April 15, 2016 (Vol. 36, No. 8)
To Focus Your Resources and Develop the Next Winning Drug, Try More Rigorous Assays and Models
It may sound harsh, but drug candidates that are bound to be poor performers should be identified sooner rather than later—and ruthlessly eliminated from development. Granted, early in development, drugs tend to wade into the shallow end of the pharmacokinetic pool, and it can be hard to tell which drugs will thrash and flounder later in development, e.g., deep into clinical trials or even clinical use. That’s why a little cruelty early in development can, in the long run, be the kindest thing, really.
By quickly exposing a drug’s weaknesses, drug developers can avoid coddling a loser that is destined to waste financial resources—or worse. New prescription drugs have about a one in five chance of causing serious adverse reactions in patients.
Typically, a drug candidate’s prospects are assessed by means of ADME/Tox, the profiling of the drug’s absorption, distribution, metabolism, and elimination properties, as well as its potential for toxicity. Yet even ADME/Tox can fail to identify problems before they impose serious losses.
ADME/Tox has certainly been a priority since the sequencing of the human genome, a period in which the number of drugs in development has increased 62%. But even though research and development costs have doubled during this time, the average number of new drugs approved by the Food and Drug Administration (FDA) continues to decline.
More rigorous ADME/Tox assessments are needed. Such assessments require careful analysis, exact planning, and maybe a little luck. In this article, we’ll not dwell on luck, except to say that we agree with Benjamin Franklin, who called diligence the mother of good luck. As for diligence, and what it means in the context of ADME/Tox, we’ll have the benefit of several expert views. These views make up the balance of this article, and they clarify how ADME/Tox can provide data critical for selecting preclinical candidates.
Liz Roquemore, Ph.D., technology manager, cell applications, GE Healthcare, provided a perspective. “In recent years, it has become increasingly clear that there is an advantage to obtaining ADME/Tox data at high throughput from more relevant cell/tissue models as early in the drug discovery process as possible,” she says. “Undesirable properties and potential toxicities can be identified and corrected before too much effort and expense have been invested.”
“This ‘fail early’ approach is anticipated to reduce the number of costly program failures and decrease the risks/costs associated with toxic and/or ineffective drugs progressing to the clinic,” Dr. Roquemore adds. “In order to be successful at this stage, the methodologies need to be relatively high throughput to enable screening approaches, since there are more candidates to contend with at this point.”
Relevant methodologies include high-throughput pharmacokinetics (the examination of biochemical properties) as well as high-throughput microscopy and image analysis (the detailed phenotyping of drug effects at the cellular level), both of which may be accomplished with GE Healthcare products. Specifically, high-throughput pharmacokinetics and high-content analysis (HCA) can be pursued with Biacore and IN Cell Analyzer products, respectively.
According to Dr. Roquemore, cell-based and other in vitro assays could all be run individually and on diverse platforms (both imaging and nonimaging formats). Yet there may be advantages to more consolidated approaches.
“HCA approaches enable many such assays to be combined in a single imaging run,” she points out. “This provides a significant speed advantage while also enabling more robust correlation of results across endpoints.”
“With the addition of fluorescent dyes and immunomarkers, image-based HCA assays enable simultaneous assessment of a host of different toxicity indications,” Dr. Roquemore continues. “These include assessment of cell viability, mitochondrial toxicity, calcium flux, apoptosis (caspase, annexin V, etc.), and oxidative stress, to name a just few. By following multiple toxicity endpoints in the context of the same cell background, it is possible to build up distinct profiles for different drug treatments.
“This approach provides insight into mechanisms of toxicity and also has great predictive potential, as ‘libraries’ of phenotypic profiles can be established for reference compounds of known toxicity/mechanisms. IN Cell Analyzer platforms are capable of automating the HCA process, and can be flexibly configured, for example, with optional liquid handling, environmental control, and objective choices for specific drug discovery applications.”
3D Liver Models
James J. Cali, Ph.D., research director, Promega, describes the value of in vitro methods for evaluating the ADME/Tox profiles of drug candidates: “It all depends on how well in vitro methods predict in vivo outcomes. Recent improvements to the predictive value of in vitro toxicity testing come from advances in 3D cell culture and assay chemistries.”
“For example,” Dr. Cali points out, “3D liver microtissues improve on 2D hepatocyte cultures for predicting liver toxicity. We apply drugs to the microtissues and detect toxicities using our cell-viability assay (CellTiter-Glo® 3D), which that we formulated for optimal performance with 3D cultures.”
Dr. Cali also suggests that an especially predictive area of in vitro ADME work focuses on the role of hepatic cytochrome P450 (CYP) enzymes as mediators of adverse drug-drug interactions: “Typically, a known CYP substrate is used as a probe with hepatocytes, liver microsomes, or recombinant CYPs (rCYPs). The system is then interrogated with drug candidates to detect their capacity to induce CYP activity in hepatocytes or inhibit the activity (in microsomes or rCYPs).
“CYP induction in vitro predicts induction in vivo where enhanced activity toward a co-administered drug accelerates its clearance and reduces efficacy,” explains Dr. Cali. “In vitro, CYP inhibition predicts the potential for toxic accumulation of a co-administered drug due to reduced CYP-dependent clearance.”
Pathways of Elimination
ADME/Tox assessments also need to consider drug-drug interactions (DDIs), which necessarily involve the “E” in ADME/Tox. “Understanding the pathways of drug elimination from the body,” says Chris Patten, Ph.D., research and development program manager, Corning Life Sciences, “is critical for reducing the chances that a new drug candidate will be involved in a DDI in patients.”
According to Dr. Patten, a thorough understanding of all routes of elimination is required by regulatory agencies for new drug approval. To emphasize this point, Dr. Patten point out that “drugs with a single route of elimination, either by a metabolic enzyme (for example, CYP) or drug transporter, are more at risk for a DDI, and will typically not be approved for patient use.”
In the past, Dr. Patten recalls, pharma faced many technical challenges for pathway identification or reaction phenotyping: “Mistakes in identification could be very costly. Insufficient knowledge of the pathways of elimination was a common cause of candidate failure in clinical trials.
“Nowadays, however, accurate pathway identification can be achieved using relatively inexpensive in vitro ADME products such as recombinant enzymes and drug transporters, human and animal tissue fractions, chemical- and antibody-based enzyme inhibitors, and enzyme/transporter probe substrates specific for a single enzyme or drug transporter.”
Elimination analysis can be accomplished with various in vitro ADME/Tox products. Such products from Corning include Supersomes™, TransportoCells™ and UltraPool™ 150-donor-HLM (human liver microscomes).
Dr. Patten also notes that the FDA and the European Medicines Agency have each recently published guidance documents on the use of in vitro ADME products for analyzing drug candidate ADME/Tox properties: “New pharma companies should review the guidance documents. Personnel from these companies are encouraged to attend the many available workshops that provide training on the latest in vitro ADME/Tox technologies.”
Screening assays are becoming more complex, says Laura Moriarty, Ph.D., marketing manager, drug discovery and development, Bio-Rad Laboratories. Also, higher throughput and multiple parameters are often being interrogated at the same time to speed up time-to-answer.
“At the cellular level, important data include cell viability, cell proliferation, and cell death,” she informs. “At the molecular level, understanding if a drug affects gene expression gives insights into how the drug will be handled in vivo.”
Dr. Moriarty indicates that some laboratories are screening drug candidates in vitro. These laboratories, she says, are “looking at the change in expression of cytochrome P450 genes, which produce enzymes known to metabolize specific drug classes.”
According to Dr. Moriarty, streamlining workflow via automation makes the whole process more efficient. In support of this point, she cites a Bio-Rad-specific example: “Our company’s semi-automated gene expression analysis workflow, which involves the CFX Real-Time PCR system, CFX Automation System II, and PrimePCR™ Pathway Plates, streamlines a researcher’s quantitative polymerase chain reaction (PCR) workflow.”
Researchers also can employ Western blot analysis to examine protein expression changes to assess ADME/Tox effects. Dr. Moriarty says it’s important to utilize a Western blotting workflow and antibodies that provide a truly validated quantitative protein analysis.
Finally, Dr. Moriarty asserts that multiplexing is becoming increasingly important: “For those researchers who want to analyze multiple proteins simultaneously, multiplex immunoassay technology allows for the measurement of multiple analytes or markers of toxicity simultaneously in a single sample. This is especially important when working with precious samples or those with only small volumes.”
Multiplex ADME/Tox assays, suggests Dr. Moriarty, include the Bio-Plex Pro™ RBM Kidney Toxicity Assays. These assays, she insists, “constitute a highly relevant set of biomarkers for early detection and characterization of kidney toxicity and injury.”
Amar Thyagarajan, Ph.D., senior product manager, Taconic Biosciences, advises that companies should employ both in vitro and in vivo models for ADME/Tox. “Animal models enable multiparametric measurements of ADME of a drug in a living system and can provide insights into drug disposition in humans,” he says. “They can be critical in evaluating metabolism pathways, clearance rates, identifying active metabolites, and assessing safety of drugs.”
Dr. Thyagarajan also notes that researchers can employ genetically humanized models to address mechanistic questions related to drug metabolism: “For example, genetically humanized models can be used to address which CYP450 enzymes and drug transporters may be critical for in vivo metabolism and disposition of a drug, identify contributions of liver versus gut in drug metabolism, and which metabolism pathway may lead to formation of active metabolites.”
Dr. Thyagarajan adds that improvements in genetically engineered animals have enhanced their their ability to reflect metabolic complexities, as well as their suitability for ADME/Tox analysis relevant to humans.
“A recent study reported development of a mouse in which 33 mouse drug metabolism genes were replaced with their human counterparts to create one of the most complex humanized models available,” he points out. “Such models have been used to model human drug metabolism and DDIs in the mouse.”
Companies should pay close attention to changing guidelines in the industry as well, according to Dr. Thyagarajan. In particular, he cites the International Council on Harmonization S1, which provides guidelines for carcinogenicity testing of compounds.
At present, explains Dr. Thyagarajan, the standard for such testing is the two-year rodent study. Yet changes are being proposed that may lead to revised criteria. For example, alternative approaches, such as the rasH2 model from Taconic, could be used to add value to the existing approach to carcinogenic risk assessment. It is even possible, suggests Dr. Thyagarajan, that requirements for the two-year study could be waived, helping study sponsors save time and money.
Dr. Thyagarajan, like the other experts cited in this article, recognizes that drug development faces rising regulatory pressures and an increasing cost-containment focus. These factors, all the experts agree, put great emphasis on obtaining robust ADME/Tox data as early as possible during drug development.
Examining Tissue Distribution and Pharmacokinetics of Lipidated Chemerin
Targeting delivery of peptide therapeutics to specific cellular locations still poses significant complications for drug development. Plasma membrane targeting can be achieved by addition of lipid modifications, or lipidation. While lipidation can improve drug efficacy by increasing bioavailability and peptide stability, it also changes the pharmacokinetic (PK) and pharmacodynamic (PD) properties, making it necessary to characterize the lipidated version.
Researchers in the Kopin laboratory at Tufts Medical Center and the Kumar laboratory at Tufts University are studying the impact of lipidation for targeting peptide hormone receptors, such as the chemerin receptor, a G protein-coupled receptor involved in modulation of inflammation and neuropathic pain.
In a 2014 study published in the Journal of Biological Chemistry, the group was able to demonstrate that a synthethic, lipidated version of the chemerin C-terminus decreased allergic airway inflammation and neuropathic pain in vivo.
To examine the PK behavior and tissue distribution of systemically administered lipidated chemerin and with an ultimate goal of developing lipidated chemerin into a new biologic product, the Kopin lab partnered with Biogen, a global biotechnology company, and WIL Research, a contract research organization specializing in discovery support services such as ADME, bioanalytical chemistry, and pharmacokinetics.
“Advancing efforts around targeted drug delivery of new medications is core to finding the next true breakthrough in treating disease, and we have been fortunate to work with partners such as Tufts and Biogen to understand the power of lipidation,” says Prathap Shastri, PK study director at WIL Research. WIL Research designed and executed studies to determine the pharmacokinetics and tissue distribution of the lipidated chemerin using 125I-radiolabeling and quantitative imaging techniques. Taken together with the in vivo studies, the data support lipidated chemerin as a viable therapeutic for drug development.