A pharmaceutical study on certain aminosalicylates master defense

A Pharmaceutical Study on Certain
Aminosalicylates
By
Ibrahim Al Sharabi
Under the supervision of
Prof. Dr. Ahmed Abd Elbary Abd Elrahman
Professor of Pharmaceutics and Industrial Pharmacy
Ex- Dean of faculty of pharmacy-Cairo University.
Dr. Ahmed A. Aboelwafa
Lecturer of Pharmaceutics
Cairo University

Inflammatory bowel diseases
(IBDs)
• Affect 4 million people worldwide.
• Symptoms include:
Diarrhea, abdominal pain, hematochezia, abdominal mass,
malnutrition and abdominal distension.
Types
Ulcerative Colitis (UC):
Inflammation and ulcers only
in the mucosa of the colon
Crohn’s disease (CD):
Inflammation of all layers and
sites of the GIT

Ulcerative Colitis
• Anatomic Extent of Disease
Distal
(Proctitis)
Left-Sided
(Distal Colitis)
Pancolitis
(Extensive)
18%40% 40%

UC and Aminosalicylates
• 5-ASA products remain the first line treatment for patient with UC.
• 5-ASA is thought to work topically on the inflamed mucosa.
• 5-ASA is extremely absorbed from the proximal small intestine and
then extensively metabolized to inactive metabolite (Low
availability at the distal part of the GIT – systemic side effects.)
• Large doses of 5-ASA are required in induction and maintenance
therapy of UC (up to 4.8 g/d).
• large number of tablets (up to 12/day in 3 or 4 divided doses).
• Rate of adherence to maintenance treatment ≈ 40%.
• High rate of relapse.
• Un treated UC is an important risk factor for colonic cancer.

Commercially applied colon
targeting techniques for 5-ASA
Prodrugs Modified release formulations
• Delayed release products
– Salofalk®
– Asacol®
• Extended release products
– Pentasa®
O
NHS
O
Sulfasalazine
Olsalazine
Inert Carrier
Balsalazide

In-vivo distribution of 5-ASA Formulations
Pulsatile type oral
preparations
Conventional
sustained release
preparations
Enteric coated – extended
release preparations
Lialda (Mezavant)
Apriso

Chapter 1
Preformulation Studies

Physicochemical properties of 5-ASA
• Name: 5-Aminosalicylic acid(5-ASA), m-aminosalicylic acid,
fisalamine, mesalamine or mesalazine.
• Molecular formula is C7H7N03 and molecular weight of
153.14.
• Melting Point :283°C
• Very slightly soluble in water, practically insoluble in organic
solvents.
• The drug powder exhibits poor flowability.

Experimental
Spectral study
Determination of aqueous
solubility of
5-ASA at different pH values
Compatibility study
(DSC and FTIR)

Stability indicating assay of 5-ASA.
Chromatographic Condition:
• Mobile phase: methanol-phosphate buffer (pH 4.6) (20:80,
v/v).
• Flow rate : 0.8 ml/min.
• Detection wavelength : 220 nm.
• Injection volume : 5 l.
• Method validation:
– linearity
– Precision
– Accuracy
– Specificity

Results and discussion-Spectral study
λmax = 330λmax = 303
λmax = 299

5-ASA calibration curves at different pH
y = 0.024x + 0.000
R² = 0.999
y = 0.024x + 2E-06
R² = 0.999
y = 0.021x + 0.000
R² = 0.999
0
0.2
0.4
0.6
0.8
1
0 4 8 12 16 20 24 28 32 36
Absorbance
Concentration µg/ml
pH 1.2 pH 4.5 pH 7.4

Aqueous solubility of 5-ASA at different pH
values
9.99
1.73
9.23
0
2
4
6
8
10
12
0 1 2 3 4 5 6 7 8
Solubility(mg/ml)
pH

DSC of 5-ASA, pharmaceutical excipients and
their 1:1 w/w physical mixtures.

FT-IR of 5-ASA, pharmaceutical excipients and their 1:1 w/w
physical mixtures.

Stability Indicating assay of 5-ASA
y = 85157x
R² = 0.994
0
5000000
10000000
15000000
0 25 50 75 100 125 150
AUP
Concentration (µg/ml)

Chromatogram obtained following injection of 5-ASA 1(100µg/ml) in
phosphate buffer (pH 4.5) kept in ambient conditions for 72 hr

Conclusion
• 5-ASA has a pH dependent solubility profile.
• 5-ASA was shown to be physically and chemically
compatible with most of all of the tested excipients.

Chapter 2
Formulation and evaluation of 5-ASA
matrix tablets

Formulation Design
• pH dependent polymer
dissolving at pH > 6
(Eudragit® L and
Eudragit® S).
Delaying drug
release
• Hydrophobic polymers
Eudragit® RS.
• Hydrophilic polymer
Carbopol®
Release
sustainment • Superdisintegrant
Disintegrating
• Minimizing the
level of excipients
High loading
1200 mg 5-ASA
pH dependent polymer
Sustained release polymer
Superdisintegrant

Batches
Carbopol
(%)
Eudragit RS
(%)
Eudragit S
(%)
Eudragit L
(%)
Crospovidone
(%)
Croscarmellose Na
(%)
F1 - 2.5 - - - -
F2 - 5 - - - -
F3 - 10 - - - -
F4 2.5 - - - - -
F5 5 - - - - -
F6 10 - - - - -
F7 2.5 - 2.5 - - -
F8 2.5 - 5 - - -
F9 2.5 - 10 - - -
F10 20 - 5 - - -
F11 25 - 5 - - -
F12 20 - - 5 - -
F13 10 - 20 - - -
F14 10 2 18 - - -
F15 10 2 10 8 - -
F16 5 5 - - - -
F17 5 5 - - - 5
F18 - 5 - - 5 -
F19 10 5 - - - -
F20 10 5 - - 5 -
Composition of matrix tablets formulations

Experimental
Preparation of
tablets
Characterization of tablets
Hardness
Friability
Drug content
In-vitro release
studies and kinetics
analysis

In-vitro release studies
pH1.2
pH4.5
pH7.4
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16
Time (h)
350 ml
600 ml
900 ml
50 rpm

Kinetic Analysis
• Zero order kinetics : Mt/M∞ = kt
• Higuchi model : Mt/M∞ = k(t)0.5 + C
• Korsmeyer-Peppas: Mt/M∞ = ktn + C. Mt/M∞

Results and discussion
• Hardness (8-12 kg)
• Friability (<0.5%)
• Drug content (± 6.5 %)

0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
F1
F2
F3
In-vitro release studies
In-vitro release profile of 5-ASA from matrix tablets with varying
proportion of Eudragit® RS.
↑ Eudragit® RS  ↓ DR

0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
Ideal Targeted Profile
F4
F5
F6
In-vitro release profile of 5-ASA from matrix tablets with varying proportion of
Carbopol®®.
↑ Carbopol®  No significant
effect

0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
F16
F17
F18
F19
F20
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
F7
F8
F9
F10
F11
F12
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
F13
F14
F15
There was no sufficient retardation in precolonic stage of release studies even
with inclusion of high con. Of pH dependent polymers

Conclusion
• Incorporation of pH dependent polymers (Eudragit®
S and Eudragit® L) and Carbopol® in the used levels
was insufficient to achieve the targeted release profile
proposed in this study.
• Application of coat may be necessary to prevent
abrupt initial release of 5-ASA .

Chapter 3
Once Daily, High Dose 5-ASA Controlled Release
Tablets for Colonic Delivery: Optimization Of
Formulation Variables Using Box-Behnken Design.

1200 mg 5-ASA
pH dependent polymer
Sustained release polymer
Superdisintegrant

Experimental Design
Formulation variables
Level used
(-1) (0) (+1)
X1= Carbopol® content (%) 0 4 8
X2= Eudragit® RS content (%) 1 5.5 10
X3= Croscarmellose Na content (%) 0 1 2
Responses variables Constraints
Y1 = Release (%) after 6 hr 7.5% ≤ Y1 ≤ 22.5%
Y2 = Release (%) after 10 hr 42.5% ≤ Y2 ≤ 57.5 %
Y3 = Release (%) after 14 hr 72.5% ≤ Y3 ≤ 87.5%

Composition of Box-Behnken formulations
Run
Factors
X1 X2 X3
1 0 5.5 2
2 4 5.5 1
3 0 5.5 0
4 8 10 1
5 4 1 2
6 8 5.5 0
7 4 5.5 1
8 4 1 0
9 4 5.5 1
10 8 1 1
11 4 10 2
12 4 10 0
13 0 10 1
14 8 5.5 2
15 0 1 1

Preparation of
granules to be
compressed
Evaluation of granules to
be compressed
• Angle of Repose
• The percentage
compressibility (Carr's
Index).
• The Hausner ratio
Preparation
of 5-ASA
core tablets
Evaluation of
5-ASA core
tablets.
• Hardness.
• Friability .
• Drug content.
Coating of the
core tablets
and in-vitro
release studies
Preparation and Characterization of
experimental runs

Results and discussion
• Granules
– Angles of repose between 27 and 31 suggesting a
reasonable flow.
– Carr’s index values indicate fair flow properties for all
formulations while Hausner ratios suggest a low inter-
granular friction .
• Core tablets
– Drug content (± 5% of the theoretical amount).
– Hardness was set to be in the range of (10-11).
– Friability (< 0.9%).

In-vitro release profiles of all formulations.
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAreleased
Time (hr)
Ideal targeted release
Run1
Run2
Run3
Run4
Run5
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAreleased
Time (hr)
Run6
Run7
Run8
Run9
Run10
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAreleased
Time (hr)
Run11
Run12
Run13
Run14
Run15

Observed responses from randomized
runs in Box-Behnken design
Run
Responses
Y1 Y2 Y3
1 6.8 32.7 53.3
2 11.0 44.7 84.3
3 3.0 11.3 20.5
4 2.0 13.6 44.7
5 17.2 65.4 95.1
6 4.5 20.2 49.2
7 10.5 66.4 90.3
8 12.1 63.2 93.5
9 9.0 69.2 92.7
10 7.9 39.0 83.5
11 11.0 44.7 84.3
12 2.0 6.6 15.6
13 3.5 12.2 18.3
14 4.3 46.9 92.1
15 16.0 60.1 92.2

Summary of statistical analysis
Source Y1 Y2 Y3
Sum of squares P > F Sum of squares P > F Sum of
squares
P > F
(a) Model analysis
Mean vs. total 953.44 25918.13 69559.39
Linear vs.
mean
203.43 0.0115 3817.85 0.0609 8647.99 0.0087
2FIa vs. linear 18.75 0.7118 455.62 0.8094 1457.86 0.3927
Quadratic vs.
2FI
81.69 0.0487 3517.22 0.0023 3032.47 0.0092
Cubic vs.
quadratic
23.69 0.0667 238.23 0.0899 385.41 0.0628
Residual 1.12 15.44 17.04
Total 1282.11 33962.49 83100.16
(b) Lack of fit
Linear 124.13 0.0395 4211.07 0.0163 4875.74 0.0156
2FI 105.38 0.0311 3755.45 0.0122 3417.88 0.0148
Quadratic 23.69 0.0667 238.23 0.0899 385.41 0.0628
Cubic 0.000 0.000 0.000
Pure error 1.12 15.44 17.04
R2 Ra
2 PRESS R2 Ra
2 PRESS R2 Ra
2 PRESS
(c) R2 analysis
Linear 0.619 0.5150 258.03 0.475 0.3313 6764.99 0.637 0.5401 8608.39
2FI 0.676 0.4330 524.30 0.531 0.1797 10468.81 0.746 0.5561 10430.84
Quadratic 0.925 0.7886 381.63 0.968 0.9117 3846.43 0.970 0.9168 6204.88
Cubic 0.997 0.9762 ND b 0.998 0.9866 ND 0.999 0.9912 ND

Effect of the contents of Eudragit® RS (X1) and croscarmellose Na (X2) on 5-ASA
release at 6h using response surface plot and its contour plot at 4% Carbopol®
content.
sign-Expert® Software
ease at 6 hours
Design points above predicted value
Design points below predicted value
17.21
2.01
= B: Eudragit RS
= C: Croscarmellose Na
tual Factor
Carbopol = 4.00
1.00
3.25
5.50
7.75
10.00 0.00
0.50
1.00
1.50
2.00
2
6
10
14
18
Releaseat6hours
B: Eudragit RS
C: Croscarmellose Na

Effect of the contents of Eudragit® RS (X1) and croscarmellose Na (X2) on 5-ASA
release at 10h and 14 h using response surface plot and its contour plot at 4%
Carbopol® content.
1.00
3.25
5.50
7.75
10.00 0.00
0.50
1.00
1.50
2.00
6
24
42
60
78
Releaseat10hours
B: Eudragit RS
Design-Expert® Software
Release at 14 hours
Design points above predicted value
Design points below predicted value
96.12
15.6042
X1 = A: Carbopol
X2 = C: Croscarmellose Na
Actual Factor
B: Eudragit RS = 5.50
0.00
2.00
4.00
6.00
8.00
0.00
0.50
1.00
1.50
2.00
20
40.25
60.5
80.75
101
Releaseat14hours
A: Carbopol

Optimization of drug release
Overlay plot showing the optimized parameters suggested by the
software to get the responses in the required range.

Validation of the optimized formulation
Responses (predicted, %) Observed (%) Predicted error (%)
Y1 (7.58) 8 5.54
Y2(45.54) 46.2 1.45
Y3(78.83) 82.3 4.40
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAreleased
Time (hr)
Optimized formulation
ODMP
ƒ2 = 67.7

Release rate kinetics for the Box-Behnken
and the optimized formulations
Run
r2
Mechanism
Correlation time
span (h)
t20% t50% t80%
Zero order Diffusion Peppas
Run1 0.9809 0.9965 0.9743 Diffusion (6-16) 7.60 13.48 22.16
Run2 0.9993 0.9960 0.9979 Zero order (5-14) 7.01 10.49 13.98
Run3 0.9746 0.9853 0.9844 Diffusion (5-16) 14.40 45.72 97.96
Run4 0.9974 0.9900 0.9960 Zero order (8-16) 9.74 15.16 20.57
Run5 0.9961 0.9966 0.9910 Diffusion (5-16) 5.99 8.51 12.05
Run6 0.9855 0.9806 0.9894 Peppas (6-16) 9.50 15.04 19.79
Run7 0.9928 0.9861 0.9921 Zero order (5-12) 6.22 9.56 12.91
Run8 0.9856 0.9618 0.9921 Peppas (5-12) 8.12 10.44 12.10
Run9 0.9845 0.9776 0.9892 Peppas (5-12) 6.99 9.97 12.51
Run10 0.9991 0.9929 0.9965 Zero order (6-14) 7.36 10.76 14.17
Run11 0.9961 0.9715 0.9989 Peppas (5-14) 7.30 10.76 13.77
Run12 0.9576 0.9016 0.9813 Peppas (5-16) 14.97 22.72 28.62
Run13 0.9878 0.9923 0.9805 Diffusion (5-16) 15.03 51.33 112.94
Run14 1.0000 0.9977 0.9798 Zero order (6-12) 7.42 10.38 13.34
Run15 0.9956 0.9798 0.9970 Peppas (5-12) 6.68 9.67 12.33
Optimized
formulation
0.9974 0.9917 0.9898 Zero order (5-14) 7.12 10.47 13.82

Accelerated stability study for the optimized
formulation
• No Physical changes.
– No cracking.
– No change in color.
• No degradation.
• No significant change in
release profile.
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
Zero Time
3 Months
6 Months

Conclusion
• Box-Behnken design was successfully used to evaluate and optimize
the formulation variables.
• The optimized hydrophilic-hydrophobic, high loading 5-ASA matrix
tablets demonstrated a zero order drug release kinetics potentially
suitable for once daily administration.
• The optimized formulation, containing 5.72 % Carbopol®, 9.77%
Eudragit® RS and 1.45 % croscarmellose sodium in addition to
other excipients, was fabricated utilizing the simple wet granulation
technique and produced a release profile comparable to that of the
marketed product.

Chapter 4
Development, Evaluation and Optimization of
Novel Colonic Targeting 5-ASA Microspheres

Rational for formulating 5-ASA into
microspheres
• Controlling rate and site of drug release while providing:
– Less variations in gastric emptying.
– Better distribution in the GIT.
– Less liability of dose dumping.

0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16
Time (h)
Formulation Design
Drug Particles
pH1.2
pH4.5
pH7.4

5-ASA and
Mg stearate
Polymers
dissolved in
acetone
30 min
5 min
• Preparation of microspheres.
Experimental

Liquid paraffin
4⁰C premixed
with 2% span
Acetone
2000 rpm for 5 min1000 rpm for 4-6 hr

Morphological
and particle
size analyses
Production
yield
Encapsulation
efficiency
DSC and
FTIR
studies
In-vitro
release
studies and
kinetics
analysis
Characterization of microspheres

Composition of microspheres formulations
Formulation
5-ASA
(mg)
Drug: ES100
ratio
ES 100
(%)
Drug :EC
Ratio
Mg Stearate
(%)
Internal :
external
phase ratio
Surfactant
(%)
Microparticles prepared with varying polymer to internal phase ratio
MF1 500 1:1 2 - 5 1:2 2
MF2 500 1:1 1 - 5 1:2 2
MF3 500 1:1 0.667 - 5 1:2 2
Microparticles prepared with different internal-to-external phase ratio
MF2 500 1:1 1 - 5 1:2 2
MF4 500 1:1 1 - 5 1:4 2
MF5 500 1:1 1 - 5 1:6 2
MF6 500 1:1 1 - 5 1:8 2
Microparticles prepared with varying drug to ES 100 ratio
MF7 500 1:0.25 1 - 5 1:2 2
MF8 500 1:0.5 1 - 5 1:2 2
MF9 500 1:0.7 1 - 5 1:2 2
MF2 500 1:1 1 - 5 1:2 2
MF10 500 1:2 1 - 5 1:2 2
Microparticles with varying drug to polymeric blends ratio
MES70EC10 500 1:0.7 1 1:0.1 5 1:2 2
MES70EC15 500 1:0.7 1 1:.0.15 5 1:2 2
MES70EC20 500 1:0.7 1 1:0.2 5 1:2 2
MES85EC10 500 1:0.85 1 1:0.1 5 1:2 2
MES85EC15 500 1:0.85 1 1:0.15 5 1:2 2
MES185EC20 500 1:0.85 1 1:0.2 5 1:2 2
MES100EC10 500 1:1 1 1:0.1 5 1:2 2
MES100EC15 500 1:1 1 1:0.15 5 1:2 2
MES100EC20 500 1:1 1 1:0.2 5 1:2 2

Effects of varying proportion of Eudragit® S to internal phase
Formulation Physical Appearance Particle Size (µm) Yield (%) Entrapment Efficiency (%)
MF1 Spherical, discrete 83.35±22.74 84.45 ± 2.45 82.15 ± 2.54
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
MF1 MF2
MF3

Effects of internal phase to external phase ratio.
MF5 Irregular in shape 151.68±56.61 91.54 ± 2.96 79.25 ± 1.22
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
MF2 MF4
MF5 MF6

Effects of varying proportion of Eudragit® S to drug .
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time (hr)
MF2
MF7
MF8
MF9

Physical characterization of EC-Eudragit® S formulations.
Formulation Physical Appearance Particle Size(µm) Yield (%) Entrapment Efficiency (%)
MES70EC10 Spherical, discrete 151.29±84.54 91.00 ± 3.45 84.15 ± 2.93

DSC and FTIR studies of EC-Eudragit® S formulations.

In-vitro release profiles of Eudragit® S-EC formulations
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time
MES70EC10
MES85EC10
MES100EC10
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time
MES85EC15
MES100EC15
MES70EC15
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAReleased
Time
MES70EC20
MES85EC20
MES100EC20

Kinetic analysis of EC-Eudragit® S formulations.
Formulation
r2
Mechanism
Correlation
time span
(hr)
t20% t50% t80%
Zero order Diffusion Peppas
MES70EC10 0.894 0.923 0.844 Diffusion (4-8) 5.02 8.30 13.84
MES70EC15 0.948 0.960 0.903 Diffusion (4-6) 5.27 8.14 12.67
MES70EC20 0.931 0.945 0.925 Diffusion (4-6) 4.24 6.72 11.88
MES85EC10 0.952 0.972 0.930 Diffusion (5-12) 6.63 10.74 16.76
MES85EC15 0.944 0.968 0.931 Diffusion (5-12) 6.50 10.45 16.25
MES85EC20 0.989 0.996 0.985 Diffusion (5-12) 6.30 10.08 15.66
MES100EC10 0.980 0.993 0.952 Diffusion (5-12) 6.87 11.07 17.13
MES100EC15 0.956 0.973 0.927 Diffusion (5-10) 6.46 10.19 15.59
MES100EC20 0.945 0.958 0.921 Diffusion (5-8) 5.82 8.88 13.44

Scanning electron micrographs of microspheres from batch MES100EC15
before drug release (330X)

before drug release (140X)

Scanning electron micrographs of inner part of the microspheres from batch
MES100EC10 before drug release (1000X)

after drug release (200X)

after drug release (2000X)

Accelerated Stability Study for selected
formulation MES100EC10
• No Physical changes.
– No change in color.
• No degradation.
• No significant change in
release profile.
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14 16 18
%5-ASAreleased
Time (hr)
Zero Time
3 Months
6 Months

Conclusion
• Oil/oil solvent evaporation method resulted in preparation
of microspheres suitable for colon targeting with 5-ASA.
• Pulsatile type colon targeting microspheres could be
successfully achieved with formulation MF2 however such
formulation could not suitable sustained release purposes.
• Controlled release profile for 10hr after a lag time of 4hr
was achieved with formulation MES100EC10 which seems
potential for once daily dosing purposes.
• Acceptable spherical morphology and size characters were
observed for both of these formulations.

A pharmaceutical study on certain aminosalicylates master defense

A pharmaceutical study on certain aminosalicylates master defense

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