Intestinal absorption of S-nitrosothiols: Permeability and transport mechanisms

Abstract

S-Nitrosothiols, a class of NO donors, demonstrate potential benefits for cardiovascular diseases. Drugs for such chronic diseases require long term administration preferentially through the oral route. However, the absorption of S-nitrosothiols by the intestine, which is the first limiting barrier for their vascular bioavailability, is rarely evaluated. Using an in vitro model of intestinal barrier, based on human cells, the present work aimed at elucidating the mechanisms of intestinal transport (passive or active, paracellular or transcellular pathway) and at predicting the absorption site of three S-nitrosothiols: S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-l-cysteine (NACNO) and S-nitroso-N-acetyl-d-penicillamine (SNAP). These S-nitrosothiols include different skeletons carrying the nitroso group, which confer different physico-chemical characteristics and biological activities (antioxidant and anti-inflammatory). According to the values of apparent permeability coefficient, the three S-nitrosothiols belong to the medium class of permeability. The evaluation of the bidirectional apparent permeability demonstrated a passive diffusion of the three S-nitrosothiols. GSNO and NACNO preferentially cross the intestinal barrier though the transcellular pathway, while SNAP followed both the trans- and paracellular pathways. Finally, the permeability of NACNO was favoured at pH 6.4, which is close to the pH of the jejunal part of the intestine. Through this study, we determined the absorption mechanisms of S-nitrosothiols and postulated that they can be administrated through the oral route.

Introduction

Nitric oxide (NO) is a gaseous mediator with a short half-life (less than 5 s [1]). Due to its radical nature and oxidative activity, NO is involved in various signalling pathways among different cellular types and physiological systems. NO is continuously synthesised by oxydoreductases, i.e. the three endothelial, inducible or neuronal isoforms of NO synthases. The decrease in NO bioavailability, linked to vascular endothelium dysfunction and oxidative stress, plays a major role in ageing and cardiovascular chronic diseases like atherosclerosis, angina pectoris and stroke. As a result, the restoration of NO bioavailability, using among NO donors the physiologically occurring S-nitrosothiols, is a therapeutic key to treat cardiovascular diseases [2], [3], [4], [5], [6], [7]. S-Nitrosothiols are formed by S-nitrosation – i.e. formation of a covalent bond between NO and a reduced thiol function of a cysteine residue belonging to high or low molecular weight proteins or peptides. In vivo, S-nitrosothiols like S-nitrosoalbumin, S-nitrosohemoglobin and S-nitrosoglutathione (GSNO) are the physiological forms of NO storage and transport [8]. Indeed, the formation of the S-NO bond extends NO half-life from 45 min up to several hours [9], [10] and limits the oxidative/nitrosative stress induced by NO oxidation into peroxynitrite ions (ONOO^–) [11]. Despite the therapeutic potential of S-nitrosothiols, their half-life linked to their physico-chemical instability (heat, light, metallic cations,…) and/or enzymatic (redoxines or, for GSNO only, γ-glutamyltransferase) degradation, is too short for chronic diseases treatment [12].

Nowadays, many preclinical studies focused on cardiovascular therapeutics using S-nitrosothiols [6], [13], [14], [15], [16], [17]. For example, daily intraperitoneal administration of S-nitroso-N-acetyl-l-cysteine (NACNO) for two weeks shows anti-atherosclerotic effects in mice [13]. However, compared to the oral route, the intraperitoneal administration is less suitable for chronic treatments. GSNO administration through the oral route in a context of stroke [14] results in neuroprotective effects: GSNO maintains the blood-brain barrier integrity, reduces peroxynitrite formation and stabilises several deleterious factors via S-nitrosation [13], [14], [15], [16]. Despite such beneficial effects following oral administration, to the best of our knowledge, no study evaluated the mechanisms of intestinal absorption of GSNO and other S-nitrosothiols. Only Pinheiro et al. [17] demonstrated that oral administration of nitrite and nitrate ions (stable NO derived species) to rats increased the concentration of circulating S-nitrosothiols, thus produced antihypertensive effects. This study indirectly proves the intestinal absorption of S-nitrosothiols without elucidated the underlying mechanisms. However, the understanding of the intestinal absorption mechanisms of S-nitrosothiols is a prerequisite to control the dose and the kinetic of NO reaching its action sites.

To predict the intestinal absorption of drugs, the Biopharmaceutical Classification System (BCS) [18] defines four classes based on the physico-chemical properties (solubility) and intestinal permeability of drugs. The intestinal permeability of a drug is characterised, using in vitro or ex vivo models, by apparent permeability coefficient (Papp) from low permeability (<1 × 10⁻⁶ cm.s⁻¹) to high permeability (≥10 × 10⁻⁶ cm.s⁻¹) including also a medium permeability class [19]. Thus far, only one of our studies was interested in the improvement and the prolongation of GSNO intestinal absorption by proposing alginate/chitosan nanocomposite formulation [20]. Using an in vitro intestinal barrier model of differentiated Caco-2 cells, we showed low intestinal permeability for GSNO with a Papp of 0.83 × 10⁻⁷ cm.s⁻¹. The nanocomposite formulation delayed GSNO absorption up to 24 h (1 h for free GSNO) and multiplied by four the Papp value (3.41 × 10⁻⁷ cm.s⁻¹) even if GSNO stayed in the low class of permeability [20]. This study showed the ability for GSNO to cross the intestinal barrier model and the possibility to modulate its kinetics of absorption. This opens new therapeutic applications in the treatment of chronic pathologies linked to a decrease of NO bioavailability.

Intestinal absorption of low molecular weight molecules is mainly driven by their physico-chemical properties such as lipophilicity, correlated with the octanol/water partition coefficient, expressed as a logarithmic value (log P), and the ionisation constant (pKa). For S-nitrosothiols, the log P value is driven by the skeleton carrying NO. GSNO, NACNO and S-nitroso-N-acetyl-d-penicillamine (SNAP), the three main S-nitrosothiols described in the literature, are characterised by calculated log P value of −2.70, −0.47 and 1.08, respectively [2]. The skeleton carrying NO presents also different therapeutic properties linked with its chemical structure. GSNO is a physiological S-nitrosothiol [21], present in the cytosol at a high concentration especially in erythrocytes [22], platelets and cerebral tissue. Its reduced glutathione (GSH) skeleton shows an antioxidant chemical structure thanks to its thiol functional group and forms, with the glutathione disulphide (GSSG), the intracellular redox buffer. NACNO and SNAP are synthetic S-nitrosothiols. NACNO with its N-acetyl-l-cysteine (NAC) skeleton possesses also an antioxidant activity in accordance with its chemical structure (thiol function). Furthermore, NAC is already used in human medicine as a mucolytic agent (oral administration) or as the antidote in acetaminophen intoxication [23]. SNAP shows in addition to its antioxidant properties (thiol function), an anti-inflammatory skeleton, N-acetyl-d-penicillamine (NAP) is used in the treatment of Wilson’s disease (Trolovol®) and rheumatoid arthritis.

In this study, using an in vitro cell model of intestinal barrier, we propose to elucidate the intestinal transport mechanisms of S-nitrosothiols and NO in relation to their physico-chemical properties. Three different conditions were studied, i) permeability from the apical to the basolateral compartment, ii) permeability from the basolateral to the apical compartment to highlight an active transport such as drug influx/efflux, or a passive diffusion, and iii) permeability from an acidified apical compartment, mimicking the luminal intestinal pH of the jejunum, the major site of amino acid absorption [24].