A report on a recent AAPS webinar.
By Cathy Yarbrough
Microsampling can significantly lessen the volume of blood and plasma/serum that is collected and analyzed to determine circulating concentrations of therapeutic drugs, metabolites, and biomarkers in preclinical and clinical research, said Neil Spooner, Ph.D., C.Chem., FRSC, one of two presenters at the recent webinar, Microsampling—How It Can Influence Drug Development.
With conventional sampling methods, the sample volume can total about 200mL. Microsampling can reduce sample volume by 50 percent to ≤100mL, said Spooner, founder and director of Spooner Bioanalytical Solutions in Hertford, U.K.
Available microsampling technologies range from dried blood spot (DBS) to capillary microsampling for obtaining plasma samples. Under development are additional microsampling techniques including patient-centric, wearable, fully automated technologies, including a device to collect and store serial blood samples.
FEWER LAB ANIMALS
A key driver of the biopharmaceutical industry’s widespread adoption of microsampling in preclinical research are the 3Rs (replacement, reduction, and refinement) principles, said Spooner. Microsampling can reduce the number of rodents in preclinical research by 30 to 40 percent, particularly in toxicokinetic (TK) studies that use satellite animals to reduce the number of repeated conventional blood sampling procedures conducted on main study animals.
Repeated conventional blood sampling has been shown to negatively affect lab animals. A 2014 study1 found a statistically significant decrease (p<0.001) in hemoglobin, hematocrit, and red blood cell count as well as an increase in reticulocytes in rodents subjected to repeated conventional sampling. In contrast, a slight toxicologically insignificant decrease in hemoglobin concentration characterized the microsampled animals.
By eliminating satellite animals, microsampling can significantly reduce R&D costs and improve the quality of research data by allowing scientists to conduct all TK studies on main study animals. In addition to enabling direct correlation of exposure with pharmacodynamic and toxicological outcomes, microsampling allows scientists to identify additional pharmacokinetic (PK) and TK time points and investigate the influence of biomarkers, metabonomics, and comedications.
IN CLINICAL RESEARCH
Microsampling, particularly fingerstick DBS, also can play a role in clinical research, particularly studies involving pediatric and critically ill patients, in which blood volumes are a clinical or ethical concern. In DBS, a small amount of the patient’s blood is spotted onto an absorbent card or other specialized paper material. As soon as the sample has dried, the DBS card is mailed to the bioanalysis laboratory where it is “sub-punched” to create a small disc of fixed-diameter. The disc is then added to an extraction solvent to encourage the sample’s cellular contents to precipitate into the solution for bioanalysis. Because ambient temperature storage and shipping can be used, DBS does not require the costly cryopreservation and shipment that characterizes conventional blood samples.
Fingerstick DBS provides a convenient, low-cost way to obtain blood and plasma/serum from patients in their homes and geographically remote locations. Because it can be self-administered, fingerstick DBS can be used for therapeutic drug monitoring and patient compliance and to obtain data related to unpredictable clinical episodes such as acute migraine attacks.
NEGATIVES
The major drawback of DBS microsampling is the volumetric hematocrit (HCT) bias, which refers to blood viscosity’s effect on the accuracy of sample bioanalysis, said Spooner. The percent of HCT in a sample determines viscosity, which influences how evenly the blood spreads on the DBS card. High-HCT blood generates smaller blood spots, while low-HCT blood creates larger spots. A high or low HCT can affect the quality of the data generated by bioanalysis. Data quality can be particularly affected if the sample’s HCT varies markedly from the HCT of the control blood sample used to prepare calibrant and quality control samples, he added.
To obtain quantitative analytical results that are independent of a blood sample’s HCT, Spooner helped develop VAMS (Volumetric Absorptive Microsampling). VAMS is designed to collect a fixed volume of blood by capillary action.
Microsampling is not appropriate for all studies. “The choice of microsampling should be weighed case-by-case,” said the second webinar speaker, Prajakti Kothare, Ph.D., executive director, Quantitative Pharmacology and Pharmacometrics within Pharmacokinetics, Pharmacodynamics and Drug Metabolism at Merck.
UTILITY IN CLINICAL TRIALS
Kothare said that microsampling approaches such as DBS have the greatest potential for impact in late-stage clinical trials. In out-patient settings, DBS enables researchers to obtain patient data that otherwise could not be assessed. For example, in Merck’s first outpatient use of DBS, researchers obtained PK information on the acute migraine drug MK-1602 for exposure-response modeling. Patients in the phase 2 clinical trial of MK-1602 self-administered DBS in their homes proximal to their acute migraine attacks. In addition, during clinic visits, patient blood samples were collected by conventional venipuncture. In addition to spotting DBS cards, these samples were used for plasma analysis. During selected clinic visits, staff also performed fingerstick DBS sampling on patients. Thus, the researchers had the blood and plasma samples obtained by clinic staff to back up their bioanalysis findings from the DBS samples obtained by patients in their homes.
Before using DBS in the MK-1602 study, Merck scientists conducted in vitro and bioanalytical tests to determine the method’s feasibility and suitability for the clinical trial. Since the phase 1 study would use conventional plasma sampling, the researchers designed a robust in vivo bridging strategy that allowed quantitative interconversion of PK information between the plasma sampling and fingerstick DBS.2 “Concentration independent blood to plasma ratios and plasma protein binding insured successful bridging between plasma and DBS concentrations,” she said.
Merck first employed fingerstick DBS in its large multi-site phase 3 trial of MK-8931 for Alzheimer disease. DBS was selected because of its logistical advantages, including simplified sample preparation and ambient temperature shipping, said Kothare. These advantages likely encouraged clinical site participation and facilitated study enrollment. In the MK-8931 trial, DBS was implemented in the staged manner that characterized the MK-1602 study.3
REGULATORY ASPECT
According to both speakers, regulatory agencies support the use of DBS in preclinical toxicology studies. However, since the clinical application of DBS is still emerging, regulatory guidance on DBS in clinical studies has yet to be established. Based on Merck’s meetings with regulatory agencies prior to the MK-1602 and MK-8931 trials, Kohare said, “rational, prospective and quantitative approaches for bridging are accepted by regulators for late-stage studies.” Merck presented regulators with briefing packages that provided integrated assessments of in vitro data, bioanalytical feasibility assessments, and in vivo bridging evaluations.
Learn more about AAPS webinars.
Cathy Yarbrough is a freelance science writer.
REFERENCES
- Powles-Glover N, Kirk S, Wilkinson C, Robinson S, Stewart J. Assessment of toxicological effects of blood microsampling in the vehicle dosed adult rat. Regul Toxicol Pharmacol. 2014;68(3):325–331. doi: 10.1016/j.yrtph.2014.01.001
- Li CC, Dockendorf M, Kowalski K, et al. Population PK Analyses of Ubrogepant (MK-1602), a CGRP Receptor Antagonist: Enriching In-Clinic Plasma PK Sampling With Outpatient Dried Blood Spot Sampling. J Clin Pharmacol. 2018;58(3):294–303. doi: 10.1002/jcph.1021.
- Kothare PA, Bateman KP, Dockendorf M, et al. An Integrated Strategy for Implementation of Dried Blood Spots in Clinical Development Programs. AAPS J. 2016;18(2):519–527. doi:10.1208/s12248-015-9860-3.