The amyloid cascade is the most frequently accepted hypothesis of Alzheimer's Disease (AD). According to this hypothesis, the formation of plaques precedes the appearance of fibrillary tangles. Therapeutic agents able to inhibit the formation of plaques are therefore considered as potential disease-modifying treatments (DMT) that could prevent or limit the progression of AD. Plaques are deposits formed by aggregates of amyloid-β (Aβ)-peptides. These peptides are metabolites of amyloid precursor protein (APP) first mediated by two enzymes: β-secretase 1 (BACE1) and γ-secretase. Molecular identification of these two enzymes has stimulated the development of their inhibitors. The clinical testing of these two classes of molecules has not been successful to date. The oligomerization of Aβ-peptides into plaques is now targeted by immunological approaches such as antibodies and vaccines. Structural consideration of the Aβ-peptide sequence led to the launch of the antibody Aducanumab. Several other antibodies are in late clinical phases. Progress in the understanding of the effects of N-truncated Aβ-peptides such as pE3-42, formed by the action of recently well characterized enzymes (aminopeptidase A, dipeptidylpeptidase-4 and glutaminyl cyclase) suggests that oligomerization can be limited either by enzyme inhibitors or antibody approaches. This strategy associating two structurally interconnected mechanisms is focused in this review.

Objective: Brain renin-angiotensin system (RAS) hyperactivity is involved in the pathophysiology of hypertension and heart failure (HF). Angiotensin III/(AngIII), one of the main effector peptides of the brain RAS, exerts tonic stimulatory control over blood pressure/(BP) in hypertensive rats and plays a pivotal role in sympathetic hyperactivity and left ventricular/(LV) dysfunction in rats post-myocardial infarction/(MI). Thus, aminopeptidase A/(APA), the enzyme generating brain AngIII represents a potential therapeutic target for the treatment of these pathologies. Our aim was to evaluate the mode of action of firibastat, a brain-penetrating prodrug of the APA inhibitor, EC33, to reduce BP in hypertensive rats and prevent LV dysfunction in mice post-MI. Design and method: Firibastat and MLN4760, an angiotensin-converting enzyme2/(ACE2) inhibitor were administered by central route in hypertensive DOCA-salt rats and mean arterial BP (MABP) was recorded following insertion of a catheter into the femoral artery. Brain APA and ACE2 activities were measured 30 min post-treatment. Two days post-MI induced by the left anterior descending artery ligation, mice were randomized to receive during 4–8 weeks oral treatment with vehicle, firibastat (150 mg/kg) or the ACE inhibitor, enalapril (1 mg/kg). Cardiac function was evaluated by echocardiography Results: 1-Firibastat, when given centrally in hypertensive rats, is immediately cleaved by brain reductases, generating EC33 which inhibits brain APA activity, blocks brain AngIII formation and decreases BP. At the same time, firibastat treatment increases the activity of brain ACE2, majoring the conversion of AngII to Ang1-7, which by acting on the Mas-receptor, participate to the firibastat-induced BP decrease. 2-Chronic firibastat treatment in mice post-MI normalizes brain APA activity (normalizing brain RAS activity) and prevents sympathetic hyperactivity. It reduced LV end-diastolic pressure, LV end-systolic diameter and volume, and increased LV ejection fraction. It decreased the expression of HF and fibrosis biomarkers. Firibastat treatment prevents cardiac dysfunction and attenuates cardiac hypertrophy and fibrosis. Conclusions: Following these data, firibastat was selected for clinical development. Phase IIa/IIb clinical trials provided pharmacological proof-of-principle of firibastat efficacy for decreasing BP in hypertensive patients. The ongoing phase II study/QUORUM will determine if firibastat treatment may constitute a new therapeutic approach for preventing HF post-MI.

One of the main components of senile plaques in Alzheimer’s disease (AD)-affected brain is the Aβ peptide species harboring a pyroglutamate at position three pE3-Aβ. Several studies indicated that pE3-Aβ is toxic, prone to aggregation and serves as a seed of Aβ aggregation. The cyclisation of the glutamate residue is produced by glutaminyl cyclase, the pharmacological and genetic reductions of which significantly alleviate AD-related anatomical lesions and cognitive defects in mice models. The cyclisation of the glutamate in position 3 requires prior removal of the Aβ N-terminal aspartyl residue to allow subsequent biotransformation. The enzyme responsible for this rate-limiting catalytic step and its relevance as a putative trigger of AD pathology remained yet to be established. Here, we identify aminopeptidase A as the main exopeptidase involved in the N-terminal truncation of Aβ and document its key contribution to AD-related anatomical and behavioral defects. First, we show by mass spectrometry that human recombinant aminopeptidase A (APA) truncates synthetic Aβ1-40 to yield Aβ2-40. We demonstrate that the pharmacological blockade of APA with its selective inhibitor RB150 restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aβ-precursor protein (βAPP). Pharmacological reduction of APA activity and lowering of its expression by shRNA affect pE3-42Aβ- and Aβ1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA inhibitors and shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of pE3-42Aβ-positive plaques, APA activity is augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA is a key enzyme involved in Aβ N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with AD pathology.

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than β-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases BP in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2' subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2' subsite of human APA established various types of interactions in major part with the P2' residue but also with the P1' residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

Abstract Background Brain renin-angiotensin system hyperactivity has been implicated in the development and maintenance of hypertension. We previously showed that aminopeptidase A (APA) generates in the brain, angiotensin III, which exerts a tonic stimulatory control over blood pressure in hypertensive rats. Thus, the central injection of the specific and selective APA inhibitor, EC33 ((3S)-3-amino-4-sulfanyl-butane-1-sulfonic acid), by blocking the formation of brain angiotensin III, normalizes blood pressure in experimental models of hypertension. Therefore, brain APA appears as a potential new therapeutic target for the treatment of hypertension. We then developed RB150/firibastat, a prodrug of EC33, able of inhibiting brain APA activity and decreasing blood pressure in hypertensive rats after oral administration. Purpose However, considering the high dose of orally active RB150/firibastat required to decrease BP in spontaneously hypertensive rats (SHR) (150 mg/kg) and deoxycorticosterone acetate-salt (DOCA-salt) (50 mg/kg) rats, the aim of our work was to develop new more potent APA inhibitor prodrugs with greater bioavailability for inhibiting brain APA activity. Methods We used a salt- and volume-dependent model of hypertension, the DOCA-salt rat. For in vivo assessments of brain APA activity, brains were collected 4 hours after the oral administration. A catheter was inserted into the right femoral artery to monitor mean arterial blood pressure in alert rats. We evaluated plasma arginine-vasopressin (AVP) levels by radioimmunoassay. Rats were individually housed in metabolic cages for urine and electrolyte output measurements. Results We report here the development of a new APA inhibitor prodrug, NI956/QGC006, obtained by the disulfide bridge-mediated dimerization of NI929 ((3S,4S)-3-amino-4-mercapto-6-phenyl-hexane-1-sulfonic acid). NI929 is 10 more efficient than EC33 at inhibiting recombinant mouse APA activity in vitro. Following oral administration at a dose of 4 mg/kg in conscious DOCA-salt rats, NI956/QGC006 normalized brain APA activity and induced a marked decrease in blood pressure of −44±13 mmHg four hours after treatment (p<0.001), sustained over ten hours (−21±12 mmHg, p<0.05). Moreover, NI956/QGC006 decreased plasma AVP levels, and increased diuresis and natriuresis, that may decrease blood pressure by reducing the size of the fluid compartment. Finally, NI956/QGC006 did not affect plasma sodium and potassium concentrations. Conclusions This study shows that NI956/QGC006 is a “best-in-class” central-acting APA inhibitor prodrug, belonging to the same drug class as RB150/firibastat, supporting the strategy of brain APA inhibition for hypertension treatment. Acknowledgement/Funding ANR (Agence Nationale de la Recherche) grant to Catherine Llorens-Cortes (LabCom CARDIOBAPAI) and Quantum Genomics financial support.

Abstract The hyperactivity of the brain renin–angiotensin system (RAS) has been implicated in the development and maintenance of arterial hypertension (HTA). Our aim was to demonstrate that normalizing brain RAS hyperactivity could constitute a new therapeutic approach for HTA treatment We first demonstrated in the brain that aminopeptidase A (APA) is the enzyme generating angiotensin III (AngIII) from AngII. Then, using the specific and selective APA inhibitor, EC33 ((3S)-3-amino-4-sulfanyl-butane-1-sulfonic acid), we showed that AngIII is one of the main effector peptides of the brain RAS, exerting a tonic stimulatory control over blood pressure (BP) in hypertensive rats. This suggests that brain APA may be a potential therapeutic target for HTA treatment. We then designed RB150 {4,4-dithio[bis(3-aminobutyl sulfonic acid)]}, an orally active prodrug of EC33. RB150, given orally in conscious deoxycorticosterone acetate-salt (DOCA-salt) rats or spontaneously hypertensive rats, crosses the intestinal, hepatic and blood-brain barriers, enters the brain, where it is cleaved by brain reductases, generating two active molecules of EC33 which inhibit brain APA activity, block the formation of brain AngIII and induce a marked and sustained decrease in BP. The RB150-induced BP decrease is due to a reduced vasopressin release, which increases diuresis, reducing extracellular volume, a decrease in sympathetic tone, leading to a reduction of vascular resistances and the improvement of the baroreflex function (Figure below). RB150 was renamed firibastat by OMS. Phase Ia/Ib clinical trials showed that firibastat is clinically and biologically well-tolerated in healthy volunteers. Firibastat could constitute the first drug candidate of a new class of antihypertensive agents targeting the brain RAS, the clinical efficacy of which (Phase IIa and Phase IIb) in hypertensive patients was achieved. Acknowledgement/Funding INSERM, College de France, ANR LabCom, Quantum Genomics

Brain renin-angiotensin system hyperactivity has been implicated in the development and maintenance of hypertension. We have shown that aminopeptidase A is involved in the formation of brain angiotensin III, which exerts tonic stimulatory control over blood pressure in hypertensive deoxycorticosterone acetate-salt rats and spontaneously hypertensive rats. We have also shown that injection of the specific and selective aminopeptidase A inhibitor, (3S)-3-amino-4-sulfanyl-butane-1-sulfonic acid (EC33), by central route or its prodrug, RB150/firibastat, by oral route inhibited brain aminopeptidase A activity and blocked the formation of brain angiotensin III, normalizing blood pressure in hypertensive rats. These findings identified brain aminopeptidase A as a potential new therapeutic target for hypertension. We report here the development of a new aminopeptidase A inhibitor prodrug, NI956/QGC006, obtained by the disulfide bridge-mediated dimerization of NI929. NI929 is 10× more efficient than EC33 at inhibiting recombinant mouse aminopeptidase A activity in vitro. After oral administration at a dose of 4 mg/kg in conscious deoxycorticosterone acetate-salt rats, NI956/QGC006 normalized brain aminopeptidase A activity and induced a marked decrease in blood pressure of −44±13 mm Hg 4 hours after treatment ( P< 0.001), sustained over 10 hours (−21±12 mm Hg; P< 0.05). Moreover, NI956/QGC006 decreased plasma arginine-vasopressin levels, and increased diuresis and natriuresis, that may participate to the blood pressure decrease. Finally, NI956/QGC006 did not affect plasma sodium and potassium concentrations. This study shows that NI956/QGC006 is a best-in-class central-acting aminopeptidase A inhibitor prodrug. Our results support the development of hypertension treatments targeting brain aminopeptidase A.

Very few discoveries in the neurosciences have triggered clinical speculation and experimentation regarding the etiology of psychiatric illness to the same extent as that following identification of the opiate receptor(s) and subsequent isolation of endogenous morphine-like peptides. There is overwhelming evidence in animals and in human that opioids are involved in behaviorally relevant issues such as the modulation of pain, the response to stress, motivation, addiction, sexuality, food intake, etc., but our knowledge on the possible relation between opioids and mental illness is still very limited.These responses could be explored eitheir by using higlhy selective delta agonist or by emphasizing the effects of phasically secreted endogenous opioid peptides, enkephalin. Both approaches were investigated in particular through protection of enkephalin degradation by dual enkephalinase ihibitors DENKIs such as RB101, PL37 or PL265.

New neprilysin inhibitors containing an alpha-mercaptoketone HSC((RR2)-R-1)CO group, as zinc ligand were designed. Two parameters were explored for potency optimization: the size of the inhibitor which could interact with the S-1, S-1' or S-2' domain of the enzyme and the nature of the substituents R-1, R-2 of the mercaptoketone group. Introduction of a cyclohexyl chain in R-1, R-2 position and a (3-thiophen)benzyl group in position R-3 (compound 12n) yielded to the most potent inhibitor of this series with a Ki value of 2 +/- 0.3 nM. This result suggests that this new inhibitor interacts within the S-1, S-1' domain of NEP allowing a pentacoordination of the catalytic Zn2+ ion by the mercaptoketone moiety. (C) 2017 Elsevier Ltd. All rights reserved.

Background: The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 (P2X3 receptor antagonist), ACEA (CB1 receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). Methods: Experiments. Experiments were performed in 5–6 weeks old (26–33 g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2 × 106 cells/100 L. Then 105 cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49 ± 0.2 ◦C. Mechanical threshold values were obtained by performing the von Frey test using the “up and down” method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. Results: The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENKs, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good DOI of refers to article: http://dx.doi.org/10.1016/j.sjpain.2016.11.001. Abbreviations: ACEA, arachidonyl-2′-chloroethylamide; AEA, N-arachidonoylethanolamide, anandamide; APN, aminopeptidase N; BNI, nor-binaltorphimine; CCI, chronic onstrictive injury; CYP, cyprodime; DENKI, dual enkephalinase inhibitor; DOR, delta opioid receptor; ENK, enkephalin; FAAH, fatty acid amide hydrolase; i.p., intraperitoneal; OR, kappa opioid receptor; MOR, mu opioid receptor; NEP, neprilysin; NTI, naltrindole; Nlx-Met, naloxone methiodide; UHP, unilateral hot plate test; p.o., per os; s.c., ubcutaneous; S.E.M., standard error of mean. ∗ Corresponding author at: Pharmaleads, 11 rue Watt, 75013 Paris, France. E-mail address: bernard.roques@pharmaleads.com (B.P. Roques). 1 Joint first authors. ttp://dx.doi.org/10.1016/j.sjpain.2016.09.011 877-8860/© 2016 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Unauthenticated Download Date | 5/19/19 12:57 PM 26 S. González-Rodríguez et al. / Scandinavian Journal of Pain 14 (2017) 25–38 absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X3 receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Nav1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. Conclusions: These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. Implications: This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain. Assoc

BACKGROUND:The first line pharmacological treatment of cancer pain is morphine and surrogates but a significant pain relief and a reduction of the side-effects of these compounds makes it necessary to combine them with other drugs acting on different targets. The aim of this study was to measure the antinociceptive effect on cancer-induced bone pain resulting from the association of the endogenous opioids enkephalin and non-opioid analgesic drugs. For this purpose, PL265 a new orally active single dual inhibitor of the two degrading enkephalins enzymes, neprilysin (NEP) and aminopeptidase N (APN) was used. It strictly increased the levels of enkephalin at their sites of releases. The selected non-opioid compounds are: gabapentin, A-317491 (P2X3 receptor antagonist), ACEA (CB1 receptor antagonist), AM1241 (CB2 receptor antagonist), JWH-133 (CB2 receptor antagonist), URB937 (FAAH inhibitor), and NAV26 (Nav1.7 channel blocker). METHODS:Experiments. Experiments were performed in 5-6 weeks old (26-33g weight) C57BL/6 mice. Cell culture and cell inoculation. B16-F10 melanoma cells were cultured and when preconfluent, treated and detached. Finally related cells were resuspended to obtain a concentration of 2×106 cells/100μL. Then 105 cells were injected into the right tibial medullar cavity. Control mice were treated by killed cells by freezing. Behavioural studies. Thermal withdrawal latencies were measured on a unilatered hot plate (UHP) maintained at 49±0.2°C. Mechanical threshold values were obtained by performing the von Frey test using the "up and down" method. To evaluate the nature (additive or synergistic) of the interactions between PL265 and different drugs, an isobolographic analysis following the method described by Tallarida was performed. RESULTS:The results demonstrate the ability of PL265, a DENKI that prevents the degradation of endogenous ENKs, to counteract cancer-induced bone thermal hyperalgesia in mice, by exclusively stimulating peripheral opioid receptors as demonstrated by used of an opioid antagonist unable to enter the brain. The development of such DENKIs, endowed with druggable pharmacokinetic characteristics, such as good absorption by oral route, can be considered as an important step in the development of much needed novel antihyperalgesic drugs. Furthermore, all the tested combinations resulted in synergistic antihyperalgesic effects. As shown here, the greatest synergistic antinociceptive effect (doses could be lowered by 70%) was produced by the combination of PL265 with the P2X3 receptor antagonist (A-317491), cannabinoid CB1 receptor agonist (exogenous, ACEA and endogenous URB937-protected-AEA) and Nav1.7 blocker (NAV26) whose mechanism of action involves the direct activation of the enkephalinergic system. CONCLUSIONS:These multi-target-based antinociceptive strategies using combinations of non-opioid drugs with dual inhibitors of enkephalin degrading enzymes may bring therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain. IMPLICATIONS:This article presents synergistic antinociceptive effect produced by the combination of PL265 with non-opioid analgesic drugs acting via unrelated mechanisms. These multi-target-based antinociceptive strategies may bring therapeutic advantages by allowing the reduction of doses, which is of great interest in the chronic treatment of cancer pain.

Neuropathic pain remains difficult to treat due to the involvement of various pathophysiological mechanisms in its pathogeny. Among the different opioidergic systems the enkephalinergic one is primarily recruited via activation of delta opioid receptor (DOP) in chronic pain and of mu opioid receptor (MOP) in acute pain. To investigate the role of their endogenous ligands Met and Leu-enkephalin in neuropathic pain control, a dual inhibitor of their degrading enzymes, PL265, which acts restrictively at the level of peripheral nociceptors, was administered per os to assess its efficacy in pain prevention and alleviation using a partial sciatic nerve ligation model (PSNL) in mice. We demonstrated here that the pre-injury oral administration of PL265 (50mg/kg) during the 9 days of neuropathy development reduces thermal hyperalgesia and mechanical allodynia for two weeks after the end of treatment. The repeated administration (50mg/kg daily, during 10 days) does not induce tolerance. Therefore, protecting the enkephalins released at the peripheral level during neuropathic pain with oral PL265 seems to be a promising approach to prevent and alleviate the painful symptoms of neuropathic pain in humans without the unwanted effects of exogenous opiates such as morphine.

The peripheral endogenous opioid system is critically involved in neuropathic and inflammatory pain generation as suggested by the modulation of opioid receptors expression and enkephalins (ENKs) release observed in these painful conditions. Accordingly, an innovative approach in the treatment of these nocifensive events is to increase and maintain high local concentrations of extracellular pain‐evoked ENKs, by preventing their physiological enzymatic inactivation by two Zn metallopeptidases, the neutral endopeptidase (NEP, neprilysin, EC 3.4.24.11) and the neutral aminopeptidase (APN, EC 3.4.11.2). With this aim, new orally active dual ENKephalinase inhibitors (DENKIs) were designed as soluble prodrugs by introducing a N‐terminal cleavable carbamate in the previously described aminophosphinic inhibitors. This induces long‐lasting antinociceptive responses after oral administration, in various rodent models of inflammatory and neuropathic pain. These responses are mediated through stimulation of peripheral opioid receptors by DENKIs‐protected ENKs as demonstrated by naloxone methiodide reversion. In all tested models, the most efficient prodrug 2a (PL265) was active, at least during 150–180 min, after single oral administration of 25–50 mg/kg in mice and of 100–200 mg/kg in rats. In models of neuropathic pain, both hyperalgesia and allodynia were markedly reduced. Interestingly, combination of inactive doses of 2a (PL265) and of the anti‐epileptic drug gabapentin had synergistic effect on neuropathic pain. Pharmacokinetic studies of 2a (PL265) in rats show that the active drug is the only generated metabolite produced. These encouraging results have made 2a (PL265) a suitable candidate for clinical development.

The endogenous opioid system, essentially constituted by two opioid receptors which are stimulated by the natural internal effectors enkephalins (Met-enkephalin and Leu-enkephalin), is present at the different sites (peripheral, spinal, central) of the control of pain. We have demonstrated that the protection of the enkephalin inactivation by the two metallopeptidases (neprilysin and neutral aminopeptidase) increases their local concentration selectively induced by pain stimuli triggering analgesic responses. With the aim of increasing the orally antinociceptive responses of the previously described disulfide DENKIs ( [Formula: see text] CH(R1)CH2-S-S-CH2-C(R2R3)CONHCH(R4)COOR5), we designed new pro-drugs, in the same chemical series, with a transient protection of the free amino group by an acyloxyalkyl carbamate, giving rise to ((CH3)2CHCO2CH(CH3)OCONHCH(R1)CH2-S-S-CH2-C(R2R3)CONHCH(R4)COOR5) pro-drugs 2a-2g. These compounds were easily prepared from their parent analogs, with a good yield. They were tested per os and shown to be highly efficient in peripherally-controlled inflammatory and neuropathic pain with long lasting effects but completely inactive in the acute centrally-controlled hot plate test, a model of pain by excess of nociception. This demonstrates that DENKIs are able to relieve pain at its source thanks to the increase of enkephalin levels.

The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a δ and μ opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse.

Protecting enkephalins, endogenous opioid peptides released in response to nociceptive stimuli, is an innovative approach for acute and neuropathic pain alleviation. This is achieved by inhibition of their enzymatic degradation by two membrane-bound Zn-metallopeptidases, neprilysin (NEP, EC 3.4.24.11) and aminopeptidase N (APN, EC 3.4.11.2). Selective and efficient inhibitors of both enzymes, designated enkephalinases, have been designed that markedly increase extracellular concentrations and half-lives of enkephalins, inducing potent antinociceptive effects. Several chemical families of Dual ENKephalinase Inhibitors (DENKIs) have previously been developed but devoid of oral activity. We report here the design and synthesis of new pro-drugs, derived from co-drugs combining a NEP and an APN inhibitor through a disulfide bond with side chains improving oral bioavailability. Their pharmacological properties were assessed in various animal models of pain targeting central and/or peripheral opioid systems. Considering its efficacy in acute and neuropathic pain, one of these new DENKIs, 19-IIIa, was selected for clinical development.

Transforming growth factor-β1 (TGF-β1) protects against neuroinflammatory events underlying neuropathic pain. TGF-β signaling enhancement is a phenotypic characteristic of mice lacking the TGF-β pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-β1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of μ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of μ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-β1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-β1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-β signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.