May 1, 2007 (Vol. 27, No. 9)

Vicki Glaser Writer GEN

Advances in Microdosing, In Vitro Analysis, and Biosimulation for Optimized Predictive Studies

Realizing that a promising, effective drug has toxic side effects in humans, even if only in certain subpopulations, at certain doses, or in the presence of other drugs or co-morbidities, is a costly discovery, especially when it happens late in development.

Even as such revelations continue to occur and pharmaceutical companies are repeatedly rocked by the downfall of highly touted, novel drugs designed to fulfill an unmet medical need, the industry is pursuing strategies to uncover potential problems related to bioavailability, pharmacokinetics, or toxicity earlier in the development process—even before drug candidates enter human testing.

Pharma is focusing its efforts on a variety of innovative approaches to improve and accelerate ADME-Tox assessments, including FDA- and EMEA-endorsed Phase 0 studies that rely on microdosing using radioactively labeled drug compounds; information-rich screening and in vitro analysis strategies; and in silico-based database and biosimulation methods. All have the potential to help companies select drug candidates with the most promising efficacy and pharmacokinetic properties to take forward into human clinical trials.

The goal is clear: while it currently takes 5 to 12 years and an estimated $800 million to bring a new drug to market, new approaches are needed along the drug discovery and development pipeline to lessen the duration and cost of commercializing a novel bioactive compound. Historically, the majority of compounds that failed in development did so because of poor pharmacokinetic (PK) properties. More recently, PK and ADME testing is being done earlier and in parallel with structure-activity relationship (SAR) studies during lead optimization, sending more potent and robust compounds forward into development.

As a result, the primary reason at present for drug failure in R&D has shifted to toxicity, according to Katya Tsaioun, Ph.D., president of Apredica (, a CRO focusing on the development of preclinical ADME-tox models to reduce downstream risk in drug development. Dr. Tsaioun contends that compound screening early in development can help identify potential side effects and toxicities. She highlights hERG testing for cardiotoxicity and cytochrome P450 inhibition as a drug-drug interaction screen as examples of the emerging acceptance of and regulatory requirements for in vitro assays to evaluate ADME-tox properties.

Faulty thinking and economic pressures have combined to encourage pharmaceutical companies to make the jump from the lab bench to animal studies and from animals to humans as quickly as possible to confirm efficacy of a bioactive compound. In light of recent crash-and-burn scenarios of high-profile, promising new drugs in the clinic and even post-marketing, interest has grown in early predictive cell toxicity testing, according to Dr. Tsaioun.

It is becoming clear that “if you use multiple readouts and multiple cell lines, you get better predictability,” she says. Another advantage of relying more on in vitro studies before taking a drug candidate into animals is the need for only micrograms of compound for screening assays.

In a real-life example using screening models to predict potential liabilities of compounds in development, MaxThera (, a drug discovery company focused on developing broad-spectrum antibiotics with novel mechanisms of action to overcome the growing problem of antibiotic resistance, has partnered with Apredica. The company will apply its screening assays to detect cross-species metabolism and cross-reactivity with human host biochemical pathways and to evaluate the pharmacokinetic properties of MaxThera’s lead drug candidates.

One of the goals of this collaboration is the early determination of whether novel chemical structures will have the capability for formulation as both intravenous and oral drugs. Dual bioavailability and the ability to switch from IV to oral dosing would allow for earlier discharge of patients from the hospital with the ability to continue to provide effective antibiotic treatment outside the hospital setting. PK studies can also help determine optimal dosing. In antibiotic therapy it is especially important to maintain constant drug levels in the bloodstream and in target tissues—levels above the minimal inhibitory concentration—to minimize the risk of drug resistance.

Dr. Tsaioun describes the use of in vitro microsomal studies to obtain information on the metabolic stability of compounds. More than 70% of the metabolism of xenobiotics, for example, takes place in the liver, involving the cytochrome P450 enzymatic pathways. Liver microsomes are preparations of hepatocyte membranes that contain most of the P450 enzymes. Microsomal assays yield data on compound stability, metabolite formation, and cross-species differences in metabolism, and play a valuable role in selecting drug doses for subsequent toxicology studies. Since liver microsomes represent only a fraction of liver cells, complementary assays are also needed, as are assays based on cells from other tissues and organs where drug metabolism takes place.

In the future, efforts to make in vitro modeling strategies more predictive will depend largely on the availability of more diverse cell lines and the need for multiplexing across species and organs, in Dr. Tsaioun’s view.

Pre-IND Bridging Strategies

In March, Midwest BioResearch (MBR;, a CRO that provides drug disposition and toxicology testing services, introduced a microAmes screen that is predictive of the GLP regulatory Ames test (Salmonella/E. coli mammalian microsome reverse mutation assay), used to evaluate the mutagenicity of a compound. The screen requires 5–10 mg of sample and yields results in no more than two weeks. Like the Ames test, the microAmes incorporates five Salmonella and E. coli test strains and evaluates colony formation, cytotoxicity, and compound precipitation—parameters critical for mutagenicity assessment, according to Michael Schlosser, Ph.D., president and founder of MBR.

The microAmes screen joins MBR’s portfolio of genotoxicity tests, including a microclastogenicity screen for predicting the GLP chromosomal aberration assay, which detects changes in chromosomes that may be associated with carcinogenicity.

Characterizing the market need in pharma, Dr. Schlosser contends that while new tools for predicting and evaluating ADME-tox are important, the knowledge of how to apply those tools is where the real value lies. For example, new biomarkers for predicting toxicity have been identified, but these require “rigorous evaluation before being used to make multimillion dollar decisions,” says Dr. Schlosser. “The challenge is not necessarily in identifying a new biomarker, but ensuring that a particular toxicology biomarker correlates with expression of a classical marker of toxicity in animals, which will vary depending on the chemical class and therapeutic use of the drug.”

Companies will only benefit from new and existing predictors of ADME-tox if they use them correctly and “apply the information to new compounds in development to generate competitive advantage.” The identification of appropriate ADME-tox predictors for use in a particular drug program should be made in early lead optimization, asserts Dr. Schlosser, and ideally even before compounds are synthesized. The application of nonclinical predictors can enable lead optimization and offer companies a tremendous competitive advantage. Dr. Schlosser explains how a company, such as MBR, can work with clients to implement pre-IND bridging strategies and present timely bioanalytical data and predictive information to regulatory agencies prior to an IND filing.

From an economic perspective, ADME-tox studies done during lead selection and optimization contribute to a larger dataset to present to investors and regulatory agencies. The more that is known about a compound, its activity, and potential liabilities in disease models, including in humans—which is now possible before Phase I studies using microdosing strategies—the greater the comfort level in going forward.

“The best model for man is man,” says R. Colin Garner, Ph.D., CEO of Xceleron ( “We have a healthy skepticism of the utility of animal models and of in vitro and in silico models.” Founded in York, U.K., Xceleron established a North American base of operations in 2005.

Accelerator Mass Spectrometry

Xceleron uses Accelerator Mass Spectrometry (AMS) to detect sub-pharmacological doses of 14C-labeled drug compounds. Such microdosing or Phase 0 studies, which originated in Europe, are the basis for the FDA’s new exploratory-IND, or e-IND.

The FDA defines e-IND studies based on three main criteria: limited intent (administration of either sub-pharmacologic doses or doses expected to produce a pharmacologic but not a toxic effect); a limited number of subjects and a limited dose range for a limited period of time; and less risk to human subjects than traditional Phase I studies. E-INDs require much less preclinical support to gain approval—only an expanded acute toxicity study in one (versus two for a traditional IND) mammalian species, and no genotoxicity tests.

Regulatory agencies on both sides of the Atlantic are taking a lead in endorsing Phase 0 studies, says Dr. Garner. The EMEA released a position paper on microdosing in 2003, and the FDA provided guidance on e-INDs, which includes microdosing, in 2006.

Microdosing in combination with AMS can be used to assess human metabolism with the administration of as little as 0.5 µg of a drug substance, although a typical microdose is 100 µg. Radiolabeling of carbon or hydrogen does not change a molecule’s structure, and 14C- or 3H-labeled compounds behave the same as their nonradioactive counterparts.

AMS enables microdosing studies by providing a low limit of quantitation (20–50 x 10-18 M), highly sensitive radioactivity detection (50–200 nCi 14C per dose), and a dynamic range of six orders of magnitude, according to Michael Chansler, vp of business development at Accium BioSciences (, a CRO specializing in accelerator mass spectrometry bioanalytical services.

Chansler identifies two primary applications and advantages of microdosing. For large pharma, Phase 0 studies help companies select the best lead compound to take into clinical trials from among multiple candidates that have performed similarly in animal studies or cannot be prioritized based solely on animal and in vitro data. Phase 0 results expand the compound profile and the company’s knowledge base by adding some human data. For smaller pharmaceutical or biotechnology companies that have a promising compound coming out of preclinical development but lack the funds to initiate a full Phase I trial, the main advantage of microdosing studies is to demonstrate that the compound can pass through the human gut, be absorbed into the circulation, and reach the target tissue. This knowledge can make the compound more attractive to investors and potential partners interested in licensing the compound.

“It can take some of the PK risk off the table,” says Chansler. Accium completed installation and validation of its AMS instrument in 2006, and has established partnerships with Covance, Quintiles, and Analytical Bio-Chemistry Laboratories (ABC Labs;

The growing acceptance and implementation of Phase 0 studies is important because there is no substitute for studying the behavior of a compound in humans, agrees Noel Premkumar, Ph.D., director of regulatory strategy, preclinical/clinical at Analytical Bio-Chemistry Laboratories. If a company were trying to choose a lead compound from among a handful of viable candidates, and if all the compounds were derived from the same scaffold, shared the same target, and demonstrated an acceptable safety profile in animal studies, then the FDA would allow the company to evaluate as many as three or four compounds in a single Phase 0 microdosing trial, explains Dr. Premkumar. With the right program and study design, this can translate into time and cost savings.

In February, ABC Labs announced a collaboration with Accium BioSciences to provide 14C-labeled molecules for use in the company’s AMS-based analytical services. ABC and Accium plan to co-market a package that will provide synthesis of 14C-labeled drug substance, dose preparation for 14C-labeled drug, and AMS services.

ABC Labs has developed expertise in radiolabeling of drug compounds and is leveraging that expertise to design radiosynthesis strategies that yield labeled compounds with the highest chemical and metabolic stability at the lowest cost. The radiolabeled compounds are produced under GMP conditions and in the form required for administration to humans, whether delivered orally, intravenously, subcutaneously, or via inhalation.

Dr. Premkumar predicts that pre-IND bridging strategies and Phase 0 studies will change the drug development process, although few companies have adopted this approach yet. Instead of the current model, in which companies typically use 14C-labeled drug compound to assess ADME in late-stage preclinical testing, microdosing studies will work their way up the pipeline into lead optimization, and Phase 0 trials will act to bridge lead optimization with preclinical development.

“As sponsors continue to focus on optimizing return on their R&D dollars, more of them will use Phase 0-related toxicity studies at the lead-optimization stage,” predicts Dr. Premkumar, “rather than waiting for the preclinical or later stages of development.”

Dr. Garner says the main technology hurdles for AMS are to improve the throughput and lower the cost of the instruments. The primary obstacle to broader application of the technology is “overcoming skepticism,” notes Dr. Garner. Currently “there is probably not a sufficient body of evidence to state categorically that microdose PK will be predictive of pharmacologic dose PK.” But as the database supporting the technology continues to grow, Dr. Garner predicts that the correlation between microdose and pharmacologic dose PK, currently about 70% to 80%, will increase.

Simulating PK/PD

How do you study the pharmacokinetic and pharmacodynamic properties of a drug in a human without actually administering the drug to a patient? By generating a “virtual patient” in silico and simulating therapeutic drug delivery in the context of a particular disease model—a biosimulation strategy being developed and applied by Entelos ( based on the company’s PhysioLab® technology. Entelos’ focus is on predicting the PK and PD properties of compounds using a computer-based mathematical model of human biology. These large-scale mathematical models represent the biological mechanisms that underlie specific physiologic responses.

“The long-term vision is to develop a virtual human,” says Mikhail Gishizky, Ph.D., CSO of Entelos. “We are doing this one step at a time by building individual PhysioLab platforms focused on different disease indications that can be integrated over time.” To date, these include metabolism, cardiovascular disease, inflammatory disease, and respiratory disease.

The metabolism platform, for example, represents the tissues responsible for glucose homeostasis and lipid metabolism, including the gut, pancreas, liver, muscle, and adipose tissue. PhysioLab platforms combine a bubble-and-arrow visual format to represent interactions between tissues, cells, and biological pathways and the relationships and routes of communication between them. Embedded within these visuals are mathematical formulas that define function, activity, and interactions.

The goal, says Dr. Gishizky, is “to bring the rigor of engineering with the hypothesis testing methods used by biologists.”

For applications in drug discovery, researchers enter information and hypotheses about a compound, such as preclinical assay results, and PhysioLab platforms can then answer questions such as: does inhibiting the target produce a meaningful clinical response and what PK/PD properties must be optimized to produce the desired clinical benefit relative to the existing standard of care.

Dr. Gishizky provides an example of a collaborative project in which a customer had a compound with good efficacy, but administration of high enough doses to bring about the desired clinical effect in one preclinical model induced an adverse reaction. Using the Metabolism PhysioLab platform, Entelos was able to predict the effects of the compound in humans and demonstrated that the adverse effect would not occur because a compensatory mechanism present in humans and not in the preclinical animal model would prevent it. Subsequent clinical studies confirmed this prediction.

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