REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS

Outline
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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
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

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 can be subcategorized into:
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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
- 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 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
LIPOSOME FORMULATION
LIPOSOMES
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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

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

and Reversed phase and freeze-thaw for hydrophilic drugs.

Liposomes: Factors Affecting Performance

Release Rate and Stability
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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
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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
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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
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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
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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

o
o
o
o
o
o
o

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 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
In Vitro Drug Release

Apparatus?

Media?

Sampling methods?

Testing intervals?

Total percent release?

No standard method at present

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