Paul J. White

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides.

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.

BARRIERS TO SUCCESSFUL DELIVERY OF SHORT INTERFERING RNA AFTER SYSTEMIC ADMINISTRATION

1 RNA interference in vivo has tremendous potential, both with respect to the elucidation of protein function in animals and as a therapeutic platform in humans. In vitro, short interfering RNA (siRNA) has been shown to completely silence gene expression in mammalian cells at low picomolar concentrations. 2 Although many good publications have shown specific silencing to occur in vivo, there are few that have transferred the combination of maximal efficacy and high potency to this setting. The present review considers the biological barriers that limit the movement of siRNA from vascular lumen to target cell cytoplasm and the strategies that have been used to overcome them. 3 Intravenous administration of siRNA results in rapid, extensive removal of siRNA from the blood via renal excretion, tissue distribution and nuclease degradation. Movement across vascular capillaries appears to be a limiting factor in some cases; few examples of silencing have been reported in organs with a conventional capillary endothelium. 4 Cellular uptake and endosomal trapping are significant barriers, but can be overcome using strategies such as antibody mediated cellular uptake or polyethyleneimine‐mediated endosomal escape.

Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist

Significance The adenosine A1 receptor (A1AR) is an important target for cardioprotection, but current A1AR drugs are limited for this indication because of the occurrence of bradycardia as a major adverse effect mediated by the same receptor. To address this problem, we designed a ligand that simultaneously bridges two different sites on the A1AR; the hypothesis is that this bitopic mode would result in unique receptor conformations that will signal to desirable pathways while sparing those pathways mediating undesirable effects. This mechanism was validated in native rodent cells and isolated rat atria, providing proof of concept that the design of bitopic ligands may be a path forward to separating beneficial from harmful effects mediated by the same drug target. The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A1 GPCR (A1AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A1AR ligand (VCP746)—a hybrid molecule comprising adenosine linked to a positive allosteric modulator—specifically to engender biased signaling at the A1AR. We validate that the interaction of VCP746 with the A1AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A1AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A1AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.

Antisense oligonucleotides in cutaneous therapy.

Antisense oligonucleotides have been the subject of intense interest as research tools to elucidate the functions of gene products and as therapeutic agents. Initially, their mode of action was poorly understood and the biological effects of oligonucleotides were often misinterpreted. However, research into these gene-based inhibitors of cellular action recently has succeeded in realising their exciting potential, particularly as novel therapeutic agents. An emerging application of this technology is in cutaneous therapy. The demand for more effective dermatological drugs will ensure further development of antisense strategies in skin, with key issues being drug delivery, therapeutic target selection, and clinical applicability.

Delineating the Mode of Action of Adenosine A1 Receptor Allosteric Modulators

Despite the identification of 2-amino-3-benzoylthiophenes (2A3BTs) as the first example of small-molecule allosteric potentiators of agonist function at a G protein-coupled receptor (GPCR)—the adenosine A1 receptor—their mechanism of action is still not fully understood. We now report the mechanistic basis for the complex behaviors noted for 2A3BTs at A1 receptors. Using a combination of membrane-based and intact-cell radioligand binding, multiple signaling assays, and a native tissue bioassay, we found that the allosteric interaction between 2A3BTs and the agonists 2-chloro-N6-[3H]cyclopentyladenosine or (−)-N6-(2-phenylisopropyl)adenosine (R-PIA) or the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine is consistent with a ternary complex model involving recognition of a single extracellular allosteric site. However, when allowed access to the intracellular milieu, 2A3BTs have a secondary action as direct G protein inhibitors; this latter property is receptor-independent as it is observed in nontransfected cells and also after stimulation of another GPCR. In addition, we found that 2A3BTs can signal as allosteric agonists in their own right but show bias toward certain pathways relative to the orthosteric agonist, R-PIA. These results indicate that 2A3BTs have a dual mode of action when interacting with the A1 receptor and that they can engender novel functional selectivity in A1 signaling. These mechanisms need to be factored into allosteric ligand structure-activity studies.

Overcoming biological barriers to in vivo efficacy of antisense oligonucleotides.

Antisense oligonucleotides as a therapeutic platform have been slow to progress since the approval of the first antisense drug in 1998. Recently, there have been several examples of convincing antisense interventions in animal models and promising clinical trial data. This review considers the factors determining the success of antisense oligonucleotides as therapeutic agents. In order to produce target knockdown after systemic delivery, antisense oligonucleotides must avoid nuclease degradation, reticuloendothelial-system uptake and rapid renal excretion, and extravasate to the target cell type outside the vasculature. They then must enter the target cell, and escape the endosome-lysosome pathway so as to be free to interact with the target mRNA. We consider the significance of these limiting factors based on the literature and our own experience using systemic administration of antisense oligonucleotides.

Interaction of viruses with host cell molecular motors

Viral particles are generally too large to diffuse freely within the crowded environment of the host cell cytoplasm. They depend on mammalian cell transport systems, in particular the microtubular molecular motor dynein, to deliver their nucleic acids to the vicinity of the nucleus. An understanding of how viruses interact with dynein, and its many accessory proteins, may reveal targets for drug discovery and will unlock the toolbox required to improve the performance of synthetic gene delivery systems.

Cardioprotection induced by adenosine A1 receptor agonists in a cardiac cell ischemia model involves cooperative activation of adenosine A2A and A2B receptors by endogenous adenosine.

Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 μM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10−7M) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA; 10−7M) reduced the proportion of nonviable cells to 30.87 ± 2.49% and 35.18 ± 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 ± 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 ± 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 ± 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection.

Characterisation of opioid receptors involved in modulating circular and longitudinal muscle contraction in the rat ileum

1 The aim of the present investigation was to characterise the opioid receptor subtypes present in the rat ileum using a method that detects drug action on the enteric nerves innervating the circular and longitudinal muscles. 2 Neurogenic contractions were reversibly inhibited by morphine (circular muscle pEC50, 6.43±0.17, Emax 81.7±5.0%; longitudinal muscle pEC50, 6.65±0.27, Emax 59.7±7.8%), the μ‐opioid receptor‐selective agonist, DAMGO ([D‐Ala2,N‐Me‐Phe4,Gly5‐ol]enkephalin acetate) (circular pEC50, 7.85±0.04, Emax 97.8±3.6%; longitudinal pEC50, 7.35±0.09, Emax 56.0±6.1%), the δ‐selective agonist DADLE ([D‐Ala2,D‐Leu5]enkephalin acetate) (circular pEC50, 7.41±0.17, Emax, 93.3±8.4%; longitudinal pEC50, 6.31±0.07, Emax 66.5±5.2%) and the κ‐selective agonist U 50488H (trans‐(±)‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulphonate) (circular pEC50, 5.91±0.41, Emax, 83.5±26.8%; longitudinal pEC50, 5.60±0.08, Emax 74.3±7.2%). Agonist potencies were generally within expected ranges for activity at the subtype for which they are selective, except for U 50488H, which was less potent than expected. 3 The μ and δ receptor‐selective antagonists, CTAP (H‐D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2) and naltrindole, caused progressive, parallel rightward shifts in the DAMGO and DADLE curves, respectively. Analysis indicated conformity to theoretical simple competitive antagonist behaviour. U 50488H effects were insensitive to the κ‐selective antagonist, n‐BNI. A high concentration (1 μM) of naltrexone caused apparent potentiation of U 50488H effects. 4 CTAP pKB estimates were consistent with previously reported values for μ receptor antagonism (circular 7.84±0.17, longitudinal 7.64±0.35). However, the naltrindole pKB estimates indicated lower antagonist potency than expected (circular 8.22±0.23, longitudinal 8.53±0.35). 5 It is concluded that μ and possibly atypical δ receptors (but not κ receptors) mediate inhibition of contraction in this model. Nonopioid actions of U 50488H are probably responsible for the inhibitory effects seen with this compound.

Antisense oligonucleotide treatments for psoriasis

Antisense oligonucleotides are emerging as an exciting therapeutic strategy for treating skin diseases such as psoriasis. Potential antisense targets are proteins upregulated in psoriatic skin, in particular those associated with inflammation (intercellular adhesion molecule [ICAM]-1, IL-2 and -8), proliferation (insulin-like growth factor type I receptor [IGF-IR], epidermal growth factor) and hyperangiogenesis (vascular endothelial growth factor [VEGF]). Whereas topical application and subsequent penetration of large oligonucleotides into normal skin is problematic, the impaired barrier function of psoriatic lesions permits the uptake of antisense drugs. Studies to date indicate that topically applied antisense molecules can be delivered to target cells in the epidermis and dermis of psoriatic skin. Antisense-mediated suppression of target mRNA and protein has been demonstrated in models of human skin grafted to immunosuppressed mice and in hairless mouse models of skin inflammation. In a xenograft model of human psoriasis, treatment with repeated intradermal injections of IGF-IR antisense caused a normalisation of the epidermal hyperproliferation. This class of drug, therefore, holds much potential for the successful treatment of psoriasis in the clinical setting.