Vince Wacher

Lovastatin lactone may improve irritable bowel syndrome with constipation (IBS-C) by inhibiting enzymes in the archaeal methanogenesis pathway.

Methane produced by the methanoarchaeon Methanobrevibacter smithii ( M. smithii) has been linked to constipation, irritable bowel syndrome with constipation (IBS-C), and obesity. Lovastatin, which demonstrates a cholesterol-lowering effect by the inhibition of HMG-CoA reductase, may also have an anti-methanogenesis effect through direct inhibition of enzymes in the archaeal methanogenesis pathway. We conducted protein-ligand docking experiments to evaluate this possibility. Results are consistent with recent clinical findings. METHODS: F420-dependent methylenetetrahydromethanopterin dehydrogenase ( mtd), a key methanogenesis enzyme was modeled for two different methanogenic archaea: M. smithii and Methanopyrus kandleri. Once protein models were developed, ligand-binding sites were identified. Multiple ligands and their respective protonation, isomeric and tautomeric representations were docked into each site, including F420-coenzyme (natural ligand), lactone and β-hydroxyacid forms of lovastatin and simvastatin, and other co-complexed ligands found in related crystal structures. RESULTS: 1) Generally, for each modeled site the lactone form of the statins had more favorable site interactions compared to F420; 2) The statin lactone forms generally had the most favorable docking scores, even relative to the native template PDB ligands; and 3) The statin β-hydroxyacid forms had less favorable docking scores, typically scoring in the middle with some of the F420 tautomeric forms. Consistent with these computational results were those from a recent phase II clinical trial ( NCT02495623) with a proprietary, modified-release lovastatin-lactone (SYN-010) in patients with IBS-C, which showed a reduction in symptoms and breath methane levels, compared to placebo. CONCLUSION: The lactone form of lovastatin exhibits preferential binding over the native-F420 coenzyme ligand in silico and thus could inhibit the activity of the key M. smithii methanogenesis enzyme mtd in vivo. Statin lactones may thus exert a methane-reducing effect that is distinct from cholesterol lowering activity, which requires HMGR inhibition by statin β-hydroxyacid forms.

Mo2051 Lovastatin Improves Stool Form in Methanobrevibacter Smithii Colonized Rats With Constipation

30 adult, male Sprague-Dawley rats were placed on a high-fat diet (60.3% kcal from fat, Teklad high-fat diet TD.06414, Harlan Laboratories Inc, Madison, WI) for 7 weeks. The rats were assessed for increased M. smithii by qPCR before and after the diet, and then divided into 3 groups. Group 1 was given lovastatin in its lactone form, Group 2 was given lovastatin hydroxy acid (each 1.5 mg/rat), and Group 3 was gavaged with a placebo. Each group was gavaged daily for 10 days. Three day stool collections were performed to assess average stool wet weight and daily variability prior to commencing the highfat diet, after 7 weeks of high-fat diet, and the final days of the lovastatin gavage (still on high-fat diet). On day 10 of the gavage, rats were euthanized and DNA was extracted from contents of ligated bowel segments (duodenum, jejunum, ileum, cecum and left colon). qPCR was performed using primers for total luminal bacteria and M. smithii. RESULTS

Selection of a cut-off for high- and low-methane producers using a spot-methane breath test: results from a large north American dataset of hydrogen, methane and carbon dioxide measurements in breath.

Abstract Background: Levels of breath methane, together with breath hydrogen, are determined by means of repeated collections of both, following ingestion of a carbohydrate substrate, at 15–20 minutes intervals, until 10 samples have been obtained. The frequent sampling is required to capture a rise of hydrogen emissions, which typically occur later in the test: in contrast, methane levels are typically elevated at baseline. If methane emissions represent the principal objective of the test, a spot methane test (i.e. a single-time-point sample taken after an overnight fast without administration of substrate) may be sufficient. Methods: We analysed 10-sample lactulose breath test data from 11 674 consecutive unique subjects who submitted samples to Commonwealth Laboratories (Salem, MA, USA) from sites in all of the states of the USA over a one-year period. The North American Consensus (NAC) guidelines criteria for breath testing served as a reference standard. Results: The overall prevalence of methane-positive subjects (by NAC criteria) was 20.4%, based on corrected methane results, and 18.9% based on raw data. In our USA dataset, the optimal cut-off level to maximize sensitivity and specificity was ≥4 ppm CH4, 94.5% [confidence interval (CI): 93.5–95.4%] and 95.0% (CI: 94.6–95.5%), respectively. The use of a correction factor (CF) (5% CO2 as numerator) led to reclassifications CH4-high to CH4-low in 0.7 % and CH4-low to CH4-high in 2.1%. Conclusions: A cut-off value for methane at baseline of either ≥4 ppm, as in our USA dataset, or ≥ 5 ppm, as described in a single institution study, are both highly accurate in identifying subjects at baseline that would be diagnosed as ‘methane-positive’ in a 10-sample lactulose breath test for small intestinal bacterial overgrowth.

Initial Paradoxical Peaks on Breath Testing (First Sample High) do not Affect the Diagnostic Utility of the Spotmethane Breath Test

• A first-sample high for CH4 was observed in 12.9 % of cases but would have a small impact on the classification results (5.8 % reclassification). • The FSH has traditionally been explained biologically and may not be an artefact. However, our findings suggest that instrumentation and workflow cannot be ruled out as causative or contributory. This should be further investigated. • The data from this study give additional support to the clinical validity and usefulness of a spot (single sample) breath methane test. Previous authors suggested that the initial hydrogen peak could be neglected. If, however, only one sample is taken (spot-methane breath test), the possible impact of this phenomenon needs to be clarified and the following established: 1) Does the FSH also occur with methane? 2) If so, how does this affect its diagnostic utility of the spot-methane breath test?

Increased Pulmonary CO2 Excretion in Patients with Small Intestinal Bacterial Overgrowth (SIBO): Possible Implications for COPD

In this study, we saw a small but highly statistically significant increase in exhaled CO2-percentage in SIBO (4.28 % to 4.35 %, p < 0.0001). This increase corresponds to 0.53 mmHg, or a change in MV of 1.6 L/min. The normal MV at rest is between 5 and 8 L per minute. Bacterial CO2-production in SIBO may therefore account for 20 – 30 % of the MV needed to keep a steady state, at least in the context of the LBT. This increase should have no effect on otherwise healthy patients but could be relevant in patients with baseline hypercapnia. While further studies appear warranted, it may be reasonable to test patients with hypercapnia for SIBO as a possible contributing factor that would be amenable to intervention. 1) To investigate whether there is an increased CO2 concentration in the exhaled breath of subjects who undergo a 10-sample lactulose breath test (LBT) who have small intestinal bacterial overgrowth (SIBO) compared to those subjects who do not have SIBO by breath test criteria. 2) To correlate increased CO2 exhalation with increased minute volume and work of breathing potentially relevant in advanced lung disease.