Design and Application of pH-Responsive Liposomes for Site-Specific Delivery of Cytotoxin from Cobra Venom.

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells. Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system. Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC50 0.25 µg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%). Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment.

Revealing molecular determinants governing mambalgin-3 pharmacology at acid-sensing ion channel 1 variants.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

A pathological joint-liver axis mediated by matrikine-activated CD4+ T cells.

The knee joint has long been considered a closed system. The pathological effects of joint diseases on distant organs have not been investigated. Herein, our clinical data showed that post-traumatic joint damage, combined with joint bleeding (hemarthrosis), exhibits a worse liver function compared with healthy control. With mouse model, hemarthrosis induces both cartilage degeneration and remote liver damage. Next, we found that hemarthrosis induces the upregulation in ratio and differentiation towards Th17 cells of CD4+ T cells in peripheral blood and spleen. Deletion of CD4+ T cells reverses hemarthrosis-induced liver damage. Degeneration of cartilage matrix induced by hemarthrosis upregulates serological type II collagen (COL II), which activates CD4+ T cells. Systemic application of a COL II antibody blocks the activation. Furthermore, bulk RNAseq and single-cell qPCR analysis revealed that the cartilage Akt pathway is inhibited by blood treatment. Intra-articular application of Akt activator blocks the cartilage degeneration and thus protects against the liver impairment in mouse and pig models. Taken together, our study revealed a pathological joint-liver axis mediated by matrikine-activated CD4+ T cells, which refreshes the organ-crosstalk axis and provides a new treatment target for hemarthrosis-related disease. Intra-articular bleeding induces cartilage degradation through down-reulation of cartilage Akt pathway. During this process, the soluble COL II released from the damaged cartilage can activate peripheral CD4+ T cells, differention into Th17 cells and secretion of IL-17, which consequently induces liver impairment. Intra-articular application of sc79 (inhibitor of Akt pathway) can prevent the cartilage damage as well as its peripheral influences.

Exploration of antimicrobial and anticancer activities of L-amino acid oxidase from Egyptian Naja haje venom.

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 µmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 µg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 µg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.

Cobra (Naja naja) venom L-amino acid oxidase (NNLAAO70) induces apoptosis and secondary necrosis in human lung epithelial cancer cells.

Snake venom L-amino acid oxidases (LAAOs) are flavoenzymes with diverse physiological and pharmacological effects. These enzymes are found to showcase anticoagulant, antiplatelet, cytotoxicity and other biological effects in bite victims. However, the exact mechanism through which they exhibit several biological properties is not yet fully understood. The current study focussed on the purification of cobra venom LAAO and the functional characterization of purified LAAO. A novel L-amino acid oxidase NNLAAO70 with a molecular weight ~70 kDa was purified from the venom of an Indian spectacled cobra (Naja naja). NNLAAO70 showed high substrate specificity for L-His, L-Leu, and L-Arg during its LAAO activity. It inhibited adenosine di-phosphate (ADP) and collagen-induced platelet aggregation process in a dosedependent manner. About 60% inhibition of collagen-induced and 40% inhibition of ADP-induced platelet aggregation was observed with a 40 µg/ml dose of NNLAAO70. NNLAAO70 exhibited bactericidal activity on Bacillus subtilis, Escherichia coli, Bacillus megaterium, and Pseudomonas fluorescens. NNLAAO70 also showed cytotoxicity on A549 cells in vitro. It showed severe bactericidal activity on P. fluorescens and lysed 55% of cells. NNLAAO70 also exhibited drastic cytotoxicity on A549 cells. At 1 lg/ml dosage, it demonstrated a 60% reduction in A549 viability and induced apoptosis upon 24-h incubation. H2O2 released during oxidative deamination reactions played a major role in NNLAAO70-induced cytotoxicity. NNLAAO70 significantly increased intracellular reactive oxygen species (ROS) levels in A549 cells by six fold when compared to untreated cells. Oxidative stress-mediated cell injury is the primary cause of NNLAAO70-induced apoptosis in A549 cells and prolonged oxidative stress caused DNA fragmentation and activated cellular secondary necrosis.

Differential effects of the venoms of Russell's viper and Indian cobra on human myoblasts.

Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell's viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian 'Big Four' snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell's viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell's viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.

Thrombolysis, clotting, and genotoxic activities modulated by essential oils extracted from Lippia alba

Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action

Dendrotoxin-kappa suppresses tumor growth induced by human lung adenocarcinoma A549 cells in nude mice

Voltage-gated K+ (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-kappa (DTX-kappa), on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-kappa, reduced tumor formation in nude mice. Furthermore, treatment with DTX-kappa significantly increased protein expression of p21Waf1/Cip1, p27Kip1, and p15INK4B and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-kappa has anti-tumor effects in A549 cells through the pathway governing G1-S transition.

Diagnóstico diferencial de Naja con otros venenos de víbora / Diferencial diagnostic of Naja with other snake venoms

La idea de este trabajo es establecer un diagnóstico diferencial de Naja con otros medicamentos pero poniendo el acento en su cuadro cardíaco donde la rapidez diagnóstica es imprescindible (AU)

Actividad hemolítica de venenos de serpientes de los géneros Bothrops, Lachesis, Crotalus y Micrurus (Serpentes: Viperidae y Elapidae) / Haemolytic activity of venoms from snakes of the genera Bothrop, Lachesis, Crotalus and Micrurus (Serpentes: Viperidae and Elapidae)

Hemolytic activity of eight Peruvian snake venoms from the families Viperidae and Elapidae (Bothrops atrox, B. pictus, B. hyoprorus, B. bilineatus, B. neuwedii, Lachesis m. muta, Crotalus d. terrificus, Microrus tschudi), and three Brazilian viperids (B. jararacussu, B. alternatus and C. d. collilineatus) is described. None of the venoms cause direct lysis on washed human erythrocytes. However, all of then caused indirect hemolysis provided that the incubation medium contains an exogenous source of lecithin. Venom of Micrurus tschudi was the most hemolytic (HD50 2.8 ug/ml) while that of B. bilineatus was the least (HD50 681.3 ug/ml). Only six of eleven venoms showed parallel curves of hemolytic activity, and the HD50 varied from 198 to 681 ug/ml and the following decreasing order of hemolytic activity was obtained: L. muta, C. d. terrificus, C. d. collilineatus, B. hyoprorus, B. bilineatus, B. alternatus

Biochemical and behavioral effects of intraseptal microinjection of fasciculin, an irreversible acetylcholinesterase inhibitor

We examined the effect, in rats, of an intraseptal microinjection of fasciculin (FAS), an irreversible peptide acetylcholinesterase (AChE) inhibitor, on a)AChE activity measured in septum and hippocampus, b)3H-quinuclidiny benzylate (3H-QNB) and 3H-oxotremorine (3H-OXO) binding to hippocampal cholinergic muscarinic receptors, c) 3H-flunitrazepan (3H-FNZ) binding to hippocampal benzodiazepine receptors as a control for QNB and OXO binding, d) acquisition and retention in three different behavioral paradigms, i. e., water-finding (in which there is concomitant habituation to be apparatus), step-down inhibitory avoidance, and shuttle avoidance. AChE activity in septum decreased 2 days (-66%) and 5 days (-48%) after FAS microinjection; a slight reduction (-35%) occurred in the dorsal hippocampus on day 2 (P<0.05; N=6 per group); no changes in AChE activity were observed in ventral hippocampus ion day 2 or day 5. No changes in 3H-QNB, 3H-OXO, or 3H-FNZ binding constants were demonstrable in the hippocampus either 2 or 5 days after intraseptal FAS adminstration. No changes in training or test session performance in any of the three behavioral situations were observed 2-3 days after the intraseptal microinjection of FAS. The persistent inhibition of septal AChE caused by FAS microinjection into the septum is not sufficient to induce major changes either in hippocampal cholinergic muscarinic receptors, or in the learning or retention of behaviors regulated by the septum and/or hippocampus

Acidentes por serpentes peçonhentas do Brasil / Accidents by snake venoms of Brazil

O presente artigo revisa conhecimentos referentes à incidência, epidemiologia, fisiopatologia, quadro clínico, diagnóstico e prognóstico dos acidentes por serpentes peçonhentas do Brasil que säo responsáveis por mais de 20.000 acidentes e 100 óbitos por ano. Pertencem a quatro gêneros: Bothrops, Crotalus, Lachesis e Micrurus. Executando as Micrurus, todas causam distúrbios na coagulaçäo sanguínea; as Lachesis e Bothrops provocam destruiçäo tecidual na regiäo da picada, as Crotalus e Micrurus, bloqueio na placa motora e somente as Crotalus, miotoxicidade sistêmica. O quadro clínico geralmente é suficiente para o diagnóstico, exceto na distinçäo entre acidente botrópico e laquético. O tratamento com soro heterólogo específico deve ser realizado precocemente, por via endovenosa. É também importante manter os pacientes hidratados e, no caso de picados por Micrurus, prestar assistência ventilatória adequada

Informe especial: estudios tendientes a determinar las posibles propiedades antitumorales del veneno de cobra y del complejo crotoxina A y B / Special report: studies to determine the possible antitumoral properities of Cobra venom and of crotoxin complex A and B

La presente investigación fue encomendada por el CONICET (Consejo Nacional de Investigaciones Cientificas y Técnicas) para determinar las posibles propriedades antineoplásicas del veneno de Cobra (Naja Naja Siamensis) VC) y del Complejo Crotoxina a y B (CCAB), purificado a partir del veneno de Crotalus durissus terificus (cascavel sudamericana). La coordinación de las investigaciones estuvo a cargo de los Dres. Baldi y Mordoh, y la ejecución de la parte experimental fue llevada a cabo por los restantes autores. Se utilizaron diversos sistemas experimentales: 1) lúneas celulares in vitro: de origen murino, normales (3T3 A31), o transformadas (M-A31, Ki-A31 y BP-A31) o de origen humano (adenocarcinoma mamario: MCF-7 y T47D). el efecto de las drogas fue determinado a 1, 10 y 100 ng/ml de VC y CCAB, solas o en combinación. En ninguna de las líneas celulares mencionadas se observaron cambios significativos en la velocidad de crecimiento o en la morfología celular; 2) desarrollo in vivo del Sarcoma 180 (S180) en ratones Balb/c: el VC a dosis de 21 ó 26 ng/g (inyecciones i.p. a los días 10, 11, 20, 21, 30 y 31 post-inoculo tumoral) no afectaron el crecimiento ni la sobrevida de los animales. Dosis de CCAB de 6 ng/g o 9 ng/g ]con el mismo esquema de inoculación anterior) no afectaron el crecimiento de s180 hasta los 25 días de desarrollo tumoral. Entre los días 25 y 30 la dosis de 9 ng/g determinó una evolución tumoral más rápida. La combinación de 13 ng/g VC y 4,5 ng/g CCAB con iguales tiempos de inyección no afectaron el desarrollo tumoral. La histología de los tumores de los animales tratados y controles no evidenció cambios significativos; 3) desarrollo in vivo del carcinoma de Ehrlich en ratones Swiss: se probó el efecto de CCAB (9 ng/g inyectados i.p. a los días 8, 9, 16, y 17) sin obtenerse modificación del desarrollo tumoral;...(AU)

Informe especial: estudios tendientes a determinar las posibles propiedades antitumorales del veneno de cobra y del complejo crotoxina A y B / Special report: studies to determine the possible antitumoral properities of Cobra venom and of crotoxin complex A and B

La presente investigación fue encomendada por el CONICET (Consejo Nacional de Investigaciones Cientificas y Técnicas) para determinar las posibles propriedades antineoplásicas del veneno de Cobra (Naja Naja Siamensis) VC) y del Complejo Crotoxina a y B (CCAB), purificado a partir del veneno de Crotalus durissus terificus (cascavel sudamericana). La coordinación de las investigaciones estuvo a cargo de los Dres. Baldi y Mordoh, y la ejecución de la parte experimental fue llevada a cabo por los restantes autores. Se utilizaron diversos sistemas experimentales: 1) lúneas celulares in vitro: de origen murino, normales (3T3 A31), o transformadas (M-A31, Ki-A31 y BP-A31) o de origen humano (adenocarcinoma mamario: MCF-7 y T47D). el efecto de las drogas fue determinado a 1, 10 y 100 ng/ml de VC y CCAB, solas o en combinación. En ninguna de las líneas celulares mencionadas se observaron cambios significativos en la velocidad de crecimiento o en la morfología celular; 2) desarrollo in vivo del Sarcoma 180 (S180) en ratones Balb/c: el VC a dosis de 21 ó 26 ng/g (inyecciones i.p. a los días 10, 11, 20, 21, 30 y 31 post-inoculo tumoral) no afectaron el crecimiento ni la sobrevida de los animales. Dosis de CCAB de 6 ng/g o 9 ng/g ]con el mismo esquema de inoculación anterior) no afectaron el crecimiento de s180 hasta los 25 días de desarrollo tumoral. Entre los días 25 y 30 la dosis de 9 ng/g determinó una evolución tumoral más rápida. La combinación de 13 ng/g VC y 4,5 ng/g CCAB con iguales tiempos de inyección no afectaron el desarrollo tumoral. La histología de los tumores de los animales tratados y controles no evidenció cambios significativos; 3) desarrollo in vivo del carcinoma de Ehrlich en ratones Swiss: se probó el efecto de CCAB (9 ng/g inyectados i.p. a los días 8, 9, 16, y 17) sin obtenerse modificación del desarrollo tumoral;...

Coral snake venoms: mode of action and pathophysiology of experimental envenomation

As cobras corais säo representantes da familia Elapidae nas Américas. Classificam-se em dois gêneros Micruroides e Micrurus. O gênero Micrurus compreende a quase totalidade das espécies de cobra coral e todas as que causam acidentes no homem. Podem-se fazer as seguintes generalizaçöes quanto aos efeitos produzidos por suas peçonhas e a algumas propriedades destas. As peçonhas das cobras corais säo neurotóxicas, causando perda da força muscular e morte por paralisia respiratória. Näo provocam edema local e necrose assim como näo produzem coagulaçäo sanguínea ou hemorragias. A atividade proteolítica das peçonhas de cobras corais é pequena ou nula. Exercem atividade fosfolipase A**2. Näo induzem efeitos nefrotóxicos. Os componentes tóxicos da peçonha das Elapidae säo as neurotoxinas pré-sinápticas, as neurotoxinas pós-sinápticas, as cardiotoxinas e fosfolipases A**2 com atividade mionecrótica ou semelhante à das cardiotoxinas. O modo de açäo das peçonhas de Micrurus frontalis, M. lemniscatus, M. corallinus e M. fulvius foi investigado em preparaçöes neuromusculares isoladas e é aqui exposto. Mostra-se que enquanto as peçonhas de M. frontalis e M. lemniscatus devem conter apenas toxinas que atuam através de ligaçäo com os receptores colinérgicos da placa terminal (neurotoxinas pós-sinápticas), a de M. corallinus atua também na junçäo neurovascular inibindo a liberaçäo de acetilcolina pelos impulsos nervosos e a de M. fulvius induz despolarizaçäo da membrana das fibras musculares. Relatam-se também os efeitos produzidos pelas peçonhas de M. corallinus e M. fulvius in vivo em cäes e os provocados pela peçonha de M. frontalis em cäes e macacos

Mecanismo de acción de miotoxinas aisladas de venenos de serpientes / Mechanism of action of myotoxins isolated from snake venoms

Desde los puntos de vista bioquímico y farmacológico, las miotoxinas aisladas de venenos de serpientes se ubican en cuatro grupos: (1) Fosfolipasas A miotóxicas, (2) miotoxinas básicas de bajo peso molecular, (3) cardiotoxinas de venenos elapídeos y (4) miotoxinas hemorrágicas. Las fosfolipasas miotóxicas notexinam taipoxina, crotoxina y miotoxina de Bothrops asper afectan inicialmente la integridad de la membrana plasmática, induciéndose un influjo de calcio que culmina con la muerte celular. Las miotoxinas básicas de bajo peso molecular crotamina y miotoxina a actúan específicamente en los canales de sodio del sarcolema, induciendo un influjo de sodio que trae como consecuencia despolarización y contracción muscular y vacuolización del retículo sarcoplásmico. Las cardiotoxinas son polipéptidos básicos capaces de desorganizar la estructura de las membranas, siendo su acción miotóxica una consecuencia de la alteración drástica que las mismas inducen en el sarcolema del músculo esquelético. Finalmente, dos componentes hemorrágicos (toxina hemorrágica b y viriditoxina) poseen actividad miotóxica, habiendose sugerido que este efecto es una consecuencia de la isquemia tisular resultante de la acción hemorrágica de estos componentes