Pharmacokinetics and pharmacodynamics of CHF5074 after short-term administration in healthy subjects.

CHF5074 has been shown to inhibit brain ß-amyloid deposition and attenuate memory deficits in different transgenic mice models of Alzheimer disease. We evaluated the safety, pharmacokinetics, and pharmacodynamics of 3 ascending dose regimens of CHF5074 (200, 400, and 600 mg/d for 14 d) in a double-blind, placebo-controlled, parallel group study involving 48 healthy subjects. Plasma, urine, and cerebrospinal fluid (CSF) samples were collected for measuring drug and main metabolite concentrations and potential biomarkers of pharmacodynamic activity (ß-amyloid1-40, ß-amyloid1-42, soluble CD40 ligand, and tumor necrosis factor-α). All subjects completed the study, and no serious or severe adverse events were reported. The maximum tolerated dose was close to 600 mg/d with mild diarrhea being the most frequent adverse event at this dose. CHF5074 reached peak plasma levels 2 to 3 hours after drug administration and then was slowly eliminated (t(1/2z)=30 h) in the urine as glucoronide. Systemic exposure to the drug appeared to be dose-proportional with a 2-fold accumulation ratio at steady state. Metabolite plasma levels peaked at 4 to 5 hours and accounted for about 25% of the parent compound. Drug levels in the CSF were dose-proportional. The drug dose-dependently lowered the levels of the soluble CD40 ligand, a marker of microglia activation, in both plasma and CSF samples.

Blood microsampling in cynomolgus monkey and evaluation of plasma PK parameters in comparison to conventional sampling.

Microsampling, a reduced volume sampling method, has successfully gained attention at the International Conference on Harmonization (ICH) level and established benefits support its use in Toxicokinetic (TK) studies. These improved sampling techniques are less invasive and in large animal species improve animal welfare (refinement). To evaluate if the plasma concentrations of drugs were influenced by the blood sampling method, the traditional method from femoral vein and microsampling from tail vein in Cynomolgus monkeys were compared. The pharmacokinetic parameters (Cmax, Tmax and AUC) of four drugs (selected based on acid-base and volume of distribution properties) in non-human primate were correlated. The plasma samples were quantified using standard LC-MS/MS methods, qualified to evaluate the precision and accuracy before the analysis of real samples. The results reported in this work demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. The data show that the exposure of drugs tested after blood collection using standard procedure from femoral vein and microsampling from tail vein is correlated and is not influenced by acid-base characteristics and volume of distribution.

A review of analytical methods for the determination of 5-fluorouracil in biological matrices.

5-Fluorouracil (5-FU) is a cytostatic agent that has been widely used in the treatment of various solid tumours for more than 20 years, and is still considered to be among the most active antineoplastic agents in advanced colorectal cancer and malignancies of the head and neck. A large number of non-chromatographic and chromatographic methods for the quantitation of 5-FU, related prodrugs and their metabolites in biological matrices have been developed in the last 30 years to support preclinical and clinical studies. However, 5-FU monitoring has not been widely used, at least not in the USA, and certainly not outside the clinical research setting, given the absence of simple, fast and inexpensive testing methods for 5-FU monitoring. Recent developments with testing based on liquid chromatography-tandem mass spectrometry and a nanoparticle antibody-based immunoassay may facilitate routine monitoring of 5-FU in daily clinical practice. In this review the advantages and disadvantages of the bioanalytical methods developed and used for 5-FU, its metabolites and related prodrugs are discussed.

Development and validation of bioanalytical methods for LNA-i-miR-221 quantification in human plasma and urine by LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, has proved favorable efficacy and safety profiles in the preclinical studies, leading to being approved for use in clinical trials by regulatory authorities. The objective of this study was to develop and validate LC-MS/MS methods to quantify LNA-i-miR-221 in human plasma and urine. Chromatographic separation was performed with a gradient system on HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase. LNA-i-miR-221 was detected on tandem mass spectrometer with electrospray ionization source in negative ion mode. The methods showed good linearity within the calibration range of 50-25000 ng/mL and 50-50000 ng/mL for human plasma and urine, respectively. The methods proved to be accurate, precise and selective in both human matrices. These validated methods are reliable and are currently in use to support a first-in-human clinical trial of LNA-i-miR-221 in patients affected by refractory multiple myeloma and advanced solid tumors.

Development and validation of a highly sensitive gradient chiral separation of pramipexole in human plasma by LC-MS/MS.

AIM: Development of a high-sensitivity chiral LC-MS/MS method was required to evaluate a combination of pramipexole (S-PPX) and its enantiomer dexpramipexole (R-PPX) in a proposed clinical trial. The previously available methods suffered from low sensitivity for the (S)-enantiomer in the presence of the more abundant (R)-enantiomer. Based on the projected dosing regimen in the clinical trial, a 5000-fold improvement in sensitivity was required for the (S)-enantiomer. METHODOLOGY: Spiked human plasma samples were extracted by liquid-liquid extraction using ethyl acetate and injected onto a CHIRALPAK ID column under pH gradient conditions. CONCLUSION: An improved analytical method was developed and validated with a final LLQ for (S)-PPX of 0.1 ng/ml in the presence of 2000 ng/ml of (R)-PPX.

Hydrophilic interaction liquid chromatography-APCI-mass spectrometry determination of 5-fluorouracil in plasma and tissues.

A simple and fast analytical method using hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry was developed to analyse 5-fluorouracil (5-FU) in plasma and tissues. The HILIC system overcomes problems reported in obtaining satisfactory retention of 5-FU with other types of HPLC systems. After addition of internal standard (IS) (5-Chlorouracil (5-CU)), plasma proteins were precipitated with acetonitrile, and tissue samples homogenised with a micro-dismembrator. The analysis was performed using a polymer-based column (Ashaipak NH2) and the compounds were eluted under gradient conditions at 1 ml/min using a mobile phase containing a mixture of ammonium formate and acetonitrile. MS detection used a API 4000 mass spectrometry with heated nebulizer source and multiple reaction monitoring operated in the negative ion mode. The mass transitions of 5-FU and its internal standard were 129 m/z-->42m/z and 145 m/z-->42 m/z, respectively. The lower limits of quantitation in plasma and tissues were about 5 ng/ml and 10 ng/g, respectively, using 25 microl of plasma and 50mg of tissue. Good linearity, accuracy and precision were obtained in all matrices tested. The suitability and robustness of the method for in vivo samples were confirmed by analysis of mouse plasma, muscle and tumour from animals dosed with 5-FU.

Ion-suppression effects in liquid chromatography-tandem mass spectrometry due to a formulation agent, a case study in drug discovery bioanalysis.

Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has become the technology of choice for bioanalysis, due to its high selectivity and high sample throughput. However, concerns have grown that this technique may be subject to errors due to "invisible" interferences, in particular ion-suppression. Investigations on ion-suppression from formulation agents have only been published to a limited extent. Such effects can be of particular importance in pre-clinical discovery studies where drugs may be formulated with large amount of solubilisers and bioanalysis may use fast generic methods. In a preliminary pharmacokinetic study we observed strong ion-suppression from a polysorbate co-solvent, which, if undetected, would have given highly erroneous pharmacokinetic results and possibly could have led to the inappropriate elimination of a promising drug candidate. Different chromatographic methods were tested indicating that the separation step was essential in controlling these effects. A method based on matrix dilution is proposed to check for these effects during the use of discovery support methods, where full validation is not practical. Some excipients commonly used in formulations are polydispersed polymers, for which very limited pharmacokinetic information is available. Further investigation is needed to better understand the mechanisms of ion-suppression and the kinetics of the suppressing species to allow the development of new LC/MS/MS based analytical strategies, which will not be subject to such ionisation interferences.

Assessment of normal and tumor tissue uptake of MAG-CPT, a polymer-bound prodrug of camptothecin, in patients undergoing elective surgery for colorectal carcinoma.

PURPOSE: MAG-camptothecin (MAG-CPT) is the lead compound of a novel drug delivery system in which an active cytotoxic moiety, camptothecin (CPT), is covalently linked to a soluble polymeric carrier (MAG) to form an inactive prodrug. The mechanism of action of CPT remains unaltered, but the delivery system is thought to allow the carrier-bound drug to accumulate in tumor tissues and release the active CPT locally. This proof-of-concept clinical study was designed to determine whether MAG-CPT was preferentially delivered to or retained in tumor tissue compared to adjacent normal tissue or plasma, and to estimate the degree of intratissue release of CPT. METHODS: This was an open, non-randomized study in ten adult patients scheduled for elective surgery for colorectal cancer. Patients received a single dose of 60 mg/m2 (CPT equivalent) of MAG-CPT 24 h, 3 days or 7 days prior to surgery. Plasma, tumor, and adjacent normal tissue samples were collected simultaneously at the time of surgery and analyzed for MAG-bound and released CPT concentrations. RESULTS: MAG-bound and free CPT concentrations in plasma, tumor, and normal tissue achieved equilibrium by 24 h after dosing, declining in parallel up to 7 days after dosing. MAG-bound CPT was delivered to similar levels to tumor and normal tissue. At 24 h after dosing, the mean+/-SD MAG-bound CPT concentrations were 861+/-216 ng/g in tumor and 751+/-215 ng/g in adjacent normal tissue, and free CPT concentrations were lower in tumor than in normal tissue (12.2+/-4.7 ng/g and 21.9+/-6.7 ng/g, respectively). At 24 h after dosing, mean+/-SD ratios of MAG-bound and free CPT in tumor and plasma were 0.13+/-0.03 and 0.22+/-0.09, respectively, and the ratios did not change for up to 7 days after dosing, indicating a lack of preferential retention of MAG-bound CPT or release of free CPT in tumor. These results are in marked contrast to previous data from animal tumor xenograft studies, where MAG-CPT levels were higher in tissue than in plasma at 3 and 7 days after a single i.v. dose. CONCLUSIONS: Delivery of CPT to the target tumor tissue is achievable by means of the MAG-CPT polymer-bound delivery system, with the equilibrium between plasma and tumor tissue concentrations of released CPT being established within 24 h after dosing. However, preferential retention of MAG-bound or released CPT in the tumor relative to normal tissue or plasma was not detected during the 7 days after dosing. The methods employed in our study could be of use in making "go/no-go" decisions on further development of anticancer drugs.

Quantitative determination of paclitaxel in human plasma using semi-automated liquid-liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry.

This paper describes a high-throughput sample preparation procedure combined with LC-MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid-liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected in positive ion mode using the mass transition m/z 854.6-->286.2 and m/z 831.6-->263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1-1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.

Recommendations on bioanalytical method stability implications of co-administered and co-formulated drugs by Global CRO Council for Bioanalysis (GCC).

An open letter written by the Global CRO Council for Bioanalysis (GCC) describing the GCC survey results on stability data from co-administered and co-formulated drugs was sent to multiple regulatory authorities on 14 December 2011. This letter and further discussions at different GCC meetings led to subsequent recommendations on this topic of widespread interest within the bioanalytical community over the past 2 years.

Conference report: the 3rd Global CRO Council for Bioanalysis at the International Reid Bioanalytical Forum.

The 3rd Global CRO Council Closed Forum was held on the 3rd and 4th July 2011 in Guildford, United Kingdom, in conjunction with the 19th International Reid Bioanalytical Forum. In attendance were 21 senior-level representatives from 19 CROs on behalf of nine European countries and, for many of the attendees, this occasion was the first time that they had participated in a GCC meeting. Therefore, this closed forum was an opportunity to increase awareness of the aim of the GCC and how it works, share information about bioanalytical regulations and audit findings from different agencies, their policies and procedures and also to discuss some topics of interest and aim to develop ideas and provide recommendations for bioanalytical practices at future GCC meetings in Europe.