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Insight & Intelligence : Dec 9, 2013
In Vivo Screening for New Therapeutics and Biomarkers Gains Traction
Novel models and strategies could make drug discovery easier.!--h2>
Drug discovery efforts would be greatly facilitated by screening tools that incorporate the complexities of human disease, scientists say. They note that attempts to recapitulate true molecular diversity of tumors, much less genuine physiological interactions, with drugs in a cell culture system may often produce misleading results. As a case in point, investigators at Johns Hopkins showed in 2011 that, in head and neck squamous cell carcinoma cell lines, methylation differs significantly from that of primary tumors and xenografts.
The investigators evaluated DNA extracted from primary head and neck squamous cell carcinomas (HNSCC), xenografts grown from these primary tumors in nude mice, HNSCC-derived cell lines, normal oral mucosal samples, and minimally transformed oral keratinocyte-derived cell lines using Illumina Infinum Humanmethylation 27 genome-wide methylation microarrays.
They found greater than 2,200 statistically significant methylation differences between cancer cell lines and primary tumors and when comparing normal oral mucosa to keratinocyte cell lines, but no statistically significant promoter methylation differences between primary tumor xenografts and primary tumors.
While this study demonstrates that tumor-derived xenografts are highly accurate representations of promoter methylation in primary tumors, it also shows that cancer-derived cell lines have significant drawbacks for discovery of promoter methylation alterations in primary tumors.
These findings also support use of primary tumor xenografts for the study of methylation in cancer, drug discovery, and the development of personalized cancer treatments, scientists concluded.
While these scientists support the use of xenografts as more representative of cancer cell epigenetic changes, other investigators continue to work on in vivo models that they hope will more closely mimic what happens to a drug candidate under true physiological conditions. Some new trends have emerged, particularly the use of in vivo systems for drug screening. These systems range from fruit flies to whole animals. Companies that have developed sophisticated in vivo models for development have also attracted the recent attention of larger drug discovery and development services companies.
Under the Crown
Crown Bioscience of Santa Clara, CA, acquired the shares of U.K.-based Preclinical Oncology Services (PRECOS), a preclinical R&D service provider with a specific focus on oncology in 2013. PRECOS, formerly affiliated with the University of Nottingham, says it has unique clinically relevant models, which reflect the patient situation for each aspect of cancer progression encompassing precancerous lesions, primary tumors, and metastasis.
“The acquisition represents the next step in expanding the range of specialized services we offer pharmaceutical and biopharmaceutical customers worldwide,” said Jean-Pierre Wery, president of Crown Bioscience.
Lianhai Zhang, and colleagues working at Peking University and Crown BioSciences reported the identification of predictive biomarkers for responsiveness to the antibody cetuximab using “avatar mice” or “xenopatients”, mice with patient-derived tumor xenografts (PDXs) that mirror a patient’s histopathological profile. Large collections of the avatars reflect, they say, the diversity of tumor in patient populations.
The investigators had established a large collection of cancer PDXs by transplanting surgically removed tumor tissues from patients into immunocompromised BALB/c nude mice via subcutaneous inoculation, including many gastric cancer PDXs (GC-PDXs), to assess drug activities.
This study investigated the activity of cetuximab in 20 GC-PDX models. After therapeutic responders and nonresponders were identified following discovery of predictive biomarkers including genomic and gene expression analysis, sequencing of key oncogenes was carried out. And the expressions of candidate biomarkers were validated by quantitative PCR, immunohistochemistry, and fluorescence in-situ hybridization (FISH).
In this instance the use of an in vivo model revealed a series of predictive biomarkers that could eventually, the investigators concluded, prove useful in identifying human GC patients responsive to the antibody cetuximab.
And in a further commitment to developing in vivo drug test systems, Crown said they had an exclusive two-year collaboration agreement with Horizon Discovery, a provider of research tools to support the development of personalized medicines. The relationship continues a previous collaboration between the two companies.
Horizon will share its X-MAN (X-Gene Mutant and Normal) cell lines with Crown to provide in vivo animal services to third parties. Crown will use Horizon’s cell lines to provide animal model development, study execution, and business marketing to its clients. “The suite of in vivo models developed with Crown will ideally complement our extensive range of in vitro drug discovery tools and services. We are confident this partnership will yield some exciting results,” commented Darrin Disley, CEO of Horizon Discovery.
In Vivo Pharmacokinetics
For rapid and economical identification of novel, bioavailable antitumour chemicals, say Lee Willoughby and colleagues at the Peter MacCallum Cancer Centre and the University of Melbourne, the use of appropriate in vivo tumor models suitable for large-scale screening is key. In this case, Drosophila provides an in vivo pharmacokinetics assessment model.
Using a Drosophila Ras-driven tumor model, the investigators showed that tumor overgrowth can be curtailed by feeding larvae with chemicals that have the in vivo pharmacokinetics essential for drug development and known efficacy against human tumor cells.
The scientists developed an in vivo 96-well plate chemical screening platform to carry out large-scale chemical screening with the model. In a proof-of-principle pilot screen of 2,000 compounds, they identify the glutamine analogue, acivicin, a chemical with known activity against human tumor cells, as a potent and specific inhibitor of Drosophila tumor formation. RNAi-mediated knockdown of candidate acivicin target genes implicates an enzyme involved in pyrimidine biosynthesis, CTP synthase, as a possible crucial target of acivicin-mediated inhibition.
In June 2013, Maria Belen Jiminez Diaz, Ph.D., working with a team at GlaxoSmithKline in Spain, reported that they had evaluated an in vivo screening strategy aimed at increasing the speed and efficiency of drug discovery projects in malaria.
The new in vivo screening concept was developed based on human disease parameters, the investigators said—that is, parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR). These parameters were allometrically down-scaled into P. berghei-infected mice.
Mice with an initial parasitemia were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication or induce parasite clearance.
In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo effects.
High-throughput, high-content in vivo screening, the investigators concluded, could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines, and proposed a global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task.
In the journal Neuropsychopharmacology in 2008, Richard Houghten, Ph.D., and F. Ivy Carroll, Ph.D., proposed that an approach to circumventing high attrition rates during drug development would be to use in vivo models directly in the discovery phase to identify candidates. These screens could identify compounds with desired biological profiles while simultaneously eliminating those compounds with poor absorption, distribution, metabolism, and elimination (ADME)/pharmacokinetic properties.
The scientists note that, although combinatorial chemistry and high-throughput screening are universally utilized tools for drug discovery and development, the drug discovery process remains extremely slow and enormously expensive. Drug candidates resulting from many combinatorial approaches have also often tended not to have drug-like properties and thus have a high inherent rate of attrition in the later stages of drug development because of poor physicochemical properties.
Conotoxins and Antinociception
As a case in point, Christopher J. Armishaw, Ph.D., and colleagues at the Torrey Pines Institute for Molecular Studies in Port St. Lucie, Florida, studied the potential utility of marine cone snail venoms, conotoxins, as potential antinociceptive agents. Marine cone snails contain large, naturally occurring combinatorial libraries of disulfide-constrained peptide neurotoxins known as conotoxins, which have considerable potential for the development of analgesics.
The scientists, working in Dr. Houghten’s lab at Torrey Pines, developed a synthetic combinatorial strategy that probes the hypervariable regions of conotoxin frameworks to discover novel analgesic agents by utilizing high-diversity mixture-based positional-scanning synthetic combinatorial libraries (PS-SCLs).
They hypothesized that direct in vivo testing of these mixture-based combinatorial library samples during the discovery phase would facilitate the identification of novel individual compounds with desirable antinociceptive profiles while simultaneously eliminating many compounds with poor activity and side effects like impaired locomotion and respiration.
A PS-SCL was designed based on the α-conotoxin RgIA-ΔR n-loop region and consisted of 10,648 compounds systematically arranged into 66 mixture samples. Mixtures were directly screened in vivo using the mouse 55°C warm-water tail-withdrawal assay, which allowed deconvolution of amino acid residues at each position that confer antinociceptive activity. A second generation library of 36 individual α-conotoxin analogues was synthesized using systematic combinations of amino acids identified from PS-SCL deconvolution and further screened for antinociceptive activity.
The assay led to the identification of three lead compounds that produced dose-dependent antinociception without significant respiratory depression or decreased locomotor activity. The results, the researchers say, represent a unique approach for rapidly developing novel lead α-conotoxin analogues as low-liability analgesics with promising therapeutic potential.
And although scientists expect that three-dimensional micro-organoid systems will play an increasing role in drug testing and therapeutics over the next decade, in vivo systems for HTS will continue to be explored to get closer to true, contextual drug responses.
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