Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases

Article abstract of DOI:10.1007/s00726-009-0429-2

N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N′-(3-ethylamino-propyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K_m(APAO) = 10 μM, k_cat(APAO) = 1.1 s ^-1 and K_m(SMO) = 28 μM, k_cat(SMO) = 0.8 s^-1, respectively], metabolized BnEtSPM to EtSPD [K_m(APAO) = 0.9 μM, k_cat(APAO) = 1.1 s^-1 and K_m(SMO) = 51 μM, k_cat(SMO) = 0.4 s^-1, respectively], and metabolized DBSPM to BnSPD [K_m(APAO) = 5.4 μM, k _cat(APAO) = 2.0 s^-1and K_m(SMO) = 33 μM, k_cat(SMO) = 0.3 s^-1, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K_m(APAO) =16 μM, k_cat(APAO) = 1.5 s ^-1; K_m(SMO) = 25 μM, k_cat(SMO) = 8.2 s ^-1; BnSPM K_m(APAO) = 6.0 μM, k_cat(APAO) = 2.8 s^-1; K_m(SMO) =19 μM, k_cat(SMO) = 0.8 s^-1, respectively]. Surprisingly, EtSPD [K_m(APAO) = 37 μM, k_cat(APAO) = 0.1 s^-1; K_m(SMO) =48 μM, k_cat(SMO) = 0.05 s^-1] and BnSPD [K_m(APAO) = 2.5 μM, k_cat(APAO) = 3.5 s^-1; K_m(SMO) =60 μM, k_cat(SMO) = 0.54 s^-1] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could playanimportant roleinpolyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues. Springer-Verlag 2009.

Full text of DOI:10.1007/s00726-009-0429-2

Amino Acids (2010) 38:369–381
DOI 10.1007/s00726-009-0429-2
ORIGINAL ARTICLE
Metabolism of N-alkylated spermine analogues
by polyamine and spermine oxidases
•
Merja R. Hakkinen Mervi T. Hyvonen Seppo Auriola Robert A. Casero Jr
•
•
•
¨
¨
Jouko Vepsalainen Alex R. Khomutov Leena Alhonen Tuomo A. Keinanen
•
•
•
¨ ¨
¨
Received: 3 July 2009 / Accepted: 24 September 2009 / Published online: 10 December 2009
Ó Springer-Verlag 2009
Abstract N-alkylated polyamine analogues have
potential as anticancer and antiparasitic drugs. However,
their metabolism in the host has remained incompletely
defined thus potentially limiting their utility. Here, we have
studied the degradation of three different spermine ana-
logues N,N0-bis-(3-ethylaminopropyl)butane-1,4-diamine
(DESPM), N-(3-benzyl-aminopropyl)-N0-(3-ethylamino-
propyl)butane-1,4-diamine (BnEtSPM) and N,N0-bis-
(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and
related mono-alkylated derivatives as substrates of
recombinant human polyamine oxidase (APAO) and
spermine oxidase (SMO). APAO and SMO metabolized
DESPM to EtSPD [Km(APAO) = 10 lM, kcat(APAO)
1.1 s-1 and Km(SMO) = 28 lM, kcat(SMO) = 0.8 s-1, respec-
tively], metabolized BnEtSPM to EtSPD [Km(APAO)
0.9 lM, kcat(APAO) = 1.1 s-1 and Km(SMO) = 51 lM,
kcat(SMO) = 0.4 s-1
respectively], and metabolized
=
=
,
DBSPM to BnSPD [Km(APAO) = 5.4 lM, kcat(APAO)
=
2.0 s-1 andKm(SMO) = 33 lM,kcat(SMO) = 0.3 s-1, respecti-
vely]. Interestingly, mono-alkylated spermine derivatives
were metabolized by APAO and SMO to SPD [EtSPM
¨
¨ ¨
M. R. Hakkinen (&) ꢀ J. Vepsalainen
Laboratory of Chemistry, Department of Biosciences,
Biocenter Kuopio, University of Kuopio,
P.O.Box 1627, 70211 Kuopio, Finland
e-mail: merja.hakkinen@uku.fi
Km(APAO) = 16 lM, kcat(APAO) = 1.5 s-1
;
Km(SMO) =
25 lM, kcat(SMO) = 8.2 s-1; BnSPM Km(APAO) = 6.0 lM,
kcat(APAO) = 2.8 s-1
0.8 s-1, respectively]. Surprisingly, EtSPD [Km(APAO)
37 lM,
kcat(APAO) = 0.1 s-1
kcat(SMO) = 0.05 s-1] and BnSPD [Km(APAO) = 2.5 lM,
kcat(APAO) = 3.5 s-1
Km(SMO) = 60 lM, kcat(SMO)
;
Km(SMO) = 19 lM,
kcat(SMO)
=
=
;
Km(SMO) = 48 lM,
¨
¨
M. T. Hyvonen ꢀ L. Alhonen ꢀ T. A. Keinanen
Department of Biotechnology and Molecular Medicine,
A.I. Virtanen Institute for Molecular Sciences,
Biocenter Kuopio, University of Kuopio,
;
=
0.54 s-1] were metabolized to SPD by both the oxidases.
Furthermore, we studied the degradation of DESPM,
BnEtSPM or DBSPM in the DU145 prostate carcinoma cell
line. The same major metabolites EtSPD and/or BnSPD were
detected both in the culture medium and intracellularly after
48 h of culture. Moreover, EtSPM and BnSPM were detected
from cell samples. Present data shows that inducible SMO
parallelwith APAOcouldplayanimportant roleinpolyamine
based drug action, i.e. degradation of parent drug and its
metabolites, having significant impact on efficiency of these
drugs, and hence for the development of novel N-alkylated
polyamine analogues.
P.O.Box 1627, 70211 Kuopio, Finland
S. Auriola
Department of Pharmaceutical Chemistry,
Biocenter Kuopio, University of Kuopio,
P.O.Box 1627, 70211 Kuopio, Finland
R. A. Casero Jr
Department of Oncology, Johns Hopkins University
School of Medicine, Baltimore, MD 21231, USA
R. A. Casero Jr
Sidney Kimmel Comprehensive Cancer Center at Johns
Hopkins, Baltimore, MD 21231, USA
A. R. Khomutov
Keywords Polyamines ꢀ N-alkylated polyamine
analogues ꢀ Flavin-dependent amino-oxidoreductases ꢀ
Spermine oxidase ꢀ Polyamine oxidase
Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, Vavilov Street 32,
Moscow 119991, Russia
123

¨
M. R. Hakkinen et al.
370
N,N0-bis-(2,3-butadienyl)-1,4-
butanediamine
Abbreviations
ACN
MDL 72527
Acetonitrile
APAO
(Acetyl)polyamine oxidase (exo-N4-amino)
NMR
Nuclear magnetic resonance
N1,N12-diacetylspermine
N1,N12-diacetylspermidine
N1,N12-Diacetylspermine
N1-AcSPD
N1-AcSPM
N1,N12-
DiAcSPM
PA
[EC 1.5.3.11]
BnDAP
N1-benzyl-propane-1,3-diamine
N1-benzyl-2,2,3,3-2H4-propane-1,3-
diamine
BnDAP-4D
BnEtSPM
N-(3-benzylaminopropyl)-N0-(3-
Polyamine
ethylaminopropyl)butane-1,4-diamine
PUT
Putrescine, butane-1,4-diamine
1,1,2,2,3,3,4,4-2H8-butane-1,4-diamine
(PAOh1), Spermine oxidase [EC 1.5.3.-]
Spermidine, N1-(3-aminopropyl)butane-
1,4-diamine
BnEtSPM-8D N-(3-benzylamino-1,1,2,2-2H4-propyl)-N0-
(3-ethylamino-1,1,2,2-2H4-propyl)butane-
1,4-diamine
PUT-8D
SMO
SPD
BnNH2
Benzylamine
a,a-2H2-benzylamine
SPD-2D
SPM
N1-(3-amino-1,1-2H2-propyl)butane-1,4-
BnNH2-2D
BnSPD
N1-(3-benzylaminopropyl)butane-1,4-
diamine
diamine
Spermine, N,N0-bis-(3-aminopropyl)
butane-1,4-diamine
N-(3-amino-1,1,2,2-2H4-propyl)-N0-(3-
aminopropyl)butane-1,4-diamine
Selected reaction monitoring
Spermidine/spermine N1-acetyltransferase
[EC 2.3.1.57]
BnSPD-4D
BnSPM
N1-(3-benzylamino-1,1,2,2-2H4-
propyl)butane-1,4-diamine
N-(3-aminopropyl)-N0-(3-
SPM-4D
benzylaminopropyl)butane-1,4-diamine
N-(3-aminopropyl)-N0-(3-benzylamino-
1,1,2,2-2H4-propyl)butane-1,4-diamine
Collision energy
SRM
BnSPM-4D
SSAT
CID
STD
QC
QL
Standard
DAP
Propane-1,3-diamine
Quality control
DAP-2D
DBSPM
1,1-2H2-propane-1,3-diamine
N,N0-bis-(3-benzylaminopropyl)butane-
1,4-diamine
Qualifier ion
QT
Quantifier ion
DBSPM-8D
DENSPM
DESPM
N,N0-bis-(3-benzylamino-1,1,2,2-2H4-
propyl)butane-1,4-diamine
Diethylnorspermine, N,N0-bis-(3-
ethylaminopropyl)-propane-1,3-diamine
N,N0-bis-(3-ethylaminopropyl)butane-1,4-
diamine
Introduction
The polyamines (PA) spermidine (SPD), spermine (SPM) and
their diamine precursor putrescine (PUT) are small organic
bases, which are ubiquitous in mammalian cells (Tabor and
Tabor 1984). Their intracellular levels are strictly regulated
by several enzymes and the cell membrane transport system
(Seiler 2004). Dysregulated polyamine metabolism has been
associated with neoplastic transformation and cancer cell
growth (Pegg 1988; Pegg and Feith 2007). Parasitic diseases
are an enormous health problem, especially in many devel-
oping countries, requiring cost effective drugs. Fortunately,
the polyamine metabolic pathway has been found to contain
several potential drug targets in parasites (Heby et al. 2007;
Kaiser et al. 2006; Muller et al. 2008; Reguera et al. 2005).
Thus, targeting differences inpolyamine metabolism between
host and parasite, or healthy and malignant tissue may offer a
selective advantage and avoid significant host toxicity.
Therefore, polyamine metabolism or their cellular targets
offers a rational basis for drug design against both parasitic
diseases and diseases caused by uncontrolled proliferation
(Casero Jr and Marton 2007; Heby et al. 2007).
DESPM-4D
N,N0-bis-(3-ethylamino-1,1-2H2-
propyl)butane-1,4-diamine
EtDAP
N1-ethylpropane-1,3-diamine
N1-ethyl-3,3-2H2-propane-1,3-diamine
N1-(3-ethylaminopropyl)butane-1,4-
diamine trihydrochloride
EtDAP-2D
EtSPD
EtSPD-2D
EtSPM
N1-(3-ethylamino-1,1-2H2-propyl)butane-
1,4-diamine
N-(3-aminopropyl)-N0-(3-
ethylaminopropyl)butane-1,4-diamine
N-(3-aminopropyl)-N0-(3-ethylamino-
1,1-2H2-propyl)butane-1,4-diamine
US, Food and Drug Administration, USA
Heptafluorobutyric acid
EtSPM-2D
FDA
HFBA
HPLC
IS
High pressure liquid chromatography
Internal standard
LC-MS/MS
Liquid chromatography-electrospray
ionization-tandem mass spectrometry
123

Metabolism of N-alkylated spermine analogues
371
Various polyamine analogues and polyamine synthesis
inhibitors have been investigated as chemopreventive,
chemotherapeutic or antiparasitic agents (Pegg et al. 1995;
Seiler 2003; Williams 1997). It is known that relatively
modest structural modifications in the PA backbone or
incorporation of terminal N-alkyl substituent(s) may evoke
significant differences in their chemical and biological
behaviour (Bergeron et al. 1997; Byers et al. 1990). Some of
the N-alkylated polyamine analogues have already shown
promising results in various model systems of chemother-
apy (Casero Jr and Marton 2007; Seiler 2003). N-ethyl
substituted polyamines, like diethylnorspermine (DENS-
PM) have displayed cytotoxic activity and are potential
therapeutics for cancer, while N-benzyl substituted poly-
amines have shown to be effective in the treatment for
malaria in a mouse model (Bitonti et al. 1989; Casero Jr and
Marton 2007; Edwards et al. 1991). Close structural PA
analogues mimic their natural counterparts in regulatory
functions, deplete natural polyamines in cells and most
importantly block the compensatory uptake of polyamines
(Bergeron et al. 1997). Unfortunately, most of the clinical
trials have failed due to apparent toxicity and/or low efficacy
of the studied compounds. One reason for low cell response
to antitumour polyamine analogues may be the catabolism
of the active drug by different oxidases (Lawson et al. 2002).
Interestingly, at the same time, the biological activity of
specific polyamine analogues has been shown to originate
from the active catabolism of cellular polyamines by SMO
to cytotoxic hydrogen peroxide (Pledgie et al. 2005).
Vujcic et al. 2003; Wu et al. 2003). By contrast, SMO
possesses very different substrate specificities for the nat-
ural polyamines as compared with APAO. It prefers non-
acetylated polyamines, efficiently oxidizing SPM, less
efficiently N1-AcSPM, and does not use SPD as a substrate
(Wang et al. 2003). In addition to APAO and SMO, also
other oxidases (such as mono and diamine oxidases) can
metabolize polyamines by selectively removing primary
N-terminal amine, which will lead to subsequent spontane-
ous cleavage at the exo side of the N4-amine of SPM (Fig. 1)
(Lee and Sayre 1998; Seiler 2004; Agostinelli et al. 2004).
Although APAO and SMO may significantly affect the
efficacy of polyamine-based drugs, metabolic studies with
purified enzymes and N-alkylated PA analogues have been
very limited (Lawson et al. 2002; Wang et al. 2003, 2005a; Wu
et al. 2003). A key for detailed metabolic studies has been the
development of a novel accurate LC-MS/MS method for
determining polyamines, N-alkylated analogues and their
metabolites from biological samples. Using LC-MS/MS
method we were able to verify the complicity of APAO-
¨
mediated degradation of DESPM (Hakkinen et al. 2007, 2008).
In this study we have thoroughly investigated the substrate
properties
of
N,N0-bis-(3-ethylaminopropyl)butane-1,
4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N0-(3-eth-
ylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N0-bis-
(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and
their predicted secondary metabolites with APAO and SMO.
In addition, experiments were performed to evaluate the cel-
lular accumulation, catabolism and excretion of the metabo-
lites of N,N0-bis-alkylated spermine analogues in DU145
prostate cancer cells. Results of this study provide important
information of the substrate properties of these two catabolic
enzymes that will benefit future drug design.
APAO and SMO are two of the key enzymes of poly-
amine catabolism. APAO was characterized in 1977 when
¨
¨
¨ ¨
Holtta purified rat liver APAO enzyme (Holtta 1977).
Since then, APAO has been purified from very few other
sources and the human enzyme was cloned in 2003 (Libby
and Porter 1987; Tsukada et al. 1988; Wu et al. 2003).
SMO (PAOh1) was initially cloned and characterized in
2001 (Wang et al. 2001). Cloning of both the enzymes has
made them readily available for recombinant protein pro-
duction that has facilitated further testing of their substrate
Materials and methods
Reagents
¨
properties (Jarvinen et al. 2006a; Wang et al. 2003, 2005a).
Ultra-gradient HPLC-grade acetonitrile (ACN) was from
During polyamine catabolism, SPM and SPD are first
acetylated by spermidine/spermine N1-acetyltransferase
(SSAT) and subsequently oxidized by APAO to produce
SPD and PUT, respectively, and to yield stoichiometric
amounts of hydrogen peroxide and 3-acetamidopropanal.
APAO prefers acetylated polyamines over non-acetylated
polyamines, and its natural substrates are N1,N12-DiAcSPM,
N1-AcSPM and N1-AcSPD (Seiler 1995; Wang et al.
2005a). In addition to the natural polyamines, some ter-
minally N-alkylated polyamines are degraded by APAO,
and some of the otherwise metabolically stable polyamine
analogues are catabolized when certain aldehydes are
J.T. Baker, heptafluorobutyric acid (HFBA, [99%) from
Fig. 1 Endo- and exo-cleavage sites of N-alkylated PA analogue are
shown with arrows. R1, R2 = Et and/or Bn
¨
present in the reaction mixture (Jarvinen et al. 2005, 2006a;
123

¨
M. R. Hakkinen et al.
372
Fluka, benzonitrile from Lancaster, and succinonitrile-d4
and benzylamine from Aldrich. Formic acid, glycine and
NaOH were from Sigma. Ultrapure water was prepared
using a Milli-Q Gradient system (Millipore, Milford, MA,
USA). Human recombinant APAO and SMO were pro-
earlier (Bergeron et al. 2001); 13C NMR (D2O): d 133.4
(2C), 132.8 (4C), 132.7 (2C), 132.2 (4C), 54.1 (2C), 49.9
(2C), 47.4 (2C), 46.8 (2C), 25.6 (2C), 25.5 (2C).
N,N0-bis-(3-Benzylamino-1,1,2,2-2H4-propyl)butane-1,4-
diamine tetrahydrochloride (DBSPM-8D) was prepared
N1-benzyl-bis-N1,N3-(2-nitrobenzenesulphonyl)-
¨
duced as described earlier (Jarvinen et al. 2005). APAO
from
2
¨
and SMO were stored frozen in 20% glycerol at -80°C,
until use. Some loss of activity was detected during stor-
age, thus 250 lM SPM or N1-AcSPD activity controls in
triplicates were always included for all the analyses. Pro-
pane-1,3-diamine dihydrochloride (DAP, 98%) was from
Aldrich. Butane-1,4-diamine dihydrochloride (PUT, 98%),
2,2,3,3- H4-propane-1,3-diamine (Hakkinen et al. 2009)
(1.13 g, 2.1 mmol) and 1,4-diiodobutane (310 mg,
¨
1 mmol) using the methods described in (Hakkinen et al.
2009) to give DBSPD-8D (318 mg, 59%) as a colourless
solid. 1H NMR (D2O): d 7.56–7.47 (10H, m), 4.29 (4H, s),
3.20 (4H, s), 3.17–3.08 (4H, m), 1.85–1.73 (4H, m); 13C
NMR (D2O): d 133.3 (2C), 132.7 (4C), 132.6 (2C), 132.2
(4C), 54.1 (2C), 49.8 (2C), 46.6 (4C, s ? m), 25.6 (2C),
24.7 (2C, quintet J = 19 Hz).
N1-(3-aminopropyl)butane-1,4-diamine
trihydrochloride
(SPD, 98%), N,N0-bis-(3-aminopropyl)butane-1,4-diamine
tetrahydrochloride (SPM,[95%) were from Sigma. All the
deuterated reference compounds except those described
¨
below were prepared as previously described (Hakkinen
Instrumentation
et al. 2009). Benzylamine hydrochloride (BnNH2) was
prepared from benzylamine and recrystallized from etha-
nol-ethyl acetate.
a,a-2H2-Benzylamine hydrochloride (BnNH2-2D) was
prepared from benzonitrile (1 g, 9.7 mmol) and LiAlD4
(815 mg, 19.4 mmol) using the previously described
Liquid chromatography separations, MS/MS detection and
analysis of the compounds were achieved with Agilent
6410 Triple Quad LC/MS equipped with Agilent 1200
Series Binary Pump SL pumping system and Agilent 1200
Autosampler. Data acquisition and analysis were per-
formed using an Agilent MassHunter Workstation software
(Agilent Corporation, MA, USA). 1H and 13C NMR spectra
were measured on a Bruker Avance (Bruker, Rheinstetter,
Germany) 500 DRX spectrometer as described earlier
¨
methods (Hakkinen et al. 2009) to give BnNH2-2D as
colourless crystals. (974 mg, 69%). 1H NMR (D2O): d
7.57–7.43 (5H, m); 13C NMR (D2O): d 135.3, 132.1 (4C),
131.7, 45.4 (quintet, J = 22 Hz).
1,1,2,2,3,3,4,4-2H8-Butane-1,4-diamine dihydrochloride
(PUT-8D) was prepared from succinonitrile-d4 (200 mg,
2.38 mmol) by catalytic deuteration essentially as descri-
bed in (Smith and Daves Jr 1978) to give PUT-8D as
colourless crystals. (158 mg, 39%). 13C NMR (D2O): d
41.0 (2C, quintet, J = 22 Hz), 25.6 (2C, quintet,
J = 19 Hz)
(Hakkinen et al. 2009).
¨
Analytical conditions
Liquid chromatography-electrospray ionization-tandem
mass spectrometry experiments were based on selected
reaction monitoring (SRM) analysis and were performed
N1-(3-Benzylamino-1,1,2,2-2H4-propyl)butane-1,4-diam-
ine trihydrochloride (BnSPD-4D) was prepared from
N1-benzyl-bis-N1,N3-(2-nitrobenzenesulfonyl)-2,2,3,3-2H4-
essentially as described earlier (Hakkinen et al. 2008). The
¨
chromatographic separations were carried out using a
Phenomenex Gemini reversed phase C18 column (3 lm,
˚
50 mm 9 2 mm, 110 A) protected with a Phenomenex
¨
propane-1,3-diamine (Hakkinen et al. 2009) (808 mg,
¨
1.5 mmol) and N-(4-iodobutyl)phthalimide (Jarvinen et al.
2006b) (545 mg, 1.65 mmol) using method described in
C18 guard column (4 mm 9 2 mm). A gradient solvent
system consisting of 0.1% (v/v) HFBA in water (solvent A)
and 0.1% (v/v) HFBA in ACN (solvent B) was used as
¨
(Hakkinen et al. 2009) to give BnSPD-4D (193 mg, 37%) as
1
a colourless solid. H NMR (D2O): d 7.55–7.48 (5H, m),
¨
before (Hakkinen et al. 2008), but the gradient was
4.29 (2H, s), 3.20 (2H, s), 3.16–3.10 (2H, m), 3.10–3.03 (2H,
m), 1.85–1.73 (4H, m); 13C NMR (D2O): d 133.3, 132.7
(2C), 132.6, 132.2 (2C), 54.1, 49.8, 46.6 (2C, s ? m), 41.7,
26.7, 25.6, 24.7 (quintet, J = 20 Hz).
N,N0-bis-(3-Benzylaminopropyl)butane-1,4-diamine
tetrahydrochloride (DBSPM) was prepared from N1-ben-
zyl-bis-N1,N3-(2-nitro-benzenesulfonyl)propane-1,3-diamine
increased from 2 to 50% B over 12 min at a flow rate of
0.2 ml min-1. Two time segments were used, turning point
being 7.5 min. In the first time segment (0–7.5 min) dwell
times were set to 100 (10 ion transitions), and after 7.5 min
dwell time were 20 (30 ion transitions). The precursor and
the product spectra of each polyamine were recorded
¨
similarly as described earlier (Hakkinen et al. 2007).
¨
(Hakkinen et al. 2009) (1 g, 1.87 mmol) and 1,4-diiodo-
Fragmentor voltage value was set to 60 V for DAP, DAP-
2D, PUT, PUT-8D, BnNH2 and BnNH2-2D, and to 90 V
for rest of the analytes. Precursor ions, selected product
ions for quantification and qualification and collision
butane (276 mg, 0.89 mmol) using methods described in
¨
(Hakkinen et al. 2009) to give DBSPM (211 mg, 45%) as a
colourless solid. 1H NMR chemical shifts as described
123

Metabolism of N-alkylated spermine analogues
373
Table 1 LC-MS/MS properties of polyamines used in this study
Amine
standard
Precursor
[M ? H]?
QT
CID
(eV)
QL
CID
(eV)
Deuterated internal
standard
Precursor
[M ? H]?
QT
CID
(eV)
Retention
time
DAP
75
89
58
72
5
5
–
–
DAP-2D
77
97
60
80
5
5
4.4
4.6
5.7
6.7
7.8
8.2
8.3
8.9
9.1
9.3
9.4
9.9
10.1
10.8
PUT
–
–
PUT-8D
EtDAP
BnNH2
SPD
103
108
146
174
165
203
231
259
236
293
321
383
86
5
58
15
20
10
15
20
15
10
20
15
20
20
20
EtDAP-2D
BnNH2-2D
SPD-2D
105
110
148
176
169
207
233
263
240
297
329
391
88
5
91
10
15
15
5
65
93
10
10
15
5
72
112
86
114
88
EtSPD
BnDAP
SPM
72
EtSPD-2D
BnDAP-4D
SPM-4D
148
129
157
157
148
219
219
219
91
152
133
159
159
152
223
223
223
5
112
129
112
112
112
112
112
5
EtSPM
DESPM
BnSPD
BnSPM
BnEtSPM
DBSPM
10
10
15
10
10
15
EtSPM-2D
DESPM-4D
BnSPD-4D
BnSPM-4D
BnEtSPM-8D
DBSPM-8D
10
10
15
10
10
15
Chemical structures with abbreviated names are shown in Table 2, and the chemical names are given in ‘‘Materials and methods’’
QT quantifier ion, QL qualifier ion, CID collision energy
energy values for all analytes used in the quantitative SRM
analysis are given in Table 1. DAP and PUT have only one
product ion.
weighted linear or quadratic (for SPD and EtSPM) least-
squares regression model. Assay validation was performed
according to the FDA guideline for bioanalytical method
validation (FDA 2001) essentially as described earlier
¨
(Hakkinen et al. 2008).
Preparation of standards and quality controls
Standard working solutions (concentrations of 0.03, 0.1,
0.3, 1, 3, 10, 30 and 60 lM) and working solutions for
quality control (QC) samples (concentrations of 0.05, 0.2, 2
and 20 lM) in 90 mM glycine–NaOH–formic acid buffer,
and internal standard (IS) working solution containing
1 lM of each 14 deuterated amine in water, were prepared
Enzyme kinetic analyses with human recombinant
APAO and SMO
The kinetic studies were carried out twice in triplicates at
four to seven different (SMO 25–250 lM and 100–
1,000 lM; APAO 10–1,000 lM) substrate concentrations.
The enzyme stock solution was diluted with 50 mM sodium
phosphate buffer (pH 8.0) (containing 0.1% Triton X-100)
before kinetic studies to yield 0.1–2.0 lg/10 ll of APAO or
SMO for each reaction mixture. Reactions were carried out
in a total volume of 180 ll in the 100 mM glycine-NaOH at
pH 9.5 buffer and were allowed to proceed for 2–60 min at
37°C before addition of 20 ll of 50% (v/v) formic acid. All
kinetic determinations included a T1/2 reference (incubated
half of the reaction time of actual samples to monitor lin-
earity of reaction) and the reaction mixture without enzyme
supplements both with the highest studied drug concentra-
tion treated identically as analysed sample to exclude non-
enzymatic degradation of studied compounds. Moreover,
with some selected reaction mixtures preincubation with
100 lM MDL 72,527 was used to inactivate SMO in order
to rule out any nonenzymatic degradation of tested drug.
LC-MS/MS was used to determine the concentrations of the
polyamines and their analogues as described above. Prior to
LC-MS/MS analysis, concentrated samples ([50 lM) were
¨
essentially as described earlier (Hakkinen et al. 2008).
Two types of calibration standards and QC samples
were prepared using these working solutions by adding
50 ll of each STD working solution or QC working solu-
tion, 50 ll of 1 lM IS working solution and 25 ll 0.5%
HFBA (calibration curve 1 for enzyme kinetic analyses) or
10% HFBA (calibration curve 2 for cell culture experi-
ments). Additional HFBA was used to maintain the chro-
matographic performance of samples containing
sulphosalicylic acid. Samples were transferred into Agilent
polypropylene vial inserts for the LC-MS/MS analysis. The
calibration curves included also a blank sample and a
¨
‘‘zero’’ sample as described earlier (Hakkinen et al. 2008).
Calibration curves and assay validation
Calibration curves were constructed from accurate con-
centrations of working solutions of each analyte in x-axis,
and peak-area ratio sample vs. IS in y-axis using 1/x
123

¨
M. R. Hakkinen et al.
374
¨
diluted with 90 mM glycine–NaOH–formic acid buffer and
all samples were passed through a 0.22-lm filter. LC-MS/
MS samples were prepared similarly to calibration stan-
(Hakkinen et al. 2009). All of the 14 amines tested were
quantifiable in a single run (Fig. 2). The dynamic range for
each analyte was 0.03–60 lM, except for DAP which was
0.03–10 lM. The correlation coefficients (R2) were always
[0.995 for all analytes. Inter-day accuracy of the assay for
all analytes ranged between 88.8 and 109.8% and the inter-
day precision of the method was always\13.4%.
¨
dards and QC samples (Hakkinen et al. 2008).
SMO activity at different pH in Bis-Tris propane buffer
Fixed 500 lM SPM in 170 mM Bis-Tris propane at pH
7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 was used with 0.2 lg of SMO
in total volume of 180 ll, incubated for 5 or 2.5 min at
37°C water bath. Reactions carried out twice in triplicates
were stopped by adding 20 ll of 50% sulphosalicylic acid
containing 100 lM 1,7-diaminoheptane and the SPD con-
tent was determined by using HPLC method described
SMO activity at various pH
SMO activity was the highest between pH 8.5 and 9.5
(Fig. 3). Therefore, all the kinetic determinations were
carried out at pH 9.5 being near optimal for both APAO
¨ ¨
and SMO (Holtta 1977; Royo and Fitzpatrick 2005).
¨
earlier (Hyvonen et al. 1992).
Terminally N,N0-bis-alkylated polyamine analogues as
substrates for recombinant human APAO and SMO
Cell culture
The prostate carcinoma cell line DU145 was obtained from
American Type Culture Collection, USA. The cells were
growninDulbecco’sModifiedEagle’sMediumsupplemented
with 10% fetal bovine serum and 50 lg ml-1 gentamycin
under conditions of ?37°C and 10% CO2. The cells were
harvested by trypsinization, counted electronically (Coulter
Counter model Z1) and divided to six well plates. Cells were
allowed to attach and after 24 h, medium supplemented with
or without 50 lM DESPM, BnEtSPM or BnSPM was added.
Samples were collected at 24 and 48 h post-treatment and
analysed for polyamines and N-alkylated analogues or their
metabolites. LC-MS/MS samples were prepared similarly as
calibration standards for calibration curve 2, and QC samples.
Assays were carried out twice in triplicates.
As shown in Table 3, DESPM was catabolized to EtSPD
by SMO and APAO. Minor N4-endo cleavage pathways
resulting in formation of EtDAP was detected with both
oxidases. Moreover, very minor de-ethylation takes place
with APAO. BnEtSPM was likewise catabolized to EtSPD.
However, in the case of SMO some competing debenzy-
lation occurred and evolved EtSPM was readily being
cleaved to SPD. Moreover, some other cleavage site
products were detected that were formed from secondary
metabolites by both the oxidases. DBSPM was mainly
metabolized to BnSPD by APAO. However, DBSPM was
metabolized to BnSPM and BnSPD by SMO (Table 3).
Metabolism of predicted secondary metabolites
by APAO and SMO
Statistical analyses
As shown in Table 3, EtSPM was readily degraded to SPD by
both oxidases. APAO metabolized EtSPM variably, other
competing pathways being less efficient. BnSPM was readily
metabolized to SPD by APAO and SMO. APAO showed
again more versatile substrate usage in comparison with
SMO. Benzylamine is formed either from direct N1-endo or
N4-exo cleavage and subsequent chemical decomposition of
benzylaminopropanal under alkaline conditions (Fig. 1).
BnSPD was debenzylated to yield SPD by both oxidases.
EtSPD was catabolized to SPD inefficiently by both the
oxidases. Interestingly, EtDAP was not metabolized by either
of the oxidases, but APAO was able to debenzylate BnDAP
to DAP with reasonable catalytic efficiency (Table 3).
The data are expressed as the mean S.D. A software
package, GraphPad Prism 4.03 (GraphPad Software, Inc.,
San Diego, CA, USA) was used for the analyses of enzyme
kinetic data using Michaelis–Menten equation with non-
linear fitting (Mr of 55,382 for APAO and Mr of 68,000 for
SMO were used in calculations).
Results
LC-MS/MS method used for polyamine and polyamine
analogue quantification
Polyamine quantification was based on the previously
¨
described LC-MS/MS method (Hakkinen et al. 2007, 2008).
Metabolism of N,N0-bis-alkylated polyamine analogues
in DU145 cells
Structures and abbreviations for the studied polyamines are
shown in Table 2 together with their deuterium-labelled
derivatives used in the LC-MS/MS measurements as the ISs
The effects of 50 lM analogues on intracellular polyamine
levels in DU145 cells are shown in Table 4. DESPM
123

Metabolism of N-alkylated spermine analogues
375
Table 2 Structures of the amine compounds in the study and their deuterated analogues as internal standards in LC-MS/MS quantification
Structure and abbreviation
Internal standard (IS)
D
D
H2N
H2N
H2N
NH2
DAP
PUT
SPD
SPM
DAP-2D
PUT-8D
SPD-2D
SPM-4D
H2N
NH2
D
D
D
D
NH2
NH2
H2N
D
D
D
D
D
D
NH2
N
H
NH2
H2
N
N
H
D
D
H
N
H
N
NH2
NH2
H2N
N
H
H2N
N
H
D
D
D
D
D
D
D
N
H
NH2
EtDAP
EtSPD
EtSPM
EtDAP-2D
EtSPD-2D
EtSPM-2D
N
H
NH2
NH2
D
N
H
N
H
NH2
N
H
N
H
H
N
D
H
N
NH2
NH2
N
H
N
H
N
H
N
H
H
N
H
N
D
H
N
H
N
DESPM
BnNH2
DESPM-4D
BnNH2-2D
BnDAP-4D
BnSPD-4D
BnSPM-4D
BnEtSPM-8D
N
H
N
H
N
H
N
H
D
D
D
D
NH2
NH2
D
D
N
H
NH2
N
NH2
BnDAP
BnSPD
BnSPM
BnEtSPM
H
D
D
D
D
D
D
D
D
D
D
D
NH2
D
N
H
N
H
NH2
N
H
N
H
H
N
D
H
N
NH2
NH2
N
H
N
H
N
H
N
H
D
H
N
H
N
D
D
H
N
H
N
N
H
N
H
N
H
N
H
D
D
D
D
D
H
N
H
N
D
H
N
H
N
DBSPM
DBSPM-8D
N
H
N
H
N
N
H
H
D
D
D
D
accumulated readily inside the cells and most efficiently
depleted the natural polyamine pools. EtSPD was detected
as the major metabolite in conjunction with trace of EtS-
PM. BnEtSPM accumulated as efficiently as DESPM
inside the cells, but did not deplete polyamine pools as
efficiently as the treatment with DESPM did. EtSPD and
EtSPM were detected as the major metabolites and some
BnSPD accumulated during 48 h in the BnEtSPM-treated
cells. DBSPM was taken up least efficiently among the
studied analogues. Moreover, it distorted intracellular
polyamine pools less efficiently in comparison with DES-
PM and BnEtSPM. BnSPM and BnSPD were detected as
the major metabolites from DBSPM. In culture medium
from cell samples treated with DESPM or BnEtSPM, only
EtSPD was detected together with intact drugs (data not
shown). BnSPD and a trace amount of BnDAP (48 h time
point) were detected as the metabolites of DBSPM treat-
ment. Total excretion of each metabolite from the cells was
less than 0.2 lM (equals to 400 pmol/metabolite in 2 ml of
medium) in culture medium within 48 h with all the drugs
studied. Some PUT and SPD, at less than 0.8 lM con-
centrations, were detected in control and drug supple-
mented culture media in 24 and 48 h samples (2 ml
medium/well, data not shown). Interestingly, no SPM was
123

¨
M. R. Hakkinen et al.
376
Fig. 2 Overlaid RP-LC-MS/
MS SRM chromatograms of all
the tested 14 amines, which
were able to quantify in a single
run. Interday accuracy for all
was between 88.8 and 109.8%
and precision \13.4%
been clearly shown that human and murine recombinant
¨
APAO is capable of metabolizing DENSPM (Hakkinen
et al. 2008; Vujcic et al. 2003; Wu et al. 2003). Some
unsymmetrically N-substituted norspermine analogues are
substrates of APAO (Wang et al. 2005a). In the case of
SMO, studies have shown the affinity of many of the
studied analogues to the enzyme and many of them have
been thought to be inhibitors, not substrates (Vujcic et al.
2002; Wang et al. 2003). The assay method used has been
based on fluorometric detection of hydrogen peroxide in a
dual enzyme assay system (Wang et al. 2003). The fluo-
rometric detection method is sensitive, but lacks the ability
to distinguish which drug in a combination of analogues/
polyamine is used as a substrate and how it is being
metabolized in the reaction mixtures. However, other
methods are valid for testing substrate properties of tested
compounds and can be used in inhibition studies. A recent
application of using 13C NMR for the detection of oxidase-
mediated catabolism of labelled tracer drug in vitro offers
means to characterize different reaction products (Bacchi
et al. 2009). NMR is a feasible method but lacks sensi-
tivity, and mixtures of polyamine analogues and reaction
products are sometimes hard to interpret accordingly. We
have recently developed a novel LC-MS/MS method that
was exploited to reveal versatile cleavage of DESPM by
APAO and shed some light on earlier controversy related
to degradation pathways of diethylated SPM analogues
Fig. 3 Reaction velocities (lmol/min) for SMO were determined by
using 500 lM SPM as a substrate at different pH in 170 mM Bis-Tris
propane buffer
detected in any of the culture media samples (data not
shown). Moreover, SPD was present at 2.5–5 times higher
concentrations in drug-treated medium samples in com-
parison with control medium samples (data not shown).
This could imply that SPM is metabolized to SPD prior to
being excreted from the cells.
Discussion
N-alkylated PA-analogues have been shown to be metab-
olized by cellular oxidase(s) which are sensitive to MDL
72,527 (Bergeron et al. 1995, 1997; Bitonti et al. 1990;
Bolkenius and Seiler 1989). The oxidase responsible was
thought to be APAO but recent cloning and characteriza-
tion of SMO has complicated the picture of how these
drugs are being metabolized in vivo and in vitro. It has
¨
(Hakkinen et al. 2007, 2008). Now, we have further
extended these studies to analyse the metabolism of three
different SPM analogues in vitro and in situ.
The LC-MS/MS method proved to be functional for
analyses of N-alkylated polyamine analogues and their
metabolites in in vitro enzyme assay mixtures. Moreover,
the method was sufficiently sensitive for quantifying
123

Metabolism of N-alkylated spermine analogues
377
Table 3 Kinetic values of N-alkylated polyamine analogues and their predicted secondary metabolites for recombinant polyamine and spermine
oxidases
APAO
Km
SMO
Km
Substrate
Metabolites
DAP
Vmax
36.9
kcat
Vmax
NA
kcat
NA
N
H
NH2
84
0.62
NA
PUT
18.5
209
0.4
2.5
NA
5.0
6.0
12
0.31
3.49
NA
0.33
32.2
0.30
13.2
44.9
NA
54
60
0.01
0.54
0.01
0.22
0.75
NA
NH2
N
H
N
H
SPD
BnDAP
BnNH2
SPD
NA
75
26.1
166
0.44
2.77
0.06
0.12
0.09
0.05
2.02
0.09
0.01
0.01
0.00
1.46
0.03
0.04
0.02
1.07
0.07
0.08
0.07
1.06
0.08
0.09
NA
9.8
19
H
N
NH2
N
H
N
H
SPM
3.79
7.51
5.59
3.17
121
NA
NA
8.3
34
BnSPD
BnNH2
BnDAP
BnSPD
BnSPM
PUT
20
NA
NA
1.4
6.3
5.4
17
9.79
4.53
17.8
16.7
0.28
2.83
0.09
490
NA
0.16
0.08
0.30
0.28
0.00
0.05
0.00
8.17
NA
H
N
H
N
N
H
N
H
33
5.45
0.38
5.90
0.23
87.6
1.73
2.51
1.48
64.2
4.05
4.66
4.17
63.5
5.00
5.96
NA
117
47
36
NH2
N
H
N
H
SPD
37
48
EtDAP
SPD
48
79
16
25
H
N
SPM
11
NA
NA
NA
28
NH2
N
H
N
H
EtSPD
EtDAP
EtSPD
EtDAP
EtSPM
EtSPM
EtSPD
EtDAP
BnNH2
SPD
25
NA
NA
4.7
10
NA
NA
45.6
1.56
NA
0.76
0.03
NA
H
N
H
N
N
H
N
H
6.6
17
33
NA
651
51
1.9
0.9
0.2
1.0
NA
NA
8.28
21.1
1.73
8.89
8.44
4.95
0.14
0.35
0.03
0.15
0.14
0.08
27
H
N
H
N
N
H
N
H
49
5.7
34
BnSPD
NA
NA
BnDAP
NA
NA
NA
1.51
28
0.02
Kinetic data was obtained as described in ‘‘Materials and methods’’ by using human recombinant APAO or SMO. The abbreviations and
structures are shown in Table 2. Apparent Km(lM), Vmax (lmol/min/lmol protein), kcat (1/s)
NA not applicable. Reference activity for SMO by using 250 lM SPM as a substrate was 460 62 lmol/min and for APAO 270 15 lmol/
min by using 250 lM N1-AcSPD as a substrate
123

¨
M. R. Hakkinen et al.
378
Table 4 Intracellular polyamine and analogue levels in DU145 cells
Cell number PUT
9106 (pmol/106 (pmol/106
cells) cells)
SPD
SPM
(pmol/106
cells)
Analogue
(pmol/106
cells)
EtSPM
BnSPM
EtSPD
BnSPD
(pmol/106 (pmol/106 (pmol/106 (pmol/106
cells)
cells)
cells)
cells)
Control 0 h
0.63 0.03 396 65 2,409 320 1,916 138 NA
1.06 0.08 246 16 2,194 68 2,226 72 NA
2.29 0.11 117 18 1,626 224 1,759 140 NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
31
NA
Control 24 h
Control 48 h
DESPM 24 h
DESPM 48 h
NA
NA
1.01 0.02 ND
1.50 0.06 ND
179 12
35
488 30
111 14
1,543 74
777 72
3,799 270 ND
3
6
3
2
NA
4
3,706 351 3.7 0.3 NA
41
NA
BnEtSPM 24 h 0.97 0.08 ND
BnEtSpm 48 h 1.72 0.08 ND
430 41
282 19
3,794 202 18
3,929 144 16
1
1
ND
ND
50
34
ND
29
5.1 0.3
14 1.6
12 0.3
DBSPM 24 h
DBSPM 48 h
1.04 0.05 98
0.86 0.07 34
9
5
1,359 119 2,419 149 962 39
822 50 1,824 52 1047 72
NA
NA
3
2
NA
NA
36
NA not applicable, ND not detectable
polyamines and their N-alkylated analogues in cell and
medium samples, i.e. different sample matrix. The validity
of the assay is based on the use of deuterated reference
compounds with LC-MS/MS analysis of each compound.
Advantages over existing HPLC methods are the increased
sensitivity, and exact quantification and identification of
retaining EtSPM and DESPM the reaction velocity is
slower when compared with N1-AcSPM and N1,N12-Di-
¨
AcSPM, respectively (Jarvinen et al. 2006a). BnSPM was
metabolized to SPD by both oxidases, APAO being more
efficient than SMO. Some other competing pathways were
also present. EtSPD was a very weak substrate for both
oxidases and was de-ethylated to SPD. However, BnSPD
was very efficiently debenzylated by APAO to yield SPD,
and with a 15% reaction velocity by SMO in comparison to
APAO. Moreover, APAO was capable in debenzylating
BnDAP to DAP with a reasonable catalytic efficiency. SPD
has been shown to be an inhibitor for SMO, but it seems
that the aromatic N-terminal substituent evokes catalytic
activity with SMO.
¨
each analyte (Hakkinen et al. 2008).
APAO- and SMO-mediated catabolism of N-alkylated
polyamine analogues was mediated through the same
major catabolic pathways. Both enzymes showed ability
for versatile cleavages of the studied substrates in the pilot
studies with DESPM, BnEtSPM or DBSPM. Thus, a sys-
temic study with all predicted metabolites of DESPM,
BnEtSPM or DBSPM was required to dissociate direct
metabolism from the metabolism of secondary metabolites.
SMO and APAO metabolized DESPM to EtSPD, thus
mimicking the degradation of N1,N12-DiAcSPM by APAO.
The aromatic benzyl end of BnEtSPM was strongly pre-
ferred by SMO and APAO, resulting in formation of EtS-
PD. However, with SMO there was an active debenzylation
pathway present, and evolved EtSPM was effectively fur-
ther metabolized to SPD. In the case of SMO, N1-exo and
N4-exo cleavage were equally active when DBSPM was
used as a substrate. Furthermore, some BnDAP was
detected indicating the presence of N4-endo cleavage.
APAO strongly preferred N4-exo cleavage yielding BnSPD
with DBSPM as substrate and other competing pathways
were less than 5% active when compared to the catalytic-
centre activity of the major pathway.
The metabolite formation was linear with all tested
metabolites except for BnNH2, which may also be formed
via further chemical degradation of produced benzylami-
nopropanal liberating acrolein in the incubation solutions
of high pH. This was noted as an increased reaction
velocity of the formation of BnNH2 after a longer (29)
incubation time in comparison to velocities obtained with
T1/2 reference samples. Thus, rapid acidification of reaction
mixture stabilizes benzylaminopropanal and prevents its
chemical decomposition to BnNH2 and acrolein. The pre-
vious data rules out N1-endo cleavage as a source of
BnNH2.
APAO is constitutively expressed in various tissues but
SMO activity has been shown to be inducible by some of
the N-alkylated polyamine analogues in certain cell lines
(Wang et al. 2001, 2005b; Devereux et al. 2003). Signifi-
cant APAO activity has been determined from post mortem
human specimens (Suzuki et al. 1984) and furthermore
SMO and APAO expression could be estimated by using
virtual Northern analysis of EST databases as described in
(Vujcic et al. 2003). Evaluation of the previous data
strongly supports our view that the role of N-alkylated
EtSPM was metabolized very efficiently to SPD by
SMO. It has been shown that SMO is able to degrade
N1-AcSPM and it seems that ethyl substituent that retains
the amine as positively charged strongly accelerates reac-
tion velocity (Wang et al. 2003). Similarly, SPD was a
major metabolite with APAO, but some other competing
pathways were also detected. Interestingly, with charge
123

Metabolism of N-alkylated spermine analogues
379
analogue degradation in determining the drug effect should
be systematically studied. The basal activity of
431 92 pmol mg-1 h-1 for SMO in DU145 cell has
charged amines have been synthesized (Bergeron et al.
1997; Casero Jr and Marton 2007). Alterations in carbon
backbone could have a profound effect on their substrate
properties with APAO and SMO and should be systemat-
ically studied in future. This work may require synthesis of
appropriate reference compounds for exact quantification
of reaction products by using LC-MS/MS. However, for
screening of reaction mixtures just to detect any of the
metabolites derived from the tested drugs could be carried
out without internal reference compounds.
¨
been determined earlier (Hyvonen et al. 2007). We did not
determine the induction of SMO or APAO activity by
N-alkylated analogues in our current study. Cell culture
study was included to test the applicability of LC-MS/MS
method for quantification of PA, N-alkylated PA analogues
and their metabolites from different sample matrix than in
vitro enzyme assay mixture.
Recently, SMO was shown to be responsible for drug-
induced apoptosis acting as the primary source of hydrogen
peroxide, and thus, playing an important role in polyamine
catabolism-mediated cytotoxic response (Pledgie et al.
2005). Our present data clearly shows that inducible SMO
is capable of metabolizing N-alkylated polyamine ana-
logues. This is not in agreement with earlier publications
showing that none of the studied N,N0-bis-alkylated PA
analogues were metabolized by SMO (Vujcic et al. 2002;
Wang et al. 2003). Discrepancy could be explained by
structural difference (3-3-3) or (4-4-4), instead of (3-4-3)
carbon backbone in conjunction with different conditions
in in vitro assay system. We did use the same cDNA
described in (Vujcic et al. 2002) for production of
recombinant SMO. However, Vucjic et al. did not purify
enzyme instead they used cell lysates that could have
caused DESPM behave like an inhibitor of SMO, not
substrate like EtSPM. Thus, some apparent toxicity or low
efficacies of the polyamine-based drugs in some cell lines
may be explained by the ability of these two enzymes
metabolize these drugs and their metabolites. It should be
noted that some of the metabolites generated from
N-alkylated PA analogues by SMO or APAO could be
substrates for other cellular oxidases, e.g. mono- or dia-
mine oxidase. Although cellular APAO and SMO activities
might have a significant impact on drug efficacy, kinetic
studies of DESPM, BnEtSPM or DBSPM and their pre-
dicted secondary metabolites using purified polyamine
oxidases have not been done until now. It is clear that
present data should be taken into a consideration when
novel N-alkylated polyamine analogues are being devel-
oped and used for the treatment of proliferative disorders or
parasitic diseases. Further studies are clearly needed in
order to assess the role of APAO and SMO in polyamine
analogue-mediated drug response. Large versatility in their
substrate properties and flexible cleavage of their substrates
could implicate that SMO and APAO may have other
natural substrates in addition to natural polyamines (Bacchi
et al. 2009; Lentini et al. 2007). It should be noted that the
studied analogues resembled SPM (3-4-3) carbon back-
bone. Many analogues based on norspermine (3-3-3) or
homospermine (4-4-4) carbon backbone and several ana-
logues having a variable length carbon chain between
In summary, here we show for the first time that both
APAO and SMO are capable of metabolizing several
N-alkylated polyamine derivatives. This suggests that they
might have a role in drug-mediated cytotoxic response,
especially after the natural polyamines have been depleted.
Although, exact mechanism of drug action of N-alkylated
PA-analogues remains obscure, polyamine metabolism is
still an attractive target for drug design. It is evident that
further studies including substrate properties on analogues
with APAO and SMO are required.
¨ ¨
Acknowledgements We thank Ms. Helena Vepsalainen and Ms.
Maritta Salminkoski, Department of Biosciences, Laboratory of
Chemistry, University of Kuopio, for their help with LC-MS/MS
sample preparation and in the synthesis work. Ms. Anne Karppinen
and Ms. Tuula Reponen at A. I. Virtanen Institute, University of
Kuopio, are acknowledged for their help with enzyme and cell
experiments. This work was supported by Academy of Finland
(projects 1241851 and 1287022), NIH (USA) CA984544, and the
Russian Foundation for Basic Research (project 08-04-917775).
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