Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes
Introduction
Targeted or non-targeted soluble copolymers based on poly [N-(2-hydroxypropyl)methacrylamide] (PHPMA) are used as carriers for anti-cancer [3], [4], [5] and immunosuppressive drugs [6]. In our previous studies, drugs have been covalently linked to the PHPMA carrier through a proteolytically cleavable bond [1]. Following cellular uptake via fluid phase endocytosis (in the case of non-targeted conjugates) or receptor-mediated endocytosis (in the case of targeted conjugates), the linker is cleaved by lysosomal enzymes allowing intracellular drug release [7]. Cytostatic and cytotoxic effects of targeted or non-targeted polymeric prodrugs based on HPMA copolymers have been reported in vitro and in vivo [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. DOX-PHPMA copolymer conjugate containing a GlyPheLeuGly spacer (PK1) has been evaluated in Phase I clinical trial in the U.K. [14] and a Phase II clinical study is underway. Preliminary data on a galactosamine-targeted conjugate (PK2) have recently been published [15].
Generally, drugs can be released from their polymeric carrier by the action of proteolytic hydrolases [1] or by spontaneous hydrolysis [16] which, in certain cases, can result in pH-controlled drug release [17]. Pharmacological activity of DOX-PHPMA conjugates in which the drug is bound to the polymeric carrier through proteolytically cleavable bond depends on the activity, specificity and concentration of lysosomal proteases [7].
Recently, we have developed a new system in which doxorubicin is bound to the polymer carrier via hydrolytically unstable hydrazone bond formed by a reaction of the carbonyl group of DOX with the hydrazide group in the side chain of the polymer [18]. Such DOX–PHPMA conjugate is stable in aqueous solution at pH 7.4 (i.e., in the blood stream) and DOX is released at pH 5–6.0 (i.e. in the endosomes and lysosomes).
In this paper we have addressed the following questions; (a) what is the in vitro pharmacological efficacy of HC conjugates compared to PK1 conjugates, (b) how does the composition of oligopeptide side chains of the PHPMA influence the cytotoxicity of the conjugates, and (c) what is the anti-proliferative activity of PK1 and HC conjugates in the target cells with a limited content of lysosomes such as the erythromyeloid leukemia cell line K 562.
Section snippets
Synthesis of conjugates containing doxorubicin bound to GlyPheLeuGly oligopeptide side chains of water-soluble poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA)-based copolymer carrier through a proteolytically-cleavable bond (PK1 conjugates)
The synthesis was repeatedly described [1], [10], [13]. Fig. 1 shows the chemical structure of the PK1 conjugate.
Synthesis of conjugates containing doxorubicin bound to GlyPheLeuGly or GlyGly oligopeptide side chains of water-soluble poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA)-based copolymer carrier through a hydrolytically-cleavable bond (HC conjugates)
The synthesis is described in detail by Etrych et al. [18]. Fig. 2 shows the chemical structure of a HC conjugate with the hydrazone bond between the copolymer carrier and doxorubicin.
Cancer cell lines
The following cell lines were used. Mouse origin: EL4 T-cell lymphoma, BCL1 B-cell leukemia, 38C13 B-cell lymphoma, P388 leukemia; human origin: SW 480 colorectal carcinoma (primary), SW620 colorectal
Results and discussion
Weight- and number-average molecular weights of polymers, Mw and Mn, were estimated by size exclusion chromatography with RI detector and light-scattering detector (DAWN DSP-F, Wyatt Technology Co.). Composition of HPMA copolymers and conjugates was estimated by amino acid analysis (LDC amino acid analyser), by UV/VIS spectrophotometry and the content of hydrazide groups in sample 8 (8.6 mol%) was determined by modified TNBS method [18]. The content of side chains terminated in p-nitrophenyl
Conclusion
Conjugates with hydrolytically-releaseble doxorubicin (HC conjugates), exert in vitro considerably higher cytotoxic effect than PK1 conjugates with proteolytically-releasable doxorubicin. They are highly effective also in the erythromyeloid leukemia cell line K 562 with a limited content of lysosomes where the anti-proliferative activity of PK1 conjugates is very low. The higher cytotoxic activity of HC conjugates is most probably related to a faster release of free doxorubicin already in the
Acknowledgments
This research was supported by the Ministry of Health (Grant 5050-3) and by the Grant Agency of the Czech Republic (Grant 307/96/K226). We thank Ms. Hana Semorádová and Ms. Helena Mišurcová for their excellent technical assistance.
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2020, Advanced Drug Delivery ReviewsCitation Excerpt :Therefore, acid-labile chemical bonds that are likely to be cleaved in acidic pH will release APIs in tumor tissues, but these linker bonds should be stable at neutral pH so as to ensure stability during circulation. Many studies validated these findings [49,51,53,59,62,63]. For example, Ulbrich and his group at the Institute of Macromolecular Chemistry, Czech Republic, developed an HPMA polymer-conjugated doxorubicin, and we also subsequently prepared a pirarubicin conjugate with a hydrazone bond that had a superior EPR effect and demonstrated rapid release of an API (doxorubicin or pirarubicin) in the tumor environment.