Rafael Stuani Floriano

The ex vivo neurotoxic, myotoxic and cardiotoxic activity of cucurbituril-based macrocyclic drug delivery vehicles

The cucurbituril family of drug delivery vehicles have been examined for their tissue specific toxicity using ex vivo models. Cucurbit[6]uril (CB[6]), cucurbit[7]uril (CB[7]) and the linear cucurbituril-derivative Motor2 were examined for their neuro-, myo- and cardiotoxic activity and compared with β-cyclodextrin. The protective effect of drug encapsulation by CB[7] was also examined on the platinum-based anticancer drug cisplatin. The results show that none of the cucurbiturils have statistically measurable neurotoxicity as measured using mouse sciatic nerve compound action potential. Cucurbituril myotoxicity was measured by nerve-muscle force of contraction through chemical and electrical stimulation. Motor2 was found to display no myotoxicity, whereas both CB[6] and CB[7] showed myotoxic activity via a presynaptic effect. Finally, cardiotoxicity, which was measured by changes in the rate and force of right and left atria contraction, was observed for all three cucurbiturils. Free cisplatin displays neuro-, myo- and cardiotoxic activity, consistent with the side-effects seen in the clinic. Whilst CB[7] had no effect on the level of cisplatins neurotoxic activity, drug encapsulation within the macrocycle had a marked reduction in both the drugs myo- and cardiotoxic activity. Overall the results are consistent with the relative lack of toxicity displayed by these macrocycles in whole animal acute systemic toxicity studies and indicate continued potential of cucurbiturils as drug delivery vehicles for the reduction of the side effects associated with platinum-based chemotherapy.

Pharmacological study of a new Asp49 phospholipase A(2) (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations.

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4-6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 μg/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 ± 1 mV (basal) to -66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.

Biochemical, pharmacological, and structural characterization of new basic PLA2 Bbil-TX from Bothriopsis bilineata snake venom.

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25–37°C. Maximum PLA2 activity required Ca2+ and in the presence of Cd2+, Zn2+, Mn2+, and Mg2+ it was reduced in the presence or absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro.

In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1-30 μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15-90 min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120 min (from 80 ± 15 to 206 ± 25U/ml, n=6, p<0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30 μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30 min followed by significant inhibition at ≥ 90 min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22 °C instead of 37 °C delayed the onset of blockade, as did inhibition of venom PLA(2) activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA(2).

Functional and structural studies of a Phospholipase A2-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism

BACKGROUND One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA2-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA2-like proteins and to advance the current knowledge of their action mechanism. METHODS Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction. RESULTS We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA2-like proteins undergo to exert their function. CONCLUSIONS Our findings show that zinc ions are inhibitors of PLA2-like proteins and suggest two different mechanisms of inhibition for these ions. GENERAL SIGNIFICANCE Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA2-like proteins.

Neutralising ability of Terminalia fagifolia extract (Combretaceae) against the in vitro neuromuscular effects of Bothrops jararacussu venom

Abstract The ability of Terminalia fagifolia hydroalcoholic extract (Tf-HE) to neutralise the paralysis and myotoxicity induced by Bothrops jararacussu venom was assayed using mouse phrenic nerve-diaphragm (PND) preparation and two varieties of chick biventer cervicis (BC) preparations. Tf-HE 100 μg/mL and 500 μg/mL were tested against 40 and 200 μg of venom/mL in PND and BC preparations, respectively, using pre- and post-venom incubation treatments. The effects of Tf-HE against the myotoxicity caused by venom were evaluated via histological analysis (PND) and creatine kinase (CK) release (BC). Tf-HE was able to reverse the venom paralysis in both preparation types. The contractures to exogenous ACh in BC preparations showed that Tf-HE may act on extrinsic, preserving those intrinsic postsynaptic receptors. There was a positive correlation between CK and morphological changes. The high non-hemolytic saponin content can explain the Tf-HE efficacy against the toxic effects of B. jararacussu venom in vertebrate neuromuscular preparations.

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A2 toxins (PLA2) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA2 toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.

A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa)

ABSTRACT Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A2 (PLA2) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA2 and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 &mgr;g/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 &mgr;M), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 &mgr;g/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 &mgr;g/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh‐induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post‐synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer the potential to go further in the identification of their components and biological targets. HIGHLIGHTSE. bizona and P. neuwiedii venom induced partial neuromuscular blockade in chick biventer cervicis preparations.E. bizona (Double‐banded coral snake mimic) venom (30 &mgr;g/ml) potentiated the ACh‐induced contracture without changing the twitch tension.P. neuwiedii (Neuwieds false boa) venom myotoxicity is higher than E. bizona venom and is likely due to proteolytic activity.The venom of both species showed low phospholipase A2 activity.The neuromuscular blockade observed is likely induced by muscle damage in consequence of their high proteolytic activity.

Prospective study of a Bothrops jararacussu venom batch (Bj2015) – phospholipase A2 activity, immunogenicity, neurotoxicity, and myotoxicity parameters

Abstract Bothrops jararacussu venom’s (Bj2015) batch was biomonitored quarterly for one year to assess phospholipase A2 (PLA2) activity, immunogenicity, neurotoxicity, and myotoxicity. In silico models were applied to evaluate losses using decay model and recoveries by predictive trend analysis. Mice were immunized with Bj2015. Antibodies were detected by double-immunodiffusion and total protein and albumin were measured. Neuromuscular blockade-induced by 40 μg mL−1 venom solution was carried out using mouse nerve phrenic-diaphragm preparation. Resulting muscles were submitted to light microscopy to evaluate the myotoxicity. PLA2 activity of 0.1 mg mL−1 Bj2015 was measured using 4-nitro-3-(octanoyloxy)benzoic acid as substrate. Over time, greater losses occurred in neurotoxicity than PLA2, but not in myotoxicity and immunogenicity. Concluding, the neurotoxicity decrease can be related to enzymatic losses, including PLA2. Depending on the purpose of use, the collected venom responds on a long time, avoiding unnecessary new collections, improving life quality of animals in captivity and increasing their longevity.