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Feature Articles : Sep 1, 2007 ( )
Accelerator MS Is a Powerful New Tool
Technology Has Broad Applicability in Biomedical Research!--h2>
To meet investor expectations and to achieve annual growth rates of 10%, each major pharma company must launch four new chemical entities (NCEs) per year, each with average annual sales of $350 million. However, the industry launches fewer than one NCE per year per company, and only a quarter of launched products achieve the desired level of sales. Underlying this deficit is an expensive and lengthy clinical development process. Fewer than 5% of screened compounds enter preclinical development, and only 2% of these enter clinical testing. Of all drugs that enter Phase I, 80% will fail in development.
Increased length and complexity of trials is a major contributor to the escalating cost of bringing drugs to the market. Eliminating poor candidates early will help to reduce attrition rates during clinical trials. Technological innovations, applied at the earliest stages of discovery, could be a powerful tool for improving the entire development process. One such emerging technology, accelerator mass spectrometry (AMS), is poised to revolutionize early clinical studies. AMS enables detection of attomole quantities of radiolabeled drugs and allows collection of human ADME data before or during Phase I trials. This data could lead to early abandonment of less-promising compounds, reducing overall development risk by as much as 30%.
Early Days of AMS
Plants and animals that are in equilibrium with the biosphere have natural 14C in them from atmospheric sources. The amount of 14C varies depending on the time that the organism existed on Earth. For example, currently living organisms have about 100 attomoles (amol) of 14C per mg of carbon, but a bone of a person who has been dead for 10,000 years contains only 30 amol (30% Modern).
Radiometric counting is used to measure 14C atoms decays. By determining the number of 14C decays that take place in a certain time, one can calculate the number of 14C atoms in the sample. Calculation of 14C concentration allows calculation of the age of the material. This method carries an inherent and high degree of uncertainty and requires long measurement times. An ancient bone or artifact often had to be destroyed to be carbon dated using decay counting. These inefficiencies gave impetus to development of AMS for radiocarbon dating.
AMS detects 14C on the background of 13C and 12C, present in infinitely higher amounts. The natural abundance of 14C is about one atom per trillion (1012) atoms of 12C. An AMS device essentially consists of two linear accelerators joined end-to-end, with the joint section charged to a very high positive potential of 0.5–3 million volts or higher. The process begins with the sample first being combusted to CO2 and subsequently reduced to elemental carbon. As carbon atoms are accelerated through the AMS instrument, they undergo various changes at the atomic level that in the end enables differentiation between high-energy 14C radioisotopes and molecular debris.
The AMS technique literally extracts and counts the 14C atoms in the sample and at the same time determines the amount of the stable isotopes, 13C and 12C. For archeological measurements, only a few milligrams and only a few hours are required to calculate 14C concentration in a given sample.
Even though it has its origins in radiocarbon dating, the real excitment about AMS lies in the possibilities it offers for completely new kinds of measurements and sample materials.
Changing the Drug Discovery Process
“I became interested in AMS as an ultrasensitive analytical technique during my tenure as cancer researcher at the University of York,” says Colin Garner, Ph.D., CEO at Xceleron (www.xceleron.com). “In 1993, in collaboration with Lawrence Livermore National Lab, we ran the very first human microdosing study, evaluating the fate of potential carcinogens. It quickly became clear that AMS could have broad applications in biomedical research and, specifically, in human clinical trials. Recognizing this potential, several large pharma companies funded the purchase of the original Xceleron AMS instrument.”
Xceleron became the first company to commercialize AMS technology for clinical research. The company has positioned itself as a provider of a comprehensive early drug development portfolio where AMS serves as a principal analytical tool. Xceleron is capable of managing the entire clinical development project, including registration, preclinical toxicology, clinical studies, and bioanalysis.
Microdosing is a key feature, according to Dr. Garner. The EMEA/FDA defines microdosing as 1/100th of a pharmacological dose based on animal data/in vitro systems or a dose that does not exceed 100 micrograms. Microdosing regulations have been adopted in Europe, and in 2006, the FDA issued the guideline for exploratory IND studies (or Phase 0) that use sub-pharmacological doses of drugs.
“We have learned well how to treat cancer in mice and rats but we still can’t cure people. Costly clinical development pitfalls result from inconsistent data derived from animal models and from poor correlation between human and animal pharmacokinetics (PK) data. Microdosing could be used to obtain early PK data in the ultimate target species with only minimal toxicology. Thus, poor candidates could be failed earlier,” continues Dr. Garner. “Microdosing increases patient safety and eliminates an unnecessary sacrifice of laboratory animals.”
Since its inception, Xceleron has analyzed over 200 NCEs. The company leads the European Union Microdose AMS Partnership Programme (EUMAPP; www.eumapp.com). This 30-month, $4 million project aims to demonstrate the reliability of microdosing for predicting drug’s PK, to develop in silico PK predicting models based on microdosing data, and to certify AMS as a technology of choice for microdosing studies.
New Generation of Accelerators
Vitalea Science (www.vitaleascience.com)established an AMS operation in the U.S. in 2004 through a collaboration with the Swiss Institute of Technology. This collaboration led to the development of a next-generation AMS instrument, bioMICADAS AMS.
“BioMICADAS is the subcompact of the AMS world. It requires only 200 kVolts for operation,” says Stephen Dueker, Ph.D., president of Vitalea. “It includes fully optimized features for GLP bioanalysis such as high-throughput, continuous sample loading; barcode tracking; and operational safety features. Exceptional ease-of-use will make BioMICADAS the new industry standard.” The instrument will be placed into Vitalea’s expanded facility in the fall of 2007.
“U.S. companies are still learning how to incorporate valuable Phase 0 studies in their portfolio,” continues Dr. Dueker. “For now, we are helping the industry to perform hybrid Phase I/microADME studies. Early ADME data could be easily obtained by co-administering a microdose of 14C-labeled drug with a pharmacological dose during Phase I trials. This means reduction in radiation exposure and better decision making for a minimal extra cost.”
Hybrid studies with microdoses of 14C-labeled drugs provide more accurate and early data for mass balance, metabolic profiling, and absolute bioavailability studies, reports Dr. Dueker. “This design empowers Phase I as never before with an immensely richer dataset. This is a must-have application,” he adds.
Vitalea co-markets its services with several CROs and has a comprehensive partner network for procurement of radiolabels, bioanalytical support, and clinical access. The company is developing a test to examine anti-HIV drugs’ efficacy in pediatric populations and a diagnostic test for vitamin B12 absorption.
The latter is aimed to supplant the existing Schilling Urinary Excretion test, a rather imprecise method requiring high doses of radioactive cobalt. If developed, the 14C-based test will greatly improve the diagnosis of vitamin B12 malabsorption, which is associated with such serious conditions as dementia, Crohn’s disease, and stomach ulcers, according to Dr. Dueker.
“We are still educating the market,” says Dr. Dueker, “but AMS could prove to be a disruptive technology, enabling experiments that could not be conceived before.” The high specificity and sensitivity of AMS may be critical for some proteomic applications such as binding of 14C-labeled compounds to proteins or tissues under different experimental conditions, including co-administration of several drugs, diet, and exercise. While the practice of immunoassaying has experienced a transition from radioisotopic labeling to nonisotopic labeling over the last two decades, this trend may be reversed with the advent of AMS.
Molecular Biology Space
In addition to enhanced sensitivity, another advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity. With AMS, the sensitivity of an immunoassay is constrained by the affinity constant of the antibody and not the detection system. In a radioimmunoassay (RIA) using 14C-labeled pesticide atrazine, the detection limit of atrazine was about 10 times lower than the Ka of the capture antibody and the radioactivity used in this assay was 1,000 times lower than in conventional RIA. Knowing a true Ka of an antibody by using AMS would help the understanding of antibody properties and antigen interactions and assist with antibody design and engineering.
AMS could also be applied to elucidation of the role of modified nucleosides in cancer initiation and progression or in viral cycles, for gene-therapy studies, and for the evaluation of novel DNA delivery technologies. In 2005, Vitalea completed an AMS-based study on metabolism of the anti-retroviral compound AZT. This drug is a deoxythymidine analog, which causes termination of elongating DNA chain.
“We were able to quantify the uptake and retention of the orally administered AZT in white blood cells and, specifically, into genetic material of these cells,” explained Le Thuy Vuong, co-founder of Vitalea. “Such data could not have been obtained by any other method and is particularly valuable for a pediatric population.”
“AMS, being the most sensitive analytic technique ever developed, allows us to follow the fate of large biological molecules such as peptides and DNA,” continues Dr. Garner. Radioactive precursors could be supplied directly into cell culture growth media. Addition of 14C deoxyguanosine leads to incorporation of the label into a growing DNA chain. An antibody can be labeled in vivo using radioactive amino acids added to the hybridoma growth media.
Xceleron used AMS to trace CAT-192 (metelimumab) labeled in vivo with 14C. Even though the incorporation of the tracer into the antibody was only 1.4%, AMS was able to detect intact and denatured antibodies, as well as antibody fragments and metabolites. Moreover, the limit of detection was about 1 ng/mL serum, which is ~15 times lower than ELISA, says Dr. Garner.
The success of this study predicts that it may be possible to detect disease-specific biomarkers using ELISA with 14C-labeled antibodies. “We are exploring AMS applications for population monitoring, diagnostics, and pharmacogenomics,” adds Dr. Garner. “And that’s only scratching the surface.”
“We are interested in expanding AMS applications into genomic and proteomic areas,” says Ali Arjomand, Ph.D., president and scientific director at Accium Biosciences (www.acciumbio.com). “A lightly labeled 14C biological, for example, is eventually broken down in the body and its 14C nucleotides or 14C amino acids are recycled. By isolating and quantifying a specific 14C-labeled product, AMS can help to integrate the biochemical, genomic, and proteomic processes involved in a particular disease, eventually leading to discovery of validated biomarkers.”
Even though AMS does not identify the exact metabolite structure but only concentration of the label, it is possible to split HPLC fractions between classical MS and AMS. This way, both the structure and the concentration of the metabolite could be determined. Moreover, AMS could also track interactions between drugs, their metabolites, and cellular machinery such as transporters involved in internalization and excretion of the chemical moieties.
Early Drug Discovery Process
Accium BioSciences operates a contract AMS analytical services facility in the U.S. Through its network of CROs and a recently announced collaboration with Analytical Bio-Chemistry Laboratories (www.abclabs.com; ABC), Accium strives to provide a seamless process for AMS-based studies. “We aim to create a continuum between drug synthesis and clinical trials results,” says Michael Chansler, vp of business development. “To maximize the success of the study, we actively participate in clinical trial design, advising on the best dose, sample collection, volume, and even shipping conditions.”
ABC is a cGMP-compliant drug development CRO with a particular expertise in non-routine analytical services and chemical synthesis. Accium and ABC plan to co-market a package that includes synthesis, dose preparation, and AMS services. “We participate closely in the design of the radioactive molecule,” continues Dr. Arjomand. “14C is usually located in the core of the molecule to escape immediate cleavage. Beyond that, the labeling strategy is designed to meet the specific intent of our pharmaceutical clients.”
The company emphasizes a particular AMS utility in profiling multiple compounds in parallel studies, which allows early prioritization and selection for further clinical development. AMS is also indispensable in studies with targeted therapeutic agents administered in low concentrations due to their high potency.
According to the company, an AMS clinical study can be planned and conducted within six months. Accium’s compact accelerator mass spectrometer is designed specifically for carbon-only studies, accelerating the atoms to 0.5 million volts. The company operates under GLP guidelines and has introduced rigorous QA procedures to verify validity of generated data. “By providing a turnkey AMS service,” continues Chansler, “we help our clients keep their focus on their research while we deliver the ultrasensitive AMS detection.”
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