Mass balance and metabolism of the antimalarial pyronaridine in healthy volunteers.

This was a single dose mass balance and metabolite characterization study of the antimalarial agent pyronaridine. Six healthy male adults were administered a single oral dose of 720 mg pyronaridine tetraphosphate with 800 nCi of radiolabeled (14)C-pyronaridine. Urine and feces were continuously collected through 168 h post-dose, with intermittent 48 h collection periods thereafter through 2064 h post-dose. Drug recovery was computed for analyzed samples and interpolated for intervening time periods in which collection did not occur. Blood samples were obtained to evaluate the pharmacokinetics of total radioactivity and of the parent compound. Total radioactivity in urine, feces, and blood samples was determined by accelerator mass spectrometry (AMS); parent concentrations in blood were determined with LC/MS. Metabolite identification based on blood, urine, and feces samples was conducted using a combination of LC + AMS for identifying radiopeaks, followed by LC/MS/MS for identity confirmation/elucidation. The mean cumulative drug recovery in the urine and feces was 23.7 and 47.8 %, respectively, with an average total recovery of 71.5 %. Total radioactivity was slowly eliminated from blood, with a mean half-life of 33.5 days, substantially longer than the mean parent compound half-life of 5.03 days. Total radioactivity remained detectable in urine and feces collected in the final sampling period, suggesting ongoing elimination. Nine primary and four secondary metabolites of pyronaridine were identified. This study revealed that pyronaridine and its metabolites are eliminated by both the urinary and fecal routes over an extended period of time, and that multiple, varied pathways characterize pyronaridine metabolism.

Accelerator mass spectrometry for quantification of micro- and therapeutic-dose diclofenac in microdialysis samples.

Background: Microdialysis sampling after drug microdosing may provide tissue pharmacokinetic data early in clinical drug development. However, low administered doses and small sample volumes pose an analytical challenge, particularly for highly protein-bound drugs. Materials & methods: Carbon-14 [14C]diclofenac was used as a model drug to assess the technical and analytical feasibility of in vivo microdialysis after microdose administration in an in vitro setup. Results: [14C]diclofenac dialysate concentrations were accurately quantified with accelerator MS. [14C]diclofenac dialysate recoveries were similar in the presence and absence of therapeutic diclofenac concentrations but were considerably decreased when albumin was added to the immersion solution, suggesting high protein binding. Conclusion: These results demonstrate the feasibility of combining microdosing and microdialysis to assess tissue pharmacokinetics.

An Investigation of the Metabolism and Excretion of KD101 and Its Interindividual Differences: A Microtracing Mass Balance Study in Humans.

The absorption, metabolism, and excretion (AME) profiles of KD101, currently under clinical development to treat obesity, were assessed in humans using accelerator mass spectrometry (AMS) after a single oral administration of KD101 at 400 mg and a microdose of 14 C-KD101 at ~ 35.2 µg with a total radioactivity of 6.81 kBq. The mean total recovery of administered radioactivity was 85.2% with predominant excretion in the urine (78.0%). The radio-chromatographic metabolite profiling showed that most of the total radioactivity in the plasma and the urine was ascribable to metabolites. The UDP-glucuronosyltransferase (UGT), including UGT1A1, UGT1A3, and UGT2B7, might have contributed to the interindividual variability in the metabolism and excretion of KD101. The microtracing approach using AMS is a useful tool to evaluate the AME of a drug under development without risk for high radiation exposure to humans.

Accelerator mass spectrometry can be used to assess vitamin A metabolism quantitatively in boys in a community setting.

A survey indicated that high-dose vitamin A (HD-VA) supplements had no apparent effect on vitamin A (VA) status, assessed by serum retinol concentrations, of Zambian children lt 5 y of age. To explore possible reasons for the lack of response, we quantified absorption, retention, and urinary elimination of either a single HD-VA supplement (209.8 micromol; 60 mg) or a smaller dose of stable isotope (SI)-labeled VA (17.5 micromol; 5 mg), which was used to estimate VA pool size, in 3- to 4-y-old Zambian boys (n = 4 for each VA dose). A tracer dose of [(14)C(2)]-labeled VA (0.925 kBq; 25 nCi) was coadministered with the HD-VA supplement or SI-labeled VA, and 24-h stool and urine samples were collected for 3 and 7 consecutive days, respectively, and 24-h urine samples at 4 later time points. Accelerator MS was used to quantify (14)C in stool and urine. Estimates of absorption, retention, and the urinary elimination rate (UER) were 83.8 +/- 7.1%, 76.3 +/- 6.7%, and 1.9 +/- 0.6%/d, respectively, for the HD-VA supplement and 76.5 +/- 9.5%, 71.1 +/- 9.4%, and 1.8 +/- 1.2%/d, respectively, for the SI-labeled VA. Mean estimates of absorption, retention, and the UER did not differ by size of the VA dose administered. Estimated absorption and retention were negatively associated with reported fever (r = minus 0.83; P = 0.011). The HD-VA supplement and SI-labeled VA were adequately absorbed, retained, and utilized in apparently healthy Zambian preschool-age boys; absorption and retention may be affected by recent fever.

Human ADME for YH12852 using wavelength scanning cavity ring-down spectroscopy (WS-CRDS) after a low radioactivity dose.

Aim: Human 14C radiotracer studies provide information-rich data sets that enable informed decision making in clinical drug development. These studies are supported by liquid scintillation counting after conventional-sized 14C doses (50-200 µCi) or complex accelerator mass spectrometry (AMS) after microtracer-sized doses (∼0.1-1 µCi). Mid-infrared laser-based 'cavity ring-down spectroscopy' (CRDS) is an emerging platform for the sensitive quantitation of 14C tracers. Results & methodology: We compared the total 14C concentrations in plasma and urine samples from a microtracer study using both CRDS and AMS technology. The data were evaluated using statistical and pharmacokinetic modeling. Conclusion: The CRDS method closely reproduced the AMS method for total 14C concentrations. With optimization of the automated sample interface and further testing, it promises to be an accessible, robust system for pivotal microtracer investigations.

Nanotracing and cavity-ring down spectroscopy: A new ultrasensitive approach in large molecule drug disposition studies.

New therapeutic biological entities such as bispecific antibodies targeting tissue or specific cell populations form an increasingly important part of the drug development portfolio. However, these biopharmaceutical agents bear the risk of extensive target-mediated drug disposition or atypical pharmacokinetic properties as compared to canonical antibodies. Pharmacokinetics and bio-distribution studies become therefore more and more important during lead optimization. Biologics present, however, greater analytical challenges than small molecule drugs due to the mass and selectivity limitation of mass spectrometry and ligand-binding assay, respectively. Radiocarbon (14C) and its detection methods, such as the emerging 14C cavity ring down spectroscopy (CRDS), thus can play an important role in the large molecule quantitation where a 14C-tag is covalently bound through a stable linker. CRDS has the advantage of a simplified sample preparation and introduction system as compared to accelerator mass spectrometry (AMS) and can be accommodated within an ordinary research laboratory. In this study, we report on the labeling of an anti-IL17 IgG1 model antibody with 14C propionate tag and its detection by CRDS using it as nanotracer (2.1 nCi or 77.7 Bq blended with the therapeutic dose) in a pharmacokinetics study in a preclinical species. We compare these data to data generated by AMS in parallel processed samples. The derived concentration time profiles for anti-IL17 by CRDS were in concordance with the ones derived by AMS and γ-counting of an 125I-labeled anti-IL17 radiotracer and were well described by a 2-compartment population pharmacokinetic model. In addition, antibody tissue distribution coefficients for anti-IL17 were determined by CRDS, which proved to be a direct and sensitive measurement of the extravascular tissue concentration of the antibody when tissue perfusion was applied. Thus, this proof-of-concept study demonstrates that trace 14C-radiolabels and CRDS are an ultrasensitive approach in (pre)clinical pharmacokinetics and bio-distribution studies of new therapeutic entities.

Opportunities in low-level radiocarbon microtracing: applications and new technology.

14C-radiolabeled (radiocarbon) drug studies are central to defining the disposition of therapeutics in clinical development. Concerns over radiation, however, have dissuaded investigators from conducting these studies as often as their utility may merit. Accelerator mass spectrometry (AMS), originally designed for carbon dating and geochronology, has changed the outlook for in-human radiolabeled testing. The high sensitivity of AMS affords human clinical testing with vastly reduced radiative (microtracing) and chemical exposures (microdosing). Early iterations of AMS were unsuitable for routine biomedical use due to the instruments' large size and associated per sample costs. The situation is changing with advances in the core and peripheral instrumentation. We review the important milestones in applied AMS research and recent advances in the core technology platform. We also look ahead to an entirely new class of 14C detection systems that use lasers to measure carbon dioxide in small gas cells.

Plasma beta-carotene and retinol concentrations of children increase after a 30-d supplementation with the fruit Momordica cochinchinensis (gac).

BACKGROUND: In rural Vietnam, vitamin A deficiency is a concern. Among the indigenous fruit and vegetables, Momordica cochinchinensis (gac) fruit has been identified as having the highest beta-carotene concentration. Locally, it is mixed with rice in a preparation called xoi gac. OBJECTIVE: The purpose of this study was to assess this beta-carotene- rich rice preparation as a source of provitamin A for children in rural Vietnam. DESIGN: Preschoolers (n = 185) participated in a 30-d controlled supplementation trial. Children with low hemoglobin concentrations were assigned to 1 of 3 groups: a fruit group, who received xoi gac that contained 3.5 mg beta-carotene per serving; a powder group, who received rice mixed with 5.0 mg synthetic beta-carotene powder; and a control group, who received rice without fortification. RESULTS: The mean increase in plasma beta-carotene concentrations in the fruit and powder groups was significantly greater than that in the control group (P < 0.0001). After supplementation, the mean plasma retinol concentration in the fruit group was significantly higher than that in the control (P = 0.006) and powder (P = 0.0053) groups. Among the children with initial hemoglobin concentrations

Variability in conversion of beta-carotene to vitamin A in men as measured by using a double-tracer study design.

BACKGROUND: The vitamin A activity of beta-carotene is variable and surprisingly low in women. The reasons for this are not well understood. The vitamin A activity of beta-carotene in men is still uncertain. Contributions of dietary factors compared with individual traits are largely unknown. OBJECTIVE: Our objective was to measure the intrinsic variability in the vitamin A activity of beta-carotene among healthy, well-fed men living in a controlled environment. DESIGN: We used a double-tracer test-retest design. We dosed 11 healthy men orally with 30 micromol hexadeuterated (D6) retinyl acetate (all-trans-19,19,19,20,20,20-[2H6]retinyl acetate) and then with 37 micromol D6 beta-carotene (19,19,19,19',19',19'-[2H6]beta-carotene) 1 wk later. Doses were taken with breakfasts containing 16 g fat. We measured D6 retinol, D6 beta-carotene, and trideuterated (D3) retinol (derived from D6 beta-carotene) concentrations in plasma. Areas under the plasma concentration x time since dosing curves (AUCs) were determined for D6 retinol, D6 beta-carotene, and D3 retinol. RESULTS: All men had detectable D6 retinol concentrations in plasma. The mean (+/-SE) absorption of D6 beta-carotene in all subjects was 2.235 +/- 0.925%, and the mean conversion ratio was 0.0296 +/- 0.0108 mol retinol to 1 mol beta-carotene. Only 6 of 11 men had sufficient plasma concentrations of D6 beta-carotene and D3 retinol that we could measure. The mean absorption of D6 beta-carotene in these 6 subjects was 4.097 +/- 1.208%, and the mean conversion ratio was 0.0540 +/- 0.0128 mol retinol to 1 mol beta-carotene. CONCLUSION: The vitamin A activity of beta-carotene, even when measured under controlled conditions, can be surprisingly low and variable.

Quantitation of in vivo human folate metabolism.

BACKGROUND: A quantitative understanding of human folate metabolism is needed. OBJECTIVE: The objective was to quantify and interpret human folate metabolism as it might occur in vivo. DESIGN: Adults (n = 13) received 0.5 nmol [(14)C]pteroylmonoglutamate (100 nCi radioactivity) plus 79.5 nmol pteroylmonoglutamate in water orally. (14)C was measured in plasma, erythrocytes, urine, and feces for >/=40 d. Kinetic modeling was used to analyze and interpret the data. RESULTS: According to the data, the population was healthy and had a mean dietary folate intake of 1046 nmol/d, and the apparent dose absorption of (14)C was 79%. The model predictions showed that only 0.25% of plasma folate was destined for marrow, mean bile folate flux was 5351 nmol/d, and the digestibility of the mix (1046 + 5351 nmol/d) was 92%. About 33% of visceral pteroylmonoglutamate was converted to the polyglutamate form, most of the body folate was visceral (>99%), most of the visceral folate was pteroylpolyglutamate (>98%), total body folate was 225 micromol, and pteroylpolyglutamate synthesis, recycling, and catabolism were 1985, 1429, and 556 nmol/d, respectively. Mean residence times were 0.525 d as visceral pteroylmonoglutamate, 119 d as visceral pteroylpolyglutamate, 0.0086 d as plasma folate, and 0.1 d as gastrointestinal folate. CONCLUSIONS: Across subjects, folate absorption, bile folate flux, and body folate stores were larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate was an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. It successfully quantitated folate metabolism, encouraging further rigorous testing.

Phytochemical research using accelerator mass spectrometry.

Vegetables and fruits provide an array of microchemicals in the form of vitamins and secondary metabolites (phytochemicals) that may lower the risk of chronic disease. Tracing these phytochemicals at physiologic concentrations has been hindered by a lack of quantitative sensitivity for chemically equivalent tracers that could be used safely in healthy people. Accelerator mass spectrometry is a relatively new technique that provides the necessary sensitivity (in attomoles) and measurement precision (<3%) towards 14C-labeled phytochemicals for detailed kinetic studies in humans at dietary levels.

New frontiers-accelerator mass spectrometry (AMS): Recommendation for best practices and harmonization from Global Bioanalysis Consortium Harmonization Team.

The technique of accelerator mass spectrometry (AMS) is applicable to the analysis of a wide range of trace elemental isotopes. However, in the context of the pharmaceutical industry, it is invariably used to measure radiocarbon ((14)C). There are two broad modes of application: analysis of total (14)C sometimes termed "direct AMS" and analysis of specific (14)C-labelled analytes in a variety of matrices following some method of isolation. It is the latter application which is within the remit of the GBC team, and the team has made efforts to propose harmonized recommendations for the validation of AMS when used in a regulatory bioanalytical mode, i.e. the quantification of specific analyte(s) using liquid chromatography with off-line detection by AMS now known as "LC + AMS". The GBC team has reached a position where they have agreed to many aspects, but also differ on some aspects of what constitutes a bioanalytical assay validation in support of clinical studies using this technology. The detail of most of this will be covered under separate publication(s), but for the purposes of this paper, we have outlined the points of consensus. The purpose of this article is not to provide a roadmap for validation of LC + AMS assays, but to highlight agreements amongst the industry representative experts and the practitioners, as well as identifying specific areas essential for establishing assay quality but where additional discussion is required to reach agreement.

Pharmacokinetic analysis of 14C-ursodiol in newborn infants using accelerator mass spectrometry.

Pharmacokinetic studies in the neonatal population are often limited by the small volume of blood that can be collected. The high sensitivity of (14) C-accelerator mass spectrometry (AMS) enables pharmacokinetic studies to be conducted with greatly reduced sample volumes. We demonstrated the utility of AMS in infants by studying the plasma pharmacokinetic behavior of nanogram doses of (14) C-ursodiol administered as a non-perturbing microdose or as a microtracer with therapeutic doses of non-labeled ursodiol in infants. Five non-cholestatic infants were administered 3 consecutive oral microdoses of (14) C-ursodiol: 8 ng (1.0 nCi), 26 ng (3.3 nCi), and 80 ng (10 nCi) 48 hours apart. Three additional infants with cholestasis were administered a single 80 ng (10.0 nCi) oral dose of (14) C-ursodiol together with a therapeutic dose of 40 mg/kg of non-labeled ursodiol. A pharmacokinetic model describing ursodiol concentrations was developed using nonlinear mixed-effects modeling. The pharmacokinetics of ursodiol in this pilot study were best described by a two-compartment model with first-order elimination. This study demonstrates the feasibility and utility of microdose and microtrace methodology in pediatric research.

Overcoming bioanalytical challenges in an Onglyza(®) intravenous [(14)C]microdose absolute bioavailability study with accelerator MS.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Quantifying exploratory low dose compounds in humans with AMS.

Accelerator Mass Spectrometry is an established technology whose essentiality extends beyond simply a better detector for radiolabeled molecules. Attomole sensitivity reduces radioisotope exposures in clinical subjects to the point that no population need be excluded from clinical study. Insights in human physiochemistry are enabled by the quantitative recovery of simplified AMS processes that provide biological concentrations of all labeled metabolites and total compound related material at non-saturating levels. In this paper, we review some of the exploratory applications of AMS (14)C in toxicological, nutritional, and pharmacological research. This body of research addresses the human physiochemistry of important compounds in their own right, but also serves as examples of the analytical methods and clinical practices that are available for studying low dose physiochemistry of candidate therapeutic compounds, helping to broaden the knowledge base of AMS application in pharmaceutical research.

Accelerator mass spectrometry best practices for accuracy and precision in bioanalytical (14)C measurements.

Accelerator mass spectrometers have an energy acceleration and charge exchange between mass definition stages to destroy molecular isobars and allow single ion counting of long-lived isotopes such as (14)C (t½=5370 years.). 'Low' voltage accelerations to 200 kV allow laboratory-sized accelerator mass spectrometers instruments for bioanalytical quantitation of (14)C to 2-3% precision and accuracy in isolated biochemical fractions. After demonstrating this accuracy and precision for our new accelerator mass spectrometer, we discuss the critical aspects of maintaining quantitative accuracy from the defined biological fraction to the accelerator mass spectrometry quantitation. These aspects include sufficient sample mass for routine rapid sample preparation, isotope dilution to assure this mass, isolation of the carbon from other sample combustion gasses and use of high-efficiency biochemical separations. This review seeks to address a bioanalytical audience, who should know that high accuracy data of physiochemical processes within living human subjects are available, as long as a (14)C quantitation can be made indicative of the physiochemistry of interest.

Early human ADME using microdoses and microtracers: bioanalytical considerations.

Quantitative assessment of metabolites of drug candidates in early-phase clinical development presents an analytical challenge when methods, standards and assays are not yet available. Radioisotopic labeling, principally with radiocarbon ((14)C), is the preferred method for discovering and quantifying the absolute yields of metabolites in the absence of reference material or a priori knowledge of the human metabolism. However, the detection of (14)C is inefficient by decay counting methods and, as a result, high radiological human (14)C-doses had been needed to assure sensitive detection of metabolites over time. High radiological doses and the associated costs have been a major obstacle to the routine (and early) use of (14)C despite the recognized advantages of a (14)C-tracer for quantifying drug metabolism and disposition. Accelerator mass spectrometry eliminates this long-standing problem by reducing radioactivity levels while delivering matrix-independent quantitation to attomole levels of sensitivity in small samples or fractionated isolates. Accelerator mass spectrometry and trace (14)C-labeled drugs are now used to obtain early insights into the human metabolism of a drug candidate in ways that were not previously practical. With this article we describe some of our empirically based approaches for regualted bioanalysis and offer perspectives on current applications and opportunities for the future.

Use of accelerator mass spectrometry to measure the pharmacokinetics and peripheral blood mononuclear cell concentrations of zidovudine.

The remarkable sensitivity of accelerator mass spectrometry (AMS) is finding many new applications in pharmacology. In this study AMS was used to measure [(14)C]-Zidovudine (ZDV) concentrations at the drug's site of action (peripheral blood mononuclear cells, PBMCs) following a dose of 520 ng (less than one-millionth of the standard daily dose) to a healthy volunteer. In addition, the pharmacokinetics of this microdose were determined and compared to previously published parameters for therapeutic doses. Microdose ZDV pharmacokinetic parameters fell within reported 95% confidence intervals or standard deviations of most previously published values for therapeutic doses. Blood, urine, stool, saliva, and isolated PBMCs were collected periodically through 96 h postdose and analyzed for ZDV and metabolite concentrations. The results showed that ZDV is rapidly absorbed and eliminated, has one major metabolite, and is sequestered in PBMCs. (14)C mass balance assessments indicated a significant portion of ZDV remained after 96 h with a much prolonged elimination half-life. Results of this study demonstrate the usefulness of microdosing and AMS as a tool for studying the pharmacokinetic characteristics, including PBMC concentrations, of ZDV and underscore the value of AMS as a tool with which to perform pharmacokinetic and mass balance studies using trace amounts of radiolabeled compound.

Human vitamin B12 absorption measurement by accelerator mass spectrometry using specifically labeled (14)C-cobalamin.

There is a need for an improved test of human ability to assimilate dietary vitamin B(12). Assaying and understanding absorption and uptake of B(12) is important because defects can lead to hematological and neurological complications. Accelerator mass spectrometry is uniquely suited for assessing absorption and kinetics of carbon-14 ((14)C)-labeled substances after oral ingestion because it is more sensitive than decay counting and can measure levels of (14)C in microliter volumes of biological samples with negligible exposure of subjects to radioactivity. The test we describe employs amounts of B(12) in the range of normal dietary intake. The B(12) used was quantitatively labeled with (14)C at one particular atom of the dimethylbenzimidazole (DMB) moiety by exploiting idiosyncrasies of Salmonella metabolism. To grow aerobically on ethanolamine, Salmonella enterica must be provided with either preformed B(12) or two of its precursors, cobinamide and DMB. When provided with (14)C-DMB specifically labeled in the C2 position, cells produced (14)C-B(12) of high specific activity (2.1 GBq/mmol, 58 mCi/mmol) (1 Ci = 37 GBq) and no detectable dilution of label from endogenous DMB synthesis. In a human kinetic study, a physiological dose (1.5 microg, 2.2 kBq/59 nCi) of purified (14)C-B(12) was administered and showed plasma appearance and clearance curves consistent with the predicted behavior of the pure vitamin. This method opens new avenues for study of B(12) assimilation.

A parallel processing solid phase extraction protocol for the determination of whole blood folate.

We describe an improved whole blood folate analysis method that facilitates increased throughput compared to our previous method (Dueker et al. (2000) Anal. Biochem. 283, 266). Improvements include three items: first, a buffered solvent exchange to remove interfering amino acids, especially phenylalanine whose esters may interfere with the analysis because their retention times on the gas chromatography are close to those of the para-aminobenzoic acid (pABA) isotopomers; second, substituting an NH2 solid phase extraction step for an HPLC step permits the batch parallel processing of samples; third, replacing trifluoroacetyl derivatives of ethyl-esterified pABA isotopomers with heptafluorobutyl derivatives, which are better resolved on the GC column. The method measures pABA, a stable degradation product of folate. This simplifies sample handling and purification. Relative standard deviations are typically 5% or less and a single operator can process samples in batches of 40. Results from our GCMS method correlate (R = 0.98) with the Lactobacillus casei assay for whole blood folate. The modifications will facilitate the development of high throughput methods for whole blood folate. Our method holds promise for epidemiological and clinical studies, where accurate whole blood folate concentrations are needed. Because it is internally standardized, interlaboratory variation should be minimal.