Palmitoylethanolamide, a naturally occurring disease-modifying agent ...

Inflammopharmacol (2014) 22:79–94 DOI 10.1007/s10787-013-0191-7

Inflammopharmacology

REVIEW

Palmitoylethanolamide, a naturally occurring disease-modifying agent in neuropathic pain Stephen D. Skaper • Laura Facci • Mariella Fusco Maria Federica della Valle • Morena Zusso • Barbara Costa • Pietro Giusti

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Received: 2 August 2013 / Accepted: 10 September 2013 / Published online: 1 November 2013 Ó Springer Basel 2013

Abstract Persistent pain affects nearly half of all people seeking medical care in the US alone, and accounts for at least $80 billion worth of lost productivity each year. Among all types of chronic pain, neuropathic pain stands out: this is pain resulting from damage or disease of the somatosensory nervous system, and remains largely untreatable. With few available treatment options, neuropathic pain represents an area of significant and growing unmet medical need. Current treatment of peripheral neuropathic pain involves several drug classes, including opioids, gabapentinoids, antidepressants, antiepileptic drugs, local anesthetics and capsaicin. Even so, less than half of patients achieve partial relief. This review discusses a novel approach to neuropathic pain management, based on knowledge of: the role of glia and mast cells in pain and neuroinflammation; the body’s innate mechanisms to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation. The discovery that palmitoylethanolamide, a member

S. D. Skaper (&) L. Facci M. Zusso P. Giusti Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Largo ‘‘E. Meneghetti’’ 2, 35131 Padua, Italy e-mail: [email protected] M. Fusco Epitech Group Srl, Scientific Information and Documentation Center, Saccolongo, PD, Italy M. F. della Valle CeDIS (Scientific Information and Documentation Center), Innovet Italia Srl, Saccolongo, PD, Italy B. Costa Department of Biotechnology and Bioscience, University of Milan-Bicocca, Milan, Italy

of the N-acylethanolamine family which is produced from the lipid bilayer on-demand, is capable of exerting antiallodynic and anti-hyperalgesic effects by down-modulating both microglial and mast cell activity has led to the application of this fatty acid amide in several clinical studies of neuropathic pain, with beneficial outcome and no indication of adverse effects at pharmacological doses. Collectively, the findings presented here propose that palmitoylethanolamide merits further consideration as a disease-modifying agent for controlling inflammatory responses and related chronic and neuropathic pain. Keywords Neuropathic pain Mast cells Microglia Neuro-immune interaction Natural products Palmitoylethanolamide

Neuropathic pain: background and currently available therapeutics (and why we need something better) Neuropathic pain (NP), as defined by the International Association for the Study of Pain, results from damage or disease affecting the somatosensory system (Jensen et al. 2011). Depending on localization of the lesion or disease, peripheral or central NP may take place. The former can be caused by various diseases (e.g., diabetes mellitus, herpes zoster, human immunodeficiency virus infection), medical interventions (e.g., chemotherapy, surgery), and injuries (e.g., brachial plexus avulsion). In addition, pain associated with particular disorders, i.e. osteoarthritis and pelvic diseases correlated to chronic pain, exhibits both neuropathic and inflammatory components (McDougall and Linton 2012; George et al. 2012). Central NP states are most often caused by stroke, spinal cord injury or multiple sclerosis (Kerstman et al. 2013). Individuals suffering from NP

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experience compromised health and quality of life worse compared to patients with severe chronic diseases. In peripheral NP, or painful neuropathy, the chronic pain is not a symptom of injury, but rather the pain is itself the disease process. Data emerging from recent epidemiological studies show that NP is more common than previously thought, with a significant health and economic impact (Smith and Torrance 2012). Approximately, 3–4.5 % of the global population is affected by NP, with the rate of incidence increasing as the global population ages (Toth et al. 2009; Smith and Torrance 2012). Indeed, between 2000 and 2050, the proportion of the world’s population over 60 years will double from about 11–22 %. A wide range of analgesic medications are currently marketed. Tricyclic antidepressants, dual serotonin/norepinephrine reuptake inhibitors, calcium channel a2-c ligands (i.e., gabapentin and pregabalin), and topical lidocaine are recommended as first-line treatment options on the basis of the results of randomized clinical trials, with opioid analgesics and tramadol generally as second-line treatments (Dworkin et al. 2010). Despite the numerous treatment options for relieving NP, less than half of all patients experience clinically meaningful pain relief, which is more often than not only partial (Mao et al. 2011). Patients frequently experience major adverse effects which lead to discontinuation of treatment. Clearly, there is a strong need for novel analgesics that are more effective and safer than existing drugs when used over a long period (Nightingale 2012; O’Connor 2009; Langley et al. 2013). Many investigators now believe that new strategies for improving pain management should focus on developing drugs targeting the underlying mechanism(s) of pain, rather than pain intensity only (Varrassi et al. 2011; Dworkin et al. 2010; Dworkin 2012). An important development in this direction has been the discovery that initiation and maintenance of NP involve communication between neurons and non-neuronal immunocompetent cells, such as mast cells and microglia, together with a cascade of proand anti-inflammatory cytokines (Ren and Dubner 2010; Calvo et al. 2012; Sacerdote et al. 2013). Lesion or disease of the somatosensory nervous system not only profoundly alters the function of primary sensory neurons and their central projection pathways, but is associated also with a robust immune response at virtually every level of the somatosensory system. Mast cells are immune cells particularly located within tissues at the boundary of the external environment, in close proximity to blood vessels and nerve endings (Tsai et al. 2011). A close association between mast cells and neighboring sensory nerves has long been recognized (Levy et al. 2012; Forsythe and Bienenstock 2012); the former are found also within the endoneurial compartment of peripheral nerves (Mizisin and Weerasuriya 2011). Mast cells can modify sensory

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transmission via a wide spectrum of mediators, including (but not limited to) biogenic amines such as histamine and serotonin, cytokines (interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) in particular), enzymes, lipid metabolites, ATP, neuropeptides, nerve growth factor (NGF), and heparin—most of which can interact with sensory nerve terminals. Sensory neurons, in turn, by releasing neuropeptides may provoke mast cell activation/ degranulation (Tore and Tuncel 2009). In the absence of a tight physiological control, mast cell-nerve terminal activity results in nociceptor sensitization, reduced pain threshold at the site of inflammation and, ultimately, dysfunctional pain signaling and hyperalgesia (Demir et al. 2013; Moalem and Tracey 2006; Zuo et al. 2003). Persistent increased responsiveness of nociceptors can also sensitize spinal cord neurons, leading to central sensitization (Dirckx et al. 2013; Skaper and Facci 2012). Glia are important mediators of pain processes at the spinal level (Milligan and Watkins 2009; Wilkerson and Milligan 2011). Sensitization of central somatosensory neurons is thought to be responsible for the symptoms of chronic NP states (Tsuda et al. 2013). Microglia, the brain’s resident macrophages, interact with these neurons at the site of injury or disease as well as remotely. Microglia can be activated through engagement of a number of constitutively expressed cell surface molecules, and respond also to pro-inflammatory signals released from peripheral cells of immune origin—including mast cells (Skaper et al. 2012). A bidirectional cross talk between brain mast cells and microglia has been suggested (Bulanova and Bulfone-Paus 2010; Yuan et al. 2010; Skaper et al. 2012; Zhang et al. 2012). Activated microglia contribute to pain states by releasing pro-inflammatory cytokines, chemokines, and proteases. Further, systemic inflammation can give rise to signals that communicate with the brain and lead to changes in metabolism and behavior, including the expression of a pro-inflammatory phenotype by microglia (Cunningham 2013). Astrocytes, the most abundant glial cell type involved in neuroinflammation (Milligan and Watkins 2009), also play a major role in pain facilitation and are a fundamental contributor to NP (Gosselin et al. 2010). Upon their activation, the extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways become activated, resulting in the release of IL-1b, IL-6, TNF-a and prostaglandin E2 (Milligan and Watkins 2009; Nicotra et al. 2012). Chronic astrocytic activation in nerve injury results in down-regulation of glutamate transporter 1 and the glutamate-aspartate transporter, ultimately resulting in decreased glutamate uptake and increased excitatory transmission (Sung et al. 2003; Tawfik et al. 2006). The above findings thus support the notion that controlling mast cell-glia reactivity can provide an attractive

Palmitoylethanolamide

therapeutic avenue for treating NP (Skaper and Facci 2012). Targeting immune cells might offer as well the possibility to develop a novel disease-modifying approach that promotes nature’s inherent anti-inflammatory mechanisms by dialing into this ‘built-in’ program to resolve inflammation (Tabas and Glass 2013).

81 H

OH N

O

LEA

H

OH N

Palmitoylethanolamide: an N-acylethanolamine with anti-inflammatory and pain-relieving properties Given the risks posed by inflammation to the organism, it would not be unreasonable to expect that nature may have endowed us with the capacity for auto-defense. Indeed, we now know that pathways exist which are capable of generating molecules involved in such protective mechanisms, being activated following different types of tissue damage or stimulation of inflammatory responses and nociceptive fibers. Chronic inflammatory processes such as those sustaining NP are opposed by a program of resolution that includes the production of lipid mediators able to switch off inflammation (Serhan and Savill 2005; Buckley et al. 2013). Given that chronic inflammatory conditions may lower the levels or actions of these molecules (Zhu et al. 2011), it can be argued that administration of such lipid mediators might provide an avenue ‘‘to commandeer Nature’s own anti-inflammatory mechanisms and induce a ‘‘dominant’’ program of resolution’’ (Tabas and Glass 2013). Within this context consider the N-acylethanolamines (NAEs), a class of naturally occurring mediators composed of a fatty acid and ethanolamine—the so-called fatty acid ethanolamines (FAEs). Principal FAE family members include the endocannabinoid N-arachidonoylethanolamine (anandamide), and its congeners Nstearoylethanolamine, N-oleoylethanolamine and N-palmitoylethanolamine (PEA, or palmitoylethanolamide) (Pacher et al. 2006). The NAEs (Fig. 1) are formed from N-acylated phosphatidylethanolamine (NAPE) by several enzymatic pathways (Hansen and Diep 2009; Tsuboi et al. 2013; Ueda et al. 2013), the major one utilizing a membrane-associated NAPE-phospholipase D (NAPE-PLD) to generate the corresponding NAE and phosphatidic acid (Leung et al. 2006; Okamoto et al. 2004). N-palmitoylphosphatidyl-ethanolamine is converted into PEA by this enzyme (Petrosino et al. 2010). Hydrolysis of NAEs in the mammalian brain relies on: (i) fatty acid amide hydrolase (FAAH) localized in the endoplasmic reticulum, which breaks down the NAE into its component fatty acid and ethanolamine (Ahn et al. 2008; Cravatt et al. 1996; Schmid et al. 1983); (ii) NAE-hydrolyzing acid amidase (NAAA) localized in lysosomes with structural homology to ceramidase and belonging to the family of choloylglycine hydrolases (Tsuboi et al. 2007). The predominantly

O

MEA

H

OH N

O

PEA

H

OH N

O

SEA

H

OH N

O

OEA

H

OH N

O

AEA

Fig. 1 Chemical structures of the N-acylethanolamines. LEA lauroylethanolamide (C12:0), MEA myristoylethanolamide (C14:0), PEA palmitoylethanolamide (C16:0), SEA stearoylethanolamide (C18:0), OEA oleoylethanolamide (C18:1); AEA anandamide (C20:4)

macrophagic NAAA hydrolyzes only NAEs having \18 carbon atoms, i.e., PEA, but not N-oleoylethanolamine and N-stearoylethanolamine. In contrast, FAAH can hydrolyze all three NAEs. The potential beneficial actions of FAEs first began to be realized some decades ago when Coburn and Moore (1943) unexpectedly found that dried chicken egg yolk had antipyretic properties. Another decade passed before the lipid fraction from egg yolk was identified as the bioactive fraction (Coburn et al. 1954) and PEA as the active ingredient (Kuehl et al. 1957). The therapeutic applications of PEA remained largely unexplored, however, until its anti-inflammatory (Mazzari et al. 1996), pain-relieving

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(Calignano et al. 1998), and anticonvulsant (Lambert et al. 2001) properties began to emerge. These past 15 years have seen a rather remarkable rise in the number of studies published on PEA anti-inflammatory and pain-relieving actions (Petrosino et al. 2010; Skaper 2013). It has been proposed that a key role of PEA may be to maintain cellular homeostasis when faced with external stressors provoking, for example, inflammation (Solorzano et al. 2009; Skaper and Facci 2012). PEA is produced/ hydrolyzed by microglia and mast cells (Bisogno et al. 1997; Muccioli and Stella 2008); it down-modulates mast cell activation (Facci et al. 1995; Cerrato et al. 2010), and controls microglial cell behavior (Franklin et al. 2003; Lorıá et al. 2008; Luongo et al. 2013). Through controlling these targets, PEA acts as a disease-modifying rather than a symptom-modifying agent, since it exerts its effects on the ‘‘roots of the problem’’, i.e., on cellular targets involved in the generation and maintenance of pain. PEA levels are altered in brain areas involved in nociception as well as in spinal cord following NP induction (Petrosino et al. 2007), in other conditions associated with pain development (Franklin et al. 2003; Jhaveri et al. 2008; Lorıá et al. 2008), and in muscle dialysates from women with chronic widespread pain and chronic neck-/shoulder pain (Ghafouri et al. 2011, 2013). Collectively, the above studies suggest that PEA maintains cellular homeostasis by acting as a mediator of resolution of inflammatory processes, including pain. Yet, it is quite possible that pathological settings may be encountered where PEA production is insufficient to deal with the ensuing inflammatory cascade. In such cases, provision of exogenous PEA might prove to be therapeutically beneficial. This supposition appears to be borne out for NP, as discussed in the following subsections.

S. D. Skaper et al.

bladder and NGF-induced bladder hyper-reflexia (Jaggar et al. 1998; Farquhar-Smith and Rice 2001, 2003; Farquhar-Smith et al. 2002). Moreover, PEA administered 1 h before or even after carrageenan reduced inflammatory parameters in a time-dependent manner (Costa et al. 2002; Conti et al. 2002), and abolished carrageenan-evoked thermal hyperalgesia (Conti et al. 2002); SR144528

PEA: preclinical studies Numerous preclinical studies support the view of PEA as an endogenous anti-inflammatory agent. In one of the first such reports, orally given PEA dose-dependently (0.1–10 mg/kg) reduced carrageenan-induced hyperalgesia, as well as carrageenan-, formalin- and dextran-induced edema (Mazzari et al. 1996). In later studies, intraplantar injection of PEA (50 lg) suppressed both early and late phase responses to formalin. This effect was reversed by the cannabinoid CB2 receptor antagonist/inverse agonist SR144528, suggesting the participation of CB2 and/or CB2-like receptors (Calignano et al. 1998, 2001; Jaggar et al. 1998). The anti-nociceptive effect of subcutaneous PEA and involvement of a CB2-like receptor was recently confirmed using the CB2 receptor antagonist AM630 (Romero et al. 2012, 2013a, b). PEA attenuated the hyperreflexic response following turpentine instillation into the

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Fig. 2 Representative light micrographs of mast cells stained with toluidine blue (940) in the sciatic nerve longitudinal section from mice submitted (CCI) or not (sham) to the chronic constriction injury (CCI) of the sciatic nerve as neuropathic pain model: sham/vehicle (a), CCI/vehicle 8 days after the surgery (b), CCI/PEA 8 days after the surgery (c). Non-active mast cells were intact and stained intensely with toluidine blue. Degranulated mast cells were recognized by the extensive loss of dye and for the presence of residual cytoplasmatic granules (black arrow), while degranulating mast cells had an oblong shape and the intense blue (black arrowhead). The images reveal that PEA (10 mg/kg p.o. for 1 week) treatment delayed mast cell recruitment and protected mast cell against degranulation


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blocked the anti-edema and anti-hyperalgesic effects of PEA (Conti et al. 2002). In a mouse visceral pain model mediated by the prostaglandin system, intraperitoneally administered PEA exerted anti-nociceptive effects in a CB2 receptor-independent fashion (Haller et al. 2006). The case for a CB2 receptor-independent site of PEA action remains equivocal; however, given new studies claiming that the CB2 receptor antagonist AM630 prevents the peripheral anti-nociceptive effect of PEA in the rat hindpaw following intraplantar injection of prostaglandin E2 (Romero et al. 2013a, b). The anti-nociceptive PEA effect observed after intraplantar injection points to a local peripheral site of action in modulating pain responses. However, intracerebroventricular administration of PEA, 30 min before carrageenan injection, markedly reduced mechanical hyperalgesia up to 24 h following the inflammatory insult—suggesting a centrally mediated component for PEA in controlling inflammatory pain (D’Agostino et al. 2009). The attenuation of hyperalgesia by PEA was associated with prevention of IjB-a degradation and p65 nuclear factor-jB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral hyperalgesia (D’Agostino et al. 2009). Microinjection of PEA in the ventrolateral periaqueductal gray of male rats reduced the ongoing activity of ON and OFF cells in the rostral ventromedial medulla and produced an increase in the latency of the nociceptive reaction (the periaqueductal gray—rostral ventromedial medulla pathway is a key circuit in pain processing) (de Novellis et al. 2012). In line with the latter finding, intrathecal administration of PEA significantly decreased both pain-related scores in the

formalin test and IL-1b expression in rat spinal cord (Naderi et al. 2012). The anti-inflammatory and pain-relieving properties of PEA have been observed in a model of granuloma-induced hyperalgesia: locally given, PEA dose-dependently reduced the expression and release of NGF, mast cell degranulation, prevented nerve fiber formation and sprouting, reduced mechanical allodynia and normalized sensory ganglia changes (de Filippis et al. 2011). Importantly, PEA displays pain-relieving properties in models of NP. For example, it inhibited resiniferatoxin-induced calcitonin gene-related peptide and somatostatin release in vivo without affecting basal neuropeptide concentrations, and counteracted neuropathic hyperalgesia following partial sciatic nerve injury (Helyes et al. 2003). These results fit with evidence that calcitonin gene-related peptide and somatostatin derived from the capsaicin-sensitive subpopulation of sensory afferents contribute to the development of neuropathic hyperalgesia (Helyes et al. 2003). In a chronic constrictive sciatic nerve injury mouse model of NP, PEA treatment (10 mg/kg), starting the first postlesion day, relieved both thermal hyperalgesia and mechanical allodynia by acting on nociceptive pathway receptors (Costa et al. 2008). Here, PEA delayed mast cell recruitment and prevented their degranulation, abolished the NGF increase in sciatic nerve, and preserved nerve fiber integrity while reducing microglia activation in the spinal cord (Bettoni et al. 2013; Figs. 2, 3). Similar results were reported by Di Cesare Mannelli et al. (2013). The subcutaneous daily treatment with 30 mg/kg PEA for 14 consecutive days from the day after surgery reduced hypersensitivity to noxious and non-noxious stimuli,

Fig. 3 Representative images of transversal sections of dorsal horn of the lumbar (L4–L5) spinal cord from mice submitted (CCI) or not (sham) to the chronic constriction injury (CCI) of the sciatic nerve as neuropathic pain model. Images show the ipsilateral site of dorsal horn on 8th day after the surgery immunostained with F4/80 antibody and counterstained with hematoxylin. Experimental group: sham/ vehicle (left panel), CCI/vehicle 8 days after the surgery (middle panel), CCI/PEA 8 days after the surgery (right panel). Activated

microglia stained deep pink. Original magnification 940. Insert shows F4/80 positive cells. We found an increased expression of F4/ 80-positive cells, indicative of the activation of microglia, in the spinal cord of CCI mice treated with vehicle 8 days after the sciatic nerve injury as compared to healthy mice. PEA (10 mg/kg p.o. for 1 week) treatment strongly reduced microglia activation as demonstrated by the representative section

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Table 1 Preclinical studies demonstrating the effects of PEA on pain behaviors, inhibition of somatosensory activation and correlated events Model

PEA: administration route and dosage

PEA biological action

References

Decreased mechanical hyperalgesia;

Mazzari et al. (1996)

Pain caused by inflammation i.pl. canageenan in rat paw

10 mg/kg os

Reduced paw edema 10 mg/kg os

Decreased thermal hyperalgesia;

Conti et al. (2002)

Reduced paw edema i.c.v. 0.1–1 lg

Reduced mechanical hyperalgesia Reduced expression of cyclooxygenase-inducible nitric oxide synthase in sciatic nerves

D’Agostino et al. (2007, 2009)

Attenuated the development of paw edema i.pl. formalin in mouse and rat paw

0.001–100 mg/kg, i.pl., i.v., i.p.

Inhibited formalin-evoked nociception in mice;

5–10 kg/kg i.a.

Attenuated the behavioral response during the second phase of the formalin test Jaggar et al. (1998)

20 mg/kg s.c. 1, 10 lg i.t.

Prevented formalin-induced firing of spinal cord nociceptive neurons in rats Reduced early and late phases of formalin-induced nociception

Calignano et al. (1998, 2001)

Suppressed formalin-induced firing in rat spinal cord nociceptive neurons

LoVerme et al. (2006)

Decreased pain score

Naderi et al. (2012)

Reduced the activation of interleukin-1b in the spinal cord Turpentine infusion into the urinary bladder

10–30 mg/kg i.a.

Attenuated the viscero-visceral hyper-reflexia

Jaggar et al. (1998)

10–30 mg/kg i.p.

Reduced thermal hyperalgesia

Farquhar-Smith and Rice (2001)

Attenuated bladder hyper-reflexia

Farquhar-Smith et al. (2002)

NGF infusion into the urinary bladder 1.5–2.5 mg/kg i.a.

Reduced Fos expression in spinal cord i.pl. NGF

10–25 mg/kg i.pl.

Decreased thermal hyperalgesia Reduced neutrophil accumulation

Farquhar-Smith and Rice (2003)

Capsaicin-induced release of substance P

Systemic perfusion 0.05–0.5 lM

Inhibited the activation of C fibers

Yoshihara et al. (2005)

Acetic acid, kaolin and magnesium sulfate-evoked writhing

1–20 mg/kg i.p.

Reduced the number of writhing

Calignano et al. (2001)

Magnesium sulfate-evoked writhing

1–100/kg s.c.

Reduced the number of writhing


Phenyl-p-quinone induced visceral pain

2–8 mg/kg i.p.

Inhibited stretching movements

Haller et al. (2006)

i.pl. PGE2 in rat paw

5–20 lg i.pl.

Reduced mechanical hyperalgesia

Romero et al. (2012, 2013a, b)

Chronic granulomatous inflammation

in situ 100 ll of 200, 400, 800 lg/ml

Prevented nerve fiber formation and sprouting;

De Filippis et al. (2011)

Reduced mechanical allodynia and inhibits sensory ganglia activation; Reduced mast cell recruitment and activation Reduced NGF expression and release

Neuropathic pain Chronic constriction injury of sciatic nerve

100 lg/kg, i.p.

Inhibited of mechanonociceptive hyperalgesia

Helyes et al. (2003)

Reduced sensory neuropeptide release 10 mg/kg i.p.

Reduced thermal hyperalgesia and mechanical allodynia

Costa et al. (2008)

Inhibited nuclear factor-kB activation in spinal cord Normalized the production of trophic factors in the lumbar spinal cord 10 mg/kg i.p.

Reduced thermal hyperalgesia and mechanical allodynia

Bettoni et al. (2013)

Preserved nerve morphology Reduced mast cell recruitment and activation in sciatic nerve Reduced microglia activation in the spinal cord Normalized the production of trophic factors in sciatic nerve 30 mg kg s.c.

Reduced the hypersensitivity to a mechanical noxious stimulus and to a nonnoxious mechanical stimulus;

Di Cesare Mannelli et al. (2013)

Preserved nerve morphology Reduced: Endoneural edema Immune cell infiltration Pro-inflammatory mediators Sciatic nerve ligation in Swiss mice

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5–30 mg/kg i.p.

Reduced mechanical and thermal hyperalgesia



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Table 1 continued Model

i.pl. formalin-induced long-term effects

PEA: administration route and dosage

PEA biological action

5–10 mg/kg, i.p. 1 lg/ll Reduced mechanical allodynia and thermal hyperalgesia in situ Abolished spontaneous and evoked neuronal activity changes induced by formalin

References

Luongo et al. (2013)

Normalized glial activation in spinal cord No pain 3–6 nmol intra-PAG

Increased the latency of the nociceptive reaction to thermal stimulus Inhibited the ongoing activity of the ON cells

de Novellis et al. (2012)

Reduced the ongoing activity of the OFF cells In vitro F11 cells CHO-expressing TRPV1

1–30 lM

Increased [Ca2?]i in F11 cells Activated and desensitized TRPV1 channels heterologously expressed in CHO cells

DRG cell–fibrosarcoma cell coculture

10 lM

Decreased the amplitude of the depolarization-evoked Ca2? transient in DRG neurons co-cultured with fibrosarcoma cells

Ambrosino et al. (2013) Khasabova et al. (2012)

F11 cells, fusion of mouse neuroblastoma cell line N18TG-2 with embryonic rat dorsal root ganglion (DRG) neurons; CHO, Chinese hamster ovary cells; i.t., intrathecal; i.pl., intraplantar; i.v., intravenous; i.p., intraperitoneal; os, oral, s.c., subcutaneous; i.c.v., intracerebroventricular; NGF, nerve growth factor; PAG, ventrolateral periaqueductal gray

preserved nerve morphology and reduced endoneurial edema, immune cell infiltration and pro-inflammatory mediators (Di Cesare Mannelli et al. 2013). Interestingly, the effect of PEA on mechanical allodynia and thermal hyperalgesia was seen 7 days after peripheral injection of formalin (Luongo et al. 2013). Electrophysiological analysis revealed a significant increase in duration and frequency, and a rapid decrease in onset of evoked activity of the spinal nociceptive neurons 7 days after formalin. PEA normalized formalin-induced glial cell activation in the spinal cord and led to an increased expression of glial IL-10 (Luongo et al. 2013). These preclinical studies are summarized in Table 1. Collectively, the above findings clearly demonstrate that mast cells and glia are targets of PEA in NP.

PEA: clinical studies The pain-relieving properties of PEA in human diseases associated with chronic pain go back almost 2 decades (Jack 1996). A number of recent clinical studies have confirmed the pain-relieving properties of micronized and ultra-micronized PEA in chronic pain; details of compound form and preparation will be described in a later section. The largest study of PEA in chronic pain involved patients affected by lumbosciatalgia, mainly caused by nerve root compression (Guida et al. 2010). This multicenter, doubleblind, randomized, controlled study employed 2 doses of micronized PEA (300 mg/day or 300 mg BID for 3 weeks) vs placebo. Treatment with micronized PEA (NormastÒ) for 21 days dose-dependently reduced pain intensity

evaluated with the VAS scale; the effect was associated with a consistent improvement of health status of patients as evaluated by the Roland–Morris Disability Questionnaire (Guida et al. 2010). PEA treatment in chronic lumbosciatalgia was accompanied by a significant reduction in non-steroidal anti-inflammatory drug use (Canteri et al. 2010). More recently, the pain-relieving effect of micronized PEA was demonstrated in a large number of (more than 600) patients suffering from chronic pain caused by different etiopathogeneses, including nerve root compression, osteoarthritis, postherpetic neuralgia, diabetic polyneuropathy, pain associated with chemotherapyinduced neuropathy, among others—suggesting the PEA effect to be independent of pain etiopathogenesis (Gatti et al. 2012). In multiple myeloma patients undergoing treatment with thalidomide/bortezomib and having painful neuropathy, a 2-month treatment with micronized PEA (300 mg BID) led to a reduction of pain intensity paralleled by a partial improvement of neurophysiological function of all myelinated fiber groups (Truini et al. 2011). PEA treatment also improved electrophysiological parameters in patients with carpal tunnel syndrome (Conigliaro et al. 2011) also when occurring in patients with diabetes (Assini et al. 2010). Chronic pain associated with diabetic neuropathy is reported responsive to treatment with micronized PEA (Schifilliti et al. 2013), as is pudendal neuralgia (Calabro` et al. 2010), and chronic pain associated with vestibulodynia (Murina et al., 2013)—endometriosis (Indraccolo and Barbieri 2010; Cobellis et al. 2011; Giugliano et al. 2013), or adolescent dysmenorrhea (Fulghesu et al. 2010). In patients afflicted by chronic temporomandibular joint

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123 610 600 mg bid for 3 weeks ? 600 mg/day for next 4 weeks ± standard analgesics

Open (ultra-micronized PEA ± analgesics)

Radiculopathy (331)

Open micronized PEA ± acupuncture)

Open ultra-micronized PEA ? oxycodone

Cervicobrachial or sciatical pain

Low back pain

Other diseases (51)

Oncologic (22)

FBSS (76)

Diab. Neuropathy (32)

Herpes Zoster (44)

Osteoarthritis (54)

111 1st arm: 300 mg/day 9 3 weeks Score pain reduction 2nd arm: 600 mg/day for Reduced exposure to anti-inflammatory or 3 weeks analgesic drugs

Double-blind, randomized, two doses of micronized PEA vs placebo

Lumbosciatica

20 PEA 600 mg/bid ?oxycodonea for 30 days

30 300 mg/bid for 8 weeks

2nd arm: 600 mg/day for 3 weeks

Reduces disability

Score pain reduction

Reduced pain impact on employment

Reduced pain impact on emotional state

Reduced chronic pain score


Reduced disability

636 1st arm: 300 mg/day 9 3 weeks; Score pain reduction

Double-blind, randomized, two doses of micronized PEA vs placebo

Post surgery pain relief

PEA effects

24 900 mg/day for Greater score pain reduction 7 days; ? 300 mg/bid for next Better maximum mouth opening 7 days vs ibuprofen 1,800 mg/ Greater tolerability day for 14 days

30 300 mg/bid for 15 days (6 before, 9 after surgery)

Regimen of PEA administration

Chronic pain Lumbosciatica

Single-blind, randomized, splitmouth. Micronized PEA

n

Double-blind randomized; micronized PEA vs ibuprofen

Lower third molar extraction

Study design

Osteoarthritis Temporomandibular joint

Postsurgery pain

Source of pain (n = patient number)

Desio (2011)

Crestani et al. (2013)

Gatti et al. (2012)

Canteri et al. (2010)

Guida et al. (2010)

Marini et al. (2012)

Bacci et al. (2011)

References

Table 2 Clinical studies demonstrating the pain-relieving effect of micronized and ultra-micronized PEA, concomitant reduction in disability and/or improvement of neurological functions and quality of life

86 S. D. Skaper et al.

Open ultra-micronized PEA ? pregabalin

Diabetic neuropathy (11)


47 400 mg/bid (?polydatin 20 mg/ bid) for 3 months

Open (case series) micronized PEA

Double-blind, randomized parallel-group, micronized PEA vs placebo

Prospective micronized PEA

Endometriosis

Endometriosis

Endometriosis:

Open micronized PEA Case report micronized PEA

Case report micronized PEA Open controlled ultra-micronized PEA ? physiotherapy. vs physiotherapy Open micronized PEA

Primary dysmenorrhea

Pudendal neuralgia

Occipital neuralgia

Post-stroke patients

Chemotherapy-induced neuropathy

Ovary (28)

Recto-vaginal septum (19)

61 400 mg/tid (? 40 mg/tid polydatin) for 3 months vs celecoxib 200 mg/bid for 7 days

Group-controlled, randomized, two doses of micronized PEA vs no-treated patients

Carpal tunnel syndrome

Improved sensory conduction velocity


Reduces disability


Reduces disability


PEA effects

Dyschezia

Dysmenorrheal

Dyspareunia

CPP

Decreased pelvic pains:

Dysmenorrhea

Dyspareunia

CPP


Decreased use of analgesics

Dysmenorrhea

Dyspareunia

CPP


Reduced discomfort

20 300 mg/bid for 2 months

20 600 mg/bid for 60 days ? 600 mg/day for next 30 days

600 mg/bid

gradually decreasing to 300 mg/ day for 1 year

1 300 mg/tid

Increased amplitude of foot-LEPs, suralSNAPs, peroneal-CMAPs

Pain score reduction

Spasticity reduction

Pain intensity reduction

pain

Reduction of Burning

Resolution of pain

20 400 mg/bid (? polydatin 40 mg/ Decreased pelvic pain bid) for 6 months

4 200 mg/bid (?polydatin 20 mg/ bid) for 3 months

2nd arm: 600 mg/bid for 30 days Minor Tinel’s sign presence

26 1st arm: 300 mg/bid for 30 days Reduced median nerve latency time

50 600 mg/bid for 60 days

Group-controlled, randomized, micronized PEA vs standard care

Diabetic neuropathy ? carpal tunnel syndrome

31 PEA 600 mg/ bid ? carbamazepina for 45 days

Open ultra-micronized PEA ? carbamazepin

30 PEA 600 mg/bid ?pregabalina for 45 days

n

Trigeminal neuralgia

postherpetic neuralgia (19)

Study design

Source of pain (n = patient number)

Table 2 continued

Truini et al. (2011)

Parabita et al. (2011)

Calabro` and Bramanti (2013)

Fulghesu et al. (2010) Calabro` et al. (2010)

Giugliano et al. (2013)

Cobellis et al. (2011)

Indraccolo and Barbieri (2010)

Conigliaro et al. (2011)

Assini et al. (2010)

Desio, et al. (2010)

Desio (2010)

References

Palmitoylethanolamide 87

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Schifilliti et al. (2013) Pain relief;

Reduced neuropathic symptoms

a

See text for dosage

PEA: mechanism(s) of action

CPP chronic pelvic pain

Neuropathic pain

pain, micronized PEA elicited a progressive decrease of pain intensity; in contrast, ibuprofen showed faster pain relief in the first week but no further decrease in the following week. The PEA effect was associated with a greater improvement in maximum mouth opening at treatment end, as compared to ibuprofen (Marini et al. 2012). PEA also reduced postoperative pain (Bacci et al. 2011). In situations where PEA treatment is in association with drugs for NP (e.g. carbamazepine, pregabalin and oxycodone) (Desio et al. 2010; Desio 2010, 2011), the former induced pain relief even when the two drugs were given at doses below their respective therapeutic thresholds. This apparent additive or synergic drug–drug effect could conceivably reflect PEA action on non-neuronal cells such as mast cells and microglia. PEA alleviates also the NP associated with lumbosciatica (Domıńguez et al. 2012) and multiple sclerosis (Kopsky and Hesselink 2012). Interestingly, a just published study claims that occipital neuralgia does respond to PEA oral treatment (Calabro` and Bramanti 2013). Table 2 summarizes these clinical studies.

30 300 mg/bid for 60 days Open micronized PEA Diabetic polyneuropathy

Pain score reduction

Group-controlled, randomized, 118 300 mg/bid for 30 days micronized PEA ?standard analgesic therapies vs standard analgesic therapies Lumbosciatica

Quality life improvement

Persistent reduction of pain intensity 1 900 mg daily for 5 weeks Case report: micronized PEA ? acupuncture Multiple sclerosis

PEA effects Source of pain (n = patient number)

Table 2 continued

Study design

n


Kopsky and Hesselink (2012) Domıńguez et al. (2012)

S. D. Skaper et al.

References

88

The mechanism(s) underlying PEA action in NP remain to be fully defined. PEA, however, appears to function as an anti-inflammatory and pain-reliever with more than one ‘‘modus operandi’’, and able to modulate the ‘‘endocannabinoidome’’ in a safer and more therapeutically efficacious way (Maione et al. 2013). In 2005, the transcription factor peroxisome proliferator activated receptor alpha (PPAR-a) was identified as one possible target mediating the anti-inflammatory actions of PEA (LoVerme et al. 2005a, b). In PPAR-a-null mice, nociception elicited by intraplantar injection of formalin or intraperitoneal magnesium sulfate was insensitive to PEA (LoVerme et al. 2006). Moreover, the CB2 receptor antagonist SR144528 blocked the anti-nociceptive effects of PPAR-a agonists by interacting with a site distinct from the CB2 receptor, further clarifying the anti-nociceptive action of PEA (LoVerme et al. 2006). Acute intracerebroventricular administration of PEA modulated carrageenan-induced paw edema in mice in a PPAR-adependent manner (D’Agostino et al. 2007). In the case of PPAR-a activation by PEA, there appear to be both shortterm (non-nuclear) anti-hyperalgesic actions, possibly mediated by Ca2?-activated K? channels, and long-term (nuclear) anti-inflammatory effects (LoVerme et al. 2006; Romano and Lograno 2012; de Novellis et al. 2012). PEA effects on the periaqueductal gray—rostral ventromedial medulla mentioned above pathway were prevented by a selective PPAR-a antagonist (de Novellis et al. 2012). Newly published data suggest that PEA may interact also


89

Table 3 Particle size profile of naive PEA compared to micronized and ultra-micronized PEA Particle size

Naive PEA

Micronized PEA

Ultra-micronized PEA

100–700 microns 100 %

Absent

Absent

[14 microns

–

Trace

Absent

\10 microns \6 microns

– –

About 96 % 80 %

100 % 99.9 %

\2 microns

–

Not determined 59.6 %

\1 microns

–


\0.6 micron

–


with other members of the PPAR family to elicit its antiinflammatory activity (Costa et al. 2008; Paterniti et al. 2013). PPARs are found on both dorsal root ganglion sensory neurons and glial cells. As such, PEA may activate these receptors and modulate both the perception and transmission of peripheral pain signaling and spinal amplificatory pain mechanisms—thereby exerting its activity in different types and phases of pain (Khasabova et al. 2012). PEA potentiates anandamide actions at cannabinoid receptors in a CB2 receptor antagonist-sensitive fashion (while itself having no appreciable affinity for either CB1 or CB2 receptors) and anandamide desensitization of transient receptor potential cation channel subfamily V member 1 (TRPV1) channels (‘‘entourage’’ effects); direct activation and desensitization of TRPV1 are also important mechanisms in modulation of pain signaling (Ambrosino et al. 2013; De Petrocellis et al. 2001). Not to be overlooked, as well, is the involvement of de novo neurosteroid synthesis in the modulation of pain behavior by PEA (Raso et al. 2011; Sasso et al. 2012). Collectively, these results suggest that PEA may meet the need of a multi-target approach to tackle the so far unsolved problem of untreatable chronic and neuropathic pain (Maione et al. 2013).

Molecular PEA: size does matter in pharmaceutical technology The clinical studies described above with micronized and ultra-micronized PEA in chronic pain employed one of several PEA-based products commercially available in Italy and several other European countries under the tradenames NormastÒ, PelvilenÒ and GlialiaÒ (Epitech Group Srl, Italy). These products were developed to be used as ‘Dietary Foods for Special Medical Purposes’, intended to assist in the dietary management of individuals with chronic pain associated with particular disease states, in agreement to Commission Directive 1999/21/EC. In particular, PEA contained in the afore-mentioned formulations is a patented, pharmaceutical grade compound, whose particles have been subjected to the so-called fluid jet micronization process. It is important to bear in mind that: (i) PEA is a crystalline and highly lipophilic compound; (ii) the low dissolution rate of poorly water-soluble drugs in biological fluids is the rate-limiting step in their absorption (Rao et al. 2011; Sareen et al. 2012) which, in turn, is the limiting factor for a pharmacological response (Sareen et al. 2012). Particle size is thus a crucial pharmaceutical technology issue. PEA used in the aforementioned compositions was subjected to different kinds of micronization to obtain particles with a defined size profile (Table 3), being completely different and statistically lower (6–10 lm at most) in comparison to naive PEA (in the 100–700 lm range) (Fig. 4). Moreover, the ultramicronization process yields a different crystalline structure with higher energy content. The smaller particle size (with higher surface-to-volume ratio) combined with increased potential energy contributes to better solubility (Rao et al. 2011; Sareen et al. 2012). These characteristics result in better diffusion and distribution of micronized and ultra-micronized PEA compared to the naive form, and thus superior biological efficacy. In the previous section

Fig. 4 Photomicrographs of naı¨ve (a) and ultra-micronized (b) PEA obtained by scanning electron microscopy. Bar 10 microns in each panel

123

90

dealing with clinical trials, all references to PEA intend the micronized or ultra-micronized form.

Concluding remarks NP remains one of the most challenging of all neurologic diseases and represents an enormous unmet medical need across the globe (Institute of Medicine, Relieving Pain in America: A Blueprint for Transforming Prevention, Care, Education, and Research, June 2011). Approximately, 4.7 million people suffer from painful neuropathy in the USA alone, and few available treatment options are available today. Clearly, we still have much to learn about signaling mechanisms that regulate neuroinflammation and NP. As discussed in this review, a knowledge of mast cell—glia communication may open new perspectives for designing therapies to target NP, as well as other pathologies of a neuroinflammatory nature, by differentially modulating the activation of non-neuronal cells normally controlling neuronal sensitization—both peripherally and centrally (Skaper and Facci 2012). The capacity of PEA to modulate the protective responses of animals during inflammation and pain led to the hypothesis that endogenous PEA may be a component of the complex homeostatic system controlling the basal threshold of both inflammation and pain. PEA has basically no adverse effects at pharmacological doses, while possessing a double therapeutic effect (i.e., anti-inflammatory and pain-relieving). PEA, its analogs, and agents that inhibit specifically its degradation are likely to result in the development of new therapeutics for the treatment of NP, as well as other pathological conditions where inflammation (both central and peripheral) is a factor. Acknowledgments The authors wish to thank Gabriele Marcolongo for his valuable input in providing details of the micronization/ultramicronization process for PEA, and the associated figures and data. L. Facci was supported by Fondazione CARIPARO ‘‘Progetto Dottorati di Ricerca’’ Anno 2009. This study was supported in part by MIUR, PON ‘Ricerca e Competitivita` 2007–2013’ project PON01_02512. Conflict of interest MF is an employee of Epitech Srl; MFdV is a scientific consultant for Innovet Italia Srl.

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