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Mark Sutherland

Publications and source records attributed to Mark Sutherland.

9 recordsLinked to original sources
PubMed2006

A cleavable molecular adapter reduces side effects and concomitantly enhances efficacy in tumor treatment by targeted toxins in mice.

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

Alanine Transaminase↗
PubMed2006

The saponin-mediated enhanced uptake of targeted saporin-based drugs is strongly dependent on the saponin structure.

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.

Animals↗
PubMed2005

Influence of protein transduction domains on target-specific chimeric proteins.

Direct targeting to the cytoplasm and nucleus using protein transduction domains (PTD) has been described to be efficient but non-cell-type-specific, and only has clinical relevance when the molecule is active exclusively in the diseased cell. The use of PTDs is an attractive mechanism to improve drug delivery. In this work, we designed recombinant proteins that contain epidermal growth factor as ligand to render uptake target cell-specific. We evaluated the potential of several PTDs to induce the cytosolic uptake of the catalytic domain of diphtheria toxin by measuring cytotoxicity. Although PTD-dependent membrane transfer is very low, the proteins exhibited concentration-dependent cytotoxic activity. Higher binding at 4 degrees C compared to 37 degrees C suggests that uptake by the PTDs MTS and TLM occurs via an endocytic pathway. Non-specific binding is predominantly a function of the PTD and greatly increases by substitution of a non-polar glycine with a negatively charged glutamate in the PTD HA2.

Amino Acid Sequence↗
PubMed2005

Combined application of saponin and chimeric toxins drastically enhances the targeted cytotoxicity on tumor cells.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.

Animals↗
PubMed2004

Method for developing an optimal emergence profile using heat-polymerized provisional restorations for single-tooth implant-supported restorations.

This article describes a method for creating an improved emergence profile with single-tooth, implant-supported restorations. An easily trimmed silicone gingival substitute is used to allow polymerization of acrylic resin provisional restorations to achieve control of the emergence profile. Gingival trauma is minimized by eliminating intraoral use of monomer and minimizing surgical procedures. Provisional restorations can be assessed to ensure the contour is acceptable and the trimmed gingival substitute can be used to fabricate a similar profile in the definitive prosthesis. The provisional restorations may be used instead of standard prefabricated healing abutments to guide the healing contours of the peri-implant gingival tissue.

Acrylic Resins↗
PubMed2003

A cleavable adapter to reduce nonspecific cytotoxicity of recombinant immunotoxins.

One of the problems associated with the administration of immunotoxins is hypersensitivity reaction such as vascular leak syndrome. This may be prevented by decreasing the plasma half-life. To improve immunotoxins with respect to reduced side effects, we have previously described the development of a cleavable adapter. This adapter links the toxic moiety and ligand that are usually directly coupled. In our study, the cytotoxicity of saporin linked either directly or via the adapter to epidermal growth factor (EGF) was evaluated in vitro. The immunotoxins exhibited similar cytotoxic activity towards A-431 and HER14 cells (IC(50) < 10 nM). The supernatant from 6 hr cultures of HER14 cells incubated in the presence of the adapter-containing immunotoxin exhibited a significantly reduced cytotoxicity as compared to the directly coupled immunotoxin. Western blotting revealed that the adapter was cleaved, thus supporting our proposal that cleavable adapters may reduce nonspecific effects. A similar reduced half-life was detected in platelet-poor plasma. In contrast MCF-7 cells remain unaffected by the immunotoxins. This was shown to be due to the absence of detectable EGF-receptor in comparison to A-431 and HER14 cells as determined by Western blotting. Furthermore, we could show that the adapter does not exert an effect on the N-glycosidase activity of saporin. These results suggest that the use of cleavable adapters may be a useful tool in immunotoxins for reducing the killing of surrounding noncancerous cells due to nonspecific binding.

Adenine↗
PubMed2002

A colorimetric assay for the quantitation of free adenine applied to determine the enzymatic activity of ribosome-inactivating proteins.

Adenine quantitation is required for a variety of applications. To date, the prevalent method for quantifying free adenine, in a variety of applications, is the detection of fluorescent-derivatized adenine by HPLC. For the present study, we developed a high-throughput, nonradioactive, enzyme-based colorimetric adenine quantitation assay that is performed in one multireaction incubation step. The assay does not require adenine derivatization and is designed for microplates. The key step is the conversion of adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally produce three inorganic phosphate ions per adenine molecule. Phosphate is quantitated by the color-generating phosphorylysis of a particular purine derivate. Ribosome-inactivating proteins that release adenine from polynucleotides are often used to investigate intracellular protein trafficking and are important for the design of immunotoxins. We therefore used ricin, dianthin, saporin, and a variety of saporin fusion proteins to show that this method is suitable for quantifying adenine release using different substrates. The measured rate of adenine release and substrate specificity are comparable to those determined by HPLC and radioactive detection techniques.

Adenine↗
PubMedDate not supplied

Changes in vascular dynamics of the adult rat testis leading to transient accumulation of seminiferous tubule fluid after administration of a novel 5-hydroxytryptamine (5-HT) agonist.

The aim of the present study was to evaluate the possible mechanisms of testicular toxicity of GR40370X, a follow-up 5-hydroxytryptamine (5-HT) receptor agonist. Administration to adult male rats of a single (toxic) dose of 750 mg/kg GR40370X induced marked distension of seminiferous tubules and an associated increase in testis weight at 12-24 h with a gradual recovery to normal by 96 h. Seminiferous tubule distension was due to expansion of the lumen, which occurred at all stages of the spermatogenic cycle and was accompanied by vacuolation of the cytoplasm of elongating spermatids. Seminiferous tubule distension was preceded/accompanied by distension of the efferent ducts and rete testis with maximal changes evident at 24-48 h. These changes could not be explained by increases in seminiferous tubule fluid or interstitial fluid production, as both were reduced (15-20%), rather than increased, by treatment. Examination of the vasculature after treatment with 750 mg/kg GR40370X revealed significant changes that were maximal at 4 h and thus preceded rete/testicular changes. Veins of the mediastinal venous plexus, which overlies the rete, were constricted and arteriovenous anastomoses in the spermatic cord were shut/constricted, as determined (indirectly) by measurement of the dilution of outflowing testicular venous blood by incoming arterial blood. The latter effect of GR40370X could be blocked by co-administration of minoxidil, a vasodilator. Vascular effects of GR40370X had normalised by 24-48 h. It was also noted that administration of a toxic dose of GR40370X significantly lowered blood levels of LH and testosterone, though these changes were considered to be incidental and not involved in the other changes described above. None of the above changes were induced by a pharmacologically active dose (1 mg/kg) of GR40370X. It is concluded that the mechanism of testicular toxicity induced by 750 mg/kg GR40370X results from primary effects on the vasculature of the testis/neighbouring region, which in turn lead to impaired fluid resorption from the efferent ducts and rete and thence to accumulation of seminiferous tubule fluid in the rete and testis.

Animals↗