The use of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) as a specific CYP2D6 probe in human liver microsomes.

Recently, a novel nonfluorescent probe 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin (AMMC), which produces a fluorescent metabolite AMHC (3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-hydroxy-4-methylcoumarin) was used with microsomes containing recombinant enzymes (rCYP) to monitor CYP2D6 inhibition in a microtiter plate assay. This article describes the studies that were performed in human liver microsomes (HLM) to establish the selectivity of AMMC toward CYP2D6. Metabolism studies in HLM showed that AMMC was converted to one metabolite identified by mass spectrometry as AMHC. Kinetic studies indicated an apparent K(m) of 3 microM with a V(max) of 20 pmol/min. mg of protein for the O-demethylation reaction. The O-demethylation of AMMC in HLM was inhibited significantly in the presence of a CYP2D6 inhibitory antibody. Using a panel of various HLM preparations (n = 12), a good correlation (r(2) = 0.95) was obtained between AMMC O-demethylation and bufuralol metabolism, a known CYP2D6 substrate, but not with probes for the other major xenobiotic metabolizing CYPs. Finally, only rCYP2D6 showed detectable metabolism in experiments conducted with rCYPs using AMMC at a concentration of 1.5 microM (near K(m)). However, at a concentration of 25 microM AMMC, rCYP1A also contributed significantly to the formation of AMHC. Knowing the experimental conditions under which AMMC was selective for CYP2D6, a microtiter assay was developed to study the inhibition of various compounds in HLM using the fluorescence of AMHC as an indication of CYP2D6 activity. The inhibition potential of various chemicals was found to be comparable to those determined using the standard CYP2D6 probe, bufuralol, which requires high-performance liquid chromatography separation for the analysis of its CYP2D6-mediated 1'-hydoxylated metabolite.

Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe.

The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (approximately 1-2 nmol/(min. mg protein)) to give the fluorescent compound DFH. The involvement of CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC(50) curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability <10% and interday variability indicated less than a 2-fold variation in the IC(50) values) and is now routinely used in our laboratory to evaluate CYP3A inhibition of NCEs.

Soft-tissue complications of intra-arterial chemotherapy for extremity sarcomas.

High-grade sarcomas have a high rate of local recurrence as well as distant metastases. This has led to the development of intra-arterial chemotherapy (IAC) as part of a multimodal approach to control local disease and/or reduce the extent of surgical resection. Intra-arterial catheters are positioned by an interventional radiologist into the feeding vessels of the tumor. Adriamycin and 5-fluorodeoxyuridine are infused intra-arterially. Cisplatinum, with or without granulocyte colony stimulating factor, is given systemically. Patients usually experience acute self-limited soft-tissue inflammation in the treated area. In our experience of 118 patients, 3 patients experienced soft-tissue necrosis that required excision and reconstruction. The first was treated for synovial sarcoma of a metatarsal. After IAC with Adriamycin, she sloughed the skin, subcutaneous tissue, and some of the posterior compartment musculature of her calf. This tissue was debrided. A gastrocnemius flap and skin graft were used for coverage. She is free of disease and ambulatory. The second patient was treated with IAC Adriamycin for a radial head chondrosarcoma. She developed soft-tissue slough, which became infected with Pseudomonas. She required extensive debridement of the skin, subcutaneous tissue, and muscle, and was subsequently reconstructed with a latissimus flap and a split-thickness skin graft (STSG). She later developed a local recurrence requiring amputation. The latissimus was elevated and used to cover the distal stump. She also is free of disease. The third patient was treated with IAC Adriamycin for Ewing's sarcoma of the right femur. This was complicated by fat necrosis and persistent pain. Subsequent radiotherapy only worsened her symptoms. She underwent wide excision and muscle flap/STSG repair, which relieved her pain. She is currently ambulatory and free of disease. In conclusion, as the use of IAC continues, its complications may become more common. Our experience with this previously unknown entity is illustrated and therapeutic options are discussed.