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Advanced Drug Delivery Reviews 46 (2001) 169185

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SMANCS and polymer-conjugated macromolecular drugs:


advantages in cancer chemotherapy q
Hiroshi Maeda*
Department of Microbiology, Kumamoto University School of Medicine, Honjo 2 -2 -1, Kumamoto 860, Japan

Abstract
This review discusses the development and therapeutic potential of prototype macromolecular drugs for use in cancer
chemotherapy, in particular the development and use of SMANCS, a conjugate of neocarzinostatin and poly(styrenecomaleic acid). The various topics covered include a brief description of the chemistry and polymer conjugation, the binding
of the conjugate to albumin and the biological behaviour in vitro and in vivo after arterial injection in animals, including
plasma half-life, and the lipid solubility of SMANCS in medium chain triglycerides and Lipiodol, a lipid contrast medium
suitable for use in X-ray-computed tomography. The biological response-modifying effects and the tumor-targeting
mechanism of SMANCS and other macromolecular drugs are also discussed. The latter mechanism is accounted for in terms
of a tumor enhanced permeability and retention (or EPR) effect. A principal advantage in the use of SMANCS or other
macromolecular drugs is the potential for a reduction or elimination of toxicity. Macromolecular drugs such as a pyran
copolymerNCS conjugate show a marked reduction in bone marrow toxicity normally associated with the use of NCS. This
is believed to be due to a hypothetical bloodbone marrow barrier which, relative to NCS, restricts or limits access of the
macromolecular drug to the bone marrow. In addition, the clinical possibilities for SMANCS are discussed, including the
suggestion that angiotensin II-induced hypertension has clinical potential in improving the selective delivery of macromolecular drugs (i.e. SMANCS) to tumors. Aqueous SMANCS formulations have been tested in pilot studies in patients
with solid tumors of the ovary, esophagus, lung, stomach, adrenal gland and in the brain. Formulations based on
SMANCS / Lipiodol have been shown to be effective both as a diagnostic tool and for therapeutic use in solid tumors where
the formulations are given arterially via a catheter. In a pilot study in primary unresectable hepatoma, an objective reduction
in tumor size was observed for about 90% of cases when an adequate amount of the macromolecular drug was administered.
A patient receiving such treatment with no active liver cirrhosis and tumor nodules / lesion confined within one liver segment
might expect to have a 90% chance of survival after treatment for at least 5 years. 2001 Elsevier Science B.V. All rights
reserved.
Keywords: Poly(styrene-co-maleic acid / anhydride)neocarzinostatin conjugate; Protein modification; Enhanced permeability and retention
effect; Neocarzinostatin; Pyranneocarzinostatin conjugate

Abbreviations: BRM, biological response-modifying; CT, computed tomography; DIVEMA, divinyl ether and maleic acid; EPR,
enhanced permeability and retention; IgG, immunoglobulin G; LD 50 , lethal dose for 50% subjects; NCS, neocarzinostatin; SBTI, soybean
trypsin inhibitor; SMA, poly(styrene-co-maleic acid anhydride); SMANCS, conjugate of neocarzinostatin and poly(styrene-comaleic acid
anhydride; TB, tumor / blood
q
The article was originally published in Advanced Drug Delivery Reviews 6 (1991) 181202.
*Tel.: 181-96-373-5098; fax: 181-96-362-8362.
E-mail address: msmaedah@gpo.kumamoto-u.ac.jp (H. Maeda).
0169-409X / 01 / $ see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0169-409X( 00 )00134-4

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H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

Contents
1. Introduction ............................................................................................................................................................................
2. Chemistry of SMA conjugation with NCS.................................................................................................................................
3. Improvements in stability in vivo and in vitro by polymer conjugation and reduced immunogenicity.............................................
3.1. In vivo stability................................................................................................................................................................
3.2. Immunogenicity ...............................................................................................................................................................
3.3. In vitro stability ...............................................................................................................................................................
4. Tumor-targeting mechanism of SMANCS and other macromolecular compounds: EPR effect ......................................................
4.1. Elevated vascular permeability and hypervasculature in tumor tissue ...................................................................................
4.2. Factors involved in extravascular permeability in tumor tissue.............................................................................................
4.3. Enhanced drug delivery to tumor tissue by using angiotensin II-induced hypertension...........................................................
4.4. Decreased drug delivery to the bone marrow by polymer conjugation ..................................................................................
5. Oily formulation of SMANCS..................................................................................................................................................
6. Clinical outlook for SMANCS .................................................................................................................................................
6.1. SMANCS / Lipiodol..........................................................................................................................................................
6.2. Side-effects .....................................................................................................................................................................
6.3. Aqueous formulation of SMANCS ....................................................................................................................................
7. Conclusion .............................................................................................................................................................................
Acknowledgements ......................................................................................................................................................................
References ..................................................................................................................................................................................

1. Introduction
Antitumor proteins such as neocarzinostatin
(NCS) [1], actinoxanthin and macromomycins are an
interesting group of antitumor agents. Their highly
potent activity surpasses that of the widely used
conventional antitumor agents such as 5-fluorouracil,
adriamycin and cis-platinum at the minimal effective
concentration. For instance, NCS can inhibit tumor
cell growth at the nanomolar range, whereas many of
the low-molecular-weight compounds do so at the
micromolar or millimolar range [2].
The major limitation of NCS or macromomycin
for wide clinical use is severe toxicity, primarily
bone marrow suppression [3]. In addition, the very
short half-life (t 1 / 2 ) of NCS in mice of about 1.9 min
translates to a requirement in clinical usage for a
meticulously manipulated infusion velocity, on the
basis of a computer-simulated pharmacological compartment model for more effective plasma concentrations and less systemic toxicity [4].
An interesting earlier finding that differentiated
NCS from low-molecular-weight substances is its
predominant accumulation in the regional lymph
nodes when administered subcutaneously [5]. This
finding is quite important because the lymphatic
system is the route by which tumor cells frequently
metastasize and most therapeutic failures in cancer

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treatment occur because of lymphatic metastasis, as


well as hematogenous metastasis, to secondary sites
or organs. Therefore, if we could effectively target
the anticancer agents to the lymphatics, the agents
would be of potential value. The problem with NCS,
however, is its extremely short half-life both in vivo
(e.g. in blood) and in vitro, as described above.
I postulated that if one could make NCS more
lipophilic and structurally stable, it could be directed
more effectively to the lymphatics [5,6]. For this
purpose I found poly(styrene-co-maleic acid / anhydride) (SMA) to be an ideal compound to modify
NCS because of the anhydride group, which would
react with the amino group and because the styrene
side chain would confer potential hydrophobicity to
the molecule. I knew from my previous chemical
modification study of NCS that succinylation of the
two free amino groups by reaction with succinic
anhydride would not affect biological activity of
NCS [7]. Thus, succinic anhydride and SMA were
similarly reacted with NCS, as described below.
In an earlier study of the conjugation of SMA with
NCS, we used a SMA of about 6 kDa [8]. We
subsequently found that a smaller sized SMA (1.5
kDa) would be almost as effective [9], because the
smaller SMA conjugate, like the larger conjugate,
can also bind effectively to plasma albumin (see
below) [34]. During the past 12 years the conjugate

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

designated as SMANCS has been developed as a


potential anticancer agent for use against various
solid tumors in humans.

2. Chemistry of SMA conjugation with NCS


A more extensive account of this chemistry has
been given previously [9]. As shown in Fig. 1, SMA
contains alternating linkages of styrene and maleic
acid. We used SMA in which about 3050% of the
maleic acid is in reactive anhydride form and half of
the free carboxyl groups are butylated; this design
was found to give substantial hydrophobicity [9,34].
SMA can react with the free amino groups of NCS,
one at Ala-1 and the other at Lys-20, at pH 8.5 in
about 1020 h in the presence of excess SMA
(1020 mol / mol of NH 2 ) and complete blockage of
the free amino groups was achieved via carbamide
links. The bond between maleyl and amino group is

171

not so stable at high (.9) or low (,4) pHs or in the


presence of sulfate and other ionic groups.
The nature of the alkyl group and the extent of
alkyl esterification of the carboxyl groups are important factors in determining the binding affinity of
SMANCS to albumin, its lipid solubility and the
biological function of the covalently attached drug.
The degree of polymerization of styrene-maleic acid
and the different types of (maleyl carboxylate ester)
alkyl groups (such as n-butyl-, ethyl- or methyl-),
and the free carboxyl group itself, significantly affect
the binding affinity of the conjugate to albumin [34].
SMANCS with methyl ester SMA and SMANCS
with free carboxyl SMA do not bind albumin. Other
conjugates of NCS with hydrophilic copolymers of
divinyl ether and maleic acid (DIVEMA or pyrancopolymer) also do not bind to albumin [11].
As for its biological activity such as biological
response-modifying (BRM) effect, other than an
effect of inhibition of DNA synthesis [2], SMANCS

Fig. 1. (A) Structure of SMA, poly(styrene-co-maleic acid / anhydride) half butyl ester and (B) diagrammatic representation of the reaction
with NCS to produce the conjugate SMANCS.

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H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

is capable of activating macrophages, natural killer


cells and T-cells and it induces interferon-g [1113].
When the abilities of different types of SMA (e.g.
butyl, ethyl, free maleyl residues) to activate macrophages were compared, the n-butyl ester derivative
was found to be the most potent [11]. SMA with
longer alkyl chains such as n-pentyl and hexyl were
of low aqueous solubility. Thus, n-butyl ester was
chosen as the most desirable alkyl residue.

3. Improvements in stability in vivo and in vitro


by polymer conjugation and reduced
immunogenicity

3.1. In vivo stability


Most small, simple proteins of less than 40 kDa
have a limited plasma half-life when injected into the

circulation (Table 1). For instance, superoxide dismutase (bovine, Cu 21 , Zn 21 30 kDa) has a plasma
t 1 / 2 of 4 min, whereas that of NCS (12 kDa) is only
1.8 min. Human recombinant interferon (25 kDa),
which is devoid of carbohydrate, has a t 1 / 2 of less
than 10 min. The rapid clearance of these small
proteins is primarily due to renal clearance because
of their small size. Polymer conjugation or chemical
modification has been shown to prolong the plasma
t 1 / 2 of many proteins (see Table 1). The in vivo t 1 / 2
of SMANCS is ten times longer than that of NCS
(see Table 1). However, most proteins, even large
ones like albumin, which has a plasma t 1 / 2 of more
than 34 days, have much shorter t 1 / 2 value after
extensive chemical modification or denaturation, i.e.
25 min to a few hours. Rapid clearance in the latter
instances may be attributed to action of the bodys
own scavenger system and / or phagocytic macrophages. These data indicate that molecular size (.50

Table 1
Plasma clearance time of various proteins and their polymer conjugates or modified proteins
Protein

Type of polymer
or modification

NCS
SMANCS

None
Poly(styrene-comaleic
acid)NCS
None
Cross-linked
None
Dextran
DTPA / NH 2 / 51 Cr d
None
SMA conjugate
None
PEG
None
Evans Blue dye

Formaldehyde / 125 I
None
PEG 2 -linked
DTPAd
Iodination / 125 I
Iodination / 125 I

Ribonuclease
Ribonuclease dimer
Soybean trypsin inhibitor (SBTI a)
DextranSBTI
Ovomucoid
Cu 21 , Zn 21 superoxide dismutase (SOD)
SODSMAb
Bilirubin oxidase
PEG c bilirubin oxidase
Serum albumin, mouse
Serum albumin, mouse
Formaldehydeconjugated human
serum albumin
L-Asparaginase
L-AsparaginasePEG
Immunoglobin G, mouse
a 2 -Macroglobulin, human
a 2 -Macroglobulinplasmin complex
a

SBTI, Kunitz type.


SMA, styrene-maleic acid / anhydride copolymer.
c
PEG, polyethylene glycol.
d
DTPA, diethylene triamine pentaacetic acid.
e
Human albumin in humans: 19 days.
b

Molecular
mass (kDa)

t1 / 2

t 1 / 10

Test
animal

Refs.

1.8 min

15 min

Mouse

[10,56]

16
13.7
27
20
127
29
30
40
50
70
68

19 min
5 min
18 min
,2.0 min
.20 min
5 min
4 min
.300 min
,10 min
5.0 min
34 days e
2h

5h
30 min
5h
3 min
.80 min
34 min
30 min
.10 h
1.8 min
48 h

30 h

Mouse
Mouse
Mouse
Rabbit
Rabbit
Mouse
Rat
Rat
Rat
Rat
Mouse
Mouse

[10,56]
[57]
[57,58]
[59]
[59,60]
[56]
[56]
[59]
[61]
[61]
[56]
[20,56]

653(28)

150
18034
18032

25 min
1.53.4 h
56 h
60 h
140 h
2.5 min

4h

11 days

22 days
20 min

Rat
Rat
Mouse
Rat
Mouse
Mouse

[62]
[63]
[63]
[56]
[64]
[64]

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H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

kDa), electric character (neutral or negative preferred) and biocompatibility are important parameters for a prolonged plasma t 1 / 2 .
The luminal surface of the blood vessel wall and
the cell membrane contain many anionic components
such as heparan sulfate, chondroitin sulfate, hyaluronate and proteoglycan and they will interact with
polycationic polymers readily. Thus, it might be
predicted that polycationic conjugates would have an
extremely short plasma t 1 / 2 in vivo, and in fact,
polycations have been shown to have a short plasma
t 1 / 2 experimentally [14]. Furthermore, many carbohydrate-binding receptors or proteins are known to
be present in the liver and other organs and in
plasma. Thus, care is needed in the use of such
carbohydrate ligands in drug targeting because of
such interaction and clearance.

3.2. Immunogenicity
Many proteins may elicit immunological reaction
in vivo. However, NCS with its very short plasma
half-life does so very little [4,15] (see also Table 1).
When SMANCS was examined in conventional
guinea pigs for its antigenicity and anaphylaxisinducing activity it was found that such effects were
much less evident than with NCS [16].

3.3. In vitro stability


Proteolytic enzymes in serum seem to cause
degradation or inactivation of NCS and hence the
addition of protease inhibitors prolongs the biological activity t 1 / 2 of NCS [17]. We found that in blood
or plasma in vitro, the biological activity t 1 / 2 of
SMANCS was ten-fold greater than that of NCS in
vivo. In various physically harsh environments,
SMANCS is much more stable than NCS. Exposure
of NCS at a concentration of 1 mg / ml to 378C for 24
h results in a 90% loss of activity [18]. The time
required for the biological activity to fall to 1% of
the original activity of NCS at 568C, in buffer at pH
8.4, is about 6 h, compared with 60 h for SMANCS.
In addition, NCS is relatively unstable to denaturation at low pH (below pH 4.0), whereas SMANCS is
relatively stable [18].
Greatly improved stability is observed in a lipid
milieu for SMANCS but not for NCS. Incubation of

173

SMANCS at 1 mg / ml with Lipiodol did not affect


its stability at room temperature after 100 h, whereas
incubation at 378C caused a 40% decrease in stability
in 120 h [18]. Ultraviolet and X-ray irradiation
caused a greater decrease in stability in a water than
in a lipid environment.

4. Tumor-targeting mechanism of SMANCS and


other macromolecular compounds: EPR effect
[19]

4.1. Elevated vascular permeability and


hypervasculature in tumor tissue
The unique character of tumor vasculature has not
been fully utilized for cancer chemotherapy and little
attention has been paid to this subject in drug design
or tumor targeting. During our search for tumoritropic macromolecular anticancer agents, it became
apparent that vascular properties of tumor tissue
indeed differed greatly from those of normal tissue,
as described before [1923], namely: (a) tumor
angiogenesis resulted in hypervasculature [24,25],
which can be demonstrated clinically by angiography; (b) enhanced permeability was noted in tumor
tissues, which was induced by tumor-derived factors,
as described below [2628]; (c) the lymphatic system operated poorly, with thus little drainage recovery via the lymphatics [20,29] (Fig. 2); (d) tumor
vasculature showed architectural and functional incompleteness [2123].
It is known that macromolecules such as plasma
proteins are retained within the blood circulation in
the normal healthy state and do not leak out of the
vascular endothelial compartment into the interstitial
space; namely, a substance of molecular size above
50 kDa will not leak out into the normal tissue (see
Fig. 2) [19]. However, this description is not strictly
valid in the vasculature of inflammatory tissue
[10,30,31] and, more important in tumor tissue (see
Fig. 2). Experimental data provided by Peterson and
Appelgren [32], Shibata et al. [33] and ourselves
(Matsumura and Maeda [19,20] and Iwai et al. [29])
showed that radiolabeled fibrinogen, albumin, a 1 acid glycoprotein, SMANCS and other macromolecules were retained more in both solid tumors and
granulomas than in normal organ or tissue. Similarly,

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H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

Fig. 2. Diagrammatic representation of normal and tumorous vascular structures and transport of various substances into or out of
capillaries. Note the much enhanced leakage of macromolecules in tumorous tissue but no lymphatic recovery. Furthermore, there is no
backward flow of macromolecules into the blood capillary (from Ref. [19]).

the macromolecular anticancer agent SMANCS (16


kDa), as well as albumin, immunoglobulin (IgG) and
lipid, accumulated preferentially in tumor relative to
normal tissue (Figs. 2 and 3; see also Refs.
[19,20,29]). The small protein NCS (12 kDa) and the
small glycoprotein ovomucoid (29 kDa) did not do
so at all. SMANCS, which is itself a small protein
but which can bind non-covalently to albumin as

described, behaves like a large protein of about 80


kDa in vivo [34].
It is known that in normal inflammatory tissue,
proteins and lipids will leak out of the blood vessels
(at postcapillary venules) into the interstitial space,
be recovered via the lymphatics, and then return to
the general circulation via the thoracic duct [6]. In
tumor tissue, SMANCS and plasma albumin (or IgG)

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

175

Fig. 3. Tissue distribution and plasma concentration of SMANCS and NCS. (A) Tumor and tissue distribution 24 h after intravenous (i.v.)
injection of 51 Cr-labelled drugs in S-180 tumor bearing mice. About 1310 5 cpm / 0.2 ml was injected (see Refs. [20,56]). Note biological
activity of NCS is extremely short though radioactivity is detectable. Star marks on bars indicate these values are reduced to 1 / 10 of actual
values. (B) Intratumor accumulation of various antitumor agents. SMANCS exhibits a much higher and more prolonged tumor concentration
than mitomycin C (MMC) and NCS. All drugs were given as an i.v. bolus at 10 mg / kg to rabbits bearing VX-2 tumor in the liver. Assay
was by antibacterial activity (Micrococcus luteus) which was standardized by separate standard curves and experiments (from Ref. [16]). (C)
Plasma concentration of NCS and SMANCS in human after an i.v. bolus injection. The concentration was determined by radioimmunoassay
for NCS (modified after data in Ref. [65] for four patients) and immunological assay for SMANCS (unpublished Phase I study data,
Yamanouchi Pharmaceutical Co. Ltd., Tokyo).

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H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

can penetrate between the vascular endothelial cells,


move into the interstitial space and thus accumulate
in the tumor tissue [20]. Another important finding is
that these substances are much less likely to be
recovered via the lymphatic system, as observed in
normal tissue. Thus, these macromolecules and lipids
are retained in the tumor tissue more effectively.
This phenomenon is termed the enhanced permeability and retention (EPR) effect. A comparison
of SMANCS, NCS and mitomycin C is shown in
Fig. 3, in which a high concentration of SMANCS
was found in the VX2 tumor in the liver of rabbits.
SMANCS was retained for a longer period in the
solid tumor (the EPR effect). We have also shown
that proteins and lipids were not cleared from the
tumor for a long time, which indicates little operative
lymphatic drainage, in great contrast to normal tissue
[19,20,29,35,36,50].
During radiological detection of solid tumors by
using radioemitting gallium, the EPR effect appears
to be the prime mechanism in which gallium is
complexed with the iron-binding protein transferrin
(approximately 80 kDa) [37]. A large proportion of
transferrin is known to circulate as apoprotein in
blood, where gallium can be bound. The time course
required for good contrast in clinical radioscintigraphy is 4872 h, which is similar to the time period
required for notable accumulation of radiolabeled
albumin and IgG in the tumor tissue [20].

4.2. Factors involved in extravascular permeability


in tumor tissue
Vascular permeability means transport or leakage
of plasma proteins and other macromolecules and
lipids out of the circulatory lumen into either the
interstitial space or other compartments, such as
pleural or abdominal cavities. Thus, if this phenomenon can be utilized for drug delivery, the method
has great implications for macromolecular therapeutics. The enhanced extravascular permeability in
tumor tissue now appears to be affected by more
than two different vascular permeability factors.
These factors can affect normal as well as tumor
vessels and they are known to be produced in the
tumor compartment and affect extravascular leakage
of plasma components such as albumin. One factor,
discovered by Senger et al. [26], called tumor

vascular permeability factor has been described. It is


a protein of about 40 kDa and is produced by many
types of tumor cells. The other factors include
bradykinin moiety found by us (Maeda et al. [27]
and Matsumura et al. [28]): bradykinin and its
[hydroxypropyl 3 ]-bradykinin (Hyp 3 -bradykinin), in
which the third amino acid proline in bradykinin is
replaced by hydroxyproline. These bradykinins seem
to be actively produced by a serine protease cascade
[28]: plasminogen activatorplasminHageman
factor(pre)kallikreinhigh-molecular weight kininogenbradykinin (Fig. 4): Because bradykinin is
a potent permeability and pain-inducing factor, this
finding gives a logical explanation for tumor pathology and the above-described EPR effect (see Fig. 2).
Hyp 3 -bradykinin exhibits almost the same activity
and potency as bradykinin [27,38].
Kinin generation and degradation can be manipulated by various inhibitors, which can affect such
agents as kallikrein or kininases, respectively (see
Fig. 4) [28]. The kininases (I and II) are known to be
identical with carboxypeptidase N and angiotensinconverting enzyme, respectively. When mice bearing
ascitic tumors received intraperitoneal injection of
soybean trypsin inhibitor (SBTI, Kunitz type), ascites
formation was significantly suppressed; in this system kallikrein was effectively inhibited by SBTI and
thus little bradykinin was formed [28]. As a consequence, effective suppression of permeability in the
abdominal compartment resulted in less formation of
ascitic fluid. Thus, by suppressing kinin generation
upstream with SBTI in the abdominal compartment,
one can inhibit vascular leakage and thus ascitic fluid
accumulation [28].
When kininases and hence kinin degradation were
inhibited downstream by kininase inhibitors, the
local concentration of kinin is increased and permeability increases. Thus, use of kininase inhibitors
(angiotensin-converting enzyme inhibitors), which
are commonly used as antihypertensive drugs, would
potentially enhance drug delivery to the tumor
compartment in combination with macromolecular
therapeutics [28].
We recently found greatly elevated free bradykinin
contents in rodent and human ascitic fluid of gastric,
ovarian and other cancers [39,40]. High levels of
bradykinin have also been observed in the pleural
effusion of lung cancer patients. In these ascitic and

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

177

Fig. 4. Kinin-generating and -degrading cascade and points of inhibition by various inhibitors. CPNI, carboxypeptidase N inhibitor. ACEI,
angiotensin converting enzyme inhibitor (from Ref. [28]).

pleural fluids, the elevated level of Hyp 3 -bradykinin


compared with that of bradykinin was found to be
unique to cancer patients [39,40], which exists as the
Hyp 3 -form already in the zymogen (kininogen). It
should also be mentioned, however, that the Hyp 3 bradykinin level is also elevated in some non-malignant chronic diseases such as Crohns disease. The
ratio of elevated Hyp 3 -bradykinin to total of kinin
plus kininogen may be of diagnostic value [39,40].
Bradykinin can initiate prostaglandin synthesis via
activation of phospholipase A 2 [41,42]. Because
prostaglandin E 2 is known to enhance vascular
permeability, it is thus reasonable to assume that
prostaglandin E 2 may facilitate this process in tumor
tissue via bradykinin. Another factor, leukotriene, is
found in most inflammatory tissues, but its presence
needs to be confirmed in tumor tissue. Plateletderived growth factor and possibly other factors such
as tumor necrosis factor also facilitate tumor vascular
permeability. Serotonin is noticeably elevated only in
carcinoid syndrome; it is induced by bradykinin [43].
Greenbaum et al. [44] previously reported a vascular
enhancing factor named leukokinin, which was obtained by acidifying the tumor ascites to pH 4 it
was not otherwise detectable. The generation of
leukokinin can be suppressed by pepstatin, an inhibitor of pepsin and other acid proteases and accord-

ingly ascites formation was decreased by injecting


pepstatin. However, the exact chemical nature of
leukokinin remains to be elucidated.

4.3. Enhanced drug delivery to tumor tissue by


using angiotensin II-induced hypertension
It has been reported by Suzuki et al. [21] that
blood flow in normal blood vessels remains constant
even under elevated blood pressure which can be
induced by administration of angiotensin II, which
causes the diameter of the vascular lumen to decrease. In contrast, tumor blood vessels expand
passively under hypertensive conditions and thus
blood flow increases in parallel with the blood
pressure caused by angiotensin II. Thus, in the
angiotensin II-induced hypertensive state, drug delivery to the tumor can be increased, but to normal
tissues, it can be reduced including the bone marrow
and the kidney (see Section 4.4). Unresponsiveness
of the tumor blood vessels indicates the absence of
receptors for angiotensin II or contractive smooth
muscle. The latter possibility was supported by a
histological study of tumor blood vessels; namely,
this architectural defect of the vessels was reported
[22]. This was also demonstrated in carcinogeninduced colon cancer in rats [23].

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H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

In a preliminary clinical study initiated by the


Tohoku University Cancer Chemotherapy Group,
several advantages of angiotensin II-induced hypertensive chemotherapy compared with normotensive
chemotherapy were found as follows: a higher
response rate, more rapid onset of tumor size reduction, less toxicity and better host tolerance [66]. It
appears that this method of chemotherapy will yield
better results for macromolecular drugs because
these will not traverse freely through blood capillaries from the interstitial space of a tumor. As is
clear from the earlier discussion, the EPR effect of
macromolecular agents such as SMANCS will be
more prominent under the induced hypertensive state
than under normotensive conditions. Low-molecularweight antitumor agents will not show this differentiation due to free diffusion. Similar results are
emerging for other macromolecular anticancer
agents.

4.4. Decreased drug delivery to the bone marrow


by polymer conjugation
In general, in vivo toxicity of biocompatible
polymer conjugated anticancer drugs is less than that
of the original agents [4448]. This is the case with
the pyran copolymer-conjugated NCS (pyranNCS)
(23 kDa), namely, the LD 50 of this conjugate was
1.7-fold higher than that of NCS. Because the doselimiting toxicity of NCS is primarily marrow suppression as discussed earlier [3], the hematotoxicity
of the two drugs was compared [49]. Although both
pyranNCS and NCS showed the same cytotoxicity
against in vitro cultured cells, the in situ toxicity of
pyranNCS against bone marrow cells assayed in
vivo / in vitro was much lower (about 30%) than that
of NCS (Fig. 5). Thus, pyranNCS may accumulate
less in the bone marrow than does NCS. Furthermore, the distribution of pyranNCS was less in the
spleen compared with that of NCS. In addition,
pyranNCS accumulated very little in the liver. This
trend became more apparent under the hypertensive
condition induced by angiotensin II as discussed
above (unpublished results). The vascular permeability barrier in the brain is well known. A
permeability barrier may also exist for pyranNCS
which limits its access from the blood to the bone

Fig. 5. Dosesurvival curves for bone marrow stem cells, as


colony-forming unit in culture (CFU-C) in situ. Pyran copolymerconjugated NCS (pyranNCS) or NCS was injected intravenously
into normal A / Jax mice. After 24 h, bone marrow cells were
separated and CFU-C / femur values were determined on day 7.
Each experimental point is a mean; vertical bars indicate 6S.E.M.
(n55). (d), pyranNCS; (s), NCS (see details in Ref. [49]).

marrow and spleen, and also to a lesser extent to


other organs.1
We have reported that the LD 50 of SMANCS in
vivo was about 23 times of that of NCS [9,16],
which indicates reduced marrow toxicity because of
increased molecular weight. There is no detailed
study of SMANCS and bone marrow toxicity, so it is
difficult to compare SMANCS with pyranNCS in
this regard.
It was hypothesized above that the good EPR
effect and longer plasma t 1 / 2 of SMANCS are based
on its relatively large apparent molecular size, namely, if a 1:1 complex is formed its physical mass of 84
kDa which results from SMANCS (15.5 kDa) binding to serum albumin (68 kDa) non-covalently as
described [34]. Serum albumin binding may also
account for the high biocompatibility of SMANCS.

Editors note: Access from the blood capillaries to the interstitium in the brain is limited by a morphologically distinct
endothelium, the bloodbrain barrier. On the current evidence, it
is unlikely that any such physical barrier is responsible for the
restriction in permeability of the pyranNCS conjugate from the
blood to the bone marrow.

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

Thus, it will exhibit less bone marrow toxicity and


less general toxicity, because of limited vascular
permeability.

5. Oily formulation of SMANCS


There are at least two beneficial oily formulations:
one is a formulation of SMANCS in a lipid contrast
medium (Lipiodol) (SMANCS / Lipiodol) [29,35,
36,50], the other is an oily formulation with medium
chain triglyceride developed for oral administration
[51]. Lipiodol can be replaced by other edible
vegetable oils [50,51]. Lipiodol, a product of the
Laboratoire Guerbet, Paris, is the ethyl ester of
iodinated poppy seed oil, which contains about 37%
iodine (w / w). This agent is used for lymphography
because of selective recovery into the lymphatics and
hence detection by X-ray. SMANCS / Lipiodol can
usually be administered via the tumor-feeding arteries (e.g. the hepatic artery for either hepatoma or
pancreatic cancer; the bronchial artery for lung or
bronchial cancer) [29,35,36,52]; the renal artery for
renal cancer [35,53]; intracavitary (pleural or abdominal) administration is also used. Extremely
selective targeting efficiency of the drug can be
accomplished by this method (Table 2). This type of
targeting is by far the most efficient of any other
targeting method known including monoclonal antibody tagged with anticancer agents, for which the
tumor / blood (T / B) ratio of the drug is 210. The
T / B ratio of SMANCS / Lipiodol is greater than 2500
(see Table 2).
Oily formulations of SMANCS can also be prepared with other biocompatible lipids, as stated
above and they exhibit the same beneficial pharmacological properties as Lipiodol, although X-ray
systems cannot detect them [50]. We found that a
semisynthetic medium chain triglyceride is a potential candidate for such a formulation [50].
The oily formulations become quite stable against
heat or radiation [18]. Most of the hydrolytic enzymes, such as proteases and esterases, will probably
not degrade the polymer conjugates in the lipid
media because these enzymes require water molecules for hydrolysis which are not available in this
condition. In any event the arterial injection of
SMANCS or other anticancer agents such as oily or

Table 2
Accumulation of

14

179

C-iodinated fatty acid a

Organs and tissues

Tumor
Liver (adjacent)
Liver (remote)*
Small intestine
Lung
Kidney
Stomach
Heart
Large intestine
Spleen
Bladder
Brain
Muscle
Skin
Mesenteric lymph node
Cervical lymph node
Thymus
Serum
Plasma cells
Bone marrow
Urine (excreted)
Urine (vesical)
Bile

Radioactivity DPM / g
(310 3 )
15 min

3 days

1252.58
56 625
28.95
1.06
2.66
1.61
10.97
2.65
0.35
2.39
0.28
,0.1
,0.1
,0.1
0.15
0.22
0.22
0.58
0.86
,0.1

,0.1
70.91

130.94
17.02
6.89
4.44
2.02
2.57

1.72
1.06
3.28
1.31
0.38
0.46
1.42
2.21
1.61
0.93
1.03
1.57
2.97
1.14
1.06
1.78

Intrahepatic arterial dose: 0.3 ml. Tumor implanted in the liver


of rabbits. From Ref. [29] with permission. To and * from tumor.

Lipiodol formulations is becoming a new focus in


cancer therapy.
A possible future use for oily formulations is oral
drug administration, because of increased stability
against various hydrolytic enzymes and against acid
pH in the stomach and, most importantly, because of
an increased intestinal absorption rate [51]. We tested
an oily formulation for possible oral use (SMANCS
with medium chain triglyceride) and found that its
plasma concentration was about 11-fold higher than
SMANCS in phosphate-buffered saline in terms of
area under the concentration curve [51].2 Most
proteins and peptides are expected to be less likely to
reach the intestine in an active / intact form because

Editors note: 0.044% of the administered dose was detected in


the blood 5 h after oral administration of the SMANCS oily
formulation.

180

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

many digestive enzymes degrade polypeptides. However, even many substances of large molecular size
are known to be absorbed from the intestine although
much less effectively (see Ref. [51]). Lipids are
known to be absorbed effectively as particles
(chylomicrons) from the intestine. Thus the effective
intestinal uptake of lipids and their protection against
hydrolytic enzymes would encourage the development of oral formulations in the future.

6. Clinical outlook for SMANCS

6.1. SMANCS /Lipiodol


We initiated this clinical trial at Kumamoto University Medical School, Japan, in 1982 with patients
with primary hepatoma, for whom there was no
promising therapeutic method available. We have just
completed a multi-institutional Phase II study of
more than 200 cases in Japan in 1990 (unpublished
results). Our early pilot study for unresectable hepatoma showed an unprecedented response rate when
an adequate amount of the drug was administered.
Both the dose and the tumor image can be quantified
accurately and optimized easily by X-ray computed
tomography (CT) because of the visibility of
Lipiodol under X-ray. By this procedure, about 90%
of the patients showed an objective reduction both in
tumor size and in amount of marker protein for
hepatoma such as a-fetoprotein.
A CT scan is done before the drug is injected into
the patient. A second scan is then done 3 days after
arterial injection of SMANCS / Lipiodol in cases of
metastatic liver cancer or cancer of the kidney; in
case of primary liver cancer a CT scan should be
done 710 days after drug injection, and then at
intervals of every month or so. Usually, 0.25 mg (in
0.25 ml Lipiodol) SMANCS per cm 2 of the cut
surface area of tumor is required to cover more than
50% (grade III) or 100% (grade IV) of the tumor
image. The clinical dose of SMANCS / Lipiodol
(usually containing 1 mg of SMANCS per ml of
Lipiodol) for hepatoma ranged from 1 to 10 ml and
was more frequently 35 ml. For lung cancer, the
range was 0.52 ml, more frequently 1 ml per dose.
The prognosis of the patient receiving intra-arterial
SMANCS / Lipiodol depends on the severity of liver

cirrhosis and the spread of the tumor. For instance, if


the patient has no active liver cirrhosis and the tumor
has not spread to more than two segments of the
liver, he or she would most likely have a 90%
chance of survival for at least five years after
treatment. In contrast, very few patients live more
than 6 months with conventional therapy (Fig.
6A,D).3 These patients usually receive more than
four injections. Most tumors are reduced to less than
50% of their original size after 6 months or more.
The other advantage of arterial injection of
SMANCS / Lipiodol is extremely high sensitivity in
detecting tumors. Namely, tumors as small as 34
mm in diameter can be detected by CT scan or by
plain abdominal film because of the high electron
density of Lipiodol [19,29,35,36,5255]. Table 3
provides a comparison of diagnostic data: each
patient underwent the same diagnostic tests and the
number of tumor foci detected by each method were
compared. Because of the selectivity of Lipiodol
(SMANCS / Lipiodol) for tumor, the present X-ray
method is more sensitive than all others [54].
Renal cancer, for which the drug is given via the
renal artery, or metastatic liver cancer may require a
higher viscosity (Lipiodol Ultrafluid F) and higher
concentration (1.52.0 mg) of SMANCS per ml of
Lipiodol for a good tumor response.
For metastatic liver cancer, the interval between
the first arterial injection and the second is shorter
than a month; more frequent injections may be
needed. For primary liver cancer (hepatocellular
carcinoma), the initial interval between injections is
1 month; then intervals are prolonged to 1.5, 2 or 3
months or longer, depending on the retention of the
drug, which can easily be determined by CT scan.
Many patients need several injections over a period
of more than a year.

6.2. Side-effects
As shown in Table 4, a major side-effect of this
treatment, observed in almost 50% of the patients, is

Editors note: If a direct comparison is made between patients of


all groups as in Fig. 6A, 40% of patients undergoing SMANCS /
Lipiodol therapy are still alive after 3 years.

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

181

Fig. 6. Survival of patients with unresectable hepatocellular carcinoma (HCC) treated with intra-arterial administration of SMANCS /
Lipiodol. (A) Comparison of survival of patients after conventional therapy and after SMANCS / Lipiodol. (B) Survival of HCC patients
classified according to metastatic spread of tumor in the liver. (C,D) Survival of HCC patients according to the classification of tumor
metastasis and the extent of liver cirrhosis (child class A, B and C). All SMANCS treated patients are inoperable and / or in advanced stage.
Mets, metastasis (data: courtesy of Dr T. Konno and SMANCS Pilot Study Group, Kumamoto University).

Table 3
Numbers of detected primary tumor lesions and nodules by various diagnostic methods
Method

Angiography
CT, ordinary
Scintigraphy
Ultrasonography
CT, SMANCS / Lipiodol
No. of cases a
for above methods
a

Number of tumor lesions detected in hepatoma (multiple type)


Major
lesion

Daughter
nodules

Hepatoma,
massive type

17
15
14
18
23

5
0
0
0
31

.9
14
0
3
35

15

All patients underwent the same diagnostic procedures.

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

182

Table 4
Side-effects of arterial administration of SMANCS / Lipiodol
Effect

Change

No. (%) of
patients effected

Duration of effect

1. Fever

38398C

112 / 233 (48)

Transitory (310 days)

56 / 229 (24)

Transitory (,20 min)

2. Abdominal pain
3. Liver function
GOT a value

GPT a value

4. White blood
cell count
a

Elevated
Unchanged
Decreased
Elevated
Unchanged
Decreased
Increased
Unchanged
Decreased

52 / 204
130 / 204
21 / 204
36 / 204
143 / 204
25 / 204

(25)
(64)
(10)
(18)
(70)
(12)

66 / 159 (42)
65 / 159 (41)
28 / 159 (17)

Transitory

Transitory

Transitory

GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase.

low-grade fever, which usually lasts no more than a


few days and can easily be controlled by conventional antifever agents. In addition, dull abdominal pain
may occasionally occur shortly after injection into
the hepatic artery, which lasts no more than 20 min.
This pain may be associated with angiography, in
which contrast medium is used. Toxicity to the liver
or the kidney is minimal. A hematological effect is
not usually observed. Some deviations in Table 4 are
usually transitory. For injection into the bronchial
artery, care must be taken to avoid the flow of agents
into the spinal artery.

during a 3-week period. Slow intravenous doses of 4


mg / day resulted in a decrease in white blood cells
and / or platelets after 3 consecutive weeks, although
this effect is reversible.
It is conceivable that angiotensin II-induced hypertensive chemotherapy will be most effective for
this type of macromolecular drug because these
drugs are not subject to reverse flow out of the tumor
tissue and into the blood capillary and thus their
accumulation by the EPR effect is facilitated
(Maeda, H. and Li, J.Q., unpublished results).

6.3. Aqueous formulation of SMANCS

7. Conclusion

This formulation has been tested only on a pilot


study basis in our group for various human solid
tumors in such organs as the ovary, the esophagus,
the lung, the stomach and the adrenal gland and for
various brain tumors; it was found to be effective
against all these tumors. It does not seem to be
effective against malignant melanoma and metastatic
tumor in the bone. However, metastatic lung cancer
arising from primary kidney cancer showed an
effective response rate (.40%), according to the
Gunma University Medical School (Yamanaka and
Kobayashi, unpublished results).
Preliminary Phase I data showed that a daily dose
of 2 mg / 60 kg of body weight by slow intravenous
infusion did not produce any remarkable toxicity

Two directions in antitumor drug development are


the main concern of this review: polymer conjugates
and oily formulations. The former improves the
plasma t 1 / 2 , offers a higher accumulation in the
tumor tissue than in normal tissue (EPR effect),
solves immunological problems (prevents immunogenicity in certain cases), and adds a biological
response-modifying (BRM) effect. In addition, general bone marrow toxicity can be reduced. Use of the
oily formulation (SMANCS / Lipiodol) provides the
most selective targeting method, with a catheter and
can produce as much as 2500-fold higher drug
concentration in the tumor compared with the concentration in blood. Clinically, a definite response
with few side-effects is expected for many solid

H. Maeda / Advanced Drug Delivery Reviews 46 (2001) 169 185

tumors. Furthermore, diagnostic advantages are


great. All parameters thus favor these two directions
as a novel approach for cancer chemotherapy. There
is one common focal point: tumor vasculature, which
has different pathophysiological and pathohistological characteristics compared with the normal counterpart, as discussed in the text. For this reason,
macromolecular anticancer agents and lipid-modified
agents will gain more attention in the future. Small
water-soluble agents cannot accomplish this objective of selective tumor targeting in vivo so effectively.

Acknowledgements
I wish to thank Drs T. Konno, Y. Matsumura, J.
Takeshita, M. Ueda, T. Matsumoto and all my other
colleagues who were involved in the SMANCS
project and I also wish to extend my gratitude to
Judith Gandy and Yuko Tsuda who provided invaluable editorial / secretarial assistance.

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