Approaches to intravenous clinical pharmacokinetics: Recent developments with isotopic microtracers.

Obtaining pharmacokinetic data from the intravenous route for drugs intended for oral administration has traditionally been expensive and time consuming because of the toxicology requirements and challenges in intravenous formulations. Such studies are necessary, however, particularly when regulator agencies request absolute bioavailability data. A method has emerged whereby the drug administered intravenously is isotopically labeled and dosed at a maximum of 100 µg concomitantly with an oral administration given at a therapeutically relevant level. The intravenous administration has been termed a microtracer and obviates intravenous toxicology requirements as well as simplifying formulations. The study design also essentially removes issues of nonlinear pharmacokinetics that may occur when oral and intravenous doses are administered separately. This review examines the methodology and the literature to date, including those studies intended for regulatory submission. The method has been extended to the study of prodrug-to-active drug kinetics and to obtaining clearance, volume of distribution, and absolute bioavailability at steady-state conditions.

A historical perspective on radioisotopic tracers in metabolism and biochemistry.

Radioisotopes are used routinely in the modern laboratory to trace and quantify a myriad of biochemical processes. The technique has a captivating history peppered with groundbreaking science and with more than its share of Nobel Prizes. The discovery of radioactivity at the end of the 19th century paved the way to understanding atomic structure and quickly led to the use of radioisotopes to trace the fate of molecules as they flowed through complex organic life. The 1940s saw the first radiotracer studies using homemade instrumentation and analytical techniques such as paper chromatography. This article follows the history of radioisotopic tracers from meager beginnings, through to the most recent applications. The author hopes that those researchers involved in radioisotopic tracer studies today will pause to remember the origins of the technique and those who pioneered this fascinating science.

The expanding utility of microdosing.

The concept of microdosing has been around for more than a decade. It consists of the subpharmacologic administration of an investigational drug (1% of the pharmacologic dose or 100 µg, whichever is lower) to human subjects to attain pre-phase 1 pharmacokinetics (PK) in humans. The major concern with microdosing has been the potential for nonlinear PK between doses, but methods are emerging to evaluate the potential for nonlinear PK prior to conducting a study. Currently, approximately 80% of drugs tested by the oral route and 100% by the intravenous route have exhibited scalable PK between a microdose and a therapeutic dose (within a factor of 2). Over the past few years microdosing has found utility in pediatrics, protein-based therapeutics, and a new application known as intra-arterial microdosing that focuses more on localized pharmacodynamics than PK. Compared with other PK predictive methods, such as physiologically based pharmacokinetic modeling, allometry, and in vitro-in vivo extrapolation, microdosing appears to provide a significantly better understanding of PK prior to phase 1, albeit within what is currently a limited database.

Microdosing and drug development: past, present and future.

INTRODUCTION: Microdosing is an approach to early drug development where exploratory pharmacokinetic data are acquired in humans using inherently safe sub-pharmacologic doses of drug. The first publication of microdose data was 10 years ago and this review comprehensively explores the microdose concept from conception, over the past decade, up until the current date. AREAS COVERED: The authors define and distinguish the concept of microdosing from similar approaches. The authors review the ability of microdosing to provide exploratory pharmacokinetics (concentration-time data) but exclude microdosing using positron emission tomography. The article provides a comprehensive review of data within the peer-reviewed literature as well as the latest applications and a look into the future, towards where microdosing may be headed. EXPERT OPINION: Evidence so far suggests that microdosing may be a better predictive tool of human pharmacokinetics than alternative methods and combination with physiologically based modelling may lead to much more reliable predictions in the future. The concept has also been applied to drug-drug interactions, polymorphism and assessing drug concentrations over time at its site of action. Microdosing may yet have more to offer in unanticipated directions and provide benefits that have not been fully realised to date.

Absolute oral bioavailability and metabolic turnover of ß-sitosterol in healthy subjects.

The metabolic turnover, absolute oral bioavailability, clearance, and volume of distribution for ß-sitosterol were measured in healthy subjects. [(14)C]ß-Sitosterol was used as an isotopic tracer to distinguish pulse doses from dietary sources and was administered by both oral and intravenous routes. The administered doses of [(14)C]ß-sitosterol were in the region of 3 to 4 µg, sufficiently low as not to perturb the kinetics of ß-sitosterol derived from the diet. Because the plasma concentrations of [(14)C]ß-sitosterol arising from such low doses were anticipated to be very low, the ultrasensitive isotope ratio analytical method of accelerator mass spectrometry was used. The limit of quantification for [(14)C]ß-sitosterol was approximately 0.1 pg/ml, the oral absolute bioavailability was just 0.41%, clearance was 85 ml/h, volume of distribution was 46 L, and the turnover was 5.8 mg/day. Given the steady-state concentrations of ß-sitosterol (2.83 µg/ml), then the dietary load was calculated to be approximately 1400 mg/day.

Meeting the MIST regulations: human metabolism in Phase I using AMS and a tiered bioanalytical approach.

The metabolites in safety testing and ICH-M3 guidance documents emphasize the importance of metabolites when considering safety aspects for new drugs. Both guidances state that relevant metabolites should have safety coverage in humans (although the guidelines have different definitions of relevant metabolites). Not having safety coverage for important metabolites in humans may cause significant delay in the overall pharmaceutical development program. This article discusses the regulatory background regarding safety and metabolites, as well as outlines an integrated strategy taken by one pharmaceutical company, Lundbeck A/S. Lundbeck uses metabolite exposure data from first-in-man studies, obtained using an accelerator MS approach followed by a two-tiered bioanalytical investigation. This enables early availability of key data on this aspect and, overall, represents a powerful risk mitigation strategy.

Predicting drug candidate victims of drug-drug interactions, using microdosing.

OBJECTIVE: The aim of this crossover human male volunteer study was to investigate the utility of microdosing in the investigation of drug-drug interactions. METHODS: A mixture of midazolam, tolbutamide, caffeine and fexofenadine were administered as a microdose (25 µg each) before and after administration of a combined pharmacological dose of ketoconazole (400 mg) and fluvoxamine (100 mg) to inhibit P-glycoprotein and metabolism by cytochrome P450 (CYP) 1A2, CYP3A4 and CYP2C9. RESULTS: When administered alone, pharmacokinetics for all four microdosed compounds scaled well with those reported for therapeutic doses and with previously performed microdose studies. The pharmacokinetics of each compound administered as a microdose were significantly altered after the administration of ketoconazole and fluvoxamine, showing statistically significant (p < 0.01) 12.8-, 8.1- and 3.2-fold increases in the area under the plasma concentration-time curve from time zero to infinity (AUC(∞)) for midazolam, caffeine and fexofenadine, respectively. A 1.8-fold increase (not statistically significant) in AUC(∞) was observed for tolbutamide. The changes in pharmacokinetics mediated by ketoconazole and fluvoxamine were quantitatively consistent with previously reported, non-microdose, drug-drug interaction data from studies including the same compounds. CONCLUSION: The initial data reported here demonstrate the utility of microdosing to investigate the risk of development drugs being victims of drug-drug interactions.

Comparative pharmacokinetics between a microdose and therapeutic dose for clarithromycin, sumatriptan, propafenone, paracetamol (acetaminophen), and phenobarbital in human volunteers.

A clinical study was conducted to assess the ability of a microdose (100 µg) to predict the human pharmacokinetics (PK) following a therapeutic dose of clarithromycin, sumatriptan, propafenone, paracetamol (acetaminophen) and phenobarbital, both within the study and by reference to the existing literature on these compounds and to explore the source of any nonlinearity if seen. For each drug, 6 healthy male volunteers were dosed with 100 µg (14)C-labelled compound. For clarithromycin, sumatriptan, and propafenone this labelled dose was administered alone, i.e. as a microdose, orally and intravenously (iv) and as an iv tracer dose concomitantly with an oral non-labelled therapeutic dose, in a 3-way cross over design. The oral therapeutic doses were 250, 50, and 150 mg, respectively. Paracetamol was given as the labelled microdose orally and iv using a 2-way cross over design, whereas phenobarbital was given only as the microdose orally. Plasma concentrations of total (14)C and parent drug were measured using accelerator mass spectrometry (AMS) or HPLC followed by AMS. Plasma concentrations following non-(14)C-labelled oral therapeutic doses were measured using either HPLC-electrochemical detection (clarithromycin) or HPLC-UV (sumatriptan, propafenone). For all five drugs an oral microdose predicted reasonably well the PK, including the shape of the plasma profile, following an oral therapeutic dose. For clarithromycin, sumatriptan, and propafenone, one parameter, oral bioavailability, was marginally outside of the normally acceptable 2-fold prediction interval around the mean therapeutic dose value. For clarithromycin, sumatriptan and propafenone, data obtained from an oral and iv microdose were compared within the same cohort of subjects used in the study, as well as those reported in the literature. For paracetamol (oral and iv) and phenobarbital (oral), microdose data were compared with those reported in the literature only. Where 100 µg iv (14)C-doses were given alone and with an oral non-labelled therapeutic dose, excellent accord between the PK parameters was observed indicating that the disposition kinetics of the drugs tested were unaffected by the presence of therapeutic concentrations. This observation implies that any deviation from linearity following the oral therapeutic doses occurs during the absorption process.

AMS method validation for quantitation in pharmacokinetic studies with concomitant extravascular and intravenous administration.

A technique has emerged in the past few years that has enabled a drug's intravenous pharmacokinetics to be readily obtained in humans without having to conduct extensive toxicology studies by this route of administration or expand protracted effort in formulation. The technique involves the intravenous administration of a low dose of (14)C-labelled drug (termed a tracer dose) concomitantly with a non-labelled extravascular dose given at therapeutically levels. Plasma samples collected over time are analysed to determine the total parent drug concentration by LC-MS (which essentially measures that arising from the oral dose) and by LC followed by accelerator mass spectrometry (AMS) to determine the (14)C-drug concentration (i.e., that arising from the intravenous dose). There are currently no published accounts of how the principles of bioanalytical validation might be applied to intravenous studies using AMS as an analytical technique. The authors describe the primary elements of AMS when used with LC separation and how this off-line technique differs from LC-MS. They then discuss how the principles of bioanalytical validation might be applied to determine selectivity, accuracy, precision and stability of methods involving LC followed by AMS analysis.

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.

A combined accelerator mass spectrometry-positron emission tomography human microdose study with 14C- and 11C-labelled verapamil.

BACKGROUND AND OBJECTIVE: In microdose studies, the pharmacokinetic profile of a drug in blood after administration of a dose up to 100 µg is measured with sensitive analytical techniques, such as accelerator mass spectrometry (AMS). As most drugs exert their effect in tissue rather than blood, methodology is needed for extending pharmacokinetic analysis to different tissue compartments. In the present study, we combined, for the first time, AMS analysis with positron emission tomography (PET) in order to determine the pharmacokinetic profile of the model drug verapamil in plasma and brain of humans. In order to assess pharmacokinetic dose linearity of verapamil, data were acquired and compared after administration of an intravenous microdose and after an intravenous microdose administered concomitantly with an oral therapeutic dose. METHODS: Six healthy male subjects received an intravenous microdose [0.05 mg] (period 1) and an intravenous microdose administered concomitantly with an oral therapeutic dose [80 mg] of verapamil (period 2) in a randomized, crossover, two-period study design. The intravenous dose was a mixture of (R/S)-[14C]verapamil and (R)-[11C]verapamil and the oral dose was unlabelled racaemic verapamil. Brain distribution of radioactivity was measured with PET whereas plasma pharmacokinetics of (R)- and (S)-verapamil were determined with AMS. PET data were analysed by pharmacokinetic modelling to estimate the rate constants for transfer (k) of radioactivity across the blood-brain barrier. RESULTS: Most pharmacokinetic parameters of (R)- and (S)-verapamil as well as parameters describing exchange of radioactivity between plasma and brain (influx rate constant [K(1)] = 0.030 ± 0.003 and 0.031 ± 0.005 mL/mL/min and efflux rate constant [k(2)] = 0.099 ± 0.006 and 0.095 ± 0.008 min-1 for period 1 and 2, respectively) were not statistically different between the two periods although there was a trend for nonlinear pharmacokinetics for the (R)-enantiomer. On the other hand, all pharmacokinetic parameters (except for the terminal elimination half-life [t1/2;)]) differed significantly between the (R)- and (S)-enantiomers for both periods. The maximum plasma concentration (C(max)), area under the plasma concentration-time curve (AUC) from 0 to 24 hours (AUC(24)) and AUC from time zero to infinity (AUC(∞)) were higher and the total clearance (CL), volume of distribution (V(d)) and volume of distribution at steady state (V(ss)) were lower for the (R)- than for the (S)-enantiomer. CONCLUSION: Combining AMS and PET microdosing allows long-term pharmacokinetic data along with information on drug tissue distribution to be acquired in the same subjects thus making it a promising approach to maximize data output from a single clinical study.

Addressing metabolite safety during first-in-man studies using ¹4C-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: current and the future.

The concept of microdosing has been around for approximately 10 years. In this time there have been an increasing number of drugs reported in the literature where the pharmacokinetics at a microdose have been compared with those observed at a therapeutic dose. Currently, approximately 80% of the microdose pharmacokinetics available in the public domain have been shown to scale to those observed at a therapeutic dose, within a twofold difference. Microdosing is now being extended into areas of drug development other than purely pharmacokinetic prediction. Microdosing has been applied to the study of drug-drug interactions by giving human volunteers a microdose of the candidate drug before and after the administration of a drug known to inhibit or induce certain enzymes, such as the cytochrome P450s. Early data on the metabolism of a drug candidate can be obtained by administering a (14)C-drug to human volunteers and comparing the plasma concentration-time curves for total (14)C and unchanged parent compound. Full metabolic profiles can be generated as an early indication of the drug's metabolism in humans, prior to Phase 1 clinical studies. Microdosing is also being applied to situations where the concentration of a drug in cell or tissue types is key to its efficacy. The application of microdosing as a tool in drug development is therefore widening into new and previously unforeseen fields.

Pharmacokinetics of fexofenadine: evaluation of a microdose and assessment of absolute oral bioavailability.

A human pharmacokinetic study was performed to assess the ability of a microdose to predict the pharmacokinetics of a therapeutic dose of fexofenadine and to determine its absolute oral bioavailability. Fexofenadine was chosen to represent an unmetabolized transporter substrate (P-gP and OATP). Fexofenadine was administered to 6 healthy male volunteers in a three way cross-over design. A microdose (100microg) of (14)C-drug was administered orally (period 1) and intravenously by 30min infusion (period 2). In period 3 an intravenous tracer dose (100microg) of (14)C-drug was administered simultaneously with an oral unlabelled therapeutic dose (120mg). Plasma was collected from all 3 periods and analysed for both total (14)C content and parent drug by accelerator mass spectrometry (AMS). For period 3, plasma samples were also analysed using HPLC-fluorescence to determine total drug concentration. Urine was collected and analysed for total (14)C. Good concordance between the microdose and therapeutic dose pharmacokinetics was observed. Microdose: CL 13L/h, CL(R) 4.1L/h, V(ss) 54L, t(1/2) 16h; therapeutic dose: CL 16L/h, CL(R) 6.2L/h, V(ss) 64L, t(1/2) 12h. The absolute oral bioavailability of fexofenadine was 0.35 (microdose 0.41, therapeutic dose 0.30). Despite a 1200-fold difference in dose of fexofenadine, the microdose predicted well the pharmacokinetic parameters following a therapeutic dose for this transporter dependent compound.

A pharmacokinetic evaluation of five H(1) antagonists after an oral and intravenous microdose to human subjects.

AIMS: To evaluate the pharmacokinetics (PK) of five H(1) receptor antagonists in human volunteers after a single oral and intravenous (i.v.) microdose (0.1 mg). METHODS: Five H(1) receptor antagonists, namely NBI-1, NBI-2, NBI-3, NBI-4 and diphenhydramine, were administered to human volunteers as a single 0.1-mg oral and i.v. dose. Blood samples were collected up to 48 h, and the parent compound in the plasma extract was quantified by high-performance liquid chromatography and accelerator mass spectroscopy. RESULTS: The median clearance (CL), apparent volume of distribution (V(d)) and apparent terminal elimination half-life (t(1/2)) of diphenhydramine after an i.v. microdose were 24.7 l h(-1), 302 l and 9.3 h, and the oral C(max) and AUC(0-infinity) were 0.195 ng ml(-1) and 1.52 ng h ml(-1), respectively. These data were consistent with previously published diphenhydramine data at 500 times the microdose. The rank order of oral bioavailability of the five compounds was as follows: NBI-2 > NBI-1 > NBI-3 > diphenhydramine > NBI-4, whereas the rank order for CL was NBI-4 > diphenhydramine > NBI-1 > NBI-3 > NBI-2. CONCLUSIONS: Human microdosing provided estimates of clinical PK of four structurally related compounds, which were deemed useful for compound selection.

Biotransformation profiling of [(14)C]ixabepilone in human plasma, urine and feces samples using accelerator mass spectrometry (AMS).

Ixabepilone (BMS-247550, Ixempra is a semi-synthetic analog of the natural product epothilone B and marketed for its use in the treatment of cancer. A conventional human ADME study using decay counting methods for (14)C detection could not be conducted due to the radiolytic instability of [(14)C]ixabepilone at a typical specific activity (generally 1-10 microCi/mg). However, [(14)C]ixabepilone was sufficiently stable at low specific activity (1-2 nCi/mg). These low levels required the use of accelerator mass spectrometry (AMS) for radioactivity detection. The metabolic fate of this compound was investigated in eight patients following single intravenous doses of [(14)C]ixabepilone (70 mg, 80 nCi; specific activity: 1.14 nCi/mg). Metabolite profiles in pooled urine, feces and plasma samples were determined by HPLC-AMS analysis. The major radioactive component in urine and plasma was [(14)C]ixabepilone. Feces had a small amount of ixabepilone. There were numerous other drug-related components in both urine and fecal extracts (each <6% of the administered dose). LC/MS analysis of plasma, urine and feces samples showed the presence of three degradants of ixabepilone and several oxidative metabolites (M+16, M+14 and M-2 metabolites). This study demonstrates the utility of AMS in investigating the metabolite and excretion profiles of [(14)C]ixabepilone following administration to humans.

New ultrasensitive detection technologies and techniques for use in microdosing studies.

In a microdosing study, subpharmacologically active doses of drug are given to human volunteers at an early stage of development in order to obtain preliminary pharmacokinetic data. The very low doses of drug administered (≤100 µg) consequently lead to very low concentrations of drug appearing in the body and therefore highly sensitive analytical techniques are required. There are three such analytical technologies currently used in microdosing studies: PET, liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and accelerator mass spectrometry (AMS). Both PET and AMS employ radioisotopic tracers. PET is an imaging technique and AMS is an extremely sensitive isotope ratio method, able to measure drug concentrations in the ag/ml range. LC-MS/MS does not require the presence of an isotopic tracer and its sensitivity is in the pg/ml range. This review examines each of these three analytical modalities in the context of performing microdosing studies.

The utility of microdosing over the past 5 years.

BACKGROUND: Microdosing studies (human Phase 0) are used to select drug candidates for Phase I clinical trials on the basis of their pharmacokinetic properties, using subpharmacologic doses (maximum 100 microg). There are questions as to whether pharmacokinetic data obtained at these low doses will predict those at the clinically relevant dose. OBJECTIVE: To review the current literature on microdosing and assess how well microdose data have predicted the pharmacokinetics obtained at a therapeutic dose. METHODS: All data published in the peer reviewed literature comparing pharmacokinetics at a microdose with a therapeutic dose were reviewed, excluding those studies aimed at imaging. CONCLUSIONS: Of the 18 drugs reported, 15 demonstrated linear pharmacokinetics within a factor of 2 between a microdose and a therapeutic dose. Therefore, data that support the utility of microdosing are beginning to emerge.

Biomedical accelerator mass spectrometry: recent applications in metabolism and pharmacokinetics.

BACKGROUND: Accelerator mass spectrometry (AMS) is a sensitive isotope ratio technique used in drug development that allows for small levels of 14C-drug to be administered to humans, thereby removing regulatory hurdles associated with radiotracer studies. AMS uses innovative study designs to obtain pharmacokinetic and metabolism data. OBJECTIVE: This review addresses the metabolism and pharmacokinetics relevant to cases where therapeutic drug concentrations are achieved in humans. METHODS: The review focuses on two study designs: i) administration of tracer 14C-drug intravenously with a simultaneous non-labelled extravascular therapeutic dose to obtain the pharmacokinetic parameters of clearance, volume of distribution and absolute bioavailability without extensive intravenous toxicology safety studies or formulation development; and ii) use of low levels of 14C-drug during Phase I studies to investigate the quantitative metabolism of the drug in humans early in drug development, as required by the new FDA guideline issued in February 2008. RESULTS/CONCLUSIONS: Early knowledge about a drug's clearance, volume of distribution, absolute bioavailability and metabolism can affect the development of a new drug candidate.

Positron emission tomography for use in microdosing studies.

Positron emission tomography (PET) imaging using microdoses of radiolabeled drug tracers is gaining increasing acceptance in modern clinical drug development. This approach is unique in that it allows for direct quantitative assessment of drug concentrations in the tissues targeted for treatment, thereby bridging the gap between pharmacokinetics and pharmacodynamics. Current applications of PET in anticancer, anti-infective and central nervous system drug research are reviewed herein. Situated at the interface of preclinical and clinical drug testing, PET microdosing is a powerful and highly innovative tool for pharmaceutical development.