Preparation and in vitro characterization of chitosan nanoparticles containing Mesobuthus eupeus scorpion venom as an antigen delivery system

Hydrophilic nanoparticles have been widely investigated in recent years as delivery systems for therapeutic macromolecules such as antigens. In the present study Mesobuthus eupeus venom-loaded chitosan nanoparticles were prepared via ionic gelation of tripolyphosphate (TPP) and chitosan. The optimum encapsulation efficiency (91.1 percent) and loading capacity (76.3 percent) were obtained by a chitosan concentration of 2 mg/mL, chitosan-to-TPP mass ratio of 2 and M. eupeus venom concentration of 500 µg/mL. The average nanoparticle size at optimum conditions was determined by Zetasizer (Malvern Instruments, UK). The nanoparticle size was about 370 nm (polydispersity index: 0.429) while the zeta potential was positive. Transmission electron microscope (TEM) imaging showed a spherical, smooth and almost homogenous structure for nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. The in vitro release of nanoparticles showed an initial burst release of approximately 60 percent in the first ten hours, followed by a slow and much reduced additional release for about 60 hours. It is suggested that the chitosan nanoparticles fabricated in our study may provide a suitable alternative to traditional adjuvant systems.(AU)

N-methyl-D-aspartate receptors are involved in lithium-induced state-dependent learning in mice.

We have previously shown lithium-induced state-dependent learning in a step-down inhibitory avoidance task. In the present study, the effects of intracerebroventricular injections of N-methyl-D-aspartate (NMDA) receptor agents on the lithium-induced state-dependent learning have been investigated. A single-trial step-down inhibitory avoidance task was used to assess memory in male Naval Medical Research Institute (NMRI) mice. The results showed that post-training lithium (10 mg/kg) decreased the step-down latency on the test day, which was reversed by pre-test administration of the same dose of the drug; indicating state-dependent learning induced by lithium. Pre-test administration of NMDA (0.0001, 0.001 and 0.01 microg/mouse, intracerebroventricular) could also substitute for pre-test lithium to reverse the decrease of the step-down latency induced by post-training lithium. Furthermore, pre-test co-administration of an ineffective dose of NMDA (0.00001 microg/mouse, intracerebroventricular.) with lower doses of lithium (1.25, 2.5 and 5 mg/kg, intraperitoneally.) synergistically reversed the decrease of the step-down latency. On the contrary, pre-test injections of NMDA receptor antagonist D-AP5 (0.25, 0.5, 1 and 2 microg/mouse, intracerebroventricular.) disrupted state-dependent learning induced by lithium. The results suggest that NMDA receptors may be involved, at least partly, in the lithium-induced state-dependent learning.

Antinociceptive effect of intracerebroventricular administration of cholecystokinin antagonist in nerve-ligated mice.

In the present study we investigated the effects of intracerebroventricular injection of caerulein, the cholecystoki nin receptor agonist and proglumide, the receptor antagonist on morphine response in the sciatic nerve ligation in mice. Subcutaneous administration of morphine induced antinociception in the both intact and ligated mice, however, the response of the opioid was lower in the ligated mice as compared with the intact animals. Caerulein induced antinociception in the non-ligated but not in the nerve-ligated animals. Combination of caerulein with morphine elicited higher response in ligated animals, however, the response induced in ligated animals was much more prominent. Proglumide alone did not elicit any response in both animal groups. The antagonist decreased the response of caerulein in the nonligated mice. Low doses of proglumide in the combination with caerulein induced antinociception in the ligated mice. We conclude that cholecystokinin receptor mechanism(s) may alter morphine resistance induced by nerve ligation.

Role of cholecystokinin receptors in induction of antinociception in hot-plate test.

In the present study, the antinociceptive effect of cholecystokinin receptor agonists in the hot-plate test in mice has been evaluated. Subcutaneous administration of cholecystokinin octapeptide (cholecystokinin-8; 0.001, 0.005, 0.01, 0.05, and 0.1 mg/kg), unsulfated cholecystokinin octapeptide (cholecystokinin-8U; 0.1 mg/kg) or caerulein (0.25 mg/kg) produced antinociception. Administration of the cholecystokinin tetrapeptide (cholecystokinin-4; 0.25, 0.5 and 1.0 mg/kg) had no effect in the hot-plate test. Subcutaneous injection of the selective cholecystokinin receptor antagonists, MK-329 (0.125, 0.25 and 0.5 mg/kg) or L-365,260 (0.125, 0.25 and 0.5 mg/kg), produced no antinociceptive response. When the animals were pretreated with the cholecystokinin receptor antagonists or naloxone (0.5 and 1 mg/kg), a significant decrease in the antinociceptive response induced by cholecystokinin-8 and caerulein was obtained. The results indicate that single administration of cholecystokinin receptor agonists could produce an antinociceptive effect which is probably mediated via cholecystokinin receptors. With respect to the results obtained from morphine and naloxone administration, it is concluded that there may be an interaction between cholecystokinin and opiate mechanisms.

Cholecystokinin and morphine-induced hypothermia.

The effects of cholecystokinin-8 sulfate (CCK-8), cholecystokinin-8 unsulfate (CCK-8U), cholecystokinin-4 (CCK-4), caerulein and morphine on mice core body temperature have been studied in the present work. Subcutaneous injection of different doses of caerulein (0.05, 0.1 and 0.5 mg/kg), CCK-8 (0.05, 0.1 and 0.25 mg/kg) and morphine (10, 20 and 30 mg/kg) induced hypothermia. CCK-8U and CCK-4 did not elicit any response. The hypothermic response induced by caerulein, a CCK-related decapeptide but not morphine was decreased by selective CCK(A) receptor antagonist MK-329. However, the hypothermia induced by morphine but not caerulein was reduced by opioid antagonist naloxone. When morphine plus caerulein was administered a higher hypothermia was induced. Pretreatment of animals with L-365 260, a selective CCK(B) receptor antagonist did not alter the hypothermia induced by the drugs. The response induced by combination of the both drugs was decreased by MK-329. Administration of CCK antagonists MK-329 and L-365 260 to mice did not exert any effect on temperature. It is concluded that the CCK(A) receptor mechanism may be involved in the hypothermic effect of CCK agonists or morphine, while opioid receptor mechanism is not involved in CCK receptor agonists' response.

Effect of nicotine on apomorphine-induced licking behaviour in rats.

In the present study, the dopaminergic receptor agonist apomorphine (0.1, 0.25 and 0.5 mg/kg) induced a dose-dependent licking in rats. Nicotine administration (0.025-250 microg/kg) altered the apomorphine-induced licking. The lower doses of nicotine (0.05 and 0.5 microg/kg) increased while the higher dose of the drug (250 microg/kg) reduced the apomorphine response. The antimuscarinic drug atropine (2.5 and 5 mg/kg) reduced the effects of apomorphine or nicotine plus apomorphine. The central nicotinic receptor antagonist mecamylamine (0.05, 0.25 and 0.5 mg/kg) also reduced the response induced by apomorphine or nicotine plus apomorphine. However, the peripheral nicotinic receptor antagonist hexamethonium (2.5, 5 and 10 mg/kg) reduced the response induced by nicotine plus apomorphine but not that elicited by apomorphine alone. The results indicate that the nicotinic receptor mechanism(s) may interact with apomorphine-induced licking in rats. Although central nicotinic and cholinergic mechanisms may be involved in the licking induced by apomorphine, peripheral nicotinic mechanism may be involved in the nicotine-induced increased apomorphine effect.

Effects of CCK antagonists on GABA mechanism-induced antinociception in the formalin test.

In this work, the influences of CCK receptor antagonists on antinociception induced by the GABA receptor agonist, baclofen, and the GABA uptake inhibitor, THPO, in the formalin test have been studied. GABA-B agonist baclofen (0.75, 1.25 and 2.5 mg/kg), THPO, a GABA uptake inhibitor (1 and 2 mg/kg) and morphine (1.5, 3 and 6 mg/kg) induced antinociception in both phases of the formalin test in mice. The selective CCK receptor antagonists, L-365,260, MK-329 (0.05, 0.125 and 0.25 mg/kg) and non-selective CCK receptor antagonist, proglumide (2.5, 5, 10 and 20 mg/kg) induced antinociception only in high doses. The CCK receptor antagonists potentiated baclofen (0.75, 1.25 and 2.5 mg/kg) or THPO (1 and 2 mg/kg) responses. It may be concluded that the CCK receptor mechanism may interact with GABA-function in its antinociceptive effect.

Cholecystokinin receptor mechanism(s) and morphine tolerance in mice.

In a previous work, the effects of cholecystokinin receptor agonists on tolerance to morphine antinociception were evaluated. In the present study, the influence of cholecystokinin antagonists on the inhibition of tolerance to morphine antinociception by cholecystokinin agonists has been investigated. Maximum tolerance to morphine antinociception was obtained by morphine administration (50 mg/kg) to mice once daily for 4 days. The cholecystokinin receptor agonists caerulein (0.005 mg/kg) or cholecystokinin-8 (0.01 mg/kg) but not unsulfated cholecystokinin-8 (0.01 mg/kg) decreased the development of tolerance to morphine (9 mg/kg). The cholecystokininA receptor antagonist MK-329 (1 mg/kg) or the cholecystokininB receptor antagonist L-365,260 (0.25, 0.5 and 1 mg/kg) also diminished the tolerance to morphine antinociception. When animals were challenged with different doses of MK-329 (0.25, 0.5 and 1 mg/kg) against cholecystokinin-8 (0.01 mg/kg), caerulein (0.005 mg/kg) or unsulfated cholecystokinin-8 (0.01 mg/kg) on day 4 in tolerant mice, different response were obtained. Higher doses of MK-329 (1 mg/kg) caused a small decrease in attenuation of the morphine tolerance induced by cholecystokinin-8 and caerulein. Low doses of L-365, 260 diminished the effect of cholecystokinin-8 on morphine tolerance. Conversely high doses of the drug potentiated the response of caerulein (0.005 mg/kg). When animals were treated with MK-329 or L-365,260 before unsulfated cholecystokinin-8, reduction of the tolerance to morphine antinociception was obtained. These data indicate that both cholecystokinin receptors may modulate morphine tolerance.

Interactions between antinociception induced by cholecystokinin antagonists and GABA agonists in the tail-flick test.

In this work, the influences of cholecystokinin receptor antagonists L-365,260, MK-329 and proglumide on antinociception induced by baclofen and GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo [4,5-c]pyridin-3-ol (THPO) in the tail flick test has been studied. Baclofen and THPO induced antinociception in the tail flick test. Morphine, and the CCK receptor antagonists, MK-329, L-365,260 and proglumide also induced antinociception. The CCK receptor antagonists potentiated antinociceptive response induced by both baclofen and THPO. It may be concluded that cholecystokinin receptor mechanism(s) may interact with antinociception induced by GABA receptor mechanism(s).

On the mechanism(s) of cholecystokinin (CCK): receptor stimulation attenuates morphine dependence in mice.

In the present study, effect of cholecystokinin (CCK) agonists and on dependence to morphine in mice has been investigated. The influence of dopaminergic, adrenergic, cholinergic and serotonergic on attenuation of naloxone-induced jumping in morphine-dependent mice by CCK agonists were also considered. Mice were treated subcutaneously with morphine (50, 50 and 75 mg/kg) three times daily (10 a.m. 1 p.m. and 4 p.m.) for 3 days, and a last dose of morphine (50 mg/kg) was administered on the 4th day. Withdrawal syndrome (jumping) was precipitated by naloxone (5 mg/kg) which was administered intraperitoneally 2 hr after the last dose of morphine. To study effects of CCK receptor agonists, 10 injection of morphine (3 administrations each day) for dependence and a dose of 5 mg/kg of naloxone for withdrawal induction were employed. The CCK agonists CCK-8 (0.001-0.1 mg/kg), unsulfated CCK-8 (CCK-8U; 0.001-0.1 mg/kg) and caerulein (0.00001-0.01 mg/kg) were able to prevent withdrawal signs precipitated by naloxone (5 mg/kg). Sulpiride and pimozide increased response induced by CCK-8 agonists. The dopamine antagonists also attenuates jumping by themselves. SCH 23390 did not alter the CCK-8 effect, but decreased the jumping by itself. Phenoxybenzamine, propranolol, methysergide and atropine did not change the caerulein effect significantly. However, single administration of atropine increased and methysergide decreased jumping. It is concluded that CCK mechanism(s) may be involved in morphine dependence, and dopaminergic mechanism(s) may interact with CCK in attenuation of naloxone-induced jumping.

Effects of caerulein and CCK antagonists on tolerance induced to morphine antinociception in mice.

Different groups of mice received one daily dose (50 mg/kg) of morphine subcutaneously (SC) for 3, 4 or 5 days to develop tolerance to the opioid. The antinociceptive response of morphine (9 mg/kg) was tested in the hot-plate test 24 h after the last dose of the drug. Tolerance to morphine was obtained in all groups. The group of mice that received morphine for 4 days was employed for the rest of the experiments. Pretreatment of animals with a single dose of caerulein (0.025, 0.05, and 0.1 mg/kg, SC) 30 min prior to receiving morphine (50 mg/kg; during the development of tolerance to the opioid) on day 1, 2, 3, 4 or 5 of morphine administration potentiate antinociception induced by morphine (test dose of 9 mg/kg). The dose of 0.05 mg/kg of caerulein, used 30 min before morphine administration on day 3, was also used to evaluate the effects of antagonists on caerulein-induced decrease in tolerance. The selective cholecystokinin (CCK) receptor antagonists, MK-329 [1-methyl-3-(2 indoloyl)amino-5-phenyl-3H-1,4-benzodiazepin-2-one; 0.25 and 0.5 mg/kg] or L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H- 1,4-benzodiazepin-3-yl)-N-(3-methyl-phenyl)urea: 0.25 and 0.5 mg/kg] decreased potentiation of morphine response induced by caerulein. MK-329 or L-365,260, when were injected 35 min before morphine injection during the development of tolerance and on day 3, decreased the tolerance to morphine. A single administration of MK-329 or L-365,260 (in the absence of caerulein) 35 min and 48 h before the test dose of morphine (9 mg/kg) potentiated the antinociception of morphine in nontolerant animals. In conclusion, CCK mechanism(s) may interact with morphine tolerance.

Caerulein may potentiate morphine-induced antinociception by cholecystokinin-A and/or cholecystokinin-B receptor mechanisms.

1. The effects of a cholecystokinin agonist and antagonist on morphine antinociception in the tail-flick test have been evaluated. 2. The administration of different doses of caerulein (0.01, 0.05 and 0.1 mg/kg) 30 min prior to morphine (1.5, 3 and 6 mg/kg) increased the antinociception induced by morphine in mice. 3. In animals pretreated with cholecystokinin antagonists MK-329 (0.125 and 0.25 mg/kg) and L-365,260 (0.125 and 0.25 mg/kg), the antinociceptive effect of morphine was not changed. However, high doses (0.5 mg/kg) of each antagonist potentiated the morphine response. 4. Low doses of cholecystokinin antagonists (0.125 and 0.25 mg/kg), that did not cause antinociception, when employed in combination with caerulein (0.05 mg/kg) decreased the response of morphine plus caerulein. 5. It is concluded that the cholecystokinin agonist caerulein potentiated the morphine response by stimulation of cholecystokinin-A and/or cholecystokinin-B receptors.

Effects of cholecystokinin receptor agonist and antagonists on morphine dependence in mice.

In the present study, the effect of cholecystokinin agonists and antagonists on dependence to morphine in mice has been investigated. Mice were treated subcutaneously with morphine (50, 50 and 75 mg/kg) three times daily for 2-4 days, and a last dose of morphine (50 mg/kg) was administered on day 3, 4 or 5. Withdrawal syndrome (jumping) was precipitated by naloxone (2.5, 5 and 10 mg/kg) which was administered intraperitoneally 2 hr after the last dose of morphine. To study the effects of cholecystokinin receptor agonists or antagonists, 10 injection of morphine (3 administrations each day) for dependence and a dose of 5 mg/kg of naloxone for withdrawal induction were employed. Cholecystokinin-8 (0.001-0.01 mg/kg), low doses of the cholecystokinin agonists caerulein (0.00001 and 0.0001 mg/kg) and, unsulfated cholecystokinin (but not high doses) as well as the antagonists MK-329 (0.5-1 mg/kg) and L-365,260 (0.5-1 mg/kg) elicit reduction of the nalaxone-induced jumping. The inhibition of jumping induced by caerulein was reduced with the selective cholecystokinin antagonists MK-329 and L-365,260. It is concluded that cholecystokinin mechanism(s) may be involved in morphine dependence, that the agonists may act on a presynaptic receptors and that the antagonists may work on postsynaptic receptors.

Effects of 5-HT receptor antagonists on morphine-induced tolerance in mice.

The effect of 5-HT receptor antagonists on tolerance to morphine antinociception was studied in mice. Slow release morphine suspension was injected subcutaneously (s.c.) in order to produce tolerance. When different doses of morphine (3, 6 and 9 mg/kg) were administered on the 4th day after injection of slow-release morphine suspension, tolerance to the test doses of morphine was observed. The tolerance obtained was decreased by pretreatment with the non-selective 5-HT receptor antagonist methysergide (1 and 2 mg/kg) or the 5-HT2 receptor antagonist ritanserin (1 and 2 mg/kg). When the 5-HT receptor antagonists were used on the 2nd and 3rd day after injection of slow-release morphine suspension or on the 4th day (60 min before last dose of morphine), a maximum reduction in morphine tolerance was observed on the 3rd day. Pretreatment of animals with metergoline (1 and 2 mg/kg) or mianserin (1 and 2 mg/kg) also decreased the tolerance to morphine. It may be concluded that at least a 5-HT2 receptor mechanism is involved in tolerance to morphine antinociception.

Caerulein changes morphine-induced antinociception depending on pretreatment times.

1. In the present study, the effect of caerulein (CLN) on morphine analgesia has been tested. 2. Different doses of morphine produced antinociception in a dose dependent manner. When animals were pretreated with CLN 5 min before morphine administration, the morphine response was decreased. However, when CLN was injected 30 or 60 min prior to morphine, the drug effect was increased. 3. Naloxone, sulpiride, metergoline or proglumide decreased the potentiation of morphine response caused by CLN. CLN, proglumide, sulpiride, metergoline and naloxone alone did not exert any effect on tail-flick latency. 4. It is concluded that, depending on pretreatment times of CLN administration the response of morphine was decreased or increased and D-2 dopamine, serotonin and/or CCK receptors may be involved in the potentiation of morphine response induced by CLN.

CCK receptor activation may prevent tolerance to morphine in mice.

Different groups of mice were treated with morphine (50 mg/kg s.c.) once daily for 1, 2, 3, 4 or 5 days, in order to develop tolerance to the drug. The antinociceptive effect of morphine (9 mg/kg s.c.) was tested 24 h after each dose of the drug administration. Tolerance to morphine reached its peak on the 4th day. Daily pretreatment of animals for a period of 4 days with different doses of cholecystokinin octapeptide (CCK-8; 0.001, 0.01, 0.05 and 0.1 mg/kg s.c.), caerulein (0.0001, 0.001, 0.005 and 0.01 mg/kg s.c.) but not unsulfated cholecystokinin octapeptide (0.001, 0.01 or 0.1 mg/kg s.c.) 30 min before daily administration of morphine (50 mg/kg s.c.) prevented the development of tolerance. A group of animals received a single dose of caerulein (0.005 mg/kg), CCK-8 (0.01 mg/kg) or unsulfated CCK-8 (0.01 mg/kg) 30 min before morphine injection (50 mg/kg s.c.) on the 3rd or 4th day. In these animals, which were tested for antinociception on the 5th day, tolerance to the drug (3, 6 and 9 mg/kg s.c.) was also decreased by caerulein, CCK-8 but not unsulfated CCK-8. In a group of mice in which peptides were administered 30 min prior to the doses of morphine (3, 6 or 9 mg/kg s.c.) on the 5th day, similar results were obtained. The results of the present study indicate that activation of both CCKB and CCKA receptors may prevent the development of tolerance to morphine, and the sulfate group in the CCK-8 molecule may be essential for the tolerance inhibition.