Name: Tahir
Roll no: 22-80088
Section: A
Subject: Pharmaceutical Quality
Management
Submitted to: Dr. Khurram Rehman
Assignment
Recent drug release studies using rat
cecal dissolution media
“Polymeric nanocomposite:
Development, characterization, ex vivo
and in vivo evaluation for Ulcerative
Colitis”
Ulcerative Colitis is basically a bowel
disease in which there is mucosal
inflammation, rectal bleeding, pain in
abdomen and diarrhea.
The inflammation occurs mostly in colon
and start from rectum towards cecum.
For treating and management various
immunomodulatory drugs,
aminosalycylates, ant –biotic and protein
kinase inhibitors are administered. But
flares occur despite the treatment in
most cases so no remission can be
achieved most of the time. Colorectal
cancer may also develop due to
uncontrolled addition of epithelial cells
in response to rapid loss.
Nano sized particles were first
experimented for better therapeutic
effect because of their reach to inflamed
areas but there was a drawback later
found which is that they release before
they reach the target which not only
reduce its efficacy but induce toxicity. To
rectify that we need a drug delivery
system that reach the target. For this,
mesalazine (MSZ) loaded chitosan
nanoparticles and Chitosanmontmorillonite nano-composites (CHMMT-NCPs).
 Chillate (CH) can protect the drug
release in the upper part of GIT.
 MMT is a clay mineral and absorb
dietary toxins which causes
gastrointestinal disturbance.
Formulation of CH-NPs
 Take a beaker and add TPP aqueous
solution in CH solution.
 At room temperature, stir the
solution for 60 min with magnetic
stirrer.
 Then add mesalazine or FITC in CH
solution so that the drug or FITC is
filled in CH-NPs.
 Untrapped mesalzine and FITC was
removed by centrifuge process at
30,000rpm for 30 min.
 Wash the dosage form for 3 times to
remove any extremities.
 The formulation is then preserved
until its use.
Formulation of CH-MMT-NCPs
 Separately prepare CH solution and
MMT aqueous dispersion.
 Take CH solution that contains
mesalazine and tweem80.
 Add MMT aqueous dispersion in it
and stir.
 Now add and stir TPP solution in CHMMT-NCPs so that mesalazine will
fully dispersed in the dosage form.
 Sonicate MMT after dispersing it in
distilled water for 24h.
 Add and stir certain amount of
mesalazine and Tween80 in MMT.
 Add and stir MMT at 5000 rpm for 2h
after adding it to CH solution.
 Sonicate it for 15min.
 Under constant stirring mix TPP
solution in CH-MMT dropwise.
 Centrifuge the Nano composite that
form.
 The formulation is ready, preserve it
until use.
Nano constructs Characterization
 We determine the zeta potential by
particle size analyzer which is
diffraction based.
 Transmission electron microscope
(TEM) helped in analyzing its
construction. Acceleration voltage
was set was set at 100kV.
 Photographs of Nano constructs were
taken.
Drug entrapment determination
 First we do the centrifugation of
medium containing Nano constructs
at 25000x g at 10 Celsius for 10 min
to get separated Nano constructs
and now we can determine drug
entrapment percentage.
 We use mobile phase to perform
HPLC in isocratic mode for certain
time.
 Mobile phase is basically composed
of acetic acid, water and acetonitrile.
 We observe that basic correction was
supernanant of unloaded Nano
particles.
 The equation for drug entrapment
efficacy is as follows
DEE%= AB/A
 Where A is total amount of
Mesalazine.
 Free amount of MSZ in supernatant is
represented by B.
FTIR
 FTIR is Fourier Transform Infrared
Spectroscopy.
 It has range from 400 – 4000c/m.
 First we put the MSZ that was
released from CH-MMT-NCPs and
CH-NPs in sealed glass at 37 Celsius.
 Then weigh the formulation at
about 100 mg and place in
dissolution media of 100 ml.
 Shake the vial and withdraw the
samples in a periodic manner for
24h.
 Replace the withdrawn sample with
colonic media.
 Centrifuge the samples at 2000 rpm
for standard time.
 HPLC method is used to analyze the
samples efficacy.
DSC studies
 The purpose of DSC in this process
was to determine the physical
activity of the drug.
 Take certain amount of a drug
sample.
 Add in the aluminum pan at certain
temp. (50-300 Celsius).
 Set the heat rate at 10 Celsius/min.
 Observe the drug’s characteristics.
General in vitro drug release studies
The ph, enzymatic activity,
microflora and other variables should
be same. CH-NPs and CH-MMT-NCPs
are some of the formulations tested
for in vitro. 100 ml dissolution
medium is used. Stir it up at 100 rpm
at 37 celsius. MNZ loaded NPs are
placed or dispersed in 10 ml of PBS
ph 7.4 and that dispersion is then put
in the bag known as dialysis
membrane bag and consider
molecular cutoff at 5kDa.
This bag is then tied and dispersed in
100 ml of PBS solution. After some
certain time 2ml of dffusion medium
is removed and 2 ml of fresh PBS ph
7.4 is added. The samples that are
withdrawn are assayad for
mesalazine using method known as
HPLC.
Rat Cecal content (in vitro)
The same dosage forms that are CHNPs and CH-MM-NCPs containg
mesalazine are tested for rat cecal.
30 min before the experiment rat is
sacrificed and cecum is separated and
added into the PBS ph 7.4. along with
CO2. The desired cocentrations of
cecum are taken and added in PBS ph
7.4. Firstly mesalazine with the above
dosage forms are added in the glass
vials then 100 mg weighed
formulation is added in 100 ml
dissolution media containing cecal
solution. Shake the vial and samoles
are withdrawn 1ml each periodically
for 24 hours. Then the sample is
centrifuged at 2000 rpm just for 10
minutes and then analyzed for
mesalazine by HPLC.
Cellular uptake studies
Both the doage forms that are CHNPs and CH-MMT-NCPs are put in a
growth medium for different times.
Add certain amount of PBS solution
containing 2.5mg/ml of trypsin.
Incubate for short period of time and
let the cells harvest.it is done by
treating it with probe sonicator and 1
ml PBS solution. Then centrifuge the
resultant that is cell lysate at 10,000
rpm for like 10 min and analyze it by
FACS.
Confocal laser scanning microscopy
Now the above dosage forms are
tested under confocal laser scanning.
Delvery system is loaded by FITC. The
staining was done by especially using
DAPI dispersing in PBS. By viewing we
get the dosage forms at lambda ex.
488nm and lambda em. 536-624 nm.
When see the DAPI fluorescent it was
at lambda ex. 385nm and lambda em.
400-480 nm.
Colitis model
In colitis model mouse is
administered with TNBS interarectally
to induce inflammation at the cecal
site. Then the mouse is set to be in a
certain place for two days without
treating. Then administer mesalazine
either in either or with Nano
formulations orally. Make the
concentration 0.1ml. To check the
efficacy, sacrifice the mouse and
separate cecum or colon and observe
the changes.
Pathophysiological parameters
Some of the signs of rectal
inflammation might consist of rectal
bleeding, weight loss and stool
consistency. Activity of neutrophils in
the inflammated area by
myeloperoxidase and enzymatic
activity is also determined separately.
Histological studies
In this studies we observe how much
inflammation has occurred and how
much tissue has been damaged. For
this microscopic examination is
conducted on the tissue of the colon
after staining with formalin and eosin
and then observe under microscope
and print the picture.
Results and discussions
The two dosage forms CH-MMT-NCPs
and CH-NPs are studied.
Optimization and characterization
 The dosage form CH-MMT-NCPs
has the particle size of around
287.47nm and for CH-NPs is
267.82nm. it is to be noted that
they contain mesalazine too.
 When we talk about percentage
drug entrapment for CH-MMT-
NCPs it is 86.49 % and for CH-NPs
it is 70.45%.
 CH-MMT-NCPs has zeta potential
32.5 and for CH-NPs has 23.6.
FT-IR spectrum
 For CH-NPs the spectrum has a peak
at 3440.2c/m and then shifts at
3416.2c/m and becomes wide. It has
an impression that hydrogen bond is
enhanced, while N-H bending is at
1633.1 and 1555.0c/m which shows
electrostatic interactions among the
particles.
 For CH-MMT-NCPs the spectrum has
a peak 1597.8c/m. A higher peak is
also shown at 3450-3200c/m and
then decreases which shows the
interaction between CH and MMT
which proves good dispersion among
the particles.
Differential scanning calorimetry (DSC)
 In CH-MMT-NCPs, MMT has
endothermic peak below 100 Celsius
representing moisture. CH at 70
Celsius representing loss of humidity
whereas MSZ that is fused with this
dosage form has a peak at 280 Celsius
where then it disappears which
represents physical mixture.
In vitro results
 From the in vitro studies of drug
release we carried out the difference
between the two dosage forms
containing mesalazine for the
treatment of Urinary Colitis.
 CH-NPs may release some in the
gastric area or in the pouch while CHMMT-NCPs has sustained release
because of the strong bond between
negatively charged MMT and
positively charged MSZ.
 In CH-MMT-NCPs, CH has low
solubility in gastric area makes it to
avoid any release.
Cytotoxicity results
 When we talk about CH and CH-NPs,
CH has GI percentage of 84.29% and
CH-NPs has 93.64%.
 When we talk about MMT and CHMMT-NCPs at around 500
microgram/ml MMT was at 36.19%
and CH-MMT-NCPs was at 23.68%.
 CH has low toxicity but at high dose,
dose dependent toxicity may be
reported.
 MMT has low toxicity because of the
structure multiple layers.
 It was studied through SRB assay.
Cellular uptake results
 From this study we gather several
data
 We used Colo 205 cells and studied
through FTIC used as fluorescent
marker.
 Study taken in 1h period was plain
FITC shows 7.45%, CH-NPs-FTIC
shows 36.89% and CH-MMT-NCPs
shows 58.9% in 1h incubation.
 In 3h incubation period plain FTIC
shows 19.86%, CH-NPs-FTIC shows
49.53% and CH-MMT-NCPs-FTIC
shows 83.77%.
 MMT bonding with CH may increase
cellular adhesion and adsorption
because of increased viscosity
among the particles.
 The particles become more compact
by developing hydrogen bond,
London-vander-Wals forces among
them.
CLSM
 By seeing the images of Colo205
through CLSM we conclude that
 We can see that CH-MMT-NCPs
has increased uptake and
fluorescence as compared to CHNPs.
 It is to be noted that both the
dosage forms had more uptake
than plain FITC.
Ulcerogenic results
 By studying ulcerogenic
activity we conclude that
 The sample shows decreased
inflammation within 24-48h
administered with MSZ and
Nano constructs.
 It is to be noted that we
observed low clinical activity
by CH-NPs comparing MSZ.
 While the sample that was
administered with CH-MMT-
NCPs shows excellent
recovery.
 By administrating 10 times
higher doses of CH-MMTNPCs, we see very minute
activity of myeloperoxidase
Histological results
 From the histological studies
we conclude that
 By examination of CH-MMTNCPs it shows significant
relief from urinary colitis, it
can be clearly observed that
inflammation was decreased
and ulceration was also
reduced. Epithelial
regeneration can also be
seen.
 We also conducted light
microscopy and it was
observed that CH-MMTNCPs administration causes
reduction cellular
infiltration, Lymphocyte
infiltration than other
dosage form administered.
 CH-MMT-NCPs when
examined by CLSM, it shows
more uptake and
fluorescent intensity than
CH-NPs.
 When we observe the in
vitro studies we conclude
that CH-MMT-NCPs was very
effective in releasing
mesalazine to the target
tissues and treat urinary
colitis than CH-NPs and plain
MSZ.
Conclusions
 When we talk about the
dosage form CH-MMT-NCPs,
it protects the drug in the
GIT especially where more
danger of release that is the
upper part.
 It is made such that it only
releases the drug when it is
metabolized by the enzymes
in colon.
 In vivo studies also shows
that the efficacy of CHMMT-NCPs. There are
number of reasons for that.
 It forms a protective layer
over inflamed mucosa of
colon.
 They have anti-inflammatory
activity including increase in
the levels of mucin and
adsorption of luminal
antigens.
 They act from the luminal
side, hence reduces the
effects of intestinal
inflammation.
 So it is concluded that
Chitosan Nano particles (CHNPs) is less effective in drug
release than Chitosanmontmorillonite
nanocomposites (CH-MMTNCPs).
 Also it has more healing
efficacy than CH-NPs and
mesalazine solution.
 It is noted that CH-MMTNCPs showed decreased
ulcer index that is around
3.80 while comparing with
CH-NPs that is around 7.03
and mesalazine solution
index is around 9.85.
 So to conclude it is evident
that CH-MMT-NCPs
containing the drug has
more efficacy than CH-NPs
and drug solution.
 Source cited
(Arvind Gulbake, 2016)
REFERENCE LINK
1. https://www.tandfonlin
e.com/doi/abs/10.1080/0
0914037.2015.1119690