Burgess, June 28, 20011 REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS Diane J. Burgess, Ph.D. Professor of Pharmaceutics University of Connecticut Office

1Burgess, June 28, 2001

REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS

Diane J. Burgess, Ph.D.Professor of Pharmaceutics University of Connecticut

Office of Testing and Research

CDER, FDA

Burgess, June 28, 2001 2

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


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

LIPOSOMESLiposomes 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.

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.


LiposomesLocalized 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


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


.APPROVED LIPOSOME PRODUCTS:– Doxil Daunorubicin 1995– Daunoxome Daunorubicin

1996– Ambisome Amphotericin B 1997– Depocyt Cytarabine 1999

APPROVED LIPID COMPLEX PRODUCTS:– Ambelcet Amphotericin B 1995– Amphotec Amphotericin B 1997


SELECTION OF DELIVERY SYSTEMLiposomes – 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


LIPOSOME FORMULATIONLIPOSOMESLiposomal 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


LIPOSOME FORMULATION

Processing methods:Extrusion, ultrasonication and microfluidization for hydrophobic drugs and

Reversed phase and freeze-thaw for hydrophilic drugs.


Liposomes: Factors Affecting Performance Release Rate and Stability

Phase transition temperature (Tg) 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


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


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


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


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


CRITICAL FACTORS IN LIPOSOME PREPARATIONJ

• Particle size • Method of manufacture• Lipid types

• Phase transition temperature• Polymerization• Interfacial charge• Steric stabilization• Sterilization


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.


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 um) - Blockage of capillaries Size affects RES uptake and tissue targeting

Stability: shelf-live and in vivo Dose dumping (via protein binding) Sterility


LIPOSOME CHARACTERIZATION StabilIty –

Drug Lipids Liposome

Phase transition temperature Percent drug loading Percent free drug Drug release rate/stability Particle size Morphology (lamellarity) Sterility


STERILITY

Terminal sterilization?

Aseptic processing

Must consider both internal and external sterility


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.


LIPOSOME DESTABILIZATION

Protein binding

Membrane fusion


Drug Release from LiposomesRelease 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


In Vitro Drug Release

Apparatus? Media? Sampling methods? Testing intervals? Total percent release?

No standard method at present


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


Liposome Performance – In Vitro Release and Stability

• Release 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.


Current Methods of In Vitro Testing of Liposome Systems

• Membrane Diffusion Technique

• Sample and Separate Technique

• In Situ Technique

• Continuous Flow Technique


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


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


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


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


Media and Methods that can affect Release

• Solvents• pH• Temperature• Agitation• Enzymes• Cell culture• Sink conditions• Volume• Sampling interval


In Vivo Factors Affecting Drug Release


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


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


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

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Burgess, June 28, 20011 REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS Diane J. Burgess, Ph.D. Professor of Pharmaceutics University of Connecticut Office