Joachim Caron

Anti-HIV efficacy and biodistribution of nucleoside reverse transcriptase inhibitors delivered as squalenoylated prodrug nanoassemblies.

Due to their hydrophilic nature, most nucleoside reverse transcriptase inhibitors (NRTIs) display a variable bioavailability after oral administration and a poor control over their biodistribution, thus hampering their access to HIV sanctuaries. The limited cellular uptake and activation in the triphosphate form of NRTIs further restrict their efficacy and favour the emergence of viral resistance. We have shown that the conjugation of squalene (sq) to the nucleoside analogues dideoxycytidine (ddC) and didanosine (ddI) leads to amphiphilic prodrugs (ddC-sq and ddI-sq) that spontaneously self-organize in water as stable nanoassemblies of 100-300 nm. These nanoassemblies can also be formulated with polyethylene glycol coupled to either cholesterol (Chol-PEG) or squalene (sq-PEG). When incubated with peripheral blood mononuclear cells (PBMCs) in vitro infected with HIV, the NRTI-sq prodrugs enhanced the antiviral efficacy of the parent NRTIs, with a 2- to 3-fold decrease of the 50% effective doses and a nearly 2-fold increase of the selectivity index. This was also the case with HIV-1 strains resistant to ddC and/or ddI. The enhanced antiviral activity of ddI-sq was correlated with an up to 5-fold increase in the intracellular concentration of the corresponding pharmacologically active metabolite ddA-TP. The ddI-sq prodrug was further investigated in vivo by the oral route, the preferred route of administration of NRTIs. Pharmacokinetics studies performed on rats showed that the prodrug maintained low amounts of free ddI in the plasma. Administration of (3)H-ddI-sq led to radioactivity levels higher in the plasma and relevant organs in HIV infection as compared to administration of free (3)H-ddI. Taken together, these results show the potential of the squalenoylated prodrugs of NRTIs to enhance their absorption and improve their biodistribution, but also to enhance their intracellular delivery and antiviral efficacy towards HIV-infected cells.

A new nanomedicine based on didanosine glycerolipidic prodrug enhances the long term accumulation of drug in a HIV sanctuary.

New nanomedicines could improve drug accumulation in HIV sanctuaries and ameliorate their antiretroviral efficiency. In this view, we propose herein a combined strategy based on a biomimetic prodrug of ddI and its formulation in well-characterized lipid nanoobjects. The glycerolipidic prodrug of ddI (ProddINP) has been synthesized and its bulk structure was characterized. An appropriate formulation of this prodrug has been designed using a rational approach combining different physicochemical techniques. The high incorporation ratio of the prodrug into dipalmitoylphosphatidylcholine (DPPC) bilayers was determined by DSC. Then two liposome preparation methods were compared, with respect to size, incorporation yield and molecular/supramolecular organization of vesicles. The best liposomal formulation of ProddINP has been checked to keep intact the anti-HIV activity of ddI. This formulation was finally compared to ddI after oral route in rat. The animal experiments evidenced the increase of ddI blood half life (3-fold) and its enhanced accumulation as prodrug form at 24h in numerous organs and especially intestine after administration of ProddINP in comparison with free drug. Finally, the tested liposomal formulation of ProddINP seems to be a promising approach to eradicate HIV infection from intestinal sanctuaries where the virus can concentrate.

“Squalenoylcurcumin” Nanoassemblies as Water‐Dispersible Drug Candidates with Antileishmanial Activity

Curcumin, a natural polyphenolic compound, showed antiparasitic potential, including trypanocidal and leishmanicidal activity, in several in vitro and in vivo models. The molecule is well tolerated in humans. However, it is insoluble in water and displays poor oral bioavailability as a result of low absorption. New derivatives of curcumin were prepared by esterification of one or two of its phenolic groups with 1,1′,2‐tris‐norsqualenic acid. These “squalenoylcurcumins” were formulated as water‐dispersible nanoassemblies of homogeneous size, and they proved to be stable. Squalenoylcurcumins were inactive against Trypanosoma brucei brucei trypomastigotes, even as nanoassemblies, in contrast with curcumin. However, against Leishmania donovani promastigotes, the activities of the squalenoylcurcumins and their nanoassemblies were enhanced relative to that of curcumin. In L. donovani axenic and intramacrophagic amastigotes, they showed activity in the range of miltefosine, with good selectivity indexes. In regard to their dispersibility in water and to the safety of curcumin, these nanoassemblies are promising candidates for preclinical study toward the treatment of visceral leishmaniasis.

Combined antitumoral therapy with nanoassemblies of bolaform polyisoprenoyl paclitaxel/gemcitabine prodrugs

We report here the design and synthesis of novel bolaform prodrugs in which the two headgroups are constituted of gemcitabine and/or paclitaxel anticancer drugs bound together through short polyisoprenoyl spacers. Thanks to the unique auto-assembling properties of the polyisoprenoyl linker, these bolaform prodrugs were found to self-assemble in aqueous media into 100–200 nm nanoassemblies. The synthesis of the prodrugs and the characterization of the nanoassemblies by DLS, TEM and cryoTEM are described. These gemcitabine/paclitaxel nanodevices with high drug payloads displayed improved in vitro activities on several human and murine cancer cell lines comparatively to nanoassemblies of the corresponding squalenoyl drugs alone or in combination.

Preactivated Oxazaphosphorines Designed for Isophosphoramide Mustard Delivery as Bulk Form or Nanoassemblies: Synthesis and Proof of Concept

Oxazaphosphorines are alkylating agents used in routine clinical practices for treatment of cancer for many years. They are antitumor prodrugs that require cytochrome P450 bioactivation leading to 4-hydroxy derivatives. In the case of ifosfamide (IFO), the bioactivation produces two toxic metabolites: acrolein, a urotoxic compound, concomitantly generated with the isophosphoramide mustard; and chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains. To improve the therapeutic index of IFO, we have designed preactivated IFO derivatives with the covalent binding of several O- and S-alkyl moieties including polyisoprenoid groups at the C-4 position of the oxazaphosphorine ring to avoid cytochrome bioactivation favoring the release of the active entity and limiting the chloroacetaldehyde release. Thanks to the grafted terpene moieties, some of these new conjugates demonstrated spontaneous self-assembling properties into nanoassemblies when dispersed in water. The cytotoxic activities on a panel of human tumor cell lines of these novel oxazaphosphorines, in bulk form or as nanoassemblies, and the release of 4-hydroxy-IFO from these preactivated IFO analogues in plasma are reported.

Abstract 5702: NanoOxaza, pre-activated oxazaphosphorines prodrugs designed for drug delivery strategy with wide anticancer activity

The oxazaphosphorines belong to the alkylating drug class with a wide spectrum of antineoplasic activity. Ifosfamide (IFO) demonstrated activity against various tumour types, from soft tissue sarcomas to lymphomas both in adult and paediatric patients. Oxazaphosphorines are metabolised by cytochromes, leading to ring opening and subsequent production of active nitrogen mustards, which display DNA cross-links. Associated toxicities of IFO are due to 1) acrolein, a urotoxic metabolite concomitantly generated with the release of the alkylating mustard and 2) chloroacetaldehyde, a metabolite derived from oxidation of side chains of the molecule via the action of cytochrome P450 (CYP) known to be responsible for neurotoxicity and nephrotoxicity (Kerbusch et al., 2001). To circumvent the oxidative related-toxicities, we have designed pre-activated oxazaphosphorines. (Paci et al., 2001) through electrochemical oxidation and engraftment of various moieties such as O-alkyl groups (fatty acids, alcohols, polyols, or sugars) and S-alkyl (thionyl) groups. Actually, IFO is activated by substitution at position 4 with labile moieties leading to the release of the cytotoxic entity without enzyme activity. These moieties can bring tissue specificity or physico-chemical characteristics leading to nanoassemblies for drug delivery purposes. Some of these pre-activated prodrugs may be constitutive of drug delivery systems, such as nanoparticles, aiming to address alkylating moieties to their target. We present here the use of squalene conjugation as squalene-based drug delivery systems have been favourably used for drug delivery (Couvreur et al. 2006). The designed compounds, SQ-IFO and SQ-thioIFO, have then two interesting properties. First, they are pre-activated formulations of IFO with C4-oxidation allowing to by-pass the CYP activation with the direct release of the alkylating mustard. The second property is the described capacity of squalenic derivatives for self-assembly in aqueous medium leading to nanoparticles. The SQ-IFO and SQ-thioIFO nanoparticles have been fully characterized with a mean diameter of 182 nm when prepared at 1mg/mL in 5% Dextrose. The cytotoxic activity of these nanoassemblies has been studied in vitro on a panel of 9 human and murine cancer lines (A549, MCF-7, B16F10, M109, MiaPaCa-2, KB 3.1, SK-N-MC, UW 479, IGR-OV1). Squalenoyl IFO derivatives present high activity in vitro while IFO has no activity on these cells even it is used in clinical practices to treat these cancers. This proves the pre-activated property of these new compounds and the lack of CYP activation needs. Their therapeutic activity is currently studied in vivo on human Ewing sarcoma xenografts in mice after i.v. administration. These new formulations of IFO could constitute promising candidates for both oral and IV administration in a wide panel of tumours. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5702. doi:1538-7445.AM2012-5702