume or less, preferably 15% or less, more preferably 10% or less.

Typically, 25% to 70%, preferably 35% to 60%, more preferably 45% to 55% of the total pore volume is made up of pores having a mean diameter of 2 nm or less.

Typically, 80% or more, preferably 85% or more, more preferably 90% or more of the remainder of the total pore volume is made up of pores having a mean diameter of from 30 nm to 500 nm, preferably 30 nm to 300 nm, more preferably 50 to 200 nm.

Typically, the total pore volume as measured by nitrogen adsorption is from 0.5 to 2.5 cm³g^-1, preferably 1.0 to 2.0 cm³g^-1, more preferably 1.2 to 1.8 cm³g^-1. In one embodiment the total pore volume as measured by nitrogen adsorption is 1.3 to 1.8 cm³g^-1. In one embodiment the total pore volume as measured by nitrogen adsorption is 1.3 to 1.4 cm³g^-1.

Typically, the pore volume attributable to micropores having a mean diameter of 2 nm or less is 0.2 cm³g^-1 or more, preferably 0.2 to 0.5 cm³g^-1, more preferably 0.3 to 0.4 cm³g^-1.

Typically, the bulk density of the porous carbon particles is 0.10 gem'³ or more, preferably 0.15 gem'³ or more, more preferably 0.20 gem'³ or more. Particles having a higher bulk density result in a reduced overall volume of carbon required for oral administration, which is beneficial e.g. for patient compliance. In one embodiment the bulk density of the porous carbon particles is 0.10 gem'³ to 0.30 gem'³, preferably 0.15 gem'³ to 0.25 gem'³ , more preferably 0.18 gem'³ to 0.22 gem'³.

In contrast, in one embodiment, in the porous carbon at least 20% of the total pore volume is made up of pores having a mean diameter of from 2 to 200 nm.

Preferably, at least 20% of the total pore volume of the porous carbon particles is made up of pores having a mean diameter of from 20 to 200 nm, preferably from 30 to 200 nm, preferably from 30 to 150 nm, more preferably from 50 to 120 nm, or from 60 to 100 nm. The contribution of pores of these sizes to the total pore volume is preferably greater than 25%, more preferably greater than 30%. Suitably, pores of the aforementioned sizes make up from 25 to 75% of the total pore volume, preferably from 30 to 60%, preferably 30 to 50%, more preferably 30 to 40% of the total pore volume.

The porous carbon particles for use in the present invention may also comprise micropores having a mean diameter of from 0.6 to 2 nm. The contribution of such micropores to the total pore volume may be up to 50%, for example from 5 to 30%.

The porous carbon particles may also comprise larger macropores, having a diameter of greater than 200 nm, for example greater than 500 nm. The contribution of such macropores having a diameter of greater than 200 nm to the total pore volume may be up to 74%, for example from 25 to 70%.

Preferably, the total volume of pores having a mean diameter of from 30 to 150 nm is from 0.2 to 2.0 cm³/g, preferably 0.5 to 1.5 cm³/g.

When the particles additionally comprise micropores, the total volume of micropores having a mean diameter of from 0.6 to 2 nm is preferably from 0.01 to 1.5cm³/g.

When the particles additionally comprise larger macropores, the total volume of macropores having a mean diameter of greater than 200 nm is preferably from 0.2 to 2.0 cm³/g, preferably from 0.2 to 1.0 cm³/g.

In one particularly preferred embodiment the porous carbon particles for use in the present invention have the properties set out below.

• Micropore Pore size 0.5-2nm

• BET surface area 700 to 2000, preferably 1000-1500m²/g

• Micropore Pore volume 0.1 to 1.1 cm³/g, preferably 0.3 to 1.0cm³/g

• Meso/small macropore size 30-500nm, preferably 50-300nm

• meso/small macropore volume 0.8 to 2.5cm³/g

• Total pore volume 0.9 to 3.5 cm³/g, preferably 1.1 to 2.0 cm³/g

• Proportion of micropores (%volume) 27% to 29%

In one embodiment, the porous carbon particles for use according to the present invention have at least 20% of the total pore volume of the porous carbon particles made up of pores having a mean diameter of from 20 to 200 nm, and 20% to 90% of the total pore volume is made up of pores having a mean diameter of 2 nm or less, but less than 75% of the remainder of the total pore volume is made up of pores having a mean diameter of from 30 nm to 500 nm.

In one embodiment, the porous carbon particles for use in the present invention have 20% to 90% of the total pore volume made up of pores having a mean diameter of 2 nm or less, and 75% or more of the remainder of the total pore volume made up of pores having a mean diameter of from 30 nm to 500 nm, but at least 80% of the total pore volume of the porous carbon particles made up of pores which do not have a mean diameter of from 20 to 200 nm.

Carbon porosity can be measured using mercury porosimetry (e.g. using an automatic mercury intrusion porosimeter such as the PoreMaster® mercury intrusion porosimeter (Quantachrome Instruments)) and/or gas sorption analysis (e.g. using an Autosorb gas sorption analyser (Quantachrome Instruments)).

Mercury porosimetry measures pores greater than 2 nm, particularly greater than 20 nm, and gas sorption analysis is used to measure micropores and mesopores and generally provides an effective measure of porosity for pores having a mean diameter of 0.5 nm to 50 nm, and so it may be necessary to use both methods, especially to measure particles having bimodal porosity as described above. Above 50 nm results obtained by the nitrogen technique may not agree with those obtained by the mercury techniques. In the case of a discrepancy in results for pores of mean diameter greater than 50 nm, the results obtained by mercury should be used.

Figure 2 shows a measure of the pore volume in the larger, small macropores of particles according to the present invention as provided by mercury porosimetry. Micropores are not visible in Figure 2 because mercury porosimetry measures pores greater than 2 nm. Figure 1 shows the evolution of the nitrogen pore volume in the <2 nm and 50-500 nm pore range for the TE7 carbon as a function of burn off (degree of activation).

The change in mercury pore volume with activation is shown in Figure 4. When the mercury pore volume is reported on a cm³gm^_1 basis there is an apparent large increase in pore volume with activation. However this primarily reflects the density decrease with activation. On a volume basis, which is a better reflection of the structure change, the small macropore volume remains constant for all levels of activation, i.e. only the micropore volume is enhanced by activation.

Results obtained by mercury porosimetry may show results at higher pore diameters which correspond to voids between porous carbon particles, and do not reflect the size of pores within the carbon particles. Thus, there will be an effective macropore volume equivalent to -35% of the volume of the beads attributable to voids, where the void size is -20% of the bead size. Mercury porosimetry results of 15% of bead size or more, e.g. 20% of bead size or more, can therefore be disregarded when considering porosity. For example, carbon particles of 250-500 pm in size may have an interparticle void size reflected in the mercury data of around 50-100 pm.

Typically, the large mesopore/small macropore volume on a weight basis, determined by mercury porosimetry is 0.60 cm³gm^_1, preferably higher than 1.1 cm³gm' ¹ and more preferably higher than 1.5 cm³gm^_1.

The gas sorption analysis technique used to measure micropores is typically nitrogen sorption analysis.

The micro pores in the carbons can be increased by activation and the change in surface area and pore volume with activation is shown in table 2. Preferably, the porous carbon particles have a specific surface area as measured by a BET (Brunauer-Emmett- Teller) method of at least 700 m²/g. The specific surface area may be in excess of 900 m²/g, typically in excess of 1000 m²/g. In one embodiment the specific surface area is over 1200 m²/g. Suitable specific surface areas are in the range of 1000 to 2500 m²/g, preferably 1400 to 2000 m²/g. In one embodiment the specific surface area is from 700 m²/g to 2000 m²/g, typically 900 m²/g to 1400 m²/g, preferably 1000 m²/g to 1200 m²/g. In another embodiment the specific surface area is 1200 m²/g or less, e.g. 700 to 1200 m²/g, 900 to 1200 m²/g or 1000 to 1200 m²/g.

Preferably, the porous carbon particles have a mean diameter of from 2 to 2000 pm, for example from 50 to 2000 pm, from 200 to 1600 pm, or from 100 to 1000 pm. Suitable particles may thus have a mean diameter of, for example, from 200 to 600 pm, preferably 250 to 500 pm. Other suitable particles may have mean diameters of 1000 to 2000 pm, preferably 1000 to 1500 pm. However, particles having a mean diameter of 1000 pm or less are preferred. The particle size can be measured using laser diffraction (e.g. using a Malvern particle sizer (Malvern Instruments)).

Preferably, the porous carbon particles are in the form of spherical particles.

In one embodiment, the porous carbon particles may be surface-modified in order to alter their adsorption capacity for biological molecules.

The porous carbon particles may be in the form of uncoated particles. Such uncoated porous carbon particles have proven biocompatibility. Alternatively, the particles may be coated in order to control their release and adsorption properties. For example, the particles may be coated with a film that will allow predominant release into the large bowel.

The porous carbon particles for use in the present invention may be produced by any suitable method. Suitable methods are described, for example in WO 02/12380.

Preparation of Phenolic Resin Derived Spherical Carbon Beads with Bimodal Porosity

In relation to this invention there are two types of macropores. In macroporous beads they are located within beads and formed by pore-formers. Their size is typically 30-500nm, preferably 50 - 300 nm

Typically a precursor resin formulation is used which comprises a significant proportion of pore former, e.g. 250 parts ethylene glycol or other pore former to 100 parts of resin-forming components although high porosity can also be achieved through the use of additives such as urea in combination with the ethylene glycol.

US2008025907A1 (Tennison et al.,) the disclosure of which is incorporated herein by reference) discloses making a mesoporous resin by condensing a nucleophilic component which comprises a phenolic compound or a phenol condensation prepolymer with at least one electrophilic cross-linking agent selected from formaldehyde, paraformaldehyde, furfural and hexamethylene tetramine in the presence of a pore-former selected from the group consisting of a diol (e.g. ethylene glycol), a diol ether, a cyclic ester, a substituted cyclic ester, a substituted linear amide, a substituted cyclic amide, an amino alcohol and a mixture of any of the above with water to form a resin. The pore-former is present in an amount effective to impart macroporosity to the resin (e.g. at least 150 parts by weight of the pore former being used to dissolve 100 parts by weight of the total resin forming components, i.e. nucleophilic component plus electrophilic component), and it is removed from the porous resin after condensation by cascade washing with water or by vacuum drying.

The resulting resin may be carbonised by heating in an inert atmosphere to a temperature of at least 600°C to give a material having a bimodal distribution of pores, the pore structure as estimated by nitrogen adsorption comprising micropores and mesopores or macropores. The value for the differential of pore volume with respect to the logarithm of pore radius (dV/dlogR) for the mesopores is greater than 0.2 for at least some values of pore size in the range 20-500 A. The mesoporous carbon may have a BET surface area of 250-700m2/g without activation. It may be activated by heating it at high temperature in the presence of carbon dioxide, steam or a mixture thereof, e.g. by heating it in carbon dioxide at above 800°C. It may then have surface areas of up to 2000 m2/g and even higher e.g. 1000-2000m2/g. As used herein the term "BET surface area" is determined by the Brunauer, Emmett, and Teller (BET) method according to ASTM D1993-91, see also ASTM D6556-04. For the purposes of the current invention it is preferred to use carbon dioxide.

Phenolic resins - nucleophilic component

Resins for making carbonaceous material can be prepared from any of the starting materials disclosed in US2008025907A1. Nucleophilic components may comprise phenol, bisphenol A, alkyl phenols e.g. cresol, diphenols e.g. resorcinol and hydroquinone and aminophenols e.g. m-amino-phenol.

It is preferred to use as nucleophilic component a phenolic novolac or other similar oligomeric starting material which because it is already partly polymerized makes polymerization to the desired resin a less exothermic and hence more controllable reaction. The preferred novolacs have average molecular weights (AMW) in the range of from 300 to 3000 prior to cross-linking (corresponding to a DP with respect to phenol of about 3-30). Where novolac resins are used, they may be solids with melting points in the region of 100°C. Novolac resins of MW less than 2000 and preferably less than 1500 form crosslinked resins which on carbonisation tend to produce carbons with desired pore size distributions using lower amounts of pore former. Novolacs are thermally stable in that they can be heated so that they become molten and cooled so that they solidify repeatedly without structural change. They are cured on addition of cross-linking agents and heating. Fully cured resins are infusible and insoluble.

Whilst commercial novolacs are largely produced using phenol and formaldehyde, a variety of modifying reagents can be used at the pre-polymer formation stage to introduce a range of different oxygen and nitrogen functionalities and crosslinking sites. These include but are not limited to: -

(a) Dihydric phenols e.g. resorcinol and hydroquinone. Both are more reactive than phenol and can lead to some cross-linking at the pre-polymer production stage. It is also possible to introduce these compounds at the cross-linking stage to provide different cross-linking paths. These also increase the oxygen functionality of the resins. (b) Nitrogen containing compounds that are active in polycondensation reactions, such as urea, aromatic (aniline, m-amino phenol) and heteroaromatic (melamine) amines. These allow the introduction of specific types of nitrogen functionality into the initial polymer and final carbon and influence the development of the mesoporous structure of both the resins and the final carbons. Like hydroquinone and resorcinol, all the nitrogen containing nucleophilic modifying reagents which can be used possess two or more active sites and are more reactive in condensation reactions than phenol or novolacs. It means that they are first to react with primary cross-linking agents forming secondary cross-linking agents in situ.

The nucleophilic component may be provided alone or in association with a polymerization catalyst which may be a weak organic acid miscible with the novolac and/or soluble in the pore former e.g. salicylic acid, oxalic acid or phthalic acid. Whilst these can be used in the current invention the use of phenol alone preferred to minimise the concentration of more hydrophilic sites.

The concentration of novolac in the pore former may be such that when combined with the solution of cross-linking agent in the same pore former the overall weight ratio of pore former to (novolac + cross-linking agent) is at least 150: 100 by weight. The actual ratios of novolac:pore former and cross-linking agent:pore former are set according to convenience in operation e.g. in the case of the process disclosed in WO 2008/043983 (Tennison) by the operational requirements of a bead production plant and are controlled by the viscosity of the novolac:pore former solution such that it remains pumpable and by the ratio of cross-linking agent:pore former such that the cross-linking agent remains in solution throughout the plant

Cross-linking agents for phenolic resins

The cross-linking agent is normally used in an amount of from 5 to 40 parts by weight (pbw) per 100 parts by weight of the nucleophilic components e.g. novolac,. It may be, for example, an aldehyde e.g. formaldehyde or furfural, it could be hexamethylenetetramine (hexamine), or hydroxymethylated melamine.

Hexamine is preferably used as cross-linking agent. It is preferably used for cross-linking novolac resin at a proportion of 10 to 25 pbw e.g. about 15 to 20 pbw hexamine per 100 pbw of novolac. This ensures formation of the solid resin with maximal cross-linking degree and ensures the stability of the macropore structure during subsequent removal of the pore former Pore-formers

The pore former also acts as solvent. Thus, the pore former is preferably used in sufficient quantities to dissolve the components of the resin system, the weight ratio of pore former to the total components of the resin system resin being preferably at least 1.5: 1. Below this level the resulting resins have essentially no macroporosity.

Details of suitable pore formers are given in US2008025907A1 (Tennison). The pore former may be, for example, a diol, a diol-ether, a cyclic ester, a substituted cyclic or linear amide or an amino alcohol e.g. ethylene glycol, 1,4-butylene glycol, di ethylene glycol, triethylene glycol, y-butyrolactone, propylene carbonate, dimethylformamide, N-methyl-2-pyrrolidinone and monoethanolamine, ethylene glycol being preferred, and where the selection is also limited by the thermal properties of the solvent as it should not boil or have an excessive vapour pressure at the temperatures used in the curing process.

It is thought that the mechanism of meso- and macropore generation is due to a phase separation process that occurs during the cross-linking reaction. In the absence of a pore former, as the linear chains of pre-polymer undergo cross-linking, their molecular weight initially increases. Residual low molecular weight components become insoluble in the higher molecular weight regions causing a phase separation into cross-linked high molecular weight domains within the lower molecular weight continuous phase. Further condensation of light components to the outside of the growing domains occurs until the cross-linked phase becomes essentially continuous with residual lower molecular weight pre-polymer trapped between the domains. In the presence of a low level of pore former the pore former is compatible with, and remains within, the cross-linked resin domains, (e.g., <120 parts/100 parts Novolac for the Novolac-Hexamine-Ethylene Glycol reaction system), whilst the remainder forms a solution with the partially cross-linked polymer between the domains. In the presence of higher levels of pore former, which exceed the capacity of the cross-linked resin, the pore former adds to the low MW polymer fraction increasing the volume of material in the voids between the domains that gives rise to the mesoporosity and/or macroporosity. In general, the higher the pore former content, the wider the mesopores, up to macropores, and the higher the pore volume.

This phase separation mechanism provides a variety of ways of controlling the pore development in the cross-linked resin structures. These include chemical composition and concentration of the pore former; chemical composition and quantity of the cross-linking electrophilic agents, presence, chemical nature and concentration of modifying nucleophilic agents, chemical composition of phenolic nucleophilic components (phenol, novolac), the presence of water within the solvent and concentration of any curing catalyst if present.

Production of resin precursor and carbon in bead form

In US2008025907A1, production of the resin in both powder and bead form is disclosed. Production of the bead form may be by pouring a solution of a partially cross-linked pre-polymer into a hot liquid such as mineral oil containing a dispersing agent and stirring the mixture. The pre-polymer solution forms into beads which are initially liquid and then, as curing proceeds, become solid. The average bead particle size is controlled by several process parameters including the stirrer type and speed, the oil temperature and viscosity, the pre-polymer solution viscosity and volume ratio of the solution to the oil and the mean size can be adjusted between 5 and 2000pm. The beads can then be filtered off from the oil. In a preparative example, industrial novolac resin is mixed with ethylene glycol at an elevated temperature, mixed with hexamine and heated to give a viscous solution which is poured into mineral oil containing a drying oil, after which the mixture is further heated to effect curing. On completion of curing, the reaction mixture is cooled, after which the resulting porous resin is filtered off, and washed with hot water to remove pore former. The cured beads are carbonized to porous carbon beads which have a pore structure as indicated above, and may be activated as indicated above. The beads can be produced with a narrow particle size distribution e.g. with a D90:D10 of better than 10 and preferably better than 5.

US2010/0086469 Al (Tennison) describes and claims a process for producing discrete solid beads of polymeric material e.g. phenolic resin having a porous structure, which process may produce resin beads on an industrial scale without aggregates of resin building up speedily and interrupting production. The process comprises the steps of: (a) combining a stream of a polymerizable liquid precursor e.g. a novolac and hexamine as cross-linking agent dissolved in a first polar organic liquid e.g. ethylene glycol with a stream of a liquid suspension medium which is a second non-polar organic liquid with which the liquid precursor is substantially or completely immiscible e.g. transformer oil containing a drying oil; (b) mixing the combined stream to disperse the polymerizable liquid precursor as droplets in the suspension medium e.g. using an in- line static mixer; (c) allowing the droplets to polymerise in a laminar flow of the suspension medium so as to form discrete solid beads that cannot agglomerate; and (d) recovering the beads from the suspension medium.

Dispersion medium

For bead production, the pore former comprises a polar organic liquid e.g. ethylene glycol chosen in combination with dispersion medium which is a non-polar organic liquid so as to form a mainly or wholly immiscible combination, the greater the incompatibility between the pore former which forms the dispersed phase and the dispersion medium, the less pore former becomes extracted into the dispersion medium. The pore former desirably has a greater density than the dispersion medium with which it is intended to be used so that droplets of the pore former containing dissolved resinforming components will pass down a column more rapidly than a descending flow of dispersion medium therein. Both protic and aprotic solvents of different classes of organic compounds match these requirements and can be used as pore formers, both individually and in mixtures. In addition to dissolving the reactive components and any catalyst, the pore former should also, in the case of phenolic resins, be compatible with water and/or other minor condensation products (e.g. ammonia) which are formed by elimination as polymerization proceeds, and the pore former is preferably highly miscible with water so that it can be readily removed from the polymerized resin beads by washing.

The dispersion medium is a liquid which can be heated to the temperature at which curing is carried out e.g. to 160°C without boiling at ambient pressure and without decomposition and which is immiscible with ethylene glycol and with the dissolved components therein. It may be hydrocarbon-based transformer oil which is a refined mineral oil and is a by-product of the distillation of petroleum. It may be composed principally of C15-C40 alkanes and cycloalkanes, have a density of 0.8-0.9 depending upon grade and have a boiling point at ambient pressure of 260-330°C, also depending upon grade. Transformer oil has a viscosity of about 0.5 poise at 150°C which is a typical cure temperature. Transformer oil or other dispersion medium may be used in volumes 3-10 times the volume of the combined streams of nucleophilic precursor and crosslinking agent e.g. about 5 times.

Dispersing agents Preferred dispersing agents which are dissolved in the dispersion medium before that medium is contacted with the reaction mixture to be dispersed therein to retard droplet coalescence are either sold as drying oils e.g. Danish oil or are produced by partially oxidizing naturally occurring precursors such as tung oil, linseed oil etc. The dispersing agents are consumed as the process proceeds, so that if the dispersion medium is recycled, dispersing agent in the recycled oil stream should be replenished. The dispersing agent is conveniently supplied as a stream in solution in the dispersion medium e.g. transformer oil and e.g. in an amount of 5-10%v/v where Danish oil is used which contains a low concentration of the active component to give final concentration of the dispersant in the dispersion medium 0.2 - 1% v/v. Higher dispersant concentrations would be used in the case of oxidised vegetable oils.

Solvent Removal from resin beads and granular materials

The resin beads or granules formed as described above must first be treated to remove the pore former after which they can be carbonised and activated. The pore former can be removed either by water washing or vacuum drying. The beads can be treated directly . If water washing is used this preferably uses at least a two stage process using hot water at ~80C. This is preferably carried out using a cascade washing process where the water from the second stage, which contains a relatively low level of the pore former, is recycled to the first washing stage. The waste water from the first stage, which contains a high level of the pore former can either be disposed of or the pore former can be recovered by distillation. Vacuum drying can be carried out using any commercially available vacuum dryers although it is preferred that this should use a stirred or moving bed rather than a static tray system.

Carbonisation and Activation of Resin Structures

In US 2010/0098615A1 (Tennison, the disclosure of which is incorporated herein by reference) there is provided a process for carbonizing and activating bead or granular polymeric material and especially the solid beads of polymeric material resulting from the process of US2010/0086469, which comprises supplying the material to an externally fired rotary kiln maintained at carbonizing and activating temperatures, the kiln having a downward slope to progress the material as it rotates, the kiln having an atmosphere free of oxygen provided by a counter-current of carbon dioxide or steam, and annular weirs being provided at intervals along the kiln to control progress of the material.

Alternatively the resin beads can be carbonised and activated on a smaller scale using a batch furnace. Here the carbonisation and activation may be carried out as separate steps where the carbonisation takes place in carbon dioxide at -800C and the activation in carbon dioxide at between 850 and 950C or in steam at between 700 and 850C.

For the purposes of this invention it is preferred to use carbon dioxide as the activating medium although it is also possible to use other media.

Use of Porous Carbon Particles in the Treatment of Disease

The porous carbon particles described above are useful in the treatment or prevention of disease by modulating the microbiome of the gut.

“Treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting the disease or condition (e.g., decreasing one or more symptoms resulting from the disease or condition and/or diminishing the extent of the disease or condition); b) slowing or arresting the development of one or more clinical symptoms associated with the disease or condition (e.g., stabilizing the disease or condition, preventing or delaying the worsening or progression of the disease or condition and/or preventing or delaying the spread of the disease or condition); and/or c) relieving the disease, that is, causing the regression of clinical symptoms (e.g., ameliorating the disease state, providing partial or total remission of the disease or condition, enhancing effect of another medication, delaying the progression of the disease, increasing the quality of life and/or prolonging survival).

“Prevention” or “preventing” means any treatment of a disease or condition that causes the clinical symptoms of the disease or condition not to develop.

“Subject” refers to an animal, such as a mammal (including a human), that has been or will be the object of treatment, observation or experiment. The methods described herein may be useful in human therapy and/or veterinary applications. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

Modulation of the microbiome of the gut may include the reversal of changes in the microbiome abundance at the family and genus levels. This effect on the microbiome may subsequently result in modulating gut health, permeability, bacterial translocation and thus systemic inflammation.

Administration of the porous carbon particles described herein may restore homeostasis of the gut. Thus, the porous carbon particles may remove toxins and unwanted elements of the gut to restore gut homeostasis. This may in turn reduce gut dysbiosis and modulate the microbiome of the gut.

The porous carbon particles described herein have been shown to modulate the microbiome of the gut and thereby reduce systemic inflammation. The porous carbon particles described herein are therefore useful in treating inflammatory diseases or disorders.

Inflammatory diseases may include those in which the disease is caused by inflammation or those in which the disease results in ongoing inflammation in the subject. Inflammatory disorders may include those in which the subject’s immune system causes inflammation by mistakenly attacking the subject’s own cells or tissues.

The inflammatory disease or disorder may be selected from coronary heart disease, obesity, Alzheimer’s disease, dementia, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary airways disease, encephalitis, allograft rejection, Graves' disease, Hashimoto's thyroiditis, autoimmune uveoretinitis, giant cell arteritis, asthma, atherosclerosis, regional enteritis, granulomatous enteritis, distal ileitis, regional ileitis, terminal ileitis, dermatitis, insulindependent diabetes mellitus, non-insulin-dependent diabetes mellitus, diverticulitis, fibromyalgia, multiple sclerosis, pernicious anemia, sarcoidosis, sarcopenia, scleroderma, systemic lupus erythematous, nephritis, diseases associated with cholestasis, and Parkinson’s disease.

Preferably, the disease is selected from Alzheimer’s disease, dementia, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary airways disease, encephalitis, allograft rejection, Graves' disease, Hashimoto's thyroiditis, autoimmune uveoretinitis, giant cell arteritis, asthma, atherosclerosis, regional enteritis, granulomatous enteritis, distal ileitis, regional ileitis, terminal ileitis, dermatitis, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, diverticulitis, fibromyalgia, multiple sclerosis, pernicious anemia, sarcoidosis, sarcopenia, scleroderma, systemic lupus erythematous, nephritis and Parkinson’s disease. Administration of the porous carbon particles described herein may protect organs distant from the gut or liver from inflammation. This may be achieved by reducing systemic inflammation. For the present purposes, organs distant from the gut or liver are organs in a subject which are not the subject’s gut or liver. The organs distant from the gut or liver may include the brain and the kidneys.

Administration of the porous carbon particles described herein may protect organs distant from the gut or liver from inflammation that would otherwise have damaged the organs distant from the gut or liver to a greater extent without administration of the porous carbon particles described herein. The inflammation may be as a result of an inflammation disease or disorder, which may include Alzheimer’s disease, dementia, Parkinson’s disease, insulin-dependent diabetes mellitus, non- insulin-dependent diabetes mellitus, atherosclerosis or nephritis.

Administration of the porous carbon particles described herein may affect gut bacterial bile metabolism via modulation of the gut microbiome. Bile acids are synthesized from cholesterol in the liver and further metabolized by the microbiome of the gut into secondary bile acids, thus modulating the signalling properties of bile acids via the nuclear famesoid X receptor and the G protein-coupled membrane receptor 5. Bile acids also affect the composition of the gut microbiome. The porous carbon particles described herein modulate the gut microbiome, thus altering the affects of bile acid on the gut microbiome composition and the metabolism of bile acids by the gut microbiome. Administration of the porous carbon particles described herein may therefore impact diseases associated with cholestasis in this way. Administration of the porous carbon particles described herein may also reduce systemic inflammation and organ damage resulting from diseases associated with cholestasis.

The diseases associated with cholestasis include inherited diseases of cholestasis, intrahepatic cholestasis of pregnancy, biliary tract diseases such as primary sclerosing cholangitis, primary biliary cholangitis, drug induced cholestasis, ischemic cholangiopathy, post-transplant cholangiopathies and chronic rejection post-liver transplantation.

Administration of the porous carbon particles described herein may be of use in treating coronary heart disease or obesity via modulation of the microbiome of the gut. The modulation of the microbiome of the gut has been shown to reduce systemic inflammation, which is a key driver of coronary heart disease and obesity. Intervention in these diseases by administration of the porous carbon particles described herein may therefore take place via this mechanism of action.

Administration of the porous carbon particles described herein may prevent radiation-induced organ injury. The main cause of radiation-induced organ injury depletion of tissue stem cells and progenitor cells, and damage to vascular endothelial microvessels. Recent research has shown that the recovery of stromal stem cells following radiation exposure remains chronically impaired by pro-inflammatory cytokines/chemokines, long-lived free radicals and reactive oxygen species. Reduction in systemic inflammation resulting from the administration of the porous carbon particles described herein may therefore prevent radiation-induced organ injury.

The radiation source may be from medical imaging tests that use x-rays or involve giving radioactive substances, radiation accidents and fallout from previous nuclear weapons testing. In one embodiment, the radiation may be nuclear radiation. In another embodiment, the subject is exposed to the radiation by radiation therapy.

The porous carbon particles described herein may be used to prevent the deleterious effects of ageing. Such deleterious effects of ageing may be driven by inflammation. Common deleterious effects of ageing include hearing loss, cataracts and refractive errors, back and neck pain and osteoarthritis, chronic obstructive pulmonary disease, diabetes, depression and dementia.

The porous carbon particles described herein may also be used in a method of promoting healthy ageing. Indicators of healthy ageing may include survival to a specific age, being free of chronic diseases, autonomy in activities of daily living, wellbeing, good quality of life, high social participation, only mild cognitive or functional impairment, and little or no disability.

The porous carbon particles described herein may be administered in combination with one or more further therapeutic agents. The porous carbon particles and the one or more further therapeutic agents may be administered simultaneously, separately or sequentially.

Suitable further therapeutic agents may be selected from:

(i) an antibiotic;

(ii) an ammonia-lowering agent, such as lactulose, L-ornithine L-aspartate, L-omithine phenyl acetate or Rifaximin;

(iii) faecal microbial transplantation;

(iv) a corticosteroid; and/or (v) drugs that target diseases selected from Alzheimer’s disease, dementia, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary airways disease, encephalitis, allograft rejection, Graves' disease, Hashimoto's thyroiditis, autoimmune uveoretinitis, giant cell arteritis, asthma, atherosclerosis, regional enteritis, granulomatous enteritis, distal ileitis, regional ileitis, terminal ileitis, dermatitis, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, diverticulitis, fibromyalgia, multiple sclerosis, pernicious anemia, sarcoidosis, sarcopenia, scleroderma, systemic lupus erythematous, nephritis and Parkinson’s disease.

The further therapeutic agent may cause an increase in systemic inflammation when administered on its own. However, when administered in combination with the porous carbon particles described herein, the further therapeutic agent may cause a reduced increase in systemic inflammation or no increase in systemic inflammation.

The present invention also relates to a method of treating or preventing a disease, the method comprising administering porous carbon particles as defined herein to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease. Preferably the disease is selected from coronary heart disease, obesity, Alzheimer’s disease, dementia, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary airways disease, encephalitis, allograft rejection, Graves' disease, Hashimoto's thyroiditis, autoimmune uveoretinitis, giant cell arteritis, asthma, atherosclerosis, regional enteritis, granulomatous enteritis, distal ileitis, regional ileitis, terminal ileitis, dermatitis, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, diverticulitis, fibromyalgia, multiple sclerosis, pernicious anemia, sarcoidosis, sarcopenia, scleroderma, systemic lupus erythematous, nephritis and Parkinson’s disease.

The porous carbon particles of the present invention may be administered in a variety of dosage forms. Thus, the porous carbon particles may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The porous carbon particles may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The porous carbon particles may also be administered rectally, for example in the form of a suppository. A physician will be able to determine the required route of administration for each particular patient. Preferably, the porous carbon particles are administered orally or rectally. When administered orally or rectally, the porous carbon particles act intraluminally in the gut, since they are non-absorbable. Preferably, the porous carbon particles are administered orally, for example in free-flowing form (suitably provided in a sachet) or tablet form.

In another embodiment, the porous carbon particles may be used in a method of treating blood extracorporeally, by passing blood through a medical device containing the carbon particles before it is returned to the body, wherein the blood is from an individual having an inflammation-related disease. This method may be achieved by any suitable means. Blood which has been treated in this way may be returned to the individual for therapeutic purposes, or may be used for another purpose. For example, blood may be treated in this way prior to transfusion into a different individual.

The formulation of the porous carbon particles will depend upon factors such as the nature of the exact agent, whether a pharmaceutical or veterinary use is intended, etc. An agent which is to be used to treat disease may be formulated for simultaneous, separate or sequential use.

The porous carbon particles are typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, com starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar-coating, or filmcoating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Formulations for oral administration may be formulated as controlled release formulations, for example they may be formulated for controlled release in the large bowel.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

The dose of the porous carbon particles may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen.

Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 2 g per kg of body weight, according to the age, weight and conditions of the individual to be treated, the type and severity of the degeneration and the frequency and route of administration. Daily dosage levels may be, for example, from 0.5 to 15 g, preferably from 1 to 15 g, more preferably from 4 to 12 g or 8 to 15 g, more preferably from 8 to 12 g, more preferably from 10 to 12 g, or if appropriate higher daily dosages such as 10 to 100 g, preferably 20 to 80 g, may be used. Preferably, the daily dosage is administered all in one unit dose.

A physician will be able to determine the required time schedule of administration for any particular patient. Best practice dictates that a dose of the porous carbon particles should be administered to a patient (i) at least 4 hours after the patient has consumed a meal to ensure that the consumed food has cleared the stomach, and/or (ii) at least 6 hours before the patient is administered with any further therapeutic agent.

All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains.

All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be clear to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims.

Certain aspects of the invention

Certain aspects of the invention are disclosed below.

1. Porous carbon particles for use in a method of preventing or treating disease, wherein: said porous carbon particles comprise micropores of diameter 2 nm or less and mesopores/small macropores of diameter 30 nm to 500 nm, but substantially no mesopores of diameter greater than 2 nm and less than 30nm, and substantially no large macropores of diameter greater than 500 nm; and said method comprises administering the porous carbon particles to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease.

2. Porous carbon particles for use according to aspect 1, wherein mesopores of diameter greater than 2 nm and less than 30nm and large macropores of diameter greater than 500 nm together make up 10% or less of the total pore volume of the porous carbon particles.

3. Porous carbon particles for use according to aspect 1 or 2, wherein 5 to 30% of the total pore volume of the porous carbon particles is made up of pores having a mean diameter of from 0.6 to 2 nm.

4. Porous carbon particles for use according to any one of the preceding aspects, wherein 85% or more of the pore volume of the porous carbon particles which is made up of pores having a mean diameter greater than 2nm is made up of pores having a mean diameter of from 30 nm to 500 nm, preferably wherein 90% or more of the pore volume of the porous carbon particles which is made up of pores having a mean diameter greater than 2nm is made up of pores having a mean diameter of from 30 nm to 500 nm, preferably from 50 nm to 300 nm, preferably from 50 nm to 200 nm.

5. Porous carbon particles for use according to any one of the preceding aspects, wherein the total pore volume is from 0.5 to 2.5 cm³g^-1.

6. Porous carbon particles for use any one of the preceding aspects, wherein the volume of pores having a mean diameter of from 0.5 to 2 nm in the porous carbon particles is 0.1 to 1.1 cm³g^-1 and the volume of pores having a mean diameter of from 30 to 500 nm is 0.8 to 2.5 cm³g^-1. 7. Porous carbon particles for use according to any one of the preceding aspects, wherein the total specific surface area as measured by a BET (Brunauer-Emmett- Teller) method is greater than 700 m²/g.

8. Porous carbon particles for use according to aspect 7, wherein the total specific surface area is greater than 1000 m²/g.

9. Porous carbon particles for use according to aspect 8, wherein the total specific surface area is from 1400 to 2000 m²/g.

10. Porous carbon particles for use according to any one of the preceding aspects, wherein the particles are in the form of spherical particles.

11. Porous carbon particles for use according to any one of the preceding aspects, wherein the particles are administered orally or rectally.

12. Porous carbon particles for use according to aspect 18, wherein the particles are administered orally in free-flowing form or in tablet form.

13. Porous carbon particles for use according to any one of the preceding aspects, wherein the porous carbon particles are coated in order to control their release and adsorption properties.

14. Porous carbon particles for use according to aspect 13, wherein the porous carbon particles are coated with a film that will allow predominant release into the large bowel.

15. Porous carbon particles for use according to any one of the previous aspects, wherein the disease is an inflammatory disease or disorder.

16. Porous carbon particles for use according to aspect 15, wherein the modulation of the microbiome of the gut results in a reduction in systemic inflammation, thereby treating or preventing the disease.

17. Porous carbon particles for use according to aspect 15 or 16, wherein the inflammatory disease or disorder is selected from coronary heart disease, obesity, Alzheimer’s disease, dementia, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, encephalitis, allograft rejection, Graves' disease, Hashimoto's thyroiditis, autoimmune uveoretinitis, giant cell arteritis, asthma, chronic obstructive pulmonary airways disease, atherosclerosis, regional enteritis, granulomatous enteritis, distal ileitis, regional ileitis, terminal ileitis, dermatitis, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, diverticulitis, fibromyalgia, multiple sclerosis, pernicious anemia, sarcoidosis, sarcopenia, scleroderma, systemic lupus erythematous, nephritis, diseases associated with cholestasis and Parkinson’s disease.

18. Porous carbon particles for use according to any one of the preceding aspects, wherein the administration of the porous carbon particles protects organs distant from the gut or liver from inflammation.

19. Porous carbon particles for use according to any one of aspects 1 to 15, wherein the porous carbon particles are for use in preventing radiation-induced organ injury.

20. Porous carbon particles for use according to aspect 19, wherein the radiation is nuclear radiation.

21. Porous carbon particles for use according to aspect 19, wherein the subject is exposed to the radiation by radiation therapy.

22. Porous carbon particles for use according to any one of aspects 1 to 15, wherein the porous carbon particles are for use in preventing the deleterious effects of ageing or promoting healthy ageing.

23. A method of promoting healthy ageing, the method comprising administering porous carbon particles as defined in any one of aspects 1 to 15 to a subject, thereby modulating the microbiome of the gut and promoting healthy ageing.

24. A method of treating or preventing a disease, the method comprising administering porous carbon particles as defined in any one of aspects 1 to 15 to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease.

25. Use of porous carbon particles as defined in any one of aspects 1 to 15 in the manufacture of a medicament for modulating the microbiome of the gut, thereby treating or preventing disease.

26. A product containing: (a) porous carbon particles as defined in any one of aspects 1 to 15; and (b) one or more further therapeutic agents, for simultaneous, separate or sequential use in the treatment of a subject suffering from or susceptible to a disease.

27. A product for use according to aspect 26, wherein the further therapeutic agent is selected from: i. an antibiotic; ii. an ammonia-lowering agent, such as lactulose, L-ornithine L-aspartate, L-ornithine phenyl acetate or Rifaximin; iii. faecal microbial transplantation; iv. a corticosteroid; and/or v. drugs that target diseases defined in aspect 17.

Examples

The following Examples illustrate the invention:

Materials and Methods

Carbon materials

Preparative Example 1

A solution of 100 parts by weight of industrial Novolac resin with an average molecular weight 700-800D (Hexion Specialty Chemicals) in ethylene glycol was heated to 90-95°C and thoroughly mixed for 2-5 minutes with a solution of 15 - 20 parts by weight of hexamethylenetetramine (hexamine) in ethylene glycol heated to the same temperature. The resulting clear solution was poured in a stream into 2.5-6 fold volume of stirred hot (150-155°C) low viscosity mineral oil (insulating oil or transformer oil) containing 0.2 - 1% (v/v) of a dispersing agent which was an industrial drying oil (Danish oil), a major component being polyunsaturated (oxidised) vegetable oils. The temperature of the mixture fell to 135-140°C, and the mixture was reheated to 150- 155°C over a period of 15-20 minutes. Typically curing occurred within 1-2 minutes at around 140°C followed by substantial evolution of gas. The further heating to 150- 155°C for 15-20 minutes ensured the completion of curing. The mixture was cooled and the resulting beads were separated from the oil by filtration or centrifugation Ethylene glycol was removed from the resin either by multiple hot water extraction or by drying in vacuum (120°C at 50 mm Hg). In the above procedure, compared to Example 3 of WO 02/12380, the hexamine content has been increased to 15-20pbw per lOOpbw of novolac from the previously exemplified 9 pbw, and the temperature of the oil into which the resin solution is poured is increased from 115-120°C to 150-155°C, and “flash” cure is brought about rather than a “slow” cure as previously exemplified.

Water- washed wet, dried or vacuum-dried resin beads were heat treated to produce carbon materials. A typical procedure comprised but is not restricted to carbonisation in a flow of carbon dioxide with temperature ramping from ambient to 800°C at 3°C/min, classification by particle size and further “physical” activation of selected fraction in carbon dioxide flow at 900°C. Many variations of this routine known in the art may also be applied. The degree of activation in these samples was approximately 30%.

The meso/macro pore size distribution in the resulting carbons is pre-determined by the porosity of the resin-precursor, which is controlled by the content of the solvent/pore former and the degree of activation of the resulting carbon. Table 1 below gives details of four resin compositions that are precursors to the meso/macro porous carbons, as illustrated by nitrogen porosimetry and mercury porosimetry tests of the activated materials. The predominantly micro - macroporous materials, TE7 and TE8 have a very similar macropore structure. The TE3 and TE5 materials, which have smaller pores in the meso/macro domain are for illustration and give inferior performance in the adsorption of the larger molecules such as TNFa.

The particle size distribution of resulting resin beads depends on various parameters including but not restricted to the type of stirring tool, stirring rate, viscosity of the resin solution, concentration of the dispersing agent, resin solution to oil ratio and temperature of the dispersion. Though the distribution is typically broad the size of the predominant fraction could effectively be shifted between ~10 micron and ~1 mm.

Table 1

Preparative Example 2: Activation of the Carbonised Beads

The resin beads may be activated in either carbon dioxide or steam. Carbon dioxide is more controllable whilst steam is preferred for larger scale preparations on a cost basis. Activation in carbon dioxide occurs at around 900C with the degree of bum off controlled by the residence time in the furnace. Steam activation preferably takes place at around 700C. In both cases the conditions are not critical and the temperature and time can be adjusted to give the required degree of activation as known by anyone skilled in the art. The effect of carbon dioxide activation on the pore structure of the TE8 beads is shown in Figure 3 with the pore distribution summarised in table 2. The increase in the micro pore volume can be seen from Figure 3. It can also be seen from table 2 that the pore volume in the micropores (<2nm) is very low (0.1cm3/g) in the unactivated sample which also corresponds to the low BET surface area (534m2/g). Activation to at least 30% bum off significantly increases both the micropore volume and the surface area. The preferred materials for the biomedical adsorption have areas of at least 1000m2/g, micropore volumes in excess of 0.3cm3/g.

Table 2 Pore structures for CO2 Activated TE8 Carbons

The change in the macropore volume of the carbon with activation is an artefact of the nitrogen adsorption method but should be >0.5cm3/g. The larger pore structures should be measured using mercury porosimetry. These are shown for TE7 and TE8 activated to 40% bum off in Figure 2. The larger pores at >38000nm are due to interparticle voids between the beads, not to any internal porosity within the beads. The TE7 and TE8 particles have pore diameter peaks at 88 nm and 91 nm respectively, surface area of 1499 m²/g, pore volume of 1.36 cm³/g, bulk density of 0.2 g/cm³, particle size of 240-500 pm and 40% degree of activation. The absence of pores both above the main peak (>300nm) is readily apparent as is the essential absence of the pores in the mesopore (2-50nm) domain for both materials. Mercury cannot provide data in pores below ~6nm due to the pressure involved.

Materials for studies

Activated carbon samples according to the invention were acquired from MAST Carbon International Ltd, UK. The activated carbon samples are referred to as Yaq-001.

Scanning Electron Microscopy analysis Yaq-001 samples were mounted on aluminium stubs using carbon adhesive tape and coated with a 2nm thick layer of platinum using a Quorum Q150TES coater (Quorum Technologies, UK). The surface as well as internal morphology was examined using a Zeiss Sigma field emission field emission scanning electron microscope (FESEM) (Carl Zeiss Microscopy, Germany) at an accelerating voltage of 5 kV at 100, 500 and 50000x magnifications.

Pore size distribution by Mercury Porosimetry

Mercury porosimetry analysis was performed using a mercury porosimeter PoreMaster (Quantachome Instruments, USA). Prior to the analysis, all materials were dried in a vacuum oven at 110 °C under 800 bar vacuum for 3 hours. The meso- (2- 50nm) and macro- (diameter >50 nm) pore size distributions of the Yaq-001 samples were determined by mechanical intrusion of mercury. Data was analysed using PoreWin 6.0 software (Quantachrome Instruments, USA).

Unless otherwise specified the porous carbon particles used in the following in vitro and in vivo studies were Yaq-001.

In vitro studies

Adsorption studies

Adsorption of biomolecules of varying molecular weight (albumin, myoglobin and caffeine). For each of the molecular weight size markers the kinetics of adsorption was studied compared with that of no carbon control.

Materials for the adsorption studies

Activated carbon samples Yaq-001 (MAST Carbon International Ltd, UK), bovine serum albumin (Sigma-Aldrich, catalogue Number A6003), myoglobin from equine skeletal muscle (Sigma-Aldrich, M0630), caffeine (Sigma-Aldrich C0750), bicinchoninic acid solution (Sigma-Aldrich, catalogue number B9643), copper (II) sulfate pentahydrate 4% solution (Sigma-Aldrich, catalogue number C2284), phosphate buffer solution, pH= 7.4.

Albumin (66. 7 kDa) adsorption kinetics For the kinetics study 0.1g of Carbon adsorbent was pre-wetted into eppendorf tubes (15 mL capacity) with PBS under vacuum for 3 hours at -1000 mbar pressure. Pre-wetting solutions were removed by micropipette and 5 mL of bovine albumin solution (2 mg/mL) was added to each tube. Samples were incubated at 37°C on orbital shaker for 24h at 120 rpm. The albumin concentration was measured by collecting supernatants following centrifugation for 2 minutes at 4000 rpm at 0, 0.5, 1, 2, 3, 5 and 7 hrs. Albumin concentration was measured using a BCA method according to manufacturer’s instructions. The BCA working reagent was prepared by mixing 50 parts of reagent A to 1 part reagent B to give a light green colour. Albumin standards concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5 and 2 mg/mL in PBS were used to prepare a standard curve to calculate unknown albumin concentration. 2 mL of BCA working reagent was mixed with 0.05 mL of albumin sample, standard or blank. The solutions were Incubated at 37 °C for 30 minutes, transferred into a cuvette and absorbance was measured using a Jenway 6705 UV/Vis spectrophotometer at 562 nm by UV spectrophotometry.

Myoglobin (16.7 kDa) adsorption kinetics

For the kinetics study, 0.1g of carbon adsorbent samples were pre-wetted in triplicate into 15 mL eppendorf tubes with PBS under vacuum for 3 hours at -1000 mbar pressure. Pre-wetting solution was removed, 5 mL of myoglobin solution (500 ug/mL) was added and samples were incubated at 37°C on orbital shaker for 24h at 120 rpm. Supernatant were collected at 0, 0.5, 1, 2, 3, 5 and 7 hrs, centrifuged for 2 minutes at 4000 rpm and transferred to a sterile universal tube for measurement of myoglobin concentration. 3 mL of supernatant sample from controls and experimental samples was placed in a cuvette and absorbance was measured using a Jenway 6705 UV/Vis spectrophotometer at a wavelength of 409 nm. A standard curve was prepared using myoglobin standard ranging from 0-500pg/mL.

Caffeine (0.194kDa) adsorption kinetics

For the kinetic study, 0.1g of carbon adsorbent samples were pre-wetted in triplicate into 15 mL eppendorf tubes with PBS under vacuum for 3 hours at -1000 mbar pressure. 5 mL of a 10 mg/mL caffeine solution was added into each tube and incubated at 37°C on an orbital shaker for 24h at 120 rpm. 3 mL of supernatant was collected into clean centrifuge tubes at 0, 0.5, 1, 2, 3, 5 and 7 hrs following centrifugation for 2 minutes at 4000 rpm for measurement of residual concentration. The caffeine concentration was measured using a Jenway 6705 UV/Vis spectrophotometer (Bibby Scientific Ltd, UK) at a wavelength of 273 nm. A standard curve was prepared using a concentration range of 0 to 10 mg/mL. Standards and samples were diluted in methanol (1 : 100) and 3 mL of supernatant was added to a quartz cuvette for analysis.

Endotoxin adsorption

All carbon samples were sterilised at 200°C for 3 hours. 0.1 g sample was weighed into pyrogen free glass vials in triplicate. 4 mL spiked endotoxin solution (10 EU/mL) in SIF was added to each carbon sample and incubated on a shaking plate at 37°C for 90 minutes. 450 pL of sample was collected at 0, 15, 30, 45, 60 and 90 minutes for the kinetic study. The standards (50, 5, 0.5, 0.05, 0.005 EU/mL) were made according to manufacturer’s instructions in endotoxin free glass tubes using a 5mL volume for each dilution and vortexing well between each dilution. 0.1 ml of standard, sample or control in duplicate was added to the wells of a 96 well plate. 0.1 mL LAL reagent was prepared and immediately added to each well. The plate was placed in an incubating microplate reader (Biotek Instruments, USA) that measures absorbance at 405 nm and monitored over time using Gen5 2.0 software.

Bile acids

For the adsorption kinetics study, series of carbon adsorbent samples (0, 0.025, 0.05, 0.1, 0.25 & 0.5g) were pre-wetted in triplicate into 15 mL Eppendorf tubes with PBS under vacuum for 3 hours at -1000 mbar pressure. Pre-wetting solution was removed, 5 mL of bile acid spiked simulated intestinal fluid (SIF) solution [Cholic (150 pMol/L), Deoxy cholic (200 pMol/L), Lithocholic acid (800 pMol/L), Chendeoxycholic acid (800 pMol/L), Sodium glycholate (800 pMol/L) and Sodium glycochenodeoxycholate (354 pMol/L)] was added and samples were incubated at 37°C on orbital shaker for 24h at 120 rpm. Supernatants were collected after 24 hrs, centrifuged for 2 minutes at 4000 rpm and transferred to a sterile universal tube for measurement of bile acid concentration. 3 mL of supernatant sample from controls and experimental samples was placed in a cuvette and absorbance was measured using a Jenway 6705 UV/Vis spectrophotometer at a wavelength of 405 nm. A standard curve was prepared using respective bile acid standards ranging from 0-1000 pMol/L. Bacterial growth in the presence of Yaq-001

The effect of activated carbons on the kinetics of bacterial growth was studied for Staph aureus and E. coli. 5 mL of overnight culture prepared by standard technique was transferred to a 15 mL falcon tube and centrifuged at 4000 G for 10 minutes at 21 °C. The bacterial pellet was re-suspended in 5 mL of phosphate buffered saline (PBS). 0.1 mL was added to 100 mL of Tryptone soya broth (TSB) to achieve a final concentration of ~ 106 CFU/mL. The bacterial cultures supplemented with 10 mg/mL of activated carbon samples were incubated for 6 hours in triplicate at 37°C whilst shaking at 150rpm. 1 mL aliquots were withdrawn from the culture medium and the optical density at 600nm was measured every 30 minutes in triplicate. Comparison to the growth curves obtained in the absence of carbon samples and amoxicillin antibiotic (control). The carbon beads did not alter the bacterial growth kinetics of E. coli and Staph aureus cultures over 6 hours.

In vivo studies

All animal experiments were conducted according to Home Office guidelines under the UK Animals in Scientific Procedures Act 1986. Male Sprague-Dawley rats (body weight 280-300g) were used (Charles River Laboratories UK Ltd.). All rats were housed in the unit and given free access to standard powdered rodent chow and water, with a light/dark cycle of 12 hours, at a temperature of 19°C to 23°C and humidity of approximately 50%.

Bile duct Ligation Model

Under halothane anesthesia 194 male Sprague-Dawley rats (weights 260 +/-20 g, age 8-10 weeks) underwent bile duct-ligation (BDL) or Sham biliary surgery. Rats were pair-fed powered chow -I- pre-hydrated Yaq-001 carbon (250-500pm) at a dose of 0.4g/100g body weight per day from two weeks after bile duct ligation until completion of the experiment at 4 weeks from initial surgery. Intraperitoneal Klebsiella lipopolysaccharide (LPS) (0.33 mg/kg) was administered to 4 subgroups 6 hours prior to completion of study. The following groups were studied: Sham (n=36), Sham+Yaq- 001 (n=30), Sham+LPS (n=9), Sham+LPS+ Yaq-001 (n=10), BDL (n=37), BDL+Yaq- 001 (n=44), BDL+LPS (n=16), BDL+LPS+ Yaq-001 (n=12).

Haemodynamic measurements and sample collection Under halothane anaesthesia (5ml/min induction 2ml/min maintenance) an internal carotid catheter (0.96 outer diameter Portex fine-bore polythene tubing, Scientific Laboratory Supplies Ltd., Nottingham, UK) was inserted as previously described. The catheter was held in place for the duration of the study by both proximal and distal holding sutures. The catheter was transduced and mean arterial pressure determined. A laparotomy was then performed under sterile conditions and a catheter placed in the portal vein. Arterial and portal venous catheters were transduced. Concomitant arterial and portal venous plasma was collected aseptically into lithium heparin and EDTA tubes until a state of exsanguination was achieved. Liver, brain, kidneys and colon were collected and stored in formalin and cryotubes and stored at - 80°C.

Biomarkers

Plasma levels of alanine transaminase (ALT), alkaline phosphatase (ALP), total bilirubin (TBIL), albumin, bile acids, creatinine, urea, ammonia, endotoxin, bacterial DNA, D-lactate and brain water were detected.

Biochemical analysis

Biochemical profile was determined using standard techniques (COBAS).

D-lactate Assay

Plasma was collected in EDTA tubes from rats in different groups and analyzed using the D-Lactate Assay Kit (Colorimetric, ab83429) according to the manufacturer’s protocols.

Brain water percentage

The entire brain was weighed immediately after sacrifice using an electronic balance to determine the wet weight. The brain was then dried in an oven at 100°C for 24 hours to obtain the dry weight. The BW content was then calculated according to the formula: BW content (%) = (Wet weight - Dry weight) / (Wet weight) x 100

Ammonia levels

Standard operating procedures for ammonia measurement that involved collection of the sample in cooled EDTA tubes, rapid sample transport to the laboratory on ice and spectrophotometric assays. Plasm arterial and portal venous ammonia was detected by using Fujifilm Dri-Chem NX500 (Fujifilm Corporation, Japan) instrument and related cartridges.

Bacterial DNA Isolation from Plasma lOO l of fresh or thawed plasma was added to 600pl of chilled Nuclei Lysis Solution and homogenized for 10 seconds. This was followed by a 15-30 minute incubation step at 65°C. 3pl of RNase solution was added to the lysate, mixed and incubated for 15-30 minutes at 37°C. The solution was then cooled to room temperature, 200pl of Protein Precipitation Solution added and subsequently chilled on ice for 5 minutes. This was then centrifuged at 13000-16000g for 4 minutes. The supernatant was transferred to a fresh tube containing 600pl of room temperature isopropanol and mixed gently by inversion. The samples were then centrifuged at 13000-16000g for 1 minute, supernatant removed and 600pl of room temperature 70% ethanol added. Following mixing and a further centrifugation step, the ethanol was aspirated and pellet allowed to air dry for 15 minutes. The DNA was then re-suspended in lOOpl of DNA Rehydration Solution overnight at 4°C.

DNA Amplification and Sequencing (Plasma samples)

2pl of DNA template was added to a reaction mix containing: lOmmol/L Tris buffer (pH 8.3), 50 mmol/L KC1, 1.5mmol/L MgC12, 200 mol/L of each deoxynucleoside triphosphate, 50 pmol of primers 5_- AGAGTTTGATCATGGCTCAG-3_ and 5 ACCGCGACTGCTGCTGGCAC-3 , 1.25 U BioTaq (Bioline, London, England) to complete a final volume of 50pl. A 35-cycle PCR was then run in GeneAmp 9700 (Applied Biosystems, Foster City, CA) using the following profile: 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds. The total PCR reaction volume was then filtered with QIAquick Spin Columns (QIAquick PCR Purification Kit; QIAGEN) to remove rests of primers. 5mcl of purified products were then analyzed by 2% agarose gel electrophoresis and UV visualization. A band of about 540 base pairs was obtained from different bacterial cultures corresponding to the specific amplification of the prokaryotic 16S ribosomal RNA gene.

Histological analysis

Liver tissue was processed in accordance with standard protocol and Haematoxylin and Eosin together with Sirius Red staining was performed. Sirius red staining was quantified using computer assisted digital image analysis. Collagen proportionate area was determined using Zeiss KS300 image analysis software.

TUNEL assay

Enzymatic in situ labeling of cell death was assessed by In Situ Cell Death Detection Kit, POD (11684817910, Roche, Basel, Switzerland). Briefly, PFFE liver slides were dewaxed (3x xylene, 5 min) and rehydrated (Ethanol, 95%, 90%, 80%, 70% and ddH20, 3 min each). After a wash in PBS, the tissues were surrounded by permanent histology pen and incubated with 150 pL Proteinase K (03115887001, Roche, Basel, Switzerland) solution 20 pg/ml in 10 mM Tris/HCl, pH 7.4-8 for 30 min. Following, the slides were incubated with 0.1% Triton X-100, 0.1% sodium citrate (T8787, 1613859, Sigma-Aldrich, Saint Louis, MO, USA), freshly prepared for 8 min. The slides were rinsed twice with PBS and incubated with 100 pL TUNEL reaction mixture for 60 min at -37°C in a humidified atmosphere in the dark. The slides were washed again in PBS twice and incubated with 100 pl Converter-POD in a humidified chamber for 30 min at 37°C. The samples were washed with PBS and 100 pL of POD substrate was added for 10 min. Nuclei were counterstained with hematoxylin (HHS16, Sigma-Aldrich, Saint Louis, MO, USA). The slides were dehydrated and mounted.

Isolation of Non-Parenchymal Cell Fraction from Rodent Liver Tissue

Perfused liver tissue was dissected with a scalpel and homogenized in Hanks balanced salt solution (with calcium and magnesium - collagenase 0.01% and DNAse I (0.01%). The homogenate was transferred to a 50ml Falcon tube and incubated at 37°C prior to filtration through a cell strainer (100mm for rat tissue). This was then centrifuged at 500rpm for 5 minutes at 4°C and the supernatant subsequently centrifuged at 2000rpm for 10 minutes at 4°C. The supernatant was discarded and the pellet re-suspended in PF4 (HBSS with no calcium or magnesium, DNAse I 0.01%, bovine serum albumin (0.25%)) and centrifuged at 2000rpm for 10 minutes at 4°C. The pellet was then re-suspended in 3.9ml of RPMI 1640 and mixed gently with 2.1ml (RPMI and Optiprep 22%). RPMI was then layered on top followed by a 25 minute centrifugation step at 2800rpm without brake at 4°C. The non-parenchymal cells were isolated from the interface, re-suspended in an equivalent volume of PF4 and centrifuged at 2000rpm at 4°C for 10 minutes.

The pellet was re-suspended in 5ml of Red Cell Lysis Buffer (BioLegend) and incubated for 5 minutes at 4°C with occasional shaking. The reaction was then stopped by addition of 10 ml of PBS. This was then centrifuged at 2000rpm for 10 minutes at 4°C, the supernatant discarded and the cells re-suspended in 3ml of culture media. The cells were counted and adjusted to a concentration of 10⁷ cells/ml. IxlO⁶ cells were used in all subsequent assays.

Whole Blood Preparation

2ml of blood was collected from portal vein and arterial blood. 40ml of RBC lysis buffer was added to each 2ml sample, vortexed and incubated for 15 minutes at room temperature. Following centrifugation at 1500rpm (450g) for 5 minutes the supernatant was discarded and the pellet re-suspended with 1ml of complete culture media (RPMI (Gibco), 10% FBS, 200mM L-Glutamine) .The cell number using the nucleoCounter method and adjusted to lxl0⁷cell/ml.

Kupffer Cell Population studies

2ul of Fc blocker (anti-CD32 antibody) was added to 10⁶ cells (non-parenchymal cell fraction) and incubated for 5 minutes at 4°C. The cells were then co-incubated with anti-CD163 antibody for 30 minutes at 4°C in the dark. The cells were washed with 1ml of FACS buffer, centrifuged and re-suspended in 100ml FACS buffer solution and analyzed immediately on a Becton Dickinson LSR II flow cytometer.

Cell population assay

2ul of Fc blocker (anti-CD32 antibody) was added to 10⁶ cells and incubated for 10 minutes at 4°C. The cells were then co-incubated with the following primary antibodies for 20 minutes at 4°C: PE-CDl lb (0.2mg/ml), Alexa647-CD43 (0.5mg/ml), FITC-HIS48 (200ug/ml). After incubation, 2 ml of FACS buffer was added to each tube, centrifuged at 1500rpm (450xg) for 5 minutes at 4°C and supernatant discarded. This last step was then repeated with 1ml FACS solution and the pellet re-suspended the pellet in lOOul of FACS buffer. 5ul of DAPI solution was added into the each tube prior to analysis.

Kupffer Cell and Portal Venous ROS studies

20ug/ml of E.coli endotoxin was added to lx 10⁶ non-parenchymal cells sample and incubated for 30 minutes at 37°C. ROS inducer at a final concentration of 200- 500uM was used as a positive control. The samples were then centrifuged at 500g for 5 minutes and the supernatant discarded. The cells were re-suspended in 5ml of wash buffer, centrifuged at 500g for 5minutes and the supernatant removed. The cells were re-suspended in 500ul of ROS detection solution and incubated for 30 minutes at 37°C in the dark. Following centrifugation, the cells were re-suspended in lOOul of FACS buffer, Fc blocker added (1 :25) and incubated for lOminutes at 4°C. Anti-CD163 antibody was added and the cells incubated for 30 minutes at 4°C in the dark (Kupffer cells). The cells were then washed with 1ml of FACS buffer, centrifuged and resuspended in 100ml FACS buffer solution. All samples were kept at 4°C and analyzed immediately using a FACS LSR II machine. Data was analyzed using FlowJo software.

Cytokine analysis Plasma cytokine levels were measured in EDTA anti -coagulated plasma using a Bio-Plex Pro rat cytokine assay kit (R & D systems) and a Bio-Plex Magpix instrument (Bio-Rad Laboratories Ltd., Watford, UK) according to the manufacturer's instructions. The cytokines measured were IL-ip, IL-6, IL-10 and TNF-a.

Endotoxin measurement

The chromogenic limulus amoebocyte lysate kinetic assay (Charles River Laboratories) was used for the detection of endotoxin. Portal venous plasma (lOOmcl) was diluted 1 : 10 with endotoxin-free water and incubated at 75°C for 30 minutes. lOOmcl of sample and lOOmcl of LAL reagent were mixed in a 96-well plate and analyzed at 405nm with spectrophotometer using the Endoscan V software. Results are expressed as EU/ml.

Statistical Analysis

All groups of results were tested for normality and subsequently analyzed by parametric or non-parametric analysis as appropriate. P-value <0.05 was considered statistically significant. Software used included Graphpad Prism 9.0 (GraphPad software, Inc., San Diego, CA).

Results

In Vitro studies

Example 1: Investigating whether Yaq-001 exhibits the appropriate pore size distribution to bind endotoxin

Electron microscopy and mercury porosimetry were performed on Yaq-001 samples. The Yaq-001 beads exhibited a consistent macroporous structure with a bead diameter within the 250-500pm range and internal nanoporosity was present at high magnification (Figure 5). Mercury porosimetry showed that Yaq-001 had a consistent pore size distribution plot in the meso-macroporous range from 30-200 nm (Figure 6).

Example 2: Investigating whether Yaq-001 has ability to adsorb larger biomolecules in addition to smaller biomolecules

Having established that Yaq-001 was comprised of a wide range of porosities including meso-macroporous domains, it was determined whether this was associated with ability to adsorb larger biomolecules such as albumin (66.5kDa) and myoglobin (16.7kDa) in addition to smaller biomolecules such as caffeine (0.194kDa) as these are measures of macro, meso and microporous binding respectively (Figure 7). Yaq-001 rapid adsorbed all 3-types of biomolecules. Adsorption studies of endotoxin (18kDa) were subsequently conducted. The concentration of endotoxin detected in the SIF solution initially spiked with 10 EU ml'¹, however, it declined to 2.5 EU ml'¹ detected value at time 0. The concentration of endotoxin adsorbed from the simulated intestinal fluid (SIF) solution was 1.5 EU ml'¹ after 30 minutes of incubation and remained constant for the next 90 minutes. Therefore, after 30 minutes, Yaq-001 sample adsorbs 60% (from 2.5 to 1.5 EU mL'¹) spiked endotoxins. The control solution, on the other hand, did not detect any endotoxin and maintained a steady 0 EU ml'¹ concentration over time. Yaq-001 samples achieved endotoxin adsorptive equilibrium at 30 minutes, demonstrating its ability to remove endotoxins rapidly (Figure 7). Furthermore, Yaq- 001 was also found to rapidly adsorb a range of bile acids including sodium glycochenodeoxy cholate hydrate, sodium glycocholate hydrate, lithocholic acid, chendeoxycholic acid, cholic acid and deoxycholic acid (Figure 8).

Example 3: Investigating whether Yaq-001 affects bacterial growth kinetics

Having established that Yaq-001 was a potent endotoxin adsorbent, its effects on bacterial growth kinetics were evaluated. Luminescence is an indirect measure of cell viability and cell number via the determination of bacterial adenosine triphosphate. Optical density values were comparable between Yaq-001 and the no adsorbent control for both species over 72 hours. Direct co-incubation of Yaq-001 with bacterial suspensions of either Escherichia coli or Staphylococcus aureus indicated that Yaq-001 does not affect bacterial growth kinetics for either species following direct contact (Figure 9).

In Vivo studies

Example 4: Yaq-001 results in attenuation of liver injury and portal pressure in bile-duct ligated (BDL) rats

To evaluate the in vivo effect of oral Yaq-001 administration, the 4-week BDL rat model was used as a model of advanced fibrosis (Figure 10). Animals were treated with Yaq-001 for the last 2 weeks (0.4g/100g body weight/day). The four groups were as follows: Sham (n=30); Sham+Yaq-001 (n=23); BDL (n=30); BDL+Yaq- 001(n=37).This model is known to exhibit features of bacterial translocation, portal hypertension, immune and organ dysfunction and thus were the ideal models to ascertain efficacy of Yaq-001. 120 animals in all the four groups were alive at the end of 4 weeks. BDL rat was associated with a significant reduction in 4-week body weight compared to Sham controls [(median (IQR) 428g (396-450) vs 471g (455-487), (p<0.0001)] and 2-week treatment with Yaq-001 resulted in a significant increase in final body weight compared to untreated-BDL rats (Figure 11) [(median (IQR) 439g (IQR 410.8-472.5) vs 428g (396-450), (p=0.045)].

Yaq-001 treatment was associated with a significantly lower plasma ALT in BDL rats compared to untreated-BDL controls (Figure 12) [(median (IQR) 62.5 lU/ml (IQR 47.9-77.8) vs 82.8 (61.7-102.5)) (p=0.012)]. A considerable reduction in ALT was also observed between Yaq-001 -treated and untreated- Sham controls [(median (IQR) 33.7 lU/ml (30.9-41.3) vs 46.9 lU/ml (43.0-51.5)) (p=0.007)]. BDL rat was also associated with a remarkable increase in ALP, TBIL and a significant reduction in albumin compared to Sham controls (Figure 13A, B, C). However, Yaq-001 had no significant effect on these biological markers. Yaq-001 -treated Sham rats had a significantly lower bile acid concentration compared to untreated- Sham rats [(median (IQR) 14.3 (4.4-31.7) vs 106.7 (17.0-195.5) (p=0.042) (Figure 14E). There was no significant difference of bile acid concentration in Yaq-001 -treated BDL versus untreated-BDL animals.

MAP was lower in BDL animals compared to Sham controls (Figure 14F) [(median (IQR) 84.7 mm Hg (67.5-101.00) vs 110.4 mm Hg (104.2-122.0)), (p=0.0002)]. No significant difference in MAP was observed between untreated or Yaq- 001 -treated BDL groups. Yaq-001 therapy was associated with a significant reduction in portal pressure compared to untreated controls in BDL rats (Figure 12) [((median (IQR) 11.1 mm Hg (10.30-11.72) vs 12.4 mm Hg (10.8-13.3)), (p=0.015)].

TUNEL assay was performed on liver tissue to ascertain degree of liver cell death. Significantly higher staining was observed in the liver tissue of BDL compared to Sham rats (Figure 12A) (p<0.0001). A significant reduction in TUNEL assay was observed in Yaq-001 -treated BDL rats compared to untreated-BDL rats (p=0.025). Collagen proportionate area (CPA) was determined on Picrosirius Red (PSR) stained liver tissue to ascertain degree of fibrosis. A significantly higher CPA was observed in untreated-BDL rats compared to Sham (Figure 15) indicative of increased collagen deposition in the model. No significant differences were observed between Yaq-001 treatment and untreated controls in either Sham or BDL groups (p=0.211).

Example 5: Effect of Yaq-001 on organ dysfunction, endotoxemia and bacterial translocation in BDL rats

Brain

Arterial ammonia concentrations were significantly increased in BDL rats compared to Sham rats (p<0.0001), which was significantly reduced by Yaq-001 treatment (Figure 16) [(median (IQR) 143.9 pmol/L (126.0-167.8) vs 185.8 pmol/L (149.7-227), (p=0.003)]. Portal venous ammonia concentrations were also significantly increased in BDL compared to Sham rats (p=0.0002), which was significantly reduced [(median (IQR) (230.5 pmol/L (176.0-290.1) vs 341.0 pmol/L (273.2-435.7), (p=0.001)] with Yaq-001 therapy (Figure 16).

Kidneys

BDL animals had higher plasma creatinine compared with Sham group (p=0.064), which was significantly reduced on treatment with Yaq-001 [(median (IQR) 29.8pmol/L (28.0-31.9) vs 33.0pmol/L (28.3-36.8), (p=0.047)] (Figure 17). Urea was significantly higher in BDL group compared with Sham (p=0.044), which was significantly reduced with Yaq-001 treatment [(median (IQR) (4.9 mmol/L (4.3-5.5) vs 5.3 mmol/L (4.6-6.1), (p=0.042)] (Figure 17).

Gut permeability, Endotoxemia and Bacterial DNA

Plasma D-lactate levels were measured, which is produced by the microbiome and its presence in the plasma reflects gut permeability. Significantly increased D- lactate was observed in BDL rats compared to Sham rats (p=0.024) (Figure 18G), which was significantly reduced in Yaq-001 -treated compared to untreated-BDL rats [(median (IQR) (295.1pmol/L (261.4-384.2) vs 567.3 pmol/L (314.4-683.2), (p=0.02)].

BDL rats exhibited marked endotoxemia as shown by significantly higher portal venous endotoxin (p<0.0001) and arterial endotoxin concentrations compared to Sham rats (p<0.0001) (Figure 18H). Yaq-001 treatment significantly reduced portal venous and arterial endotoxin concentrations [(median (IQR) 0.54 lU/ml (0.45-0.63) vs 1.30 lU/ml (1.04-1.48), (p<0.0001)] and [(median (IQR) (0.40 lU/ml (0.34-0.51) vs 0.78 lU/ml (0.62-1.08), (p=0.004)] respectively (Figure 18H). Portal venous bacterial DNA was detectable in significantly higher number of BDL rats compared to Sham rats (p<0.05), which was markedly reduced in Yaq-001 administered BDL rats (p=0.08) (Figure 18H). Yaq-001 had no effect in arterial bacterial DNA positivity.

Example 6: Effect of Yaq-001 on multiorgan function in a model of ACLF

This experiment was performed to determine whether Yaq-001 prevents the progression of cirrhotic rats to multiorgan failure and associated mortality following a precipitating injury induced by LPS. The four groups were as follows: Sham+LPS (n=9); Sham+LPS+ Yaq-001 (n=10); BDL+LPS (n=16); BDL+LPS+Yaq-001(n=12) (Figure 19A).

Survival

Animals were sacrificed either at coma stages (considered as a surrogate for mortality) or at 6-hours post LPS. Yaq-001 treatment significantly reduced time to coma of BDL+LPS rats compared to untreated rats (Figure 19B) (p<0.01), which was associated with reduction in severity of liver injury, portal hypertension, brain edema and ammonia, kidney injury and markers of systemic inflammation.

Liver

Yaq-001 was associated with significantly lower ALT in BDL+LPS rats compared to untreated rats (Figure 19C) [(median (IQR) 66.4 lU/ml (54.1-76.7) vs 104.7 (78.8-113.1), (p=0.002)]. No significant effect of Yaq-001 was observed on ALP, TBIL and albumin (Figure 20A, B, C). The severity of fibrosis measured using CPA and the body weight were unchanged (Figure 21D, E).

Systemic and Portal hemodynamics

No significant difference in MAP was observed between untreated or Yaq-001- treated BDL+LPS groups (Figure 21F). Yaq-001 therapy was associated with a significant reduction in portal pressure compared to untreated controls in LPS-treated BDL rats [(median (IQR) 11.6 mm Hg (7.8-13.4) vs 15.6 mm Hg (13.3-18.4), (p=0.003)], (Figure 19C).

Brain

Yaq-001 administration significantly reduced brain water compared with untreated- BDL+LPS rats [median (IQR) (78.9% (78.1-80.0) vs 80.3% (79.4-80.5), (p=0.014)]. Arterial and portal venous ammonia concentrations were significantly increased in BDL+LPS rats compared to Sham+LPS rats (Figure 22), which was significantly reduced in Yaq-001 -treated animals [(median (IQR) arterial: 232.0 pmol/L (199.0-278.0) vs 321.0 pmol/L (288.0-333.0) (p=0.007); portal venous: 333 pmol/L (282.0-386.5) vs 416.0 pmol/L (384.1-451.7), (p=0.032)], (Figure 22) compared to untreated-BDL+LPS rats.

Kidneys

Creatinine concentrations were significantly higher in BDL+LPS animals compared with Sham+LPS group (p=0.019) which was significantly reduced in Yaq- 001-treated BDL+LPS groups [median (IQR) (29.6 pmol/L (26.7-33.6) vs 40.5pmol/L (27.2-55.3), (p=0.02)], (Figure 23). Although urea levels were significantly higher in BDL+LPS group compared with Sham+LPS group (p=0.004), it did not change significantly with Yaq-001.

Markers of systemic inflammation

BDL+LPS group had a significantly higher plasma IL-ip concentrations compared to Sham+LPS (p<0.01), which was significantly reduced with Yaq-001 therapy (median (IQR) 93.54 (19.22-472.1) vs 1374 (56.02-3141), (p<0.05). No significant differences were observed with IL-6, TNF-a or IL- 10 concentrations between any of the groups.

Example 7: Effect of Yaq-001 on peripheral blood cells and Kupffer cells

Significant expansions in total leucocyte, neutrophil and monocyte counts in the artery and portal vein were observed with BDL rats compared to Sham controls (Figure 24) (artery: p=0.0009, p=0.0004, p=0.016; portal vein: p=0.0001, p=0.0002, p=0.018 respectively). Yaq-001 therapy resulted in a significant reduction in neutrophil count (1.8x l0⁷ (0.8-2.2) vs 3.3 * 10⁷ (2.5-4.3) (p=0.03)) with non-significant downward trends observed in total leucocyte and monocyte counts in portal vein compared to untreated- BDL rats (7.0x l0⁷ (3.2-9.4) vs 11.4x l0⁷ (8.3-13.7), (2.3x l0⁷ (1.1-3.2) vs 3.0x l0⁷ (1.6- 6.1)) (p=0.078, p=0.378). Yaq-001 significantly decreased the circulating total leucocyte and neutrophil levels in the artery (4.3 x lO⁷ (4.0-5.1) vs 7.6x l0⁷ (6.0-12.3), (1.4X 10⁷ (1.2-1.4) vs 2.2x l0⁷ (2.0-3.4)) (p=0.04, p=0.03).

In order to determine whether Yaq-001 impacts on response of peripheral inflammatory cells and Kupffer cells to generate ROS to LPS, ex vivo studies using isolated cells incubated with LPS were performed. An increase in constitutive and LPS- induced ROS production in monocyte/macrophage and neutrophil populations was observed in untreated-BDL rats compared to Sham controls. This is indicative of a heightened primed state to endotoxin challenge. Yaq-001 therapy was associated with significantly lower LPS-induced ROS production in CD 163" Kupffer cells in BDL rats (Figure 25) (3.2% (2.4-5.2) vs 9.3% (7.1-10.7)) (p=0.006) and portal venous CD43¹¹¹ monocyte populations of BDL rats (14.5% (14.3-22.0) vs 29.6% (27.2-34.7)) (p=0.01).

Example 8: Effect of Yaq-001 on gene expression profiles in the liver, gut, brain and kidney in BDL rats

Having established that Yaq-001 attenuates liver injury, improves portal pressure and reduces multiorgan dysfunction in two models, a multiorgan transcriptomic analysis was performed to determine the molecular mechanisms by which this was occurring. The four groups were as follows: Sham (n=3), Sham+Yaq- 001(n=3), BDL (n=3) and BDL+Yaq-001(n=4). In the volcano plots, vertical dashed lines indicate the threshold for 1.2-fold change whereas horizontal dashed line indicates adjusted p=0.05 and p=0.1 thresholds.

Methodology - RNA isolation and NanoString gene expression analysis

Liver, colon, brain and kidneys from three rats in Sham, Sham-Yaq-001, BDL groups and four in BDL- Yaq-001 group were randomly selected to perform transcriptome analysis. Total RNA was isolated and then cleaned up by using QIAzol Lysis Reagent (79306 Qiagen, CA, USA), and RNeasy Mini Kit (74104 Qiagen), respectively, according to the manufacturer’s protocol. RNA integrity was analyzed with the Agilent RNA 6000 Nano chip on the Agilent 2100 Bioanalyzer and only samples with R.I.N. above 8 were kept. For NanoString gene expression analysis, we used 100 ng RNA for inflammation profiling with a Customized NanoString nCounter CDR-RatCirrh-21590 panel including 167 genes (NanoString Technologies). Each 5pl RNA sample was hybridized with 8 pl nCounter Reporter probe in hybridization buffer, and 2pl nCounter Capture probes at 65°C for 16-30 h. Excess probes were removed through a twostep magnetic bead-based purification procedure using the nCounter Prep Station (NanoString Technologies). Specific target molecule abundance was quantified using the nCounter Digital Analyzer to count individual fluorescent barcodes, whereby the corresponding target molecules were assessed. Each assay involved a high-density scan encompassing 280 visual fields. Images of the immobilized fluorescent reporters in the sample cartridge were acquired using a CCD camera, and then data were collected using the nCounter Digital Analyzer. Data analysis was performed using nSolver4.0 software (NanoString Technologies). The mRNA profiling data were normalized against housekeeping genes and analyzed using R software (www.rproject.org). Liver

Analysis of liver tissue showed 82 DEGs at the threshold of 1.2-fold change and p=0.1 in four groups. Compared with Sham group, expression of 62 genes were upregulated and 15 genes were downregulated in BDL. These significantly changed genes were associated with inflammation (representative genes such as C-X-C motif chemokine ligand 16, interferon gamma receptor 1 and toll like receptor 1), cell death (representative genes such as BCL2 apoptosis regulator and caspase 1), and cell senescence (representative genes such as transforming growth factor beta 2). Compared to untreated-BDL group, the expression of 7 genes (representative genes such as C-C motif chemokine ligand 24 and bone morphogenetic protein 1) were upregulated and 12 genes (representative genes such as toll like receptor 3, transforming growth factor beta 2 and caspase 1) were downregulated in the Yaq-001 -treated BDL group, indicating the potential role of Yaq-001 in reducing inflammation, cell death and cell senescence. Furthermore, 2 genes were upregulated, and 4 genes downregulated in Sham+ Yaq-001 group in comparison to Sham group.

Functional analysis demonstrated that BDL rats had enriched pathways related to inflammation (cytokine-cytokine receptor interaction, Th 17 cell differentiation, Thl and Th2 cell differentiation, cell adhesion molecules, leukocyte transendothelial migration, neutrophil extracellular trap formation), cell senescence (TGF-beta signaling pathway, cellular senescence), cell death (necroptosis, cytosolic DNA-sensing pathway, apoptosis), TLR signaling (Toll-like receptor signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway) and other related signaling in comparison with Sham. These results were consistent with previous studies and further indicated the important role of biological processes in the progression of cirrhosis (Figure 26A-C). Yaq-001 treatment could target the altered pathways comparing with untreated-BDL group, indicating potential mechanisms in preventing the progression of cirrhosis. Meanwhile, Yaq-001 treatment also changed the pathways in liver when compared to Sham group, demonstrating the effect in rats even without cirrhosis or in the early stage (Figure 26A-C)

Colon

To elucidate the transcriptional changes upon Yaq-001 treatment in colon, 43 DEGs were identified from the colonic tissue as shown in heatmap. The volcano plots demonstrated that 5 genes correlated with inflammation (representative genes such as C-X-C motif chemokine ligand 1 and C-C motif chemokine ligand 7) and cell death (representative genes such as Fos proto-oncogene and RELA proto-oncogene) were found to be upregulated, and 15 genes (representative genes such as TNF superfamily member 10, vascular endothelial growth factor A, interleukin 15) were downregulated in BDL compared with Sham group. Moreover, the expression of 10 genes (representative genes such as vascular endothelial growth factor A and C-X-C motif chemokine ligand 10) were upregulated, and 13 genes (representative genes such as RELA proto-oncogene and Fos proto-oncogene) were downregulated with Yaq-001 treatment compared to untreated-BDL group. Only 1 gene was upregulated in Sham+ Yaq-001 group, and 16 genes were downregulated with Yaq-001 compared with untreated- Sham group.

Functional analysis also indicated that inflammation (cytokine-cytokine receptor interaction, Thl7 cell differentiation, Thl and Th2 cell differentiation), cell senescence (TGF-beta signaling pathway), cell death (necroptosis, cytosolic DNA-sensing pathway, apoptosis, natural killer cell mediated cytotoxicity), TLR signaling (Toll-like receptor signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway) and intracellular signaling were associated with BDL in comparison with Sham (Figure 27A-C). Compared with the untreated-BDL group, Yaq-001 targeted the altered pathways, indicating the potential mechanisms in the prevention of gut dysfunction and permeability. Besides, Yaq-001 treatment of Sham animals also changed similar pathways in colon when compared to Sham group (Figure 27A-C).

Brain

To elucidate the transcriptional changes induced by Yaq-001 treatment in the brain, 17 DEGs were identified from the brain tissue. Compared with Sham group, expression of 2 genes were upregulated and 13 genes were downregulated in BDL groups. These significantly changed genes were associated with inflammation (representative genes such as interleukin 23 subunit alpha, C-C motif chemokine receptor 5), cell death (representative genes such as interleukin 18, interferon regulatory factor 7 and RNA sensor RIG-I), and cell senescence (representative genes such as transforming growth factor beta 1 and transforming growth factor beta 2). Compared to the untreated-BDL group, the expression of 5 genes (representative genes such as interleukin 18, transforming growth factor beta 2 and toll like receptor 7) were upregulated and 2 genes (representative genes such as interferon regulatory factor 7 and interleukin 23 subunit alpha) were downregulated in the Yaq-001 -treated BDL group. Functional analysis demonstrated that BDL rats had enriched pathways related to inflammation (cytokine-cytokine receptor interaction, Th 17 cell differentiation), cell senescence (TGF-beta signaling pathway), cell death (cytosolic DNA-sensing pathway), TLR signaling (Toll-like receptor signaling pathway, NF-kappa B signaling pathway) and intracellular signaling in comparison with Sham (Figure 28A-C). Yaq-001 treatment could target cytokine-cytokine receptor interaction, cytosolic DNA-sensing pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and Neutrophil extracellular trap formation pathways compared to untreated-BDL group (Figure 28A-C). Yaq-001 treatment also changed the pathways including TGF-beta signaling pathway and cytokine-cytokine receptor interaction in brain when compared to Sham group (Figure 28A-C).

Kidneys

To elucidate the transcriptional changes induced by Yaq-001 treatment on kidneys, 30 DEGs were identified from kidney tissue. The volcano plots demonstrated that 9 genes correlated with inflammation (representative genes such as toll like receptor 8, intercellular adhesion molecule 1 and integrin subunit alpha M) were found to be upregulated, and 10 genes (representative genes such as interleukin 1 receptor associated kinase 1, bone morphogenetic protein 7 and C-X-C motif chemokine ligand 11) were downregulated in BDL compared with Sham group. The expression of 5 genes (representative genes such as adiponectin and C-C motif chemokine ligand 21) were upregulated, and 4 genes (representative genes such as toll like receptor 3 and signal transducer and activator of transcription 1) were downregulated with Yaq-001 treatment compared to untreated-BDL group. 5 genes were upregulated in Sham+Yaq-001 group, and 3 genes were downregulated with Yaq-001 compared with untreated-Sham group.

Functional analysis also indicated that inflammation (cytokine-cytokine receptor interaction, Th 17 cell differentiation, cell adhesion molecules, neutrophil extracellular trap formation, Chemokine signaling pathway) and TLR signaling (Toll-like receptor signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway) was associated with BDL in comparison with Sham (Figure 29A-C). Compared with the untreated-BDL group, Yaq-001 targeted the altered pathways, indicating the potential mechanisms in the prevention of renal dysfunction. Besides, Yaq-001 treatment of the Sham animals also changed similar pathways in kidneys when compared to Sham group (Figure 29A-C). Example 9: Effect of Yaq-001 on the microbiome profile

Having characterized the effects of Yaq-001 on the transcriptome in different organs, the effects of non-absorbable Yaq-001 on the microbiome composition were characterized with 16S studies.

Analysis of faecal microbiota was performed as follows. DNA was extracted from 200 mg of faecal pellets, one pellet per animal, and DNA was combined within treatment groups. Total DNA was extracted using an initial bead-beating step and the QIAamp DNA stool mini kit (Qiagen, West Sussex, UK). Universal 16SrRNA primers, designed to amplify from highly conserved regions corresponding to those flanking the V4 region, i.e. the forward primer Fl (5'-AYTGGGYDTAAAGNG) and a combination of four reverse primers R1 (5'-TACCRGGGTHTCTAATCC), R2 (5'- TACCAGAGTATCTAATTC), R3 (5'-CTACDSRGGTMTCTAATC) and R4 (5'- TACNVGGGTATCTAATC) (RDP’s Pyrosequencing Pipeline: http://pyro.cme.msu.edu/pyro/help.jsp) were used for Taq-based PCR amplification. Sequencing was performed on a Roche 454 GS-FLX using Titanium chemistry by the Teagasc 454 Sequencing Platform (Teagasc, Fermoy, Ireland). Resulting reads were quality trimmed, clustered, aligned and checked for chimeras using the Qiime suite of tools. The reads for the major phyla were averaged for each group and expressed as a percentage of the total number of reads for that particular group.

Differential abundant analysis was performed based on the bacterial abundance between groups, the bacteria with the p value of pairwise test lower than 0.5 (Wilcoxon Rank Sum Test, p<0.5, Sham vs BDL and BDL vs BDL-C) or the bacteria with greater than four-fold variation between groups (log2FC>2, Sham vs BDL and BDL vs BDL-C) were selected into visualization at both genus and family level.

At the family level, an abundance of 7 bacteria was identified that were significantly altered at the threshold of 2-fold change or p=0.05 in pairwise groups comparing BDL with Sham group (Figure 30). Additionally, on the genus level, 20 bacteria with significantly altered abundance were identified (Figure 31). These changes were reversed with Yaq-001 treatment compared to untreated-BDL rats. The Venn diagram plot of the changes in the microbiome with Yaq-001 at family and genus level are shown in Figure 32C-D.

To further investigate the potential importance of the changes in the microbiome induced by Yaq-001, the changes were correlated with all significantly changed DEGs and the top 20 DEGs in the four organs. Circos plot indicated significant correlation between them (Figure 31A).

The Spearman’s rank correlation coefficient was calculated between these datasets. For this purpose, raw metagenomics abundance data was correlated with raw gene expression data or raw metabolite abundance data across samples using the spearmanr function as part of the scipy package in python3.10.

Circos plot indicated significant correlation between them. Porphyromonadaceae, which is significantly different between groups, was observed to positively correlate with three DEGs - transforming growth factor beta 2 and caspase 1 in liver tissue, and fos proto-oncogene in colon tissue. Also, this genus was negatively correlated with five DEGs-transforming growth factor beta 2, Interleukin 18 and C-C motif chemokine receptor 5 in brain tissue, C-X-C Motif Chemokine Ligand 10 in colon tissue and C-C Motif Chemokine Ligand 24 in kidney tissue.

Discussion

This study explored the role of a novel therapeutic agent, Yaq-001, which is a highly engineered, multi porous, insoluble, non-absorbable carbon adsorbent that is completely excreted following oral ingestion, in inflammation, using models of cirrhosis and ACLF. The study showed that despite never leaving the gut, administration of Yaq-001 impacted positively on markers of gut permeability, brain and kidneys in rodent models of advanced fibrosis and ACLF. These pleiotropic effects of Yaq-001 were associated with restoration of the composition of the microbiome, reduction in the severity of endotoxemia and ammonia, which impacted on the severity of inflammation, cell death, intracellular signaling and LPS sensitivity.

Many lines of investigation provide evidence that bacterial translocation, for instance of bacterial LPS, plays a key role in driving systemic inflammation and resultant organ failure in many diseases. Indeed, selective gut decontamination using norfloxacin or rifaximin are considered the current standard of care for secondary prophylaxis of patients with, for example, spontaneous bacterial peritonitis and hepatic encephalopathy respectively. However, the use of these antibiotic strategies induces the risk of antibiotic resistance. Furthermore, antibiotics result in endotoxin generation and have a variable influence on endotoxin kinetics dependent on antibiotic class. The data presented here provide an alternative non-antibiotic strategy, Yaq-001, which has the potential to diminish translocation and improve organ injury, as well as reduce systemic inflammation. The studies demonstrate in in vitro studies that Yaq-001 has the optimal pore size distribution to bind intraluminal factors that are relevant to the acceleration of systemic inflammation. Yaq-001 has a high affinity for free endotoxin without significant effect on bacterial growth kinetics.

Endotoxemia has also been implicated in the pathogenesis of both innate and adaptive immune dysfunction resulting in a dysregulated systemic inflammatory response syndrome, which is strongly associated with the progression of many disease states and disorders. Yaq-001 treatment reduced the severity of endotoxemia as shown by significantly decreased arterial/portal venous, endotoxin levels and portal venous bacterial DNA positivity. Furthermore, Yaq-001 therapy attenuated systemic inflammation, as evidenced by reduced numbers of leucocyte and neutrophils in circulation. In addition, significant improvements in LPS-induced ROS production were observed in trafficking portal venous monocytes suggesting that Yaq-001 had attenuated the primed state of monocyte/macrophage populations within the gut-liver axis in association with the reduction in portal venous endotoxin. This observed reduction in LPS-induced ROS production may be important in explaining the reduction in plasma IL-ip in LPS-treated BDL rats.

Accumulating evidence demonstrates that gut microbiome is important in modulating gut health, permeability, bacterial translocation and systemic inflammation. In this study, BDL was associated with evidence of marked dysbiosis characterized by changes in the microbiome abundance at the family and genus levels, which were reversed by Yaq-001 treatment. In particular, the abundance of Porphyromonadaceae and Barnesiella were significantly elevated in BDL rats and significantly decreased with Yaq-001 treatment. This is consistent with previous studies, which reported that Porphyromonadaceae is a pro-inflammatory microbiome that has been positively correlated with hepatic encephalopathy. Abundance of Barnesiella and Porphyromonadaceae has also been associated with advanced liver cancer.

These effects on the microbiome and the severity of endotoxemia were associated with a reduction in plasma D-lactate, which is produced by microorganisms and its presence in the circulation is a marker of gut permeability. Transcriptomic analysis of colonic tissue demonstrated evidence of upregulation of genes associated with necroptosis, apoptosis and inflammation in the colon in BDL animal compared with controls. Functional analyses pointed to cytokine-cytokine receptor interaction, toll-like receptor signaling pathways, TNF signaling pathway, chemokine signaling pathway and IL- 17 signaling pathway as the top 5 significantly changed pathways in BDL rats, which were significantly impacted following administration of Yaq-001. The data allow us to hypothesize that the reduction in intestinal permeability, endotoxemia and systemic inflammation were possibly mediated by Yaq-001 -induced reduction in gut inflammation, cell death and cell signaling.

Yaq-001 significantly reduced the severity of liver injury and portal hypertension in both models of advanced fibrosis and ACLF. Of note, Yaq-001 therapy did not impact negatively on mean arterial pressure suggesting that the hemodynamic effects are confined to the portal circulation. The lack of significant difference in collagen proportionate area between untreated and Yaq-001 -treated BDL groups suggests that the reduction in portal pressure is not a function of changes in fixed intrahepatic resistance but modulation of the inflammatory component of portal hypertension at a sinusoidal level. The most marked reduction in portal pressure was observed in LPS-treated BDL rats suggesting that Yaq-001. The importance of inflammation in driving severity of portal hypertension has been highlighted previously and proposes Yaq-001 as a novel treatment for portal hypertension. Reduction in ALT levels and TUNEL assay of the liver tissue confirmed a reduction in cell death in the hepatocytes of BDL rats treated with Yaq-001. Like portal pressure, the severity of liver injury was marked attenuated in the LPS treated BDL animals suggesting that Yaq-001 has a particular effect on in vivo endotoxin sensitivity This hypothesis was studied in isolated Kupffer cells, which confirmed that LPS-induced ROS production was significantly impacted by Yaq-001 treatment suggesting that diminished translocation of bacterial products results in diminished Kupffer cell priming and therefore, LPS- induced ROS production.

Transcriptomic analysis of liver tissue demonstrated that the upregulated genes, CXCL16, CASP1 and TGF-P2 in BDL rats was prevented by Yaq-001 administration. In line with our findings, a previous study reported that silencing of CXCL16 alleviates hepatic ischemia reperfusion injury and CXCL16 variant is also associated with Hepatitis B virus related acute liver failure. CASP1 mediates pro-inflammatory cytokine release and pyroptotic cell death in cirrhosis and its inhibition has been shown to prevent ACLF. TGF-P2 is an important mediator of cellular senescence and induces hepatic fibrosis indicating potential role for Yaq-001. Yaq-001 significantly impacted on many of the pathways related to inflammation and TLR signaling that were elevated in BDL rats. Of note, Yaq-001 also modified necroptosis and cytosolic DNA-sensing pathways representing cell death in liver. Both pyroptosis and necroptosis are known to be activated by LPS and are immunogenic forms of cell death that can trigger further cell death and lead to systemic inflammation. The effect of Yaq-001 on these cell death pathways serves to explain the effect of Yaq-001 in reducing liver injury as targeting both forms of cell death are potential therapeutic targets for ACLF.

Yaq-001 therapy significantly impacted on time to coma of ACLF rats compared to untreated controls, which was associated with reduced brain water. Yaq-001 also significantly lowered ammonia levels both in the portal vein and the systemic circulation supporting its ammonia adsorptive property in the gut. Transcriptomic analysis of brain tissue showed that IL18, TGF-P2, CCR5 and IL23a were dysregulated in BDL rats and these were corrected by Yaq-001 providing insight into potential mechanism of the neuroprotective effect of Yaq-001. IL18 is released during pyroptosis by activation of the inflammasome complex in neuroinflammatory and neurodegenerative diseases. The effect of Yaq-001 on TGF-P2 suggests that it may impact on senescence, which is known to be associated with hepatic encephalopathy. CCR5 has been implicated in neuroprotection and is novel therapeutic target in stroke. The impact of Yaq-001 on IL23a indicates possible reduction in neuroinflammation. Yaq-001 impacted in particular on inflammatory pathways such as cytokine-cytokine receptor interaction, cytosolic DNA-sensing, toll-like and NOD-like receptor signaling and neutrophil traps, which were abnormally expressed in BDL rats.

In both the animal groups with cirrhosis and with ACLF, Yaq-001 treatment was associated with reduced renal dysfunction measured using creatinine levels. Transcriptomic analysis of kidney tissue showed that CCL24 was downregulated in BDL rats, which was prevented in the Yaq-001 -treated animals. CCL24 protects renal function in the development of early diabetic nephropathy by exerting an antiinflammatory effect. Yaq-001 impacted, in particular on the cytokine-cytokine receptor interactions and chemokine and toll-like signaling pathways, which were abnormal in the BDL rats.

The BDL animals tend to become sarcopenic and lose weight, which was significantly abrogated in the Yaq-001 -treated animals. The mechanisms underlying this are multifactorial. Yaq-001 reduced ammonia significantly, which has been shown to induce sarcopenia. Weight loss in cirrhosis is also attributed to an increased catabolic state in the context of systemic inflammatory response and thus the observed improvement in body weight may reflect the diminished catabolic state with reduced inflammation. These data also further emphasize the safety profile of the intervention and suggest that during the study period, there were no deleterious effects of the nutritional status but with this study it is not possible to comment on an effect on micronutrients and vitamins.

The data correlating the changes in the microbiome induced by administration of Yaq-001 with changes in the gene expression of multiple relevant pathways is particularly important as it achieves the beneficial effects in the distant organs such as the liver, brain and kidneys with demonstrable changes without leaving the gut. Without wishing to be bound by theory, one possibility is that LPS adsorption changes the milieu of the gut allowing proliferation of more autochthonous microbial profile, which impacts on gut inflammation that reduces gut permeability leading to a reduction in endotoxemia, systemic and organ inflammation, organ priming and improvement of organ function and LPS-sensitivity. In this study, each of these changes have been described but whether this is happening in sequence has not been studied.

Example 10: Randomised., Placebo-Controlled Trial of Oral Yaq-001

In this double-blinded, randomised, placebo-controlled, 28 patients with diuretic-responsive cirrhotic ascites (Child-Pugh 7-11) were randomised 1 : 1 to 4g nocte of Yaq-001 or equivalent placebo. The primary endpoint of safety and tolerability was met. Secondary endpoints included blood endotoxin, organ function and nutritional status in which no significant difference was observed. Trends to reductions in whole blood ROS, leucocyte count, CRP, IL6, CXCL10, plasma D-lactate, faecal IL17a and TNFa were observed in the active group. Reductions in pathogenic bacterial species were observed in Yaq-001 -treated vs placebo control.

Introduction

Multiple lines of evidence have implicated the microbiome in. Bacterial products, such as endotoxin, translocate from the gut lumen to the liver and systemic circulation driving a dysregulated inflammatory response resulting in organ injury. Current interventional strategies to modulate this process are limited to oral antibiotics with the attendant risk of superinfection and antimicrobial resistance and as such, are administered chronically for secondary prophylaxis of hepatic decompensation. Safer, alternative therapeutic strategies which may be utilised either as primary or secondary prophylaxis, remain an unmet clinical need. Yaq-001 is a novel synthetic carbon with a high adsorptive capacity for biologically relevant pathogenic mediators such as endotoxin and cytokines. Yaq-001 has been tailored to a pore size distribution relevant to the adsorption of gut-derived pathogenic factors related to the pathogenesis of liver disease. The bimodal porosity of Yaq-001 is within the meso- macroporous (> 2 nm) and microporous (< 2 nm) range. The larger meso-macropores allow for removal of larger biological relevant molecules such as bacterial endo- and exo-toxins together with inflammatory cytokines. The micropores adsorb other pathogenic mediators such as acetaldehyde and indoles. The large surface area allows for favourable adsorption kinetics. When administered orally, Yaq-001 is non-absorbable with no effect on bacterial growth kinetics in vitro. Pre- clinical studies in models of cirrhosis demonstrated oral Yaq-001 administration was associated with reduced endotoxin sensitivity, portal pressure, inflammatory indices, and markers of liver injury.

This study sought to evaluate the safety and tolerability of Yaq-001 in the context of a first-in-man double-blind randomised placebo-controlled study. Patients with a history of diuretic responsive ascites with a Child Pugh score of 7-11 were screened and randomised 1 : 1 to 4g nocte of Yaq-001 or equivalent placebo. 14 patients were included in each arm and treated for 12 weeks. In addition to safety assessments, additional blood, urine and stool samples were collected at baseline, month 1 and 3. The primary endpoints were safety and tolerability. Secondary endpoints were blood endotoxin, organ function and nutritional status. Exploratory end points relating to microbiome composition and functionality, local and systemic inflammatory indices and markers of intestinal barrier permeability were also assessed.

No serious adverse events or decompensation frequency were observed in either arm. Adverse events were predominantly gastrointestinal in nature, but no significant differences were observed between either group. Trends to reductions in systemic and faecal inflammatory indices were observed in the Yaq-001 -treated group at week 12 compared to baseline values. In contrast, an upward trend in faecal D-lactate, indicative of an improvement of microbial metabolism, was observed.

This first in man study met its primary end point of demonstrating safety and tolerability of Yaq-001 carbon. The data suggests a proof of mechanism that endotoxinbinding Yaq-001 improves intestinal and systemic inflammation and promotes improvement in gut health. Future efficacy studies are warranted to further investigate these findings. Methods

Study Design and Participants

The Carbalive-Safety Study is a multicentre, double blind randomised, placebo- controlled trial of oral Yaq-001 in stable decompensated cirrhosis.

Inclusion Criteria: Participants aged 18 years or above with a clinical diagnosis of diuretic-responsive cirrhotic ascites (Child-Pugh score = 7-11 inclusive) and written informed consent were recruited. A histological diagnosis of cirrhosis was not required for inclusion but abstinence from alcohol for at least 4 weeks prior to screening was required.

Patients were randomized 1 : 1 to receive 4g of oral Yaq-001 or equivalent placebo nocte for 12 weeks.

Endpoints and Assessments

Primary Endpoints: The main objective of this clinical investigation is to assess the safety and tolerability of Yaq-001 throughout the three months’ treatment period. Safety assessments were performed on Day 1, Weeks 1, 4, 8 and 12 and comprised a physical examination, clinical laboratory tests, urinalysis, 12-lead ECG and an assessment of reported and observed adverse events. Local laboratory tests included Clinical laboratory tests: haematology, coagulation, clinical chemistry, and urinalysis. Core laboratory analyses were performed at baseline, week 1 and week 12. ECGs were analysed independently by Broomwell Healthwatch, one of the National Institute for Clinical Excellence (NICE) recommended remote ECG interpretation consultancy services for cardiovascular disease.

Secondary Endpoints: The secondary endpoints in the study were: changes in blood endotoxin activity, organ function and nutritional status.

EAA Assay: The Endotoxin Activity Assay (EAA) was performed by in duplicate on whole blood collected in BD EDTA tubes in accordance with the manufacturer’s instruction at baseline, 1-week, 4-week, 8-week and 12-week visits. Samples were analysed within 2 hours of collection. Results were included for analysis if the CV % was less than 20. Changes in organ function (kidney, liver, brain, intestinal and immune function)'. These were assessed by: Child-Pugh and MELD scores and clinical laboratory tests at baseline, 4-week and 12-week visits. Changes in nutritional status: were determined by the Royal Free Hospital Global Assessment tool at each safety assessment together with micronutrient analysis at baseline, week 4 and 12. Vitamin B 12, D and folate were measured in the local clinical laboratories. Vitamins A, E and KI were analysed using Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) and trace elements Copper, Zinc and Selenium were analysed using inductively coupled plasma-mass-spectrometry (ICP-MS).

Core Laboratory Analyses

Luminex Assay: The Luminex Assay Protocol was performed in accordance with the manufacturer’s instructions. In brief, 50mcl of sample/ standard and 50mcl of Microparticle Cocktail are added to a 96 well plate, sealed and incubated for 2 hours at room temperature on a horizontal orbital microplate shaker. The plate was then washed three times with wash buffer and 50pL of diluted Biotin Antibody Cocktail added to all wells. The plate was sealed and incubated for 1 hour at room temperature. 50pL of Streptavidin-PE was then added to all wells and incubated for 30 minutes at room temperature. Following washing with buffer, the microparticles were then resuspended in Wash Buffer and incubated for 2 minutes at room temperature. The plate was then read within 90 minutes using Bio-Plex® MAGPIX™ Multiplex Reader and analysed on xPONENT software.

Faecal cytokine analysis and D-lactate assays were performed as previously described (PMID: 32838247).

Bile Acid Analysis Cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid and their glycine and taurine conjugated species were identified and quantified by ultra-performance liquid chromatography - quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Mass spectrometry analysis was performed using Xevo G2-XS Qtof system from Waters using an electrospray interface. BAs were separated by elution gradient mode with a mobile phase composed of a mixture of ammonium acetate buffer 15 mM, pH 8.0 (Solvent A) and acetonitrile: methanol = 75:25 v/v (Solvent B). The separation was obtained with a total run time of 6 minutes. All the chromatograms were acquired in electrospray negative ionization with the mass spectrometer operating in multiple reactions monitoring mode.

Analysis of SCFA in stool: Prior to analysis, eppendorf tubes were weighed and approximately lOOmg of stool was added to individual tubes. These stool samples were then homogenized in ImL of buffer (0.1 M Tris, 0.15M NaCl, IM urea, lOmM CaCh, 0.1M citric acid monohydrate, 5g/l bovine serum albumin (BSA) and 0.25mM thimerosal, pH 8.0) using a plastic homogenizer. Sample preparation and derivatization 20mL of stool sample and 15mL of Internal standard mix was added to a fresh glass vial. The volume in the vial was adjusted to lOOmL with d.H2O. 500mL of 0. IM TBA (tetrabutylammonium bisulfate in 0. IM KH2PO4 (pH 7.4) was added to each vial followed by 500mL of 2% PFBBR (Pentafluorobenzyl bromide) in dichloromethane . Samples were then sonicated for 60 minutes. Samples were extracted into 2mL of hexane and centrifuged at 1500rpm for 5 minutes at 4°C. The organic phase was moved to a fresh glass vial and evaporated under N2 gas to approximately lOOmL. lOOmL of sample was then transferred to a glass autosampler vial and run via GCMS. Samples were analysed on Rxi®-5Sil MS fused silica, 30m x 0.25mm I.D, 0.25pm film thickness), Inlet Temperature: 280°C, MS Transfer line: 250°C, Split ratio: 1 : 10, carrier gas (Helium) flow rate: 0.9ml/min, ion source: 225°C, chemical ionization (Methane 2ml/min). Oven temperature was programmed from 45°C to 200°C at 10°C/min, then to 300°C at 30°C/ min. Negative ions were detected in selected ion monitoring: The remaining stool sample was dried overnight using a vacuum centrifuge and dry weight was calculated.

Metagenomics analysis

Metagenomic sequence data: Samples from the UK, sequenced using Illumina HiSeq 2000, were analysed. All metagenomes passed over half the quality metrics in FastQC 0.11.3 with pass rates calculated in MultiQC.

Gene and Species abundance profding: The raw reads for all samples were trimmed using AlienTrimmer 0.4.0 with parameters -k 10 -1 45 -m 5 -p 40 -q 20 and Illumina contaminant oligonucleotides [REF], Trimmed samples were mapped against the IGC2 using Bowtie integrated into the METEOR pipeline [REF] against a catalog that contains MSPs of the human gastrointestinal microbiota built by binning co- abundant genes of the IGC2 catalog [REF] with MSPminer [REF], Human contaminant sequences were removed from all samples by discarding reads that mapped against a human reference genome. Downsizing of the gene count tables was done using the MOMR downsizeMatrix function to eliminate the sequencing depth fluctuations. Normalization of the downsized gene count tables was done using the MOMR normFreqRPKM function and according to the FPKM strategy. MSP abundances were estimated using mean abundance of 100 ‘marker’ genes (highly correlated within the MSP). Less than 10% of ‘marker’ genes were assumed to have species abundance of 0. MSPs functional annotation: IGC2 catalog was annotated for the Antibiotic Resistant Determinants (ARD) described in the Mustard database. Protein sequences were aligned against 9462 ARD sequences using blastp 2.7.1+ (option -evalue = 10^A-5).

Best-hit alignments were filtered for identity < 95% and bidirectional alignment coverage > 90\% (at query and subject level), giving a list of ARD candidates belonging to 30 families.

Annotation of the carbohydrate-active enzymes (CAZymes) of the IGC2 catalog was performed by comparing the predicted protein sequences to those in the CAZy database and to Hidden Markov Models (HMMS) built from each CAZy family, following a procedure previously described for other metagenomics analysis. Proteins of IGC2 catalog were also annotated to KEGG orthologs using Diamond agains KEGG database. Best-hit alignments with e-value > 10\^A-5.

NMR analysis

Aliquots of plasma (from blood collected into lithium heparin tubes) and urine were stored frozen at -80°C until NMR analysis. All samples were gently thawed in preparation for NMR analysis. Urine and serum samples were prepared for NMR analyses using standardised protocols. NMR data were acquired using a Bruker 600MHz (AVANCE NEO) NMR spectrometer and a ^^C/^N TCI Prodigy (nitrogen- cooled) probe. Pulse-collect (urine, serum) and spin-echo (serum) ID NMR data sets were acquired using PURGE water suppression and the PROJECT spin-echo sequences. Paired NMR data were compared using principal component analyses (PC A) (Wiley Sciences Solution KnowItAll® Informatics, Metabolomics Edition vl7.0, and MetaboAnalyst 4.0).

Statistical Analysis

All statistical analyses of study data were carried out using SAS v9.3 or a later version.

For categorical variables, summary tabulations of the number and percentage of subjects within each category (with a category for missing data) of the parameter are presented. Percentage calculations are based on non-missing data unless otherwise specified. For continuous variables, the number of subjects, mean, standard deviation (SD), standard error of the mean (SEM), median, 25th and 75th quartiles, minimum, and maximum values is presented.

Longitudinal summaries of continuous variables include absolute values, change, and percentage change from baseline at each post-baseline visit. Unless indicated otherwise 95% Cis are provided for efficacy endpoints by treatment group for the final.

Demographics and baseline characteristics

Demographics and baseline characteristics are summarized and presented by treatment group and overall, for the Safety and PP Populations. For continuous measures, the number of subjects, mean, SD, median, QI, Q3, minimum, and maximum are presented. For categorical measures, number and percentage of subjects are presented. No inferential statistical comparisons were performed.

Analysis populations

There are two analysis populations for this trial: Safety Population - The safety population includes all randomized subjects who took at least one dose of the investigational product. All safety analysis and summaries were performed on the Safety population. Per-Protocol Population - The per-protocol population (PP) includes all the subjects who satisfied the eligibility criteria, took at least 70% of the treatment doses (2 sachets/day for Cohort 1) and did not present major protocol non-compliances. The Safety Population is the main study population. All measures of efficacy including laboratory tests are also performed on the PP population.

Results

Patient characteristics

A total of 34 subjects were screened for this study at 8 European centres for Cohort 1 within the CARB ALIVE Consortium between February 2019 and March 2020. 28 subjects met the study entry criteria and were randomized to either active or placebo groups. 6 subjects screened did not meet the study entry criteria. Dosing was not initiated in 2 subjects randomized to placebo due to withdrawal of consent. During the 12- week treatment portion of the study, 13 enrolled active subjects completed the study, per protocol. 10 placebo-treated subjects completed the study, per protocol.

8 subjects were screened at 1 UK and 3 Spanish centres for the second dosing cohort of 8g. The study was terminated prematurely due to the coronavirus pandemic.

Baseline characteristics are shown in table 3. The patients were well matched with regards to age, sex, ethnicity, body mass index and stage of cirrhosis. Median age was 58.5 years in both treatment groups with a male percentage of 75% in the placebo compared to 71.4% in the active group. In accordance with study entry criteria, all subjects had cirrhosis with diuretic-responsive ascites and Child-Pugh score of 7-11 inclusive. Median Child Pugh Score in both groups was 7 with a median MELD score of 13.2 and 12.6 in placebo and active groups respectively.

Table 3

Safety and Tolerability

No deaths or serious adverse events were reported in the study. Treatment- emergent adverse events (TEAEs) are shown in table 4, the most frequent of which were gastrointestinal in nature in both groups. Of these, only constipation and diarrhoea were evaluated by the clinical investigator as possibly related to the investigational product. The frequency of constipation in placebo and active groups was 25% (3/12) and 14% (2/13) in placebo and Yaq-001 -treated groups respectively. The frequency of diarrhoea in placebo and active groups was 17% (2/12) and 0% (0/13) in placebo and Yaq-001 -treated groups respectively. One placebo-treated subject withdrew from the study due to diarrhoea.

Table 4 The majority of the TEAEs reported across both treatment groups (40/51) were not considered by the clinical investigator to be related to treatment (32/38 active; 8/13 placebo) and were mild in intensity. Systemic antibiotics were administered for the following TEAEs: Amoxicillin (acute bronchitis); Clarithromycin (acute bronchitis); Phosphomycin (urinary tract infection). None of these infections were related to the administration of the investigational product. There were no treatment-related clinically significant laboratory abnormalities.

With regards to nutritional status, no significant differences were observed in either treatment group with regards to Royal Free global assessment status, vitamin B12, A, E, folate or trace elements. Median vitamin A, zinc and baseline vitamin D concentrations were below the limit of normal range but no differences between treatment groups were observed. No changes were observed in any of the micronutrient parameters with treatment in either group. With regards to treatment compliance, of the 14 subjects enrolled in the Yaq-001 treatment group, 13 (93%) completed 12 weeks of therapy. The median duration of exposure was 83 (6 - 94) days. 10 of the 12 (83%) subjects who received placebo completed the course of treatment. The median duration of exposure was 83 (14 -86) days. Compliance in the active and placebo groups was 92.9% and 66.7% respectively.

Inflammatory Indices

A trend to reduction in total leucocyte count and c-reactive protein from baseline to week 12 was observed in the active-treatment group (median % change: -11.82%, - 6.06% respectively) (Figure 33). Median percentage changes in leucocyte count and C- reactive protein in the placebo group were +2.85% and 0.00% respectively. Using the solution estimates, the Endotoxin Activity Assay values decreased (mean % change: - 2.42%), while in the placebo group values increased (mean % change: +29.55%). A trend toward reduction in whole blood reactive oxygen species production was observed in the active-treated group after 12 weeks compared to placebo (mean % change: - 37.3% vs. +34.3%) (Figure 33).

Non-significant reductions in IL-6, CXCL10 (Figure 33) and TNFa (Figure 34) were observed after 12 weeks in the active group compared to placebo (IL-6: -10.0% vs +5.7%; CXCL10: -17.2% vs +20.5%; -7.1% vs -2.8%). A median reduction in IL-10 concentrations was observed in the active of -21.2% compared to the placebo group of - 11.0% following 12 weeks of treatment (Figure 34). No differences between groups or treatment time intervals were observed in the following plasma values: CCL2, CD14, CD163, IL-lbeta, IL-23, IL-33, IL-4, IL-7, IL-8, IL-12p70, IL1B reporter assay, L- Selectin, Lipocalin-2, Myeloperoxidase, TIMP-1, endotoxin as measured by the LAL assay) and LBP. No differences between groups or treatment time intervals were observed with serum IL- 18 or N-GAL.

A non-significant reduction in faecal pro-inflammatory cytokines IL- 17 A, TNF a, IFN-y, IL-21 towards healthy control levels was observed with active treatment (Figure 34). A trend towards reduction in faecal IL-10 from baseline at Week 12 was observed in the active treatment group (median change: - 8%) in contrast to a upward trends in the placebo group (median change: +17.8%).

Markers of Endotoxaemia

Endotoxin Activity Assay ratios at baseline, weeks 4 and 12 are shown in Figure 36. Using solution estimates, EAA values decreased (mean change: -2.42%) with Yaq-001 treatment in contrast to the placebo group in which EAA values increased (mean change: +29.55%). No significant differences between time points or groups were observed. The median percentage changes in Bactericidal Permeability Increasing (BPI) protein from baseline at week 4 were +11.3 (-75.2- +36.1)% in the placebo compared to -30.8 (-64.9 — 0.2)% the active group (p<0.05). At week 12, the median percentage changes were -6.6 (-44.3-+29.1)% and -19.7 (-53 ,5-+17.5)% in placebo and active groups respectively (p<0.05). The median percentage changes in Lipopolysaccharide binding protein (LBP) protein from baseline at week 4 were +2.42(- 11.9-+16.2)% in the placebo compared to -9. l(-13.7-+7.9)% the active group (p<0.05). At week 12, the median percentage changes were -0.5 (-8.4-+53.6)% and +9.6 (-13.8- +21.0)% in placebo and active groups respectively (p<0.05). The median percentage changes in Endotoxin concentration as measured by the LAL assay from baseline at week 4 were +4.1 (-27.2-+332.6)% in the placebo compared to -4.4(-36.1-+139.9)% the active group (p<0.05). At week 12, the median percentage changes were 29.3 (-58.3- +76.4)% and -7.6 (-64.8-+465.0)% in placebo and active groups respectively (p<0.05) (Figure 36).

Markers of Gut Barrier Integrity

A decrease from baseline in the lactulose and rhamnose permeability assays was seen in the active treatment group (median change: - 98.4%) while an increase was seen in the placebo group (+111.7%) (Figure 35). Of note, most of the values were within the normal range of <0.05. Faecal D-lactate trended upwards in both the Yaq-001 treated and placebo treated subjects but was more marked in the placebo group (median change + 4.9% v +76.9%, respectively). Conversely, plasma D-lactate trended downwards with Yaq-001 treatment (baseline median: 245 nMol/ml; week 12 median: 217 nMol/ml) while this increased in the placebo group (baseline median: 237 nMol/ml; week 12 median: 247 nMol/ml.

Taxonomic Profile of the Faecal Microbiome

Metagenomic analysis of stool was performed at baseline, week 4 and week 12. No significant difference in bacterial diversity was observed. To investigate the importance gut microbial species play in the observed changes to the clinically relevant metadata, faecal metagenomics analysis was conducted. Abundance of metagenomic species biomarkers of liver cirrhosis (PMID: 25079328) were found to be reduced by Yaq-001. Differential abundance analysis demonstrated that Yaq-001 treatment was associated with a reduction in Veillonella and Streptococcus spp (Figures 37 and 38) and an increase in Roseburia spp. Significant findings following correlation analyses between bacterial species and endotoxin, ammonia and D-lactate of the entire population are represented in Figures 37-38. Significant correlations were observed between Veillonella and Streptococcus spp and endotoxin concentrations.

Associations with compositional changes in gut bacterial species and changes in faecal and serum bile acid profde

Significant correlations between faecal bile acids and bacterial species of the entire population are represented in the Circo plots in Figure 40. Bacterial species and bile acid glycol-lithocholic acid (G-LCA) previously described to be altered in cirrhosis are highlighted in Figures 39A-B. G-LCA was non-significantly reduced at week 4 and 12 in both groups although most markedly in Yaq-001 -treated groups compared to placebo. That G-LCA was significantly negatively associated with Roseburia spp.

Whilst no significant differences in short chain fatty acid concentrations in stool (Figure 42) or plasma (Figure 41) were observed between groups, bacterial species that were significantly negatively correlated with serum butyrate were decreased in abundance four weeks after treatment by Yaq-001 compared to placebo control (Figure 41). No significant differences were observed in either groups with regards to plasma or urinary metabolites on principal component analysis (Figures 41 and 42). NMR analysis demonstrated a non-statistically increase in urinary hippurate levels following 4 weeks of Yaq-001 treatment compared to placebo

Discussion

The gut microbiome constitutes an important therapeutic target as translocated bacterial products drive a dysregulated systemic inflammatory response, central to pathogenesis. Current interventions targeting the gut microbiome are limited by antibiotic resistance and superinfection and thus alternative strategies remain an unmet clinical need. Oral Yaq-001 carbon represents a novel therapeutic approach to sequester bacterial toxins and metabolites without exerting an antibiotic effect. In vivo studies have demonstrated favourable effects on organ injury, immune activation status and haemodynamic profile. The results of this first-in-man randomised, placebo-controlled trial suggest that oral Yaq-001 at a dose of 4g nocte is well tolerated with a favourable safety profile. Despite the rapid adsorption kinetics for bacterial toxins and metabolites, Yaq-001 treatment exerted no significant effect on micronutrient levels or impact on macronutrient profile as assessed by the gold standard Royal Free Global Assessment tool.

Multiple markers of inflammation were assessed in this study as exploratory endpoints to ascertain whether oral Yaq-001 administration, via modulation of the gut microenvironment, had the potential to impact on systemic inflammation. Leucocyte count and C-reactive protein are standard clinical indices of inflammation and in cirrhosis for example have been identified as independent predictors of mortality (PMID: 23474284, PMID: 25463539, PMID: 24950482). Furthermore, markers of oxidative injury in the systemic circulation have been implicated in the pathogenesis of advanced cirrhosis (PMID: 34888354) for example. In this study we observed a reduction in total leucocyte count and C-reactive protein at Week 12 from baseline in the actively treated group compared to an increase in the placebo-treated group. Whole blood reactive oxygen species production was markedly, although non-significantly, reduced in the YaqOOl -treated group also in contrast to placebo control. Similar trends were observed in pro-inflammatory cytokines IL-6, TNFa and CXCL10, all of which have been found to be predictive of adverse clinical outcomes in decompensated cirrhosis (PMID: 27483394) for example.

A disturbance in inflammatory profile has also been demonstrated at the intestinal barrier interface in addition to that of the systemic circulation (PMID: 32838247). Faecal cytokine profiling represents an innovative approach to investigate the localised intestinal cytokine micro-environment. Existing data reveal that acute decompensation in cirrhosis is associated with a highly inflamed and permeable gut barrier. In light of the propensity of oral Yaq-001 to adsorb inflammatory cytokines, we hypothesised that oral Yaq-001 might be able to modulate the faecal inflammatory profile towards healthy control levels. Faecal IL-17A is a cytokine with a well-known pro-inflammatory role, but is also involved in promoting epithelial proliferation, crucial for both wound closure and replacing cells lost through homeostatic and likely pathological shedding. Active treatment with Yaq-001 was associated with an overall reduction from baseline in IL-17A concentrations compared to placebo control suggestive of a reduction in inflammation at the gut-barrier interface. Concentrations of faecal pro-inflammatory cytokines TNF-a, IFN-y, IL-ip and IL-21 have been shown to be increased in subjects with cirrhosis, and their values are reflective of increased gut inflammation. A trend to reduction in luminal pro-inflammatory cytokines IL-17A, TNF a, IFN -y, IL-21 to healthy control levels was observed with active treatment. Anti- inflammatory faecal cytokine IL- 10 known to be increased in subjects with decompensated cirrhosis and reflecting response to injury was non-significantly reduced from baseline at Week 12 towards healthy controls in the active treatment group. Taken together, these data are suggestive of a less inflammatory gut barrier milieu and provide preliminary support that Yaq-001 potentially reduces the severity of gut inflammation with the potential to improve intestinal permeability also.

Integrity of gut barrier interface, a key factor driving the translocation of bacterial products from the gut was evaluated by several methods in this study. Urinary excretion of lactulose and rhamnose was used as a measure of small intestinal permeability and expressed as the lactulose:rhamnose ratio. A decrease from baseline in the lactulose: rhamnose ratios was observed in the active treatment group and an increase was seen in the placebo group Importantly, most of the values were within normal range of <0.05 reflective of more stable disease.

Gut-derived bacterial toxins have long since been recognised as an important driver of dysregulated inflammatory response, in particular endotoxin, derived from gram negative organisms. In this study we utilised the endotoxin activity assay, an FDA-approved measure of endotoxin in whole blood. This method has been proposed as superior method of endotoxin measurements compared to conventional assay such as the Limulus amebocyte lysate (LAL) test with the inherent limitations of endogenous inhibitors present in biological fluids. The EAA assay utilises a chemiluminescent method to measure whole blood production of reactive oxygen species with and without endotoxin stimulation. The ratio increases with endotoxin concentration and reference ranges indicative of low, intermediate, and high risk of sepsis have been established by the manufacturer. Using the solution estimates, EAA values decreased with Yaq-001 treatment in contrast to the placebo group in which EAA values increased. Taken together with changes in BPI and LBP, these observations are suggestive of improvements in bacterial translocation rates compared to the placebo population however these observations did not achieve statistical significance.

Direct effects on the faecal microbiome by Yaq-001 were assessed by metagenomic and metabolomic analysis. No significant difference in bacterial diversity, as evidenced by the Shannon index, was observed with either Yaq-001 treatment or placebo groups indicating that Yaq-001 does not exert an antibiotic effect and distinguishes it mechanistically from other microbiome targeting treatments. Favourable shifts in bacterial species previously described as over-represented in the cirrhotic microbiome and associated with adverse clinical outcome, Veillonella spp. and Roseburia spp., were observed with treatment suggestive of a less hostile environment in the gut. This was further evidenced by the correlations observed in Yaq-001 -treated patients in which a phenotypic change was observed.

It is increasingly understood that microbial metabolites in addition to bacterial ligands contribute to disease status in cirrhosis. Bacteria play a key role in bile salt metabolism both in deconjugation and 7a-dehydoxylation reactions. Disturbances in the microbiome can therefore influence the nature of the bile acid pool. Previous studies have observed higher glyco-lithocholic acid concentrations with more progressive disease (REF). In this study, we observed marked reduction in faecal glyco-lithocholic acid associated with an increase in Roseburia spp. Trends towards reduction in faecal SCFAs butyrate, acetate and propionate were observed in the active arm which may represent either enhanced gut metabolism induced by Yaq-001 via impact on gut inflammation or direct removal by Yaq-001. No clinically significant differences were observed between treatment groups when urine and plasma was analysed by ¹HNMR spectroscopy. Taken together, these data suggest that Yaq-001 likely has no deleterious effect on microbial diversity but may favourably impact faecal, but not plasma, SCFA, suggesting potential beneficial effects on gut metabolism. The trend towards reduction of urinary hippuric acid and plasma ammonia suggests possible beneficial systemic metabolic effects.

D-lactate is a metabolic by-product of fermentation of dietary carbohydrate by the gut and metabolised very slowly by the liver. As a result of this such, elevated levels are an indicator of bacterial translocation, due to a breach in the gut epithelial barrier. A trend towards an increase in faecal D-lactate was observed in both the Yaq-001 treated and placebo treated subjects but was more marked in the placebo group. Plasma D- lactate showed a trend towards reduction in the active group with an increase observed in the placebo group. The D-lactate data suggest that Yaq-001 may reduce the severity of gut permeability, whilst improving microbial metabolism.

Conclusion

In summary, this clinical study demonstrates that oral treatment with Yaq-001 is safe and well tolerated in a cohort of out-patient decompensated cirrhotic patients and thereby meeting the primary end point of the study. Despite the small patient cohort assessed and low dose of interventional product, multiple pathological inflammatory indices, taxonomic and functional profile of the gut microbiome were seen to be favourably modified following 12-weeks of Yaq-001 treatment compared to placebo. These data suggest that oral Yaq-001 exhibits features of a novel class of therapy to modulate the pathological microbiome which is not associated with the deleterious effects of direct anti-microbial therapy. Thus, the data presented here provide compelling evidence for the potential of Yaq-001 as a safe and novel therapy targeting the microbiome, bacterial translocation and gut permeability that impacts on systemic inflammation and organ function in diseased states.

Claims

2. Porous carbon particles for use according to claim 1, wherein mesopores of diameter greater than 2 nm and less than 30nm and large macropores of diameter greater than 500 nm together make up 10% or less of the total pore volume of the porous carbon particles.

3. Porous carbon particles for use according to claim 1 or 2, wherein 5 to 30% of the total pore volume of the porous carbon particles is made up of pores having a mean diameter of from 0.6 to 2 nm.

4. Porous carbon particles for use according to any one of claims 1 to 3, wherein 85% or more of the pore volume of the porous carbon particles which is made up of pores having a mean diameter greater than 2nm is made up of pores having a mean diameter of from 30 nm to 500 nm, preferably wherein 90% or more of the pore volume of the porous carbon particles which is made up of pores having a mean diameter greater than 2nm is made up of pores having a mean diameter of from 30 nm to 500 nm, preferably from 50 nm to 300 nm, preferably from 50 nm to 200 nm.

5. Porous carbon particles for use according to any one of claims 1 to 4, wherein the total pore volume is from 0.5 to 2.5 cm³g^-1.

6. Porous carbon particles for use according to any one of claims 1 to 5, wherein the total pore volume is from 1.0 to 2.0 cm³g^-1.

7. Porous carbon particles for use according to any one of claims 1 to 6, wherein the bulk density of the porous carbon particles is 0.10 gem'³ to 0.30 gem'³.

8. Porous carbon particles for use according to any one of claims 1 to 7, wherein the bulk density of the porous carbon particles is 0.15 gem'³ to 0.25 gem'³.

9. Porous carbon particles for use according to any one of claims 1 to 8, wherein the specific surface area is from 700 m²/g to 2000 m²/g.

10. Porous carbon particles for use according to any one of claims 1 to 9, wherein the specific surface area is from 900 m²/g to 1400 m²/g.

11. Porous carbon particles for use according to any one of claims 1 to 10, wherein the volume of pores having a mean diameter of from 0.5 to 2 nm in the porous carbon particles is 0.1 to 1.1 cm³g^-1 and the volume of pores having a mean diameter of from 30 to 500 nm is 0.8 to 2.5 cm³g^_1.

12. Porous carbon particles for use according to any one of claims 1 to 11, wherein the particles are administered orally or rectally, preferably wherein the particles are administered orally in free-flowing form or in tablet form.

13. Porous carbon particles for use according to any one of claims 1 to 12, wherein the porous carbon particles are coated in order to control their release and adsorption properties, preferably wherein the porous carbon particles are coated with a film that will allow predominant release into the large bowel.

14. Porous carbon particles for use according to any one of claims 1 to 13, wherein the disease is an inflammatory disease or disorder.

15. Porous carbon particles for use according to claim 14, wherein the modulation of the microbiome of the gut results in a reduction in systemic inflammation, thereby treating or preventing the disease.

16. Porous carbon particles for use according to claim 14 or 15, wherein the inflammatory disease or disorder is selected from coronary heart disease, obesity, Alzheimer’s disease, dementia, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary airways disease, encephalitis, allograft rejection, Graves' disease, Hashimoto's thyroiditis, autoimmune uveoretinitis, giant cell arteritis, asthma, atherosclerosis, regional enteritis, granulomatous enteritis, distal ileitis, regional ileitis, terminal ileitis, dermatitis, insulindependent diabetes mellitus, non-insulin-dependent diabetes mellitus, diverticulitis, fibromyalgia, multiple sclerosis, pernicious anemia, sarcoidosis, sarcopenia, scleroderma, systemic lupus erythematous, nephritis, diseases associated with cholestasis and Parkinson’s disease.

17. Porous carbon particles for use according to any one of claims 1 to 15, wherein the administration of the porous carbon particles protects organs distant from the gut or liver from inflammation.

18. Porous carbon particles for use according to any one of claims 1 to 13, wherein the porous carbon particles are for use in preventing radiation-induced organ injury.

19. Porous carbon particles for use according to claim 18, wherein the radiation is nuclear radiation.

20. Porous carbon particles for use according to claim 18, wherein the subject is exposed to the radiation by radiation therapy.

21. Porous carbon particles for use according to any one of claims 1 to 17, wherein the porous carbon particles are for use in preventing the deleterious effects of ageing or promoting healthy ageing.

22. A method of treating or preventing a disease, the method comprising administering porous carbon particles as defined in any one of claims 1 to 13 to a subject in need thereof, thereby modulating the microbiome of the gut and preventing or treating the disease.

23. Use of porous carbon particles as defined in any one of claims 1 to 13 in the manufacture of a medicament for modulating the microbiome of the gut, thereby treating or preventing disease.

24. A product containing:

(a) porous carbon particles as defined in any one of claims 1 to 13; and

(b) one or more further therapeutic agents; for simultaneous, separate or sequential use in the treatment of a subject suffering from or susceptible to a disease.

25. A product for use according to claim 24, wherein the further therapeutic agent is selected from:

(i) an antibiotic;

(iii) faecal microbial transplantation;

(iv) a corticosteroid; and/or

(v) drugs that target diseases defined in claim 16.