Development of a simple and reliable method for α-amanitin detection in rat plasma and its application to a toxicokinetic study.

RATIONALE: α-Amanitin is a highly toxic peptide widely found in species of poisonous mushrooms. The matrix effect has been a major obstacle for accurate determination of α-amanitin in plasma samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, the strategy to eliminate the matrix effect of α-amanitin with a one-step dilution approach after deproteinization was applied. METHODS: Rat plasma samples were processed by protein precipitation with methanol followed by a nine-fold dilution with pure water. The matrix effect value of α-amanitin was 19.7%-22.2% by protein precipitation and then changed to 87.5%-88.7% after dilution. α-Amanitin and the internal standard (roxithromycin) were analyzed on an ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm, 1.7 µm) column within 3.0 min by gradient elution. RESULTS: The linear ranges were 0.90-600 ng/mL with a correlation coefficient r >0.9958. A lower limit of quantification (LLOQ) of 0.90 ng/mL was achieved using only 50 µL of rat plasma. The intra- and inter-day precisions for the analyte ranged from 3.2% to 7.5% and 3.1% to 7.1%, respectively, and the accuracy ranged from -5.3% to -8.0%. CONCLUSIONS: The matrix effect of α-amanitin was reduced by sample dilution after plasma deproteinization. A reliable LC/MS/MS method for the determination of α-amanitin in rat plasma was developed. This method was successfully applied for a toxicokinetic study of rats after intravenous injection of α-amanitin with a subacute toxicity dose at 0.10 mg/kg.

Pharmacokinetics of α-amanitin in mice using liquid chromatography-high resolution mass spectrometry and in vitro drug-drug interaction potentials.

The aim of this study was to determine pharmacokinetics of α-amanitin, a toxic bicyclic octapeptide isolated from the poisonous mushrooms, following intravenous (iv) or oral (po) administration in mice using a newly developed and validated liquid chromatography-high resolution mass spectrometry. The iv injected α-amanitin disappeared rapidly from the plasma with high a clearance rate (26.9-30.4 ml/min/kg) at 0.1, 0.2, or 0.4 mg/kg doses, which was consistent with a rapid and a major excretion of α-amanitin via the renal route (32.6%). After the po administration of α-amanitin at doses of 2, 5, or 10 mg/kg to mice, the absolute bioavailability of α-amanitin was 3.5-4.8%. Due to this low bioavailability, 72.5% of the po administered α-amanitin was recovered from the feces. When α-amanitin is administered po, the tissue to plasma area under the curve ratio was higher in stomach > large intestine > small intestine > lung ~ kidneys > liver but not detected in brain, heart, and spleen. The high distribution of α-amanitin to intestine, kidneys, and liver is in agreement with the previously reported major intoxicated organs following acute α-amanitin exposure. In addition, α-amanitin weakly or negligibly inhibited cytochrome P450 and 5'-diphospho-glucuronosyltransferase enzymes activity in human liver microsomes as well as major drug transport functions in mammalian cells overexpressing transporters. Data suggested remote drug interaction potential may be associated with α-amanitin exposure.

Enhancing the Pharmacokinetics and Antitumor Activity of an α-Amanitin-Based Small-Molecule Drug Conjugate via Conjugation with an Fc Domain.

Herein we describe the design and biological evaluation of a novel antitumor therapeutic platform that combines the most favorable properties of small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Although the small size of SMDCs, compared to ADCs, is an appealing feature for their application in the treatment of solid tumors, SMDCs usually suffer from poor pharmacokinetics, which severely limits their therapeutic efficacy. To overcome this limitation, in this proof-of-concept study we grafted an α-amanitin-based SMDC that targets prostate cancer cells onto an immunoglobulin Fc domain via a two-step "program and arm" chemoenzymatic strategy. We demonstrated the superior pharmacokinetic properties and therapeutic efficacy of the resulting Fc-SMDC over the SMDC in a prostate cancer xenograft mouse model. This approach may provide a general strategy toward effective antitumor therapeutics combining small size with pharmacokinetic properties close to those of an ADC.

A cost-effective LC-MS/MS method for identification and quantification of α-amanitin in rat plasma: Application to toxicokinetic study.

α-Amanitin is the main lethal component of amanita mushrooms, and data on its toxicokinetics are few. The aim of this study was to develop a sensitive and cost-effective method to identify α-amanitin and investigate its toxicokinetic parameters using liquid chromatography-triple quadrupole tandem mass spectrometry. The colchicine was used as the internal standard (IS). The compounds were extracted from plasma samples by protein precipitation with acetonitrile (containing 1% formic acid). The analysis was performed through multiple reactions monitoring. The molecular ions and fragment ions of α-amanitin could be used as characteristic ions to perform qualitative analysis of α-amanitin. The assay was successfully validated by selectivity, linearity, matrix effect, precision and accuracy, recovery and stability according to the U.S. Food and Drug Administration Guidance, and applied to study the toxicokinetic profile of α-amanitin in rats after a single intraperitoneal administration.

Dermal absorption and toxicity of alpha amanitin in mice.

The fungus Amanita phalloides is known to contain two main groups of toxins: amanitins and phallotoxins. The amanitins group effectively blocks the RNA polymerase II enzyme found in eukaryotic cells. As alpha amanitin has a lethal effect on the majority of eukaryotic cells, it can be valuable as an antiparasitic or antifungal drug. It can be used externally against ectoparasites. It is critical that percutaneous applications of the alpha amanitin toxin are not harmful to the recipient. In this study, the absorption and the toxicity of percutaneous and intraperitoneal (ip) applications of 1 mg/kg alpha amanitin to mice were compared. Potential skin, liver and kidney toxicities were investigated through pathological examination. HPLC analysis was used to determine the amount of the toxin. No toxicity or toxin were found in the skin, liver, or kidneys of the mice in the control group. Interestingly, the percutaneous application group also showed no toxicity, and the toxin was not present in this group. After 24 h, Councilman-like bodies and pyknotic cells were observed in the mice in which alpha amanitin was applied intraperitoneally, demonstrating the presence of toxicity. Peak levels of alpha amanitin (µg/mL) in the liver, kidney, and blood in the ip application group were measured at 3.3 (6 h), 0.2 (6 h) and 1.2 (1 h), respectively. The results demonstrated that the toxin was not absorbed through the skin of the mice and that the percutaneous application of alpha amanitin did not have any toxic effects. Thus, alpha amanitin may be administered percutaneously for therapeutic purposes.

The enterohepatic circulation of amanitin: kinetics and therapeutical implications.

BACKGROUND: Amatoxin poisoning induces a delayed onset of acute liver failure which might be explained by the prolonged persistence of the toxin in the enterohepatic circulation. Aim of the study was to demonstrate amanitin kinetics in the enterohepatic circulation. METHODS: Four pigs underwent α-amanitin intoxication receiving 0.35 mg/kg (n=2) or 0.15 mg/kg (n=2) intraportally. All pigs remained under general anesthesia throughout the observation period of 72 h. Laboratory values and amanitin concentration in systemic and portal plasma, bile and urine samples were measured. RESULTS: Amanitin concentrations measured 5h after intoxication of 219±5ng/mL (0.35 mg/kg) and 64±3 (0.15 mg/kg) in systemic plasma and 201±8ng/mL, 80±13ng/mL in portal plasma declined to baseline levels within 24h. Bile concentrations simultaneously recorded showed 153±28ng/mL and 99±58ng/mL and decreased slightly delayed to baseline within 32 h. No difference between portal and systemic amanitin concentration was detected after 24h. CONCLUSIONS: Amanitin disappeared almost completely from systemic and enterohepatic circulation within 24 h. Systemic detoxification and/or interrupting the enterohepatic circulation at a later date might be poorly effective.

[Toxicity of alpha-amanitin on mice in vivo].

OBJECTIVE: To study the toxic effect of alpha-amanitin from Amanita fungi on mice in vivo. METHODS: The LD50 of alpha-amanitin was determined with intravenous and intraperitoneal injections of alpha-amanitin. The mice were then injected intravenously with LD50 dose of alpha-amanitin, and the viscera index was evaluated. The peripheral blood samples were collected 24 hours later to measure the hemogram and biochemical indicators. The histological changes of internal organs were examined. The alpha-amanitin in organ tissues were measured. RESULTS: The intraperitoneal and intravenous LD50 doses of alpha-amanitin were 0.742 mg/kg and 0.327 mg/kg bodyweight, respectively. The WBC, RBC and Hb decreased significantly, whereas the serum BUN and Crea increased significantly. The serum ALT, AST, TBIL and DBIL increased to 24.0, 9.6, 26.3 and 37.0 times of the levels of controls, respectively, 24 hour after the injection. The viscera indexes of liver and kidney also increased significantly, and focal necrosis was found in the tissue slices 48 hours after the injection. The poison compound alpha-amanitin was detected in liver and kidney tissue homogenates by RP-HPLC 48 hours after the injection. CONCLUSION: Serum BUN, Crea, ALT, AST, TBIL and DBIL are sensitive indicators for the toxicity of alpha-amanitin in vivo. The pathological changes of liver and kidney are very serious. The cyclopeptide alpha-amanitin could reside in the two organs for a long last toxic effect.