Using Lipids To Discover New Drugs

Karen — Fri, 25 May 2012 08:31:16 +0000

What if you could differentiate compounds that had the ability to affect innate immunity from those that could not, and from those that could affect adaptive immunity, in a single human cell-based screen? If you had the ability to do this within a few minutes, and be able to compare results of new therapeutic pipeline compounds to marketed ones so you could pick out the ones with greatest competitive potential?

Until now, most researchers have been using single or multi-cytokine activity assays, using ELISA or RT-PCR, or measuring TNFα or TNFα receptor levels to screen for compounds with the potential for anti-infectious or anti-inflammatory applications. You have to do a whole battery of follow-on experiments to find out just what your potential drug candidate might be triggering further downstream, and from there, its potential application. So what if we had a way to precisely monitor and differentiate the downstream effects of TNFα receptor activation, and see these effects within minutes of adding the compound to the screen? A pretty useful early screening tool?

In looking for a better, more direct way to measure TNFα receptor activity, we turned to lipid bioanalysis of human primary cell models, using mass spectrometry. Recent evidence suggests that lipids are effectors of TNFα receptor activation; indeed TNFα triggers lipid metabolism, which might be the pathophysiological basis of atherosclerosis, diabetes and coronary heart diseases, for starters. The complexity of TNFα-mediated lipid metabolism and the roles of various lipid metabolites in distinct signalling pathways is becoming increasingly better understood, and this presents an opportunity to use this information to design better ways to find and validate new drug candidates.

A closer look at lipid metabolism suggests that they could be ideal biomarkers for drug discovery because they have the potential to differentiate drugs that affect innate from those that affect adaptive immunity signalling pathways, for example. Apparently, different lipids are involved in each. This could be invaluable for stratifying compounds that might be useful anti-infectious, or anti-inflammatory agents for example. So, by measuring changes in certain lipids, we were hoping to find dynamic, sensitive and differentiating biomarkers of immune/inflammatory response. This appears to be the case.

Assessing the dose response on HAoEC (Human Primary Aorta Endothelial Cells) with TNFα in the presence/absence of an anti-TNFα compound reveals a lipid profile that is clearly directly responsive to the TNFα receptor activity. These lipids fall back to base line when anti-TNF-alpha is added – showing that lipidomics can be used to effectively demonstrate a receptor-mediated process.

Differences in Ceramide levels measured in HAoEC activated with TNFα (blue bars) and activated with TNFα in the presence of anti-TNFα antibodies (red bars).

We’re now monitoring the effects of different compounds on lipid profiles to find those that have effects on the TNFα receptor, and to group them according to the various pathways that they might initiate, on the basis of which lipids they increase. We’re also able to compare the lipid profiles of new drugs with marketed ones, and to see what happens when we add potential anti-inflammatory compounds to human cells that have been stimulated with LPS to trigger inflammation.

For more information on how we’re using lipidomics in drug discovery, see our REACT-Lipid service, or just give us a call on +33 (0) 4 50 43 84 91.

Lipids As Biomarkers in Immune Mediated Inflammatory Diseases

Karen — Thu, 03 May 2012 09:56:11 +0000

A very attractive feature of lipids as potential biomarkers in drug discovery and development, is that they can be readily measured in in vitro systems, and non-invasively in the clinic, from easily accessible samples such as plasma, tears and skin patches.

Recent literature underlines the potential of lipid biomarkers for use in immune mediated inflammatory diseases. Here at CellMade, we have identified and patented (pending) different lipid profiles for monitoring the modulation of cytokine receptors, for potential use in drug discovery screens and in clinical diagnostics.

What’s the Evidence So Far?

1. Lipids As Key Modulators in Innate Immunity

Lipid remoduling is essential to regulate phagosome signaling. This is shown by the modulating effects of extracellular factors (like IL-4) on lipid remodeling and protein recruitment during phagocytosis ¹
Link between dietary fatty acid uptake, inflammation and immunity.²
Macrophage membrane lipid composition is critical to gene expression and cytokine synthesis against microorganism infection ²
Statins, by modulating sterol synthesis, increase the number of microbial extracellular traps used by neutrophils and macrophages in innate immunity.⁴,⁵,⁶
Lipid production in the response to pathogen challenge:link between LPS-induced lipogenesis and inflammasome-mediated inflammatory response in macrophages.⁷,⁸,⁹
Oxidized phospholipids are mediators of angiogenesis in endothelial cell inflammation. ¹⁰,¹¹

2. Lipids As Key Modulators in Adaptive Immunity

The sphingolipid system influences the host protection and immunity against virus infections.¹²
Stimulating endothelial cells in vitro causes changes in lipid profiles. In our own experiments, when peripheral blood mononuclear cells (PBMCs) are stimulated with LPS they secrete TNFα that, by binding to the TNFα receptor, stimulates human aorta endothelial cells (HAoECs) to express COX2 and the subsequent production of PGE2. After longer periods of co-culture this also results in changes in lipid profiles. Download our poster to learn more: CellInsight PBMC-EC

References
1. Sandra de Keijzer and al. IL-4 alters Early Phagosome phenotype by modulating class I PI3K dependent lipid remodeling and protein recruitment. Plos One (2011) Vol.6 (Issue 7), pp 1-13. ↩
2. Axel Schoeniger and al. The impact of membrane lipid composition on Macrophage activation in the immune defense against Rhodococcus equi and Pseudomonas aeruginosa. Int.J.Mol.Sci. (2011) Vol.12, pp 7510-7528. ↩
3. Axel Schoeniger and al. The impact of membrane lipid composition on Macrophage activation in the immune defense against Rhodococcus equi and Pseudomonas aeruginosa. Int.J.Mol.Sci. (2011) Vol.12, pp 7510-7528. ↩
4. Samia Burridge. Lipids in immunity: statins set up bacterial traps. Lipidomics Gateway (2010) (doi: 10.1038/lipidmaps.2010.36). ↩
5. Chow, O.A. and al. Statins enhance formation of phagocyte extracellular traps. Cell Host & Microbe (2010) (doi: 10.1016/j.chom.2010.10.005). ↩
6. Brinkmann, V. and Zychlinsky, A. Benefical suicide: why neutrophils die to make NETs. Nature Reviews Micrpbiology (2007) Vol.5,pp 577-582. ↩
7. Olive Leavy. Innate Immunity: linking lipids and inflammasomes. Nature Reviews Immunology(2011) Vol.11, pp368-369. ↩
8. Im SS and al. Linking lipid metabolism to the innate immune response in macrophages through sterol regulatory element binding protein-1a. Cell Metab. (2011) vol.13,pp 540-549. ↩
9. Nitya Krishnan and al. Mycobacterium tuberculosis lineage influences innate immune response and virulence and is associated with distinct cell envelope lipid profiles. Plos One (2011) Vol. 6 (Issue 9), pp 1-16. ↩
10. Yvone Bordon. Innate immunity: a new vein of TLR biology. Nature Reviews Immunology (2010) Vol.10, pp 748-749. ↩
11. West X.Z. and al. Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands. Nature (2010), Vol.467, pp 972-976. ↩
12. Young-Jin Seo and al. Does cytokine signaling link sphingolipid metabolism to host defense and immunity against virus infections? Cytokine Growth Factor Rev. (2011)Vol.22 (No.1), pp 55-61. ↩

A New Way To Evaluate Inflammatory Effects Of Drug Candidates

Karen — Wed, 02 May 2012 07:30:36 +0000

The global inflammatory therapeutics market was estimated at $57.8 billion in 2010, representing a cumulative annual growth rate of 7.6% between 2002 and 2010. – GBI Research.

Estimates for the future Compound Annual Growth Rate (CAGR) of this market are around 5.8% between 2010 and 2017, to record a sales value of $85.9 billion. A key driver for this growth is the emergence of a strong pipeline of new anti-inflammatory drugs.¹

Developers of any new anti-inflammatory drugs aiming to enter this fast-growing market need a reliable platform to enable them to select safer and more efficacious drug candidates. Being able to quickly and reliably identify and characterize anti-inflammatory effects during compound library screening and preclinical studies is evermore critical, in order to effectively focus the considerable resources needed to progress compounds through clinical development.

A good way to provide specific and quantitative selection criteria is to assess the effects of new compounds on inflammasome components ² in human primary cell-based models, and compare the results with benchmark standard of care therapeutics. Human primary cells offer distinct advantages over traditional cell culture assays, including the ability to assess cells from relevant cell types from patients as well as offering the opportunity to observe the “normal” situation in healthy individuals. Because they haven’t been cultured over numerous passages, they provide a more realistic representation of the human situation in vivo, which will inevitably improve the quality of selection decisions, and ultimately reduce clinical stage failure rates. ³

We have found that combining cytokine/chemokine analysis with lipidomics profiling in human primary cell models offers an effective way to assess the effects of anti-inflammatory drugs, as well as to indicate undesirable pro-inflammatory effects as part of a compound safety screen.

Recent evidence strongly suggests that activation and deactivation of the TNF alpha-receptor, monitored by lipid profiles measurements on cells and tissue, is directly relevant to inflammatory processes that underlie infectious diseases ⁴ as well as the onset of chronic diseases such as atherosclerosis, Crohn’s disease, type 2 diabetes ⁵.

Whilst cytokines can appear very quickly after an inflammatory stimulus, they do so predominantly via the central P38 MAP kinase signalling pathway which is by no means limited to inflammation. Lipids, on the other hand, are linked to alternative pathways and specifically respond to changes in TNF alpha receptor activation⁶. Therefore, using both cytokines and lipids to monitor the effects of compounds in cell models provides a way to monitor inflammatory effects more dynamically, sensitively and specifically than any one alone.

If you want to learn more about how we monitor inflammatory effects in primary human cell models, please just call us on +33 (0) 4 50 43 84 91 for a no-obligation, scientific discussion about whether this could be a useful approach for your project.

Launching Inflammatory Cell Biology Services

Karen — Mon, 27 Feb 2012 13:04:03 +0000

Support For Biomarker & Drug Discovery

February 2012: CellMade, Chappes, France: CellMade, a specialist provider of inflammatory cell biology products and services for biomarker and drug discovery, announces the launch of its new website. Reflecting the company’s leading position in using human cell-based technology to better inform research decisions, the new site features free poster and protocol downloads as well as updated information on the CellInsight™ platform and REACT-lipid™ service. www.cellmade.com looks set to become a primary resource for drug discovery and preclinical researchers working in inflammatory diseases.

Proprietary human primary co-culture models, primary human cells and stem cells, coupled with cytokine secretion assays and lipid bioanalysis, enables the CellMade team to identify and characterise the best biomarkers of early inflammatory response for drug discovery projects. These are critical tools to help companies to evaluate the safety and efficacy of their new drugs. In addition, the company uses its unique technology to develop companion tests for personalized medicine.

Ronald Bronsaer, Chief Executive Officer, CellMade said: “There is a growing need for effective companion tests for anti-inflammatory drugs, as patients and FDA concerns grow regarding the costs and unwanted side effects of the TNF-alpha antagonists. CellMade technology is being used to identify people who would benefit from such treatments, and to giving early warning signs of toxicity before clinical symptoms develop. The new website is a perfect vehicle to showcase our expertise and specialist knowledge in this area.”

To find out more about inflammatory cell biology support, and to contact CellMade about a specific discovery project, visit: www.cellmade.com

End

Jos Paquaij

Editor — Fri, 27 Jan 2012 13:14:31 +0000

Chief Scientific Officer… Read the rest

Going Back to Basics For Drug Discovery R&D – Human Primary Cells

Editor — Sun, 15 Jan 2012 15:26:55 +0000

Because of the difficulties and high costs associated with obtaining human tissue and cell samples for drug research purposes, pharmaceutical R&D has come to rely on non-human biology-based testing and computational prediction methods for drug target research, and safety and efficacy screening of potential new therapies. This has led to significant unreliability in predicting drug behaviour in man, contributing to high failure rates in clinical development.

For this reason, CellMade has focused on enabling drug discovery researchers to gain easy access to human primary cells and co-culture systems, and to use these systems to identify biomarkers that could be reliable indicators of drug behaviour in vivo. By generating data from primary human cells in early drug discovery, we can enable better candidate selection decisions, improve the design of preclinical studies and help to de-risk clinical development stages.

There is a growing need for effective companion tests for anti-inflammatory drugs, as patients and FDA concerns grow regarding the costs and unwanted side effects of the TNF-alpha antagonists. This move towards more “personalised medicine” is one area where Cellmade’s technology could be used to identify people who would benefit from such treatments, in addition to giving early warning signs of toxicity before clinical symptoms develop.