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
170
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
170
171
172
172
173
173
173
173
176
177
178
179
180
180
180
182
182
183
183
171
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.
172
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
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]
12
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].
173
174
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]).
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).
176
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]).
178
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.
Table 2
Accumulation of
14
179
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
180
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.
6.2. Side-effects
As shown in Table 4, a major side-effect of this
treatment, observed in almost 50% of the patients, is
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
Daughter
nodules
Hepatoma,
massive type
17
15
14
18
23
5
0
0
0
31
.9
14
0
3
35
15
182
Table 4
Side-effects of arterial administration of SMANCS / Lipiodol
Effect
Change
No. (%) of
patients effected
Duration of effect
1. Fever
38398C
56 / 229 (24)
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
7. Conclusion
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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185