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  • Polymer conjugates of doxorubicin bound through an amide and hydrazone bond: Impact of the carrier structure onto synergistic action in the treatment of solid tumours.

Polymer conjugates of doxorubicin bound through an amide and hydrazone bond: Impact of the carrier structure onto synergistic action in the treatment of solid tumours.

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences (2014-03-19)
Tomáš Etrych, Vladimír Subr, Richard Laga, Blanka Ríhová, Karel Ulbrich
ABSTRACT

In this study, we describe the synthesis, physico-chemical characterisation and results of the in vitro and in vivo evaluation of the biological behaviour of N-(2-hydroxypropyl)methacrylamide-based (HPMA) copolymer conjugates bearing doxorubicin (DOX) partly bound via a pH-sensitive hydrazone and partly via enzymatically degradable amide bonds, each contributing to a different anti-tumour mechanism of action of the polymer-doxorubicin conjugate. The following two types of HPMA copolymer drug carriers designed for passive tumour targeting were synthesised and compared: the linear non-degradable copolymer and the biodegradable high-molecular-weight (HMW) diblock copolymer. The HMW diblock copolymer carrier containing a degradable disulphide bond between the polymer blocks showed a rapid degradation in a buffer containing glutathione within the first few hours of incubation. In contrast to the conjugate with the amide bond-bound DOX requiring the presence of lysosomal enzymes to release DOX, the polymer-drug conjugate with the DOX bound via a hydrazone bond released DOX by pH-sensitive hydrolysis, which was significantly faster in a buffer of pH 5.0 (intracellular pH) than pH 7.4, mimicking the conditions in the bloodstream. The significant and comparable in vivo anti-tumour activity of the diblock HMW conjugate and an equimolar mixture of the conjugates differing in the DOX attachment method along with the development of cancer resistance during treatment with these conjugates demonstrated the high potential of these compounds in the development of new nanomedicines suitable for the treatment of solid tumours.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Doxorubicin hydrochloride, suitable for fluorescence, 98.0-102.0% (HPLC)
Sigma-Aldrich
2,2′-Dithiodipyridine, ≥99.0% (GC)
Sigma-Aldrich
tert-Butyl carbazate, 98%
Sigma-Aldrich
Hydrazine hydrate, reagent grade, N2H4 50-60 %
Sigma-Aldrich
2,2′-Dithiodipyridine, powder
Doxorubicin hydrochloride, European Pharmacopoeia (EP) Reference Standard
Supelco
DL-Dithiothreitol solution, 1 M in H2O
Sigma-Aldrich
Doxorubicin hydrochloride, 98.0-102.0% (HPLC)
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
USP
Doxorubicin hydrochloride, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Hydrazine monohydrate, N2H4 64-65 %, reagent grade, ≥97%
Sigma-Aldrich
Methyl 6-aminohexanoate hydrochloride, ≥99.0% (AT)
Sigma-Aldrich
Methacryloyl chloride, 97%, contains ~200 ppm monomethyl ether hydroquinone as stabilizer
Supelco
Trifluoroacetic acid, analytical standard
Sigma-Aldrich
Amino-2-propanol, 93%
Sigma-Aldrich
Aldrithiol-2, 98%
Sigma-Aldrich
Picrylsulfonic acid solution, 5 % (w/v) in H2O, BioReagent, suitable for determination of primary amines
Sigma-Aldrich
Triisopropylsilane, 98%
Sigma-Aldrich
Trifluoroacetic acid, ≥99%, for protein sequencing
Sigma-Aldrich
Trifluoroacetic acid, suitable for HPLC, ≥99.0%
Sigma-Aldrich
Trifluoroacetic acid, ReagentPlus®, 99%
Supelco
(±)-1-Amino-2-propanol, analytical standard
Sigma-Aldrich
Trifluoroacetic acid, puriss. p.a., suitable for HPLC, ≥99.0% (GC)
Sigma-Aldrich
Hydrazine hydrate solution, puriss. p.a., 24-26% in H2O (RT)