Development of a gut microbe–targeted nonlethal therapeutic to inhibit thrombosis potential

May combine two claims.

Safety screening of IMC and FMC showed no hERG inhibition, no mitochondrial toxicity in HepG2 cells, no adverse HK-2 viability effects, and no AMES signals up to 1000 μg/well.

“Screening of IMC and FMC in a battery of pharmacological safety tests failed to show: (i) inhibition in hERG channel function; (ii) toxicity to mitochondria in HepG2 cells cultured in glucose- or galactose-supplemented media; (iii) adverse effects on the viability of HK-2 cells in culture; or (iv) adverse signals during AMES testing at levels up to 1000 μg/well”

May combine two claims.

A single oral dose of FMC or IMC caused almost complete reduction in plasma TMA and TMAO for a sustained period, with FMC trough persisting over 3 days and IMC over 2 days.

“Both FMC and IMC induced an almost complete reduction in plasma TMA and TMAO levels following a single oral dose (via gavage) for a sustained period, with FMC demonstrating a striking trough that persisted for over 3 days (Figure 3b right), and IMC for over 2 days post-gavage”

May combine two claims.

Phenylcholine inhibited P. mirabilis CutC/D with IC50=150 nM, and its inhibition was reversible after dialysis.

“reversible inhibition of CutC/D activity following dialysis was observed with an alternative, potent inhibitor developed, phenylcholine (IC50=150 nM against P. mirabilis CutC/D)”

May combine two claims.

FMC was the most potent halomethylcholine examined, with IC50=900 pM against P. mirabilis CutC/D, and docking predicted it had the tightest active-site interaction.

“the fluorinated choline analogue, fluoromethylcholine (FMC), was the most potent halomethylcholine examined (IC50=900 pM against P. mirabilis CutC/D), and in docking calculations, was predicted to have the tightest interaction with the active site”

May combine two claims.

IMC acts kinetically as a mixed mechanism inhibitor, decreasing Vmax and increasing KM.

“Increasing concentrations of IMC similarly decreased Vmax, but also increased KM (Supplementary Figure 5), indicating IMC acts kinetically as a mixed mechanism inhibitor, with elements of both competitive and non-competitive inhibition.”

May combine two claims.

High-dose DMB did not fully restore choline diet-associated TMAO elevations, increased platelet aggregation, and shortened in vivo thrombus formation to chow-fed levels.

“despite the high dose of DMB provided, choline diet-associated TMAO elevations, increased platelet aggregation, and shortened in vivo thrombus formation were not fully rescued compared to chow-fed mice”

May combine two claims.

Halomethylbetaines act as CutC/D TMA lyase inhibitors but are about 2-3 orders of magnitude weaker than their choline counterparts.

“the halomethylbetaines were found to act as CutC/D TMA lyase inhibitors but approximately 2–3 orders of magnitude weaker than their choline counterparts”

May combine two claims.

Direct incubation of FMC and IMC with mouse or human platelet-rich plasma, and of their metabolites FMB and IMB with human platelet-rich plasma, did not affect platelet aggregometry.

“FMC and IMC were directly incubated with PRP from both mice and humans (with or without agonist), and no effects on platelet aggregometry responses were observed. Their primary in vivo metabolites, FMB and IMB, also did not affect platelet aggregometry responses in human PRP”

May combine two claims.

Prolonged 15-20 week exposure to IMC in male and female mice produced no adverse effects on renal function, liver function, or CBC with differential.

“In alternative studies in both male and female mice with more prolonged exposures (15–20 weeks) to IMC, no adverse effects were observed, including normal indices of renal function, liver function, and CBC with differential”

May combine two claims.

Most FMC and IMC remained in the gut luminal compartment, with only nominal FMB or IMB in feces.

“analyses of feces revealed the majority of FMC and IMC remain in the gut luminal compartment, with only nominal levels of FMB or IMB present in fecal samples”

May combine two claims.

Choline supplementation produced 10-fold higher plasma TMAO and enhanced aggregometry response to submaximal ADP in platelet-rich plasma.

“choline supplementation resulted in 10-fold higher plasma TMAO levels (p<0.0001) and enhanced aggregometry response to submaximal levels of ADP (1 μM) in PRP (p<0.0001; Figure 1a).”

May combine two claims.

With d9-choline co-administered, FMC and IMC suppressed d9-TMA (EC50=0.008 and 0.5 mg/kg) and d9-TMAO (EC50=0.01 and 0.2 mg/kg).

“both FMC and IMC demonstrated remarkable capacity to suppress production of d9-TMA (EC50=0.008 and 0.5 mg/kg, respectively; Supplementary Figure 8) and d9-TMAO (EC50=0.01 mg/kg and 0.2 mg/kg, respectively; Table 1, Figure 3a right).”

May combine two claims.

The TMAO-enhancing effect on stimulus-dependent platelet aggregation was also seen with washed platelets from high-choline-fed mice and was greatest at submaximal agonist levels.

“the TMAO enhancing effect on stimulus-dependent platelet aggregation was also observed with washed platelets from mice fed the high choline diet (p=0.0002), and was greatest at submaximal levels of agonist”

May combine two claims.

Fecal choline levels significantly increased after FMC or IMC administration, while plasma choline levels were not significantly affected.

“Analyses of fecal samples revealed significant increases in choline levels following FMC or IMC administration, which before addition of the inhibitor were extremely low (Supplementary Figures 9,10). Importantly, no significant effects on plasma choline levels were observed”

May combine two claims.

FMC was more potent than IMC against polymicrobial human fecal cultures, with EC50 values of 7.9 nM and 1.6 μM, respectively.

“FMC was also more potent than IMC against our polymicrobial, human fecal cultures (EC50=7.9 nM and 1.6 μM, respectively; Table 1).”

May combine two claims.

DMB significantly reduced TMAO levels and ADP-dependent platelet aggregation in platelet-rich plasma from choline-supplemented mice.

“DMB significantly reduced both TMAO levels (p<0.0001) and stimulus-dependent (ADP) platelet aggregation (p=0.003) in PRP recovered from choline-supplemented mice”

May combine two claims.

DMB attenuated the choline diet-induced rise in TMAO and rate of clot formation.

“provision of DMB attenuated the choline diet-induced rise in TMAO and rate of clot formation (p=0.002; Figure 1b,c).”

May combine two claims.

Four hours after a single oral gavage, FMC and IMC selectively accumulated in the large intestine to millimolar levels (2-6 mM), with virtually complete elimination of luminal TMA and a marked increase in microbial choline.

“within the large intestine (cecum and colon), the anatomic location where the majority of choline TMA lyase harboring commensals reside49, we observed the selective accumulation of each inhibitor to the millimolar level (2–6 mM), virtually complete elimination in detectable luminal TMA (product), and a similar marked increase in intestinal microbial choline (substrate) levels”

• cites [49] Koeth, R. A. et al. γ-Butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of l-carnitine to TMAO. Cell Metab. 20, 799–812 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

FMC and IMC completely suppressed choline diet-enhanced TMAO generation and the accompanying enhancement in ADP-dependent platelet aggregation.

“Results confirmed complete suppression of choline diet-enhanced TMAO generation and accompanying enhancement in ADP-dependent platelet aggregation response with provision of either FMC (p=0.0001) or IMC (p=0.001; Figure 4a).”

May combine two claims.

FMC and IMC dose-dependently suppressed TMA production (EC50=4.5 and 31 mg/kg) and systemic TMAO (EC50=3.4 and 45 mg/kg), with FMC about an order of magnitude more potent.

“both FMC and IMC showed dose-dependent suppression in both TMA production (EC50=4.5 and 31 mg/kg, respectively; Supplementary Figure 8) and systemic TMAO levels, with FMC demonstrating potency an order of magnitude greater than IMC (EC50=3.4 and 45 mg/kg, respectively; Table 1, Figure 3a right).”

May combine two claims.

FMC and IMC were detectable at low μM levels only for the first few hours in plasma, while their halomethylbetaine oxidation products were about 10-fold more abundant and peaked 2 hours post-gavage.

“FMC and IMC were detectable at low (μM) levels only for the first few hours in plasma, but their halomethylbetaine oxidation products, fluoromethylbetaine (FMB) and iodomethylbetaine (IMB), were ~10-fold more abundant, reaching their peak level 2 hours post-gavage”

May combine two claims.

Resveratrol was largely ineffective in the screening assays and had no effect on plasma TMAO in choline-fed mice.

“resveratrol was largely ineffective in each of the screening assays employed (Figure 1e) and showed no effect on plasma TMAO levels in vivo when administered to mice on a choline-supplemented diet (p=0.91; data not shown)”

• cites [43] Chen, M. L. et al. Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota. MBio 7, e02210–e02215 (2016).Not checked yet

  Not checked in the free run.

May combine two claims.

Halomethylbetaine levels in all intestinal segments were significantly lower than their halomethylcholine counterparts, and there was no significant effect on gut luminal betaine or TMAO.

“Levels of halomethylbetaines in all intestinal segments were significantly lower than their halomethylcholine counterparts, and there was no significant effect on gut luminal betaine or TMAO levels”

May combine two claims.

A high-choline diet was associated with increases in Dehalobacterium and Adlercreutzia and reductions in Bifidobacterium, and these proportions were associated with TMAO levels and time to vessel occlusion; IMC reversed these shifts.

“Provision of a high-choline diet was associated with both significant increases (e.g. Dehalobacterium and Adlercreutzia) and reductions (e.g. Bifidobacterium) in the proportions of several genera, which were each also significantly associated with both TMAO levels and time to vessel occlusion; moreover, these shifts in proportions were reversed with the addition of IMC”

May combine two claims.

Dietary choline supplementation and IMC exposure each induced significant rearrangements in cecal microbial composition.

“Principal Coordinates Analysis of microbial taxa revealed distinct clusters, indicating both dietary choline supplementation and IMC exposure each induced significant (p=0.001) rearrangements in microbial composition”

May combine two claims.

Neither FMC nor IMC prolonged occlusion time beyond that observed with normal chow.

“neither FMC (p=0.85) nor IMC (p=0.73) led to a prolongation in occlusion time beyond that observed with normal chow.”

May combine two claims.

FMC and IMC completely blocked choline diet-induced increases in plasma TMAO and in vivo rate of clot formation.

“provision of either FMC (p<0.0001) or IMC (p<0.0001) completely blocked choline-supplemented diet-induced increases in both plasma TMAO and in vivo rate of clot formation”

May combine two claims.

Inhibitor treatment reversed the choline diet-induced increases in platelet responses to agonists and in thrombus formation in vivo.

“reversing choline diet-induced increases in platelet responses to agonists and thrombus formation in vivo.”

May combine two claims.

IMC reversed choline diet-dependent increases in TMAO and platelet adherence to collagen under physiological shear.

“Provision of IMC reversed choline diet-dependent increases in both TMAO levels and platelet adherence to collagen matrix to levels observed in chow-fed mice”

May combine two claims.

IMC and FMC altered the composition of the mouse cecal microbiota, especially when mice received a choline-supplemented diet, and reversed some choline-driven changes in taxon proportions.

“It is also notable that both IMC and FMC induced a shift in mouse cecal microbiota composition, particularly on a choline-supplemented diet, reversing choline-induced changes in the proportions of certain taxa.”

May combine two claims.

Inhibiting gut microbial TMA production lowered plasma TMAO levels for extended periods while systemic drug exposure remained limited.

“by inhibiting gut microbial production of TMA, we suppressed plasma TMAO levels for prolonged periods with limited systemic exposure of the drugs within the host”

May combine two claims.

The inhibitors did not kill cultured bacteria in nutrient-rich broth and did not reduce microbial fitness after prolonged exposure.

“even if the inhibitors are non-lethal to cultured bacteria in nutrient-rich broths and fail to demonstrate reduction in microbial fitness with prolonged exposures.”

May combine two claims.

TMAO increases platelet responsiveness and in vivo thrombosis potential in animal models.

“enhances both platelet responsiveness and in vivo thrombosis potential in animal models”

Probably not a substantial claim: may not pass S3 (load-bearing).

A link between dietary phosphatidylcholine, gut microbiota-dependent TMAO generation, and cardiovascular disease pathogenesis was first described less than a decade ago.

“a link between dietary phosphatidylcholine, a nutrient common in a Western diet, gut microbiota-dependent generation of the metabolite trimethylamine N-oxide (TMAO), and cardiovascular disease (CVD) pathogenesis, was first described7.”

• cites [7] Wang, Z. et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 472, 57–63 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

TMAO alters calcium signaling in platelets, enhancing responsiveness and in vivo thrombosis potential in animal models.

“recent studies reveal TMAO alters calcium signaling in platelets, enhancing responsiveness and in vivo thrombosis potential in animal models15.”

• cites [15] Zhu, W. et al. Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell 165, 111–124 (2016). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Antiplatelet agents reduce cardiovascular events and mortality, but their wider use is limited by increased bleeding risk and nonadherence.

“Use of antiplatelet agents has become a cornerstone for the treatment of CVD because of substantial reduction in CVD events and mortality37,38. However, more widespread use of antiplatelet agents has been limited by the increased risk of bleeding, which also leads to nonadherence39–41.”

• cites [37] Bobadilla, R. V. Acute coronary syndrome: focus on antiplatelet therapy. Crit. Care Nurse 36, 15–27 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [38] Levine, G. N. et al. 2016 ACC/AHA guideline focused update on duration of dual antiplatelet therapy in patients with coronary artery disease: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J. Thorac. Cardiovasc. Surg. 152, 1243–1275 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [39] Jennings, L. K. Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb. Haemost. 102, 248–257 (2009). sourceNot checked yet

  Not checked in the free run.

• cites [40] Manchikanti, L. et al. Assessment of bleeding risk of interventional techniques: a best evidence synthesis of practice patterns and perioperative management of anticoagulant and antithrombotic therapy. Pain. Physician 16, SE261–SE318 (2013).Not checked yet

  Not checked in the free run.

• cites [41] Cohen, M. Expanding the recognition and assessment of bleeding events associated with antiplatelet therapy in primary care. Mayo Clin. Proc. 84, 149–160 (2009). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In atherosclerosis-prone ApoE−/− mice on a choline-supplemented diet, DMB lowered plasma TMAO and attenuated macrophage cholesterol accumulation, foam cell formation, and atherosclerotic lesion development.

“When given to atherosclerosis-prone ApoE−/− mice on a choline-supplemented diet, plasma TMAO levels were significantly lowered, and concurrently, macrophage cholesterol accumulation, foam cell formation and atherosclerotic lesion development were attenuated35.”

• cites [35] Wang, Z. et al. Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Cell 163, 1585–1595 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

A non-lethal, mechanism-based inhibitor aimed at the gut microbial TMAO pathway, engineered to concentrate in the microbial compartment, can lower thrombosis while reducing host systemic exposure.

“Herein we show that a mechanism-based non-lethal inhibitor of the gut microbial TMAO pathway designed to selectively accumulate within the gut microbial compartment, can serve as a new therapeutic approach for attenuating thrombosis while simultaneously limiting systemic exposure in the host.”

• AssumptionRests on an assumption: The mouse models used for platelet function and thrombosis predict human clinical effects of the inhibitors.

May combine two claims.

DMB inhibits microbial choline TMA lyase activity in vitro and in vivo.

“We recently reported the use of a natural product, 3,3-dimethyl-1-butanol (DMB), as a tool drug that inhibits microbial choline TMA lyase activity in vitro and in vivo35.”

• cites [35] Wang, Z. et al. Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Cell 163, 1585–1595 (2015). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

IMC and FMC display characteristics consistent with covalent inhibition, being time-dependent, irreversible, and non-competitive.

“both IMC and FMC display numerous characteristics consistent with a covalent mode of inhibition (time-dependent, irreversible, and non-competitive inhibition)”

May combine two claims.

IMC and FMC are non-competitive, time-dependent, and irreversible, which are hallmarks of mechanism-based inhibitors.

“Kinetic analyses demonstrate that IMC and FMC are non-competitive, time-dependent and irreversible, hallmarks of mechanism-based inhibitors.”

May combine two claims.

Inhibiting microbial TMAO production would not push platelet function below normal and therefore would not increase bleeding risk.

“inhibition in microbial TMAO generation would not hypothetically suppress platelet function “below normal” and thus heighten bleeding risks.”

Classified as probably not a substantial claim. May combine two claims.

Inhibitors that are poorly absorbed, act as suicide substrates on gut microbial catabolic pathways tied to disease pathogenesis, and are potentially generalizable for achieving therapeutic outcomes in the host.

“The present studies suggest development of poorly absorbed suicide substrate inhibitors of gut microbial catabolic pathways mechanistically linked with disease pathogenesis deserves further investigation as a potentially generalizable approach to achieve therapeutic outcomes in the host.”

• AssumptionRests on an assumption: The mouse models used in these studies predict human TMAO metabolism, thrombosis, and therapeutic response.
• AssumptionRests on an assumption: The inhibitor-induced changes in mouse cecal microbiota composition represent effects on the human gut microbiome.
• AssumptionRests on an assumption: The absence of systemic toxicity and bleeding effects in mice predicts safety in humans.

Probably not a substantial claim: may not pass S3 (load-bearing).

Fluorine substitution can confer beneficial properties to suicide substrate inhibitors and their pharmaceutical use.

“fluorine substitution can confer properties beneficial to the chemistry of suicide substrate inhibitors and their potential use as pharmaceuticals56.”

• cites [56] Clarke, D. D. Fluoroacetate and fluorocitrate: mechanism of action. Neurochem. Res. 16, 1055–1058 (1991). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Investigators were not blinded to mouse group allocation during animal husbandry tasks.

“Investigators were not blinded to mouse group allocation during the performance of animal husbandry requirements for experiments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Investigators who performed quantitative analyses of endpoints were blinded to group allocation, with samples identified only by code.

“Investigators performing quantitative analyses of endpoints (e.g., plasma, urine, or fecal metabolite levels) were blinded to group allocation with samples labeled by code only.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Many drugs cause shifts in gut microbial community composition.

“many drugs induce shifts in microbial community composition57,58.”

• cites [57] Imhann, F. et al. Proton pump inhibitors affect the gut microbiome. Gut 65, 740–748 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [58] Rogers, M. A. M. & Aronoff, D. M. The influence of non-steroidal anti-inflammatory drugs on the gut microbiome. Clin. Microbiol. Infect. 22, 178.e1–178.e9 (2016). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

In a study of more than 1,000 clinically used drugs tested against many human gut commensals, nearly one quarter had antibiotic-like side effects.

“In a recent examination of over 1,000 clinically used drugs and their impact on a broad selection of human gut commensals, nearly a quarter demonstrated antibiotic-like side effects59.”

• cites [59] Maier, L. et al. Extensive impact of nonantibiotic drugs on human gut bacteria. Nature 555, 623–628 (2018). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

A study of metformin suggested that a shift in microbial community structure contributed in part to its beneficial metabolic effects.

“And in a recent elegant study with metformin, a shift in microbial community structure was suggested to contribute in part to its observed beneficial metabolic effects60.”

• cites [60] Wu, H. et al. Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nat. Med. 23, 850–858 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Inhibiting microbial choline use will lower TMA/TMAO generation and may improve choline availability for the host.

“These results suggest that inhibition in microbial choline utilization will not only suppress TMA/TMAO generation, but also may help enhance choline bioavailability for the host.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All animals in a given experiment were included in the analyses.

“Results from all animals in a given experiment were included in the analyses.”