TRANSPORT STUDIES IN SUBMICRONSIZED TRIPHASIC EMULSION

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REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS

• Diane J. Burgess, Ph.D.
• Professor of Pharmaceutics
• University of Connecticut
• Office of Testing and Research
• CDER, FDA

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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 (diacylphosphatidylcholin
  e), 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.

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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.

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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
• 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

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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)
• 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

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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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