1. Burgess, June 28, 20011 REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS Diane J. Burgess, Ph.D. Professor of Pharmaceutics University of Connecticut Office of Testing and Research CDER, FDA

2. Burgess, June 28, 20012 Outline What are liposomes? What are they used for? What drugs? Why liposomes? Liposome formulation Liposome characterization Safety concerns Performance concerns – In vitro release testing – stability

3. Burgess, June 28, 20013 Outline Continued Purpose of in vitro release tests? Design of in vitro release test Accelerated/stress tests Method variables affecting release Methods under development In vivo factors affecting release In vivo data and models? IVIVC? Research proposal

4. LIPOSOMES Liposomes are colloidal, lipid vesicles consisting of one or more self-assembled lipid bilayers enclosing a similar number of aqueous compartments. Lipids, such as lecithin (diacylphosphatidylcholine), are amphiphilic molecules. Due to the bulky nonpolar part of the molecule they do not pack into spherical micelles in aqueous phase but rather self-assemble into bilayers which tend to self-close at low concentrations into spherical structures.

5. LIPOSOMES Contd. Liposomes can be subcategorized into: Small unilamellar vesicles (SUV), 25 to 100 nm in size that consist of a single lipid bilayer Large unilamellar vesicles (LUV), 100 to 400 nm in size that consist of a single lipid bilayer Multilamellar vesicles (MLV), 200 nm to several microns, that consist of two or more concentric bilayers Vesicles above 1 μm are known as giant vesicles.

6. Burgess, June 28, 20016 Liposomes Localized and rate controlled delivery: Improved therapeutic response – Achieve appropriate tissue or blood levels Reduced adverse reactions – Less drug administered – Targeted drug release Lower dosing frequency – Improved patient compliance – Simpler dosing regimens – Lower cost per dose Utilization of otherwise un-useable compounds – Poorly soluble drugs

7. Burgess, June 28, 20017 Drug Candidate Selection Known therapeutics with clear toxicity and pharmacokinetic profiles – Potent compounds – Not “Narrow Therapeutic Index” drugs Problems associated with the current dosage forms – First pass effects or poor absorption – Gastric irritation – Rapid clearance Medical need for improved delivery Drugs compatible with manufacturing conditions

8. Burgess, June 28, 20018. APPROVED LIPOSOME PRODUCTS: – Doxil Daunorubicin1995 – DaunoxomeDaunorubicin1996 – AmbisomeAmphotericin B1997 – DepocytCytarabine1999 APPROVED LIPID COMPLEX PRODUCTS: – AmbelcetAmphotericin B1995 – AmphotecAmphotericin B1997

9. Burgess, June 28, 20019 SELECTION OF DELIVERY SYSTEM Liposomes – targeted delivery. They can deliver agents directly into cells. Routes: i.v., s.c., i.m., topical, pulmonary Microspheres - can provide continuous drug delivery over periods of months to years. Systemic and localized. i.m., s.c., oral, pulmonary Emulsions - can be used to make highly water insoluble compounds bioavailable. i.v., oral, topical

10. Burgess, June 28, 200110 LIPOSOME FORMULATION LIPOSOMES Liposomal composition determines the properties (e.g. surface charge, rigidity and steric interactions) and the in vitro and in vivo performance. Both water soluble and water insoluble drugs may be encapsulated Processing methods affect particle size, percentage drug entrapment, stability and release rates

11. Burgess, June 28, 200111 LIPOSOME FORMULATION Processing methods: E xtrusion, u ltrasonication and microfluidization for hydrophobic drugs and Reversed phase and freeze-thaw for hydrophilic drugs.

12. Burgess, June 28, 200112 Liposomes: Factors Affecting Performance Release Rate and Stability Phase transition temperature (T g ) effects membrane changes from ordered solid to disordered fluid and is dependent on the length and degree of saturation of the hydrocarbon chains. Cholesterol - disordering of the ordered phase and ordering of the disordered phase eventually leading to an elimination of the phase transition. High stability and low leakage Surface charge and steric interaction : RES targeting/avoiding RES uptake

13. Burgess, June 28, 200113 Types of Liposomes Conventional Liposomes - Prepared form natural neutral and anionic lipids and have nonspecific interactions with their environment - Relatively unstable, have low carrying capacities, and tend to be “leaky” to entrapped drug substances - May literally fall apart on contact with plasma, particularly those of high fluidity, - Choleterol is often added to increase plasma stability

14. Burgess, June 28, 200114 Types of Liposomes Non-conventional Liposomes - Small sized (≤ 100 nm), surface modified to overcome some of the short comings of conventional liposomes - Modified to reduce negative charge, decrease fluidity and cause steric hinderance to phagocytosis - Properties altered (e.g. by incorporation of cholesterol) - Polymerized liposomes more stable and less “leaky” - Polyetheylene glycol, “pegylated” liposomes, avoid uptake by the mononuclear phagocytic cells

15. Burgess, June 28, 200115 TYPES OF LIPOSOMES Target specific ligands, such as antibodies, immunoglobulins, lectins and oligosaccharides attached to the surface to actively target to specific sites in the body Targeting via particle size Liposomes prepared with cationic and fusogenic lipids are currently being utilized in gene therapy to deliver DNA into target cells

16. Burgess, June 28, 200116 TYPES OF LIPOSOMES Highly reactive liposomes - readily undergo phase transition in particular situation – sensitive to pH, ions, heat and light – For example, pH-sensitive liposomes can undergo phase transition in acidic conditions resulting in increased membrane fluidity and loss of encapsulated materials

17. Burgess, June 28, 200117 CRITICAL FACTORS IN LIPOSOME PREPARATIONJ Particle size Method of manufacture Lipid types Phase transition temperature Polymerization Interfacial charge Steric stabilization Sterilization

18. Burgess, June 28, 200118 Liposomes: Factors Affecting Performance Liposome preparations can be stored: frozen, in liquid form and as a freeze dried powder. Reconstitution of liposomes may affect particle size and size distribution.

19. Burgess, June 28, 200119 SAFETY CONCERNS: LIPOSOME FORMULATION  Lipid toxicity (RBC lysis)  Type and concentration  % Lyso-lipids  Presence of protein and lipoprotein for natural lipids  Residual solvent  Overload of RES  Particle size  ( tail above 1 u m) - Blockage of capillaries  Size affects RES uptake and tissue targeting  Stability: shelf-live and in vivo  Dose dumping (via protein binding)  Sterility

20. Burgess, June 28, 200120 LIPOSOME CHARACTERIZATION  StabilIty –  Drug  Lipids  Liposome  Phase transition temperature  Percent drug loading  Percent free drug  Drug release rate/stability  Particle size  Morphology (lamellarity)  Sterility

21. Burgess, June 28, 200121 STERILITY  Terminal sterilization?  Aseptic processing  Must consider both internal and external sterility

22. Burgess, June 28, 200122 STABILITY  Active  Inactives (especially the lipids)  Liposome as a whole need  Any change in particle size can affect targeting, RES uptake, safety and efficacy.  In vivo stability of whole liposome is particularly important for targeted liposomes, since they should remain stable in the plasma without loss of contents until uptake at the target site.

23. Burgess, June 28, 200123 LIPOSOME DESTABILIZATION  Protein binding  Membrane fusion

24. Burgess, June 28, 200124 Drug Release from Liposomes Release profiles are application dependent.  Targeted liposomes should remain intact until delivery at site  Other (short term CR and solubilization) release during appropriate time scale. Release controlled by  Fluidity/stability (lipids/co-lipids)  Condition sensitivity of lipids  Size  MLV or a SUV  Physicochemical properties of drug  Drug/lipid interaction

25. Burgess, June 28, 200125 In Vitro Drug Release  Apparatus?  Media?  Sampling methods?  Testing intervals?  Total percent release?  No standard method at present

26. Burgess, June 28, 200126 Liposome Performance – In Vitro Release and Stability Separation of liposomes from dissolution media complicates testing Current USP methods designed for oral and transdermal routes In vitro tests need to take into account the expected in vivo performance of liposomes

27. Burgess, June 28, 200127 Liposome Performance – In Vitro Release and Stability R elease test for a targeted liposome would need to show – 1) liposome is stable until uptake at the site – 2) liposome releases drug at the site (based on the mechanism of release in vivo ). Release test for an immediate release liposome would need to show – Drug is released immediately in conditions mimicking human plasma.

28. Burgess, June 28, 200128 Current Methods of In Vitro Testing of Liposome Systems Membrane Diffusion Technique Sample and Separate Technique In Situ Technique Continuous Flow Technique

29. Burgess, June 28, 200129 Development of In Vitro Release and Stability Methods for Liposomes Purpose: methods to be used in setting regulatory specifications for these products for quality control (QC) purposes to differentiate between “good” and “bad” batches. Tests design will vary depending on the intended in vivo performance of liposomes

30. Burgess, June 28, 200130 Purpose of In Vitro Release Test? Quality control and safety evaluation – Batch to batch – Manufacturing process changes – Substantiation of label claims – Evaluation of potential dose dumping – Assessment of in vivo stability “Real time” vs accelerated/stress test In vitro - in vivo correlation

31. Burgess, June 28, 200131 Design of In Vitro Release Method Select media and apparatus to achieve reproducible results – Attempt to overcome limitations of existing methods – Miniaturize methods Prepare formulation variants with different in vivo performance Test formulation variants in vitro and in vivo Modify in vitro test if not discriminatory – Determine in vivo factors that effect release Modify in vitro methods to obtain IVIV relationship

32. Burgess, June 28, 200132 Accelerated In Vitro Release Methods These tests should be predictive of “real time” in vitro tests Drug release mechanism should not be altered Accelerated test should not simply dissolve the liposome

33. Burgess, June 28, 200133 Media and Methods that can affect Release Solvents pH Temperature Agitation Enzymes Cell culture Sink conditions Volume Sampling interval

34. Burgess, June 28, 200134 In Vivo Factors Affecting Drug Release

35. Burgess, June 28, 200135 In Vivo Factors Delivery System Independent (Type I) Delivery System Dependent (Type II) – Barriers to drug diffusion: fluid viscosity, – tissue barriers (e.g. connective tissue) – Drug partitioning at the site – Available volume at the site – Motion at Site – Enzymatic degradation of delivery system – Protein adsorption – Phagocytosis – Inflammatory response

36. Burgess, June 28, 200136 In Vivo Data Systemic delivery, then plasma levels may be suitable Localized delivery, plasma levels will be low and unrepresentative. Requires tissue levels Use animal models in method development Use Biomarkers

37. Burgess, June 28, 200137 In Vivo Data Use animal model to help design in vitro test Establish relationship between in vitro data and animal in vivo data Establish a relationship between animal in vivo data and human PK, biomarkers, PD response Develop relationship between in vitro data Human data

38. Burgess, June 28, 200138