Markus Walles

Verapamil drug metabolism studies by automated in-tube solid phase microextraction

Verapamil is a common calcium antagonist described with antianginal, antihypertensive and antiarrythmic properties. The metabolites of verapamil have also shown pharmacological properties and therefore sample preparation and analysis techniques capable of metabolic screening for verapamil are important. In-tube SPME is a relatively new method integrating sample extraction, concentration and introduction into one single step without the use of organic solvents. The capability of in-tube SPME in bioanalysis has been reviewed but there has been no application described in the field of drug metabolism. Since automation and interfacing of in-tube SPME coupled to liquid chromatography-mass spectrometry (LC-MS) is possible, we confirm in this study that it is a powerful method to monitor the main metabolites of verapamil in various biological matrices like plasma, urine and cell culture media. Further, we show that it could also be used in routine pharmacokinetics measurements. An in-tube SPME LC-MS method was developed to extract and analyze the metabolic profile of verapamil from biological matrices. The detection limit for verapamil, gallopamil, norverapamil and PR22 were 52, 53, 65 and 83 ng/ml (UV detection) and 5, 6, 6 and 8 ng/ml (MS detection), respectively. The precision of the method was calculated in various biological matrices and the average % R.S.D. (N=5) for verapamil, gallopamil, norverapamil and PR22 was 3.9, 3.7, 3.8 and 4.3% (MS detection), respectively. The linear dynamic range was determined to be 100-800 ng/ml (UV detection) with a total sample preparation and analysis time of 34 min.

Verapamil: identification of novel metabolites in cultures of primary human hepatocytes and human urine by LC-MS n and LC-NMR

1. Verapamil is a well-known and world-wide prescribed calcium antagonist, but it suffers from extensive first-pass metabolism. Although it has been marketed for many years, a complete understanding of its biotransformation in humans is still lacking. 2. The metabolism of verapamil was therefore investigated in cultures of primary human hepatocytes and in extracts of human urine after oral dosing. Identification of metabolites was done with LC-MS n and LC-NMR (600 MHz) to obtain in-depth information on its biotransformation products and definitive proof of the proposed chemical structures of metabolites. 3. Hyphenation of LC-MS n and LC-NMR was shown to be a powerful and effective platform for the identification of metabolites. Indeed, 21 Phase I and 16 Phase II metabolites were identified. Basically, all the Phase II metabolites (glucuronides) and 11 of the Phase I (oxidative) metabolites were not reported previously. 4. New insight into verapamils biotransformation pathway is provided as well as evidence about its true complexity of metabolic disposal.

Verapamil: new insight into the molecular mechanism of drug oxidation in the human heart.

Verapamil is a commonly prescribed cardiovascular drug, but surprisingly its metabolism in the target tissue of pharmacotherapy is basically unknown. We therefore investigated its biotransformation in human heart tissue and correlate the production of metabolites with the gene expression of major drug metabolising enzymes. Using electrospray LC-MS-MS and LC-MS3 experiments, a total of nine metabolites were observed in incubation experiments with verapamil and microsomes isolated from the human heart tissue, and this included a carbinolamine-, N-formyl-, ahemiacetale-, and formate-intermediate of N-demethyl- and O-demethylverapamil. We also observed a hydroxylation product at the benzylic position of atom C-7 (M9). Metabolites M5-M9 are novel and were not observed in previous studies with liver or other human tissues. A fine example of the considerable metabolic competence of human heart is the formation of M1-M4, e.g. dealkylverapamil, norverapamil and isomers of O-demethylverapamil, which were believed to be exclusively produced by the liver.

Inhalation Studies with the Göttingen Minipig

A method for inhalative exposure of minipigs to aerosols and gases has been developed. Minipigs are exposed via mask inhalation to the test substance using a computer-controlled exposure system that permits simultaneous exposure of groups of four animals in parallel to different controlled dose levels. We studied inhalation treatment of verapamil, a cardiovascular drug, and show good absorption and favorable pharmacokinetics when compared with iv drug application. The results shown in this study encourage inhalation studies with the Göttingen minipig.A method for inhalative exposure of minipigs to aerosols and gases has been developed. Minipigs are exposed via mask inhalation to the test substance using a computer-controlled exposure system that permits simultaneous exposure of groups of four animals in parallel to different controlled dose levels. We studied inhalation treatment of verapamil, a cardiovascular drug, and show good absorption and favorable pharmacokinetics when compared with iv drug application. The results shown in this study encourage inhalation studies with the Göttingen minipig.