A Practical View on the Use of
Phospholipids in Pharmaceutical
Formulations
chemical, regulatory and application aspects
J. Zirkel
16.09.2013
Definition of Lecithin
USP:
“Lecithin is a complex mixture of acetone-insoluble
phosphatides, which consist chiefly of phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine and
phosphatidylinositol, combined with various amounts of
other substances such as triglycerides, fatty acids and
carbohydrates, as separated from the crude vegetable oil
source. It contains not less than 50% of “AcetoneInsoluble Matter“.
Regulatory and Safety Aspects (I)
World Health Organization (WHO)
•
No limit placed on the oral intake of lecithin
•
No ADI value (Acceptable Daily Intake) for lecithin as a food additive
(WHO, Food Additives Series, 1974, No. 5, p.234)
•
Soybean and egg lecithin (unsaturated and saturated) are non-toxic even
when given parenterally
By injection of lipid emulsions up to 12 g of
phospholipids are administered per day!
Regulatory and Safety Aspects (II)
European Commission
•
Lecithin is a food additive (E322) “generally permitted for use in foodstuffs
...“
Annex I of directive 95/2/EC of 20 February 1995
http://ec.europa.eu/food/fs/sfp/addit_flavor/flav11_en.pdf
•
No ADI value has been fixed for lecithin in Europe; the material may be
used „quantum satis“
http://ec.europa.eu/food/fs/sfp/addit_flavor/flav15_en.pdf
Regulatory and Safety Aspects (III)
United States Food and Drug Administration (FDA)
• GRAS (Generally Recognized As Safe) affirmation for commercial
lecithin (21 CFR § 184.1400 revised as of April 1, 2004)
• FDA Center for Drug Evaluation and Research lists various
approved drug products containing different phospholipids types as
inactive ingredients
Phospholipids – Production and Quality Aspects
Sources for lecithin and phospholipids
Vegetable Sources
Animal Sources
• Soybean
• Egg yolk
• Rapeseed
• Milk
• Sunflower
• Bovine brain (mainly PS)
• Wheat Germ
• Krill
• Flaxseed
Synthesis
Phospholipids – Structure and Properties
Phosphatidylcholine (PC)
Zwitterionic
at physiological pH
Monoacyl PC (LPC)
Zwitterionic
at physiological pH
Phosphatidylethanolamine (PE)
Negatively charged
at basic pH
Phosphatidylglycerol (PG)
Phosphatidylinositol (PI)
Phosphatidylserine (PS)
Negatively charged
at physiological pH
Soy Phospholipids – Production and Quality Aspects
Phospholipid 70 % PC
Purification
Crude lecithin
10-15% PC
PC
Purification
Phospholipid
≥ 70% PC
Phosphatidylcholine
≥ 90% PC
Soy Phospholipids – Production and Quality Aspects
TLC chloroform/methanol/water 65/25/4 (v/v/v) and copper sulfate
Ref. Std. PC 20% PC 45 % PC 70 % PC 76%
PE,PI, LPC
PC 94 %
PC 98 % Ref. Std.
N-Acyl PE, PA
Soy Phospholipids – Production and Quality Aspects
HPLC (normal phase, ELSD)
PC 20 %
PC 45 %
Soy Phospholipids – Production and Quality Aspects
HPLC (normal phase, ELSD)
PC 70 %
PC 76 %
Soy Phospholipids – Production and Quality Aspects
HPLC (normal phase, ELSD)
PC 94 %
PC 98 %
Soy Phospholipids – Production and Quality Aspects
Phospholipid composition of the lecithin, phospholipid and PC fractions
Compound
PC
LEC PC 20
LEC PC 45
PL PC 70
PE
PI
PA
LPC
LPE
N-Acyl-PE
Glycolipids
Triglycerides (TG)
Free Fatty Acids
(FFA)
( )
( )
D,L-α-Tocopherol
(Toc)
Sterols
: High,
( )
: Medium,
: Low, ( ): Negligible, n.d.: not detectable
PL PC 76
PC 94
PC 98
( )
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Soy Phospholipids – Production and Quality Aspects
Fatty acid composition of the lecithin, phospholipid and PC fractions
Peak
Name
C14:0
C16:0
C18:0
C18:1
C18:2
C18:3
C20:0
C22:0
Sum
Parameter LEC PC 20
LEC PC 45
PL PC 70
PL PC 76
PC 94
PC 98
Area %
Area %
Area %
Area %
Area %
Area %
Area %
Area %
Area %
0.06
16.05
3.35
9.48
63.26
5.99
0.08
0.12
98.39
0.06
14.88
3.53
10.27
63.51
5.82
0.08
0.10
98.25
0.06
14.66
3.69
11.66
62.72
5.21
0.08
0.09
98.17
0.07
14.61
3.69
12.37
62.59
4.69
0.07
0.07
98.16
0.06
12.36
3.33
9.71
66.45
6.09
0.06
0.06
98.12
0.06
21.12
4.65
9.89
56.69
5.01
0.13
0.41
97.96
Soy PC contains about 40 % DLPC
Phase transition temperature < 0 C
Soy Phospholipids – Production and Quality Aspects
Monographs for Soybean Lecithins
Ph. Eur.
[60]
USP 33 /
NF 28 [61]
DAB [62]
Temp.
Pharm.
Monogr.
Chinese
Pharmaco
peia [64]
IP [65]
JPE [66]
Soya
Lecithin
Lecithin
(crude
veget. oil)
Soybean
Lecithin
[63]
Title
Soya
Lecithin
Monograph No.
2316
Country/Region
Identification
Europe
AcetoneInsoluble
Matter [%]
≥ 60
Lecithin
Oil-Free
Soya
Lecithin
Phospholi
pid from
Soybeans
VFS 423051-98
CAS No.
8002-43-5
USA
Germany
Russia
China
India
Japan
Yellow
YellowishWhite
Yellow
Yellow
white
Precipitate
Precipitate Precipitate Precipitate
Precipitate
≥ 50
≥ 90
≥ 50
≥ 60
Soy Phospholipids – Production and Quality Aspects
Monographs for Soybean Lecithins
Ph. Eur.
[60]
USP 33 /
NF 28 [61]
DAB [62]
Temp.
Pharm.
Monogr.
Chinese
Pharmac
opeia [64]
IP [65]
JPE [66]
≤ 0.3
≤ 0.3
≤ 0.3
≤ 36
≤ 40
[63]
TolueneInsoluble Matter
[%]
HexaneInsoluble Matter
[%]
PC content [%]
≤ 0.3
≤ 0.3
≤ 0.3
≤ 0.3
≥ 20≤31.6
≥ 75
LPC content [%]
≤ 3.5
LPE content [%]
≤ 0.5
Acid value [mg
KOH] or [mg
NaOH]
≤ 35
≤ 36
≤ 35
≤ 20
≤ 30
Soy Phospholipids – Production and Quality Aspects
Monographs for Soybean Lecithins
Ph. Eur.
[60]
USP 33 /
NF 28 [61]
DAB [62]
Temp.
Pharm.
Monogr.
Chinese
Pharmac
opeia [64]
IP [65]
JPE [66]
[63]
Iodine value
[cg iodine/g
sample]
Peroxide value
Water (KarlFischer or
LOD*)[%]
Heavy Metals
[ppm]
Arsenic [ppm]
Lead [ppm]
≤ 10
≤ 2.0
≤ 10
≤ 1.5
≤ 20
76-85
≥ 75
≤ 10
≤ 1.0
≤ 7.0
≤5
≤ 1.5
≤ 10
≤ 1.5
≤ 10
≤ 2.0
≤5
≤ 20
≤ 20
≤ 20
≤2
≤ 10
≤2
≤ 10
Hydrogen. Soy PC – Production and Quality Aspects
PC
Hydrog. PC
H2 / Cat.
Phosphatidylcholine
Hydrogenated
Phosphatidylcholine
Hydrogen. Soy PC – Production and Quality Aspects
TLC chloroform/methanol/water 65/25/4 (v/v/v) and copper sulfate
Hydrogen. Soy PC – Production and Quality Aspects
HPLC (normal phase, ELSD)
PC
SEDEX 85-I I
SMP-SPC-3 256375-a2
900
Name
800
700
600
mV
500
400
300
200
100
HPLC einfügen
0
-100
0
2
4
6
8
10
12
14
Minutes
16
18
20
22
24
26
28
HPLC (reversed phase, UV)
K-2000
SPC-3 525600-2130483-1-b
Name
20
18
DSPC
16
14
10
8
6
PSPC
mAU
12
4
2
0
-2
0
1
2
3
4
5
6
7
8
9
10
Minutes
11
12
13
14
15
16
17
18
19
20
Hydrogen. Soy PC – Production and Quality Aspects
Phospholipid composition of hydrogenated soy phosphatidylcholine
Component
HSPC
PC
n.l.t. 98.0 %
L PC
n.m.t. 0.5 %
PE
n.m.t. 0.1 %
N-Acyl-PE
n.m.t. 0.5 %
Hydrogen. Soy PC – Production and Quality Aspects
Fatty acid composition of the hydrogenated soy PC
Peak Name
Parameter
HSPC
C16:0
Area %
13
C18:0
Area %
86
C18:1
Area %
0,3
C20:0
Area %
0,3
C22:0
Area %
0,1
HSPC contains about 27 % PSPC and 73 % DSPC
Phase transition temperature > 50 C
Hydrogen. Soy PC– Production and Quality Aspects
Monographs for hydrogenated soy PC
• None
• HSPC is listed in the Inactive Ingredient Guide by the FDA
Center for Drug Evaluation
• Requirements also described in
• Draft Guidance on Doxorubicin Hydrochloride (FDA, Feb.
2010) referring to
• Draft guidance for industry: Liposome Drug Products
(FDA 2002)
• Reflection paper on the data requirements for intravenous
liposomal products developed with reference to an
innovator liposomal product (EMA, 21. February 2013)
Egg Phospholipids – Production and Quality Aspects
Egg Yolk
Extraction
Chromatography
Purified Egg Phospholipid
PC > 70 %
Chromatography
Egg Phosphatidylcholine
PC
Egg Phospholipids – Production and Quality Aspects
HPTLC chloroform/methanol/water 65/25/4 (v/v/v) and copper sulfate
Ref. Std PC 75 % PC 80 % PC 98 % Ref. Std.
PE, PC
ESM, LPC
Egg Phospholipids – Production and Quality Aspects
HPLC
(ELSD)
PC 75 %
E PC
PC 80 %
Egg Phospholipids – Production and Quality Aspects
Phospholipid composition of the lecithin, phospholipid and PC fractions
Component
PC
PE
SM
L PE
L PC
PL PC 75
72.0 %
17.0 %
2.0 %
1.0 %
2.0 %
PL PC 80
81.0 %
8.5 %
2.0 %
0.3 %
2.0 %
PC 98
99.0 %
0.0 %
0.4 %
0.0 %
0.0 %
Egg Phospholipids – Production and Quality Aspects
Fatty acid composition of the lecithin, phospholipid and PC fractions
Peak
Parameter PL PC 75 PL PC 80 PC 98
Name
C14:0
Area %
0.15
0.14
0.18
C16:0
Area %
30.76
30.9
34.31
C18:0
Area %
15.28
14.02
11.59
C18:1 n9
Area %
24.31
27.85
27.48
C18:2 n6
Area %
16.29
15.29
16.28
C20:4 n6
Area %
5.63
4.83
3.59
C22:4 n6
Area %
0.29
0.30
0.17
C22:5 n3
Area %
0.15
0.15
0.10
C22:6 n3
Area %
2.20
1.81
1.75
Egg PC contains about 40 % POPC, 10 % SOPC, 26 % PLPC and 13 % SLPC
Phase transition temperature < 0 C
Egg Phospholipids – Production and Quality Aspects
Monographs for Egg Phospholipids
JPE
Ch. Pharm. Stand.
USP
Purified Yolk
Lecithin
Egg Yolk Lecithin
Egg
Phospholipids
PC
-
n.l.t. 72.0 %
Report value
PE
-
n.m.t. 18.0 %
Report value
LPC
-
< 3.5 %
n.m.t. 3.0 %
3.5-4.4 %
3.5-4.1 %
-
Nitrogen content
1.6-2.0 %
1.75-1.95 %
-
Acid value
n.m.t. 25
-
n.m.t. 20
-
195-212
-
Title
Phosphorus
content
Saponification
value
Egg Phospholipids – Production and Quality Aspects
Monographs for Egg Phospholipids
JPE
Ch. Pharm. Stand.
USP
Iodine value
60-82
65-73
-
Limit of fat
-
-
n.m.t. 7 %
Peroxide value
-
n.m.t. 3.0 mEq per n.m.t. 3 mEq per
kg
kg
Water
n.m.t. 4.0 %
-
n.m.t. 6 %
Heavy metals
n.m.t. 5 ppm
-
n.m.t. 10 ppm
Arsenic
n.m.t. 2 ppm
-
-
-
-
n.m.t. 100 cfu
n.m.t. 6 EU/g
per g
n.m.t. 6 EU/g
Microbial limits
Endotoxins
-
Synth. Phospholipids – Production and Quality Aspects
Glycerophosphocholine (GPC)
Esterification with Fatty Acid
Phosphatidylcholine
e.g. DMPC
Biochemical Modification
Phosphatidylglycerol
e.g. DMPG
Synth. Phospholipids – Production and Quality Aspects
TLC chloroform/methanol/water/NH3 65/25/4 (v/v/v) and copper sulfate
DPPC
Ref. Std.
PC
FFA
LPC LPC
Synth. Phospholipids – Production and Quality Aspects
HPLC (normal phase, ELSD)
PC
SEDEX 85-II
SMP-DSPC 565256-A-b
450
Name
400
350
300
mV
250
200
150
100
50
0
-50
0
2
4
6
8
10
12
14
Minutes
16
18
20
22
24
26
28
Synth. Phospholipids – Production and Quality Aspects
Phospholipid composition of synthetic phospholipids e.g. DPPC
Component
DPPC
PC
n.l.t. 99.0 %
L PC
n.m.t. 0.5 %
Unidentified concomittant components
n.m.t. 0.5 %
Free fatty acids
n.m.t. 0.3 %
Synth. Phospholipids – Production and Quality Aspects
Fatty acid composition of the synthetic phospholipids e.g. DPPC
Peak Name
Parameter
DPPC
C16:0
Area %
> 98
Phase transition temperature depending on fatty acid composition and polar head
Synth. Phospholipids – Production and Quality Aspects
Monographs for synthetic phospholipids
• None
• Various synthetic phospholipids listed in the Inactive
Ingredient Guide by the FDA Center for Drug Evaluation
• Requirements also described in
• Draft Guidance on Doxorubicin Hydrochloride (FDA,
Feb. 2010) referring to
• Draft guidance for industry: Liposome Drug
Products (FDA 2002)
• Reflection paper on the data requirements for
intravenous liposomal products developed with
reference to an innovator liposomal product (EMA, 21.
February 2012)
Chemical Stability – Phospholipids
Storage
Oxidation
Hydrolysis
In vivo
Beside chemical degradation,
enzymatic degradation:
Phospholipases C,D, A1, A2 etc.
Phospholipids – Production and Quality Aspects
Suitability for Pharmaceutical Administration
• Reproducible quality
• Composition
• Low content of Lyso-PL and FFA
• Low peroxide value (POV)
• Residual raw material
• Residual intermediates
• Possible synthetic by-products
• Residual solvent
• Low microbial burden
• Low endotoxin content
Phospholipids – Production and Quality Aspects
Requirements for the manufacturer of phospholipids
• cGMP
• Certified according ICH Q7
• ISO 9001
• Drug Master Files
• Registration Documents
Parenteral Applications
Parenteral Drug
Delivery Systems
Lipid Emulsions for
Total Parenteral Nutrition
Role of Phospholipids in Parenteral Delivery
• Solubilization of poorly soluble drugs
• Coating of drug particles (suspensions)
• Encapsulation of hydrophilic and lipophilic drugs (liposomes,
emulsions)
• Modification of drug release (e.g. prolongation of circulation)
• Drug targeting (e.g. antibodies grafted on liposomes)
• Stabilization of sensitive drugs (prevent chemical,
photochemical or oxidative degradation)
• Reduction of side effects
Most Popular Delivery Vehicles
Mixed Micelle
5 – 10 nm
Emulsion
150 – 350 nm
Lipophilic Drug
Hydrophilic Drug
Bile Salt
Liposome
20 – 10,000 nm
Diacyl PL (PC)
Fatty Acids
Lipid Emulsions
Emulsion
Mixed Micelle
150 – 350 nm
5 – 10 nm
Liposome
20 – 10,000 nm
Emulsifier – Lipid Emulsions
• Purified egg phospholipids are the state of the art emulsifier
for parenteral lipid emulsions
• Fat-free egg phospholipid with 80% phosphatidylcholine (PC)
Egg phospholipid with 80 % PC
Summary and Conclusions – Lipid Emulsions
• Egg PLs are the emulsifiers of choice as they are:
of natural origin
non-irritant
non-toxic
compatible with the other ingredients
They provide a perfect (phospholipid) composition to stabilize
the emulsion.
They are used worldwide for more than 50 years.
Mixed Micelles
Emulsion
150 – 350 nm
Mixed Micelle
5 – 10 nm
Liposome
20 – 10,000 nm
Pharmaceutical Use of Mixed Micelles
• Technology developed for diazepam
solubilization to replace organic solvents in injectables
• Approximately equimolar mixture of soybean phosphatidylcholine
(SPC) and sodium glycocholate or other bile salts
• SPC to overcome the hemolytic effect of the bile salt
• Mixed micelles are not hemolytic, low toxicity
Parenteral Application – Mixed Micelles
• Purified soybean phosphatidylcholine (SPC) with > 94% PC
is the state of the art product for parenteral mixed micelles
Soy phosphatidylcholine
Liposomes
Emulsion
150 – 350 nm
Mixed Micelle
5 – 10 nm
Liposome
20 – 10,000 nm
Pharmaceutical Use of Liposomes
• Solubilization (iv)
• Active targeting (iv)
• Passive targeting (iv)
• Delaying elimination of drugs (iv)
• Slow release (sc/im/it)
• Adjuvant (sc/im)
Approved Phospholipids – Liposomes
• Purified egg phospholipids
• Hydrogenated soybean phosphatidylcholine
• Synthetic phospholipids as e.g. DMPC, DSPC,
DOPC, DSPG, DSPE-PEG 2000
Properties and Benefits of Suspensions
• Possible to formulate drugs that are insoluble in water and in oil
• Formulation of drugs with high melting points
• Drugs in their crystalline state can be applied
• Can function as parenteral sustained release system
(drug depot formation)
•
High physical stability
Approved Phospholipids – Suspensions
• Soybean phospholipids
• Various synthetic PLs
Summary and Conclusions – Suspensions
• Natural soybean PL (n.l.t. 75% PC) or synthetic phospholipids
are applied
• Non-toxic, safe for i.v. use in humans
• Phospholipids stabilize the suspensions
• Prevent the particles from agglomeration
• Low production costs
Pulmonary Applications
Metered Dose Inhaler
Nebulizer
Mouth piece
Cap
Dry Powder Inhaler
Approved Phospholipids for Pulmonary Applications
• Soybean Lecithin
• DPPC
• DSPC
Phospholipids either used as active or as
excipient
Phospholipids as Excipients in
Oral Pharmaceutical Applications
Application in Oral Formulations
• PLs can enhance the water solubility and permeability of APIs
leading to improved drug absorption and bioavailability
• PLs can stimulate the lymphatic transport of APIs
• PLs are able to reduce local side effects of NSAID drugs
• PLs can protect APIs in the gastric fluid
• PLs can retard the release of APIs in the intestinal fluid
• (Active)
Soy derived lecithins and phospholipids are more or less
exclusively used in oral formulations
Phospholipids as Excipients in
Dermal Pharmaceutical Applications
Outline
•
Skin Delivery Systems
•
Penetration / Permeation Enhancer
•
Improved Drug Retention
•
Improvement of Drug Stability
•
Active (Wound Healing)
Recommended Topical Phospholipids
• Monoacyl plant phosphatidylcholine
• Soybean phospholipids
• Soybean phosphatidylcholine
• Hydrogenated soybean phosphatidylcholine
Summary and Conclusions
Phospholipids used in the different applications
natural
phospholipids
egg
parenteral
emulsion
liposome
mixed micelles
suspension
pulmonary
oral
dermal
hydrogenated
phospholipids
soy
synthetic
phospholipids
Summary and Conclusions
• Phospholipids are used in a broad range of pharmaceutical
formulations for decades.
• Phospholipids are considered as excipients by the authorities.
• All kind of phospholipids (natural, hydrogenated and synthetic)
are used and accepted by authorities worldwide.
• In the overall phospholipid market synthetic phospholipids play
compared to natural phospholipids a minor role.
• Natural phospholipids are based on renewable sources.
Summary and Conclusions
• All side products are used while producing natural
phospholipids.
• In purified natural phospholipids used in parenterals all
substances present are known including their toxicological
aspects. Neither chemicals nor toxic solvents are used for the
production.
• In natural phospholipids as well as in synthetic phospholipids
via the GPC-route the natural configuration is ensured.
• Synthetic phospholipids can be possibly replaced by natural
phospholipids.
Summary and Conclusions
• Natural phospholipids are accepted by all authorities
worldwide.
• Most drugs (including the parenterals) have been produced by
using natural phospholipids.
• Natural phospholipids are less expensive and available in
large volumes.
• Phospholipids provide promising solutions in the development
of new pharmaceutical formulations not only as they are bodyown and biodegradable.