Pharmacodynamic Approach for Proving Equivalence of Two Ophtalmic Solutions Containing Brimonidine Tartarate 0 . 2 % in Healthy Volunteers A

Pharmacodynamic Approach for Proving Equivalence of Two Ophtalmic Solutions Containing Brimonidine Tartarate 0.2% in Healthy Volunteers A Ruska Valcheva Hristova1, Irenna Stojanova Demircheva1, Andrey Dimitrov Petrov2, Emil M. Gatchev2, Ursula Th yroff -Friesinger3, Wolfram Hagen Richter4, Rossen Krumov Koytchev4 A 1 Medical University of Sofi a, Clinic of Ophthalmology, University Hospital ”Tsaritsa Joanna-ISUL”, Bulgaria 2 Medical University of Sofi a, Department of Clinical Pharmacology and Th erapeutics, University Hospital ”Tsaritsa Joanna-ISUL”, Bulgaria 3 Hexal AG, Holzkirchen, Germany 4 Cooperative Clinical Drug Research and Development AG, Hoppegarten, Germany


INTRODUCTION
Brimonidine tartrate is a relatively selective alpha-2 adrenergic agonist for ophthalmic use.Th e chemical name of brimonidine tartrate is 5-bromo-6-(2-imidazolidinylideneamino) quinoxaline L-tartrate.It has a molecular weight of 442.24 as the tartrate salt, and is soluble in water (5.6 mg/ml) at pH 6.5 [1].
Brimonidine is indicated for the lowering of intraocular pressure in patients with open-angle glaucoma or ocular hypertension.It has a peak ocular hypotensive eff ect occurring at two hours post-dosing [1].Fluorophotometric studies in animals and humans suggest that brimonidine tartrate has a dual mechanism of action by reducing aqueous humor production and increasing uveoscleral outfl ow.A meta-analysis of randomized clinical trials performed by van der Valk et al. (2005) demonstrates that brimonidine is comparable to other agents like beta-blockers, prostaglandin analogs and carbonic anhydrase inhibitors regarding its IOP-lowering eff ect [2].
In humans, systemic metabolism of brimonidine is extensive.It is metabolized primarily by the liver.Urinary excretion is the major route of elimination of the drug and its metabolites and approximately 87% of an orally-administered radioactive dose was eliminated within 120 hours, with 74% found in the urine [1].

AIM
Th e purpose of the present study was to evaluate the pharmacodynamic equivalence of two brimonidine tartarate 0,2% ophthalmic preparations in healthy volunteers.

METHODS
Th is paper is part of commercial phase-1 clinical trial, sponsored by Hexal AG, Holzkirchen, Germany, who gave the permission for publication.

Study design
Th e test drug (Brimonidine Tartrate Ophthalmic Solution 0.2%) was manufactured by Sandoz Canada Inc. Alphagan® ophthalmic solution 0.2% manufactured by Allergan Ltd., United Kingdom was used as a reference product.
Th e study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments, and in accordance with regulation documents [3][4][5][6][7].Th e study was reviewed and approved by the ethics committee of the University Hospital "Tsaritsa Joanna-ISUL", Bulgaria and by the Bulgarian Drug Agency.
Th e study was performed at the Clinic of Clinical Pharmacology and Th erapeutics and the Clinic of Ophthalmology, University Hospital "Tsaritsa Joanna-ISUL", Medical University of Sofi a, Bulgaria.
Th e most important inclusion criteria were as follows: age between 18 and 55 years, physically and mentally healthy, IOP between 16 and 21 mmHg (mean value of 3 measurements) at screening, and normal body weight (BMI between 19 and 27 kg/m2).
All participants gave their informed consent to participate in writing prior to inclusion in the study and aft er a complete written and verbal explanation of the nature, scope and possible consequences.A total number of 45 volunteers gave their informed consent for participation in the study and underwent the screening examination.Th irty six subjects met all inclusion and none of the exclusion criteria and were judged eligible for the study.Nine subjects did not meet one or more inclusion criteria or met one or more exclusion criteria.Th irty six volunteers were randomized in the study and all of them completed the study according to the protocol.
Th e results of all 36 study completers were subjected to statistical evaluation.

Procedures
Th e clinical part of the study included a 12 hours overnight stay (on day 0) and further 6 hours confi nement aft er dosing (on day 1) at the healthy volunteer unit of the clinical center for drug administration, measurement of intraocular pressure and assessment of local tolerability.Two visits were performed for entry and fi nal examination (up to 7 days before and up to 7 days aft er dosing).
Th e volunteers spent the night before dosing at the healthy volunteer unit.In the morning on day 1 the volunteers received a standard breakfast between 7:00 and 7:30 a.m.Th e trial medication was administered between 9:15 and 10:50 a.m.: a single dose of 1 drop of the test product in the conjunctival sac of one eye and 1 drop of the reference drug in the conjunctival sac of the other eye.Th e administration of the study medication was performed always by the same investigator for all volunteers aft er completion of IOP measurement and was timely related to the pre-treatment measurement of IOP.Th e measurement of IOP was performed three times on each eye within 3 minutes, always by the same investigator and using the same tonometer for the same volunteer, always starting with the right eye, followed by the left eye.Th e mean value of the three measurements was taken for evaluation.Th e resulting schedule was as follows: -0 min: administration of local anaesthetic eye drops into the fi rst (right) eye -2 min: fi rst measurement of IOP of the fi rst To reduce the possible systemic absorption, the lacrimal sac was compressed at the medial canthus (punctal occlusion) for one minute immediately following the instillation of each drop.
Th e sequence of administration was the same for all volunteers: the right eye was dosed fi rst, followed by the left eye.Th e allocation of test and reference product to the left or to the right eye was randomized.
Another measurement of IOP of both eyes was performed two hours post dosing within a period of 9 minutes, following the same procedure as described above.
Th e measurement of IOP was performed by means of a Goldmann applanation tonometer.Immediately before each measurement of IOP fl uorescein was applied using sterile, individually packed Fluorescein strips (Haag-Streit AG, Switzerland).Th e tonometer was calibrated according to the instructions of the manufacturer.
Th e present trial was performed under observer-blinded conditions.Th e investigator responsible for the measurement of IOP and the evaluation of local tolerability and vital signs measurement (always the same person for the same volunteer) was held blind regarding the identity of test and reference product.For achieving blindness the dosing was performed in a separate room by a separate investigator responsible only for drug dosing.
Th e local tolerability was assessed before dosing and 15 min, 1, 2 (before the IOP measurement), and 6 hours post dose in each eye by rating following symptoms: blurred vision, ocular burning, epiphora and hyperemia.
Eye motility examination was performed by means of saccades assessment (by asking the subject move his eye quickly to a target at the far right, left , top and bottom) and slow tracking assessment (using the so called 'follow my fi nger' test).
Pupillary function examination included 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.
Visual acuity measurement was performed using 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.
Th e evaluation of the anterior eye was performed by means of a slit lamp biomicroscope (Reichert).
Th e evaluation of the fundus was performed by means of the biomicroscope using corresponding lenses.
All additional ophthalmologic examinations were performed at screening visit for check of exclusion criterion and at the fi nal visit and evaluated by the investigator as "normal" or "abnormal".

Statistical analysis
A total number of thirty-six male and female Caucasians were planned for inclusion.Th e sample size was calculated in respect of the primary target parameter (absolute decrease in IOP 2 hours post dose) and an estimated equivalence margin of 1.5 mmHg.
Th e resulting sample size was n =32.Taking into account possible drop outs, 4 volunteers more than the number needed according to statistical considerations were planned for enrolment.Th e total number of volunteers to be enrolled and treated was thus 36.
Th e pharmacodynamic equivalence of both products was evaluated by calculating the two-sided 95% confi dence interval for the diff erence (test-reference) of the primary target parameter absolute decrease in IOP 2 hours post dose.Th e confi dence intervals were determined by means of analysis of variance (ANOVA).Th e ANOVA model included treatment, administration pattern (which product was administered in which eye), and subject within pattern as factors.Th e 95% confi dence interval was then compared with the predefi ned clinical acceptance range of ± 1.5 mmHg.
Th e relative (as percentage of baseline) decrease in IOP 2 hours post dose of both products was evaluated as a secondary target parameter in the present study.
Th e safety parameters in this trial included the evaluation of local tolerability and vital signs (heart rate, blood pressure), clinical and laboratory examinations (carried out in a certifi ed local clinical laboratory) at the beginning and at the end of the trial as well as the registration of adverse events and/or adverse drug reactions during the total duration of the trial.

RESULTS
All 36 volunteers who were randomized completed the study according to protocol.Th e demographic data of the volunteers are presented in Table 1.Th e results of the individual IOP measurements on day 1 (pre-dose and 2 h post dose) are presented in Table 2. Th e primary and secondary target parameters are summarized in Table 3.
Th e mean value of the primary target parameter "absolute decrease in intraocular pressure 2 hours post dose" was 4.60 ± 1.26 mmHg for the test formulation and 4.40 ± 0.89 mmHg for the reference formulation (Figure 1).Th e mean value of the secondary target parameter "relative decrease in intraocular pressure 2 hours post dose" was 27.89% ± 7.48% for the test formulation and 26.70% ± 5.25% for the reference formulation (Figure 2).Th e twosided 95% confi dence interval calculated for the diff erence (test-reference) of the absolute decrease in intraocular pressure 2 hours post dose was between -0.275 and 0.670 mmHg and thus entirely within the pre-defi ned acceptance range of ± 1.5 mmHg ( served before administration of trial medication (baseline event).All 8 non serious AE's were followed by a complete restitution.

DISCUSSION
Th e market share of generic drugs has grown substantially in recent years.For example since the passage of the Waxman-Hatch Act in the tions was good and revealed no clinically signifi cant diff erences between both products.A total number of 8 non serious adverse events (AEs) were registered in 6 volunteers in the of the trial: blurred vision, ocular burning, epiphora and ocular hypeaemia.Two AEs were observed in the eye treated with the test drug; 5 AEs were observed in the eye treated with the reference drug and 1 event was ob- Pharmacokinetic bioequivalence trials are normally performed for the registration of generic products.Pharmacodynamic parameters can be used for demonstrating bioequivalence of locally applied, locally acting products as in such cases pharmacokinetic bioequivalence is not a suitable approach since plasma drug levels are not relevant for local effi cacy [3].
Th is refers to products locally applied to the eye for the treatment of glaucoma such as beta blocking agents, carboanhydrase inhibitors, etc..When performing a pharmacodynamic bioequivalence trial several important aspects have to be taken into account.In most of the cases pharmacodynamics equivalence trials are performed in patients using a clinical endpoint.In rare cases trials in healthy volunteers might also be feasible provided that the drug tested in the trial fulfi lls the following requirements: a) it has a pronounced and measurable pharmacodynamics eff ect in healthy volunteers and b) this eff ect is representative for the clinical effi cacy in patients.Irrespective whether a pharmacodynamic bioequivalence trial is performed in patients or in healthy volunteers the limits within which a generic product is still defi ned as being "equivalent" to the reference product have to be a) justifi ed based on their clinical relevance and b) prospectively defi ned in the study protocol.It has been demonstrated that brimonidine signifi cantly reduces intraocular pressure in healthy volunteers [8][9].Th is makes an equivalence trial in healthy subjects possible.
Th e equivalence limit of 1.5 mmHg was chosen as the largest medically justifi able deviation of the test formulation compared to the reference formulation based on a recommendation published in an article in the Ophthalmology Times [10].Th e same equivalence limit was also used by other authors [11][12], when comparing the effi cacy of diff erent formulations of timolol for the lowering of intraocular pressure, as well as by the authors of the current publication in two other clinical trials [13][14], for evaluating the pharmacodynamics equivalence of products containing dorzolamide and the combination of dorzolamide plus timolol.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 most frequently used study design in bioequivalence trial is cross-over design in which the same volunteer receives both study drugs in two diff erent periods separated by an appropriate wash-out period.Data from the literature show that in the case of eye drops 586 Volume administration of topical carbonic anhydrase inhibitor and beta-blockers in one of both eyes has practically no eff ect on the contralateral eye [11][12].A feasible alternative to a cross-over design is thus dosing of both eyes simultaneously: one of both eyes receives the test drug and the other one the reference product.Such study design was used by the same working group when evaluating the pharmacodynamics equivalence of dorzolamide [14].Th e chosen design of the study was considered to be adequate to determine the pharmacodynamic target parameters of the test and reference preparations.As the maximum IOP lowering eff ect of both products was expected about 2 hours post dose, the assessment was performed at that time, considering that the sensitivity of the comparison (i.e. the ability to show diff erence between both products) would be the highest.
Both products caused a pronounced and almost identical decrease of IOP: 4.60 ± 1.26 mmHg aft er the test drug and 4.40 ± 0.89 mmHg aft er the reference product.Th ese fi ndings are well comparable to literature data.It has been demonstrated that the topical administration of brimonidine reduces the intraocular pressure in healthy volunteers by roughly 3 to 5 mmHg compared to pre-dose values [16][17][18].
Th e two-sided 95% confi dence interval was calculated of the diff erence (test-reference) of the absolute decrease in IOP 2 hours post dose and then compared with the predefi ned confi dence limits of ±1.5 mmHg.Th e calculated confi dence interval was between -0.275 and 0.670 mmHg and thus within the acceptance range.Th e 95% confi dence intervals are based on the data of 36 study completers.
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 preparation with respect to their safety profi le: both products were well tolerated and showed no signs of systemic reactions.
Th e fi ndings regarding the pharmacodynamic parameter (lowering of IOP) are coherent and demonstrate the therapeutic equivalence of the test product (Brimonidine Tartrate Ophthalmic Solution 0.2%) with the reference product (Alphagan® ophthalmic solution 0.2%).Th ey can thus be considered as interchangeable for the treatment of patients with ocular hypertension.

CONCLUSION
Brimonidine Tartrate Ophthalmic Solution 0.2% was pharmacodynamically equivalent to the reference preparation (Alphagan®) with respect both to effi cacy and safety.

LIMITATIONS OF THE STUDY
Th e main limitation of the present study is the fact that it was performed in healthy volunteers with normal intraocular pressure.
(right) eye -3 min: second measurement of IOP of the fi rst (right) eye -4 min: third measurement of IOP of the fi rst (right) eye -5 min: administration of local anaesthetic eye drops into the second (left ) eye -7 min: fi rst measurement of IOP of the second (left ) eye -8 min: second measurement of IOP of the second (left ) eye -9 min: third measurement of IOP of the second (left ) eye -15 min: administration of study medication (1 drop in the conjunctival sac) to the fi rst (right) eye -20 min: administration of study medication (1 drop in the conjunctival sac) to the second (left ) eye.

Table 4
). Th e local tolerability of both prepara-

Table 1 .
Demographic data of the volunteers

Table 2 .
Results of the individual IOP measurements on day 1 (pre-dose and 2 hours post dose)

Table 4 .
95% confi dence interval for the diff erence of the primary target parameter absolute decrease in IOP 2 hours post dose (n=36)