POLYMER CARRIERS FOR TARGETED DRUG DELIVERY AND
CONTROLLED DRUG RELEASE
K. Ulbrich1, M. Pechar1,
T. Etrych1, M. Jelínková2, M.
Kovář2, B. Říhová2
1Institute of Macromolecular Chemistry, Academy
of Sciences of the Czech Republic, Heyrovský Sq. 2, 162 06 Prague 6, Czech
Republic
2Institute of
Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083,
142 20 Prague 4, Czech Republic
INTRODUCTION
Water-soluble conjugates of synthetic copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with anti-cancer drugs and
antibodies provide a potential drug delivery system facilitating specific drug
delivery to model tumors in mice [1, 2]. In the system, an anti-cancer drug is attached to the polymer through
a biodegradable spacer susceptible
to enzymatic hydrolysis enabling intracellular drug release at a controlled rate, and a specific
antibody is attached as a homing device, recognising specific receptors
expressed on the surface of the target cells. Water-soluble drug carriers based
on nondegradable copolymers of N-(2-hydroxypropyl)methacrylamide
(PHPMA), or biodegradable multiblock poly(ethylene glycol)s (PEG) polymers were
designed as lysosomotropic drug delivery systems. Various structures of the
carriers and polymer drugs have been synthesised and studied. In the classic
structure, anti-cancer drug doxorubicin (DOX) and a targeting antibody are attached
to the hydrophilic PHPMA- or PEG-based backbone via enzymatically degradable oligopeptide
sequences randomly distributed along the polymer chain. Such conjugates provide
a potential and powerful drug delivery system facilitating specific drug
delivery to tumor cells or model tumors inoculated in mice [3]. Despite its significant
anti-cancer activity, the system has some drawbacks, such as not well-defined
branched structure, high molecular weight and broad distribution of molecular
weights. These drawbacks are minimised in a star structure of the conjugate. In
the star system, a number of semitelechelic PHPMA chains, bearing DOX attached through
biodegradable GFLG spacers, are linked to the central antibody molecule via
amide bonds formed by the reaction of terminal reactive ester groups of the
polymer with ε-amino groups of lysine residues in the antibody. Both
classic and star systems enable
drug release only in secondary lysosomes after contact with lysosomal enzymes. Biological activity of all the DOX
conjugates depends on the detailed structure of the conjugate and the ability
of the system to release active drug in the target cells. A prerequisite for biological
activity of the classic and star conjugates is the presence of lysosomal
enzymes at the polymer drug target.
In the third system under study - hydrazone
conjugates (HC), DOX is attached to the biodegradable PEG or nondegradable PHPMA
backbone via a spacer containing hydrazone group. The hydrazone conjugates are
stable under physiological conditions (blood circulation, pH 7.4), but
hydrolytically degradable in mildly acidic environment (e.g., in endosomes and
lysosomes, pH ~ 5 ‑ 6). The systems can circulate in the blood stream
for a long time and are specifically activated (DOX is released) in the target
cells, including those not exhibiting enzymatic activity.
RESULTS AND DISCUSSION
Polymer carriers were used for conjugation with
DOX and selected monoclonal and polyclonal antibodies differing in their
specificity for antigen (B1 mAb, anti-Thy1,2 mAb, anti-EL4 mAb, IgG).
Lysosomotropic conjugates. In addition
to classic systems, the star conjugates differing in the degree of antibody
substitution and the length of the polymer chain were synthesised. All the antibody
conjugates of star structure had lower molecular weights and a significantly
narrower molecular weight distribution than the conjugates of classic
structure. The rate of release of DOX from the classic conjugates incubated in
the presence of lysosomal enzyme cathepsin B strongly depended on the structure
of the oligopeptide spacer, being the highest for the conjugate containing the GFLG
spacer. DOX release from the star conjugate was almost faster by a factor of
two than from those of classic structure. The star conjugates showed a lower
binding activity to cancer cells in vitro,
but their cytostatic activity measured by [3H]thymidine
incorporation was higher by a factor of three than that observed with classic
conjugates. Cytostatic activity of non-targeted and IgG-modified (with irrelevant
immunoglobulin) PHPMA conjugates was more than hundred times lower compared with
the star conjugates targeted with specific monoclonal antibody.
Both classic and star types of
antibody-targeted conjugates shoved a considerably stronger anti-tumor in vivo activity than non-targeted PHPMA-DOX
conjugates or free doxorubicin, but the star conjugates had a remarkably higher
anti-tumor effect than the classic systems. A single intravenous injection of
100 mg of doxorubicin in the form of the
star conjugate on day 11 completely cured 5 out of 9 experimental animals
whereas the classic structure of the targeted conjugate administered in the
same way only increased the survival of experimental mice to 138 % relative to control.
These results show that the star structure of the antibody-targeted PHPMA-DOX
conjugate is highly suitable for targeted drug delivery, possessing better
characteristics, a higher cytostatic activity in vitro and a stronger anti-tumor potential in vivo than the classic conjugates undergoing clinical evaluation
at present [4,5].
Hydrazone conjugates. Release of
DOX from the conjugates incubated in a buffer at pH 5 was faster by a factor of
ten than that at pH 7.4. The effect of pH on DOX release was more pronounced
for PEG-based conjugates. The rate of drug release depended on the structure
and length of the spacer (based on cis-aconitic
acid, glycine, β-alanine, 4-aminobenzoic acid, 6-aminohexanoic acid or oligopeptides).
At pH 5, the highest rate was obtained for the conjugate containing the 6-aminohexanoic
acid-hydrazone spacer and the slowest for the conjugates containing the cis-aconityl spacer. The presence of
lysosomal enzyme cathepsin B in incubation media increased the rate of DOX
release from the conjugate with the GFLG spacer while the DOX release from the conjugates
with amino acid or diglycine spacers remained unchanged [6]. Cytotoxicity of the
conjugates for treated cells (T-splenocytes, EL-4 lymphoma cells) depended on
the detailed structure of the spacer and the used antibody. The hydrazone conjugates
were more cytotoxic than those with ester bonds and the cytotoxicity of the antibody-targeted
conjugates was comparable with that of free DOX (IC50 < 0.1 μg/mL).
In vivo anti-tumor activity of the conjugates
compared with free DOX was significantly higher, exhibiting extensive
inhibition of tumor growth and 40 - 60 % of long-term survivors (there were no
survivors after treatment of mice with free DOX). The efficiency of the antibody-targeted
conjugates was the highest.
CONCLUSION
Application of conjugates of cancerostatics
with synthetic water-soluble polymers and antibodies as site-specific
anti-cancer drugs has been studied. Biological tests, considerable anti-tumor
activity in mice and preliminary evaluation in human demonstrate that the
polymer drug rank among the most promising candidates for successful application
in human cancer chemotherapy.
ACKNOWLEDGEMENT
The authors thank the Léčiva Praha Co. for
supporting this work. It was also supported by the Grant Agency of the Czech
Republic (grant No. 305/02/1425) and by the Grant Agency of Academy of Sciences
of the Czech Republic (grant No. A4050201).
LITERATURE
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