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Background: We investigated potential for hypersensitivity reactions after repeated sugammadex administration and explored the mechanism of hypersensitivity., Methods: In this double-blind, placebo-controlled study (NCT00988065), 448 healthy volunteers were randomised to one of three arms to receive three repeat i.v. administrations of either sugammadex 4 mg kg -1 , 16 mg kg -1 , or placebo. Primary endpoint was percentage of subjects with hypersensitivity (assessed by an independent adjudication committee). Secondary endpoint of anaphylaxis was classified per Sampson and Brighton criteria. Exploratory endpoints included skin testing, serum tryptase, anti-sugammadex antibodies [immunoglobulin (Ig) E/IgG], and other immunologic parameters., Results: Hypersensitivity was adjudicated for 1/148 (0.7%), 7/150 (4.7%), and 0/150 (0.0%) subjects after sugammadex 4 mg kg -1 , 16 mg kg -1 , and placebo, respectively. After sugammadex 16 mg kg -1 , one subject met Sampson criterion 1 and Brighton level 1 (highest certainty) anaphylaxis criteria; two met Brighton level 2 criteria. After database lock it was determined that certain protocol deviations could have introduced bias in the reporting of hypersensitivity signs/symptoms in a subject subset. Objective laboratory investigations indicated that potential underlying hypersensitivity mechanisms were unlikely to have been activated; the results suggest that most of the observed hypersensitivity reactions were unlikely IgE/IgG-mediated., Conclusion: Dose-dependent hypersensitivity or anaphylaxis reactions to sugammadex were observed when administered without prior neuromuscular blocking agent. Laboratory investigations do not suggest prevalent allergen-specific IgE/IgG-mediated immunologic hypersensitivity. Because it could not be fully excluded that estimates of hypersensitivity/anaphylaxis incidence were unbiased, an additional study was conducted to characterise the potential for hypersensitivity reactions and is described in a companion report., Clinical Trial Registration: http://www.clinicaltrials.gov NCT00988065; Protocol number P06042., (Copyright © 2018 British Journal of Anaesthesia. All rights reserved.)

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

Hydroxypropyl-β-cyclodextrin (HPβCD) is a complexation agent used to enhance drug solubilization and formulation stability. Although its toxicity is well characterized, its cardiovascular effects are less known. To investigate them, HPβCD was infused intravenously over 10 min in anesthetized dogs (10-40% (w/v, i.e. 200-800 mg/kg) in non-denervated animals and at 40% in denervated animals). HPβCD increased renal arteriolar resistance and decreased renal blood flow at all doses, almost immediately after infusion start, more drastically in females. A less pronounced increase in total peripheral resistance occurred in females only due to sex difference in sympathetic tone. Pulmonary hemodynamic parameters remained unaffected, suggesting that the renal effect was rather selective. As a consequence of the increased systemic blood pressure, heart rate decreased in normal animals without direct effect on cardiac conductance. This effect was abolished in denervated animals. This suggests that autonomous nervous feedback loops are functional in normal animals and that HPβCD has no direct chronotropic effect. In conclusion, systemic and renal hemodynamic changes should be considered as potential background effects at 200-400 mg/kg. At higher doses (800 mg/kg), changes are more pronounced and could mask/exacerbate hemodynamic response of drug candidate; such doses should be avoided in nonclinical safety studies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Sugammadex, a thiolated γCD derivative used as an antagonist of steroidal blockers, was studied with regard to its tendency to self-associate in aqueous solution. Three independent methods - permeation through semi-permeable cellophane membranes, dynamic light scattering, and sedimentation equilibrium analytical ultracentrifugation - were used for this purpose. The results were in agreement with each other and showed no evidence of self-association in a wide sugammadex concentration range from 0.25 to 100mg/ml., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A concept relating the lipophilicity of chemicals with their genotoxicity on a chromosomal level had been generated by Schultz and Onfelt (Chem Biol Interact 126:97-123, 2000). It was shown that aneuploidy in Chinese hamster V79 cells was elicited by lipophilic chemicals at concentrations related to their hydrophobicity (log P), whereas toxicants with a specific mode of action acted at concentrations consistently lower than predicted based on log P. We have now combined available data sets on aneuploidy/micronucleus formation with procedures used in QSAR modelling, in order to find new molecular descriptors for modelling non-specific chromosomal genotoxicity, and to optimise combinations thereof. Molecular structures of 26 chemicals, including steroids, were converted into single 3D models using Corina (version 3.20), and 11 descriptors of molecular properties were calculated. The data of 16 compounds assigned to a non-specific mode of action were imported into the QSAR module of the software package Cerius(2) (version 4.10). Applying genetic function approximation (GFA), linear equations were set up relating molecular descriptors with experimental concentrations at which doubling of micronuclei occurred in V79 cells (exp -log C). The number of variables (molecular descriptors) was limited to a maximum of three, and linear and quadratic terms were allowed. Based on the descriptions provided by the GFA procedure, log P was the most suitable single property to describe non-specific genotoxicity [r (2 ) = 0.88], confirming the original concept of Schultz and Onfelt. Using more descriptors (up to three in combination) resulted in an optimization of correlations up to r (2 )= 0.97. Such optimal correlation coefficients were obtained by combinations (a) of the numbers of hydrogen bond acceptors, the polar surface and total surface areas of molecules on one hand, and by (b) the dipole moment, polar surface and total surface descriptors on the other hand. In essence, the relation of polar surface to the total molecular surface appears pivotal to determine the non-specific chromosomal genotoxicity of lipophilic compounds.

As part of regulatory safety testing program, a 13-week oral toxicity study with a new antipsychotic drug candidate was performed in beagle dogs. During this study, dark red/brown feces were recorded in treated dogs and increases in liver parameters (alanine aminotransferase, alkaline phosphatase, bilirubin) were measured biochemically. At the end of the study, livers of high-dose (50 mg/kg) animals were (mottled) dark brown, sometimes with pale foci. Histopathological examination of these livers showed dark globular pigment deposits in the hepatocellular cytoplasm and within the bile canaliculi. Varying numbers of inflammatory cell infiltrates were additionally present in association with the deposits. These pigment deposits showed birefringency with characteristic "Maltese Cross"-like structures under polarized light. Electronmicroscopy revealed the typical, so-called "sunburst" pattern with radiating double-lined crystalline structures. These morphologic characteristics strongly indicated at the presence of porphyrins, which was definitely confirmed biochemically. Published reports of drug-induced hepatic porphyria in dogs are rare. The possible underlying mechanism in the dog and man is discussed.

Hormonal steroids have a widespread use in medicine and their side effects are continuously debated. The possible genotoxic activity of steroids has been the subject of many investigations. The natural estrogens estradiol, estrone and estriol are generally negative in the ICH core battery of tests, but several positive results have been obtained when using additional endpoints of genotoxicity. The genotoxic activity of the 4-hydroxy metabolites of estradiol and estrone is well established. The synthetic steroidal estrogens have a comparable profile of negative and positive test results. Cyproterone acetate and some of its analogues have a special position within the group of progestins. Their genotoxic potential has been established. Other progestins are generally negative in the routine tests. Anti-glucocorticoids, anti-progestins, corticosteroids, androgens, anabolics and anti-androgens appear to be devoid of genotoxic activities. The genotoxic potential of estradiol, estrone and cyproterone acetate with its analogues may play no role under normal physiological and therapeutic conditions. The metabolic conditions that are needed for the formation of DNA-reactive metabolites and oxygen radicals may not be present in humans. Epidemiological cancer data seem to support this view. The importance of thresholds in the dose-effect-relationship of genotoxicity data and their use in risk assessment is discussed.

To investigate the involvement of K+ efflux in apoptotic cell shrinkage, we monitored efflux of the K+ congener, 86Rb+, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in 86Rb+ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed 86Rb+ efflux and cell shrinkage induced by the anti-CD95 antibody. A wide range of inhibitors against most types of K+ channels could not inhibit CD95-mediated efflux of 86Rb+, however, the uptake of 86Rb+ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of 86Rb+ in Jurkat cells was sensitive to ouabain (a specific Na+/K(+)-ATPase inhibitor), demonstrating Na+/K(+)-ATPase dependent K+ uptake. Ouabain induced significant 86Rb+ efflux in untreated cells, as well as it seemed to compete with 86Rb+ efflux induced by the anti-CD95 antibody, supporting a role for Na+/K(+)-ATPase in the CD95-mediated 86Rb+ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na+/K(+)-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.

Although human JURKAT T lymphocytes induced to undergo apoptosis with anti-Fas/APO-1 antibody were observed to rapidly lose reduced glutathione (GSH), increased concentrations of oxidized products were not detectable. Unexpectedly, the reduced tripeptide was instead quantitatively recovered in the incubation medium of the cells. As GSH loss was blocked by bromosulfophthalein and dibromosulfophthalein, known inhibitors of hepatocyte GSH transport, a specific export rather than nonspecific leakiness through plasma membranes is proposed to be responsible. Apoptosis was delayed when GSH-diethylesters were used to elevate intracellular GSH, although the high capacity of the activated efflux system quickly negated the benefit of this treatment. Stimulation of GSH efflux provides a novel mechanism whereby Fas/APO-1 ligation can deplete GSH. We speculate that it enhances the oxidative tonus of a responding cell without requiring an increase in the production of reactive oxygen species.

In the current paradigm for apoptotic cell death, the activity of a family of proteases related to interleukin 1-beta converting enzyme (ICE) orchestrates the multiple downstream events (such as cell shrinkage and chromatin degradation) that comprise apoptosis. A variety of stimuli can induce this type of cell death. One of the most reproducible inducers is mild oxidative stress, although it is unclear how an oxidative stimulus activates ICE-like proteases. Oxidative modification of proteins and lipids have also been observed in cells undergoing apoptosis in response to non-oxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. However, attempts to consistently detect a requirement for reactive oxygen species in apoptosis have been inconclusive. Recent experiments revealing that apoptosis is typically accompanied by a depletion of intracellular reduced glutathione (GSH) are also discussed. In JURKATT lymphocytes treated with antibodies to the Fas/APO-1 surface receptor, this depletion results from an accelerated efflux of the reduced thiol rather than any intracellular oxidation. As GSH is the most abundant cytosolic reductant, we propose that its efflux may provide a non-oxidative mechanism by which the reducing environment of apoptotic cells is lost. An increase in oxidative damage to proteins and lipids would then result even in the absence of an increase in the production of oxidants. This may explain the seemingly contradictory findings that increased oxidative stress is not required for apoptosis even though antioxidants often inhibit the process and peroxidised products accumulate in apoptotic cells.

A variety of stimuli can induce cells to undergo apoptotic death. One of the most reproducible inducers is mild oxidative stress, be it via exposure to hydrogen peroxide, redox-cycling quinones or thiol-alkylating agents. Oxidative modifications of proteins and lipids have also been observed in cells undergoing apoptosis in response to non-oxidative stimuli such as glucocorticoids or topoisomerase II inhibitors. This suggests that some unidentified oxidative changes occur during apoptosis in many, if not all, cases. However, recent experiments demonstrating apparently normal apoptosis even when cells are cultured at low oxygen tensions show that reactive oxygen species cannot be essential mediators of this type of cell death. Experiments revealing that apoptosis is typically accompanied by a depletion of intracellular reduced glutathione (GSH) are also discussed. As GSH depletion will lower a cell's capacity to buffer against endogenous oxidants, we propose that it contributes to the increased oxidative damage commonly observed to accompany apoptosis. In addition, it may set a time limit on continued mitochondrial function (and thus indirectly on total ATP levels and membrane integrity) in apoptotic cells, and thereby explain the often observed 'secondary necrosis' of cells undergoing apoptosis in vitro.

Autophosphorylation is thought to be an essential step in the insulin receptor signal transduction cascade. Previous studies have shown that thiol alkylation of the receptor can block this receptor autophosphorylation, whereas an oxidative environment can increase this process. Since the toxicity of quinones can be related to two mechanisms-redox cycling resulting in oxidative stress, and arylation of cellular nucleophilic groups-the effects of 1,4-naphthoquinone and menadione on insulin receptor autophosphorylation were investigated. The results show that these two quinones have a dual effect: lower concentrations leading to oxidative stress increase insulin receptor autophosphorylation, whereas higher concentrations cause a thiol depletion and inhibit the normal insulin receptor autophosphorylation.