Adverse effects by artificial grapefruit seed extract products in patients on warfarin therapy.

OBJECTIVE: Grapefruit seed extract (GSE) is promoted as a natural product with antibacterial and antiviral properties. The aim of this study was to investigate the composition of some commercially available GSE products and evaluate their effect in vitro on two cytochrome P450 enzymes, CYP2C9 and CYP3A4. METHODS: A couple on lifelong treatment with warfarin and continuous regular follow-ups took some drops of a GSE product for 3 days. The female patient experienced a minor subcutaneous haematoma 3 days later, and her international normalised ratio (INR) value was 7.9. This was reported to the Swedish Medical Products Agency (MPA) as a spontaneous post-marketing report concerning adverse drug reactions/interactions. The composition of the GSE products was determined by proton and carbon-13 nuclear magnetic resonance spectroscopy (NMR). The inhibitory effect of the GSE products on the cytochrome P450 enzymes was tested in an in vitro baculosome assay. RESULTS: The NMR analysis showed that all three investigated GSE products contained the synthetic preservative benzethonium chloride (BTC) in addition to glycerol and water. No authentic GSE extract was found in any of the three GSE products analysed. Furthermore, BTC was found to be a potent inhibitor of CYP3A4 and CYP2C9 activity in vitro. CONCLUSION: Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.

Benzethonium chloride: a novel anticancer agent identified by using a cell-based small-molecule screen.

PURPOSE: This study aims to identify a novel therapeutic agent for head and neck cancer and to evaluate its antitumor efficacy. EXPERIMENTAL DESIGN: A cell-based and phenotype-driven high-throughput screening of approximately 2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal cancer) and GM05757 (primary normal human fibroblast) lines. The "hit" compound was assayed for efficacy in combination with standard therapeutics on a panel of human cancer cell lines. Furthermore, its mode of action (using transmission electron microscopy and flow cytometry) and its in vivo efficacy (using xenograft models) were evaluated. RESULTS: Benzethonium chloride was identified as a novel cancer-specific compound. For benzethonium (48-hour incubation), the dose required to reduce cell viability by 50% was 3.8 micromol/L in FaDu, 42.2 micromol/L in NIH 3T3, 5.3 micromol/L in C666-1, and 17.0 micromol/L in GM05757. In vitro, this compound did not interfere with the effects of cisplatin, 5-fluorouracil, or gamma-irradiation. Benzethonium chloride induced apoptosis and activated caspases after 12 hours. Loss of mitochondrial membrane potential (DeltaPsiM) preceded cytosolic Ca2+ increase and cell death. In vivo, benzethonium chloride ablated the tumor-forming ability of FaDu cells, delayed the growth of xenograft tumors, and combined additively with local tumor radiation therapy. Evaluation of benzethonium chloride on the National Cancer Institute/NIH Developmental Therapeutics Program 60 human cancer cell lines revealed broad-range antitumor activity. CONCLUSIONS: This high-throughput screening identified a novel antimicrobial compound with significant broad-spectrum anticancer activity.

Identification of benzethonium chloride in commercial grapefruit seed extracts.

Commercial grapefruit seed extracts (GSE) were extracted with chloroform. The solvent was evaporated, and the resulting solid was subsequently analyzed by high-performance liquid chromatography, electrospray ionization mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and elemental analysis (by proton-induced X-ray emission [PIXE] analysis). The main constituent was identified as benzethonium chloride, a synthetic antimicrobial agent commonly used in cosmetics and other topical applications. This compound comprised 8.03% (n = 2) of the liquid GSE sample. Higher amounts of benzethonium chloride were found in powder GSE samples.