Enhancement Of Bioavailability
Of Atorvastatin Calcium by Micro
and Nanoparicle preparationDesign and Evaluation
Dr.K.Senthilkumaran
INTI International University
College



S.Dinesh kumar, M.Nappinnai
Department of Pharmaceutics,
C.L.Baid Metha College of Pharmacy,
Chennai, India.
Introduction

Bioavailability :is the true rate and
extent of therapeutically active drug,
which reaches the systemic
circulation and is available at the site
of action from an administered
dosage form.


Bioavailability studies provide other
useful pharmacokinetic information
related to distribution, elimination
and metabolism of drugs in the body.
Bioavailability data may also provide
information indirectly about some
properties of a drug substance - such
as membrane permeability

Absolute bioavailability of a drug is 1
(or 100%) indicates complete
absorption of drug.
SIGNIFICANCE OF BIOAVAILABILITY
STUDIES


Development of a suitable dosage
form for a new drug entity
Determination of influence of
excipients, patient related factors, on
the efficiency of absorption.


Development of new formulations of
the existing drugs.
Development of suitable dosage form
for a new drug.
METHODS FOR ASSESSING
BIOAVAILABILITY


Pharmacokinetic methods: based on the
plasma concentration of the drug and on
the assumption that the pharmacokinetic
profile reflects the therapeutic
effectiveness of a drug
Pharmacodynamic methods: involves
direct measurement of drug effect on a
pathophysiologic process as a function of
time.
REASONS FOR POOR BIOAVAILABILITY


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Poor aqueous solubility of the drug
Poor stability of the dissolved drug at
physiological pH.
Poor permeation through the
biomembranes
Extensive presystemic metabolism.
METHODS TO ENHANCE
BIOAVAILABILITY






To enhance the dissolution rate
of a drug:
Micro encapsulation
Use of surfactants
Solute-solvent complexations
Solid dispersions
Molecular encapsulations with
cyclodextrins
In the present study



Atorvastatin calcium is reported to
have low bioavailability of 14%
The low systemic availability is
attributed to presystemic clearance
in gastro-intestinal mucosa
The drug is rapidly absorbed after
oral administration and maximum
plasma concentrations occur within 2
hours.
SCOPE OF THE PRESENT
WORK


Atorvastatin calcium is formulated as
colloidal particles for oral drug
delivery with an aim to improve its
bioavailability.
Biodegradable polymer poly- lactide
co- glycolide was used to prepare the
colloidal particles.
Approaches


Incorporation of the drug into
colloidal particles may protect it from
the first pass effect, which may lead
to an increased bioavailability
Prolonging the release may also
improve the bioavailability.

Size reduction of the final
formulation in colloidal particles
improve absorption leading to
improvement in bioavailability.
METHODOLOGY


Preparation of colloidal particles of
atorvastatin by using poly lactideco-glycolide PLGA-resomer RG
50:50 H by solvent evaporation
techniques.
Determination of drug content, drug
loading and encapsulation efficiency
of prepared colloidal particles.



Determination of drug – polymer
interaction by FT-IR
In vitro release studies of
dissolution release kinetic analysis.
In vivo release study of atorvastatin
calcium colloidal particles


Determination of pharmacokinetic
parameters.
In vivo pharmacodynamic study
DRUG PROFILE

Structure:
Molecular formula: (C 33 H 34 F N 2O 5) 2 Ca. 3 H 2 O
Molecular weight: 1209.42
Description: White to off-white crystalline powder.
Solubility :Atorvaststin calcium is very slightly soluble in water,
freely soluble in methanol.
Dose: 10 -40mg daily
PREPARATION OF COLLOIDAL PARTICLES OF
ATORVASTATIN CALCIUM
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
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Atorvastatin calcium was prepared into
colloidal particles with polylactide – coglycolide resomer (RG 50:50H) by solvent
evaporation method.
100 mg of PLGA was weighed and
dissolved in a mixture of dichloro methane
and ethanol in ratio (4:1)
100 mg of atorvastatin calcium was added
to the polymer solution and mixed well.

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The above mixture was added drop wise
to the (0.4%) preheated 40oC) polyvinyl
alcohol in distilled water.
Tween 80 was used for emulsion formation
and stabilization.
This mixture was continuously
homogenized to yield a oil in water
emulsion.


The emulsion was stirred continuously for
3 hours to allow the organic solvent to
evaporate and the hardening of
atorvastatin calcium colloidal particles to
be completed.
The colloidal particles of atrovastatin
calcium, were recovered by refrigerated
centrifugation/membrane filtration/
lyophilisation
The formulation trials of
Drug :polymer
S.No
Formulation
Drug :
Polymer
1
FI
1:1
2
F II
1:2
3
F III
1:3
4
F IV
1:4
5
FV
1:5
6
F IV
1:6
Harvesting Method
Methods of
preparation
SO
C
M
L
HO
C
M
L
SO
C
M
L
HO
C
M
L
SO
C
M
L
HO
C
M
L
SO
C
M
L
HO
C
M
L
SO
C
M
L
HO
C
M
L
SO
C
M
L
HO
C
M
L
Percentage yield of colloidal particles
Percentage yield of colloidal particles
S.No.
Formulation
Drug : Polymer
SO
HO
C
L
C
L
1
F1
1:1
50
45
65
76
2
F2
1:2
55
44
64
75
3
F3
1:3
48
40
65
70
4
F4
1:4
49
42
75
73
5
F5
1:5
50
59
70
71
6
F6
1:6
50
45
69
70
Determination of Drug content in
colloidal particles


Determination the amount of
atorvastatin calcium present in the
100 mg colloidal particles was
calculated by using standard
calibration graph of atrovastatin
calcium in methanol.
The same procedure was triplicated
for each formulation
Drug content of Colloidal Particles
Amount of Drug in 100 mg of colloidal particles
S.No.
Formulation all homogenized sample
(HO)
1
Centrifugation
(mg)
Lyophilisation
(mg)
F1
60
55
2
F2
62
59
3
F3
61
60
4
F4
75
78
5
F5
65
74
6
F6
72
70
Figure 1 Drug content of Colloidal particles
100
90
Centrifuged
80
Lyophilised
Percentage
70
60
50
40
30
20
10
0
F1
F2
F3
F4
Formulations
F5
F6

From the results of analysis of drug
content, drug loading, drug
encapsulation formulation F4 was
selected for further studies.
Particle size analysis


The particle size of formulated
colloidal micro particles were
analyzed by Particle size analyzer.
FTIR spectra of the drug, PLGA and
the drug loaded PLGA colloidal
particles were obtained.
In vitro release studies
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The sample equivalent to 100 mg of
atorvastatin calcium was placed in 10 ml
of phosphate buffer pH 7.2 it was
maintained at 37°C and magnetically
stirred at 100rpm.
2 ml of sample aliquot was with drawn at
different time intervals and filtered
through a 0.45 mm filter


The dissolution media was then
replaced with 2 ml of fresh buffer.
The atorvastatin calcium
concentration was determined by
HPLC using ODS, C18 column with
flow rate of 1ml/min at 250 nm
In vitro Dissolution study of colloidal particles
S. No.
Sampling time
in course
Concentration mcg/ml
(from HPLC
chromatogram)
Concentration in
sampling fluid (2 ml)
mcg
Cumulative
amount of drug
release
mg
Cumulative drug
release percentage
%
1
15 mins
4.50
450
2.25
2.88
2
30 mins
6.53
3265
16.77
21.51
3
45 mins
8.82
4410
25.76
33.03
4
1 hr
10.00
5000
33.12
41.19
5
2 hr
11.19
5595
41.10
52.69
6
3 hrs
11.93
5965
48.54
62.24
7
6 hrs
12.69
6345
56.41
72.32
8
8 hrs
13.63
6815
65.11
83.47
9
24 hrs
20.26
8104
78.36
100
In vitro drug release study of colloidal
particles
Figure 4
In vitro release study of F4
120
100
Percentage
80
60
40
20
0
15 mins 30 mins 45 mins
1 hr
2 hr
Time
3 hrs
6 hrs
8 hrs
24 hrs
In vivo Pharmacokinetic Study of
atorvastatin colloidal particles



Procedure was carried with the
approval from animal ethical
committee
CPCSEA/IAEC approved detail 10/14CLBMCP/2005-2006.
Male rabbits weighing about 1 kg to
1.5 kg were used in this study


The animals were housed under
standard environmental conditions
(23°C  2°C, 55  5% relative
humidity; 12 hours light/ dark cycle).
Prior to oral administration, the
rabbits were starved for 24 hours
and are allowed free access to tap
water only.


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The animals (12) were divided into
two groups of 6 animals in each
group.
Group- I received standard drug of
atorvastatin calcium 2.4mg/kg dose
Group – II received F4 HO, L
particles equivalent to 2.4 mg/kg


1ml blood sample were with drawn
from the marginal ear vein of the
rabbit at regular time interval.
The serum samples were separated
by centrifugation and estimated by
using HPLC method.
Comparison of In vivo
pharmacokinetic release study
S.No.
Sampling time
Atorvastatin Calcium
Formulation F4 atorvastatin
(raw material)
colloidal particle mcg/ml
1
30mts
40.9
61.6
2
1hr
71.28
98.8
3
2hr
121.6
129.7
4
4hr
110.3
118.56
5
8hr
70.5
85.54
6
24hr
21.3
35.23
Figure 5 In vivo pharmacokinetic release study
Plasma Drug Concn. (mcg/ml)
140
Standard
120
Test-F4
100
80
60
40
20
0
0
30 min
1 hr
2 hr
4 hr
Time (hrs)
6 hr
8 hr
24 hr
In vivo pharmacokinetic
parameters
Units
Standards
Test – F4
C max
mcg/ml
121.6
129.7
t max
Hrs
2.0
2.0
Mcg-hr/ml
1917
2449
(mcg-hr* hr/ml
28570
42406
Hr
14.9
17.3
Parameters
AUC0-
AUMCO-
MRT
Pharmacodynamic Study




Procedure was carried with approval
from animal ethical committee
CPCSEA/IAEC approved detail 10/15CLBMCP/2005-2006.
The rats were divided into two
groups A and B
Group A and B received high fat diet
(HFD)
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group A was treated with standard
drug
group B received the formulation F4
colloidal particles of atorvastatin
calcium orally, administered.
At the end of experimental period all
the animals were fasted over night
and blood was with drawn.

The total lipids (TL), total
cholesterol (TC), triglycerides (TG),
phospholipids (PL) and HDl.
Cholesterol were estimated in the
serum, using commercially available
standard kits.
In vivo Pharmocodynamic study
S.No
Parameters
Standard Atorvastatin calcium (Raw
material) mg/dl
Test F4 – Formulation Colloidal
particles mg/dl
1
TC
67.1 + 1.7
60.5+ 2
2
TG
55+ 4.6
46.7 + 1.5
3
HDL
20.9+ 3
14.5+ 1.0
4
LDL
37.4+ 1.0
32.5+ 2.2
5
VLDL
10.2 + 1
9.1 + 0.6
Figure 6 In vivo Dynamic Study
100
90
Standard
80
Test
Percentage
70
60
50
40
30
20
10
0
TC
TG
HDL
Parameters
LDL
VLDL
SUMMARY

In the present study an attempt has
made to increase the bio availability
of the drug by preparing colloidal
particles using different drug to
polymer ratio to determine the
suitable formation.


Atorvastatin calcium colloidal
particles was prepared by using
emulsification solvent evaporation
technique to achieve maximum drug
content.
The Prepared Colloidal particle were
evaluated for drug content, drug
loading and encapsulation efficiency.



F4Ho, L particles showed smooth
spherical shape.
Therefore F4Ho, L were subjected to
particle size analysis
The in vitro drug release from F4Ho,
L shows the maximum release
studies after 24 hrs.


In vivo pharmacokinetic study results
revealed that the formulation F4 shows
better Cmax, tmax, AUC, AUMC and MRT,
than the standard drug.
The in vivo pharmacodynamic study shows
significance decrease in lipidaemic
parameters in rats with formulation F4
than these administered with standard
atorvastatin calcium.


To conclude, colloidal particles of
atorvastatin calcium improved
bioavailability of the drug.
The relative Bio-availability is 1.27
and it is proved by pharmacokinetic
and pharmacodynamic studies.

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