Drug
Metabolism Letters
ISSN: 1872-3128
Drug Metabolism Letters
Volume 1, Number 4, December 2007
Contents
Inhibition of Calcium Oxalate Nephrotoxicity with
Cymbopogon Schoenanthus (Al-Ethkher) Pp. 241-244
S.S. Al-Ghamdi, A.A. Al-Ghamdi and A.A. Shammah
[Abstract]
Modelling and Measuring Redox Cycling and
Cytotoxicity of Quinones Pp. 245-253
L. Hughes, J. Wingate, R. Griffith and R.J. Aitken
[Abstract]
Endothelial Cell-Based Methods for the Detection of
Cyanobacterial Anti-Inflammatory and Wound-Healing Promoting
Metabolites Pp. 254-260
C. Wiesner, J. Kopecky, M. Pflueger, H. Hundsberger, B.
Entler, C. Kleber, J. Atzler, P. Hrouzek, D. Stys, A. Lukesova,
W. Schuett and R. Lucas
[Abstract]
The N-Terminal of Human UGT1A6 Is on the Outside,
as Evidenced by ELISA with Autoantibody in Autoimmune Hepatitis
Sera Pp. 261-266
H. Mori, M. Shinoda and T. Mizutani
[Abstract]
Clinically Significant Proteinuria Following the Administration
of Sirolimus to Renal Transplant Recipients Pp. 267-271
A.S. Perlman, E.H. Kim, B. Kallakury, J.A. Light and J.
Moore, Jr.
[Abstract]
Metabolite Interference in Pharmacokinetic Studies
Pp. 272-275
M.F. Moghaddam, J.-H. Fan, Y. Tang, O. Khatsenko, J.C.
Katz and M.A. Shirley
[Abstract]
Allelic Variations in CYP2D6 Gene and Susceptibility to Cervical
Cancer Pp. 276-280
S. Wajid, S.H. Naqvi, A. Juneja M. Bharadwaj and A.B.
Mitra
[Abstract]
Comprehensive Identification of Cytochrome P450
Isoforms from Solubility-Based Fractionated Rat Liver Microsomes
Pp. 281-286
H. Yamanaka, M. Takeyoshi, Y. Minobe, Y. Yakabe, M. Takatsuki
and H. Sato
[Abstract]
Mass Defect Filtering on High Resolution LC/MS Data as a Methodology
for Detecting Metabolites with Unpredictable Structures: Identification
of Oxazole-Ring Opened Metabolites of Muraglitazar Pp.
287-292
D. Zhang, P.T. Cheng and H. Zhang
[Abstract]
LC-MS/MS-Based Approach for Obtaining Exposure Estimates
of Metabolites in Early Clinical Trials Using Radioactive
Metabolites as Reference Standards Pp. 293-298
D. Zhang, N. Raghavan, T. Chando, J. Gambardella, Y. Fu,
D. Zhang, S.E. Unger and W.G. Humphreys
[Abstract]
In Vitro Metabolism of Leflunomide by Mouse
and Human Liver Microsomes Pp. 299-305
E.C.Y. Chan and L.-S. New
[Abstract]
A New Method to Measure P-gp (ABCB1) Activity
Pp. 306-310
M. Masuda and T. Mizutani
[Abstract]
Glucocorticoid Receptor Functions in HeLa Cells Are
Perturbed by 2,3,8,9-tetrachlorodibenzo-p-dioxin
(TCDD) Pp. 311-314
R. Vrzal, J. Ulrichová, Z. Dvorák and P.
Pávek
[Abstract]
Abstracts
[Back to top]
Inhibition of Calcium Oxalate Nephrotoxicity with
Cymbopogon Schoenanthus (Al-Ethkher)
S.S. Al-Ghamdi, A.A. Al-Ghamdi and A.A. Shammah
We investigated the effects of Cymbopogon schoenanthus
herb on experimental induced kidney stones in male Wistar
albino rats. Oxalate nephrotoxicity was experimentally induced
by 200 mg single dose of glycolic acid given orally (gavage).
Rats were divided into three groups, glycolic acid, glycolic
acid plus Cymbopogon schoenanthus, and control (D. water).
Urine analysis of blood urea nitrogen (BUN), creatinine, and
calcium revealed significant differences comparing to the
control. In addition, significant pathological changes were
found in the kidney revealed by histopathological studies.
Daily oral treatment with Cymbopogon schoenanthus (1 ml of
the extract) significantly corrected the incidence of nephrotoxicity,
BUN, creatinine, and calcium level differences. Moreover,
optimization studies showed highly potent diuretic activity
of Cymbopogon schoenanthus. After three days of experiments,
four rats treated with the glycolic acid only died. The rest
of animal survived and looked healthy.
[Back to top]
Modelling and Measuring Redox Cycling and
Cytotoxicity of Quinones
L. Hughes, J. Wingate, R. Griffith and R.J. Aitken
The roles of alkylation and redox cycling in quinone
toxicity were investigated. In general the more cytotoxic
quinones produced the highest responses in an assay monitoring
redox activity. No evidence of alkylation of high molecular
weight protein thiols was detected. We conclude quinone toxicity
is dominated by redox cycling.
[Back to top]
Endothelial Cell-Based Methods for the Detection
of Cyanobacterial Anti-Inflammatory and Wound-Healing Promoting
Metabolites
C. Wiesner, J. Kopecky, M. Pflueger, H. Hundsberger, B.
Entler, C. Kleber, J. Atzler, P. Hrouzek, D. Stys, A. Lukesova,
W. Schuett and R. Lucas
Acute lung injury is accompanied by an increased endothelial
chemokine production and adhesion molecule expression, which
may result in an extensive neutrophil infiltration. Moreover,
a destruction of the alveolar epithelium and capillary endothelium
may result in permeability edema. As such, the search for
novel anti-inflammatory substances, able to downregulate these
parameters as well as the tissue damage holds therapeutic
promise. We therefore describe here the use of human endothelial
cell-based in vitro assays for the detection of anti-inflammatory
and wound-healing metabolites from cyanobacteria.
[Back to top]
The N-Terminal of Human UGT1A6 Is on the Outside,
as Evidenced by ELISA with Autoantibody in Autoimmune Hepatitis
Sera
H. Mori, M. Shinoda and T. Mizutani
Sera from AIH (autoimmune hepatitis) type 2 patients contain
an autoantibody against the UGT1A subtype, called anti-LKM3.
Previously, we reported that sera in AIH type 1 patients contained
autoantibodies against drug-metabolizing enzymes (Shinoda
et al. (2004) Autoimmunity, 37, 473). In this report,
we showed that AIH-1 sera did not react with some peptides
in the C-terminal half of the UGT1A subtype but reacted with
a peptide P1(33-42) among several common peptides in the N-terminal
half of the UGT1A subtype. This result suggests that the P1
site (33-42) presents on the outside of the UGT1A molecule
to be recognized by lymphocytes of the immune system to produce
an autoantibody. To detect a key recognition site on peptide
P1(33-42), we studied the reactivity of two peptides, M1(28-37)
and M2(38-47), containing the N-terminal and C-terminal half
of peptide P1. Peptide M2 did not react with AIH-1 serum but
peptide M1 did. Thus, the common peptide sequence 33-37 in
the positive peptide M1(28-37) and P1(33-42) is a key recognition
sequence. Next, we studied the reactivity of some other synthetic
peptides, in which some amino acids in the sequence 33-37
in peptide M1 changed to Ala. The peptides changing to Ala
(PQ33-34AA) or (DGS35-37AAA) did not react with AIH-1 sera.
Meanwhile, these AIH sera did not inhibit the glucuronidation
of p-nitrophenol by UGT1A6, suggesting that the key sequence
33-37 might not be contained in active sites of glucuronidation
by UGT1A6. In conclusion, sera from AIH-1 patients reacted
with the amino acids in the sequence 33-37 (PQDGS) of the
N-terminal of UGT1A6.
[Back to top]
Clinically Significant Proteinuria Following the Administration
of Sirolimus to Renal Transplant Recipients
A.S. Perlman, E.H. Kim, B. Kallakury, J.A. Light and J.
Moore, Jr.
Background: Sirolimus is the latest immunosuppressive agent
used to prevent rejection, and may have less nephrotoxicity
than calcineurin inhibitor (CNI)-based regimens. To date there
has been little documentation of clinically significant proteinuria
linked with the use of sirolimus. We have encountered several
patients who developed substantial proteinuria associated
with sirolimus use. In each patient, the close temporal association
between the commencement of sirolimus therapy and proteinuria
implicated sirolimus as the most likely etiology of the proteinuria.
Methods: We analyzed the clinical and laboratory information
available for all 119 patients transplanted at the Washington
Hospital Center between 1999-2003 for whom sirolimus was a
component of their immunosuppressant regimen. In these patients,
the magnitude of proteinuria was assessed on morning urine
samples by turbidometric measurement or random urine protein:creatinine
ratios, an estimate of grams of proteinuria/day. Laboratory
results were compared between prior, during and following
sirolimus use.
Results: Twenty-eight patients (24%) developed increased proteinuria
from baseline during their post-transplantation course. In
21 patients an alternative cause of proteinuria was either
obvious or insufficient data was available to be conclusive.
In 7 of the 28 patients there was a striking temporal association
between the initiation of sirolimus and the development of
nephrotic-range proteinuria. Proteinuria correlated most strongly
with sirolimus therapy when compared to other demographic
and clinical variables. In most patients, discontinuation
of sirolimus resulted in a decrease, but not resolution, of
proteinuria.
Conclusions: Sirolimus induces or aggravates pre-existing
proteinuria in an unpredictable subset of renal allograft
recipients. Proteinuria may improve, but does not resolve,
when sirolimus is withdrawn.
[Back to top]
Metabolite Interference in Pharmacokinetic Studies
M.F. Moghaddam, J.-H. Fan, Y. Tang, O. Khatsenko, J.C.
Katz and M.A. Shirley
Our policy of conducting biotransformation studies with extended
chromatography prior to pharmacokinetic bioanalyses allowed
us to quickly detect an unusual, cis/trans metabolite
in rat plasma that was inseparable using a short chromatographic
method. We caution investigators that short methods invite
unknown isobaric metabolites to cause inaccuracies in plasma
concentration measurements.
[Back to top]
Allelic Variations in CYP2D6 Gene and Susceptibility
to Cervical Cancer
S. Wajid, S.H. Naqvi, A. Juneja M. Bharadwaj and A.B.
Mitra
Epidemiological studies have identified a number of risk
factors that contribute to the development of cervical cancer
precursors and cervical cancer. These include infection with
certain oncogenic types of human papillomaviruses (HPVs) and
other socio-economic factors. Tobacco smoking is an independent
risk-factor for cervical neoplasia. It has been found that
polymorphism at loci that encode carcinogen-metabolizing enzyme
such as cytochrome P450 2D6 (CYP2D6) catalyzing the detoxification
of carcinogens may determine susceptibility to cervical cancer.
Therefore, it is likely that an understanding of these allelic
differences is important for determining an individual’s
risk of cancer and susceptibility to potentially toxic agents.
The aim of the present study was to elucidate the role of
CYP2D6 polymorphism and susceptibility to squamous cell carcinoma
of the uterine cervix in Indian population. Therefore, the
genotype frequencies at this locus in females suffering with
low-grade CIN, high-grade CIN and squamous cell carcinoma
were compared. The control group consisted of 77 females with
normal cervical cytology and the cases comprised of 61 mild/moderate
dysplasia, 48 severe dysplasia and 45 cases of squamous cell
carcinoma of uterine cervix. The individuals were divided
into poor metabolizers (PM) and extensive metabolizers (EM)
on the basis of their ability to metabolize certain drugs
and carcinogens. Comparison of the frequency distribution
for the combination of CYP2D6 EM genotype and smoking between
mild/moderate and severe dysplasia was statistically significant
(p=0.047) suggesting that women with cervical intraepithelial
neoplasia I/II (CIN I/ CIN II) and CYP2D6 EM genotype who
smoke appears to have more chances for the lesions to progress
to CIN III. Whereas, frequency distribution for the same combination
between severe dysplasia and squamous cell carcinoma failed
to attain any statistical significance suggesting that CIN
III with CYP2D6 EM genotype has less chance to progress to
cervical cancer. Increased frequency of CYP2D6 EM and tobacco
smoking show strong association with CIN III, indicating that
not all lesions with the histopathological high grade CIN
are premalignant. Conversely some squamous cell carcinomas
may not be preceded by CIN.
[Back to top]
Comprehensive Identification of Cytochrome P450 Isoforms from
Solubility-Based Fractionated Rat Liver Microsomes
H. Yamanaka, M. Takeyoshi, Y. Minobe, Y. Yakabe, M. Takatsuki
and H. Sato
Cytochrome P450 isoforms from male rat liver microsomes
were comprehensively identified using nano liquid chromatography
tandem mass spectrometry (nanoLC-MS/MS). The enrichment of
P450, an endomembrane-anchored heme protein, was achieved
by solubility-based protein fractionation, and greatly improved
the total number of identified P450 isoforms. LC-MS/MS analysis
of fractions resulted in the identification of total 36 P450
isoforms. The combination of proteomic analysis and the solubility-based
fractionation would provide powerful tool for the expression
analysis of the superfamily proteins having great similarities
between the amino acids sequences.
[Back to top]
Mass Defect Filtering on High Resolution LC/MS Data as a Methodology
for Detecting Metabolites with Unpredictable Structures: Identification
of Oxazole-Ring Opened Metabolites of Muraglitazar
D. Zhang, P.T. Cheng and H. Zhang
This study describes the application of the mass defect filter
method for the detection of two unpredicted oxazolering opened
metabolites of muraglitazar in the feces of humans following
oral administration. Unlike other muraglitazar metabolites,
these metabolites formed little to no protonated ions, and
the NH4+
or Na+ adduct ions that were
formed were weak and not discernible from fecal interferences
even after background subtraction. With mass defect filtering
on high resolution LC/MS data, the resulting total ion chromatogram
and the simplified mass spectra allowed for the identification
and characterization of these metabolite ions, and their structures
were confirmed by synthesis.
[Back to top]
LC-MS/MS-Based Approach for Obtaining Exposure Estimates
of Metabolites in Early Clinical Trials Using Radioactive
Metabolites as Reference Standards
D. Zhang, N. Raghavan, T. Chando, J. Gambardella, Y. Fu,
D. Zhang, S.E. Unger and W.G. Humphreys
An LC-MS/MS-based approach that employs authentic radioactive
metabolites as reference standards was developed to estimate
metabolite exposures in early drug development studies. This
method is useful to estimate metabolite levels in studies
done with non-radiolabeled compounds where metabolite standards
are not available to allow standard LC-MS/MS assay development.
A metabolite mixture obtained from an in vivo source
treated with a radiolabeled compound was partially purified,
quantified, and spiked into human plasma to provide metabolite
standard curves. Metabolites were analyzed by LC-MS/MS using
the specific mass transitions and an internal standard. The
metabolite concentrations determined by this approach were
found to be comparable to those determined by valid LC-MS/MS
assays. This approach does not requires synthesis of authentic
metabolites or the knowledge of exact structures of metabolites,
and therefore should provide a useful method to obtain early
estimates of circulating metabolites in early clinical or
toxicological studies.
[Back to top]
In Vitro Metabolism of Leflunomide
by Mouse and Human Liver Microsomes Pp.
E.C.Y. Chan and L.-S. New
Leflunomide was found to be metabolized predominantly
to A77-1726 and two novel hydroxylated metabolites, M1 and
M2, in microsomes while A77-1726 was only biotransformed to
M1. M1 and M2 were proposed to be the hydroxylated α
-cyanoenol form of A77-1726 and the hydroxylated 5
methyl-isoxazole form of leflunomide, respectively.
[Back to top]
A New Method to Measure P-gp (ABCB1) Activity
M. Masuda and T. Mizutani
We have developed an easy and sensitive method to measure
P-glycoprotein (P-gp) activity using [γ-32P]ATP
and charcoal. This method utilizes the nature of adsorption
of organic phosphate to charcoal. The standard assay method
is as follows: the reaction mixture (20 μl)
of 1 mM [γ-32P]ATP
(1 Ci/mol), 2.5 μg
P-gp membranes, and the drug was incubated for 30 min, and
50 μl
of 20% charcoal suspension in 0.1 M phosphate buffer at pH7.3
was then added to the mixture. The solution was centrifuged
and the supernatant (20 μl)
in duplicate containing inorganic 32P
was spotted onto filter paper, and radioactivity was measured
by radio-image analyzer. This method can reduce the amount
of P-gp membrane compared to the conventional method utilizing
coloring of the inorganic phosphate-molybdate reaction. This
method is also applicable to other ATP-binding cassette (ABC)
transporters in phosphate buffer.
[Back to top]
Glucocorticoid Receptor Functions in HeLa Cells Are Perturbed
by 2,3,8,9-tetrachlorodibenzo-p-dioxin (TCDD)
R. Vrzal, J. Ulrichová, Z. Dvorák and P.
Pávek
We used 2,3,8,9-tetrachlorodibenzo-p-dioxin
(TCDD) and dexamethasone (DEX) to examine their effects on
aryl hydrocarbon receptor (AhR) and glucocorticoid receptor
(GR) in HeLa cells. TCDD (5 nM), DEX (100 nM) and their combination
down-regulated GR after 24 h. DEX reversed AhR mRNA increase
and AhR protein decrease caused by TCDD. Since AhR-GR cross-talk
occurs in cell-type and species-specific manner, the presented
data may serve as the basis in the understanding of mechanisms
underlying mutual interactions between AhR and GR.
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