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.

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.

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.

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.

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.

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.

[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.

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.

A garlic derivative, ajoene, inhibits platelet deposition on severely damaged vessel wall in an in vivo porcine experimental model.

Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic. In vitro, ajoene reversibly inhibits platelet aggregation as well as the release reaction induced by all known agonists. We used a well characterized perfusion chamber to study the in vivo effects of ajoene on platelet deposition onto a highly thrombogenic, severely damaged arterial wall, obtained by stripping off the intimal layer and exposing tunica media. Platelet-vessel wall interaction and the effect of ajoene was studied under flow conditions of high and low local shear rate that mimics laminar blood flow in small and medium size arteries (1690 sec-1 and 212 sec-1). Our results indicate that administration of ajoene to heparinized animals, significantly prevents thrombus formation at local low blood shear rate. Ajoene does not inhibit binding of vWF to GPIb, therefore, it does not affect platelet adhesion. In fact, although ajoene impairs fibrinogen and vWF (less efficient) binding to GPlIb/IIIa, it does not totally inhibits platelet deposition to the substrates at any of the shear rates used in this study. Our present results, under in vivo flow conditions and in the presence of physiological calcium levels, suggest that ajoene may be potentially useful for the acute prevention of thrombus formation induced by severe vascular damage, mainly in arterial sites with local low shear rates.

Inhibition of phosphatidylcholine biosynthesis and cell proliferation in Trypanosoma cruzi by ajoene, an antiplatelet compound isolated from garlic.

Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 microM ajoene, while 100 microM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 microM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 microM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.

Effect of ajoene on dimorphism of Paracoccidioides brasiliensis.

Ajoene, a compound derived from ethanolic garlic extracts, inhibits the growth of the dimorphic pathogenic fungus Paracoccidioides brasiliensis, yeast cells being more sensitive to its action than mycelial cultures. Sulphydryl protector groups were able to cancel the inhibitory effect on the yeast cells but not on the mycelial cultures. The dimorphic process was also blocked when mycelia were placed to transform to yeast. Synthesis in vitro of cell wall glucan was not affected by ajoene. We discuss the possible involvement of ajoene on sulphydryl metabolism of P. brasiliensis.

Effect of ajoene, the major antiplatelet compound from garlic, on platelet thrombus formation.

Ajoene, (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide, is a potent antiplatelet compound isolated from alcoholic extracts of garlic (Allium sativum). Ajoene reversibly inhibits in vitro platelet aggregation as well as release reaction induced by all known agonists. We used a well characterized cylindrical perfusion chamber to study the effect of ajoene on platelet deposition onto physiological substrates such as pig aortic subendothelium and tunica media as a model of mildly and severely damaged vessel wall respectively. Experiments were performed under flow conditions of high and low shear rate that mimic laminar blood flow in small and medium size arteries (1690 sec-1 and 212 sec-1). Our results indicate that ajoene prevents thrombus formation both at low and high shear rate in citrated whole blood. The inhibitory effect of ajoene on platelet-thrombus formation seems to be dependent on its inhibition of fibrinogen binding, since significantly higher concentrations of ajoene are needed to affect von Willebrand factor binding to GPIIb/IIIa receptors. Further, ajoene does not impair Ristocetin-induced platelet agglutination, mediated by GPIb. Our results suggest that ajoene may be useful for the acute prevention of thrombus formation induced by vascular damage.

Inhibition of growth of the dimorphic fungus Paracoccidioides brasiliensis by ajoene.

Ajoene, a garlic-derived compound that prevents platelet activation, inhibited the growth of Paracoccidioides brasiliensis, a fungal pathogen for humans, by affecting the integrity of the fungal cytoplasmic membrane. This action may be the basis for the study of ajoene as a possible specific antifungal drug.

Reversible prevention of platelet activation by (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide (ajoene) in dogs under extracorporeal circulation.

Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), an organosulfur compound derived from garlic inhibits platelet activation induced ex vivo by all known agonists. The effect of ajoene on the thrombocytopenia associated with the circulation of blood through extracorporeal devices such as dialyzers or oxygenators was studied under the following conditions: Ex vivo using fresh, heparinized human blood, circulating through a dialyzer or oxygenator, and in vivo, in dogs subjected to extracorporeal circulation. In both experimental conditions ajoene proved very efficacious in preventing platelet loss (60-65% loss in controls vs. 15-20% loss in the presence of ajoene, p less than 0.01). Moreover, recuperation of platelet function was achieved after 3-4 h in the in vivo experiments.

In vivo platelet hyperreactivity, another risk factor for patients under continuous ambulatory peritoneal dialysis.

A high mortality rate due to thromboembolic accidents has been described in patients undergoing chronic haemodialysis. This type of complications, although recognized, has not been appropriately evaluated in continuous ambulatory peritoneal dialysis patients. The present study demonstrates that continuous ambulatory peritoneal dialysis patients present in vivo platelet hyperreactivity, as evidenced by enhanced platelet responses to epinephrine ex vivo and an increased MDA/MDAa index which traduces a decreased threshold for activation of the arachidonate pathway and subsequent thromboxane production. Since the etiopathogeny of this platelet abnormality seems to be related to abnormalities in lipid metabolism, compounds such as fish oil must be beneficial in the management of this risk factor.