Pharmacodynamic Equivalence Study of Two Preparations of Eye Drops , Containing Dorzolamide in Healthy Volunteers A

Pharmacodynamic Equivalence Study of Two Preparations of Eye Drops, Containing Dorzolamide in Healthy Volunteers A Emil M. Gatchev1, Andrey D. Petrov1, Russka W. Hristova2, Irenna S. Demircheva2, Ursula Th yroff -Friesinger3, Wolfram H. Richter4, Rossen K. Koytchev4 A 1 Medical University of Sofi a, Department of Clinical Pharmacology and Th erapeutics, University Hospital “Tsaritsa Joanna-ISUL”, Bulgaria 2 Medical University of Sofi a, Clinic of Ophthalmology, University Hospital “Tsaritsa Joanna-ISUL”, Bulgaria 3 Hexal AG, Holzkirchen, Germany 4 Cooperative Clinical Drug Research and Development AG, Hoppegarten, Germany A SUMMARY


INTRODUCTION
Dorzolamide 20mg/ml eye drops (dorzolamide hydrochloride (CAS: 120279-96-1) is a topical carbonic anhydrase inhibitor indicated for the reduction of elevated intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension [1].Th e drug decreases elevated intraocular pressure, whether or not associated with glaucoma, by reducing aqueous humor secretion.Elevated intraocular pressure is a major risk factor in the pathogenesis of optic nerve damage and glaucomatous visual fi eld loss.Th e higher the level of intraocular pressure, the greater the likelihood of glaucomatous fi eld loss and optic nerve damage.Dorzolamide hydrochloride is an inhibitor of human carbonic anhydrase II.Inhibition of carbonic anhydrase in the ciliary processes of the eye decreases aqueous humor secretion, presumably by slowing the formation of bicarbonate ions with subsequent reduction in sodium and fl uid transport.Unlike miotics, dorzolamide reduces intraocular pressure without the common adverse eff ects of miotics such as night blindness, accommodative spasm and pupillary constriction.Unlike topical beta-blockers, dorzolamide has minimal or no eff ect on pulse rate or blood pressure.Unlike oral carbonic anhydrase inhibitors, topical administration of dorzolamide hydrochloride allows the drug to exert its eff ects directly in the eye at substantially lower doses and therefore with less systemic exposure [2].In clinical studies, this resulted in a reduction in IOP without the acid-base disturbances or alterations in electrolytes characteristic of oral carbonic anhydrase inhibitors.Th e maximum eff ect has been observed approximately 2 h post dose.It has been demonstrated that the topical administration of dorzolamide reduces the intraocular pressure in healthy volunteers by 3.5 to 7 mmHg compared to pre-dose values [3].Th e main pharmacodynamic eff ect of dorzolamide can be thus evaluated in healthy volunteers aft er administration of a single dose [4].
Th e aim of the present study was to assess the pharmacodynamic equivalence (lowering of intraocular pressure) of two preparations of eye drops containing 20 mg dorzolamide 1 ml eye drops.

Study Preparations
Th e test formulation in the present study (dorzolamide 20mg/ml eye drops) was manufactured by the company Hexal AG, Holzkirchen, Germany.Packaging and labeling was done according to the national and international requirements and GMP.Th e reference product (Trusopt®, Chibret Pharmaceutische GmbH) was purchased in a pharmacy.
Th e study was reviewed and approved by the LEC (local ethics committee) of the University Hospital "Tsaritsa Joanna-ISUL", Bulgaria.
Th e study was performed at the Medical University of Sofi a, Department of Clinical Pharmacology and Th erapeutics and the Clinic of Ophthalmology, University Hospital "Tsaritsa Joanna-ISUL", Bulgaria.
Th e inclusion criteria pre-defi ned in the study protocol provided for the inclusion of thirty-eight male and female Caucasians (18 female/18 male) within the age range of 18 to 55 years, who were physically and mentally healthy as judged by means of a medical and standard laboratory examination, with intraocular pressure between 16 and 21 mmHg (mean value of 3 measurements) at screening with a normal body weight assessed by the BMI (accepted range 19 to 27 kg/m 2 ).
Prior to being enrolled into the study, the volunteers gave their informed written consent to participate in the study in response to a complete written and verbal explanation of the nature, scope and possible consequences of the study, which was done by the clinical investigator.
All enrolled subjects met all inclusion and none of the exclusion criteria and were judged eligible for the study, based on medical history, demographic data, medication history, physical examination, vital signs and clinical laboratory tests.
A total number of 36 volunteers were enrolled in the study and all of them completed the study according to the protocol.

Study Design
Th e study was designed as a monocentric, observer-blinded, randomized, single-dose, single period study, with duration of hospitalization of approximately 6 h aft er dosing on day 1.

Study procedure
Th e volunteers were hospitalized for approximately 12 h overnight stay and further 6 h confi nement on the day of dosing.Aft er consumption of a standard breakfast in the morning on day 1 between 7:00 and 7:30 a.m., all volunteers received between 9:00 and 11:00 a.m. the study medication: a single dose of 1 drop eye drops containing 20 mg dorzolamide in 1 ml eye drops in the conjunctival sac of the right or the left eye.Th e allocation of test and reference product to the left or to the right eye was randomized.Th e sequence of randomized administration was the same for all volunteers: the right eye was dosed fi rst, followed by the left eye.
Th e same investigator performed the study drug administration always for all volunteers, aft er the pre-dose measurement of intraocular pressure was completed.Measurement of intraocular pressure (IOP) of both eyes (by a blinded observer) was performed on day 1 two hours post dosing by means of Goldmann applanation tonometry.
Th e administration of study medication was related to the measurement of intraocular pressure.Th e administration was performed always by the same investigator for www.hophonline.orgall volunteers aft er measurement of intraocular pressure was completed: 1.Administration of Alcaine® eye drops (a topical anesthetic ophthalmic solution containing proparacaine hydrochloride 0.5%, manufactured by Alcon Manufacturing, Ltd., Herts, UK and acquired at the local pharmacy) to the right eye, followed two minutes later by three consecutive measurements of intraocular pressure of the right eye. 2. Administration of Alcaine® eye drops, to the left eye, followed two minutes later by three consecutive measurements of intraocular pressure of the left eye.

Measurement of intraocular pressure
Measurement of intraocular pressure (IOP): the intraocular pressure was measured by means of Goldmann applanation tonometry on both anesthetized eyes (between 9 a.m. and 11 a.m.) at screening and on the right eye only on day 1 and at the fi nal examination.Alcaine eye drops were used to anesthetize the cornea.Th e anesthetic was applied 2 min before the examination.Th e measurement of intraocular pressure was performed three times on each eye within 4 min, always by the same investigator and the same tonometer for the same volunteer.Th e mean value of the three measurements was taken for evaluation.

Additional ophthalmologic procedures
Eye motility: Saccades were assessed by having the volunteer move his eye quickly to a target at the far right, left , top and bottom.Slow tracking was assessed by the 'follow my fi nger' test, in which the examiner's fi nger traces an imaginary "H", which touches upon the six cardinal fi elds of gaze.Th is procedure is suitable to test the inferior, superior, lateral and medial rectus muscles of the eye, as well as the superior and inferior oblique muscles.
Pupil function: An examination of pupillary function includes inspecting the pupils for equal size (1 mm or less of diff erence may be normal), regular shape, reactivity to light, and direct and consensual accommodation.Th e results of the examination could be described as: pupils equal and regular; reactive to light; accommodate (direct and consensual).A swinging-fl ashlight test was used to evaluate the reactivity to light.In a normal reaction to the swinging-fl ashlight test, both pupils constrict when one is exposed to light.As the light is being moved from one eye to another, both eyes begin to dilate, but constrict again, when light has reached the other eye.
Visual acuity: Visual acuity is the eye's ability to detect fi ne details and is the quantitative measure of the eye's ability to see an in-focus image at a certain distance.Visual acuity was measured with a Snellen chart.Th e standard defi nition of normal visual acuity (20/20 or 6/6 vision) is the ability to resolve a spatial pattern separated by a visual angle of one minute of arc.
Slit lamp biomicroscopy: Th e evaluation of the anterior eye was performed by means of slit lamp biomicroscopy.Evaluation of the fundus: Th e evaluation of the fundus was performed by means of the biomicroscope using corresponding lenses.
Th ese additional ophthalmologic examinations were performed and evaluated by the investigator as "normal" or "abnormal"; in case of abnormal, the investigator had to comment.Th e tests were performed at entry (screening) visit for check of exclusion criterion and at the fi nal visit.

Statistical analysis
In order to investigate the pharmacodynamic equivalence of both products, the 95% confidence interval was calculated for the diff erence (test-reference) of the primary target parameter absolute decrease in intraocular pressure 2 h post dose.Th e confi dence intervals were determined by means of a parametric (ANO-VA) statistical method.Th e ANOVA model included treatment, administration pattern, period and subject within pattern as factors.Th is confi dence interval was then compared with the corresponding clinical acceptance range (±1.5 mmHg).
Th e secondary target parameter of the present study was to evaluate the relative (as percentage of baseline) decrease in intraocular pressure 2 h post dose of both products.In addition, the safety of both preparations was evaluated based on safety clinical and laboratory examinations and registration of local tolerability, vital signs (heart rate, blood pres-

RESULTS
A total number of 36 volunteers completed the study according to protocol.Th e results of the IOP measurements on day 1 (pre-dose and 2 h post dose) are presented in Table 1.Th e primary and secondary target parameters of dorzolamide, administered as 1 drop in the conjunctival sac of the right or left eye of the test formulation or 1 drop in the conjunctival sac of the right or left eye of the reference formulation of the 36 volunteers who were subjected to pharmacodynamic and statistical evaluation are summarized in Table 2.
Th e mean value of the primary target parameter "absolute decrease in intraocular pressure 2 h post dose" was 3.10 ± 1.23 mmHg for the test formulation and 3.23 ± 1.15 mmHg for the reference formulation.Th e mean value of the secondary target parameter, relative decrease in intraocular pressure 2 h post was 17.65% ± 6.75% for the test formulation and 18.39% ± 5.96% for the reference formulation.A graphical presentation is given in Figure 1.
For the analysis of pharmacodynamic bioequivalence, the 2-sided 95% confi dence interval was calculated for the diff erence (test-reference) of the absolute decrease in intraocular pressure 2 h post dose and then compared with the predefi ned acceptance range of ±1.5 mmHg.Th e diff erence observed has been 0.125 mmHg.Th e calculated confi dence interval was between -0.65 -0.40 mmHg and thus well within the acceptance range.Th e 95% confi dence are presented in Table 3.
Th e local tolerability of both preparations was assessed on day 1 before dosing (before the IOP measurement) as well as 15 min, 1 h, 2 h (before the IOP measurement), and 6 h post dose in each eye by rating following symptoms: blurred vision, ocular burning, epiphora and hyperemia.
Th e two preparations were similarly well tolerated without any signs of clinically signifi cant adverse eff ects.A to-  Th e main objective of the present study was to assess the pharmacodynamic equivalence (lowering of intraocular pressure) of two formulations of eye drops containing 20 mg dorzolamide in 1 ml eye drops.Th e chosen design of the study was adequate to determine the pharmacodynamic target parameters of the test and reference formulation.
No changes to the protocol were done aft er the start of the study and no major deviations from the protocol were observed.
A total number of 36 volunteers completed the study according to the protocol.Th e results of all these volunteers were analyzed.Th e 95% confi dence intervals are based on the data of 36 study completers.
All clinical work was performed according to GCP guidelines, local requirements and the current Declaration of Helsinki.
Both products caused a pronounced and almost identical decrease of the intraocular pressure: 3.10 ± 1.22 mmHg aft er administration of the test formulation and 3.23 ± 1.15 mmHg aft er administration of the reference formulation.Th ese fi ndings are well comparable to literature data.In [3] it has been demonstrated that the topical administration of dorzolamide reduces the intraocular pressure in healthy volunteers by 3.5 to 7 mmHg compared to pre-dose values.
For the analysis of pharmacodynamic bioequivalence, the 2-sided 95% confi dence interval was calculated of the diff erence (testreference) of the absolute decrease in intraocular pressure 2 h post dose and then compared with the predefi ned acceptance range of ±1.5 mmHg.Th e diff erence observed has been 0.125 mmHg.Th e calculated confi dence interval was between -0.653 -0.403 mmHg and thus well within the acceptance range.Th e equivalence limit of 1.

DISCUSSION
It has been shown [10] that administration of topical carbonic anhydrase inhibitor in one of both eyes has practically no eff ect on the [11].Th e same equivalence limit was also used by other authors [4], [12] when comparing the effi cacy of diff erent formulations of timolol for the lowering of intraocular pressure.A diff erence of 1.5 mmHg can be therefore regarded as a generally accepted border of clinical signifi cance in glaucoma research.Th e maximum eff ect was expected at 2 h post dose.Th erefore, the assessment has been made at that time, considering that the sensitivity of the comparison would be the highest.

CONCLUSION
All fi ndings regarding the pharmacodynamic parameter (lowering of intraocular pressure) are coherent and demonstrate the therapeutic equivalence of the test formulation (dorzolamide 20mg/ml eye drops) with the reference formulation.Dorzolamide 20mg/ml eye drops can be considered as interchangeable with the reference formulation for the treatment of ocular hypertension.
Th e assessment of local tolerability together with the recording of vital signs and adverse events revealed no diff erence between the test and the reference formulation with respect to their safety profi le.Both products were very well tolerated.
Th e test formulation dorzolamide 20mg/ml eye drops showed comparable results obtained at a time probably equal to the maximum eff ect concerning the primary target parameter lowering of intraocular pressure 2 h post dose and showed no diff erence concerning the safety profi le and tolerability when compared to the reference formulation and is therefore considered to be therapeutically equivalent.

3 .
Administration of study drugs (either 1 drop of the test formulation or 1 drop of the reference formulation in the conjunctival sac of the right eye) 15 minutes aft er Alcaine® administration to the right eye. 4. Administration of study drugs (either 1 drop of the test formulation or 1 drop of the reference formulation in the conjunctival sac of the left eye) 15 minutes aft er Alcaine® administration to the left eye.Following sequence of procedures was followed: -time 0 min: administration of ALCAINE® eye drops to the right eye -time 2 min: fi rst measurement of intraocular pressure of the right eye -time 3 min: second measurement of intraocular pressure of the right eye -time 4 min: third measurement of intraocular pressure of the right eye -time 5 min: administration of ALCAINE® eye drops to the left eye -time 7 min: fi rst measurement of intraocular pressure of the left eye -time 8 min: second measurement of intraocular pressure of the left eye -time 9 min: third measurement of intraocular pressure of the left eye -time 15 min: administration of study drug (1 drop in the conjunctival sac) to the right eye -time 20 min: administration of study drug (1 drop in the conjunctival sac) to the left eye.
tal number of 22 non-serious adverse events were registered in 20 volunteers in the course of the study: 11 AEs were observed in the eye treated with the test formulation; 11 AEs were observed in the eye treated with the reference formulation.Seventeen AEs were of mild, three AEs of moderate and two AEs were of severe severity.Almost all AE fell into the category of localized ocular reactions (hyper-contralateral eye.A feasible alternative to a cross-over design was thus dosing of both eyes simultaneously: one of both eyes received the test drug and the other one the reference product.Th e study was planned and conducted as a monocentric, observer-blinded, randomized, single-dose study in healthy volunteers.

Volume 3 •Figure 1 .
Figure 1.Comparative bar chart of absolute IOP decrease 2 h post dose

Figure 2 .
Figure 2. Comparative bar chart of relative IOP decrease 2 h post dose