Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

MRAP2 Inhibits ß-Arrestin-2 Recruitment to the Prokineticin Receptor 2.

Melanocortin receptor accessory protein 2 (MRAP2) is a membrane protein that binds multiple G protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, including prokineticin receptors. These GPCRs are expressed both centrally and peripherally, and their endogenous ligands are prokineticin 1 (PK1) and prokineticin 2 (PK2). PKRs couple all G-protein subtypes, such as Gαq/11, Gαs, and Gαi, and recruit ß-arrestins upon PK2 stimulation, although the interaction between PKR2 and ß-arrestins does not trigger receptor internalisation. MRAP2 inhibits the anorexigenic effect of PK2 by binding PKR1 and PKR2. The aim of this work was to elucidate the role of MRAP2 in modulating PKR2-induced ß-arrestin-2 recruitment and ß-arrestin-mediated signalling. This study could allow the identification of new specific targets for potential new drugs useful for the treatment of the various pathologies correlated with prokineticin, in particular, obesity.

Olfactory Neuron Prokineticin-2 as a Potential Target in Parkinson's Disease.

OBJECTIVE: The objective of this study was to outline the dynamics of prokineticin-2 pathway in relation to clinical-pathological features of Parkinson's disease by examining olfactory neurons of patients. METHODS: Thirty-eight patients (26 de novo, newly diagnosed) and 31 sex/age-matched healthy controls underwent noninvasive mucosa brushing for olfactory neurons collection, and standard clinical assessment. Gene expression levels of prokineticin-2, prokineticin-2 receptors type 1 and 2, and prokineticin-2-long peptide were measured in olfactory neurons by real-time polymerase chain reaction (PCR); moreover, the prokineticin-2 protein and α-synuclein species (total and oligomeric) were quantified by immunofluorescence staining. RESULTS: Prokineticin-2 expression was significantly increased in Parkinson's disease. De novo patients had higher prokineticin-2 levels, directly correlated with Movement Disorder Society-Sponsored Revision of the Unified Parkinson Disease Rating Scale (MDS-UPDRS) part III motor score. In addition, oligomeric α-synuclein was higher in Parkinson's disease and directly correlated with prokineticin-2 protein levels. Total α-synuclein did not differ between patients and controls. INTERPRETATION: Prokineticin-2 is a chemokine showing neuroprotective effects in experimental models of Parkinson's disease, but translational proof of its role in patients is still lacking. Here, we used olfactory neurons as the ideal tissue to analyze molecular stages of neurodegeneration in vivo, providing unprecedented evidence that the prokineticin-2 pathway is activated in patients with Parkinson's disease. Specifically, prokineticin-2 expression in olfactory neurons was higher at early disease stages, proportional to motor severity, and associated with oligomeric α-synuclein accumulation. These data, consistently with preclinical findings, support prokineticin-2 as a candidate target in Parkinson's disease, and validate reliability of olfactory neurons to reflect pathological changes of the disease. ANN NEUROL 2023;93:196-204.

MRAP2a Binds and Modulates Activity and Localisation of Prokineticin Receptor 1 in Zebrafish.

The prokineticin system plays a role in hypothalamic neurons in the control of energy homeostasis. Prokineticin receptors (PKR1 and PKR2), like other G-protein-coupled receptors (GPCRs) are involved in the regulation of energy intake and expenditure and are modulated by the accessory membrane protein 2 of the melanocortin receptor (MRAP2). The aim of this work is to characterise the interaction and regulation of the non-melanocortin receptor PKR1 by MRAP2a in zebrafish (zMRAP2a) in order to use zebrafish as a model for the development of drugs targeting accessory proteins that can alter the localisation and activity of GPCRs. To this end, we first showed that zebrafish PKR1 (zPKR1) is able to interact with both zMRAP2a and human MRAP2 (hMRAP2). This interaction occurs between the N-terminal region of zPKR1 and the C-terminal domain of zMRAP2a, which shows high sequence identity with hMRAP2 and a similar propensity for dimer formation. Moreover, we demonstrated that in Chinese hamster ovary (CHO) cells, zMRAP2a or hMRAP2 are able to modulate zPKR1 activation induced by zebrafish PK2 (zPK2) resulting in an impaired ERK and STAT3 activation.

A Progressive Build-up of Perineuronal Nets in the Somatosensory Cortex Is Associated with the Development of Chronic Pain in Mice.

Chronic pain is sustained by a maladaptive form of neuronal plasticity occurring in all stations of the pain neuraxis, including cortical regions of the pain matrix. We report that chronic inflammatory pain induced by unilateral injection of complete Freund's adjuvant (CFA) in the hindpaw of male mice was associated with a progressive build-up of perineuronal nets (PNNs) in the contralateral somatosensory cortex (SSC), medial prefrontal cortex (mPFC), and reticular thalamic nucleus. In the SSC, the density of PNNs labeled by Wisteria floribunda agglutinin (WFA) was increased at both 3 and 7 d following CFA injection, but only after 7 d in the mPFC. The number of parvalbumin (PV)-positive interneurons enwrapped by WFA+/PNNs was also increased in all three brain regions of mice injected with CFA. Remarkably, PNN degradation induced by intracortical infusion of chondroitinase-ABC significantly reduced mechanical and thermal pain, and also reversed the increased frequency of IPSCs recorded in layer 5 pyramidal neurons of the contralateral SSC in CFA-injected mice. These findings suggest a possible relationship between cortical PNNs and nociceptive sensitization, and support the hypothesis that PNNs maintain their plasticity in the adult life and regulate cortical responses to sensory inputs.SIGNIFICANCE STATEMENT The brain extracellular matrix not only provides structural support, but also regulates synapse formation and function, and modulates neuronal excitability. We found that chronic inflammatory pain in mice enhances the density of perineuronal nets (PNNs) in the somatosensory cortex and medial prefrontal cortex. Remarkably, enzymatic degradation of PNNs in the somatosensory cortex caused analgesia and reversed alterations of inhibitory synaptic transmission associated with chronic pain. These findings disclose a novel mechanism of nociceptive sensitization and support a role for PNNs in mechanisms of neuronal plasticity in the adult brain.

Substance P and Prokineticin-2 are overexpressed in olfactory neurons and play differential roles in persons with persistent post-COVID-19 olfactory dysfunction.

Persistent olfactory dysfunction (OD) is one of the most complaining and worrying complications of long COVID-19 because of the potential long-term neurological consequences. While causes of OD in the acute phases of the SARS-CoV-2 infection have been figured out, reasons for persistent OD are still unclear. Here we investigated the activity of two inflammatory pathways tightly linked with olfaction pathophysiology, namely Substance P (SP) and Prokineticin-2 (PK2), directly within the olfactory neurons (ONs) of patients to understand mechanisms of persistent post-COVID-19 OD. ONs were collected by non-invasive brushing from ten patients with persistent post-COVID-19 OD and ten healthy controls. Gene expression levels of SP, Neurokinin receptor 1, Interleukin-1ß (IL-1ß), PK2, PK2 receptors type 1 and 2, and Prokineticin-2-long peptide were measured in ONs by Real Time-PCR in both the groups, and correlated with residual olfaction. Immunofluorescence staining was also performed to quantify SP and PK2 proteins. OD patients, compared to controls, exhibited increased levels of both SP and PK2 in ONs, the latter proportional to residual olfaction. This work provided unprecedented, preliminary evidence that both SP and PK2 pathways may have a role in persistent post-COVID-19 OD. Namely, if the sustained activation of SP, lasting months after infection's resolution, might foster chronic inflammation and contribute to hyposmia, the PK2 expression could instead support the smell recovery.

Special Issue "GPCR: Roles in Cell Development and Disease".

We are pleased to present the following Special Issue of the International Journal of Molecular Sciences (IJMS), entitled "GPCR: Roles in Cell Development and Disease" [...].

Non-Peptide Agonists and Antagonists of the Prokineticin Receptors.

The prokineticin family comprises a group of secreted peptides that can be classified as chemokines based on their structural features and chemotactic and immunomodulatory functions. Prokineticins (PKs) bind with high affinity to two G protein-coupled receptors (GPCRs). Prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) are involved in a variety of physiological functions such as angiogenesis and neurogenesis, hematopoiesis, the control of hypothalamic hormone secretion, the regulation of circadian rhythm and the modulation of complex behaviors such as feeding and drinking. Dysregulation of the system leads to an inflammatory process that is the substrate for many pathological conditions such as cancer, pain, neuroinflammation and neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The use of PKR's antagonists reduces PK2/PKRs upregulation triggered by various inflammatory processes, suggesting that a pharmacological blockade of PKRs may be a successful strategy to treat inflammatory/neuroinflammatory diseases, at least in rodents. Under certain circumstances, the PK system exhibits protective/neuroprotective effects, so PKR agonists have also been developed to modulate the prokineticin system.

Serum prokineticin-2 in prepubertal and adult Klinefelter individuals.

The prokineticin-2 (PROK2) is a small peptide belonging to the prokineticin family. In humans and rodents this chemokine is primarily involved in the control of central and peripheral reproductive processes. Klinefelter's syndrome (KS) is the first cause of male genetic infertility, due to an extra X chromosome, which may occur with a classical karyotype (47, XXY) or mosaic forms (46, XY/47, XXY). In affected subjects, pubertal maturation usually begins at an adequate chronological age, but when development is almost complete, they display a primary gonadal failure, with early spermatogenesis damage, and later onset of testosterone insufficiency. Thus, the main aim of the present study was to investigate the serum levels of PROK2 in prepubertal and adult KS patients, comparing them with healthy subjects. We showed for the first time the presence of PROK2 in the children serum but with significant changes in KS individuals. Indeed, compared with healthy subjects characterized by PROK2 serum elevation during the growth, KS individuals showed constant serum levels during the sexual maturation phase (higher during the prepubertal phase but lower during the adult age). In conclusion, these data indicate that in KS individuals PROK2 may be considered a biomarker for investigating the SK infertility process.

Nrf2 Pathway in Huntington's Disease (HD): What Is Its Role?

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease that occurs worldwide. Despite some progress in understanding the onset of HD, drugs that block or delay symptoms are still not available. In recent years, many treatments have been proposed; among them, nuclear transcriptional factor-2 (Nrf2) enhancer compounds have been proposed as potential therapeutic agents to treat HD. Nrf2 triggers an endogenous antioxidant pathway activated in different neurodegenerative disorders. Probably, the stimulation of Nrf2 during either the early phase or before HD symptoms' onset, could slow or prevent striatum degeneration. In this review, we present the scientific literature supporting the role of Nrf2 in HD and the potential prophylactic and therapeutic role of this compound.

Arginine 125 Is an Essential Residue for the Function of MRAP2.

MRAP2 is a small simple transmembrane protein arranged in a double antiparallel topology on the plasma membrane. It is expressed in the paraventricular nucleus of the hypothalamus, where it interacts with various G protein-coupled receptors, such as the prokineticin receptors, and regulates energy expenditure and appetite. The aim of this work was to analyze the functional role of the specific arginine residue at position 125 of MRAP2, which affects protein conformation, dimer formation, and PKR2 binding. Results obtained with the MRAP2 mutants R125H and R125C, which are found in human patients with extreme obesity, and mouse MRAP2, in which arginine 125 is normally replaced by histidine, were compared with those obtained with human MRAP2. Understanding the mechanism by which MRAP2 regulates G protein-coupled receptors helps in elucidating the metabolic pathways involved in metabolic dysfunction and in developing new drugs as specific targets of the MRAP2-PKR2 complex.

High-fat diet impairs duodenal barrier function and elicits glia-dependent changes along the gut-brain axis that are required for anxiogenic and depressive-like behaviors.

BACKGROUND: Mood and metabolic disorders are interrelated and may share common pathological processes. Autonomic neurons link the brain with the gastrointestinal tract and constitute a likely pathway for peripheral metabolic challenges to affect behaviors controlled by the brain. The activities of neurons along these pathways are regulated by glia, which exhibit phenotypic shifts in response to changes in their microenvironment. How glial changes might contribute to the behavioral effects of consuming a high-fat diet (HFD) is uncertain. Here, we tested the hypothesis that anxiogenic and depressive-like behaviors driven by consuming a HFD involve compromised duodenal barrier integrity and subsequent phenotypic changes to glia and neurons along the gut-brain axis. METHODS: C57Bl/6 male mice were exposed to a standard diet or HFD for 20 weeks. Bodyweight was monitored weekly and correlated with mucosa histological damage and duodenal expression of tight junction proteins ZO-1 and occludin at 0, 6, and 20 weeks. The expression of GFAP, TLR-4, BDNF, and DCX were investigated in duodenal myenteric plexus, nodose ganglia, and dentate gyrus of the hippocampus at the same time points. Dendritic spine number was measured in cultured neurons isolated from duodenal myenteric plexuses and hippocampi at weeks 0, 6, and 20. Depressive and anxiety behaviors were also assessed by tail suspension, forced swimming, and open field tests. RESULTS: HFD mice exhibited duodenal mucosa damage with marked infiltration of immune cells and decreased expression of ZO-1 and occludin that coincided with increasing body weight. Glial expression of GFAP and TLR4 increased in parallel in the duodenal myenteric plexuses, nodose ganglia, and hippocampus in a time-dependent manner. Glial changes were associated with a progressive decrease in BDNF, and DCX expression, fewer neuronal dendritic spines, and anxiogenic/depressive symptoms in HFD-treated mice. Fluorocitrate (FC), a glial metabolic poison, abolished these effects both in the enteric and central nervous systems and prevented behavioral alterations at week 20. CONCLUSIONS: HFD impairs duodenal barrier integrity and produces behavioral changes consistent with depressive and anxiety phenotypes. HFD-driven changes in both peripheral and central nervous systems are glial-dependent, suggesting a potential glial role in the alteration of the gut-brain signaling that occurs during metabolic disorders and psychiatric co-morbidity.

Trypanosoma cruzi trans-sialidase induces STAT3 and ERK activation by prokineticin receptor 2 binding.

Tc85, as other members of trans-sialidase family, is involved in Trypanosoma cruzi parasite adhesion to mammalian cells. Particularly, Tc85 acts through specific interactions with prokineticin receptor 2, a G-protein coupled receptor involved in diverse physiological and pathological processes. In this manuscript, through biochemical analyses, we demonstrated that LamG, a Tc85 domain, physically interacts with the prokineticin receptor 2. Moreover, expressing prokineticin receptor 1 and 2 we demonstrated that LamG specifically activates prokineticin receptor 2 through a strong coupling with Gαi or Gαq proteins in yeast strains and inducing ERK and NFAT phosphorylation in CHO mammalian cells. To demonstrate a Tc85 physiological role in T. cruzi infection of the nervous system, we evidenced a strong STAT3 and ERK activation by LamG in mice Dorsal Root Ganglia. L173R is the most common prokineticin receptor 2 mutation reported in Kallmann syndrome and it is a founder mutation. Our results demonstrated that in cells co-expressing prokineticin receptor 2 mutant (L173R) and wild-type, LamG is unable to induce signal transduction. The L173R mutation in heterozygosity may allow for a selective advantage due to increased protection from T. cruzi infection. SIGNIFICANCE OF THE STUDY: The Chagas' disease affecting millions of people worldwide is caused by an eukaryotic microorganism called T. cruzi. Pharmacological treatment for patients with Chagas' disease is still limited. Indeed, the small number of drugs available shows important side effects that can be debilitating for patient health. In order to replicate and produce new parasites T. cruzi uses a complex of different proteins produced by both the parasite and the human host cells. So, understanding the molecular details used by T. cruzi to be internalised by different types of human cells is an important step towards the development of new drugs for this disease. Prokineticin receptors are relevant for host-parasite interaction. To characterise the signal transduction cascade induced by their activation may help to understand the molecular details of cell infection, leading to novel therapeutic alternative for this debilitating disease.

Targeting prokineticin system counteracts hypersensitivity, neuroinflammation, and tissue damage in a mouse model of bortezomib-induced peripheral neuropathy.

BACKGROUND: Neuropathy is a dose-limiting side effect of many chemotherapeutics, including bortezomib. The mechanisms underlying this condition are not fully elucidated even if a contribution of neuroinflammation was suggested. Here, we investigated the role of a chemokine family, the prokineticins (PKs), in the development of bortezomib-induced peripheral neuropathy (BIPN), and we used a PK receptor antagonist to counteract the development and progression of the pathology. METHODS: Neuropathy was induced in male C57BL/6J mice by using a protocol capable to induce a detectable neuropathic phenotype limiting systemic side effects. The presence of allodynia (both mechanical and thermal) and thermal hyperalgesia was monitored over time. Mice were sacrificed at two different time points: 14 and 28 days after the first bortezomib (BTZ) injection. At these times, PK system activation (PK2 and PK-Rs), macrophage and glial activation markers, and cytokine production were evaluated in the main station involved in pain transmission (sciatic nerve, DRG, and spinal cord), and the effect of a PK receptors antagonist (PC1) on the same behavioral and biochemical parameters was assessed. Structural damage of DRG during BTZ treatment and an eventual protective effect of PC1 were also evaluated. RESULTS: BTZ induces in mice a dose-related allodynia and hyperalgesia and a progressive structural damage to the DRG. We observed a precocious increase of macrophage activation markers and unbalance of pro- and anti-inflammatory cytokines in sciatic nerve and DRG together with an upregulation of GFAP in the spinal cord. At higher BTZ cumulative dose PK2 and PK receptors are upregulated in the PNS and in the spinal cord. The therapeutic treatment with the PK-R antagonist PC1 counteracts the development of allodynia and hyperalgesia, ameliorates the structural damage in the PNS, decreases the levels of activated macrophage markers, and prevents full neuroimmune activation in the spinal cord. CONCLUSIONS: PK system may be a strategical pharmacological target to counteract BTZ-induced peripheral neuropathy. Blocking PK2 activity reduces progressive BTZ toxicity in the DRG, reducing neuroinflammation and structural damage to DRG, and it may prevent spinal cord sensitization.

Multimodal antidepressant vortioxetine causes analgesia in a mouse model of chronic neuropathic pain.

Vortioxetine is a multimodal antidepressant that potently antagonizes 5-HT3 serotonin receptors, inhibits the high-affinity serotonin transporter, activates 5-HT1A and 5-HT1B receptors, and antagonizes 5-HT1D and 5-HT7 receptors. 5-HT3 receptors largely mediate the hyperalgesic activity of serotonin that occurs in response to nerve injury. Activation of 5-HT3 receptors contributes to explain why selective serotonin reuptake inhibitors, such as fluoxetine, are not indicated in the treatment of neuropathic pain. Here, we studied the analgesic action of vortioxetine in the chronic constriction injury model of neuropathic pain in mice. Vortioxetine was injected once a day for 27 days at doses (10 mg/kg, intraperitoneally) that determine >90% 5-HT3 receptor occupancy in the central nervous system. The action of vortioxetine was compared to the action of equal doses of the serotonin-noradrenaline reuptake inhibitor, venlafaxine (one of the gold standard drugs in the treatment of neuropathic pain), and fluoxetine. Vortioxetine caused a robust analgesia in chronic constriction injury mice, and its effect was identical to that produced by venlafaxine. In contrast, fluoxetine was inactive in chronic constriction injury mice. Vortioxetine enhanced mechanical pain thresholds in chronic constriction injury mice without changing motor activity, as assessed by the open-field and horizontal bar tests. None of the three antidepressants caused analgesia in the complete Freund's adjuvant model of chronic inflammatory pain. These findings raise the attractive possibility that vortioxetine can be effective in the treatment of neuropathic pain, particularly in patients with comorbid depression and cognitive dysfunction.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia.

BACKGROUND: Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity. METHODS: Diarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα-/- mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα-/- mice. RESULTS: HIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα-/- mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation. CONCLUSIONS: EGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

N-acetyl-cysteine, a drug that enhances the endogenous activation of group-II metabotropic glutamate receptors, inhibits nociceptive transmission in humans.

BACKGROUND: Emerging research seeking novel analgesic drugs focuses on agents targeting group-II metabotropic glutamate receptors (mGlu2 and mGlu3 receptors). N-Acetylcysteine (NAC) enhances the endogenous activation of mGlu2/3 receptors by activating the glial glutamate:cystine membrane exchanger. Here, we examined whether NAC inhibits nociceptive responses in humans and animals. We tested the effect of oral NAC (1.2 g) on thermal-pain thresholds and laser-evoked potentials in 10 healthy volunteers, according to a crossover, double-blind, placebo-controlled design, and the effect of NAC (100 mg/kg, i.p.) on the tail-flick response evoked by radiant heat stimulation in mice. RESULTS: In healthy subjects, NAC treatment left thermal-pain thresholds unchanged, but significantly reduced pain ratings to laser stimuli and amplitudes of laser-evoked potentials. NAC induced significantly greater changes in these measures than placebo. In the tail-flick test, NAC strongly reduced the nocifensive reflex response to radiant heat. The action of NAC was abolished by the preferential mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.). CONCLUSIONS: Our findings show for the first time that NAC inhibits nociceptive transmission in humans, and does the same in mice by activating mGlu2/3 receptors. These data lay the groundwork for investigating the therapeutic potential of NAC in patients with chronic pain.