In-vitro dissolution testing: Method development and importance of ...

In-vitro dissolution testing:
Method development and importance
of biorelevant test conditions
Sandra Klein
Ernst Moritz Arndt University, Greifswald, Germany
Sandra.Klein@uni-greifswald.de
EUFEPS BABP Network Open Discussion Forum
Revision of BE Requirements for Modified Release Products
February 23‐24 • 2011 •Barcelona •Spain

Modified release (MR) formulations
• intended to release drugs in a controlled and pre-determined
fashion and/or to target to selective sites in the gastrointestinal
(GI) tract
• release drug over a long time period (12 hrs, 24 hrs, ..)
• typically contain much higher doses than IR formulations
• give release patterns which in turn can offer a lot of benefits
� dose dumping
� insufficient drug release

Typical MR formulations
Coated dosage forms
1. Delayed release formulations
2. Extended Release Formulations
Matrix systems
Extended Release Formulations
⇒ erodible or non-erodible matrices
Osmotic systems
⇒ monolithic dosage forms vs. multiparticulates

Possible release profiles
% Release
time [h]

CPMP/QWP/604/96
Development and validation of dissolution methods
In-vitro dissolution test must be capable of:
• discriminating between batches
• showing batch to batch consistency
• determining stability of the relevant release characteristics
⇒ various conditions (media pH: 1-6.8/8; apparatus, agitation etc.)
⇒ for additional details reference is made to Ph.Eur.
⇒ sink conditions should be ensured
⇒ as possible methods should be predictive for the in-vivo
performance of the formulation

Test methods Ph.Eur.
Ph. Eur.: 2.9.3 Drug release of solid dosage forms
• Extended Release Articles
• Delayed Release (Enteric Coated) Articles
• Apparatus 1 (Basket)
• Apparatus 2 (Paddle)
• Apparatus 3 (Reciprocating Cylinder)
• Apparatus 4 (Flow through cell)

Test conditions Ph.Eur.
Extended Release Dosage Articles
⇒ Apparatus 1 and Apparatus 2
• simple buffer media

Specifications Ph.Eur.
Extended Release Dosage Articles
• Specification of drug release at 3 or more test-time points
• Time point 1:
to check for „dose dumping“, @ ~ 20-30 % drug released
• Time point 2:
to characterize the release profile, @ ~ 50 % drug released
• Time point 3:
to check for complete drug release, @ > 80 % drug released

Test conditions Ph.Eur.
Delayed-Release (Enteric Coated) Articles
⇒ Apparatus 1 and Apparatus 2
• Procedure:
Unless otherwise directed in the individual monograph:
Method A (Half change) or Method B (Full change)
⇒ 2 hrs acid stage (pH 1)
⇒ 45 min buffer stage (pH 6.8)

In vitro characterization of MR dosage forms
Ph.Eur. methods
• primarily use of apparatus I and II
• simple dissolution media
• useful for quality control purposes
• not useful for examining biopharmaceutical properties of the
dosage form
• often not predictive for the in vivo performance of a MR dosage
form

CPMP/QWP/604/96
Development and validation of dissolution methods
In-vitro dissolution test must be capable of:
• discriminating between batches
• showing batch to batch consistency
• determining stability of the relevant release characteristics
⇒ various conditions (media pH: 1-6.8/8; apparatus, agitation etc.)
⇒ for additional details reference is made to Ph.Eur.
⇒ sink conditions should be ensured
⇒ as possible methods should be predictive for the in-vivo
performance of the formulation

In vitro characterization of MR dosage forms
Biorelevant dissolution tests
• simulation of the gastrointestinal passage of the dosage form
• prediction of in vivo absorption using in vitro data
How to simulate a gastrointestinal passage ???

GI passage
Stomach
• pH: fasted: 1-3 fasted
fed: 3-7
• passage time: fasted: 0.5-2h
fed: several hours

GI passage
Duodenum
• pH = 4-6
• bile- and pancreatic secretion
• short passage time (< 10 min)

GI passage
Jejunum
• pH = 6-7

GI passage
Ileum
• pH = 7-7.5
• quantitative absorption of bile salts
(active transport)

GI passage
Colon
• pH = 5-7
• great number and variety of bacteria
• individual passage times differ largely

Criteria for dissolution test design
Media to simulate gastrointestinal passage
• pH-value / pH profiles
• volume
• composition
Dissolution equipment
• apparatus
• hydrodynamic conditions
Passage times
• multiple units ↔ single unit dosage form
Food effects
• fasted ↔ fed state conditions

Dissolution equipment
Flow through cell Reciprocating cylinder / BioDis
• a series of media can be used → variable pH–values
• variable passage times
• variable motility patterns
• variable hydrodynamics / flow rates
• complex media → food effects

Case examples
Impact of gastrointestinal pH on drug release
⇒ pH gradient
• site and extent of drug release from Mesalazin DR formulations
Impact of gastrointestinal contents on drug release
⇒ biorelevant media gradient
• food effects on drug release from Theophyllin ER formulations

Site specific drug delivery
Therapy of of inflammatory bowel diseases
• oral mesalazin treatment
⇒ high concentrations of drug at the inflamed areas
⇒ avoid drug release and absorption in the proximal GI tract
• mesalazin is not absorbed in the colon
Crohn´s disease Ulcerative colitis

Formulation concepts of marketed dosage
forms
Product Dosage from Polymer type* Polymer
brand name
Release pH
Claversal ® coated tablet MA:MM (1:1) Eudragit ® L > pH 6
Salofalk ® coated tablet MA:MM (1:1) Eudragit ® L > pH 6
Asacolitin ® coated tablet MA:MM (1:2) Eudragit ® S > pH 7
Pentasa ® IR tablet containing
coated microgranules
Salofalk ®
Granustix ®
Claversal ®
Micropellets
coated microgranules coating: MA:MM (1:1)
core: MM neutral esters
Surelease ® pHindependent
Eudragit ® L
Eudragit ® NE
coated microgranules coating: MA/MM (1:1 & 1:2) Eudragit ® L
Eudragit ® S
*MA = methaycrylic acid, MM = methacrylate esters
> pH 6
> pH 6-7
S. Klein et al., J. Pharm. Pharmacol. 2005: 57 (6) 709-720

Compendial method: SIF pH 6.8 vs. pH 7.5
Release [%]
100
80
60
40
20
0
Claversal 500 mg
Salofalk 500 mg
Asacolitin 400 mg
pH 6.8 pH 7.5
0 60 120 180 240 300
Time [min]
Release [%]
100
80
60
40
20
0
Claversal 500 mg
Salofalk 500 mg
Asacolitin 400 mg
0 60 120 180 240 300
Time [min]
S. Klein et al., J. Pharm. Pharmacol. 2005: 57 (6) 709-720

pH-gradient „preprandial“
GI-segment pH Media
single unit
dosage forms
multiple unit
dosage forms
Stomach 1.8 Simulated Gastric Fluid (SGFsp)* 60 min 30 min
Proximal Jejunum 6.5 Phosphatpuffer pH 6.5 15 min 45 min
Distal Jejunum 6.8 Simulated intestinal fluid pH 6.8 (SIFsp) USP 24 15 min 45 min
Proximal Ileum 7.2 Phosphatpuffer pH 7.2 R (Ph. Eur 1997) 30 min 45 min
Distal Ileum 7.5 Simulated intestinal fluid pH 6.8 (SIFsp) USP 23 120 min 45 min
Proximal Colon 6.5 Phosphatpuffer pH 6.5 360 min 360 min
Proximal Colon 6.5 Phosphatpuffer pH 6.5 240 min 240 min
Distal Colon 6.8 Simulated intestinal fluid pH 6.8 (SIFsp) USP 24 360 min 360 min
Distal Colon 6.8 Simulated intestinal fluid pH 6.8 (SIFsp) USP 24 240 min 270 min
* pH modified
Passage time
S. Klein et al., J. Pharm. Pharmacol. 2005: 57 (6) 709-720

pH-gradient
Release [%]
100
80
60
40
20
0
0 60 120 180 240 300 360 420 480
Time [min]
Claversal 500 mg
Salofalk 500 mg
Asacolitin 400 mg
S. Klein et al., J. Pharm. Pharmacol. 2005: 57 (6) 709-720

Colonic delivery
pH- and time-based multi-unit colonic delivery system
� oral therapy of Ulcerative colitis and other colon related
diseases
Outer layer: Eudragit FS
Inner layer: Eudragit RL+RS
Mesalazin / Caffein core
M.W. Rudolph et al., Eur J Pharm Biopharm 2001: 51(3) 183-190

“Fraction released”
Release [%]
100
80
60
40
20
0
0 60 120 180 240 300 360 420 480
Time [min]
Salofalk 500 mg
Asacolitin 400
mg
Salofalk
Granustix 500
mg
Prototype
S. Klein et al., J. Contr. Rel. 2008, 130: 216-219

Plasma profile � “fraction absorbed”
Plasmakonzentration [µmol/L]
9
8
7
6
5
4
3
2
1
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Zeit [h]
Calculation
Fraction absorbed [%]
100
80
60
40
20
0
0 120 240 360 480
Zeit [min]
S. Klein et al., J. Contr. Rel. 2008, 130: 216-219

LEVY-Plot
fraction absorbed [%]
100
� prospective IVIVC
� predictive in-vitro method
80
60
40
20
0
y = 0,6104x + 1,4488
R 2 = 0,9948
0 20 40 60 80 100
fraction released [%]
S. Klein et al., J. Contr. Rel. 2008, 130: 216-219

„Extended release“ formulations
Theophylline
• treatment of chronic asthma
• narrow therapeutic index
Therapeutic requirements
• plasma levels in the therapeutic range
• reduce serum level fluctuations
⇒ risk for dose dumping?
⇒ impact of food on drug release?

Theophylline ER beads – food effect
Medium-fat breakfast High-fat breakfast
A. Karim et al., Clin. Pharmacol. Ther. 38, 642-647 (1985)

Theophylline ER tablets – food effect
High-fat breakfast
A. Karim et al., Clin. Pharmacol. Ther. 38, 642-647 (1985)

Compendial method: Paddle – SIF pH 6.8
Release [%]
100
80
60
40
20
0
Contiphyllin 300 mg
Tromphyllin 300 mg
0 60 120 180 240 300 360 420 480
Time [min]

Dissolution media
1. Simulation of pH-conditions in the upper GI tract
• buffer solutions – pH gradient
2. Simulation of the physiological GI contents
• biorelevant dissolution media – biorelevant pH gradient
Stomach
Small
intestine
Colon
preprandial
FaSSGF
FaSSIF
SCoF
postprandial
Milk, Ensure ® Plus
FeSSIF
SCoF

Fasted State Simulated Gastric Fluid
„Biorelevant“ conditions in the fasted stomach
Na taurocholate 80 µmol/L
Lecithin 20 µmol/L
Pepsin 0.1 mg/mL
NaCl 34.2 mmol/L
HCl conc. ad pH 1.6
Demineralized water ad 1000 mL
pH 1.6
Surface tension 42.6 mN/m
Osmolality 121 mOsm/kg
M Vertzoni et al. Eur J Pharm Biopharm. 60:413-417 (2005)

Milk
Objective: Simulation of the initial gastric conditions after a light
breakfast
Whole milk, 3,5 % fat
(per 100 g)
269 kJ
64 kcal
3,5 g fat
3,3 g proteins
4,7 g carbohydrates
Ref.: CMA (www.cma.de)
P. Macheras et al. Int. J. Pharm. 33: 125-136 (1986)
S. Klein et al. J. Pharm. Pharmacol. 56: 605-610 (2004)

Ensure ® Plus
Objective: to simulate initial composition after a standardized
standardized (FDA) high fat breakfast „worst case“
Standard Breakfast FDA Division of Biopharmaceutics
2 slices of toasted white bread with butter
2 eggs fried in butter
2 slices of bacon
2 ounces of hash-browned ( fried shradded ) potatoes = 56,7 g
8 ounces of whole milk
Carbohydrate: 58g, 232 kcal, 972 kJ, 24 %
Protein: 33g, 132 kcal, 552 kJ, 14 %
Fat: 67 g, 603 kcal, 2533 kJ, 62 %
Klein et al. J. Pharm. Pharmacol. 56: 605-610 (2004)

Fed State Simulated Gastric Fluid
„Biorelevant“ conditions in the fed stomach some time after a meal
NaCl 237.02 mmol/L
Acetic acid 17.12 mmol/L
Sodium acetate 29.75 mmol/L
Demineralized water ad 1000 mL
Milk:acetate buffer 1:1
HCl conc. ad pH 5.0
pH 5.0
Osmolality 400 mOsm/kg
Buffer capacity 25 mEq/L/pH
E Jantratid et al. Pharm Res. 25 (7):1663–76 (2008)

Fasted State Simulated Intestinal Fluid
NaH 2 PO 4
3.438 g
Na taurocholat 3 mmol/L
Lecithin 0.75 mmol/L
NaCl 6.186 g
NaOH ad pH 6.5
Demineralized water ad 1 Liter
pH 6.5
Osmolality 270 + 10 mOsm/kg
Buffer capacity 10 + 2 mEq/L/pH

Fed State Simulated Intestinal Fluid
Glacial acetic acid 8.65 g
Na taurocholate 15 mmol/L
Lecithin 3.75 mmol/L
NaCl 11.874 g
NaOH ad pH 5.0
Demineralized water ad 1 Liter
pH 5.0
Osmolality 635 + 10 mOsm/kg
Buffer capacity 76 + 2 mEq/L/pH

Simulated Colonic Fluid
Simulation of the pH-values and ions in the proximal colon
1 molar acetic acid solution 170 mL
1 molar NaOH solution 157 mL
Demineralized water ad 1 Liter
pH 5.8
Osmolality 295 mOsm/kg
Buffer capacity 29 mEq/L/pH
Fotaki et al , Eur. J. Pharm. Sci. 24 : 115-122, 2005

Dissolution media
1. Compendial buffers
• Simulation of the pH conditions in the GI tract
2. Biorelevant media
• Simulation of the physiological GI milieu
� Fasted state
Stomach: FaSSGF
Small intestine: FaSSIF
Colon: SCoF
� Fed State
→ FaSSIF–Gradient
Stomach: Milk / Ensure ® Plus
Small intestine: FeSSIF → FeSSIF–Gradient
Colon: SCoF
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

FaSSIF / FeSSIF gradients
Concentration of bile components: FaSSIF gradient
GI-section pH Biorelevant media Sodium taurocholate Lecithin
Proximal Jejunum 6.5 FaSSIF 3 mmol/l 0.75 mmol/l
Distal Jejunum 6.8 FaSSIF a,b
3 mmol/l 0.75 mmol/l
Proximal Ileum 7.2 FaSSIF a,b
1.5 mmol/l 0.375 mmol/l
Distal Ileum 7.5 Blank FaSSIF a
-------- --------
a pH modified, b Concentration of bile components modified
Concentration of bile components: FeSSIF gradient
GI-section pH Biorelevant media Sodium taurocholate Lecithin
Proximal Jejunum 5.0 FeSSIF 15 mmol/l 3.75 mmol/l
Distal Jejunum 6.5 FeSSIF a,b
15 mmol/l 3.75 mmol/l
Proximal Ileum 6.5 FeSSIF a,b
7.5 mmol/l 1.875 mmol/l
Distal Ileum 7.5 Blank FaSSIF a
-------- --------
a pH modified, b Concentration of bile components modified,
c Phosphate buffer
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Biorelevant pH-gradient methods
GI-section
GI-section
Compendial media pH Biorelevant media pH Tablets Pellets
Stomach SGFsp* 1.8 FaSSGF 1.8 60 30
Proximal Jejunum Blank FaSSIF 6.5 FaSSIF 6.5 15 45
Distal Jejunum Blank FaSSIF 6.8 FaSSIF* 6.5 15 45
Proximal Ileum Blank FaSSIF 7.2 FaSSIF*/** 7.2 30 45
Distal Ileum Blank FaSSIF* 7.5 Blank FaSSIF* 7.5 120 45
Proximal Colon SCoF 5.8 SCoF 5.8 240 240
* pH modified, ** Concentration of bile components modified
Compendial media pH Biorelevant media pH Tablets Pellets
Stomach Acetate buffer / SGFsp*5.0 / 2.0
®
Ensure Plus (Milk) 6.5 (120/120) 240 120
Proximal Jejunum Blank FeSSIF 5.0 FeSSIF 5.0 45 45
Distal Jejunum Blank FaSSIF 6.5 FeSSIF* 6.5 45 45
Proximal Ileum Blank FaSSIF 6.5 FeSSIF*/** 6.5 45 45
Distal Ileum Blank FaSSIF* 7.5 Blank FaSSIF* 7.5 45 45
Proximal Colon SCoF 5.8 SCoF 5.8 --- 240
* pH modified, ** Concentration of bile components modified
Media and pH-values preprandial pH-Gradient
Media and pH-values postprandial pH-Gradient
Passage time [min]
Passage time [min]
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Contiphyllin 300 mg tablets
Release [%]
100
80
60
40
20
0
0 60 120 180 240 300 360 420 480
Time [min]
Preprandial pH profile: � = compendial buffers, � = biorelevant media
Postprandial pH profile: � = compendial buffers, � = biorelevant media
biorelevant
media
compendial
media
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Tromphyllin 300 mg tablets
Release [%]
100
80
60
40
20
0
?
0 60 120 180 240 300 360 420 480
Time [min]
Preprandial pH profile: � = compendial buffers, � = biorelevant media
Postprandial pH profile: � = compendial buffers, � = biorelevant media
biorelevant
media
compendial
media
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Drug release in the postprandial stomach
Release [%]
120
100
80
60
40
20
0
Tromphyllin 300 mg fed state stomach
Contiphyllin 300 mg fed state stomach
High-fat breakfast
17,48
15,62
40,81
30,35
60,89
39,51
77,76
43,70
0 60 120 180 240
Time [min]
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Drug release in the postprandial state
Release [%]
100
80
60
40
20
0
Contiphyllin 300 mg fed state milk
Tromphyllin 300 mg fed state milk
Light breakfast
0 60 120 180 240
Time [min]
300 360 420 480
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Drug release in the postprandial state
Milk and simple buffer media
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Drug release in the postprandial state
Ensure ®
Plus
Milk
Klein S. , Dissolution Technologies, 16 (3): 28-40, 2009

Importance of biorelevant test conditions
• pH-gradient and biorelevant pH-gradient methods allow a
better prediction of the potential in-vivo behaviour of MR
dosage forms
• pH values and passage times can be easily adapted to
different patient subgroups or a best / worst case scenario
• composition of media can be even better adapted to
physiological conditions
• methods can be adapted to check for alcohol-dependent
dose dumping
• hydrodynamic conditions need to be examined in more detail

Thank you !!!