Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors - Benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy.

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

[18F]FDG and [11C]PK11195 PET imaging in the evaluation of brown adipose tissue - effects of cold and pharmacological stimuli and their association with crotamine intake in a male mouse model.

This study aimed to evaluate the role of positron emission tomography (PET) with [11C]PK11195 and [18F]FDG in the characterization of brown adipose tissue (BAT). METHODS: Male C57BL/6 mice were studied with the glucose analogue [18F]FDG (n = 21) and the TSPO mitochondrial tracer [11C]PK11195 (n = 28), without stimulus and after cold (6-9 °C) or beta-agonist (CL316243) stimuli. PET studies were performed at baseline and after 21 days of daily treatment with crotamine, which is a peptide described to induce adipocyte tissue browning and to increase BAT metabolism. Tracer uptake (SUVmax) was measured in the interscapular BAT and translocator protein 18 kDa (TSPO) expression was evaluated by immunohistochemistry. RESULTS: The cold stimulus increased [18F]FDG uptake compared to no-stimulus (5.21 ± 1.05 vs. 2.03 ± 0.21, p < 0.0001) and to beta-agonist stimulus (2.65 ± 0.39, p = 0.0003). After 21 days of treatment with crotamine, there was no significant difference in the [18F]FDG uptake compared to the baseline in the no-stimulus group and in the cold-stimulus group, with a significant increase in uptake after CL stimulus (baseline: 2.65 ± 0.39; 21 days crotamine: 4.77 ± 0.81, p = 0.0003). Evaluation of [11C]PK11195 at baseline shows that CL stimulus increases the BAT uptake compared to no-stimulus (4.47 ± 0.66 vs. 3.36 ± 0.68, p = 0.014). After 21 days of treatment with crotamine, there was no significant difference in the [11C]PK11195 uptake compared to the baseline in the no-stimulus group (2.94 ± 0.58, p = 0.7864) and also after CL stimulus (3.55 ± 0.79, p = 0.085). TSPO expression correlated with [11C]PK11195 uptake (r = 0.83, p = 0.018) but not with [18F]FDG uptake (r = 0.40, p = 0.516). CONCLUSIONS: [11C]PK11195 allowed the identification of BAT under thermoneutral conditions or after beta3-adrenergic stimulation in a direct correlation with TSPO expression. The beta-adrenergic stimulus, despite presenting a lower intensity of glycolytic activation compared to cold at baseline, allowed the observation of an increase in BAT uptake of [18F]FDG after 21 days of crotamine administration. Although some limitations were observed for the metabolic changes induced by crotamine, this study reinforced the potential of using [11C]PK11195 and/or [18F]FDG-PET to monitor the activation of BAT.

Biophysical and pharmacological characterization of a full-length synthetic analog of the antitumor polypeptide crotamine.

Crotamine is a polypeptide isolated from the venom of a South American rattlesnake. Among the properties and biological activities of crotamine, the most extraordinary is its ability to enter cells with unique selective affinity and cytotoxic activity against actively proliferating cells, such as tumor cells. This peptide is also a cargo carrier, and anticipating commercial application of this native polypeptide as a potential theranostic compound against cancer, we performed here a side-by-side characterization of a chemically synthesized full-length crotamine compared with its native counterpart. The structural, biophysical, and pharmacological properties were evaluated. Comparative NMR studies showed structural conservation of synthetic crotamine. Moreover, similarly to native crotamine, the synthetic polypeptide was also capable of inhibiting tumor growth in vivo, increasing the survival of mice bearing subcutaneous tumor. We also confirmed the ability of synthetic crotamine to transfect and transport DNA into eukaryotic cells, in addition to the importance of proteoglycans on cell surface for its internalization. This work opens new opportunities for future evaluation of chimeric and/or point-mutated analogs of this snake polypeptide, aiming for improving crotamine properties and applications, as well as possibly diminishing its potential toxic effects. KEY MESSAGES: • Synthetic crotamine showed ex vivo and in vivo activities similar to native peptide. • Synthetic crotamine structure conservation was demonstrated by NMR analysis. • Synthetic crotamine is able to transfect and transport DNA into eukaryotic cells. • Synthetic crotamine shows tumor growth inhibition in vivo. • Synthetic crotamine increases survival of mice bearing tumor.

Peripheral biomarkers allow differential diagnosis between schizophrenia and bipolar disorder.

Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders that pose important challenges for diagnosis by sharing common symptoms, such as delusions and hallucinations. The underlying pathophysiology of both disorders remains largely unknown, and the identification of biomarkers with potential to support diagnosis is highly desirable. In a previous study, we successfully discriminated SCZ and BD patients from healthy control (HC) individuals by employing proton magnetic resonance spectroscopy (1H-NMR). In this study, 1H-NMR data treated by chemometrics, principal component analysis (PCA) and supervised partial least-squares discriminant analysis (PLS-DA), provided the identification of metabolites present only in BD (as for instance the 2,3-diphospho-D-glyceric acid, N-acetyl aspartyl-glutamic acid, monoethyl malonate) or only in SCZ (as isovaleryl carnitine, pantothenate, mannitol, glycine, GABA). This may represent a set of potential biomarkers to support the diagnosis of these mental disorders, enabling the discrimination between SCZ and BD, and among these psychiatric patients and HC (as 6-hydroxydopamine was present in BD and SCZ but not in HC). The presence or absence of these metabolites in blood allowed the categorization of 182 independent subjects into one of these three groups. In addition, the presented data suggest disturbances in metabolic pathways in SCZ and BD, which may provide new and important information to support the elucidation and/or new insights into the neurobiology underlying these mental disorders.

Pharmacological characterization of crotamine effects on mice hind limb paralysis employing both ex vivo and in vivo assays: Insights into the involvement of voltage-gated ion channels in the crotamine action on skeletal muscles.

The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10-25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.