Mark E. Hayes

Pharmacokinetics and in vivo drug release rates in liposomal nanocarrier development

Liposomes represent a widely varied and malleable class of drug carriers generally characterized by the presence of one or more amphiphile bilayers enclosing an interior aqueous space. Thus, the pharmacological profile of a particular liposomal drug formulation is a function not only of the properties of the encapsulated drug, but to a significant extent of the pharmacokinetics, biodistribution, and drug release rates of the individual carrier. Various physicochemical properties of the liposomal carriers, the drug encapsulation and retention strategies utilized, and the properties of the drugs chosen for encapsulation, all play an important role in determining the effectiveness of a particular liposomal drug. These properties should be carefully tailored to the specific drug, and to the application for which the therapeutic is being designed. Liposomal carriers are also amenable to additional modifications, including the conjugation of targeting ligands or environment-sensitive triggers for increasing the bioavailability of the drug specifically at the site of disease. This review describes the rationale for selecting optimal strategies of liposomal drug formulations with respect to drug encapsulation, retention, and release, and how these strategies can be applied to maximize therapeutic benefit in vivo.

Development of ligand-targeted liposomes for cancer therapy.

The continued evolution of targeted liposomal therapeutics has resulted in new agents with remarkable antitumour efficacy and relatively mild toxicity profiles. A careful selection of the ligand is necessary to reduce immunogenicity, retain extended circulation lifetimes, target tumour-specific cell surface epitopes, and induce internalisation and subsequent release of the therapeutic substance from the liposome. Methods for assembling targeted liposomes, including a novel micellar insertion technology, for incorporation of targeting molecules that efficiently transforms a non-targeted liposomal therapeutic to a targeted one, greatly assist the translation of targeted liposome technology into the clinic. Targeting strategies with liposomes directed at solid tumours and vascular targets are discussed. The authors believe the development of ligand-targeted liposomes is now in the advanced stage and offers unique and important advantages among other targeted therapies. Anti-HER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials, the results of which should provide new insights into the promise of ligand-targeted liposomal therapies.

Development of a highly stable and targetable nanoliposomal formulation of topotecan.

Topotecan (TPT), a highly active anticancer camptothecin drug, would benefit from nanocarrier-mediated site-specific and intracellular delivery because of a labile lactone ring whose hydrolysis inactivates the drug, poor cellular uptake resulting from both lactone hydrolysis and a titratable phenol hydroxyl, and the schedule-dependency of its efficacy due to its mechanism of action. We have encapsulated topotecan in liposomes using transmembrane gradients of triethylammonium salts of polyphosphate (Pn) or sucroseoctasulfate (SOS). Circulation lifetimes were prolonged, and the rate of drug release in vivo depended on the drug load (T(1/2)=5.4 h vs. 11.2 h for 124 and 260 g TPT/mol PL, respectively) and the nature of intraliposomal drug complexing agent used to stabilize the nanoliposome formulation (T(1/2)=11.2 h vs. 27.3 h for Pn and SOS, respectively). Anti-EGFR and anti-HER2-immunoliposomal formulations dramatically increased uptake of topotecan compared to nontargeted nanoliposomal topotecan and poorly permeable free topotecan in receptor-overexpressing cancer cell lines, with a corresponding increase in cytotoxicity in multiple breast cancer cell lines and improved antitumor activity against HER2-overexpressing human breast cancer (BT474) xenografts. We conclude that stabilization of topotecan in nanoliposomes significantly improves the targetability and pharmacokinetic profile of topotecan, allowing for highly active formulations against solid tumors and immunotargeting to cancer-overexpressing cell surface receptors.

Anti-CD166 single chain antibody-mediated intracellular delivery of liposomal drugs to prostate cancer cells.

Targeted delivery of small-molecule drugs has the potential to enhance selective killing of tumor cells. We have identified previously an internalizing single chain [single chain variable fragment (scFv)] antibody that targets prostate cancer cells and identified the target antigen as CD166. We report here the development of immunoliposomes using this anti-CD166 scFv (H3). We studied the effects of a panel of intracellularly delivered, anti-CD166 immunoliposomal small-molecule drugs on prostate cancer cells. Immunoliposomal formulations of topotecan, vinorelbine, and doxorubicin each showed efficient and targeted uptake by three prostate cancer cell lines (Du-145, PC3, and LNCaP). H3-immunoliposomal topotecan was the most effective in cytotoxicity assays on all three tumor cell lines, showing improved cytotoxic activity compared with nontargeted liposomal topotecan. Other drugs such as liposomal doxorubicin were highly effective against LNCaP but not PC3 or Du-145 cells, despite efficient intracellular delivery. Post-internalization events thus modulate the overall efficacy of intracellulary delivered liposomal drugs, contributing in some cases to the lower than expected activity in a cell line–dependent manner. Further studies on intracellular tracking of endocytosed liposomal drugs will help identify and overcome the barriers limiting the potency of liposomal drugs. [Mol Cancer Ther 2007;6(10):2737–46]

Improved Pharmacokinetics and Efficacy of a Highly Stable Nanoliposomal Vinorelbine

Effective liposomal formulations of vinorelbine (5′ nor-anhydro-vinblastine; VRL) have been elusive due to vinorelbines hydrophobic structure and resulting difficulty in stabilizing the drug inside the nanocarrier. Triethylammonium salts of several polyanionic trapping agents were used initially to prepare minimally pegylated nanoliposomal vinorelbine formulations with a wide range of drug release rates. Sulfate, poly(phosphate), and sucrose octasulfate were used to stabilize vinorelbine intraliposomally while in circulation, with varying degrees of effectiveness. The release rate of vinorelbine from the liposomal carrier was affected by both the chemical nature of the trapping agent and the resulting drug-to-lipid ratio, with liposomes prepared using sucrose octasulfate displaying the longest half-life in circulation (9.4 h) and in vivo retention in the nanoparticle (t1/2 = 27.2 h). Efficacy was considerably improved in both a human colon carcinoma (HT-29) and a murine (C-26) colon carcinoma model when vinorelbine was stably encapsulated in liposomes using triethylammonium sucrose octasulfate. Early difficulties in preparing highly pegylated formulations were later overcome by substituting a neutral distearoylglycerol anchor for the more commonly used anionic distearoylphosphatidylethanolamine anchor. The new pegylated nanoliposomal vinorelbine displayed high encapsulation efficiency and in vivo drug retention, and it was highly active against human breast and lung tumor xenografts. Acute toxicity of the drug in immunocompetent mice slightly decreased upon encapsulation in liposomes, with a maximum tolerated dose of 17.5 mg VRL/kg for free vinorelbine and 23.8 mg VRL/kg for nanoliposomal vinorelbine. Our results demonstrate that a highly active, stable, and long-circulating liposomal vinorelbine can be prepared and warrants further study in the treatment of cancer.

Building and Characterizing Antibody-Targeted Lipidic Nanotherapeutics

Immunoliposomes provide a complementary, and in many instances advantageous, drug delivery strategy to antibody-drug conjugates. Their high carrying capacity of 20,000-150,000 drug molecules/liposome, allows for the use of a significantly broader range of moderate-to-high potency small molecule drugs when compared to the comparably few subnanomolar potency maytansinoid- and auristatin-based immunoconjugates. The multivalent display of 5-100 antibody fragments/liposome results in an avidity effect that can make use of even moderate affinity antibodies, as well as a cross-linking of cell surface receptors to induce the internalization required for intracellular drug release and subsequent activity. The underlying liposomal drug must be effectively engineered for long circulating pharmacokinetics and stable in vivo drug retention in order to allow for the drug to be efficiently delivered to the target tissue and take advantage of the site-specific bioavailability provided for by the targeting arm. In this chapter, we describe the rationale for engineering stable immunoliposome-based therapeutics, methods required for preparation of immunoliposomes, as well as for their physicochemical and in vivo characterization.

Pharmacokinetics, tumor accumulation and antitumor activity of nanoliposomal irinotecan following systemic treatment of intracranial tumors

AIM We sought to evaluate nanoliposomal irinotecan as an intravenous treatment in an orthotopic brain tumor model. MATERIALS & METHODS Nanoliposomal irinotecan was administered intravenously in the intracranial U87MG brain tumor model in mice, and irinotecan and SN-38 levels were analyzed in malignant and normal tissues. Therapy studies were performed in comparison to free irinotecan and control treatments. RESULTS Tissue analysis demonstrated favorable properties for nanoliposomal irinotecan, including a 10.9-fold increase in tumor AUC for drug compared with free irinotecan and 35-fold selectivity for tumor versus normal tissue exposure. As a therapy for orthotopic brain tumors, nanoliposomal irinotecan showed a mean survival time of 54.2 versus 29.5 days for free irinotecan. A total of 33% of the animals receiving nanoliposomal irinotecan showed no residual tumor by study end compared with no survivors in the other groups. CONCLUSION Nanoliposomal irinotecan administered systemically provides significant pharmacologic advantages and may be an efficacious therapy for brain tumors.

Abstract 3912: MM-310, a novel EphA2-targeted docetaxel nanoliposome

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Taxanes are widely used to treat solid tumors either in the curative or palliative setting, in first or later lines of therapy. Analysis of docetaxel dose-response relationship strongly suggests that a higher dose would lead to high response, however will also lead to higher toxicity. This is likely related to the lack of organ and cellular specificity of docetaxel leading to high exposures in normal tissues and the relatively short circulation half-life which indirectly requires higher doses. With the goal of addressing the pharmacokinetic limitations of free docetaxel and the lack of cellular specificity, we developed a novel docetaxel-based nanoliposome (MM-310), targeted against Ephrin receptor A2 (EphA2) which is overexpressed in a wide range of tumors. MM-310 provides sustained release of docetaxel following accumulation in solid tumors. Preclinical models have demonstrated that MM-310 leverages tumor-specific accumulation through the enhanced permeability and retention effect, and cellular specificity through active targeting of EphA2 with specific scFv antibody fragments conjugated to the surface of the liposomes. Pharmacokinetic and biodistribution studies were performed in mice and rats to compare MM-310 to free docetaxel. Chronic tolerability studies were performed in rodent and non-rodent models with focus on overall animal health, as well as hematologic toxicities. Several cell-derived models of breast, lung and prostate xenografts were used to evaluate the differences betweenf MM-310 and free docetaxel. MM-310 had a significantly longer half-life than free docetaxel with prolonged exposure at the tumor site. In chronic tolerability studies, MM-310 was found to be 6-7 times better tolerated than free docetaxel with a maximum tolerated dose of at least 120 mpk, compared to 20 mpk for free docetaxel and no detectable hematological toxicity. At equitoxic dosing, MM-310 50 mpk showed greater activity than docetaxel 10 mpk in several breast, lung and prostate xenograft models. In conclusion, we developed a novel EphA2 targeted docetaxel nanoliposome with prolonged circulation time and slow and sustained drug release kinetics, to enable organ and cellular targeting. MM-310 was able to overcome hematologic toxicities observed upon treatment with free docetaxel in rodent and non-rodent models. MM-310 was also able to induce tumor regression or control tumor growth in several cell derived xenograft models, and was found to be more active than free docetaxel in most models. Citation Format: Dmitri B. Kirpotin, Suresh Tipparaju, Zhaohua Richard Huang, Walid S. Kamoun, Christine Pien, Tad Kornaga, Shinji Oyama, Ken Olivier, James D. Marks, Alexander Koshkaryev, Sarah S. Schihl, Gerald Fetterly, Birgit Schoeberl, Charles Noble, Mark Hayes, Daryl C. Drummond. MM-310, a novel EphA2-targeted docetaxel nanoliposome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3912.

SPECT-CT study of directed drug delivery using /sup 111/In-labeled liposomes in a murine mammary carcinoma model

Liposomal drugs offer the promise of an improved therapeutic index due to improvements in the specific delivery of anti-cancer agents to tumors. The presented work concentrates on imaging the tumor uptake of /sup 111/In-labeled liposomes noninvasively as a specific tumor drug delivery carrier in a murine cancer model. The tumor uptake of liposomes has been imaged using a microSPECT/microCT small animal dedicated scanner prototype constructed at our laboratory. The imaging system consists of a high resolution SPECT (700 /spl mu/m) and high resolution CT (70 /spl mu/m). The SPECT subsystem consists in specially designed CZT gamma cameras shielded for energies up to 250 keV. The mice were injected with liposomes and scanned at the time of maximum tumor uptake (24 h). The total activity of the mouse samples was of the order of 250 uCi, and with tumor sizes of 1000-1500 mm/sup 3/. The imaging geometries in the CT and SPECT acquisitions were selected to obtain high magnification and high efficiency to image the tumor located within the torso. The SPECT and CT projections were taken sequentially. The acquired images show that necrotic tumors can de imaged with high resolution to observe liposome inhomogeneous uptake.