![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Center of Drug Research and Medical Biotechnology, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany
(Received October 28, 2002; accepted March 20, 2003)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). This results in low drug bioavailability and considerable
variability of therapeutic plasma levels
(Piovan et al., 1995).
Metabolism of verapamil leads to pharmacological inactivation and, thus,
patients require frequent dosage of this particular drug. Although the
therapeutic benefit of verapamil is well documented
(Weaver, 1986), the
tissue-specific metabolism may impact its mode of action in cardiovascular
diseases. Indeed, verapamil is initially metabolized into two break-down
products, namely norverapamil and
D-6171. These
metabolites are subject to further metabolism by the cytochrome P450 system to
form additional secondary metabolites. Tracy et al.
(1999) reported several
cytochrome P450 isoforms to be involved in the metabolism of verapamil, and
this includes CYP3A4, CYP3A5, CYP2C8, CYP2C18, CYP2D6, and CYP2E1.
Verapamil acts primarily on heart tissue and is transported via coronary vessels to heart muscle cells. An important barrier to overcome is the endothelium, and it is highly interesting to explore the contribution of human coronary arterial endothelial cells in the heart-specific metabolism of verapamil.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Quality Assurance. Expression of the endothelial-specific surface
protein PECAM-1 was determined by fluorescence-activated flow cytometry as
described previously (Thum et al.,
2000).
RNA and cDNA. RNA was isolated from endothelial cells using a total
RNA Isolation kit (Macherey-Nagel, Duren, Germany) according to the
manufacturer's recommendation. Quality and quantity of isolated RNA were
checked using capillary electrophoresis (Agilent Bioanalyzer 2100) following
the manufacturer's instructions. Total RNA (2 µg) from each sample was used
for reverse transcription, as described previously
(Thum and Borlak, 2000). The
resulting cDNA was frozen at–20°C until further experimentation.
Semiquantitative Reverse Transcription-PCR. PCRs were carried out
with a thermal cycler (T3; Biometra, Göttingen, Germany) using the
following PCR conditions: denaturation at 95°C for 45 s, annealing at
72°C for 60 s, and extension at 72°C for 60s. A total of 30–36
PCR cycles were carried out. Detailed oligonucleotide sequence information was
published previously (Thum and Borlak,
2000) and can be obtained from the authors. DNA contamination was
checked for by direct amplification of RNA extracts prior to conversion to
cDNA. Contamination of RNA extracts with genomic DNA could be excluded. PCRs
were done within the linear range of amplification, and amplification products
were separated using a 1.5% agarose gel and stained with ethidium bromide.
Gels were photographed on a transilluminator (Kodak Image Station 440; see
Fig. 1).
|
Protein Expression. Western immunoblotting was done as follows.
Total protein extracts (100 µg) from a cultured endothelial cell line
(EAhy926) and microsomal extracts from human liver tissue (50 µg) were
denaturated at 95°C for 5 min, followed by SDS-polyacrylamide gel
electrophoresis on 12% polyacrylamide gels, and blotted onto a polyvinylidene
difluoride membrane [PerkinElmer Life Sciences (Germany) GmbH,
Rodgau-Jügesheim, Germany] at 350 mA for 2 h in a buffer containing 400
mM glycine, 50 mM Tris (pH 8.3). Nonspecific binding sites were blocked with
Rotiblock (Roth, Karlsruhe, Germany) in 1x TBS buffer. After
electroblotting of proteins, membranes were incubated with polyclonal
antibodies for CYP2C and CYP2E1 (Chemicon, Hofheim, Germany; dilution
1:1,000–1:2,000) for 1 h and washed three times with 1x TBS buffer
containing 0.1% Tween 20 (Roth). Subsequently, the membranes were incubated
with a 1:10,000 diluted anti 
-sheep antibody (Chemicon) for 1 h at room
temperature followed by three successive washes with 1x TBS buffer
containing 0.1% Tween 20 (Roth). Immunoreactive proteins were visualized with
a chemiluminescence reagent kit [PerkinElmer Life Sciences (Germany) GmbH]
according to the manufacturer's instructions, and bands were scanned with the
Kodak Image Station CF 440 and analyzed using the Kodak 1D 3.5 imaging
software (Eastman Kodak, Rochester, NY).
Chemicals. Chemicals were obtained from the following companies: AcCN (Malinckrodt-Baker, Deventer, Holland), MeOH (Mallinckrodt Baker, Deventer, Holland), NH4Ac (Merck, Darmstadt, Germany), acetic acid (Fluka, Buchs, Switzerland), norverapamil [5-N-(3,4-dimethoxyphenethyl)amino-2-(3',4'-dimethoxyphenyl)-2-isopropyl-valeronitrile] (Sigma/RBI, Natick, MA), and verapamil [5-N-(3,4-dimethoxyphenethyl)methylamino-2-(3',4'-dimethoxyphenyl)-2-isopropyl-valeronitrile] (lot 56H0925; Sigma-Aldrich, Steinheim, Germany). O-Demethylverapamil [5-N-(3,4-dimethoxyphenethyl)methylamino-2-(3'-methoxy-4'-hydroxyphenyl)-2-isopropylvaleronitrile, D-703] was a kind gift of W. L. Nelson (University of Washington, Seattle, WA).
LC/MS Analysis. For solid phase extraction, a lipophilic cartridge (RP8 Select B; Merck) was used and conditioned with methanol, followed by equilibration with water. The sample was loaded onto the cartridge without any organic solvent and washed with 3% (v/v) methanol to separate any sample matrix. Verapamil and its basic metabolites were eluted with methanol. Eluants were evaporated to dryness and reconstituted in 200 µl of acetonitrile/ammonium acetate (0.01 M, pH 6.0, 50:50, v/v). Aliquots of 20 µl were injected onto the LC/MS system.
LC/MS analyses were done on a Waters (Milford, MA) LC instrument (pumps 590) coupled to an ion trap mass spectrometer (Esquire from Bruker Daltonics, Bremen, Germany) operated with positive ion electrospray conditions in the full scan and, when possible, in the MSn mode. The nebulizer pressure was set to 40 psi and the dry gas temperature to 350°C, while +3 kV were applied to the nebulizing capillary. Full mass spectra were acquired by scanning the mass range of m/z 100 to 700. Collision-induced dissociation spectra were obtained from the protonated molecules (M + H)+. Highperformance liquid chromatographic analysis was carried out with a gradient elution of ammonium acetate buffer (0.01 M, pH 6.0) and acetonitrile using a flow of 0.2 ml/min. The gradient started with 25% acetonitrile and was raised to 50% within 15 min, and further, to 75% acetonitrile from 15 to 30 min. From 30 to 55 min, delivery of acetonitrile remained constant and was decreased to 25% from 55 to 60 min. Separation of verapamil and its metabolites was achieved on a 250 x 2 mm RP select B column with a particle size of 4 µm (Merck).
Recovery. Recovery experiments were done in quadruplicate. Solid phase extraction was done as described above, and the resultant eluent was reduced in volume to 200 µl. Measurement of these extracts was done as described above, and recoveries of 85 ± 4% and 81 ± 5% for verapamil and norverapamil, respectively, were estimated.
Quantification. Abundances were calculated from the peak ratios of the metabolites relative to verapamil (see Table 1). This allows a semiquantitative estimate of metabolites.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). As shown
in Fig. 1a, we found CYP1A1,
CYP2A6/7, CYP2A13, CYP2B6/7, CYP2C8, CYP2E1, CYP2J2, and cyclophilin
(housekeeping gene) to be expressed in cultures of human coronary arterial
endothelial cells, but transcript levels of CYP1A2, CYP2C9, CYP2C18, CYP2C19,
CYP2D6, CYP2F1, CYP2S1, CYP3A4, CYP3A5, CYP3A7, and CYP4B1 were below the
limit of detection. Protein expression of CYP2C and CYP2E1 was evidenced by
Western immunoblotting. As shown in Fig.
1b, the polyclonal antibodies recognized epitopes of CYP2C and
CYP2E1 in extracts of cultures of the endothelial cell line EAhy926 and
microsomal extracts of human liver (positive control). Noticeably, the
polyclonal antibody used to probe for CYP2C detected major isoforms of this
protein (CYP2C8/9/18/19).
Metabolite Identification. Identification of metabolites was done
essentially as described previously
(Walles et al., 2001).
Analysis of extracts from culture supernatants of primary human coronary
arterial endothelial cells are highly suggestive for metabolism of verapamil
to proceed via production of D-617 (1.4%) and norverapamil (1%; see
Table 1). Next to the
O-demethylated products minor amounts of D-702 (0.2%), D-703 (0.2%),
and D-620 (0.05%) were produced additionally.
Metabolite M1 (D-620). In the full-scan MS mode, a protonated molecule, (M + H)+ = 277, was observed, which points to oxidative dealkylation of the lower substituted phenylalkyl moiety with simultaneous demethylation (M = 178 Da less than verapamil). Further fragmentation of m/z = 277 in the MS2 mode produced two abundant ions of m/z = 234 (loss of the isopropyl group) and m/z = 260 (loss of ammonia), thus demonstrating oxidative N-demethylation (see Table 1).
Metabolite M3 (D-617). The (M + H)+ of m/z = 291 provided evidence for oxidative dealkylation of the lower substituted phenylalkylamine moiety (M = 164 Da less than verapamil). Fragmentation in the MS2-mode (see Table 1) produced two abundant ions of m/z = 248 (loss of the isopropyl group) and m/z = 260 (N-C cleavage of the higher substituted moiety), which supported the structure of M3 shown in Fig. 2.
|
Metabolite M6 (norverapamil). The protonated molecule
(M+H)+ of m/z = 441 pointed to oxidative
dealkylation. Fragmentation in the MS2 mode (see
Table 1) produced an abundant
ion of m/z = 165 and additional ions at
m/z = 398, 289, and 151, which are typical for oxidative
N-demethylation. The prominent ion of m/z = 165 can
be explained by N-alkyl cleavage with charge transfer, whereas the
m/z = 289 evidenced C-C cleavage in the -position and
subsequent proton transfer. The fragment of m/z = 398 is
formed by loss of isopropyl, m/z = 151, by C-C cleavage in
the -position to the nitrogen and charge transfer. The structure of
metabolite M6 was confirmed with a synthetic reference compound.
Metabolite M7 (D-702). The protonated molecule (M + H)+
of m/z = 441 was produced by oxidative desmethylation of a
methoxy group. Fragmentation in the MS2 mode (see
Table 1) produced an abundant
ion of m/z = 291. The prominent ion at m/z
= 291 stems from a N-alkyl cleavage with proton transfer. The other
fragment ions of m/z = 303 (C-C cleavage in -position
to the nitrogen), m/z = 248 and 260 (loss of the isopropyl
group or methylamine from m/z 291) confirmed
O-demethylation in position 21 or 23. It was shown previously that
O-demethylation of D-702 occurred in position 21
(Nelson et al., 1988).
Metabolite M9 (D-703). The (M + H)+ of
m/z = 441 is the result of oxidative dealkylation, e.g.,
loss of a methyl group. Fragmentation in the MS2 mode (see
Table 1) led to abundant ions
of m/z = 289, 165, and 151, which pointed to oxidative
demethylation in position C-31 or C-33. The prominent ions of
m/z = 289 and 151 are C-C cleavages in the -position
to the nitrogen (the former accompanied by proton transfer). The ions of
m/z = 165 are in support of oxidative demethylation at
position 31 or 33. It was shown previously that O-demethylation of
D-703 occurs in position 31 (Nelson et al.,
1988). The structure of M9 was confirmed with a synthetic
reference compound.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) and
arachidonic acid (Fisslthaler et al.,
1999). Additionally, Salvemini et al.
(1993) showed endothelial
cells to metabolize glyceryl trinitrate to nitric oxide, and Brittebo and
Brandt (1994) demonstrated
metabolic activation of the food mutagen
3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole in cultures of
endothelial cells of mice.
Recently, we showed rat aortic endothelial cells to express several genes
that code for drug-metabolizing enzymes
(Thum et al., 2000), but there
is limited information on the expression and activity of P450 monooxygenases
in human endothelium. We now demonstrate human coronary arterial endothelial
cells to contribute to verapamil's bio-transformation and link gene and
protein expression of two major cytochrome P450 monooxygenases, viz. CYP2C8
and CYP2E1, to verapamil's disposition. Importantly, we demonstrate the
metabolism of verapamil to proceed via N-desalkylation, i.e.,
production of D-617 and norverapamil. These metabolites were previously
identified in assays with human or rat liver microsomes
(Nelson et al., 1988). The
latter investigators also reported that less than 3% of verapamil is excreted
as unchanged drug in humans. It is well established that verapamil undergoes
extensive first-pass metabolism (McEvoy,
2001). Its intrinsic hepatic clearance was also studied
(Iwatsubo et al., 1997), and
from the above-cited studies, we conclude that human coronary arterial
endothelial cells may only contribute to about 3% of the overall metabolism of
verapamil. Consequently, it is unlikely that coronary arterial endothelial
cells play a major role in the overall systemic biotransformation of
verapamil, but tissue-specific metabolism of verapamil may be important for
local drug response. Further studies should investigate the intra- and
extracellular distribution of metabolites.
Tracy et al. (1999)
demonstrated CYP3A4, CYP3A5, and CYP2C8 to be key players in the metabolism of
verapamil, and our observation of CYP2C8 and CYP2E1 to be expressed in
cultures of primary human coronary arterial endothelial cells fits well to the
overall production of metabolites reported in this study. Nonetheless,
expression of CYP3A4 and CYP3A5 was below the limit of detection; this
illustrates, further, tissue specificity in the expression of isoforms of P450
monooxygenases and their contribution toward the oxidation of drugs and other
xenobiotics. Some of the P450 isoforms identified by us are key players in the
metabolic activation of polycyclic aromatic hydrocarbons and certain
components of cigarette smoke. It will therefore be of interest to
investigate, further, whether activity of these isoforms (CYP1A1, CYP2A6/7,
CYP2E1) can be causally related to the inflammatory reactions and
cardiovascular disease frequently observed in exposed animals
(Zhang et al., 2001). In
addition, CYP2C8 is highly implicated in the metabolic turnover and production
of vasodilative agents including 11,12-epoxyeicosatrienic acid, and further
studies are now on the way to determining the relevance of CYP2C8 genetic
polymorphisms in the overall control of vascular tonus. Next to verapamil,
other cardiovascular drugs are substrates for endothelial CYP2E1 or CYP2C,
including the ß-blocking agent carvedilol
(Oldham and Clarke, 1997) and
the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor, fluvastatin
(Scripture and Pieper,
2001).
In conclusion, endothelium is a metabolically active tissue and should be investigated, particularly when tissue-specific metabolism and/or metabolic inactivation is being considered.
|
Footnotes |
|---|
Address correspondence to: Prof. Dr. Jürgen Borlak, Fraunhofer Institute of Toxicology and Experimental Medicine, Center for Drug Research and Medical Biotechnology, Nicolai-Fuchs-Str. 1, D-30659 Hannover, Germany. E-mail: Borlak{at}item.fraunhofer.de
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. Fang, J. Gu, F. Xie, M. Behr, W. Yang, E. D. Abel, and X. Ding Deletion of the NADPH-Cytochrome P450 Reductase Gene in Cardiomyocytes Does Not Protect Mice against Doxorubicin-Mediated Acute Cardiac Toxicity Drug Metab. Dispos., August 1, 2008; 36(8): 1722 - 1728. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Haorah, B. Knipe, J. Leibhart, A. Ghorpade, and Y. Persidsky Alcohol-induced oxidative stress in brain endothelial cells causes blood-brain barrier dysfunction J. Leukoc. Biol., December 1, 2005; 78(6): 1223 - 1232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Hukkanen, P. Jacob III, and N. L. Benowitz Metabolism and Disposition Kinetics of Nicotine Pharmacol. Rev., March 1, 2005; 57(1): 79 - 115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Borlak, I. Schulte, and T. Thum ANDROGEN METABOLISM IN THYMUS OF FETAL AND ADULT RATS Drug Metab. Dispos., June 1, 2004; 32(6): 675 - 679. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
