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Vol. 298, Issue 2, 805-811, August 2001
with a Liver-Affinity
Polysaccharide Based on Metal Coordination in Mice
Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto, Japan (Y.S., F.M., T.C.); Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan (Y.T.); and Faculty of Medical Engineering, Suzuka University of Medical Science, Mie, Japan (Y.I.)
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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Frequent and high-dose i.v. injections of interferon-
(IFN-) have been used clinically to treat patients with viral
hepatitis despite various side effects. Because side effects are caused by the systemic effects of IFN-, the purpose of this study was to
target the drug specifically to the liver, thus reducing the adverse
events. A chelating residue, diethylenetriaminepentaacetic acid (DTPA),
was introduced to pullulan, a water-soluble polysaccharide with a high
affinity for the liver. Murine IFN- could be coordinately conjugated
with the DTPA-pullulan by simple mixing in an aqueous solution
containing zinc ion (Zn2+). Intravenous injection of the
IFN--DTPA-pullulan conjugate with Zn2+ coordination
enhanced liver induction of an antiviral enzyme, 2',5'-oligoadenylate
synthetase (2-5AS), to a greater extent than that by free IFN-,
although the 2-5AS levels in the liver depended on the mixing ratio of
the IFN-/DTPA residue of DTPA-pullulan/Zn2+. In
addition, the duration of the liver 2-5AS induction by the IFN--DTPA-pullulan conjugate with Zn2+ coordination was
longer than that by free IFN-. The liver targeting of IFN- by
DTPA-pullulan with Zn2+ coordination may be a promising IFN therapy.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Because interferon (IFN) does not have organ-specific affinity and its in vivo half-life is short, patients with viral hepatitis may not always achieve a therapeutic effect when it is injected as a bolus. One possible way to enhance the in vivo efficacy of IFN is to target it to the site where the therapeutic activity is desired. This targeting would allow us to decrease the dose necessary to achieve the desired effect, thereby reducing the side effects caused by frequent and high-dose injections.
Various drug carriers have been used to enhance liver targeting of
drugs. For example, it has been shown that following i.v. injection,
particulate carriers incorporating a drug are mainly captured by the
reticuloendothelial system in the liver, resulting in drug targeting of
the liver (Senior, 1987
; Juliano, 1988). More specific drug targeting
of the liver has been attempted by using asialoglycoprotein receptors
of liver cells (Ashwell and Harford, 1982; Duncan et al., 1983; Lu et
al., 1994; Hirabayashi et al., 1996). On the other hand, liver
targeting of drugs with positively charged, water-soluble polymers is
based on free extravasation of most water-soluble substances from the
vascular system of the liver as well as on negative charges on the
liver cell surface (Hashida and Takakura, 1994). Thus, polymers have
been used as the carrier to allow drugs to target to the liver based on
such anatomical and biochemical characteristics of the liver.
Pullulan, a linear, nonionic polysaccharide with a repeated unit of
maltotriose condensed through 
-1,6 linkage, has been used
extensively as a food additive and in the pharmaceutical industry.
Pullulan was found to accumulate in the liver at significantly higher
amounts than other water-soluble polymers (Yamaoka et al., 1993, 1994,
1995).
2',5'-Oligoadenylate synthetase (2-5AS) is one of the major
IFN-inducible intracellular enzymes, and it plays a critical role in
mediating antiviral and immunomodulating actions of IFN (Goodbourn et
al., 2000). Indeed, in the clinical field 2-5AS activity in the serum
is considered to be the most sensitive marker of the effectiveness of
exogenously administered IFN (Shindo et al., 1988; Moritz et al., 1992;
Giannelli et al., 1993). Moreover, it has been shown that IFN
administration potently enhances the 2-5AS production in mouse liver
(Asada-Kubota et al., 1998). Recently, we succeeded in inducing 2-5AS
in the mouse liver more efficiently than free IFN- through its
chemical conjugation with pullulan (Xi et al., 1996). This potent
induction of 2-5AS is due to the specific targeting of IFN to the liver
with pullulan. However, the chemical coupling involves a multistep,
complicated process that is poorly reproducible and loses a
considerable amount of the drug activity, making it difficult to use
IFN-polymer chemical conjugates clinically despite their high
pharmacologic efficacy.
In the present study, therefore, we took advantage of metal
coordination to bind IFN to pullulan instead of the chemical coupling. Metal coordination has been applied to metal chelate affinity chromatography for protein separation (Porath et al., 1975). It was
reported that Zn2+-chelating affinity
chromatography is used to separate IFN- with its biologic activity
maintained (Heine et al., 1981; Sulkowski, 1985). For metal chelation
in this study, we introduced DTPA residues to pullulan (DTPA-pullulan).
As expected, simple mixing of IFN- and DTPA-pullulan in an aqueous
solution containing Zn2+ ions resulted in
formation of an IFN--DTPA-pullulan conjugate with
Zn2+ coordination. After i.v. injection of the
conjugate in mice, the liver 2-5AS was induced more strongly than that
by free IFN-.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
An aqueous solution of recombinant murine IFN-
(mol. wt. = 18,000; 3 × 107 IU/mg of
protein) was kindly supplied by Toray Industries Inc. (Kamakura,
Japan). Pullulan (mol. wt. = 200,000) and DTPA anhydride were purchased
from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), and Dojindo
Laboratories (Kumamoto, Japan), respectively.
Preparation of DTPA-Pullulan. DTPA anhydride (662.0 mg) and 4-aminopyridine (22.6 mg) were added to 500 ml of dehydrated dimethyl sulfoxide containing 1 mg/ml pullulan. The reaction solution was agitated at 40°C for 24 h to introduce DTPA residues to the hydroxyl groups of pullulan, followed by dialysis against double distilled water for 2 days and freeze-drying to obtain DTPA-introduced pullulan (DTPA-pullulan). The amount of DTPA residues introduced was 1.38 µmol/mg of pullulan as measured by a conventional conductometric titration. Briefly, the conductivity of DTPA-pullulan was measured in 0.01 M NaOH aqueous solution to theoretically calculate the introduced DTPA amount considering the presence of four carboxyl groups for one DTPA residue.
Preparation of IFN--DTPA-Pullulan Conjugate with
Zn2+ Coordination.
The IFN- original solution was
diluted with double distilled water to a concentration of 2 × 105 IU/ml (104 IU/50 µl
or 1.85 × 10
11 mol/50 µl). The IFN-
solution (50 µl) was mixed with 40 µl of DTPA-pullulan aqueous
solution with various molar ratios of IFN-/DTPA residue: 50, 500, 5,000, and 50,000. Then, 10 µl of various concentrations of
ZnCl2 aqueous solution in 0.01 M HCl was added to
the mixed solutions of IFN- and DTPA-pullulan with resulting
IFN-/Zn2+ molar ratios of 0.5, 1, 5, 50, 100, 1,000, 5,000, and 50,000 (100 µl). From extra samples, an aliquot of
the solution (25 µl) was used for the following gel filtration
chromatography (GFC). Following addition of 1.5 µl of saline or 1.5 µl of various concentrations of NaOH aqueous solution ranging from
0.01 to 1.0 M to adjust at pH 7.0, the resulting solution (101.5 µl)
was left at 25°C for 15, 60, or 120 min under gentle agitation to
allow the IFN- to conjugate to the DTPA-pullulan with
Zn2+ coordination. Following dilution with 98.5 µl of saline (final volume: 200 µl), it was used for the animal experiments.
GFC for IFN--DTPA-Pullulan Conjugate with Zn2+
Coordination.
The mixed solution of IFN-, DTPA-pullulan, and
Zn2+ containing 105 IU/ml
IFN- (25 µl) was subjected to GFC system (Tosoh, Tokyo, Japan)
equipped with TSK G4000SWXL (7.8-mm i.d. × 300 mm; Tosoh) column at 40°C and at a flow rate of 1.0 ml/min in 0.05 M
phosphate-buffered solution containing 0.5 M NaCl (pH 7.0). The GFC
peak was fluorescently detected at the excitation and emission
wavelengths of 278 and 348 nm, respectively (FS-8020; Tosoh). As
controls, free IFN-, the DTPA-pullulan, Zn2+,
and the mixtures including two of these three components were used.
In Vivo Assessment of IFN Activity.
Specific, pathogen-free,
inbred, female BALB/c mice (three mice/group), aged 6 weeks, were used
in this study. All mice received an i.v. injection of free IFN-
(102-105 IU) or
IFN--DTPA-pullulan conjugate containing IFN-
(10-104 IU) with or without
Zn2+ chelation in a volume of 200 µl. The
injection dose of the conjugate was expressed on the basis of the dose
of IFN used for pullulan conjugation. The livers were removed from the
mice at 1, 2, 3, and 4 days after injection, frozen in liquid nitrogen
immediately after saline washing, and stored at 
85°C until assay to
detect 2-5AS. The lung and spleen were simultaneously obtained 1 day after injection. All animal experiments were conducted in accordance with the United States National Institutes of Health guidelines for the
care and use of experimental animals.
Western Blotting for Mouse 2-5AS.
After cervical
dislocation, the organs were removed and homogenized with modified
lysis buffer (10 mM HEPES-KOH, 50 mM KCl, 3 mM
Mg(OAc)2, 0.3 mM EDTA, 10% glycerol, 0.01%
NaN3, 0.5% Triton-X 100, 100 µM
phenylmethylsulfonyl fluoride, 7 mM 2-mercaptoethanol, pH 7.5). After
centrifugation at 20,800g for 5 min at 4°C, the supernatants were used as the organ samples. The protein content in the
samples was determined by UV absorption at a wavelength of 280 nm, and
then the concentration was adjusted to 40 µg/µl by the lysis
buffer. Then the samples were diluted to the concentration of 20 µg/µl by adding the same volume of sample buffer (2% SDS, 0.125 M
Tris-HCl, 20% v/v glycerol, 1% 2-mercaptoethanol, 0.004% bromophenol
blue). After boiling for 5 min, these tissue lysates (300 µg of
protein) were electrophoresed in sodium dodecyl sulfate on 12%
polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA) by semidry blotting. The
transfer buffer used was Tris (25 mM), glycine (190 mM), and methanol
(20%). Transfer was carried out at 15 V for 50 min. The membrane was
blocked in 5% nonfat dry milk in phosphate-buffered saline for 1 h at room temperature, incubated for 1.5 h at 37°C with a rat
monoclonal antibody to mouse 42-kDa 2-5AS (Sokawa et al., 1994;
Asada-Kubota et al., 1995, 1998), diluted 1:40 with the blocking
solution. The membrane was washed and incubated with peroxidase-conjugated goat anti-rat IgG (Zymed, San Francisco, CA),
diluted 1:10,000 with the blocking solution for 1.5 h at 37°C.
The membrane was then washed and incubated with enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 1 min, and then it was exposed to an X-ray
film for 30 to 60 min. After Western blot analysis, each lane
was quantified using densitometry and NIH Image software.
Data Analysis. Data are expressed as the mean ± S.E. of the three samples. Statistical analysis was first performed by a one-way analysis of variance. When significant F values were obtained, the Fisher's protected least-significant difference was performed to determine which means were significantly different from one another, with a two-tail p value of <0.05 considered significant.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Conjugation of IFN- to the DTPA-Pullulan with Zn2+
Coordination.
Figure 1 shows GFC
patterns of IFN- before and after mixing with the DTPA-pullulan in
the presence or absence of Zn2+. The molar ratio
of IFN-, the DTPA residue of DTPA-pullulan, and
Zn2+ was 1:500:5.
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was added, irrespective
of the presence or absence of Zn2+ mixing. When
the 25 µl of the solution containing over 106
IU/ml of free IFN- was used, a small peak at the retention time around 14 min was observed (data not shown). When IFN- was mixed with DTPA-pullulan and Zn2+ ions, a peak appeared
at a retention time around 6 min at 15 min after mixing, and thereafter
neither the retention time nor the area changed until 3 h after
mixing. There was also a peak at a similar retention time for the
mixture of IFN- and DTPA-pullulan solution without
Zn2+, although the peak area was smaller than
that of the IFN/DTPA-pullulan/Zn2+ mixture. No
peak was detected for the DTPA-pullulan or Zn2+
alone or the mixture of DTPA-pullulan and Zn2+.
Effect of Mixing Conditions of IFN--DTPA-Pullulan Conjugate with
Zn2+ Coordination on 2-5AS.
Figure
2 shows the effects of various mixing
conditions with IFN-, DTPA-pullulan, and Zn2+
on the liver levels of 2-5AS. The 42-kDa 2-5AS was induced by IFN
injection, although the 2-5AS level was higher with administration of
IFN--DTPA-pullulan conjugate than with free IFN-. The extent of
2-5AS induction depended on the IFN-/DTPA residue ratio, and was
maximum at the ratio of 1:500 or 1:5000 (Fig. 2A). When the IFN-/DTPA residue ratio was fixed at 1:500, a maximum level of the
conjugate-induced 2-5AS was observed at the
IFN-/Zn2+ ratio of 1:5 (Fig. 2B). Based on
these findings, the conjugate prepared at the IFN-/DTPA residue of
the DTPA-pullulan/Zn2+ ratio of 1:500:5 was used
for the following experiments.
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and DTPA-pullulan
around 4 to 5, the pH effect on the conjugate-induced 2-5AS level was
examined. The level of liver 2-5AS induced by the
Zn2+-coordinated IFN--DTPA-pullulan conjugate
with low pH (pH 4-5) was similar to that induced by the conjugate
neutralized by NaOH aqueous solution (data not shown). The
Zn2+-coordinated conjugation of IFN- and
DTPA-pullulan was performed using IFN- aqueous solution containing 1 mg/ml of human serum albumin. A comparison study with the conjugate
prepared by albumin-free IFN- revealed no difference in the
induction ability of liver 2-5AS between the two types of conjugates
(data not shown).
Figure 3 shows the effects of
duration of the mixing time of the IFN- and DTPA-pullulan with
Zn2+ on the induction of liver 2-5AS. Similar
induction levels were observed for all the conjugates prepared with
different mixing times. Thus, the mixing time was fixed at 15 min
thereafter unless otherwise indicated. When IFN--DTPA-pullulan
conjugate was left for different time periods at room temperature after
dilution with saline before injection, there was no difference in the
induction of liver 2-5AS at least for 1 h (data not shown).
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2-5AS Induction in Various Tissues by i.v. Injection of Free
IFN- and IFN--DTPA-Pullulan Conjugate with Zn2+
Coordination.
Figure 4 shows 2-5AS
levels in the lung, spleen, and liver of mice 1 day after i.v.
injection of free IFN- and IFN--DTPA-pullulan conjugate with
Zn2+ coordination. Free IFN- injection
significantly enhanced induction of 2-5AS in the three organs.
Intravenous injection of the IFN--DTPA-pullulan conjugate with
Zn2+ coordination enhanced the 2-5AS induction
more significantly than the same dose of free IFN- selectively in
the liver.
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Effects of Doses of Free IFN- and IFN--DTPA-Pullulan
Conjugate with Zn2+ Coordination on 2-5AS in the
Liver.
Figure 5 shows the effects of
the various doses of free IFN- and IFN--DTPA-pullulan conjugate
with Zn2+ coordination on 2-5AS in the liver.
Saline, DTPA-pullulan alone, the mixture of DTPA-pullulan and
Zn2+, or the Zn2+ aqueous
solution alone did not induce 2-5AS in the liver. The simple mixture of
IFN- with DTPA-pullulan in the absence of Zn2+
augmented the effect of free IFN- on the induction of liver 2-5AS,
although the extent was less than that by
Zn2+-coordinate conjugate. These findings
indicate that conjugation with the DTPA-pullulan through
Zn2+ chelation was essential to enhance the liver
2-5AS level by IFN- administration.
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Time Course of 2-5AS Induction after i.v. Injection of Free IFN-
and IFN--DTPA-Pullulan Conjugate with Zn2+
Coordination.
The 2-5AS levels were the highest at 1 day after
injection of either free IFN- or IFN--DTPA pullulan conjugate,
and decreased thereafter. However, the 2-5AS level induced by the
IFN--DTPA-pullulan conjugate with Zn2+
coordination was significantly higher than that by free IFN- for the
first 2 days (Fig. 6).
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The present study demonstrates clearly that conjugation of IFN-
with DTPA-pullulan induced the antiviral enzyme 2-5AS selectively in
the liver. In a previous report, it was shown that chemically conjugated pullulan-IFN- accumulated specifically in the liver (Xi
et al., 1996). Thus, although the tissue distribution of
IFN--DTPA-pullulan was not determined here, it appears reasonable to
speculate that Zn2+-mediated coordinate
conjugation of IFN- and DTPA-pullulan also achieves liver targeting
of IFN-, resulting in enhanced liver-specific induction of 2-5AS.
The GFC peak of IFN- shifted to a shorter retention time after
mixing with DTPA-pullulan and Zn2+. This peak
shift can be attributed to an increase in the apparent molecular size
of the IFN- molecule caused by conjugation with DTPA-pullulan. A
similar change in the IFN- peak in a mixture of IFN- and
DTPA-pullulan without Zn2+ may also be due to
metal coordination. It is conceivable that trace metal ions originally
present in the mixing solution allowed IFN- to couple to
DTPA-pullulan by way of metal coordination. However, this coordination
appeared so poor without Zn2+ that a significant
amount of IFN- may have been separated from the conjugate and
adsorbed to the GFC column (Heine et al., 1981). Consequently, the peak
was smaller than that of IFN--DTPA-pullulan conjugate with
Zn2+ coordination. The absence of a
chromatographic change after a 3-h conjugation time indicates that the
metal coordinate conjugate was formed as rapidly as 15 min after
mixing, followed by no further change in respect of molecular size of
the conjugate or its amount. The induction of liver 2-5AS was also not
influenced by the mixing time (Fig. 3).
Intravenous injection of IFN--DTPA-pullulan conjugate with
Zn2+ coordination was more efficient than
injection of free IFN-, with respect to the amount of IFN-
necessary to induce liver 2-5AS as well as to the duration of 2-5AS
induction. This appears to be due to the enhanced and prolonged liver
accumulation of IFN- by metal coordinate conjugation with
DTPA-pullulan. It is likely that the Zn2+
coordination bond enabled IFN- to conjugate to DTPA-pullulan firmly
enough to carry it to the liver without dissociation in the body.
Binding of an IFN- molecule to its cell surface receptor leads to
initiation of the subsequent intracellular response (Johnson et al.,
1994). Once an IFN- molecule in the free form binds to the receptor,
the intracellular response is initiated while it is internalized in the
cell to be metabolized (Kushnaryov et al., 1985, 1986). On the other
hand, the IFN- molecule conjugated to DTPA-pullulan is also bound to
the receptor, but the subsequent internalization and metabolization
might not occur because it is bound coordinately to pullulan. Thus, the
IFN- molecule might be released from the receptor without
internalization and might rebind to another receptor. It is possible
that such concentration and subsequent rebinding to liver cells may
have enabled IFN- to prolong the duration of 2-5AS induction.
Mixing with DTPA-pullulan in the absence of Zn2+
also enhanced induction of liver 2-5AS by IFN-, although the extent
was much lower than that by the coordinate conjugate. The GFC study
suggested that formation of a conjugate of IFN- and DTPA-pullulan
takes place even without the addition of Zn2+.
However, it is likely that the stability of the conjugation in the body
is so low without Zn2+ coordination that free
IFN- is easily released into the blood after i.v. injection. This
also indicates that the presence of Zn2+ is
indispensable for liver targeting of IFN- through conjugation of
IFN- with DTPA-pullulan. However, the targeting ability depended on
the molecular ratio of the Zn2+/DTPA residue of
the DTPA-pullulan in coordinate conjugation. When the ratio is small,
the amount of Zn2+ is too small to form a
conjugate of IFN- and DTPA-pullulan. On the other hand, a large
amount of Zn2+ might allow both IFN- and
DTPA-pullulan to form inter- and intramolecular aggregates, resulting
in reduced conjugation between IFN- and DTPA-pullulan molecules
(Litzinger et al., 1995). As a result, there would be an optimal
IFN/DTPA residue/Zn2+ ratio to form the
IFN--DTPA-pullulan conjugate for maximum induction of liver 2-5AS.
The lethal dose (LD) of ZnCl2 was reported to be
60 to 90 mg/kg when it was administered to rats intravenously (Bluner,
1950). The conjugate, including IFN- (104 IU)
prepared as the IFN-/DTPA residue of
DTPA-pullulan/Zn2+ (at a ratio of 1:500:5),
contains less than 2.0 × 105 mg
ZnCl2. Thus, the dose of
ZnCl2 administered in this study was much lower
than the LD.
The LD50 value of pullulan is >14.3 g/kg
following oral administration to mice, and pullulan has no bacterial
mutagenicity (Kimoto et al., 1997). Because the amount of pullulan
injected is much lower than the LD50, the
pullulan content seems to be sufficiently low so as not to cause any
complications. However, since the route of administration was different
from that in our study, the side effects of pullulan must be checked
carefully before clinical application. Toxicologic and immunologic
studies are currently under way.
The present coordination method may be practical for clinical IFN
pharmaceuticals that contain human serum albumin as a stabilizer. Indeed, Zn2+-coordinated conjugate was formed in
this study using an aqueous solution of IFN- containing human serum albumin.
Recently, polyethylene glycol-conjugated IFN- (PEG-IFN-) has been
reported to prolong the half-life of IFN- in the blood (Takacs et
al., 1999; Lukaszewski and Brooks, 2000). However, PEG-IFN- does not
appear to actively target IFN- to the liver. Therefore,
IFN--DTPA-pullulan conjugate may be superior to PEG-IFN- in terms
of its specific targeting of the liver.
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Acknowledgments |
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We are grateful to Dr. Yoshihiro Sokawa, Kyoto Institute of Technology, Kyoto, Japan, for helpful advice on Western blotting experiments and supply of antibody to mouse 2-5AS.
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Footnotes |
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Accepted for publication April 12, 2001.
Received for publication December 27, 2000.
This work was supported by a grant of Research for the Future Program from the Japan Society for the Promotion of Science (JSPS-RFTF 97I0201).
Address correspondence to: Tsutomu Chiba, M.D., Ph.D., Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, 54 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507. E-mail: cteya{at}kuhp.kyoto-u.ac.jp
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Abbreviations |
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IFN, interferon; 2-5AS, 2',5'-oligoadenylate synthetase; DTPA, diethylenetriaminepentaacetic acid; GFC, gel filtration chromatography; LD, lethal dose; PEG, polyethylene glycol.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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receptor-mediated endocytosis and nuclear membrane binding.
Proc Natl Acad Sci USA
82:
3281-3285
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, saline; 
, free IFN-
,
IFN-
