Drug Discovery World Winter11

Drug Discovery

on ion channels (including hERG) and trans- porters; isolated animal tissues including rabbit wedge for ECG and QT elongation studies; or whole animal experiments using rats, dogs, or monkeys for ECGs, hemodynamic studies, and pathological evaluation of repeat dose studies to identify cardiotoxic compounds.

Recent progress in manufacturing cardiomy- ocytes has been made using pluripotent stem cell technology. In 2009, Cellular Dynamics International (CDI) introduced the first human iPS cell-derived product, iCell&#x2122; Cardiomyocytes, while GE/Geron corporation released embryonic stem cell-derived cardiomyocytes in 2010. These human cells have the potential to reduce or replace the assay systems and animal models mentioned above. Both platforms are a pan-cardiac popula- tion consisting of ventricular, atrial and nodal cells. Extensive characterisation of these cells has demonstrated molecular, electrophysiological, and cardiotoxic function similar to native heart cells27 (Ma et al, in preparation). In addition to providing large numbers of reproducible cardiomyocytes for use in traditional toxicity testing, the in vivo func- tionality of pure populations of iPS cell-derived cardiomyocytes will enable the development of potentially more targeted, sensitive, and relevant biochemical and electrophysiological cardio-specif- ic testing paradigms. Hepatocytes: Liver toxicity is another major cause of drug withdrawal from the market. Current sys- tems for measuring hepatotoxicity include primary human hepatocytes (PHH) or human hepatocyte cell lines such as HepG2s. Both of these have major draw backs. Primary human hepatocytes must be shipped either fresh immediately after isolation or frozen, and they display batch-to-batch variation. Additionally, they are not stable in culture for more than a few days and lose P450 expression. Yet PHH remain the gold standard. Cell lines such as HepG2 express little to no P450 enzymes, the major drug metabolising enzymes, making these cell lines inappropriate model systems28. What is needed is a reliable cell supply with batch-to-batch consistency. In addition, these cells must express appropriate markers and enzymes including the P450 enzymes and Phase I and Phase II xenobiotic metabolising enzymes, thus demonstrating intrinsic hepatocyte metabolism including oxygen con- sumption and ATP utilisation, exhibiting appropri- ate cell morphology and polarisation, and having the ability to identify known hepatotoxic com- pounds. Stem cell-derived hepatocytes have been reported to exhibit many, but not yet all, of these phenotypic and functional characteristics24,29-30.

Drug Discovery World Winter 2010/11

Small molecule screening Traditional small molecule screens are performed using purified protein or recombinant cell lines over-expressing the target of interest. The industry uses such systems because they are easy to develop and configure to their platforms, are relatively inexpensive, and have a track record of success (Macarron et al, in preparation). Recombinant sys- tems are not ideal, however. The target of interest is over-expressed in a non-native cell where splice variants or post-translational modifications may not occur; iPS cells have the potential to change that. A recent example comes from a paper by McNeisch et al31 where neurons expressing a- amino-3-hydroxyl-5-methyl-4-isoxazoleproprion- ate (AMPA) subtype glutamate receptors were gen- erated from mouse iPS cells. This is a particularly challenging target because there are four subtypes, multiple RNA splice variants, and interactions with transmembrane regulatory proteins, all giving rise to a significant number of potentially expressed receptors. McNeisch et al31 successfully generated a sufficient quantity of cells to run a high-throughput screen of several million com- pounds and identified novel small molecule AMPA potentiators. Such an approach might readily be applied to other targets including ion channels, GPCRs, and receptor tyrosine kinases where full functionality requires multimers of subtypes, chap- erones, and protein complexes.

In vitro disease models

Induced pluripotent stem cell technology provides a unique opportunity to generate &#x2018;disease pheno- types in a dish&#x2019; for use as in vitro model systems and substrates for small molecules screens. While it is unlikely that an in vitro system can adequately represent all aspects of a multi-faceted disease, the ability to generate any human tissue cells from any genotype or disease provides unparalleled access for studies. There are an increasing number of examples where iPS cells have been derived from patients, but as of now only a few show measura- ble phenotypic endpoints (Table 2). The notable examples include iPS cell-derived motor neurons from patients with spinal muscular atrophy (SMA), where the derived neurons demonstrated a significantly reduced level of survival motor neu- ron 1 (SMN1) gene expression, and appropriate response to drugs known to increase SMN1 gene expression accurately mimicked the phenotype observed in SMA patients32. Another example comes from Lee et al33 who studied familial dysau- tonomia (FD), a fatal neurodegenerative disease of sensory and autonomic neurons. iPS cell-derived

Continued from page 34

9 Kim, D, Kim, CH, Moon, JI, Chung, YG, Chang, MY, Han, BS, Ko, S, Yang, E, Cha, KY, Lanza, R and Kim, KS. (2009).

Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell (6):472-6. 10 Fusaki, N, Ban, H, Nishiyama, A, Saeki, K and Hasegawa, M. (2009). Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85(8):348-62. 11Warren, L, Manos, PD, Ahfeldt, T, Loh, YH, Li, H, Lau, F, Ebina, W, Mandal, PK, Smith, ZD, Meissner, A, Daley, GQ, Brack, AS, Collins, JJ, Cowan, C, Schlaeger, TM and Rossi, DJ. (2010). Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7(5):618-30. 12 Aasen, T, Raya, A, Barrero, MJ, Garreta, E, Consiglio, A, Gonzalez, F, Vassena, R, Bilic, J, Pekarik, V, Tiscornia, G, Edel, M, Bou&#xE9;, S and Izpis&#xFA;a Belmonte, JC. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26(11):1276-84. 13 Park, IH, Arora, N, Huo, H, Maherali, N, Ahfeldt, T, Shimamura, A, Lensch, MW, Cowan, C, Hochedlinger, K and Daley, GQ. (2008). Disease- specific induced pluripotent stem cells. Cell 134(5):877-86. 14 Sun, N, Panetta, NJ, Gupta, DM, Wilson, KD, Lee, A, Jia, F, Hu, S, Cherry, AM, Robbins, RC, Longaker, MT and Wu, JC. (2009). Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci U S A 106(37):15720-5.

Continued on page 37 35

Page 1 | Page 2 | Page 3 | Page 4 | Page 5 | Page 6 | Page 7 | Page 8 | Page 9 | Page 10 | Page 11 | Page 12 | Page 13 | Page 14 | Page 15 | Page 16 | Page 17 | Page 18 | Page 19 | Page 20 | Page 21 | Page 22 | Page 23 | Page 24 | Page 25 | Page 26 | Page 27 | Page 28 | Page 29 | Page 30 | Page 31 | Page 32 | Page 33 | Page 34 | Page 35 | Page 36 | Page 37 | Page 38 | Page 39 | Page 40 | Page 41 | Page 42 | Page 43 | Page 44 | Page 45 | Page 46 | Page 47 | Page 48 | Page 49 | Page 50 | Page 51 | Page 52 | Page 53 | Page 54 | Page 55 | Page 56 | Page 57 | Page 58 | Page 59 | Page 60 | Page 61 | Page 62 | Page 63 | Page 64 | Page 65 | Page 66 | Page 67 | Page 68 | Page 69 | Page 70 | Page 71 | Page 72 | Page 73 | Page 74 | Page 75 | Page 76 | Page 77 | Page 78 | Page 79 | Page 80