Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex ...reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs.
Background and purpose: The p38 kinase regulates the release of proinflammatory cytokines including tumour‐necrosis factor‐α (TNFα) and is regarded as a potential therapeutic target in rheumatoid ...arthritis (RA). Using the novel p38 inhibitor Org 48762‐0, we investigated the therapeutic potential of p38 inhibition and compared this to anti‐mouse (m)TNFα antibody treatment in murine collagen‐induced arthritis (CIA).
Experimental approach: Pharmacological profiles of Org 48762‐0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)‐induced inflammation in mice. The effects of Org 48762‐0 and of mTNFα‐neutralization on established arthritis were examined in murine CIA.
Key results: Org 48762‐0 potently inhibited p38α kinase with a high degree of kinase selectivity. In cellular assays, Org 48762‐0 reduced LPS‐induced TNFα release. Oral administration of Org 48762‐0 in mice showed drug‐like pharmacokinetic properties and inhibited LPS‐induced cytokine production. These pharmacological characteristics of Org 48762‐0 prompted a comparison of therapeutic efficacy with mTNFα‐neutralization in CIA. Org 48762‐0 and anti‐mTNFα antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762‐0 treatment only. Further, micro‐computed tomographical and histopathological analyses confirmed the protective effects of Org 48762‐0 on joint damage.
Conclusions and implications: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFα produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.
The rationale fo the development of prodrugs relies upon delivery of higher concentrations of a drug to target cells compared to administration of the drug itself. In the last decades, numerous ...prodrugs that are enzymatically activated into anti-cancer agents have been developed. This review describes the most important enzymes involved in prodrug activation notably with respect to tissue distribution, up-regulation in tumor cells and turnover rates. The following endogenous enzymes are discussed: aldehyde oxidase, amino acid oxidase, cytochrome P450 reductase, DT-diaphorase, cytochrome P450, tyrosinase, thymidylate synthase, thymidine phosphorylase, glutathione S-transferase, deoxycytidine kinase, carboxylesterase, alkaline phosphatase, beta-glucuronidase and cysteine conjugate beta-lyase. In relation to each of these enzymes, several prodrugs are discussed regarding organ- or tumor-selective activation of clinically relevant prodrugs of 5-fluorouracil, axazaphosphorines (cyclophosphamide, ifosfamide, and trofosfamide), paclitaxel, etoposide, anthracyclines (doxorubicin, daunorubicin, epirubicin), mercaptopurine, thioguanine, cisplatin, melphalan, and other important prodrugs such as menadione, mitomycin C, tirapazamine, 5-(aziridin-1-yl)-2,4-dinitrobenzamide, ganciclovir, irinotecan, dacarbazine, and amifostine. In addition to endogenous enzymes, a number of nonendogenous enzymes, used in antibody-, gene-, and virus-directed enzyme prodrug therapies, are described. It is concluded that the development of prodrugs has been relatively successful; however, all prodrugs lack a complete selectivity. Therefore, more work is needed to explore the differences between tumor and nontumor cells and to develop optimal substrates in terms of substrate affinity and enzyme turnover rates fo prodrug-activating enzymes resulting in more rapid and selective cleavage of the prodrug inside the tumor cells.
For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in ...chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.
Several selenocysteine Se-conjugates have been shown to possess potent chemopreventive activity in animal models for chemical carcinogenesis. As a mechanism of action, β-elimination reactions to form ...chemopreventive selenols, ammonia, and pyruvate has been proposed. The enzymes involved in these β-elimination reactions, however, have been partially elucidated. Next to cysteine conjugate β-lyases, as yet unidentified non-pyridoxal-5‘-phosphate-dependent enzymes also appear to be involved in cytosolic β-elimination reactions. In the present study, it was investigated whether amino acid oxidases contribute to the bioactivation of selenocysteine Se-conjugates. Using purified l-amino acid oxidase from Crotalus adamanteus as a model enzyme, significant β-elimination activities were indeed observed upon incubation with Se-methylselenocysteine (K m, 195 μM; k cat, 48 min-1), Se-allylselenocysteine (K m, 608 μM; k cat, 34 min-1), Se-phenylselenocysteine (K m, 107 μM; k cat, 57 min-1) and Se-benzylselenocysteine (K m, 59 μM; k cat, 13 min-1). For all selenocysteine Se-conjugates tested, the rate of pyruvate formation was comparable to that of hydrogen peroxide, one of the products of oxidative deamination. The fact that addition of catalase did not alter pyruvate formation indicated that the β-elimination reaction observed was not mediated by selenoxidation/syn-elimination due to the hydrogen peroxide formed via the oxidative deamination pathway. Using d-amino acid oxidase from porcine kidney and d-SeCys conjugates similar results were obtained. To delineate whether mammalian l-amino acid oxidases are also able to catalyze β-elimination of selenocysteine Se-conjugates, rat renal cytosol was fractionated and screened for β-elimination and oxidative deamination activities. One of the fractions isolated displayed oxidative deamination activity with several amino acids and cysteine S-conjugates. With selenocysteine Se-conjugates as substrates, however, this fraction displayed both oxidative deamination and β-elimination activities, when incubated in the presence of aminoxyacetic acid to block contribution of pyridoxal-5‘-phosphate-dependent enzymes. The potential significance of this novel bioactivation route for the chemopreventive activity of selenocysteine Se-conjugates is discussed.
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