Why would anyone need a regulated bioanalytical assay that measures plasma concentrations far below the potency of a drug? This question came up as our Early Phase Team was preparing for the Workshop on Recent Issues in Bioanalysis (WRIB), which will be held April 9-13 in Philadelphia. If you plan to be there or have questions about early phase trial issues, please fill out a general inquiry form and specify to be in contact with Michele Malone, Senior Director, Early Phase Services/Bioanalytical Sciences, Worldwide Clinical Trials.
At first glance, it may seem logical to avoid this type of supersensitive assay in early phase clinical trials. Pushing the limit of quantitation too low results in false positives, such as anomalous exposures in predose subjects or control animals in good laboratory practice (GLP) studies. If the objective is to ensure safety and establish pharmacokinetics (PK) over efficacious concentrations, then a supersensitive method is unnecessary and can lead to poorer quality data in early phase studies.
However, a supersensitive human plasma assay may be needed to measure free fractions in protein binding and renal-impaired clinical studies or in ascending-dose studies to assess safety and PK proportionality. Another use is IV microdosing to establish absolute oral bioavailability (%F), an Australian requirement.
Clinical formulation development is critical, particularly for Biopharmaceutics Classification System (BCS) II compounds due to their low solubility and high permeability. Dissolution rate-limited absorption reduces therapeutic exposure, requiring the right selection of crystalline form, particle size, and formulation.
Our Guide to Microdosing in Early Phase Clinical Studies:
• Was this our best formulation?
• Is PK from my pentagastrin-induced dog model sufficient to predict PK in first in human (FIH) studies?
• When do we stop testing other formulations?
Unless absolute %F is known, pharmaceutical development personnel do not know if an optimum human formulation has been developed. Few companies develop both oral (PO) and intravenous (IV) products simultaneously, and extensive IV toxicology studies preclude IV dosing.
The FDA introduced the exploratory investigational new drug (eIND) as a means to ensure that drug developers had tools to answer these microdosing questions. Two technologies, accelerator mass spectrometry (AMS) and positron emission tomography (PET), were noted as enabling technologies. Guidance defined a microdose as ≤ 100 micrograms (mcg). The microdose in an eIND clinical study minimizes the toxicology work that would otherwise be needed for an IV dose.
Stable-Labeled and Radiolabeled Microdosing Strategies
The dose can be unlabeled, stable-labeled, or radiolabeled. If only an unlabeled drug is available, then an IV microdose is administered, followed by washout and oral dose at therapeutic levels. A better clinical study design reduces variability when the labeled IV microdose is given simultaneously with unlabeled PO dose. The question of stable-labeled vs. radiolabeled depends upon your measurement tool. If your bioanalytical lab is capable of building regulated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays with sub or low picogram per milliliter (pg/mL) lower limits of quantitation (LLOQs), then this is the most direct and cost-effective route. An assay with a low pg/mL LLOQ will cover five half-lives for IV microdosed drugs of moderate-to-high volumes of distribution (≤ 50 L/kg). Detection sensitivity is entirely dependent upon the LC-MS/MS method.
If you have not built a supersensitive assay, then consider a radiolabeled IV dose with fractionation LC-AMS analysis. AMS requires the synthesis of a high specific activity 14C-radiolabeled drug, extensive specimen preparation, and expensive instrumentation to achieve a supersensitive method. It has the added advantage that IV metabolites may also be quantified, albeit at added expense due to additional fractionation and AMS analysis. AMS has been used in eIND clinical studies to define IV PK and determine absolute bioavailability. Detection sensitivity is limited by specific activity, not by the method. Limits are generally ~0.01 disintegration per minute (DPM), which translates to low pg/mL concentrations for a radiolabel with high isotope incorporation.
A radiolabeled microtracer (≤ 1 uCi) study can be given as either a microdose or a therapeutic dose. When a microtracer is given at therapeutic doses, AMS is likely to be used in a clinical absorption, metabolism, and elimination (AME) study. Traditional AME studies use liquid scintillation counting and 100 uCi doses or what is limited by human dosimetry assessments (FDA regulations 21 CRF 361.1). When given as both a microtracer and an IV microdose in an eIND study, both %F and IV metabolism may be determined.
Accelerated Mass Spectrometry Provides Microdosing Alternative in Early Phase Clinical Trials
In early AMS studies, Malcolm Roland established the Consortium for Resourcing and Evaluating AMS Microdosing (CREAM) trials and illustrated the predictability of PK in PO microdose studies to higher therapeutic doses. For drugs limited by dissolution or having saturated transport, over-prediction of ascending PO doses was a challenge. Several pharma companies have used radiolabeled AMS studies in eINDs to assess drug candidates with varying degrees of success. Many have never tried. They saw the eIND as a dead end and predicted human PK with their best allometric scaling or SimCyp™ predictions from animals and in vitro studies. The use of AMS to establish IV PK in humans has recently seen resurgence with issuance of the 2014 Australian requirement. Oddly, the use of AMS in an eIND IV PK has proven to be more useful than projecting microdose PK to therapeutic doses.
A good clinic will have a track record of performing successful AME studies using both microtracer and traditional radiolabeled doses. Worldwide Clinical Trials has an eight-year history of successful clinical microtracer studies with AMS laboratories and has built more than 2,300 regulated assays in its 28-year history. We can assist in the selection of LC-MS/MS or AMS as an enabling technology to determine absolute %F.