Hydrastine pharmacokinetics and metabolism after a single oral dose of goldenseal (Hydrastis canadensis) to humans.

The disposition and metabolism of hydrastine was investigated in 11 healthy subjects following an oral dose of 2.7 g of goldenseal supplement containing 78 mg of hydrastine. Serial blood samples were collected for 48 hours, and urine was collected for 24 hours. Hydrastine serum and urine concentrations were determined by Liquid Chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters for hydrastine were calculated using noncompartmental methods. The maximal serum concentration (Cmax) was 225 ± 100 ng/ml, Tmax was 1.5 ± 0.3 hours, and area under the curve was 6.4 ± 4.1 ng ⋅ h/ml ⋅ kg. The elimination half-life was 4.8 ± 1.4 hours. Metabolites of hydrastine were identified in serum and urine by using liquid chromatography coupled to high-resolution mass spectrometry. Hydrastine metabolites were identified by various mass spectrometric techniques, such as accurate mass measurement, neutral loss scanning, and product ion scanning using Quadrupole-Time of Flight (Q-ToF) and triple quadrupole instruments. The identity of phase II metabolites was further confirmed by hydrolysis of glucuronide and sulfate conjugates using bovine ß-glucuronidase and a Helix pomatia sulfatase/glucuronidase enzyme preparation. Hydrastine was found to undergo rapid and extensive phase I and phase II metabolism. Reduction, O-demethylation, N-demethylation, hydroxylation, aromatization, lactone hydrolysis, and dehydrogenation of the alcohol group formed by lactone hydrolysis to the ketone group were observed during phase I biotransformation of hydrastine. Phase II metabolites were primarily glucuronide and sulfate conjugates. Hydrastine undergoes extensive biotransformation, and some metabolites may have pharmacological activity. Further study is needed in this area.

Characterization of transgenic Gfrp knock-in mice: implications for tetrahydrobiopterin in modulation of normal tissue radiation responses.

AIMS: The free radical scavenger and nitric oxide synthase cofactor, 5,6,7,8-tetrahydrobiopterin (BH4), plays a well-documented role in many disorders associated with oxidative stress, including normal tissue radiation responses. Radiation exposure is associated with decreased BH4 levels, while BH4 supplementation attenuates aspects of radiation toxicity. The endogenous synthesis of BH4 is catalyzed by the enzyme guanosine triphosphate cyclohydrolase I (GTPCH1), which is regulated by the inhibitory GTP cyclohydrolase I feedback regulatory protein (GFRP). We here report and characterize a novel, Cre-Lox-driven, transgenic mouse model that overexpresses Gfrp. RESULTS: Compared to control littermates, transgenic mice exhibited high transgene copy numbers, increased Gfrp mRNA and GFRP expression, enhanced GFRP-GTPCH1 interaction, reduced BH4 levels, and low glutathione (GSH) levels and differential mitochondrial bioenergetic profiles. After exposure to total body irradiation, transgenic mice showed decreased BH4/7,8-dihydrobiopterin ratios, increased vascular oxidative stress, and reduced white blood cell counts compared with controls. INNOVATION AND CONCLUSION: This novel Gfrp knock-in transgenic mouse model allows elucidation of the role of GFRP in the regulation of BH4 biosynthesis. This model is a valuable tool to study the involvement of BH4 in whole body and tissue-specific radiation responses and other conditions associated with oxidative stress.

Validation of a liquid chromatography-tandem mass spectrometric assay for the quantitative determination of hydrastine and berberine in human serum.

A high throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of berberine and hydrastine in human serum, after oral administration of goldenseal (Hydrastis canadensis L.), was developed using simple acetonitrile treatment of serum samples. Noscapine served as the internal standard. Lower limit of quantification for both analytes was 0.1 ng mL(-1) using positive ion electrospray tandem mass spectrometry (MS/MS). The intra-day (n=5) accuracy and precision of the method for hydrastine was 82+/-8.8%, 97.9+/-2.4% and 96.2+/-3.3%, respectively. The inter-day (n=4) accuracy and precision for hydrastine was 90.0+/-15.17%, 99.9+/-7.1% and 98+/-6.54%, respectively. For berberine quantitation intra-day accuracy and precision was 96.0+/-8.4%, 92.5+/-4.7% and 94.4+/-3.7%, respectively. The respective values for inter-day quantitation were 91.0+/-8.4%, 94.3+/-4.7% and 94.4+/-3.7%. The analytical recovery for hydrastine was 82.4-96.2% and for berberine it was 94.4-96.0%. The analytes and noscapine were stable for 24h at room temperature (CV 5-10%). Matrix ion effects were studied by post-column infusion of hydrastine and berberine, calculation of calibration curve slope precision was obtained using serum from five different subjects, and by comparison of the response of methanol standards and extracted serum samples. The method was further validated by determination of serum pharmacokinetics of hydrastine and berberine after administration of a single oral dose of goldenseal extract containing 77 mg of hydrastine and 132 mg of berberine.

Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine.

Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, kappa light chain, K(d) = 11 nM) and found to have similar ligand affinity (K(d) = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer ( approximately 75%) and dimer ( approximately 25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n = 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [(3)H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [(3)H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t(1/2lambdaz) of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t(1/2lambdaz) (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.