pharmaceuticals
Article 

Effectsof GPR18 LigandsonBodyWeightand Metabolic 
Parameters in a Female Rat Model of Excessive Eating 

Magdalena Kota´nska1,* , Kamil Mika 1, Małgorzata Szafarz 2, Monika Kubacka 1 , ChristaE. Müller 3 , 
Jacek Sapa 1 and Katarzyna Kie´c-Kononowicz4 






Citation: Kota ´nska, M.; Mika, K.; 
Szafarz, M.; Kubacka, M.; Müller, 
C.E.; Sapa,J.;Kie´c-Kononowicz,K. 
Effects of GPR18 Ligands on Body 
Weight and Metabolic Parametersina 
Female Rat Model of Excessive 
Eating. Pharmaceuticals 2021, 14, 270. 
https://doi.org/10.3390/ph14030270 

Academic Editor:Félix Carvalho 

Received:2February 2021 
Accepted: 13 March 2021 
Published: 16 March 2021 

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Copyright: © 2021 by the authors. 
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4.0/). 

1 Department of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University, Medical College, 
9Medyczna Street, 30-688 Kraków, Poland; kamil.mika@doctoral.uj.edu.pl (K.M.); 
monika.kubacka@uj.edu.pl (M.K.); jacek.sapa@uj.edu.pl (J.S.) 

2 Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Medical College, 
9Medyczna Street, 30-688, Krakow, Poland; malgorzata.szafarz@uj.edu.pl 
3 Pharmaceutical&Medicinal Chemistry, Pharmaceutical Institute, PharmaCenter Bonn, Universityof Bonn, 
An der Immenburg4, D-53121 Bonn, Germany; christa.mueller@uni-bonn.de 
4 DepartmentofTechnology and BiotechnologyofDrugs, Facultyof Pharmacy, Jagiellonian University, 
Medical College,9Medyczna Street, 30-688 Kraków, Poland; katarzyna.kiec-kononowicz@uj.edu.pl 

* Correspondence: magda.dudek@uj.edu.pl;Tel./Fax: +48-12-6205530 
Abstract: GPR18 has been proposed to play a role in the progression of metabolic disease and 
obesity. Therefore, the aim of this study was to determine the effects of selective GRP18 ligands (the 
antagonists PSB-CB5 and PSB-CB27 and the agonist PSB-KK1415) on body mass and the development 
of metabolic disorders commonly accompanying obesity. Experiments were carried out on female 
Wistar rats. In order to determine the anorectic activity of the investigated ligands, their effect on food 
and water intakeina modelof excessive eating was assessed. Lipidprofile, glucoseand insulin levels 
as well as alanine aminotransferase, aspartate aminotransferase, and 
-glutamyl transpeptidase 
activity in plasma were also evaluated. Potential side effects were examined in rat models of pica 
behavior and conditioned taste aversion. Animals treated with different ligands gained significantly 
less weight than rats from the obese control group. Effects of GPR18 antagonists on food intake and 
body weight were specific and unrelated to visceral illness, stress or changes in spontaneous activity. 
However, the GPR18 agonist is likely to affect body weight by inducing gastrointestinal disorders 
suchas nausea. Thepresentedpreliminarydata supporttheideathatthe searchfor selectiveGPR18 
antagonists for the treatment of obesity might be promising. 

Keywords: GPR18 ligands; anorectic activity; palatable diet; excessive eating model; PSB-CB5 

1. Introduction 
The endogenous cannabinoid system consists of substances acting as neuromodulators 
i.e., endocannabinoids, theirreceptors and enzymes that areresponsible for their biosynthesisand
 degradation[1]. Endocannabinoidsbindto severalreceptors, includingCB1,CB2, 
GPR18, GPR55,and GPR119,peroxisomeproliferator-activatedreceptorsand transientreceptor
 potential vanilloidtype1(TRPV1)[2–4].Theyaresynthesizedfrom arachidonicacid 
and other polyunsaturated fatty acids. Anandamide (AEA) and 2-arachidonoylglycerol 
(2-AG) were the first endocannabinoids discovered; they are most abundant in the human 
brain[5]. AEA partially, whereas 2-AG fully activate both CB1 and CB2 cannabinoid receptors[
6,7]. Other endocannabinoids include 2-arachidonoylglyceryl ether (noladin ether), 
O-arachidonoylethanolamine (virodhamine), and N-arachidonoyl-dopamine[8]. 

GPR18,identifiedin1997,hasbeenreportedtobeactivatedbyD9-tetrahydrocannabinol 
(THC), AEA[9], and N-arachidonylglycine—the endogenous metaboliteof AEA[10]and 
resolvin D2[11]. However, there are contradicting reports on several proposed putative 
GPR18 agonists[12–15], and the only one which is generally confirmed is THC; therefore,
 GPR18 has been considered asa thirdcannabinoidreceptor[9]. GPR55isproposed 

Pharmaceuticals 2021, 14, 270. https://doi.org/10.3390/ph14030270 https://www.mdpi.com/journal/pharmaceuticals 


Pharmaceuticals 2021, 14, 270 2of16 

tobe activated endogenouslyby lysophosphatidylinositol[16]. GPR119is activatedby 
monounsaturated fatty acid analogues of AEA and 2-AG, N-oleoylethanolamine and 
2-oleoylglycerol[17],butdoesnot appeartorespondto plant-derivedorsynthetic cannabinoids.
 GPR18 and GPR55, however, have been suggested to be targets for some of these 
agents[2]. 

Endocannabinoidsarelipid messengers involvedin overallbodyweight controlby 
interfering with multiple central and peripheralregulatory mechanisms that coordinate energy
 homeostasis[18]. They influence the energy metabolism through central modulation of 
caloric intake, as well as peripheral changes in nutrienttransport, cellular metabolism, and 
energy storage[19]. However,inordertotreat overeating, even though pharmacological 
blockadeof cannabinoidreceptortype1(CB1)byits inverse agonist rimonabant admittedly 
suppressed feedingit alsoresultedin psychiatric sideeffects. Therefore,research within 
the last decade focused on deciphering the underlying cellular and molecular mechanisms 
of central cannabinoid signaling that control feeding as well as other behaviors, with the 
overall aim being the identification of specific targets to develop safe pharmacological 
interventions for the obesity treatment[20]. 

Since some researchers suggest the possible role of GPR18 in the progression of 
metabolic disease and obesity,thisreceptor and itsligands becamea new potential therapeutic
 target[10]. So far, only few (mostly nonselective) GPR18 agonists[15]or antagonists 
have been described[21–23]. Ourgroups haverecently developed potent and selective 
GPR18 agonists, e.g., compound PSB-KK1415. Additionally, several selective antagonists 
of GRP18 were obtained[22,23]—e.g., PSB-CB27 and PSB-CB5—the last one being the first 
potent and strongly preferential GPR18 antagonist, now commercially available (product 
name CID-85469571) from several companies. Since previously known GPR18 antagonists 
also antagonizeGPR55[24],ithasbeendifficultto separatetheeffectsofthesetworeceptor 
targets. Due to the recent availability of selective GPR18 ligands these compounds may become
 usefultoolsinresearch focusedonthe mechanismsof actionofthisreceptor.Theaim 
of our work was to determine the effects of the above-mentioned GRP18 ligands (PSB-CB5, 
PSB-CB27, and PSB-KK1415) on body mass and selected metabolic disorders commonly 
accompanying obesity,ina female rat modelof excessive eating. The contributionof the 
receptor itself on the development of obesity and hyperglycemia, as well as its influence 
on the lipid profile have also been taken into account. The research project was carried 
out using a rat model of excessive eating which perfectly illustrates the excess caloric 
intake from the excessively available tasty products rich in sugar and fat. In this model, 
the animals have access not only to high-calorie foods such as peanuts, cheese, milk with 
increased fat content, and chocolate, but also to the standardfeed. However, feeding is 
not in any way forced, as in other models where animals are fed only a high-fat diet, or 
when they are temporarily deprived of food (binge eating models). The experience related 
tothis model suggeststhatgreaterdifferences(requiringless animalspergroupinorder 
to obtain significant results) in the severity of disturbances in body weight and selected 
metabolic parametersare observedin female rats[25–27]. Therefore,to maketheeffect 
more noticeable, and in line with the principle of reducing the number of animals, female 
rats were selected. 

2. Results 
2.1. Effectof Diet orTested Compounds on BodyWeight, Amountof Intraperitoneal Adipose 
Tissue, Caloric, andWater Intakes 
Animals fed a palatable diet gained more weight than rats from the control group, 
starting from the second day of the experiment. Throughout the experiment (21 days), 
control rats gained only 18.32% weight, while the rats from the control obese group gained 
36.40%(Figure 1A).Attheendof experiment, animalsfedapalatable feedandtreated with 
all tested compounds, namely PSB-KK1415 (GPR18 agonist), PSB-CB27, or PSB-CB5 (GPR18 
antagonists), or rimonabant (CB1 antagonist), weighted significantly less than animals 
from the obese controlgroup. However, the rats’ body weight in all these groups was still 
significantly higher thanin the controlgroup fed witha standardfeed (Figure 1A,B). 


Pharmaceuticals 2021, 14, 270 3of16 


Figure 1. Effect of diet or long-term administration of the GPR18 ligands or rimonabant, on body weight of female 
Wistar ratsin the modelof excessive eating.(A)Sumofbody weight changes.(B)Changes of body weight in individual 
weeks. Result are means ± 
SEM, n = 6. Multiple comparison against the vehicle-treated control group(*) or against the 
vehicle-treated obese control group (^) or against the vehicle-treated obese control group in second week (#) or against the 
PSB-KK1415-treated group in first week (o) or against the PSB-CB27-treated group in first week (+) were performed by 
one-wayANOVATukeyposthoc. Significantdifferencesare denotedby*,^,o p<0.05; **,^^p<0.01; ***,^^^,###,+++p<0.001. 

2.2. Effectof Diet orTested Compounds on theWeightof SelectedOrgans 
Animals from the control obese group had a statistically higher amount of peritoneal 
fatpads(about50%more)than controlrats(Figure 2A). However, animalsfromalltreated 
groups had significantly less peritoneal fat pads than controlobese rats (Figure 2A). Additionally,
 the heart weight was lower in rats treated with all tested compounds compared to 
the controlgroup feda standardfeed, and lowerin thegroupsreceiving the antagonists 
as compared to the control group fed palatable feed (Figure 2B). The kidney mass was 
significantly higher in the obese control animals and rats treated with PSB-KK1415 vs. 
control animals (Figure 2C). There were no statistical differences between the studied 
groupsin the liver weight (Figure 2D). 

2.3. Effectof Diet orTested Compounds on Caloric andWater Intakes 
Calorieintakeinallgroupswassignificantlyhigherthaninthecontrolgroup(Figure3A), 
and in the case of PSB-KK1415 treatment the difference was also significant vs. the control 
obese rats. Significantly less water was consumed by obese control animals, which could 
probably be explained by the fact that they had also access to milk. However, the rats fed 
palatable feed and treated with PSB-KK1415, PSB-CB5, or rimonabant drank significantly 
more water than obese control animals. Only animals treated with PSB-CB27 consumed a 
similar amountof water as control animals fedapreferential feed (Figure 3B). 


Pharmaceuticals 2021, 14, 270 4of16 


Figure 2. Effect of diet or long-term administration of the GPR18 ligands or rimonabant, on weights of various organs 
of femaleWistar rats in the model of excessive eating.(A)Peritoneal adipose tissue.(B)Heart.(C)Kidneys.(D)Liver. 
Results are the means ± 
SEM, n = 6. Multiple comparisons against the vehicle-treated control group (*) or against the 
vehicle-treatedobesecontrolgroup(^)wereperformedbyone-wayANOVATukeyposthoc; Significantdifferencesare 
denotedby *,^ p<0.05; ***,^^^p<0.001. 


Figure 3. Effectof long-term administrationoftheGPR18ligandsor rimonabant,onfood(A)or water intake(B)of female 
Wistar rats fed a palatable feed. Results are the means± 
SEM, n = 6. Multiple comparisons against the vehicle-treated 
controlgroup(*)oragainstthe vehicle-treatedobese controlgroup(^)were performedby one-wayANOVATukeyposthoc; 
Significant differences are denoted by **,^^ p<0.01; ***,^^^p<0.001. 


Pharmaceuticals 2021, 14, 270 5of16 

2.4. Influenceof Diet orTested Compounds on Plasma Glucose and Insulin Levels 
All animals fed palatable feed had significantly higher blood glucose levels than 
control rats. Additionally, animals treated with PSB-KK1415 had blood glucose levels 
even higher than ratsfrom the obese controlgroup (Figure 4A).An agonistof GPR18— 
PSB-KK1415, one of antagonists—PSB-CB5, and rimonabant increased levels of insulin 
in plasma of rats fed a palatable feed, comparing to the level of insulin in control obese 
animals. However,only the increase observed in PSB-KK1415 treated group was statistically 
significant. Treatment with another GPR18 antagonist, compound PSB-CB27, did not 
influencetheinsulinlevelwhichstayedsimilartotheone observedinplasmaofcontrol 
obese rats (Figure 4B). 


Figure 4. Effect of administration of the GPR18 ligands or rimonabant on plasma glucose(A), insulin(B), triglyceride 
(C), total cholesterol(D)or HDL-cholesterol(E)level or alanine aminotransferase(F), aspartate aminotransferase(G), 
-glutamyl transpeptidase, and(H)activityof femaleWistar rats feda palatable feed. Results are the means± 
SEM, n =6. 
Multiple comparisons against the vehicle-treated control group (*) or against the vehicle-treated obese control group (^) 
were performedby one-way ANOVATukey post hoc; Significant differences are denotedby *,^ p< 0.05, **,^^ p< 0.01, 
***,^^^ p<0.001. 


Pharmaceuticals 2021, 14, 270 6of16 

2.5. Influenceof Diet orTested Compounds on LipidProfile 
The level of triglyceride in blood was higher in obese rats than in the control group, 
but the HDL-cholesterol level was lower (Figure 4C,E). Rats treated for three weeks with 
rimonabant or test compounds, except PSB-KK1415, and fed palatable diet had slightly 
lower levels of triglycerides in plasma than animals from the obese control group. There 
was no statistical significance between levels of triglycerides in these groups and the control 
group. However, in the PSB-KK1415-treated group the triglyceride level was significantly 
higher thanin the controlgroup (Figure 4C). There were no significant differencesin total 
cholesterol plasma levels between all studiedgroups (Figure 4D).Groups fedapreferential 
feed and treated with all test compounds had a higher level of HDL-cholesterol than the 
obese control rats, and in the cases of PSB-KK1415 or rimonabant administration it was 
even higher thanin the control animals (Figure 4E). 

2.6. Influenceof Diet orTested Compounds on Alanine Aminotransferase (AlAT), Aspartate 
Aminotransferase (AspAT), and g-GlutamylTranspeptidase (GGT) Activityin Rats Plasma 
Asignificantly higher level of AlATactivity was observed in plasma of rats treated 
with PSB-KK1415orPSB-CB5comparedtothelevelsinbothcontrolgroups(Figure 4F). 
A significantly higher level of AspAT activity was observed in plasma of rats treated 
with PSB-KK1415 or PSB-CB5 compared to the level in control groups fed palatable feed 
(Figure 4G). The GGT activity was significantly lower in plasma of rats treated with 
PSB-KK1415comparedtothelevelsinthecontrolgroupfed palatablefeed(Figure 4H). 

2.7. InfluenceofSingleandChronic AdministrationoftheTested Compoundson Locomotor 
Activity of Rats Fed Palatable Diet and Housed in Pairs in Home Cages 
All tested compounds at the administered doses had no effect on locomotor activity 
afterbothsingleandchronic administrationtoratsfedpreferentialfeed(Figure 5). 

2.8. Effects onVisceral Illness via Measurementof Kaolin Intake (Pica Behavior Model) 
Significantly lower weight gain over the 24-h period of the study was observed 
in the groups treated with PSB-KK1415 or PSB-CB27 vs. control group treated with 
1%Tween 80 (negative control). The positive control group was given CuSO4 and was 
characterizedby the lossof weight, thegreatest intakeof kaolin and the smallest intakeof 
feed(Figure 6A). Animalsreceiving PSB-KK1415 or rimonabant consumed significantly 
more kaolin than rats from the negative control group, however, less than rats from the 
positive control group. While, animals receiving PSB-CB27 or PSB-CB5 consumed kaolin 
in the amount comparable to the negative control group (Figure 6B). Animals from all 
groupsreceiving tested compounds ate significantly less than animalsfrom controlgroup 
that received only vehicle and significantly more than rats from group treated with CuSO4 
(Figure 6C). There were no statistically significant differences in water intake between 
groups(Figure 6D). AnimalsfromthegroupstreatedwithCuSO4or PSB-CB27 excreted 
significantlylessfeceswithin24haftercompound administrationcomparedtothecontrol 
group (Figure 6E). 


Pharmaceuticals 2021, 14, 270 7of16 


Figure 5. Influenceof the GPR18 ligands or rimonabant on locomotor activity aftera single dose(A,C,E,G)and chronic 
treatment(B,D,F,H). Locomotoractivityofratsfed palatablefeedduring18hperiodaftertreatmentwith tested compounds 
(5mg/kgb.w.,i.p.), rimonabant(1mg/kgb.w.),or vehicle. Activityisdirectlyrelatedtoentranceto variousareasofthe 
cage. Results are the means ± 
SEM, n =6(multiple t-test). Significant differences are denotedby^ p<0.05. 


Pharmaceuticals 2021, 14, 270 8of16 


Figure 6. Effectofsingle administrationoftheGPR18ligandsorrimonabant,onbodyweight(A), kaolin intake(B), food 
intake(C), water intake(D), the amountof feces(E)of femaleWistar ratsin the Pica behavior model. Theeffectof multiple 
(sevendays) administrationoftheGPR18ligandsor rimonabant,onbodyweight(F), sucrose intake(G), and fluid intake 
(H)of femaleWistar ratsin conditioned tested aversion model. Results are the means± 
SEM, data for two animals are 
reared together, n = 6. Multiple comparisons against the vehicle-treated control group (*) or against the vehicle-treated obese 
controlgroup(^)were performedby one-wayANOVATukeyposthoc. Significantdifferencesare denotedby *,^ p<0.05; 
^p<0.01; ***,^^^p<0.001. 

2.9. Effects onVisceral Illness via Measurementof Saccharin Intake 
PSB-CB5 and PSB-CB27 did not induce any significant conditioned taste aversion 
measured by saccharin preference following a seven-day conditioning period in which 
rats were administered (i.p.) above mentioned compounds. In contrast, PSB-KK1415, the 
reference compound LiCl (40 mg/kg b.w.) and rimonabant caused a significant reduction 
of saccharine consumption. Slight, however significantly lower weight gain over the 24-h 
period of the study was observed in the groups treated with PSB-KK1415, PSB-CB27, LiCl, 
or rimonabant (Figure 6F,G). 


Pharmaceuticals 2021, 14, 270 9of16 

3. Discussion 
Obesity may be, at least partially, linkedto dysregulation of the endocannabinoid 
system in both central and peripheral tissues, affecting appetite as well as glucose and 
lipid metabolism[28–30]. Therefore,inorderto determinethe pharmacological activity 
of GPR18 ligands we have chosen the model of excessive overeating with preferential 
feed, in which animals have unrestricted access to the standard diet with addition of 
products such as peanuts, cheese, milk, and chocolate. The availability of such diet results 
in increased caloric intake and development of hyperglycemia, lipid disorders, and obesity 
with accompanying increase of the amount of peritoneal fat[25,26,31]. Indeed, in our study, 
within 21 days of being on such a diet rats were overweight and developed the abovementioned
 metabolic disorders. By treating animals with the selective GPR18 ligands we 
aimed to determine if this receptor could be, to some extent, related to dysregulation of 
the endocannabinoid system in obesity. The particular aim of this study was to identify 
whether the administration of selective GPR18 ligands would prevent induction of at least 
some pathological disorders accompanying obesity.We also wanted to determine whether 
these compounds could act anorectically and which ligand—agonist or antagonist—would 
have the most beneficial effect. 

Endocannabinoids and non-selective GPR18 agonists, such as AEA or 2-arachidonoyl 
glycerol increase food intake and consequently body weight by activating central endocannabinoidreceptors[
28,32,33]. It was hypothesized[34]that the highefficacyof 
cannabinoid receptor inverse agonists such as rimonabant in reducing body weight of 
obese animalsand humans(regardlessofthefood intake inhibition),isduetothe occurrence
 of a general up-regulation of the endocannabinoid system in obesity, not only at 
the central[34],butalsoatthe peripheral[32,35,36]levels. Additionally,overreactivityof 
the endocannabinoid system can increase accumulation of fat and reduce glucose uptake 
in human fat cells, subsequently increasing the risk of insulin resistance and impaired 
glucose tolerance[28]. These data let us believe that GPR18 antagonists could exert beneficial
 effects in reducing both, body weight and symptoms of metabolic disorder in obese 
animals. Surprisingly, animals treated for 21 days with different ligands (GPR18 agonist, 
antagonists and CB1 antagonist) gained significantly less weight than rats from the control 
obese group. However, this effect was observed only in the thirdweek of treatment except 
for thereference compound (rimonabant), which significantly slowed down the animals’ 
weight gain already from the first week of administration. Additionally, in all these animals 
significantly smaller amount of peritoneal adipose tissue was observed than in the control 
obese rats. 

After obtaining such results, it became obvious that we had to rule out other possible 
effects exerted by the tested compounds, such as sedation or gastrointestinal disorders, 
which could also contribute to the slowing down the weight gain and reduction of the 
amount of peritoneal fat in experimental animals. Stress as well as changes in the spontaneous
 activity could be very disadvantageous and distort an assessment of the impact of 
tested compounds on body weight[37]. Sickness, gastrointestinal malaise, or drug-induced 
toxicity can also be the source of weight loss causing changes in food consumption or 
acting through some other mechanisms (e.g., malabsorption)[38]. 

The effect of tested compounds on the spontaneous activity was investigated in the 
groupof animalsfeda palatable feed.Inordertoreducestress, rats were housedinpairs 
in home cages. The activity monitoring system was imperceptible to the animals and did 
not affect their behavior in any way. None of the tested compounds had a statistically 
significant effect on rats’ mobility after both single and chronic administration. Thus, the 
changes in activity could be ruled out as the potential cause of reduced weight gain. 

Somedrugs mayreduce food intakebyproducing gastrointestinal malaise, whichis 
difficultto detectsolelybythechangesinanimal behavior.Ratsandmicelackthe emetic 
response, which distinguishes them from humans. However, the persistent eating of inedible
 substances by rodents can be used to evaluate illness-response behavior analogous to 
vomiting in other species[38,39]. Thus, in our study, animals beside preferential feed had 


Pharmaceuticals 2021, 14, 270 10 of 16 

also access to kaolin clay. Administration of GPR18 antagonists PSB-CB27 or PSB-CB5 did 
not result in the excessive intake of clay (compared to the negative control group receiving 
vehicle only) which proved that in these animals’ disorders of the gastrointestinal tract, 
such as visceral irritation or nausea, did not occur. On the other hand, CuSO4, used asa 
positive control, extensivelyreduced the food and water intake and significantly increased 
the amount of consumed kaolin clay, indicating the stomach upset. Unfortunately, the 
administration of PSB-KK1415 (GPR18 agonist) caused analogous changes in animals’ 
behavior. Thus, in rats treated chronically with this compound, disorders of the gastrointestinal
 tract could be, at least partially,responsible for thereduced weight gain. Aftera 
single PSB-KK1415 administration rats consumed less food than ones from the negative 
controlgroup, however,the amountofexcreted fecesinbothgroupswas similarwhatmay 
also suggest that PSB-KK1415 has a potential ability to increase the intestinal passage. 

Sucrose preference test is often used to examine possible side effects such a nausea or 
malaise. In most cases, this test is performed in acute settings. However, the most relevant 
question, mainly from a therapeutic point of view, is whether the support of potential 
conditioned tasted aversion(CTA) willbe sustained duringrepeated administration[40]. 
The results of seven-day subchronic CTA test showed that compounds PSB-CB5 and 
PSB-CB27 did not cause gastrointestinal disturbances. In contrast, PSB-KK1415 (same 
as rimonabant) caused an adverse reaction of similar intensity to the one produced by 
the reference compound (lithium chloride), that clearly indicated development of the 
gastrointestinal disorders in these animals. This adds further support to the conclusion 
that effects of GPR18 antagonists PSB-CB5 or PSB-CB27 on food intake and body weight 
are specific and unrelated to visceral illness, stress, or changes in the spontaneous activity. 
In contrast, GPR18R agonist compound PSB-KK1415 is likely to affect body weight by 
inducing gastrointestinal disorders, such as nausea. 

An appropriate insulin sensitivity and the activation of its downstream machinery 
isa fundamentalprerequisitetoallowadequatefuelsupplytothefatcells[41],therefore, 
impairedglucose transportand insulinresistancearethe sourceoffat storage disorders. 
Inversely, the excess of free fatty acids released by visceral adipose tissue is the cause of 
“lipotoxicity”. Increased fat oxidation in the muscles causes inhibition of glycolysis, and in 
the liver contributestothe intensificationof gluconeogenesis, whichrequires compensatory 
secretion of insulin by ß 
cells. In both cases, plasma glucose, triglyceride, and insulin 
levels are elevated. In the presented study such metabolic changes were observed in rats 
treated with PSB-KK1415 (GPR18 agonist). Animals receiving this compound, despite 
lower weight gain, consumed significantly more kcal even than control obese animals. 
The body’s response was only partially analogous to that of proposed endogenous GPR18 
agonists—AEA or 2-arachidonoyl glycerol[28,32,33]and the proposed exogenous agonist— 
O-1602[42]all of which caused both increase in the food intake and weight gain. However, 
it shouldbe emphasizedthatthe abovementioned compoundsarenot,ornotonly,targeting 
GPR18incontrastto PSB-KK1415thatisaselectiveGPR18agonist.Moreover,intheprocess 
of developing metabolic disorders such as diabetes, after the initial period of weight loss, 
usually gain of weight occurs, so it is possible that longer studies would show a similar 
trend after chronic PSB-KK1415 administration. 

Animals treated with PSB-KK1415 also drank more water than rats from both control 
groups, despite the fact that animals eating the preferential feed also consumed milk. 
Taking into account that the typical symptoms of developing diabetes are polydipsia and 
weight loss, as well as elevated glucose levels, it can be concluded that the administration of 
PSB-KK1415 may cause the development of pre-diabetes or diabetes and insulin resistance. 
Previously, Ikeda et al. showed that 2-arachidonoyl glycerol causes increased insulin 
releasefromratisletcells[43]. However,to confirmourhypothesis further studies,suchas 
glucose tolerance and insulin sensitivity tests after PSB-KK1415 administration are needed. 

Fatty liver is usually associated with elevated levels of AlATand AspATin plasma[44]. 
Yet, studies suggest that AlATlevels can fluctuate, therefore, measurement ata single time 
pointis consideredtobe insufficienttodraw conclusionsregardingthe liver function[45]. 


Pharmaceuticals 2021, 14, 270 11 of 16 

In the present study, no high changes in the activity of all three tested liver enzymes (AlAT, 
AspAT, and GGT) were observed. While some changes were statistically significant, they 
could not be considered clinically alarming. 

In conclusion, the present study has addressed a number of critical issues in the 
evaluation of the concept of using GPR18 ligands to decrease food intake and lower body 
weight gain. Animals treated with different ligands, both agonists and antagonists, gained 
significantly less weight than ratsfrom the controlgroup fed palatable feed.However, the 
effects of GPR18 antagonists on food intake and body weight were specific and unrelated 
to visceral illness, stress or changes in the spontaneous activity, while the GPR18 agonist 
was likely to affect body weight by inducing gastrointestinal disorders, such as nausea. 
Rats treated with GPR18 antagonists had also slightly lower levels of triglycerides in 
plasma than animals from the obese control group. Thus, the presented preliminary data 
support the idea that selective GPR18 antagonism represents a promising avenue for 
the further research, and that the development of selective GPR18 antagonists for the 
treatmentof obesity mightresultin valuable andpromising outcomes. Currently, the most 
urgent issue seemstobethe identificationofa potential candidates with betterefficacy 
and superior pharmacological activity than current drugs towards the normalization of 
lipid and carbohydrate disorders as well as insulinresistance,a metabolic condition often 
accompanying obesity. 

4. Materials and Methods 
4.1. Animals andTested Compounds 
Experiments were carried out on femaleWistar rats with the initial body weightin 
the rangeof 165–175g(six weeks old: Jagiellonian University Medical College, Krakow, 
Poland). The animals were housed in plastic cages in constant temperature facilities 
exposedto12–12light-darkcycle.Waterandfoodwere availablead libitum. Controland 
experimental groups consisted of six to eight animals each. 

Tested compounds suspendedin1%Tween80 were administered intraperitoneally 
(i.p.) onceadayfor21daysatthedoseof5mg/kgb.w. (PSB-KK1415; PSB-CB27; PSB-CB5) 
or1 mg/kg b.w. (rimonabant—reference compound). The dose of tested compounds 
was chosen based on preliminary locomotor activity studies (unpublished observations). 
One non-sedative dose common to all compounds was selected. There were also two 
control groups, one fed a palatable diet (control obese group) and one fed a standarddiet 
(control group). 

4.2. Drugs, Chemical Reagents, and Other Materials 
Heparin was purchased from Polfa Warszawa S.A. (Warsaw, Poland), thiopental 
sodium from Sandoz International (Stryków,Poland),Tween80from Sigma-Aldrich (Darmstadt,
 Germany) and rimonabant was from AK Scientific, Inc., USA. 

GPR18 ligands: agonist (PSB-KK1415 with undisclosed structure) and antagonists: 
(PSB-CB5-(Z)-2-(3-(4-chlorobenzyloxy)benzylidene)-6,7-dihydro-2H-imidazo [2,1-b][1,3]thiazin-
3(5H)-one) and (PSB-CB-27-(Z)-2-(3-(6-(4-chlorophenoxy)hexyloxy)benzylidene)-6,7dihydro-
2H-imidazo[2,1-b][1,3]thiazin-3(5H)-one) were synthesized at the Department of 
Technology andBiotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical
 College, Krakow, Poland according to a procedure described previously[20,21]. Agonist:
 (PSB-KK-1415 hGPR18 - 
EC50 = 0.0191(M), -arrestinrecruitment assay; hGPR55 - 
EC50>10(M)), antagonist (PSB-CB5 - 
IC50 = 0.279(M) in -arrestin recruitment assay 
as inhibitorof GPR18 activationby THC, hGPR55,IC50>10(M); hCB1,Ki>10(M); 
hCB2,Ki = 4.03(M)) and antagonist (PSB-CB27 - 
IC50 = 0.650(M), full inhibition of THC 
induced activation of GPR18 in -arrestinrecruitment assay, hGPR55,IC50>10(M); hCB1, 
Ki>10(M); hCB2,Ki>10(M)). The compounds were discoveredin the laboratoryof 

C.E. Müller, University of Bonn, Germany. 

Pharmaceuticals 2021, 14, 270 12 of 16 

4.3. EffectofTested Ligands on BodyWeight, Locomotor Activity, and Food andWater Intake 
In order to determine the anorectic activity of tested ligands, its effect on food and 
water intakein the modelof excessive eating was assessed[25,26]. Rats were housedin 
groups of three. Five groups of six rats were fed diet consisting of milk chocolate with 
nuts, cheese, salted peanuts, and 7% condensed milk and also had access to standardfeed 
(Labofeed B, Morawski Manufacturer Feed, Poland) and water ad libitum for three weeks. 
All tested compoundswere suspendedin1%Tween80and administeredi.p. onceaday 
(from the first day of experiment) for 21 days. First experimental group was given vehicle 
(1%Tween 80)—obese controlgroup, second wasgiven PSB-KK1415 (GPR18 agonist)at 
thedoseof5mg/kgb.w.,thirdgroupwasgiven PSB-CB27(GPR18 antagonist)atthedose 
of5mg/kgb.w., fourthgroupwasgiven PSB-CB5(GPR18 antagonist)alsoatthedoseof 
5mg/kg b.w. and the fifth group was given rimonabant (CB1 antagonist) at the dose of 
1mg/kg b.w. The lastgroupof rats was feda standardfeed for3weeksad libitum and 
wasgiveni.p.a vehicle(1%Tween 80)—controlgroup. Intakesoffoodand waterwere 
evaluated three times per week, and body weights were measured daily immediately prior 
to the administration of the investigated compounds. 

Palatable diet contained: 100gpeanuts—614 kcal; 100mL condensed milk—131 kcal; 
100gmilk chocolate with hazelnuts—195 kcal; 100gGreek cheese—270 kcal. The standard 
diet contained 100gfeed—280 kcal. 

The locomotor activity of rats was measured on the first and 20th day of experiment
 withaTraffiCage (TSE-Systems, Germany) radio-frequency identification system 
(RFID)[46]. The animals had subcutaneously implanted transmitter identification, which 
enabled the presence and time spent in different areas of the cage to be recorded, and then 
data were collected with a special computer program. Locomotor activity was monitored 
for18hafter single andrepeated administrationof the tested compounds. 

On the 22nd day, 20 min after i.p. administration of heparin 600 U/rat, animals were 
sacrificed. Peritoneal fat, liver, kidneys, and heart were weighed, and plasma was collected 
for further analysis. Schemeof experimentis showedin Figure 7A. 

4.4. InfluenceofTested Compounds on LipidProfile, Glucose and Insulin Levels asWell as AlAT, 
AspAT, and GGT Activity in Plasma 
To determinethelipidprofile, glucose levelandtheAlAT,AspAT,andGGT activityin 
plasma standardenzymatic and spectrophotometric tests (Biomaxima S.A. Lublin, Poland) 
were used.To determine the insulin levelin plasma, an ELISA test (FineTest,Wuhan Fine 
Biotech Co., Ltd., China) was used, performed in two replicates (standard curve range: 
15–480 ng/mL (0.1 mIU/L–40 mIU/L), sensitivity: 0.05 mIU/L, intra-assay: CV < 8%, 
inter-assay: CV<10%). 

4.5. Effects onVisceral Illness via Measurementof Kaolin Intake (Pica Behaviour) 
To exclude the possibility that the suppressionof food intakeby tested compounds 
was caused by visceral illness, Pica behavior was evaluated. The method was based on 
the worksbyTakeda[39],Yamamoto[47], and Kota´nska[26]. The experiment lasted eight 
days. In addition to free access to feed, animals had also free access to the white kaolin. 
For the first few days animals were accustomed to the presence of kaolin in their cages. On 
the sixth day, food and kaolin were removed for 24 h, so the animals would be more or 
less at the same level of hunger and have a similar amount of feces in the digestive tract, 
since at the end of experiment feces were weighted to assess the level of intestinal passage. 
Onthe seventhday animalswerei.p. given: tested compoundsatadoseof5mg/kgb.w. 
or rimonabantatadoseof1mg/kgb.w.or vehicle (negative controlgroup)ora solution 
ofCuSO4atadoseof6mg/kgb.w.(1/3LD50;LD50 =18mg/kgforaratatthisrouteof 
administration) (positive controlgroup). The standardfeed and kaolin weregiven back 
to the cages. The amount of approved food, water drunk, kaolin consumed and excreted 
feces was determined after 24 h. Animals were also weighed prior to the administration of 
the tested compound and after24h. Schemeof the experimentis showedin Figure 7B. 


Pharmaceuticals 2021, 14, 270 13 of 16 


Figure 7. Aschematic diagram of chronic administration of tested compounds in model of excessive 
eating. PSB-KK1415 or PSB-CB27 or PSB-CB5 (5 mg/kg b.w.) or rimonabant (1 mg/kg b.w.) were 
administrated intraperitoneally (i.p.) to rats for 22 consecutive days. Control groups received 
1% Tween 80(A).Aschematic diagramofpica behavior test. Rats weretreated with single doseof 
tested compounds(5 mg/kg b.w., i.p.) or rimonabant(1 mg/kg b.w., i.p.). Controlgroupreceived 
1%Tween80 or CuSO4(6 mg/kg b.w., i.p.)(B).Aschematic diagramof conditioned tasted aversion 
test.Ratsweretreatedwithrepeated (seven-times)doseof tested compounds(5mg/kgb.w.,i.p.)or 
rimonabant(1mg/kgb.w.,i.p.). Controlgroupreceived1%Tween80orLiCl(40mg/kgb.w.,i.p.). 
The measurementperiodbeganonthe seventhdayof administrationand lastedforthenext24h(C). 


Pharmaceuticals 2021, 14, 270 14 of 16 

4.6. Effects onVisceral Illness via Measurementof Sucrose Intake (ConditionedTasted 
Aversion; CTA) 
Aseven-day subchronic CTAparadigm was adopted in order to examine whether 
tested compounds inducedCTAfollowingrepeated administration. The method was based 
on the worksby Malmlöf[40]with minor modification. The experiment lasted seven days. 
Animals were divided into six groups: one group received daily i.p. injections of vehicle 
(negative control group), a second group received a solution of lithium chloride (LiCl, 
40 mg/kg b.w., i.p., positive controlgroup),a thirdgroup wasgiven rimonabant(1 mg/kg 
b.w., i.p.) and subsequent groups received the tested compounds (5 mg/kg b.w., i.p.). Rats 
were allowed to adapt for two days, they were given water in two identical bottles instead 
of one. Immediately following the first dose, and throughout the five-day injection period, 
the water in both bottles was replaced with freshly-made saccharin solution (1 g/L). This 
supply was withdrawn exactly 48hbefore the last dosing (immediately before the 6th 
dose), andboth bottles were again filled with water for another24hperiod. Exactly before 
the last dosing, rats were given access to one bottle filled with saccharin, and another 
filled with water. This two-bottle preference test was maintained for 24 h. During this 
time, saccharin intake, water intake, total fluid intake, the ratio saccharin/total fluid were 
registered. Body weight and the amount of consumed feed were also measured. The 
volumeof saccharinesolution consumedinrelationtothetotalfluidintakewasusedasa 
measure of CTA, induced by treatment, and was expressed as a percentage of the saccharin 
intake. The schemeof the experimentis shownin Figure 7C. 

4.7. Data Analysis and Statistical Procedures 
Statistical calculations were carried out with the GraphPad Prism6program (Graph-
Pad Software, San Diego, CA, USA). The results were given as arithmetic means with 
standard errors of the mean (SEM). The normality of data sets was determined using the 
Shapiro–Wilk test. The statistical significance was calculated using one-way ANOVAposthocTukey’s
 Multiple ComparisonTest or Multiplettests (locomotor activity). Differences 
were considered statistically significant at: *p. 
0.05, **p. 
0.01, ***p. 
0.001. 

Author Contributions: Conceptualization: M.K. (Magdalena Kota ´nska); methodology: M.K. (Magdalena
 Kota ´nska); validation: M.K. (Magdalena Kota ´nska) and K.M.; formal analysis: M.K. (Magdalena
 Kota´nska) and K.M.; investigation: K.M. and M.K. (Magdalena Kota´nska);resources: M.K. 
(Magdalena Kota ´nska), K.K.-K.; data curation: M.K. (Magdalena Kota ´nska); writing—original 
draft preparation: M.K. (Magdalena Kota ´nska); writing—review and editing: M.S., M.K. (Monika 
Kubacka), C.E.M. and K.K.-K.; visualization: M.K. (Magdalena Kota ´nska) and M.S.; supervision: 
C.E.M., J.S., and K.K.-K.;project administration: M.K. (Magdalena Kota´nska); funding acquisition: 
M.K.(Magdalena Kota ´nska) and K.K.-K. All authors have read and agreed to the published version 
of the manuscript. 

Funding: ThisworkwassupportedbystatutoryfundsfromtheFacultyofPharmacy,JagiellonianUniversityMedicalCollege,
Krakow,Poland(N42/DBS/000045,N42/DBS/000138,N42/DBS/000039). 

Institutional ReviewBoard Statement: All experiments were conducted according to the guidelines 
of the Animal Use and Care Com-mittee of the Jagiellonian University and were approved for 
realization (PermissionNo 150/2018, dateof approval: 18.07.2018). Theresearch complies with the 
commonly-accepted ‘3Rs’. 

Informed Consent Statement: Not applicable. 

DataAvailability Statement: The data presented in this study are available on request from the 
corresponding author. 

Acknowledgments: The authors wish to gratefully acknowledge Joanna Knutelska and Maria Kaleta 
for their technical assistance and Adrian Olczyk for creating a computer program to process the data 
from theTraffiCage. 

Conflicts of Interest: The authors declare no conflict of interest. 


Pharmaceuticals 2021, 14, 270 15 of 16 

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