Chronic Treatments and Side Effects of Mixed Inhibitors of NEP and APN

The main advantage of modifying the concentration of endogenous peptides by use of peptidase inhibitors is that pharmacological effects are induced only at receptors tonically or phasically stimulated by the natural effectors. Moreover, in contrast to exogenous agonists or antagonists, chronic administration of mixed enkephalin-degrad-ing enzymes inhibitors does not induce changes in the synthesis of its peptide precursors, as well as in the secretion of the active peptide (20).

As expected from their mechanims of action, the mixed inhibitors are devoid of the main drawbacks of morphine (i.e., respiratory depression (21), constipation and physical and psychic dependence [22,23] [Fig. 3]). This is due mainly to the weaker, but more specific, stimulation of the opioid-binding sites by the tonically or phasically released endogenous opioids, thus minimizing receptor desensitization or down-

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Fig. 2. (A) Dual inhibitors of NEP and APN. Two strategies have been followed: RB 101, association by a disulfide bond of a potent APN inhibitor with a potent NEP inhibitor, and RB 3001, a true dual NEP/APN inhibitor. (B) Differences in the duration of antinociceptive responses (hot-plate test in mice) provided by RB 101 ▲ (10 mg/kg iv) and RB 3001 O (25 mg/kg iv). *p <0.05 and **p <0.01 compared with control.

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Fig. 2. (A) Dual inhibitors of NEP and APN. Two strategies have been followed: RB 101, association by a disulfide bond of a potent APN inhibitor with a potent NEP inhibitor, and RB 3001, a true dual NEP/APN inhibitor. (B) Differences in the duration of antinociceptive responses (hot-plate test in mice) provided by RB 101 ▲ (10 mg/kg iv) and RB 3001 O (25 mg/kg iv). *p <0.05 and **p <0.01 compared with control.

regulation that usually occurs after ubiquitous activation of opioid receptors by exogenous agonists. This limited opioid receptor occupation by the endogenous peptides is in agreement with in vivo binding studies, demonstrating that the increase in tonically released endogenous enkephalins is too low to saturate opioid receptors (24).

Moreover, chronic morphine induces a hypersensitivity of noradrenaline-containing neurons in the locus coeruleus, considered one of the main causes of the withdrawal syndrome (25-27). In this brain region a very low tonic release of enkephalins was observed (28), and this is probably one of the main reasons why the withdrawal syndrome is almost absent after chronic treatment by peptidase inhibitors as compared to exogenous opioids. Moreover, because of their higher intrinsic efficacy, enkephalins need to occupy fewer opioid receptors than does morphine to generate the same pharmacological responses (29).

Fig. 3. (A) Antinociceptive dose-response curves recorded in the hot-plate test (i.e., jump response) 10 min after iv administration of morphine to mice chronically pretreated with saline O, RB 101 ■ (80 mg/kg), or morphine • (3 mg/kg), ip twice daily, for 4 d. (B) Antinociceptive dose-response curves recorded in the hot-plate test (jump response) 10 min after iv administration of RB 101 to mice chronically pretreated with vehicle □, RB 101 ■ (80 mg/kg), or morphine • (3 mg/kg), ip, twice daily for 4 d. (C) Comparison of the withdrawal symptoms induced by naloxone after chronic treatment with morphine (6 mg/kg) or RB 101 (160 mg/kg), injected ip, twice daily for 5 d. (D) Comparison of the psychic dependence induced by chronic morphine (6 mg/kg) or RB 101 (160 mg/kg), injected ip, in the place preference test. **p < 0.01, as compared with other groups.

Fig. 3. (A) Antinociceptive dose-response curves recorded in the hot-plate test (i.e., jump response) 10 min after iv administration of morphine to mice chronically pretreated with saline O, RB 101 ■ (80 mg/kg), or morphine • (3 mg/kg), ip twice daily, for 4 d. (B) Antinociceptive dose-response curves recorded in the hot-plate test (jump response) 10 min after iv administration of RB 101 to mice chronically pretreated with vehicle □, RB 101 ■ (80 mg/kg), or morphine • (3 mg/kg), ip, twice daily for 4 d. (C) Comparison of the withdrawal symptoms induced by naloxone after chronic treatment with morphine (6 mg/kg) or RB 101 (160 mg/kg), injected ip, twice daily for 5 d. (D) Comparison of the psychic dependence induced by chronic morphine (6 mg/kg) or RB 101 (160 mg/kg), injected ip, in the place preference test. **p < 0.01, as compared with other groups.

Several studies have used the expression of immediate early genes as markers for neuronal activity in an attempt to differentiate the effects of exogenously administered opioids from tonically released endogenous opioid peptides. By measuring c-Fos expression generated by either noxious thermal stimuli or chronic pain, it was shown that dual inhibitors of NEP and APN reduced this expression to a maximum of 60%, as compared to 90% for morphine (30-32), and a lack of tolerance to repeated administration of RB 101 was observed (33).

The moderate degree or lack of tolerance or physical dependence observed following chronic treatment with mixed inhibitors could also be due to weaker intracellular modifications. Indeed, opioid side effects are complex processes involving multiple cellular targets including receptors involved at central or peripheral levels on respiration, cardiovascular control, intestinal functioning, and so on (34). The lack of undesirable effects might also be related to differences in the mechanisms of receptor endocytosis following receptor activation by enkephalins or by alcaloids or synthetic heterocyclic agonists (35). Similarly, different desensitization of opioid receptors may occur, which could be caused by an alteration in adenylyl cyclase functioning or expression, phosphorylation and/or dimerization of receptors, or changes in G-protein subunit concentrations.

The biochemical mechanisms involved in psychic dependence that could result in craving remain still unclear. However, it is well established that dopaminergic neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens play a major role in this process (36). The failure of RB 101 to induce psychic dependence in various animal models used to predict the addictive properties of a given compound (23) probably results from a lower recruitment of opioid receptors and weaker modifications of intracellular events for endogenous enkephalins than for morphine. This hypothesis is supported by the weaker changes in dopamine release in the nucleus accumbens after administration in the VTA of a dual NEP/APN inhibitor, compared with a <x-opioid agonist (37), and by the apparent absence of effects on the levels of dopamine and metabolites in the nucleus accumbens following administration of a selective NEP inhibitor (38).

Accordingly, when injected in the VTA of rats, mixed inhibitors, unlike morphine, have no effect or only slightly decrease the rate of intracranial self-stimulation, a response strongly triggered by addictive drugs (39).

Constipation Prescription

Constipation Prescription

Did you ever think feeling angry and irritable could be a symptom of constipation? A horrible fullness and pressing sharp pains against the bladders can’t help but affect your mood. Sometimes you just want everyone to leave you alone and sleep to escape the pain. It is virtually impossible to be constipated and keep a sunny disposition. Follow the steps in this guide to alleviate constipation and lead a happier healthy life.

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