Optimized synthesis, characterization and in vitro systematic evaluation of adamantane-doxorubicin prodrugs sensitive to pH in breast cancer cells
Graphical abstract
Introduction
Dox is one of the most effective chemotherapeutics for the treatment of leukaemia and a broad variety of solid tumors [1]. However, its adverse effects, specifically dose-dependency, myelosuppression and lethal cardiotoxicity limit its use and the outcomes of the therapy [[2], [3], [4]]. Prodrugs are pharmacologically inactive chemical entities that, after their administration, are biotransformed into pharmacologically active species [5]. Targeted prodrug approach is one of the new trends in the treatment of cancer. An important strategy to achieve the local activation of prodrugs involves the use of pH sensitive conjugates [6]. Therefore, antineoplastic prodrugs come up as one of the strategies to increase selectivity, improve therapeutic indexes and reduce toxicity to normal tissues. The integration of prodrugs with nanotechnology-based drug delivery strategies has given rise to nanosystems improving prodrug stability and increasing drug availability. Moreover, multifunctional platforms containing prodrugs have been designed for targeted and smart stimuli-triggered drug release [7,8]. In these platforms the prodrug can be covalently attached to the nanocarrier or one of its components (building block or polymer scaffold), or it can be simply loaded into the system.
The design of Dox prodrugs is an approach that has been continuously explored to overcome Dox side effects. Furthermore, a number of nanocarriers containing Dox prodrugs have been investigated [7]. These delivery nanosystems include from simple liposomes and micelles to sophisticated platforms displaying multiple functionalities i.e. theranostics, response to external or internal stimuli and intracellular targeting [9,10].
A considerable number of works report the in vitro and in vivo effectiveness of Dox delivery smart platforms [[11], [12], [13]], however, none of them has been approved. At their best, some have reached Phase II clinical studies but any has even proceeded to Phase III, due to their structural complexity that makes very difficult to obtain them in a reproducible manner and to scale up the synthetic processes, which are generally expensive and involve several steps. In addition, is very difficult to predict the interaction of the multiple components with target and biological barriers. In summary, these platforms are too intricate to be translational [14,15].
The release of a drug is a quite complex process. It is unrealistic to simplify the delivery mechanisms for all the nanocarriers and make them to fulfill the targeting, cell attachment, internalization, and the release requirements. Therefore, a better understanding of drug delivery systems is crucial to make a good choice of the linker and the triggering mechanism that will generate the active drug. Their processes are fundamental to develop clinically achievable nanoformulations. This fact has motivated us to focus our research on the rational design of Dox prodrugs delivery systems through the systematic study of all their components at physicochemical and biological levels, keeping in mind the following requirements: a) an easy and affordable synthesis; b) feasible materials to be used as excipients and suitable for scaling them up; c) structurally simple systems.
To start our investigation, we selected three Dox prodrugs formed by the conjugation of Dox to an adamantyl moiety using different linkers: amide (Ad-a-Dox), hydrazone (Ad-h-Dox), and ester (Ad-e-Dox). The engineering of drug delivery systems by supramolecular design has gained great attention since reversible and tunable features that characterize supramolecular materials can afford carrier systems with controlled and predictable drug release. Cyclodextrin (CD) is a well-known structural unit used in the design of supramolecular host-guest drug delivery nanosystems.
Given that adamantane is a guest that fits perfectly into the CD cavity [[16], [17], [18]], it has been frequently employed in prodrug design and in the construction of CD-based supramolecular platforms. Dox is not the exception and there are several works reporting Ad-Dox prodrugs loaded into CD-based supramolecular platforms [[19], [20], [21]]. With the aim to be sure about the potential success of CD-based platforms, we focused the present work exclusively on the study of Ad-Dox systems. Firstly, we report novel and simple synthetic procedures for the obtainment of Ad-a-Dox and Ad-e-Dox. Moreover, we have optimized of the synthesis of Ad-h-Dox, and studied whether the activation can occur under in vitro acidic conditions (Fig. 1), acidic environment in tumor tissue) as well as their cytotoxicity in the estrogen receptor-positive (MCF-7) and the triple-negative breast cancer cells (TNBC). To our knowledge, this is the first time that these Dox prodrugs are systematically studied and evaluated against TNBC.
Section snippets
Materials
All starting materials were commercially available, reagent grade, and were used without any further purification. Reactions were monitored by analytical TLC on pre-coated silica gel 60 F254 plates (Aldrich). Infrared spectra were recorded on a Nicolet FT-5SX spectrophotometer. 1H NMR spectra were recorded on a Bruker spectrometer (400 MHz). Chemical shifts are reported in ppm (δ) and the signals are described as singlet (s), doublet (d), triplet (t), quartet (q), broad (br), multiplet (m), and
Synthesis
Nowadays it is mandatory to develop affordable, reproducible and quantitative synthetic methods in order to exploit them, that is why we discuss a cost-effective method to obtain Ad-a-Dox. The amide bond formation is fundamentally important in organic synthesis [24,25] as well as in the drug delivery field. COMU, an effective reagent in solution, is one of the coupling agents which arised from the group of salts based on uronium/aminium, phosphonium salts and iminium [26]. Hence, we
Conclusions
We report a novel synthetic procedure to obtain Ad-a-Dox and Ad-e-Dox prodrugs as well as the optimization of the synthesis of Ad-h-Dox. All methods include a few steps and an easy work-up. In addition, they showed to be affordable and reproducible. Herein, we have demonstrated that acid conditions are required for the hydrolysis of Ad-h-Dox and therefore its bioactivation can occur intracellularly.
Ester and amide bonds have been successfully used in prodrugs design. In our case, the outcomes
Acknowledgements
We are grateful to Gerardo Cedillo for his help with 1H and 13C NMR spectroscopy and Miguel Canseco for his assistance in the characterization of the compounds. We thank CONACYT (Projects 253155 and 279380) for financial support. I. González-Méndez thanks Posgrado en Ciencias Químicas UNAM and CONACYT for scholarship and financial support, respectively. YRA is grateful to Programa de Apoyo a la Investigación y el Posgrado (PAIP 5000-915), Facultad de Química UNAM and Materials Research Institute
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2020, Bioorganic and Medicinal ChemistryCitation Excerpt :Recently, adamantane (Ad) linked small molecules have attracted great interest since it favors the formation of stable inclusion complexes (ICs) within the βCD cavity, which makes easier to control the therapeutic load in these carriers.12–15 Previously, pH-sensitive prodrugs bearing a covalent bond (ester, amide or hydrazone) between Ad and Dox (Ad-Dox) were constructed by our research group to control the drug release (Fig. 1), taking advantage of the biological events that are specific for cancer cells, such as low oxygen rates or pH changes.16–22 Since more complex DDS have been constructed with apparent lack of reproducibility, it is time to take a pause in the design of these super-complex systems and evaluate its components, to understand better their mechanism and get reproducible systems.23–27
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