Black Tea Theaflavin Detoxifies Metabolic Toxins in the Intestinal Tract of Mice.

SCOPE: This study is to determine the in vivo efficacy of black tea theaflavin (TF) to detoxify two metabolic toxins, ammonia and methylglyoxal (MGO), in mice METHODS AND RESULTS: Under in vitro conditions, TF is able to react with ammonia, MGO, and hydrogen peroxide to produce its aminated, MGO conjugated, and oxidized products, respectively. In TF-treated mice, the aminated TF, the MGO conjugates of TF and aminated TF, and the oxidized TF are searched using LC-MS/MS. The results provide the first in vivo evidence that the unabsorbed TF is able to trap ammonia to form the aminated TF; furthermore, both TF and the aminated TF have the capacity to trap MGO to generate the corresponding mono-MGO conjugates. Moreover, TF is oxidized to dehydrotheaflavin, which underwent further amination in the gut. By exposing TF to germ-free (GF) mice and conventionalized mice (GF mice colonized with specific-pathogen-free microbiota), the gut microbiota is demonstrated to facilitate the amination and MGO conjugation of TF. CONCLUSION: TF has the capacity to remove the endogenous metabolic toxins through oxidation, amination, and MGO conjugation in the intestinal tract, which can potentially explain why TF still generates in vivo efficacy while showing a poor systematic bioavailability.

Dual purpose secondary compounds: phytotoxin of Centaurea diffusa also facilitates nutrient uptake.

Traits that allow more efficient foraging for a deficient resource could increase the competitiveness of a species in resource-poor habitats. Considering the metal-nutrient mobilization ability of many allelochemicals, it is hypothesized that, along with the reported toxic effect on the neighbors, these compounds could be directly involved in resource acquisition by the allelopathic plant. Using nutrient manipulation treatments in hydroponic culture, this hypothesis was tested using Centaurea diffusa, an invasive species that produces the putative phytotoxin 8-hydroxyquinoline (8HQ). The exudation of 8HQ by C. diffusa was very limited and transient. It was further shown that: C. diffusa utilizes 8HQ for its own acquisition of iron, a nutrient deficient in many of its alkaline, invaded habitats; there possibly exists a unique mechanism for the uptake of the 8HQ-complexed iron (Fe) in C. diffusa, which is novel to the nongraminaceous species; although phytotoxic at very low concentrations, the toxic effect of 8HQ showed a conditional response in the presence of metals, and was significantly reduced when 8HQ was complexed with copper (Cu) and Fe. This study, in addition to elucidating one of the possible adaptive mechanisms conferring competitive advantage to C. diffusa, also outlines measures to negate the phytotoxicity of its putative allelochemical. The results indicate that the exudation of 8HQ by C. diffusa could be primarily for nutrient acquisition.

Phase I trial of r viscumin (INN: aviscumine) given subcutaneously in patients with advanced cancer: a study of the European Organisation for Research and Treatment of Cancer (EORTC protocol number 13001).

Safety of aviscumine by subcutaneous route was assessed in patients with advanced cancer refractory to chemotherapy. Patients with progressive disease received escalating doses twice weekly. Treatment of the accrued 26 patients (10 colorectal cancer (CRC), 6 soft tissue sarcoma (STS), 5 melanoma (MM), 5 others) was well tolerated without substance-related grade 3 or 4 toxicities. Grade 1/2 toxicities were predominantly injection site reactions. Aviscumine lacked dose-limiting toxicity (DLT) up to a maximal dose of 10 ng/kg. An increase of interleukin-1 beta and interferon-gamma from baseline was seen in the patient's plasma between the 1st and 11th injection. Highest release of both cytokines was in the dose range of 4-5.9 ng/kg. Interferon-gamma was not detected after doses higher than 6 ng/kg. Eight patients (5 CRC, 1 MM, 1 STS, 1 RCC) had disease stabilisation for 79-250 days (median122 days) associated with an increase of interleukin (IL)-1 beta and interferon (IFN)-gamma. Aviscumine was well tolerated and appeared to possess clinical activity at a biologically active dose between 4 and 6 ng/kg.

Using the continual reassessment method: lessons learned from an EORTC phase I dose finding study.

Many clinicians often do not feel comfortable with the Continual Reassessment Method (CRM). This article reviews its implementation, showing the characteristics, advantages and limitations of this method in Phase I studies as an alternative to the classical 'Fibonacci' escalation schema. A two center, dose escalation phase I study of rViscumin was carried out. Thirty-seven patients were included at 14 different dose-levels (10 to 6400 ng/kg). The complete clinical results are presented elsewhere. A 2-step CRM design enables one to speed-up the study and most importantly to obtain an accurate estimate of the maximum tolerated dose (MTD). Different management issues related to a multicenter study are illustrated and we show how the method can go wrong when severe toxicity, or dose limiting toxicity (DLT), is not considered by the clinician as being sufficient to limit dose escalation (here a grade 3 asthenia related to the drug). This would have affected any dose finding methods. We believe that CRM is a good alternative to the standard method from both a statistical and a practical point of view but further methodological research is necessary to address the issues related to the composite nature of the endpoint.

Phase I trial of intravenous aviscumine (rViscumin) in patients with solid tumors: a study of the European Organization for Research and Treatment of Cancer New Drug Development Group.

BACKGROUND: Aviscumine is an Escherichia coli-derived recombinant type II ribosome-inactivating protein with potent antitumor activity in vitro and in vivo. It is the recombinant counterpart of natural mistletoe lectin-I. The current study was performed to determine the safety profile, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the intravenous (i.v.) administration of aviscumine in cancer patients. Translational research included the evaluation of pharmacokinetics and monitoring of plasma cytokine and anti-aviscumine antibody induction after administration of the drug. PATIENTS AND METHODS: Aviscumine was given twice weekly as a 1 h central i.v. infusion in patients with advanced, refractory progressive, solid malignant tumors who had not been previously exposed to natural mistletoe preparations. They had histologically or cytologically verified disease, were > or =18 years old, had an Eastern Cooperative Oncology Group performance status < or =2 and adequate bone marrow, liver and renal function. DLT was defined as any non-hematological grade 3-4 toxicity (National Cancer Institute Common Toxicity Criteria version 2.0), neutrophil count <500/microl for > or =7 days, febrile neutropenia or thrombocytopenia grade 4. The MTD was defined as the dose at which >20% of patients experienced DLT during the first treatment cycle. The Continual Reassessment Method was used to determine the number of patients required per dose level. RESULTS: Forty-one fully eligible patients (19 male, 22 female) with a median age of 56 years (range 37-74) were enrolled. Colorectal, ovarian, renal cell and breast cancer were the most common tumor types. Dose levels of aviscumine ranged from 10 to 6400 ng/kg. The median number of cycles was two (range one to eight). Common clinical toxicities in cycle 1 were fatigue, fever, nausea, vomiting and allergic reactions. Fatigue grade 3 was dose limiting in one of six patients at 4000 ng/kg and reversible grade 3 liver toxicity (elevation in alkaline phosphatase, transaminases and/or gamma-glutamyltransferase) occurred in one of 10 patients at 4800 ng/kg and in two of five patients at 6400 ng/kg. The best response (RECIST criteria) was stable disease in 11 patients, lasting for two to eight cycles. The pharmacokinetic evaluation revealed a short alpha half-life of 13 min and linear kinetics on dose levels > or =1600 ng/kg. Aviscumine stimulated the immune system with a release of cytokines such as interleukin (IL)-1beta, IL-6 and interferon-gamma, and induced immunoglobulin (Ig) G- and/or IgM-anti-aviscumine antibodies of uncertain clinical relevance. CONCLUSIONS: The recommended dose for further clinical trials is 5600 ng/kg twice weekly. Based on the short half-life of the recombinant protein observed in this trial, the exploration of prolonged infusion schedules of aviscumine is warranted.

Characterization of uremic toxin transport by organic anion transporters in the kidney.

BACKGROUND: Harmful uremic toxins, such as indoxyl sulfate (IS), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoleacetate (IA), and hippurate (HA), accumulate to a high degree in uremic plasma. IS has been shown to be a substrate of rat organic anion transporter 1 (rOat1) and rOat3. However, the contribution of rOat1 and rOat3 to the renal uptake transport process of IS and other uremic toxins in the kidney remains unknown. METHODS: The cellular uptake of uremic toxins was determined using stable transfectants of rOat1/hOAT1 and rOat3/hOAT3 cells. Also, the uptake of uremic toxins by rat kidney slices was characterized to evaluate the contribution of rOat1 and rOat3 to the total uptake by kidney slices using inhibitors of rOat1 (p-aminohippurate) and rOat3 (pravastatin and benzylpenicillin). RESULTS: Saturable uptake of IS, CMPF, IA, and HA by rOat1 was observed with Km values of 18, 154, 47, and 28 micromol/L, respectively, whereas significant uptake of IS and CMPF, but not of IA or HA, was observed in rOat3-expressing cells with Km values of 174 and 11 micromol/L, respectively. Similar parameters were obtained for human OAT1 and OAT3. Kinetic analysis of the IS uptake by kidney slices revealed involvement of two saturable components with Km1 (24 micromol/L) and Km2 (196 micromol/L) values that were comparable with those of rOat1 and rOat3. The Km value of CMPF uptake by kidney slices (22 micromol/L) was comparable with that of rOat3, while the corresponding values of IA and HA (42 and 33 micromol/L, respectively) were similar to those of rOat1. PAH preferentially inhibited the uptake of IA and HA by kidney slices, while pravastatin and benzylpenicillin preferentially inhibited the uptake of CMPF. The effect of these inhibitors on the uptake of IS by kidney slices was partial. CONCLUSIONS: rOat1/hOAT1 and rOat3/hOAT3 play major roles in the renal uptake of uremic toxins on the basolateral membrane of the proximal tubules. Both OAT1 and OAT3 contribute almost equally to the renal uptake of IS. OAT3 mainly accounts for CMPF uptake by the kidney, while OAT1 mainly accounts for IA and HA uptake.

Effect of excipients on the stability and aerosol performance of nebulized aviscumine.

Pulmonary delivery is an attractive alternative route to deliver protein drugs that are currently delivered by injection. Inhalation therapy via nebulizers is a well accepted way for pulmonary application of proteins considering the formulation difficulties of MDIs or DPIs. This research presents the effect of variable excipients on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization. Aviscumine formulations containing 100 mmol/L Tris buffer, 0.1% (w/v) Polysorbate 80, 0.01% (w/v) Na(2)-EDTA and 8% (w/v) Hydroxyethyl starch have been lyophilized and reconstituted with a buffered isotonic solution pH 8. The aviscumine activity was determined by a binding assay directly after reconstitution and after nebulization with a PariBoy air-jet nebulizer, a Multisonic and a Systam ultrasonic nebulizer. The stabilization of aviscumine by the addition of variable buffer salts to the reconstitution medium, such as 50, 100, and 200 mmol/L Tris buffer, 20 and 100 mmol/L phosphate buffer, and 20 and 100 mmol/L Tricine buffer, was studied. About 50% of aviscumine activity was lost after 20 min nebulization time without any additives. Nevertheless, higher buffer concentrations confer greater stability. About 70% of the aviscumine activity could be retained by the addition 0.03% octanoyl-N-methylglucamide and 100 mmol/L Tricine to the reconstitution medium.

Evidence for stimulation of tumor proliferation in cell lines and histotypic cultures by clinically relevant low doses of the galactoside-binding mistletoe lectin, a component of proprietary extracts.

The toxic galactoside-specific lectin from mistletoe, a component of proprietary extracts with unproven efficacy in oncology, exhibits capacity to trigger enhanced secretion of proinflammatory cytokines at low doses (ng/ml or ng/kg body weight) and reductions of cell viability with increasing concentrations. To infer any tumor selectivity of this activity, cytofluorimetric and cell growth assays with a variety of established human tumor cell lines were performed. Only quantitative changes were apparent, and the toxicity against tumor cells was within the range of that of the tested fibroblast preparations from 5 donors. No indication for any tumor selectivity was observed. In kinetic studies with 8 sarcoma and 4 melanoma lines, this evidence for quantitative variability of the response in interindividual comparison was further underscored. At 50 pg lectin/ml x 10(5) cells, even a growth-stimulatory impact was noted in 5 of 12 tested cases. To mimic in vivo conditions with presence of cytokine-secreting inflammatory and stromal cells, exposure to the lectin was extended to histotypic cultures established from 30 cases of surgically removed tumor. As salient result, 5 specimens from 4 of the 8 tested tumor classes responded with a significant increase of [3H]-thymidine incorporation relative to controls during the culture period of 72 hours, when the lectin was present at a concentration in the described immunomodulatory range (1 ng/ml). A relation of this activity to the extent of the actual proliferative status of the reactive samples could not be delineated. Therefore, a non-negligible percentage of the established tumor cell lines (e.g., 3 from 8 sarcoma lines) can be markedly stimulated by the lectin at a very low dose and with dependence on the cell type. Furthermore, the feasibility to elicit a significant growth enhancement is likewise documented for human tumor explants in 16.6% of the examined cases. In view of the uncontrolled application of lectin-containing extracts in alternative/complementary medicine, the presented results on unquestionably adverse lectin-dependent effects in two culture systems call for rigorous examination of the clinical safety of this unconventional, scientifically entirely experimental treatment modality.

Heat shock treatment protects human HL-60 cells from apoptosis induced by lectin II isolated from Korean mistletoe, Visum album var. Coloratum.

Mistletoe lectin II (ML II) isolated from Korean mistletoe (Viscum album var. Coloratum), an effective therapeutic agent for cancers, is known to induce cell death via apoptosis. In the present study, we found the protective effect of heat shock treatment of human leukemia HL-60 cells against ML II-induced apoptosis. Exposure of HL-60 cells to ML II for 4 h resulted in apoptosis of the cells, which was evaluated by examining "DNA ladder" formation and DNA fragmentation assay. The DNA fragmentation was significantly reduced in the cells subjected to heat shock treatment by incubation at 42 degrees C for 1 h and subsequently allowed to recover for 2-16 h at 37 degrees C, prior to exposure to ML II. HL-60 cells transfected with heat shock protein (hsp) 70 gene exhibited resistance to ML II-induced apoptosis very similar to that seen when untransfected cells were heat-shocked. These results indicate that ML II-induced apoptosis in HL-60 cells is inhibited by heat shock treatment, at least in part, via a hsp 70-mediated mechanism.

The immunomodulatory beta-galactoside-specific lectin from mistletoe: partial sequence analysis, cell and tissue binding, and impact on intracellular biosignalling of monocytic leukemia cells.

Nanogram quantities of the beta-galactoside-specific lectin from mistletoe (ML-I) that is composed of two different types of subunits exhibit immunomodulatory potency and enhance cytokine secretion in vitro and in vivo. Partial sequence analysis of the carbohydrate-binding B chain revealed a ragged N-terminus and overall homologies to the B subunit of Ricin D and Ricin E. Two evolutionarily neutral substitutions were apparent in the otherwise identical N-terminal sequences of the two toxic chains within the lectin preparation. On the basis of the influence of chemical modification by group-specific reagents on ligand binding, the lectin was biotinylated with biotinyl-N-hydroxysuccinimide ester to allow monitoring of cell binding. Monocytic leukemia cells (THP-1) specifically bound the lectin with positive cooperativity at low lectin concentrations. Radiolabelled lectin could be found in several organs and in an experimental solid tumor in biodistribution in mice. Its presence in a notable amount in spleens is especially noteworthy with respect to the already reported immunomodulation. To determine intracellular responses that precede the lectin-dependent augmentation of cytokine secretion, phosphorylation of proteins and phospholipids as well as Ca(2+)-mobilization were assessed in THP-1 cells. Quantitative increases of [32P]-phosphate incorporation were determined for a 28 kDa protein and for phosphatidylinositol-4,5-biphosphate. Similarly, the fluorescence activity of the intracellular Ca(2+)-indicator fluo-3 is elevated by approximately 25% after lectin treatment. Apparently, cell binding of the lectin is followed by modulation of biosignalling processes.

Relationship between internalization kinetics and cytotoxicity of mistletoe lectin I to L1210 leukaemia cells.

We have taken two different approaches to the study of the entry of mistletoe lectin I (MLI) into murine L1210 leukaemia cells. As detected by cellular protein synthesis and DNA synthesis inhibition, the lectin was cytotoxic to L1210 leukaemia cells. Inhibition of [3H]leucine and [3H]thymidine incorporation into L1210 cells by MLI was found dose dependent in a concentration range from 10(-16) to 10(-12) mol/ml. The kinetics of cellular protein synthesis inhibition by MLI was concentration dependent, too. Using preembedding electron microscopy, the binding and intracellular routing of the gold-labelled lectin (MLI.Au15) were studied. MLI was internalized into L1210 leukaemia cells by two different pathways: via coated pits to coated vesicles and via long enclosed invaginations of the plasma membrane.