Hepatic effects of novel selenazolidine prodrugs of selenocysteine

1
Hepatic effects of novel selenazolidine prodrugs
of selenocysteine developed as potential cancer
chemopreventive agents.
Tarek Aboul-Fadl, Wael M. El-Sayed, Tenley
Schofield, Jonathan Constance, John G.
Lamb, Jeanette C. Roberts, and Michael R.
Franklin. Departments of Medicinal Chemistry and
Pharmacology and Toxicology, University of Utah,
Salt Lake City, UT 84112. College of Pharmacy,
University of Wisconsin, Madison.

• Abstract
• Novel selenozolidines have been synthesized
  as potential cancer chemopreventive agents and
  their hepatic effects investigated. The prodrugs
  (2a-g) were synthesized by the reaction of
  selenocysteine (1) with the appropriate carbonyl
  derivative.
•  
• The lipophilicity of these compounds
  (expressed as Clog P values) ranged from -3.062
  (2a) to -0.512 (2f) but hepatic effects appeared
  unrelated to this parameter.
• Male CF1 mice were treated daily for 7 days with
  equi-selenium (1.25 mg Se/kg) doses of each
  agent, by either the intraperitoneal (ip) or
  intragastric (ig) route and the effects compared
  with those of selenocystine. Hepatic parameters
  were determined 24 hours after the last dose. In
  general, few significant (p lt0.05) changes were
  seen with ig as compared to ip administration and
  the synopsis below documents the latter.
• Slight liver toxicity (elevated sALT) was only
  observed with 2b and 2c. Elevated glutathione
  S-transferase activity was observed with 2a, 2b,
  2c, and 2g. For 2c, this was accompanied by
  elevations in mRNAs of Gst-alpha and Gst-mu, but
  for 2a and 2b, only changes in Gst-alpha and
  Gst-mu respectively were observed. A decrease in
  glutathione peroxidase activity was observed for
  compounds 2e, 2f, and 2g and an increase in
  quinone oxidoreductase activity was observed for
  compounds 2a, 2c, and 2d. Except for quinone
  oxidoreductase mRNA with 2a, the enzyme activity
  changes were not accompanied by similar changes
  in mRNA. Increases in Ugt mRNAs were observed for
  2a and 2c, but these did not result in changes in
  UDP-glucuronosyltransferase (UGT) activity.
  Compounds 2a and 2c elicited mRNA increases in
  Ugt1a1 and Ugt1a9. In addition, compound 2c
  caused an increase in Ugt2b5. Thioredoxin
  reductase activity was elevated by 2a, 2c, and
  2d. Thioredoxin reductase and glutathione
  S-transferase activities were the only parameters
  that were altered with selenocystine treatment.

2.2.3. UDP- Glucuronosyl Transferase (UGT)
Activity.
The various substituents (R) at the C-2
position of the ring provide a way to fine-tune
pharmacokinetic parameters and selenocysteine
release mechanisms. Furthermore, they will allow
the development of critical structure-activity
relationships for cancer chemoprevention. Based
on the sulfur analog, thioproline, selenaproline
(2a, SCA) will require the action of
mitochondrial proline dehydrogenase to release
selenocysteine (Scheme 2). OSCA, 2c, was designed
to require the action of the enzyme
5-oxoprolinase to release selenocysteine, while
MSCA, 2b, and other derivatives with various
substituents at the C-2 position (2d-g) were
designed to release the amino acid after
non-enzymatic ring opening and hydrolysis.
Analogs of MSCA in which the methyl group is
replaced by a more lipophilic entity (phenyl,
n-butyl, or cylcohexyl) have since been
synthesized to provide a graded range of
lipophilicities (ClogP values of -1.884, -0.956,
and -0.512 respectively compared with -2.543 for
MSCA). Higher lipophilicity may enhance the
passage of the prodrugs through cell membranes
and, therefore, their absorption from the diet
and passage into cells of target organs.
Table 5 The Effect of Selenocysteine
Prodrugs on Hepatic Cytosolic Glutathione
Peroxidase Activity.
BSCA and PhSCA, both ig, depressed UGT
activity (4-nitrophenol as substrate).
Elevations in mRNAs following ChSCA (Ugt1a9)
given ig and SCA (Ugt1a1 and Ugt1a9) and OSCA
(Ugt1a1, Ugt1a9, and Ugt2b5) given ip, did not
result in elevated activity (Table 3).
Table 3 The Effect of
Selenocysteine Prodrugs on Hepatic Cytosolic UDP-
Glucuronosyl transferase

sig () Plt0.05, sig (-) Plt0.05 from corn oil
ip treated animals but not significantly
different from untreated.
1. Introduction
Selenium, an essential nutritional trace
element, is critical to the normal physiology of
many mammalian species, including humans. Among
its interesting biochemical properties, selenium
is generally regarded as having cancer
chemopreventive activity. Epidemiological
studies have suggested that an increased
incidence of cancer may be associated with low
serum selenium levels.1 Furthermore, clinical
trials showed that supplemental selenium reduced
the incidence and mortality of several types of
human cancers, including gastric cardia,
esophageal,2 liver,3 and prostate cancers.4 The
SELECT Trial, a phase III clinical trial to
explore the efficacy of selenium (and vitamin E)
in the prevention of prostate cancer, is
currently in progress.5 Moreover, selenium
compounds have been shown to have antitumorigenic
activities in several animal models.6 Selenium
compounds appear to achieve cancer
chemoprevention by multifaceted mechanisms,
depending on dosage and chemical form of selenium
and the nature of the carcinogenic insult. These
mechanisms include alteration of carcinogen
metabolism, carcinogen-DNA interaction, gene
methylation or expression, induction of
apoptosis, cytotoxicity of selenium metabolites,
regulation and redox modification of critical
enzymes, regulation of DNA synthesis, cell
proliferation and the cell cycle, inhibition of
angiogenesis, and overcoming oxidative stress.7
Several organic and inorganic selenium compounds
(Figure 1) have been investigated as selenium
supplements. Their safety and efficacy differs
markedly because of their differential metabolic
processing by the body. Selenocysteine is
metabolized by a single enzymatic cleavage step,8
that releases the selenium without unwanted side
reactions or negative attributes associated with
other inorganic and organic compounds. However,
selenocysteine is chemically unstable and
difficult to handle, which hinders its
usefulness. Accordingly, the current work
utilized the prodrug approach to design forms of
selenocysteine with enhanced physicochemical
properties and reduced toxicity.
3. Conclusion
sig () Plt0.05 ,  sig (-) Plt0.05 from
untreated (Selenocystine, SCA, MSCA, OSCA) and
corn oil treated (BSCA, PhSCA, ChSCA,
PhOHSCA) controls

• Although the novel selenazolidine prodrugs of
  selenocysteine were without direct
  hepatotoxicity, they elicited changes in many
  enzymes related to xenobiotic metabolism and
  protection against reactive intermediates.
• The biological response often differed when the
  compound was administered by the ip or ig route.
  Overall, a greater number of significant changes
  were seen when the compounds were given ip.
• SCA and OSCA, and to a more limited extent,
  BSCA, were the agents eliciting the most
  responses. For SCA and OSCA, particularly
  following ip administration, elevation of
  activities of several enzymes could be linked to
  higher levels of mRNAs.
• In future studies, the ability of the
  prodrugs to reduce the lung carcinogenicity of
  NNK in a mouse model will be compared to their
  ability to elicit increases in the "protective
  enzymes" in the present study.

2.2. Hepatic Effects of the Selenocysteine
Prodrugs 2.2.1. Serum Alanine Aminotransferase
Activity (sALT).
2.2.4. Cytosolic Quinone Oxidoreductase (QOR)
and Thioredoxin Reductase (TRR)Activities
No compound, administered either ip or ig, was
significantly hepatotoxic. A slight elevation in
sALT activity was by MSCA and OSCA given ip
(Table 1).
SCA, OSCA, and BSCA were the only three
compounds that significantly affected cytosolic
quinone oxidoreductase and thioredoxin reductase
activities. Both enzyme activities were
increased by SCA, OSCA and BSCA when the
compounds were given ip. OSCA also increased
both enzyme activities when given ig. SCA given
by the ig route elevated only thioredoxin
reductase activity. With the exception of the
quinone oxidoreductase transcript by SCA and the
thioredoxin reductase transcript by OSCA, both
given ip, the increases in enzyme activity were
not accompanied by significant elevations in
mRNAs of the enzymes (Table 4).
Table 1 Examination of
Selenocysteine Prodrugs as Hepatotoxins.
Table 4 The Effect of Selenocysteine
Prodrugs on Hepatic Cytosolic Reductase Activities
4. References
1.  S.D. Mark, Y. L .Qiao, S.M. Dawsey, Y.P. Wu,
H . Katki, E. W.Gunter, J. F. Fraumeni, Jr.,
W.J. Blot, Z.W. Dong and P.R.Taylor, J Natl
Cancer Inst, 92,1753 (2000).   2.  P.R. Taylor,
B. Li, S.M. Dawsey, J.Y. Li, C.S. Yang, W. Guo
and W. J. Blot, Cancer Res, 54,
2029s(1994).   3.  S.Y. Yu, Y.J. Zhu, W.G. Li,
Q.S. Huang, C.Z. Huang, Q.N. Zhang and C. Hou,
Biol Trace Elem Res, 29, 289(1991).   4. L. C.
Clark, B. Dalkin, A. Krongrad, G.F. Combs Jr.,
B.W. Turnbull, E.H. Slate, R. Witherington, J.H.
Herlong, E. Janosko, D. Carpenter, C. Borosso, S.
Falk and J. Rounder, Br J Urol, 81,
730(1998).   5.      http//www.clinicaltrials.gov
  6.   B.S. Reddy, T.T. Wynn, K. El-Bayoumy, P.
Upadhyaya, E. Fiala and C.V. Rao, Anticancer
Res, 16,1123(1996).   7.   R. Gopalakrishna and
U. Gundimeda, Nutr Cancer, 40,55
(2001).   8.    N. Esaki, T. Nakamura, H. Tanaka
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J.C. Roberts, H.T. Nagasawa, R.T. Zera, R.F.
Fricke and D.J. Goon, J Med Chem,

(ig) intragastric, (ip) intraperitoneal
2.2.2. Glutathione S-Transferase Activity (GST)
? NADH dependent , ?? NADPH dependent sig ()
Plt0.05 from untreated (Selenocystine, SCA, MSCA,
OSCA) and corn oil treated (BSCA, PhSCA, ChSCA,
PhOHSCA) controls
GST activity (1-chloro-2,4-dinitrobenzene as
substrate) was significantly elevated by
selenocystine, SCA, and OSCA, given ip and ig,
and by MSCA and PhOHSCA given ip. SCA elevations
were accompanied by significant elevations in
Gst-pi (2.4 kb) mRNA. SCA ip also elevated
Gst-alpha mRNA. OSCA given ip elevated Gst-alpha
and Gst-mu (5.3kb) mRNAs, while MSCA by the same
route only elevated the Gst-mu transcript.
Selenocystine did not elevate any Gst transcript.
Elevation in Gst-alpha and Gst-mu(2.3kb) by
BSCA, ip, and Gst-mu (2.3kb) by ChSCA ig were not
accompanied by increases in GST activity (Table
2). Increased GST activity with PhOHSCA occurred
in the absence of any significant elevation of
Gst transcript.
2.2.5. Glutathione peroxidase (GPx) Activity
2. Results
2.1. Chemistry
Glutathione peroxidase activity was
elevated by corn oil given ip, an elevation that
was accompanied by an increase in the glutathione
peroxidase 3kb mRNA. When given ig, corn oil
also increased the 1.6 kb GPx transcript, but by
this route corn oil did not produce a significant
elevation in activity. With BSCA, PhSCA, ChSCA
and PhOHSCA in corn oil (ip), the increase in
activity and mRNA seen with corn oil vehicle
alone was nullified (Table 5).
The design of the current prodrugs of
selenocysteine is based on an analogous system
developed for prodrugs of the thiol amino acid,
cysteine.9 The prodrugs were prepared by the
condensation of selenocysteine and the approprate
carbonyl compounds, as described in Scheme 1. The
structures of the target compounds were verified
on the basis of elemental and spectral methods of
analysis.
Table 2 The Effect of Selenocysteine Prodrugs
on Hepatic Cytosolic Glutathione S-transferase

• Acknowledgement

This study was supported by NIH grant R01 GM05891
sig () Plt0.05 from untreated (Selenocystine,
SCA, MSCA, OSCA) and corn oil treated (BSCA,
PhSCA, ChSCA, PhOHSCA) controls