Liposomal nano-drugs based on amphipathic weak acid steroid prodrugs for treatment of inflammatory diseases

<p><b>Background:</b> Steroids are the most efficacious anti-inflammatory agents. However, their toxicities and side-effects compromise their clinical application. Various strategies and major efforts were dedicated for formulating viable liposomal glucocorticosteroids (GCs), so far none of these were approved.</p> <p><b>Objectives:</b> To evaluate these approaches for formulating GC-delivery systems, especially liposomes, and with focus on the Barenholz Lab experience.</p> <p><b>Methods:</b> We developed PEGylated nano-liposomes (NSSL) remotely loaded with water-soluble amphipathic weak acid GC-prodrugs. Their remote loading results in high, efficient and stable loading to the level that enables human clinical use. We characterized them for their physical chemistry and stability. We demonstrated their therapeutic efficacy in relevant animal models and studied their pharmacokinetics (PK), biodistribution (BD) and pharmacodynamics advantages over the free pro-drugs.</p> <p><b>Results:</b> Our steroidal nano-drugs demonstrate much superior PK, BD, tolerability and therapeutic efficacies compared to the free pro-drugs and to most drugs currently used to treat these diseases. These nano-drugs act as robust immune-suppressors, affecting cytokines secretion and diminishing hemorrhage and edema.</p> <p><b>Conclusions:</b> The combination of improved physical-chemistry, PK, BD, tolerability and therapeutic efficacy of these steroidal nano-drugs over the pro-drugs “as-is” support their further clinical development as potential therapeutic agents for treating inflammatory diseases.</p

Liposome characterization.

<p>*No phase transition is explained by the high mole % of cholesterol.</p

Biodistribution of doxorubicin following PLD and PLDTS administration.

<p>Biodistribution of doxorubicin following PLD and PLDTS administration.</p

Therapeutic Efficacy of Combining PEGylated Liposomal Doxorubicin and Radiofrequency (RF) Ablation: Comparison between Slow-Drug-Releasing, Non-Thermosensitive and Fast-Drug-Releasing, Thermosensitive Nano-Liposomes

<div><p>Aims</p><p>To determine how the accumulation of drug in mice bearing an extra-hepatic tumor and its therapeutic efficacy are affected by the type of PEGylated liposomal doxorubicin used, treatment modality, and rate of drug release from the liposomes, when combined with radiofrequency (RF) ablation.</p><p>Materials and Methods</p><p>Two nano-drugs, both long-circulating PEGylated doxorubicin liposomes, were formulated: (1) PEGylated doxorubicin in thermosensitive liposomes (PLDTS), having a burst-type fast drug release above the liposomes’ solid ordered to liquid disordered phase transition (at 42°C), and (2) non-thermosensitive PEGylated doxorubicin liposomes (PLDs), having a slow and continuous drug release. Both were administered intravenously at 8 mg/kg doxorubicin dose to tumor-bearing mice. Animals were divided into 6 groups: no treatment, PLD, RF, RF+PLD, PLDTS, and PLDTS+RF, for intra-tumor doxorubicin deposition at 1, 24, and 72 h post-injection (in total 41, mice), and 31 mice were used for randomized survival studies.</p><p>Results</p><p>Non-thermosensitive PLD combined with RF had the least tumor growth and the best end-point survival, better than PLDTS+RF (p<0.005) or all individual therapies (p<0.001). Although at 1 h post-treatment the greatest amount of intra-tumoral doxorubicin was seen following PLDTS+RF (p<0.05), by 24 and 72 h the greatest doxorubicin amount was seen for PLD+RF (p<0.05); in this group the tumor also has the longest exposure to doxorubicin.</p><p>Conclusion</p><p>Optimizing therapeutic efficacy of PLD requires a better understanding of the relationship between the effect of RF on tumor microenvironment and liposome drug release profile. If drug release is too fast, the benefit of changing the microenvironment by RF on tumor drug localization and therapeutic efficacy may be much smaller than for PLDs having slow and temperature-independent drug release. Thus the much longer circulation time of doxorubicin from PLD than from PLDTS may be beneficial in many therapeutic instances, especially in extra-hepatic tumors.</p></div

Cryo-TEM micrographs of PLDTS (a) and PLD (b) liposomal formulations at 25°C.

<p>Clearly seen are the ellipsoid liposomes containing the loaded doxorubicin, similar to what was previously observed for doxorubicin-loaded liposomes (Doxil/Myocet) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092555#pone.0092555-Barenholz1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092555#pone.0092555-Lasic2" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092555#pone.0092555-Swenson1" target="_blank">[62]</a>.</p

Cryo-TEM micrographs of PLDTS (a) and PLD (b) liposomal formulations at 25°C.

<p>Clearly seen are the ellipsoid liposomes containing the loaded doxorubicin, similar to what was previously observed for doxorubicin-loaded liposomes (Doxil/Myocet) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092555#pone.0092555-Barenholz1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092555#pone.0092555-Lasic2" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092555#pone.0092555-Swenson1" target="_blank">[62]</a>.</p

Doxorubicin biodistribution following PLD.

<p>(a) Total doxorubicin recovered in evaluated most relevant tissues; (b) Doxorubicin fraction in plasma, free and liposomal forms; (c) Doxorubicin fraction in liver; and (d) Fraction of intra-tumoral doxorubicin, with or without RF treatment. n = 4–7 per group. Statistically significant difference (p≤0.05).</p

Mice tumor model therapeutic efficacy studies using PLD and PLDTS with and without RF ablation.

<p>(a) Effect of the various treatments on tumor volume during 90 days duration, and (b) survival of mice by using surrogate end point.</p

Cardinal Role of Intraliposome Doxorubicin-Sulfate Nanorod Crystal in Doxil Properties and Performance

The uniqueness of Doxil can be attributed, to a large extent, to its intraliposomal doxorubicin-sulfate nanorod crystal. We re-examine these nanocrystal features and their mechanism of the formation by studying pegylated liposomal doxorubicins (PLDs) of the same lipid composition, size distribution, and extraliposome medium that were prepared at different ammonium sulfate (AS) concentrations. This study includes a comparison of the thermotropic behavior, morphology, and in vitro ammonia-induced doxorubicin release (relevant to Doxil’s in vivo performance) of these PLDs. In this study, we confirm that a transmembrane ammonium gradient is critical for doxorubicin remote loading, and we demonstrate that the intraliposomal concentration of sulfate counteranions and ammonium ions determine to a large extent the physical state and stability of the PLDs’ remote loaded doxorubicin. “Fully-developed” intraliposome doxorubicin-sulfate nanorod crystals (as defined by cryogenic transmission electron microscopy imaging) develop only when the ammonium sulfate (AS) concentration used for PLD preparation is ≥150 mM. Less than 10% of PLDs prepared with 100 mM AS show fully developed nanorod crystals. Intraliposomal AS concentration ≥200 mM is required to support the stable nanocrystallization in PLDs. The presence of nanocrystals and their melting enthalpy and phase transition co-operativity strongly affect the ammonia-induced doxorubicin release of PLDs. A quick, biphasic release occurs for PLDs that lack the nanorod crystals or have crystals of poor crystallinity, whereas PLDs prepared with ≥200 mM AS show a monophasic, zero-order slow release. This study also demonstrates that after remote loading, residual intraliposomal ammonium concentration and the transmembrane pH gradient related to it also play an important role in doxorubicin-sulfate intraliposomal crystallization and ammonia-induced doxorubicin release

Coencapsulation of alendronate and doxorubicin in pegylated liposomes: a novel formulation for chemoimmunotherapy of cancer

<p>We developed a pegylated liposome formulation of a dissociable salt of a nitrogen-containing bisphosphonate, alendronate (Ald), coencapsulated with the anthracycline, doxorubicin (Dox), a commonly used chemotherapeutic agent. Liposome-encapsulated ammonium Ald generates a gradient driving Dox into liposomes, forming a salt that holds both drugs in the liposome water phase. The resulting formulation (PLAD) allows for a high-loading efficiency of Dox, comparable to that of clinically approved pegylated liposomal doxorubicin sulfate (PLD) and is very stable in plasma stability assays. Cytotoxicity tests indicate greater potency for PLAD compared to PLD. This appears to be related to a synergistic effect of the coencapsulated Ald and Dox. PLAD and PLD differed in <i>in vitro</i> monocyte-induced IL-1β release (greater for PLAD) and complement activation (greater for PLD). A molar ratio Ald/Dox of ∼1:1 seems to provide an optimal compromise between loading efficiency of Dox, circulation time and <i>in vivo</i> toxicity of PLAD. In mice, the circulation half-life and tumor uptake of PLAD were comparable to PLD. In the M109R and 4T1 tumor models in immunocompetent mice, PLAD was superior to PLD in the growth inhibition of subcutaneous tumor implants. This new formulation appears to be a promising tool to exploit the antitumor effects of aminobisphosphonates in synergy with chemotherapy.</p