Comparison of AzaSite® and Azithromycin 1% for Bacterial Conjunctivitis Christopher Crean 1, Jason L. Vittitow 1, Richard C. Zink 1, Lori Richards 1, Rozemarijn S. Verhoeven 1, Kendall D. Powell 2 and RK Brazzell 1 1 Inspire Pharmaceuticals, Inc., Durham, NC 2 Enthalpy Analytical, Inc. Durham, NC Christopher Crean 1, Jason L. Vittitow 1, Richard C. Zink 1, Lori Richards 1, Rozemarijn S. Verhoeven 1, Kendall D. Powell 2 and RK Brazzell 1 1 Inspire Pharmaceuticals, Inc., Durham, NC 2 Enthalpy Analytical, Inc. Durham, NC Research Sponsored by:

2 PurposePurpose  To study the ocular distribution of 1 % azithromycin in an aqueous formulation and 1 % azithromycin in DuraSite® (AzaSite®).  To compare the tissue distribution and pharmacokinetics of AzaSite® after a single administration and after the approved seven-day course treatment.

3 This study was designed as a terminal sample collection study in pigmented rabbits. At the indicated times after drug administration, 40 µL of PBS were applied to the ocular surface of anesthetized animals and 20µL was collected for estimation of tear drug concentrations. Ocular tissues were removed in the following order: inferior and superior bulbar conjunctiva, inferior and superior palpebral conjunctivas followed by cornea and eyelids. Samples were analyzed by LC/MS/MS. MethodsMethods Sampling times: 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120 and 144 h Single administration: Multiple administration: Sampling times: Days 1-6 : Predose (0), 0.5, 1 h Day 7: 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120 and 144 h Day Sampling Time Period Day

4 ResultsResults The azithromycin conjunctiva and cornea tissue concentrations are higher for the mucoadhesive formulation. Concentration of azithromycin (mean ± SE) in conjunctiva and cornea is plotted on a log-linear graph in nanograms of drug per gram of tissue over time, where 1000 nanograms is equal to 1 microgram. The conjunctiva plotted in this graph is an average plot of the four conjunctiva quadrants. ( … ) The dotted line in the graph represents the MIC 50 of azithromycin for typical bacterial pathogens.

5 ResultsResults Seven days of treatment with azithromycin in DuraSite increases the observed conjunctiva and cornea tissue concentrations. Concentration of azithromycin (mean ± SE) in conjunctiva and cornea is plotted on a log-linear graph in nanograms of drug per gram of tissue over time, where 1000 nanograms is equal to 1 microgram. The conjunctiva plotted in this graph is an average plot of the four conjunctiva quadrants. (…) The dotted line in the graph represents the MIC 50 of azithromycin for typical bacterial pathogens.

6ResultsResults The azithromycin conjunctiva tissue concentrations reach a stable minimum concentration by day 2. Concentration of azithromycin (mean ± SE) in conjunctiva is plotted in nanograms of drug per gram of tissue over time, where 1000 nanograms is equal to 1 microgram. The conjunctiva plotted in this graph is an average plot of the four conjunctiva quadrants. Azithromycin reaches steady state tissue levels by day 2 that are a minimum of 15 fold higher than the typical minimum inhibitory concentration (MIC 50 ) level for bacterial pathogens. Azithromycin is cleared from conjunctiva with a 40 hour average half-life. Eye Drop Application

7ResultsResults The azithromycin cornea tissue concentrations reach a stable minimum concentration by the end of day 2. Eye Drop Application Concentration of azithromycin (mean ± SE) in cornea is plotted in nanograms of drug per gram of tissue over time, where 1000 nanograms is equal to 1 microgram. Azithromycin reaches steady state tissue levels by day 2 that are a minimum of 30 fold higher than the typical minimum inhibitory concentration (MIC 50 ) level for bacterial pathogens. Azithromycin is cleared from cornea with a 43 hour average half-life.

8ResultsResults The azithromycin eye lid tissue concentrations reach a stable minimum concentration by the start of day 3. Eye Drop Application Concentration of azithromycin (mean ± SE) in eye lids is plotted in nanograms of drug per gram of tissue over time, where 1000 nanograms is equal to 1 microgram. Azithromycin reaches steady state tissue levels by day 2 that are a minimum of 40 fold higher than the typical minimum inhibitory concentration (MIC 50 ) level for bacterial pathogens. Azithromycin is cleared from eye lids with a 125 hour average half-life.

9ResultsResults Azithromycin resides on the ocular surface at higher levels and for a longer period of time when formulated in DuraSite®. Eye Drop Application Concentration of azithromycin (mean ± SE) in tears is plotted in micrograms per milliliter.

10 Azithromycin Calculated Peak and Total Concentration Levels Total exposure is reported as mean±SE area under the curve (AUC) in h*ng/g for the tissues and h*ng/mL for tear in log format for visual purposes; while peak concentration is reported in mean±SE ng/g for the tissues and ng/mL for tear. The reported AUC values for the single dose studies are from 0-144h while the multi dose AUC values are after the last eye drop from h. Peak concentrations (Cmax) are reported after the initial eye drop from the single dose studies and the last eye drop (9th) on Day 7 for the multi dose study. Within a given tissue there was no appreciable difference between the different quadrants of conjunctiva or the inferior or superior eye lids. The eye lids were not analyzed during the single dose studies. All tissues exhibit higher concentrations of azithromycin in a mucoadhesive formulation and after multiple administrations.

11 Pharmacokinetic Parameters of Azithromycin Treatment Group Tissue Type AUC 0-t (h*ng/g) a Cmax (ng/g) a Tmax (h)T ½ (h)CL/F (g/h) a Vz/F (g) a 1% Azithromycin with DuraSite® (AzaSite®) Single Eye Drop Cornea837,31940, Average Conjunctiva 737,717108, Tears3,016,25710,539, % Azithromycin without DuraSite® Single Eye Drop Cornea196,4868, Average Conjunctiva 79,2279, Tears154,750893, % Azithromycin with DuraSite® (AzaSite®) Multiple Eye Drops Cornea16,402,463252, Averaged Conjunctiva 3,101,108177, Tears392,0211,217, Average Eye Lids 8,146,912180, a For tears and aqueous humor, units for C max (ng/mL), AUC 0-t (h*ng/mL), CL/F (mL/h) and Vz/F (mL) were different Noncompartmental pharmacokinetic parameters for the different matrices were calculated with the WinNonlin pharmacokinetic software from Pharsight, Inc., utilizing the sparse sampling option. The time course of analysis varied depending on the treatment paradigm; single administration time period was from h, while the multi-dose period was evaluated from h post initial eye drop or h after the last eye drop.

12 ConclusionsConclusions Azithromycin differs pharmacokinetically from a number of antibiotics in its ability to achieve and maintain high tissue concentrations after a single and multiple eye drops and sustain the exposure within the tissue for some time after the last eye drop due to its extended half-life. Pharmacodynamically it differs from other macrolide antibiotics (e.g. erythromycin) in its ability to kill both gram positive and negative bacteria. Also, potential clinical effectiveness of azithromycin is further enhanced by its formulation within Durasite®, which increases tissue exposure 5-18 fold over a non-mucoadhesive formulation. This data provides evidence that azithromycin administered from a mucoadhesive formulation has levels at steady state; in conjunctiva at least 15-fold above the average MIC, in cornea at least 30-fold above the average MIC, in eye lids at least 40-fold above the average MIC. persists in all tissues analyzed 6 days after the last eye drop