REGULATORY SCIENCE OF LIPOSOME

DRUG PRODUCTS

Burgess, June 28, 2001

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

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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

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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.

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 m are known as giant vesicles.

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

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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
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APPROVED LIPOSOME PRODUCTS:

.
Daunorubicin

Doxil
Daunoxome Daunorubicin
Ambisome Amphotericin B
Depocyt
Cytarabine

1995
1996
1997
1999

APPROVED LIPID COMPLEX PRODUCTS:

Ambelcet Amphotericin B
Amphotec Amphotericin B
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1995
1997
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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

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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

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LIPOSOME FORMULATION
Processing methods:
Extrusion, ultrasonication and
microfluidization for hydrophobic drugs and
Reversed phase and freeze-thaw for
hydrophilic drugs.

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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

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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

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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

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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
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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

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CRITICAL FACTORS IN LIPOSOME PREPARATIONJ

Particle size
Method of manufacture
Lipid types
Phase transition temperature

Polymerization
Interfacial charge
Steric stabilization
Sterilization

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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.

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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

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LIPOSOME CHARACTERIZATION
StabilIty
Drug
Lipids
Liposome

Phase transition temperature

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

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STERILITY
Terminal sterilization?
Aseptic processing
Must consider both internal and
external sterility

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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.

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LIPOSOME DESTABILIZATION
Protein binding
Membrane fusion

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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

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In Vitro Drug Release

Apparatus?
Media?
Sampling methods?
Testing intervals?
Total percent release?
No standard method at present

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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
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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.
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Current Methods of In Vitro Testing

of Liposome Systems
Membrane Diffusion Technique
Sample and Separate Technique
In Situ Technique
Continuous Flow Technique
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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
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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

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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

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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
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Media and Methods that can affect Release

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

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In Vivo Factors Affecting Drug

Release

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In Vivo Factors
Delivery System Independent (Type I)
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

Delivery System Dependent (Type II)

Enzymatic degradation of delivery system
Protein adsorption
Phagocytosis
Inflammatory response
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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
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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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