Multipoint dilution hemofiltration: A new technology for maximum convective clearance.

Convection-based renal replacement therapies (RRTs) have the potential to improve patient outcomes when compared to diffusion-based RRT such as hemodialysis (HD), but have limited clearance rates. We propose and characterize multipoint dilution hemofiltration (MPD-HF), a purely convective blood purification technology which removes the fundamental filtration limit associated with convective RRT resulting in clearance rates on par with HD. In MPD-HF, filtration of liquid and solutes occurs along the length of the hollow fibers that convey the blood, and substitution fluid is pushed into the fibers at multiple points along their length. Since multiple filtration and dilution steps are contained within one pass of the blood through the hollow fiber, the fraction of fluid that can be filtered may be increased to allow a high clearance rate that removes a wide range of toxins. In vitro tests yielded an average steady-state filtrate fraction of 68%, exceeding commercial HDF cartridge filtrate fractions by a factor of approximately 3. The molecular weights of molecules cleared spans up to the cutoff of 66 kDa for albumin.

Pharmacokinetics of Toxin-Derived Peptide Drugs.

Toxins and venoms produced by different organisms contain peptides that have evolved to have highly selective and potent pharmacological effects on specific targets for protection and predation. Several toxin-derived peptides have become drugs and are used for the management of diabetes, hypertension, chronic pain, and other medical conditions. Despite the similarity in their composition (amino acids as the building blocks), toxin-derived peptide drugs have very profound differences in their structure and conformation, in their physicochemical properties (that affect solubility, stability, etc.), and subsequently in their pharmacokinetics (the processes of absorption, distribution, metabolism, and elimination following their administration to patients). This review summarizes and critically analyzes the pharmacokinetic properties of toxin-derived peptide drugs: (1) the relationship between the chemical structure, physicochemical properties, and the pharmacokinetics of the specific drugs, (2) the major pharmacokinetic properties and parameters of these drugs, and (3) the major pharmacokinetic variability factors of the individual drugs. The structural properties of toxin-derived peptides affect their pharmacokinetics and pose some limitations on their clinical use. These properties should be taken into account during the development of new toxin-derived peptide drugs, and for the efficient and safe use of the clinically approved drugs from this group in the individual patients.

Detoxifying symbiosis: microbe-mediated detoxification of phytotoxins and pesticides in insects.

Covering: up to 2018 Insects live in a world full of toxic compounds such as plant toxins and manmade pesticides. To overcome the effects of these toxins, herbivorous insects have evolved diverse, elaborate mechanisms of resistance, such as toxin avoidance, target-site alteration, and detoxification. These resistance mechanisms are thought to be encoded by the insects' own genomes, and in many cases, this holds true. However, recent omics analyses, in conjunction with classic culture-dependent analyses, have revealed that a number of insects possess specific gut microorganisms, some of which significantly contribute to resistance against phytotoxins and pesticides by degrading such chemical compounds. Here, we review recent advances in our understanding on the symbiont-mediated degradation of natural and artificial toxins, with a special emphasis on their underlying genetic basis, focus on the importance of environmental microbiota as a resource of toxin-degrading microorganisms, and discuss the ecological and evolutionary significance of these symbiotic associations.

Elimination of a Viscumin-Ferromagnetic Nanoparticles Conjugate from the Tumor Nodule in Mice.

External magnetic field is characterized by low toxicity and existence of magnetic properties, which contributes to an interest in the development of products from ferromagnetic nanoparticles (FNP) for antitumor therapy. Previously we synthesized a conjugate of ferromagnetic magnetite nanoparticles and viscumin (mistletoe lectin I, MLI), which exhibits the antitumor activity. Studying the pharmacological properties of this conjugate (FNP-MLI) was directed to the evaluation of FNP-MLI elimination after intratumor injection in mice. The elimination rate of FNP-MLI was much lower than that of native plant MLI. The presence of FNP-MLI was not accompanied by undesired changes in the tumor tissue. The use of a FNP-MLI conjugate allowed us to prolong the time of MLI presence in tissues without increasing the dose of exogenous lectin. These features contribute to the prolongation of an immunomodulatory effect of MLI.

Biodistribution of Viscumin after Subcutaneous Injection to Mice and In Vitro Modeling of Endoplasmic Reticulum Stress.

Viscumin (mistletoe lectin I, MLI) in concentrations of 10-11-10-7 M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

Characterization of a Chinese hamster ovary cell mutant having a mutation in elongation factor-2.

Retroviral insertional mutagenesis provides an effective forward genetic method for identifying genes involved in essential cellular pathways. A Chinese hamster ovary cell line mutant resistant to several bacterial ADP-ribosylating was obtained by this approach. The toxins used catalyze ADP-ribosylation of eukaryotic elongation factor 2 (eEF-2), block protein synthesis, and cause cell death. Strikingly, in the CHO PR328 mutant cells, the eEF-2 substrate of these ADP-ribosylating toxins was found to be modified, but the cells remained viable. A systematic study of these cells revealed the presence of a structural mutation in one allele of the eEF-2 gene. This mutation, Gly717Arg, is close to His715, the residue that is modified to become diphthamide. This Arg substitution prevents diphthamide biosynthesis at His715, rendering the mutated eEF-2 non-responsive to ADP-ribosylating toxins, while having no apparent effect on protein synthesis. Thus, CHO PR328 cells are heterozygous, having wild type and mutant eEF-2 alleles, with the latter allowing the cells to survive even in the presence of ADP-ribosylating toxins. Here, we report the comprehensive characterization of these cells.

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.

Delivery methods for peptide and protein toxins in insect control.

Since the introduction of DDT in the 1940s, arthropod pest control has relied heavily upon chemical insecticides. However, the development of insect resistance, an increased awareness of the real and perceived environmental and health impacts of these chemicals, and the need for systems with a smaller environmental footprint has stimulated the search for new insecticidal compounds, novel molecular targets, and alternative control methods. In recent decades a variety of biocontrol methods employing peptidic or proteinaceous insect-specific toxins derived from microbes, plants and animals have been examined in the laboratory and field with varying results. Among the many interdependent factors involved with the production of a cost-effective pesticide--production expense, kill efficiency, environmental persistence, pest-specificity, pest resistance-development, public perception and ease of delivery--sprayable biopesticides have not yet found equal competitive footing with chemical counterparts. However, while protein/peptide-based biopesticides continue to have limitations, advances in the technology, particularly of genetically modified organisms as biopesticidal delivery systems, has continually progressed. This review highlights the varieties of delivery methods currently practiced, examining the strengths and weaknesses of each method.

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.

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.

Effect of neomycin on the hydrolysis and toxicity of vicine and convicine in rats.

This study in the rat established the effects that a broad-spectrum and poorly absorbed antibiotic, neomycin sulfate, had on the in vitro and in vivo hydrolysis of vicine and convicine by the intestinal microflora, and on vicine- and convicine-induced depletion of blood glutathione and the resulting toxicity. The in vitro studies demonstrated that digesta from the caecum and large intestine were highly effective in hydrolysing vicine and convicine, whereas digesta from the same sections of the gastro-intestinal tract of neomycin-treated rats were much less effective (P < 0.0001). The in vivo studies showed that the total amount of vicine and convicine excreted in the urine and faeces was much greater in neomycin-treated rats compared with controls (P < 0.05), indicating the ability of neomycin to increase the amount of glycosides, particularly that of vicine, excreted in the faeces. The ability of glycosides to decrease the concentration of glutathione in blood (P < 0.05) and to increase rat mortality was greatly reduced in rats that were treated with neomycin, particularly in those treated ip with the toxin. Thus, the results demonstrated that neomycin reduced the rate at which vicine and convicine were hydrolysed in the lower section of the gastro-intestinal tract, and that neomycin treatment was associated with a reduced toxicity of the glycosides.

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.