Purpose: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme catalyzing the metabolic degradation of the anticancer drug 5-fluorouracil (5-FU). Population studies of DPD activity in peripheral blood mononuclear cells (PBMC) were reported in healthy volunteers and cancer patients. Although these studies were done in mainly Caucasian and African American populations, only a little information is available for a Japanese population. Experimental Design: One hundred fifty healthy Japanese volunteers were screened for a population distribution of PBMC-DPD activity. Genetic analysis of a volunteer with very low DPD activity was carried out by reverse transcriptase-PCR and genomic sequencing. Bacterially expressed recombinant mutant DPD proteins were purified and characterized. Results: Mean and median values of PBMC-DPD activity for 5-FU reduction in the study population were 0.173 and 0.166 nmol/min/mg protein, respectively. A 57-year-old female volunteer (proband in this study) had very low DPD activity (0.014 nmol/min/mg protein) with a very low level of expression of DPD protein. Two novel nucleotide substitutions, at nucleotide positions 1097 (1097G > C) and 2303 (2303C > A), resulting in amino acid substitutions at positions 366 (G366A) and 768 (T768K), respectively, were identified.The G366A mutation caused not only a marked decrease in the affinity of the enzyme to cofactor NADPH but also reducedV max for 5-FUreducing activity to f0.5. T768K mutant lost its activity much faster than did wild DPD. Conclusions: We found one healthy volunteer (0.7% of the population) with very low PBMC-DPD activity due to heterozygosity for a mutant allele of the DPYD gene in a population of 150 Japanese.Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases, uracil and thymine (1), and is also known to be the key enzyme catalyzing the metabolic degradation of the anticancer drug 5-fluorouracil (5-FU; refs. 1, 2). 5-FU has been commonly and widely used as a chemotherapeutic agent for the treatment of cancer of the gastrointestinal tract, breast, and head and neck (3). More than 85% of the given 5-FU is catabolized by DPD (4). The clinical importance of DPD has been shown with the identification of severe or lethal toxicity in patients given 5-FU who are deficient in or have low levels of DPD activity in their peripheral blood mononuclear cells (PBMC; refs. 5 -7).The importance of the role of DPD in 5-FU chemotherapy also has been shown by studies with competitive and irreversible DPD inhibitors (8, 9). Recently, we reported a possible mechanism for the 18 acute deaths of Japanese patients in 1993 that were caused by interactions between oral 5-FU prodrugs and the new oral antiviral drug, sorivudine [1-h-D-arabinofuranosyl-(E )-5-(2-bromovinyl)uracil], which was being used for treatment of herpes zoster (9 -13). Our study indicated that these patients were most likely to have extremely low levels of hepatic DPD activity ...

Garlic extract, the ethanol-soluble fraction of garlic, prevented formation of thiobarbituric-acid-reactive substances and fluorescent substances during lipid peroxidation of rat liver microsomes. Lipid peroxidation increased the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene labelled to the microsomes while this increase was prevented by the garlic extract. It thus seems probable that the garlic extract serves to maintain membrane fluidity. These effects were dependent on its concentration and particularly prominent on exceeding a certain concentration of garlic extract. These results suggest its possible role of protecting the membranes from lipid peroxidation.

A highly sensitive simultaneous quantitative method for a cassette cold-microdosing study on celiprolol and atenolol was developed with liquid chromatography-tandem mass spectrometry. The method utilizes a combination of solid-phase extraction (SPE) with strong cation exchange (SCX) cartridge columns and reversed-phase chromatography with an ODS analytical column. SCX-SPE cartridge columns (100 mg sorbent) were used for a selective extraction of celiprolol, atenolol and metoprolol (internal standard) from 500 μL of human plasma samples. Turbo-ion spray at positive mode was employed for the ionization of the drug compounds. Quantitation was performed on a triple quadrupole mass spectrometer by selected reaction monitoring with the transitions of m/z 380 to m/z 251 for celiprolol and m/z 267 to m/z 145 for atenolol. Separation of analytes was achieved on an ODS column (100 mm length × 2.1 mm id, 3 μm) by a gradient elution with 10 mM formic acid and methanol by varying their proportion at a flow rate of 0.2 mL/min. The method was validated in the range of 1-250 pg/mL for celiprolol and 2.5-250 pg/mL for atenolol and was successfully applied to the elucidation of pharmacokinetic profiling in a cold cassette microdosing study of the β-blockers.

Abstract. The L2 Global Harmonization Team on large molecule specific assay operation for protein bioanalysis in support of pharmacokinetics focused on the following topics: setting up a balanced validation design, specificity testing, selectivity testing, dilutional linearity, hook effect, parallelism, and testing of robustness and ruggedness. The team additionally considered the impact of lipemia, hemolysis, and the presence of endogenous analyte on selectivity assessments as well as the occurrence of hook effect in study samples when no hook effect had been observed during pre-study validation.

Lapatinib was well tolerated at doses of 900-1600 mg/day in Japanese solid tumor patients. Overall, our findings were similar to those of overseas studies.