Vinpocetine and Piracetam Exert Antinociceptive Effect in Visceral Pain Model in Mice

Vinpocetine and piracetam exert antinociceptive effect in visceral pain model in mice

Omar M.E. Abdel Salam

Department of Pharmacology, National Research Centre, Tahrir St., Dokki, Cairo, Egypt

Correspondence: Omar M.E. Abdel Salam, e-mail: [email protected]

Abstract: The effect of vinpocetine or piracetam on thermal and visceral pain was studied in mice. In the hot plate test, vinpocetine (0.9 and 1.8 mg/kg), but not piracetam, produced a reduction in nociceptive response. Vinpocetine (0.45–1.8 mg/kg, ip) or piracetam (75–300 mg/kg, ip) caused dose-dependent inhibition of the abdominal constrictions evoked by ip injection of acetic acid. The effect of vinpocetine or piracetam was markedly potentiated by co-administration of propranolol, guanethidine, atropine, naloxone, yohimbine or prazosin. The marked potentiation of antinociception occurred upon a co-administration of vinpocetine and baclofen (5 or 10 mg/kg). In contrast, piracetam antagonized antinociception caused by the low (5 mg/kg), but not the high (10 mg/kg) dose of baclofen. The antinociception caused by vinpocetine was reduced by sulpiride; while that of piracetam was enhanced by haloperidol or sulpiride. Either vinpocetine or piracetam enhanced antinociception caused by imipramine. The antinociceptive effects of vinpocetine or piracetam were blocked by prior administration of theophylline. Low doses of either vinpocetine or piracetam reduced immobility time in the Porsolt’s forced-swimming test. This study indicates that vinpocetine and piracetam possess visceral antinociceptive properties. This effect depends on activation of adenosine receptors. Piracetam in addition inhibits GABA-mediated antinociception.

Key words: vinpocetine, piracetam, thermal pain, visceral pain, mice

Introduction [16], to indicate this category of drugs that enhance memory, facilitate learning and protect memory pro- cesses against conditions which tend to disrupt them. Piracetam, the first of the pyrrolidone derivatives Despite the fact that piracetam and vinpocetine (2-oxopyrrolidine), is a widely used drug in the treat- have been used in clinical practice for around three ment of cerebral stroke [20], dementia or cognitive decades, their precise mechanism of action within the impairment in the elderly [41], and myoclonus epi- brain remains largely unknown. Enhancement of oxi- lepsy [10]. The drug reverses hippocampal membrane dative glycolysis, anticholinergic effects, positive ef- alterations in Alzheimer’s disease [8]. Vinpocetine, fects on the cerebral circulation, effects on membrane a synthetic derivative of the alkaloid vincamine hav- physics, and also reduction of platelet aggregation ing antianoxic, antiischemic and neuroprotective and improvement in erythrocyte function are likely to properties, is frequently used in the therapy of cere- account for a cognition-enhancing effect and a neuro- bral disorders of vascular origin [11]. Although both protective effect of piracetam in stroke [36]. On the drugs belong to distinct chemical classes, they share other hand, vinpocetine is a potent vasodilator at the the name nootropic, the term introduced by Giurgea cerebral vascular bed, increasing cerebral blood flow,

680 Pharmacological Reports, 2006, 58, 680691 Antinociception by vinpocetine and piracetam Omar M.R. Abdel Salam

that increases the regional cerebral glucose uptake vided ad libitum. All experimental procedures were and, to a certain extent, glucose metabolism in the conducted in accordance with the guide for the care so-called peri-stroke region as well as in the relatively and use of laboratory animals and were reviewed by intact brain tissue [40]. Vinpocetine has been found to the Local Animal Care and Use Committee. The interfere with various stages of the ischemic cascade: doses of vinpocetine or piracetam used in the study adenosine triphosphate (ATP) depletion, activation of were based upon the human dose after conversion to voltage-sensitive Na+- and Ca++-channels, glutamate that of rat [31]. and free radicals release [17]. Apart from their cognitive enhancing properties, Hot-plate assay both drugs are likely to possess other important pharmacological properties. Gastric protective effects The hot-plate test was performed by using an elec- have been described for vinpocetine [28], whereas pi- tronically controlled hot plate (Ugo Basile, Italy) racetam has been suggested to exert anti-inflammatory heated to 52 ± 0.1°C). The cut-off time was 30 s. properties [27]. Prolonged consumption of piracetam Groups of mice (n = 6/group) were given vinpocetine resulted in a 3-fold decrease in beta-endorphin con- (0.45, 0.9 or 1.8 mg/kg, sc, 0.2 ml, n = 6/group) or pi- centration in plasma of rats [39], but its ip administra- racetam (75, 150 or 300 mg/kg, sc, 0.2 ml) or saline tion (100 mg/kg) inhibited gamma aminobutyric acid (control), 30 min prior to testing. The experimenter (GABAergic) antinociception caused by baclofen was blind to the dose. Latency to lick a hind paw or [13]. Nefiracetam, a new member of the piracetam jump out of the apparatus was recorded for the control group of cognition enhancers, reversed the thermal hyper- and drug-treated groups. algesia observed in nerve-injured mice and the thermal hyperalgesia observed in streptozotocin-induced diabetic Abdominal constriction assay mice [33]. Vinpocetine is capable of blocking NaV1.8 sodium Separate groups of 6 mice each were given vinpocet- channel activity, which suggests a potential additional ine (0.45, 0.9 or 1.8 mg/kg, ip, 0.2 ml), piracetam (75, utility of the drug in various sensory abnormalities 150 or 300 mg/kg, ip, 0.2 ml), indomethacin arising from abnormal peripheral nerve activity [44]. (20 mg/kg, ip, 0.2 ml) or saline (0.2 ml, ip, control). The above data suggest that these nootropic drugs are Unless otherwise indicated in the text, drugs or saline likely to modulate nociception and might prove useful were administered 30 min prior to an ip injection of in certain types of pain. In the light of the above and 0.2 ml of 0.6% acetic acid [22]. Each mouse was then since the nootropic agents vinpocetine and piracetam placed in an individual clear plastic observational are widely prescribed for the pharmacotherapy of chamber, and the total number of writhes experienced memory and neurodegenerative disorders, it, thereby each mouse was counted for 30 min after acetic fore, looked pertinent to investigate and compare the acid administration by an observer who was not aware pharmacological effects of piracetam and vinpocetine of the animals’ treatment. Afterwards, animals were in two experimental pain models in mice: the acetic sacrificed by CO2 exposure. acid-induced writhing response, a model of visceral Further experiments were designed in an attempt to inflammatory pain (chemonociception), and the tail- elucidate the mechanisms by which vinpocetine or pi- flick test, a model of supraspinal analgesia. Moreover, racetam exerts its anti-nociceptive effect. The dose of the effect of the two drugs on locomotor activity and 1.8 mg/kg of vinpocetine or 300 mg/kg of piracetam their potential antidepressant effect were evaluated. was then selected to be used in the subsequent experiments. Thus, we examined the effects of co-administration of the a-1 adrenoceptor antagonists prazosin (2 mg/kg, Materials and Methods ip) and doxazosin (4 or 16 mg/kg, ip), a-2 adrenoceptor antagonist yohimbine (4 mg/kg, ip), b-adrenocep- Animals tor antagonist, propranolol (2 mg/kg, ip), muscarinic acetylcholine receptor antagonist atropine (2 mg/kg Male albino Swiss mice 20–22 g of body weight were ip), non-selective opioid receptor antagonist naloxone used. Standard laboratory food and water were pro- (5 mg/kg ip), GABA agonist baclofen (5 or 10 mg/kg)

Pharmacological Reports, 2006, 58, 680691 681 and ATP-gated potassium channel blocker glibencla- Briefly, each mouse was placed individually in a glass mide (5 mg/kg, ip) on antinociception caused by vin- cylinder (diameter 12 cm, height 24 cm) filled with pocetine or piracetam. Drugs were administered water to a height of 12 cm. Water temperature was 30 min prior to the abdominal constriction assay. maintained at 24–25°C. The animal was forced to The effect of adrenergic neuron blockade with swim for 6 min and the duration of immobility was guanethidine (16 mg/kg,ip) co-administered with ei- measured. The mouse was considered as immobile ther vinpocetine or piracetam was also investigated. when it stopped struggling and moved only to remain The abdominal constriction assay was carried out floating in the water, keeping its head above water 30 min after the administration of vinpocetine or pi- [32]. racetam. In two separate sets of experiments, the floating In addition, the effect of the non-selective adeno- time was recorded after treatment with different doses sine receptor antagonist theophylline (20 mg/kg ip)or of vinpocetine (0.45–1.8 mg/kg, sc) or piracetam the nonsteroidal anti-inflammatory drug indometha- (37.5–300 mg/kg, sc) and compared to that of imi- cin (20 mg/kg, ip) was examined. Theophylline or pramine (15 mg/kg, sc). The control group received indomethacin was administered 30 min prior to vin- saline. In the third set of experiments, the possible pocetine or piracetam (60 min prior to the abdominal modulation of the antidepressant effect of imipramine constriction assay). (15 mg/kg, sc) by either vinpocetine (1.8 mg/kg, sc) In further experiments, the effect of co- or piracetam (300 mg/kg, sc) or the possible effect of administration of the centrally acting dopamine D2 combined vinpocetine (1.8 mg/kg, sc) and piracetam receptor antagonists, sulpiride (10 or 20 mg/kg, ip) (300 mg/kg, sc) administration was investigated. and haloperidol (0.5, 1.5 or 3 mg/kg, ip) or D2 recep- ip tor agonist bromocryptine (1.5 or 3 mg/kg, ) on vin- Drugs pocetine (1.8 mg/kg, ip) or piracetam (300 mg/kg, ip) antinociception was examined. Lastly, the effect of Vinpocetine (Vinporal, Amrya. Pharm. Ind., Cairo, vinpocetine or piracetam on antinociception caused ARE), piracetam (Nootropil, Chemical Industries De- by the tricyclic agent imipramine was studied. velopment; CID, Cairo, ARE), guanethidine, propranolol hydrochloride, yohimbine hydrochloride, na- Rota-rod test loxone hydrochloride (Sigma, St. Louis, USA), imipramine hydrochloride, bromocryptine (Novartis Motor performance in mice was measured as the la- Pharma, Cairo, ARE), amitriptyline hydrochloride, tency to fall from an accelerating rota-rod located haloperidol, indomethacin (Kahira Pharm & Chem. over plates connected to an automatic counter (Ugo IND Co., Cairo, ARE), glibenclamide (Hoechst Ori- Basile, Varese, Italy). Mice were trained to remain on ent, Cairo, ARE), atropine sulfate, baclofen (Misr a rotating rod for 2 min as the rod rotated toward the Pharm Co., Cairo, ARE), domperidone (Janssen- animal. After the 2-min training period, the mice were Cilag, Switzerland), clozapine (APEX Pharma, Cairo, administered vehicle (saline) or drugs (sc) and 30 min ARE) were used. Analytical-grade glacial acetic acid later were placed on the rotating rod as it accelerated (Sigma, St. Louis, USA) was diluted with pyrogen- from 4 to 40 rpm over 5 min and the time they could free saline to provide a 0.6% solution for ip injection. remain on the accelerating rod was recorded [25]. The All drugs were dissolved in isotonic (0.9% NaCl) sa- cut-off time was 600 s. The time was measured from line solution immediately before use. the start of the acceleration period. The test was re- peated 2 h after vehicle or drug injection. Six animals Statistical analyses were used per dose and for the controls. Data are expressed as the mean ± SE. Differences be- Porsolt’s forced-swimming test tween vehicle control and treatment groups were tested using one- and two-way ANOVA followed by In this test, a commonly used tool for screening of po- Duncan’s multiple comparison test. When there were tential antidepressants, the floating time, which is only two groups, a two-tailed Student’s t-test was considered as the measure of despair, is decreased by used. A probability value less than 0.05 was consid- the administration of antidepressant drugs [32]. ered statistically significant.

682 Pharmacological Reports, 2006, 58, 680691 Antinociception by vinpocetine and piracetam Omar M.R. Abdel Salam

Results

Effect of vinpocetine or piracetam on thermal nociception

The mean reaction time in the hot plate test was significantly delayed at 30 min after the administration of vinpocetine at doses of 0.9 mg/kg or 1.8 mg/kg, compared with basal (pre-drug) values, denoting decreased nociception (p < 0.05, one way ANOVA). Pi- racetam given at doses of 75–300 mg/kg failed to affect the nociceptive response (Fig. 1).

Fig. 2. Effect of different doses of vinpocetine (0.45, 0.9 or 1.8 mg/kg), piracetam (75, 150 or 300 mg/kg), indomethacin (20 mg/kg) or saline (control) on abdominal constrictions caused by EF injection of dilute acetic acid in mice. Drugs or saline (control) were administered EF 30 min prior to testing. Data are expressed as the mean ± SE and percent inhibition (%) compared to the control animals. * p < 0.05 LI. control, n = 6

Hot-plate latency (s)

mice in a dose-dependent manner. The degree of inhibition was 42.3, 53, 63.9% for vinpocetine at 0.45, 0.9 or 1.8 mg/kg (Fig. 2A) and 48.1, 51.3, 76.8% for piracetam at 75, 150 or 300 mg/kg, respectively (Fig. 2B). In comparison, the non-steroidal anti-inflammatory drug indomethacin (20 mg/kg) decreased the number of abdominal constrictions by 54.9%.

Investigation of the mechanism of visceral Fig. 1. Effect of different doses of vinpocetine (0.45, 0.9 or 1.8 mg/kg) antinociception by vinpocetine or piracetam or piracetam (75, 150 or 300 mg/kg) in the hot-plate test of thermal pain. Basal and 30-min latencies were determined for each treatment group (saline, vinpocetine or piracetam). The first column rep- Figures 3–5 show the effect of the a-2 adrenoceptor resents the basal (pre-drug) latencies and the second column repre- antagonist yohimbine (4 mg/kg) and a-1 adrenoceptor sents the 30 min-(post-drug)values in each treatment group. Saline or drugs were administered EF 30 min prior to testing. Data are ex- antagonists prazosin (2 mg/kg) and doxazosin pressed as the mean ± SE. Hot-plate latencies comparisons were (16 mg/kg) on the nociceptive responses. The admini- made between baseline paw withdrawal latencies (pre-drug values) as compared with the respective post-drug values. * p < 0.05 LI.ba- stration of prazosin or doxazosin caused a marked re- sal (pre-drug value), n = 6 duction in the number of abdominal constrictions. The administration of yohimbine or prazosin at the above-mentioned doses significantly enhanced the Effect of vinpocetine or piracetam on visceral antinociceptive action of vinpocetine or piracetam in nociception the abdominal constriction assay. When administered at 16 mg/kg, doxazosin given alone or combined with Either vinpocetine (0.45, 0.9 or 1.8 mg/kg) or pi- either vinpocetine or piracetam markedly inhibited racetam (75, 150 or 300 mg/kg) inhibited the number the abdominal constrictions by 94.8–90.2% (Fig. 4). of writhes induced by ip acetic acid administration in To delineate whether there is any synergism between

Pharmacological Reports, 2006, 58, 680691 683 Fig. 5. Effect of doxazosin (4 mg/kg) on antinociception caused by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg) in the abdominal constriction assay. Drugs or saline (control) were administered EF 30 min prior to testing. Data are expressed as the mean ± SE, * p < 0.05 Fig. 3. Effect of yohimbine (4 mg/kg, EF) or prazosin (2 mg/kg, EF)on compared to control group. The plus sign (+) indicates significant antinociception caused by vinpocetine (1.8 mg/kg, EF) or piracetam change from the other treatment groups, n = 6 (300 mg/kg, EF) in the abdominal constriction assay. Drugs or saline (control) were administered 30 min prior to testing. Data are expressed as the mean ± SE and percent inhibition (%) compared to the control animals. * p < 0.05 LI. control and between different groups as shown in the figure. The plus sign (+) indicates significant difference from the yohimbine-treated group, n = 6

Fig. 6. Effect of atropine (2 mg/kg) or propranolol (2 mg/kg) on antinociception caused by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg) in the abdominal constriction assay. Drugs or saline (control) were administered EF 30 min prior to testing. Data are expressed as the mean ± SE and percent inhibition (%) compared to Fig. 4. Effect of doxazosin (16 mg/kg) on antinociception caused by the control animals. * p < 0.05 compared to control and between dif- vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg) in the abdominal ferent groups as shown in the figure. The plus sign (+) indicates sig- constriction assay. Drugs or saline (control) were administered EF nificant change from the atropine-treated group. The (#) sign indi- 30 min prior to testing. Data are expressed as the mean ± SE, * p < 0.05 cates significant difference from the vinpocetine + atropine or vinpo- compared to control group, n = 6 cetine + propranolol-treated groups, n = 6

684 Pharmacological Reports, 2006, 58, 680691 Antinociception by vinpocetine and piracetam Omar M.R. Abdel Salam

/kg

Glibenclamide 5 mg

Glibenclamide + Piracetam

Glibenclamide + Vinpocetine

Fig. 7. Effect of guanethidine (16 mg/kg) on visceral antinociception Fig. 8. (A) Effect of naloxone (5 mg/kg) on antinociception caused by caused by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg). Drugs vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg) in the abdominal or saline (control) were administered EF 30 min prior to testing. Data constriction assay. * p < 0.05 LI. control. The plus sign (+) indicates are expressed as the mean ± SE and percent inhibition (%) com- significant change from the naloxone-treated group. The (#) sign in- pared to the control animals. * p < 0.05 compared to control and be- dicates significant difference from the vinpocetine + naloxone or pi- tween different groups as shown in the figure. The plus sign (+) indi- racetam + naloxone-treated groups. (B) Effect of gibenclamide cates significant change from the guanethidine-treated group, n = 6 (5 mg/kg) on visceral antinociception caused by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg). * p < 0.05 compared to control. The plus sign (+) indicates significant change from the vinpocetine-treated group. Drugs or saline (control) were administered EF 30 min prior to testing. Data are expressed as the mean ± SE doxazosin and either nootropic, doxazosin was ad- and percent inhibition (%) compared to the control animals, n = 6 ministered at a lower dose of 4 mg/kg. Figure 5 shows that doxazosin at the dose of 4 mg/kg was able to enhance the piracetam-induced antinociception. ciception by vinpocetine or piracetam (Fig. 8B). The The effect of vinpocetine was markedly potentiated non-steroidal anti-inflammatory drug indomethacin by co-administration of the beta-adrenergic antagonist given 30 min prior to vinpocetine or piracetam in- propranolol or muscarinic receptor antagonist atro- creased the antinociceptive response (Fig. 9). The vin- pine, with 99.5% inhibition of the abdominal constric- pocetine- or piracetam-induced antinociception was, tion response. Propranolol or atropine also enhanced the piracetam effect (Fig. 6). Antinociception caused however, inhibited by pretreatment with the non- by vinpocetine or piracetam was, in addition, en- selective adenosine receptor antagonists theophylline hanced by co-administration of the adrenergic neuron administered at the dose of 20 mg/kg (Fig. 9). blocking agent guanethidine. Guanethidine by itself The antinociception induced by the nootropic drugs elicited a significant reduction in the number of ab- vinpocetine and piracetam was also examined in the dominal constrictions as compared to the control presence of the dopamine D2 receptor antagonists, group (Fig. 7). sulpiride and haloperidol or the dopamine D2 receptor Potentiation of antinociception caused by vinpocet- agonist bromocryptine. As shown in Figure 10, the ine or piracetam was also observed when the non- antinociception caused by vinpocetine was reduced specific opioid receptor antagonist naloxone was ad- by sulpiride; while that of piracetam was enhanced by ministered along with the nootropics. Naloxone ad- sulpiride. The administration of either vinpocetine or ministered alone increased the writhing response (Fig. piracetam with haloperidol at 1.5 or 3 mg/kg, almost 8A). The potassium channel blocker gibenclamide ad- abolished the writhing response. However, haloperi- ministered ip 30 min prior to testing inhibited dol administered at 1.5 or 3 mg/kg by itself inhibited abodominal constrictions but did not affect antino- the writhing response by 93.1 and 98.4%, respec-

Pharmacological Reports, 2006, 58, 680691 685 Fig. 9. Effect of theophylline (20 mg/kg) or indomethacin (20 mg/kg) on antinociception caused by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg). Theophylline or indomethacin was administered ip 30 min prior to vinpocetine or piracetam (60 min prior to the abdominal Fig. 11. Effect of haloperidol (0.5 mg/kg) on antinociception caused constriction assay). Data are expressed as the mean ± SE and percent by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg). Drugs or saline inhibition (%) compared to the control animals. * p < 0.05 compared to (control) were administered EF 30 min prior to testing. Data are ex- control and between different groups as shown in the figure, n = 6 pressed as the mean ± SE and percent inhibition (%) compared to the control animals. * p < 0.05 compared to control. The plus sign (+) or the sign (#) indicates significant change from the other treatment groups, n = 6

Fig. 10. Effect of sulpiride (10 or 20 mg/kg) and haloperidol (1.5 or 3 mg/kg) on antinociception caused by vinpocetine (1.8 mg/kg) or pi- Fig. 12. Effect of bromocryptine (1.5 or 3 mg/kg) on visceral antino- racetam (300 mg/kg). Drugs or saline (control) were administered EF ciception caused by vinpocetine (1.8 mg/kg) or piracetam 30 min prior to testing. Data are expressed as the mean ± SE and per- (300 mg/kg). Drugs or saline (control) were administered EF 30 min cent inhibition (%) compared to the control animals. * p < 0.05 com- prior to testing. Data are expressed as the mean ± SE and percent in- pared to control. The plus sign (+) indicates significant change from hibition (%) compared to the control animals. * p 0.05 compared to the sulpiride (20 mg/kg)-treated group. The (#) sign indicates signifi- control. The plus sign (+) indicates significant change from the pi- cant difference from the sulpiride (10 mg/kg)-treated group, n = 6 racetam or bromocryptine (1.5 mg/kg)-treated groups, n = 6

686 Pharmacological Reports, 2006, 58, 680691 Antinociception by vinpocetine and piracetam Omar M.R. Abdel Salam

Effect of vinpocetine or piracetam in rota-rod test

Vinpocetine (0.45, 0.9 or 1.8 mg/kg) or piracetam (75, 150 or 300 mg/kg) did not produce any significant changes on the rota-rod performances of the mice. Both controls and either vinpocetine- or piracetam- treated mice remained on accelerating rotating rod during the acceleration period (5 min) and for 5 min thereafter (data not shown).

Effect of vinpocetine or piracetam in Porsolt’s forced-swimming test

The floating time, was reduced by 19.9 and 20.8% after treatment with low doses of vinpocetine (0.45 mg/kg) or piracetam (37.5 mg/kg), respectively, but was markedly reduced by 63.4% after treatment with imipramine (15 mg/kg) (Fig. 14A, B). The co- Fig. 13. (A) Effect of baclofen (5 mg/kg) on antinociception caused by vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg). * p < 0.05 com- administration of either vinpocetine (1.8 mg/kg) or pi- pared to control. The plus sign (+) indicates significant change from the piracetam + baclofen (5 mg/kg)-treated group. The (#) sign indicates significant difference from the vinpocetine-treated group, n = 6. (B) Effect of imipramine (15 mg/kg) alone or co-administered with either vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg) or vinpocetine (1.8 mg/kg) + piracetam (300 mg/kg) on visceral nociception. * p < 0.05 compared to control. The plus (+) sign indicates significant difference from the vinpocetine + piracetam-treated group, n = 6. Drugs or saline (control) were administered EF 30 min prior to testing. Data are expressed as the mean ± SE and percent inhibition (%) compared to the control animals, n = 6

immobility (s)

f tively, vs. the saline-treated control group (Fig. 10).

Thus, the effect of a lower dose of haloperidol Duration o (0.5 mg/kg,ip) was examined. Figure 11 shows that the piracetam effect was enhanced by a lower dose (0.5 mg/kg) of the dopamine D2 receptor antagonist haloperidol. On the other hand, the administration of the D2 receptor agonist bromocryptine (1.5 or 3 mg/kg) reduced the number of abdominal constrictions in a dose-dependent manner by 30.1 and 61.3%, respectively (Fig. 12). A marked and significant antinociception was still observed when either nootropic was co-administered with bromocryptine (Fig. 12). Figure 13A shows that marked potentiation of Fig. 14. Porsolts forced-swimming test. The graph shows three sets of experiments. In parts A & B the effect of different doses of antinociception occurred upon a co-administration of vinpocetine (0.453.6 mg/kg) or piracetam (37.5300 mg/kg) were vinpocetine and GABA agonist baclofen (5 or 10 mg/kg). investigated and compared to that of imipramine (15 mg/kg) or saline. * p < 0.05 compared to saline control. The plus (+) sign In contrast, piracetam antagonized antinociception indicates significant difference from the imipramine-treated group. caused by the low (5 mg/kg), but not the high The (#) sign indicates significant difference from the vinpocetine (0.45 mg/kg)-treated group. In part C the effect of combined (10 mg/kg) dose of baclofen (Fig. 13A). administration of either vinpocetine (1.8 mg/kg) or piracetam (300 mg/kg) with imipramine (15 mg/kg) or the effect of vinpocetine When we examined the effect of vinpocetine or pi- (1.8 mg/kg) combined with piracetam (300 mg/kg) was studied. racetam on the imipramine-induced inhibition of the * p < 0.05 compared to saline control. The plus (+) sign indicates significant difference from the vinpocetine + piracetam-treated writhing response, we observed that either nootropic en- group. Data are expressed as the mean ± SE and percent inhibition hanced antinociception caused by imipramine (Fig. 13B). (%) compared to the control animals, n = 6

Pharmacological Reports, 2006, 58, 680691 687 racetam (300 mg/kg) with imipramine (15 mg/kg) had induce a potent analgesia in the rat; spinal M1 and/or no significant effect on the antidepressant effect of M3 receptor subtypes [26] likely mediate these ef- imipramine (Fig. 14C). Meanwhile, a decrease in im- fects. Activation of spinal muscarinic receptors inhib- mobility time by 31.8% was noted in mice treated its dorsal horn neurons through inhibition of the gluta- with a combination of vinpocetine (1.8 mg/kg) and pi- matergic synaptic input [23] and potentiation of syn- racetam (300 mg/kg), suggesting an additive antide- aptic GABA release in the spinal cord [43]. Also, pressant effect of either nootropic (Fig. 14C). M1-muscarinic agonists increased the pain threshold in the mouse acetic acid writhing test and in rat paw pressure test [1]. In contrast, atropine, a cholinergic muscarinic antagonist, has been reported to produce analgesia when administered at low doses of 1–100 µg/kg, Discussion while hyperalgesia was observed with 5 mg/kg of the agent. The analgesia caused by very low doses of at- The present study provides evidence that the ropine was attributed to amplification of cholinergic nootropic drugs vinpocetine and piracetam exert transmission by a selective blockade of presynaptic pain-modulating effects. In the hot-plate test of ther- muscarinic autoreceptors [15]. In the present study, mal pain, vinpocetine showed antinociceptive effect. atropine administered ip at 2 mg/kg increased the Vinpocetine is capable of blocking NaV1.8 sodium number of writhes in the abdominal constriction as- channel activity, which is likely to be responsible for say, yet amplified the antinociceptive action of vinpo- the analgesic properties of the drug [44]. In this model cetine or piracetam. of supraspinal analgesia, piracetam failed to alter no- The sympathetic nervous system has also been im- ciception. In another study, nefiracetam, a new mem- plicated in the modulation of nociceptive transmis- ber of the piracetam group of cognition enhancers, re- sion. Certain pain conditions involve the sympathetic versed the thermal hyperalgesia in mouse models of nervous system, e.g., visceral pain due to abdominal neuropathic pain [33]. It should be noted, however, and pelvic cancers; ischemic pain from peripheral that members of the 2-oxopyrrolidine group though vascular disease, arterial spasm or frostbite and oth- structurally close to piracetam differ markedly in their ers. Sympathetcomy or intravenous regional sympa- pharmacological properties and clinical effects. thetic block with guanethidine can be carried out to On the other hand, either vinpocetine or piracetam reduce pain [35]. Afferent nociceptive fibers from the clearly inhibited the abdominal constrictions induced viscera accompany sympathetic nerves to enter the in mice by injection of dilute acetic acid into the peri- spinal cord at the dorsal horn. Transmission of pain toneal cavity, a model of visceral inflammatory pain, impulses through the spinal cord can be inhibited by brought about by the release of prostaglandins [2]. descending reflexes that originate in the noradrener- Vinpocetine and piracetam did not impair motor per- gic neurons of the mesencephalic periductal gray mat- formance in the rota-rod test, thus ruling out the con- ter, and the pontine locus ceruleus, thereby altering founding influence of a possible sedative effect. The ascending transmission mediating visceral sensation inhibition of adrenergic, cholinergic and opioidergic [4]. Behavioral, neurophysiological and clinical evi- systems appears to facilitate vinpocetine- or pi- dence shows that most forms of gastrointestinal pain racetam-induced antinociception. are mediated by activity in visceral afferent fibers The induction of pain behaviors in animals relies running in sympathetic nerves [5]. Sympathetic block upon a stimulus applied to a nociceptive neuron and interrupts these pathways and also the efferent the activation of a pain pathway and reflexes. Several viscero-visceral reflexes so that ischemia and spasm drugs induce analgesia or antinociception by interfer- are relieved [9]. Voltage-gated sodium channels ing with the neuronal pathways involved in the receipt (Nav1.7) have been found predominantly in sensory and transmission of nociceptive information from the and sympathetic neurons [42]. Sympathectomy pro- periphery to higher centers in the central nervous sys- duced in rats by administering 6-hydroxydopamine tem. In this context, the spinal cholinergic system and reduced spontaneous activity of sacral spinal cord muscarinic receptors are important for modulation of neurons and attenuated visceral nociceptive responses nociception. Spinal muscarinic agonists, such as car- due to colorectal distension [19]. Duarte et al. [7] ob- bachol and the cholinesterase inhibitor neostigmine, served a significant inhibition of writhing in the acetic

688 Pharmacological Reports, 2006, 58, 680691 Antinociception by vinpocetine and piracetam Omar M.R. Abdel Salam

acid abdominal constriction assay with the adrenergic ciceptive behavior in the abdominal constriction assay neuron blocker guanethidine (27% at 30 mg/kg, sc). when administered alone and was thus expected to re- In the present study, a 30% reduction in the nocicep- duce antinociception by the two nootropic drugs. tive response was observed with 16 mg/kg of ip Voltage-gated potassium channels can also contribute guanethidine. The latter is taken up into adrenergic to the sensitization of primary afferents observed in neurons, where it binds to the storage vesicles and gastrointestinal pain states [6]. The possible involve- prevents release of the neurotransmitter in response to ment of ATP-gated potassium channels in the media- a neuronal impulse, which results in generalized de- tion of antinociception induced by vinpocetine or pi- crease in sympathetic tone. In the present study, racetam is ruled out, in view of the inability of gliben- guanethidine potentiated antinociception when co- clamide, an ATP-sensitive potassium channels administered with either nootropic. A decrease in blocker to reduce their antinociceptive effect. writhing response was also observed after a non- Adenosine is an inhibitory neuromodulator that can selective beta-adrenoceptor antagonist propranolol. increase nociceptive thresholds in response to noxious The latter enhanced antinociception elicited by pi- stimulation [34] and blockade of adenosine receptors racetam, whereas its administration with vinpocetine by theophylline, a non-selective adenosine receptor almost abolished the writhing response. Based upon antagonist at A1 and A2 was shown to induce hyperal- these results, it might be suggested that a decrease in gesia [30]. The antinociceptive effects of vinpocetine sympathetic tone facilitates antinociception caused by or piracetam were reversed by prior administration of vinpocetine or piracetam. the adenosine receptor blocker theophylline. There- Descending brainstem systems influence nocicep- fore, it is suggested that an adenosine sensitive tive stimuli spinally while other systems modulate no- mechanism is likely to be responsible for the visceral ciceptive input supraspinally. The administration of antinociceptive properties of both nootropic drugs. a -2 adrenoceptor agonists produces anti-nociception Baclofen, a prototypical agonist of GABAB recep- in rodents by inhibiting synaptic transmission in the tors, alters nociception at the level of the spinal cord spinal cord dorsal horn and there is an evidence of de- by acting on GABAB receptors located on primary af- scending noradrenergic system, the stimulation of ferent terminals and is known to produce analgesia in which results in the activation of spinal a-2-adrenergic man and animals [18]. In the present work, baclofen receptors and antinociception [37]. Visceral nocicep- markedly inhibited the nociceptive response in the ab- tion induced by ip administered 2% solution of acetic dominal constriction assay. The drug potentiated acid was reduced dose-dependently by prazosin, an antinociception elicited by vinpocetine. In contrast, a-1-adrenoceptor antagonist and clonidine, an a-2- piracetam antagonized antinociception caused by the adrenoceptor agonist [21]. Results of the present low dose (5 mg/kg), though not the high dose study indicated that visceral nociception was mark- (10 mg/kg) of baclofen. The prevention of baclofen edly inhibited by the administration of a-1 antago- antinociception is in accordance with other studies. nists. The administration of a-1 or a-2 antagonists Piracetam (and also aniracetam) antagonized GABA- amplified the vinpocetine or piracetam antinocicep- ergic antinociception caused by baclofen [13]. tion. Piracetam exerts GABAB antagonistic effect Dopamine D2 receptors are involved in nociceptive [13]. There is evidence to suggest that spinally pro- and analgesic mechanisms [12, 38] and there is evi- jecting noradrenergic A7 neurons in the dorsolateral dence to suggest that dopamine may acts tonically in pontine tegmentum may be under tonic inhibitory the cortex to inhibit nociception [3]. In the present control by GABA neurons [29]. Removal of inhibi- study, haloperidol itself dose-dependently inhibited tory GABAB mechanisms by piracetam might thus fa- the nociceptive behavior. In contrast, sulpiride, a highly cilitate a descending noradrenergic pathway so as to selective D2-dopamine receptor antagonist, increased alter nociception. This hypothesis, however, cannot the nociceptive response. It is likely that different explain the potentiating effect of a-1 or a-2 antago- pharmacological receptor binding profiles of the two nists on vinpocetine antinociception. antagonists might be associated with different effects Unexpected also was the finding that the visceral on visceral nociception. Haloperidol is a partially se- antinociceptive effects of vinpocetine or piracetam lective dopamine D2 receptor antagonist [24]. The in- were enhanced by co-administration the opioid recep- volvement of other dopamine receptors as well is tor antagonist naloxone. The latter increased the no- likely to account for the antinociceptive effect of ha-

Pharmacological Reports, 2006, 58, 680691 689 loperidol seen in the present study. Haloperidol at intestinal disorders. In: Gastroenterology Clinics of NA: 0.5 mg/kg potentiated antinociception caused by pi- Gastrointestinal Motility in Clinical Practice, vol. 25. Ed. Camilleri M, Saunders, Philadelphia, 1996, 247–258. racetam. On the other hand, sulpiride reduced antino- 5. Cervero F: Neurophysiology of gastrointestinal pain. ciception caused by vinpocetine, but enhanced that of Baillieres Clin Gastroenterol, 1988, 2, 83–99. piracetam. Blockade of D2 receptors thus potentiates 6. Cervero F, Laird JM: Role of ion channels in mecha- piracetam antinociception, but might reduce vinpocet- nisms controlling gastrointestinal pain pathways. Curr ine antinociception. These results suggest alteration Opin Pharmacol, 2003, 3, 608–612. 7. Duarte ID, Nakamura M, Ferreira SH: Participation of of dopaminergic neurotransmission by modifying the the sympathetic system in acetic acid-induced writhing D2-receptors which can alter antinociception induced in mice. Braz J Med Biol Res, 1988, 21, 341–343. by the two nootropic drugs. 8. Eckert GP, Cairns NJ, Muller WE. Piracetam reverses Antidepressant drugs such as clomipramine or imi- hippocampal membrane alterations in Alzheimer’s dis- pramine can also modulate visceral nociception [14]. ease. J Neural Transm, 1999, 106, 757–761. 9. Eisenberg E, Carr DB, Chalmers TC. Neurolytic celiac Data from the present study indicate that vinpocetine plexus block for treatment of cancer pain: a meta-analysis. or piracetam are likely to enhance visceral antino- Anesth Analg, 1995, 80, 290–295. ciception caused by imipramine. Interestingly, when 10. Fedi M, Reutens D, Dubeau F, Andermann E, D’Agost- examined in the forced-swimming test in mice, ino D, Andermann F: Long-term efficacy and safety of a commonly used tool for screening of potential anti- piracetam in the treatment of progressive myoclonus epi- lepsy. Arch Neurol, 2001, 58, 781–786. depressants [32], low doses of vinpocetine and also 11. Fischhof PK, Moslinger-Gehmayr R, Herrmann WM, piracetam displayed antidepressant-like activity. Friedmann A, Russmann DL: Therapeutic efficacy of In summary, vinpocetine, but not piracetam re- vincamine in dementia. Neuropsychobiology, 1996, 34, duced thermal nociception. The two nootropics, how- 29–35. 12. Frussa-Filho R, Rocha JB, Conceicao IM: Effects of do- ever, displayed marked antinociceptive effect on vis- ip paminergic agents on visceral pain measured by the ceral pain caused by injection of acetic acid in mouse writhing test. Arch Int Pharmacodyn Ther, 1996, mice. This effect depended on the activation of adeno- 331, 74–93. sine receptors. Cholinergic muscarinic blockade with 13. Galeotti N, Ghelardini C, Bartolini A: Piracetam and ani- atropine, adrenorecptor blockade or opioid antago- racetam antagonism of centrally active drug-induced antinociception. Pharmacol Biochem Behav, 1996, 53, nism with naloxone potentiated vinpocetine or 943–950. piracetam-induced antinociception. An interaction at 14. Ghelardini C, Galeotti N, Bartolini A: Antinociception the level of dopamine D2 receptors by the two induced by amitriptyline and imipramine is mediated by nootropic agents is also suggested. alpha2A-adrenoceptors. Jpn J Pharmacol, 2000, 82, 130–137. 15. Ghelardini C, Malmberg-Aiello P, Giotti A, Malcangio M, Bartolini A: Investigation into atropine-induced antinociception. Br J Pharmacol, 1990, 101, 49–54. Acknowledgment: 16. Giurgea C: The “nootropic” approach to the pharmacol- The author acknowledges Wael Zedan and Mohamed Abd-Elaziz ogy of the integrative activity of the brain. Cond Reflex, for their technical assistance in the experiments. 1973, 8, 108–115. 17. Hadjiev D: Asymptomatic ischemic cerebrovascular disorders and neuroprotection with vinpocetine. Ideggyogy Sz, 2003, 56, 166–172. References: 18. Hara K, Saito Y, Kirihara Y, Sakura S: The interaction between gamma-aminobutyric acid agonists and dilti- azem in visceral antinociception in rats. Anesth Analg, 1. Bartolini A, Ghelardini C, Fantetti L, Malcangio M, 2004, 98, 1380–1384. Malmberg-Aiello P, Giotti A: Role of muscarinic recep- 19. Kalmari J, Niissalo S, Konttinen YT, Pertovaara A: tor subtypes in central antinociception. Br J Pharmacol, Modulation of visceral nociceptive responses of rat spi- 1992, 105, 77–82. nal dorsal horn neurons by sympathectomy. Neuroreport, 2. Berkenkopf JW, Weichman BM: Production of prostacy- 2001, 12, 797–801. clin in mice following intraperitoneal injection of acetic 20. Kessler J, Thiel A, Karbe H, Heiss WD: Piracetam im- acid, phenylbenzoquinone and zymosan: its role in the proves activated blood flow and facilitates rehabilitation writhing response. Prostaglandins, 1988, 36, 693–709. of poststroke aphasic patients. Stroke, 2000, 31, 2112–2116. 3. Burkey AR, Carstens E, Jasmin L: Dopamine reuptake 21. Korzeniewska-Rybicka I, Plaznik A: Role of serotoner- inhibition in the rostral agranular insular cortex produces gic and noradrenergic systems in a model of visceral antinociception. J Neurosci, 1999, 19, 4169–4179. pain. Pol J Pharmacol, 2001, 53, 475–480. 4. Camilleri M, Saslow SB, Bharucha AE: Gastrointestinal 22. Koster R, Anderson M, De Beer EJ: Acetic acid for anal- sensation: mechanisms and relation to functional gastro- gesic screening. Fed Proc, 1959, 18, 412.

690 Pharmacological Reports, 2006, 58, 680691 Antinociception by vinpocetine and piracetam Omar M.R. Abdel Salam

23. Li DP, Chen SR, Pan YZ: Role of presynaptic muscarinic 35. Serpell M: Role of the sympathetic nervous system in and GABA(B) receptors in spinal glutamate release and pain. Anaesth Intensive Care Med, 2005, 6, 52–55. cholinergic analgesia in rats. J Physiol, 2002, 543, 807–818. 36. Shorvon S: Pyrrolidone derivatives. Lancet, 2001, 358, 24. Melis MR, Succu S, Mascia MS, Argiolas A, Bernard B: 1885–1892. PD-168077, a selective dopamine D4 receptor agonist, 37. Tanabe M, Takasu, K, Kasuya N: Role of descending induces penile erection when injected into the paraven- noradrenergic system and spinal alpha2-adrenergic re- tricular nucleus of male rats. Neurosci Lett, 2005, 379, ceptors in the effects of gabapentin on thermal and me- 59–62. chanical nociception after partial nerve injury in the 25. Millan MJ, Bervoets K, Rivet J-M, Widdowson P, Re- mouse. Br J Pharmacol, 2005, 144, 703–714. nouard A, Le Marouille-Girardon S, Gobert A: Multiple 38. Taylor BK, Joshi C, Uppal H: Stimulation of dopamine alpha -adrenergic receptor subtypes. II. Evidence for D2 receptors in the nucleus accumbens inhibits inflam- a role of rat R )-ARs in the control of nociception, motor matory pain. Brain Res, 2003, 987, 135–143. behaviour and hippocampal synthesis of noradrenaline. 39. Vakulina OP, Iasnetsov VV, Isachenkov VA, Krylova IN, J Pharmacol Exp Ther, 1994, 270, 958–972. Tishkova IP, Bragin EO, Popkova EV: Effects of pi- 26. Naguib M, Yaksh TL: Antinociceptive effects of spinal racetam on pain sensitivity and levels of beta-endorphin cholinesterase inhibition and isobolographic analysis of in blood and cAMP in the cerebral cortex of rats (Rus- the interaction with mu and alpha 2 receptor systems. sian). Biull Eksp Biol Med, 1990, 109, 163–165. Anesthesiology, 1994, 80, 1338–1348. 40. Vas A, Gulyas B, Szabo Z, Bonoczk P, Csiba L, Kiss B, 27. Nikolova M, Petrova L, Dushkova R: Effect of piracetam Karpati E, Panczel G, Nagy Z: Clinical and non-clinical in models of experimental inflammation. Int J Tissue Re- investigations using positron emission tomography, near act, 1984, 6, 17–21. infrared spectroscopy and transcranial Doppler methods 28. Nosalova V, Machova J, Babulova A: Protective action on the neuroprotective drug vinpocetine: a summary of of vinpocetine against experimentally induced gastric evidences. J Neurol Sci, 2002, 203–204, 259–262. damage in rats. Arzneimittelforschung, 1993, 43, 981–985. 41. Waegemans T, Wilsher CR, Danniau A, Ferris SH, Kurz 29. Nuseir K, Proudfit HK: Bidirectional modulation of no- A, Winblad B: Clinical efficacy of piracetam in cognitive ciception by GABA neurons in the dorsolateral pontine impairment: a meta-analysis. Dement Geriatr Cogn Dis- tegmentum that tonically inhibit spinally projecting nora- ord, 2002, 13, 217–224. drenergic A7 neurons. Neuroscience, 2000, 96, 773–783. 42. Wood JN, Boorman JP, Okuse K, Baker MD: Voltage- 30. Paalzow GH: Noradrenaline but not dopamine involved gated sodium channels and pain pathways. J Neurobiol, in NMDA receptor-mediated hyperalgesia induced by 2004, 61, 55–71. theophylline in awake rats. Eur J Pharmacol, 1994, 252, 43. 87–97. Zhang HM, Li DP, Chen SR: M2, M3, and M4 receptor 31. Paget GE, Barnes JM. Toxicity tests. In: Evaluation of subtypes contribute to muscarinic potentiation of GA- Drug Activities Pharmacometics. Eds. Laurence DR, BAergic inputs to spinal dorsal horn neurons. J Pharma- Bacharach AL, Academic Press, London, New York, col Exp Ther, 2005, 313, 697–704. 44. 1964, 1–135. Zhou X, Dong XW, Crona J, Maguire M, Priestley T: 32. Porsolt RD, Le Pichon M, Jalfre M: Depression: a new Vinpocetine is a potent blocker of rat NaV1.8 animal model sensitive to antidepressant treatments. tetrodotoxin-resistant sodium channels. J Pharmacol Exp Nature, 1977, 266, 730–732. Ther, 2003, 306, 498–504. 33. Rashid MH, Ueda H: Nonopioid and neuropathy-specific analgesic action of the nootropic drug nefiracetam in mice. J Pharmacol Exp Ther, 2002, 303, 226–231. 34. Sawynok J: Adenosine receptor activation and nocicep- Received: tion. Eur J Pharmacol. 1998, 347, 1–11. April 6, 2006; in revised form August 22, 2006.

Pharmacological Reports, 2006, 58, 680691 691