Chemical composition and biological effects of kratom (Mitragyna speciosa): In vitro studies with implications for efficacy and drug interactions.

The safety and efficacy of kratom (Mitragyna speciosa) for treatment of pain is highly controversial. Kratom produces more than 40 structurally related alkaloids, but most studies have focused on just two of these, mitragynine and 7-hydroxymitragynine. Here, we profiled 53 commercial kratom products using untargeted LC-MS metabolomics, revealing two distinct chemotypes that contain different levels of the alkaloid speciofoline. Both chemotypes were confirmed with DNA barcoding to be M. speciosa. To evaluate the biological relevance of variable speciofoline levels in kratom, we compared the opioid receptor binding activity of speciofoline, mitragynine, and 7-hydroxymitragynine. Mitragynine and 7-hydroxymitragynine function as partial agonists of the human µ-opioid receptor, while speciofoline does not exhibit measurable binding affinity at the µ-, δ- or ƙ-opioid receptors. Importantly, mitragynine and 7-hydroxymitragynine demonstrate functional selectivity for G-protein signaling, with no measurable recruitment of ß-arrestin. Overall, the study demonstrates the unique binding and functional profiles of the kratom alkaloids, suggesting potential utility for managing pain, but further studies are needed to follow up on these in vitro findings. All three kratom alkaloids tested inhibited select cytochrome P450 enzymes, suggesting a potential risk for adverse interactions when kratom is co-consumed with drugs metabolized by these enzymes.

Physiologically based pharmacokinetic modeling framework for quantitative prediction of an herb-drug interaction.

Herb-drug interaction predictions remain challenging. Physiologically based pharmacokinetic (PBPK) modeling was used to improve prediction accuracy of potential herb-drug interactions using the semipurified milk thistle preparation, silibinin, as an exemplar herbal product. Interactions between silibinin constituents and the probe substrates warfarin (CYP2C9) and midazolam (CYP3A) were simulated. A low silibinin dose (160 mg/day × 14 days) was predicted to increase midazolam area under the curve (AUC) by 1%, which was corroborated with external data; a higher dose (1,650 mg/day × 7 days) was predicted to increase midazolam and (S)-warfarin AUC by 5% and 4%, respectively. A proof-of-concept clinical study confirmed minimal interaction between high-dose silibinin and both midazolam and (S)-warfarin (9 and 13% increase in AUC, respectively). Unexpectedly, (R)-warfarin AUC decreased (by 15%), but this is unlikely to be clinically important. Application of this PBPK modeling framework to other herb-drug interactions could facilitate development of guidelines for quantitative prediction of clinically relevant interactions.CPT Pharmacometrics Syst. Pharmacol. (2014) 3, e107; doi:10.1038/psp.2013.69; advance online publication 26 March 2014.

Optimization of expression of human sulfite oxidase and its molybdenum domain.

The conditions for the heterologous expression of both untagged and His-tagged human sulfite oxidase in Escherichia coli have been optimized. Maximum production of active enzyme requires expression in a mob- cell strain at low levels of the inducer. Using these conditions, 3.9-5.6 mg of untagged and 15 mg of His-tagged sulfite oxidase were isolated per liter of cell culture. These represent significantly higher levels than previously reported for any molybdopterin-containing protein. High levels of enzyme activity and molybdenum incorporation were maintained despite the increase in yield, and no significant differences in kinetic properties were observed between the tagged and untagged sulfite oxidase. Additionally, the molybdenum domain of sulfite oxidase was expressed in a stable, active form as a His-tagged protein. The molybdenum domain was also expressed in the presence of tungstate to enable examination of the molybdopterin-tungsten form of sulfite oxidase.

Isolated sulfite oxidase deficiency: review of two cases in one family.

OBJECTIVE: The authors describe two cases of isolated sulfite oxidase deficiency found in one family. This is a rare autosomal-recessive disorder presenting at birth with seizures, severe neurologic disease, and ectopia lentis. It can be easily missed with metabolic screening; however, the finding of lens subluxation stresses the importance of ophthalmic assessment in making the diagnosis. DESIGN: Two observational case reports. INTERVENTION/METHODS: Ophthalmic assessment, biochemical assay for specific urinary and plasma metabolites, magnetic resonance imaging, and gene sequencing were used to make the diagnosis of the disease in the proband. The diagnosis was subsequently recognized in a previously affected sibling after the postmortem neuropathology was reviewed. Mutation analysis was performed on cultured fibroblasts from the proband to identify and categorize the specific mutation responsible for the disease in the family. From this, future prenatal detection of sulfite oxidase deficiency is possible. MAIN OUTCOME MEASURES: The diagnosis of sulfite oxidase deficiency was established in this family, enabling appropriate genetic counseling and recurrence risk estimation. RESULTS: Point mutations were found in both alleles of the sulfite oxidase gene in the proband. The first is a 623C-->A mutation, which predicts an A208D substitution, and the second is a 1109C-->A, which predicts an S370Y substitution. Both residues A208D and S370Y are critical for sulfite oxidase activity. CONCLUSIONS: Isolated sulfite oxidase deficiency is a rare heritable disease for which mutation analysis can allow accurate prenatal screening. It often is difficult to diagnose by clinical presentation alone, but the critical finding of lens subluxation accompanying seizures and diffuse neurologic disease in an infant should alert the physician to the diagnosis.

Human sulfite oxidase R160Q: identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme.

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Production and purification of N-terminal half-transferrin in Pichia pastoris.

Human serum transferrin, the major iron transport protein in humans, is a monomeric glycoprotein that is composed of two homologous domains; the N-terminal domain is formed by amino acids 1-331 and the C-terminal domain is formed by amino acids 338-679. Each domain is capable of binding one iron atom concomittantly with a carbonate anion; however, the two homologous iron binding sites are not chemically equivalent. The cDNA sequence coding for the N-terminal domain has been cloned and overexpressed in the methylotrophic yeast, Pichia pastoris. The transformants secrete a protein of approximately 38 kDa (the size expected for N-terminal half-transferrin), its N-terminal sequence agrees with the predicted sequence, and the protein reacts with anti-human serum transferrin antibodies. The purified protein appears to be properly folded and can bind iron as demonstrated by its spectral properties and urea-PAGE mobility. It is estimated that N-terminal half-transferrin represents approximately 90% of all protein secreted into the culture medium and that it is expressed at levels exceeding 50 mg/l. This study demonstrates that N-terminal half-transferrin can easily be expressed in the simple host system, Pichia pastoris, and that the purified protein is capable of reversibly binding iron.