Ewa Witkowska

Influence of new deltorphin analogues on reinstatement of cocaine-induced conditioned place preference in rats.

The aim of this study was to investigate whether the δ-opioid receptors are involved in the rewarding and reinstatement effect of cocaine in the conditioned place preference (CPP) test. Male Wistar rats were conditioned with cocaine (5 mg/kg) or saline in a biased CPP procedure. The intracerebroventricular (i.c.v.) administration of naltrindole (5 nmol), δ-opioid receptor antagonist but not β-funaltrexamine (5 nmol), or nor-binaltorphimine (10 nmol), μ-opioid and κ-opioid receptor antagonists, respectively reversed the expression of the cocaine CPP. The i.c.v. administration of new analogues of deltorphins with potent agonist activity at δ-opioid receptors, such as cyclo(Nδ, Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) at the dose of 10 and 20 nmol and deltorphin II N-(ureidoethyl)amide (DK-4) at the dose of 10 and 20 nmol reinstated the rewarding effect of cocaine after extinction sessions in the CPP test. Naltrindole (5 nmol, i.c.v.) abolished the reinstated effect of DK-4 (10 nmol). In addition, DEL-6 and DK-4 induce anxiolytic-like effects in the elevated plus-maze test. However, neither peptide given alone either produced a rewarding effect in the CPP test, or influenced the locomotor activity and motor coordination, thus suggesting that these effects of peptides did not influence the results obtained in the reinstatement procedure of CPP. In conclusion, our results show that δ-opioid receptors play a dominant role in cocaine reward and reinstatement of cocaine seeking behavior in the CPP test.

Enkephalin derivative, cyclo[Nε,Nβ-carbonyl-D-Lys2, Dap5] enkephalinamide (cUENK6), induces a highly potent antinociception in rats

The aim of the study was to evaluate whether the newly synthesized analog of enkephalin, cyclo[N(epsilon),N(beta)-carbonyl-D-Lys(2), Dap(5)] enkephalinamide (cUENK6), a highly potent mu- (guinea pig ileum assay) and delta-receptors (mouse vas deferens assay) ligand, induces an antinociceptive effect in the hot-plate test and tail-immersion test after intracerebroventricular administration. Our study indicated that this peptide at the dose of 0.25 nmol produced comparable but at the dose of 0.5 nmol stronger than morphine (13 nmol), antinociceptive effect in both tests. Furthermore, rats with developed tolerance to morphine indicated cross-tolerance to antinociceptive effects of cUENK6. The antinociceptive effects of cUENK6 and morphine were inhibited by non-selective opioid receptor antagonist--naloxone. More detailed study indicated that the delta-opioid receptor antagonist - naltrindole very strongly and, to the lower extent, mu-opioid antagonist - beta-funaltrexamine (beta-FNA), inhibited antinociceptive effect of cUENK6 in the tail-immersion test. Nor-binaltorphimine (nor-BNI), a kappa-opioid receptor antagonist, did not influence this effect. These data suggest the dominant role of delta-opioid receptors as compared with mu-receptors in mediation antinociceptive effect of cUENK6. Furthermore, we found that cUENK6 is much more effective in inhibiting pain in the hot-plate (ED(50)=0.0792 nmol) than in the tail-immersion (ED(50)=0.3526 nmol) test. However, cUENK6 at the antinociceptive doses induced hypolocomotion, and although this effect is observed after administration of opioid agonists in rats as a one phase of their biphasic action (inhibition followed by activation), in our study it was not naloxone-reversible. Therefore, our study suggests that not only opioid receptors may be involved in behavioral effects of cUENK6.

Vanillin Biosynthetic Pathways

Vanilla extract is widely used in the food and the confectionery industry. Vanillin is the most abundant component of vanilla extract. An understanding of the biosynthetic pathway of vanillin may be important for regulating the production of the compound in plants. It is agreed that vanillin (C6-C1) is a product of phenylpropanoid (C6-C3) compounds. One school of thought suggests that vanillin is formed from ferulic acid that undergoes chain shortening. It was shown that inVanilla planifoliatissue cultures the benzoate derivative pathway operates using phenylpropanoid substrates, leading from trans-cinnamic acid to flavor compounds. The branch point between the C6-C3and C6-C1pathways most likely occurs at the level of p-coumaric acid. The tissue possesses constitutive 3-0-methyltransferase, capable of methylation of externally added 3,4-dihydroxybenzaldehyde. Aromatic C6-C1aldehydes are reduced to the corresponding alcohols by a specific alcohol dehydrogenase and stored.

Design, synthesis and in vitro biological evaluation of a small cyclic peptide as inhibitor of vascular endothelial growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR (vascular endothelial growth factor receptor), but it is also suggested that NRP-1 in tumour cells may serve as a separate receptor for VEGF165. Therefore molecules interfering with VEGF165 binding to NRP-1 seem to be promising candidates as new anti-angiogenic and anti-tumour drugs. Here, we report the design, synthesis, biological evaluation and molecular modelling of the small cyclic peptide, which shows a good inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18μM). The reported compound could be considered as one of the smallest cyclic peptides (MW=510) interfering with VEGF165/NRP-1 binding presented up to now.

Antinociceptive effects of two deltorphins analogs in the tail-immersion test in rats.

The antinociceptive effects of analogs of deltorphins: cyclo(Nδ,Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) after intracerebroventricular (i.c.v.) administration were investigated in the tail-immersion test in rats. Morphine, the most commonly used μ-opioid receptors (MOR) agonist, was employed as a reference compound. The contribution of the MOR, δ-(DOR) and κ-opioid receptors (KOR) in antinociceptive effects of the deltorphins analogs was studies using selective antagonists of these receptors. The results indicated that DK-4 (5, 10 and 20 nmol) and DEL-6 (5, 10 and 20 nmol) were the most effective in alleviating thermal pain at the dose of 20 nmol. The antinociceptive potency of DEL-6 at the dose of 20 nmol was approximately equal but DK-4 at the dose of 20 nmol was less effective than morphine at the dose of 13 nmol. DOR antagonist - naltrindole (NTI, 5 nmol) very strongly and, to the lower extent MOR antagonist - β-funaltrexamine (β-FNA, 5 nmol), inhibited antinociceptive effect of DK-4 (20 nmol). In turn, β-FNA was more potent than NTI in inhibition of the antinociceptive effects of DEL-6. Co-administration of DEL-6 and morphine at doses of 5 nmol, which do not produce measurable antinociception, generated additive antinociceptive effect. Chronic intraperitoneal (i.p.) injection of morphine (9 days) displayed a marked analgesic tolerance to the challenge dose of morphine and a slight cross-tolerance to challenge doses of DEL-6 and DK-4, given i.c.v. These findings indicate that the new deltorphin analogs recruit DOR and MOR to attenuate the nociceptive response to acute thermal stimuli.

Structure-activity relationship study of a small cyclic peptide H-c[Lys-Pro-Glu]-Arg-OH: a potent inhibitor of Vascular Endothelial Growth Factor interaction with Neuropilin-1

Inhibition of angiogenesis is one of the most promising approaches in anticancer therapy. It was recently suggested that Neuropilin-1 (NRP-1) in tumour cells may serve as a separate receptor for Vascular Endothelial Growth Factor-165 (VEGF165) which is one of the main pro-angiogenic agents in the organism. Therefore molecules inhibiting VEGF165 binding to NRP-1 could be potential candidates for new antiangiogenic and anticancer drugs. Here we present a structure-activity relationship study of the peptide H-c[Lys-Pro-Glu]-Arg-OH which showed high inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18μM) in our previous study. We report the design, synthesis, in vitro assays and docking analysis of four small cyclic peptides (14-,15-membered ring) and one bigger cyclic compound (30-membered ring). Our study shows that both the ring size and configuration of amino acid residues present in the structure are crucial for high inhibitory effect.

Enkephalin analog, cyclo[Nε,Nβ-carbonyl-D-Lys2,Dap5] enkephalinamide (cUENK6), inhibits the ethanol withdrawal-induced anxiety-like behavior in rats

An analog of enkephalin, cyclo[N(ε),N(β)-carbonyl-D-Lys(2),Dap(5)] enkephalinamide (cUENK6), is predominantly a functional agonist of μ-opioid receptors (MOPr) and, to a lesser extent, of δ-opioid receptors (DOPr) in vitro. The aim of the present study was to determine whether cUENK6 could affect ethanol withdrawal-induced anxiety-like behavior in the elevated plus maze (EPM) test in rats. An anxiety-like effect of withdrawal was predicted to occur in the EPM test 24 h after the last ethanol administration (2 g/kg, intraperitoneally [i.p.]; 15% w/v once daily for 9 days). Ethanol withdrawal decreased the percent of time spent by rats in the open arms and the percent of open-arms entries. cUENK6 (0.25 nmol), given by intracerebroventricular (i.c.v.) injection, significantly reversed these anxiety-like effects of ethanol withdrawal and elevated the percent of time spent by rats in the open arms and the percent of open-arms entries. These effects of cUENK6 were significantly inhibited by the DOPr antagonist naltrindole (NTI) (5 nmol, i.c.v.), but not by the MOPr antagonist β-funaltrexamine (β-FNA) (5 nmol, i.c.v.). The preferential DOPr agonist [Leu(5)]-enkephalin (LeuEnk) (2.7 and 5.4 nmol, i.c.v.) and the MOPr agonist morphine (6.5 and 13 nmol, i.c.v.) reduced the anxiety-like effects of ethanol withdrawal. cUENK6 at the dose of 0.25 nmol did not disturb locomotor activity in the EPM, in contrast to cUENK6 at the dose of 0.5 nmol, and morphine at 6.5 and 13 nmol. However, similarly to LeuEnk, cUENK6 induced the anxiolytic-like effects in naïve rats. Thus, our study suggests that cUENK6 reduced ethanol withdrawal-induced anxiety-like behavior by activation of δ-opioid receptors rather than μ-opioid receptors.

The influence of the new enkephalin derivative, cyclo[Nε,Nβ-carbonyl-d-Lys2,Dap5] enkephalinamide (cUENK6), on reinstatement of ethanol-induced conditioned place preference in rats

The aim of the present study was to determine whether a new cyclic analog of enkephalin, cyclo[N(ε),N(β)-carbonyl-d-Lys(2),Dap(5)] enkephalinamide (cUENK6), a preferential μ-(MORs), and, to a lower extent, a δ-opioid receptor (DORs) agonist in vitro, could reinstate ethanol-induced conditioned place preference (CPP). In our work, male Wistar rats were first conditioned either with ethanol (10% w/v, 0.5g/kg, intraperitoneally (i.p.)) or 0.9% NaCl in a biased CPP procedure. The intracerebroventricular (i.c.v.) administration of DORs antagonist (naltrindole, 2.5 and 5nmol) or MORs antagonist (β-funaltrexamine, 5 and 10nmol), but not the κ opioid receptor (KORs) antagonist (norbinaltorphimine, 5 and 10nmol) was then administered and inhibited the expression of ethanol-induced CPP. After the extinction session, i.c.v. administration of cUENK6 at the dose of 0.125, 0.25 and 0.5nmol occurred, and was found to reinstate the ethanol-induced CPP similar to that of the priming injection of ethanol. However, the reinstated effect of cUENK6 (0.25nmol) was strongly abolished by administration of naltrindole and, to lesser extent, by β-funaltrexamine. Furthermore, the preferential MORs agonist-morphine (13nmol, i.c.v.) and the DORs agonist-[Leu(5)]-enkephalin (2.7 and 5.4nmol, i.c.v.) also reinstated the ethanol-induced CPP. cUENK6 given alone at the dose of 0.25nmol before the testing phase had no effect in animals that received 0.9% NaCl during the conditioning phase and also did not influence their locomotor activity. These data suggest that the effects of cUENK6 did not have an impact on the results obtained in the reinstatement procedure of CPP. Overall, the data support the idea that both MORs and DORs are normally involved in the expression and reinstatement of ethanol conditioned seeking behavior - as indexed by CPP in rats.

Involvement of delta and mu opioid receptors in the acute and sensitized locomotor action of cocaine in mice

Analogs of deltorphins, such as cyclo(Nδ, Nδ-carbonyl-d-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) are functional agonists predominantly for the delta opioid receptors (DOR) in the guinea-pig ileum and mouse vas deferens bioassays. The purpose of this study was to examine an influence of these peptides (5, 10 or 20 nmol, i.c.v.) on the acute cocaine-induced (10mg/kg, i.p.) locomotor activity and the expression of sensitization to cocaine locomotor effect. Sensitization to locomotor effect of cocaine was developed by five injections of cocaine at the dose of 10mg/kg, i.p. every 3 days. Our results indicated that DK-4 and DEL-6 differently affected the acute and sensitized cocaine locomotion. Co-administration of DEL-6 with cocaine enhanced acute cocaine locomotion only at the dose of 10 nmol, with minimal effects at the doses 5 and 20 nmol, whereas co-administration of DK-4 with cocaine enhanced acute cocaine-induced locomotion in a dose-dependent manner. Similarly to the acute effects, DEL-6 only at the dose of 10 nmol but DK-4 dose-dependently enhanced the expression of cocaine sensitization. Pre-treatment with DOR antagonist - naltrindole (5 nmol, i.c.v.) and mu opioid receptor (MOR) antagonist, β-funaltrexamine abolished the ability of both peptides to potentiate the effects of cocaine. Our study suggests that MOR and DOR are involved in the interactions between cocaine and both deltorphins analogs. A distinct dose-response effects of these peptides on cocaine locomotion probably arise from differential functional activation (targeting) of the DOR and MOR by both deltorphins analogs.

Are mu-opioid receptors (MORs) involved in the anxiety-like effect of ethanol withdrawal in rats?

Withdrawal from alcohol leads to a negative emotional state including an increase of anxiety, hyperirritability or depressed mood. The withdrawal-induced anxiety has been reported to be a risk factor for relapse [Lucht et al., Eur Addict Res, 2002]. Strong evidence indicates that enkephalins, endogenous opioid peptides with delta-opioid receptors (DORs) affinity, reveal anxiolytic-like effects [Perine et al., Br J Pharmacol, 2006] and reduce withdrawal-induced anxietylike behavior in the ethanol dependent rats [van Rijn et al., J Pharmacol Exp Ther, 2010]. A cyclic analog of enkephalin, cyclo[N ,N -carbonyl-D-Lys , Dap ] enkephalinamide (cUENK6) showed predominant affinity to MORs and, to a lesser extent to DORs in vitro [Pawlak et al., J Pept Sci, 2001]. The aim of the present study was to determine whether cUENK6 could affect an anxiety-like behavior measured during withdrawal from chronic ethanol administration in male Wistar rats. Ethanol dependence was induced according to the previously established method [Kotlinska and Bochenski, Eur J Pharmacol, 2008]. Our study indicated that ethanol decreased the time spent by rats in the open arms. This effect was reversed by intracerebroventricular (icv) administration of cUENK6 (0.0625, 0.125 and 0.25 nmol) at the doses of 0.0625 and 0.25 nmol. Our study suggests that MORs could participate in an anxiety-like behavior during withdrawal from chronic ethanol administration.