Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 
https://doi.org/10.1186/s40360-021-00539-1
RESEARCH Open Access
Assessment of NSAIDs as potential
inhibitors of the fatty acid amide hydrolase
I (FAAH-1) using three different primary
fatty acid amide substrates in vitro
Julius T. Dongdem1,2* , Gideon K. Helegbe1 , Kwame Opare-Asamoah3 , Cletus A. Wezena4 and
Augustine Ocloo5
Abstract
Background: Pain relief remains a major subject of inadequately met need of patients. Therapeutic agents
designed to treat pain and inflammation so far have low to moderate efficiencies with significant untoward side
effects. FAAH-1 has been proposed as a promising target for the discovery of drugs to treat pain and inflammation
without significant adverse effects. FAAH-1 is the primary enzyme accountable for the degradation of AEA and
related fatty acid amides. Studies have revealed that the simultaneous inhibition of COX and FAAH-1 activities
produce greater pharmacological efficiency with significantly lowered toxicity and ulcerogenic activity. Recently, the
metabolism of endocannabinoids by COX-2 was suggested to be differentially regulated by NSAIDs.
Methods: We analysed the affinity of oleamide, arachidonamide and stearoylamide at the FAAH-1 in vitro and
investigated the potency of selected NSAIDs on the hydrolysis of endocannabinoid-like molecules (oleamide,
arachidonamide and stearoylamide) by FAAH-1 from rat liver. NSAIDs were initially screened at 500 μM after which
those that exhibited greater potency were further analysed over a range of inhibitor concentrations.
Results: The substrate affinity of FAAH-1 obtained, increased in a rank order of oleamide < arachidonamide <
stearoylamide with resultant Vmax values in a rank order of arachidonamide > oleamide > stearoylamide. The
selected NSAIDs caused a concentration-dependent inhibition of FAAH-1 activity with sulindac, carprofen and
meclofenamate exhibiting the greatest potency. Michaelis-Menten analysis suggested the mode of inhibition of
FAAH-1 hydrolysis of both oleamide and arachidonamide by meclofenamate and indomethacin to be non-
competitive in nature.
Conclusion: Our data therefore suggest potential for study of these compounds as combined FAAH-1-COX inhibitors.
Keywords: Arachidonamide, Affinity, FAAH-1, Hydrolysis, Oleamide, Arachidonamide, Stearoylamide, Inhibition,
NSAIDs, Mode
* Correspondence: julius.dongdem@uds.edu.gh; jtdongdem@gmail.com
1Department of Biochemistry and Molecular Medicine, School of Medicine,
University for Development Studies, Tamale-Campus, Tamale, Ghana
2School of Life Sciences, University of Nottingham Medical School, NG7 2UH
Nottinghamshire, UK
Full list of author information is available at the end of the article
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Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 2 of 11
Introduction COX cascade by a wide range of stimuli are prostaglan-
Several therapeutic agents have been designed to address dins (PGD2, PGE2, PGF2α and PGI2). AA embedded in cell
different forms of pain, yet pain relief remains an area of membranes as esters of phospholipids is the precursor of
significant unmet patient need [1, 2]. Drugs adminis- prostaglandins (PGs). AA is made available by action of
tered to treat pain and inflammation presently have low several enzymes including cPLA2/sPLA2, αβ Hydrolase 4
to moderate efficiencies with significant untoward side and GDE [24]. Once induced, COX, LOX and cytochrome
effects such as gastrointestinal bleeding, ulceration, renal P450 enzymes convert available AA to various eicosa-
dysfunction, nausea and vomiting. noids. These eicosanoids are known essential physiological
Fatty acid amide hydrolase I (FAAH-1) has been pro- and pathophysiological mediators implicated in a wide
posed as a promising target for the discovery of drugs to scope of therapeutic interest such as in inflammation,
treat pain, inflammation and other pathologies [3, 4]. pain, cancer, glaucoma, male sexual dysfunction, osteo-
FAAH-1 is the primary enzyme that is responsible for porosis, cardiovascular disease, labour, asthma, etc [25]
the degradation of N –Arachidonoyl ethanolamide Selected NSAIDs have also been reported to inhibit
(Anandamide, AEA) and related fatty acid amides which FAAH-1 activity from mouse and rat preparations [26].
constitute a group of biologically active endogenous am- Studies in animal models have revealed that the
ides [5, 6]. Inhibition of FAAH-1 results in the accumu- simultaneous inhibition of COX and FAAH-1 activities
lation of AEA and other endocannabinoid-like molecules produce greater pharmacological efficiency with
in the central and peripheral nervous systems where they significantly lowered toxicity and ulcerogenic activity as-
act as ligands of cannabinoid (CB1 and CB2) receptors. sociated with COX inhibitors [27, 28]. More recently,
Similar to Δ9 -tetrahydrocannabinol (THC), AEA is a the metabolism of endocannabinoids by COX-2 was sug-
partial agonist at both CB1 and CB2 transmembrane re- gested to be differentially regulated by NSAIDs resulting
ceptors - members of the G-protein-coupled receptor in antinociceptive effects mediated via cannabinoid re-
superfamily [7–9] however, in contrast to THC, AEA ceptors [29–32]. Apart from catalysing the formation of
also stimulates the transient receptor potential vanilloid PGs from AA, COX-2 also catalyses the formation of
receptor type 1 (TRPV1) [10–12]. AEA exhibits cannabi- prostaglandin-glycerol esters and prostaglandin ethanol-
mimetic effects at the cannabinoid receptors [13]. Palmi- amines from 2-arachidonoyl glycerol (2-AG) and AEA
toyl ethanolamide has also been reported to be active at respectively [30, 33–35]. Since COX-2 is a significant
peroxisome proliferator-activated receptors (PPARs) as target of NSAIDs, COX-2 inhibition can reduce this
well as vanilloid receptors. The primary fatty acid amides mechanism of endocannabinoid metabolism to enhance
(PFAMs) such as oleamide, arachidonamide, stearoyla- their concentrations in vivo [36, 37]. Moreover, rapid re-
mide, stearoyl ethanolamide, palmitamide, etc.) are also versible inhibitors of COX-2 selectively inhibit the oxy-
important molecules controlling sleep, angiogenesis, genation of 2-AG and AEA with much higher potencies
locomotion, convulsions and inhibition of gap junction for AA, a phenomenon referred to as substrate selective
formation among several other functions [14–18]. effect [30, 38]. The fact that selected NSAIDs inhibit
Although the major current strategy for drug develop- AEA and 2-AG metabolism via FAAH-1 and COX in-
ment is to design compounds that are selective for a hibition in vivo, suggests that at the appropriate concen-
given target, compounds that target more than one trations, NSAIDs may co-regulate the activity of both
biochemical process may have superior efficacies with COX and FAAH-1 enzymes which make them better
better safety profiles compared with standard selective suitable therapeutic agents [39, 40]. Since cannabinoids
compounds. This can be achieved by administering the possess anti-inflammatory, antinociceptive, analgesic,
drugs either separately or in single tablets made of more anti-tumour and immunosuppressive properties, inhibi-
than one active ingredient. The disadvantage in both tors of endocannabinoid degrading enzymes (FAAH-1,
cases is the potential for a large pharmacokinetic vari- FAAH-2, NAAA, COX-2, LOX, MAGL) may be of
ability that is equivalent to the concomitant administra- therapeutic significance via augmentation of endocanna-
tion of separate drugs. The alternative to avoid these binoid and endocannabinoid-like molecule accumulation
drawbacks is to develop drugs that target more than one in vivo. Based on this previous knowledge, it is essential
molecular mechanism [19]. Inhibition of COX-1 and -2 to conduct further investigations on the ability of other
at the first committed step of prostanoid and other NSAIDs to inhibit FAAH-1 deamination of endocanna-
eicosanoid biosynthesis from arachidonic acid (AA) binoid and endocannabinoid-like molecule substrates
underlies the analgesic action of non-steroidal anti- (e.g. oleamide, arachidonamide, stearoylamide and stear-
inflammatory drugs (NSAIDs) [20–23]. NSAIDs consti- oyl ethanolamide among others) for the reason that
tute a class of chemically diverse compounds that provide NSAIDs with both inhibitory capabilities (on COX and
analgesic, antipyretic and anti-inflammatory effects. The FAAHs) will synergistically enhance therapeutic effica-
fatty acid metabolic end-products of the induction of the cies. The aim of this study therefore, was to assess
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 3 of 11
pharmacological profiles of FAAH-1 with regards to po- after which 200 μl of each dilution was added to 1 ml of
tential substrates and inhibitors. The investigation was solution A (100 ml of 2% sodium carbonate and 1ml
specifically designed to assess the potency of selected each of both sodium potassium tartrate and copper
NSAIDs on the hydrolysis of oleamide, arachidonamide sulphate). The solutions were mixed and allowed to
and stearoylamide by FAAH-1. stand at room temperature. After 10 min, 100 μl of dilute
Folin Ciocalteau’s reagent 1:1 ddH2O was added and
Materials and methods mixed immediately. The absorbance of each sample was
FAAH-1 activity was studied in rat liver homogenate. read at a wavelength of 700 nm following incubation at
room temperature for 1 h. Relative absorbance of each
Preparation of rat liver homogenate sample was entered into GraphPad prism and analysed.
Liver obtained from male Wister rats (150–250 g, The protein concentration of preparations were interpo-
Charles River Laboratories, Wilmington, USA) which lated from the standard (Fig. SS1), using non-linear,
had been stored at − 40 °C was thawed. A volume of 6 second order polynomial (quadratic) graph of the
ml/g wet weight of rat liver was homogenized in 0.2M standards.
potassium phosphate buffer, pH 7.4 using a hand held
homogenizer (Ultra-turrax) (Merck KGaA, Darmstadt, Statistical analysis
Germany). The resulting mixture was centrifuged at 250 Data obtained were entered into a Microsoft Excel 2010
g for 10 min after which the pellet obtained was re- spread sheet and analysed with GraphPad Prism computer
homogenised and centrifuged as aforementioned. The software programme (GraphPad Software Inc., San Diego,
supernatants were combined and centrifuged at 20,000 g CA USA). Effect of 500 μM concentration of NSAIDs
for 30 min, after which the membrane containing pellet (SIGMA-ALDRICH, Poole, UK) on each enzyme activity
was re-suspended in 1:1w/v 0.2M potassium phosphate was analysed by removing the baseline line. Each specific
buffer, pH 7.4, and stored in 1 ml aliquots at − 40 °C. activity was then plotted as percentage of control. Specific
activity obtained at each inhibitor concentration for the
Assay of FAAH-1 activity concentration-inhibition curves were normalized and
FAAH-1 activity was assayed essentially as described analysed using the inbuilt log (inhibitor) versus response
previously [41]. Briefly, rat liver homogenate was pre- variable slope (robust fit) and were constrained at the bot-
incubated at 37 °C with shaking (50 × 10 rpm) for 10 min tom (= 0.0%). Each specific activity was then plotted as
in 0.2 M phosphate buffer, pH 7.4 in 96-well microtitre percentage of the control. To determine the mode of
plates (Thermo Scientific Inc., Waltham, USA) prior to inhibition, Vmax values were initially extrapolated from the
substrate addition and incubation at 37 °C for 30 min. (NH4)2SO4 standard curve plotted using the inbuilt
The 100 μl total assay reaction mixtures were halted second order polynomial (quadratic) Michaelis-Menten
with an equivalent volume of o-phthaldehyde (OPA) de- enzyme kinetics. These values were then adjusted using
veloping solution (0.4M potassium phosphate buffer, the protein concentrations of the preparations obtained
pH 11.5) and incubated further at 37 °C for 15 min be- from the Lowry protocol [42] (Fig. SS1 and SS2).
fore assessing fluorescence using a FLUOstar Galaxy
(Excitation 390 nm, Emission 450–10 nm) (BMG LAB- Results and discussion
TECH GmbH, Ortenberg, Germany). Substrate blank Affinity of oleamide, arachidonamide and stearoylamide
and a control containing 0.2M phosphate buffer, pH 7.4, at FAAH-1
were incorporated into the experiments. Several drugs are inhibitors of the most relevant en-
Subsequently, the influence of ethanol concentrations zymes since blocking these enzymes can kill a pathogen
on the ability of particular NSAIDs e.g. indomethacin or correct a metabolic imbalance. To characterise an en-
(SIGMA-ALDRICH, Poole, UK) was assessed by varying zyme in the presence of inhibitors however, a good kin-
the volume of inhibitor solution added, using both abso- etic description of its activity is essential. Here, the
lute ethanol and buffer blanks to account for back- ability of rat liver to hydrolyse oleamide, stearoylamide
ground influences on enzymatic activity. and arachidonamide was assessed by Michaelis-Menten
analysis (Fig. 2). The resultant Michaelis-Menten con-
Protein assay stant (Km) and maximum velocity (Vmax) values obtained
Homogenate protein content was measured by modifica- are summarized in Table 1. The substrate affinity of
tions of the method described [42] using 200 μl of differ- FAAH-1 increased in a rank order of oleamide < arachi-
ent concentrations of bovine serum albumin (0, 25, 50, donamide < stearoylamide with resultant Vmax values in
100, 150, 200, 300 μg/ml) as standard and 200 μl of 0.5 a rank order of arachidonamide > oleamide > stearoyla-
M NaOH as blank (Fig. SS1). Briefly, 50 μl of each mem- mide (Fig. 1, Table 1). The kinetic values for FAAH-1
brane fragment in 5ml of 0.5M NaOH was prepared, hydrolysis of oleamide obtained are consistent with
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 4 of 11
Table 1 Km and Vmax values determined for rat liver FAAH-1 hydrolysis of three different fatty acid amides
FAAH-1 kinetics Substrate
Oleamide Arachidonamide Stearoylamide
Km (μM) 177.2 ± 15.5 44.9 ± 7.0 4.6 ± 0.8
Vmax (nmol/min/mg protein) 8.9 ± 1.1 10.1 ± 3.0 2.5 ± 0.6
Data are mean ± SEM (Standard Error of the Mean) of four separate preparations (n = 4) conducted in triplicate
previous observations. Similar K and V values of and V of 8.9 ± 1.1 nmol.min− 1m max max .mg protein
− 1 obtained
129 μM and 15 nmol.min− 1.mg protein− 1 from oleamide in our findings (Table 1) [41]. An affinity of 104 μM and
hydrolysis by FAAH-1 in rat liver preparations and a Km a Vmax of 5.7 nmol.min
− 1.mg protein− 1 for rat liver
value of 179 μM with FAAH-1 in rat brain were previ- FAAH-catalysed oleamide hydrolysis has been reported
ously obtained compared with Km of 177.2 ± 15.5 μM [44]. Additionally, an affinity of 37 ± 7 μM at pH 9 for rat
Fig. 1 Hydrolysis of oleamide (a), arachidonamide (b) and stearoylamide (c) by rat liver FAAH-1 activity. Rat liver FAAH-hydrolytic activity of each
primary amide substrate in vitro, was assayed by quantification of ammonia released after hydrolysis. Ammonia generated in the presence of sulphite
ions is reacted with alkaline o-phthaldehyde (OPA) to generate the stable fluorescent isoindole derivative (1-sulphonatoisoindole) which is quantified
by fluorescent spectroscopy [41, 43]. Four separate experiments with three replicates on the same microtiter plate were conducted for each substrate
using different rat liver preparations. Data are mean ± SEM (Standard Error of the Mean) of four separate preparations (n = 4) conducted in triplicate
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 5 of 11
recombinant FAAH-catalysed oleamide hydrolysis has sulphone, indomethacin and dipyrone were moderate in-
also been reported [45]. hibitors of oleamide hydrolysis and inhibited FAAH-1
FAAH-1 has the ability to hydrolyse a wide range of activity to between 50 and 70% of control. Tolmetin, sal-
unsaturated and, to a lesser extent, saturated PFAMs icyluric acid, salicylic acid (diluted in 0.2M potassium
and other fatty acids e.g. oleamide and palmitoyl ethano- phosphate buffer) evoked weak inhibitory ability of
lamide [46, 47]. In our findings, FAAH-1 capacity (Vmax) FAAH-1 activity to between 70 and 100% of control.
was 12% higher for arachidonamide compared with olea- Acetaminophen and acetyl salicylic acid appeared to en-
mide and 75% higher than that for stearoylamide. This hance enzyme activity.
confirms the propensity of FAAH-1 to turn over polyun- Acetaminophen is reported to be metabolised to N-
saturated PFAMs particularly with cis double bonds at arachidonoylaminophenol (AM404) via FAAH-1 [50].
higher rates than monounsaturated and saturated AM404 then inhibits FAAH-1 activity and prevents AEA
PFAMs and is consistent with literature (Fig. 1) [48, 49]. metabolism. Thus, FAAH-1 is active until concentra-
tions of AM404 are high enough to inhibit its function.
Screening of NSAIDs as potential inhibitors of oleamide, AEA accordingly activates platelets, however, the process
arachidonamide and stearoylamide hydrolase activity is unaffected by acetyl salicylic acid, thus it is possible it
Following pilot experiments that revealed indomethacin did not affect rat liver FAAH-1 activity [51]. The differ-
to have an IC50 ~ 500 μM, 16 selected NSAIDs were ences in reaction of FAAH-1 to specific compounds (e.g.
screened at 500 μM (Fig. 2) for ability to inhibit FAAH-1 ketorolac or ibuprofen) might be due to differences in
in order to assess pharmacological profiles of rat liver structures, their sites of binding to FAAH-1 and how
FAAH-catalysed hydrolysis of the three PFAMs assayed this affects substrate entry and binding at the catalytic
at a concentration ≥ Km value determined [41, 43]. NSAI sites [52–55].
Ds were randomly selected based on availability and
considering what had not been reported while using a Effect of vehicle controls on FAAH activity
few that had been reported against FAAH-1 as reference As the NSAIDs are differently soluble in aqueous com-
standards. Meclofenamic acid exhibited complete inhib- pared to organic solution, the effect of a range of con-
ition of FAAH-1 activity when oleamide was used as centrations of the vehicle ethanol was assessed using
substrate. Sulindac, diclofenac, carprofen, ketorolac and indomethacin as a reference compound. Indomethacin
diflunisal exhibited a higher degree of inhibition of rat evoked a concentration-dependent inhibition of FAAH-1
liver FAAH-1 activity by inhibiting oleamide hydrolysis activity in pIC50 values between 15, 20 or 25% ethanol
to below 50% of control (Fig. 2). Ibuprofen, sulindac concentrations (Fig. 3). Tukey’s multiple comparisons
Fig. 2 Effect of 500 μM concentration of NSAIDs on rat liver FAAH-1 oleamide hydrolase activity. Data are mean ± SEM (Standard Error of the
Mean) of four separate preparations (n = 4) conducted in triplicate
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 6 of 11
Fig. 3 Effect of 15, 20 and 25% ethanol on the inhibition of rat liver oleamide hydrolase activity by indomethacin. Data are mean ± SEM
(Standard Error of the Mean) of four separate preparations (n = 4) conducted in triplicate
test with single pooled variance, p = 0.7250, p < 0.05 as assayed exhibited very similar potencies (pIC50 values)
significantly different, CI = 95% indicated no significant against activity of FAAH-1. The inhibition exhibited by
difference between pIC50 values obtained (Table 2). This the selected NSAIDs to FAAH-1 activity (Fig. 4, Table 3)
implies that, within the experimental limits, ethanol had is consistent with earlier studies although under different
no effect on the inhibitory function of indomethacin, conditions [26, 39, 56, 57]. The rank order of potency
albeit with a reduced capacity for basal oleamide displayed by NSAIDs screened at 500 μM was not
hydrolysis of 95 ± 1, 78 ± 1 and 76 ± 4% of control for exactly the same when the pIC50 values were examined.
15, 20 and 25% assay ethanol respectively consistent Earlier findings indicate that NSAID inhibition of
with earlier reports that butanol reduced FAAH-1 FAAH-1 activity is pH dependent [58] with a pH
activity by 30 to 50% but did not affect the enzyme optimum of ~ 9 [46, 59–63]. The rank order of NSAIDs
response to inhibitors [40]. reported for potency against rat brain FAAH-1 activity
at pH 7.4 was; indomethacin (pIC50 = 4.18) ≈ carprofen
Concentration-dependence of rat liver FAAH-1 oleamide (pIC50 = 4.10) > ibuprofen (pIC50 = 3.1) and is similar to
hydrolase inhibition our findings however, indomethacin was less effective
NSAIDs selected on the basis of the greater levels of than carprofen and more potent than ibuprofen [52].
inhibition at 500 μM were examined over a range of Other studies found apparently biphasic pH dependence
concentrations in absolute ethanol, from 4.0 × 10− 6 to of FAAH AEA metabolism using brain microsomes [64].
1.024 × 10− 3 M (Fig. 4). These exhibited concentration-
dependent inhibition of FAAH-1 oleamide hydrolase ac- Mode of inhibition of FAAH-1 metabolism by
tivities. The order of inhibitory potency against rat liver meclofenamic acid and indomethacin
FAAH-1 hydrolysis of oleamide was sulindac > carprofen To date, little has been reported on the mode of inhibition
> meclofenamic acid > sulindac sulphone > indometh- of NSAIDs on FAAH-catalysed hydrolysis of endocanna-
acin > diflunisal > ibuprofen > valdecoxib > ketorolac > binoids and endocannabinoid-like molecules [26, 52].
diclofenac > dipyrone (Table 3). The remaining NSAIDs Hence, meclofenamic acid and indomethacin were se-
lected for further mechanistic investigation as the former
evoked the greatest inhibition and the latter has previously
Table 2 Potency of indomethacin in the presence of different been examined extensively in the literature [26].
concentrations of ethanol Michaelis-Menten analysis indicated no significant
15% EtOH 20% EtOH 25% EtOH changes in substrate affinity (Km) values but with de-
pIC50 3.4 ± 0.1 3.5 ± 0.1 3.4 ± 0.1 creasing Vmax values (Fig. 5, Table 4), thus indicative of
FAAH-1 activity (%) 95 ± 1 78 ± 1 76 ± 4% non-competitive type inhibition of FAAH activity by the
Data are mean ± SEM (Standard Error of the Mean) of four separate preparations two inhibitors (meclofenamic acid and indomethacin).
(n = 4) conducted in triplicate This finding is consistent with similar findings that
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 7 of 11
Fig. 4 Concentration-dependence of rat liver oleamide hydrolase activity inhibition. Data are mean ± SEM (Standard Error of the Mean) of four
separate preparations (n = 4) conducted in triplicate
FAAH is mechanistically allosteric in nature which is associated with NSAIDs [19]. There is evidence to
often associated with a non-competitive mode of inhib- support the controversy that dual-action FAAH-COX
ition, thus FAAH might also likely exhibit a non- inhibitors may be more useful in this aspect. In vitro
competitive mode of inhibition against these NSAIDs evidence suggests that the metabolism of AEA by
[58, 65, 66]. Unlike aspirin which is an irreversible in- COX-2 might be the most predominant degradation
hibitor of COX enzymes, most other NSAIDs are revers- pathway after blocking the major FAAH metabolic
ible competitive inhibitors of the COX enzymes [67]. pathway. Combinations of URB597 and diclofenac
Previously scientists [38] found that meclofenamic acid have demonstrated synergistic analgesic interactions
and ibuprofen are also potent inhibitors of COX-2 sug- [27, 69]. Also, in vivo synergistic effect was achieved
gestive of the potential for the design of a dual targeting by administration of a combination of AEA and rofe-
inhibitor possibly in combination with URB597 an un- coxib. Local injection of AEA with NSAID (ibuprofen or
competitive FAAH inhibitor [68], which may reduce the rofecoxib) generated higher amounts of fatty acid ethano-
loading dose of NSAIDs with resultant fewer side effects. lamides [70]. Synergistic effects have also been reported
after a systematic administration of URB597 and diclofe-
Therapeutic application of novel multi-target (FAAH/COX) nac in a mouse model of visceral pain [71]. Meclofenamic
analgesics acid, carprofen and indomethacin are among the most
In vivo increases in the levels of AEA resulting from potent inhibitors of the COX enzymes and at the same
FAAH-1 inhibition potentiates actions of COX inhibitors time FAAH-1 from our study [72–75]. Our in vitro results
[19, 31] suggesting that, compounds that inhibit both support the possibility of combined therapeutic agents be-
FAAH and COX enzymes can be as effective as NSAIDs ing explored. This suggests that, a combination of FAAH
but with a reduced COX inhibitor ‘load’, consequently inhibitors such as URB597 and the NSAIDs with dual in-
with accompanying reduction in the adverse effects hibitory capability may have greater utility to treat pain
with reduced NSAID load and may have enhanced effica-
Table 3 Potencies of NSAIDs as inhibitors of rat liver oleamide cies and safety profiles.
hydrolase activity
NSAID pIC50 (M) NSAID pIC50 (M) Conclusion
Sulindac 3.65 ± 0.08 Ibuprofen 3.01 ± 0.06 We established inhibitory potencies of NSAIDs against
rat liver FAAH-1 using oleamide, arachidonamide and
Carprofen 3.58 ± 0.09 Valdecoxib 3.00 ± 0.15
stearoylamide as substrates. Substrate affinity of FAAH-
Meclofenamic acid 3.57 ± 0.06 Ketorolac 2.91 ± 0.07 1 increased in a rank order of oleamide < arachidona-
Sulindac sulphone 3.35 ± 0.03 Diclofenac 2.90 ± 0.07 mide < stearoylamide with resultant Vmax values in a
Indomethacin 3.28 ± 0.03 Dipyrone 2.77 ± 0.07 rank order of arachidonamide > oleamide > stearoyla-
Diflunisal 3.15 ± 0.04 mide. Our Findings confirmed the propensity of FAAH-
Data are mean ± SEM (Standard Error of the Mean) of four separate preparations 1 to turn over polyunsaturated PFAMs particularly with
(n=4) conducted in triplicate cis double bonds at higher rates than monounsaturated
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 8 of 11
Fig. 5 Mode of inhibition of rat liver FAAH-1 hydrolysis of (a) oleamide and (b) arachidonamide by meclofenamic acid and indomethacin. Data
are mean ± SEM (Standard Error of the Mean) of four separate preparations (n = 4) conducted in triplicate
and saturated PFAMs. In the presence of meclofenamate inhibitors against FAAH-1 activity though more research
or indomethacin, Michaelis-Menten analysis suggested a is required for conclusive evidence. Even though, there
reduction in the Vmax of oleamide and arachidonamide was no indication of any selective action of NSAIDs,
hydrolysis, without significant alteration in substrate af- these results suggest potential for study of these com-
finity, indicative of a non-competitive action of these pounds as combined FAAH-COX inhibitors.
Table 4 Mode of inhibition of rat liver FAAH-1 oleamide hydrolysis by indomethacin and meclofenamate
FAAH-1 Kinetics Km (μM) Vmax (nmol/min/mg protein) Substrate
Control 18.4 ± 3.5 4.6 ± 0.5 Oleamide
+ 200 μM indomethacin 24.4 ± 3.3 3.8 ± 0.4 Oleamide
+ 100 μM meclofenamate 22.7 ± 1.4 2.9 ± 0.1 Oleamide
Control 19.8 ± 2.0 8.4 ± 1.2 Arachidonamide
+ 200 μM indomethacin 21.6 ± 2.8 7.2 ± 0.9 Arachidonamide
+ 100 μM meclofenamate 23.4 ± 3.7 6.7 ± 1.0 Arachidonamide
Data are mean ± SEM of triplicate assessments conducted on five transient transfects (n = 5)
Dongdem et al. BMC Pharmacology and Toxicology            (2022) 23:1 Page 9 of 11
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1Department of Biochemistry and Molecular Medicine, School of Medicine, 20. Garavito RM, Malkowski MG, DeWitt DL. The structures of prostaglandin
University for Development Studies, Tamale-Campus, Tamale, Ghana. 2School endoperoxide H synthases-1 and -2. Prostaglandins Other Lipid Mediat.
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