.JPG?ad=18503&adview=true)
[Cancer Research 60, 6416-6420, November 15, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
Antitumor Activity of Tumor Necrosis Factor-
Conjugated with Polyvinylpyrrolidone on Solid Tumors in Mice1
Haruhiko Kamada,
Yasuo Tsutsumi,
Yoko Yamamoto,
Tetsunari Kihira,
Yoshihisa Kaneda,
Yu Mu,
Hiroshi Kodaira,
Shin-ichi Tsunoda,
Shinsaku Nakagawa and
Tadanori Mayumi2
Faculty of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 [H. Ka., Y. T., Y. Y., T. K., Y. K., Y. M., H. Ko., S. N., T. M.] and National Institute of Bioscience and Human Technology, Ibaraki 305-8566 [S-i. T.], Japan
 |
ABSTRACT
|
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We attempted the development of a novel polymer conjugation to
further improve the therapeutic potency of antitumor cytokines compared
with PEGylation for clinical application. Compared with native tumor
necrosis factor (TNF)-
in vitro, specific
bioactivities of polyvinyl-pyrrolidone (PVP)-modified TNF-s
(PVP-TNF-s) were decreased by increasing the degree of PVP
attachment. PVP-TNF- fraction 3, Mr
101,000, had the most effective antitumor activity of the
various PVP-TNF-s in vivo.
PVP-TNF- fraction 3 had >200-fold higher antitumor effect
than native TNF-, and the antitumor activity of PVP-TNF- fraction
3 was >2-fold higher than that of MPEG-TNF-
(Mr 108,000), which had the highest
antitumor activity among the polyethylene glycol (PEG)-conjugated
TNF-s. Additionally, a high dose of native TNF- induced toxic
side effects such as body weight reduction, piloerection, and tissue
inflammation, whereas no side effects were observed after i.v.
administration of PVP-TNF- fraction 3. The plasma half-life of
PVP-TNF- fraction 3 (360 min) was about 80- and 3-fold longer than
those of native TNF- (4.6 min) and MPEG-TNF- (122 min),
respectively. The mechanism of increased antitumor effect in
vivo caused the prolongation of plasma half-life and increase
in stability. These results suggested that PVP is a useful polymeric
modifier for bioconjugation of TNF- to increase its antitumor
potency, and multifunctionally bioconjugated TNF- may be a
potentiated antitumor agent for clinical use.
|
INTRODUCTION
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TNF3
- was originally identified as an endotoxin-induced factor able to
induce hemorrhagic necrosis of tumors in mice (1)
. The
antitumor effect of TNF- is known to result not only from its direct
cytotoxicity against tumor cells but also from activation of antitumor
effector immune cells in the blood, such as macrophages, cytotoxic
lymphocytes, and neutrophils (2
, 3)
, and furthermore from
specific damage to tumor blood vessels (4, 5, 6)
. Clinical
applications of TNF- have been attempted as novel antineoplastic
agents to tumors instead of traditional antitumor drugs (7
, 8)
. However, because of its very low stability and pleiotropic
action in vivo, attempts to use TNF- as a systemic
anticancer agent in humans failed because of the appearance of severe
systemic side effects, such as a fever and decreased blood pressure the
same as an endotoxin-like shock, before therapeutic doses could be
reached (9
, 10)
. Accordingly, clinical applications of
TNF- for cancer therapy are still limited despite high expectations
(11
, 12)
.
In general, it is difficult to use bioactive proteins, such as
cytokines, for therapeutic use because of poor stability and short
half-lives. These proteins are rapidly cleared from blood by the liver,
kidney, and others organs, and the rate of clearance depends on
the size of molecule and degree of proteolysis. Plasma proteases cause
degradation and rapid loss of biological activity; therefore, achieving
a clinical effect is still a problem. To overcome these problems,
soluble-polymer technology (i.e., bioconjugation) attempts
to bind polymers for protection against degradation by the host
(13)
. Bioconjugation, forming a stable linkage between the
protein and polymer, can prolong the half-life and preserve biological
activity. Therefore, therapeutically useful proteins would be modified
with various polymers to improve their pharmacokinetic properties and
may acquire many advantages after polymer conjugation
(14)
.
Generally, the more polymer chains attached per protein molecule,
the greater the extension of half-life, but specific activity may be
proportionally reduced (15)
. The increased plasma
half-lives of proteins are attributable to several mechanisms,
increased size of protein conjugates surpasses the limit for glomerular
filtration (Mr 70,000) and decreased
proteolysis because of polymer steric hindrance. Previously, we
conjugated TNF- with PEG to increase stability in vivo
and selectively obtain antitumor activity for clinical use
(16, 17, 18)
. Optimal PEGylated TNF- effectively increased
the protease resistance and circulation time by sterically blocking
proteolysis and decreasing clearance via glomerular filtration
(19)
. These effects were attributed to increases in steric
hindrance and in molecular weight of bioconjugated proteins, both of
which were attributable to attachment of polymers. As a result,
optimally bioconjugated cytokines showed much better therapeutic
effects than native (unconjugated) cytokines. However, to design
conjugated cytokines with greater efficacy and safety, i.e.,
to selectively enhance desirable therapeutic activities of cytokines
without increasing their side effects, it is necessary to closely
regulate their behavioral characteristics in vivo, taking
into account their mechanism of action.
Thus, it is necessary to develop polymeric modifiers with useful
DDS functions, which can regulate their behavioral characteristics
in vivo, such as targeting capability and controlled
release. The molecular structure of PEG as a general polymeric modifier
does not readily allow the addition of such new functions, and other
polymeric modifiers to which these new functions can be added are
required. Recently, we evaluated the behavior of various water-soluble
polymers, and PVP, to which useful functional groups can be introduced
by radical copolymerization, was found to be retained in the blood
better than
PEG.4
PVP, which was widely used as a plasma expander until clinical studies
revealed a lack of efficacy, is a water-soluble polymer available in
molecular weights ranging from a few thousand to several hundred
thousand. Therefore, PVP may be a better polymeric modifier to increase
the half life of cytokines by conjugation.
In this study, we conjugated TNF- with PVP (PVP-TNF-) to increase
its half-life and selectively increase its therapeutic potency as a
systemic antitumor agent. The results of this study will provide
fundamental information enabling us to design useful bioactive protein
derivatives showing selective localization in the vascular space
against tumors and various cardiovascular diseases and to develop
better polymeric modifiers with DDS functions to enhance the
therapeutic activity and safety of conjugated bioactive proteins.
|
MATERIALS AND METHODS
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Materials.
Natural human TNF- (1.78 x 106
JRU/mg TNF- in 0.2 M phosphate buffer, pH 7.2) was
kindly supplied by Hayashibara Biological Laboratories (Okayama,
Japan). N-Vinyl-2-pyrrolidone was purchased from Wako
(Osaka, Japan), 4–4'-azobis-4-cyanovaleric acid was from Aldrich
(Milwaukee, WI), and 3-mercaptopropionic acid was from Dojindo
Laboratories (Kumamoto, Japan). Other reagents and solvents were of
analytical grade.
Synthesis and Activation of PVP.
The terminal COOH-bearing PVP was synthesized from
N-vinyl-2-pyrrolidone (27 mmol) by radical polymerization in
dimethyl formamide (7 ml) with the aid of 4,4'-azobis-(4-cyanovaleric
acid) (1.2 mmol) as a radical initiator, and 3-mercaptopropionic acid
(2.7 mmol) as a chain transfer agent at 60°C for 6 h. Resultant
PVPs with an average molecular weight of
Mr 6,000 [polydispersity
(Mr/average
Mr), 1.14] were separated and
purified by high-performance liquid chromatography (GF-HPLC; TSK Gel
G4000PwxL; Toso Co., Ltd., Tokyo, Japan). The terminal COOH group of
synthetic PVP was activated by the
N-hydroxysuccinimide/dicyclohexyl carbodiimide method.
Conjugation of TNF- with Activated PVP.
TNF- was reacted with a 60-fold molar excess of activated PVP
at room temperature for 10 min, and then 
-amino caploic acid (5-fold
molar excess against activated PVP) was added to stop the reaction. The
resultant PVP-TNF- was separated into five fractions of various
molecular weights by gel filtration chromatography (TSK Gel G3000SwxL;
Tosoh Co., Ltd.). The average Mr of
PVP-TNF-s were estimated by gel filtration chromatography analysis,
and degree of PVP-attachment to TNF- was calculated from the average
Mr (protein standard; Gel Filtration
LMW Calibration kit; Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom). The specific bioactivities in vitro of
PVP-TNF-s were measured by L-M cell cytotoxicity assay, according to
the method described by Yamazaki et al. (20)
and were expressed in terms of JRU defined previously
(20)
.
Screening and Evaluation of Antitumor Effects in
Vivo.
S-180 cells (5 x 105 cells) were
implanted intradermally in the abdomen of male ddY mice (5 weeks of
age; SLC, Hamamatsu, Japan). After 7 days (tumor size, 8–9 mm in
diameter), native TNF- and PVP-TNF-s were administered in a
single i.v. injection. Antitumor effects of PVP-TNF-s in
vivo were screened by determining mean tumor volume calculated
from the formula described by Haranaka et al.
(21)
. The in vivo antitumor potencies of native
TNF-, MPEG-TNF-, in which 56% of the TNF- lysine amino groups
were conjugated with PEG, and PVP-TNF- fraction 3 in which 40% of
TNF- lysine amino groups were conjugated with PVP, were evaluated as
follows. Meth-A fibrosarcoma cells (4 x 105 cells) were inoculated intradermally into the
abdomen of 5-week-old female BALB/c mice (SLC). Seven days later (tumor
size, 7–8 mm in diameter), native TNF-, MPEG-TNF-, and
PVP-TNF- fraction 3 were i.v. administered by a single injection.
Drug efficacy against Meth-A tumors was expressed by a score of tumor
hemorrhagic necrosis according to the method of Carswell et
al. (1)
. Briefly, the maximal necrotic response
(score 3) indicates that 
50% of the tumor mass is necrotic, the
moderate response (score 2) indicates 25–50% necrotic, the minimal
response (score 1) indicates <25% necrotic, and no response (score 0)
indicates no visible necrosis.
Assessment of Plasma Clearance.
Native TNF-, MPEG-TNF-, and PVP-TNF-s were radiolabeled with
125I by the lactoperoxidase method, yielding
125I-native TNF-,
125I-MPEG-TNF-, and
125I-PVP-TNF-s with specific activities of
4.44 µCi/mg protein. The biological activities of
125I-radiolabeled TNF- and its derivatives
were indistinguishable from those of nonradiolabeled TNF- and its
derivatives (data not shown). Their pharmacokinetic profiles in blood
circulation after i.v. injection into female BALB/c mice (5 weeks of
age) were studied at a dose of 31.6 ng of protein/mouse. Blood was
collected from the tail vein at various time points, and radioactivity
was measured in each sample.
Statistical Analysis.
The hemorrhagic necrosis scores and tumor volume were statistically
evaluated by the Student t test.
|
RESULTS
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Preparation of PVP-TNF-s.
Natural human TNF- was conjugated with activated PVP (average
Mr 6000;
Mr/average
Mr, 1.14) via amide bonds between
amino groups of TNF- and N-hydroxyscuccinimide groups of
PVP at the end of the main chain. The rate of PVP attachment to TNF-
increased with increasing reaction time and molar mass of PVP to
TNF- (data not shown). The resulting TNF--conjugated PVPs were
purified from native TNF- and separated into five fractions of
various molecular sizes by gel filtration-high performance liquid
chromatography (protein standard). Table 1
shows the average Mr, degree of PVP
modification of these separated PVP-TNF-s, and their activities
compared with that of native TNF-. The activities of PVP-TNF-s
decreased with increasing of molecular weight and degree of PVP
modification (PVP attachment to TNF-). This result was also observed
when TNF- was conjugated with PEG (Fig. 1)
, and this profile of changes in the bioactivity of PVP-TNF-s was
similar to that observed with modification of TNF- with PEG (average
Mr, 5000;
Mr/average
Mr, 1.32) reported previously
(17)
.
Differing Antitumor Activities of Various PVP-TNF-s in
Vivo.
The antitumor effects of PVP-TNF-s on S-180 solid tumors were
compared with those of native TNF- and MPEG-TNF- by single i.v.
injection (Fig. 2)
. The antitumor effects were evaluated by a score of hemorrhagic
necrosis 24 h after sample administration. Native TNF-
inhibited tumor growth dose dependently, but marked side effects
(e.g., sudden death, transient decrease in body weight, and
others) were observed in all mice administered native TNF- at a dose
of 10,000 JRU. Therefore, 10,000 JRU of TNF- was found to be the
maximal applicable dose. All PVP-TNF-s were injected i.v. at a dose
of 1,000 JRU. PVP-TNF- fraction 1 (average
Mr, 134,000) and fraction 5 (average
Mr, 74,000) slightly observed tumor
necrosis. PVP-TNF- fraction 2 (average
Mr, 117,000) and fraction 4 (average
Mr, 84,000) had showed antitumor
effects comparable with that of native TNF- at a dose of 10,000 JRU.
The tumor growth- inhibitory effect of PVP-TNF- fraction 3 (average
Mr, 101,000), in which 40% of the
total lysine amino groups of TNF- were coupled with PVP, was
markedly higher than that of native TNF- at a dose of 10,000
JRU/mouse and induced complete regression in two of seven mice (data
not shown). No side effect was observed, such as decrease in body
weight, in any mice administered PVP-TNF-s.
Antitumor Effect of PVP-TNF- Fraction 3 in Vivo.
PVP-TNF- fraction 3 showed the best characteristics among the
PVP-TNF-s examined. To clarify the usefulness of PVP as a polymeric
modifier and PVP-TNF- fraction 3 as a systemic antitumor agent, we
compared the antitumor potency of PVP-TNF- fraction 3 to those of
native TNF- and MPEG-TNF- with scheduled i.v. injections on
Meth-A solid tumors. Control mice (saline or PVP alone) showed no
antitumor effect and hemorrhagic necrosis, whereas native TNF-
showed suppression of tumor growth in a dose-dependent manner (Fig. 3)
. However, three of eight mice administered native TNF- at a dose of
10,000 JRU died within 24 h, and the remaining mice developed
piloerection and tissue inflammation (e.g., erythema) and
showed a decrease in body weight (data not shown). This dose of native
TNF- completely inhibited tumor growth up to day 28 after tumor
inoculation, but tumor growth was observed in one of eight mice after
that. PVP-TNF- fraction 3 and MPEG-TNF- had markedly increased
their antitumor potencies compared with native TNF-. As shown in
Table 2
, PVP-TNF- fraction 3 and MPEG-TNF- at a dose of 200 JRU showed
the maximal antitumor effects without any toxic side effects (such as
sudden death and others) and had antitumor effects superior to that of
native TNF- at a dose of 10,000 JRU. On the other hand, only 50 JRU
of PVP-TNF- fraction 3 was needed to exhibit a marked antitumor
potency, and tumor growth was completely inhibited for the observation
period, as in 10,000 JRU native TNF- and 100 JRU MPEG-TNF-. These
results indicated that PVP-TNF- fraction 3 was approximately 200-
and 2-fold more potent an antitumor agent than native TNF- and
MPEG-TNF-, respectively.
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Table 2 Antitumor effect of PVP-TNF- by scheduled
administrationa on survival days
after Meth-A tumor inoculation
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Pharmacokinetics of PVP-TNF-s.
The pharmacokinetics of native TNF-, MPEG-TNF-, and
PVP-TNF-s were examined (Fig. 4)
. Native TNF- rapidly disappeared from the circulation, and
its plasma half-life was only 3.0 min. This half-life corresponds with
that reported previously. This rapid clearance of native TNF- was
found to be attributable to proteolysis, renal excretion, and broad
distribution to various tissues (data not shown). In contrast, the
plasma clearance of PVP-TNF- fraction 3 was markedly decreased
relative to those of native and MPEG-TNF-, and its plasma half-life
(365 min) was about 80- and 3-fold longer than those of native TNF-
and MPEG-TNF- (123 min), respectively.
|
DISCUSSION
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The distribution of TNF- from blood into the adversely
affected tissue, such as the liver, causes toxic side effects (8
, 22)
. The improved retention of TNF- in the vascular
space and the resultant decrease in transfer of TNF- to these
tissues is expected to reduce the side effects of TNF- therapy.
Thus, the improvement in circulation time may selectively enhance its
antitumor action without increasing its side effects, resulting in
augmentation of its bioavailability. To assess the usefulness of PVP as
a polymeric modifier for protein conjugation, we evaluated the
therapeutically potency of PVP-TNF-s compared with that of PEG
conjugate (PEG-TNF-) in vivo. The results, shown in Table 1
, indicated that the bioactivity in vitro was decreased
with increasing the degree of modification. These findings suggested
two hypotheses: (a) PVP chain TNF- sterically inhibited
TNF-receptor binding of PVP- TNF-s; and (b) lysine amino
residues of TNF- play an important role in its bioactivity. We
assumed that decreased activity in vitro was caused by
steric hindrance, inhibiting the binding with TNF receptor. In fact,
lysine 11 of TNF- is known to fulfill a structural role
(23)
. PVP was reacted with -amino groups; therefore, if
this lysine 11 has been conjugated with the polymer, the bioactivity of
TNF- was almost diminished or vanished. Although this lysine has not
been conjugated, if the polymers conjugate in the neighborhood of
lysine 11, it is assumed the activity of bioconjugated TNF- is
reduced because of the steric hindrance.
In Fig. 2
, the antitumor activities in vivo were
evaluated by hemorrhagic necrosis between fractionated PVP-TNF-s.
The results showed that PVP-TNF- fraction 3,
Mr 101,000, had the highest antitumor
activity in vivo. MPEG-TNF-, which showed the highest
antitumor activity of the PEG-bioconjugated TNF-, has a
Mr 108,000. This finding suggests that
the optimal molecular weight of bioconjugated TNF- is
Mr 
100,000 to increase the
bioactivity in vivo. However, the optimal molecular weight
was limited in this case to that of conjugated TNF- with PEG or PVP.
Our previous study showed that DIVEMA-TNF- (24
, 25)
,
which has a Mr 63,000, showed the most
marked antitumor activity. DIVEMA, which is known as a biological
response modifier (26
, 27)
, has many reactive anhydride
residues that form amide bonds with TNF-. PEG or PVP are reacted
only at the end point of the main chain. It is assumed that the optimal
bioconjugated conditions would exist because of the selected polymeric
modifiers.
In Fig. 3
, to investigate the antitumor effect of PVP-TNF- fraction
3, which showed the highest effects in more detail, the comparison
between native- and MPEG-TNF-s was determined by scheduled
administration. The results showed that the antitumor effect was almost
same for doses of 10,000 JRU native TNF- and dosage of 50 JRU of
PVP-TNF- fraction 3; thus, the antitumor effect increased 200-fold
over the native TNF-. Complete regression was observed in only half
the mice by administration of native TNF- at 10,000 JRU, whereas
administration of PVP-TNF- fraction 3 only 200 JRU inhibited tumor
growth completely. In the case of native TNF- administration, the
maximal dose was most effective but caused side effects
(e.g., sudden death and others.), whereas the
minimal dose was not effective for tumor growth inhibition. However,
this problem did not arise with PVP-TNF-s; therefore, we are
confident that PVP-TNF-s, especially PVP-TNF- fraction 3, will be
beneficial for cancer therapy. The reduction in administered dose was
possible by increasing the stability of TNF- in vivo and
eliminating the high initial dose that caused side effects. This
increase in circulation time of PVP-TNF- fraction 3 was found to be
caused by an increase in stability attributable to the shielding of
proteolytic cleavage sites of TNF- by the PVP chain and reduction of
the renal clearance attributable to increased molecular size through
attached PVP. PVP has no toxicity when given i.v. administration at
this dose and has not shown antitumor effects on tumor-bearing mice.
Additionally, after i.v. administration of PVP, experience confirms
that polymers with a Mr <20,000 are
completely eliminated through the kidneys (28)
.
Therefore, it is assumed that the increasing antitumor effect of
PVP-TNF- fraction 3 is not caused by direct action of PVP.
PVP-TNF- fraction 3 was mainly localized in the vascular space. In
general, it has been well known that macromolecules are accumulated and
retained in the tumor tissue effectively. This phenomenon is termed the
"enhanced permeability and retention" effect (29)
, and
many macromolecular anticancer agents, such as synthetic
polymer-conjugating drugs and polymeric micelle-containing drugs and
others, have been reported (30
, 31)
. We have reported
previously that the tumor distribution of MPEG-TNF- was markedly
enhanced compared with native TNF- and gradually increased over time
(19)
. About 9-fold more MPEG-TNF- was distributed to
the tumor than native TNF-. Thus, we come to the conclusion that the
marked increase in the antitumor potency of MPEG-TNF- resulted from
the enhanced tumor accumulation. PVP-TNF- fraction 3
(Mr 101,000) has the same molecular
size as MPEG-TNF- (Mr 108,000).
Thus, it is assumed that the concentration PVP-TNF- fraction 3 would
be higher than that of native TNF- in tumor site.
As described in the introduction, antitumor effects of TNF- were
attributable not only to direct cytotoxicity against tumor cells but
also to specific injury of the tumor vascular and effective activation
of antitumor immune cells. TNF- selectively enhanced the vascular
permeability of tumor vessels. The enhancement of TNF- half-time may
lead to a decrease in its distribution to the liver and spleen, which
are the major sites of side effects, and would selectively increase its
antitumor effects. Therefore, in Fig. 4
we are confident that the
increased antitumor potency of PVP-TNF- fraction 3 relative to
native TNF- and MPEG-TNF- may be attributed to increased
half-life. It is important to determine why the circulation time of
PVP-TNF- fraction 3 was much longer than that of MPEG-TNF-,
although their molecular sizes were almost the same. We found
previously that the mean residence time in blood of PVP6,000 is about
seven times longer than that of PEG5,000. Thus, we assumed that the
behavior of these conjugated TNF-s in vivo was affected
by the behavioral characteristics of attached polymeric
modifiers.
In this study, we showed that the conjugation of TNF- to PVP
selectively increases its antitumor effects, and PVP-TNF- fraction 3
may be useful as a more potent antitumor therapeutic agent than
PEGylated TNF-. In addition, we demonstrated that PVP is a more
useful polymeric modifier than PEG. These findings indicated that the
conjugation of TNF- with PVP effectively increased its antitumor
potency without adverse effects. We are currently synthesizing various
PVP derivatives by radical copolymerization. PEG has been used as a
polymeric modifier, but it is difficult to introduce functions, such as
targeting capability and controlled release, into PEG. Therefore, we
examined other candidates as polymeric modifiers for introduction of
these new functions. PVP can be easily introduced various reactive
residues by radical copolymerization. Furthermore, PVP, used as a
suspending agent, binder disintegrant, and tablet lubricant for
preparation of various medicines (32)
, is a highly
biocompatible, amphiphatic, nontoxic, and nonimmunogenic polymer
similar to PEG. In addition, we found that the plasma half-life of PVP
itself was longer than that of PEG after i.v. injection.
In the process of hemorrhagic necrosis in tumor vessels (33
, 34) , the vascular permeability is selectively increased
(35
, 36) , promoting transport from blood to tumor tissue
(37)
. We are attempting to introduce functions into PVP
such as immunopotential action of polydivinylether-maleic anhydride
(DIVEMA) and targeting capability to specific tissues for obtaining
useful TNF- derivatives. We developed a novel polymeric modifier
that can be used to introduce some useful DDS functions for controlling
the behavior of conjugated bioactive proteins in vivo to
further increase their therapeutic activities and safety by isolation
of desirable activities for clinical use.
|
FOOTNOTES
|
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by a grant from the Uehara
Memorial Foundation, in part by Grants-in-Aid for Cancer Research and
for Scientific Research from the Ministry of Education, Science, Sports
and Culture of Japan on Priority Areas (Cancer/Biotarget), for
Scientific Research and for Encouragement of Young Scientists, and in
part by Health Sciences Research Grants for Research on Health Sciences
focusing on Drug Innovation from the Ministry of Health and Welfare.
This study supported in part by Cancer Research Fellowships of the
Japan Society for the promotion of Science for Young
Scientists. 
2 To whom requests for reprints should be
addressed, at Faculty and Graduate School of Pharmaceutical Sciences,
Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone:
81-6-6879-8175; Fax: 81-6-6879-8179; E-mail: mayumi{at}phs.osaka-u.ac.jp
3 The abbreviations used are: TNF, tumor necrosis
factor; PVP, polyvinylpyrrolidone; PEG, polyethylene glycol; MPEG,
middle fraction of PEG; DIVEMA, divinyl ether and maleic anhydride
copolymer; JRU, Japan reference unit(s); IL, interleukin; DDS, drug
delivery system.
4 Unpublished data.
Received 12/13/99.
Accepted 9/19/00.
|
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ABSTRACT
INTRODUCTION
