Cannabinoid receptor agonists from Conus venoms alleviate pain-related behavior in rats.

Cannabinoid (CB) receptor agonists show robust antinociceptive effects in various pain models. However, most of the clinically potent CB1 receptor-active drugs derived from cannabis are considered concerning due to psychotomimetic side effects. Selective CB receptor ligands that do not induce CNS side effects are of clinical interest. The venoms of marine snail Conus are a natural source of various potent analgesic peptides, some of which are already FDA approved. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. HEK293 cells expressing CB1 receptors were treated with venom extracts and CB1 receptor internalization was analyzed by immunofluorescence. Results showed C. textile (C. Tex) and C. miles (C. Mil) samples as the most potent. These were serially subfractionated by HPLC for subsequent analysis by internalization assays and for analgesic potency evaluated in the formalin test and after peripheral nerve injury. Intrathecal injection of C. Tex and C. Mil subfractions reduced flinching/licking behavior during the second phase of formalin test and attenuated thermal and mechanical allodynia in nerve injury model. Treatment with proteolytic enzymes reduced CB1 internalization of subfractions, indicating the peptidergic nature of CB1 active component. Further HPLC purification revealed two potent antinociceptive subfractions within C. Tex with CB1 and possible CB2 activity, with mild to no side effects in the CB tetrad assessment. CB conopeptides can be isolated from these active Conus venom-derived samples and further developed as novel analgesic agents for the treatment of chronic pain using cell based or gene therapy approaches.

Effect of mouse strain and gender on LD(50) of yessotoxin.

The aim of the present study was to determine whether the intraperitoneal LD(50) for yessotoxin (YTX) in mice varies with strain or gender. Thirty-six male and 36 female mice, of body weight 16-20 g, from each of the strains ICR (CD-1), Swiss (CFW-1) and NMRI were employed. They were not fasted before YTX treatment. At each dose, nine mice were injected with YTX solutions at 1.0 mL/20 g body weight, and observed for 24h. Symptoms and time to death were recorded. Within each mouse strain and gender arm, the study was performed as a basic four level Response Surface Pathway designed trial with nine mice at each dose level. YTX was isolated from a culture of Protoceratium reticulatum. The LD(50) values for female and male mice, respectively, were estimated as 380 and 462 microg/kg for the ICR, 269 and 328 microg/kg for the Swiss, and 314 and 412 microg/kg for the NMRI strains. The increases in LD(50) from female to male mice were found to be 22% for ICR, 22% for Swiss and 31% for NMRI. The largest difference in LD(50) among mouse strains was detected between the ICR and Swiss strains, where the deviation was 41% in both females and males. The difference between mouse strains was found significant (p = 0.03). For all three strains, females were more susceptible than males, with a difference in LD(50) of 1.2-1.3-fold. The largest difference between the least- and most-susceptible strain was 1.4-fold for both females and males. The largest difference in LD(50), 1.7-fold, was observed between female Swiss and male ICR mice. The difference between genders was not significant (p = 0.12). These results indicate that other factors, like handling of the animals, and the source and handling of the toxin, may significantly influence the outcome of studies on acute toxicity since the reported differences in LD(50) vary by a factor of about seven.

Short-term oral toxicity of homoyessotoxins, yessotoxin and okadaic acid in mice.

A short-term toxicity study after 7 days oral daily administration of yessotoxin (YTX; 2 mg/kg/day), homoYTX (1 mg/kg/day), 45-hydroxy-homoYTX (1 mg/kg/day) and of the main diarrhoetic shellfish toxin okadaic acid (OA; 1 mg/kg/day) was carried out in mice. Symptoms, lethality, food consumption, body and organ weights, gross pathology and histopathology of the main organs and tissues, leukocytes formula as well as plasmatic levels of transaminases, lactate dehydrogenase and creatinine phosphokinase were evaluated. Heart tissue was studied also hystochemically for the presence of apoptotic nuclei and by transmission electron microscopy. No mortality, signs of toxicity or cumulative effects were induced by the repeated oral exposure to YTXs. Only ultrastructural changes in the cardiac muscle cells near the capillaries, such as package of rounded mitochondria and alteration of the cells boundary were observed, without any increase of lactate dehydrogenase, an index of cardiac damage. OA induced diarrhoea, body weight loss, reduced food consumption, and the death of 2/5 mice after 5 days. Necroscopy and/or light microscopy analysis revealed toxic effects mainly at forestomach (ulceration and hyperplasia), liver and, indirectly to body weight loss of mice, atrophic signs in the lymphoid organs and exocrine pancreas. Electron microscopy of heart tissue showed alterations of mitochondria and fibers in myocardiocytes, although no apoptotic change was recorded.

Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice.

The acute toxicity of yessotoxin (YTX), homoyessotoxin (homoYTX) and 45-hydroxy-homoyessotoxin (45-OH-homoYTX) has been studied in comparison to that of okadaic acid (OA), the main diarrhogenic toxin, both after intraperitoneal (i.p.) and oral administration. After i.p. administration, homoYTX and YTX showed similar lethality (LD(50)=444 microg/kg and 512 microg/kg), higher than that of OA (LD(50)=225 microg/kg), while 750 microg/kg of 45-OH-homoYTX did not cause death. OA induced the already known toxic signs: before death, mice were motionless and cyanotic; small intestine and liver damage were shown at post-mortem. Mice treated with YTX and homoYTX were restless and jumped before death; necroscopy did not show major changes. After oral treatment, 2 mg/kg of OA induced diarrhoea and body weight loss, causing 4/5 deaths; necroscopy and/or histology revealed degenerative lesions to small intestine, forestomach and liver (confirmed by increased plasma transaminase), but no myocardium alterations. On the contrary, the oral treatment with YTX (1 and 2 mg/kg) and its derivatives (1 mg/kg) did not cause any death or signs of toxicity, except some ultrastructural myocardiocyte alterations, adjacent to capillaries, such as cytoplasmic protrusions (YTX, 1 and 2 mg/kg), fibrillar alteration (YTX, 1 mg/kg) or mitochondria assemblage (45-OH-homoYTX). Altogether, our data show that YTX and its derivatives are less toxic than OA after acute oral and i.p. treatments, at doses which may represent up to 100 times of the possible human daily intake.

Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice.

Currently, yessotoxin is regulated among the toxins in the diarrhetic shellfish poisoning (DSP) complex. Yessotoxin is equally acutely toxic towards mice upon intraperitoneal injections as those algal toxins giving diarrhea, but is not diarrheagenic. Its presence in mussels may therefore lead to overestimation of risk of DSP in consumers when the standard mouse bioassay is used. Arguments are presented for the use of analytical methods instead of the mouse bioassay for the diarrheagenic DSP toxins and yessotoxin. Yessotoxin was found to be more than ten times less toxic to mice via the oral route, compared with intraperitoneal injections. Even at 10mg/kg body weight, the highest dose ever tested orally, yessotoxin did not kill the mice. By means of light microscopy of several organs, moderate changes were only observed in the heart. Ultrastructural studies revealed swelling of heart muscle cells leading to separation of the organelles. Effects were most pronounced close to the capillaries. The pathological changes were clearly dose dependent, and the lowest oral dose where any effects were seen was 2.5mg yessotoxin per kg.

Inhibition by omega-conotoxin GVIA of adrenal catecholamine release in response to endogenous and exogenous acetylcholine.

Effects of the N-type voltage-dependent Ca2+ channel (VDCC) blocker, omega-conotoxin GVIA, and the L-type VDCC blockers, nifedipine and verapamil, on adrenal catecholamine release were examined in anesthetized dogs. These blockers were infused into the adrenal gland through the phrenicoabdominal artery. Splanchnic nerve stimulation at 1 and 3 Hz produced frequency-dependent increases in epinephrine and norepinephrine output determined from adrenal venous blood. Infusion of omega-conotoxin GVIA (0.4 micrograms/min) significantly inhibited the splanchnic nerve stimulation-evoked increases in epinephrine and norepinephrine output. Furthermore, increases in epinephrine and norepinephrine output induced by intraarterial injection of acetylcholine (3 micrograms) into the adrenal gland also were inhibited by omega-conotoxin GVIA (0.4 micrograms/min). Further inhibition of splanchnic nerve stimulation- or exogenous acetylcholine-induced increases in catecholamine output was observed even after the cessation of omega-conotoxin GVIA infusion. Neither nifedipine (1 microgram/min) nor verapamil (10 micrograms/min) affected the splanchnic nerve stimulation-evoked increases in catecholamine output, whereas they inhibited the exogenous acetylcholine-evoked catecholamine release. These results suggest that N-type VDCCs located in adrenal medullary cells may contribute to the release of adrenal catecholamines in response to endogenous and exogenous acetylcholine in the dog.

Intracerebroventricular injection of neosurugatoxin induces a prolonged blockade of brain nicotinic acetylcholine receptors.

The intracerebroventricular injection of neosurugatoxin (2 x 3.2 micrograms) significantly reduced the specific binding of [3H]nicotine but not of [3H]cismethyldioxolane in the cerebral cortex, hippocampus and thalamus of rats 3 days after the toxin injection. Scatchard analysis of [3H]nicotine binding in the rat cerebral cortex revealed that neosurugatoxin can selectively decrease the maximal binding sites (Bmax) for [3H]nicotine by approximately 38%. A significant decrease in the Bmax value for [3H]nicotine was also observed in this brain region 7 days after the neosurugatoxin injection. Thus, the intracerebroventricular injection of neosurugatoxin can induce a prolonged blockade of brain nicotinic acetylcholine receptors.