Berberine as a chemical and pharmacokinetic marker of the butanol-extracted Food Allergy Herbal Formula-2.

RATIONALE: Food Allergy Herbal Formula-2 (FAHF-2) provided protection against peanut anaphylaxis in a murine model and induced beneficial immune-modulation in humans. Butanol-refined FAHF-2, B-FAHF-2, retained safety and efficacy in the peanut allergic murine model at only 1/5 of FAHF-2 dosage. One compound, berberine, was isolated and identified in vitro as a bioactive component present in FAHF-2 and B-FAHF-2. The aim of this study was to investigate berberine as a chemical and pharmacokinetic marker of B-FAHF-2. METHODS: The consistency of constituents between B-FAHF-2 and FAHF-2 was tested. Peanut allergic C3H/HeJ mice were orally administered with 1mg of berberine or B-FAHF-2 containing an equivalent amount of berberine, and the ability to protect against peanut anaphylaxis and pharmacokinetic parameters were determined. Human intestinal epithelial cells (Caco-2) were cultured with berberine with or without the nine individual herbal constituents in B-FAHF-2, and the absorbed berberine levels were determined. RESULTS: Berberine is one of the major components in B-FAHF-2 and FAHF-2 formula. In a peanut allergic mouse model, B-FAHF-2, but not berberine, protected mice from anaphylaxis reactions. Pharmacokinetic profiles showed that the Cmax of B-FAHF-2 fed mice was 289.30±185.40ng/mL; whereas berberine alone showed very low bioavailability with Cmax value of 35.13±47.90ng/mL. Caco-2 cells influx assay showed that 7 of 9 herbal constituents in B-FAHF-2 increased berberine absorption at rates ranging from 18 to 205%. CONCLUSIONS: B-FAHF-2 remarkably increased the bioavailability of berberine. Berberine can be used as chemical and pharmacokinetic marker of B-FAHF-2. Other herbal components in B-FAHF-2 may facilitate the absorption of berberine.

C-X-C motif chemokine 12 influences the development of extramedullary hematopoiesis in the spleens of myelofibrosis patients.

Myelofibrosis (MF) is characterized by the constitutive mobilization of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) and the establishment of extramedullary hematopoiesis. The mechanisms underlying this abnormal HSC/HPC trafficking pattern remain poorly understood. We demonstrated that both splenic and peripheral blood (PB) MF CD34(+) cells equally share a defective ability to home to the marrow, but not to the spleens, of NOD/LtSz-Prkdc(scid) mice. This trafficking pattern could not be attributed to discordant expression of integrins or chemokine receptors other than the downregulation of C-X-C chemokine receptor type 4 by both PB and splenic MF CD34(+) cells. The number of both splenic MF CD34(+) cells and HPCs that migrated toward splenic MF plasma was, however, significantly greater than the number that migrated toward PB MF plasma. The concentration of the intact HSC/HPC chemoattractant C-X-C motif chemokine 12 (CXCL12) was greater in splenic MF plasma than PB MF plasma, as quantified using mass spectrometry. Functionally inactive truncated products of CXCL12, which are the product of proteolytic degradation by serine proteases, were detected at similar levels in both splenic and PB MF plasma. Treatment with an anti-CXCL12 neutralizing antibody resulted in a reduction in the degree of migration of splenic MF CD34(+) cells toward both PB and splenic MF plasma, validating the role of CXCL12 as a functional chemoattractant. Our data indicate that the MF splenic microenvironment is characterized by increased levels of intact, functional CXCL12, which contributes to the localization of MF CD34(+) cells to the spleen and the establishment of extramedullary hematopoiesis.

Phase 1 study of intravenous rigosertib (ON 01910.Na), a novel benzyl styryl sulfone structure producing G2/M arrest and apoptosis, in adult patients with advanced cancer.

Rigosertib (ON 01910.Na), a synthetic novel benzyl styryl sulfone, was administered to 28 patients with advanced cancer in a Phase I trial in order to characterize its pharmacokinetic profile, determine the dose-limiting toxicities (DLT), define the recommended phase II dose (RPTD) and to document any antitumor activity. Patients with advanced malignant neoplasms refractory to standard therapy were given escalating doses of rigosertib (50, 100, 150, 250, 325, 400, 650, 850, 1,050, 1,375, 1,700 mg/m(2)/24h) as a 3-day continuous infusion (CI) every 2 weeks. An accelerated Fibonacci titration schedule with specified decreases for toxicities was used for escalation until grade ≥2 toxicity occurred. Intrapatient dose escalation was allowed if toxicity was grade ≤2 and the disease remained stable. Plasma pharmacokinetics (PK) and urinary PK assessments were studied in the 1st and 4th cycles. Twenty-nine patients (12 men and 17 women; age 36-87 y with a median of 63 y) were registered, but one died before study drug was given. Twenty-eight patients received a median of 3 cycles of therapy. Most common grade ≥2 toxicities attributable to rigosertib included fatigue, anorexia, vomiting and constipation. DLTs included muscular weakness, hyponatremia, neutropenia, delirium and confusional state. Risk factors for severe toxicities include pre-existing neurological dysfunction or advanced gynecologic cancer after pelvic surgery. Rigosertib pharmacokinetics showed rapid plasma distribution phases and urinary excretion. Elevations in plasma Cmax and AUC due to decreases in plasma clearance were associated with acute grade ≥3 toxicities. Of 22 evaluable patients, 9 (41%) achieved a best overall response of stable disease; all other patients (n=13; 59%) progressed. The median progression-free survival time was 50 days (95% confidence interval [CI]: 37-80 days). Nine (41%) patients survived for over 1 y. In summary, prolonged IV infusions of rigosertib were generally well tolerated. Nine (41%) patients achieved stable disease and 9 (41%) patients survived for over 1 year. The RPTD appears to be 850 mg/m(2)/24hr CI x 3 days. (ClinicalTrials.gov identifier: NCT01538537).

Determination of the glucuronide metabolite of ON 013100, a benzylstyrylsulfone antineoplastic drug, in colon cancer cells using LC/MS/MS.

ON 013100, (E)-2,4,6-trimethoxystyryl-3-hydroxy-4-methoxybenzyl sulfone, is a potent kinase inhibitor whose phosphate form is in Phase I clinical trials in lymphoma and acute lymphoid leukemia. The objectives were to: (a) investigate the possible presence of the glucuronide metabolite of the drug in two representative colon cancer cell lines, a drug resistant (colo-205) and a drug sensitive (colo-320); (b) quantify the glucuronide metabolite and the unchanged drug in the cells after treatment with ON 013100. The glucuronide was synthesized and a selective LC/MS/MS method was developed and validated for the characterization and quantification of the metabolite. The glucuronide metabolite (570.6 Da) was found in the drug-resistant cells upon a 1h incubation with ON 013100 (20 µg/ml). After treatment with the drug, the concentration of the metabolite gradually decreased from 0.84 µg/ml at 0 h through 0.21 µg/ml at 6h to below detection limit of 8.0 ng/ml at 9 h. No glucuronide metabolite was detected in the drug-sensitive cells. The concentrations of intact ON 013100 in the drug-resistant cells gradually decreased from 0.41 µg/ml (0 h) to 0.06 µg/ml (9 h). The corresponding concentrations of the intact drug in the drug-sensitive cells were from 2.88 µg/ml to 0.94 µg/ml.

Discontinuous drug binding to proteins: binding of an antineoplastic benzyl styryl sulfone to albumin and enzymes in vitro and in phase I clinical trials.

Sodium (E)-{N-[2-methyloxy-5-(2',4',6'-trimethoxy-styrylsulfonyl) methylenephenyl]amino}acetate (C(21)H(24)NNaO(8)S, ON 01910.Na) is a novel, synthetic benzyl styryl sulfone, currently in phase I clinical trials in cancer patients. Our objective was to use electrospray mass spectrometry to determine, in intact complexes, the number of drug molecules bound to albumin and selected enzymes. Native and recombinant albumin incubated with the drug, at various molar ratios, revealed simultaneous and discontinuous progression of drug binding, yielding intact albumin-drug complexes containing up to 22 drug molecules. Comparable complex protein-drug patterns were obtained for several enzymes, e.g., carbonic anhydrase. Intact albumin-ON 01910 complexes were also found in all patient samples. The drug-binding profiles were comparable, but not identical, for increasing sampling times and different doses (400-1700 mg/m(2)). We concluded that the techniques developed are capable of detecting the simultaneous formation of intact protein-drug complexes and of determining the number of drug molecules bound to proteins. The results enhance our hypothesis that drug binding may lead to conformational changes in proteins that, in turn, account for the exclusion of specific binding complexes and may influence protein behavior and activity. Application of these techniques reveals new insights about the nature of the antineoplastic drug ON 01910 in patient plasma, and the information obtained may have significance in understanding drug delivery to tumors.

The effect of CXCL12 processing on CD34+ cell migration in myeloproliferative neoplasms.

Primary myelofibrosis (PMF) and polycythemia vera (PV) are chronic myeloproliferative neoplasms. PMF and, to a lesser degree, PV are characterized by constitutive mobilization of hematopoietic stem cells (HSC) and progenitor cells (HPC) into the peripheral blood (PB). The interaction between the chemokine CXCL12 and its receptor CXCR4 plays a pivotal role in determining the trafficking of CD34(+) cells between the bone marrow (BM) and the PB. PMF, but not PV, is associated with downregulation of CXCR4 by CD34(+) cells due to epigenetic events. Both PV and PMF patients have elevated levels of immunoreactive forms of CXCL12 in the BM and PB. Using electrospray mass spectrometry, the PB and BM plasma of PV and PMF patients was shown to contain reduced amounts of intact CXCL12 but significant amounts of several truncated forms of CXCL12, which are lacking in normal PB and BM plasma. These truncated forms of CXCL12 are the product of the action of several serine proteases, including dipeptidyl peptidase-IV, neutrophil elastase, matrix metalloproteinase-2 (MMP-2), MMP-9, and cathepsin G. Unlike CXCL12, these truncates either lack the ability to act as a chemoattractant for CD34(+) cells and/or act as an antagonist to the action of CXCL12. These data suggest that proteolytic degradation of CXCL12 is characteristic of both PV and PMF and that the resulting truncated forms of CXCL12, in addition to the reduced expression of CXCR4 by CD34(+) cells, lead to a profound mobilization of HSC/HPC in PMF.

The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor.

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Resveratrol in raw and baked blueberries and bilberries.

Resveratrol in the fruits of bilberry (Vaccinium myrtillus L.), the lowbush "wild" blueberry (Vaccinium angustifolium Aiton), the rabbiteye blueberry (Vaccinium ashei Reade), and the highbush blueberry (Vaccinium corymbosum L.) were measured using a new assay based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS assay provided lower limits of detection than previous methods for resveratrol measurement, 90 fmol of trans-resveratrol injected on-column, and a linear standard curve spanning >3 orders of magnitude. The recoveries of resveratrol from blueberries spiked with 1.8, 3.6, or 36 ng/g were 91.5 +/- 4.5, 95.6 +/- 6.5, and 88.0 +/- 3.6%, respectively. trans-Resveratrol but not cis-resveratrol was detected in both blueberry and bilberry samples. The highest levels of trans-resvertatrol in these specimens were 140.0 +/- 29.9 pmol/g in highbush blueberries from Michigan and 71.0 +/- 15.0 pmol/g in bilberries from Poland. However, considerable regional variation was observed; highbush blueberries from British Columbia contained no detectable resveratrol. Because blueberries and bilberries are often consumed after cooking, the effect of baking on resveratrol content was investigated. After 18 min of heating at 190 degrees C, between 17 and 46% of the resveratrol had degraded in the various Vaccinium species. Therefore, the resveratrol content of baked or heat-processed blueberries or bilberries should be expected to be lower than in the raw fruit. Although blueberries and bilberries were found to contain resveratrol, the level of this chemoprotective compound in these fruits was <10% that reported for grapes. Furthermore, cooking or heat processing of these berries will contribute to the degradation of resveratrol.

Cytotoxic dammarane glycosides from processed ginseng.

Steaming ginseng at high temperature increased its cytotoxicity to SK-Hep-1 hepatoma cancer cells. HPLC separation and fractionation followed by MTT assay revealed that ginsenosides Rg3, Rg5, Rk1, Rs5, and Rs4 are the active principles. Their 50% growth inhibition concentration (GI50) values were 41, 11, 13, 37, and 13 microM, respectively. Cisplatin had a GI50 of 84 microM in the same assay conditions.