INTRODUCTION:
Ophthalmic drug delivery is one of the most interesting
and challenging endeavours facing the pharmaceutical scientist...The anatomy,
physiology and biochemistry of the eye render this organ exquisitely impervious
to foreign substances...The challenge to the formulator is to circumvent the
protective barriers of the eye without causing permanent tissue damage...The
primitive ophthalmic solutions, suspensions and ointment dosage forms are clearly no longer sufficient to combat some
present virulent diseases.1.2
Good corneal penetration is required to improve the
bioavailability of the drug in the eye,which in turn can be fulfilled by
prolonged contact time with corneal tissue. Iontophoresis, prodrugs, ion pair
formation and cyclodextrins have all been used as means of enhancing ocular
drug absorption.3 An ideal topical ophthalmic formulation would
enhance bioavailability by sustaining drug release, while remaining in contact
with the front of eye for prolonged periods of time.4
Recently, much research has been dedicated to
mucoadhesive polymers i.e. macromolecules. This approach relies on vehicles
containing polymers that adhere via non-covalent bonds to conjunctival mucin,
thus ensuring contact of the medication with the `precorneal tissues until
mucin turnover causes elimination of the polymer.5 Mucoadhesive
polymers are usually hydrocolloids with numerous hydrophilic functional groups
such as carboxyl, hydroxyl, amide and sulphate. These groups can establish
electrostatic interactions, hydrophobic interactions, Van der Waals
intermolecular interactions and hydrogen bonding with mucus substrates. For
many polymers, hydrogen bonding appears to play a significant role in
mucoadhesion, thus the presence of water seems to be a prerequisite for a
majority of mucoadhesive phenomena.6-8 Various synthetic,
semi-synthetic and naturally occurring polymers (hydroxypropyl cellulose,
polyacrylic acid, high-molecular weight (<200,000) polyethylene glycols,
dextrans, hyaluronic acid, poly galacturonic acid, xyloglucan, etc.) have been
evaluated for mucoadhesion.9-12Active investigations on mucoadhesive
polymers as ingredients for ophthalmic vehicles are now underway in many
laboratories and will hopefully lead to the development of more efficient
ocular delivery systems.
Fig-1: Frequency polygon representing the particle size
distribution of group A of ocular microparticles
Fig -2: Frequency polygon representing the particle
size distribution of group B of ocular microparticles
Chitosan is a natural polysaccharide
comprising copolymers of glucosamine and N-acetylglucosamine. Due to its
excellent biocompatibility and biodegradability, and unique polymeric cationic
character, chitosan had been widely exploited in the pharmaceutical industry
for its potential in the development of drug controlled release systems10-
Microparticles have an average particle size greater than 1μm and may be
microcapsules or microspheres. Upon topical instillation, the particles reside
in the ocular cul-de-sac, and the drug is released from the particles through
diffusion or polymer degradation.13-15Techniques for the manufacture
of particles include: denaturation or cross-linking of macromolecules in
emulsion; interfacial polymerization; formation of emulsions and solvent
removal; solution enhanced dispersion by supercritical fluids and spray drying.16,
17
Ketorolac tromethamine (KT), a nonsteroidal
anti-inflammatory drug (NSAID), is indicated for the short-term management of
severe acute pain that requires analgesia at the opioid level. It is one of the
commonly used drugs for the treatment of pain that is inexpensive, safe, and
well tolerated. Ketorolac (free acid) is sparingly soluble in water and,
therefore, it is marketed in the form of tromethamine salt, which increases its
solubility in water.18
Ketorolac is a nonsteroidal anti-inflammatory
drug (NSAID) that is chemically related to indomethacin and tolmetin. Ocular
administration of ketorolac reduces prostaglandin E 2 levels in
aqueous humor, secondary to inhibition of prostaglandin biosynthesis. Ketorolac ophthalmic may be administered in conjunction
with other ophthalmic medications, such as antibiotics, beta-adrenergic
blocking agents, carbonic anhydrase inhibitors, cycloplegics, and mydriatics19,20
Fig-3: Frequency polygon representing the particle size
distribution of group C of ocular microparticles
Table -1 : Composition of various batches of Ocular
Microparticles
|
Batch no
|
Chitosan (3ml)
|
TPP(3+3 ml)
|
Span 80
|
|
A1
|
2%
|
2%
|
1ml
|
|
A2
|
1.8%
|
2%
|
1ml
|
|
A3
|
1.6%
|
2%
|
1ml
|
|
A4
|
1.4%
|
2%
|
1ml
|
|
A5
|
1.2%
|
2%
|
1ml
|
|
A6
|
1.0%
|
2%
|
1ml
|
|
B1
|
2%
|
1.5%
|
1ml
|
|
B2
|
1.8%
|
1.5%
|
1ml
|
|
B3
|
1.6%
|
1.5%
|
1ml
|
|
B4
|
1.4%
|
1.5%
|
1ml
|
|
B5
|
1.2%
|
1.5%
|
1ml
|
|
B6
|
1.0%
|
1.5%
|
1ml
|
|
C1
|
2%
|
1.0%
|
1ml
|
|
C2
|
1.8%
|
1.0%
|
1ml
|
|
C3
|
1.6%
|
1.0%
|
1ml
|
|
C4
|
1.4%
|
1.0%
|
1ml
|
|
C5
|
1.2%
|
1.0%
|
1ml
|
|
C6
|
1.0%
|
1.0%
|
1ml
|
TPP= Sodium tripolyphosphate
Fig 4-8: Morphology of Ocular microparticles
prepared by modified emulsification-ionotropic gelation method and observed
by digital optical microscopy
In this study chitosan microparticles of
ketorolac tromethamine were prepared by modified emulsification ionic gelation
method, using sodium tripolyphosphate as the ionic cross linking agent21-24. Thus, an attempt is made in the present investigation’s to
use chitosan as a mucoadhesive polymer and prepare microparticles that
reside in the ocular cul-de-sac, and the drug is released from the particles
through diffusion. Chitosan microparticles prepared
are in the size range of 1-14 µm there by these are small enough to remain
undetectable by the eyes and big enough to entrap drug efficiently. The data
obtained from in vitro release studies was fit into various kinetic
models to study the release mechanism and release kinetics. Biological response
was measured in rabbits by means of prostaglandin E2-induced
rabbit ocular inflammation model.
Material and methods:
Ketorolac tromethamine was a gift sample
from Ranbaxy lab, Gurgaon, Chitosan was obtained from Central institute of
Fisheries technology, Kochi, India. Sodium tripolyphosphate was purchased from
Sigma Aldrich Chemie., Germany. Ether, Liquid Paraffin, GAA were procured from
S.D Fine Chem. Ltd., Mumbai, India. Span 80 was received from Loba Chemie. Pvt.
Ltd., Mumbai, India, and all other chemical and reagents used were of
analytical grade
Preparation of microparticles:
Chitosan microparticles were prepared using
modified emulsification-ionotropic gelation method. The drug containing
chitosan solution (in 1% acetic acid) was injected through 22 gauze syringe
needle into liquid paraffin containing Span-80, as the emulsifying agent. The
addition of chitosan was accompanied with stirring at 8000 rpm using high speed
stirrer. The cross linking of dispersed chitosan droplets was accomplished by
adding sodium tripolyphosphate solution, with continuous stirring at 8000 rpm
for 3 h. The chitosan microparticles so obtained were collected by centrifugation,
and then washed four times with solvent ether. After the final wash the
microparticles were allowed to dry in air. Dry powder thus obtained was
collected and stored in a desiccator. .
Fig: 10-13 Scoring of extent of lid closure of rabbit eye.
Evaluation
of Ocular microparticles:
a.
Determination of
Mean Particle Size and Particle Size Distribution:
Particle size analysis of drug-loaded chitosan
microspheres was performed by optical microscopy using a compound microscope. A
small amount of dry microspheres was suspended in purified water (10 ml). A
small drop of suspension thus obtained was placed on a clean glass slide. The
slide containing chitosan microspheres was mounted on the stage of the
microscope and diameter of at least 300 particles was measured using a
calibrated ocular micrometer. The process was repeated for each batch prepared.
b.
Determination of
Uniformity Index:
Uniformity
Index (UI) was determined by the following formula:
Where, Dw and Dn are weight
average diameter and number average diameter, respectively, and are calculated
as follows:
Where, Ni is the number of particles with Di diameter
values of UI ranging from 1.0 to 1.1 and 1.1 to 1.2 indicate monodisperse
and nearly monodisperse
c. Morphological Study of Microspheres:
The morphology of the
prepared microparticles was studied and photographed using biological
microscope from Nico, Japan which equipped with digital camera connected
to PC set with imaging software. The microparticles were dispersed with liquid
paraffin in a microscope slide and samples were observed microscopically.
Fig -14 Comparison of effect of ketorolac ocular film
with Eye drop formulation on PGE2 induced PMN migration in tears of rabbit
Fig – 15 in vitro release profile of the
prepared microparticles with batch code A1, A2, A3, A4, A5, A6
d. Percentage Yield:
The percentage yield of the microparticles
obtained is calculated by the formula:-
e. Entrapment efficiency:
25 gm of microspheres were weighed and
suspended in 10 ml of the methanol, as the drug is soluble in methanol. Only
the free drug which was present in microspheres got extracted in the methanol.
The suspension was shaken vigorously for 1 hour on the vortex mixer and kept
for 24 hours with intermittent shaking. After this the suspension was taken and
centrifuged for 15 to 20 minutes at 5000 rpm and the supernatant was collected
and analyzed at λ max 319 nm.Entrapment efficiency was
calculated by the formula:-
% Entrapment
Efficiency =Total amount of drug- Free drug x 100
Total
amount of drug
In vitro drug release studies:
The in vitro release of drug was carried out by
using water bath shaker maintained at 37 0C with a shaker frequency of 50 times per minute. 25
mg of the microparticles were suspended in 5 ml phosphate buffer solution (SPB,
pH 7.4) in 5 ml vial. A 4 ml of release media was withdrawn at different time
intervals and centrifuged for 5000x g for 30 minutes. The supernatant was
collected and was analyzed at 323 nm using UV spectrophotometer. The sedimented
microparticles were redispersed in the same volume of release medium so as to
guarantee the sink conditions for the Ketorolac tromethamine
Fig – 16 in
vitro release profile of the prepared microparticles with batch code B1,
B2, B3, B4, B5, B6
a.
Drug Kinetic
Analysis:
To determine the kinetics of release rate of ketorolac
tromethamine from chitosan microparticles the released data was plotted for
cumulative drug release vs. time (zero order), log cumulative release vs. time
(first order), cumulative release vs. sqrt. time (Higuchi) and log cumulative
release vs. log time (Korsmeyer and peppas model). The curves obtained were
regressed, the values of R2 for various kinetic models tested to
describe the drug release from the microparticles is shown in Table 3.
b.
In vivo anti-inflammatory study:
An in vivo study of the optimized formulation of
ketorolac tromethamine microparticles was done using PGE2 induced
ocular inflammation in rabbit’s model. The protocol of the experiment was
designed and approval of Institutional Animal Ethics Committee (IAEC) was
taken. The detailed protocol is as follows:-
Procedure:
Six albino rabbits of either sex weighing 1-1.5 kg were
divided randomly into two groups of three each. Animals were housed in
institutional animal room under standard conditions, with 24 hour light /dark
cycle. During the housing period, they had free access to food and tap water.
Inflammation was produced by administration of the 1µg / ml of PGE2
[Dinoprostone®], (Astra Zeneca India Ltd) in rabbit eyes. Left eye
of all the rabbits served as controls. The right eye of the rabbits in the
First group was treated with marketed Ketorolac eye drop, 0.5% w/v [Keto drops (Cipla)] and those of second
group were treated with microparticles. After 10 minutes of administration of
the drugs in respective eyes 50µl of PGE2 (1 µg/ml in normal saline)
was instilled in both eyes of all rabbits. After the treatment all the eyes
were evaluated for the signs of inflammation as follows:
a) Lid closure extent:
Scoring of the lid closure is done as
Fully opened : 0 Two third open : 1
One third open : 2 Fully closed: 3
Fig – 17 in vitro release profile of the
prepared microparticles with batch code C1, C2, C3, C4, C5, C6
b) PMN migration:
Two drops of normal saline were instilled into inferior
cul-de-sac of rabbit's eye, and after gentle mixing 50 µl of the tear fluid was
withdrawn at different time intervals following PGE2 instillation
and tear fluid PMN was counted in Neubaeuer haemocytometer, tear fluid so
withdrawn into WBC pipette till the mark of 0.5 and then Turke’s fluid was
drawn into the pipette till the mark of 1.0.
Composition of Turke’s fluid (WBC diluting fluid):
No. of cells /
mm3 of tear fluid =
Cells counted X fluid dilution X chamber depth
Area of Chamber Counted
|
|
Formula used:
Where, Fluid dilution = 20 Depth Factor
=10 Area of chamber counted = 4
Results andDiscussion:
The shape and morphology of the
microparticles prepared by modified emulsification ionic gelation method was
examined by digital optical microscopy Under optical digital microscopy, the
microparticles appeared spherical ,discrete in nature .(Fig-4-9)
Mean particle size and uniformity index of different
batches of chitosan microparticles is shown in Table 2 and Particle size was
found to be in the range of 8.12 µm to 3.42 µm It was observed that on
decreasing the concentration of chitosan and TPP from 2% to 1%, the particle
size of microparticles is decreased. Fig 1-3 shows the frequency polygon
representing the particle size distribution in various batches. For a
suspension dosage form to be used in eye it must be micro fine i.e. no particle
should be greater than 50 µm and 90% of the particles should be less than 10
µm. These microparticles fulfill this requirement and are expected not to cause
any irritation to the eye. The results of percentage yield of different batches
of chitosan microparticles are shown in Table 2 The percentage yield of
microparticles was found to vary between 78.2% to 86.8%. It was may be due to
the reason that 3-4 time washing with solvent ether was given to microparticles
which would have reduce the yield to such extent. The results of the entrapment
efficiency of different batches of chitosan microparticles are shown in
Table 2 The entrapment efficiency of all the batches
were in the range of 64.49 -80.42%.
The results of in vitro release of ketorolac
tromethamine from the ocular microparticles are shown in Table 3 All the
batches prepared were able to sustain the drug release for more than 18 hours.
The study was carried for up to 18 hours. Among the group A, different Batches
A1, A2, A3, A4, A5 and A6 released 67.7%, 68.4%, 69.1%, 70.72%, 71.2 % and
73.7% ketorolac tromethamine respectively at the end of 18 hours. Among the
group B, batches B1, B2, B3, B4, B5 and B6 released 68%, 69.6%, 70.4%, 71.1%,
74.3% and 75% ketorolac tromethamine, respectively, at the end of 18 hours.
Among the group C, batches C1, C2, C3, C4, C5 and C6 released 69.5%, 70.7%,
71.2%, 73.8%, 77% and 80.04% ketorolac tromethamine, respectively at the end of
18 hours.
To determine the release rate kinetics of ketorolac
tromethamine from chitosan microparticles the drug release data was plotted for
cumulative drug release vs. time (zero order), log cumulative release vs. time
(first order), cumulative release vs. sqrt.time (Higuchi) and log cumulative
release vs. log time (Korsemeyer and Peppas model). The curves obtained were
regressed and the values of R2 for various kinetic models given in
Table 3. Groups for in vitro drug release it was observed that the
release of ketorolac tromethamine from the microparticles go on increasing as
the concentration of chitosan and TPP was decreased. This may be due to the
-less entrapment and less cross-linking and smaller particle size of
microparticles as we go an decreasing the concentration of chitosan and TPP.
The value of R2 in most of cases is higher for zero order than other
models. From the results it can be inferred that release of the ketorolac tromethamine
from the prepared chitosan microparticles follows ‘Zero order kinetics’. The
value of (0.43 < n < 0.85) the release exponent of Korsemeyer and Peppas
model indicates that the release mechanism of ketorolac tromethamine from the
microparticles is anomalous i.e. combination of diffusion controlled and
swelling controlled release.
Topical instillation of prostaglandins induces ocular
inflammation and polymorphonuclearleukocytes (PMN) migration in tear fluid.
Hence PGE2 induced lid closure and PMN migration in rabbit was used to evaluate
anti-inflammatory effect of ocular microspheres of ketorolac tromethamine .The
scoring of extent of lid closure of rabbit eye has been shown in the Fig 11-13
and. Table 5 shows the comparison of the ketorolac ocular microspheres with
control showing that lid closure in the eye with the ocular microspheres had a
less prominent effect for First1hr ,after that their was slight decrease in the
lid closure with time showing the sustained release of the drug .As from the
results the lid closure was decreasing continuously with time, so the drug
release was sustained, after hours it may had released the sufficient amount of
drug which was needed to counteract with the inflammation .Up to 8 hrs the
lid was opened showing completely open eye. This may be due to the reason
that as the microspheres were mucoadhesive in nature so they must have adhered
to the eye surface showing drug release for a longer time, which is an
advantage over the eye drops which get drained out of the eye after 1-2 hours.
Fig14 draws a comparison of PMN count in the tear of
rabbit in all the eyes, It showed no increase in First 1 hr after that there
was increase in the PMN count up to 3hr and after ward it decreased. In the
Ocular formulations the PMN counts were observed to be less than the control
eyes. In the eye drops the PMN count decreased till 3-4 hours after that it did
not showed much decrease in the PMN count, rather it showed the increase in the
PMN count, where as in the case of ocular microspheres it showed the increase
in the PMN count in the beginning but after that it showed a continuous
decrease in the PMN count due to the sustained effect of the microspheres.
Conclusions:
The decreasing concentrations of chitosan and TPP in microparticles
lead to decrease in particle size. Also there is les entrapment, less cross
linking at reduced concentration of TPP in microparticles, which result in
increased release of Ketorolac tromethamine from microparticles. The release
mechanism shows a combination of diffusion and swelling controlled release. The
batch C6 formulated was found to give the best release with 80% at the end of
18 hours and having smallest particle size.
REFERENCES:
1.
Eriksen SP. “Ophthalmic Drug Delivery Systems”. American
Pharmaceutical Association, Washington, 1980;.55–70.
2.
Lang JC. “Ocular drug
delivery: conventional ocular formulations”. Adv. Drug Delivery Revs, 1995; 16:39–43.
3.
Le Bourlais CA,
Treupel-Acar L, Rhodes CT, Sado PA, Leverge R. “New Ophthalmic Drug Delivery
Systems”. Drug Dev. and Indust. Pharm. 1995; 21:19-59.
4.
Kaur IP, Kanwar M.
“Ocular Preparations: The formulation approach”. Drug Dev. Indust. Pharm.,2002;
28: 473-493.
5.
Robinson JC.
“Ophthalmic Drug Delivery Systems”. Marcel Dekker : New York, US. 1993; 29-57.
6.
Krishnamoorthy R and
Mitra AK. “Ophthalmic Drug
Delivery systems”. M Dekker, Inc., New York, 1993; 199–222.
7.
Hägerström H, Paulsson M
and Edsman K. “Evaluation of mucoadhesion for two polyelectrolyte gels in
simulated physiological
conditions using a rheological method”. Eur. J. Pharm. Sci., 2000; 9: 301–33.
8.
Lele BS and Hoffman AS.
“Insoluble ionic complexes of polyacrylic acid with a cationic drug for use as
a mucoadhesive,ophthalmic drug delivery system”. J. Biomater. Sci. Polym. Ed. 2000; 13: 19–31.
9.
Davies NM.
“Biopharmaceutical considerations in topical ocular drug delivery”. Clinical
and Experimental Pharmacology and Physiology,2000;27: 558-562.
10.
Kang JC, Sticmka MM.
“Biological barriers to ocular delivery in ocular therapeutics and drug
delivery”. A multidisciplinary approach”, Technomic Publishing Company,1996:
51-132.
11.
Banker GS, Rhodes CT.
“Modern Pharmaceutics”. Marcel Dekker Inc., New York, Vol. 72: 415-476
12.
Ding S. “Recent
developments in ophthalmic drug delivery”. PSTT,1998; 1: 328-335.
13.
Kas HS. “Chitosan:
properties, preparation and application to microparticulate systems”. J.
Microencap.1997 ;14: 687-711
14.
Illium R. “Chitosan and
its use as a pharmaceutical excipients”. Pharm. Res.1998 15: 1326-1331.
15.
Skaugrud O. “Chitosan-
New biopolymer for cosmetics and drugs”. Drug Cosmetic Ind. 1991;148: 24-29
16.
Zimmer K, Kreuter J.
“Microspheres and nanoparticles used in ocular drug delivery systems”. Adv.
Drug Del. Rev., 16, pp. 61-73
17.
Joshi A.
“Microparticulates for ophthalmic drug delivery”. J Ocul Pharmacol. 1994; 10
(1): 29-45.
18.
Bartfield JM, Kern AM,
Robak RN, Snyder HS, Baevsky RH. “Ketorolac tromethamine use in a
university-based emergency department”.Acad Emerg Med. 1994; 1:532-538.
19.
Rooks WH. “The
pharmacological activity of ketorolac tromethamine”, Pharmacotherapy .1990 ;
10: 30-32
20.
Gupta AK, Sumit M,
Majumdar DK, Amarnath M. Ketorolac entrapped in polymeric micelles:
preparation, characterization and ocular anti-inflammatory studies”. Int. J
Pharm. 2000; 209: 1-14.
21.
Shu XZ, and Zhu KJ.
Chitosan / gelatin microspheres prepared by modified eulsification and
ionotropic gelation ethod . J. Micro Cap.2001; 18(2) : 237-245
22.
Sasaki H, Yamamura K,
Mukai T, Nishida K, Nakamura M. “Enhancement of ocular drug penetration”.
Critical Review in Therapeutics Drug Carriers Systems.1999; 16: 85-146.
23.
Mi FL, Sung HW, Shyu SS. “Synthesis and characterization of a novel
chitosan-based network prepared using naturally occurring crosslinker”. J.
Polym. Sci. Part A Polym. Chem.2000; 38: 2804–2814.
24.
Dal Pozzo A, Vanini L,
De Benedittis A. “Preparation and characterization of poly(ethylene
glycol)-crosslinked reacetylated chitosans”. Carbohydr. Polym.2000;42 : 201–206