Drug discovery and development is a high-cost/high-risk endeavor. On average, the process to bring a candidate from conception to market takes 12–15 years and costs, at current estimates, $1.8 billion. Whether or not a drug is a blockbuster, late-stage attrition due to on- or off-target toxicity can be devastating for a company.
At the recent “Predictive Human Toxicity and ADME/Tox Studies” conference sponsored by the Mondial Research Group, the overriding theme was the importance of assessing the druggability of test compounds early in the development cycle to avoid costly late-stage attrition. By identifying ADMET issues early, pharma has the opportunity to increase the probability of success, decrease overall costs, and reduce the time to market.
Early Assessment of Toxic Potential
The missionary work has been accomplished. Companies are incorporating in vitro models to assess ADMET profiles earlier in the developmental process.
By way of example, UCB Pharma
understands the value proposition. Six years ago, Franck Atienzar, Ph.D., was brought in to head the newly formed in vitro toxicology unit at UCB with the charter to establish a set of validated assays and protocols to monitor the toxic characteristics of all small molecule compounds in the drug discovery pipeline.
Dr. Atienzar’s team works closely with each of the R&D groups in the different therapeutic areas to monitor all compounds in the pipeline by putting them through a rigorous battery of cytotoxicity, genotoxicity, and phospholipidosis tests in vitro. The derived profiles are used to rank the compounds and select the best candidates for further development. They also work with R&D teams to find ways to reduce toxicity while maintaining or improving potency without loss of specificity. Compounds that make it through to lead optimization are then subjected to an in vivo toxicogenomics screen.
The team has established cell-based assays to assess the cytotoxic potential of drug candidates using cell lines derived from human and rat hepatocytes. After overnight incubation, the equilibrated cells in 96-well plates are exposed to different concentrations of the test compound for 2 and 24 hours. A multiplexed evaluation of each well provides a measure of cell viability, membrane integrity (LDH levels), energy levels (ATP measurement), and apoptosis (caspase activity) to determine the LC50 value.
Primary human hepatocytes are the gold standard but suffer from problems with availability, variability, and price. The current standard protocol uses HepG2 cells, an established cell line derived from human hepatocarcinoma cells. The downside is that HepG2 cells don’t metabolize compounds to the level seen in primary cultures. Thus, the cells provide a good measure of the toxic potential of the mother compound but don’t enable a measure of the impact of compound metabolites.
“We are constantly on the search for new cell lines and cell types that will provide improved results from this analysis,” explained Dr. Atienzar. “We are currently investigating the use of human cryopreserved hepatocytes.”
Genotoxicity is the measure of the mutagenic potential of the NCE. Compounds that show a high mutagenic potential are pulled from the development pipeline, unless medicinal chemists can alter the structure to reduce the mutagenic potential without losing potency and on-target specificity. Dr. Atienzar’s group uses a battery of tests to monitor genotoxicity, including VitoTox, GreenScreen, and Ames II.
Both VitoTox and GreenScreen are high-throughput assays that look at mutagenic potential by monitoring the induction of DNA-repair enzymatic activity in bacteria and yeast, respectively. The results from the HTS assays are confirmed using the Ames II test to monitor mutagenic potential by looking at the ability of bacterial cells to grow on a selective media. All three in vitro tests are predictive of a miniAmes test outcome, the gold standard for regulatory approval by the FDA.
Phospholipidosis (PLD) is the accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies, an adverse effect of many drug candidates. To avoid the need for costly animal studies, Dr. Atienzar identified 11 biomarker genes that are present in HepG2 cells in response to compounds that are known to induce PLD. By measuring the gene-expression levels of these biomarkers, Dr. Atienzar defined an index, PI, that shows a dose response to PLD-inducing compounds but not to negative compounds.
Compounds that UCB selects for lead optimization are also subjected to an in vivo toxicogenomics screen. To do this, the team harvests tissue samples from rats exposed to the test compounds for analysis using the ToxFX Analysis Suite from Affymetrix
. The data output provides toxicological drug signatures and identifies biological pathways to predict safety problems or unanticipated off-target effects in liver, heart, or kidney.
The array data derived from 2,073 probes sets represents a nonredundant union of heart, kidney, and liver classifier genes; nonredundant genes from toxicologically important pathways; a comprehensive set of cytochrome P450 genes; xenobiotic metabolism genes; and genes involved in stress-signaling, adaptive response, cell cycle control, DNA repair, inflammation, and tissue repair.
Filling the Gap
What are you to do if you don’t have an internal team to provide you with the analysis to guide your drug development process? Fortunately, there are a number of CROs that have such expertise. I had the opportunity to speak to a few of them directly. CeeTox
takes a cell-based approach when looking at toxicity potential using a number of different cell models to assess not only hepatocellular-based toxic potential but also cardiotoxicity.
The company has introduced the use of neonate rat cardiomyocytes cultured in 96-well plates to monitor the potential for cardiotoxicity of a drug candidate. In addition to monitoring cellular proliferation and morphological changes, it monitors biochemical markers (e.g., troponin and glutathione) released into the spent media and gene-expression profiles for hypertrophy, apoptotic induction, and oxidative stress. A really cool feature of these cells is that they actually have a beat resonance that enables the determination of arrhythmia as another indicator in the toxicological profile.
I spoke with CeeTox’ CEO, Jim McKim, Ph.D., to learn what else he had in his bag of tricks to provide an early success profile of the drug candidates he screens. “Overall, CeeTox provides a unique and robust assessment of the risk for adverse events that may be associated with new drug candidates.”
“We have developed a proprietary algorithm that incorporates multiple endpoint analysis of key biochemical functions, dose-response profiles, Pgp interaction, solubility, metabolic activation and stability, drug-drug interaction factors, and in vivo validation to provide an estimate of the sustained blood concentration in a rat, 14-day, repeat-dose study where toxicity would first be expected to occur (Ctox).”
Because the Ctox value represents a threshold for toxicity, the expectation is that approved drugs will have a maximum therapeutic plasma concentration value below the Ctox value. Indeed 97% of the approved drugs do not reach or exceed the Ctox value. Compounds with Ctox values (mM) in the range of 1–20 have the highest probability of in vivo toxicity and should be carefully evaluated before moving forward. Compounds in this category should be reviewed for potency, expected plasma concentrations, duration of exposure, and therapeutic endpoint.
Compounds with values in the range of 21–50 are flagged with caution based on their impact on chronic markers of toxicity that have been associated with drugs that have been withdrawn from the market for toxicity. Nafezadone is an example of one such drug, according to CeeTox. Compounds with values in the 51–300 range have the lowest probability of in vivo toxicity. The company continues to add known drug compounds to its database to improve the predictive power of its in vitro testing portfolio.
“We combine the results of nine different biochemical assays to predict the point at which toxicity will first occur in vivo and give detailed information on subcellular sites of toxicity, all designed to expedite the lead-optimization process,” said Dr. McKim. “We provide in vitro assays that evaluate CYP enzyme induction, metabolic activation, and CYP inhibition to evaluate the drug-drug interaction potential of a compound. We also evaluate cardiotoxicity, lipidosis, endocrine interactions, and antitumor candidates.”
Drug permeability is the key first step, without which all other parameters tested in vitro are moot. Validated in vitro models to assess drug absorption in the intestine have been around for sometime now. PAMPA technology has been applied to the measure of passive diffusion of drug candidates through biological lipid membranes coated on filter plates. Though more challenging to set up, the cell-based assay using CaCO-2 cells allows for the additional measure of permeation based on active transport via phosphoglycoproteins (PgP) and other active transporters in the cell membranes.
The development of validated in vitro models for assessing drug absorption across the blood-brain barrier, however, has been slower. The need to develop these models has been driven by the failure of in silico methods to offer accurate predictions as well as the cost and low throughput of animal models. A cell-based assay method, similar to the CaCO-2 assay, has been developed based on plating bovine brain microvessel endothelial cells and the capillary-enriched fraction from bovine brain homogenates isolated from fresh cow brains on filter plates. Given the challenges of growing primary cells and obtaining sufficient source material to support this methodology, an alternative would be welcome.
Hinnerk Boriss, Ph.D., CEO of Sovicell
, introduced that alternative, Transil® Brain Absorption kit, at the conference. Sovicell’s Transil technology platform is a bead-based system formatted in 96-well plates for the ease-of-use measure of membrane permeability and serum-protein binding (albumin and a1-acid glycoprotein). The Transil Intestinal Absorption kit for the modeling of intestinal absorption has been on the market for a long time.
“While clearly an essential determinant for the whole class of drugs that have a CNS therapeutic focus,” noted Dr. Boriss, “we also developed the Transil Brain Absorption kit to provide the ability to assess the potential for off-target effects of drugs that have a different therapeutic target.”
Also new on the market is the ability to assess inhalation routes of drug delivery, an expedient and relatively noninvasive route of delivery for respiratory therapeutics, CNS drugs, and pain analgesics. In speaking with Katya Tsaioun, Ph.D., president of CRO Apredica
, I learned that the company has an in vitro assay protocol in its portfolio to screen drug candidates for permeation of nasal and lung membranes.
Apredica’s intranasal and lung absorption and toxicity assays are performed using MatTek’s
EpiAirway System. The system consists of normal, human-derived tracheal/bronchial epithelial cells that have been cultured to form a pseudo-stratified, highly differentiated model that closely resembles the epithelial tissue of the respiratory tract. Test agent is added to one side of the tissue, and permeability is assessed using HPLC or LC/MS.
Another important service at Apredica is its hERG and Drug-Drug Interaction Screening Assays provided in collaboration with ChanTest
. At issue is the observation that most pharmaceuticals are metabolized by only a small number of liver enzymes. CYP 3A4 metabolizes 40% of all currently marketed drugs. Since most patients take multiple drugs, the potential that a drug is safe when given alone but accumulates to toxic levels when given in combination with drugs metabolized by the same overused enzyme is a significant possibility.
The classic example of this is Seldane, which was pulled from the market in 1998. The hERG-450™ screening assay is available in two protocols: the rapid screen, which combines a fluorescent-based CYP 3A4 inhibition screen with the hERG channel screen; and the profile screen, which screens CYP 3A4, 1A2, 2C9, 2C19, and 2D6 combined with a panel of ion channel assays including hERG.
“We formed Apredica to provide the biotech and small pharma market segment with consulting and service-based support for all its early ADME Tox screening needs,” shared Dr. Tsaioun. “We provide our customers with a complete workup from physico-chemical characterization of the drug candidates to in vivo PK and bioanalysis studies.”
Considering Reproductive Health
And finally, an area that is not often considered in the discussion of in vitro ADMET testing, assays to screen compounds for their influence on the reproductive cycle and embryonic development. I spoke with Rita Cortvrindt, CEO of EggCentris, about her company’s intricate in vitro bioassays that closely mimic the in vivo physiology of the target systems of the reproductive system. EggCentris has developed a number of multiparametric in vitro bioassays that are applicable to screening chemical and pharmaceutical compounds.
“Our primary assay, the follicle bioassay, provides for the simultaneous evaluation of folliculogenesis, steroidogenesis, and oogenesis,” explained Cortvrindt. “The assay mimics in vitro the physiological processes of the ovary and allows the prediction of the impact on female health and fertile capacity.”
Coupled with the in vitro maturation assay to evaluate the meiotic process of the oocyte and the in vitro fertilization assay to evaluate perturbation of the conception process, EggCentris evaluates all aspects of oocyte developmental competence.
To look at the impact of a compound on embryo development, the company also developed the Mouse Embryo Peri-implantation Assay and performs the Embryonic Stem Cell test to analyze preimplantation embryo development and predict embryo toxicity, respectively.
“These assays were developed applying the 4R principle for laboratory animal use: replacement, reduction, refinement, and relevance,” reported Cortvrindt. “Replacement: in vivo studies done by a battery of in vitro bioassays; reduction: bioassays well designed to maximize the output and minimize the need of animals for primary tissue cultures; refinement: tissues are treated ex vivo, avoiding animal exposure and distress; and relevance: the bioassays deliver relevant information that correlates with the in vivo physiology.”
The in vitro bioassays are also valuable tools to assess the bioactivity of new compounds for fertility treatment and anticonception or to assess the impact on the steroid pathway. They can replace or add information to ovulation induction studies to assess ovarian responsivity to gonadotropins or to embryo transfer studies to assess embryo quality and uterine receptivity.
As a contract research laboratory, EggCentris approaches the complex processes of the reproductive function with a battery of in vitro bioassays. Not all processes, however, can as yet be modeled in vitro. If the established bioassays are not adequate to resolve the question, then new, more appropriate in vitro models are developed, or carefully designed in vivo studies are included in the study project.