Experimental paracoccidioidomycosis: alternative therapy with ajoene, compound from Allium sativum, associated with sulfamethoxazole/trimethoprim.

Ajoene has been described as an antithrombotic, anti-tumour, antifungal, antiparasitic and antibacterial agent. This study deals with the efficacy of ajoene to treat mice intratracheally infected with Paracoccidioides brasiliensis. The results indicate that ajoene therapy is effective in association with antifungal drugs (sulfametoxazol/trimethoprim), showing a positive additive effect. Ajoene-treated mice developed Th1-type cytokine responses producing higher levels of IFN-gamma and IL-12 when compared to the infected but untreated members of the control group. Antifungal activity of ajoene involves a direct effect on fungi and a protective pro-inflammatory immune response. Reduction of fungal load is additive to chemotherapy and therefore the combined treatment is mostly effective against experimental paracoccidioidomycosis.

Ajoene, el principal compuesto activo derivado del ajo (Allium sativum), un nuevo agente antifúngico / Ajoene the main active compound of garlic (Allium sativum): a new antifungal agent

Desde tiempos inmemoriables el ajo ha transitado junto al hombre por los caminos de la tierra, múltiples son los beneficios que la humanidad ha obtenido de sus propiedades medicinales y mágico-religiosas, pero fue en las últimas décadas cuando el estudio de sus propiedades permitió establecer una base racional para sus potencialidades clínicas. Se conoció la existencia de la alicina, el ajoene los tiosulfinatos y una gama de compuestos organosulfurados como componentes esenciales de este bulbo y se comprobó, para muchos de ellos, una diversidad de efectos que abarcan desde propiedades antitrombóticas, antitumorales, antiparasitarias y antifúngicas. Sin embargo, el ajoene, un producto de gran estabilidad, que se origina de la ruptura y la reparación no enzimática de la alicina, y qe puede también ser obtenido sintéticamente, demostró poseer la mayor actividad biológica en todos los sistemas estudiados. En este artículo nos referiremos a las propiedades antifúngicas del ajoene y a las potencialidades que posee esta novel molécula para ser utilizada en la terapéutica de las infecciones micóticas(AU)

The curative properties of garlic in medicine have been known for a long time. But, it was only in the last three decades when garlic properties were seriously investigated confirming its potential as therapeutic agent. Allicin, ajoene, thiosulfinates and a wide range of other organosulphurate compounds, are known to be the constituents linked to the garlic properties. Regarding the biochemical properties of these compounds, ajoene [(E,Z)-4,5,9 Trithiadodeca 1,6,11 Triene 9-oxide] is stable in water, and it can be obtained by chemical synthesis. There is evidence that some of the garlic constituents exert a wide variety of effects on different biological systems. However, ajoene is the garlic compound related to more biological activities, as showed in in vitro and in vivo systems. Those studies found that ajoene has antithrombotic, anti-tumoral,antifungal, and antiparasitic effects. This study deals with a recently described antifungal property of ajoene, and its potential use in clinical trails to treat several fungal infections(AU)

Suceptibilidad in vitro al ajoene de aislados de candida albicans C. parapsolosis y C. krusei: obtenidos de pacientes con onicomicosis y su relación con el tratamiento tópico / Suceptibility in vitro to the ajoene of isolated of candida albicans C. parapsolosis and C. krusei obtained from patient with onycomycosis and their relationship with tropical treatment

Ajoene es un compuesto órganosulfurado, obtenido originalmente de extractos alcohólicos del ajo, y presenta una actividad antimicótica que ha sido ampliamente demostrada. En este trabajo estudiamos la suceptibilidad in vitro al ajoene de levaduras aisladas de pacientes con onicomicosis, siguiendo directrices de NCCLS M27-A con modificaciones (RPMI 2 por ciento G y uso de un hemocitómetro). Se determinaron la CMI y CI de ajoene, fluconazol y terbinafina. Los valores del CMI oscilaron entre (2,34 - 70,2 µM/ml), y la CI entre (0,26 - 7,08 µM/ml), y se relacionaron con la actividad in vivo mostrada por este compuesto. Para ello fueron seleccionados 8 pacientes con diagnóstico clínico y micológico de onicomicosis. Seis recibieron ajoene solución 0,4 por ciento y fluconazol 150 mg/semanales por 4 meses, con controles clínicos y micológicos a los 30, 60 y 90 días. Todos los pacientes tratados con ajoene mostraron después de cuatro meses de tratamiento, una importante mejoría de los síntomas, y siete de los ocho tratados mostraron cura micológica

Estudio de la suceptibilidad in vitro de aislados de microsporum canis al ajoene terbinafina y griseofulvina: utilizando el método de microdilución / Suceptibility in vitro of microsporum canis to ajoene terninafine and griseofulvin: evaluatiom of a microdilution test

Ajoene es un agente seguro y eficaz en el tratamiento de algunas dermatofitosis; ha mostrado su actividad sobre aislados de microsporum canis, prefilándose como una potencial droga para el tratamiento tópico de la tinea capitis; por ello consideramos relevante determinar la suceptibilidad in vitro de seis aislados de microsporum canis, obtenidos de pacientes con dicha patología, a los antifúngicos ajoene, terbinafina y griseofulvina, utilizando procedimientos establecidos por el NCCLS M38-A, con algunas modificaciones. Para el compuesto ajoene los valores de la CIM y CI estuvieron entre 30-204 µM y 1,45-2,96 µM; las CIMs y CI de terbinafina se localizaron entre 10-30 µM y 0,02-0,12 µM para la griseofulvina entre 10-30 µM y 1,08-3,91 µM, respectivamente. M. canis fue suceptible a ajoene de manera dosis-dependiente, pudiendo este compuesto constituir una alternativa eficiente para el tratamiento de la tinea capitis

Ajoene and 5-fluorouracil in the topical treatment of Cladophialophora carrionii chromoblastomycosis in humans: a comparative open study.

Ajoene and 5-fluorouracil (5-FU) are compounds that have shown in-vitro activity against Cladophialophora carrionii, an important etiologic agent of chromoblastomycosis. An open comparative trial was conducted to assess safety and effectiveness of topical ajoene and 5-FU in the treatment of localized chromoblastomycosis. Thirty-seven patients with a clinically and mycologically confirmed diagnosis were randomly distributed into two groups allocated to ajoene (0.5% gel; n = 19) or 5-FU (1% cream; n = 18). Topical treatment was applied to localized lesions (< or = 2.5-cm diameter) once a day, with occlusion, for 12-16 weeks. Complete clinical and mycological remission was achieved in 14/19 patients (74%) treated with ajoene and 14/18 patients (78%) treated with 5-FU. All 5-FU-treated patients developed a post-treatment scar at the site of the lesion, while ajoene-treated patients showed only a slight depigmentation of the skin. The differences observed in cure rate between ajoene and 5-FU are not statistically significant. Follow-up of all patients for 4 years revealed no relapses in the ajoene-treated group, while one patient in the 5-FU-treated group had a relapse 6 months after the end of therapy. This trial represents the first clinical use of ajoene in the control of a deep mycosis.

Intravascular hemolysis increases atherogenicity of diet-induced hypercholesterolemia in rabbits in spite of heme oxygenase-1 gene and protein induction.

Free radical mediated oxidation of apoB lipoproteins in the arterial intima appears to contribute to atherogenicity of the entrapped particles. A plausible pathogenic mechanism for oxidation is the one induced by heme leaking from erythrocytes that is then carried into the arterial wall by its high affinity for lipoproteins. In the intima, in the presence of H(2)O(2) secreted by macrophages, heme can be a potent oxidant. To study the role of heme as a promoter of oxidative stress damage in vivo we used a model of intravascular hemolysis (IVH) caused by phenylhydrazine in rabbits with and without diet-induced moderate hypercholesterolemia (MHC). Evaluation of the antioxidant status of plasma indicated that at the end of the treatment period this was compromised by the MHC-IVH. After 10 weeks the animals with combined MHC-IVH showed more of the aorta surface covered by lesions (27%+/-8, mean (SD) than the animals with only MHC (11%+/-7), in spite of having similar plasma levels of VLDL+LDL lipoproteins. The animals with only IVH, as well as the controls, showed minimal lesions (<1%). Heme oxygenase (HO-1) expression in aorta and other tissues was markedly increased in the group with MHC-IVH and it was correlated with the extent of IVH. The data suggest that the oxidative stress associated with IVH potentiates the atherogenicity of moderate hypercholesterolemia and that in spite of a strong induction of HO-1 this is not sufficient to counteract the atherogenicity of the combined condition.

Efficacy of ajoene in the treatment of tinea pedis: a double-blind and comparative study with terbinafine.

Ajoene, an organosulfur compound originally isolated from garlic, has been shown to be effective in short-term treatment of tinea pedis. We compare the safety and effectiveness of twice-daily topical application during 1 week of 0.6% and 1% ajoene and 1% terbinafine in the treatment of tinea pedis. Seventy soldiers from the Venezuelan Armed Forces, with clinical and mycologic diagnosis of tinea pedis, were included in this study. However, only 47 were available for final evaluation. The patients were randomly distributed into 3 treatment groups: 0.6% ajoene, 1% ajoene, and 1% terbinafine. Clinical follow-up shows a rapid decline in the signs and symptoms in all groups. Efficacy of the treatments, measured as mycologic cure, 60 days after the end of the therapy was 72% for 0.6% ajoene, 100% for 1% ajoene, and 94% for 1% terbinafine. This represents the first demonstration of the therapeutic application of an inhibitor of phospholipid biosynthesis in human dermatophytosis.

Ateroesclerosis: ¿infección dentro de la multifactorialidad? / Atherosclerosis: infection within multifactorial disease?

No disponibe (AU)

Draculin, the anticoagulant factor in vampire bat saliva, is a tight-binding, noncompetitive inhibitor of activated factor X.

The kinetic mechanism of action of Draculin on activated Factor X (FXa) is established. Draculin inhibits activated Factor X within seconds of incubation at near equimolar concentration (2-6 times on molar basis). Fitting the data to the equation for a tight-binding inhibitor gives a value for K(i)(K(d)) = 14.8+/-1.5 nM. The formation of the Draculin-FXa complex can be explained by a two-step mechanism, where for the first, reversible step, k(on) = 1.117 (+/- 0.169, S.E.M.) x 10(6) M(-1)s(-1) and k(off) = 15.388 (+/- 1.672) x 10(-3) s(-1), while for the second, irreversible step, which is concentration-independent, k(2) = 0.072 s(-1). K(d) obtained from k(off)/k(on) = 13.76 nM. Lineweaver-Burk plot shows a noncompetitive behavior. This noncompetitive mode of inhibition of Draculin is supported by the observation that Draculin, at concentrations giving complete inhibition, does not impair binding of p-aminobenzamidine to FXa. Moreover, under the same conditions, Draculin induces <14% decrease of the fluorescence intensity of the p-aminobenzamidine-FXa complex. We conclude that Draculin is a noncompetitive, tight-binding inhibitor of FXa, a characteristic so far unique amongst natural FXa inhibitors.

Membrane-bound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase.

Human plasma platelet activating factor acetylhydrolase (pPAF-AH) is a phospholipase A(2) that specifically hydrolyzes the sn-2 ester of platelet activating factor (PAF) and of phospholipids with oxidatively truncated sn-2 fatty acyl chains. pPAF-AH is bound to lipoproteins in vivo, and it binds essentially irreversibly to anionic and zwitterionic phospholipid vesicles in vitro and hydrolyzes PAF and PAF analogues. Substrate hydrolysis also occurs in the absence of vesicles, with a maximum rate reached at the critical micelle concentration. A novel pre-steady-state kinetic analysis with enzyme tightly bound to vesicles and with a substrate that undergoes slow intervesicle exchange establishes that pPAF-AH accesses its substrate from the aqueous phase and thus is not an interfacial enzyme. Such a mechanism readily explains why this enzyme displays dramatic specificity for phospholipids with short sn-2 chains or with medium-length, oxidatively truncated sn-2 chains since a common feature of these lipids is their relatively high water solubility. It also explains why the enzymatic rate drops as the length of the sn-1 chain is increased. pPAF-AH shows broad specificity toward phospholipids with different polar headgroups. Additional results are that PAF undergoes intervesicle exchange on the subminute time scale and it does not undergo transbilayer movement over tens of minutes.

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A(2): structure-function studies with vesicle, micelle, and membrane assays.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

Ajoene in the topical short-term treatment of tinea cruris and tinea corporis in humans. Randomized comparative study with terbinafine.

Ajoene (CAS 92284-99-6), an organic trisulphur originally isolated from garlic, has an antimycotic activity which has been widely demonstrated both in vitro and in vivo. The objective of this work was to compare the safety and effectiveness of ajoene (0.6%, gel) with terbinafine (CAS 91161-71-6) (1%, cream) for the treatment of tinea corporis and tinea cruris. The patients selected were 60 soldiers with clinical and mycological diagnosis of either dermatophytosis. They were distributed at random in two treatment groups, one treated with ajoene at 0.6% and the other with terbinafine at 1%. All patients were evaluated clinically and mycologically 30 and 60 days after completion of the treatment, which was considered effective when clinical signs and symptoms had disappeared and the mycological cultures were negative. Thirty days after treatment, the percent healing rate was 77 and 75 for the groups treated with ajoene and terbinafine, respectively. Sixty days after treatment, the healing rate 73% and 71% for the groups treated with ajoene and terbinafine, respectively. These results and those obtained in previous studies confirm that ajoene is a new agent for the topic treatment of superficial mycoses, and for the first time show the therapeutic usefulness of an inhibitor of phospholipids biosynthesis in eukaryotes.

Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule.

Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.

Selective in vitro protection of SIVagm-induced cytolysis by ajoene, [(E)-(Z)-4,5,9-trithiadodeca-1,6,11-triene-9 oxide].

We studied the effect of synthetic ajoene on simian immunodeficiency virus (SIVagm)-mediated cell fusion and subsequent virus-induced cytolysis. Our data indicate that this compound is a strong antifusion agent with a 50% syncytium inhibitory concentration (SIC50%) value of about 2.9 microM. We suggest that ajoene interacts with the cell-specific integrin molecules and sterically hinders the association between fusion (or other co-receptors) and the CD4-gp120 complex at the cell surface of SIV-infected cells. Although ajoene was maximally effective in suppressing syncytium formation during the early period (ie, up to 6 h) of the fusion process, when the compound was recurrently added to the co-cultures, the inhibitory effect was regained and further cell death was markedly delayed. This indicates that ajoene was also effective after the initial cell-to-cell contact stage. These data suggest that ajoene may be a promising approach for the treatment of SIV/human immunodeficiency virus (HIV) infections.

Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells.

The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A2 (cPLA2) in human platelets and HeLa cells. Tryptic digests of 32P-phosphorylated and -immunoprecipitated cPLA2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-alpha or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stress-activated protein kinase (p38(mapk)), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38(mapk) inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA2 expressed in a baculovirus/insect cell system to cPLA2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA2 in different mammalian cells stimulated with multiple agonists.

[Activity of ajoene on dermatophytes, Candida albicans and Malassezia furfur.]. / Efectos del ajoeno sobre dermatofitos, Candida albicans y Malassezia furfur.

The sensitivity in vitro of an isolate of Trichophyton rubrum and another of Trichophyton mentagrophytes to ajoene. This compound inhibited the growth of both isolates, showing an minimal inhibitory concentration (MIC) of 60 microg/ml and a minimal fungicidal concentration (MFC) of 75 microg/ml. In vivo, the ajoene cream at 0.4% used once a day and every five days in 38 patients (thirty dermatophytosis and eight Candida intertrigo cases) achieved a low percentage of cures (23.3% and 12.5%, respectively). However, an excellent clinic response was obtained in eight patients with pityriasis versicolor, with a cure in 87.5% of the cases.

Gossypol modification of Ala-1 of secreted phospholipase A2: a probe for the kinetic effects of sulfate glycoconjugates.

Gossypol is shown to covalently modify secreted phospholipase A2 (PLA2) in the aqueous phase, but not at the interface. A rapid initial noncovalent binding of gossypol is followed by a slow covalent modification of the alpha-amino group of Ala-1 by stoichiometric amounts of gossypol. The rate of modification increases in the presence of calcium, but occupancy of the substrate binding site does not alter the rate. Pancreatic PLA2 is modified at the alpha-amino group of the N terminus to form a Schiff base, which can be stabilized by reduction with borohydride. Residual activity of the gossypol-modified PLA2 from several different sources is about 10%, indicative of impaired catalytic turnover. The half-time for the inactivation is about 10 min, and it is more than 100-fold longer for PLA2 at the interface. Gossypol promotes binding of PLA2 to the interface, and the binding of PLA2 to the interface promotes only the noncovalent binding of gossypol, but not the covalent modification. Gossypol, in conjunction with spectroscopic and kinetic protocols, is used to characterize the kinetic effects of sulfated glycoconjugates, heparin and artery wall proteoglycans, with human inflammatory and pancreatic PLA2.1 The conjugates do not interfere with the binding of PLA2 to the interface or with the catalytic cycle at the interface. The conjugates do not influence the kinetics of modification of PLA2 by gossypol in the aqueous phase, and the enzyme at the interface is not modified in the presence of the conjugates. The conjugates bind to PLA2 at the interface with only a modest effect on the interfacial catalytic turnover without dislodging the bound enzyme. Complex kinetic effects induced by the conjugates are shown to be due to sequestration of PLA2 in the aqueous phase as a high-molecular mass complex, which dissociates with added NaCl.

In vitro suppression of HIV-1 replication by ajoene [(e)-(z)-4,5,9-trithiadodeca-1,6,11-triene-9 oxide].

Studies were performed to establish whether synthetic ajoene exhibited differential inhibitory activity against human immunodeficiency virus (HIV)-1 (IIIB) and to clarify the mechanism of its antiviral effects. Our results demonstrate that ajoene protected acutely infected Molt-4 cells against HIV-1 and blocked further destruction of CD4 T-cells in vitro. Ajoene showed dose-dependent inhibition, with 50% cytotoxic concentration (CTC50%) and 50% effective inhibitory concentration (EIC50%) values of 1.88 microM and about 0.35 microM, respectively, when the test compound was added before or after HIV-1 infection and incubation carried out at 37 degrees C for 4 days. Ajoene proved relatively more active than dextran sulfate in blocking HIV-1 virus-cell attachment. The mode of anti-HIV action of ajoene can be ascribed to the inhibition of early events of viral replication, particularly virus adsorption.

Efficacy of ajoene, an organosulphur derived from garlic, in the short-term therapy of tinea pedis.

The present report shows the efficacy of ajoene, a garlic-derived organic trisulphur, for short-term therapy of tinea pedis. The use of ajoene as a 0.4% (w/w) cream resulted in complete clinical and mycological cure in 27 of 34 patients (79%) after 7 days of treatment. The remaining seven patients (21%) achieved complete cure after seven additional days of treatment. All patients were evaluated for recurrence of mycotic infections 90 days after the end of treatment, yielding negative cultures for fungus. These results show that ajoene is an alternative, efficient and low-cost antimycotic drug for short-term therapy of tinea pedis. The fact that ajoene can be easily prepared from an alcoholic extract of garlic may make it suitable for Third World public health care.

Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus).

From the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1-4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa. When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.