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Clinical Cancer Research Vol. 10, 2545-2550, April 2004
© 2004 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Design of a pH-Sensitive Polymeric Carrier for Drug Release and Its Application in Cancer Therapy

Haruhiko Kamada, Yasuo Tsutsumi, Yasuo Yoshioka, Yoko Yamamoto, Hiroshi Kodaira, Shin-ichi Tsunoda, Takayuki Okamoto, Yohei Mukai, Hiroko Shibata, Shinsaku Nakagawa and Tadanori Mayumi

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: In this study, to optimize the polymeric drug delivery system for cancer chemotherapy, we developed a new pH-sensitive polymeric carrier, poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD], that could gradually release native form of drugs with full activity, from the conjugates in response to changes in pH. We examined the usefulness of PVD as a polymeric drug carrier.

Experimental Design: PVD was radically synthesized with vinylpyrrolidone and 2,3-dimethylmaleic anhydride, which is known to be a pH-reversible amino-protecting reagent. Conjugates between PVD and other drugs, such as Adriamycin (ADR), were prepared under the slightly basic conditions (pH 8.5). The drug-release pattern and the antitumor activity of PVD were examined.

Results: At pH 8.5, the release of the drugs from the conjugate was not observed. In contrast, PVD could release fully active drugs in the native form in response to the change in pH near neutrality, and gradually released drugs at neutral pH (7.0) and slightly acidic pH (6.0). The drug-release pattern in serum was almost similar to that observed during these physiological conditions. The PVD-conjugated ADR showed superior antitumor activity against sarcoma-180 solid tumor in mice, and it had less toxic side effects than free ADR. This enhancement in the antitumor therapeutic window may be due to not only the improvement of plasma half-lives and tumor accumulation of ADR, but also its controlled and sustained release from the conjugates in vivo.

Conclusions: These results indicate that PVD is an effective polymeric carrier for optimizing cancer therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major limitation of antitumor agents, typified by Adriamycin (ADR, doxorubicin), used in clinical applications, is its severe toxicity, such as bone marrow suppression and cardiotoxicity (1, 2, 3, 4) . This is caused by the high and frequent dose of antitumor agents, which have a very short half-life and a wide tissue distribution. The chemical conjugation of antitumor agents with water-soluble polymeric carriers has been found recently to overcome these drawbacks. The conjugation of low molecular weight antitumor agents to water-soluble polymeric carriers, such as N-(2-hydroxypropyl)methylacrylamide, divinylether-co-maleic anhydride, styrene-co-maleic anhydride, dextran, and polyethylene glycol (PEG), offers a potential mechanism to improve cancer chemotherapy (5, 6, 7, 8, 9, 10) . Distribution of the conjugates, which have a higher molecular weight, is usually restricted to the intravascular space after i.v. injection due to the low permeability in most organs with a continuous capillary bed. In contrast, these macromolecular conjugates preferentially accumulate in solid tumors due to the enhanced permeability and retention effect (11 , 12) . As a result, the polymeric drug delivery system (DDS) may selectively expand the therapeutic windows of antitumor agents.

However, there is a restriction on the clinical application of this polymeric DDS for cancer chemotherapy. For instance, after the ADR that is taken up into the tumor cells intercalates between double strands of DNA, its antitumor activity is induced by inhibition of DNA replication and topoisomerase activity in the tumor cells (13) . However, the intercalation of polymer-conjugated ADR between double strands of DNA is based on macromolecular interactions, which are sterically hindered by the attached polymeric carrier. The introduction of polymeric carriers to antitumor agents, including ADR, is generally targeted at ionic functional groups in the antitumor agent, some of which may play an important role for their cytotoxic activity. Additionally, the conjugates with larger molecular size are hardly taken up into tumor cells through various transporters, because these transporters carry low molecular weight antitumor agents. Thus, for obtaining in vivo antitumor effects, a sufficient amount of antitumor agents is required to be released from the conjugates, because polymer-conjugated anticancer drugs themselves seldom show antitumor activity. However, in most cases, the conjugate between an antitumor agent and a polymeric carrier is formed through stable covalent bonding. As a result, the antitumor therapeutic effects of these conjugates have often not been observed in their clinical trials. To overcome these problems, a relatively unstable linker was used for the conjugation between an antitumor agent and a polymeric carrier. Most of the antitumor agents released from the conjugates have a linker fragment. Furthermore, these modified antitumor agents show much lower specific activities than original antitumor agents in their native form, because the linker fragment is attached to an active functional group of the antitumor agents (14 , 15) . Thus, it is necessary to develop a novel polymeric DDS for optimization of cancer chemotherapy.

Dimethylmaleic anhydride (DMMAn) with a double bond in its structure is used as a pH-reversible protective reagent of amino groups in proteins and chemical compounds (16 , 17) . DMMAn binds to an amino group by forming an amide bond through its acid anhydride group over pH 8, and then reversibly dissociates from the amino group for change in pH to near slightly acidic from neutral. Thus, if a polymeric carrier with this function of DMMAn is synthesized, it will release a native drug in response to changes in pH. It is known that pH of both tumor and inflammatory tissues are slightly acidic unlike normal tissues (18) . Thus, the conjugates between an antitumor agent and the polymeric modifier with the function of DMMAn may show superior plasma half-life and tumor accumulation compared with unconjugated antitumor agents and, therefore, effectively release the antitumor agents in native form in the tumor tissues.

In this study, to optimize the polymeric DDS for cancer chemotherapy, poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD] was radically synthesized with vinylpyrrolidone and DMMAn. ADR was used as a model antitumor agent and conjugated with PVD along with other drugs. Fully active drugs were gradually released from the conjugates in response to the change in pH. The PVD-conjugated ADR showed superior antitumor activity against S-180 sarcomas in mice and had less side effects than free ADR. These results indicate that PVD is an effective polymeric carrier for cancer therapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
DMMAn was purchased from AKROS (Aichi, Japan). Other reagents and solvents were obtained from standard sources.

Synthesis of PVD.
PVD was synthesized by the radical polymerization method using 4,4'-azobis-4-cyanovaleric acid as a radical initiator. Briefly, DMMAn and vinylpyrrolidone were mixed in a ratio of 1:20 in a glass tube containing dimethyl formamide and incubated at 60°C for 6 h. The resulting copolymer was precipitated in dry diethyl ether, collected immediately after filtration, and dried under vacuum three times. The molecular weight was determined by gel-filtration chromatography (GFC; TSKgel G4000PW, TSKgel {alpha}-3000 columns; Tosoh, Tokyo, Japan). Polyvinylpyrrolidone (PVP) was also similarly synthesized. These polymers were separated into several fractions by GFC to obtain polymers with a narrow molecular weight distribution.

Conjugation of Lucifer Yellow Cadaverine (LYC) with PVD.
An amino group of water-soluble fluorophore LYC (1.27 mg/300 µl of borate buffer; pH 8.5) was conjugated with PVD (6 mg) by forming an amide bond through its acid anhydride group. After the conjugation reaction for 30 min, the unconjugated free LYC was removed using 10-PG columns (Bio-Rad, Hercules, CA). PVD-modified LYC (PVD-LYC) was separated and purified by GFC. The release of free LYC from the PVD-LYC was not observed at pH 8.5. The solution of PVD-LYC was prepared to desired pH values and incubated at 37°C. Samples collected after various incubation times, went through desalting columns, and were analyzed for ratios between conjugated LYC and free LYC by measuring fluorescence intensity at 530 nm (emission) with excitation at 424 nm. The release of LYC from the conjugate in the serum was also measured by the same method after mixing it with an equal volume of mouse serum.

Toxicity of PVD.
Monkey renal tubular (LLC-MK2) cells were seeded in 96-well plates at a concentration of 1.2 x 104 cells/well. After incubation for 24 h at 37°C, LLC-MK2 cells were incubated with PVD, PVP, PEG, and Polybrene at different concentrations. The plates were incubated for 24 h at 37°C, and cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Dojindo, Kumamoto, Japan) assay as described by Mosmann (19) with minor modifications. Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg/ml; 10 µl) was added into each well, and the cells were incubated at 37°C for 4 h. The resulting formazan product was dissolved by the addition of 100 µl of 10% SDS (Wako Pure Chemical Ind. Co., Ltd., Osaka, Japan) and 15 mM HCl. The solution was read on a microplate reader at 595 nm test wavelength with a reference wavelength of 655 nm.

Pharmacokinetic Analysis.
PVD and PVP were dissolved in dimethyl formamide and activated for 24 h at room temperature with dicyclohexyl carbodiimide and N-hydroxysuccinimide, respectively. The polymers were incubated with thyramine hydrochloride for 24 h at 4°C, dialyzed in water, and lyophilized. Polymer-thyramine conjugates were radiolabeled by the chloramine-T method. 125I-labeled polymer was purified by GFC. The clearance profiles of PVD and PVP (10 µg/mouse; 1 x 106 cpm/200 µl in saline) were studied after i.v. injection into the tail veins of ddY mice. Blood was collected from the tail vein at intervals, and radioactivity was measured. GFC analysis confirmed that >95% of the radioactivity in circulating blood 3 h after i.v. injection was derived from intact 125I-PVD and 125I-PVP. Mice were housed in metabolic cages to collect urine and sacrificed 3 h after treatment to evaluate tissue distribution.

Conjugation of ADR with PVD.
PVD was dissolved in N-methyl-2-pyrrolidone at a concentration of 80 mg/4 ml. Then ADR (20 mg) was added to this solution. After the addition of triethylaminde (30 ml), the mixture was additionally incubated overnight at room temperature. After the reaction, distilled water was added, and pH of the mixture was adjusted to 8.5 by the addition of 0.2 M NaH2PO4. Unconjugated ADR was removed by ultrafiltration using a PM-30 (Amicon). The PVD-modified ADR (PVD-ADR) was then purified by dialysis. The amount of conjugated ADR was estimated by measuring its absorbance at 340 nm.

In Vivo Antitumor Activity of PVD-ADR.
In vivo antitumor effects of PVD-ADR were assessed by using ddY mice bearing mouse sarcoma-180 solid tumors. PVD-ADR and free ADR were injected i.v. to the mice at different doses, 60, 200, and 600 µg/mouse (ADR-equivalent dose) on days 7, 9, and 11 after tumor inoculation. The tumor volume was measured by a standard method as described elsewhere (20 , 21) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chemical Structure of PVD.
For the radical synthesis of PVD, DMMAn was introduced at a ratio of 5% to the amount of vinylpyrrolidone. The average molecular weight of PVD was Mr 6,000 [Polydispersity (molecular weight/the number average molecular weight) = 1.14]. PVD was synthesized by radical copolymerization. Reflecting the properties of DMMAn, drugs were conjugated with PVD by forming an amide bond through its acid anhydride group of DMMAn to amino groups of drugs at more than pH 8.0 and released at lower than pH 7.0 (Fig. 1)Citation .



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  		Fig. 1. Chemical structures and characteristics of poly(vinylpyrrolidone-co-dimethylmaleic anhydride).

 
The pH-Sensitive Drug (LYC) Release from PVD-Conjugated LYC.
The release of LYC as a model drug, which is a fluorophore with an amino group, from PVD-LYC was assessed. The release of LYC from PVD-LYC was time dependent, and the release speed increased with an increase in the pH (Fig. 2)Citation . At pH 8.5, a weak basic condition, almost no release or <1% release of LYC was observed. However, ~6% of the total LYC was released at pH 6.0 after 24 h incubation. Additionally, ~3% of the LYC was gradually released at pH 7.0 after 24 h of incubation. When PVD-LYC was incubated in the serum, results similar to the LYC release pattern at pH 6.0 were obtained.



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  		Fig. 2. pH-sensitive controlled release of lucifer yellow cadaverine (LYC) from the conjugates between poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD] and LYC. The release of LYC from the PVD-conjugated LYC (PVD-LYC) was assessed at indicated time intervals. Small molecule fractions collected after desalting gel-filtration chromatography were assigned as free LYC released from the macromolecular conjugates of PVD-LYC. The concentration of free LYC was measured by fluorescence intensity at 530 nm (excitation, 424 nm). The amounts of free LYC, relative to that of initiate PVD-conjugated LYC, are shown on the vertical axis. {blacktriangleup}, pH 6.0; {blacksquare}, pH 7.0; •, pH 8.5; {diamondsuit}: serum.

 
Cytotoxicity of PVD.
The in vitro cytotoxicity of PVD was assessed for clarifying its usefulness as a polymeric drug carrier. PVP and PEG had no effect on LLC-MK2 cells, whereas the cationic polymer, Polybrene, which is known to be an antitumorigenic agent, showed considerable cytotoxicity (Fig. 3)Citation . In contrast, PVD was not cytotoxic at concentrations up to 3 mg/ml. Evidently, PVD as well as PVP and PEG had no cytotoxic effect on human endothelial cells (data not shown).



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  		Fig. 3. Cytotoxicity of various polymeric carriers against LLC-MK2 cells. LLC-MK2 cells were incubated with various polymeric carriers for 24 h. Viability of the cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Each value is mean (n = 3); bars, ± SD. {blacktriangleup}, polyethylene glycol; {triangleup}, polyvinylpyrrolidone; •, Polybrene; {circ}, poly(vinylpyrrolidone-co-dimethylmaleic anhydride).

 
Pharmacokinetics of PVD after i.v. Administration.
The pharmacokinetics of PVD and PVP after i.v. administration were studied for 3 h (Fig. 4)Citation . PVP was separated and purified by GFC to adjust the molecular weight and polydispersity of PVD. PVD was effectively retained in blood as compared with PVP, although PVP was found previously to have the longest plasma half-life and to be the most suitable polymeric drug carrier for localizing the conjugated drug in blood. The half-lives of PVD and PVP were ~10 min and 30 min, respectively.



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  		Fig. 4. Blood residency of polyvinylpyrrolidone (PVP) and poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD] after their i.v. administration. 125I-labeled PVP and 125I-labeled PVD were i.v. administered to normal ddY mice, and the radioactivity in blood was measured. The curved line was drawn by using the least-squares method based on measured values at indicated time intervals. Mice were used in groups of 4. Each value is mean; bars, ± SE. {circ}, PVP; •, PVD.

 
In Vivo Antitumor Effect of PVD-ADR.
To clarify the usefulness of PVD as a polymeric drug carrier for optimization of cancer chemotherapy, we synthesized PVD-ADR and compared its antitumor potency with that of ADR alone (Fig. 5Citation and 6Citation , Table 1Citation ). Through GFC analysis, we confirmed that PVD-ADR was composed of one PVD molecule and one ADR molecule. As shown in Fig. 6Citation , all of the mice administered with free ADR at a dose of 600 µg/mouse and 200 µg/mouse died within 6–10 days after their i.v. administration (within 13 days or 17 days after tumor inoculation) because of toxicity of ADR. There was a marked weight loss in these mice after the high dose of free ADR (data not shown). In mice treated with free ADR at a dose of 60 µg/mouse, although tumor growth was slightly inhibited without causing sudden death or weight loss (Figs. 5Citation and 6)Citation , complete tumor regression, defined as disappearance of tumor without regrowth within 100 days, was not observed (Table 1)Citation . Similar results were also observed in mice administered with a mixture of PVD and free ADR due to side effects (data not shown). In contrast, the antitumor activity of PVD-ADR at a dose of 60 µg ADR/mouse was more effective than that of free ADR at 60 µg/mouse (Fig. 5)Citation . Tumor growth was remarkably and completely inhibited by PVD-ADR at a dose of 200 µg ADR/mouse and 600 µg ADR/mouse (Fig. 5)Citation . Complete tumor regression was observed in 75%, 25%, and 25% of mice treated with PVD-ADR at a dose of 600 µg ADR/mouse, 200 µg ADR/mouse, and 60 µg ADR/mouse, respectively (Table 1)Citation . During the experimental period, all doses of PVD-ADR were well tolerated, and no loss in body weight was observed (Fig. 6)Citation .



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  		Fig. 5. Antitumor effects of Adriamycin (ADR) and poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD]-ADR on sarcoma-180 solid tumors. The ADR and PVD-ADR were i.v. administered to sarcoma-180 solid tumor-bearing mice on days 7, 9, and 11, after tumor inoculation. Data were expressed as relative tumor volume by the following equation: Relative tumor volume = mean tumor volume at a given time/mean tumor volume on day 7. Mice were used in groups of 4. Each value is mean; bars, ± SE. Intact mice, {diamondsuit}. Open symbol, free ADR-treated mice. {circ}, 600 µg/mouse/day; {triangleup}, 200 µg/mouse/day; {square}, 60 µg/mouse/day. Closed symbol, PVD-ADR-treated mice. •, 600 µg/mouse/day; {blacktriangleup}, 200 µg/mouse/day; {blacksquare}, 60 µg/mouse/day.

 


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  		Fig. 6. Survival time of the sarcoma-180 tumor-bearing mice treated with Adriamycin (ADR) and poly(vinylpyrrolidone-co-dimethylmaleic anhydride) [PVD]-ADR. The ADR and PVD-ADR were i.v. administered to sarcoma-180 solid tumor-bearing mice on days 7, 9, and 11, after tumor inoculation. Mice were used in groups of 4. Survival time of treated-mice after i.v. administration of ADR and PVD-ADR were shown as the survival rate (%). Open symbol, free ADR. {circ}, 600 µg/mouse/day; {triangleup}, 200 µg/mouse/day; {square}, 60 µg/mouse/day. Closed symbol, PVD-ADR. •, 600 µg/mouse/day; {blacktriangleup}, 200 µg/mouse/day; {blacksquare}, 60 µg/mouse/day.

 

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  		Table 1 Antitumor effects of ADRa and PVD-ADR on sarcoma-180 solid tumors

Complete regression was defined when tumor was not regrown for >100 days.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, to optimize the polymeric DDS for cancer chemotherapy, we attempted to develop a novel polymeric carrier that could release a native form of drugs in response to changes in pH near the neutrality. As described in the "Introduction," there are certain characteristics needed by the polymeric drug carrier: (a) to be excellent in blood residency for effectively obtaining the enhanced permeability and retention effect in tumors; (b) to gradually release the fully active form (native form) of antitumor agents; and (c) to efficiently release the native antitumor agents under the slightly acidic conditions, if possible, because it is known that the pH of tumor tissues is slightly lower than that of normal tissues (18) . From such a viewpoint, some polymeric carriers, typified by divinylether-co-maleic anhydride and styrene-co-maleic anhydride, were developed (6 , 7) . Some maleic anhydride, that is one of the acid anhydride, were contained in the structure of these polymeric carriers, and the antitumor agents were conjugated with these polymeric carriers via the formation of amide bonds between the amino group of antitumor agents and the acid anhydride groups. However, the amide bonds formed through maleic anhydride are very stable near neutral pH, and the antitumor agents are released from the conjugates under strong acidic conditions (<pH 3). As a result, the antitumor therapeutic effects of these conjugates have often not been observed in their clinical trials. In contrast, DMMAn binds to an amino group by forming an amide bond through its acid anhydride group over pH 8, and then reversibly dissociates from the amino group on change in pH to slightly acidic from neutral. However, there are no reports about DMMAn-introduced carrier.

For enhancing the blood residency of drugs, PVP was found to be one of the most useful polymeric carriers, because the plasma half-life of PVP itself was much longer than those of PEG and other polymeric modifiers (21 , 22) . In fact, PVP-conjugated tumor necrosis factor (TNF)-{alpha} showed a higher half-life than PEG-conjugated TNF-{alpha} despite having the same molecular size (21) . As a result, PVP-conjugated TNF-{alpha} had a more potent antitumor effect than PEG-conjugated TNF-{alpha}, without any toxic side effects. This phenomenon has also been observed in PVP-conjugated interleukin 6 and leukemia inhibitory factor (22) . Furthermore, PVP can be introduced in various useful functional groups by radical copolymerization (23) . Additionally, the safety of PVP has been clinically confirmed. Using PVP as a backbone polymer, we synthesized PVD as a novel polymeric carrier for the development of antitumor polymeric DDS. Reflecting the property of DMMAn, the conjugate between PVD and drugs (LYC) could gradually release the native drugs directly (nonlinker) by responding to a change in pH near neutrality (Fig. 2)Citation . As shown in Fig. 3Citation , the safety of PVD appears to be similar to PEG and PVP, which are used clinically. Additionally, the plasma half-life of PVD was longer than that of parental PVP (Fig. 4)Citation . These results strongly suggested that PVD may be useful as a polymeric drug carrier for cancer chemotherapy.

We show here that the conjugation of ADR with PVD increases its antitumor activity while decreasing its nonspecific toxicity (Figs. 5Citation and 6Citation ; Table 1Citation ). Overall, the therapeutic window is markedly increased. These results have important clinical implications for the use of antitumor chemotherapeutic agents in patients. The expansion of the therapeutic window is probably due to the following reasons. It is known that vascular permeability of macromolecules into solid tumors and its retention in tumor tissues are enhanced compared with normal tissues. This is generally called the enhanced permeability and retention effect (11 , 12) . Thus, this enhanced permeability and retention effect may effectively accumulate the PVD-ADR in tumors. Additionally, as it is known that pH of tumor tissues is slightly lower than that of normal tissues, the PVD-ADR is likely to release free ADR more efficiently in tumor tissues (18) . But detailed studies on the pharmacokinetics of PVD-ADR are necessary to clarify the mechanism of its wider therapeutic window, and these are currently under investigation.

To improve the therapeutic bioavailability of bioactive proteins, bioactive proteins have been conjugated with water-soluble polymers such as PEG (24) . PEGylation of proteins increases their molecular size and enhances steric hindrance, both of which are dependent on PEG attached to the protein. This results in the improvement of the plasma half-lives of proteins and stability against proteolytic cleavage as well as a decrease in its immunogenicity. We also reported that PEGylation of proteins, such as TNF-{alpha}, interleukin 6, and immunotoxin, could enhance therapeutic potency and could reduce undesirable side effects (21, 22, 23 , 25, 26, 27) . However, there is a restriction to this approach, because PEGylation is frequently accompanied with a significant loss of specific activity of a protein. Lysine amino groups of proteins are often used for PEGylation because they are highly reactive. This PEGylation, however, is nonspecific and occurs at the NH2 terminus as well as at all of the internal lysine residues in proteins, some of which may be in or near their active site. Resultant PEGylated proteins are heterogeneous and show significantly lower bioactivity. These problems remarkably limit the clinical application of PEGylated proteins. The present study shows that PVD effectively releases fully active drugs in the native form at neutral pH ranges and is a safe drug carrier that has no cytotoxicity. Additionally, PVD may be a suitable polymeric carrier for the prolongation of plasma half-life of drugs as well as PVP, rather than PEG. Thus, we are now attempting to design the PVD-conjugated cytokines for promotion of protein therapies against solid and metastatic tumors.

In conclusion, we developed a new polymeric carrier, PVD, that could gradually release native drugs from the conjugate in response to changes in pH near neutrality. The PVD-ADR showed superior antitumor activity and had less side effects than free ADR. This enhancement of the antitumor therapeutic window may be due to not only the improvement of plasma half-lives and tumor accumulation of ADR, but also its controlled and sustained release from the conjugates in vivo. These results indicate that PVD is an effective polymeric carrier for cancer therapy.


   FOOTNOTES
 
Grant support: Grant-in-Aid for Scientific Research (No. 15680014) from the Ministry of Education, Science and Culture of Japan, Health Sciences Research Grants for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation (KH63124), and Takeda Science Foundation.

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.

Note: H. Kamada, Y. Tsutsumi, and Y. Yoshioka contributed equally to the work.

Requests for reprints: Yasuo Tsutsumi, Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-0-6-6879-8178; Fax: 81-0-6-6879-8178; E-mail: tsutsumi{at}phs.osaka-u.ac.jp

Received 11/ 7/03; revised 12/25/03; accepted 12/31/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Praga C, Beretta G, Labianca R. Cardiac toxicity from antitumor therapy. Oncology (Basel), 37: 51-8, 1980.
  2. Wadler S, Fuks JZ, Wiernik PH. Phase I and II agents in cancer therapy: I. anthracyclines and related compounds. J Clin Pharmacol, 26: 491-509, 1986.[Abstract]
  3. Bristow MR, Billingham ME, Mason JW, Daniels JR. Clinical spectrum of anthracycline antibiotic cardiotoxicity. Cancer Treat Rep, 62: 873-9, 1978.[Medline]
  4. Sparano JA. Doxorubicin/taxane combinations: cardiac toxicity and pharmacokinetics. Semin Oncol, 26: 14-9, 1999.[Medline]
  5. Seymour LW, Ulbrich K, Steyger PS, et al Tumour tropism and anti-cancer efficacy of polymer-based doxorubicin prodrugs in the treatment of subcutaneous murine B16F10 melanoma. Br J Cancer, 70: 636-41, 1994.[Medline]
  6. Yamamoto H, Miki T, Oda T, et al Reduced bone marrow toxicity of neocarzinostatin by conjugation with divinyl ether-maleic acid copolymer. Eur J Cancer, 26: 253-60, 1990.
  7. Maeda H. SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy. Adv Drug Deliv Rev, 46: 169-85, 2001.[CrossRef][Medline]
  8. Senter PD, Svensson HP, Schreiber GJ, Rodriguez JL, Vrudhula VM. Poly(ethylene glycol)-doxorubicin conjugates containing ß-lactamase-sensitive linkers. Bioconjug Chem, 6: 389-94, 1995.[Medline]
  9. Matsumoto S, Yamamoto A, Takakura Y, Hashida M, Tanigawa N, Sezaki H. Cellular interaction and in vitro antitumor activity of mitomycin C-dextran conjugate. Cancer Res, 46: 4463-8, 1986.[Abstract/Free Full Text]
  10. Kabanov AV, Okano T. Challenges in polymer therapeutics: state of the art and prospects of polymer drugs. Adv Exp Med Biol, 519: 1-27, 2003.[Medline]
  11. Maeda H, Sawa T, Konno T. Mechanism of tumor-targeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS. J Control Release, 74: 47-61, 2001.[CrossRef][Medline]
  12. Kopecek J, Kopeckova P, Minko T, Lu ZR, Peterson CM. Water soluble polymers in tumor targeted delivery. J Control Release, 74: 147-58, 2001.[CrossRef][Medline]
  13. Denny WA. DNA-intercalating ligands as anti-cancer drugs: prospects for future design. Anticancer Drug Des, 4: 241-63, 1989.[Medline]
  14. Duncan R, Spreafico F. Polymer conjugates. Pharmacokinetic considerations for design and development. Clin Pharmacokinet, 27: 290-306, 1994.[Medline]
  15. Duncan R, Seymour LW, O’Hare KB, et al Preclinical evaluation of polymer-bound doxorubicin. J Control Release, 19: 331-46, 1992.[CrossRef]
  16. Butler PJ, Harris JI, Hartley BS, Leberman R. Reversible blocking of peptide amino groups by maleic anhydride. Biochem J, 103: 78-9, 1967.
  17. Butler PJ, Harris JI, Hartley BS, Lebeman R. The use of maleic anhydride for the reversible blocking of amino groups in polypeptide chains. Biochem J, 112: 679-89, 1969.[Medline]
  18. Wike-Hooley JL, Haveman J, Reinhold HS. The relevance of tumour pH to the treatment of malignant disease. Radiother Oncol, 2: 343-66, 1984.[Medline]
  19. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65: 55-63, 1983.[CrossRef][Medline]
  20. Haranaka K, Satomi N, Sakurai A. Antitumor activity of murine tumor necrosis factor (TNF) against transplanted murine tumors and heterotransplanted human tumors in nude mice. Int J Cancer, 34: 263-7, 1984.[Medline]
  21. Kamada H, Tsutsumi Y, Yamamoto Y, et al Antitumor activity of tumor necrosis factor-{alpha} conjugated with polyvinylpyrrolidone on solid tumors in mice. Cancer Res, 60: 6416-20, 2000.[Abstract/Free Full Text]
  22. Tsunoda S, Kamada H, Yamamoto Y, et al Molecular design of polyvinylpyrrolidone-conjugated interleukin-6 for enhancement of in vivo thrombopoietic activity in mice. J Control Release, 68: 335-41, 2000.[Medline]
  23. Kamada H, Tsutsumi Y, Sato-Kamada K, et al Synthesis of a poly(vinylpyrrolidone-co-dimethyl maleic anhydride) co-polymer and its application for renal drug targeting. Nat Biotechnol, 21: 399-404, 2003.[CrossRef][Medline]
  24. Youngster S, Wang YS, Grace M, Bausch J, Bordens R, Wyss DF. Structure, biology, and therapeutic implications of pegylated interferon {alpha}-2b. Curr Pharm Des, 8: 2139-57, 2002.[CrossRef][Medline]
  25. Tsunoda S, Ishikawa T, Watanabe M, et al Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol, 112: 181-8, 2001.[Medline]
  26. Tsutsumi Y, Onda M, Nagata S, Lee B, Kreitman RJ, Pastan I. Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) improves antitumor activity and reduces animal toxicity and immunogenicity. Proc Natl Acad Sci USA, 97: 8548-53, 2000.[Abstract/Free Full Text]
  27. Yamamoto Y, Tsutsumi Y, Yoshioka Y, et al Site-specific PEGylation of a lysine-deficient TNF-{alpha} with full bioactivity. Nat Biotechnol, 21: 546-52, 2003.[CrossRef][Medline]




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