Gasotransmitters

1. Gasotransmitters in AIRD

2. Outline of presentation
• Introduction
• History
• Properties, Structure & Function
• Role of Gasotransmitters in AIIRD
• Conclusion

3. Gaseous signaling molecules
• Gaseous molecules
• Endogenously or received from exogenous environement
• Transmit chemical signals which induce certain physiological or biochemical changes
in the organism, tissue or cell.
• Include 𝑂2, C𝑂2, NO, CO, H2S, S𝑂2, 𝑂2, Hydrogen cyanide, ammonia,methane,
hydrogen, ethylene etc..
• Subfamily of endogenously synthesised gaseous
signaling molecules, including NO, CO, H2S.Gasotransmitters
Introduction

4. Introduction
• Terminology first introduced by Wang in 2002.
• Family of regulatory & signaling molecules:
• Naturally occurring atmospheric gases in the prebiotic world.
• Involved in the origin of life when atmospheric O2 was low.
• By-products of industrialization that were considered pollutants.
• Toxic to humans at high concentrations.
• At low concentrations they function as important signaling molecules
in biological systems.
Gaso-transmitters
Term “gaso-transmitter” is a misnormer:
• Gases at STP, but act as solutes in the aqueous environment of the intra and extracellular fluids.

5. History
1987, Robert Furchgott, Louis Ignarro, and Ferid Murad.
Discovered NO as the labile factor released from the vascular endothelium
mediating ACh induced vasodilation.
• Showed for first time that an endogenously produced gas can act as signaling
molecule in biological systems.
1998: Nobel Prize in Physiology or Medicine.
Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor.Nature. 1987;327:524526.

6. • Produced during heme metabolism
• CO relaxes vascular vessels lowers blood pressure, and protects from
ischemia/reperfusion damage
• Involved in neurotransmission
1990s, CO was discovered to be a putative signaling molecule.
• Regulating neurotransmission and neuromodulation.
In 2002, Wang discovered 𝐻2S – as the third gaso-transmitter.
• Not classical signaling molecules
• Regulators of cellular function via complex chemical interactions with each other
and target proteins.
Present concept:
History…
Wang, R (2002). "Two's company, three's a crowd - Can H2S be the third endogenous gaseous transmitter?". FASEB Journal. 16 (13): 1792–1798.

7. Characteristic Properties
Low molecular weight molecules
• (NO-30Da; CO-28Da; 𝐻2S −34Da)
It is endogenously and enzymatically generated and its production is regulated
Not stored. Short half-life. Amphiphilic.
It is freely permeable to membranes, without the requirement of specific receptors or transporters .
Specific functions at physiologically relevant concentrations. Thus, manipulating the endogenous
levels of this gas evokes specific physiological changes.
Functions of this endogenous gas can be mimicked by its exogenously applied counterpart
Cellular effects may or may not be mediated by second messengers, but should have specific
cellular and molecular targets.
Wang, R., 2014. Gasotransmitters: growing pains and joys. Trends Biochem. Sci. 39, 227–232

8. Structure and Chemical Properties
Chemical
properties
NO is a radical with an unpaired electron
CO is biologically inactive.
𝐻2S in solution is a weak acid that partially dissociates to form an equilibrium
between 𝐻2S and HS2.
NO and 𝐻2S can participate in redox reactions, and can react with each other
and their respective metabolites.

9. Synthesis of Gasotransmitters
CBS: cystathionine β-synthase
CSE: cystathionine γ-lyase
HO: Heme oxygenase
NOS: NO synthase
CO, NO and H2S are produced in the mammalian tissues by endogenous enzymes.

10. Ligands and Signal Transduction Pathways
These gases can form coordination chemistries with
prosthetic metal groups (heme or non-heme proteins)
Via protein modifications such as S-nitrosylation and
S-sulfhydration.
• NO and CO generally interact with ferrous iron
• 𝐻2S with ferric iron.

11. Receptors and Signal Transduction Pathways
NO and CO binds sGC (heme protein receptor) to generate the
second messenger cGMP.
• Functional interdependence of the gasotransmitters.
The ability of CO to bind to sGC is dependent on NO concentration,
as NO binds to sGC with higher affinity.
𝐻2S does not bind to sGC but inhibits cGMP PDEs that augments
intracellular cGMP concentration.

12. Biological functions
Affect the function of all cells that express target proteins.
Regulate fundamental intracellular functions such as cellular respiration and ATP synthesis.
• NO and CO are inhibitory.
• 𝐻2S is bifunctional, stimulatory at low conc. and inhibitory at high conc.
Both 𝐻2S and CO are strongly implicated in O2 sensing, as their respective intracellular
concentrations are dependent on the O2 concentration.
Control vast array of physiological functions.
• Regulation of the cardiovascular, nervous, gastrointestinal, excretory, and immune systems.
Regulating many cellular functions.
• Cytoprotection, apoptosis, proliferation, inflammation, and gene transcription

13. Gaseous transmitters in non-immune disease
• Gaseous transmitters in cardiovascular system:
• NO in maintaining vascular tone and atherogenic properties.
• Endothelial dysfunction and various pathologies
• Gaseous transmitters in urogenital tract:
• Pathophysiology of erectile function and lower urinary tract symptoms.
• Gaseous transmitters in pulmonary system:
• Bronchial hyper-reactivity and pulmonary inflammation.
• Gaseous transmitters in cancer:
• Hydrogen sulfide: development and progression of human melanoma.

14. Gasotransmitters in AIRD
How do we prevent autoimmunity?
Physiological mechanisms that down-regulate and turn off immune responses.
(innate and acquired immune system)
Hormones
(corticosteroi
ds, D3
vitamin)
Endogenous
antagonists of
cytokines, such
as soluble
receptors
Naturally occurring
autoantibodies
Anti-inflammatory
cytokines(Th2,Th3)
•IL-10, IL-13, TGF-
beta and IL-35
Proteins: IDO-1
and heme
oxygenase 1
Classical immune
checkpoints, such
as CTLA4 and
PD1 and PD2
More recent evidence has shown that additional regulation of immune responses is mediated by a family of gases normally
produced in the body such NO, H2S, and CO.
( Motterlini and Otterbein, 2010; Wallace et al., 2015; Wallace and Wang, 2015)

15. Gasotransmitter: Carbon Monoxide
CO is physiologically produced during heme metabolism in the phagocytic system of RES.
Catalyzed by HO enzymes, encoded by the HMOX genes.
Three isoforms: HO-1, HO-2 and HO-3
HO-1 is the inducible isoform and levels increase following cellular stress.
HO-2 and HO-3 isoforms are constitutively expressed.
In the promoter region of
HMOX1 gene, found
binding sites for several
transcription factors.
• Nuclear factor-kB (NF-kB)
• Activator protein-1 (AP-1)
• c-myc
• IL-6 response elements.
Several factors can induce
HO-1
• heat shock proteins
• oxidized lipids
• nitric oxide
• Radiation
• hydrogen peroxide
• Hypoxia, exogenous CO
• lipopolysaccharides (LPS)
• Cytokines: IL-1, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ.
Chauveau, C., Rémy, S., Royer, P.J., Hill, M., Tanguy-Royer, S., Hubert, F.-X., Tesson, L., Brion, R., Beriou, G., Gregoire, M., Josien, R., Cuturi, M.C., Anegon,
I., 2005. Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression. Blood 106, 1694–1702.

16. Gasotransmitter: Carbon Monoxide
CO is freely diffusible across cellular membranes and rapidly bind intracellular targets:
• sGC
• heme-containing potassium channels
• NO synthase
• NADP/NADPH oxidase.
Foresti, R., Shurey, C., Ansari, T., Sibbons, P., Mann, B.E., Johnson, T.R., Green, C.J., Motterlini, R., 2005. Reviewing the use of carbon monoxide-releasing
molecules (CORMs) in biology: implications in endotoxin-mediated vascular dysfunction. Cell. Mol. Biol. (Noisy-Le-Grand) 51, 409–423.
Positively controls:
• Heme containing proteins
• sGC: increased cGMP
• NOS increased NO
Inhibitory control:
• NADPH oxidase, thus modulating the
production of superoxide
• mitochondrial cytochrome c.
Bcoz of its ability to bind metal-containing proteins, exert several physiological effects
• inhibition of platelet aggregation
• anti-proliferative action on smooth muscle
• neurotransmission
• vasodilation

17. Carbon Monoxide in Immunity
Human and rat immature DC express HO-1, decreases upon DC maturation.
Overexpression of HO-1 in DC inhibits LPS-induced maturation and pro-inflammatory functions,
modulating the suppressive capacity of regulatory T-cells.
CO mediates the effects of HO-1 in DC.
Rémy, S., Blancou, P., Tesson, L., Tardif, V., Brion, R., Royer, P.J., Motterlini, R., Foresti, R., Painchaut, M., Pogu, S., Gregoire, M., Bach, J.M., Anegon, I., Chauveau,
C., 2009. Carbon monoxide inhibits TLR-induced dendritic cell immunogenicity. J. Immunol. 182, 1877–1884
Anti-inflammatory effect

18. Effects of CO exposure in Macrophages
Physiological levels of CO selectively inhibit LPS induced increase of TNF-α, IL-1β and MIP-1b in
the Raw264.7 cell line.
CO increases the production of the anti-inflammatory cytokine, IL-10.
These effects were mediated by the p38 kinase in a cGMP independent manner.
In an in vivo model of endotoxemia, CO reduced the levels of serum TNF-a levels and increased
IL-10 production in a dose dependent manner.
These effects were attributed to MAP kinase 3(MKK3), since CO had no effects in LPS-injected
MKK3 -/- mice.
Choi, A.M.K., Otterbein, L.E., Bach, F.H., Alam, J., Soares, M., Tao Lu, H., Wysk, M., Davis, R.J., Flavell, R.A., 2000. Carbon monoxide has anti-inflammatory effects
involving the mitogen-activated protein kinase pathway. Nat. Med. 6, 422–428
Anti-inflammatory effect

19. Effects of CO exposure in T-cells
Kapturczak et al., 2004: Splenocytes from HO-1 deficient mice produce more Th1-type cytokines,
such as IL-1β, IFN-γ, IL-6, TNF-α, after polyclonal stimulation of T-cells.
Pae et al., 2004: Among the byproducts of HO-1, CO is the only one to exert an antiproliferative
effect following anti-CD3 plus anti-CD28 Abs stimulation.
CO blocks the cell cycle entry of T-cells, independently of the GC/cGMP pathway, and inhibits the
production of IL-2.
JurkaT-cells when exposed to CO:
• Up-regulation of the pro-apoptotic FADD
• Activation of caspase-8, -9, and -3
• Down-regulation of the anti-apoptotic BCL-2 protein
• Fas/CD95-induced apoptosis.

20. Effects of CO exposure in T-cells
In Treg cells, Foxp3 and HO-1 genes are constitutively co-expressed in CD4+CD25+ Treg cells.
Inhibition of HO-1 activity by Zinc protoporphyrin (ZnPP) results in the abrogation of the
suppressive function of CD4+CD25+ Treg cells.
Choi, B.-M., Pae, H.-O., Jeong, Y.-R., Kim, Y.-M., Chung, H.-T., 2005. Critical role of hemeoxygenase-1 in Foxp3-mediated immune suppression. Biochem. Biophys.
Res. Commun. 327, 1066–1071.

21. On the other hand, it has also been reported that HO-1-/- Treg cells are as effective as HO-1+/+
Treg cells in inhibiting proliferation of effector T-cells in vitro (George et al., 2008).
Absence of HO-1 in APCs is associated with reduced suppressive activity of Treg cells on effector
T-cells.
• HO-1 via CO exerts a suppressive effect on CMI via CO
• HO-1 activity in APCs is more important for Treg-mediated suppression
than the absence of HO-1 in Treg cells themselves.
Conclusion:
Effects of CO exposure in T-cells

22. In vivo preclinical studies have shown that administration of exogenous CO improves
several immune-inflammatory and autoimmune conditions.
• administration of CO by inhalation of the gas
• administration of HO-1 inducers
• metallo-organic carbonyl compounds.
• Other methods of CO delivery include:
• hemoglobin-based CO carriers, such as CO-MP4, CO-saturated
RBCs.
• CO-saturated solutions
• CO intraperitoneal injection.
To provide exogenous CO,
three major approaches have
been applied:
Motterlini, R., Otterbein, L.E., 2010. The therapeutic potential of carbon monoxide. Nat. Rev. Drug Discov. 9, 728–743
Hu, H., Sun, Q., Ye, Z., Sun, X., 2016. Characteristics of exogenous carbon monoxide deliveries. Med. Gas Res. 6, 96
Carbon monoxide-releasing molecules (CORMs)

23. Carbon monoxide-releasing molecules (CORMs)
CO-Releasing Molecules release safe and therapeutically effective quantities of CO.
The first in class compounds were the commercially available transition metal carbonyls, CORM1
and CORM-2.
Subsequently, CORM-3, a ruthenium-based carbonyl and CORM-A1, a boron containing carboxylic
acid, that releases CO with a half-life of 21 min under physiological conditions, were synthesized.
CORMs represent specific and practical pharmaceutical alternatives to administer gaseous
molecules in a safe and accurate way.

24. Carbon monoxide-releasing molecules
Autoinflammatory/autoimmune preclinical models ameliorated by exogenous CO administration

25. Two Phase II clinical trials of inhaled CO in doses up to 250 ppm
in COPD (NCT00122694) and IPF (NCT01214187) are ongoing.
Carbon monoxide-releasing molecules (CORMs)

26. Biology of NO
• 3 isoforms: nNOS, iNOS and eNOS.
• The nNOS and eNOS enzymes are constitutively expressed.
• iNOS is mainly upregulated by inflammatory stimuli, and depends on an
intracellular calcium rise.
• A fourth NOS enzyme, mithocontrial NO synthase (mtNOS), has been identified in
rat liver.
NO is mainly synthesized from L-arginine by NOS enzymes
NO is also generated in tissues by reduction of nitrite to NO under acidic and reduced
conditions that occur during ischemia.
Allows restoration of the physiological levels of NO when its production is dysregulated, as
it may happen during inflammation and atherosclerosis.

27. NO and immune system
NO once produced in the cytoplasm, rapidly diffuses across the cell membranes and promptly reacts
with free radicals such as O2- to generate peroxinitrite (ONOO) and reactive nitrogen species (RNS).
The primary mode of action: soluble cGMP signaling cascade in cell target.
S-nitrosylation-dependent mechanisms are also implicated.
NO can act as oncosuppressor or oncogenic factor.
NO can be both pro-inflammatory and immunosuppressive.
• Depends on the concentrations of NO
• Type of immune responses and cellular target
• Immunological environment during NO production.
Eg: iNOS-expressing MDSCs Inhibit CD4+T cell proliferation in collagen-induced arthritis, whereas constitutive
NOS enzymes are involved in the pathogenic mechanisms of the disease (García-Ortiz and Serrador, 2018)

28. NO and immune system
• High levels of NO are produced
• Promote killing of intracellular pathogens and cytotoxicity towards tumor cells.
M1-polarized macrophages
• express high amount of arginase I
• Diminished NO production.
M2 macrophages
Expression of iNOS and the subsequent production of NO/RNS contribute to the
immunosuppressive activity of myeloid derived suppressor cells.

29. NO and immune system
• promote Th1 polarization and upregulation of IL-12 R beta 2.
Low concentrations of NO
• inhibit the IL-2-mediated signaling pathway, thus imparing CMI.
• impairs the polarization toward the Th17 phenotype via nitration of tyrosine
residues in RORγt.
High concentrations of NO
The NO-p53-IL-2-OX40 survivin signaling pathway has been implicated in the generation
of a novel subset of Tregs, named NO-Treg.

30. NO and humoral immunity
• required for IgA humoral responses
• inhibits the antiviral IgG2a response.
In B-cells iNOS
• Increased the IgM and IgG3 responses
• Increased MZB-cell numbers
• Increased peritoneal B1b B-cells
• Increase in serum levels of B-cell-activating factor (BAFF)/BLyS.
• T-cell-dependent antibody responses are only slightly increased.(Giordano et al.,
2014).
In iNOS-knockout mice
Giordano, D., Draves, K.E., Li, C., Hohl, T.M., Clark, E.A., 2014. Nitric oxide regulates BAFF expression and T cell-independent antibody responses. J. Immunol. 193, 1110–1120

31. NO and neuroinflammation
NO exerts a dichotomic role in neuroinflammation.
NO produced by iNOS
contributes to EAE and
MS
lipid peroxidation
oligodendrocytes damage
activation of matrix metalloproteinases
disruption of blood-brain barrier
(Aboul-Enein et al., 2006;Cross et al., 1997; Maeda et al., 1998; Mitrovic et al., 1995; Redford et al., 1997; Smith and Lassmann, 2002).
Exogenous NO can promote apoptosis of encephalitogenic T-cells.
NO strongly inhibits CXCL-12 gene expression in a p38 dependent manner and protects rats from
developing EAE
Zettl, U.K., Mix, E., Zielasek, J., Stangel, M., Hartung, H.P., Gold, R., 1997. Apoptosis of myelin-reactive T cells induced by reactive oxygen and nitrogen intermediates in vitro. Cell.
Immunol. 178, 1–8

32. NO and GUT inflammation
Pathogenic role for NO has been reported for IBD (Boughton-Smith, 1994).
Rectal biopsies from patients with active UC - have higher levels of citrulline, a co-product of NO
synthesis (Middleton et al., 1993a).
Upregulation of iNOS in colonic epithelial cells has been correlated with active UC. (Middleton et al.,
1993a, 1993b; Ribbons et al., 1995).
No significant increase in NO synthase observed in the inflamed colonic specimens from patients
suffering from active Crohn's disease (CD).
Locally delivered NO donors or inducers of NO production may be useful in CD (Boughton-Smith, 1994)

33. Nitric oxide donor drugs
Classic NO donor drugs are organic nitrates and nitrite esters
• nitroglycerin, amyl nitrite, isosorbide-5-mononitrate, nicorandil and pentaerythritol tetranitrate.
• mimic NO activity via unknown mechanism
• Used for the treatment of ischemic coronary disease.
S-nitrosothiols
• new class of NO donor drugs that produce NO and perform trans-nitrosylation reactions.
• Some are naturally occurring molecules: S-nitroso-glutathione, S-nitrosoalbumin and S-nitrosocysteine.
The exposure of nucleophilic molecules to NO produces NO–nucleophilic complexes, called diazeniumdiolates
or NONOates.
• Are ‘true’ NO donors with spontaneous breakdown in solution
• NO release independent of plasma components
• Stable in solid form and solubility in aqueous solution
• Predictable level of therapeutic NO release.
• Ideal as NO-releasing drug.

34. Nitric oxide donor drugs
• Covalent attachment of an NO moiety to the parental compound.
• Combine the anti-inflammatory and anti-nociceptive effects of the NSAID with the vasodilator,
antimicrobial and immune-modulator effects of NO.
• NO-NSAIDs exhibit less adverse effects, such as GI toxicity, while retaining the desired activities of their
parent compounds.
NO-releasing nonsteroidal anti-inflammatory drugs (NO-NSAID):
• These agents exhibited enhanced anti-inflammatory activity in several preclinical settings.
Mesalamine, acetaminophen and prednisolone, have been modified similarly.

35. NO-ASPIRIN
NO–aspirin: Aspirin linked via an ester bond to a spacer that is bound to an NO-releasing moiety.
Compared with aspirin, NO-aspirin exerts anti-inflammatory activities that involve regulation of caspase-1 and NF-κB.
Fiorucci, S., Del Soldato, P., 2003. NO-aspirin: mechanism of action and gastrointestinal safety. Dig. Liver Dis. 35 (Suppl 2), S9–S19

36. NO-NSAIDs
Naproxcinod
• A randomized, double-blind, parallel-group, multicenter study to evaluate the effects of naproxen
vs naproxcinod in patients with osteoarthritis of the hip.
• Efficacy of naproxcinod for treating the signs and symptoms of hip osteoarthritis was similar to
that of naproxen with significantly lower effects than the parental compound on systolic blood
pressure (Baerwald et al., 2010).
• Trial was terminated in light of a Phase III study where the compound missed its primary
endpoints as compared to naproxen (ClinicalTrials.gov Identifier: NCT00504127).
Noflurbiprofen
• Shown in a Phase I study to have increased anti-inflammatory and immunomodulatory properties with a safer profile than
the parental compound (Zacharowski et al., 2004).
• Ameliorated disease severity when administered in prophylactic regime in a model of EAE.
• Reduction of the number of CNS-infiltrating T-cells
• Decreased ability of auto-reactive T-cells to proliferate
• Increase in the number of Treg cells in the spleen, and a decrease in axonal demyelination (Furlan et al., 2004).

37. Hydrogen sulfide biology
H2S is a colourless, water-soluble, gas with a smell like rotten egg.
Considered as a toxic gas and an environmental hazard
Produced in substantial quantities in mammalian tissues and it can be detected at significant levels
H2S is involved in the regulation of several biological processes:
•Neuromodulation
•angiogenesis
•Vasodilatation
•protection from ischemia/reperfusion injury.
Production of H2S has been demonstrated in mouse brain, liver and colon tissues (Linden et al., 2010).
Another source of H2S is represented by the enterobacterial flora (Flannigan et al., 2011).

38. Hydrogen sulfide and the immune system
H2S exerts pro- and anti-inflammatory effects in immune cells.
• via binding of ferric iron, zinc, or copper residues of metalloproteins and sulfhydration of
protein cysteine residues.
H2S promotes NFYB activity by sulfhydration reaction.
High concentrations of the H2S donor, NaHS promotes the release of TNF-α and IL-1 from IFN-γ-
stimulated U.937 cells(monocytic cell line), in a NFκB dependent manner (Zhi et al., 2007).
H2S donors have been shown to reduce TNF-α release following LPS exposure in RAW 264.7
cells(murine macrophage) (Huang et al., 2016).

39. Hydrogen sulfide and the immune system
H2S signaling appears to be a key element in the process of T-cell activation
Exogenously administered H2S, at physiological levels enhances T-cell activation.
• as it increases CD69 expression, IL-2 expression, and CD25 levels. (Miller et al., 2012)
Ability of T-cells to generate H2S by CBS and CSE is switched on as a consequence of T-cell
activation.
Suppression of CBS and CSE expression inhibits T-cell activation and T-cell proliferation, restored by
administration of exogenous H2S. (Miller et al., 2012).

40. On the contrary, at supra-physiological concentrations, H2S inhibit proliferation of cytotoxic CD8+ T-
cells (Mirandola et al., 2007).
Both natural and induced Treg cells express higher amounts of CBS and CSE in comparison to other
CD4+ T-cell subsets.
• Inhibition of CBS and CSE activity in mice leads to a reduction in the number of Foxp3+ Treg
cells, suggesting a role for these enzymes in maintenance of Treg cell.
IL-10-deficient mice show impaired synthesis of H2S in the colon and develop colitis.
• Exogenous administration of H2S upregulates IL-10 expression and IL-10 administration
restored normal colonic H2S synthesis
• A regulatory interaction between IL-10 and H2S. (Gemici et al., 2015)
Hydrogen sulfide and the immune system

41. Hydrogen sulfide in AIIRD
In a model of carrageenan-induced paw edema
• 40% increase in H2S-synthesizing activity was observed in paw homogenates (Bhatia et al.,
2005a, 2005b)
In a mouse model of pancreatitis
• circulating levels of sulfide were reported to increase from 23 uM to 31 uM (Bhatia et al.,
2005a, 2005b).
In a mouse model of endotoxic shock
• liver and kidney homogenates showed an increase in sulfide between 30% and 60% as
compared with healthy control animals (Collin et al., 2005; Li et al., 2005).
Twofold increase in sulfide levels was described in a polymicrobial sepsis mouse model (Zhang et
al.,2006).
In a mouse model of streptozotocin-induced diabetes
• H2S formation was significantly increased in homogenates of pancreas and liver
• Reversed with insulin treatment (Yusuf et al., 2005).

42. Hydrogen sulfide in AIIRD
Whiteman et al. (2010), demonstrated that H2S levels correlate to
inflammation and disease activity in RA.
• Plasma H2S levels:
• positively correlated with disease activity score and white cell count
• Synovial fluid H2S levels
• significantly higher than plasma H2S in each RA patient
• negatively correlated with white cell count.
Increased synovial fluid H2S was also found in patients with PsA,
ReA and septic arthritis (Whiteman and Winyard, 2011).

43. Hydrogen sulfide donors
• The increasing data on the biological relevance of H2S support its therapeutic use in several pathological conditions (Wallace et al.,
2015). The potential properties of H2S in improving pathological processes have been originally tested using H2S donors such as Sodium
hydrogen sulfide (NaHS), Na2S, N-acetylcysteine or Lawesson's reagent.
Zanardo and collegues reported that NaHS and Na2S inhibited aspirininduced leukocyte adherence in mesenteric venules, likely via activation of ATP-sensitive K+
channels, and that NaHS, Lawesson's reagent, and N-acetylcysteine suppressed leukocyte infiltration in an air pouch model (Zanardo et al., 2006). In addition, the
authors reported that carrageenan-induced paw edema was suppressed by NaHS and Na2S to the same extent as by diclofenac and that it was enhanced by an
inhibitor of H2S synthesis. However, such H2S donors release H2S too quickly, and for this reason, they may have detrimental effects in patients and exert only
short-lived effects (Caliendo et al., 2010).
On a side note, a number of marketed therapeutics have shown to exert their therapeutic effects via release of H2S. It is the case of anethole trithione, marketed
under the brand names Sialor and Surfarlem, used for xerostomia, and Zofenopril, an angiotensin converting enzyme inhibitor that once absorbed, undergoes
hydrolysis to a sulfhydrylcontaining active metabolite with potent anti-oxidant properties and beneficial cardiovascular effects, attributed to the release of H2S
(Bucci et al., 2014; Chopra et al., 1992; DeForrest et al., 1989).
• Several natural compounds have shown to possess therapeutic effects due to the release of H2S, including garlic, and the isothiocyanates, sulforaphane, erucin and
iberin (reviewed in Kashfi and Olson, 2013). Garlic was shown to be able to prevent renal, hepatic, cardiac, cerebral and pulmonary ischemia-reperfusion injury
and to possess anti-cancer and anti-diabetes properties (reviewed in Kashfi and Olson, 2013). Sulforaphane is able to protect vascular smooth muscle cells and
endothelial cells from oxidative and inflammatory stress and to suppress angiogenesis, as well as to protect against ischemia reperfusion injury, hemorrhage and
serotonin-induced toxicity (reviewed in Kashfi and Olson, 2013). Among synthetic H2S donors such as morpholin-4-ium 4 methoxyphenyl(morpholino)
phosphinodithioate (GYY4137), decrease the production of iNOS, nitrate/nitrite, prostaglandin 2 (PGE2), TNF-α, IL-1β, IL-6, NF-κB, expression of inducible nitric
oxide synthase (iNOS), and cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells and when administered to rats prior to LPS injection, significantly reduced
nitrite/nitrate, C-reactive protein, L-selectin and lung myeloperoxidase activity, as well as, plasma creatinine/alanine amino transferase levels (Liet al., 2008).

44. • More recently, H2S hybrid molecules have been developed by linking sulfide moieties to existing compounds, such as NSAID, with
interesting results in several animal models (Szabó, 2007). Antibe Therapeutics has developed an H2S-releasing derivative of
naproxen,
ATB-346 that has gone under extensive phase 1 trial, completed in 2015 and a phase 2 trial in osteoarthritis patients, completed in
2016 (http://www.antibethera.com/pipeline/atb-346/). ATB-346 inhibited alveolar bone loss and inflammation in rats with
ligature-induced periodontitis, attenuated nociceptive responses, inflammatory, cellular and biochemical changes in a rat model of
zymosan-induced arthritis and reduced edema and pain score, as well as leukocyte infiltration, in rats with carrageenan-induced
synovitis, showing a better toxic and pharmacodynamic profile than naproxen (Dief et al., 2015; Ekundi-Valentim
et al., 2013; Herrera et al., 2015).
• In the Phase 2 trial, ATB-346 improved Western Ontario and McMaster Universities Osteoarthritis Index
(WOMAC) pain scale and significantly decreased arthritis index score (http://www.antibethera.com/pipeline/atb-346/).
A beneficial effect of H2S in IBD has been suggested by the demonstration of improved anti-inflammatory activity of a H2S-
releasing derivative of mesalamine, in an experimental colitis model (Fiorucci et al., 2007). S-mesalamine (ATB-429) was more
effective than mesalamine in reducing mucosal injury and disease activity, as determined by body weight variation, fecal blood,
and diarrhea, as well as colonic granulocyte infiltration (Fiorucci et al., 2007). S-mesalamine also decreased the mRNA for TNF-α,
IFN-γ, IL-1, IL-2, IL-12 and RANTES (Fiorucci et al., 2007).
• Also, Distrutti and collegues reported that in the same model, S-mesalamine modulated the expression of colonic proinflammatory
mediators, COX-2 and IL-1β (Distrutti et al., 2006). A summary of in vivo immunoinflammatory/autoimmune preclinical
models ameliorated by exogenous H2S administration is

45. NO–H2S releasing hybrids
• Based on the encouraging data coming from the NO-NSAID and SNSAID, it was more recently proposed that
an NSAID releasing both NO and H2S could be more potent than either one of the two.
• This was followed by the development of four NOSH molecules having aspirin as scaffold.
• Nitrate (-ONO) was used as NO-donating moiety and linked to the aspirin through an aliphatic spacer, while
either 5-(4-hydroxyphenyl)-3H-1, 2-dithiole-3-thione (ADTOH), or 4-hydroxy benzothiazamide (TBZ) or lipoic
acid were directly coupled to aspirin, as H2S releasing moieties (Chattopadhyay et al., 2012; Kodela et al.,
2012).
• Among the compounds developed, it was shown that NOSH-1 (NOSH-aspirin, NBS-1120) dose-dependently
releases H2S and NO both in vitro and in vivo (Chattopadhyay et al., 2012; Kodela et al., 2012).
• In the rat carrageenan paw edema model, NOSH-1 exerted anti-inflammatory effects comparable to those of
the parental compound, by reducing paw edema and local PGE2 levels.
• On the other hand, administration of ASA to rats increased the pro-inflammatory cytokine, TNF-α, in plasma,
but this increase was substantially lower in the NOSH-1 treated rats (Kodela et al., 2012). It was later shown
that NOSH-aspirin inhibits acetic acid-induced writhing response and carrageenan-induced inflammatory
hyperalgesia in a dose-dependent manner and to a higher extent than the same doses of the parental
molecule (Fonseca et al., 2015).

46. Conclusions
Research on gasotransmitters is a growing field of science.
Key players in both health and diseases.
Key effectors in both innate and adaptive immune responses.
Encouraging results from preclinical animal models provide the basis for therapeutic
approaches of infectious, autoimmune, and chronic inflammatory diseases.
Better understanding of the molecular mechanisms and the development of novel
donors will be critical to propel investigations on possible clinical applications of these
molecules in the management of inflammatory diseases.