Advanced Drug Delivery Reviews 61 (2009) 290–302

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Therapeutic strategies by modulating oxygen stress in cancer and inflammation☆

Jun Fang a,⁎, Takahiro Seki b, Hiroshi Maeda a,⁎
a
Department of Microbiology & Oncology, Faculty of Pharmaceutical Sciences, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan
b
Innovative Collaboration Organization, Kumamoto University, Kumamoto, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Oxygen is the essential molecule for all aerobic organisms, and plays predominant role in ATP generation,
Received 14 November 2008 namely, oxidative phosphorylation. During this process, reactive oxygen species (ROS) including superoxide
Accepted 13 February 2009 anion (O•− 2 ) and hydrogen peroxide (H2O2) are produced as by-products, while it seems indispensable for
Available online 26 February 2009
signal transduction pathways that regulate cell growth and reduction–oxidation (redox) status. However,
during times of environmental stress ROS levels may increase dramatically, resulting in significant damage to
Keywords:
Reactive oxygen species
cell structure and functions. This cumulated situation of ROS is known as oxidative stress, which may,
Cancer however, be utilized for eradicating cancer cells.
Oxidative stress It is well known that oxidative stress, namely over-production of ROS, involves in the initiation and progression
Inflammation of many diseases and disorders, including cardiovascular diseases, inflammation, ischemia–reperfusion (I/R)
EPR effect injury, viral pathogenesis, drug-induced tissue injury, hypertension, formation of drug resistant mutant, etc.
Tumor-targeting Thus, it is reasonable to counter balance of ROS and to treat such ROS-related diseases by inhibiting ROS
Macromolecular therapeutics production. Such therapeutic strategies are described in this article, that includes polymeric superoxide
Oxidation therapy
dismutase (SOD) (e.g., pyran copolymer-SOD), xanthine oxidase (XO) inhibitor as we developed water soluble
form of 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP), heme oxygenase-1 (HO-1) inducers (e.g.,
hemin and its polymeric form), and other antioxidants or radical scavengers (e.g., canolol).
On the contrary, because of its highly cytotoxic nature, ROS can also be used to kill cancer cells if one can
modulate its generation selectively in cancer. To achieve this goal, a unique therapeutic strategy was developed
named as “oxidation therapy”, by delivering cytotoxic ROS directly to the solid tumor, or alternatively inhibiting
the antioxidative enzyme system, such as HO-1 in tumor. This anticancer strategy was examined by use of O•− 2
or H2O2-generating enzymes (i.e., XO and D-amino acid oxidase [DAO] respectively), and by discovering the
inhibitor of HO-1 (i.e., zinc protoporphyrin [ZnPP] and its polymeric derivatives). Further for the objective of
tumor targeting and thus reducing side effects, polymer conjugates or micellar drugs were prepared by use of
poly(ethylene glycol) (PEG) or styrene maleic acid copolymer (SMA), which utilize EPR (enhanced
permeability and retention) effect for tumor-selective delivery. These macromolecular drugs further showed
superior pharmacokinetics including much longer in vivo half-life, particularly tumor targeted accumulation,
and thus remarkable antitumor effects.
The present review concerns primarily our own works, in the direction of “Controlling oxidative stress:
Therapeutic and delivery strategy” of this volume.
© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
1.1. ROS and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
1.1.1. Endogenous defense system against ROS in cancer cells as a unique anticancer target . . . . . . . . . . . . . . . . . . . . 291
1.1.2. Another side of ROS in cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
1.2. Other diseases related with ROS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
1.3. Concerns in ROS-mediated anticancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Controlling Oxidative Stress: Therapeutic and Delivery Strategies”.
⁎ Corresponding authors. Fang is to be contacted at Tel.: +81 96 326 4137; fax: +81 96 326 4114. Maeda, Tel.: +81 96 3226 4114; fax: +81 96 326 4114.
E-mail addresses: fangjun@ph.sojo-u.ac.jp (J. Fang), hirmaeda@ph.sojo-u.ac.jp (H. Maeda).

0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2009.02.005
 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302 291

2. Therapeutic strategies by inhibiting ROS generation: the use of antioxidative agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
2.1. Xanthine oxidase inhibitors and SOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
2.1.1. Pyrazolopyrimidine derivatives (AHPP): inhibitors of XO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
2.1.2. SOD and its polymer conjugate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
2.2. Heme oxygenase (HO). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
2.3. NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
2.4. Canolol, a ROS scavenging phenols from plant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
3. Oxystress inducing anticancer therapy: oxidation therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
3.1. Poly(ethylene glycol) conjugated xanthine oxidase (PEG–XO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
3.2. Poly(ethylene glycol) conjugated D-amino acid oxidase (PEG–DAO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
3.3. HO inhibitor: zinc protoporphyrin IX (ZnPP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
3.3.1. Polymer conjugated ZnPP (PEG–ZnPP) and SMA–micelle of ZnPP (SMA–ZnPP) . . . . . . . . . . . . . . . . . . . . . . . 299
3.3.2. PEG–ZnPP/SMA–ZnPP for photodynamic therapy (PDT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
3.3.3. Other aspects of ZnPP and its polymer derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
3.3.4. New insight into the mechanisms of PEG–ZnPP/SMA–ZnPP dependent cytotoxicity . . . . . . . . . . . . . . . . . . . . . 299
3.4. Caution for oxidation therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

1. Introduction ROS from aerobic respiration or ambient oxygen-derived ROS, thus

reduce the potential insults of ROS. When an antioxidative enzyme
Reactive oxygen species (ROS) is a collective term of oxygen- defective mutant (e.g. SOD (−)) is generated, the growth of the bacteria
derived species, including not only the oxygen radicals (superoxide in aerobic condition becomes suppressed. However, its growth in
anion radical O•− •
2 , hydroxyl radicals OH etc.) but also some non-radical anaerobic condition is normal [7]. So-called facultative bacteria can
derivatives of O2 (hydrogen peroxide H2O2, singlet oxygen, alkyl adjust their metabolism depending on oxygen pressure. Similar
peroxide etc.). They are generally very small molecules and are highly phenomenon could be found in cancer cells. Cancer cells, particularly
reactive due to the presence of unpaired valence shell electrons. ROS is in the center area of the tumor nodule or mass, where ambient oxygen
potentially hazardous and it can be a by-product of cellular pressure is low, i.e. hypoxic conditions, they adapt more like anaerobic
metabolism. It also involves cell development, growth, survival, cell bacteria, having low levels of mitochondrial oxidative phosphorylation.
killing, aging, drug metabolism, pathogenesis of viral infection and This has been well known as Warburg-effect for a long time [8]. Under
development of cancer [1,2]. During the production of ROS, molecular such hypoxic conditions, they produce ATP mainly by glycolysis, or even
oxygen usually acts as electron acceptors to become oxygen free fermentation of amino acids. Recently, it has also been reported that
radicals. In aerobic life, where molecular oxygen is ubiquitous, ROS acetyl-CoA synthetase played important roles in producing ATP for
becomes the primary mediators of free radical reactions in cells, which
are generated during the production of ATP by aerobic metabolism in
mitochondria. The leakage of electrons from mitochondria, during the
electron-transport steps of ATP production, generates ROS, e.g. O•− •
2 . O2
−
is converted by superoxide dismutase (SOD) to generate H2O2, from
which further •OH is generated in a reaction catalyzed by Fe2+ or Cu2+
ions. In addition to these ROS, oxides of nitrogen (•NO, •NO2) are also
free radicals, and they are frequently called reactive nitrogen species
(RNS), and play important roles in biology similar to ROS.
ROS is a group of highly reactive molecules, which can react quickly
and damage various types of biomolecules, including proteins [3], DNA
[4] and lipids [5]. Under physiological conditions, to overcome the
potential toxicity of ROS, cells have evolved a series of antioxidative
defense systems to counteract these highly dangerous and extremely
reactive insults. These defense systems include intracellular SOD,
catalase and glutathione peroxidase that eliminate O•− 2 and H2O2.
There are also other enzymes or compounds that contribute to
scavenging free radicals (e.g., heme oxygenase-1 (HO-1), ascorbate,
tocopherol and glutathione) (Fig. 1), by which the cellular insults
caused by ROS are reduced to a nontoxic level [6].
Fig. 1. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism
and the antioxidant defense system in aerobic organisms. Oxidative free radicals, such
1.1. ROS and cancer as superoxide (O•− •
2 ), hydroxyl radicals ( OH), hydrogen peroxide (H2O2), nitric oxide
(NO), are generated during metabolism of cells, whereas, antioxidative defenses
1.1.1. Endogenous defense system against ROS in cancer cells as a unique including superoxide dismutase (SOD), heme oxygenase-1 (HO-1), catalase, glu-
tathione peroxidase, thioredoxin and various antioxidants (e.g., glutathione, Vitamin C,
anticancer target
E, canolol) serve as scavengers of these ROS and RNS. The balance between ROS (RNS)
As discussed above, the balance of ROS formation and antioxidative generation and antioxidative defenses is important for the versatile behaviors of cells.
defense level is crucial for cell survival and growth, and it is very Under physiological conditions, ROS is well controlled to tolerable levels, which will not
important for the cell to remove ROS properly for it to remain viable and harm the cells but it is rather involved in the process of cell development and growth.
maintain its vital function. Namely, normal cellular function will be On the contrary, imbalance of the ROS generating vs defending system, either by
overproduction of ROS or by deficits in antioxidative systems, will increase the level of
altered depending on this balance, which will in turn affect the fate of ROS, leading to cellular damages because of its highly toxic nature, which is the cause of
the cell. For example, most aerobic bacteria have adequate antioxidative many diseases. ONOO−, peroxynitrite; NOS, nitric oxide synthase; Arg, arginine. NAC,
systems, including SOD and catalase, which can eliminate hazardous N-acetylcysteine. Reproduced from Ref. [124] (modified) with permission.
292 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
tumor cells [9]. Namely, generation of ATP in the hypoxic tumor cells is
not an oxygen-dependent phenomenon. Parallel with these facts, it is
very intriguing and more important that cancer cells are more
frequently deficient in most crucial antioxidative enzymes, such as
catalase, glutathione peroxidase and SOD [10–13]. This means a high
vulnerability of tumor cells to ROS will be observed [14,15]. In fact, many
conventional anticancer drugs including vinblastine, doxorubicin,
camptothecin, cisplatin and inostamycin, exhibit antitumor activity via
ROS generation if not totally [16]. Accordingly, a unique antitumor
strategy named “oxidation therapy” was developed by delivering excess
oxidative stress into tumor cells or targetedly disrupting the antiox-
idative defense systems of tumor cells [17–20].
1.1.2. Another side of ROS in cancer
With regard to ROS and cancer, however, it should be noted that
the biological effects of ROS in cancer are multiple and non-linear.
Namely high levels of oxidative stress exhibit cytotoxicity as
mentioned above, inhibiting cell proliferation and leading to apopto-
tic/necrotic cell death; whereas low or intermediate levels of
oxidative stress are most effective in DNA damage, causing mutation,
inflammatory reaction and promoting proliferation of cells, and
ultimately inducing carcinogenesis via initiation, progressing to
cancer development [21]. In deed, convincing evidence indicated
that ROS is an endogenous class of carcinogens by triggering the
mutation of the cells [22–24]. Chronic infection of Helicobacter pylori,
hepatitis C virus, human papilloma virus (HPV) and their carcinogen-
esis can be explained by this mechanism. Namely, pathological levels
of ROS induce increased damage to DNA, which accompanies trigger of
mutagenesis via DNA base-modifications and mismatch repair [21]. In
addition, RNS including nitric oxide (NO) and its derivatives such as
peroxynitrite (ONOO−) play important roles in the development of
cancer and viral infection. Maeda and other researchers found that
during the course of viral and bacterial infections, production of NO is
enhanced through up-regulation of inducible nitric oxide synthase
(iNOS) at the site of infection or inflammation, production of O•− 2 in
Fig. 2. Schematic illustration of enhanced permeability and retention (EPR)-effect of macromolecules in solid tumors. Distribution of low molecular weight compounds (upper panel)
and high molecular weight compounds (lower panel) in tumors and inflammatory tissues are represented in time-dependent manner. Low molecular weight compounds rapidly
disappear from blood stream with very short plasma half-lives and no tumor (tissue) accumulation, whereas high molecular weight compounds gradually accumulate in solid tumor
(inflammatory tissues) by EPR. Reproduced from Ref. [124] (modified) with permission.
parallel manner is also known in the same time course either by
xanthine oxidase (XO) activation or NADPH oxidation to NO
generation [25–28]. NO and O•− 2 , react immediately and more reactive
derivative, ONOO− is generated, which plays probably more impor-
tant role in carcinogenesis [29–32], and also metastasis by activating
matrix metalloproteinases (MMPs) [33,34]. We further reported the
increased viral and bacterial mutation under such condition of
extensive ONOO− generation [35–37].
Moreover, oxidative stress can stimulate the expansion of mutated
cell clones by modulating genes related to proliferation and triggering
redox-responsive signaling cascades such as epidermal growth factor
(EGF), tyrosine phosphorylation, protein kinase C (PKC) [38], and
transcription factors that regulate inflammation and apoptosis
including NF-κB, Activator protein 1 (AP-1) and NF-E2-related factor
(Nrf2) [39–41]. In addition, apoptosis signal-regulating kinase 1
(ASK1), which is a member of the mitogen-activated protein kinase
kinase kinase (MAPKKK) family, plays crucial roles in ROS (e.g., H2O2)-
mediated cellular responses [42,43]. Namely, ROS such as H2O2
activates ASK1, which subsequently actives both p38 and c-Jun N-
terminal kinase (JNK) pathway, inducing a wide variety of cellular
responses such as proliferation, differentiation, senescence, and
apoptosis [42–44]. ASK family, which consists of ASK1 and newly
characterized ASK2, may probably be involved in ROS-mediated car-
cinogenesis and other human diseases, via MAPK signaling cascades
by triggering apoptosis and inflammation [44].
In regards to these signal transduction pathways, it has been
reported by Sawa et al. more recently that 8-nitroguanosine 3′,5′-cyclic
monophosphate (8-nitro-cGMP) that is a NO dependent nitrated
derivative of cGMP, plays an important role in signal transduction via
the S-guanylation of a redox-sensor signaling protein Kelch-like ECH-
associated protein 1 (Keap1) [45]. The S-guanylation accompanying
inactivation of Keap1 by 8-nitro-cGMP will result in nuclear export of
Nrf2 and activation of transcriptional activity of Nrf2, which subse-
quently induces the expression of antioxidant enzymes such as HO-1
[41] and regulates the progression of inflammation. Accordingly, 8-
 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
Table 1
Factors involved in enhanced permeability and retention (EPR)-effect of macromolecules in
solid tumors.
1 Active angiogenesis and high vascular density in tumora
2 Defective vascular architecture, e.g.
Lack of smooth muscle cells
Reduced number of receptors for angiotensin II
Large gaps in endothelial cell–cell functions
Anomalous vascular conformation (branching or stretching)
3 Extensive production of vascular mediators that facilitate extravasation a, including
Bradykinin
Nitric oxide
VPF and VEGFb
Prostaglandins
Matrix metalloproteinases, collagenase
peroxynitrite etc.
4 Impaired lymphatic clearance of macromolecules and lipids from interstitial tissue
(resulting in their retention)
See Refs. [61–66] and text for details.
a
These phenomena are also found in inflammatory tissues.
b
VPF, vascular permeability factor; VEGF, vascular endothelial growth factor.
nitro-cGMP seems to be a second messenger of NO, which may thus be
closely associated with ROS/RNS-induced inflammatory pathological
process and the subsequent carcinogenesis.
Accordingly, it is critical to control the amount of ROS carefully in
so-named oxidation therapy, because insufficient induction of ROS
will inversely lead to increased growth of tumors.
1.2. Other diseases related with ROS
Besides cancer, ROS is known to be implicated in many diseases,
because of its high reactivity and cytotoxicity. Under pathological
conditions such as acute and chronic infection and inflammation,
overproduction of these highly reactive metabolites will induce lethal
damage to cellular integrity and survival [46–48], resulting in
reversible or irreversible tissue injury. These pathological changes,
namely ROS-related diseases and disorders include microbial infec-
tions, inflammation, atherosclerosis, diabetes, ischemia–reperfusion
(I/R) injury, neurologic disorders, Parkinson disease, hypertension,
anticancer drug-induced tissue injury, smoking-related diseases, and
aging [20,46–52]. Thus, it is reasonable to develop therapeutics for
these ROS-related diseases by suppressing ROS generation. Indeed,
this therapeutic rationale has been challenged by many research
groups, with the use of various antioxidative agents of low molecular
weight nature as well as enzymes, such as SOD and catalase. Further,
inhibitors of ROS generating enzyme XO, or inducers of antioxidative
enzyme HO-1 were being considered along this line [46,47,53–60].
These therapeutics, however, are opposite in mechanism to the
anticancer strategy of ROS-dependent cytotoxicity as described above.
Although it seems to be controversial, it was found experimentally
practical if one can control the production of ROS at the local
pathological site, i.e. inducing the ROS generation in cancer tissue
selectively. For the treatment of inflammation and other ROS-related
diseases, suppression or elimination of ROS production can be a
remedy. Our approaches toward these goals are discussed below.
1.3. Concerns in ROS-mediated anticancer therapy
One crucial problem to be solved for anticancer strategy by ROS is
how to target the ROS production in cancer tissues selectively;
otherwise unexpected or systemic side effects will occur. Of great
importance, this problem could be effectively solved by enhanced
permeability and retention (EPR)-effect using macromolecular drugs,
polymeric micelles or nanoparticles [61–64] (Fig. 2). EPR effect is a
universal phenomenon seen in solid tumor for macromolecular drugs
in that the macromolecular or polymeric drugs will accumulate and
remain selectively in solid tumor tissues, and also inflammatory
293
tissues due to anatomical characteristics and pathophysiological
reaction of these tissues. Namely, vascular mediatory factors such as
bradykinin and NO play a key role in extravasation [62–64] (Table 1).
Thus EPR effect is now becoming a gold standard for anticancer drug
design. More recent reviews of EPR effect are seen in Refs [64,65].
In addition to the tumor tropic mechanism by EPR effect, macro-
molecular therapeutics (e.g., PEGylated proteins) will offer better
pharmacokinetics (i.e., longer plasma half-life), which is thus used for
various other diseases such as inflammation [64–66]. The macro-
molecule-based ROS-mediated therapy will thus be warranted for the
clinical application.
In this review, the therapeutic rationales of ROS will be discussed,
and some candidate agents, especially those with high therapeutic
response (e.g., PEGylated D-amino acid oxidase [DAO], PEGylated zinc
protoporphyrin [ZnPP]) based on the EPR effect are introduced below.
2. Therapeutic strategies by inhibiting ROS generation: the use of
antioxidative agents
Antioxidative agents include small ROS scavengers, inhibitors of ROS
generating enzymes, as well as antioxidative enzymes. The first
antioxidative agent applied in clinic is 3-methyl-1-phenyl-2-pyrazolin-
5-one (MCI-186, edaravone, Radicut®), a small molecular ROS scavenger,
which was approved for acute brain infarction in Japan, 2001 [67,68]. In
our group, we focused on inhibitors of XO (O•−2 generating enzyme), as
well as SOD and recently HO-1, which will be discussed in details below.
2.1. Xanthine oxidase inhibitors and SOD
2.1.1. Pyrazolopyrimidine derivatives (AHPP): inhibitors of XO
O•−
2 could be produced from several different oxidase enzymes,
including XO, NADPH-dependent oxidases, lipoxygenase, and NO
synthases, among which XO/xanthine is one of the major sources of
O•−
2 generation in vascular systems and in most inflammatory lesions
[47,59,69,70]. It is therefore conceivable that suppression of O•− 2
production with the use of XO inhibitors may be beneficial to the host
as a therapeutic means.
In this regard, allopurinol is a well-known XO inhibitor, which is a
structural analogue of alloxanthine among pyrazolopyrimidine deriva-
tives (Fig. 3). Allopurinol is used to treat gout that is caused by high
levels of uric acid in the body. However, one problem inherent to the use
of allopurinol is that the therapeutic effect of allopurinol is not dose-
dependent: at higher dose it becomes the substrate for XO, which will in
turn produce O•− 2 and thus exacerbate the disease unless adequate dose
regimen is fulfilled [59,71]. This delicate dose optimization of allopurinol
will limit its wide application for the treatment of O•−
2 driven diseases.
Instead of allopurinol, we recently found a much potent XO
inhibitor, 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP),
Fig. 3. Chemical structures of xanthine and its analogues. (A), xanthine; (B), hypoxanthine;
(C), allopurinol; (D), AHPP; (E), alloxanthine.
294 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
which is more useful in terms of biochemistry (Fig. 3D). AHPP showed
an apparent Ki value of 0.17 µM, which is about 3-times lower than
that of allopurinol (Ki of 0.5 µM), and it cannot be the substrate of XO
at higher concentration [59].
The therapeutic effect of AHPP was first examined in a sponta-
neously hypertensive rat (SHR) model, in which we hypothesized that
the mechanism of hypertension is attributed to O•− 2 generated by XO
in the vascular endothelium that would scavenge NO because of the
rapid reaction between them [56]. Instead, NO reacts with O•− 2 at
diffusion rate-limited manner, six-fold faster than the removal of O•− 2
by Cu, Zn–SOD [72,73]. Thus consumption of NO would result in
hypertension [72,74]. Namely, vascular endothelial XO may counteract
against NO-induced vasorelaxation indirectly by O•− 2 generation. Thus
inhibition of XO activity may result in an antihypertensive effect
(vasodilatation) by increasing the level of NO in vascular system. This
offers a new strategy against hypertension. As expected, AHPP
significantly augmented NO-mediated relaxation of aortic rings from
both rabbits and SHR in a dose-dependent manner; i.v. injection of
AHPP reduced the blood pressure of SHR to 70% of the initial blood
pressure, which is almost the level of normal rats [59] (Fig. 4). These
data strongly suggest the therapeutical potential of AHPP.
Fig. 4. Effect of AHPP on the blood pressure of spontaneous hypertensive rats (SHR).
(A) shows the antihypertensive effect of AHPP after i.v. administration in SHR, and the
effect of L-NAME on reduction of hypertension induced by AHPP in SHR is shown in
(B). XO inhibitors (e.g., AHPP) were injected via the jugular vein (at 0 time), after
which mean arterial blood pressure was continuously measured. Data are means ± SE
(n = 8). ⁎P b 0.05; †P b 0.025; ‡P b 0.01. L-NAME, Nω-nitro-L-arginine methyl ester (NOS
inhibitor). Reproduced from Ref. [59] with permission.
The main problem we encountered is, however, the very poor
solubility of AHPP, which is hardly soluble in physiological solutions,
making its therapeutic application impossible. To overcome this
drawback, a styrene maleic acid copolymer (SMA, Mw ~ 1280)
conjugated AHPP (SMA–AHPP) was prepared, which shows relatively
high water-solubility, meanwhile exhibits superior pharmacokinetic
parameter in vivo due to the macromolecular nature of the conjugates
[75]. Similar to AHPP, SMA–AHPP shows potent XO inhibitory activity,
and most important, remarkable antihypertensive effect by both i.v. or
oral administration in SHR [75]. In addition, XO is induced in many
infectious and inflammatory diseases such as influenza virus infection
and inflammatory bowel disease. O•− 2 is then generated there, which is
the major pathological factor in these diseases [46–48,76]. Therefore
the effect of SMA–AHPP was examined in a mouse colitis model,
which is induced by oral administration of dextran sodium sulfate
(DSS). Our preliminary data showed that the symptom, as well as the
survival rate of mice of this colitis model were greatly improved by
SMA–AHPP (unpublished data).
2.1.2. SOD and its polymer conjugate
Great interests have been focused on SOD for therapeutic use,
including Cu, Zn–SOD and Mn–SOD as the major scavenger of O•− 2 [21].
However, because of their poor pharmacological properties, such as an
extremely rapid plasma clearance time (e.g. plasma half-life b5 min in
mice), instability, and immunogenicity in vivo, the clinical application
of SOD as a therapeutic agent was very limited. The clinical application
of SOD is still problematic and remains unclear. The double-blind
controlled clinical trial has not reported positive effect of SOD in a
chronic inflammatory or autoimmune disease in humans. And it
seemed that SOD is not fully protective for myocardial reoxygenation
injury in open-chest dogs [21]. Many other trials of native/
recombinant SOD did not show efficacy in clinic. The reasons about
these problems may be explained by the following issue: SOD itself
catalyses dismutation of O•− 2 to yield H2O2, which will be then
converted to •OH via Fenton reaction by Cu2+ or other metals, and
more important Cu2+ is released from Cu, Zn–SOD [77]. This Cu2+
mediated generation of •OH may result in adverse effects of SOD
therapy. In this context, when combined with catalase, the effect of
SOD is greatly improved [21,53]. In addition, this SOD-medicated •OH
generation may be inhibited by chemical modification of SOD with Cu2+
chelating capacity, for example our findings from pyran copolymer–
SOD indicate that chelating effect of dicarboxylate of pyran may
suppress this reaction [65,78].
To improve these pharmacological and biochemical problems, a
wide variety of Cu, Zn–SOD conjugates were developed initially,
including pyran copolymer–SOD, polyethylene glycol (PEG)–SOD,
Ficoll–SOD, lecithinized SOD, styrene maleic acid copolymer (SMA)–
SOD, and gelatin or albumin conjugated SOD complexes. They all have
longer circulation half-lives than unconjugated SOD, high stability and
less immunogenicity [21,79,80].
In our laboratory, in the past 20 years we have studied on the role
of O•−
2 in various diseases, especially in virus infections, and developed
a pyran copolymer–SOD conjugate as most useful among others [46–
48]. In the pathogenesis of various viral diseases including influenza,
herpes simplex virus and Sendai virus, and bacterial infections
[27,41,46,47], source of O•−2 production is mainly from i) host's own
macrophages and granulocytes (neutrophils) in the “respiratory
burst”, and ii) XO, which is elevated enormously in serum and tissues
after influenza virus infection [46–48]. Namely, XO is 30–400 folds
higher in the lung of virus-infected mouse than healthy control mice
[48]. Therefore, treatment was undertaken by use of SOD or XO
inhibitors. For this purpose, pyran copolymer–SOD conjugate was
developed in 1989 [46,47]. Compared with native SOD, pyran
copolymer–SOD showed much improved pharmacological properties,
e.g., prolonged plasma half-life, 6 h vs about 4 min of native SOD,
while retaining about 80% of the enzyme activity [79]. Daily i.v.
 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
injection of pyran copolymer–SOD conjugate at the dose of 200 U/
mouse from day 5 to 8 after influenza virus infection protected 95% of
the mice from death while less than 5% survival without this
treatment (Fig. 5). It should be noted that, in this mouse model,
there was no detectable virus while the disease reached maximum as
judged by consolidation score of the lung and mortality as well as
infiltration of plasma cells in the alveolar compartment [46]. These
studies thus demonstrated for the first time that the real cause of this
viral disease was O•− 2 , but not the virus per se. Along this line, we
further developed a gelatin–SOD conjugate, which showed significant
suppressive effective against collagen-induced arthritis in mouse
model, which indicated that O•− 2 is also involved in the arthritis [80].
Meanwhile, many other research groups reported similar ther-
apeutic effect in various diseases including I/R injury, by use of various
SOD conjugates [21]. Among these, lecithinized Cu, Zn–SOD, which
was developed by Igarashi R et al. [81] exhibited promising
therapeutic efficacy against various ROS-related diseases, such
as ischemic injury, neuron injury, inflammation and HIV infection
[81–85], and it is now under the clinical trail for ulcerative colitis and
interstitial pneumonitis.
2.2. Heme oxygenase (HO)
HO is a key enzyme to regulate the intracellular heme level, which
catalyzes the initial and rate-limiting step of heme degradation,
resulting in the formation of biliverdin, carbon monoxide (CO), and
free iron [60,86]. Biliverdin is subsequently reduced by biliverdin
reductase to produce bilirubin, a potent and crucial antioxidant (Fig.
6). Three isoforms have been identified for mammalian HO: HO-1, HO-
2 and HO-3 [60]. HO-1 is the inducible form enzyme, which is however
highly expressed in liver and spleen under physiological condition, and
interestingly in most cancer cells; HO-2 is the constitutive isoform,
expressed mainly in brain and testis. HO-3 is a recently identified
isoform, which has been detected in many organs; however, its activity
is very low compared to other two isoforms, and thus its physiological
role remains unclear.
Among these isoforms, HO-1 plays most important role in cell
growth and cell death, by protecting cells against harmful oxidative
stress and apoptosis [60]. HO-1 also belongs to a member of the heat
shock protein family (Hsp-32), whose expression is triggered by a
Fig. 5. Therapeutic effect of SOD and pyran–SOD conjugate on influenza virus-infected
mice. Male ddY mice, aging 4–6 weeks, were used in the experiments. Mice received the
influenza virus by inhalation of virus aerosol at 2 × LD50 dose. Ten mice were used in
each treated group and 20 mice in the control group. SOD (200 and 1000 U per mouse)
and pyran–SOD conjugate (200 U per mouse) were given i.v. injection once daily for
four consecutive days from 5 days after virus infection. ●, Control; ○, SOD (200 U per
mouse); e, SOD (1000 U per mouse); ▲, pyran–SOD conjugate (200 U per mouse). The
mortality rate of mice did not increase after day 15. Mice surviving on day 15 were
considered as being cured. Reproduced from Ref. [46] with permission.
295
Fig. 6. Schematic representation of cytoprotective role of HO-1 and mechanisms of
antitumor effect of ZnPP and its polymer (micelle) derivatives. Reproduced from Ref.
[124] with permission.
variety of stress-inducing stimuli including hypoxia, heavy metals, UV
irradiation, ROS and RNS such as H2O2 and NO [60,87,88]. It has been
reported that HO-1 stumulates cell growth and the proliferation of
various cells including epidermal keratinocytes, vascular endothelium
and coronary endothelial cells, via its cytoprotective and antiapoptotic
effect [60]. Studies with HO-1-deficient mice show embryonic loss and
subsequent death significantly. Further chronic inflammation in the
kidney and the liver was observed in HO-1−/− adult mice, which
further support this role of HO [60]. The antioxidative and anti-
apoptotic effect of HO-1 is proposed to be related to multiple
mechanisms: i) decreasing the prooxidant level (heme); ii) increasing
the antioxidant level (bilirubin); iii) producing the antioxidative,
antiapoptotic molecule CO; iv) inducing ferritin, which removes
and detoxifies free ferric ion, a potent source of •OH generation; and
v) preventing over-stimulation of the immune response [60,89,90]
(Fig. 6).
Because of the antioxidative and antiapoptotic effect, potent
cytoprotective role of HO-1 was found subsequently to be involved
in various diseases and disorders, including atherosclerosis, hyperten-
sion, acute renal injury, toxic nephropathy, transplant rejection,
endotoxic shock, chronic obstructive lung disease, gastrointestinal
diseases, and Alzheimer's disease [60]. These facts suggest HO-1 as
a possible therapeutic target in these diseases. Indeed, inhibition of
HO-1 by specific HO inhibitor such as ZnPP or tin protoporphyrin
has resulted in the worsening of the diseases [60]. On the
other hand, administration of HO-1 inducers, such as cobalt proto-
porphyrin, hemin, and trinitrobenzene sulfonic acid or HO-1 gene
transfection showed significant therapeutic effect in many diseases
such as I/R injury, cisplatin nephrotoxicity and hypoxia-induced lung
injury [60].
Regarding the agent related to HO-1, hemin is a potent HO-1
inducer, and it has been used for acute intermittent porphyria in USA
and Europe as an orphan drug, however, it encountered with technical
difficulties and complications, and is associated with a high risk of
thrombophlebitis and coagulation disturbances [91]. A water soluble
form of hemin, heme arginate (Normosang®) was then developed,
however, its plasma half-life is relatively short (~10.8 h in human after
i.v. injection) mostly due to binding to hemopexin [91]. To overcome
these problems and to obtain better pharmacokinetics, we synthe-
sized PEG conjugated hemin (PEG–hemin), which became water
soluble molecules and behaved as water soluble micelles (unpublished
data). We thus anticipate a much longer plasma half-life of PEG–hemin
as shown in many other PEGylated agents, such as PEG–interferon
[92,93]. In our preliminary experiments, addition of PEG–hemin to
296 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302

cultured cells significantly induced the HO-1 expression; i.v. injection
of PEG–hemin markedly alleviated the degree of liver injury in a rat I/R
model, as evaluated by the value of liver enzymes in plasma (AST, ALT,
LDH) (unpublished data). Thus it became apparent that PEG–hemin
might be a potentially useful agent for various ROS-related diseases,
which warrants further investigations.
2.3. NO
NO produced in biological systems exerts a variety of pathophy-
siological roles [94,95]. Excessive formation of NO at local site such as
cancer and inflammation have pro-inflammatory effects such as
edema formation involving enhanced vascular permeability, inflam-
matory cell infiltration, and cytotoxicity, possibly through the
subsequent formation of ONOO− and other RNS [28]. In our
laboratory, we found NO is produced at the site of viral and bacteria
infections, by inducible NO synthase (iNOS) that is primarily
expressed in inflammatory leukocytes, from L-arginine as substrate.
Inhibition of NO production by using NOS inhibitors or knockdown of
iNOS greatly improved the histopathological changes and suppressed
disease progression [25–27,35]. However, NO also was found
beneficial when it exists at low or moderate levels, including anti-
inflammatory activities, antiapoptotic and antioxidative effects
[96,97]. NO has been reported to exhibit remarkable cytoprotective
effect for I/R injuries [95,98]. Lack of NO may also contribute the
pathogenesis of I/R because decrease of NO can trigger neutrophil
adherence, platelet aggregation and exudates into the ischemia area,
which exacerbates reperfusion injury. The cytoprotective and bene-
ficial effects of NO (or nitrite) is extensively reviewed in this special
issue of the journal by Gladwin et al.
Regarding the cytoprotective effect of NO in I/R injuries, we
developed a S-nitrosated α1-protease inhibitor (S-NO-α1-PI), which
has stable endogenous NO releasing activity, with long plasma half-
life and superior pharmacokinetics [99]. S-NO-α1-PI treatment
significantly suppressed the elevation of liver enzymes (AST, ALT,
LDH), neutrophil accumulation and apoptosis of liver cells in rat I/R
model. Moreover, it improved the impaired hepatic blood flow to a
great extent [100] (Fig. 7). Similarly, another NO donor, S-nitrosated
albumin, showed remarkable cytoprotective effect against I/R injury
of rat liver and bacterial infections [100]. These data suggest the
feasibility of these well designed NO donors for clinical application.
2.4. Canolol, a ROS scavenging phenols from plant
Many flavonoids and phenolic compounds show potent antiox-
idant activity [21]. Along this context, we recently isolated a phenolic

Fig. 7. Therapeutic effects of S-NO-α1-PI in a rat liver ischemia–reperfusion (I/R) model. Changes in serum levels of AST, ALT and hepatic blood flow are shown in (A) and (B),
respectively. (C) shows the neutrophil infiltration in rat liver after I/R, with or without S-NO-α1-PI treatment, and (D) shows apoptosis of hepatic cells induced by I/R in the presence
or absence of S-NO-α1-PI. ⁎P b 0.05, ⁎⁎P b 0.01. Reproduced from Ref. [96] with permission.
 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
Fig. 8. Chemical structure of canolol (Ref. [101]).
compound from crude canola (rape seed) oil, 4-vinyl-2,6-dimethox-
yphenol named canolol (Fig. 8), which exhibited potent antioxidant
activity [101]. Scavenging potency of canolol against alkylperoxyl
radical (ROO•) is much higher than well-known antioxidants, such as
α-tocopherol, vitamin C, β-carotene, rutin, and quercetin [102].
Canolol also showed strong scavenging capacity against the endo-
genous mutagen, ONOO−, thus greatly suppressing bacterial muta-
tion, protecting DNA from oxidative damage, as well as inhibiting
oxidation of lipids and proteins [102]. Moreover, canolol exerted
cytoprotective activity against ONOO− induced cytotoxicity in human
kidney epithelial cells HEK293 and human bronchial epithelial cells
HBE140 (unpublished data). Canolol can also suppress the induction
of iNOS and various inflammatory cytokines such as interleukin-1β
(IL-1β), tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ) and
cyclooxygenase-2 (COX-2) [103]. As a consequence, gastric carcino-
genesis was markedly suppressed in H. pylori-infected Mongolian
gerbils [103]. Interestingly, the viable H. pylori count was not changed
by feeding with the canolol containing diet. These results suggest that
the effect of canolol is to ameliorate the inflammation and
carcinogenesis caused by ROS, but it dose not kill the bacteria. This
is the same mechanisms of pyran–SOD induced protection against
viral infection [46]. In addition, we recently found that canolol showed
remarkable therapeutic effect in a murine colitis model induced by
orally administration of DSS (unpublished data). These data suggest
the potential of canolol as a cancer preventive agent via anti-
inflammatory effect.
Regarding the activity of canolol, it should be noted that canolol
directly scavenges ROS (ONOO−, O•− 2 and ROO∙ etc.), but also
suppresses the induction of inflammatory mediators (e.g., IL-1β,
TNF-α, IFN-γ and COX-2) at mRNA level indirectly, as shown above
[103], which is the same mechanism as seen in many flavonoids and
other phenolic compounds from different plants [104,105].
3. Oxystress inducing anticancer therapy: oxidation therapy
As discussed above, because of the toxic nature of ROS and
insufficient antioxidant enzymes in cancer cells, overproduction of
ROS will eliminate unwanted cancer cell. Based on this principle, a
unique “oxidation therapy” was introduced [14,15,17–20]. One way to
achieve this anticancer therapy is to deliver ROS generating enzymes
to tumor tissues directly, such as glucose oxidase, XO and DAO, where
more selective delivery to tumor tissue can be accomplished by EPR
effect after pegylation of these oxidases [14,15,17–19]. Another
approach is to suppress the antioxidative systems in tumors using
inhibitors against for instance HO-1 which is highly upregulated in
many tumors. These antioxidative systems are important for tumor
cells to defend against the oxystress (e.g., ROS and RNS). Thus
inhibition of the antioxidant enzymes, i.e. HO-1 in tumor can also be
an anticancer therapeutic [20,106].
To exploit the oxidation therapy, we have developed several
polymer conjugates of ROS generating enzymes (XO and DAO) and
HO-1 inhibitory compounds (ZnPP). Glucose oxidase was not selected
for this tactics because its substrate glucose is available ubiquitously in
vivo that will end up no regulation in ROS generation once this
enzyme is injected in vivo. The polymer conjugates exhibited tumor-
targeting characteristics by taking advantage of EPR effect, resulting in
297
remarkable antitumor effects [14,15,106–109], which is to be dis-
cussed in details at below.
3.1. Poly(ethylene glycol) conjugated xanthine oxidase (PEG–XO)
As described earlier, XO, a major O•− 2 generating enzyme, is a
metalloflavoprotien (Mw 298 kDa, a heterodimer) that catalyzes the
oxidation of xanthine/hypoxanthine to generate O•− 2 , which is
subsequently converted to H2O2 by SOD. XO was thus considered to
be a candidate of oxidation therapy [18]. However, when XO is used as
native enzyme, its high affinity to blood vessels limits its application,
which will induce systemic vascular damage and hypertension [17,59].
This drawback can be avoided by PEGylation of XO to PEG–XO via
conjugating PEG to ɛ-amino groups of lysine residues of XO, which
play a crucial role for binding to the luminal surface of vascular wall
[14]. In addition, the EPR effect of PEG–XO and excellent pharmaco-
kinetics were demonstrated and PEG–XO activity was retained in
tumor for more than 96 h. However, for in vivo treatment we found
the infusion of the substrates (xanthine or hypoxanthine) must be
performed with adequate time interval after i.v. injection of PEG–XO,
to ensure tumoritropic accumulation of PEG–XO which will take
usually more than 24 h, otherwise the enzyme (XO) in circulation will
generate O•−2 systemically because of incoming substrate, resulting
systemic side effects. According to the above-mentioned protocol,
significant antitumor effect was achieved by PEG–XO plus subsequent
infusion of the substrate, hypoxanthine; tumor growth was remark-
ably suppressed up to at least 52 days, in which complete regression of
tumor growth was found in three of seven tumor-bearing mice.
Meanwhile, no apparent side effects were observed after this
treatment [14].
3.2. Poly(ethylene glycol) conjugated D-amino acid oxidase (PEG–DAO)
Along this line, we recently focused on H2O2-generating enzyme
DAO. DAO is a flavoprotein (Mw 39 kDa) that catalyzes the
stereoselective oxidative deamination of D-amino acids to the
corresponding α-keto acids, during which molecular oxygen is used
as an electron acceptor, and H2O2 is generated [110]. An important
merit of DAO is that the substrate (D-amino acids) exists very little
under normal circumstance, and thus making it possible to control
H2O2 production by controlling exogenous infusion of D-amino acids.
We further conjugated DAO with PEG to become macromolecules (Mw
of ~63 kDa), which exhibited tumor-selective accumulation based on
the EPR effect [15,111]. Accordingly, PEG–DAO/D-amino acids system
will yield better tumor-selective generation of H2O2 by a double
targeting effect. Namely, D-proline was administered with a time
interval (e.g., ~4 h) after PEG–DAO injection. During this lag time,
PEG–DAO is allowed to accumulate in tumor first. The subsequent
administration of D-proline will then be converted to H2O2 mostly in
tumors. As expected, significant suppression of tumor growth was
observed by this treatment, with a significant increase of the survival
rate in the tumor-bearing mice (Fig. 9A). PEG–DAO thus becomes
more ideal candidate for oxidation therapy.
In addition, DAO is a monomeric simple protein and is easy to
handle and produce by recombinant technology. Compared with DAO,
XO is difficult to manufacture by recombinant technology because it is
a relatively large molecule; it is a heterodimer with complex structure.
We recently reported production of recombinant porcine DAO using E.
coli with high yield (20 mg/l), and high enzyme activity (5.3 U/mg),
as well as reasonable stability [111].
Among the antioxidative enzymes, catalase plays a central role
against H2O2 [112]. Although some hepatomas has been found to
express high level of catalase [113], in most cases, significantly
reduced catalase activity have been reported in many tumors [10,13].
Similar findings were observed in our group by measuring the catalase
activities in tumor tissues and normal tissues, such as the liver, kidney
298 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302

Fig. 9. Antitumor effect of PEG–DAO (A), and PEG–ZnPP (B) in murine tumor models. 2 × 106 of mouse sarcoma S-180 cells (A) or colon 38 cancer cells (B) were implanted s.c. in ddY
mice or C57BL/6 mice respectively. Six to ten days after tumor inoculation, mice were treated by each agent as indicated. Inlet of (A) shows the survival rate of tumor-bearing mice
after treatment. Data are means (n = 4–8); bars, SE. ⁎, P b 0.001. Reproduced from Refs. [106,111] with permission.

and lung, in both experimental mouse tumor and chemically induced 3.3. HO inhibitor: zinc protoporphyrin IX (ZnPP)
rat tumors (Table 2). Coincided with these observations, we found a
significantly higher IC30 (the concentration of drugs when cell growth As described above, HO-1 serves as a key enzyme to defend against
is inhibited to 30% compared with untreated control cells) to PEG– oxidative stress [60,86]. More importantly, it has also been known
DAO/D-proline (H2O2) against normal cells; a great contrast to tumor upregulated and to play crucial roles in many tumors [57], including
cells (Table 3 and Ref. [101]). These findings further supported the renal cell carcinoma, prostate tumors in humans, and experimental
validity and safety of ROS-induced anticancer therapeutics, i.e. PEG– animal solid tumors, such as rat hepatoma AH136B and mouse
DAO/D-proline dependent treatment [15,111]. sarcoma S-180, to support tumor growth through its antiapoptotic and
Even though H2O2 was utilized as the antitumor principle in our antioxidative effects [60,87,88,107]. HO-1, also known as a heat shock
studies according to its potentially high cytotoxicity, it should also be protein (Hsp 32), is considered to be a survival factor of tumor cells.
noted H2O2 exists physiologically in most organisms, which is the We thus realized that impairment or inhibition of this defensive
resulting product of SOD mediated O•− 2 dismutation. With regard to molecule in tumor cells should be useful as therapeutic target, which
the physiological function of H2O2, it has been recently reported that is the second approach for oxidation therapy. Subsequently, we
H2O2 plays an important role in vasodilation, similar to the function of confirmed that this strategy worked in many experimental solid
NO [114,115]. Thus in our studies of PEG–DAO/D-proline, even though tumors, by use of a potent HO inhibitor ZnPP [87,88,106,109 and
incidental generation of H2O2 systemically may occur, it is however in
relatively low level compared to the targeted intense generation of
H2O2 in tumor that achieved by EPR effect and time-lagged infusion of Table 3
the substrate, which may be tolerated. Whereas, more important, the IC30 of PEG–DAO/D-proline against tumor cells and normal cells.
systemically generated small amount of H2O2 may increase the blood Tumor cellsa IC30 (mM)b Normal cellsa IC30 (mM)
flow of tumor because of the induced vasodilation as described above, DLD-1 1.7 BNL.CL2 11.8
which will further enhance the EPR effect, consequently improve the Sk-Hep 1.8 MDCK 10.0
therapeutic activity of PEG–DAO/D-proline. This notion however HT-29 3.1 CEF 4.8
needs further investigation to be clarified. A431 1.4 Hc 19.0
HeLa 0.9 HRPEC 40.0
CNE 1.8 CV1 8.2
ES2 1.4 HBE140 3.7
Table 2 Meth-A 3.2
Catalase activities in tumor tissues and normal tissues. SW480 0.9
Lxc 4.5
Tumor modelsa Catalase activity (Kat.f)b
KYSE510 0.3
Tumor Kidney Liver Lung Average 1.9 ± 0.4⁎ Average 13.9 ± 4.5⁎
S-180 53 7745 8495 2508
*P b 0.01.
B16 207 ± 12 10564 ± 788 7980 ± 834 1249 ± 194 a
DLD-1, HT-29, SW480, human colon cancer cells; Sk-Hep, human liver cancer cells;
DMBA induced tumor 559 ± 271 10499 8408 1017
A431, human lung cancer cells; HeLa, human cervical cancer cells; CNE, Lxc, human
Reprinted from Ref. [111] with permission. nasopharyngeal carcinoma cells; ES2, human ovarian cancer cells; Meth-A, mouse
a
S-180: mouse sarcoma S-180 tumor; B16: mouse B16 melanoma; DMBA induced fibrosarcoma cells; BNL.CL2, murine embryonic hepatocytes; MDCK, Madin–Darby
tumor: DMBA (12-dimethylbenz (a) anthracene) induced rat breast cancer. Canine Kidney cells; CEF, chick embryonic fibroblasts; Hc, human hepatic cells; HRPEC,
b
The animals bearing the tumor, which was around 10 mm diameter, were killed and human retinal pigment epithelium cells; CV1, monkey kidney cells; HBE140, human
then were subjected to reperfusion with saline containing heparin (5 U/ml). The tissues bronchi embryonic cells.
b
subsequently collected were homogenized and subjected to the catalase activity assay. IC30 was calculated by the concentration of D-proline in the presence of 10 mU/ml
Data are mean ± S.E., n = 3–6. PEG–DAO, when cell growth is inhibited to 30% compared to untreated control cells.
 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
unpublished data], indicating a potentially beneficial role of HO
inhibitor, i.e. ZnPP as a novel anticancer drug.
3.3.1. Polymer conjugated ZnPP (PEG–ZnPP) and SMA–micelle of ZnPP
(SMA–ZnPP)
ZnPP is almost insoluble in physiological aqueous solution, which
limits its practical use as drug. PEGylation of ZnPP was thus carried
out, and PEG–ZnPP showed much increased water-solubility, and
more importantly, it formed macromolecular micelles of molecular
weight more than 70 kDa in aqueous media as demonstrated by
Sephadex G-75 chromatography. Moreover, a hydrodynamic diameter
about 200 nm of PEG–ZnPP was observed indicating multimolecular
associated form of micelles [107 and unpublished data]. When PEG–
ZnPP was administered i.v. it showed a much longer plasma residence
time, 40 times of nonconjugated free ZnPP. In addition, PEG–ZnPP
preferentially accumulated in solid tumor in murine tumor models
(about 5–10 times higher concentration than normal organs) due to
EPR effect as expected [106].
PEG–ZnPP remarkably suppressed the growth of S-180 and colon
38 tumors implanted in ddY and BALB/c mice, respectively, whereas
no or very little side effect at all was noticed, if any, during this
experiment (Fig. 7B). Moreover, PEG–ZnPP treatment induced tumor-
selective suppression of HO activity significantly, even though HO-1
protein seemed to increase after this treatment, leading to apoptosis
of tumor cells probably through increased oxidative stress [106].
Besides PEG–ZnPP, we successfully prepared a highly water soluble
micellar form of ZnPP using SMA, which also revealed macromole-
cular nature with the molecular weight and average micelle size of
144 kDa and 176.5 nm, respectively, in physiological aqueous solutions
[108]. Similar to PEG–ZnPP, SMA–ZnPP showed a potent antitumor
effect in many solid tumor models including DMBA induced breast
cancer in rats and fibrosarcoma Meth-A, colon 38 cancer in mice,
whereas no apparent side effects was found even at the dose of 200
mg/kg when injected i.v. [109 and unpublished data].
3.3.2. PEG–ZnPP/SMA–ZnPP for photodynamic therapy (PDT)
It is well known that porphyrin derivatives are efficient photo-
sensitizers for photodynamic therapy (PDT), and it is among
promising and least invasive antitumor treatment modalities using
laser beam usually to generate singlet oxygen [116,117]. It is thus
feasible for ZnPP, especially its polymer (micelle) derivatives (e.g.,
PEG–ZnPP, SMA–ZnPP) to be applied in PDT, which will further elevate
the anticancer activities of these anticancer agents as discussed above.
One advantage of PEG–ZnPP/SMA–ZnPP over conventional photo-
sensitizer (i.e. photopyrin) used for PDT, is that ZnPP does not require
laser beam whereas photopyrin requires laser beam at 630 nm. In
contrast, ZnPP is active even under conventional xenon light and
endoscopic light source.
As expected, ZnPP efficiently generated highly reactive singlet
oxygen under illumination of visible light, laser, or xenon light [109].
Utilizing a tungsten–xenon light as the irradiation source, SMA–ZnPP/
PEG–ZnPP induced marked tumor regression, both in mouse tumor
xenograft and chemical induced rat beast cancers [109 and unpub-
lished data]. The light source used in this study for activating ZnPP is
the commonly used endoscopic light or bulb (Philips, TL-D 15W) used
for icteric jaundice. The laser apparatus for conventional PDT is much
more expensive (~500, 000 US dollar) than that of xenon light source,
which is used in endoscope and almost available in all hospitals and
clinics, the application of SMA–ZnPP or PEG–ZnPP under the
irradiation by endoscopic light may be more convenient for certain
cancers of such as esophagus, breast, bronchogenic origin (lung),
colon, urinary bladder, and cervicus.
3.3.3. Other aspects of ZnPP and its polymer derivatives
In combination with ROS or ROS-generation system (i.e., PEG–
DAO), preliminary study showed a synergistic effect in PEG–ZnPP and
299
conventional chemotherapeutic agents. Namely, PEG–ZnPP treated
SW480 cells in vitro became much more vulnerable to the insults
caused by ROS or ROS generating anticancer drugs doxorubicin and
camptothecin [118]. Namely, the IC50 values were reduced to 75%, 61%,
17%, and 39% for H2O2, t-butyl hydroperoxide, camptothecin and
doxorubicin, respectively, by pretreatment with PEG–ZnPP. In vivo
experiments further verified that this notion was valid with the use of
PEG–DAO/D-proline in addition to PEG–ZnPP [118].
3.3.4. New insight into the mechanisms of PEG–ZnPP/SMA–ZnPP
dependent cytotoxicity
With regard to the antitumor mechanism based on depletion of
anti-ROS defense by inhibiting HO-1 in tumor cells, PEG–ZnPP or
SMA–ZnPP was found to down regulate the oncogene BCR/ABL in
chronic myeloid leukemia (CML), which resulted in apoptosis of the
CML cells [119,120]. Moreover, both PEG–ZnPP and SMA–ZnPP were
also found effective on imatinib (Gleevec®) resistant CML as well as
acute lymphoblastic leukemia (ALL) [121,122]. These findings suggest
that HO-1 (Hsp-32) is an attractive molecular target for cancer
chemotherapy with versatile functions. Inhibition of HO-1 by PEG–
ZnPP or SMA–ZnPP not only induces the ROS-related cytotoxicity, but
also leads to the cancer cell death via other mechanisms without
detrimental toxicity to the host in vivo, which will greatly increase the
therapeutic efficacy, and expand the clinical usage of PEG–ZnPP or
SMA–ZnPP. In addition, there may be other mechanisms for PEG–
ZnPP/SMA–ZnPP actions involving intracellular zinc signaling as
described by Hirano et al. [123], in which porphyrin may serve as
the carrier of zinc.
3.4. Caution for oxidation therapy
Even though the preclinical studies of oxidation therapy showed
the promising results as shown above, it is critical and necessary to
carefully control the amount of ROS. As described earlier, the
antitumor effect of ROS is dose-dependent; however, low or
inadequate dose of ROS will trigger the progression of tumor. It is
thus necessary to deliver excess amount of ROS into tumor, e.g.,
milimolar concentration; otherwise adverse effect, namely tumor
progression, may occur. In our experiments using ROS generating
enzymes, relatively high dose of enzymes and their substrates (e.g., 2
U/mouse of PEG–DAO and 0.5 mmol/mouse of D-proline, cf. Ref. [111])
were applied, and tumor targeted delivery of ROS was of course
achieved, by using polymeric conjugate according to EPR effect and
time-lagged infusion of the substrate [111], which will further increase
the intratumor concentration of ROS, and reduce the system side
effects. In fact, at the dose level of DAO and D-proline in vivo we have
not observed toxicity such as death or significant body weight loss of
the mice. Accordingly, cautiously manipulation and development of
effective tumor-targeting system are necessary to develop the
oxidation therapy for clinical application.
4. Conclusions
ROS, a group of highly reactive molecules, exhibits vital role in
aerobic organisms as the indispensable factors of signal transduction
pathway to regulate cell growth and drug metabolism [1,2], and it is
like double-edge sword. Under physiological conditions, damages are
reduced by cellular antioxidative defenses (e.g., SOD, catalase, HO-1)
and repair mechanisms [6]. However, under pathological circum-
stances such as inflammation [46–49], over-burden of ROS will lead to
reversible or irreversible tissue injury. Namely, ROS is potentially
harmful by reacting with proteins, DNA and other vital molecules, thus
damages the survival of cells [3–5]. Consequently it will induce various
diseases, including cancer [51,52,60,124]. It was thus suggested that
inhibiting or scavenging ROS would lead to the therapeutic modality
for ROS-related diseases, utilizing either antioxidative compounds
300 J. Fang et al. / Advanced Drug Delivery Reviews 61 (2009) 290–302
(e.g., Radicut® and canolol) or antioxidant enzymes (e.g., SOD, HO-1).
However, native SOD or low Mw ZnPP (HO inhibitor) did not show
significant therapeutical potentials, but when they were made as
polymer conjugates (PEGylation or micellar formation), to exert
selective targeting and to improve their pharmacokinetics as well as
in vivo half-life, they exhibited marked effects in vivo.
On the other hand, cytotoxic ROS can also serve as a tumor
terminator if it is produced selectively in tumors. This anticancer
therapy was named as “oxidation therapy” and many examples
presented here support this strategy. One of our recent promising
developments is PEG–ZnPP, which appears to have multiple antic-
ancer mechanisms including increase of susceptibility of cancer cells
to ROS as well as modulation of signal transduction cascade.
In conclusion, all the studies of controlling ROS generation give us
clues to treat various diseases including cancer and hypertension by
modulating the ROS generation, which might be promising
approaches toward clinical application in the future, which warrants
further investigation.
Acknowledgement
The works included in this paper are supported in part by Grant-in-
Aid from the Ministry of Education, Culture, Sports, Science and
Technology of Japan (No. 20590049, No. 20015045 and No. S0801085).
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