Bufotenine, a tryptophan-derived alkaloid, suppresses the symptoms and increases the survival rate of rabies-infected mice: the development of a pharmacological approach for rabies treatment

Between 40,000-70,000 people die yearly of rabies, an incurable disease. Besides post-bite vaccination, no treatment is available for it. Methods: First, virus dilution for antiviral effects in mice was determined. Then, animals were treated as follows: control (NaCl 250 µL/animal/day); bufotenine (0.63, 1.05 and 2.1 mg in 250 µL of NaCl/animal/day); rabies (10-6,82CVS dilution); and test (10-6,82 CVS dilution and bufotenine, in the above-mentioned doses). Animals were observed daily for 21 days or until the 3rd stage of rabies infection. Twitch-tension and liposome studies were applied to understand the possible interaction of bufotenine with receptors, particularly acetylcholine. Results: Bufotenine was able to increase the survival rate of intracerebrally virus-infected mice from 15 to 40%. Bufotenine did not seem to interfere with the acetylcholine response in the skeletal muscle, indicating that its mechanism of action is not blocking the virus entrance due to nAChR antagonism. By analyzing liposomes, we could observe that bufotenine did not passively penetrates cell membranes, indicating the necessity of complementary structures to cell penetration. Conclusions: Bufotenine is a promising candidate for drug development. After further chemical modification, it might be possible to dissociate minor side effects, increase efficiency, efficacy and pharmacokinetics, yielding a true anti-rabies drug.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the viruss glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.(AU)

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...

Estudio clínico de marcadores de hipermetilación indólica en las alteraciones de la percepción / Clinical studies of markers of the indolic hypermethylation in human perception alterations

Se estudiaron los marcadores bioquímicos en 34 pacientes psicóticos frente a controles, efectuándose: dosaje de monoaminooxidasa (MAO) plaquetaria y aminooxidasa (AO) sérica, actividad transmetilante y dosaje de N,N-dimetilindolalquilaminas urinarias: bufotenina y N,N-dimetiltriptamina (DMT). Se realizaron simultáneamente tests neuropsicológicos para evaluar los parámetros psicométricos en los mismos sujetos de estudio. Los niveles urinarios de DMT y bufotenina fueron evaluados por cromatografía de gases-espectrometría de masas y por cromatografía líquida de alta resolución. Las enzimas fueron dosadas por métodos espectrofluorimétricos. Se establecieron relaciones entre los valores estadísticamente significativos de bufotenina urinaria y MAO plaquetaria, de DMT urinaria con MAO plaquetaria y con AO sérica. Los valores estadísticamente significativos de MAO plaquetaria y los de actividad de transmetilación fueron satisfactoriamente correlacionados lográndose así categorizar el 91,1% de los 34 sujetos participantes en cuatro tipos principales. La marcada disminución de MAO plaquetaria mostró concordancia con el aumento de bufotenina y DMT, y con la alteración perceptual observada en los tests neuropsicológicos. La disminución de AO sérica fue moderada, pero acorde con la actividad transmetilante registrada. Los resultados apoyan la teoría de transmetilación patológica de la esquizofrenia y muestran que estas indolalquilaminas metiladas son marcadores de estado para estas patologías.

Biochemical markers were studied in 34 psychotic patients compared to controls, e.g., dosage of platelet monoamine oxidase (MAO) and serum amine oxidase (AO), transmethylation activity, and dosage of the urinary N,N-dimethylindolealkylamines, bufotenine and N,N-dimethyltryptamine (DMT). Neuropsychological tests were simultaneously performed to evaluate psychometric parameters in the same subjects under study. Urinary levels of DMT and bufotenine were evaluated by gas chromatography-mass spectrometry and high-performance liquid chromatography. The enzymes were dosed by spectrofluorimetric methods. Relationships were established between the statistically significant values of urinary bufotenine and platelet MAO, and of urinary DMT and both platelet MAO and serum AO. The statistically significant values of platelet MAO and those of transmethylation activity were satisfactorily correlated, thus achieving the 91.1% categorization of the 34 subjects in four main types. The sharp decrease in platelet MAO was in agreement with the increase in bufotenine and DMT, and with the perceptual alteration observed in neuropsychological tests. The decrease in serum AO was moderate, but consistent with the transmethylating activity registered. The results support the pathologic transmethylation theory of schizophrenia, and show that these N,N-methylated indolealkylamines are state markers for these pathologies.

Synthesis and biological effects of N-hydroxy-methyl-5-bromoisatine and derivatives

By bromation of isatin 5-bromoisatin (I) was obtained. On boiling (I) with 4% formaldehyde solution for short period of time, a compound was isolated which has been characterized as N-hydroxymethyl-5-bromoisatin (II). By condensation of I with various H2N-B compounds, 6 derivatives (III-VIII) were formed. The structures of the obtained products were characterized by IR spectroscopy (II was characterized by MS, 1H.NMR). The synthesized compounds were tested for biological activities such as antibacterial and antifungal. Among these, two compounds (I, II) showed an antibacterial activity on 8 strains of bacteria. Compound VI showed an antibacterial activity on 3 strains of bacteria. Compound VI showed an antibacterial activity on 3 strains of bacteria.

Marcadores biologicos en Psiquiatria / Biological markers in Psychiatry

In order to determine wheter psycopathology is associated with charactheristic neurochemical changes of psychiatric patients, Noradrenergic; Dopaminergic and Serotoninergic urine compounds were quantified in 50 patients (32 females and 18 males) between 20 and 60 years old. They were classified in four groups, according to DSM-IV criteria; in MAJOR DEPRESSION (MD) 30 cases, OBSESSIVE COMPULSIVE DISEASE (OCD) 9 cases, BIPOLAR DEPRESSION (BD)4 cases and SCHIZOPHRENIA (SZ) 7 cases. The following Amine metabolites were determined in 24 hours urine samples Phenylethilamine (PEA); 3-Metho-4-Hidroxy Phenilglycol (MHPG); 5-Hidroxy-indol acetic acid (5HIAA) Homovanilic acid (HVM); Bufotenine (BU); Ometil Bufotenine (OMBU) and 3-5 Metoxy-NN-Dymethyltryptamine (MNNDMT). The results showed a dicrease of Pea levels in 43 per cent of DM; 33 per cent of OCD; 25 per cent of BP and 42 per cent SZ. MOPEG levels were disminished in 53 per cent of DM; 66 per cent of OCD; 25 per cent of BP and 85 per cent of SZ. There was an increase of HVM levels in 10 per cent of DM; 11 per cent of OCD and 25 per cent of BP. There was a dicrease of 5-HIAA levels in 10 per cent of DM while it was increased in 33 per cent and 14 per cent of SZ, BU, OMBU and NNDMT were positive in 71 per cent of SZ; 46 per cent of DM; 55 per cent of OCD and 50 per cent of BP. Our findings are consistent with the hypothesis that PEA; MHPG; 5-HIAA and HVM compounds could be "State markers"of DM and HVM of BP and SZ patients. There is further evidence to support a close interrelationship between the three systems and the urinary excretion of methylates compounds specially in SZ and in DM, OCD and BP patients.