Development of a Liposome Doxorubicin Product Drug Release Assay A barrier to the rational assessment for regulatory purposes of generic drug delivery system for parenterally administered drugs is the absence of validated mechanism-based drug release assays that reflect or predict in vivo drug release from the carrier. One such FDA licensed drug carrier is the sterically stabilized liposome product known as Doxil(R). Members of our team played important roles in the early development of Doxil(R) a liposome formulation that was specifically designed to be exceptionally stable at 37?C in the presence of serum in order to provide a sustained release of drug over the course of three to four weeks. This inherent stability is a barrier when it comes to create in vitro doxorubicin release systems. Herein we propose a mechanism-based plan to devise, assess and validate in vitro release assays (also designated as dissolution assays) that distinguish liposome formulations with variations of composition and physical properties. Based upon the in vitro release profile of the innovator product Doxil(R), we will identify assays that better correlate with Doxil(R)'s in vivo drug release profile. In the background section, we provide a critical review of liposome content release assays for liposome formulations. We use this information to create a research plan with four specific aims. In the background section, we provide a critical review of content release assays for liposome formulations.
In aim 1, we prepare and characterize a series of liposome formulations that span the range of compositions, source of materials and physical properties that can be encompassed by the product description of Doxil(R). The formulations are prepared using the technologies used in the manufacture of Doxil(R) and include modifications to the mean particle diameter, hydrogenated phosphatidylcholine lipid acyl chain composition, pegylated lipid mole percent, ammonium sulfate concentration, residual sulfate concentration in the liposome, internal pH, number of lamellae, suspending buffer composition and doxorubicin concentration. These liposome formulations will be compared in subsequent aims to the innovator product for drug retention and release characteristics.
In aim 2, we will devise and validate multiple drug release assays that define release at multiple temperatures in the presence of whole blood, serum components or synthetic materials that absorb cholesterol, lipids or doxorubicin in a traditional flow through device where the liposomes are retained by dialysis membranes or in single unit systems that require separation of the liposomes from the released drug on size exclusion columns followed by assessment in the change in liposome chemical composition or physical properties as a function of time. In this aim, we also will introduce and validate a cell-based transwell assay that involves maintaining periphery blood mononuclear cells (PBMC) in one compartment and cancer cells lines that are sensitive to doxorubicin in a second compartment. The liposomes will be introduced to the PBMC compartment and the effect of released doxorubicin on cell viability/proliferation will be monitored on cancer cells in the second compartment.
In aim 3, we will vary the drug release (dissolution) conditions and measure the extent of release and variability of release as a function of the principle components and operating conditions of the release assay. The innovator product Doxil(R) will serve as the gold standard for setting the operating conditions that lead to reproducible release of drug as a function of time until 66% of the doxorubicin content of the liposome is released. LipoDox(R) and the test formulations with the above noted variations will be assessed in the assays that provide the most consistent release and the greatest extent of release within a seven day period.
In aim 4, we will identify those drug release assays which better correlate with published in vivo human data and identify the method or methods that provide better predictions of the in vivo release profile. Our goal is to create a standard doxorubicin release assay with precisely specified conditions, devices and components so that others can readily assess if follow-on liposome doxorubicin formulations have equivalent doxorubicin release in these relevant release assays to what is measured using Doxil(R).

Public Health Relevance

Development of a Liposome Doxorubicin Product Drug Release Assay Project Relevance Our goal is to devise novel liposome drug release assays that are robust and predictive of doxorubicin in vitro release from Doxil(R)-like sterically-stabilized liposomes and to challenge these assays with liposome formulations that span the range of compositions and physical structures that met the requirements for a generic Doxil(R) formulation. Based upon the validation experiments, we will propose doxorubicin release assays that can be used to identify liposome formulations that are indistinguishable in drug release and physical properties from the innovator product Doxil(R). This study is intended to advance the regulatory review process of generic complex parenteral products that will help alleviate shortages of drugs like Doxil(R) that can be devastating for cancer patients and their families.

National Institute of Health (NIH)
Food and Drug Administration (FDA)
Research Project--Cooperative Agreements (U01)
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Special Emphasis Panel (ZFD1)
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Zoneone Pharma, Inc.
San Francisco
United States
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Yuan, Wenmin; Kuai, Rui; Dai, Zhipeng et al. (2017) Development of a Flow-Through USP-4 Apparatus Drug Release Assay to Evaluate Doxorubicin Liposomes. AAPS J 19:150-160