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Feature Articles : Dec 1, 2011 ( )
Emerging Trends for Predictive Toxicology
Novel Models Help Illuminate Safety Issues with Preclinical Drug Candidates!--h2>
Protein therapeutics is still a relatively new class of drugs. Even after several highly successful market launches (e.g., Herceptin), our knowledge of protein drug distribution remains limited. Historically, development of protein therapeutics was geared toward candidates with the highest affinity to the target antigen or ligand. This, however, may not always be the best strategy to achieve the desired efficacy and toxicity profile.
As an increasing number of protein therapeutics moves through development, pharmacokinetic evaluation of these therapeutic agents becomes more important. For chemical entities, such evaluation typically involves the determination of four critical parameters: clearance, volume of distribution, half-life, and bioavailability.
For proteins, especially those targeting solid tumors, such mechanistic studies may be particularly complicated. There are a number of issues unique to the pharmacokinetic evaluation of protein therapeutic agents. The distribution of biomolecules depends not only on the properties of the molecule itself (e.g., charge, size, binding affinity, and dose), but also on target tissue characteristics (e.g., vascular and interstitial volumes, vascular permeability, target expression level, and pressure differences between vascular and interstitial spaces).
Endogenous production of antibodies and dynamic changes in surface receptors can also affect protein drug distribution. Further complications arise from our limited ability to assay protein drugs and to distinguish between active and inactive forms in vivo. Consequently, the potential of pharmacokinetic investigations of protein therapeutics is still unclear to many scientists. On the other hand, a lack of knowledge and insight about drug disposition may result in lost opportunities to develop more efficacious clinical candidates.
At the AAPS meeting in October, several scientists talked about their novel models for early and accurate prediction of metabolism and toxicity of investigational compounds. According to Gregory P. Adams, Ph.D., co-leader, developmental therapeutics program at Fox Chase Cancer Center, “Our study was the first systematic evaluation of the effects of affinity on in vivo tumor targeting.
“We utilized a series of intact IgG molecules with varying affinities against Her2. High-affinity antibodies such as Herceptin remained close to the blood vessels, whereas lower-affinity antibodies were able to diffuse deeper into the interior of the tumor. To compare the tumor pharmacokinetics of the antibodies, the team utilized two radiolabels, I and In.
“The first isotope gives a snapshot of intact antibody in the tumor cells,” continued Dr. Adams. “The second gives a historical record of how much antibody was taken up over time. It was evident that high-affinity antibodies were internalized and degraded by tumor cells at a higher rate than low-affinity antibodies.” Inverse correlation of efficient tumor penetration with the antibody binding affinity has a tangible impact on the exposure of the tumor to the drug. Utilization of these principles allows for rational design of antibody therapeutics.
“Treatment of small tumors or delivery of cytotoxic payload may be more effective with high-affinity therapeutics. Low-affinity drugs may be used for large tumors or when it is critical to bypass the same target present on the normal tissues,” added Dr. Adams. “We utilized these principles to create a Her3/Her2 bispecific antibody. One low-affinity arm blocks the ligand-binding activity of Her3, the other arm has moderate affinity to Her2 and facilitates effective tumor targeting. The dual approach ensures deeper penetration combined with increased targeting selectivity.”
Merrimack Pharmaceuticals licensed the work and further improved on Dr. Adams’ prototype antibody, creating MM-111, a highly optimized bispecific antibody fusion protein. The two arms of the antibody are linked by a modified serum albumin.
The company initiated a Phase I of MM-111 in breast cancer patients overexpressing the Her2 receptor. The study is enrolling patients whose cancer progressed on Herceptin or other standard therapy. The efficacy goal of the study is to evaluate progression-free survival in the enrolled patients.
The ability to predict the metabolism of new drugs in humans at early stages is critical for drug development, especially in light of FDA guidance issued at the end of 2008. The Guidance for Industry: For Safety Testing of Metabolites (or MIST) requires that metabolites present in greater than 10% of total drug-related systemic exposure require additional safety testing, in case they are present disproportionately in humans as compared to study animals.
“Cultures of human hepatocytes and liver preparations are two common systems for evaluation of drug metabolites,” commented Gerhard Gross, Ph.D., head of drug metabolism at Lundbeck. “However, these systems are stationary. By using these systems we cannot adequately predict the extent of systemic exposure of drug metabolites.”
Unfortunately, because of considerable differences in metabolic pathways between laboratory animals and humans, experiments with animals do not always correctly predict what happens in humans. Therefore, the recent development of a mouse with a human liver presents the best approach for studying human pharmacotoxicity.
One such model is a uPA+/+/SCID transgenic mouse. Livers of immunodeficient (SCID) mice are damaged by targeted expression of transgene (uPA, or urokinase-type plasminogen activator). When human liver cells are injected in these animals, the human cells colonize the mouse liver. The higher the replacement rates, the more predictable this model becomes for metabolism and toxicity studies. Careful examination of histology and pharmacokinetic parameters for chimeric mice supports their use as a model for evaluation of drug AME and also drug-induced hepatotoxicity.
Lundbeck’s new investigational compound is primarily metabolized by the cytosolic enzyme aldehyde oxidase, which is preferably expressed in humans and monkeys, with significant species differences in metabolism across other species. Therefore, obtaining an accurate human PK prediction was a challenge. Testing this compound in the chimeric mouse demonstrated a significantly higher clearance rate, indicating that the model may indeed be useful for accurate human PK predictions.
Lundbeck is continuing investigations with additional compounds that showed disproportionate metabolites in humans. “The logical application of this model is in studies of human pharmacokinetics/metabolism and also human-specific toxicities. However, the model can also potentially be used for ranking compounds, specifically oncology drugs, regarding maximum tolerated doses,” according to Dr. Gross.
“Chimeric mice carrying human livers have wide applications in both preclinical and clinical areas,” commented Elizabeth Wilson, senior scientist at Yecuris. “With tremendous shortages of healthy livers, human organs grown in laboratory animals may become a viable source for transplantation. Liver cells from a patient can be used to repopulate a scaffold of animal livers, resulting in an autologous organ.”
At present, Yecuris strategically focuses on preclinical applications, developing hepatocytes for drug discovery, toxicology, and metabolism studies. Yecuris technology uniquely enables controlled replacement of mouse hepatocytes. The mice lack fumarylacetoacetate hydrolase (Fah), a critical enzyme that in normal animals breaks down the byproduct of tyrosine catabolism. In the absence of the enzyme, fumarylacetoacetate accumulates in hepatocytes, causing cell death.
Yecuris mice survive when the production of fumarylacetoacetate is blocked upstream by Nitisinone, an approved clinical drug. Once the protective drug is withdrawn, the toxic buildup slowly and selectively damages mouse liver cells. The mice lack T, B, and NK cells, facilitating engraftment of human cells. Mouse cells undergo gradual injury over several induction cycles, which provides time for even slowly dividing human adult hepatocytes to expand sufficiently to sustain liver function. Therefore, cells from any donor of any age can be used for repopulation. Moreover, the ratio between human and mouse cells can be fine-tuned to support a variety of applications.
Recently, Yecuris improved upon the initial model to include simultaneous engraftment of liver and immune systems, which represents a significantly improved tool for toxicological studies. “While high replacement levels are required for drug metabolism studies, comparative toxicology studies may need a 50/50 ratio between mouse and human cells,” continued Wilson.
A Pfizer PPARa agonist Wy-14,16443 was tested in the Yecuris model, comparing its effect on mouse and human hepatocytes in the same treated animal. Consistent with the conventional assessments, the treatment resulted in different histological profiles, gene-expression, and proliferation rates between mouse and human cells. A marked increase in hypertrophy and increased proliferation was observed in mouse cells but not in the nearby human cells.
“For such studies, it is desirable to have a mixed ratio of mouse to human cells to provide elucidation and controls of species-specific toxicity. Also, in certain infectious disease applications, it is also beneficial to have fine control over a mixed cell population to avoid death in the host species,” said John Bial, CEO. Recently Yecuris engineered FAH knockout mutations into rats and pigs and has future plans to address new organ systems.
“Chimeric mice and in vitro models complement each other in ADME/tox studies,” commented Claudia McGinnis, Ph.D., group head, NCS early and investigative toxicology at Roche. “In vitro models are essential for screening and valuable for early toxicity predictions. The key, however, is to reproduce the properties of human tissue in a complete environment to get meaningful results.”
Roche collaborates with a number of academic research centers and start-up companies to develop models closely resembling hepatic tissues and maintain the classical hepatocyte characteristics enabling in-depth mechanistic studies.
One such model Roche is using is from RegeneMed and contains three-dimensional interweaving nanofiber scaffolds for co-culturing liver-derived stromal cells and hepatocytes. Other 3-D systems developed by MIT and CellASIC add flow-through capabilities that may further aid hepatocyte functionality.
“Primary hepatic cells have limited survival, reduced functionality, and high donor-to-donor variability,” continued Dr. McGinnis. “Sometimes the donors of these cells had significant health issues. The assays with primary cells are hard to standardize. Therefore, many scientists believe that deriving hepatocytes and other tissues from embryonic or adult stem cells is the way of the future. In addition to being a more available, standardized resource, SC-derived hepatocytes can also be genetically customized or be patient-specific.”
Roche scientists have been successful in applying stem-cell based assays in the area of toxicology and developing customized strategies to aid this field. Roche has developed a stem-cell derived human cardiomyocyte assay that is highly predictive of arrhythmia.
In addition, Roche scientists have developed a specialized “hanging droplet” culture plate that allows for the generation of stem cell-derived 3-D spheroids compatible with standard laboratory robotics, which allows full automation of a previously labor intensive manual process. Roche plans to utilize the plate in high-throughput miniaturized assays to predict for teratogenicity liabilities and other endpoints.
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