Wim H. A. Dokter

Prednisolone-induced differential gene expression in mouse liver carrying wild type or a dimerization-defective glucocorticoid receptor

BackgroundGlucocorticoids (GCs) control expression of a large number of genes via binding to the GC receptor (GR). Transcription may be regulated either by binding of the GR dimer to DNA regulatory elements or by protein-protein interactions of GR monomers with other transcription factors. Although the type of regulation for a number of individual target genes is known, the relative contribution of both mechanisms to the regulation of the entire transcriptional program remains elusive. To study the importance of GR dimerization in the regulation of gene expression, we performed gene expression profiling of livers of prednisolone-treated wild type (WT) and mice that have lost the ability to form GR dimers (GRdim).ResultsThe GR target genes identified in WT mice were predominantly related to glucose metabolism, the cell cycle, apoptosis and inflammation. In GRdim mice, the level of prednisolone-induced gene expression was significantly reduced compared to WT, but not completely absent. Interestingly, for a set of genes, involved in cell cycle and apoptosis processes and strongly related to Foxo3a and p53, induction by prednisolone was completely abolished in GRdim mice. In contrast, glucose metabolism-related genes were still modestly upregulated in GRdim mice upon prednisolone treatment. Finally, we identified several novel GC-inducible genes from which Fam107a, a putative histone acetyltransferase complex interacting protein, was most strongly dependent on GR dimerization.ConclusionsThis study on prednisolone-induced effects in livers of WT and GRdim mice identified a number of interesting candidate genes and pathways regulated by GR dimers and sheds new light onto the complex transcriptional regulation of liver function by GCs.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

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.

The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers

SYD985 is a HER2-targeting antibody–drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need. Mol Cancer Ther; 14(3); 692–703. ©2015 AACR.

Chronic Prednisolone Treatment Reduces Hepatic Insulin Sensitivity while Perturbing the Fed-to-Fasting Transition in Mice

Chronic glucocorticoid use for treatment of inflammatory diseases is accompanied by severe side effects in humans (e.g. hyperglycemia and insulin resistance). The present studies were conducted to characterize consequences of chronic treatment with the synthetic glucocorticoid prednisolone on insulin sensitivity and blood glucose kinetics in mice. Prednisolone treatment increased fasting blood glucose and plasma insulin concentrations, but this apparently reduced insulin sensitivity could not be confirmed in hyperinsulinemic euglycemic clamp studies. Therefore, a novel method to study whole body glucose kinetics was used. This method revealed that prednisolone-treated mice show an increased hepatic glucose production (HGP). The increased HGP was accompanied by elevated plasma insulin concentrations, indicating reduced insulin sensitivity of hepatic glucose metabolism in prednisolone-treated mice. Compared with vehicle, prednisolone-treated mice had lower blood glucose concentrations, higher plasma free fatty acids, and higher plasma fibroblast growth factor-21 concentrations in the fed condition, i.e. mimicking a fasting situation. Next, the effects of 24-h fasting on energy metabolism were studied. Compared with controls, fasted prednisolone-treated mice had higher blood glucose concentrations and lower plasma beta-hydroxybutyrate concentrations. In conclusion, these results indicate that chronic prednisolone treatment reduces insulin sensitivity of HGP, induces a fasting-like phenotype in fed mice, and perturbs the fed-to-fasting transition.

Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.

Conjugation of ATIII-Binding Pentasaccharides to Extend the Half-Life of Proteins: Long-Acting Insulin

Tight control over plasma glucose levels in the treatment of insulin-dependent type 1 diabetes mellitus (T1DM) can be achieved by the exogenous administration of insulin. A natural overall physiological profile can be mimicked by the administration of shortand intermediate-acting analogues to control post-meal glucose excursions and a longer-acting insulin product to maintain basal levels of insulin. The very short plasma half-life of insulin poses a challenge for creating sufficiently long-acting formulations or analogues with optimal pharmacokinetic/dynamic (PK/PD) properties that provide consistent glycemic control. Approved long-acting insulin analogues for basal insulin therapy form a subcutaneous depot at the injection site (insulin glargine, detemir) and/or exhibit an extended half-life through hydrophobic interaction of fatty acid groups with serum albumin (insulin detemir). Both detemir and glargine require twice-daily administration. Several groups have reported recently on novel insulins with extended duration of action, yet there is still a need for alternatives with minimized risk of (mainly nocturnal) hypoglycemia and optimal duration of action. An expanding repertoire of methods to enhance the exposure of protein therapeutics is emerging. For instance, (bio)chemical modification (e.g. PEGylation, acylation, or glycosylation), fusion with or binding to plasma proteins (e.g. albumin, transferrin, or Fc fragments) has yielded numerous clinical protein candidates with improved PK/PD profiles. The binding of a polypeptide drug (either covalently or through a tightly binding carrier) to long-lived plasma proteins in the bloodstream enables tight control over targeted drug levels with improved exposure and distribution in the circulation that may potentially lead to lower doses, decreased side effects, and enhanced predictability. In this context, not all plasma proteins may have been explored to the full extent. Herein we demonstrate how binding to the plasma protein antithrombin III (ATIII) can be used to enhance the half-life of insulin. ATIII is a serine protease inhibitor that interrupts the blood coagulation cascade upon activation by glycosaminoglycans such as heparin, and is present in blood plasma at high concentration (~3 mm). The minimal structural requirement for ATIII affinity is a sulfated pentasaccharide (PS), the fully synthetic equivalent of which reached the market in 2002 (fondaparinux, Arixtra, Figure 1). Interestingly, the high specificity results in a much longer plasma half-life (~15 h vs. ~1 h for heparin) which can be further extended by enhancing the affinity for ATIII. The latter has resulted in the anticoagulant idraparinux (Kd=1 nm), which is now in phase III clinical development for once-weekly dosing (t1/2~120 h). In addition, the design of dual-acting antithrombotics that consist of a low-molecular-weight thrombin or GPIIb/IIIa inhibitor conjugated to the nonreducing end of a PS has revealed that the half-life of the PS largely determines the half-life of the entire PS conjugate. It occurred to us that the near linear PK behavior of ATIIIbound PS may be extended to conjugates of therapeutically relevant (non-anticoagulant) polypeptides and proteins that suffer from short plasma half-lives (see Figure 2). To avoid any undesired clinically significant ATIII-mediated anticoagulant activity, the polypeptide conjugate should have a therapeutic plasma level of <50 nm (i.e. <2% of ATIII is bound to conjugated PS, and the associated anticoagulant activity is of subtherapeutic level relative to the pharmacological activity of the polypeptide). We selected insulin as an initial target to validate this concept because: a) T1DM patients require repeated injections of long-acting (basal) insulin to complement endogenous hormonal levels in the sub-nanomolar range, b) the insulin receptor (InsR) is accessible from the circulation, and c) site-directed chemical modification of insulin does not necessarily have a deleterious effect on the biological activity. Herein we disclose our novel CarboCarrier technology for enhancing PK/PD profiles of small proteins and polypeptides at sub-anticoagulant concentrations, exemplified by the preparation and pharmacological evaluation of novel long-acting insulins conjugated to different ATIII-binding carrier PSs with varying half-lives. The N-terminal PheB1 and near C-terminal LysB29 amino functions are not essential for the bioactivity of insulin, and hence it was anticipated that the introduction of a PS at either [a] Dr. M. de Kort, B. Gianotten, J. A. J. Wisse, Dr. M. Honing, S. Vonsovic, Dr. J. C. H. M. Wijkmans, Prof. Dr. C. A. A. van Boeckel Department of Medicinal Chemistry N.V. Organon, part of Schering–Plough Corporation Molenstraat 110, PO Box 20, 5340 BH, Oss (The Netherlands) Fax: (+ 31) 412-662-546 E-mail : m.dekort@organon.com [b] Dr. E. S. Bos, G. M. T. Vogel, Dr. W. H. A. Dokter Department of Pharmacology N.V. Organon, part of Schering–Plough Corporation Molenstraat 110, PO Box 20, 5340 BH, Oss (The Netherlands) [c] Dr. M. H. M. Eppink Department of API-BT N.V. Organon, part of Schering–Plough Corporation Molenstraat 110, PO Box 20, 5340 BH, Oss (The Netherlands) [d] E. Mattaar, Dr. M.-J. Smit Department of Molecular Pharmacology N.V. Organon, part of Schering–Plough Corporation Molenstraat 110, PO Box 20, 5340 BH, Oss (The Netherlands) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.

Prednisolone-induced changes in gene-expression profiles in healthy volunteers.

BACKGROUND Prednisolone and other glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive drugs. However, prolonged use at a medium or high dose is hampered by side effects of which the metabolic side effects are most evident. Relatively little is known about their effect on gene-expression in vivo, the effect on cell subpopulations and the relation to the efficacy and side effects of GCs. AIM To identify and compare prednisolone-induced gene signatures in CD4⁺ T lymphocytes and CD14⁺ monocytes derived from healthy volunteers and to link these signatures to underlying biological pathways involved in metabolic adverse effects. MATERIALS & METHODS Whole-genome expression profiling was performed on CD4⁺ T lymphocytes and CD14⁺ monocytes derived from healthy volunteers treated with prednisolone. Text-mining analyses was used to link genes to pathways involved in metabolic adverse events. RESULTS Induction of gene-expression was much stronger in CD4⁺ T lymphocytes than in CD14⁺ monocytes with respect to fold changes, but the number of truly cell-specific genes where a strong prednisolone effect in one cell type was accompanied by a total lack of prednisolone effect in the other cell type, was relatively low. Subsequently, a large set of genes was identified with a strong link to metabolic processes, for some of which the association with GCs is novel. CONCLUSION The identified gene signatures provide new starting points for further study into GC-induced transcriptional regulation in vivo and the mechanisms underlying GC-mediated metabolic side effects.

Chronic Prednisolone Treatment Aggravates Hyperglycemia in Mice Fed a High-Fat Diet but Does Not Worsen Dietary Fat-Induced Insulin Resistance

Synthetic glucocorticoids such as prednisolone have potent antiinflammatory actions. Unfortunately, these drugs induce severe adverse effects in patients, many of which resemble features of the metabolic syndrome, such as insulin resistance. In this study, we investigated whether adverse effects of prednisolone on glucose homeostasis are aggravated in mice with compromised insulin sensitivity due to a high-fat diet by applying various methods to analyze changes in insulin sensitivity in mice. C57BL/6J mice were fed a high-fat diet for 6 wk and treated with either prednisolone (10 mg/kg · d) or vehicle for the last 7 d. Insulin sensitivity and blood glucose kinetics were analyzed with state-of-the-art stable isotope procedures in different experimental conditions. Prednisolone treatment aggravated fasting hyperglycemia and hyperinsulinemia caused by high-fat feeding, resulting in a higher homeostatic assessment model of insulin resistance. In addition, prednisolone-treated high-fat diet-fed mice appeared less insulin sensitive by detailed analysis of basal glucose kinetics. Remarkably, using hyperinsulinemic-euglycemic or hyperglycemic clamp techniques, neither hepatic nor peripheral insulin resistance was worsened in the group that was treated with prednisolone. Yet analysis of hepatic glucose metabolism revealed that prednisolone did alter glycogen balance by reducing glycogen synthase flux under hyperinsulinemic as well as hyperglycemic conditions. In addition to elevated insulin levels, prednisolone-treated mice showed a major rise in plasma leptin and fibroblast growth factor 21 levels. Our data indicate that prednisolone-induced adverse effects on glucose metabolism in high-fat diet-fed mice do not reflect impaired insulin sensitivity but may be caused by other changes in the hormonal regulatory network controlling glucose metabolism such as fibroblast growth factor 21 and leptin.

Activation of proteinase 3 contributes to Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance

Activation of inflammatory pathways is known to accompany development of obesity-induced nonalcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive proinflammatory mediators interleukin (IL)-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In this study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human α-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin-resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3-deficient mice showed strongly reduced levels of lipids in the liver after being fed a high-fat diet. Moreover, these mice were resistant to high-fat-diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1−/− mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with α-1 antitrypsin during the last 10 d of a 16-wk high-fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential.

Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide

To examine the biological characteristics of a novel glucagon‐like peptide‐1 (GLP‐1) conjugate, in which an antithrombin III (ATIII)‐binding pentasaccharide is conjugated to d‐Ala8GLP‐1 using a tetraethylene glycol linker.