2-Aminomethylene-5-sulfonylthiazole Inhibitors of Lysyl Oxidase (LOX) and LOXL2 Show Significant Efficacy in Delaying Tumor Growth.

The lysyl oxidase (LOX) family of extracellular proteins plays a vital role in catalyzing the formation of cross-links in fibrillar elastin and collagens leading to extracellular matrix (ECM) stabilization. These enzymes have also been implicated in tumor progression and metastatic disease and have thus become an attractive therapeutic target for many types of invasive cancers. Following our recently published work on the discovery of aminomethylenethiophenes (AMTs) as potent, orally bioavailable LOX/LOXL2 inhibitors, we report herein the discovery of a series of dual LOX/LOXL2 inhibitors, as well as a subseries of LOXL2-selective inhibitors, bearing an aminomethylenethiazole (AMTz) scaffold. Incorporation of a thiazole core leads to improved potency toward LOXL2 inhibition via an irreversible binding mode of inhibition. SAR studies have enabled the discovery of a predictive 3DQSAR model. Lead AMTz inhibitors exhibit improved pharmacokinetic properties and excellent antitumor efficacy, with significantly reduced tumor growth in a spontaneous breast cancer genetically engineered mouse model.

The oxidation of cysteine-containing peptides caused by perfluoroalkane sulfonyl fluorides.

Perfluorooctane sulfonyl fluoride (PFOSF) is the precursor of many fluorochemicals that are ubiquitous in the environment. However, its distribution and toxicology are rarely studied. In this work, the oxidability of PFOSF was found. PFOSF can accelerate oxidation of glutathione (GSH) to its oxidized form GSSG, and itself is reduced to a sulfinic acid. The yielded sulfinic acid was prepared and identified with high resolution mass spectrometry and NMR. Similar redox reactions were observed for PFOSF's short chain alternatives. The reduction potentials of perfluoroalkane sulfonyl fluorides (PFASFs) were determined to be -2.13 V vs. SCE with cyclic voltammetry, further demonstrating their oxidability. The peptide mixtures of GSH plus another cysteine-containing peptide were also oxidized by PFASFs to GSSG and an asymmetric disulfide GS-S-PEP. A single short-sequence PEP-SH could be oxidized to the symmetric disulfide PEP-S-S-PEP as the final product. In vitro experiments were carried out by adding PFASFs into rat liver S9 fractions. The turnover ratio of PFASFs were calculated to be about 4-10% by quantification of sulfinic acid with LC-MS/MS. Our work illustrates one of the potential metabolic pathways of PFASFs and demonstrates the oxidation of PEP-SHs by PFASFs, thus providing a preliminary exploration in the toxicology of these fluorochemicals.

Allicin Bioavailability and Bioequivalence from Garlic Supplements and Garlic Foods.

Allicin is considered responsible for most of the pharmacological activity of crushed raw garlic cloves. However, when garlic supplements and garlic foods are consumed, allicin bioavailability or bioequivalence (ABB) has been unknown and in question because allicin formation from alliin and garlic alliinase usually occurs after consumption, under enzyme-inhibiting gastrointestinal conditions. The ABB from 13 garlic supplements and 9 garlic foods was determined by bioassay for 13 subjects by comparing the area under the 32-h concentration curve of breath allyl methyl sulfide (AMS), the main breath metabolite of allicin, to the area found after consuming a control (100% ABB) of known allicin content: homogenized raw garlic. For enteric tablets, ABB varied from 36⁻104%, but it was reduced to 22⁻57% when consumed with a high-protein meal, due to slower gastric emptying. Independent of meal type, non-enteric tablets gave high ABB (80⁻111%), while garlic powder capsules gave 26⁻109%. Kwai garlic powder tablets, which have been used in a large number of clinical trials, gave 80% ABB, validating it as representing raw garlic in those trials. ABB did not vary with alliinase activity, indicating that only a minimum level of activity is required. Enteric tablets (high-protein meal) disintegrated slower in women than men. The ABB of supplements was compared to that predicted in vitro by the dissolution test in the United States Pharmacopeia (USP); only partial agreement was found. Cooked or acidified garlic foods, which have no alliinase activity, gave higher ABB than expected: boiled (16%), roasted (30%), pickled (19%), and acid-minced (66%). Black garlic gave 5%. The mechanism for the higher than expected ABB for alliinase-inhibited garlic was explored; the results for an alliin-free/allicin-free extract indicate a partial role for the enhanced metabolism of γ-glutamyl S-allylcysteine and S-allylcysteine to AMS. In conclusion, these largely unexpected results (lower ABB for enteric tablets and higher ABB for all other products) provide guidelines for the qualities of garlic products to be used in future clinical trials and new standards for manufacturers of garlic powder supplements. They also give the consumer an awareness of how garlic foods might compare to the garlic powder supplements used to establish any allicin-related health benefit of garlic.

Allicin functionalized locust bean gum nanoparticles for improved therapeutic efficacy: An in silico, in vitro and in vivo approach.

The field of nanotechnology has overgrown over the past few years and has even ventured into the field of medicine. The aim of the present study is to develop a novel allicin functionalized locust bean gum nanoparticle using the nanoprecipitation technique. The synthesized nanoparticles were characterized by dynamic light scattering, scanning electron microscopy and transmission electron microscopy. The characterization study revealed the nanoscale structure (∼100nm) of the prepared particles. In silico toxicology analysis were carried out to assess the drug-like properties and virtual toxicity of allicin. Toxicity of the prepared nanoparticles were carried out in RAW 264.7 cell lines in vitro and in vivo studies were carried out in Sprague-Dawley rats. In in vitro study, LBGAN showed a maximum toxicity of 10.51% in MTT assay, no reactive oxygen species generation on DCFDA staining and LBGAN was effective to protect the cells from apoptosis. In in vivo toxicity studies LBGAN showed no significant change in the activities of the marker enzymes like LDH, CK-MB, ALP, ACP, AST and ALT. Thus, the functionalization of nanoparticles with allicin has the benefit of providing protection and stability to the allicin, in addition to increasing its pharmacological activity.

The defense substance allicin from garlic permeabilizes membranes of Beta vulgaris, Rhoeo discolor, Chara corallina and artificial lipid bilayers.

BACKGROUND: Allicin (diallylthiosulfinate) is the major volatile- and antimicrobial substance produced by garlic cells upon wounding. We tested the hypothesis that allicin affects membrane function and investigated 1) betanine pigment leakage from beetroot (Beta vulgaris) tissue, 2) the semipermeability of the vacuolar membrane of Rhoeo discolor cells, 3) the electrophysiology of plasmalemma and tonoplast of Chara corallina and 4) electrical conductivity of artificial lipid bilayers. METHODS: Garlic juice and chemically synthesized allicin were used and betanine loss into the medium was monitored spectrophotometrically. Rhoeo cells were studied microscopically and Chara- and artificial membranes were patch clamped. RESULTS: Beet cell membranes were approximately 200-fold more sensitive to allicin on a mol-for-mol basis than to dimethyl sulfoxide (DMSO) and approximately 400-fold more sensitive to allicin than to ethanol. Allicin-treated Rhoeo discolor cells lost the ability to plasmolyse in an osmoticum, confirming that their membranes had lost semipermeability after allicin treatment. Furthermore, allicin and garlic juice diluted in artificial pond water caused an immediate strong depolarization, and a decrease in membrane resistance at the plasmalemma of Chara, and caused pore formation in the tonoplast and artificial lipid bilayers. CONCLUSIONS: Allicin increases the permeability of membranes. GENERAL SIGNIFICANCE: Since garlic is a common foodstuff the physiological effects of its constituents are important. Allicin's ability to permeabilize cell membranes may contribute to its antimicrobial activity independently of its activity as a thiol reagent.

Efficacy of low doses of amphotericin B plus allicin against experimental visceral leishmaniasis.

OBJECTIVES: To evaluate the efficacy of the combination of allicin and amphotericin deoxycholate (AmB) in the chemotherapy of Leishmania infantum infection with the final aim of reducing the dose of AmB in the chemotherapy of visceral leishmaniasis. METHODS: Hamsters were intraperitoneally (ip) infected with L. infantum (10(7) stationary phase promastigotes). On day 45 post-infection animals were treated ip with AmB (1 or 5 mg/kg/day), allicin (5 mg/kg/day) or a combination of AmB (1 mg/kg/day) + allicin (5 mg/kg/day) for 5 days. Animals were clinically and biopathologically monitored and the antibody response (IgG, IgG1, IgG2) was determined. Parasite burdens were estimated by limiting dilution and AmB biodistribution was determined by HPLC in plasma, kidney, spleen and liver. RESULTS: No clinical signs or liver and kidney alterations were observed. AmB (1 mg/kg/day) did not clear the Leishmania infection and no parasites were detected in two animals treated with 5 mg/kg/day allicin. Combination therapy (5 mg/kg allicin + 1 mg/kg AmB) reduced the L. infantum burden by >95%. Antileishmanial activity of the combination was comparable (P < 0.05) to the standard AmB treatment (5 mg/kg). CONCLUSIONS: Allicin alone (5 mg/kg/day for 5 days) significantly reduced the Leishmania burden in spleen and liver of infected hamsters. Co-administration of allicin (5 mg/kg/day for 5 days) and AmB (1 mg/kg/day for 5 days) showed a partial additive effect on the reduction of leishmanial burden in both target organs.

A review of the cardiovascular benefits and antioxidant properties of allicin.

Cardiovascular disease (CVD) is a category of chronic noncommunicable diseases causing high global mortality and has been a heavy social burden in many countries. In the search of chemicals that arise from natural food source, allicin is one such ingredient from garlic that was discovered with the potential to provide beneficial effects to the cardiovascular system. From the pharmacokinetic studies, allicin is known to be hydrophobic and can be readily absorbed through the cell membrane without inducing any damage to the phospholipid bilayer and then rapidly metabolized to exert pharmacological effects that are important to the cardiovascular system. It was found to provide cardio-protective effects by inducing vasorelaxation and alleviating various pathological conditions of CVD, including cardiac hypertrophy, angiogenesis, platelet aggregation, hyperlipidemia and hyperglycemia. Allicin was also discovered to further protect the cardiovascular system by enhancing the antioxidant status by lowering the level of reactive oxygen species and stimulating the production of glutathione. Other pharmacological benefits such as anticancer and antimicrobial activities were also discussed. It is concluded that allicin can be potentially developed into a health product for the cardiovascular system.

Efficacy of allicin in decreasing lead (Pb) accumulation in selected tissues of lead-exposed common carp (Cyprinus carpio).

The objective of this study was to evaluate the effects of allicin, the main biologically active component of garlic clove extracts, on lead levels in different common carp tissues including liver, kidney, brain, bone, and blood following experimental lead poisoning. Fish were divided randomly into five groups depending on the combination of lead acetate and allicin treatments. Lead acetate exposure (7.0 mgL(-1), 10 days) caused a significant increase in mean Pb concentrations in all examined tissues in comparison to control unexposed fish (p < 0. 001). The results showed that allicin supplementation is effective in decreasing lead accumulation in all examined tissues of common carp. The promising ameliorative effects of allicin on tissue lead levels of common carp make it a good candidate for therapeutic intervention of lead poisoning. However, more studies are required to elucidate the pharmacokinetic effects of allicin and also molecular basis of the ameliorative properties of allicin in lead poisoning.

Composition, stability, and bioavailability of garlic products used in a clinical trial.

In support of a new clinical trial designed to compare the effects of crushed fresh garlic and two types of garlic supplement tablets (enteric-coated dried fresh garlic and dried aged garlic extract) on serum lipids, the three garlic products have been characterized for (a) composition (14 sulfur and 2 non-sulfur compounds), (b) stability of suspected active compounds, and (c) availability of allyl thiosulfinates (mainly allicin) under both simulated gastrointestinal (tablet dissolution) conditions and in vivo. The allyl thiosulfinates of blended fresh garlic were stable for at least 2 years when stored at -80 degrees C. The dissolution release of thiosulfinates from the enteric-coated garlic tablets was found to be >95%. The bioavailability of allyl thiosulfinates from these tablets, measured as breath allyl methyl sulfide, was found to be complete and equivalent to that of crushed fresh garlic. S-Allylcysteine was stable for 12 months at ambient temperature. The stability of the suspected active compounds under the conditions of the study and the bioavailability of allyl thiosulfinates from the dried garlic supplement have validated the use of these preparations for comparison in a clinical trial.

Rapid uptake, metabolism, and elimination of inhaled sulfuryl fluoride fumigant by rats.

Sulfuryl fluoride (SO(2)F(2)) is a structural fumigant gas used to control drywood termites and wood-boring beetles. The pharmacokinetics and metabolism of inhaled SO(2)F(2) were evaluated in male Fischer-344 rats exposed to 30 or 300 ppm (35)S-labeled SO(2)F(2) for 4 h. Blood, urine and feces were collected during and after the exposures and analyzed for radioactivity, (35)S-labeled fluorosulfate and sulfate, and fluoride (urine and feces only). Selected tissues were collected 7 days post-exposure and analyzed for radioactivity. During and after unlabeled SO(2)F(2) exposures, blood, brain, and kidney were collected and analyzed for fluoride ion. SO(2)F(2) was rapidly absorbed, achieving maximum concentrations of radioactivity in both plasma and red blood cells (RBC) near the end of the 4-h exposure period. Radioactivity was rapidly excreted, mostly via the urine. Seven days post-exposure, small amounts of radioactivity were distributed among several tissues, with the highest concentration detected in respiratory tissues. Radioactivity associated with the RBC remained elevated 7 days post-exposure, and highly perfused tissues had higher levels of radioactivity than other non-respiratory tissues. Radioactivity cleared from plasma and RBC with initial half-lives of 2.5 h after 30 ppm and 1-2.5 h after 300 ppm exposures. The terminal half-life of radioactivity was 2.5-fold longer in RBC than plasma. Based on the radiochemical profiles, there was no evidence of parent (35)SO(2)F(2) in blood. Identification of fluorosulfate and sulfate in blood and urine suggests that SO(2)F(2) is hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride.

Allicin and allicin-derived garlic compounds increase breath acetone through allyl methyl sulfide: use in measuring allicin bioavailability.

Progress in establishing systemic pharmacological effects for fresh, crushed garlic (Allium sativum L) in humans has been hindered by (1) the inability to measure allicin bioavailability, (2) lack of direct evidence that allicin has significant systemic activity at doses of garlic normally consumed, and (3) lack of a model for an acute effect. We have addressed these problems by quantifying the increases in breath acetone and breath allyl methyl sulfide (AMS). The area under the 48 h curve was measured in humans after consumption of standardized garlic preparations, allicin, and allicin-derived compounds, at the equivalent of 7 g of crushed garlic. It was shown that the allyl thiosulfinates (mainly allicin) are solely responsible for breath AMS and increased breath acetone. Diallyl trisulfide, diallyl disulfide, ajoene, and S-allylmercaptocysteine, at isomolar dithioallyl, showed the same quantitative effects as allicin. Consumption of AMS at isomolar allyl also gave the same effects as allicin, indicating that AMS is the main metabolite of allicin and is an active metabolite. In conclusion, allicin and allicin-derived compounds are rapidly metabolized to AMS, a compound which stimulates the production of acetone and which can be used to measure the bioavailability of allicin and, hence, the ability of garlic supplements to represent fresh garlic.

In vivo metabolism of diallyl disulphide in the rat: identification of two new metabolites.

1. Diallyl disulphide (DADS), a compound formed from the organosulphur compounds present in garlic, is known for its anticarcinogenic effects in animal models. 2. The aim was to identify and analyse the metabolites produced in vivo after a single oral administration of 200 mg kg(-1) DADS to rats. The organic sulphur metabolites present in the stomach, liver, plasma and urine were measured by gas chromatography coupled with mass spectrometry over 15 days. 3. Data indicate that DADS is absorbed and transformed into allyl mercaptan, allyl methyl sulphide, allyl methyl sulphoxide (AMSO) and allyl methyl sulphone (AMSO(2)), which are detected throughout the excretion period. Overall, the highest amounts of metabolites were measured 48-72h after the DADS administration. AMSO(2) is the most abundant and persistent of these compounds. The levels of all the sulphur compounds rapidly decline within the first week after administration and disappear during the second week. Only AMSO and AMSO(2) are significantly excreted in urine. 4. These potential metabolites are thought to be active in the target tissues. Our data warrant further studies to check this hypothesis.

Allicin release under simulated gastrointestinal conditions from garlic powder tablets employed in clinical trials on serum cholesterol.

The failure of five recent clinical trials to show significant reduction in elevated serum cholesterol by a single brand of allicin-standardized garlic powder tablets is in contrast to many prior positive studies with the same brand. The hypocholesterolemic activity of garlic is mainly due to allicin, a compound that is produced by the acid-sensitive garlic enzyme, alliinase, only after tablet consumption. Therefore, the allicin-releasing ability of ten lots of these tablets--manufactured over the same years that the positive and negative clinical trials were conducted (1989-1997)--was determined under simulated gastrointestinal dissolution conditions, as defined by U.S. Pharmacopeia Method 724A. It was found that the older lots were more resistant to acid-disintegration (2.5 h vs. 1.3 h, P < 0.001) and that they released three times as much allicin (44% vs. 15 % of their potential, P < 0.001) as the newer lots. A second brand of tablets employed in a recent negative trial released no detectable amount of allicin, while a third set of tablets with high allicin release was used in a trial that gave positive effects. Hence, the persons involved in the recent negative clinical trials probably received considerably less allicin than did those in the older positive studies, possibly accounting for much of the discrepancy in the outcomes. In conclusion, clinical trials using garlic powder tablets to assess any effect of garlic that might be related to allicin, as most are, cannot be considered valid for garlic when the trial shows no effect, unless the expected allicin release from the tablets has at least been determined under standardized drug release conditions (USP 724A).

Intake of garlic and its bioactive components.

The health benefits of garlic likely arise from a wide variety of components, possibly working synergistically. The complex chemistry of garlic makes it plausible that variations in processing can yield quite different preparations. Highly unstable thiosulfinates, such as allicin, disappear during processing and are quickly transformed into a variety of organosulfur components. The efficacy and safety of these preparations in preparing dietary supplements based on garlic are also contingent on the processing methods employed. Although there are many garlic supplements commercially available, they fall into one of four categories, i.e., dehydrated garlic powder, garlic oil, garlic oil macerate and aged garlic extract (AGE). Garlic and garlic supplements are consumed in many cultures for their hypolipidemic, antiplatelet and procirculatory effects. In addition to these proclaimed beneficial effects, some garlic preparations also appear to possess hepatoprotective, immune-enhancing, anticancer and chemopreventive activities. Some preparations appear to be antioxidative, whereas others may stimulate oxidation. These additional biological effects attributed to AGE may be due to compounds, such as S-allylcysteine, S-allylmercaptocysteine, N(alpha)-fructosyl arginine and others, formed during the extraction process. Although not all of the active ingredients are known, ample research suggests that several bioavailable components likely contribute to the observed beneficial effects of garlic.

The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity.

Allicin (diallyl thiosulfinate) is the main biologically active component of the freshly crushed garlic extracts. In the present work the ability of allicin to cross through membranes (artificial and biological) was studied. Partition coefficients of allicin in water/octanol, water/hexadecane and water/phospholipids mixtures were determined. Using phospholipid vesicles loaded with hydrophilic thiols (reduced glutathione or 2-nitro-5-thiobenzoate), we observed that allicin freely permeates through phospholipid bilayers and interacts with the SH groups. The reaction rate of allicin with SH containing molecules after crossing the membrane was the same as in solution. Fast diffusion and permeation of allicin across human red blood cell membranes was also demonstrated. Allicin does not induce leakage, fusion or aggregation of membrane. The high permeability of allicin through membranes may greatly enhance the intracellular interaction with thiols.

[The pharmacokinetics of the S35 labeled labeled garlic constituents alliin, allicin and vinyldithiine]. / Untersuchungen zur Pharmakokinetik der mit 35S markierten Knoblauchinhaltsstoffe Alliin, Allicin und Vinyldithiine.

Three groups of 3 rats received oral doses (8 mg/kg) of garlic constituents (alliin, allicin and vinyldithiines (2-vinyl-[4H]-1,3-dithiine and 3-vinyl-[4H]-1,2-dithiine)) in the form of an oil macerate of the 35S-labeled substance. The measured activity was referred to 35S-alliin (35S-alliin equivalents). The blood activity levels in each group were monitored for 72 h. For 35S-allicin and the labeled vinyldithiines the excretion with the urine, feces, and exhaled air was also measured. The distribution among the organs (whole-body autoradiography) and the urinary metabolite pattern (thin-layer chromatography) were also determined. For 35S-alliin the blood activity profile differed considerably from those of 35S-allicin and the labeled vinyldithiines: both the absorption and the elimination of the radioactivity were distinctly faster than for the other garlic constituents, maximum blood levels being reached within the first 10 min and elimination from the blood being almost complete after 6 h. For the other garlic constituents the maximum blood levels were not reached until 30-60 min (35S-allicin) or 120 min (vinyldithiines) p.a. and blood levels > 1000 ng-Eq/ml were still present at the end of the study after 72 h. The mean total urinary and fecal excretion after 72 h was 85.5% (35S-allicin) or 92.3% (labeled vinyldithiines) of the dose. The urinary excretion indicates a minimum absorption rate of 65% (35S-allicin) or 73% (vinyldithiines). It is uncertain whether the 19-21% recovered in the feces was unabsorbed substance or had been excreted via the bile or intestinal mucosa. The exhaled air showed only traces of activity although the whole-body autoradiographs, after fairly long exposure (96 h), showed distinct enrichment of activity in the mucosa of the airways and pharynx. The activity is deposite mainly in the cartilage of the vertebral column and ribs. There was no detectable difference in organ distribution between 35S-allicin and the labeled vinyldithiines. All that could be established from the urinary metabolite pattern was that unchanged 35S-allicin and unchanged labeled vinyldithiines are absent. There is therefore extensive metabolization. The metabolites must have a very polar structure with acid functional groups since satisfactory separation was achievable only with acid solvent systems. Conjugates with sulfuric or glucuronic acid were not detectable. These results reveal no differences in pharmacokinetic behavior between 35S-allicin and the labeled vinyldithiines. A final verdict as to whether the metabolites, which may be pharmacologically active, are identical must await further studies designed to identify the metabolites.

Pharmacokinetics of vinyldithiins, transformation products of allicin.

The pharmacokinetic behaviour of vinyldithiins, the main constituents of oily preparations of garlic (Allium sativum L.), was investigated after oral administration of 27 mg 2-vinyl-4H-1,3-dithiin and 9 mg 3-vinyl-4H-1,2-dithiin to rats. In serum, kidney, and fat tissue, both vinyldithiins could be detected by GC-MS over a period of 24 h, whereas in liver only 1,3-vinyldithiin was found. Pharmacokinetic parameters (t1/2, ke, Cltot, AUC, and Vd) were determined using compartment models, elucidating the different pharmacokinetic behaviour of both vinyldithiins. 1,3-Vinyldithiin seems to be less lipophilic and is rapidly eliminated from serum, kidney, and fat tissue, whereas 1,2-vinyldithiin is more lipophilic and shows a tendency to accumulate in fat tissue. Experiments with liver homogenate confirmed the in vivo findings on the different degradation rates of both vinyldithiins. Allicin, the precursor of the vinyldithiins, is metabolized more rapidly in liver homogenate than the vinyldithiins.

In vitro mechanism of inhibition of bacterial cell growth by allicin.

Diallyl thiosulfinate (allicin) is the agent found in garlic which is responsible for the antibacterial and antifungal activity of extracts of this plant. The effect of bacteriostatic concentrations of allicin (0.2 to 0.5 mM) on the growth of Salmonella typhimurium revealed a pattern of inhibition characterized by: (i) a lag of approximately 15 min between addition of allicin and onset of inhibition, (ii) a transitory inhibition phase whose duration was proportional to allicin concentration and inversely proportional to culture density, (iii) a resumed growth phase which showed a lower rate of growth than in uninhibited controls, and (iv) an entry into stationary phase at a lower culture density. Whereas DNA and protein syntheses showed a delayed and partial inhibition by allicin, inhibition of RNA synthesis was immediate and total, suggesting that this is the primary target of allicin action.