A pharmacokinetic study of radiprodil oral suspension in healthy adults comparing conventional venous blood sampling with two microsampling techniques

Abstract In this phase I, single‐center, open‐label study of ten heathy adults (18‐45 years; NCT02647697), the PK, safety, and tolerability profile of radiprodil oral suspension in healthy adults were assessed, as well as two PK microsampling techniques. All participants received a single 30 mg radiprodil dose (12 mL oral suspension). Blood was collected at various time points using conventional venous sampling (intravenous catheter or venepuncture), and Mitra™ and Aqua‐Cap™ Drummond microsampling (finger‐prick and blood taken from venous blood sample tubes). Geometric mean radiprodil plasma concentrations from conventional venous samples were above the lower limit of quantification up to 48 hours after administration of a single oral dose of radiprodil. Geometric mean AUC inf and C max were 2042 h ng mL −1 and 89.4 ng mL −1, respectively. Geometric mean t ½ was 15.8 hour; median t max was 4 hour (range: 3‐6 hour). Radiprodil exposure variables for Aqua‐Cap™ Drummond sampling were similar to the conventional venous‐derived data. Conversely, radiprodil exposure variables were lower with Mitra™ sampling compared with conventional venous sampling. The geometric mean ratio (90% confidence interval) for C max of conventional venous versus Mitra™ and Aqua‐Cap™ Drummond sampling (finger‐prick blood) was 0.89 (0.85, 0.94) and 1.03 (0.97,1.08), respectively, and therefore within the conventional bioequivalence range (0.80‐1.25). Radiprodil oral suspension had an acceptable safety, tolerability, and palatability profile. The PK profile of radiprodil oral suspension was established in healthy adults, and was comparable when analyzed using conventional versus microsampling techniques. These results will support future radiprodil paediatric studies.


| INTRODUCTION
Radiprodil (UCB3491) is a selective negative allosteric modulator of NR2B-containing N-methyl-d-aspartate (NMDA) receptors and is currently under development for the treatment of infantile spasms (IS).
IS is a severe infantile seizure disorder, in which onset of seizures usually occurs within the first year of life, with a peak age of onset of 3-5 months. 1 An oral suspension formulation of radiprodil has been developed specifically for paediatric use. Before initiating any paediatric research, however, this first-in-human study for this formulation of radiprodil was carried out with the primary objective of evaluating the pharmacokinetics (PK) of radiprodil oral suspension in healthy adults. Secondary objectives were to evaluate safety and tolerability in this adult population. Exploratory objectives were to evaluate the palatability of radiprodil oral suspension and to compare conventional venous blood sampling with two microsampling techniques that sampled both finger-prick blood and blood taken from the venous blood sample tubes.
Mitra ™ volumetric absorbent microsamplers (VAMS) absorb a small sample of blood, which, after air-drying, can be used for subsequent bioanalysis. 2 Aqua-Cap ™ Drummond capillary tubes comprise a glass capillary blood sampling system for the analysis of small, exact volumes of plasma. 3 Both these techniques have the potential to overcome some of the practical challenges of evaluating PK in infants, as the blood volume required is small and can be obtained without venepuncture. It is reasonable therefore, to compare the two techniques with conventional venous blood sampling prior to paediatric development research. This is the first study with this formulation of radiprodil in humans.

| Study design and participants
This was a phase I open-label study in healthy adults (UP0027/ NCT02647697, ClinicalTrials.gov) conducted at a single center in the first quarter of 2016. The study consisted of a 21-day screening period, 3-day treatment period, and end of study (EOS) visit 9-14 days post dose.
Ten healthy male and female participants 18-45 years of age with body mass indexes between 18.0 and 30.0 kg·m − ² were enrolled. Participants entered the study center the afternoon before administration, remaining until 48 hours post administration. Each study participant received a single oral dose of radiprodil 30 mg in 12 mL suspension, via a syringe. Radiprodil was administered in the morning of Day 1 after an overnight fast of at least 12 hours.
Adverse events (AEs) were assessed throughout the study. Vital signs were measured predose, at several time points until 24 hours post dose, and at the EOS visit. A resting 12-lead electrocardiogram (in supine position after 5 minutes rest) and physical examination were performed predose, 24 hours post dose, and at the EOS visit, as were measurements for hematology, biochemistry, and urinalysis parameters. Palatability of the radiprodil suspension was evaluated using three questions on taste and texture, asked immediately following dosing.
The study was conducted according to the International Conference on Harmonisation and the Declaration of Helsinki, and the study protocol was reviewed and approved by an independent medical ethics committee (Stichting Beoordeling Ethiek Biomedisch Onderzoek, the Netherlands; Central Committee on Research involving Human Subjects number NL55708.056.15). All participants provided their informed consent in writing prior to the start of study procedures.

| Blood sampling
Blood samples (2 mL) for PK analysis were collected into EDTA tubes via intravenous catheter or direct venepuncture (conventional venous sampling) at the following time points: predose, and 0.5, 1,

| Bioanalytical methods
Solid-phase extraction was used to extract radiprodil from the plasma collected using Aqua-Cap ™ Drummond tubes and conventional sampling, all with a sample volume of 10 μL. Following extraction, all samples were analyzed using a liquid chromatographyelectrospray ionization-tandem mass spectrometry method. Separation from metabolites and interfering endogenous compounds was achieved by ultra-performance liquid chromatography using an Acquity HSS T3 column (50 × 2.1 mm, 1.8 μm particles; Waters, Hertfordshire, UK) at 40°C and gradient elution using 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B, operating at a flow rate of 0.650 mL min −1 . The lower limit of quantification (LLOQ) for radiprodil was 1 ng mL −1 and the quantification range was 1-1000 ng mL −1 . Back-calculated calibration standard concentrations and quality control sample concentrations for radiprodil in plasma samples are shown in Tables S1 and S2.

| Pharmacokinetics
Standard PK parameters were determined from the plasma radiprodil concentrations by noncompartmental analysis. PK parameters were: area under the plasma concentration versus time curve from time 0 to infinity (AUC inf ); area under the plasma concentration versus time curve from time 0 to the last quantifiable data point (AUC (0-t) ); maximum plasma concentration (C max ); terminal half-life (t ½ ); terminal elimination rate constant (λ z ); apparent total body clearance (CL/F); apparent volume of distribution (V z /F); and time to maximum plasma concentration (t max ).
The linear trapezoidal method was used for calculation of AUC values. AUC inf was calculated as AUC (0-t) + C t /λ z , where C t is the last observed quantifiable concentration, and λ z is the slope of linear regression of the natural logarithm (ln) of concentration versus time during the terminal phase of the plasma concentration versus time profile. Apparent terminal t ½ was calculated as ln(2)/λ z , CL/F was calculated as dose/AUC inf , and V z /F was calculated as (CL/F)/λ z . The analysis was performed using WinNonlin ® version 6.3 (Certara Inc, Princeton, NJ).

| Mitra ™ and Aqua-Cap ™ Drummond microsampling
Finger-prick (peripheral) blood samples were taken from the participants at the same time as the conventional venous samples using two microsampling techniques. The Mitra ™ volumetric absorbent microsamplers (Neoteryx, Torrance, CA, https://www.neoteryx.com/; Figure 1A) were dipped into the finger-prick blood spot, allowing 10 μL of blood to be adsorbed into the tip in 4-6 seconds. The tips were then allowed to air-dry for a minimum of 2 hours at 21°C, 2 allowing for subsequent assay of radiprodil in the whole blood matrix.
Aqua-Cap ™ Drummond capillary tubes (Aqua-Cap ™ Drummond Scientific Company, Philadelphia, PA, https://www.drummondsci.com/; Figure 1B) are specially designed microcapillary separation tubes that were used to draw up 70 μL of whole blood from the finger prick, and were then centrifuged to obtain the plasma for measurement of radiprodil. 3 Additional (venous) microsamples, using both Mitra ™ absorbent microsamplers (10 μL) and Aqua-Cap ™ Drummond tubes (70 μL), were taken from the conventional venous blood sample tubes, prior to centrifugation of the venous samples, in order to assess any differences between venous and peripheral collection using the microsampling techniques. The Mitra ™ samples were taken at every scheduled PK sample time point for venous sampling, whereas the Aqua-Cap ™ Drummond samples were only taken at the following time points: 2.5, 3 and 3.5 hours (the expected C max for radiprodil).

| Microsampling bioanalytical methods
Highly sensitive bioanalytical methods were established and validated in line with internationally recognized standards. 4 Two independent methods were validated for the measurements of radiprodil in plasma harvested from blood collection using Aqua-Cap ™ Drummond sampling and conventional techniques, and whole blood was collected using Mitra ™ sampling.
Radiprodil was extracted from the Mitra ™ -derived blood samples using organic extraction, and from the Aqua-Cap ™ Drummond tubesderived plasma samples using solid-phase extraction, all with a sample volume of 10 μL. Following extraction, all samples were analyzed using a liquid chromatography-electrospray ionization-tandem mass spectrometry method, as explained above. Back-calculated calibration standard concentrations and quality control sample concentrations for radiprodil in plasma and dried blood samples are shown in Tables S1-4.

| Statistical analysis
No formal sample size calculation was performed, due to the exploratory nature of the study. Geometric least squares means (LSMs) and 95% confidence intervals (CI) were calculated for AUC inf , AUC (0-t) and C max , after logarithmic transformation. Bioequivalence of the microsampling methods to the conventional venous sampling method was tested from the ratio (microsampling versus conventional venous sampling) of back-transformed geometric LSMs for AUC inf , AUC (0-t) ,and C max . Samples collected using different sampling methods were considered bioequivalent if the 90% CIs fell within the bioequivalence range of 0.80-1.25.
As an additional analysis, Bland-Altman plots were used to graphically display the level of agreement between plasma concentrations obtained using microsampling versus those obtained using conven-

| RESULTS
Ten healthy adult participants were enrolled and all completed the study. Seven participants were female, the mean age was 24.3 years (range 19-45 years), the mean body weight was 73.1 kg (range 51.8-104.2 kg), and the mean body mass index was 23.6 kg m − ² (range 18.6-28.0 kg m − ²). Nine of the ten participants were white, one was black.
All participants received a single dose of radiprodil and were included in the full analysis set and the PK per-protocol set (PK-PPS). There were no major protocol deviations.

| Pharmacokinetics
The primary objective of this study was to evaluate the PK of radiprodil oral suspension in healthy adults. Geometric mean radiprodil plasma concentrations from conventional venous samples reached a maximum at approximately 4 hours, and began to decrease at 6 hours, approaching the LLOQ 48 hours after administration (Figure 2). Radiprodil oral suspension PK parameters from conventional venous samples are summarized in Table 1. The geometric mean of AUC inf , AUC (0-t) , and C max following a single dose of radiprodil 30 mg oral suspension was 2042 h ng mL −1 , 1772 h ng mL −1 , and 89.4 ng mL −1 , respectively. The geometric mean t ½ was 15.8 hours and the median t max (range) was 4 hours (3-6 hours) ( Table 1). Inter-individual variability in the rate and extent of systemic exposure was generally low for conventional sampling at a radiprodil dose of 30 mg. The geometric coefficient of variation (geoCV%) was ≤27.1% for AUC inf and ≤28.6% for C max .

| Safety and tolerability
The secondary objectives of this study were to evaluate the safety and tolerability of radiprodil oral suspension. During the study, 12 treatment-emergent adverse events (TEAEs) were reported by eight of the ten participants. Of these, ten TEAEs in eight participants were considered to be drug related. The most frequently reported TEAEs were dizziness (4/10 participants; 40%), nausea (2/10; 20%), and somnolence (2/10; 20%). All TEAEs were mild in severity and resolved by the end of the study. There were no severe TEAEs, no serious TEAEs, no discontinuations due to TEAEs, and no deaths.
During the study there were no clinically relevant changes in hematology, biochemistry, urinalysis, or vital signs, and no clinically relevant electrocardiogram abnormalities.

| Palatability
An exploratory objective of this study was to evaluate the palatability of radiprodil oral suspension. All participants found radiprodil acceptable to swallow, with an acceptable taste, and nine of ten subjects found the texture of the formulation acceptable.

| Comparison of conventional and microsampling techniques
Two separate but simultaneous PK sample data sets were acquired for each of the two microsampling techniques (Mitra ™ and Aqua-Cap ™ Drummond), one from blood taken directly from a finger prick (peripheral; Figure 1) and one from the blood sample taken using the conventional venous method. PK parameters were not calculated for the Aqua-Cap ™ Drummond venous samples, as these were only collected at 2.5, 3, and 3.5 hours.
As with the conventional venous samples, radiprodil C max for the    (Figure 3).

| DISCUSSION
Clinical studies in infants frequently require an oral liquid suspension or solution formulation of the drug to be developed specifically for In this study, carried out in ten healthy adult volunteers, the radiprodil oral suspension formulation had an acceptable safety profile and TEAEs were all mild in severity. The taste and texture of the oral suspension were considered to be acceptable by 10/10 and 9/ 10 participants, respectively; this is a crucial property for oral liquids because palatability is a major determinant of treatment compliance in infants and children. [5][6][7] Further confirmatory studies in children of various age groups would be of value given that younger children often demonstrate different palatability preferences from older children and adults. 8 Marginally lower concentrations of radiprodil were observed over the treatment period using peripheral and venous Mitra ™ microsampling compared with Aqua-Cap ™ Drummond (peripheral and venous) microsampling and conventional venous sampling. This may be a reflection of the blood to plasma partition of radiprodil, and could be explained by the different matrices tested: plasma from samples collected using the Aqua-Cap ™ Drummond capillary tubes and conventional sampling, and whole blood samples collected using Mitra ™ volumetric absorbent samplers. However, despite these lower concentrations, the PK profile of radiprodil from samples collected via the Mitra ™ and Aqua-Cap ™ Drummond microsampling methods demonstrated bioequivalence in terms of 90% CIs of the geometric mean ratio to that obtained using the conventional venous collection techniques, regardless of the site of microsample collection. This confirms that either technique is fit for purpose for use in future clinical studies with radiprodil. These less invasive microsampling techniques have great potential to facilitate and add value to any paediatric programme. They are particularly beneficial in this population because they may allow a PK profile to be measured without the need for a venous line, and with a sample volume of no more than 70 μL; in particular, Mitra ™ microsampling only requires 10 μL of blood. In the case of Mitra ™ volumetric absorbent samplers, the microsampling technique obviates the need for centrifugation, plasma separation, and freezing (including frozen shipment), thus reducing the processing burden of blood sampling. Furthermore, during the prestudy bioanalytical method validation the impact of hematocrit was assessed and no effect was found across a supra-and infra-normal range of hematocrit levels. The phenomenon observed with dried blood spot samples (DBS), where samples spread across a DBS card in a heterogeneous manner due to variability in hematocrit, and the subsequent impact of taking a sub-section of the blood spot for analysis, has largely been resolved by Mitra ™ , since (unlike DBS), Mitra ™ collects a fixed (10 μL) volume of blood with extremely low levels of variability (<5%). This technique allows the potential for collection of samples at home or in remote areas where access to a clinic may be challenging. The key limitation of the study's relevance to infants is that it was conducted in adults. There is, however, no a priori reason why either microsampling technique should not work just as well in the infant setting.
In conclusion, the PK profile of radiprodil single-dose 30 mg oral suspension was established in healthy adults, and was comparable when analyzed using conventional versus microsampling techniques. Radiprodil had acceptable safety and palatability profiles in this population. Results from this study will therefore inform and facilitate future paediatric radiprodil studies. A study to evaluate the safety, tolerability, PK, and efficacy of radiprodil in infants 2-14 months of age with drug-resistant IS is currently ongoing (NCT02829827).

ACKNOWLEDG EMENTS
The study was sponsored by UCB Pharma. UCB Pharma was responsible for the design and conduct of the study, and collection, management and analysis of the data. UCB Pharma was involved in the preparation and review of the manuscript and covered all related costs. The authors had the final responsibility for the con-