# Presentation: 210711

## REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS

- <date/time>

**REGULATORY SCIENCE OF LIPOSOME DRUG PRODUCTS**

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

## 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**

- Burgess, June 28, 2001

## 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 **

- Burgess, June 28, 2001

## 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 μm 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**

- Burgess, June 28, 2001

## Drug Candidate Selection

**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**

- Burgess, June 28, 2001

## .

**	****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**

- Burgess, June 28, 2001

## SELECTION OF DELIVERY SYSTEM

**	**_***L***__***iposomes ***_**– 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**_

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## LIPOSOME FORMULATION

**	**** ****	**
***Processing methods:***
**	****E****xtrusion, u****ltrasonication and microfluidization  for hydrophobic drugs and **
**	****Reversed phase and freeze-thaw for hydrophilic drugs. **

- Burgess, June 28, 2001

## 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**
**	**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## CRITICAL FACTORS IN LIPOSOME PREPARATIONJ

**	****Particle size **
**	****Method of manufacture**
**	****Lipid types**
**Phase transition temperature**
**	****Polymerization**
**	****Interfacial charge**
**	****Steric stabilization**
**	****Sterilization**

- Burgess, June 28, 2001

## 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.*****	***

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## LIPOSOME CHARACTERIZATION

**	**
**StabilIty –**
**Drug**
**Lipids**
**Liposome**
**Phase transition temperature**
**Percent drug loading**
**Percent free drug**
**Drug release rate/stability**
**Particle size**
**Morphology (lamellarity)**
**Sterility**

- Burgess, June 28, 2001

## STERILITY

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

- Burgess, June 28, 2001

## 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.  **

- Burgess, June 28, 2001

## LIPOSOME DESTABILIZATION

**	**
**Protein binding**
**Membrane fusion**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## In Vitro Drug Release

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

- Burgess, June 28, 2001

## 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**** **

- Burgess, June 28, 2001

## 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.**** **

- Burgess, June 28, 2001

## Current Methods of In Vitro Testing of Liposome Systems

**Membrane Diffusion Technique**
**Sample and Separate Technique**
***In Situ***** Technique**
**Continuous Flow Technique**

- Burgess, June 28, 2001

## 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 **

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## Media and Methods that can affect Release

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

- Burgess, June 28, 2001

## In Vivo Factors Affecting Drug Release

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

- Burgess, June 28, 2001

## 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**

** **

- Burgess, June 28, 2001

## Slide 38

- Burgess, June 28, 2001