Analgesic activity of 3-mono-substituted
derivatives of dihydrofuran-2-one in experimental rodent models of pain
Kinga Sa³at1, Barbara Filipek1, Krzysztof Wiêckowski2, Barbara Malawska2
Department of Pharmacodynamics, Chair of Pharmaceutical Chemistry, Department of Physicochemical Drug Analysis, Jagiellonian University, Medical College, Medyczna 9, PL 30-688 Kraków, Poland
Correspondence:Kinga Sa³at, e-mail: salat.kinga@gmail.com
Abstract:
Three derivatives of dihydrofuran-2-one (L-PP, L-PP1, and L-SAL) were administered by intraperitoneal injection and their analge- sic activity was assayed in several models of pain. The activity of these derivatives were tested using a hot plate test, a writhing test, capsaicin- and glutamate-induced nociception, along with two models of local anesthesia, including a test for infiltration anesthesia in guinea pigs and the modified tail immersion test in mice. The results of thesein vivo experiments show that these three derivatives of dihydrofuran-2-one possess analgesic activity in rodents. The ED# values of the tested compounds are lower or comparable to the ED# values of reference compounds (acetylsalicylic acid or morphine). For the most active derivative of dihydrofuran-2-one, L-PP1 (3-[4-(3-trifluoromethylphenyl)-piperazin-1-yl]-dihydrofuran-2-one dihydrochloride), the ED# value was: 1.34 mg/kg, 0.79 mg/kg, 2.01 mg/kg and 3.99 mg/kg in the hot plate, writhing, capsaicin- and glutamate-induced pain tests, respectively.
Key words:
derivatives of dihydrofuran-2-one, hot plate, writhing, capsaicin, TRPV1, L-glutamic acid, infiltration anesthesia, tail immersion, local anesthetic.
Introduction
Treatment of pain remains an important medical prob- lem, as demonstrated by the millions of patients that visit their doctors each year seeking medication to re- lieve their pain [17]. According to the definition es- tablished by the International Association for the Study of Pain [11], pain is an unpleasant sensory and emotional experience that is associated with actual or potential tissue damage. Pain can be classified as acute or chronic based on the length of time the pain is experienced. Acute pain, resulting from tissue dam-
age, is a normal physiological response and serves a protective function. Nociceptive pain is typically acute in nature and diminishes upon healing. In con- trast to acute pain, chronic pain is an abnormal sensa- tion usually occurring after direct injury or damage to a nerve. For individuals experiencing chronic pain, their pain usually includes inflammatory and neuro- pathic pain. Neuropathic pain syndrome is a collec- tion of disorders characterized by different etiologies including viral infection, inflammation, trauma, meta- bolic disorder, and nerve damage chemically- or irradia- tion-induced [33].
Medications are prescribed to patients based on the type of pain the patient is experiencing. Opioid anal- gesics, acting mainly by influencing µ-opioid recep- tors, are used for the treatment of acute pain. For the treatment of inflammatory pain, several non-steroidal anti-inflammatory drugs (NSAIDs) are used. NSAIDs block cyclooxygenase (COX) and thus inhibit the synthesis of prostaglandins. However, both opioid an- algesics and NSAIDs can cause serious adverse side- effects. Local anesthetics are also an important group of pain-relieving drugs. Local anesthetics function by blocking nerve conduction by altering the function of voltage-gated sodium channels within the plasma membrane. By doing so, the membrane voltage-gated sodium channels are unable to transmit the painful stimulus towards the central nervous system and thus they are effective analgesics. At present, local anes- thetics and antiepileptic agents are used for the treat- ment of neuropathic pain syndromes and only a few drugs have been approved by the Food and Drug Ad- ministration (FDA) [4]. The drugs approved by the FDA include antiepileptics, such as gabapentin, pregabalin, carbamazepine, and the antidepressant drug duloxetine [23, 25, 29]. While these three main groups of analgesic drugs are commonly used to treat pain, a significant proportion of patients do not achieve full pain relief. Therefore, there is a strong medical demand for the identification of new agents that act more effectively than currently available pain-killing drugs.
Several recent reports indicate that anticonvulsants are clinically effective for the treatment of certain kinds of pain [8, 21]. Moreover, several antiepileptic agents (such as gabapentin, pregabalin, lamotrigine, tiagabine, and zonisamide) have been shown to be ef-
fective in animal models of pain [12, 13]. Previously, we reported that several derivatives of a-substituted N-benzylamides of g-hydroxybutyric acid (GHB) dis- played anticonvulsant activity [14-16, 26]. On the other hand, some derivatives of dihydrofuran-2-one (also known as g-butyrolactone, GBL), a cyclic pre- cursor of GHB [31], possess both anticonvulsant and analgesic activity [5, 9, 32]. In the present study, we have extended our investigation to new 3-monosubsti- tuted derivatives of dihydrofuran-2-one with potential analgesic activity. We designed structures containing a pharmacophoric moiety of dihydrofuran-2-one with either an arylpiperazine substituent or a benzamide group. The pharmacophoric moiety with the aryl- piperazine substituent is a very well known fragment constitute of a group of serotonin (5HT)1Areceptor ligands [19, 20]. Arylpiperazine derivatives have been reported to exert potent and efficacious analgesic ac- tivity [24]. On the other hand, a benzamide group is characteristic of NSAID drugs.
In the present study, we describe the synthesis of new derivatives of dihydrofuran-2-one and their phar- macological characteristics in the following in vivo models of pain: the hot plate test, the writing test and two methods of local anesthetic activity assessment, namely the infiltration anesthesia as well as the modi- fied tail immersion test. The dihydrofuran-2-one de- rivatives used in this study are indicated as follows:
L-PP [3-(4-phenylpiperazin-1-yl)-dihydrofuran-2-one], L-PP1 {3-[4-(3-trifluoromethylphenyl)-piperazin-1-yl]- dihydrofuran-2-one}dihydrochloride and L-SAL [2-(2- oxo-tetrahydrofuran-3-yloxy)-benzamide]. The chemi- cal structures of the dihydrofuran-2-one derivatives are shown in Figure 1.
Fig. 1. Chemical structure of the investigated dihydrofuran-2-one de- rivatives
Materials and Methods
CHEMICAL PART
The methods used for the synthesis of the desired compounds are shown in Figure 2. Melting points were determined on an Electrothermal 9300 Melting- Point Apparatus and are uncorrected. Reactions were monitored by TLC (Thin-Layer-Liquid Chromatogra- phy) on silica gel plates (5 × 8 cm, 0.25 mm; Kiseel- gel 60 F254, Merck) using the following solvent sys- tems: S1chloroform/acetone (1:1, v/v) and S2chloro- form/methanol/acetic acid (60:10:5, v/v/v) with visualization of spots under UV light. All reagents were purchased from Sigma-Aldrich Co. 1H NMR spectra were recorded with a Varian Mercury 300 spectrometer at 300 MHz with tetramethylsilane as an internal standard. The following abbreviation are used: Ph, phenyl ring; 2-, 3-, 4-, 5- or 6-Ph, indicating position of the proton in the phenyl ring; PIP, pipera- zine ring;a-, b- or g-GBL, indicating a-, b- or g-pos- ition in the lactone ring; s, singlet; d, doublet; t, trip- let; m, multiplet. Elemental analyses of C, H, and N were carried out on a Varian EL III Model Elemental Analyzer and the results are within ± 0.4 % of the theoretical values.
The 3-substituted derivatives of dihydro-2-furan- 2-one, L-PP, L-PP1, and L-SAL, were prepared by N-alkylation of 1-phenylpiperazine, and 1-(3-trifluoro- methylphenyl)-piperazine, respectively, and O-alkyl- ation of salicylamide by 3-bromo-dihydrofuran-2-one according to the procedure described previously for L-PP [14]. Compound (3-[4-(3-trifluoromethylphenyl)- piperazin-1-yl]-dihydrofuran-2-one) was converted to
its dihydrochloride salt and was used in this form for all pharmacological tests.
3-[4-(3-Trifluoromethylphenyl)-piperazin-1-yl]- dihydrofuran-2-one dihydrochloride (L-PP1)
A mixture of (3-trifluoromethylphenyl)-piperazine hydrochloride (50 mmol; 13.34 g) and anhydrous K2CO3 (150 mmol, 20.73 g) in dry acetonitrile (150 ml) was stirred at room temperature for 30 min.
Then, a solution of 3-bromo-dihydrofuran-2-one (51 mmol, 8.41 g) in dry acetonitrile (50 ml) was added drop-wise within 15 min and stirring was con- tinued for 48 h in ambient temperature. The mixture was then filtered and the filtrate was evaporated to ob- tain a crude oily residue which was purified by recrys- tallization from 2-propanol. Yield: 9.77 g (62%); m.p.
68–69°C; TLC: Rf(S1) = 0.65, Rf(S2) = 0.5;1H NMR (CDCl3, d ppm): 7.35 (t, J = 7.9 Hz, 1H, 5-Ph), 7.15–7.01 (m, 3H, 2,4,6-Ph), 4.41–4.23 (m, 2H, CH2-g), 3.59 (t, J = 9.5 Hz, 1H, CH-a), 3.29 (t, J = 5.0 Hz, 4H, CH2-3,5-PIP), 3.03 (m, 2H, CH2-2,6- PIP), 2.74 (m, 2H, CH2-2,6-PIP), 2.47–2.23 (m, 2H, CH2-b).
Anal. (C, H, N) calculated for C15H17N2O2F3; M = 314.310.
Preparation of hydrochloride salt
A solution of 3-[4-(3-trifluoromethylphenyl)-pipe- razin-1-yl]-dihydrofuran-2-one (0.395 g) in chloro- form was chilled in an ice bath and was treated with a saturated solution of hydrogen chloride in anhy- drous ethanol until acidic reaction. A small amount of diethyl ether was added and left standing overnight at 5°C and a white product precipitated. The product
Fig. 2. Synthesis scheme of the dihydrofuran-2-one derivatives. (a) MeCN and KCO!for 24 h, (b) MeCN and KCO!for 48 h
was filtered off, washed with diethyl ether and dried in vacuo. Thus, a dihydrochloride salt of 3-[4-(3- trifluoromethylphenyl)-piperazin-1-yl]-dihydrofuran- 2-one was obtained; m.p. 128–133°C. Anal. (C, H, N) calculated for C15H17N2O2F3× 2 HCl; M = 387.31.
2-(2-oxo-tetrahydrofuran-3-yloxy)-benzamide (L-SAL)
A mixture of salicylamide (2.74 g, 20 mmol) and an- hydrous K2CO3(20 mmol, 2.76 g) in acetonitrile (50 ml) was stirred at room temperature for 15 min. Then, a solution of 3-bromo-dihydrofuran-2-one (20 mmol, 3.3 g) in acetonitrile (50 mL) was added drop-wise within 15 min and stirring was continued for 24 h.
The resultant mixture was filtered and the filtrate was evaporated to obtain a brown oily residue. The crude product that precipitated after addition of 2-propanol and the little amount of n-hexane was purified by re- crystallization from 2-propanol. Yield: 2.57 g (58%);
m.p. 136–137°C; TLC: Rf(S1) = 0.50, Rf(S2) = 0.82;
1H NMR (DMSO-D6, d ppm): 7.73 (d, J = 7.6 Hz, 1H, 6-Ph), 7.54 (d,J = 10.4 Hz, 2H, NH2), 7.53–7.40 (m, 1H, 4-Ph), 7.17 (d,J = 8.3 Hz, 1H, 5-Ph), 7.14 (t, J = 7.5 Hz, 1H, 3-Ph), 5.42 (dd, 1H, J = 8.4, 9.7 Hz, CH-a), 4.47–4.37 (m, 2H, CH2-g), 2.76 (m, 1H, CH2-b), 2.40 (m, 1H, CH2-b). Anal. (C, H, N) calcu- lated for C11H11NO4; M = 221.215.
PHARMACOLOGICAL PART
Chemicals used in pharmacological tests
The investigated compounds were suspended in a 0.5% methylcellulose (MC) (Loba Chemie) solution and administered by intraperitoneal (ip) injection 30 min before the hot plate, writhing, capsaicin- and glutamate-induced pain tests. Phenylbenzoquinone (INC Pharmaceuticals, Inc. NY) was prepared as a 0.02% solution. Morphine (Morphinum hydrochlo- ricum, Polfa Kutno), acetylsalicylic acid (ASA, Pol- pharma) and mepivacaine (Meaverin 2%, Rhone- Poulenc Rorer) were used as reference drugs. Capsai- cin and L-glutamic acid (purchased from Sigma- Aldrich) were dissolved in saline and administered in- traplantarly (ipl) to the mouse paw. Control animals were given an appropriate amount of vehicle control.
Animals
Pain experiments (hot plate, writhing, capsaicin- and glutamate-induced nociception and modified tail im- mersion tests) were carried out on male, Albino Swiss mice weighing 18–30 g. All procedures were approved by the Local Ethics Committee in Cracow. They were kept in groups of 15 mice in cages at room tempera- ture (22 ± 2°C) under a light/dark cycle and had free access to food and water before the experiments. Each experimental group consisted of 6–8 animals per dose. All animals were used only once. The infiltra- tion anesthesia test was conducted in guinea pigs kept under standard conditions. Experiments were per- formed between 8 a.m. and 3 p.m.
Statistical analysis
The data are expressed as the mean ± standard error of the mean (SEM). To compare the results between two different groups of animals (the investigated com- pound groupversus the control group) in the writhing, hot plate and tail immersion tests, Student’st-test was performed. In the capsaicin- and glutamate-induced nociception, the statistical significance was assessed by means of one-way ANOVA followed by Newman- Keuls test. The difference of means was statistically significant if p < 0.005.
Methods
The hot plate test
In the hot plate test, mice were treatedip with either the compound or the standard control 30 min prior to placing the animal on the hot plate apparatus (Hot Plate 2A Type Omega) with the temperature con- trolled for 55–56°C. The time until the animal licks its back paws or jumps is recorded by the means of a stop-watch [6]. Centrally-acting analgesics (such as morphine) prolong the reaction time whereas periph- erally acting ones (such as NSAIDs) show little to no activity in this test [30].
The writhing test
In contrast to their activity in the hot plate test, NSAIDs are highly antinociceptive in the writhing test. Mice were treated with 0.25 ml of a 0.02% phen- ylbenzoquinone solution 30 min after ip administra- tion of the investigated compound or vehicle. Then, the mice were placed individually into glass beakers and 5 min were allowed to elapse. After that period, a 10 min observation was conducted for each animal and the number of characteristic writhes was counted.
The analgesic effect of the tested substances was de- termined by a decrease in the number of writhes ob- served [10].
Capsaicin-induced pain
After an adaptation period of 20 min, 20 µl of capsai- cin solution prepared in saline (1.6 µg capsaicin per mouse paw) was injected ipl into the ventral surface of the right hindpaw 30 min after the tested compound was administeredip. The animals were observed indi- vidually for 5 min following capsaicin injection. The amount of time spent licking the injected paw was re- corded with a chronometer and was considered as in- dicative of nociception [27].
Glutamate-induced pain
Glutamate-induced nociception was induced by in- jecting the animal byipl with 20 µl of an L-glutamic acid solution (30 µmol per mouse paw) under the ven- tral surface of the right hindpaw 30 min after the tested compound was administered ip. The animals were observed individually for 15 min following L- glutamic acid injection. The amount of time spent licking the injected paw was recorded with a chro- nometer and was considered as indicative of nocicep- tion [2].
Local anesthetic activity
Infiltration anesthesia (intradermal wheal test) in guinea pigs
Infiltration anesthesia was tested in guinea pigs by causing an intradermal wheal by injecting the studied compounds at a constant volume of 0.1 ml and a con- centration of 1.0% to the dorsum skin. A painful reac- tion to a prick of the skin at the center of the wheal
three times and every 5 s with 5 min intervals during the first 30 min of observation was tested. The experi- ment was continued to achieve a return of a reaction to the prick [3]. The control wheal was done by an in- tradermal injection of 0.1 ml of 0.9% NaCl. Mepiva- caine (Mep) was used as a reference drug.
The tail immersion test (modified)
The heat method used for evaluating the systemic an- algesic activity can also be used with a slight modifi- cation to determine whether a compound possesses local anesthetic activity. The method was performed by subcutaneously (sc) injecting the investigated sub- stance in a constant volume of 0.2 ml about 1 cm from the root of the mouse tail and waiting 15 min. The 3 cm distal part of the tail was immersed into temperature controlled water (50 ± 0.5°C). The reaction time (the length of time until the tail is pulled away) was meas- ured by the means of a chronometer. The observation time was limited to 20 s [7].
Results
The analgesic activity of newly synthesized sub- stances is generally evaluated first in screening tests using two basic in vivo methods. One method is the hot plate test and the second method is the writhing test. The hot plate test examines the central analgesic activity of the compound. The paws of mice and rats are very sensitive to heat at temperatures that are not damaging to the skin. The administration of centrally acting analgesics prolongs reaction time, whereas pe- ripheral analgesics (such as COX inhibitors) generally do not affect the response time [30]. In the writhing test, pain is induced by injection of an irritant (such as phenylbenzoquinone) into the peritoneal cavity of the mouse. The animal reacts with a characteristic stretching behavior called writhing. This test detects central and peripheral analgesic activity; however, some psychoactive agents (including clonidine and haloperidol) also show activity in this test [30]. Local anesthetic activity can be evaluated in rodents, as well using capsaicin and glutamate to induce pain. In the present study, only the most active compounds were used in the capsaicin- and glutamate-induced pain
Tab. 1.Analgesic activity of the dihydrofuran-2-one derivatives and morphine in the hot plate test
Compound Dose (mg/kg) Reaction time (s) to painful stimulus (± SEM)
Prolongation of the reaction time (%)
ED#
Control 0.5% MC 11.39 ± 0.76 – –
L-PP1 0.9 14.30 ± 1.79 25.55 1.34 (1.00–1.79)
1.8 20.17 ± 3.21*** 77.09
3.75 26.67 ± 2.83**** 134.15
15 29.45 ± 3.41**** 158.56
L-SAL 30 11.75 ± 1.69 3.16 –
Control 0.5% MC 10.85 ± 0.78 – –
L-PP 1.8 15.30 ± 1.40*** 41.01 4.02 (0.66–24.63)
15 17.65 ± 2.17*** 62.67
30 22.06 ± 3.04**** 103.32
Control 0.5% MC 18.4 ± 1.0 – –
Morphine 1 19.4 ± 2.1 5.4 3.39 (2.24–5.12)
3 29.9 ± 6.0* 60.9
6 30.6 ± 3.9** 66.3
Asterisks (*) indicate a significant difference compared to the methylcellulose control; * p < 0.05, ** p < 0.02, *** p < 0.01, **** p < 0.001. Each value represents the mean ± SEM obtained from eight animals. Compounds were administeredip
Tab. 2.Analgesic activity of the dihydrofuran-2-one derivatives and acetylsalicylic acid in the writhing test
Compound Dose (mg/kg) Mean number
of writhes (± SEM)
Inhibition (%) ED#
Control 0.5% MC 25.17 ± 3.2 – –
L-PP1 0.45 16.67 ± 2.60 33.77 0.79 (0.44–1.40)
1.35 5.67 ± 1.50 **** 77.47
1.8 1.17 ± 0.48 **** 95.35
L-SAL 3.75 21.20 ± 2.85 15.77 7.92 (5.01–12.50)
7.5 11.67 ± 2.25 ** 53.64
15 6.00 ± 1.82 *** 76.16
Control 0.5% MC 31.70 ± 1.38 – –
L-PP 1.8 18.50 ± 2.26 **** 41,64 2.62 (0.89–7.68)
7.5 8.67 ± 2.22 **** 72.65
15 7.20 ± 0.86 **** 77.29
Control 0.5% MC 19.20 ± 3.20 – –
Acetylsalicylic acid 30 11.20 ± 2.10 41.70 39.15 (29.1–48.4)
50 8.50 ± 1.30 ** 55.70
100 3.20 ± 1.20 **** 83.30
Asterisks (*) indicate a significant difference compared to the methylcellulose control; ** p < 0.02, *** p < 0.01, **** p < 0.001. Each value repre- sents the mean ± SEM obtained from eight animals. Compounds were administeredip
tests. The data obtained using these pain tests are shown below in tables.
The hot plate test
The hot plate test is a heat method of evaluating anal- gesic activity of central origin. Since the investigated compounds share chemical similarity with GABA (g-aminobutyric acid) we wanted to check if they would act as central analgesics. Using this test, we showed that the two piperazine derivatives of dihydrofuran-2-one, L-PP and L-PP1, significantly prolonged the nociceptive reaction time in mice in a dose-dependent manner (Tab. 1). L-PP1 was more active in this test than L-PP. L-SAL displayed mini- mal central analgesic activity that was only observed at a relatively high dose (30 mg/kg), most likely due
to its chemical similarity to salicylates. The calculated ED50values for L-PP (4.02 mg/kg) and L-PP1 (1.34 mg/kg) were similar or lower than the ED50value ob- tained for morphine (3.39 mg/kg), the reference drug used in this test (Fig. 3).
The writhing test
In this test, all the compounds acted as effective anal- gesics in mice. The results for all compounds were statistically significant. Similar to the hot plate test, L-PP1 was active at lower doses than other two com- pounds tested (Tab. 2). Interestingly, L-SAL was ac- tive in this test, which evaluates peripheral analgesic activity. These data, as suggested above, indicate that the activity of L-SAL may be attributed to the sali- cylamide fragment of its chemical structure. The
Fig. 4.The ED# values (in mg/kg) of the dihydrofuran-2-one derivatives and acetylsalicylic acid (ASA) used in the writhing test
HOT PLATE TEST
0 0.5 1 1.52 2.5 3 3.5 4 4.5
ED 50(mg/kg)
LPP1 LPP MORPH
Fig. 3.The ED# values (in mg/kg) of the dihydrofuran-2-one derivatives and morphine (MORPH) in the hot plate test
ED50 value obtained for acetylsalicylic acid (39.15 mg/kg), used as the reference drug in this test, was significantly higher than the ED50 values calculated for L-PP (2.62 mg/kg), L-PP1 (0.787 mg/kg) and L-SAL (7.92 mg/kg) (Fig. 4).
Capsaicin-induced pain
Next, we tested whether L-PP1 and L-PP were able to diminish the nociceptive reaction after capsaicin in- jection. Both of these compounds were effective in
decreasing nociceptive pain reaction. L-PP1 was more active with a calculated ED50 of 2.01 mg/kg. The ED50for L-PP could not be established. The data for this test are shown in Table 3.
Glutamate-induced pain
Since L-PP1 was the most active in the previous tests, was examined its ability to diminish the nociceptive reaction after intraplantar glutamate injection. This compound was effective at decreasing the nociceptive reaction (Tab. 4). The calculated ED50 for L-PP1 in this test was 3.99 mg/kg.
Tab. 4.Analgesic activity of the dihydrofuran-2-one derivative in the glutamate-induced pain
Compound Dose (mg/kg)
Reaction time (s) to painful stimulus (± SEM)
Prolongation of the reaction
time (%)
ED#
Control 0.5% MC 86.14 ± 8.62 – –
L-PP1 1.8 55.43 ± 7.28** 35.65 3.99
(1.87–8.49)
7.5 41.72 ± 5.29**** 51.57
30 4.17 ± 1.42**** 95.16
Asterisk (*) indicate a significant difference compared to the methyl- cellulose control; ** p < 0.02, **** p < 0.001. Each value represents the mean ± SEM obtained from eight animals. Compounds were ad- ministeredip
Tab. 3.Analgesic activity of the dihydrofuran-2-one derivatives in the capsaicin-induced pain
Compound Dose (mg/kg)
Reaction time (s) to painful stimulus
(± SEM)
Prolongation of the reaction time
(%)
ED#
Control 0.5% MC 45.90 ± 1.12 – –
L-PP1 0.225 37.95 ± 3.25* 17.32 2.01 (0.58–6.94) 1.8 20.43 ± 3.57**** 55.49
7.5 15.61 ± 2.47**** 65.99 30 7.77 ± 2.66**** 83.07
L-PP 15 32.74 ± 3.13*** 28.67 –
30 24.48 ± 2.94**** 46.67 60 24.57 ± 1.94**** 46.47
Asterisks (*) indicate a significant difference compared to the methyl- cellulose control; * p < 0.05, *** p < 0.01, **** p < 0.001. Each value represents the mean ± SEM obtained from eight animals. Com- pounds were administeredip
Fig. 5.Activity of L-PP1 and mepiva- caine in the infiltration anesthesia model
Local anesthetic activity
Infiltration anesthesia
Next, we wanted to determine the effect of the 3- substituted derivatives of dihydro-2-furan-2-one in an animal model of infiltration local anesthesia. Experi- ments performed in guinea pigs showed potent local anesthetic activity of L-PP1. This local anesthetic ac- tivity lasted up to 24 hours after the compound’s in- jection. In general, the anesthetic effect observed was similar to the activity of mepivacaine (Fig. 5).
Tail immersion test
To further test the effects of the compounds on local anesthesia, the two most active compounds, L-PP and L-PP1, were examined using the tail immersion test.
Both L-PP and L-PP1 appeared to significantly pro- long the animals’ reaction time to a heat stimulus in a concentration-dependent manner that was similar to the effect of the reference drug mepivacaine (Tab. 5).
Discussion
The transduction of a painful stimuli is a complex process that takes place in the peripheral endings of the nociceptive neuron (I neuron of the pain path- way), namely the nociceptors situated on Ad and C- fibers. Ad nerve fibers are thin and myelinated and serve as fast conduction of impulses. In turn, C-fibers are very thin and unmyelinated and are particularly sensitive to damage. C-fibers mostly function as poly- modal nociceptors receiving various types of noxious stimuli (chemical, thermal and mechanical) from the internal and external environment. C-fibers possess many different types of receptors on their surface, in- cluding peripheral opioid and transient receptor po- tential vanilloid 1 (TRPV1, formerly known as vanil- loid 1 (VR1)) receptors. The direct stimulation of VR1 results in the activation of non-selective cal- cium- and sodium-permeable cation channels result- ing in the influx of cations that generate an action po- tential within the sensory neurons [18, 34].
Tissue injury causes an increase in the levels of po- tassium, proinflammatory bradykinin, histamine, prostanoids, cytokines, substance P (SP), ATP, pro- tons, serotonin and excitatory amino acids (EAA).
These substances are responsible for the phenomenon known as peripheral sensitization of the nerve fibers.
Moreover, these substances generate the nociceptive information that is transmitted towards the spinal dor- sal horn and the cortex [34].
The proper modulation of painful stimuli takes place in the spinal dorsal horn, the spinothalamic tract and the descending antinociceptive pathways. Nor- adrenaline and serotonin are released within these pathways. Serotoninergically and noradrenergically acting coanalgetics (such as amitriptyline) also play a significant role here.
Pain perception, the endpoint of the whole nocicep- tion process, occurs in the cortex and the limbic sys- tem. These structures are responsible for the cognitive role of pain, including its realization and affective re- actions related to pain (including anxiety, irritation and aggression) [34].
Pain treatment consists of both psychological and pharmacological approaches. In our search for new, more efficacious analgesics, we identified several dihydrofuran-2-one derivatives that display antino- ciceptive activity in two basic animal models of acute pain, the hot plate test and the writhing test. The
Tab. 5.Local anesthetic activity of the dihydrofuran-2-one deriva- tives and mepivacaine in the tail immersion test
Compound Concentration (%)
Reaction time (s) to painful stimulus
( ± SEM)
Prolongation of the time reaction (%)
Control 0.5% MC 7.26 ± 2.17 –
L-PP1 1 13.11 ± 3.04 80.58
2 18.09 ± 1.91 ** 149.17
L-PP 1 14.65 ± 2.47 * 101.79
2 17.87 ± 1.43 ** 146.14
Mepivacaine 1 14.13 ± 2.67 94.63
2 15.73 ± 2.09 ** 116.67
Asterisks (*) indicate a significant difference compared to the methyl- cellulose control; * p < 0.05, ** p < 0.02. Each value represents the mean ± SEM obtained from six animals
evaluation of local anesthetic activity enabled us to determine the most likely site of action of L-PP1 is namely the peripheral nerves of sensory neurons, the Ad and C-fibers. Local anesthetic activity of L-PP1 suggests that its sodium voltage-gated channel block- ing activity is its mechanism of action. In fact, pain sensitive sodium channels, Nav1.8 and Nav1.9, are localized in small and medium-sized sensory neurons of dorsal root ganglion (DRG). Data have shown that at least half of small diameter DRG neurons possess Nav1.8 channels and these channels coexist with Nav1.9, as well as with TRPV1 receptors. Similarly, the Ad fibers also possess Nav1.8 sodium channels [22].
As mentioned above, TRPV1 receptors act as poly- modal nociceptors that are sensitive to heat (tempera- tures greater than 43°C), protons, capsaicin, endocan- nabinoid-anandamide (arachidonoylethanolamide – AEA) and other lipid mediators, including some eicosanoids generated in the lipooxygenase pathway (such as 12-(S)-HPETE, 15-(S)-HPETE, 5-(S)-HETE, LTB4) [1, 18, 28]. The fact that TRPV1 may be activated by heat suggested to us that the analgesic activity of at least one of the investigated compounds in the hot plate test (which examines thermal stimulation of the mouse paw at a temperature of 55°C) is not likely to be of central origin. Rather, the analgesic activity is a consequence of a GBL derivatives’ TRPV1 blocking activity. To prove this hypothesis, we performed an- other experiment in which pain was induced by cap- saicin administered locally into the mouse paw. The most active compounds, L-PP1 and L-PP, turned out to be effective analgesic agents in this model of pain stimulation. We hypothesize that L-PP1 and L-PP are effective analgesic agents due to their TRPV1 antago- nistic activity.
As far as phenylbenzoquinone-induced writhing is concerned, there are data suggesting that some new antagonists of TRPV1 by influencing this receptor may decrease inflammatory pain with a potency simi- lar to indomethacin. These compounds diminished capsaicin-induced pain reactions in a dose-dependent manner and moreover, they evoke hypothermia simi- lar to other TRPV1 ligands [28]. Our data suggest that L-PP1 may be one such antagonist. In the case of the two other lactones tested, L-PP and L-SAL, another mechanism of action may exist. L-PP, although active in the hot plate test, exerted weak activity when capsaicin-induced nociception was examined. There- fore, we hypothesize that the analgesic activity of
L-PP is of central origin. In regard to L-SAL, we did not observe a central nervous system antinociceptive activity using the hot plate test. As mentioned above, because of its structural similarity to salicylates, we postulate that it possesses peripheral analgesic activ- ity. However, this question remains to be addressed.
Another interesting observation was the influence of L-PP1 on glutamate-induced nociception. L-glutamic acid is a well-known potent nociceptive stimulus. The analgesic activity of L-glutamic acid is determined by its influence on ionotropic receptors resulting in a massive influx of calcium into cells. Some data sug- gest that there is a close union between capsaicin and glutamate action within sensory nerve fibers and that nociception induced by intraplantar injection of gluta- mic acid in mice is mediated by capsaicin-sensitive fi- bers [2]. Indeed, we prove that L-PP1 administered systemically (ip) before L-glutamic acid diminishes the nociceptive response to the latter.
Taking all of our results into account, L-PP1 emerged as an effective anesthetic. L-PP1 had the highest antinociceptive activity in all the tests used in the present study, making this compound the best can- didate for further investigation. The mechanism of its action is not completely clear. Our data suggest that an effect on the TRPV1 receptors should be consid- ered first. Since activation of TRPV1 receptors results in an inward calcium influx, similar to glutamate’s ac- tion on its ionotropic receptors, it is very likely that the analgesic activity of this substance is a conse- quence of inhibiting calcium currents, an effect that does not necessarily have to follow direct receptor coupling. An influence of L-PP1 on further stages of TRPV1 or glutamate receptor activation (namely the inhibition of calcium currents) must be taken into consideration, as well.
Conclusions
In the experiments presented here, we prove that 3-monosubstituted derivatives of dihydrofuran-2-one possess analgesic properties in rodents. Preliminary screening tests documented their potent central and/or peripheral analgesic activity, as well as local anesthetic activity. Using the spontaneous locomotor activity test, we show that the results presented here cannot be falsely attributed to lactones’ sedative influence (data
not shown). The most active compound, L-PP1, likely exerts its pharmacological effect through an interac- tion with TRPV1 receptors and influencing their activation pathway. In the case of the other two dihydrofuran-2-one derivatives, further examination of their distinct mechanisms of action need to be pursued.
Acknowledgments:
This study was supported by a grant of Ministry of Science and Higher Education (PhD-Research Grant No. N40502931/1910) and Jagiellonian University Collegium Medicum (W³/427/P/F).
References:
1. Alexander SPH, Mathie A, Peters JA: Guide to receptors and channels (GRAC), 2nd edition (2007 revision). Br J Pharmacol, 150, Suppl 1, S1–S168.
2. Beirith A, Santos ARS, Calixto JB: Mechanisms under- lying the nociception and paw oedema caused by injec- tion of glutamate into the mouse paw. Brain Res, 2002, 924, 219–228.
3. Buelbring E, Wajda I.: Biological comparison of local anesthetics. J Pharmacol Exp Ther, 1945, 85, 78–84.
4. Butera JA: Current and emerging targets to treat neuro- pathic pain. J Med Chem, 2007, 50, 2543–2546.
5. Canney DJ, Lu HF, McKeon AC, Yoon KW, Xu K, Hol- land KD, Rothman SM et al.: Structure-activity studies of fluoroalkyl-substitutedg-butyrolactone and g-thiobut- yrolactone modulators of GABA)receptor function.
Bioorg Med Chem, 1998, 6, 43–55.
6. Eddy N, Leimbach D: Synthetic analgesics. II. Dithien- ylbutenyl- and dithienylbutylamines. J Pharmacol Exp Ther, 1953, 107, 385–393.
7. Erenmemisoglu A, Suer C, Temocin S: Has nicotine a lo- cal anesthetic action? J Basic Clin Physiol Pharmacol, 1994, 5, 125–131.
8. Ettinger AB, Argoff CE: Use of antiepileptic drugs for nonepileptic conditions: psychiatric disorders and chronic pain. Neurotherapeutics, 2007, 4, 75–83.
9. Hadri AE, Abouabdellah A, Thomet U, Bauer R, Furt- müller R, Sigel E, Sieghart W, Dodd RH: N-Substituted 4-amino-3,3-diphenyl-2(3H)-furanones: New positive al- losteric modulators of the GABA)receptor sharing elec- trophysiological properties with the anticonvulsant lore- clezole. J Med Chem, 2002, 45, 2824–2831.
10. Hendershot LC, Forsaith J: Antagonism of the frequency of phenylbenzoquinone inducted writhing in the mouse by weak analgesic and non-analgesics. J Pharmacol Exp Ther, 1959, 125, 237–240.
11. http://www.iasp-pain.org/AM/Template.cfm?Sec- tion=Home&Template=/CM/ContentDisplay.cfm&Con- tentID=6633
12. Hunter JC, Gogas KR, Hedley LR, Jacobson LO, Kasso- takis L, Thompson J, Fontana DJ: The effect of novel
anti-epileptic drugs in rat experimental models of acute and chronic pain. Eur J Pharmacol, 1997, 324, 153–160.
13. Laughlin TM, Tram KV, Wilcox GL, Birnbaum AK:
Comparison of antiepileptic drugs tiagabine, lamotrigine, and gabapentin in mouse models of acute, prolonged, and chronic nociception. J Pharmacol Exp Ther, 2002, 302, 1168–1175.
14. Malawska B, Gobaille S: Synthesis, physicochemical and pharmacological properties of newN-substituted amides ofa-piperazine-g-hydroxybutyric acid. Phar- mazie, 1995, 50, 390–393.
15. Malawska B, Kulig K, Gajda J, Szczeblewski D, Musia³ A, Wiêckowski K, Stables JP: Design, synthesis and pharmacological evaluation ofa-substituted N-be- nzylamides ofg-hydroxybutyric acid with potential GABA-ergic activity. Part 6. Search for new anticonvul- sant compounds. Acta Pol Pharm Drug Res, 2007, 64, 127–137.
16. Malawska B, Kulig K, Œpiewak A, Stables JP: Investiga- tion into new anticonvulsant derivatives ofa-substituted N-benzylamides of g-hydroxy- and g-acetoxybutyric acid. Part 5. Search for new anticonvulsant compounds.
Bioorg Med Chem, 2004, 12, 625–632.
17. Meldrum ML: A capsule history of pain management.
JAMA, 2003, 290, 2470–2475.
18. Numazaki M, Tominaga M: Nociception and TRP chan- nels. Curr Drug Targets, 2004, 3, 479–485.
19. Obniska J, Kamiñski K, Tatarczyñska E: Impact of aro- matic substitution on the anticonvulsant activity of new N-(4-arylpiperazin-1-yl)-alkyl-2-azaspiro[4.5]decane- 1,3-dione derivatives. Pharmacol Rep, 2006, 58, 207–214.
20. Paluchowska M, Mokrosz MJ, Charakchieva-Minol S, Duszyñska B, Kozio³ A, Weso³owska A, Stachowicz K, Chojnacka-Wójcik E: Novel 4-alkyl-1-arylpiperazines and 1,2,3,4-tetrahydroisoquinolines containing diphenyl- methylamino or diphenylmethoxy fragment with differ- entiated 5-HT )/5-HT )/D receptor activity. Pol J Phar- macol, 2003, 55, 543–552.
21. Pappagallo M: Newer antiepileptic drugs: possible uses in the treatment of neuropathic pain and migrene. Clin Ther, 2003, 25, 2506–2538.
22. Priestley T: Voltage-gated sodium channels and pain.
Curr Drug Targets, 2004, 3, 441–456.
23. Rogawski MA, Loscher W: The neurobiology of antiepi- leptic drugs for the treatment of nonepileptic conditions.
Nat Med, 2004, 10, 685–692.
24. Rohet F, Rubat C, Coudert P, Couquelet J: Synthesis and analgesic effects of 3-substituted 4,6-diarylpyridazine derivatives of the arylpiperazine class. Bioorg Med Chem, 1997, 5, 655–659.
25. Sahebgharani M, Hossein-Abad AA, Zarrindast MR: On the mechanism of carbamazepine-induced antinocicep- tion in the formalin test. Int J Neurosci, 2006, 116, 1097–1113.
26. Sa³at K, Mendyk A, Librowski T, Czarnecki R, Ma- lawska B: Influence of newg-hydroxybutyric amid am- ide analogues on the central nervous system activity in mice. Pol J Pharmacol, 2002, 54, 731–736.
27. Santos ARS, Gadotti V, Oliveira GL, Tibola D, Paszcuk AF, Neto A: Mechanisms involved in the antinociception caused by agmatine in mice. Neuropharmacology, 2005, 48, 1021–1034.
28. Suh YG, Oh U: Activation and activators of TRPV1 and their pharmaceutical implication. Curr Pharm Des, 2005, 11 2687–2698.
29. Tremont-Lukats IW, Megeff C, Backonja MM: Anticon- vulsants for neuropathic pain syndromes: mechanism of action and place in therapy. Drugs, 2000, 60, 1029–1059.
30. Vogel HG: Drug Discovery and Evaluation. Pharma- cological Assays. 2nd edn., Springer-Verlag, Berlin-Hei- delberg, 2002.
31. Waszkielewicz A, Bojarski J: g-Hydroxybutyric acid (GHB) and its chemical modifications: a review of the GHBergic system. Pol J Pharmacol, 2004, 56, 43–49.
32. Williams KL, Tucker JB, White G, Weiss DS, Ferren- delli JA, Covey DF, Krause JE, Rothman SM: Lactone
modulation of theg-aminobutyric acid A receptor: Evi- dence for a positive modulatory site. Mol Pharmacol, 1997, 52, 114–119.
33. Woolf CJ, Mannion RJ: Neuropathic pain: etiology, symptoms, mechanism, and management. Lancet, 1999, 353, 1959–1964.
34. ¯ylicz Z, Krajnik M: How does pain emerge? Pain neu- rophysiology for the beginners (Polish). Pol Med Paliat, 2003, 2, 49–56.
Received:
November 13, 2008; in revised form: September 9, 2009.
