comparative incomparative in-vitro characterization of
TRANSCRIPT
Comparative in vitroComparative in-vitro characterization of liquidcharacterization of liquid liposomal formulations
Daan J.A. Crommelin
September 21, 2012BonnBonn
Utrecht Institute for Pharmaceutical SciencesUtrecht UniversityUtrecht University
The Netherlands
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9/24/2012From Gregoriadis, 1979From Gregoriadis, 1979
Issues covered
Liposomes……– introduction/positioning– components– structure– morphologyp gy– manufacturing, stability– quality control FDA EMAquality control, FDA, EMA
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Approved Liposome-based Drug Products**
Product Year Approved API Sparingly Revenue Soluble
Visudyne® 2000 Yes 90 M
DOXIL/Caelyx® 1995
AmBisome® 1990
Verteporfrin
Doxorubicin
Amphotericin B Yes 400 M $*
No 550 M $*
AmBisome® 1990
ABELCET® 1995
Definity® 2001
Amphotericin B
Amphotericin B
Octafluoropropane
Yes 400 M $
Yes
Yes
2002DepoCyte®
y
Cytarabine
p p
Myocet® 2001 Doxorubicin NoNo
DepoDur® 2004 Morphine
Daunoxome® Daunorubicin1996No
NoOctocog alfa® 2009 Factor VIII No
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Octocog alfa® 2009 Factor VIII
* Thomson Pharma** not exhaustive
No
What is in the pipeline?What is in the pipeline?
Thomson-Pharma, 2010
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Liposomes: which actives?H d hili A hi hiliHydrophilic molecules
Weak bases and weak acids
Amphiphilic molecules
Depending on the partition coefficient
Covers a wide range
hydrophilic proteins
bupivacaine ferulic acid
Covers a wide range of molecules
hydrophilic peptides
5’-fluorouracil cisplatine daunorubicin
doxorubicin
Transmembrane gradient
( pH, chemical salts)
Hydrophobic
Nucleic acid based-
drugsHydrophobic molecules
lidocaine
g
DNA
dibucaine
ibuprofenRNA
cyclosporin
paclitaxel
(Ph h )li id(Ph h )li id(Phospho)lipids(Phospho)lipidsO
C H 3
OCH 3
CH 3 O OC H
C H 2
n
+
P CCH 2
CH 2
N C H 3C H 3
C H 3O
3
P
O
CH 2 OO
n
O
+
P ECH 2
CH 2
N H 3OP
O
O
O+
CH 2
CH
C H 2OP
O
O
O
O HO HP G
O
D O T A PN C H 3C H
C H 3+
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C H 3
N i i ‘ th ti ’ f t t
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Non-ionic, ‘synthetic’ surfactants
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F t t li th f t fFactors controling the fate of liposomes in vivo after intravenous
administration:
- size of the liposomes (0.03 - 20 µm)p ( µ )- type (morphology) (unilamellar, multilamellar,
multivesicular)- charge of the bilayer (negative neutral positive)charge of the bilayer (negative, neutral, positive)- rigidity of the bilayer (gel/fluid state)- route of administration
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Evolution in Liposome Science
- 1st generationconventional liposomes- conventional liposomes
- 2nd generationimmunoliposomes- immunoliposomes
- long circulating liposomescationic liposomes- cationic liposomes
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Some pharmaceutical aspects:What are potential problems encountered in theWhat are potential problems encountered in the
development process of liposomes?
- poor quality of the raw material, the phospholipids- poor characterization of the physico-chemical propertiespoor characterization of the physico chemical properties
of the liposomes - ‘pay load’ is too low- shelf life is too short- shelf life is too short- scaling up problems occur- absence of (any) data on safety of these carrier systems
on chronic useon chronic use- ........
A l h b i h d hApparently, the above issues have not stopped the successful launching of drug-liposome products on the market....
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Sterility problemsy p
Rejected.....
Administration is exercising its enforcement discretion for the temporary importation and distribution of Sun Pharma Global'sLipodox™ (doxorubicin hydrochloride liposome injection) in the
9/24/2012 Message: manufacturing liposomes is not a trivial job.....
Lipodox™ (doxorubicin hydrochloride liposome injection) in the United States by Sun Pharma Global
9/24/2012
(Ph h )li id(Ph h )li id(Phospho)lipids(Phospho)lipidsO
C H 3
OCH 3
CH 3 O OC H
C H 2
n
+
P CCH 2
CH 2
N C H 3C H 3
C H 3O
3
P
O
CH 2 OO
n
O
+
P ECH 2
CH 2
N H 3OP
O
O
O+
CH 2
CH
C H 2OP
O
O
O
O HO HP G
O
D O T A PN C H 3C H
C H 3+
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C H 3
Liposome classification: THE li d t i t!!!THE liposome does not exist!!!
• A) Based on structural parameters
MLV :Multilamellar large vesicles - > 0.5 µmOLV: Oligolamellar vesicles - 0.1 - 1 µmUV: Unilamellar vesicles (all size range)SUV: Small unilamellar vesicles - 20-100 nmSUV: Small unilamellar vesicles 20 100 nmMUV: Medium sized unilamellar vesiclesLUV: Large unilamellar vesicles - >100 nmGUV: Giant unilamellar vesicles (vesicles with diameters > 1 µm)MVV: Multivesicular vesicles (usually large > 1µm)
B) Based on method of liposome preparation
REV: Single or oligolamellar vesicles made by reverse-phase evaporation methodMLV-REV: Multilamellar vesicles made by the reverse phase evaporation
h dmethodSPLV: Stable plurilamellar vesiclesFATMLV: Frozen and thawed MLVVET: Vesicles prepared by extrusion methods
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VET: Vesicles prepared by extrusion methodsDRV: Dehydration-rehydration vesicles
Type of Vesicle Geometry
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9/24/2012Crommelin and Storm, 1983
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Homogenizers and Extruders
• Avestin (Canada)
• APV (German)
• Microfluidics (US)• Microfluidics (US)
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Loading of liposomes withLoading of liposomes with drugsdrugs
Passive loading:
d di l d i i di• drug dissolved in organic or aqueous medium during liposome formation
loading efficiency is 5 100% depending on– loading efficiency is 5 - 100%, depending on hydrophobicity, vesicle size and preparation methodmethod
Active loading (after sizing; 11 patented methods):Active loading (after sizing; 11 patented methods):
– up to 100% loading efficiency for drugs with a pH dependent partition-coefficient
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p p p
Active Loading of DoxorubicinActive Loading of Doxorubicin Barenholz csBarenholz cs
by Ammonium Sulfate Gradient Methodby Ammonium Sulfate Gradient Method
Liposome A i
External Medium
2NH3
Aqueous Phase
Ammonia escapes
Medium2DXR-NH3Cl
2NH3 + 2H+ 2NH4+ + SO4
-2 2DXR- H3 N
+ + 2Cl-
2DXR-NH2(DXR-NH3 )2SO4 2DXR-NH2 +Gel-like Precipitate
2
2H+
Neutral form of DXR crosses
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DXR crosses bilayer
St t f D il®Structure of Doxil®
Doxorubicin
Lipid Membrane (Phospholipid +
Cholesterol)85-100 nm85-100 nm
Cholesterol)
Polyethylene Glycol
Doxil ProcessDoxil Process
From Frank MartinFrom Frank Martin
LipidsLipids
EthanolEthanolUnsized Liposome FormationUnsized Liposome Formation
Hydration
Doxil ProcessDoxil Process
Ammonium Sulfate Ammonium Sulfate BufferBuffer
Unsized Liposome FormationUnsized Liposome Formation
Extrude sequentially through capillary pore filters of decreasing pore Extrude sequentially through capillary pore filters of decreasing pore diameterdiameter
Down-Sizing
BufferSucroseSucrose
Flow Flow DialysisDialysis
FiltrateFiltrateBuffer
Exchange Ethanol Removal
Drug Loading Add Doxorubicin HClAdd Doxorubicin HCl
Sterilization
Dilution Measure Drug Conc/QS to Label StrengthMeasure Drug Conc/QS to Label Strength
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Sterilization and Fill
Sterile filter through 0.22 Sterile filter through 0.22 filterfilter
Aseptic FillAseptic Fill
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Different bilayers
Same physicochemical characteristics
Does it make a difference in vivo?vivo?
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Stability issues re aqueous liposome dispersions (shelf life preferably > 2
years)yea s)
- chemical degradationchemical degradation - hydrolysis reactions of lipids- oxydation of lipids
- physical instability- aggregation
fusion- fusion- leakage of drug
S l i i l bili bl (f ) d iSolutions to potential stability problems: (freeze) drying, careful selection of (phospho)lipids, filling conditions, ....
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(Ph h )li id(Ph h )li id(Phospho)lipids(Phospho)lipidsO
C H
OCH 3
CH 3 O OC H
C H 2
O
n
+
P CCH 2
CH 2
N C H 3C H 3
C H 3O
CH 3 O
P
O
O
CH 2
C H
OO
n
O
+
P ECH 2
CH 2
N H 3OP
O
O
O+Oxidation
CH 2
CH
C H 2OP
O
O
O
O HO HP G
Hydrolysis
O O HO H
D O T A PN C H 3C H
C H 3+
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C H 3
9/24/2012Grit and Crommelin
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GMP compliant production
+ Aseptic processing vs. end sterilisation
+ P d t ifi lid ti f filt+ Product specific validation of filters on microbial retentivity
+ Quality/origin of lipids (natural vs. synthetic)
+ Production process: type of liposomes governs type of process
To assure the quality of liposomal formulations a number of evaluationformulations a number of evaluation
tests are available:Assay/ Methodology/Analytical Target Characterization
pH pH meterOsmolarity OsmometerPhospholipid concentration Lipid phosphorus content/HPLCPhospholipid concentration Lipid phosphorus content/HPLCPhospholipid composition TLC, HPLCCholesterol concentration Cholesterol oxidase assay, HPLCDrug concentration .......g
Chemical stabilitypH pH meterPhospholipid peroxidation conjugated dienes, lipid peroxides
FA composition (GLC)Phospholipid hydrolysis HPLC, TLC, FA concentration
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Cholesterol autooxidation HPLC, TLCAntioxidant degradation HPLC, TLC
Based on Barenholz an Crommelin, 1994)
To assure the quality of liposomal formulations a number of evaluationformulations a number of evaluation
tests are available:Ph i l t bilit• Physical stabilityVesicle size distributionsubmicron range DLS micron range Coulter Counter, light microscopymicron range Coulter Counter, light microscopy
laser diffraction, GECElectrical surface potential, surface pH/zeta-potential measurements, pH
sensitive probesN b f bil SAXS NMRNumbers of bilayers SAXS, NMRPercentage of free drug GEC, IEC, protamine precipitationDilution-dependent drug release retention loss on dilutionRelevant body fluid induced leakage GEC, IEC, protamine precipitationy g , , p p p
Biological characterizationSterility aerobic and anaerobic culturesP i it bbit LAL t tPyrogenicity rabbit or LAL testAnimal toxicity monitor survival, histology, pathology
(Based on Barenholz an Crommelin, 1994)
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SAXS = small angle X-ray scattering, DLS = dynamic light scattering, GEC = gel exclusion chromatography, IEC = ion exchange chromatography, LAL = Limulus Amoebecyte Lysate, NMR = nuclear magnetic resonance, SAXS = small angle X-ray scattering, TLC = thin layer chromatography.
Particle size measurement
• Electron microscope:negative staining– negative staining
– freeze fracture
– cryo-TEM
• Dynamic light scatteringDynamic light scattering– Typical instrumentation is manufactured by
e g Malvern Instruments Ltd ( UK)e.g. Malvern Instruments Ltd. ( UK), Beckman Coulter, Inc. (USA), Nicomp (USA) and ALV GmbH Germany)
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(USA) and ALV-GmbH, Germany)
Cryo-TEM
9/24/2012Van Buul and Crommelin 2001
Electron microscope pictures of liposomepictures of liposomedispersion sequentially extruded through gmembranes: from 0.6 micrometer d t 0 2 i tdown to 0.2 micrometer
.
Bar 0.5 micrometer
Electron microscope Electron microscope pictures of liposomedispersion sequentially extruded through gmembranes: from 0.6 mm down to 0.2 mm.
.
B 0 5 i t
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Bar 0.5 micrometer
In Vitro In Vitro releaserelease
Media
Dialysis tube
Liposomes
Dialysis tube
Reverse dialysis sacDialysis sac Reverse dialysis sacDialysis sac
Media: 50 ml HEPES buffer, 10 mM, pH 7.4
Media: 125 ml HEPES buffer, 10 mM, pH 7.4
Membrane: Spectrapor Dispodialyzer 50 kDa Membrane: Spectrapor Dispodialyzer
50 kDa MWCO
Membrane: Spectrapor Dispodialyzer 50 kDa MWCO
N. Chidambaram and D.J. Burgess. AAPSPharmSci., (1999), August 31, 1999; 1(3)
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G id f I d tGuidance for Industry
Liposome Drug Products
S b i i f Ch i M f i d C lSubmission of Chemistry, Manufacturing, and Controls,Human Pharmacokinetics and Bioavailability and Labeling
Information
DRAFT GUIDANCE
This guidance document is being distributed for comment purposes only.
Comments and suggestions regarding this draft document should be submitted within 90 days of publication inthe Federal Register of the notice announcing the availability of the draft guidance. Submit comments toDockets Management Branch (HFA-305), Food and Drug Administration, 12420 Parklawn Dr., rm. 1-23,Rockville, MD 20857. All comments should be identified with the docket number listed in the notice ofavailability that publishes in the Federal Register. For questions regarding this draft document contact XXX.
U.S. Department of Health and Human Services
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U.S. Department of Health and Human ServicesFood and Drug Administration
Center for Drug Evaluation and Research (CDER)XXX, 2001 Mei-Ling Chen
FDA issues
• Bioavailability/Bioequivalence– release of active moiety from liposomes
– availability at site of actionavailability at site of action
• CMC issues– characterization of drug product
– physicochemical characteristics• size, lamellarity, free/associated
9/24/2012 Shaw/KumiShaw/Kumi
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C l iConclusions
• The liposome does not exist
• Liposomes made it to the market because:because:– Basic know how on their behavior in vivo
was collected over yearswas collected over years
– Pharmaceutical challenges were d t l tadequately met
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