Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20

Dorzolamide nanoliposome as a long action

ophthalmic delivery system in open angle
glaucoma and ocular hypertension patients

Maryam Kouchak, Mohammad Malekahmadi, Neda Bavarsad, Amal Saki

Malehi & Laleh Andishmand

To cite this article: Maryam Kouchak, Mohammad Malekahmadi, Neda Bavarsad, Amal Saki
Malehi & Laleh Andishmand (2017): Dorzolamide nanoliposome as a long action ophthalmic
delivery system in open angle glaucoma and ocular hypertension patients, Drug Development and
Industrial Pharmacy, DOI: 10.1080/03639045.2017.1386196

To link to this article: http://dx.doi.org/10.1080/03639045.2017.1386196

Accepted author version posted online: 28

Sep 2017.

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Download by: [Australian Catholic University] Date: 29 September 2017, At: 04:39
 Original Article

Clinical Science

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Dorzolamide nanoliposome as a long action ophthalmic delivery system in
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open angle glaucoma and ocular hypertension patients
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a,b
Maryam Kouchak PhD, cMohammad Malekahmadi PhD, a,b
Neda Bavarsad PhD,
d
Amal Saki Malehi PhD and eLaleh Andishmand Pharm.D
e d

a
Nanotechnology Research Center, Ahvaz Jundishapur University of Medical Sciences.
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b
Department of Pharmaceutics, School of Pharmacy, Ahvaz Jundishapur University of
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Medical Sciences
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c
Department of Ophthalmology, Imam Khomeini Hospital, Ahvaz Jundishapur

University of Medical Sciences, Ahvaz, Iran

d
Department of Biostatistics and Epidemiology, School of Public Health, Ahvaz

Jundishapur University of Medical Sciences, Ahvaz, Iran

 e
Student Research Committee , Ahvaz Jundishapur University of Medical Sciences,

Ahvaz, Iran

Address of the institutions: Imam Khomeini Academic and Medical Center, Azadegan St, Ahvaz,

Iran.

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Address of the correspondence: Student Research Committee, Ahvaz Jundishapur

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University of Medical Sciences, Ahvaz, Iran

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Short running title: Dorzolamide nanoliposome and ocular hypertension
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This study was supported by funding from Nanotechnology Research Center of Ahvaz

Jundishapur University of Medical Sciences.

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 ABSTRACT

Importance: reducing the frequency of dorzolamide eye drop administration and increasing the

duration of action.

Background: This study aims to compare the effect of dorzolamide loaded- nanoliposome with

marketed dorzolamide HCl eye drop on intraocular pressure in primary open angle glaucoma and

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ocular hypertension patients.

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Design: A randomized study was conducted in a hospital.

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Participants: Twenty patients with primary open angle glaucoma or ocular hypertension (in

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both eyes) diagnosis were recruited as participants.
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Methods: dorzolamide loaded- nanoliposome was prepared by thin layer hydration method and

characterized. Intra ocular pressure were compared between the two groups received marketed
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dorzolamide solution or dorzolamide- loaded nanoliposome.
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Main Outcome Measures: The main outcome measures include intraocular pressure in initial
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(day 0) and on days 14 and 28 and adverse effect of dorzolamide-loaded nanoliposome eye drop.
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Result: Based on the results of repeated measure, Intra ocular pressure decreased in both groups
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but these reductions in the intervention group (dorzolamide-loaded eye drop) were significantly
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higher than those in control group (p<0/05).

Conclusions: This study confirmed safety and long-lasting efficacy of dorzolamide- loaded

nanoliposome eye drop. The highly enhanced permeation through cornea can be attributed to

similarity of phospholipid bilayer of liposomes to the biological membrane and their small

particle size and positive zeta potential.

 Key words: Nanoliposome, Dorzolamide, Intraocular Pressure, Glaucoma

Introduction

Ocular hypertension means the intraocular pressure (IOP) is higher than normal. Left untreated,

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highly eye pressure can cause glaucoma. Glaucoma is a progressive optic neuropathy in which

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increased IOP lead to damage the optic nerve. This disorder can be eventually leads to blindness

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in the affected eye. So that it is the major cause of irreversible blindness in worldwide after

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diabetes mellitus (1). The goal of treatment is prevent additional optic nerve damage. Beta
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adrenoreceptor antagonists and prostaglandin analogs are used as first line treatment of open

angle glaucoma. Alpha 2 adrenoceptor agonists and carbonic anhydrase inhibitors (CAIs) are
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used as second line options (2). Dorzolamide as a CAIs decreases aqueous humor secretion

which result to decrease IOP (3). Dorzolamide 2% eye drops has been shown the highest clinical
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effect in studies and an increase in concentration has no additional clinical effect (4). Treatments
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of glaucoma include drug therapy, laser and surgery. Topical dosage forms such as eye drops are

the first choice of treatment (5).

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Poor drug delivery to posterior segment of eyes is the major problem in eye drug delivery.
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Anatomical barriers in eyes including corneal barrier, aqueous barrier and the inner and outer

blood–retinal barriers restricted the accessibility of eyes to topical drugs(6).

Liposomes are bilayer phospholipid vesicles that can encapsulate water-soluble drugs in aqueous

phase and lipid-soluble drugs in lipid phase (6). Liposomes have various advantages as drug

carrier. They can adhere to cornea (due to their phospholipid membrane) and release drugs
 gradually on the eye surface, therefore patients can benefit from sustained duration of action

following a single instillation. They are biocompatible and almost nontoxic nanocarriers with

low antigenicity (6). The nanometer size might facilitate the penetration of them into the

physiological barriers of the eye (conjunctiva and sclera) to reach more efficiently the target site

(ciliary body) (5).

In our previously study, dorzolamide loaded- nanoliposome (DRZ – nanoliposomes) formulation

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was made with lipid molar ratio of 7:4 (phosphatidylcholine:cholesterol) using thin layer

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hydration method illustrated small particle size and acceptable drug loading and these properties

remained for at least three months after preparation. The surface tension, viscosity and

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biocompatibility of the formulation were within the passable range of ocular solutions. In vivo
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evaluation of this formulation in comparison with dorzolamide solution and marketed

dorzolamide eye drops in the rabbit eyes was indicated higher and more lasting therapeutic
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effect(7). Because the nanoliposomes are able to entrap dorzolamide HCl in its hydrophilic core
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and then connect and merge with the corneal epithelial follow by gradually drug release, beside
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endocytosis of liposomes may lead to pass loaded liposome through the cornea and drug release
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occur in the anterior eye space(8).

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The aim of this study was to evaluate the safety and the IOP-lowering efficacy of DRZ –
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nanoliposome formulation in primary open angle glaucoma (POAG) and ocular hypertension

patients followed for 28 days.

 Material and methods

Material

Dorzolamide HCl obtained from Baselux (Switzerland). Phosphatidyl choline supplied by Lipoid

(Germany). Cholesterol purchased from Sigma (Germany). Fluid thioglycollate medium and

soybean casein pepton broth provided from Biolife (Italy) and Quelab (Canada), respectively.

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Methods

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The liposome was prepared with thin- layer hydration (TLH) method (7). Phosphatidyl choline

and cholesterol were dissolved in chloroform and methanol (2:1). The solvents evaporated in a

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rotavapor flask at 58o C (150rpm) and a thin film was formed on the inner side of the round
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bottom flask. Nitrogen gas was blowed in flask for 20 second to ensure that all solvents were

exited. The formed lipid film was maintained in refrigerator for 24 hour to eliminate traces of
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chloroform. The lipid film was hydrated with dorzolamide solution 2% in phosphate buffer (pH
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5.8 at 64°C) and vortexed to form nanoliposomes. The formulation was sonicated using a bath
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sonicator for 30 minutes and stored in refrigerator. The nanoliposomes were sterilized using
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0.22µm syringe filter (Minisart, Germany).

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Characterization of DRZ – nanoliposomes

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In order to determine the drug loading, the formulation was poured in Amicon filter microtube (3

KDa) and centrifuged at 11000rpm at 25 °C for 30 minutes. The absorbance of the supernatant

was measured at 254 nm using a UV spectrophotometer (Biochorm, England) for assay free

dorzolamide amount. Encapsulation efficiency (EE) was calculated indirectly as follow:

𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔−𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔

EE%= [ ] × 100
total drug
 The following parameters were analysed:

The size of nanoliposomes was measured by light scattering method using Qudix nanosizer

(South Korea). The viscosity of the formulation was measured at 25 °C using a Brookfield

viscometer (Model LVDV-II+PRO, USA) with a no. 34 Spindle at 100 rpm. The De Nouy ring

device (CSC Scientific Company, USA) was used for measurement of surface tension of the

formulation. The pH of the preparation was determined using Metttler-Toledo pHmeter

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(Switzerland). PrismaTech refractometer (Iran) was used to measure Refractive index.

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Microbiological test

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Sterility control of the preparation was done in soybean casein peptone broth for aerobic bacteria
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and fungi and fluid thioglycollate medium for anaerobic bacteria and fungi. The formulation (10

ml) was transferred into the each medium and incubated at 22.5 ± 2.5°C and at 30°C-35°C,
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respectively (9).
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Clinical trial study

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This double blind clinical study approved by Ahvaz Jundishapur University of Medical Sciences
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Ethics Committee (IR.AJUMS.REC.1394.733) and was conducted at the Imam Khomeini

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Academic and Medical Center of Ahvaz.

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Twenty patients with POAG and ocular hypertension in both eyes were divided into two groups.

The thickness of the cornea was between 530 and 540 microns and was approximately equal in the two

groups. Intervention group received DRZ–nanoliposome and control group received marketed

dorzolamide eye drop (Biosopt®). One drop was instilled in each patient’s eye 3 times daily for

4 weeks. IOP were assessed by Goldmann applanation tonometry on day 0 (before treatment)

and eight hours after instillation of eye drop on days 14 and 28.
 Patients’ selection

Study participants gave informed consent before initiation of any study-related procedures. The

patients had been avoided from using other ocular topical and antihypertensive drugs since at

least one month ago and during the study. All patients were monitored about any side effect

during the treatment. Each patient with serious side effect and also pregnant women exited from

clinical study.

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Statistical analysis

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Statistical analyses were performed using SPSS version 21 (SPSS/IBM, Inc, Chicago, IL, USA).

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Within each treatment group, the IOP before and after instillation of drops into eyes (for 14 and
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28 days) was compared with Repeated Measures test. Baseline IOP between the two groups and

the change in the parameter after treatment for the same duration time were compared with t-test.
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A P value less than 0.05 was regarded as statistically significant.
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Results
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Microbiological evaluation of the prepared formulation for 4 consecutive weeks did not
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exhibited any turbidity or microbial growth. All of the physiochemical characteristics of DRZ–
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nanoliposomes and Biosopt® such as particle size, surface tension, viscosity, pH and refractive
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index were within the acceptable range of ophthalmic solution (Table 1).
 Table 1. Particle size, pH, viscosity, surface tension, encapsulation efficiency and refractive

index of DRZ – nanoliposomes and Biosopt® (mean ± SD, n=3) eye drops

Particle size pH Viscosity Surface tension EE (%) Refractive index

(nm) (cps) (N/m)

DRZ – nanoliposome 51±3.24 5.7±0.02 44±3 0.048±0.005 48 ±2.02 1.3433 ±0.0001

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Biosopt®

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_ 5.2 ±0.01 96±3 0.07±0.005 _ 1.3418 ±0.0001

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In clinical trial study, patients in both groups complained of irritation and redness immediately

after instillation of eye drops which was resolved gradually.

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Based on the results of repeated measure test (Table 2), IOP in both groups showed a significant
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reduction (P <0.05). The lowest recorded IOP after treatment with DRZ – nanoliposomes was 10
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mmHg. It is concluded that DRZ–nanoliposomes eye drop does not cause hypotony maculopathy
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in which the IOP is below 6.5 mm Hg (10).

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 Table 2. IOP values of POAG and ocular hypertension patients before and after receiving DRZ –

nanoliposomes (intervention group) and Biosopt® (control group) eye drops

Time IOP (mmHg) (mean ± SD,n=20)

Intervention group Control group

Before study 19.1±2.42 17.65± 2

After 2 weeks 14.65± 2.6 16.50±2.32

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After 4 weeks 12.85± 2.18 14.55±2.08

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Before and two

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weeks after treatment <0.001 <0.001
(intragroup)
p-value

2 weeks and 4 weeks

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after treatment <0.001 <0.001
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 Table 3. IOP-lowering and intergroup differences in POAG and ocular hypertension patients

after treatment by DRZ – nanoliposomes (intervention group) and Biosopt® (control group) eye

drops

IOP -lowering (%) P-value

Time (mean ± SD, n=20) )Intergroup(

Intervention group Control group

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Before and two weeks after the intervention 23/26±9/24 9/25±5/76 <0.001

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Before and 4 weeks after intervention 32/60±7/90 17/48±7/62 <0.001

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2 weeks and 4 weeks after intervention 11/97±5/54 9/00±6/84 0.384

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22
intervention group
20 control group
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18
IOP (mmHg)

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0 7 14 21 28 35
Time (day)

Figure 1. IOP changes in the intervention group (DRZ – nanoliposomes eye drops) and control

group (Biosopt® eye drops)

 According to Table 3 and Figure 1, IOP reduction in the intervention group was statistically

significant further than that in control group at weeks 2 and 4 (just to receive study medication).

There were no significantly intergroup differences among IOP -lowering values during last two

weeks.

Discussion

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In this study DRZ-nanoliposome system was prepared according to our previous study using

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TLH method (7). In making this formulation, all the containers were autoclaved before working

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and the process were performed under laminar air flow. The formulation was sterilized by

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filtration method. Microbial control tests confirmed the adequacy of the used sterilization

technique. an
Fine particle size of liposomes (51 nm) implies to generate small unilamellar vesicles (SUV)
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after sonication. These kind of liposomes can connect more efficiently to the tissue resulting in

better drug delivery (11).

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The viscosity of the formulation was 44 cps that was in the proper range for ophthalmic
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formulations (25-50 cps). Low consistency solutions were not appropriate form to application in
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eyes due to quickly remove from the eye after contact with tear. Viscosity enhancers such as

cellulose derivatives can be added to the ocular formulations to increase adherence and
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persistence of the drug formulation. It is considerable that high consistency of the formulations

has undesirable effects on blinking, eye movements, eye refraction process and imaging in retina

(12). The viscosity of Biosopt® eye drop which was reported in our previous study was 96 cps

(7).
 Obtained values for surface tension, refractive index and pH of the preparation were confirmed

its safety for ophthalmic indication.

The clinical trial study showed a significant IOP-lowering after treatment of POAG and ocular

hypertension patients with DRZ-nanoliposome in comparison with Biosopt®. Nanoliposome

displayed a positive surface charge in the previous study (7) that plays an important role in the

attachment of that to the negative charge surface of corneal epithelial. It has been demonstrated

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that the most permeability of cornea belongs to cationic liposomes(13).The similarity between

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phospholipid bilayer of liposome and the cornea, small particle size and positive zeta potential

charge of them improve the corneal adhesion and permeation(7).

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All patients in each treatment group complained of irritation and redness in eyes which are the
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common side effects of dorzolamide HCl (2). These effects can be attributed to free dorzolamide
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HCl in the formulation and small particle size of the nanoliposomes (below 100nm) prevent any

additional abrasive or irritation effect in comparison with Biosopt® (11).

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The reduction of IOP in this study was lower than kouchak, et al study. This could be due to
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differences in the cases that were tested. In their study, single dose of nanoliposome formulation

was examined on rabbits healthy eyes while in this investigation, the patients with POAG were
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received multiple dose of the preparation for 4 weeks (7).

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There are many studies about increasing the efficacy of eye liposomes. Habib et al ,examined

fluconazole liposome as drug delivery system in the rabbit’s eye. They concluded that treatment

with fluconazole liposome can remove the infection in the rabbit cornea more effective than

fluconazole solution (14).

 Shinde et al, found that acetazolamide liposomes kept down IOP up to 8 hours. While the clinical

effect of marketed eye drops reached to maximum within 2 hours and finished after 4 hours (15).

Wong et al, studied the effect of single dose intraocular injection of latanoprost nanoliposome in

6 POAG and increased IOP patients. The mean IOP was reduced from 27.5 to 14.5 mmHg

within one hour and 20 % IOP-lowering remained after 3 months (5).

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Conclusion

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The prepared DRZ – nanoliposomes has acceptable physicochemical properties as an ophthalmic

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dosage form. The IOP-lowering efficacy was greater with this system than with marketed eye

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drop in respect to more intensity and extended duration of action. These results suggest that the
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nanoliposomes can improve retention time of drug in target tissue for a long period of time. It

seems that positive surface charge of the nanoliposomes assists their adhesion to negative
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corneal epithelial also the similarity of phospholipidic bilayer of liposomes to the biological

membrane and their small particle size contribute to enhanced permeation through cornea.
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The frequency of administration can be reduced by this formulation to increase patient

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compliance and improve treatment outcomes.

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Acknowledgements
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This paper is issued from Pharm D thesis of Laleh Andishmand and financial support was

provided by a grant from Nanotechnology Research Center of Ahvaz Jundishapur. We sincerely

thank the Ophthalmology Department of Imam Khomeini Hospital .

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