Accelerator MS Research Articles

How The Accelerator MS Community are Trying to Fit Into The Regulatory Framework: are The Bioanalytical Method Validation Guidances Relevant?

Xceleron Inc., 20340 Seneca Meadows Parkway, Germantown, MD 20876, USA Tel.: +44 142 353 8423 E-mail: mark.seymour@ xceleron.com Accelerator MS (AMS) was first applied in the biomedical field in the late 1990s and was initially used principally in human metabolism studies where the sensitivity of conventional radioactivity detectors was insufficient, or in human Phase 0 microdosing. The key driver for using AMS for microdosing was again sensitivity, which enabled measurement of circulating concentrations arising from doses of 100 μg or less. However, despite early predictions that microdosing would engender a paradigm shift in drug development, continuing concern regarding the predictivity of the PK data generated coupled with the continuous and continuing improvements in the sensitivity of LC–MS/MS assays has limited adoption of the approach. Furthermore, while AMS continues to be used for metabolism studies, the advantages of reduced radiation exposure to subjects and the potential to administer multiple radiolabeled doses has not driven widespread adoption. Instead, over the last 5 years, AMS has been increasingly used in enhanced Phase I PK studies, where the ability of the detector to discriminate between C-labeled and nonlabeled doses facilitates an innovative clinical study design whereby a low dose (typically 10 μg or less) of C-compound is administered intravenously contemporaneously with a pharmacologically relevant extravascular dose of the nonradiolabeled compound. This so-called IV/PK, or absolute bioavailability, study design has a number of important advantages over the conventional intravenous (i.v.)/extravascular crossover design. Scientifically, the IV/PK design is optimal: since both doses are administered at the same time there can be no variation due to temporal effects and the compound administered by both dose routes is handled by the body in exactly the same way, so the potential effect of nondose proportional kinetics does not have to be considered. Notwithstanding the latter point, the actual dose administered by the i.v. route is so low that no i.v. safety toxicology studies are required prior to human dosing. Additionally, the task of developing a suitable i.v. formulation is facilitated, particularly for poorly soluble compounds, with a simple aqueous solution typically used. While the utility of the IV/PK design is widely accepted, there is an ongoing debate as to the relative merits of the C-AMS approach compared with labeling the i.v. dose with C and analyzing using LC–MS/MS. It is undoubtedly attractive to be able to work with a stable isotope (rather than radioactive C) and to use essentially the same analytical methodology to quantify compound administered by both dose routes (discrimination being possible through the appropriate selection of ions/transitions to be monitored). On the other hand, C-labeled analogues are often used as internal standards (ISs) in LC–MS/MS assays, so a second analogue must be synthesized and the assay method must be able to distinguish between all three analogues. The sensitivity of the LC–MS/MS assay for the C-analyte is also a potential issue. It is essential that systemic concentrations arising from the i.v. dose are sufficiently low relative to those arising from the extravascular dose to avoid any possibility of perturbing the kinetics of the extravascular dose (at least a 100-fold excess should be the target). This may challenge the LLOQ of the LC–MS/MS assay, particularly for compounds with low bioavailability and/or

Iodine-129 Microdosing for Protein and Peptide Drug Development: Erythropoietin as A Case Study

Microdosing is a technique for studying the behavior of compounds in vivo at 1/100th of the dose of a test substance calculated, based on animal data, to yield a pharmacologic effect. In microdosing, use is made of accelerator MS (AMS). In this study, we investigated whether (129)I-labeling of proteins with subsequent AMS measurements is a suitable method to perform microdose studies with therapeutic proteins. We used erythropoietin (EPO) as a case study. In an animal study with (129)I-labeled EPO in Han-Wistar rats, an increase of (129)I-EPO is observed after dose administration. The half-life was found to be 2 and 5.5 h for two different EPOs. These results are in accordance with expected values. Although further research is required, (129)I-labeling of proteins seems a feasible method for AMS microdose studies with peptide and protein drugs, such as biosimilars.

Applications of Accelerator Ms in Pediatric Drug Evaluation

Accelerator MS (AMS) provides a novel method for obtaining and analyzing pharmacokinetics and pharmacodynamics in children. This paper reviews the scientific and ethical rationale for AMS in pediatric trials, the regulatory framework and general considerations with some specific examples of pediatric clinical trials using AMS. Microdosing in the context of this article refers to studies using a negligible amount (nanocuries) of (14)C as tracer, and AMS as a quantitative technique. The technology is by no means a panacea for the deficiency in pediatric clinical research; however, it lessens the challenges and provides the most quantitative tool for pediatric pharmacology studies.

Overcoming Bioanalytical Challenges in An Onglyza ® Intravenous [ 14 C]Microdose Absolute Bioavailability Study With Accelerator Ms

An absolute bioavailability study that utilized an intravenous [(14)C]microdose was conducted for saxagliptin (Onglyza(®)), a marketed drug product for the treatment of Type 2 diabetes mellitus. Concentrations of [(14)C]saxagliptin were determined by accelerator MS (AMS) after protein precipitation, chromatographic separation by UPLC and analyte fraction collection. A series of investigative experiments were conducted to maximize the release of the drug from high-affinity receptors and nonspecific adsorption, and to determine a suitable quantitation range. A technique-appropriate validation demonstrated the accuracy, precision, specificity, stability and recovery of the AMS methodology across the concentration range of 0.025 to 15.0 dpm/ml (disintegration per minute per milliliter), the equivalent of 1.91-1144 pg/ml. Based on the study sample analysis, the mean absolute bioavailability of saxagliptin was 50% in the eight subjects with a CV of 6.6%. Incurred sample reanalysis data fell well within acceptable limits. This study demonstrated that the optimized sample pretreatment and chromatographic separation procedures were critical for the successful implementation of an UPLC plus AMS method for [(14)C]saxagliptin. The use of multiple-point standards are useful, particularly during method development and validation, to evaluate and correct for concentration-dependent recovery, if observed, and to monitor and control process loss and operational variations.

Accelerator MS: its Role as A Frontline Bioanalytical Technique

Accelerator MS (AMS) is an ultrasensitive technique that can be used to quantify (14)C in biological samples. Prior to analysis, the carbon in samples is selectively isolated, with the result that the technique is independent of compound structure and nonsusceptible to matrix effects. AMS is a tracer technique and therefore can be used to quantify all compound-related material without the need to develop extraction or chromatographic separation methods. Thus AMS has some distinct advantages over conventional assay techniques, such as LC-MS/MS. AMS also complements conventional techniques, facilitating innovative, cost-effective clinical study designs. Thus, metabolism data can be obtained from early clinical trials, identifying any human metabolites that may raise safety concerns. By administration of an intravenous (14)C microtracer dose concomitantly with an extravascular dose of nonradiolabeled compound, AMS can also be used to determine absolute bioavailability and intravenous pharmacokinetic parameters without the need for intravenous toxicology or formulation development.

An AMS Method to Determine Analyte Recovery from Pharmacokinetic Studies with Concomitant Extravascular and Intravenous Administration

The absolute bioavailability, clearance and volume of distribution of a drug can be investigated by administering a very low dose of the (14)C-drug intravenously along with a therapeutic nonlabeled dose by the extravascular route (typically orally). The total drug concentration is measured by an assay such as LC-MS and the (14)C-drug is measured by accelerator MS (AMS). In another article in this issue, a method validation is proposed where AMS was used as the analytical assay. Part of the validation is to assess the recovery of the analyte being measured as this has a direct impact on its quantification. In this article, a method of internal standardisation is described where the UV response of the nonlabeled analyte, spiked in excess into the matrix being analysed, is used for internal standardization. The method allows for the recovery of analyte to be measured in each individual sample being analysed. It is important to know the recovery of a (14)C-labeled analyte when determining its mass concentration from (14)C:(12)C isotopic ratio data using AMS. A method is reported in this article that utilizes the UV response of the nonlabeled drug for internal standardization, so that the recovery for each individual sample analyzed can be ascertained.

Addressing Metabolite Safety During First-In-Man Studies Using 14 C-Labeled Drug and Accelerator Mass Spectrometry

Active drug metabolites formed in humans but present in relatively low abundance in preclinical species can lead to unpredicted adverse effects during clinical use. The regulatory guidelines in recent years have therefore required that the metabolism of a drug be quantitatively compared between preclinical species and human at the earliest practicable stage of drug development. Amongst the variety of methods available, inclusion of low radioactive doses of ¹⁴C drug in first-in-man studies coupled to the sensitive analytical technology of accelerator MS (AMS) has found utility. Measurement of ¹⁴C by AMS allows for quantification of metabolites, even if their structures are unknown, and, when used in conjunction with LC-MS, can provide both quantitative and structural data. This review examines a typical approach to using AMS and associated analytical methods in addressing the regulatory guidelines and discusses a number of possible scenarios including the question of steady state.

Microdosing: A Valuable Tool for Accelerating Drug Development and The Role of Bioanalytical Methods in Meeting The Challenge

The concept of specifically determining the clinical pharmacokinetics of a compound using a very low nonpharmacologically active dose (microdose) with an abridged safety and chemistry, manufacturing and control package is relatively new. It is not without its controversy and it is still a subject of discussion. Here, the rationale and application of this approach are examined, together with the regulatory and bioanalytical framework. There are two bioanalytical methods commonly used for human microdosing studies: LC-MS/MS and accelerator MS (AMS). Each method has advantages and disadvantages with the choice of instrumentation being closely tied to the primary objective(s) of the study. If a rapid decision is required on the appropriateness of a pharmacokinetic profile or if a choice is needed from a series of compounds, especially before radiolabeled material is available, LC-MS/MS may be preferable. However, if extreme sensitivity is required, data are required on all drug-related material and metabolites, or a simultaneous intravenous microdose is used to determine absolute bioavailability (sometimes referred to as microtracing), AMS becomes the analytical method of choice. Examples are provided of microdosing studies utilizing both of these bioanalytical techniques. It is emphasized that microdosing is only one tool in the drug developer's tool box and it should be used in the context of all available data. However, when used appropriately, microdosing is a valuable tool, bridging between lead optimization and early clinical development.