CONTEXT

Recent developments of epigenetic drugs are facing clinical limitations due to metabolic activities as well as side effects due to unspecific biodistribution. We have developed a novel delivery approach validated with several epigenetic drugs belonging to the histone deacetylase (HDAC) inhibitor family. This delivery strategy was applied to cure mesothelioma cancers.

TECHNICAL DESCRIPTION

The technology involves the preparation of “clickable” functionalities on one side and on the other side the preparation of clickable monomers (specifically norbornenyl (NB)-PEG macromonomers) (Figure 1). The click is based on the azide-alkyne cycloaddition (well-known as click reaction). When clicked together (the macromonomer and the functionalities), a library of functional macromonomer is obtained. The polymerization of a functional macromonomer with norborne gives access to functional nanoparticles. Selected different macromonomers for the polymerization (copolymerization) give access to multifunctional particles.
Functionalities can be reporter dye (fluorescent) or biomolecule releasing systems (prodrug below).

Figure 1

DEVELOPMENT STAGE

Currently a clickable rhodamine fluorescent reporter and several clickable prodrugs bearing HDAC inhibitors have been prepared and clicked to the same macromonomers to produce a library of 6 different macromonomers.

From this library several functional nanoparticles were prepared, mono or bifunctional (Figure 2, blue HDAC inhibitor, purple fluorescent dye, green pH-responsive system, orange PEO chain, black norbornene once polymerized, red click site between azido-macromonomer and alkyne-functionalities).

Figure 2

Biological models were developed to validate HDAC inhibition directly in cells using a BRET system. All compounds were tested with this assay and in all cases, free HDAC inhibitors and the polymers gave satisfying results. The fluorescent version of these nanoparticles (bearing the rhodamine group) were used to validate the biodistribution in vivo. With the two nanoparticles bearing the dye prepared to date we obtained highly selective tumor targeting in vivo without additional active targeting. (Figure 3). This was also used to demonstrate internalization by endocytosis, required for the release of the bioactive molecules based on pH response.

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Figure 3. Top: results with nanoparticles bearing rhodamine only showing left: elective tumor targeting and right: cellular internalization through endocytosis. Middle: nanoparticles bearing Vorinostat and Rhodamine A: biodistribution in organs (T:tumor, O: ovary, K:kidneys, L:liver, B: blood sample and B) quantification. Bottom: biodistribution of rhodamine-bearing nanoparticles made in an orthotopic model of peritoneal mesothelioma. A-E: distribution over time, F: quantification

BENEFITS

A first benefit of the strategy is for Vorinostat that when used free is not able in our model to increase histone acetylation, whereas when delivered in tumors an increase of histone acetylation is observed. A secondbenefit is that when used with highly nanomolar active compounds, an effective tumor reduction is obtained. (Figure 4)
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Figure 4. TOP: increased acetylation of histones in tumors with nanoparticles bearing vorinostat. BOTTOM: tumor growth reduction with nanoparticles bearing nanomolar active HDAC inhibitor. A) Quantified results, B) pancreas invasion by tumors in untreated mice, C) pancreas invasion for mice treated with inhibitor alone, D) pancreas of mice treated with nanoparticles bearing HDAC inhibitor.

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