Biotransformation of desoxypeganine by microsomal enzymes of the rabbit liver

Article abstract of DOI:10.1002/ardp.200400921

The biotransformation of the anticholinergic quinazoline alkaloid Desoxypeganine is studied by means of aerobic incubation with the non-induced supernatant obtained at 9000g from rabbit liver homogenates as enzyme source followed by an admixture of NADPH. The metabolites were identified by high-performance liquid chromatography, chemical ionisation mass spectrometry (LC-CI MS) and electron impact mass spectrometry (LC-EI MS) in comparison with synthetic reference compounds and typical ion fragments taken from literature data. C-oxidation of Desoxypeganine to the major metabolite Pegenone was observed as well as the hydroxylation of the alicyclic ring. The incubation mixture followed Michaelis-Menten kinetics characterised by K_m = 5.8 × 10^-5 mol L^-1 and V_max = 4.32 nmol Pegenone/min per mg protein or 3.37 nmol Pegenone/min per nmol CYP 450, respectively. These in vitro results demonstrate that the bioactive substance Desoxypeganine is easily oxidised to its ineffective metabolite Pegenone. This provokes a problem for correct dosage finding in formulations for the treatment of Alzheimer's disease and in the therapy of alcoholism and nicotine dependence.

Full text of DOI:10.1002/ardp.200400921

Arch. Pharm. Chem. Life Sci. 2005, 338, 49−52
DOI 10.1002/ardp.200400921
49
Biotransformation of Desoxypeganine by Micro-
somal Enzymes of the Rabbit Liver
Sascha Illgner and Rudolf Matusch
Department für Pharmazie Ϫ Zentrum für Pharmaforschung, Philipps-Universität
Marburg, Marburg, Germany
The biotransformation of the anticholinergic quinazoline alkaloid Desoxypeganine is studied by means
of aerobic incubation with the non-induced supernatant obtained at 9000g from rabbit liver homogena-
tes as enzyme source followed by an admixture of NADPH. The metabolites were identified by high-
performance liquid chromatography, chemical ionisation mass spectrometry (LC-CI MS) and electron
impact mass spectrometry (LC-EI MS) in comparison with synthetic reference compounds and typical
ion fragments taken from literature data. C-oxidation of Desoxypeganine to the major metabolite
Pegenone was observed as well as the hydroxylation of the alicyclic ring. The incubation mixture
followed Michaelis-Menten kinetics characterised by Km ϭ 5.8 ϫ 10Ϫ5 mol LϪ1 and Vmax ϭ 4.32
nmol Pegenone/min per mg protein or 3.37 nmol Pegenone/min per nmol CYP 450, respectively. These
in vitro results demonstrate that the bioactive substance Desoxypeganine is easily oxidised to its in-
effective metabolite Pegenone. This provokes a problem for correct dosage finding in formulations for
the treatment of Alzheimer’s disease and in the therapy of alcoholism and nicotine dependence.
Keywords: Desoxypeganine; Microsomes; Metabolism; Michaelis-Menten kinetic
Received: July 13, 2004; Accepted: September 1, 2004 [FP921]
Introduction
Desoxypeganine 1 was previously examined by in vivo
studies with animals. In all cases stable, long-lived urinary
and biliary metabolites were found [8, 9, 10]. Pegenone 2,
Vasicinone 3 and Isovasicinone 4 were described several
times to appear in the urine of rats receiving Desoxypegan-
ine 1 (Scheme 1).
Desoxypeganine
1 (1,2,3,9-tetrahydropyrrolo[2,1-b]-quin-
azoline; Scheme 1) is an alkaloid isolated from Peganum
harmala L. / Zygophyllaceae [1], a perennial, glabrous her-
bal plant which grows in semi-arid rangeland of the Middle
East and North Africa [2]. The substance can also be syn-
thesized in two steps via the intermediate Pegenone 2 [3, 4,
5]. Desoxypeganine 1 causes depression of monoaminoxi-
dase-A-activity and inhibition of acetylcholinesterase two
times superior than galanthamine in spite of being less toxic
[6]. These effects allow a large variety of therapeutic uses
such as nicotine dependence as well as alcoholism. Desoxy-
peganine 1 can also slow down the progression of Alzhei-
mer’s disease and improve mental cognitive capabilities.
In this study the Desoxypeganine 1 metabolites from aero-
bic incubation of 9000g supernatant of rabbit liver homo-
genates as enzyme source shall be characterized, using LC-
MS in the electron impact (EI) and positive chemical ioni-
sation (CIϩ) modes in comparison with reference com-
pounds and literature data. Since samples of microsomal
preparations were utilised under the admixture of NADPH
as coenzyme, only oxidative metabolism took place. For
further characterization the kinetic parameters of the con-
version of Desoxypeganine 1 to Pegenone 2 (1,2,3,9-tetra-
hydropyrrolo[2,1-b]-quinazoline-9-one) are reported.
In vitro studies with microsomes from different species like
rabbits or pigs are usually carried out at an early stage of
drug development in order to get preliminary information
on metabolic routes of a new drug. The advantage of these
studies is to detect also unstable and perhaps toxic meta-
bolites as ultra-short-lived or short lived intermediates
which never enter the blood circulation and therefore are
not excreted into bile or urine [7]. In addition, animal
experiments can be reduced. The biotransformation of
Results
Identification of Desoxypeganine metabolites
The incubation mixtures were first characterised by using
LC-MS in the positive chemical ionisation (CIϩ) mode in
order to detect unstable metabolites like N-oxides of Des-
oxypeganine 1 and Pegenone 2. These N-oxides were de-
scribed as compounds in plants like Nitraria komarovii L./
Zygophyllaceae [11, 12]. Although they appear as possible
Correspondence: Sascha Illgner, Institut fuer Pharmazeutische Che-
mie, Philipps-Universitaet Marburg, Marbacher Weg 6, D-35037
Marburg, Germany. Phone: ϩ49 6421 28-25899, Fax: ϩ49 6421 28-
25899, illgner@staff.uni-marburg.de
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

50
Illgner et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 49−52
amounted 10-15%. No mass peak of m/z 202 was found,
however, in the control samples without enzyme source.
Kinetics of the C-oxidation of Desoxypeganine
The formation of Pegenone 2 by incubating Desoxypegan-
ine 1 with the 9000g supernatant and NADPH followed
Michaelis-Menten kinetics. The Km and Vmax values were
calculated using the Lineweaver-Burk diagram resulting
from samples of liver preparations from three different rab-
bits (Figure 2). The Km value was 5.8 ϫ 10Ϫ5 mol LϪ1 and
the Vmax value was 4.32 nmol Pegenone/min per mg protein
or 3.37 nmol Pegenone/min per nmol CYP 450, respectively.
Discussion and conclusions
The aim of the present study was to investigate the in vitro
metabolism of Desoxypeganine 1 by the 9000g supernatant
of rabbit liver homogenate as cytochrome P-450 enzyme
source. Our results were compared with in vivo studies on
rats and we found the same three metabolites as described
in literature. The value of Vmax that we calculated fits into
the range between 1 and 15 nmol product per minute per
milligram of microsomal protein typical also for other or-
ganic substrates metabolised by cytochrome P-450 enzymes.
The determined Km value of 5.8 ϫ 10Ϫ5 mol lϪ1 reveals that
Desoxypeganine 1 belongs to a group of substrates with
high affinity to the cytochrome P-450 enzyme family [13,
14]. The fact that 78% of Desoxypeganine 1 were oxidised
to Pegenone 2 which has no anticholinergic activity (unpub-
lished data) under described conditions, and even 19% of
Desoxypeganine 1 were oxidised without any enzyme source
solely under aerobic conditions, provokes a serious problem
for pharmaceutical formulations of Desoxypeganine 1.
Since a central active compound at least should survive as
it gets over the blood-brain barrier, the quick metabolism
of Desoxypeganine 1 possibly could be the limiting factor
for the dosage in the treatment of Alzheimer’s disease or
therapy of alcoholism and nicotine dependence. The aim
therefore should be the development of suitable pharma-
ceutical formulations.
Scheme 1. Proposed main metabolic pathway for Desoxype-
ganine 1 by microsomal enzymes.
metabolites in our case none were detected in any of the
measured samples. It is well known that Desoxypeganine
1 is primarily metabolized in rats to the main metabolite
Pegenone 2 followed by hydroxylation of the alicyclic ring
system to Vasicinone 3 and Isovasicinone 4. Figure 1 shows
a typical total ion chromatogram and extracted mass chro-
matograms obtained from an incubation mixture with Des-
oxypeganine 1. Retention times, mass spectra and typical
ion fragments (EI mode) of each peak were compared with
those of standards and literature data, peaks 1-3 were
identified as Vasicinone 3, Isovasicinone 4 and Pegenone
2, respectively.
All incubation samples containing Desoxypeganine 1 or
Pegenone 2 as substrate together with rabbit liver micro-
somes in two different buffer solutions (pH 7.4 and pH 8.6),
delivered the metabolites as described before without devi-
ations. The main metabolic pathway for Desoxypeganine 1
by microsomal enzymes is summarized in scheme 1.
Experimental
Materials and methods
Quantitative analysis of Pegenone formed by
C-oxidation of Desoxypeganine
NADPH (tetrasodiumsalt) was purchased from Roche Diagnostics
GmbH (Mannheim, Germany). Boric acid, sodium hydroxide, hy-
drochloric acid, potassium dihydrogen phosphate, sodium monohy-
drogenphosphatedihydrate were obtained from Merck AG (Darm-
stadt, Germany). Sodium chloride and potassium chloride were
purchased from Roth GmbH ϩ Co (Karlsruhe, Germany). Aceto-
nitrile was HPLC-gradient grade from Merck AG (Darmstadt, Ger-
many). All other solvents and chemicals were obtained from Sigma-
Aldrich GmbH (Seelze, Germany).
After 40 min of incubation time at 37°C 78% of Desoxy-
peganine 1 were converted to Pegenone 2 by C-oxidation
considering the recovery rate. This C-oxidation also took
place in the control samples without coenzyme NADPH
(yield 42% Pegenone 2) or enzyme source (yield 19% Peg-
enone 2). The hydroxylated metabolites 3 and 4 in the total
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 49−52
Biotransformation of Desoxypeganine in vitro
51
Figure 1. Total ion chromatogram and extracted mass chromatograms obtained from an incubation mixture with Desoxy-
peganine 1. Peak 1 Vasicinone 3, Peak 2 Isovasicinone 4, Peak 3 Pegenone 2.
Figure 2. Lineweaver-Burk plot of C-oxidation of Desoxypeganine 1 measured by Pegenone 2 formation in the incubation
mixtures containing the components described under Materials and methods. Mean values are presented from three determina-
tions.
Chemistry
washed four times with phosphate buffer pH 7.4, dried with a towel,
weighed and minced with a scalpel. The minced livers were homo-
genised with 2 parts of 1.15% (w/v) KCl solution using an Ultra-
Turrax homogeniser (Ultra-Turrax type T45, Janke Kunkel GmbH
Co KG Staufen, Germany) four times for 30 s under permanent
cooling the round-bottomed flask in an ice bath. The homogenate
was transferred into plastic tubes and centrifuged for 30 min at
9000g and 2°C. The 9000g supernatant was carefully decanted and
used as enzyme source. The incubation test series and the kinetic
experiments were realised on one day storing the enzymes perma-
nently on ice. Protein concentration was determined as described
by Gornall et al. [16] and the content of cytochrome P 450 in the
supernatant was measured by using the method of Omura and Sato
Desoxypeganine 1 and Pegenone 2 are known compounds which
were synthesized by reported methods. Pegenone-N-oxide (1,2,3,9-
tetrahydropyrrolo[2,1-b]-quinazoline-9-one-4-oxide) was prepared
by treating Pegenone with 30% hydrogenperoxide, and purified by
preparative HPLC. All compounds were analyzed in the usual way.
Biological assay
Preparation of the 9000g supernatant of rabbit liver
All the following operations were carried out at 0Ϫ4°CC. The
9000g supernatant preparations were produced in most cases as de-
scribed previously [15]. The removed livers from rabbits were
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Illgner et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 49−52
[17]. The kinetic experiments were carried out with an UV-VIS
Scanning Spectrophotometer Shimadzu UV-2101 PC (Shimadzu
Corporation, Kyoto, Japan).
centrations of Pegenone 2 in unknown incubation samples directly
from this reference curve. In comparison to a calibration curve with
the same concentrations of Pegenone 2 without incubation mixture
a recovery rate of 81.4% was observed. Depending on the detection
limits of the HPLC analysis or mass spectrometry systems aliquots
up to 100 µl were used.
In vitro incubation
Incubations were carried out in 20 mL Erlenmeyer flasks at 37°C
in a shaking water bath. The incubation samples contained 1 or 2
mL of the 9000g supernatant, 1 mL cofactor solution (containing 2
mg NADPH tetrasodium salt, 0.2 mL MgCl2 0.1 M and distilled
water to a final volume of 1.0 mL), 1 mL of the substrate solution
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© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim