When microdosing was the next big thing—back in the early to mid-2000s—drug developers had great plans for it. One of the problems with drug development is that failures often happen late in the development process. Microdosing offered a way to identify potential failures earlier, before full-fledged clinical trials. Researchers would give subtherapeutic doses to humans after minimal animal testing and use those doses to figure out how the drug behaved in the body. If the drug didn’t behave as desired, they would move on to the next candidate and save themselves the trouble of doing extensive safety testing.

Some industry observers predicted that microdosing would become a standard part of the drug development process. It would give them greater confidence in the candidates that advanced to clinical trials. At that point, they could do the safety testing required for therapeutic doses, secure in the knowledge that they had a winner.

But those plans didn’t work out quite as expected. Microdosing still has a place in the drug development process, but it’s being used in ways that people didn’t anticipate.

Today microdoses—defined as 1% of a pharmacologically active dose but no more than 100 μg—are used primarily in two types of studies. Absolute bioavailability studies are performed in parallel with standard clinical trials to determine what fraction of the therapeutic dose actually reaches systemic circulation. And so-called Phase 0 studies are early human clinical trials designed to weed out drug failures early in the process, before the conventional safety and efficacy testing required for regulatory approval. People originally thought the industry would adopt microdosing for Phase 0 trials. Such trials do happen occasionally but not nearly to the extent that people had predicted. In contrast, microdose absolute bioavailability studies are on their way to becoming an industry norm. And other uses may be yet to come.

From its earliest days, microdosing has been associated with a particular analytical method—accelerator mass spectrometry. In AMS, ions are accelerated to kinetic energies that are high enough to separate individual elemental isotopes. AMS’s hallmark characteristic is its sensitivity, which is sufficient to measure low levels of rare isotopes such as carbon-14. Because the radiolabeled drugs are the only source of carbon-14, AMS allows researchers to quantify them and their metabolites in the body, but it doesn’t provide the molecular information necessary to identify the individual compounds.

But microdosing has become platform independent. “AMS was the tool that propelled microdosing, because it had the sensitivity,” says Stephen R. Dueker, chief scientific officer at Vitalea Science, an AMS services company in Davis, Calif., that was acquired last September by the German company Eckert & Ziegler.

Now microdosing can be done with any technique sensitive enough to detect the drug in biological samples. Some companies are turning to conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) for microdosing studies.

“I’m not naive enough to think that we can beat AMS in sensitivity,” says Jack Henion, chief scientific officer at Advion Bioanalytical Laboratories, a Quintiles company and an Ithaca, N.Y-based contract research organization that is developing ultratrace LC/MS/MS techniques for the analysis of biological samples collected from microdosing studies. But he does think that LC/MS/MS can surpass AMS in terms of workflow issues such as sample preparation and integration of separations.

Relative to AMS, LC/MS/MS does have other drawbacks for microdosing studies. Scientists need to be able to follow the drug as it is metabolized and excreted. That requires an assay for each metabolite—assuming that all the metabolites have been identified. Plus, the different elimination products—such as urine and feces—can respond differently in the mass spectrometer, making quantitation difficult. However, LC/MS/MS does provide molecular information about the drug and its metabolites, not just radiolabel identification.

Pharmaceutical companies are also exploring the use of LC/MS/MS for microdosing studies. For example, last September Li Sun and coworkers from Merck Research Laboratories reported that they used LC/MS/MS to quantify five HIV-1 integrase inhibitors in human plasma during a microdose clinical trial (Anal. Chem., DOI: 10.1021/ac301581h). They calculated pharmacokinetic parameters such as half-life and clearance and determined that the microdoses could be used to extrapolate to the pharmacologic doses.

CAREFUL DOSAGE [+]Enlarge Credit: Accium Biosciences

Even as the methods used for microdosing have evolved, regulatory barriers have fallen. The U.S. Food & Drug Administration cleared the way for companies to implement Phase 0 microdosing studies in 2006. But the uptake “has been much slower than expected,” says Christopher C. Kelly, a spokesman for FDA’s Center for Drug Evaluation & Research. “In our experience, there has been more enthusiasm for microdosing in Europe than in the U.S.”

One hurdle that has hindered uptake of Phase 0 studies is skepticism that the pharmacokinetic properties observed at microdoses would accurately predict those properties at therapeutic doses.

“At the beginning, people were very skeptical. They felt they knew of too many cases where small doses of drugs don’t predict the higher doses you might need for a pharmacological response,” says Malcolm Rowland, a pharmaceutical scientist at the University of Manchester, in England, and the University of California, San Francisco. “There was a lot of headwind to be overcome before people would think about using microdosing.”

European trials of microdosing conducted in the mid-2000s—the European Union Microdosing AMS Partnership Programme and the Consortium for Resourcing & Evaluating AMS Microdosing—suggest that for a majority of compounds such worries are unfounded. Rowland and Graham Lappin were involved in both studies. Lappin, formerly the chief scientific officer of Xceleron, is now reader of pharmaceutical science at the University of Lincoln, in England, and adjunct professor at Duke University. Such trials have shown microdosing to be predictive in 70 to 80% of cases, Lappin says.

“If you put microdosing in the context of the best alternative methods, then so far microdosing is much more predictive. However, when somebody does a microdose study, they always ask the same question: Can you guarantee that the microdose will give you the right answer? Of course, the answer is no, you can’t guarantee,” Lappin says. “I’m a bit bewildered why the question is still being asked to the degree that it was 10 years ago.” But the alternative methods are at best 50% predictive, he says.

A bigger hindrance to adoption is the time and money that Phase 0 trials add to drug development. “If you find that a molecule looks very promising, you still have to go back and do all the safety assessment before you start giving pharmacologic doses to humans,” Rowland says. “It doesn’t save you time if you are confident that you have got a winner.”

Where it may save time, Rowland says, is advancing backup compounds after failure of a lead compound in Phase I trials.

Although microdose trials can help prioritize candidates, the value of that information may not be enough to make the approach attractive. Pharma companies take the attitude that, “I’ve just added a whole study in the middle of my development that I never did,” says Ali Arjomand, president and chief operating officer of Accium BioSciences, a Seattle-based AMS services company. Pharmaceutical companies are more likely to take the risk of moving multiple candidates to Phase I than spend the time and money on Phase 0 trials, Arjomand says.

“Most pharmaceutical companies don’t have their own accelerator mass spectrometer, and the analysis can be quite expensive,” says Lisa Christopher, a biotransformation scientist at Bristol-Myers Squibb. “We judiciously decide whether there is a cost savings or other advantage before we do the study.”

Phase 0 microdosing “is being done on a case-by-case basis, when it answers a specific question,” Vitalea’s Dueker says. “We all expected it to be part of the regular clinical development path.” But it didn’t work out that way, he notes. “It’s not really a routine tool for any company I know.”

Though Rowland doesn’t expect Phase 0 trials to become a routine part of the drug development process, he thinks that they may be more common than people realize. “I think in reality microdosing is employed within companies, but we won’t necessarily hear about these very early-stage studies,” he says.

Indeed, Graeme Young, a scientist at GlaxoSmithKline, the only pharmaceutical company with its own AMS instruments, says that GSK has conducted several microdosing studies. “We’ve tried to use it in decision making in certain circumstances rather than use it as a scattergun approach for all projects,” he says. For example, GSK has used microdosing to identify drug-drug interactions.

Another circumstance in which microdose trials become attractive is when animal studies are giving contradictory information. “The rat might be telling you one thing, and the dog is telling you something else,” Young says. “Which one do I believe is going to be reflective of the human situation?” That’s when people start to consider microdosing, he says.

Companies may not have adopted microdosing for Phase 0 trials, but they have embraced what some have called microdosing for absolute bioavailability studies. It involves the use of an intravenous reference dose. Such studies allow scientists to determine what fraction of a therapeutic dose of their drug candidate reaches systemic circulation in a patient. (Lappin and Rowland both caution that because the pharmacokinetics in such studies is driven by the pharmacologic doses, such intravenous doses are more accurately called tracer doses than microdoses.)

In these studies, a therapeutic dose of a drug is administered via the intended route, usually oral but sometimes via another route such as inhalation or through the skin. When the drug reaches its maximum systemic concentration, an isotopically labeled microdose of the drug is administered intravenously. The IV dose is 100% bioavailable. By referring to the IV profile, researchers can calculate the fraction of drug available via its normal route of administration.

The pharmacokinetics of the IV dose is driven by the concentration already in the bloodstream from the oral dose, Lappin explains. This means that unlike Phase 0 trials, in microdose absolute bioavailability studies there are no concerns about whether the microdose pharmacokinetics scale linearly with the therapeutic dose.

Conventional absolute bioavailability studies are much harder to do. For those studies, researchers give participants an oral dose at the therapeutic concentration and quantify drug levels in plasma. After a two- or three-week “washout” period, they give the same subjects an intravenous dose, also at the therapeutic level, which sets the 100% value. The ratio of the respective integrated pharmacokinetic plots reveals the absolute bioavailability of the oral dose.

But giving such a large IV dose comes with safety and formulation concerns. The drug may not be soluble at such high concentrations. Figuring out how to make it soluble can add time and cost, Arjomand says. Then, that new formulation has to undergo additional safety and toxicity studies. “It’s basically a new drug, a new route of administration,” he says. “All these aspects have added cost, time, and risk. These absolute bioavailability studies have always been a challenge, and companies have tended to do them later if they can.”

But with microdose absolute bioavailability studies, these concerns vanish. The IV dose, which is given on top of the therapeutic dose, is small enough that the safety testing for therapeutic dose is sufficient. “Because it’s just a small fraction of what’s being absorbed orally, it’s not considered an additional toxicological concern or safety concern,” Dueker says.

The IV dose must be labeled so it can be distinguished from the pharmacologic dose. Often the IV dose is radiolabeled with carbon-14, and AMS is used to detect it. However, with enough sensitivity, LC/MS/MS can be used to detect IV doses labeled with carbon-13, says Xiaohui (Sophia) Xu, a bioanalytical scientist at Bristol-Myers Squibb.

Microdose absolute bioavailability studies have become a standardized process at BMS, Xu says. “The IV microdosing strategy is widely applied in late-stage small-molecule oral compounds,” she says.

Likewise, the absolute bioavailability study design is “gaining ground” at GSK, Young says. GSK has conducted five such studies so far. “This is the way the industry is going,” Young says. “It’s relatively inexpensive as clinical studies go. If you do it right, you can add the IV arm on a clinical study you’ve already got in place. You get a lot of information out of that study design for a relatively small investment.”


Still, Arjomand says, “we definitely are in the early days” for absolute bioavailability studies. “Some pharma companies are completely sold and are moving more and more of their compounds into this paradigm.” If regulatory authorities start requiring such studies, many more companies will have to follow the early adopters, he predicts.

Other microdosing applications could be down the road. Paul T. Henderson uses microdosing combined with AMS to determine if cancer patients are good candidates for platinum-based therapies. Henderson is a researcher at the UC Davis Medical Center and chief executive officer of Accelerated Medical Diagnostics.

For the diagnostic test, Henderson gives a patient a C-14-labeled microdose of the therapeutic one or two days before a scheduled biopsy. During the biopsy, he takes blood samples and some of the leftover biopsy material. He extracts DNA from the blood and tissue and analyzes it with AMS to see how much the drug has modified the DNA.

He and his coworkers are undertaking two clinical trials, with different drugs and patient populations, to determine the level of DNA modification that indicates a patient is a good candidate for the drug. He expects the level to be between just a few drug-DNA adducts per genome per cell.