Review Article: Inhibition of Methanogenic Archaea by Statins As a Targeted Management Strategy for Constipation and Related Disorders

Home , Methanobacteria, Methanobacteriaceae, Pseudopeptidoglycan

Alimentary Pharmacology and Therapeutics

Review article: inhibition of methanogenic archaea by statins as a targeted management strategy for constipation and related disorders

K. Gottlieb*, V. Wacher*, J. Sliman* & M. Pimentel†

*Synthetic Biologics, Inc., Rockville, SUMMARY MD, USA. † Gastroenterology, Cedars-Sinai Background Medical Center, Los Angeles, CA, USA. Observational studies show a strong association between delayed intestinal transit and the production of methane. Experimental data suggest a direct inhibitory activity of methane on the colonic and ileal smooth muscle and Correspondence to: a possible role for methane as a gasotransmitter. Archaea are the only con- Dr K. Gottlieb, Synthetic Biologics, ﬁ Inc., 9605 Medical Center Drive, rmed biological sources of methane in nature and Methanobrevibacter Rockville, MD 20850, USA. smithii is the predominant methanogen in the human intestine. E-mail: [email protected] Aim To review the biosynthesis and composition of archaeal cell membranes, Publication data archaeal methanogenesis and the mechanism of action of statins in this context. Submitted 8 September 2015 First decision 29 September 2015 Methods Resubmitted 7 October 2015 Narrative review of the literature. Resubmitted 20 October 2015 Accepted 20 October 2015 Results EV Pub Online 11 November 2015 Statins can inhibit archaeal cell membrane biosynthesis without affecting This uncommissioned review article was bacterial numbers as demonstrated in livestock and humans. This opens subject to full peer-review. the possibility of a therapeutic intervention that targets a speciﬁc aetiologi- cal factor of constipation while protecting the intestinal microbiome. While it is generally believed that statins inhibit methane production via their effect on cell membrane biosynthesis, mediated by inhibition of the HMG- CoA reductase, there is accumulating evidence for an alternative or additional mechanism of action where statins inhibit methanogenesis directly. It appears that this other mechanism may predominate when the lactone form of statins, particularly lovastatin lactone, is administered.

Conclusions Clinical development appears promising. A phase 2 clinical trial is currently in progress that evaluates the effect of lovastatin lactone on methanogenesis and symptoms in patients with irritable bowel syndrome with constipation. The review concludes with an outlook for the future and subsequent work that needs to be done.

Aliment Pharmacol Ther 2016; 43: 197–212

ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. 197 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. doi:10.1111/apt.13469 K. Gottlieb et al.

– INTRODUCTION constipation6 8 and one study that concludes that colonic Methane, which is now of great interest to climate methane production is not associated with clinical pre- researchers, plays an important if somewhat underappre- sentation in IBS patients.9 ciated role in gastrointestinal disorders. The link between Two microbiome studies correlating methanogens and this odourless gas and the intestine was first made in the stool frequency have recently become available. One early days of colonoscopy, when sparks from electrosur- study evaluated the correlation between gut microbiota gical devices sometimes led to intracolonic explosions.1 variation and stool consistency using the Bristol Stool Microbial metabolism of mannitol, a sugar alcohol that Scale (BSS) classification. Enterotypes were distinctly dis- had been commonly used for colon lavage, led to tributed over the BSS scores: Within the RB enterotype, increased concentrations of the flammable gases hydro- found in harder stool samples, the abundance of gen and methane. Modern bowel preps do not carry that Methanobrevibacter and Akkermansia was positively cor- risk. related with colon transit time.10 The results of another Methane can have inorganic (e.g. geothermal-volcanic microbiome study analysing 273 faecal samples showed methane emissions) and organic sources. The only that only patients with a constipation phenotype pre- organisms known to produce methane are methanogenic sented higher abundance of methanogenic archaea, con- archaea. Archaea are fascinating prokaryotes that were firming a link between low transit time and methane previously considered to be bacteria. However, these production capacity.11 prokaryotes, while resembling bacteria on a superficial Association does not prove causation, and it has long level, were found to have so many unique characteristics been known that intestinal dysmotility, as seen in sclero- that they were given their own name, archaea, and were derma, leads to secondary small intestinal bacterial (and, elevated to occupy a position as one of the three presumably, archaeal) overgrowth. It could be argued domains of life: archaea, bacteria, and eucarya.2, 3 that increased methane production is therefore not the Less spectacular than its explosive capacity but more cause but an effect of delayed intestinal transit. Fourteen important is recent evidence that links intestinal healthy volunteers underwent four interventions: placebo, methane production to slowed intestinal transit.4 In this sulphate supplements, or sulphate supplements with review, we will describe how large quantities of methane either senna or loperamide. With faster intestinal transit are produced in the colon and how statins may have a reduction in faecal methanogens and methane produc- therapeutic importance by inhibiting methane production was seen. The reverse effects were true with lop- tion through the selective suppression of the growth of eramide.12 However, there is experimental evidence that methanogens. methane directly affects intestinal transit and contractil- ity (methane-first hypothesis). METHANE AND CONSTIPATION-ASSOCIATED DISORDERS Experimental evidence While methane is predominantly produced in the colon, Methane and intestinal transit methanogens can also be demonstrated in the small Although 50–80% of methane in humans is passed as intestine, especially when small bowel bacterial over- flatus, the presence of methane production can be accu- growth exists.13 Pimentel and colleagues conducted a rately assessed by breath tests. A comprehensive review three-part study consisting of two experiments – one recently concluded that data from breath testing gener- in vivo (dogs) and one ex vivo (guinea pig ileum) – as ally support the association between delayed intestinal well as a retrospective review of data in irritable bowel transit and the production of methane.5 Methodological syndrome (IBS) patients who had previously undergone differences in study design precluded a formal meta-ana- breath testing and antroduodenal motility studies.4 In lysis approach for studies that correlated diagnosis of dogs, small intestinal fistulae were created and transit constipation-predominant IBS (IBS-C) or chronic idio- of a radiotracer was measured during infusion of ambi- pathic constipation with methane production, neverthe- ent air, followed by methane at a rate that was physio- less, the same report5 lists 14 studies supporting the logical (i.e. resulting in 50 ppm in exhaled air), a association between methane production and constipa- concentration that is typical for patients with irritable tion related disorders in adults. There are a few reports bowel syndrome with constipation (IBS-C). The that primarily evaluated other hypotheses that did not methane infusion resulted in decreased radiotracer show a link between methane on breath testing and recovery in each of five individual dogs compared to

198 Aliment Pharmacol Ther 2016; 43: 197–212 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. Review: methane, archaea, statins and the gut baseline. The average reduction was 59%, which was survival conditions for archaea – more methane is pro- highly significant (P < 0.001). duced further slowing down intestinal transit leading to In the second experiment, 3-cm-long terminal ileum symptomatic constipation – archaea thrive and multiply preparations from sacrificed guinea pigs were oriented in until a steady state is achieved. an oral-aboral fashion in a physiological buffer solution and stimulated before and after methane was bubbled THE ORIGIN AND PROPERTIES OF METHANE through the tissue bath. Methane exposure at a concen- PRODUCTION tration of 1000 ppm led to a significant augmentation of The five major gases in the colon are: nitrogen (23– the contractile force in response to the applied brush 80%), hydrogen (0.06–47%), carbon dioxide (5.1–29%), stroke, consistent with hyperactivity. oxygen (0.1–2.3%) and methane (0–26%).1 Finally, after retrospective review, 11 methane-produ- In addition, nitric oxide and hydrogen sulphide are cing and 12 sex-matched hydrogen-producing subjects found as well. These gases have great importance were identified who met Rome I criteria for IBS and, in through their function as gasotransmitters and compo- addition, had antroduodenal manometry data available. nents of a complex microbial ecosystem.12, 15 All the The fasting motility index, as a gross measure of forces methane in the intestine is produced by methanogenic (resistive and propulsive) in the small intestine, in bacteria of which Methanobrevibacter smithii, described methane-producing subjects was noted to be significantly in more detail below, is the most important contributor. higher (1851 Æ 861) compared with hydrogen-producing Methane is overwhelmingly produced by strictly anaero- subjects (1199 Æ 301; P < 0.05). bic archaea that produce methane from substrates such The authors concluded that methane slows small as hydrogen, carbon dioxide and certain other substrates. bowel transit and augments contractile activity in the Emissions from agriculture represent around 40% of the small bowel, and in addition, small intestinal contractile methane emissions produced by human-related activities, activity is increased in IBS patients who produce the single largest source is enteric fermentation in live- methane. They speculated that methane predisposes to stock.16 constipation because it promotes segmental (nonpropa- Archaea are a large and diverse class of prokaryotes gating contractions) and suggested that further work, that, together with bacteria and eukaryotes, make up the especially involving the colon, was warranted.4 three domains of life.17 More recently evidence for direct In similar experiments, a Korean group reproduced methane production and emission by eukaryotes such as and expanded upon the results of the above studies by plants, animals and fungi has been presented. While this including colonic motility studies and comparing the seems to have been confirmed for plants, mammalian effects of hydrogen gas with those of methane. Briefly, in endogenous (nonmicrobial) methane production remains an ex vivo experiment using guinea pig ileal, right and controversial.18 These findings, if corroborated, could, left colon segments in a peristaltic tissue bath, ileal con- however, support a gasotransmitter role for methane.19 tractile activity significantly decreased and the amplitude Various archaeal species have been shown to inhabit of peristaltic contractions increased in response to distinct human body sites such as the intestine, the oral methane insufflation, while the opposite phenomenon cavity, the vagina and, most recently, the skin.20 The was detected after hydrogen infusion. Colonic transit was majority of the so far detected archaea in humans, par- shortened by hydrogen infusion, but this effect was ticularly from the gut and mouth, represent members of blunted when methane was co-administered, especially the family Methanobacteriaceae.21 in the right colon.14 The preceding experimental and observational studies Methane phenotype support a causative role for methane in constipation- Breath tests conducted in humans register either related disorders, especially IBS-C. Ultimately the argu- methane (30–50%) or hydrogen (50–70%) but only ment that slow transit could cause overgrowth of metha- rarely both.22 This makes sense if one considers the stoi- nogenic bacteria is not necessarily a detractor from the chiometry of hydrogen removal by methanogens: The methane-first hypothesis. Perhaps a feedback loop exists production of one mole of methane removes 4 moles of + ? + where the terms cause and effect have less meaning and hydrogen (4H2 CO2 CH4 2H2O). The designa- opportunities for therapeutic intervention exist in this tion of subjects as ‘methane producers’ or ‘methane non- circle: Slow transit promotes archaeal growth – archaea producers’ based on breath testing only accounts for one produce methane – methane produces better growth and way of escape but not the large amount that is passed

Aliment Pharmacol Ther 2016; 43: 197–212 199 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. K. Gottlieb et al. directly as flatus (between 50% and 80% of the total). nected to hypoxic events leading to, or associated with, Indeed, one older report suggests that methane is not mitochondrial dysfunction. detected in breath until the methanogens reach a density In order for methane to move from its status as can- of 108 methanogenic bacteria per gram of stool.23 How- didate-gasotransmitter to accepted gasotransmitter, direct ever, as always, the situation could be more complex. A endogenous production in mammalian organisms will recent study from New Zealand using highly sensitive need to be confirmed and the biochemistry and physiol- open-circuit respiration chambers (that account for total ogy clarified. As will be reviewed in the following, animal methane emissions) identified a low- and high- regardless of the eventual outcome of these investiga- methane phenotype in sheep that was attributed to the tions, sufficient evidence is accumulating to justify explo- composition of microbial communities with methanogen ration of the methanogenesis pathway of intestinal densities that were not significantly different between the archaea as a drug target in patients with certain consti- high and low groups.24 Open-circuit respiration cham- pation related disorders. bers are not practical in humans and dividing subjects into methane producers and nonproducers by conven- METHANOGENESIS IN THE HUMAN GUT tional breath test results is both convenient and clinically Understanding the properties of methanogens in the gut meaningful. and their physiology helps us to consider how their methane production might be curtailed by statins and Methane as a gasotransmitter other compounds. Methane may be one of a series of gasotransmitters – endogenously generated, gaseous signalling molecules Archaea and methanobacteria that do not require membrane receptors for their activ- Like bacteria, archaea are prokaryotes but many of their ity. Gasotransmitters are evolutionally conserved from characteristic features are distinct from those of bacteria bacteria and archaea to plant and mammalian cells. and eukaryota. Many archaea are best known for their Nitric oxide (NO) was the first identified gasotransmit- ability to thrive in extreme environments. Others, such as ter; carbon monoxide and hydrogen sulphide have been the class called Methanobacteria (a name which was added more recently, and ammonia, hydrogen and assigned before it was recognised that they are not ‘bacte- methane have achieved candidate status.25 The discovery ria’), may live in less forbidding environments but are ‘ex- of mammalian NO metabolism (‘endothelium-derived treme’ in their metabolic capabilities. They carry out what relaxing factor’) has led to a whole class of drugs, the at first appears to be a contradiction in terms: anaerobic phosphodiesterase inhibitors.26 Drugs that release hydro- respiration. Aerobic respiration has oxygen as final elec- gen sulphide are currently being developed for their pos- tron acceptor. In contrast, anaerobic respiration relies on sible anti-neoplastic, chemoprotective and a final electron acceptor other than oxygen, in the case of chemopreventive potential.27, 28 M. smithii and other methanogenic species, carbon diox- Some intriguing findings support the candidacy of ide. Furthermore, M. smithii cannot use the conventional methane as a gasotransmitter. Boros, et al. 29 demon- carbohydrate, lipid and protein electron donors for energy fi strated that the inhalation of 2.5% CH4 signi cantly generation, instead it uses hydrogen gas. Biological ameliorated the extent of ischaemia-reperfusion damage methanogenesis is indeed an ancient ‘archaic’ process dat- in dogs. A parallel series of experiments published in the ing back 2.7 billion years when oxygen was still a trace 30 same report showed that exogenous CH4 inhibited leuco- element in the atmosphere and hydrogen was abundant. cyte infiltration in vitro, suggesting a connection between In summary, methanogens in the gut generate energy by ‘ ’ CH4 function and the immune system. Pimentel et al. breathing the exhaust of other bacteria in the gut (hydro- showed that intestinal methane gas infusion slowed gen and carbon dioxide) and producing methane as waste: + ? + 31 intestinal transit in dogs, and these experiments were 4H2 CO2 2H2O CH4. reviewed in detail above.4 Boros et al.19 carefully exam- It should be noted that methanogenic archaea are ined to which extent methane fulfils the criteria pro- not the only consumers of hydrogen (hydrogenotrophs) posed by Wang25 that characterise a gasotransmitters. in the gut. Sulphate-reducing bacteria can obtain The authors concluded that the data do not fully support energy by oxidising organic compounds or molecular the gasotransmitter concept, but suggested that methane hydrogen while reducing sulphate to hydrogen sulphide, liberation (from endogenous sources, not through the previously mentioned as a gasotransmitter. Hydrogen resident flora) may be linked to redox regulation con- sulphide is a colourless gas with the characteristic foul

200 Aliment Pharmacol Ther 2016; 43: 197–212 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. Review: methane, archaea, statins and the gut odour of rotten eggs. Hydrogen sulphide is toxic to colon by idealised theory or in vitro chemostat sys- colonocytes and can impair their metabolic function tems,33 and clinical studies will have to address this and has been implicated in the aetiology of ulcerative question. colitis.32 It is therefore conceivable that a decrease in methanogenic activity could favour sulphate-reducing M. smithii bacteria with a concomitant increase in sulphide pro- The dominant methanogen in the human intestine is duction. Indeed, competition between hydrogenotrophs M. smithii. Methanosphaera stadtmanae, which transfers has long been of interest to microbial ecologists. How- electrons from hydrogen to methanol instead of carbon ever, according to a comprehensive review of this issue, dioxide, is found less frequently (Figure 1). M. smithii is it appears impossible to fully explain the outcome of well adapted to the human intestine. It produces surface the competition among hydrogenotrophs in the human glycans resembling those found in the gut mucosa, regu-

Taxonomy of M. smithii

Kingdom Archaea

Phylum Euryarchaeota

Class Methanobacteria

Order Methanobacteriales

Family Methanobacteriaceae

Genus Methanobrevibacter (Balch and Wolfe, 1981)

Species Methanobrevibacter smithii – human feces

Methanobrevibacter arboriphilicus – eastern cotton wood tree, wetwood

Methanobrevibacter curvatus – hindgut of termite Reticulitermes flavipes

Methanobrevibacter cuticularis – hindgut of termite Reticulitermes flavipes

Methanobrevibacter filiformis – hindgut of termite, Reticulitermes flavipes

Methanobrevibacter gottschalkii – horse feces

Methanobrevibacter millerae – bovine rumen fluid

Methanobrevibacter olleyae – ovine (sheep) rumen fluid

Methanobrevibacter oralis – human oral cavity

Methanobrevibacter ruminantium – bovine rumen

Methanobrevibacter acididurans – acidogenic digester running on alcohol distillery waste

Methanobrevibacter thaueri – bovine feces

Methanobrevibacter woesei – goose feces

Methanobrevibacter wolinii – sheep feces

Figure 1 | Taxonomy of Methanobrevibacter smithii. Only two methanogenic species have so far been isolated from the human colon: M. smithii is the predominant methanogen in the human gut. Methanosphaera stadtmanae, family Methanobacteriaceae, is less abundant. Note the Methanobrevibacter species in bovine and sheep rumen and mammalian faeces. The ofﬁcial taxonomy does not necessarily correlate with current molecular phylogenetics. Data from the Integrated Taxonomic Information System (ITIS)52–64 with isolate information from the Global Catalogue of Microorganisms.65

Aliment Pharmacol Ther 2016; 43: 197–212 201 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. K. Gottlieb et al. lates the expression of adhesin-like proteins, consumes phospholipid bilayer with intercalating proteins (and fermentation products produced by saccharolytic bacte- cholesterol in animals). In organisms other than archaea, ria, effectively competes for nitrogen compounds, and – this phospholipid bilayer is composed of glycerol-3-phos- crucial for being well adapted to the gut – can grow in phate linked as an ester to fatty acids. In contrast, the presence of bile salts.34 archaeal lipids are not composed of fatty acid esters; The cytoplasm of M. smithii is enclosed by a cell instead, they are saturated, branched, repeating iso- membrane and a cell wall35 that is decorated with the prenoid subunits that attach to glycerol via an ether link- glycans (mentioned above), an arrangement that is age (Figure 2). Isoprenoids are a ubiquitous and ancient familiar to us from the Gram-positive bacteria. Indeed, class of biomolecules found in all living organisms that M. smithii stains positive with the Gram stain.34 How- exhibit a remarkable diversity of structures and func- ever, compared to bacteria, there are significant differ- tions. Isoprenoids are derived from a basic five-carbon ences, both in cell wall and in cell membrane precursor unit, isopentenyl diphosphate (IPP), and its composition. Cell wall peculiarities have relevance to isomer dimethylallyl diphosphate (DMAPP); these iso- susceptibility to common antibiotics. The cell membrane meric phosphates are the activated forms of isoprene.40 of most bacteria is synthesised by the MEP (methylery- M. smithii has a cell membrane whose phospholipid thritol 4-phosphate) pathway.36 In contrast, the iso- fraction contains isoprenoids typical for archaea, prenoid biosynthesis for the main cell membrane archaeol and caldarcheol, the latter is arranged in a components in archaea (archaeol) relies on the same monolayer rather than a bilayer in the cell enzyme that catalyses the biosynthesis of the isoprenoid membrane.41, 42 cholesterol in humans, the HMG-CoA reductase (mevalonate pathway).37 This obviously raises the possi- Archaea and humans use the MVA and bacteria the bility that statins – which are HMG-CoA reductase MEP pathway for isoprenoid biosynthesis inhibitors – could be used to interfere with the biosyn- For many years, it was believed that IPP was synthesised thesis of the archaeal cell membrane and thus to inhibit by all organisms from acetyl-CoA through the well- archaeal growth, as described by Miller and Wolin in known mevalonate (MVA) or HMG-CoA reductase 2001.38 pathway. However, it is now well established that an alternative, MVA-independent pathway is used in the Archaeal cell wall majority bacteria, algae and plants, for the biosynthesis The cell wall of archaea is composed of pseudomurein of IPP and DMAPP, the MEP pathway mentioned ear- (pseudopeptidoglycan). There are significant differences lier.40 This is relevant for two reasons: (i) Statins can between pseudomurein and murein such as the occur- interfere with the cell membrane synthesis of archaea rence of talosaminuronic acid instead of muramic acid, and will leave the overwhelming majority of the gut the presence of b (1?3) linkage instead of b (1?4) microbiome untouched. (ii) Interference with cell wall linkage of the glycan components, the partial replace- function interferes with the function of the cell mem- ment of glucosamine by galactosamine, the lack of D- brane-bound enzymes that are responsible for methano- amino acids, and the accumulation of e- and c-peptide genesis (Figure 3). bonds.35 Together these differences are responsible for resistance to cell wall antibiotics such as b-lactams and THE PHYSIOLOGY OF METHANE PRODUCTION BY to lysozyme and common proteases.35 In addition, ARCHAEA archaea are resistant to a number of other cell wall- altering antibiotics with the notable exception of squa- The importance of HMG-CoA reductase lene. They are susceptible to a variable degree to anti- HMG-CoA reductase (HMGR, 3-Hydroxy-3-methylglu- microbials that interfere with DNA, including the broad- taryl coenzyme A reductase) can be found in two differ- spectrum imidazoles (e.g. metronidazole).39 ent forms: eukaryotes and archaea use HMGR class I (membrane-bound) and a few bacteria use HMGR class Archaeal cell membrane II (cytosolic).36 As was mentioned, the majority of bacte- Perhaps, more important for our discussion is the fact ria do not employ HMGR but the enzymes of the MEP that the cell membrane of archaea contains unique polar pathway for isoprenoid biosynthesis. HMGR catalyses lipids. Recall that the limiting membrane of cells and the first step in the biosynthesis of cholesterol in humans various organelles (in eukaryotes) is composed of a fluid and also the first step in the biosynthesis of the archaeal

202 Aliment Pharmacol Ther 2016; 43: 197–212 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. Review: methane, archaea, statins and the gut

Archaeol (bilayer)

Caldarchaeol (monolayer)

Cell membrane

Archaea lipid Branched isoprene chains Ether links

Bacteria lipid

Unbranched fatty acids Ester links

100 nm

Methanobrevibacter smithii

Figure 2 | Methanobrevibacter smithii cell wall and cell membrane determine susceptibility to antibiotics and statins. The cell wall (violet) is composed of pseudomurein (and not murein as in bacteria) which makes archaea resistant to lysozyme and many antibiotics that interfere with cell wall synthesis. The cell membrane (ochre) consists of a lipid bilayer or monolayer the backbone of which composed of isoprene units that are linked to glycerol by ether bonds. In contrast, the lipid bilayer of bacteria consists of a fatty acid backbone that is linked to glycerol by an ester bond. The presence of statin-sensitive isoprene units in the cell membrane of archaea allows statins to selectively interfere with the growth of archaea while leaving the cell membrane of bacteria unaffected. While bacteria do not use isoprene units in their cell membrane they are still required elsewhere. These bacterial isoprene units are, however, synthesised by a pathway (MEP) that is not inhibited by statins. See also Figure 3.

isoprene polymers (polyprenols) archaeol and caldar- coenzymes, a multistep pathway to methane, and a num- chaeol, which are the main constituents of the M. smithii ber of unique membrane-bound enzyme complexes for cell membrane. coupling to the protein motive-force17 (Figure 4). The inhibitory effects of HMGR-inhibitors are generally Anaerobic respiration by methanogenesis thought to be related to the disruption of the cell mem- Anaerobic respiration by methane biosynthesis is more brane by interfering with isoprenoid biosynthesis, complex than many other common respiratory processes thereby disturbing the membrane-bound respiratory because it requires the biosynthesis of six unusual chain of methanogenesis; however, alternative

Aliment Pharmacol Ther 2016; 43: 197–212 203 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. K. Gottlieb et al.

MEP

O COOH Pyruvate MEP and MVA pathways O + O – for the biosynthesis GAP H O P O OH – O of isoprenoid precursors

PDH* DXS in bacteria and archaea

OH O O – DXP O P O OH – O MVA

(DXR-I) (DRL/DXR-II) O S CoA Acetyl-CoA OH O – AACT O P O OH OH – O O O [MCT/IspD] Acetoacetyl- S CoA OH O CoA O O NNH2 HMGS O P O P O O N OH OH – O – O CDP-ME O HO OH OH O HMG-CoA HOOC – – S CoA O P O CMK/IspE O O O O N NH2 (HMGR-I) (HMGR-II) O P O P O O N OH OH – O – O OH CDP-MEP HOOC HO OH OH MDS/IspF MVK – O O PMVD O P O O OH O HOOC – P MEcPP O P O O O – – O OH OH IP MVP PMVK HDS/IspG O – OO O P O OH O O – – O HOOC – O P O P O HMBPP O P O P O OH – O – O – O – O IPK [HDR/IspH] MVPP DPMD

OO (IDI-I) O O – – DMAPP O P O P O O P O P O IPP – O – O (IDI-II) – O –O

Figure 3 | Most bacteria are not affected by statins. Most bacteria produce isoprenoids by the MEP pathway (red) that is not affected by HMGR-inhibitors (statins), most other organisms including humans and archaea use the MVA pathway (blue). MEP: 2-C-methyl-D-erythritol 4-phosphate. MVA: mevalonic acid. Humans and archaea utilise the HMGR-I isoform (inhibited by statins) for isoprenoid biosynthesis. GAP, D-glyceraldehyde 3-phosphate; DXP, 1- deoxy-D-xylulose 5-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-d-erythritol; MEcPP, 2-C-methyl-D- erythritol 2,4-cyclodiphosphate; HMBPP, 4-hydroxy-3-methylbut-2-enyl diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate. HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MVP, 5-phosphomevalonate; MVPP, 5-diphosphomevalonate. The alternative steps described in archaea are shown in green (IP, isopentenyl phosphate). Enzymes catalysing other reactions besides those in the MEP pathway are between brackets. Steps catalysed by different types of enzymes (shown within parentheses) are highlighted. Enzyme acronyms are given in the paper by Perez-Gil and Rodriguez-Concepcion36 from which this illustration has been adapted. possibilities exist, and these could be relevant in explain- The ﬁnal common pathway of methanogenesis ing some of the experimental results observed with dif- Early pathways in methanogenesis funnel into the same ferent types of statins.43 A brief review of the key steps ﬁnal step catalysed by the key enzyme methyl-coenzyme in methanogenesis is therefore necessary. M reductase (mcr): methyl-coenzyme M (CH3-S-CoM)

204 Aliment Pharmacol Ther 2016; 43: 197–212 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. Review: methane, archaea, statins and the gut reacts with a second thiol coenzyme called coenzyme B through the body,48 at least in theory. Based on water- (CoB-SH) to form methane and the heterodisuphide of octanol partition coefficients, a differentiation into fat coenzyme M and coenzyme B (CoM-S-S-CoB) (Fig- soluble or hydrophobic (atorvastatin, simvastatin, fluvas- ure 4). In its active site, mcr contains a unique active tatin, lovastatin, cerivastatin) and water soluble or hydro- group, a nickel porphinoid, as prosthetic group called philic statins (rosuvastatin, pravastatin) is made. 17 coenzyme F430 (Figure 5). The characteristic auto- Lipophilic statins undergo hepatic and enteric metabo- fluorescence of methanogens, is, however, largely caused lism via cytochrome P450 (the CYP450 family of by coenzyme F (for Fluorescence) F420 [with an absorp- enzymes), whereas the water soluble statins are excreted tion maximum at 420 nm emitting at 480 nm (blue)], largely unchanged.48 Fat soluble statins are known to which participates in earlier steps of methanogenesis44 cross the blood brain barrier, whereas water soluble sta- (Figure 5). tins are often thought not to cross the barrier. However, the situation is more complicated, and this may have rel- Coenzyme F420 and NADP-dependent oxidoreductase evance for the crossing other lipid membranes.

Coenzyme F420, a hydride carrier, is found in archaea and some bacteria and has, in addition to methanogene- Lactone and hydroxyacid forms of statins sis, crucial roles in antibiotic biosynthesis, DNA repair, Another important aspect of statin chemistry is the exis- and activation of anti-tubercular compounds 45 (Fig- tence of closed-ring lactone and hydroxyacid forms (Fig- ure 5). While methanogenesis in M. smithii can proceed ure 6). All statins can be converted from one to the with carbon dioxide and hydrogen alone, there is evi- other, but simvastatin and lovastatin are the commer- dence that M. smithii can utilise the bacterial fermenta- cially available statins that come in the lactone form; tion products alcohol and methanol for methanogenesis they are prodrugs and to be active (i.e. inhibit HMGR) using an inducible NADP-dependent oxidoreductase they require opening of the lactone ring into the hydrox- 47, 49 with a coenzyme F420 prosthetic group (F420-dependent yacid form. The lactones of cerivastatin, simvastatin, NADP oxidoreductase, fno).46 Fno is mentioned here in atorvastatin, lovastatin, fluvastatin, and pravastatin, anticipation of a review of molecular docking experi- ordered according to decreasing hydrophobicity, are ments with fno that suggest an additional or alternative three to four times more lipophilic as compared to their mechanism of action for statins. acid forms.50 In other words, the lactones of lipophilic statins are best equipped to cross lipid membranes. At HOW STATINS INHIBIT METHANOGENESIS one stage or another, the lactone ring would need to be Statins are fungal secondary metabolites which inhibit broken up to inhibit HMGR; this will be discussed hydroxy-methylglutaryl coenzyme A (HMG-CoA) shortly. reductase as the first committed enzyme of cholesterol biosynthesis in humans (Figure 3, MVA pathway). Sta- Hydrophilic statins tins lower blood cholesterol by inhibiting cholesterol Pravastatin, whether lactone or acid, has the lowest oil- biosynthesis in the liver; the reduced hepatic choles- water distribution coefficients when compared with the terol concentration leads to a compensatory increase in other statins and would not normally be expected to expression of LDL receptors in liver cell membranes, cross the blood–brain barrier, however, it does, at least which enhances the clearance of the circulating LDL in mice, perhaps aided by the same organic anion cholesterol particles in the blood.47 Here we are, how- transporter polypeptide (OATP) that allows it to enter ever, interested in the methane lowering activity of sta- liver cells.49 Atorvastatin acid, lovastatin acid, and sim- tins, and to better understand the mechanism of vastatin acid, all more hydrophilic than their respective action, some statin pharmacology will need to be lactone forms, were also found to be transported into reviewed. cells by OATP2. Other transporters such as the mono- carboxylic acid transporters (MCT) may play a role as Lipid and water solubility of statins well.49 In general, statins have hydrophilic and hydrophobic regions, and are classed as amphiphilic drugs. Amphiphi- Considerations for the uptake of statins by M. smithii lic drugs do not require specific transport mechanisms to Let us now return to M. smithii. The ideal statin to get cross membranes. As they are soluble in aqueous biologi- absorbed by M. smithii would be a lactone prodrug such cal fluids and lipid membranes, they can simply diffuse as simvastatin and lovastatin. For example, the degree of

Aliment Pharmacol Ther 2016; 43: 197–212 205 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. K. Gottlieb et al.

Methanogenesis pathway

H CO2 2

MF Fdred Reduction of H O CO2 to formyl 2 Fdox O

MF C H Formyl

MP C H

H2 Reduction of F420 red formyl to methylene and H2O F then methyl 420 ox Lovastatin? C MP H2 Methylene H2 F420 red

F 420 ox Lovastatin? MP C H3 Methyl 2H CoM-SH Na+ motive force

CoM-S CH3 HS-CoB ATP Methyl reductase;

F430 complex CoM-S-S-CoB

Reduction of methyl C H4 Methane group to methane

Figure 4 | Methanogenesis pathway and potential role of lovastatin. Coenzymes F430 and F420 play crucial roles in the methanogenesis pathway (see also Figure 5). In silico molecular docking of the methanogenic enzyme F420-dependent NADP oxidoreductase (fno) showed that both lovastatin and mevastatin had higher afﬁnities for the F420 binding site on fno than did F420 itself. As such, lovastatin may act as inhibitor of fno. Fno is not shown in the diagram because it catalyses an alternative pathway of methanogenesis that utilises alcohol and methanol as substrates. However, if inhibition of fno by lovastatin can be experimentally conﬁrmed, it is likely that other enzymes, such as those in the

hydrogen-CO2-methanogenesis pathway depicted here that require the coenzyme F420 would also be inhibited by lovastatin (as suggested in the diagram). The carbon atom reduced is highlighted in green. MF, methanofuran; MP, methanopterin; CoM, coenzyme M; Fd, ferredoxin; CoB, coenzyme B. This ﬁgure was adapted from reference.66 hydrophobicity of imidazole derivatives correlates with human plasma and the liver.52 Indeed, M. smithii has a improved activity against human methanogenic predicted acetylesterase (NCBI Ref Seq archaea,51 and the situation should be analogous for sta- WP_019264088.1) which could catalyse this reaction. tins. However, if a lactone statin is given orally and Pravastatin, the most hydrophilic of all statins, would be expected to inhibit the archaeal HMGR, the lactone ring expected to be the least likely to cross the archaeal cell needs to be broken up once inside the cell – for example, wall. In view of this, it is surprising that pravastatin by a carboxylesterase or paroxonase, as is the case in remarkably inhibited the growth of M. thermautotrophi-

206 Aliment Pharmacol Ther 2016; 43: 197–212 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. Review: methane, archaea, statins and the gut

H Coenzyme F430 Coenzyme F420 O O

H O N H

– H O O O O O

H O O H N O O O H H N N – O H O ++ O P O Ni –N N H O O– H N H H N H H O O O O O– O H O H O N N O O N – O H

Figure 5 | Coenzymes in the methanogenesis pathway. The ﬁnal step in the methanogenesis pathway is catalysed by the key enzyme methyl-coenzyme M reductase (Mcr). In its active site, Mcr contains a unique active group, a nickel

porphinoid (left panel), called coenzyme F430. Coenzyme F420 (right panel) participates in two earlier steps in the methanogenesis pathway (see Figure 4) and is also responsible for the characteristic ﬂuorescence of methanogens. In

silico protein–ligand docking experiments suggest that lovastatin may have higher afﬁnity for the F420 binding site than F420 itself. cus and methanogenesis.53 Whether this can be ascribed Methanobrevibacter growth in vitro by 80–100% but did to active transport or not, is not known. not inhibit the growth of rumen bacteria responsible for In summary, the activity of statins in relation to metha- the fermentation of polysaccharides and starch in these nogens can apparently not be predicted based on simple animals (i.e. the bacteria providing methanogenic sub- pharmacological principles alone and requires empiric strates), as would be expected. In subsequent studies, a evaluation. What is, then, the experimental evidence? lovastatin concentration of 4 lmol/L caused a 50% inhibition of Methanobrevibacter growth in vitro and 100%

Experimental evidence for use of statins to inhibit inhibition of growth and CH4 production was observed methanogenesis at a lovastatin concentration of 10 lmol/L.38 Interest in statins as potential inhibitors of methanogene- The effects of lovastatin were reiterated in studies sis originated with work in ruminants and recognition using a rumen simulation technique (Rusitec). Lovastatin that the rate-limiting step in the synthesis of the iso- (150 mg/L; 371 lmol/L) supplementation to the Rusitec prene lipid membranes of archaea is catalysed by fermentation medium reduced overall CH4 production HMGR.46, 54 In the earliest reported studies, mevastatin by approximately 42% without altering bacterial counts and lovastatin were found to inhibit the growth of or nutrient fermentation (including concentrations of Methanobrevibacter species isolated from bovine rumen. short-chain fatty acids) in the medium. Garlic oil A mevastatin concentration of 5.6 lmol/L inhibited (300 mg/L) was more effective than lovastatin as an

Aliment Pharmacol Ther 2016; 43: 197–212 207 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. K. Gottlieb et al.

Lovastatin is a secondary metabolite produced during fungal growth and can be produced by cultures of Penicillium species, Aspergillus terreus, Monascus species (e.g. red yeast rice Monascus purpureus), Hypomyces, Lactone Hydroxyacid Doratomyces, Phoma, Eupenicillium, Gymnoascus and Trichoderma and is found at concentrations up to 2.8% of the dry weight of oyster mushrooms (Pleuro- tus ostreatus).58 Fermentation of rice straw (an agricultural waste pro- duct) with A. terreus produced lovastatin with a yield of 260.8 mg/kg dry matter. The lovastatin produced was found predominantly in the b-hydroxyacid form (rather than the lactone), the hydroxyacid form being the active 59 Lovastatin HMGR-inhibiting species. A methanolic extract of the fermented rice straw (FRSE) containing lovastatin (97 mg/g dry mass) significantly reduced total gas and Figure 6 | Lovastatin lactone may have a different target in archaea than the hydroxyacid. Simvastatin and CH4 production by a mixed culture of ruminant organ- lovastatin are the commercially available statins that isms in vitro but did not alter H2 production. In addi- come in the lactone form. Their cholesterol-lowering tion, the FRSE reduced the total population of effect and the impairment of archaeal membrane methanogens in the ruminant microbial culture, specifi- synthesis through inhibition of HMGR requires cally lowering Methanobacteriales and aerobic fungi. activation, i.e. the lactone ring needs to be opened to FRSE also increased the expression of the HMGR gene result in the hydroxyacid form. As can be seen, the (hmg), but had no effect on expression of methyl-coen- stereochemistry of lovastatin lactone and hydroxyacid zyme M reductase subunit A (mcrA), the key enzyme for is significantly different. Recent evidence suggests that fi 59 methanogenesis is preferentially inhibited by the the common nal pathway in methanogenesis. These fi lactone form of lovastatin. This and other evidence ndings suggest that the mechanism of action is related would suggest that lovastatin may have a different or to an impaired biosynthesis of the cell membrane iso- an additional target other than HMGR. A possible prenoids as we have discussed before. target for the lactone form are enzymes in the A subsequent study explored the effects of lovastatin- methanogenesis pathway that have F420 as coenzyme. containing FRSE on methanogenesis in more detail by See Figure 5. The 3-D was model generated with comparing the direct effects of FRSE and commercial CORINA (http://www.molecular-networks.com/). lovastatin on M. smithii. Commercial lovastatin (administered as the lactone) and FRSE (in which approximately 75% of lovastatin was the b-hydroxyacid) both fi inhibitor of CH4 production in this study (91% reduc- signi cantly inhibited CH4 production and M. smithii tion); however, garlic oil also inhibited bacterial growth, growth in vitro in a dose-dependent manner. At equiva- which likely reduced the availability of methanogenesis lent concentrations of lovastatin, the effect of FRSE on 55 substrates. Diallyl disulphide, the main ingredient of CH4 production was greater than observed for commer- garlic oil, is known to inhibit HMGR.56 However, garlic cial lovastatin, suggesting the b-hydroxyacid form of at the doses employed has unwanted effects on the gut lovastatin is more active. M. smithii morphology was sig- microbiome, and this may be one of the reasons that this nificantly altered by both commercial lovastatin and approach is apparently not being actively pursued. A FRSE, resulting in abnormal membrane formation during study comparing diallyl disulphide and lovastatin as feed mitosis and asymmetric (off-centred) cell divisions.60 It additives in sheep found that neither additive caused an is well established that the b-hydroxyacid form of lovas- absolute change in CH4 emissions; however, each treat- tatin is the active HMGR-binding form of the molecule ment modestly reduced CH4 produced per g of dietary and, as described above, HMGR catalyses the rate-limit- fibre consumed by these animals.57 ing step in the synthesis of membrane lipids in archaea. The potential expense of utilising purified statins led Increased anti-methanogenic activity of the FRSE (con- to a number of studies evaluating natural sources of sta- taining the b-hydroxyacid) relative to commercial lovas- tins as agents to mitigate ruminant CH4 production. tatin (lactone) and alteration to M. smithii membranes

208 Aliment Pharmacol Ther 2016; 43: 197–212 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. Review: methane, archaea, statins and the gut are both consistent with an inhibition of cell membrane 5 mg/g produced the maximum inhibiting effect, provid- synthesis via inhibition of HMGR. ing an average methane level of 3% of the control over However, a comparison of the effects of FRSE and time. In a final validation comparison of three lovastatin commercial lovastatin on gene expression suggests that species (5 mg/g), both lactone and lactone-diol types lovastatin may directly modulate methanogenic pro- proved to be effective. In all assessments, statins of the cesses. Consistent with the known action of statins, both hydroxyacid form were least able to inhibit methane pro- FRSE and commercial lovastatin were found to increase duction in fresh stool samples.61 HMGR gene expression. FRSE also modulated expression of several genes associated with methanogenesis, increas- CLINICAL DEVELOPMENT PROGRAMMES ing expression of mtr, mta and mcr while decreasing To summarise, there is an association between irritable expression of hmd and fno. Commercial lovastatin bowel syndrome with constipation and high breath increased mtr and mta expression and decreased fno methane levels, and there is experimental evidence sug- expression but has no effect on hmd.60 gesting that methane may slow down intestinal transit. Statins, specifically lovastatin, have been shown to lower Other potential mechanisms of action for statins in methanogenesis in human stool samples. In addition, the inhibition of methanogenesis recent research examining the effects of lovastatin in a rat If gene expression of methanogenesis enzymes is affected model of diet-induced constipation and M. smithii prolif- differently by FRSE (contains lovastatin hydroxyacid) eration showed that gavage with lovastatin significantly and lovastatin lactone, as suggested above, the question reduced the ratio of M. smithii to total bacteria in the becomes: Is this due to the lactone and hydroxyacid spe- ileum. Most importantly, there was an increase in stool cies or perhaps an unknown ingredient in FRSE? wet-weight (a proxy for soft stools in rats) noted in rats Let us assume for now that the differences are indeed receiving lovastatin lactone gavage.62 Naturally, a clinical entirely attributable to the lactone vs. hydroxyacid forms. development programme that evaluates the ability of sta- In this case one would think that the lactone is not sim- tins to suppress methane production and associated ply converted into the hydroxyacid form to exert its symptoms in humans makes sense and we have conse- effect but instead has a different target or more than one quently embarked on a proof-of-concept clinical trial. target besides the HMG-CoA reductase. Indeed, an To our knowledge, this is the only such programme in intriguing addition to the mechanistic discussion of sta- humans. This Phase 2, randomised, multi-centre, multi- tin anti-methanogenic activity was provided in a hypoth- dose study has enrolled sixty subjects with irritable bowel esis paper comparing the structures of lovastatin and syndrome with constipation who were between the ages 63 mevastatin to that of coenzyme F420. Computational of 18 and 65, with a trial duration of up to 43 days. The modelling (in silico molecular docking) of the methano- study has one placebo and two active comparator arms: genic enzyme F420-dependent NADP oxidoreductase Low Dose 21 mg and High Dose 42 mg with an assign- determined that both lovastatin and mevastatin had ment of 1:1:1. The main outcome measure is change from fi higher af nities for the F420 binding site on fno than did baseline in the area under the curve (AUC) of breath F420 itself. As such, lovastatin and mevastatin may act as CH4 production at day 7. The active drug, SYN-010, is a inhibitors of fno 43 and could inhibit other enzymes that proprietary lovastatin dual pulse dosage form. Our com- require F420 for their activity such as those that are cru- pany, Synthetic Biologics hopes to release preliminary cial to the main methanogenesis pathway (Figure 4). results towards the end of 2015. Recent experimental results seem to support a direct anti-methanogenic effect of lovastatin lactone in humans CONCLUSION AND OUTLOOK FOR THE FUTURE that is not shared by other statins. Marsh et al. assessed Many issues will need to be addressed on both basic nine statins for methane inhibition at a concentration of science and clinical science levels. Most of the knowledge 5 mg/g in homogenised human stool; lovastatin lactone gaps are apparent from this review, and we will highlight and hydroxyacid, pravastatin lactone and hydroxyacid, some which we think are particularly important. Further simvastatin lactone, mevastatin lactone, rosuvastatin research needs to be conducted to elucidate methane’s hydroxyacid, atorvastatin lactone and hydroxyacid. role as potential gasotransmitter. Like research conducted Lovastatin lactone was identified as the only effective around nitrous oxide, the payoffs may go beyond a bet- methane inhibitor, significantly inhibiting methane levels ter understanding of animal physiology. Next we need to by – 65% of the control stool. Lovastatin lactone at understand the conditions which determine the ‘methane

Aliment Pharmacol Ther 2016; 43: 197–212 209 ª 2015 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd. K. Gottlieb et al. phenotype’ in both farm animals and humans. AUTHORSHIP M. smithii is obviously tightly integrated into a network Guarantor: K. Gottlieb of microbial neighbours, and its own fate depends to a Author contributions: K.G. initiated the work on the manuscript, performed the literature searches, wrote the first and subsequent large extent on the composition of the local microbiome drafts, commissioned and revised illustrations. V. W. wrote white or symbiome. Next, we need to better understand the papers related to the subject matter which were in part used for this mechanism of action of statins as it pertains to archaeal publication. In addition, he reviewed and revised all drafts. J. S. reviewed and revised all drafts. M. P. advised on the overall content growth and methanogenesis. Research in this direction of the manuscript, reviewed and revised all drafts. could not only benefit individual humans directly by All authors approved the final version of the manuscript. reducing their own intestinal methane production but also H. sapiens as a species, if this research leads to ACKNOWLEDGEMENTS methods by which the production of the greenhouse gas The authors thank Chris Edwards, Ph.D., for critical review of the manuscript and helpful suggestions regarding organisation and read- methane can be curbed in an economical fashion. Lastly, ability. clinical studies that evaluate statins for the treatment of Declaration of personal interests: K.G. and J. S. are employees of Syn- IBS-C are currently in the proof-of-concept stage, and thetic Biologics, Inc. V. W. is a paid consultant to Synthetic Biolog- ics. M. P. is a compensated advisor to Synthetic Biologics, Inc. further clinical trials need to be conducted. Declaration of funding interests: None.

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