Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Review

The cannabinoid acids, analogs and endogenous counterparts

http://dx.doi.org/10.1016/j.bmc.2014.03.0380968-0896/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: NAgly, N-arachidonoyl glycine; AJA, ajulemic acid; THC, tetrahydrocannabinol; CBD, cannabidiol; CBN, cannabinol.⇑ Tel.: +1 508 856 2850; fax: +1 508 856 2003.

E-mail address: sumner.burstein@umassmed.edu

Please cite this article in press as: Burstein, S. H. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.03.038

Sumner H. Burstein ⇑Department of Biochemistry and Molecular Pharmacology, The University of Massachusetts Medical School, Worcester, MA 01605, USA

a r t i c l e i n f o

Article history:Received 7 February 2014Revised 15 March 2014Accepted 24 March 2014Available online xxxx

Keywords:CannabinoidD9-THC-11-oic acidAjulemic acidNAglyLipoamino acidProstaglandinLipoxin

a b s t r a c t

The cannabinoid acids are a structurally heterogeneous group of compounds some of which are endog-enous molecules and others that are metabolites of phytocannabinoids. The prototypic endogenous sub-stance is N-arachidonoyl glycine (NAgly) that is closely related in structure to the cannabinoid agonistanandamide. The most studied phytocannabinoid is D9-THC-11-oic acid, the principal metabolite ofD9-THC. Both types of acids have in common several biological actions such as low affinity for CB1anti-inflammatory activity and analgesic properties. This suggests that there may be similarities in theirmechanism of action, a point that is discussed in this review. Also presented are reports on analogs of theacids that provide opportunities for the development of novel therapeutic agents, such as ajulemic acid.

� 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Cannabinoid acids as plant constituents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Acid metabolites of the phytocannabinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.1. Acid metabolites from in vivo metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Acid metabolites from in vitro metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Actions of acid metabolites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.3.1. Psychotropic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3.2. Anti-inflammatory effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3.3. Analgesic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.4. Forensic aspects of acid metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.5. Chemical stability of acid metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Synthetic analogs of acid metabolites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.1. Ajulemic acid (AJA, HU-239, CT-3, IP-751, JBT-101) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.1.1. Analgesic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.2. Anti-inflammatory effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.3. Actions on bladder irritation and hyperactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.4. In vitro and in vivo anti-neoplastic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.5. Anti-fibrotic effects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.6. Multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.7. Suppression of osteoclastogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1.8. In vitro metabolism and metabolic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4.2. HU-235: a putative metabolite of dexanabinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3. (�)-CBD-11-oic acids and their dimethylheptyl (DMH) homologs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2 S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx

Pleas

4.4. Comparison of AJA with arachidonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Endogenous counterparts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5.1. Lipoamino acids (elmiric acids, EMA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.2. Occurrence, metabolism and tissue distribution of the lipoamino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.3. Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5.3.1. Analgesic effects of NAgly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.3.2. Anti-inflammatory actions of NAgly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.3.3. Inhibition of FAAH activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.3.4. Other actions of NAgly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.3.5. Other lipoamino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5.4. Mediation of NAgly actions by GPR-18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

6. Putative mechanisms for the anti inflammatory actions of AJA and NAgly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 007. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

References and notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

Since an earlier review on this topic published in 1999,1 therehave been a considerable number of new research findings. Thispaper represents an update of the field and also gives a more de-tailed and comprehensive treatment of this subject. The present re-view includes not only phytocannabinoid-derived acids, but alsotheir synthetic analogs and lipoamino acid counterparts, some-times called elmiric acids. In contrast to earlier beliefs, the cannab-inoid acids have a number of biological activities that are ofpotential therapeutic interest and will be discussed here.

Figure 1. Cannabinoid acids as plant constituents. (A) Tetrahydrocannabinolic acidA. (B) Tetrahydrocannabinolic acid B.

2. Cannabinoid acids as plant constituents

The penultimate intermediates in the biosynthesis of D9-THCare two molecules, tetrahydrocannabinolic acid A and tetrahydro-cannabinolic acid B, containing a carboxyl group at either the 2 or 4positions on the aromatic ring (Fig. 1).2–4 These compounds arereadily decarboxylated when heated to give D9-THC especiallywhen the plant material is consumed by smoking. It is possible thatsome of the oral preparations of Cannabis such as Bhang and Ma-jun may contain these acids. Little is known about their pharma-cology, however, several reports suggest that these compoundsmay have biological activities.5–8 Similar acids of the other phytoc-annabinoid acids such as Cannabidiolic acid, Cannabigerolic acid,Cannabidivarinic acid, Cannabichromenic acid, (5aS,6S,9R,9aR)-Cannabielsoic acid A, (5aS,6S,9R,9aR)-Cannabielsoic acid B,(1aS,3aR,8bR,8cR)-Cannabicyclolic acid, and Cannabinolic acidhave all been isolated from plant extracts.3

Figure 2. Principal route of metabolism for D9-THC in most species. Compounds A(D9-THC), B and C all showed similar biological activities. While devoid ofpsychotropic activity in mice and humans, compound D, the terminal carboxymetabolite showed NSAID-like action in several animal models, albeit at lowpotency. This prompted the synthesis of a more potent analog, ajulemic acid (videinfra).

3. Acid metabolites of the phytocannabinoids

The earliest report suggesting the existence of acid metabolitesof D9-THC was made by a Swedish group.9 They observed that ob-served that a large proportion of the urinary metabolites in therabbit were acidic in nature. However, no structural assignmentswere made. Subsequently, in a scaled up repetition of this study,large enough samples of two metabolites were isolated to allowidentifications by proton NMR and low-resolution mass spectrom-etry.10 The structures were shown to be the 10 hydroxy and 20 hy-droxy derivatives of D9-THC-11-oic acid (Fig. 2D). The occurrenceof D9-THC-11-oic acid itself as a urinary metabolite was reportedin a subsequent study that also described its synthesis and lackof psychotropic activity.11 Apparently, the possibility of otheractivities was not investigated by them or any other researchersuntil some years later (see Section 3.3). Figure 3 shows examplesof metabolites of D9-THC.

e cite this article in press as: Burstein, S. H. Bioorg. Med. Chem. (2

3.1. Acid metabolites from in vivo metabolism

The most widely studied cannabinoid acid is D9-THC-11-oicacid (Fig. 2D). It is the terminal metabolite of D9-THC (Fig. 2A) thatis generated in a three-stage process going through the hydroxyl(Fig. 2B) and aldehyde (Fig. 2C) intermediates. This route occursin humans and in every other species thus far examined.11,12 Incontrast to its precursors, it is devoid of psychotropic activity, how-ever, it does have biological actions described in Section 3.3 thatmay contribute to the pharmacological profile of D9-THC. There

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Figure 3. Examples of metabolites of D9-THC that have been detected andidentified. (A) D9-THC-11-oic acid itself. (B) Monohydroxy D9-THC-11-oic acid;arrows indicate positions of hydroxyl groups. (C–F) Side-chain degradationproducts.

Figure 4. Metabolites of other phytocannabinoids, (A) CBD-11-oic acid. (B) CBN-11-oic acid.

S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx 3

is also much interest in D9-THC-11-oic acid as the principle analytein the forensic assays for marijuana use (see Section 3.4). A morerecent review of cannabinoid pharmacokinetics contains detailedinformation on the production of D9-THC-11-oic acid in humans.13

Two other cannabinoids CBD and CBN are also metabolized simi-larly to give a carboxyl-containing product (Fig. 4).14–16

3.2. Acid metabolites from in vitro metabolism

A cytochrome P450 (P450 MUT-2) catalyzes the in vitro oxida-tion of 11-oxo-D8-THC to D8-THC-11-oic acid as shown in Fig-ure 2.17,18 This isozyme, a member of the P450 2C genesubfamily was isolated from hepatic microsomes of untreated malemice. 11-oxo-D9-THC and 11-oxo-cannabinol (11-oxo-CBN) arealso substrates for this enzyme and produce the corresponding car-boxylic acid. The data indicate that P450 MUT-2 is a major enzymefor metabolizing cannabinoids by mouse hepatic microsomes.

3.3. Actions of acid metabolites

3.3.1. Psychotropic activityD9-THC-11-oic acid was synthesized and tested for behavioral

responses in Rhesus monkeys.11 It was found to be inactive andwas not studied for any other actions. More than a decade passedbefore it was discovered that, in fact, it did have potentially impor-tant biologically activities. For example, THC-induced catalepsy inmice can be substantially inhibited by the prior administration ofD9-THC-11-oic acid, the major metabolite of THC in most speciesincluding humans.5 This suggests that the intensity and durationof psychotropic action of THC m depend to a degree on the levelsof this metabolite at its sites of action. Evidence was also reportedthat it has activity as an anti-inflammatory agent (seeSection 3.3.2).

3.3.2. Anti-inflammatory effectsSeveral reports contained data showing that THC and other can-

nabinoids, in particular, CBD, can stimulate the production of proinflammatory eicosanoids such as PGE2.19–25 Subsequently, it wasreported that its metabolite, D9-THC-11-oic acid, could inhibit thisprocess, suggesting that the acid is an anti-inflammatory agent,however, the mechanism for this action was not well character-ized.8 In any case, the findings suggested the possibility, that this

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effect may influence the in vivo actions of THC by inhibiting itsstimulatory action on cellular prostaglandin synthesis.

D8-THC-11-oic acid was active in the mouse ear edema testwhere it was about as efficacious as phenidone; however, its po-tency was less than either phenidone or indomethacin.26 It alsoshowed activity as an antagonist to platelet activating factor(PAF), which may help explain the known properties of THC as abronchodilator, antipyretic and anti-rheumatic agent. Its activityin preventing PAF-induced mortality was comparable to naproxen.

D9-THC-11-oic acid produced a 20% stimulation of phospholi-pase A2 (PLA2) activity in mouse brain synaptosomes resulting inthe increased release of free arachidonic acid from exogenouslyadded [1-14C]-phosphatidylcholine.27 This effect could provideprecursor material for the synthesis of anti-inflammatory eicosa-noids such as PGJ2 and LXA4. A more detailed study of this processis discussed for the analog AJA in Section 4.1.2.

3.3.3. Analgesic activityBoth delta D9-THC and D9-THC-11-oic acid produce analgesic

action in the mouse hot plate test done at 50 �C.28 Of special inter-est is the time course in the hot plate test when each substance isadministered orally at a dose of 20 mg/kg. At ten minutes, D9-THCis actually hyperalgesic whereas the acid is mildly analgesic. Thelatter action for both reaches a maximum at 30 min where the acidshows an effect twice as potent as D9-THC. Prior administration ofeither indomethacin or D9-THC-11-oic acid inhibits the hyperalge-sic response. In the ring test, the metabolite does not produce acataleptic state in the mouse, which eliminates catalepsy as a causefor the hot plate response.

The anti-nociceptive effect of D8-THC-11-oic acid activity wasinvestigated further with particular regard to the influence of cer-tain experimental parameters in the hot plate test.29 These in-cluded the intensity of the thermal stimulus, the composition ofthe vehicle and a possible role for Cu++. A temperature effect sim-ilar to that seen with non-steroidal anti-inflammatory drugs (NSA-IDs) was observed; at 55 �C observable anti-nociception wasproduced, however, at a surface temperature of 58 �C no drug ef-fect was seen. This suggests that opioid receptors are not involved.Non-aqueous vehicles such as peanut oil increased the potency ofD8-THC-11-oic acid possibly due to increased bioavailability. Final-ly, the substitution of purified drinking water for tap water re-duced the drug response that could be partially restored byadding Cu++ to the purified drinking water. An increase in theinhibitory effect of D8-THC-11-oic when Cu++ was added to themedia was seen in a cell culture model where the acid reducedTHC-induced prostaglandin synthesis. It has been shown thatmany anti-inflammatory agents form chelate complexes withCu++ that are more efficacious than the unchelated drug.30

3.4. Forensic aspects of acid metabolites

D9-THC-11-oic acid and its glucuronides are the principal con-stituents in urine samples following the use of marijuana. An im-proved assay that is applicable for routine urine cannabinoidtesting has recently been reported.31,32 A sensitive and specificmethod for the extraction and quantification of D9-THC, 11-OH-

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4 S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx

D9-THC, D9-THC-11-oic acid, cannabidiol, cannabinol, D9-THC-glu-curonide and D9-THC-11-oic acid-glucuronide in human urine wasdeveloped and validated. This method was tested on urine speci-mens collected from individuals participating in studies of con-trolled administration of Cannabis, and was reported to be auseful procedure for determining Cannabis use in forensic toxicol-ogy applications.

3.5. Chemical stability of acid metabolites

Data have been reported demonstrating the reactivity of D9-THC-11-oic acid in water containing free chlorine.33 It was rapidlydegraded following pseudo-first order kinetics to yield seven chlo-rine-containing products. Chlorinations involving the aromatic

Table 1Reported actions of ajulemic acid

Observed response Potency

Reduces paw edema in mice 0.1 mg/kgReduces leukocyte adhesion 0.5 mg/kgProduces analgesia 0.05 mg/kgAdjuvant-induced chronic arthritis 0.1 mg/kgReduces leukocyte migration 0.2 mg/kgDecreases chronic neuropathic pain 20 mg b.i.d.Reduction of writhing 1.2 mg/kg ivReduction of pain 4.6 mg/kg ivAnalgesia 4.4 mg/kg i.g.Reversal of hyperalgesia 0.1–1 mg/kgIncreased latency 3.3 mg/kgHypothermia 10 mg/kgReduced mechanical allodynia 10 mg/kgReduced mechanical allodynia 10 mg/kgInhibits metalloproteinases 10 lMInduction of apoptosis 1 lMDecreases secretion of IL-1b 5 lMIncreased COX-2 expression 20 lMIncreased production of LXA4 0–30 lMSuppression of bladder activity 1.44 lMInhibition of cell proliferation 5.8–16 lMAnti metastatic activity 0.1 mg/kgPrevents progression of fibrosis 1 mg/kgReduction of spastic activity 0.1 m/kg ivSuppresses osteoclastogenesis 15 lM

Figure 5. Structures of synthetic analogs of cannabinoid acids. (A) R,R-10 ,10-dimethylheptyl-D8-THC-11-oic acid (AJA). (B) S,S-10 ,10-dimethylheptyl-D8-THC-11-oic acid. (C) S,S-10 ,10-dimethylheptyl-D9-CBD-11-oic acid (HU-320); (D) R,R-10 ,10-dimethylheptyl-D9-CBD-11-oic acid.

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ring and the double bond were identified by mass spectrometry.The extent of the reaction was dependent on the level of organicmatter content in the aqueous milieu; the greater the organic mat-ter content, the lower the amount of chlorination. These resultsindicate a need for caution when handling cannabinoids in mediasuch as chlorinated drinking water, for example, in forensic assays.

A comprehensive study of D9-THC, 11-hydroxy-D9-THC, D9-THC-11-oic acid, cannabidiol, cannabinol, and D9-THC-11-oic acidglucuronide stability in urine was reported.34 They concluded thatanalysis should be conducted in frozen urine within 3 months ifnon-hydrolyzed D9-THC-11-oic acid, or D9-THC-11-oic acid glucu-ronide quantification is required.

4. Synthetic analogs of acid metabolites

4.1. Ajulemic acid (AJA, HU-239, CT-3, IP-751, JBT-101)

Ajulemic acid is a totally synthetic compound that, over theyears, has been prepared in several laboratories using differentprocedures. This has given rise to slight variations in the composi-tion and amounts of the accompanying impurities some of whichseem to have potent binding affinity for CB1. Each organizationhas assigned a code that will be indicated in parentheses afterthe name as follows. They are: HU-239, The Hebrew University;CT-3, Atlantic Pharmaceuticals; IP-751, Indevus Pharmaceuticalsand JBT-101, JB Therapeutics.

AJA (Fig. 5A) is a D8-dimethylheptyl side-chain analog of D9-THC-11-oic acid, the major metabolite of the active ingredient ofmarijuana,D9-THC. AJA was designed to have increased anti-inflam-matory properties and reduced psychotropic activity, compared tothe D9-THC parent. AJA (HU-239) was found to be effective at reduc-ing paw edema in mice induced by arachidonic acid or platelet acti-vating factor, at diminishing leukocyte adhesion in peritoneal cellsfrom AJA (HU-239) dosed mice, and at producing analgesia.35 Subse-quent studies have substantiated and expanded these findings andhave demonstrated that it can minimize the adverse effects of adju-vant-induced arthritis in rats.36 AJA (CT-3) was well tolerated innonclinical toxicity studies in mice, rats, and dogs (Atlantic Pharma-ceuticals, unpublished data). A Phase 1 clinical trial designed to

Model References

Arachidonic acid or PAF induction 35Mouse peritoneal cells 35Mouse hot plate at 55 �C 35Male rats 36Subcutaneous air pouch 36Humans with refractory pain 37p-Phenylquinone writhing test 38Formalin anti-nociception test 38Mouse tail clip test 39Inflammatory pain in the rat 40Tail flick assay 40Core temperature 40Nerve-injury induced model 41CFA-induced inflammatory pain 41Human synovial cells 49T lymphocytes 50Synovial fluid monocytes 51Human synovial cells 52Human blood or synovial cells 53Rats 47Cancer cells in vitro 54SCID-NOD mouse flank tumor Recht & Salmonsen, unpubBleomycin-induced dermal fibrosis 55Rat multiple sclerosis model 56Precursor mouse macrophages 57

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22 00

50

100

150

…………………PAF threshold

……………..Normal threshold

Ajulemic Acid [mg/kg]

With

draw

al T

hres

hold

[g]

Figure 6. Dose–response curve of AJA (CT-3) on PAF-induced mechanical allodynia.Platelet-activating factor (PAF) injected in the hind paw of rats was used as a model

S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx 5

measure the safety and pharmacokinetics of AJA (CT-3) resulted inno clinically relevant adverse events and no evidence of mari-juana-like psychoactivity (Atlantic Pharmaceuticals, unpublisheddata). In a Phase 2a trial, AJA (CT-3) showed efficacy in patients withchronic neuropathic pain.37 Thus, AJA may have significant potentialfor therapeutic benefit in the treatment of pain and chronic inflam-mation with a low potential for abuse and other adverse events. Theactions of AJA are summarized in Table 1. The enantiomer of AJAshown in Figure 5B showed greatly reduced activities suggestingreceptor mediation. A chiral analysis of the enantiomer revealedthe presence of 10–20% of AJA that was probably the source of itsactivities (Atlantic Pharma, unpublished).

4.1.1. Analgesic activityPotent anti-nociceptive effects were reported for AJA (HU-239)

in the mouse hot plate assay.35 When tested over the range of0.025–1.0 mg/kg, a bell-shaped dose–response relationship witha peak effect at 0.05 mg/kg was observed when the assay was doneat 55 �C. It was also reported that, at a dose of 0.1 mg/kg, no re-sponse in the ring test for catalepsy was seen suggesting a separa-tion of psychotropic from analgesic activities. In a subsequentstudy of AJA (CT-3), positive effects were reported in several painassays.38 These included the mouse hot plate assay at 48 �C, themouse p-phenylquinone writhing test and the mouse formalinanti-nociception test. In addition, it was observed that it did not al-ter motor function in the rotarod procedure at 4.64 mg/kg iv; againsuggesting a separation of activities. Further studies using the hot-plate (55 �C) and the tail clip tests in mice and in the tail clip test inrats showed potency similar to morphine sulfate after i.g. and ipadministration.39 In the rat, oral administration of AJA (Novartis)(0.1–1 mg/kg) produced up to 60% reversal of mechanical hyperal-gesia.40 The anti-hyperalgesic activity was prevented by the CB1antagonist SR141716A but not the CB2 antagonist SR144528 sug-gesting mediation by the CB1 receptor. They concluded that while‘it shows significant cannabinoid-like CNS activity, it exhibits asuperior therapeutic index compared to other cannabinoid com-pounds, which may reflect a relatively reduced CNS penetration’.A study comparing AJA (IP-751) with HU-210 again suggested aseparation of psychoactive properties from analgesic effects.41

Using a nerve-injury induced model of neuropathic pain and theCFA-induced model of inflammatory pain, efficacy was shown forboth compounds, however, only HU-210 was effective in the rota-rod test. They concluded ‘that ajulemic acid reduces abnormal painsensations associated with chronic pain without producing themotor side effects associated with D9-THC and other non-selectivecannabinoid receptor agonists’. A model of tonic inflammatorypain involving the injection of platelet activating factor (PAF) intothe hind paw of rats was carried out (Fig. 6, Walker et al., unpub-lished). The PAF injection control led to a drop in the withdrawalthreshold to mechanical pressure (from 83 g to 40 g). A series ofdoses of AJA (CT-3), 3.0, 4.5, 6.8, 10.1 and 15.2 mg/kg, ip wereadministered and the paw withdrawal thresholds at 40–60 minpost-drug were measured. An approximate ED-50 of 4.2 mg/kgwas estimated for the reversal of PAF-induced allodynia. A com-plete reversal was seen at around 6.8 mg/kg, above which anti-nociception was observed.

Tepper et al. have prepared highly purified AJA (JBT-101) andcompared its cannabinoid receptor binding constants with thoseobtained from other preparations.�CB2 binding did not vary greatlyamong the various samples tested, however, CB1 binding showed awide range of affinities (Ki = 5.7–628 nM). The CB1 binding activity

� Mark A. Tepper; Robert B. Zurier and Sumner H. Burstein, Highly PurifiedAjulemic Acid is a CB2 Agonist with Reduced CB1 Activity, 23rd Annual Symposiumon the Cannabinoids, International Cannabinoid Research Society, Research TrianglePark, NC, USA, 2013, P1–20.

Please cite this article in press as: Burstein, S. H. Bioorg. Med. Chem. (2

is substantially reduced in JBT-101. This finding may explain theconclusion reached by Vann et al.42 that ‘AJA, like Delta(9)-THC,exhibits psychoactive and therapeutic effects at nearly equal dosesin preclinical models, suggesting similar limitations in their putativetherapeutic profiles’. This conclusion is inconsistent with the resultsobtained in humans where another preparation of AJA (CT-3) thathas low CB1 binding was used and showed a good therapeuticprofile.37,43

In humans, a Phase 1 trial using healthy subjects did not revealany adverse events, including psychotropic effects, in an acute,high dose treatment with AJA (CT-3) (Atlantic Pharmaceuticals,unpublished data). In a Phase 2a randomized, placebo-controlled,double blind crossover trial in patients with refractory neuropathicpain, it was effective in 30% of the subjects.37The scores obtainedon the ARCI-M questionnaire indicated an absence of cannabimi-metic activity under these conditions. These data raise a questionabout the applicability to AJA of preclinical tests for cannabinoidssuch as those employed by Vann et al., for example, drug discrim-ination.65 This issue has been discussed in the literature.43–46

4.1.2. Anti-inflammatory effectsThe in vivo anti-inflammatory effects of AJA have been demon-

strated in six rodent models. These are: (1) the adjuvant-inducedchronic arthritis model,36 (2) cytokine-induced acute inflamma-tion,36 (3) arachidonic acid-induced paw edema,35 (4) PAF-inducedpaw edema,35 (5) leukocyte adhesion of mouse peritoneal cells35

and (6) interstitial cystitis.47,48

Chronic poly-arthritis was induced in rats by intradermal injec-tion of Freund’s complete adjuvant.36 AJA (CT-3) treatment(0.1 mg/kg/day in oil, p.o.) began 3 days after adjuvant injectionand was administered on Mondays, Wednesdays, and Fridays for5 weeks. Arthritis was scored in all four paws where it reducedthe numbers of severely inflamed paws, improved weight gain,and minimized arthritic damage in joints observed histologicallyand compared to untreated controls. The degree of protectionafforded by AJA (CT-3) treatment was greater in the histologicalmeasures than in the clinical measures. In a second study, acuteinflammation was induced by injecting IL-1a and TNFb into airpouches formed on the backs of mice (Fig. 6). It reduced the num-bers of leukocytes in exudates of these pouches by 42.3% (0.1 mg/kg/day) and by 65.5% (0.2 mg/kg/day).

of tonic inflammatory pain. PAF injection led to a drop in the withdrawal thresholdto mechanical pressure (83–40 g). Varying doses of AJA (3, 4.5, 6.8, 10.1, 15.2 mg/kg,ip) were administered and the paw withdrawal thresholds at 40–60 min post-drugwere plotted. An approximate ED-50 of 4.2 mg/kg was estimated for reversal ofPAF-induced allodynia. Complete reversal was seen at around 6.8 mg/kg, abovewhich anti nociception was observed (Walker et al., unpublished).

014), http://dx.doi.org/10.1016/j.bmc.2014.03.038

Figure 7. Inhibition by ajulemic acid of arachidonic acid-induced mouse pawedema. Adapted from Burstein et al. (1992). Conditions as reported.35

Figure 8. Release of free arachidonic acid is promoted by ajulemic acid (IP-751).Human synovial cells were labeled and treated as reported.52 Partial inhibitionresulted from pre treatment with the CB2 receptor antagonist SR144528 (unpub-lished data).

Figure 9. Stimulation of PGJ2 synthesis by AJA (JBT-101), THC and CBD in HL-60cells. Conditions used were as previously reported.81

6 S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx

Figure 7 shows the inhibitory action of AJA (HU-239) in themouse paw edema model of inflammation. Arachidonic acid(1.0 mg) was injected into a mouse hind paw and the volume ofthe paw was measured by fluid displacement.35 Edema inducedby either arachidonic acid or PAF was completely inhibited at adose of 0.2 mg/kg, p.o. in oil. The maximum inhibition occurredat 90 min after AJA (HU-239) administration, although significantreductions were noted at all time points tested (30, 60, 90, 120and 180 min). In a second experiment,35 reductions in the numbersof adhering peritoneal leukocytes resulted from oral administra-tion of AJA (HU-239) in oil at doses of 0.01 to 1.0 mg/kg. Cells werecollected from mice 6 h after dosing and were cultured for 18–20 hbefore adhesion was measured. Only 24% as many cells adheredafter in vivo treatment with 1.0 mg/kg than with vehicle.

MMPs (matrix metalloproteinases) are important targets foragents designed to treat inflammatory arthritis. They promote car-tilage degradation and bone erosion, and lead to joint deformitiesand crippling. The effect of AJA (CT-3) on MMP production in hu-man fibroblast-like synovial cells (FLS) and a possible role forPPAR-c was investigated.49 MMP-3 (stromelysin-1) release in FLScells that were stimulated by TNFa was reduced by 88% followingAJA (10 lM) treatment. The effect appeared to be PPAR-c indepen-dent, however the mechanism of action was not determined. Sev-eral other MMPs were also inhibited, but less so than MMP-3.

AJA (CT-3) was shown to induce apoptosis of T cells in a dose-and time-dependent manner and preceded the loss of cell viabil-ity, showing that cell loss was due to programmed cell deathrather than necrosis.50 T lymphocytes in the synovium of rheuma-toid arthritis patients are resistant to apoptosis and this is thoughtto be a factor in joint tissue injury in patients. Annexin V expres-sion, caspase-3 activity, DNA fragmentation, and microscopy wereused to demonstrate AJA-induced apoptosis. Thus, its joint sparingeffect previously reported36 may be explained in part by thiseffect.

Addition of AJA (CT-3) to human peripheral blood and synovialfluid monocytes in vitro reduced both the steady-state levels of IL-1b mRNA and the secretion of IL-1b in a concentration-dependentmanner.51 However, it had no effect on TNFa gene expression in, orsecretion from, peripheral blood monocytes. Interleukin-1beta andtumor necrosis factor-alpha are needed for the progression ofinflammation and joint tissue injury in patients with rheumatoidarthritis. Therefore, inhibition of the former supports the sugges-tion that AJA would be effective in the treatment of this disease.

The anti-inflammatory effects of AJA can best be understood atthe molecular level by its actions at several points of the arachi-donic acid cascade that regulate the expression of the eicosanoidfamily. The initial event in the cascade is the release of free arachi-donic acid from cellular phospholipid storage pools where AJA (CT-3) stimulates a robust response (Fig. 8). Treatment of human syno-vial fibroblasts with AJA (CT-3) results in a concentration depen-dent increase in COX-2 expression.52 A consequence of this is anincrease in anti-inflammatory eicosanoid production. Pretreatmentof synovial cells with the CB2 antagonist SR144528 partially inhib-its release suggesting a role for this receptor. Figure 9 shows a sim-ilar effect in the HL-60 cell line where AJA (JBT-101) is comparedwith THC that is less active and CBD that is inactive (Bursteinet al., unpublished data).

AJA (IP-751) also stimulates a product of the lipoxygenasebranch of the cascade lipoxin A4 (LXA4). Addition of AJA (0–30 lM) in vitro to human blood or synovial cells increased produc-tion of LXA4 2- to 5-fold.53 Administration of AJA (IP-751) to micewith peritonitis resulted in a 75% reduction of cells invading theperitoneum, and a 7-fold increase in LXA4 identified by mass spec-trometry. Blockade of the 12/15 LOX enzyme, which mediates LXA4

synthesis via 15-HETE production, reduced by >90% the ability ofAJA (IP-751) to enhance production of LXA4 in vitro. These results

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further suggest that AJA may be useful for conditions characterizedby chronic inflammation and tissue injury.

4.1.3. Actions on bladder irritation and hyperactivityEffects of AJA (IP-751) on bladder over activity induced by blad-

der irritation in rats have been reported.47,48 It can suppress nor-mal bladder activity and urinary frequency induced by bladdernociceptive stimuli, probably by suppression of bladder afferentactivity. These inhibitory effects of AJA (IP-751) are at least in partmediated by the CB1 receptor. These findings identify a potentialnew approach for the treatment of the painful bladder syndrome,interstitial cystitis.

4.1.4. In vitro and in vivo anti-neoplastic activityAJA (CT-3) was reported to be moderately active in inhibiting

growth in vitro against a variety of neoplastic cell lines.54 It was

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more effective than its enantiomer, HU-235, suggesting receptormediation. Moreover, CB2 but not CB1 receptor antagonistsblocked its effects. At a dose of 0.1 mg/kg, it inhibited modestlybut significantly the growth of tumors in nude mice that were sub-cutaneously implanted with U87 human glioma cells. Interestingly,the in vitro effects on cells were accompanied by the developmentof lipid droplets consisting of di and tri glycerides. The significanceof these lipids is not immediately apparent.

A subsequent experiment using a SCID-NOD mouse flank tumormodel again showed anti-neoplastic efficacy for AJA (CT-3) at adose of 0.1 mg/kg (Recht and Salmonsen, unpublished data). Onday 28 post inoculation, the vehicle treated group had a 1/5 un-healthy survivor, whereas treated mice had 3/5 tumor bearinghealthier looking survivors. More importantly, the vehicle treatedcontrol group all showed evidence of malignant ascites suggestingmetastatic disease while the AJA treated group did not developascites and generally appeared healthier. A major problem inoncology is the lack of effective ways to control metastatic disease.These preliminary findings suggest that AJA should be furtherinvestigated for possible use in this type of treatment.

4.1.5. Anti-fibrotic effectsIt has been recently reported that AJA (JBT-101) prevents pro-

gression of fibrosis in vivo and inhibits fibrogenesis in vitro bystimulating PPAR-c signaling.55 Its efficacy in pre-establishedfibrosis was studied in a modified model of bleomycin-induceddermal fibrosis and in mice over expressing a constitutively activetransforming growth factor beta (TGFb) receptor. A reduction inskin thickness and development of skin fibrosis following oraladministration of AJA (1 mg/kg) was compared to control levels(p < 0.001) with mock treated mice. They also observed that AJA(JBT-101) stimulated the expression of PPAR-c in dcSSc fibroblasts(diffuse cutaneous systemic sclerosis) from patients, increasing theproduction of an endogenous anti-inflammatory ligand PGJ2. It alsosignificantly decreased collagen neosynthesis by sclerodermafibroblasts in vitro, an effect that was reversed by co-treatmentwith the selective PPAR-c antagonist GW9662. Effective treatmentof fibrosis in scleroderma patients remains a major unmet medicalneed.

4.1.6. Multiple sclerosisA major problem for patients with the autoimmune disease

multiple sclerosis is the occurrence of spasticity. In an animal mod-el of this effect, AJA (CT-3) treatment at 0.1 m/kg iv resulted in arobust, visibly apparent reduction of spastic activity.56 Its activityat this dose was absent when the experiment was done in CB1receptor knockout mice. The authors concluded, ‘This provides fur-ther evidence for the peripheral nerve mode of action via CB1receptors’. They also studied the extent to which AJA crosses theblood brain barrier (BBB). AJA (CT-3) is a hydrophobic moleculewith a calculated log BBB brain/blood coefficient of 0.46, which isgreater than would predict CNS penetration. Cyclosporine A is awidely used immunosuppressant agent that also reduces the ac-tion of the BBB. Significant cannabimimetic effects were inducedby AJA (CT-3) following pretreatment with cyclosporine A(50 mg/kg iv) that included hypothermia and visible sedation.AJA (CT-3) induced hypothermia was studied in a series of mousestrains. Based on genetic considerations, it was concluded thatAJA is a ‘CNS-excluded cannabinoid CB1 receptor agonist’ and is amember of a novel class of agents that are safe in humans.

4.1.7. Suppression of osteoclastogenesisOsteoclasts are large multinucleated cells formed by the fusion

of hematopoietic precursors of the monocyte/macrophage lineage.In many inflammatory diseases such as periodontal disease, rheu-matoid arthritis, and metastatic cancers, excess osteoclast activity

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leads to bone resorption. Addition of AJA (IP-751) to stimulatedprecursor mouse macrophages and bone marrow cells in culturesuppresses development of multinucleated osteoclasts (osteo-clastogenesis), and prevents further osteoclast formation in cul-tures in which osteoclastogenesis had already begun.57 Thus,reduction of osteoclastogenesis may be an additional mechanismwhereby AJA prevents bone erosion in joints of rats with adjuvantarthritis.

4.1.8. In vitro metabolism and metabolic effectsPlasma samples obtained from a clinical study on 21 patients

suffering from neuropathic pain with hyperalgesia and allodynia37

were analyzed for AJA and its glucuronide. For example, plasmalevels of 599�37.2 ng/ml (mean ± R.S.D., n = 9) AJA were obtainedfor samples taken two hours after the administration of an oraldose of 20 mg AJA (CT-3). The mean AJA glucuronide concentrationat two hours was 63.8�128 ng/ml.58 No other metabolites werereported.

The in vitro metabolism of AJA (IP-751) by cryopreserved hepa-tocytes from rats, dogs, cynomolgus monkeys and humans wasstudied in which it was shown that only minor amounts of bio-transformation products were observed.59 These were primarilyside chain hydroxylation metabolites and glucuronides. Un-changed amounts of AJA detected after a two hour incubation were103%, 90%, 86% and 83% for rat, dog, monkey and human hepato-cytes, respectively. AJA (IP-751) also showed low inhibitory activ-ity against several human cytochrome P450 isozymes, CYP1A2,CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5, using human hepaticmicrosomes as the test system. These data suggest that AJA woulddisplay a high safety profile when administered to human subjects.

4.2. HU-235: a putative metabolite of dexanabinol

The S.S enantiomer of AJA, HU-235, was tested for analgesic andanti-inflammatory activity.35 This compound is a putative metabo-lite of dexanabinol so that its activities may have significance forthe actions of dexanabinol in vivo. It was observed to have moder-ate efficacy in the mouse hot plate and paw edema assays, how-ever, these findings are questionable. A subsequent chiralanalysis of this sample of HU-235 revealed that it was contami-nated with approximately 20-30% of AJA (Atlantic Pharmaceuticals,unpublished data), suggesting that the AJA was responsible for theobserved activities.

4.3. (�)-CBD-11-oic acids and their dimethylheptyl (DMH)homologs

A major, non-psychotropic constituent of Cannabis is cannabi-diol (CBD) that is similarly metabolized to give CBD-11-oic acid(Fig. 4A). This compound, its dimethylheptyl analog (HU-320,Fig. 5C) and their enantiomeric isomers were synthesized and theircannabinoid receptor binding affinities measured.60 The resultsfrom the binding experiment were surprising in that, as expected,the isomers with the R,R ‘natural’ stereochemistry were inactivehowever, unexpectedly, the S,S ‘unnatural’ isomers displayed sig-nificant affinity for CB1. No explanation for this unusual observa-tion was offered. HU-320 was studied for its anti-arthriticpotential in mice at doses of 1 and 2 mg/kg.61 The findings weresimilar to those reported earlier for AJA (Section 4.1.2) further sup-porting the hypothesis that carboxylic acid derivatives of cannabi-noids are biologically active substances with potential therapeuticapplications (Section 3.3). A comparison between HU-320, AJA andD9-THC in the mouse tetrad assay42 was done with all compoundsa single dose of 20 mg/kg in which AJA responded in similarfashion to D9-THC. This dose is 200 times that needed for a potentanti-arthritic effect as previously reported.36 Thus, the results of

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the tetrad assay in this study are probably not of any consequencefor possible therapeutic applications contrary to the conclusionreached by the authors.

4.4. Comparison of AJA with arachidonic acid

A comparison of the structure of AJA (Fig. 10B) with the longchain fatty acid arachidonic acid (Fig. 10A) gives rise to some inter-esting speculations. If the two structures are superimposed in atwo dimensional representation, a considerable overlap can result(Fig. 10C). This suggests that AJA may serve as a surrogate for ara-chidonic acid in a variety of biological events. For example, AJA hasa profound effect on the activity of COX-2 where arachidonic acidis the principle substrate. Another example is the action of PPAR-cwhere it was shown that AJA binds to62 and activates63 this impor-tant member of the nuclear receptor super family. AJA may alsosubstitute for some of the metabolites of arachidonic acid suchas the prostaglandins and the lipoxins. For example, LXA4

(Fig. 10D) has been shown to be a potent mediator in the resolutionof chronic inflammation.53,64 A second important example is PGJ2

(Fig. 10E).

5. Endogenous counterparts

An endogenous subfamily of eicosanoids, the lipoamino acids,represents possible counterparts to the cannabinoid acids. Thesesubstances are conjugates of long chain fatty acids and a-amino

A.

B.

C.

D.

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D. Lip

. Pr

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Figure 10. A comparison of the structure of AJA with the structure of arachidonicacid. A–C depicts the similarities in structure between arachidonic acid and AJA. Thestructures of lipoxin A4 and PGJ2 are shown for comparison purposes.

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acids joined by an amide linkage. They produce a variety of biolog-ical actions including the resolution of inflammation and analgesicresponses in preclinical models. The most widely studied memberof this family is N-arachidonoyl glycine (NAgly, Fig. 11), which issimilar in structure to anandamide (arachidonoyl ethanolamide),a CB1/CB2 agonist. In fact, the two are metabolically interrelated;oxidation of the hydroxyl group of anandamide leads to NAgly.65,66

This ‘endogenous counterpart’ hypothesis is further supported bystructural overlay considerations between AJA and arachidonicacid (Fig. 10C). In addition, a number of activities of the two areshared, for example, low affinity for the CB1 receptor. Several as-pects of this topic have been previously reviewed.67,68

5.1. Lipoamino acids (elmiric acids, EMA)

The lipoamino acids, which we have termed elmiric acids,67 areemerging as an important family of endogenous signaling mole-cules69 that may act as physiological regulators of pain and inflam-mation.70 The existence of these endogenous substances was firstpredicted more than 16 years ago65 when several examples weresynthesized and shown to exhibit anti-inflammatory and analgesicactivity in mice. Subsequent studies identified several naturalEMAs in rat brain extracts.71 They may be thought of as a branchof the eicosanoid super family with whom they share severalfeatures.

5.2. Occurrence, metabolism and tissue distribution of thelipoamino acids

The prototypic EMA, NAgly, shown in Figure 11, is found in ratbrain, spinal cord, and other tissues where it occurs in amountsgreater than the closely related endocannabinoid, anandamide.71

The rank order of dry weight tissue content in the rat is: spinalcord > small intestine >> kidney > glaborous skin >> brain > tes-tes > lung > liver > blood > spleen > heart. The values were ob-tained by LC/MS selected ion monitoring using lyophilizedsamples of fresh tissue samples.

Tan et al. used a targeted lipidomics approach with specificenrichment steps, nano-LC/MS/MS and high-throughput screeningof the data sets to measure the endogenous levels of lipoaminoacids in rat brain.72,73 They identified 50 different lipoamino acidsthat were present at 0.2 to 69 pmol/g of wet rat brain. They ob-served that in compounds with the same amino acid group, thosewith palmitoyl, stearoyl, and oleoyl groups were more abundantthan those with arachidonoyl or docosahexaenoyl groups. Thismatches the abundance of these fatty acids either as free acids oras phospholipids, suggesting that coupling of fatty acids with ami-no acids is a general route for lipoamino acid biosynthesis.

Evidence has been published showing that cytochrome c cata-lyzes the synthesis of NAgly from arachidonoyl coenzyme A andglycine in the presence of hydrogen peroxide.74 Other heme-con-taining proteins, hemoglobin and myoglobin, were significantly

Figure 11. The structure of NAgly. There are three regions of the molecule that areof pharmacological interest. Region 1 confers a high degree of specificity of action.Polyunsaturated residues produce molecules with analgesic and anti-inflammatoryaction whereas saturated structures, in this action, are inactive. Region 2 is relatedto metabolic stability since NAgly is degraded by FAAH (fatty acid amide hydrolase)activity. Region 3, the amino acid residue, can have an effect on the analgesic andanti-inflammatory activities depending on steric factors and the chiral nature of theamino acid.4

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less effective in promoting the synthesis of NAgly compared tocytochrome c. The formation of N-arachidonoyl serine, N-arachido-noyl alanine, and N-arachidonoyl-c-aminobutyric acid from arach-idonoyl CoA and the respective amino acids mediated bycytochrome c was reported.75 Interestingly, arachidonoyl CoAand ethanolamine were found to react spontaneously to formanandamide, independent of cytochrome c and hydrogen peroxide.

COX-2 selectively metabolizes NAgly to N-PGH2gly and N-hydroxyeicosatetraenoic-gly.76 Site-directed mutagenesis identi-fied the side pocket residues of COX-2, especially Arg-513, as crit-ical determinants of the COX-2 selectivity towards NAgly. Thesefindings suggest a possible role for COX-2 in the regulation ofNAgly synthesis and demonstrate the formation of a novel classof eicosanoids from NAgly metabolism. The lipoamino acids wereefficiently oxygenated by 12S- and 15S-lipoxygenases.77 This sug-gests that fatty acid oxygenases may play an important role in themetabolic inactivation of lipoamino acids or may convert them tobioactive derivatives.

5.3. Actions

The widespread occurrence of NAgly both in species and tissuesites within a given species suggests that it may have a variety ofphysiological and pharmacological effects. A summary of the ac-tions of NAgly is shown in Table 2. Some of the actions shown inthe table are discussed in more detail further on as are other ac-tions not shown in Table 2. There is some evidence that there areseveral proposed mechanisms that can help explain this multiplic-ity of actions. One of these relating to anti-inflammatory effects isdiscussed in Sections 5.4 and 6.

5.3.1. Analgesic effects of NAglyA preliminary report on the actions of NAgly was made in

1997.65 At that time, an increase in withdrawal time in the mousehot plate assay at 55 �C and a lack of effect in the mouse ring testfor catalepsy was observed. The latter indicated a major differencewhen compared with the closely related endocannabinoid ananda-mide and suggested that CB1 was not a target for NAgly. A morecomplete study was subsequently published supporting the earlierfindings and further suggesting that NAgly was an oxidativemetabolite of anandamide.78

Peripherally administered NAgly at 275 nmol was 50% effectivein suppressing phase 2 (tonic pain phase) of formalin-induced painbehavior.71 This suggests that NAgly can suppress formalin-in-duced hyperactivity in nociceptive afferents either directly on the

Table 2Reported actions of NAgly

Observed response Potency

Formalin-induced pain suppression 275 nmolInhibits cAMP production 10 nMResolution of inflammation 3 lMCell migration 100 nMMouse peritonitis inhibition 0.3 mg/kgInhibition of glycine transport 3.4 lMInduction of cell migration. 44 nMMacrophage apoptosis 10 lMModulates synaptic transmission 30 lMRelaxant in mesenteric arteries 30 lMInhibition of T-type Ca channels 1 lMNeuropathic pain model 700 nMRegulation of anandamide levels in vitro & in vivo 10 mg/kg & 10 lMStimulate PGJ production 3 lMInhibition of FAAH activity 8.5–50 lMIncreased [Ca++]i 10 lMInhibits store-operated Ca++ 10 lMEndothelial electrical signaling 3 lMReduces intraocular pressure 1% topical

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nerve, or indirectly by modulating their immediate interstitialenvironment. It was concluded that suppression of formalin-in-duced pain by NAgly might have relevance to postoperative andchronic pain states. A study of NAgly in a rat model of neuropathicpain, namely, intrathecal administration of NAgly (700 nmol) wasreported to reduce the mechanical allodynia induced by partialligation of the sciatic nerve.79 They also observed that HU-210,but not NAgly, produced a reduction in rotarod latency. It was sug-gested that NAgly might provide a novel approach to alleviate neu-ropathic pain. The actions of NAgly on neurons within thesuperficial dorsal horn, a key site for the actions of many analgesicagents were examined.80 The study suggested that NAgly enhancedinhibitory glycinergic synaptic transmission within the superficialdorsal horn by blocking glycine uptake via GLYT2. NAgly also de-creased excitatory NMDA-mediated synaptic transmission. Theysuggested that the study provided a cellular explanation for thespinal analgesic actions of NAgly.

5.3.2. Anti-inflammatory actions of NAglyReports from our laboratory suggest that NAgly reduces re-

sponses to inflammatory stimuli in both in vitro and in vivo mod-els.67,70,81 A mini library of lipoamino acids (elmiric acids) wassynthesized and evaluated for activity as potential anti-inflamma-tory agents in which prostaglandin production was compared witheffects on an in vivo model of inflammation (Table 3). LPS stimu-lated RAW 267.4 mouse macrophage cells were the in vitro modeland phorbol ester-induced mouse ear edema served as the in vivomodel. The prostaglandin responses were found to be stronglydependent on the nature of the fatty acid part of the molecule.Polyunsaturated acid conjugates produced a marked increase inmedia levels of PGJ2 with minimal effects on PGE production. Itis reported in the literature that prostaglandin ratios in whichthe J series predominates over the E series promote the resolutionof inflammatory conditions.82 Several of the elmiric acids testedproduced such favorable ratios suggesting their potential anti-inflammatory activity. The ear edema assay results were generallyin agreement with the prostaglandin assay findings indicating aconnection between them (Table 3). The second study was in amouse peritonitis model involving migration of pro inflammatorycell types into the peritoneal cavity following a challenge with thi-oglycollate.81 NAgly reduced migration by 60% at a dose of 0.3 mg/kg administered orally. Evidence was presented suggesting thatGPR18 played a significant role in this therapeutic response.

The nature of the fatty acid is a dominant factor in anti-inflam-matory activities both in vitro and in vivo since increasing

Model Source

Rat formalin test 71GPR18-transfected CHO cells 96RAW 264.7 cells 81BV-2 microglia 98Leukocyte migration 81Endogenous inhibitor of GLYT2 112Human endometrial HEC-1B cells 99RAW 264.7 cells 113Rat spinal cord slices 80Activation of BK (Ca++) channels 90TsA-201 cells 114Paw withdrawal threshold 79RAW 264.7 cells and rat blood 84RAW 264.7 cells 70FAAH preparations 83Rat primary b-cells 87EA.hy926, INS-1 832/13, RBL-2H3 cell lines 88EA.hy926 cells 91Mouse eye 103

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Table 3In vivo versus in vitro anti-Inflammatory effects (Burstein et al., unpublished)

Test molecule PGJ2 activitya (% Control) Activity in ear edema assayb

Palmitoyl glycine (PALgly) 100 0Oleoyl 1,1-dimethyl glycine 118 +c-Linolenoyl glycine (LINgly) 203 ++Arachidonoyl glycine (NAgly) 213 ++Arachidonoyl L-alanine 212 ++Arachidonoyl 1,1-dimethyl glycine 220 +++

a Stimulation of PGJ2 synthesis by 10 lM compound in mouse macrophage RAW cells.b Relative potency in inhibiting phorbol ester-induced ear swelling in mice. Compounds applied topically in safflower oil at 1 mg/ml.

10 S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx

unsaturation results in greater effects in both models (Table 3).Interestingly, the steric volume of the amino acid, in going fromglycine to 1,1-dimethylglycine does not seem to appreciablychange the responses in either model.

5.3.3. Inhibition of FAAH activityNAgly is a potent inhibitor of the fatty acid amide hydrolase

(FAAH), the enzyme primarily responsible for the degradation ofthe endocannabinoid N-arachidonoyl-ethanolamine (ananda-mide).83 A mini library of several N-arachidonoyl-amino acidswas synthesized including the D and L isomers of N-arachidonoylalanine. These were tested for inhibitory activity on FAAH prepara-tions from mouse, rat, and human cell lines, and from mouse or ratbrain. NAgly was the most potent compound on the rat and mouseenzymes whereas N-arachidonoyl isoleucine was active only onhuman FAAH. The data suggest that an increase in anandamide lev-els may, in part, account for the analgesic effects of NAgly in ro-dents. Data from another study suggested that it may serve as anendogenous regulator of tissue anandamide concentration.84

5.3.4. Other actions of NAglyPalmitoyl glycine (PALgly); Arachidonoyl glycine (NAgly); Pal-

mitoyl L-alanine (PAL-L-ala); Arachidonoyl L-alanine (NA-L-ala);Palmitoyl c-aminobutyric acid (PALgaba); Arachidonoyl c-amino-butyric acid (NAgaba) were tested in vitro for their effects on cellproliferation (Fig. 12).85 The results demonstrated a wide rangeof responses from 10% to 100% inhibition of proliferation. Boththe fatty acid and the amino acid regions of the molecule influ-enced the activity of the structures that were tested. No data onmechanism were reported.

The occurrence of lipoamino acids in mouse bone wasreported.86 N-oleoyl-L-serine (OS) had the highest activity in anosteoblast proliferation assay and it was found that it triggers aGi-protein-coupled receptor and Erk1/2. It also promotes osteoclastapoptosis through the inhibition of Erk1/2 phosphorylation and the

Figure 12. Structural effects of lipoamino acids on rat basophilic leukemia (RBL-2H3) cell proliferation (Adapted from Burstein et al., 2008). Cells were treated withthe lipoamino acids (10 lM) followed by treatment with LPS (10 ng/ml). Cellnumbers were obtained using the CellTiter-Glo assay. Compounds studied were:Palmitoyl glycine (PALgly); Arachidonoyl glycine (NAgly); Palmitoyl L-alanine (PAL-L-ala); Arachidonoyl L-alanine (NA-L-ala); Palmitoyl c-aminobutyric acid (PALgaba);Arachidonoyl c-aminobutyric acid (NAgaba).

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receptor activator of nuclear-j-B ligand (RANKL) expression inbone marrow stromal cells and osteoblasts. In intact mice, OS mod-erately increases bone volume density mainly by inhibiting boneresorption. Moreover, in a mouse ovariectomy model for osteopo-rosis, OS efficiently rescues bone loss by increasing bone formationand markedly limiting bone resorption.

A primary beta-cell-based functional assay to monitor intracel-lular Ca++ flux ([Ca++]i) was used to screen an assortment of com-pounds, leading to the discovery of three novel insulinsecretagogues: NAgly, 3b-(2-diethylamino-ethoxy) androstenonehydrochloride (U18666A) and 4-androstene-3,17-dione.87 NAglyincreased [Ca++]i through stimulation of the voltage-dependentCa++ channels that was dependent on the extracellular glucose le-vel. It was suggested that NAgly might be useful for the develop-ment of insulinotropic factors and drugs for treating type 2diabetes.

NAgly inhibits store-operated Ca++ entry (SOCE), a ubiquitousCa++ entry pathway regulating multiple cellular functions, in atime- and concentration-dependent manner.88 The inhibitory ac-tion on SOCE was found in the human endothelial cell lineEA.hy926, the rat pancreatic beta-cell line INS-1 832/13, and therat basophilic leukemia cell line RBL-2H3. These findings demon-strated the STIM1/Orai1-mediated SOCE machinery as a moleculartarget of NAgly that might have multiple implications in cellphysiology.

The levels of the signaling lipids anandamide, 2-arachidonoylglycerol, N-arachidonoyl glycine, N-arachidonoyl-c-amino butyricacid, and N-arachidonoyl dopamine in seven different brain areas(pituitary, hypothalamus, thalamus, striatum, midbrain, hippo-campus, and cerebellum) in male rats, and in female rats at five dif-ferent points in the estrous cycle were measured.89 The reportprovides a basis for an understanding into how differences in gen-der and hormonal status may influence mechanisms regulatingendocannabinoid production.

NAgly produces vasorelaxantion in rat small mesenteric arteriespredominantly via activation of BK (Ca++) channels.90 The data ob-tained in this study suggest that NAgly activates an unknown G(i/o)-coupled receptor that stimulates endothelial release of nitricoxide, which then activates BK (Ca++) channels in smooth muscle.It was also concluded that NAgly might activate BK (Ca++) channelsthrough G(i/o)- and nitric oxide-independent mechanisms.

A complex response relationship between the amino acid andfatty acid groups in the inhibition of cancer cell proliferation wasobserved.85 Thus, a robust inhibition was seen when arachidonoylglycine or gaba derivatives were tested; however, the palmitoylanalogs were completely inactive (Fig. 12). This is similar to the ef-fects seen in the anti-inflammatory studies (Table 3). In sharp con-trast, the L-alanine derivatives gave a reverse order of activity thatis, palmitoyl-L-ala was very active whereas arachidonoyl-L-ala wasonly moderately active. The reason for this anomaly is not imme-diately apparent.

The actions of NAgly on endothelial electrical signaling in combi-nation with vascular reactivity were recently reported.91 The data

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Table 4Stimulation of Prostaglandin J2 levels in mouse macrophage RAW 264.7 cells

Treatment Dose (lM) PGJ2 (pg/ml) SD

N-linoleoylglycine 1 <200 —N-linoleoylglycine 2.5 1843 431N-linoleoylglycine 5 3907 293N-linoleoylglycine 10 7315 1444N-arachidonoyl glycine 10 6847 625DMSO — <200 —

S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx 11

identified NCX (Na+/Ca++ exchanger) as a Ca++ entry pathway inendothelial cells and NAgly as a potent G-protein-independentmodulator of endothelial electrical signaling. The study also re-vealed a dual effect of NAgly on endothelial electrical responses. Itwas concluded that in agonist pre stimulated cells, NAgly opposeshyperpolarization and relaxation via inhibition of NCX-mediatedCa++ entry, while in unstimulated cells it promotes hyperpolariza-tion via receptor-independent activation of BKCa channels.

Lipoxin A4 has been proposed to be an important mediator inthe resolution of chronic inflammation93 Figure 13 shows a robuststimulation of LXA4 resulting from exposure to NAgly. An analogwith no anti-inflammatory activity PALgly had no effect on LXA4

synthesis. In Section 4.1.2, a similar effect is described for AJAwhere blockade of the 12/15 LOX enzyme was also reported to re-duce LXA4 levels.

5.3.5. Other lipoamino acidsN-palmitoylglycine (PALgly) was partially purified from rat lipid

extracts and it occurs in high levels in rat skin and spinal cord.94

PALgly levels were increased in fatty acid amide hydrolase (FAAH)knockout mice, suggesting enzymic regulation. PALgly has no anti-inflammatory actions, however, it potently inhibited heat-evokedfiring of nociceptive neurons in rat dorsal horn and also inducedtransient Ca++ influx in native adult dorsal root ganglion (DRG)cells and a DRG-like cell line (F-11). The nonselective calciumchannel blockers ruthenium red, SK&F96365, and La+++ blockedPALgly-induced Ca++ influx. It also stimulated the production of ni-tric oxide through calcium-sensitive nitric-oxide synthase en-zymes present in F-11 cells and was inhibited by the nitric-oxidesynthase inhibitor 7-nitroindazole.

N-linoleoyl glycine (LINgly) was tested in the mouse peritonitisassay where it showed activity in reducing leukocyte migration atdoses as low as 0.3 mg/kg.92 Peritoneal cells from analog treatedmice produced elevated levels of 15-deoxy-D13,14-PGJ2 that seemedto correlate with a decrease in peritoneal cell number. A small groupof N-linoleoyl analogs was studied for their ability to stimulate 15-deoxy-D13,14-PGJ2 production in mouse macrophage RAW cells (Ta-ble 4). The d-alanine derivative was the most active while the D-phenylalanine showed almost no response. A high degree of stereospecificity was observed when comparing the D and L alanine iso-mers; the latter being the less active suggesting that the responseis receptor mediated. LINgly is a more stable molecule than NAglyso that it might be a good substitute for the latter in future studies.

5.4. Mediation of NAgly actions by GPR-18

NAgly possesses analgesic properties but lacks the psychotropicactivity of cannabinoids such as THC or anandamide. It has been

Figure 13. Stimulation of lipoxin A4 levels in mouse macrophage RAW 264.7 cells(Burstein et al., unpublished). Cell culture and treatment conditions were aspreviously reported.81 Media were assayed for LXA4 using an enzyme Immunoassaykit. PALgly is included as a control.

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shown that NAgly has low affinity for the cannabinoid CB1 recep-tor,95 however, it appears to activate the orphan G-protein coupledreceptor (GPCR), GPR18.81,96 GPR18, a member of the GPCR superfamily, was discovered and its sequence reported in 1997.97 In asubsequent report, Kohno et al. de-orphanized GPR18 with theidentification of NAgly as a putative ligand based on functionalresponses.96 It was the only ‘hit’ out of a group of 200 bioactive lip-ids screened suggesting a high degree of structural requirementsfor functional activity. Saturable, high affinity binding has been ob-served (Fig. 14) that is consistent with the designation of NAgly asa ligand for GPR18 (Burstein et al., unpublished). There are addi-tional functional data supporting the ligand nature of NAgly and,possibly, a second molecule, called abnormal CBD.98

McHugh et al. found that NAgly is an extremely effective recrui-ter of BV-2 microglia and its effects can result in anti-inflammatoryactions in the brain.98–101 They reported that NAgly potently actson GPR18 to produce directed migration, cell proliferation and per-haps other MAPK-dependent actions. These results advance ourunderstanding of lipid-based signaling mechanisms employed bythe CNS to actively recruit microglia to sites of injury. The NAgly-GPR18 signaling pathway offers a novel approach for developingtherapeutic agents to elicit a population of regenerative microglia,or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia that contributes to and intensifies neuro-degenerative disease.

NAgly may also have an important role in peripheral sites. In asubsequent study from the same group, evidence was presentedthat NAgly, is effective in driving the migration of the human endo-metrial cell line, HEC-1B.102 They concluded that these data poten-tially explain the unusual results from previous studies in the fieldin which the effects of CB1 and CB2 were unable to account for theobserved phenomena in endometrial cells.

A recent study examined the occurrence and role of GPR-18 inthe anterior murine eye.103 GPR18 was expressed in the ciliary epi-thelium, the corneal epithelium and the trabecular meshwork.NAgly, was detected in the mouse eye at a level comparable to thatseen in the brain. It significantly reduced intraocular pressure in allof the mice tested following topical administration. Evidence wasobtained for a functional GPR18-based signaling system in themurine anterior eye, including receptors and ligands. The findingssuggested that GPR18 might serve as a target for the developmentof novel ocular hypotensive medications.

Further support for the role of GPR18 in the in vitro actions ofNAgly was obtained by the use of polyclonal antibodies as recentlyreported.81 It was found that treatment of the cells with 3 lMNAgly caused a 100% rise in PGJ concentrations in the media. Theaddition of a polyclonal antiGPR18 reduced this response by about50% suggesting that GPR18 is a mediator of this action. The furtheraddition of a GPR18 blocking peptide completely abolished theantibody action showing that this is a specific effect. These obser-vations also raise the possibility that antiGPR18 can be used toimplicate this receptor in other actions of the elmiric acids.

Using the b-arrestin PathHunter™ assay system, a newly devel-oped, generic GPCR assay format that measures b-arrestin bindingto GPCRs, an evaluation of receptor and ligand pairing for NAglyand GPR-18 was reported.104 They found no interaction between

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Figure 16. The PPAR-c inhibitor GW9662 reduces the stimulation of PGJ2 levels byNAgly in RAW cells (Burstein et al., unpublished data). Cells were cultured andtreated as previously reported.92

NAGLY [pM]

NA

gly

(p

mo

/l m

g p

rote

in)

0 100 200 300 4000

5

10

15

20

TBNSB

A

0 100 200 3000

1

2

3

4

5

NA

gly

(p

mo

l/mg

pro

tein

)

NAGLY [pM]

TB

B

Figure 14. Binding isotherm for NAgly using (A) GPR18 transfected HEK-293 cell membranes compared with (B) wild type HEK-293 membranes (Burstein et al.,unpublished). 25 mg of membranes were incubated in binding buffer (50 mM Tris, 0.5 mM EDTA, 0.1% BSA, pH 7.4) in the presence of increasing amounts of 3H-NAgly(specific activity 200 Ci/mmole). Membranes were filtered with a 96 well GF/B filter plate and washed with 500 ll wash buffer (25 mM HEPES, 1 mM CaCl2, 5 mM MgCl2,0.25 mM NaCl) using a Filtermate 196 Harvester. Filter plates were treated with 1% BSA prior to use to reduce non-specific binding. TB, total binding; NSB, non-specificbinding.

Figure 15. A proposed unified mechanism for the anti-inflammatory actions of AJAand NAgly.

12 S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx

the two in contradiction to all of the above reports showing theopposite. Another study showing GPR18 coupling in a native neu-ronal system with endogenous signaling pathways and effectorswas recently published.105 The addition of NAgly to GPR18-expressing neurons did not inhibit calcium currents but insteadpotentiated currents in a voltage-dependent manner. These resultssuggested to the authors that NAgly is not an agonist for GPR18.They concluded, ‘GPR18 signaling involves non canonical pathwaysnot examined in these studies’ and suggested that there may be ac-tions of NAgly that are mediated by it through such pathways.However, most of the experiments they reported were carriedout only at 10 lM NAgly casting doubt on the significance of thefindings. For example, several earlier reports showed inverted doseresponse relationships for NAgly102,81,68 suggesting that rather dif-ferent findings might have been obtained at lower concentrations.

In summary, the majority of evidence thus far reported sup-ports the existence of a NAgly-GPR18 mediated mechanism of ac-tion. However, when all of the reports are considered, thepossibility of more than one mechanism has been raised. Addi-tional studies are therefore needed for a better understanding ofthe molecular pharmacology of the lipoamino acids.

6. Putative mechanisms for the anti inflammatory actions ofAJA and NAgly

Although not complete, there are sufficient data to allow a pro-posed mechanism to be made for the anti-inflammatory actions ofAJA and NAgly (Fig. 15). In keeping with structural similarities(Section 4.4), there are also striking similarities in their mecha-nisms. The initial step would be the activation of one of three likelyGPCR receptors: CB1, CB2 or GPR18 (steps 1 and 2). There are datashowing that AJA does not bind significantly to CB1 but is a ligandfor CB2;�there is no evidence for AJA binding to GPR18. However, inthe case of NAgly, there is ample evidence for high affinity, satura-ble binding to GPR18 (Fig. 14), but, little is known about its effectson CB2. It was reported to have no meaningful affinity for CB1.95

There may be a role for PPAR-c in some of the actions of NAgly106

through a positive feedback step to PPAR-c.107 This possibility issuggested by the data shown in Figure 16.

NAgly induced an increase in intracellular Ca++ concentration inGPR18-transfected cells that was significantly greater than that inmock-transfected cells96 supporting step 2 of the proposed mech-anism. No such data are available for AJA. Activation of phospholip-ases is a well-known effect of increased intracellular Ca++ ionconcentration (step 3).108 With both AJA and NAgly this resultsin the mobilization and release of free fatty acids (Fig. 8 andTable 5) from their phospholipid storage sites; in particular free

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arachidonic acid (step 4), which is a substrate for a number ofeicosanoid metabolites. Step 5 indicates a selective transformationof arachidonic acid to PGD2 that is mediated by COX-2109 and PGD

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Table 5Stimulation of arachidonic acid release from RAW cells by NAglya (Adapted fromBurstein el al., 2008)

NAgly (lM) Arachidonic acid (Treated/Control) SD

1.5 1.0 0.213.0 1.2 0.136.0 1.4 0.32

12.0 1.85 0.3124.0 2.95 0.25

a Following a 2 h labeling period with 14C-arachidonic acid, the media(RPMI + 0.1% BSA) were changed and the cells treated for 1 h with NAgly in 10 ll ofDMSO. The control was 10 ll of DMSO. Release was measured by LSC on a 0.1 mlaliquot of medium. Values shown are the means ± SD. The cells were treated withnon-radioactive NAgly.

S. H. Burstein / Bioorg. Med. Chem. xxx (2014) xxx–xxx 13

synthetase.52 In an unregulated process,109 PGD2 is rapidly con-verted to PGJ2 (Fig. 9 and Table 4) a potent agent for the resolutionof chronic inflammation110 as shown in steps 7 and 8. An addi-tional metabolic pathway for free arachidonic acid is mediatedby lipoxygenases resulting in the production LXA4

53 that can alsoachieve a resolution of chronic inflammation as indicated in steps6 and 9. These mechanistic similarities indeed support the sugges-tion that the lipoamino acids are endogenous counterparts of thecannabinoid acids.

7. Conclusions

Following their discovery,10 the cannabiniod acids were deter-mined to be biologically inactive11 and for some time little was re-ported to dispute this claim. However, subsequent studies showedthey produce a number of physiological and pharmacologicalactions.1 These include anti-inflammatory activity, analgesia andinhibition of THC-induced catalepsy. The synthetic analogs showedadditional actions that include anti-neoplastic, anti-cystic, anti-fi-brotic, suppression of osteoclastogenesis and reduction of spastic-ity in multiple sclerosis.111 More recent reports describe a family ofendogenous counterparts of the acids that are structurally differentbut share many of the actions of the acids.67 Studies on mecha-nism(s) for these actions thus far show a lack of involvement ofCB1, thereby, explaining the absence of psychotropic activity forall of these substances. Data have been obtained for the involve-ment of CB2 and the orphan receptor GPR18. A role for the anti-inflammatory eicosanoids PGJ2 and LXA2 has been observed thatmay include a feedback step involving PPAR-c There is some evi-dence for inhibition of FAAH activity, which results in increasedlevels of anandamide. All of these findings indicate that potentand stable analogs would make good drug candidates for severalindications. Ajulemic acid is such a substance that is currentlyundergoing Phase 2 trials for the treatment of lupus, sclerodermaand cystic fibrosis. Further exploitation of the therapeutic potentialof the cannabinoid acids is likely to result in the discovery othercandidates and indications.

Acknowledgements

I am grateful to my many colleagues and assistants who overthe years have contributed much of the data shown here. The fol-lowing is a partial list; Robert B. Zurier, Lawrence Recht, Sheila A.Hunter, Lori Renzulli, Christian Fraioli, Catherine McQuain, RonaldRossetti, Rebecca Salmonsen, Boris Yagen, Irina Pekker, MatthiasKarst, Mark Tepper, Alonzo Ross, Li Li, Marie-Claire Daou. J. MichaelWalker, Heather Bradshaw, Susan Huang, Timothy Petros, RobertZipkin, Jeffrey Adams & John Shaw,

This publication was made possible by grant DA17969 from Na-tional Institute on Drug Abuse, National Institutes of Health,Bethesda, MD. Its contents are solely the responsibility of the

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authors and do not necessarily represent the official views of theNational Institute on Drug Abuse. Finally, thanks are due to Dr.Ethan S. Burstein for helpful discussions during the preparationof this manuscript.

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