RESEARCH ARTICLE

Aqueous carboxymethyl gum kondagogu as vehicle for oculardelivery

Ashok Kumar • Munish Ahuja

Received: 28 November 2013 / Accepted: 3 February 2014

� The Korean Society of Pharmaceutical Sciences and Technology 2014

Abstract Aqueous solutions of carboxymethyl gum

kondagogu (CMGK), an anionic bioadhesive polymer were

evaluated as vehicles for ophthalmic delivery using tro-

picamide as a model drug. Aqueous ophthalmic solution of

tropicamide (1 %, w/v) in CMGK (5 %, w/v) dispersions

were formulated. The aqueous CMGK vehicle, formulated

tropicamide eye drops and commercial tropicamide for-

mulations were assessed comparatively for ex vivo ocular

tolerance using hen’s egg chorioallantoic membrane assay.

The results indicated ocular tolerability of aqueous CMGK

vehicle. The results of comparative ex vivo corneal per-

meation study of tropicamide from the aqueous CMGK

vehicle (5 %, w/v) conducted across isolated goat cornea

revealed a no significant difference in the corneal perme-

ation of tropicamide from the CMGK vehicle based for-

mulation as compared to the commercial formulation.

Further, the results of in vivo mydriatic response study

conducted in rabbits revealed a non significant difference

in the mydriatic response of tropicamide from the aqueous

CMGK vehicle and commercial formulations. In conclu-

sion, CMGK can be used as an ocularly tolerable polymer

for formulating ophthalmic dosage forms.

Keywords Carboxymethyl gum kondagogu �Ophthalmic vehicle � Tropicamide � HET-CAM assay �Mydriatic response � Corneal permeation

Introduction

Ophthalmic drugs are generally administered as eye drops.

Controlled delivery of drug to the eye is a difficult task

since drugs administered as eye drops have low bioavail-

ability. Normal ocular protective mechanisms such as

blinking and tear drainage promote rapid clearance and

reduced bioavailability resulting in shorter duration of

pharmacological response (Maurice 1987). The ocular

residence time of most solution dosage forms ranges

between 5 to 25 min (Vandamme and Brobeck 2005; Chrai

and Robinson 1974). Only 1–10 % of topically applied

drug is absorbed (Nanjawade et al. 2007) due to drainage

through nasolacrimal duct (Sieg and Robinson 1977). One

of the approaches used to overcome the drawback of

shorter pre-corneal residence time is to use bioadhesive

dosage forms, which by virtue of their mucoadhesive

properties will be retained in the conjunctival sac providing

prolonged retention of drug for ocular absorption (Kaur

et al. 2012).

Gum kondagogu is an anionic gum obtained by tapping

from the tree of Cochlospermum gossypium DC (Family

Bixaceae). It consists of arabinose, galactose, galacturonic

acid, glucuronic acid, b-D-glucose, fructose, mannose and

rhamnose, with sugar linkage of (1 ? 2) b-D-Gal p,

(1 ? 6) b-D-Gal p, (1 ? 4) b-D-Glc p, 4-O-Me-a-D-Glc p,

(1 ? 2) a-L-Rha (Vinod et al. 2008). Carboxymethyl

functionalization of gum kondagogu increases its anionic

character reduces viscosity and improves its ionic gelling

behavior. Further, it was found that carboxymethyl gum

kondagogu (CMGK) possess 22-fold higher mucoadhesive

strength than gum kondagogu as determined by tensile test

profiles (Kumar and Ahuja 2012).

Tropicamide is a weakly basic antimuscarinic agent

which is indicated for inducing mydriasis and cyclopegia

A. Kumar � M. Ahuja (&)

Drug Delivery Research Laboratory, Department of

Pharmaceutical Sciences, Guru Jambheshwar University of

Science and Technology, Hisar 125001, Haryana, India

e-mail: munishahuja17@yahoo.co.in

123

Journal of Pharmaceutical Investigation

DOI 10.1007/s40005-014-0120-9

during eye surgery and in dilated fundoscopic examination.

It is applied topically as aqueous eye drops (0.5–1 %, w/v).

It acts by blocking the muscarinic M4 receptors thereby

dilating the pupil and preventing the eyes to accommodate

to near vision allowing thorough examination of the eye

(Bartlett and Jaanus 2007). During earlier studies bioad-

hesive polymers such as carboxymethyl cellulose, hyalu-

ronic acid, and polyacrylic acid have been tested to

improve the mydriatic response of tropicamide (Herrero-

Vanrell et al. 2000).

In an earlier study CMGK was observed to be a prom-

ising bioadhesive polymer and was evaluated for formu-

lation of bioadhesive beads for oral delivery of metformin

(Kumar and Ahuja 2012). In the present investigation the

CMGK has been tested for ex vivo ocular tolerance using

hen’s egg chorio-allantoic membrane assay (HET-CAM).

Further, it was employed as a vehicle for formulation of

tropicamide eye drops. Formulated eye drops were evalu-

ated for corneal permeation characteristics with marketed

formulation of tropicamide. Finally, the in vivo mydriatic

response was measured in rabbits.

Materials and methods

Materials

CMGK (degree of substitution 0.2) was synthesized in our

laboratory as reported earlier (Kumar and Ahuja 2012).

Tropicamide was obtained as gift sample from Optica

Pharmaceuticals (Yamunanagar, India). Sodium hydroxide

and methanol were procured from Sisco Research Labo-

ratory (Mumbai, India). Commercial formulation of eye

drop (Tropicacyl�, Sunways India Pvt. Ltd., Mumbai,

India) was purchased from local pharmacy (Hisar, India).

Ten-days old fertilized hen’s egg were purchased from

Kundan Farms (Bhiwani, India). Albino rabbits were pro-

cured from disease free small animal house of Lala Lajpat

Rai University of Veterinary and Animal Sciences (Hisar,

India). All other chemicals used were of reagent grade and

were used as received.

Preparation of tropicamide ophthalmic formulations

(1 %, w/v)

Required amount of the tropicamide was added to aqueous

dispersion of CMGK (5 %, w/v) with the aid of stirring.

The pH of the dispersion was adjusted 4–5 by adding dilute

HCl drop by drop, to completely dissolve the tropicamide

(whqlibdoc.who.int/hq/1990/2.pdf). The solution were

made isotonic by adding sodium chloride and sterilized by

autoclaving at 121 �C for 15 min, at 15 lbs/in2.

Ex vivo ocular tolerance

Ocular tolerance of tropicamide (1 %, w/v) ophthalmic

formulation prepared using CMGK (5 %, w/v) vehicle,

commercially available tropicamide (1 %, w/v) formula-

tion (Tropicacyl�), aqueous CMGK (5 %, w/v) vehicle and

control solutions of irritant (NaOH) and non-irritant (NaCl)

was assessed employing HET-CAM assay (Ahuja et al.

2006). Ten days old fertilized hen’s eggs with an air sac

and live embryo as observed by candling were used for

HET-CAM assay. Egg shells were opened and the shell

membrane was carefully removed without injuring any

blood vessel using tapered forceps. Aliquots (0.5 ml) of the

various test samples were applied over CAM and observed

for next 5 min for the signs of irritation such as haemor-

rhage, vasoconstriction and coagulation (Jimenez et al.

2010). The study was carried out in triplicate. The time of

appearance of irritation was noted and potential irritation

(PI) score were calculated as follows:

PI ¼ ð301� hÞ � 5

300þ ð301� vÞ � 7

300þ ð301� cÞ � 9

300;

ð1Þ

where h, v and c represent time of appearance of haem-

orrhage, vasoconstriction and coagulation in seconds,

respectively. The PI values were assigned as 0–0.9: non

irritant, 1–4.9: slight irritant, 5–8.9: moderate irritant, and

9–21: severe irritant.

Viscosity

Viscosity of commercial formulation of tropicamide 1 %,

w/v (Tropicacyl�) and formulated tropicamide ophthalmic

solutions (1 %, w/v) was measured using an Ostwald

viscometer.

Ex vivo corneal permeation

Corneal permeation characteristics of formulated tropica-

mide (1 %, w/v) eye drops were evaluated comparatively

with commercial ophthalmic tropicamide formulation

(Tropicacyl�) using isolated goat cornea as a model

(Yadav and Ahuja 2010). Freshly excised whole goat

eyeball was transported from the local butcher shop to the

laboratory in cold normal saline within an hour of

slaughter. Cornea was carefully excised along with

2–4 mm of scleral tissue. The tissue was cleaned and

washed with cold normal saline till free from proteins as

determined by washing with Folin–Ciocalteu’s phenol

reagent. Isolated cornea was mounted between the clamped

donor and receptor compartments of modified-Franz dif-

fusion cell, with endothelial side facing the receptor and

A. Kumar, M. Ahuja

123

epithelial side facing the donor. The receptor compartment

consisted of 11 ml of Sorensen phosphate buffer (pH 7.2)

solution maintained at 35 ± 0.5 �C under magnetic stir-

ring. The corneal area available for permeation was

0.95 cm2. One milliliter of test formulation was placed in

the donor compartment over the cornea. An aliquot (3 ml)

of the sample was withdrawn from receptor compartment

after 2 h and analyzed for the contents of tropicamide by

measuring absorbance at 257 nm in a double beam UV–

Visible spectrophotometer (Cary 5000, Varian, Australia).

The study was conducted using paired corneas i.e., one

cornea of the animal was used for the permeation of for-

mulated eye drops and the contralateral cornea was used

for commercial formulation. Corneal hydration levels were

determined by removing the scleral tissue from the cornea

at the end of experiment and weighing, followed by

overnight soaking in methanol to dehydrate and drying in

an oven at 90 �C and weighing again.

In vivo mydriatic activity

Tropicamide eye drops formulated in CMGK vehicle were

evaluated comparatively with the commercial eye drops

(Tropicacyl�) formulation for in vivo mydriatic activity

using healthy adult rabbits (Gupta et al. 2007). The pro-

tocol of the in vivo mydriatic activity was designed and an

approval of institutional animal ethics committee (CPC-

SEA Register number 436) was obtained. Three healthy

adult albino rabbits either male or female, weighing 1–

1.5 kg were used in the study. Each rabbit was acclimated

in the light of the laboratory 1 h prior to administration of

eye drops. Rabbits were placed in restraining boxes in the

normal upright position in a room with constant light, such

that their head and eye movements were allowed after

instillation of dose. One drop of the formulated eye drop

was carefully instilled into the lower cul-de-sac region of

left eye of the rabbit whereas one drop of commercial

formulation was instilled into the right eye of the rabbit.

The second and third doses of eye drops were instilled at an

interval of 15 min. Pupil diameter measurements were

taken photographically at appropriate time intervals. Pupil

diameters were measured from the photographs using rul-

ers in the Adobe photoshop. Mydriatic response intensity

(I) was calculated as follows:

I ¼ dt � do; ð2Þ

where I is the mydriatic response intensity, dt is the pupil

diameter at time t, and do is the pupil diameter at time

t = 0.

Area under the mydriatic response intensity versus time

curve (AUC) was calculated using NCSS version 9.0.2

software (NCSS LLC).

Results and discussions

CMGK was earlier found to exhibit bioadhesive property

and was employed for preparing bioadhesive beads (Kumar

and Ahuja 2012). In the present study aqueous solutions of

CMGK (5 %, w/v) have been explored as bioadhesive

vehicle for ocular delivery employing tropicamide as a

model drug. Tropicamide is a poorly water soluble, weakly

basic drug with pKa of 5.2 (Florey and Brittain 2003). Its

solutions are prepared by adjusting the pH of the solution

to acidic range (pH 4–5), which may enhance its irritation

potential. Thus, the sterile tropicamide (1 %, w/v) eye

drops were formulated using CMGK (5 %, w/v) as bio-

adhesive vehicle. CMGK apart from its bioadhesive action

also acts as viscosity modifier. Viscolizers are commonly

added to ophthalmic formulations as they are generally

believed to increase ocular bioavailability by prolonging

pre-corneal residence time (Malhotra and Majumdar 2001).

The viscosity of formulated and commercial tropicamide

ophthalmic solutions was found to be 1.079 and 4.858 cps,

respectively. However, the more viscous tropicamide

ophthalmic solution using higher concentrations of CMGK

Fig. 1 Comparative ex vivo

ocular irritation potential of

carboxymethyl gum kondagogu

(CMGK) vehicle, test and

market formulation of

tropicamide, sodium hydroxide

(irritant control), sodium

chloride (non-irritant control)

Kondagogu as vehicle for ocular delivery

123

could not be formulated as the acidic nature of tropicamide

solutions i.e., pH 4–5 resulted in precipitation of anionic

polymer above the concentration of 5 % (w/v).

The formulated tropicamide ophthalmic solutions were

comparatively evaluated for ocular tolerance with CMGK

aqueous solution (vehicle control) and Tropicacyl� (com-

mercial formulation) employing HET-CAM. HET-CAM

study is an alternate tool to Draize’s rabbit eye test for

assessment of ocular tolerance (Leupke 1985).The PI

scores (Fig. 1) of testing of CMGK (5 %, w/v) vehicle for

ocular tolerance revealed the CMGK vehicle to be non-

irritant. Further the commercial formulation (Tropicacyl�)

and the test formulation of tropicamide were also found to

be non-irritant.

The excellent ocular tolerances of CMGK vehicle and

tropicamide formulation prompted us to test the corneal

permeation characteristic of tropicamide from the formu-

lated and commercial eye drops formulation. The study

was conducted using paired corneas to minimize the bio-

logical variation. Table 1 presents the results of ex vivo

corneal permeation study of tropicamide formulations. The

results show that 3.285 ± 0.159 % of tropicamide perme-

ated from the CMGK vehicle based formulation, while

2.680 ± 1.024 % of tropicamide permeated from the

commercial formulation (Tropicacyl�).

Tropicacyl� contains chlorbutanol (0.5 %, w/v) as the

preservative and its viscosity indicates the presence of vi-

scolizer. Chlorbutanol is an alcohol based preservative with

broad-spectrum antimicrobial action. It has been reported

to disorganise the lipophilic corneal ephithelia (Noecker

2001) and has earlier been observed to enhance the corneal

permeation of ibuprofen (Gupta and Majumdar 1997). The

higher corneal hydration in Tropicacyl� treated eye drops

can be attributed to the presence of chlorbutanol. Corneal

hydration levels within the range of 75–80 % are consid-

ered normal, while 3–7 % units above the normal value

indicate damage to corneal epithelium or endothelium.

Since the corneal hydration levels in the present study are

within the range, the corneal integrity is not affected.

However, no preservative was employed in the test for-

mulation as our objective was to evaluate aqueous solu-

tions of CMGK as ophthalmic vehicle. Since, there was no

difference in the pre-corneal contact time of the formula-

tions with the corneal tissue during ex vivo corneal study,

the differences in the corneal permeation of tropicamide

from two formulations can be attributed to viscosity dif-

ferences and to the characteristic of polymers employed.

The decreased permeability from viscous vehicles can be

explained by Stoke’s–Einstein relation, which describes

diffusion coefficient as inversely related to viscosity

(Ahuja et al. 2006). Earlier studies conducted using

Table 1 Corneal permeation, viscosity and corneal hydration of test

and market formulation

Formulations Cumulative permeation

(%) 120 minaCorneal

hydration (%)aViscosity

(cps)a

Test

formulation

3.285 ± 0.159 75.71 ± 0.16 1.079

Market

formulation

2.680 ± 1.024 77.13 ± 0.76 4.858

a Values are mean ± SD (n = 3)

Fig. 2 Pupillary size increment

of rabbit eye against the

tropicamide formulation as

function of time

A. Kumar, M. Ahuja

123

different polyanionic mucoadhesive polymeric, iso-viscous

vehicles revealed different ophthalmic bioavailability

which was attributed to surface spreading characteristics.

Differing spreading characteristics of mucoadhesive poly-

mers may result in differing degree of contact at molecular

level between the mucoadhesive polymer and the mucin

coat over the corneal epithelial membrane which manifests

in different ophthalmic bioavailability (Kaur and Smitha

2002). Similar factors may be responsible for corneal

penetration of tropicamide from CMGK vehicle.

Formulated eye drops were further evaluated for their

therapeutic performance by measuring in vivo mydriatic

response in rabbits. The results of change in pupillary

diameter of eye treated with formulated and commercial

tropicamide formulations are presented in Fig. 2, which

shows the increment in pupillary size of eye treated with

tropicamide formulation as the function of time. It can be

observed from the results that it took 30 min to achieve the

steady state diameter and 180 min to achieve the maximum

pupillary response. The mean maximum mydriatic

response intensity (Imax) was 7.83 ± 2.47 mm in the

CMGK formulation while 9.33 ± 1.44 mm in commercial

formulation. Thus an average increase in the viewing area

of the pupil was 48.21 and 68.39 mm2 in eye treated with

formulation and commercial eye drop. Thus area under the

mydriatic response curve (AUC) was calculated to be

10,108.7 and 11,731.2 mm2 for CMGK and commercial

formulation treated eye, respectively. The results indicate

slightly higher Imax and AUC in commercial formulation

compared to the CMGK formulation treated eye. However

statistical analysis of the data by applying ‘paired t-test’

revealed no significant difference between AUC of com-

mercial and market formulation.

The slight differences in the mydriatic responses of two

ophthalmic formulations may be attributed to differing

viscosities. Earlier studies comparing mydriatic response of

tropicamide conducted using differing iso viscous poly-

meric vehicles showed contrary results in rabbits and

humans, which were attributed to inter-species differences

in blinking activity (Saettone et al. 1984). However, no

comparative conclusion between the mydriatic efficacies of

two formulations can be drawn due to differing viscosities

of the two formulations. Further studies incorporating vis-

cosity modifiers and antimicrobial preservatives in CMGK

vehicles are required to comment more on this aspect.

Conclusion

A bioadhesive tropicamide eye drop formulation was pre-

pared employing CMGK (5 %, w/v) as a bioadhesive

polymer. The formulation was found to be non-irritant as

evaluated by HET-CAM assay. The result of ex vivo

corneal permeation study and in vivo mydriatic response

study revealed a comparable mydriatic response between

test and commercial formulation. It can be concluded from

the results that CMGK can be employed as an ocularly

tolerable bioadhesive polymer for formulating bioadhesive

ocular dosage forms.

Acknowledgments The authors are grateful to Indian Council of

Medical Research, New Delhi for providing the Senior Research

Fellowship (SRF) to Mr. Ashok Kumar vide Grant number 45/99/

2009/PHA/BMS. All institutional and national guidelines for the care

and use of laboratory animals were followed.

Conflict of interest All authors (Ashok Kumar and Munish Ahuja)

declare that they have no conflict of interest.

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