Nicotinamide Mononucleotide Administration Triggers Macrophages Reprogramming and Alleviates Inflammation During Sepsis Induced by Experimental Peritonitis.

Peritonitis and subsequent sepsis lead to high morbidity and mortality in response to uncontrolled systemic inflammation primarily mediated by macrophages. Nicotinamide adenine dinucleotide (NAD+) is an important regulator of oxidative stress and immunoinflammatory responses. However, the effects of NAD+ replenishment during inflammatory activation are still poorly defined. Hence, we investigated whether the administration of ß-nicotinamide mononucleotide (ß-NMN), a natural biosynthetic precursor of NAD+, could modulate the macrophage phenotype and thereby ameliorate the dysregulated inflammatory response during sepsis. For this purpose, C57BL6 mice were subjected to the cecal ligation and puncture (CLP) model to provoke sepsis or were injected with thioglycolate to induce sterile peritonitis with recruitment and differentiation of macrophages into the inflamed peritoneal cavity. ß-NMN was administered for 4 days after CLP and for 3 days post thioglycolate treatment where peritoneal macrophages were subsequently analyzed. In the CLP model, administration of ß-NMN decreased bacterial load in blood and reduced clinical signs of distress and mortality during sepsis. These results were supported by transcriptomic analysis of hearts and lungs 24 h post CLP-induction, which revealed that ß-NMN downregulated genes controlling the immuno-inflammatory response and upregulated genes involved in bioenergetic metabolism, mitochondria, and autophagy. In the thioglycolate model, a significant increase in the proportion of CD206 macrophages, marker of anti-inflammatory M2 phenotype, was detected on peritoneal exudate macrophages from ß-NMN-administered mice. Transcriptomic signature of these macrophages after bacterial stimulation confirmed that ß-NMN administration limited the pro-inflammatory M1 phenotype and induced the expression of specific markers of M2 type macrophages. Furthermore, our data show that ß-NMN treatment significantly impacts NAD + metabolism. This shift in the macrophage phenotype and metabolism was accompanied by a reduction in phagolysosome acidification and secretion of inflammatory mediators in macrophages from ß-NMN-treated mice suggesting a reduced pro-inflammatory activation. In conclusion, administration of ß-NMN prevented clinical deterioration and improved survival during sepsis. These effects relied on shifts in the metabolism of organs that face up an increased energy requirement caused by bacterial infection and in innate immunity response, including reprogramming of macrophages from a highly inflammatory phenotype to an anti-inflammatory/pro-resolving profile.

Nicotinamide Mononucleotide Administration Prevents Doxorubicin-Induced Cardiotoxicity and Loss in Physical Activity in Mice.

Doxorubicin (Doxo) is a widely used antineoplastic drug with limited clinical application due to its deleterious dose-related side effects. We investigated whether nicotinamide mononucleotide (NMN) could protect against Doxo-induced cardiotoxicity and physical dysfunction in vivo. To assess the short- and long-term toxicity, two Doxo regimens were tested, acute and chronic. In the acute study, C57BL6/J (B6) mice were injected intraperitoneally (i.p.) once with Doxo (20 mg/kg) and NMN (180 mg/kg/day, i.p.) was administered daily for five days before and after the Doxo injection. In the chronic study, B6 mice received a cumulative dose of 20 mg/kg Doxo administered in fractionated doses for five days. NMN (500 mg/kg/day) was supplied in the mice's drinking water beginning five days before the first injection of Doxo and continuing for 60 days after. We found that NMN significantly increased tissue levels of NAD+ and its metabolites and improved survival and bodyweight loss in both experimental models. In addition, NMN protected against Doxo-induced cardiotoxicity and loss of physical function in acute and chronic studies, respectively. In the heart, NMN prevented Doxo-induced transcriptomic changes related to mitochondrial function, apoptosis, oxidative stress, inflammation and p53, and promyelocytic leukemia nuclear body pathways. Overall, our results suggest that NMN could prevent Doxo-induced toxicity in heart and skeletal muscle.

Safety evaluation after acute and sub-chronic oral administration of high purity nicotinamide mononucleotide (NMN-C®) in Sprague-Dawley rats.

ß-nicotinamide mononucleotide (NMN) is a natural molecule intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Preclinical evidences point to the beneficial effect of NMN administration on several age-related conditions. The present work aimed at studying mutagenicity, and genotoxicity, acute oral toxicity and subchronic oral toxicity of a high purity synthetic form of NMN (NMN-C®) following the OECD guidelines. In the experimental conditions tested, NMN-C® was not mutagenic or genotoxic. Acute toxicity assay revealed that at an oral limit dose of 2666 mg/kg, NMN-C® did not lead to any mortality or treatment-related adverse signs. Over a 90-day sub-chronic period of repeated oral administration of NMN-C® at doses of 375, 750 and 1500 mg/kg/d followed by a 28-day treatment-free recovery period, NMN-C® appeared to be safe and did not promote toxic effects as seen from body weight change, food and water consumption, feed conversion efficiency, biochemical and blood parameters as well as organ toxicity and histological examinations of main organs. In conclusion, we provide the first data highlighting the safety of short to intermediate term (sub-chronic) oral administration of NMN and our experimental results allowed to determine a No-Observable Adverse Effect Level (NOAEL) for NMN-C® to be ≥ 1500 mg/kg/d.

Cutaneous iontophoresis of µ-conotoxin CnIIIC-A potent NaV1.4 antagonist with analgesic, anaesthetic and myorelaxant properties.

Cutaneous iontophoretic delivery of µ-conotoxin CnIIIC (XEP), a potent peptide antagonist of the NaV1.4 sodium channel, was investigated using porcine ear skin and validated using human abdominal skin. Initial results demonstrated that cutaneous deposition of XEP following iontophoresis was superior to passive delivery and increased with current density. XEP deposition after iontophoresis at 0.1, 0.3 and 0.5mA/cm2 for 2h and 4h was 22.4±0.4, 34.5±1.4, 57.4±7.6µg/cm2 and 30.6±5.4, 53.9±17.2, 90.9±30.8µg/cm2, respectively (cf. corresponding passive controls - 9.8±1.1 and 16.9±1.0µg/cm2). Moreover, tape-stripping studies showed that XEP was mainly adsorbed on the skin surface when administered passively. Co-iontophoresis of acetaminophen demonstrated that XEP was present in the skin as it significantly reduced convective solvent flow as evidenced by the ∼7-fold decrease in acetaminophen permeation. Shorter duration iontophoresis (15, 30 and 60min) was performed and the effect of current density (0.1, 0.3 and 0.5mA/cm2) and concentration (0.1 and 1mM) investigated. Skin deposition of XEP was already quantifiable after iontophoresis for 15min at the lower concentration. There was no statistically significant difference between XEP deposition in porcine and human skin. Confocal laser scanning microscopy enabled post-iontophoretic visualization of FITC-labelled XEP in the epidermis.

Delivery of a lactose derivative of endomorphin 1 to the brain via the olfactory epithelial pathway.

The rapid and direct delivery of a neuroactive endomorphin 1 derivative to the brain via nasal delivery is reported. A synthetic derivative of the native opioid peptide, endomorphin 1 bearing a lactose unit on the N-terminus of the peptide has been previously reported to exhibit antinoceceptive activity similar to morphine after both intravenous and oral administration. This compound has been administered nasally to rats and appeared in the olfactory bulb within 10 min of administration with negligible levels appearing in the circulating blood or in the rest of the brain. These results indicate that the peptide is absorbed into the brain via the olfactory epithelial pathway suggesting nasal delivery may be a viable alternative route of delivery in clinical applications.

Lipophilic derivatives of leu-enkephalinamide: in vitro permeability, stability and in vivo nasal delivery.

Leu-enkephalin is an endogenous pain modulating opioid pentapeptide. Its development as a potential pharmaceutic has been hampered by poor membrane permeability and susceptibility to enzymatic degradation. The addition of an unnatural amino acid containing a lipidic side chain at the N-terminus and the modification of the C-terminus to a carboxyamide was performed to enhance the nasal delivery of the peptide. Two lipidic derivatives with varying side chain lengths (C(8)-Enk-NH(2) (1), C(12)-Enk-NH(2) (2)) and their acetylated analogues were successfully synthesised. Caco-2 cell monolayer permeability and Caco-2 cell homogenate stability assays were performed. C(8)-Enk-NH(2) (1) and its acetylated analogue Ac-C8-Enk-NH(2) (3) exhibited apparent permeabilities (mean±SD) of 2.51±0.75×10(-6)cm/s and 1.06±0.62×10(-6), respectively. C12-Enk-NH(2) (2) exhibited an apparent permeability of 2.43±1.26×10(-6) cm/s while Ac-C12-Enk-NH(2) (4) was not permeable through the Caco-2 monolayers due to its poor solubility. All analogues exhibited improved Caco-2 homogenate stability compared to Leu-Enk-NH(2) with t(½) values of: C8-Enk-NH(2) (1): 31.7 min, C(12)-Enk-NH(2) (2): 14.7 min, Ac-C8-Enk-NH(2) (3): 83 min, Ac-C(12)-Enk-NH(2) (4): 27 min. However, plasma stability assays revealed that the diastereoisomers of C8-Enk-NH(2) (1) did not degrade at the same rate, with the l isomer (t(1/2)=8.9 min) degrading into Leu-enkephalinamide and then des-Tyr-Leu-Enk-NH(2), whereas the d isomer was stable (t(1/2)=120 min). In vivo nasal administration of C(8)-Enk-NH(2) to male rats resulted in concentrations of 5.9±1.84×10(-2) µM in the olfactory bulbs, 1.35±1.01×10(-2) µM in the brain and 6.53±1.87×10(-3) µM in the blood 10 min after administration.

Oral absorption and in vivo biodistribution of alpha-conotoxin MII and a lipidic analogue.

Conotoxins are highly constrained peptide toxins that exhibit pharmaceutically relevant biological activities. We herein report the extent of absorption and profile of distribution of a native alpha-conotoxin, MII and a lipophilic analogue of MII (N-LaaMII) after intravenous (iv) and oral administration to male Sprague-Dawley rats. N-LaaMII is formed by coupling 2-amino-D,L-dodecanoic acid (Laa) to the N-terminus of MII and has previously been shown to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII while maintaining the potency in inhibition of nAChRs of the parent peptide. Both peptides crossed the GI tract after oral administration (approximately 6% after 30 m). While Laa conjugation did not significantly improve absorption, it did greatly increase the accumulation of the compound in the liver after iv administration. Neither peptide crossed the blood-brain barrier to any significant extent. This is the first study of the in vivo biodistribution of an alpha-conotoxin after oral administration.