Michaël Messaoudi

Human Opiorphin, a natural antinociceptive modulator of opioid-dependent pathways

Mammalian zinc ectopeptidases play important roles in turning off neural and hormonal peptide signals at the cell surface, notably those processing sensory information. We report here the discovery of a previously uncharacterized physiological inhibitor of enkephalin-inactivating zinc ectopeptidases in humans, which we have named Opiorphin. It is a QRFSR peptide that inhibits two enkephalin-catabolizing ectoenzymes, human neutral ecto-endopeptidase, hNEP (EC 3.4.24.11), and human ecto-aminopeptidase, hAP-N (EC 3.4.11.2). Opiorphin displays potent analgesic activity in chemical and mechanical pain models by activating endogenous opioid-dependent transmission. Its function is closely related to the rat sialorphin peptide, which is an inhibitor of pain perception and acts by potentiating endogenous μ- and δ-opioid receptor-dependent enkephalinergic pathways. Here we demonstrate the functional specificity in vivo of human Opiorphin. The pain-suppressive potency of Opiorphin is as effective as morphine in the behavioral rat model of acute mechanical pain, the pin-pain test. Thus, our discovery of Opiorphin is extremely exciting from a physiological point of view in the context of endogenous opioidergic pathways, notably in modulating mood-related states and pain sensation. Furthermore, because of its in vivo properties, Opiorphin may have therapeutic implications.

Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4–1 μM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100–200 μg/kg doses of sialorphin required the activation of μ- and δ-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

Effects of long-term administration of a cocoa polyphenolic extract (Acticoa powder) on cognitive performances in aged rats

Numerous studies have indicated that increased vulnerability to oxidative stress may be the main factor involved in functional declines during normal and pathological ageing, and that antioxidant agents, such as polyphenols, may improve or prevent these deficits. We examined whether 1-year administration of a cocoa polyphenolic extract (Acticoa powder), orally delivered at the dose of 24 mg/kg per d between 15 and 27 months of age, affects the onset of age-related cognitive deficits, urinary free dopamine levels and lifespan in old Wistar-Unilever rats. Acticoa powder improved cognitive performances in light extinction and water maze paradigms, increased lifespan and preserved high urinary free dopamine levels. These results suggest that Acticoa powder may be beneficial in retarding age-related brain impairments, including cognitive deficits in normal ageing and perhaps neurodegenerative diseases. Further studies are required to elucidate the mechanisms of cocoa polyphenols in neuroprotection and to explore their effects in man.

Antidepressant-like effects of a cocoa polyphenolic extract in Wistar-Unilever rats.

Abstract Depression is a major public health problem affecting about 12% of the world population. Drugs exist but they have many side effects. In the last few years, natural substances (e.g. flavonoids) have been tested to cure such disorders. Cocoa polyphenolic extract is a complex compound prepared from non-roasted cocoa beans containing high levels of flavonoids. The antidepressant-like effect of cocoa polyphenolic extract was evaluated using the forced swimming test in rats. Cocoa polyphenolic extract significantly reduced the duration of immobility at both doses of 24 mg/kg/14 days and 48 mg/kg/14 days, although no change of motor dysfunction was observed with the two doses tested in the open field. The results of the forced swimming test after a subchronic treatment and after an additional locomotor activity test confirm the assumption that the antidepressant-like effect of cocoa polyphenolic extract in the forced swimming test model is specific. Further, it can be speculated that this effect might be related to its content of active polyphenols.

Ethological comparison of the effects of a bovine αs1-casein tryptic hydrolysate and diazepam on the behaviour of rats in two models of anxiety

A bovine alpha s1-casein tryptic hydrolysate was previously demonstrated to display an anxiolytic-like activity in the conditioned defensive burying and in the elevated plus-maze models when i.p. injected. The present study assessed the anxiolytic-like effects of this tryptic hydrolysate after an oral administration in rats faced to the same behavioural situations using diazepam as a reference. In a first experiment, the behavioural effects of the hydrolysate in the conditioned defensive burying test were investigated at doses ranging 5-50 mg/kg. The results showed that the minimal dose required to elicit an anxiolytic-like activity is 15 mg/kg. In a second experiment, the alpha s1-casein tryptic hydrolysate (15 mg/kg, p.o.) was demonstrated to display an anxiolytic-like activity similar to diazepam (3 mg/kg, p.o.) in the conditioned defensive burying test and the elevated plus-maze. However, the ethological analysis of behaviour indicated that this hydrolysate has a different activity compared to diazepam. While diazepam induced a disinhibition state in rats, possibly related to the risk-taking behaviour observed after a benzodiazepine ingestion in humans, the tryptic hydrolysate did not display such a side effect. These results suggest that the mechanism of action of the bovine alpha s1-casein tryptic hydrolysate may differ from that of diazepam.

Effects of lifelong intervention with an oligofructose-enriched inulin in rats on general health and lifespan.

Ageing is associated with changes in physiology and morphology; nutritional strategies to decrease morbidity and to prolong life are of high interest. The aim of the study was to investigate the effects of lifelong supplementation with an oligofructose-enriched inulin on morphological and biological markers and lifespan in male and female rats. Male and female rats, age 3 months, were randomised into two groups to receive either a diet with 10 % of an oligofructose-enriched inulin (Synergy 1) or a standard diet (control) for 27 months. The rats were weighed every 2 weeks and their food intake was evaluated on four successive days every 4-6 weeks. Samples were taken at 12, 18 and 24 months of age. During the whole intervention period, male rats receiving Synergy 1 (SYN1-M) displayed lower body weight, cholesterol and plasma triacylglycerolaemia compared with the controls (Cont-M). The survival rate at 24 months of age of SYN1-M rats was 35.3 % greater than that of Cont-M rats. In female rats, the Synergy 1 supplementation (SYN1-F) group also reduced body weight, cholesterol and triacylglycerolaemia levels, but results were less consistent over the experiment. The survival rate at 24 months of age in SYN1-F rats was 33.3 % greater compared with that of the control (Cont-F) group. To conclude, lifelong intervention with Synergy 1 improved biological markers during ageing and survival rate (lifespan) of rats.

Protective effect of Acticoa powder, a cocoa polyphenolic extract, on prostate carcinogenesis in Wistar–unilever rats

The effects of Acticoa powder on prostate carcinogenesis were investigated using the N-methylnitrosourea and testosterone propionate prostate tumor model. Sixty male Wistar–Unilever rats were randomly divided in four groups of 15 rats: one control group not induced but treated with vehicle (not induced+vehicle) and three chemo-induced groups. Two weeks before prostate tumor induction and then throughout the experiment, chemo-induced rats were orally treated with Acticoa powder at 24 (chemo-induced+Acticoa powder24) or 48 (chemo-induced+Acticoa powder48) mg/kg or with vehicle (chemo-induced+vehicle), daily from Monday to Friday. Survival, body weight, food and water consumption were recorded throughout the experiment. Six rats per group were randomly killed 9 months after the prostate tumor induction for histopathological analysis of prostates. A reduction in the incidence of prostate tumors was observed for the chemo-induced+Acticoa powder48-treated group in comparison with the chemo-induced+vehicle-treated group and no tumors were observed in the chemo-induced+Acticoa powder24-treated group as in the not induced+vehicle-treated group after 9 months. The nine remaining rats per group were maintained in a long-term survival study. The life span of the chemo-induced+Acticoa powder24-treated group was significantly increased in comparison with the chemo-induced+Acticoa powder48 and the chemo-induced+vehicle-treated groups, close to the one of the not induced+vehicle-treated group. A significant reduction in the incidence of prostate tumors was also observed for the chemo-induced+Acticoa powder24 and chemo-induced+Acticoa powder48-treated groups in comparison with the chemo-induced+vehicle-treated group. In conclusion, Acticoa powder at 24 mg/kg protected rats from prostate carcinogenesis when chronically given before the initiation and promotion phases of induction.

Anxiolytic‐like effects and safety profile of a tryptic hydrolysate from bovine alpha s1‐casein in rats

The anxiolytic activity and adverse benzodiazepine‐like effects of a bovine alpha s1‐casein tryptic hydrolysate (CH) were evaluated. The effects of CH orally administered at doses of 5 and 15 mg/kg were compared with those of diazepam (DZ) at 3 mg/kg in the conditioned defensive burying test. Rats treated either with CH at 15 mg/kg or with DZ showed a decrease in anxiety. A drug‐related difference was observed in terms of duration, as the anxiolytic‐like action of CH was maintained after 7 days with twice‐daily administration, whereas that of DZ was not. CH and DZ were then evaluated for their potential effects on memory in a passive avoidance paradigm. CH‐treated rats had significantly longer latencies before entering the dark compartment where they were previously delivered a shock, indicating better retention relative to DZ‐treated rats. In the final test, CH and DZ were evaluated for place preference, an index of the possible addictive potential of these substances. DZ‐treated rats spent more time in the compartment associated with drug exposure than control rats. This effect was not found in CH‐treated rats. Thus, CH did not display side effects associated with DZ, despite its affinity for gamma‐aminobutyric acid(A) (GABAA) receptors. Specific linking of CH on GABAA receptor function involved in anxiolysis, but not on that implied in memory‐impairing effects, may be hypothesized to explain its specific activity. This profile might render it advantageous for nutritional purposes.

Behavioural and cognitive effects of oligofructose-enriched inulin in rats.

The behavioural and cognitive effects of oligofructose-enriched inulin at the doses of 5 and 10 % in the diet, orally ingested daily during 2 weeks, were investigated using a functional observational battery (FOB) and the light extinction test in male Wistar rats. Control rats received a standard diet and were tested in the same test situations. The behavioural effects were assessed 2 d before and 14 d after the beginning of the treatment period and the cognitive effects were investigated after the administration period by lever-pressing activity and learning discrimination using the light extinction test paradigm. In general, the study demonstrated that oligofructose-enriched inulin at 5 % in the diet, and particularly at 10 % in the diet, caused relaxing-like effects, stimulated and increased the general activity and interest of the rats to the test environment. In addition, both doses of oligofructose-enriched inulin showed significant effects on learning discrimination in male rats, in comparison with the control diet. These results suggest that oligofructose-enriched inulin, particularly at the dose of 10 %, improves cognitive performances in the light extinction test and the well-being of male rats using the FOB.

Structure- Activity Relationship Study and Function-Based Petidomimetic Design of Human Opiorphin with Improved Bioavailability Property and Unaltered Analgesic Activity

Human opiorphin inhibits enkephalin-inactivating ectopeptidases to produce analgesic and antidepressant-like effects in standard murine models via activation of μ and/or δ opioid pathways. It is an endogenous peptide regulator of enkephalin bioavailability. Opiorphin molecule, a QRFSR-peptide, is thus a promising prototype for the design of an improved class of analgesics. The major limitation on the clinical use of peptide drugs is their rapid degradation by circulating peptidases. Our goal was, therefore, to search for functional derivatives of opiorphin with improved metabolic stability. In order to identify the functional amino acid groups required for opiorphin inhibitory potency toward both AP-N and NEP human ectopeptidases, we used the Structure-Activity Relationship (SAR) method. From this data, a series of opiorphin derivatives was designed and selected. The best performing compound then underwent a complete metabolic profile using in vitro kinetic models. Finally, its safety profile relative to the native peptide as well as its efficacy in an in vivo rat model was evaluated. We demonstrated a tight structural selectivity in the functional interaction of opiorphin with both human NEP and AP-N targets by SAR studies. Nevertheless, we found that the addition of an N-terminal Zn-chelating group, a Cys-thiol group and the replacement of the first labile peptide bond by a polyethylene surrogate, a [CH2]6 linker,and, finally, the substitution of Ser4 by Ser-O-[CH2]8, results in a high performing C-[(CH2)6]-QRF[S-O-[CH2]8]-R peptidomimetic product. This designed opiorphin analog shows reinforced inhibitory potency toward human AP-N activity (more than 10-fold increase) and NEP activities (more than 40-fold increase) relative to the QRFSR native peptide. It also has increased metabolic stability in human plasma and yet retains full analgesic activity in the behavioral formalin-induced rat pain model. C-[(CH2)6]-QRF[S-O-[CH2]8]-R thus represents a very attractive and promising analgesic drug-candidate.