Heparanase induces inflammatory cell recruitment in vivo by promoting adhesion to vascular endothelium.

Heparanase (HPSE1) is known to be involved in mechanisms of metastatic tumor cell migration. This enzyme selectively cleaves heparan sulfate proteoglycans (HSPG), which are ubiquitously expressed in mammals and are known to be involved in regulating the activity of an array of inflammatory mediators. In the present study, we have investigated the effects of human recombinant heparanase, the inactive precursor of this enzyme (proheparanase) and enzymatically inactivated heparanase, on inflammatory cell recruitment in the rat and on human leukocyte-endothelial adhesion in vitro. Intraperitoneal injection of heparanase (500 µg) induced a significant inflammatory cell infiltrate in the rat, as assessed by peritoneal lavage 4 h later. Intravital microscopy of the mesenteric microcirculation of anesthetized rats showed an increase in rolling and adherent cells in postcapillary venules that was sensitive to heparin, a nonselective inhibitor of heparanase activity. In vitro, heparanase augmented the adhesion of human neutrophils and mononuclear cells to human umbilical vein endothelial cells in a concentration-dependent manner. Proheparanase had similar effects to the active enzyme both with respect to leukocyte accumulation in the peritoneal cavity and adhesion in vitro. However, heat-inactivated heparanase induced cell adhesion in vitro but was without effect in vivo. Together, these data indicate a role for heparanase in inflammatory cell trafficking in vivo that appears to require enzymatic activity.

Serum hepcidin but not prohepcidin may be an effective marker for anemia of inflammation (AI).

Anemia in cancer patients can result from a complex interaction of numerous factors including iron deficiency, inflammation, toxicity related to therapy and effect of cancer on the marrow. Determining effective anemia treatment can therefore be complex, requiring a combination of diagnostic tests. Research on iron metabolism has highlighted the importance of hepcidin and its potential role in development of anemia of inflammation (AI). Hepcidin is a peptide that controls iron flow, is induced by inflammation and is speculated to cause the sequestration of iron in patients with inflammation. In the present study, serum hepcidin concentration determined by LC-MS/MS was shown to correlate with inflammatory markers in patients with anemia of cancer (AoC). In the absence of a widely-available serum hepcidin detection assay, detection of prohepcidin using a commercial assay has been used for several years as a surrogate for measuring serum hepcidin concentration. Analysis of prohepcidin concentration did not reveal any correlation with hepcidin or with inflammatory markers in patient samples and our data suggest that prohepcidin may not be stable in serum. Algorithms to sub-classify AoC patients showed that hepcidin was strongly associated with the population subset with inflammation and without iron deficiency. Serum hepcidin concentrations may therefore be a good predictor of AI, useful in diagnosis of anemia etiology and in treatment determination.

Design and synthesis of conformationally constrained glucagon-like peptide-1 derivatives with increased plasma stability and prolonged in vivo activity.

A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated. The introduction of i to i + 4 glutamic acid-lysine lactam constraints in c[Glu (18)-Lys (22)][Gly (8)]GLP-1(7-37)-NH2 (6), c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-NH2 (10), and c[Glu (23)-Lys (27)][Gly (8)]GLP-1(7-37)-NH2 (11) resulted in potent functional activity and receptor affinities comparable to native GLP-1. Selected GLP-1 peptides were chemoselectively PEGylated in order to prolong their in vivo activity. PEGylated peptides [Gly (8),Aib (22)]GLP-1(7-37)-Cys ((PEG))-Ala-NH2 (23) and c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-Cys ((PEG))-Ser-Gly-NH2 (24) retained picomolar functional potency and avid receptor binding properties. Importantly, PEGylated GLP-1 peptide 23 exhibited sustained in vivo efficacy with respect to blood glucose reduction and decreased body weight for several days in nonhuman primates.

Concerns in the development of an assay for determination of a highly conjugated adsorption-prone compound in human urine.

Concerns in pre-analytical handling of urine samples are discussed using a new KDR kinase inhibitor, 3-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2-yl]-1H-quinolin-2-one (compound A), as an example of a case where high light sensitivity and low analyte recovery (high affinity for container surface) were found. The absence of these problems in plasma samples may be a result of the plasma protein content. Low recovery of the analyte from urine can be remedied by either changing the container or by using additives, such as bovine serum albumin (BSA) or non-ionic surfactant Tween-20. In the case of compound A, changing containers (polypropylene versus glass vial) or addition of BSA did bring analyte recovery up to 80%. However, the addition of 0.2% Tween-20 into urine quality controls (QCs) gave more than 95% analyte recovery, indicating effective reduction of analyte loss to the surface of containers. The urine assay using mixed-mode SPE and LC-MS/MS was not affected significantly by introducing Tween-20 into the samples. The mean SPE extraction recovery was 68.4% and matrix suppression of ionization on MS was less than 8% at all analyte concentrations. The linear range of the calibration curve was 0.5-400 ng/mL on PE Sciex API 3000 LC-MS/MS system. The assay intraday accuracy and precision were 92.1-104.8% and <4.2% (%CV), respectively. Urine QC samples, containing 0.2% Tween-20, gave excellent recovery after three cycles of freeze and thaw. Since analyte loss to its urine container surface is not unique to compound A (M. Schwartz, W. Kline, B. Matuszewski, Anal. Chim. Acta 352 (1997) 299-307; A.L. Fisher, E. DePuy, T. Shih, R. Stearns, Y. Lee, K. Gottesdiener, S. Flattery, M. De Smet, B. Keymeulen, D.G. Musson, J. Pharm. Biomed. Anal. 26 (2001) 739-752), we suggest an evaluation of the potential problem in the early stages of urine assay development to ensure reliable quantitation of analytes. The addition of Tween-20 can serve as a useful analytical tool to other analytes with similar situations.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS.

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW>4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5-->221.0) that was reflective of the mass concentration (microg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 microg/mL when extracting 200-microL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1=0.9991 and r2=0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 microg/mL for each assay. Average ranges (n=12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.

Validation of a method for the determination of AMG 579 in cerebrospinal fluid with a focus on sample collection procedures for clinical trials.

Analysis of pharmaceutical compounds in cerebrospinal fluid (CSF) may present challenges due to the combination of the low protein content in this matrix and relatively low drug concentrations, often corresponding to free drug concentrations in plasma, typically found in CSF. A 30% loss of AMG 579 was observed during preparation of quality control samples and further investigation determined that this loss was likely due to binding to collection tubes. This observation also highlighted the possibility of additional losses of AMG 579 that could occur during collection of clinical samples, such as binding to catheters used in the collection of CSF. Loss of AMG 579 in QC samples was reduced from 30% to 5% when the volume of CSF stored in 1.5 mL vials was increased from 0.06 mL to 1 mL. Modest but unavoidable losses of about 20% of AMG 579 were also found following perfusion through both silicone and polypropylene (Pharmed(®) BPT) collection catheters. Silicone tubing was used for CSF collection based on clinical site preference. An LC-MS/MS method was validated to quantify AMG 579 in human CSF to support clinical testing. The original range of the assay was 1-1000 ng/mL but the LLOQ was subsequently lowered to 0.1 ng/mL to better meet project requirements. Interday bias (% RE) and precision (% CV) were -4.2% and 12.3% at the LLOQ, and less than ± 0.9% and 8.3% for higher concentrations, respectively. The compound was stable in human CSF for at least 5h at room temperature, 55 days at -70 °C (-60 to -80 °C range), and through three freeze-thaw cycles. Careful selection of assay conditions and materials minimized losses of the compound during sample collection and storage. While these losses could not be entirely eliminated, practical sample collection and storage conditions were established to allow for analysis of AMG 579 in human clinical trials.

Glycosaminoglycans and the regulation of allergic inflammation.

Glycosaminoglycans (GAGs) are large, polyanionic molecules expressed throughout the body. The GAG heparin, co-released with histamine, is synthesised by and stored exclusively in mast cells, whereas the closely related molecule heparan sulphate is expressed, as part of a proteoglycan, on cell surfaces and throughout tissue matrices. These molecules are increasingly thought to play a role in regulation of the inflammatory response and heparin like molecules are now being seriously considered to hold potential in the treatment of inflammatory diseases such as asthma. Heparin and related molecules have been found to exert anti-inflammatory effects in a wide range of in vitro assays, animal models and in human disease. The anti-inflammatory activities of heparin are independent of the well-established anticoagulant activity of heparin, suggesting that the separation of these properties could yield novel anti-inflammatory drugs, which may be useful in the future treatment of inflammatory diseases.

Pharmacokinetics of etoricoxib in patients with hepatic impairment.

The effect of hepatic insufficiency on the pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase-2, was investigated following administration of single and multiple oral doses to mild hepatic insufficiency patients (Child-Pugh score of 5 to 6), multiple oral doses to moderate hepatic insufficiency patients (Child-Pugh score of 7 to 9), and single intravenous doses to both mild and moderate hepatic insufficiency patients. A trend of decreasing systemic clearance with increasing hepatic impairment was observed. Absorption of etoricoxib was unaffected by hepatic impairment. Binding of etoricoxib to plasma proteins was also found to be unaffected by hepatic disease. Etoricoxib was generally well tolerated by patients with mild and moderate hepatic insufficiency. Together, these results support a 60-mg once-daily dosing regimen for mild hepatic insufficiency patients and a 60-mg every-other-day dosing regimen for moderate hepatic insufficiency patients. There are no clinical or pharmacokinetic data in patients with severe hepatic insufficiency (Child-Pugh score > 9).

Single- and multiple-dose pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase-2, in man.

The single- and multiple-dose pharmacokinetics of etoricoxib, a selective inhibitor of cyclooxygenase-2, were examined in two clinical studies. Single-dose pharmacokinetics--including dose proportionality, absolute bioavailability of the highest dose-strength (120-mg) tablet, and the effect of a high-fat meal on the bioavailability of that tablet--were investigated in a two-part, open, balanced crossover study in two panels of healthy subjects (12 per panel). Steady-state pharmacokinetics were investigated in an open-label study in which 24 healthy subjects were administered 120-mg single and multiple (once daily for 10 days) oral doses of etoricoxib tablets. The pharmacokinetics of etoricoxib were found to be consistent with linearity through doses at least twofold greater than the highest anticipated clinical dose of 120 mg. Etoricoxib administered as a tablet was rapidly and completely absorbed and available; the absolute bioavailability was estimated to be 100%. A high-fat meal decreased the rate of absorption without affecting the extent of absorption of etoricoxib; therefore, etoricoxib can be dosed irrespective of food. Steady-state pharmacokinetics of etoricoxib, achieved following 7 days of once-daily dosing, were found to be reasonably predicted from single doses. The accumulation ratio averaged 2.1, and the corresponding accumulation t1/2 averaged 22 hours, supporting once-daily dosing. Etoricoxib was generally well tolerated.

Amino acid and peptide analysis using derivatization with p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether and capillary electrophoresis with electrochemical detection.

The amine derivatization reagent p-nitrophenol-2,5-dihydroxyphenylacetate bis-tetrahydropyranyl ether (NDTE) was used in conjunction with capillary electrophoresis (CE) and electrochemical detection (EC) for the pre-separation derivatization of primary amine analytes present in aqueous solution. Glycine, several dipeptides and angiotensin II were used as model analytes. A miniaturized EC detection cell was designed and fabricated, which featured a fractured-joint field decoupler with a fixed end-column carbon fiber electrode. When a series of glycine and angiotensin II calibration solutions were derivatized with NDTE followed by CE-EC determination, linear calibration plots resulted with pre-derivatization concentration limits of detection of 500 nM (106 attomoles on-column) and 6 microM (1.275 femtomoles on-column), respectively.

Bioanalytical method validation: concepts, expectations and challenges in small molecule and macromolecule--a report of PITTCON 2013 symposium.

The concepts, importance, and implications of bioanalytical method validation has been discussed and debated for a long time. The recent high profile issues related to bioanalytical method validation at both Cetero Houston and former MDS Canada has brought this topic back in the limelight. Hence, a symposium on bioanalytical method validation with the aim of revisiting the building blocks as well as discussing the challenges and implications on the bioanalysis of both small molecules and macromolecules was featured at the PITTCON 2013 Conference and Expo. This symposium was cosponsored by the American Chemical Society (ACS)-Division of Analytical Chemistry and Analysis and Pharmaceutical Quality (APQ) Section of the American Association of Pharmaceutical Scientists (AAPS) and featured leading speakers from the Food & Drug Administration (FDA), academia, and industry. In this symposium, the speakers shared several unique examples, and this session also provided a platform to discuss the need for continuous vigilance of the bioanalytical methods during drug discovery and development. The purpose of this article is to provide a concise report on the materials that were presented.

Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC-MS/MS.

An LC-MS/MS method using pre-column derivatization with phenylisothiocyanate (PITC) was developed to quantify glycine in human cerebrospinal fluid (CSF) and applied to the determination of glycine in human samples collected during clinical testing. The calibration curve range for the assay was 50-10,000 ng/mL and ¹³C2¹5N-glycine was used as an internal standard. Artificial CSF was used as a surrogate matrix for standards due to the presence of endogenous glycine in human CSF and this approach was validated with additional experiments involving either standard addition, or stable labeled glycine as an alternate calibration standard for endogenous glycine. Interday bias (% RE) and precision (% CV) were -4.2 and 12.3% at the LLOQ, and less than ±0.9 and 8.3% for higher concentrations, respectively. Glycine was stable in artificial CSF for at least 5h at room temperature, 55 days at -70 °C (-60 to -80 °C range), and through three freeze-thaw cycles.

Direct quantitative analysis of a 20 kDa PEGylated human calcitonin gene peptide antagonist in cynomolgus monkey serum using in-source CID and UPLC-MS/MS.

PEGylation is a successful strategy to improve the pharmacokinetic and pharmaceutical properties of therapeutic peptides. However, quantitative analysis of PEGylated peptides in biomatrix by LC-MS/MS poses significant analytical challenge due to the polydispersity of the polyethylene glycol (PEG), and the multiple charge states observed for both the peptide and PEG moieties. In this report, a novel LC-MS/MS method for direct quantitative analysis of 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkey serum is presented. CGRP[Cit, Cit] is an investigational human calcitonin gene peptide receptor antagonist with amino acid sequence Ac -WVTH[Cit]LAGLLS[Cit]SGGVVRKNFVPT DVGPFAF-NH(2). In-source collision-induced dissociation (in-source CID) of 20 kDa PEGylated peptide was used to generate CGRP[Cit, Cit] fragment ions, among which the most abundant b(8)(+) ion was selected and measured as a surrogate for the 20 kDa PEGylated peptide. A solid phase extraction (SPE) method was used to extract the PEGylated peptides from the biomatrix prior to the UPLC-MS/MS analysis. This method achieved a lower limit of quantitation (LLOQ) of 5.00 ng/mL with a serum sample volume of 100 µL, and was linear over the calibration range of 5.00 to 500 ng/mL in cynomolgus monkey serum. Intraday and interday accuracy and precision from QC samples were within ±15%. This method was successfully applied to a pharmacokinetic study of the 20 kDa PEGylated CGRP[Cit, Cit] in cynomolgus monkeys.

Pharmacokinetics of anti-hepcidin monoclonal antibody Ab 12B9m and hepcidin in cynomolgus monkeys.

Hepcidin is a key regulator responsible for systemic iron homeostasis. A semi-mechanistic PK model for hepcidin and a fully human anti-hepcidin monoclonal antibody (Ab 12B9m) was developed to describe their total (free + bound) serum concentration-time data after single and multiple weekly intravenous or subcutaneous doses of Ab 12B9m. The model was based on target mediated drug disposition and the IgG-FcRn interaction concepts published previously. Both total Ab 12B9m and total hepcidin exhibited nonlinear kinetics due to saturable Fc-FcRn interaction. Ab 12B9m showed a limited volume of distribution and negligible linear elimination from serum. The nonlinear elimination of Ab 12B9m was attributed to the endosomal degradation of Ab 12B9m that was not bound to the FcRn receptor. The terminal half-life, assumed to be the same for free and total serum Ab 12B9m, was estimated to be 16.5 days. The subcutaneous absorption of Ab 12B9m was described with a first-order absorption rate constant k(a) of 0.0278 h⁻¹, with 86% bioavailability. The model suggested a rapid hepcidin clearance of approximately 800 mL h⁻¹ kg⁻¹. Only the highest-tested Ab 12B9m dose of 300 mg kg⁻¹ week⁻¹ was able to maintain free hepcidin level below the baseline during the dosing intervals. Free Ab 12B9m and free hepcidin concentrations were simulated, and their PK profiles were nonlinear as affected by their binding to each other. Additionally, the total amount of FcRn receptor involved in Ab 12B9m recycling at a given time was calculated empirically, and the temporal changes in the free FcRn levels upon Ab 12B9m administration were inferred.

Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth.

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., (V600E)B-Raf A375, IC(50) on cells = 2 nmol/L; ED(50) on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor-induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients.

Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS.

INTRODUCTION: Hepcidin, a 25-amino acid peptide hormone, plays a crucial regulatory role in iron metabolism. Elevated hepcidin has been observed in response to inflammation and is speculated to be a causative factor in inflammatory anemia due to induction of functional iron deficiency. Hepcidin has been suggested as a biomarker of anemia of inflammation. An accurate assessment of human serum hepcidin is critical to understand its role in anemia. METHODS: An LC-MS/MS method was developed to quantify hepcidin in human serum using chemically synthesized hepcidin as a standard and stable isotope labeled hepcidin as internal standard. Rabbit serum was used as a surrogate matrix for standards due to the presence of endogenous hepcidin in human serum. The method was validated to FDA criteria for bioanalytical assays. RESULTS: The calibration curve was validated over the range of 2.5 to 500 ng/mL. Hepcidin was stable in serum for at least 16 h at room temperature, 90 days at -60 to -80 degrees C, and after three F/T cycles. Interday accuracy (% RE) and precision (%CV) were -11.2% and 5.6%, respectively at the LLOQ, and less than +/-7.0% and 9.2%, respectively for higher concentrations. The mean accuracy of quality control samples (5.00, 15.0, 100 and 400 ng/mL) in 21 analytical batches was between -0.7 and +2.1%, with mean precision between 5.1% and 13.4%. Hepcidin was below 2.5 ng/mL in 31 of 60 healthy subjects, while the mean concentration was less than 10 ng/mL. Sepsis and chronic kidney disease patients had mean serum concentrations of 252 ng/mL (n=16, median 121 ng/mL) and 99 ng/mL (n=50, median 68 ng/mL), respectively. CONCLUSIONS: A fully validated LC-MS/MS method has been described for the determination of hepcidin in human serum. This method was applied to the determination of hepcidin in over 1200 human samples.

Selective inhibitors of the mutant B-Raf pathway: discovery of a potent and orally bioavailable aminoisoquinoline.

The discovery and optimization of a novel series of aminoisoquinolines as potent, selective, and efficacious inhibitors of the mutant B-Raf pathway is presented. The N-linked pyridylpyrimidine benzamide 2 was identified as a potent, modestly selective inhibitor of the B-Raf enzyme. Replacement of the benzamide with an aminoisoquinoline core significantly improved kinase selectivity and imparted favorable pharmacokinetic properties, leading to the identification of 1 as a potent antitumor agent in xenograft models.

Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans.

The vanilloid receptor TRPV1 has been identified as a molecular target for the treatment of pain associated with inflammatory diseases and cancer. Hence, TRPV1 antagonists have been considered for therapeutic evaluation in such diseases. During Phase I clinical trials with AMG 517, a highly selective TRPV1 antagonist, we found that TRPV1 blockade elicited marked, but reversible, and generally plasma concentration-dependent hyperthermia. Similar to what was observed in rats, dogs, and monkeys, hyperthermia was attenuated after repeated dosing of AMG 517 (at the highest dose tested) in humans during a second Phase I trial. However, AMG 517 administered after molar extraction (a surgical cause of acute pain) elicited long-lasting hyperthermia with maximal body temperature surpassing 40 degrees C, suggesting that TRPV1 blockade elicits undesirable hyperthermia in susceptible individuals. Mechanisms of AMG 517-induced hyperthermia were then studied in rats. AMG 517 caused hyperthermia by inducing tail skin vasoconstriction and increasing thermogenesis, which suggests that TRPV1 regulates vasomotor tone and metabolic heat production. In conclusion, these results demonstrate that: (a) TRPV1-selective antagonists like AMG 517 cannot be developed for systemic use as stand alone agents for treatment of pain and other diseases, (b) individual susceptibility influences magnitude of hyperthermia observed after TRPV1 blockade, and (c) TRPV1 plays a pivotal role as a molecular regulator for body temperature in humans.

Quantitative bioanalytical methods validation and implementation: best practices for chromatographic and ligand binding assays.

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.

Evaluation of microdosing strategies for studies in preclinical drug development: demonstration of linear pharmacokinetics in dogs of a nucleoside analog over a 50-fold dose range.

The technique of accelerator mass spectrometry (AMS) was validated successfully and used to study the pharmacokinetics and disposition in dogs of a preclinical drug candidate (7-deaza-2'-C-methyl-adenosine; Compound A), after oral and intravenous administration. The primary objective of this study was to examine whether Compound A displayed linear kinetics across subpharmacological (microdose) and pharmacological dose ranges in an animal model, before initiation of a human microdose study. The AMS-derived disposition properties of Compound A were comparable to data obtained via conventional techniques such as liquid chromatography-tandem mass spectrometry and liquid scintillation counting analyses. Compound A displayed multiphasic kinetics and exhibited low plasma clearance (5.8 ml/min/kg), a long terminal elimination half-life (17.5 h), and high oral bioavailability (103%). Currently, there are no published comparisons of the kinetics of a pharmaceutical compound at pharmacological versus subpharmacological doses using microdosing strategies. The present study thus provides the first description of the full pharmacokinetic profile of a drug candidate assessed under these two dosing regimens. The data demonstrated that the pharmacokinetic properties of Compound A following dosing at 0.02 mg/kg were similar to those at 1 mg/kg, indicating that in the case of Compound A, the pharmacokinetics in the dog appear to be linear across this 50-fold dose range. Moreover, the exceptional sensitivity of AMS provided a pharmacokinetic profile of Compound A, even after a microdose, which revealed aspects of the disposition of this agent that were inaccessible by conventional techniques.