JOURNAL OF OCULAR PHARMACOLOGY
`AND THERAPEUTICS
`Volume 12. Number 4, 1996
`Mary Ann Liebert, Inc.
`
`The In Vitro and In Vivo Ocular
`
`Pharmacology of Olopatadine
`(AL-4943A), an Effective
`Anti—Allergic/Antihistaminic Agent
`
`.l.M. YANNI, DJ. STEPHENS, S.T. MILLER, LK. WEIMER,
`G. GRAFF, D. PARNELL, LS. LANG, .l.M. SPELLMAN,
`M.T. BRADY, and DA. GAMACl-IE
`
`Allergy/Inflammation Research, AICOII Laboratories, Inc. Fort Worth, Texas
`
`ABSTRACT
`
`[(z)—l1-[3—(dimethylamino)propylidene]—6,ll-dihydro-
`Olopatadine (AL—4943A; KW-4679)
`dibenzlb,e]oxepine-2 acetic acid hydrochloride] is an anti-allergic agent which inhibits mast cell
`mediator release and possesses histamine H. receptor antagonist activity. Studies were conducted to
`characterize the in vitra and in vivo pharmacological profile of this drug relevant to its topical ocular
`use. AL-4943A inhibits histamine release in a concentration-dependent fashion (ICso = 559 pM) from
`human conjunctival mast cell preparations in vitro. Histamine release was not stimulated by
`AL4943A at concentrations as high as 10 mM.
`In contrast, ketotifen stimulated histamine release
`at concentrations slightly higher than effective inhibitory concentrations. AL-4943A did not display
`any in vitro cyclooxygenase or 5-lipoxygenase inhibition. Topical ocular application of AL-4943A
`effectively inhibits antigen- and histamine-stimulated conjunctivitis
`in guinea pigs. Passive
`anaphylaxis in guinea pig conjunctiva was attenuated by AL4943A applied 30 min prior to
`intravenous or
`topical ocular antigen challenge (ED,0 values 0.0067% and 0.0170%, w/v,
`respectively). Antihistaminic activity in viva was demonstrated using a model of histamine-induced
`vascular permeability in guinea pig conjunctiva. AL—4943A applied topically from 5 min to 24 hrs
`prior
`to histamine challenge effectively and concentration-dependent]y inhibited the vascular
`permeability response, indicating the compound has an acceptable onset and a long duration of action.
`Drug concentrations 5—fold greater than those effective against histamine-stimulated conjunctival
`responses failed to inhibit vascular permeability responses induced with either serotonin or
`Platelet-Activating-Factor.
`These data indicate that
`the anti-histaminic effect observed with
`AL-4943A is specific. These anti-allergic/antihistaminic activities of AL-4943A observed in
`preclinical model systems have been confirmed in clinical trials in allergic patients.
`
`INTRODUCTION
`
`[(z)-11-[3-(dimethylamino) propylidene]—6,l l-dihydro-
`Olopatadine (AL4943A; KW-4679)
`dibenz-[b,e]oxepine-2 acetic acid hydrochloride] is an anti-allergic/antihistarninic agent synthesized
`(I) and under development by Kyowa Hakko Kogyo Co., Ltd, for the treatment of bronchial asthma,
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`allergic rhinitis and chronic urticaria. AL-4943A's anti-allergic efficacy following systemic
`administration has been demonstrated in rat and guinea pig models of immediate hypersensitivity
`(1,2,3).
`Inhibition of passive anaphylaxis persisted for 9 hrs following oral administration of
`AL-4943A (3) indicating a prolonged duration of action.
`In vitro receptor binding studies have
`demonstrated the affinity of AL-4943A for the Hl-histamine receptor (1,4,5). Additionally, these
`studies have shown selectivity of AL—4943A for the H1 receptor and demonstrated a lack of
`significant interaction with alpha adrenergic, muscarinic, doparninergic and numerous other receptors
`(5).
`
`Because of the reported long duration of anti-allergic activity, coupled with selective
`anti-histaminic activity, studies were undertaken to evaluate the utility of this compound for use in
`allergic conjunctivitis. AL-4943A's effects on human conjunctival mast cell mediator release, on
`cyclooxygenase and 5-lipoxygenase enzymes, and in models of allergen- and histamine-stimulated
`conjunctivitis following topical ocular administration were characterized. The results of those
`investigations are reported below.
`
`METHODS AND MATERIALS
`
`Inhibition of Histamine Release from RBL Rat Basophils
`
`RBL-2H3 is a rat basophilic leukemia cell line which secretes both histamine and serotonin
`upon degranulation. RBLs were passively sensitized overnight with lgE specific for dinitrophenol
`(DNP) tagged to bovine serum albumin according to Benacerraf and Levine (6). Cells were washed
`with PIPES buffer, and incubated for 15 min at 37°C with AL-4943A prior to challenge with antigen
`(DNP, 10 ng/ml). Supematants were analyzed for histamine content using a commercially available
`radioimmunoassay (RIA) system (AMAC, 1nc., Westbrook, ME).
`
`Histamine Release from Human Conjunctival Mast Cells
`
`Human conjunctival tissue was obtained from organ/tissue donors. Upper and lower palpebral
`conjunctivas were aseptically collected within 8 hrs of death (average time 4.5 hrs) and transported
`in Dexsol® corneal preservation medium (Chiron Ophthalmics, Irvine, CA). Conjunctival tissue was
`weighed and placed into a glass 20 m1 screw—cap bottle containing culture medium (RPMI 1640)
`supplemented with heat inactivated fetal bovine serum (20%), L-glutamine (2 mM), penicillin (100
`units/ml), streptomycin (100 mg/ml), amphotericin B (2.5 mg/ml), and HEPES (10 mM). Tissues and
`medium were transferred to sterile petri dishes for overnight equilibration at 37°C prior to enzymatic
`digestion.
`Modification of a previously reported method for obtaining monodispersed cell suspensions
`containing mast cells (7) was employed using human conjunctival tissue. Briefly, tissues were
`transferred to Tyrode’s buffer (in mM: 137 NaCl, 2.7 KCl, 0.35 NaHzPOM 1.8 CaClz, 0.98 MgClz,
`11.9 NaI-ICO3, 5.5 glucose) containing 0.1% gelatin (TGCM) for enzymatic treatment. Tissues were
`incubated with 200 U each of collagenase (Type IV) and hyaluronidase (Type 1-3) per gram of tissue
`for 30 min at 37° C. Following enzyme digestion, tissues were washed with an equal volume of
`TGCM over Nitex® filter cloth (Tetko, Briarcliff Manor, NY). Two digestions, completed as
`described above, were followed by additional digestion steps using 2000 U each of collagenase and
`hyaluronidase per gram of tissue for 30 min at 37°C.
`The filtrate obtained from each digestion was centrifuged (825 g, 7 min), and pelleted cells were
`resuspended in calcium/magnesium free Tyrode’s buffer (TG). Pooled cells from all digestions were
`centrifuged (825 g, 30 min) over a 1.058 g/L Percoll® cushion. Mast cell enriched cell pellets were
`resuspended and washed in TO buffer. Viability and number of mast cells were determined by vital
`dye (trypan blue) exclusion and toluidine blue 0 staining of the harvested cell suspensions. Cell
`suspensions containing 5000 mast cells were added to TGCM containing tubes. The cells were
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`treated with test compound 15 min prior to challenge with anti-human IgE (10 pg/ml). The final
`volume per reaction tube was 1.0 ml. The release reaction was terminated by the addition of ice-cold
`TGCM and centrifugation (500 g, 7 min). Supematants were collected and stored at -20°C until
`histamine analysis (described above).
`
`Inhibition of Cyclooxygenase In vitro
`
`Inhibition of cyclooxygenase activity from sheep vesicular glands by AL-4943A was assayed
`as follows: Lipid-depleted sheep vesicular gland microsomal powder (10 mg) was homogenized in
`ice-cold 50 mM phosphate buffer (pH 7.4) supplemented with 1% Tween-20, 2 pM hematin, and 5
`mM diethyldithiocarbamic acid. The solubilized microsomal preparation (10 pg) was added to the
`incubation chamber containing 3.0 ml 50 mM phosphate buffer (pH 7.4), 0.5 mM phenol, and 0.5 pM
`hematin and pre-equilibrated to 30° C. AL-4943A, dissolved in dimethyl sulfoxide (DMSO), was
`added to the incubation chamber. The mixture was stirred for 2 min before initiating the reaction by
`the addition of 30 pl of a freshly prepared aqueous solution of 10 mM ammonium arachidonate.
`Indomethacin, also in DMSO (5 30 pl), served as reference compound. Cyclooxygenase activity was
`determined polarographically by monitoring the rate of oxygen consumption, due to the conversion
`of arachidonic acid to prostaglandin Hz (8).
`
`Inhibition of 5-HETE and LTB, Formation In virra
`
`The potency of AL4943A to suppress 5-hydroxy eicosatetraenoic acid (5-HETE) and
`leukotriene l3,1 (LTB4) formation was investigated in calcium ionophore Mama—stimulated neutrophils
`isolated from rabbit peripheral blood. Neutrophils from peripheral blood were isolated by standard
`procedures. Briefly, heparinized/calcium chelated blood was obtained from five New Zealand Albino
`rabbits by heart puncture. Red cells were removed at 4°C by dextran sedimentation (9). White cells,
`contained in the supernatant fraction, were sedimented by centrifugation and contaminating red cells
`removed by hypotonic lysis. The white cell pellet obtained following hypotonic red cell lysis and
`centrifugation was resuspended in Dulbecco's phosphate buffered saline (Caz’lMg2'—free). The cell
`suspension was layered onto a 60% Histopaque-1083/40% Histopaque-l 1 l9 cushion. The neutrophil
`pellet at the bottom of the tube following centrifugation was washed and resuspended in 1/25 the
`original blood volume. Aliquots of the cell suspension were pretreated for 5 min at 37°C with either
`carrier (DMSO) or test article dissolved in DMSO.
`Immediately thereafter, CaCl2 was added to the
`cell suspension and cells stimulated by the addition of 5 pl of a mixture containing ll—“CJ-
`arachidonic acid and calcium ionophore (Anm) in DMSO. The final concentrations of CaClz,
`[l-"C]-arachidonic acid and calcium ionophore were 5.0 mM, 52 pM and 5.0 pM, respectively.
`After 3 min of incubation at 37° C, reactions were terminated by the addition of 2 volumes of acetone.
`Extraction and reversed phase (Cm-5 p) HPLC analysis of [l-“Cl-labelled arachidonic acid metabo-
`lites were conducted as previously described by Graff and Anderson (10).
`
`Passive Anaphylaxis in Conjunctiva
`
`Guinea pigs or rats (5-8/group) were passively sensitized with anti-ovalbumin serum injected
`subconjunctivally in one eye. Twenty-four (24) hrs after passive sensitization. ovalbumin (0A) was
`administered either intravenously (i.v.) or topically onto the eye.
`The anti-allergic effect of AL—4943A following i.v. antigen administration was determined as
`follows: Thirty (30) min prior to i.v. antigen challenge, the animals received 20 pl of AL-4943A or
`saline applied topically to the eye. The animals were then challenged i.v. via the marginal ear vein
`or lateral tail vein with 1.0 ml of an 0A2Evans Blue solution (100 pgzl mg, guinea pigs;
`I mg:2.5
`mg, rats). Responses were quantitated as previously described by Yanni et a]. (l l).
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`For assessment of the allergic response following topical ocular antigen challenge, 20 pl of
`ovalbumin (1.0%, w/v) was administered to the sensitized eye 5 min after topical ocular application
`of AL-4943A or saline (20 p1). During dose response studies, the order of compound administration
`was randomized. Thirty (30) min later, the reaction was quantitated, using the following scoring
`scheme (maximum score per animal = 10):
`
`Congestion (refers to palpebral and bulbar conjunctiva)
`0- Normal
`
`1 - Pink conjunctiva
`2 - Red conjunctiva
`3 — Dark red conjunctiva; petechiae present
`
`Swelling
`0- None
`
`I — Any swelling on lower lid only
`2 - Swelling upper and lower lid, lids partially closed
`3 - Lids everted, very swollen, lids at least half closed
`4- Swelling of both lids and side of face
`
`Discharge
`0- None
`
`1 - Glazed, glassy appearance
`2 - Moist lids and surrounding hair
`3 - Moist lids and surrounding hair, thicker mucous-like
`
`Histamine-induced Vascular Permeability in Guinea Pig Conjunctiva
`
`Male Dunkin Hartley Viral Antibody Free outbred guinea pigs (Charles River Labs, Portage,
`Ml), 250-350 grams, (6/group) were injected iv. via the marginal ear vein with 1.0 ml of Evans Blue
`dye (1.0 mg/ml). Forty-five (45) min post dye injection, 20 pl of test compound or saline vehicle
`was applied topically onto one eye of each experimental animal. Thirty min following topical drug
`application, the guinea pigs were anesthetized and challenged subconjunctivally with histamine (300
`ng/ 10 pl). Responses were quantitated as previously described (1 1).
`All modifications of the pretreatment interval between compound administration and histamine
`challenge are noted in the Results section.
`
`Platelet Activatin
`
`
`Conjunctiva
`
`
`
`Factor PAF- or Serotonin
`
`
`5-HT -induced Vascular Penneabilit
`
`
`
`Male Sprague Dawley rats (Harlan Sprague Dawley, Houston, TX), 125—200 grams, (6/group)
`were injected i.v. via the lateral tail vein with 1.0 ml Evans Blue dye (2.5 mg/ml). Approximately
`20 min post dye injection, 20 pl of test compound or vehicle was applied topically onto one eye of
`the experimental animal. Thirty (30) min later, each animal was anesthetized and challenged
`subconjunctivally with PAP (30 ng/lO pl) or 5-HT (100 ng/lO pl) in the treated eye. Thirty (30)
`min after challenge, the animals were killed, and responses were quantitated as noted for histamine.
`
`Communds
`
`levocabastine (Janssen
`Japan);
`AL-4943A (Kyowa Hakko Kogyo Co., Ltd., Tokyo,
`Pharmaceutica, Beerse, Belgium); histamine dihydrochloride, ovalbumin, 5-hydroxytryptamine
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`hydrochloride, Percoll®, goat lgG, ketotifen fumarate, b0vine serum albumin, dinitrofluorobenzene,
`cyproheptadine hydrochloride (Sigma Chemical Co., St. Louis, MO); Evans Blue dye (Aldrich
`Chemical Co., Milwaukee, WI); PAF (Biomol, Plymouth Meeting, PA); anti-human lgE (goat-derived
`IgG) (Cortex Biochem, San Leandro, CA).
`Immediately prior to in vivo use, all compounds were prepared as solutions or suspensions in
`saline (0.9% NaC] in water) at the concentrations noted in the Results section. All solutions and
`suspensions were prepared on a weight/volume basis to reflect the percentage of free acid or base for
`all in vivo experiments.
`
`Statistical Analyses
`
`Dunnett’s t-test (12) was used to compare the mean of each treatment group with the mean of
`the vehicle control group. Linear regression was used to analyze dose response and calculate
`effective doses (EDm).
`
`RESULTS
`
`The anti-allergic activity of AL—4943A, defined as inhibitory activity on basophil and mast cell
`degranulation, was assessed in vitro. The addition of AL-4943A 15 min before antigen challenge to
`cultured RBL cells inhibited histamine release in a concentration dependent manner. The 1C,o value
`was calculated to be 803 pM. When human conjunctiva] mast cells were treated with AL-4943A for
`15 min prior to anti-lgE challenge, histamine release was also significantly reduced in a concentration
`dependent fashion. The 1C5.) value obtained in human conjunctiva] mast cells was 559 1 277 uM.
`Dose response curves obtained using both cell types are presented in Figs. 1A and 18. The reference
`drug, ketotifen, examined in the human conjunctiva] mast cell also significantly inhibited histamine
`release. However, at concentrations 3 times greater than the maximally effective concentration,
`ketotifen caused a significant release of histamine from these cells (Fig. 18). Additional in vitro
`evaluations of AL-4943A for cyclooxygenase or 5-]ipoxygenase activity failed to detect significant
`activity (data not presented).
`Anti-allergic activity noted in vitro was confirmed in vivo using two models of passive
`conjunctiva] anaphylaxis. Significant inhibition of i.v. antigen-stimulated allergic conjunctivitis was
`observed in both rats and guinea pigs when AL-4943A was applied topically onto the eye prior to
`antigen challenge. Significant concentration dependent inhibition was noted in both species (Table
`l). AL—4943A was more efficacious when evaluated in guinea pigs compared to rats (80% inhibition
`vs. 40% inhibition, respectively, at 0.1%).
`The anti-allergic activity observed following i.v. antigen challenge was further evaluated using
`topical ocular antigen challenge of passively sensitized guinea pigs. The application of AL-4943A
`onto the eye of sensitized animals 30 min, 4 hrs or 8 hrs before allergen instillation significantly
`attenuated the allergic response (Table 2). The EDso values detemiined in these experiments were
`0.017%, 0.053% and 0.100%, respectively.
`When evaluated for topical ocular antihistaminic activity in viva, AL-4943A potently inhibited
`histamine-induced vascular permeability in the conjunctiva. The compound was applied topically 5,
`15 or 30 min, 2, 4, 8 or 24 hrs prior to histamine injection. Concentration dependent inhibition was
`observed (Table 3). The ED” values derived from these data are 0.019%, 0.004%, 0.002%, 0.0014%,
`0.0056%. 0.035% and 0.114%, respectively.
`The ability of AL—4943A to affect the response of the contralateral eye to histamine challenge
`following topical ocular administration was also assessed. AL-4943A or levocabastine (0.05%) was
`applied to one eye and histamine was subsequently injected subconjunctivally in the contralateral eye.
`The dmgs’ effects were quantified as described above. AL—4943A (0.1%) applied 30 min before
`histamine challenge failed to decrease the response in the contralateral eye (197 1 40, vehicle vs. 199
`_+_ 39, AL-4943A treated) while inhibiting the response in the treated eye by 95%. In contrast,
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`levocabastine did significantly reduce the response in the untreated eye (28%). These results indicate
`a lack of systemic effect following topical administration of AL—4943A.
`The specificity of AL-4943A noted in histamine and antigen stimulated in vivo models was
`evaluated using conjunctival vascular permeability models induced by 5-HT and PAF. AL-4943A
`applied at a concentration (0.01%) and pretreatment interval (30 min) before challenge proven to
`prevent histamine-induced responses greater than 70 % failed to significantly inhibit the response in
`the conjunctiva to either 5-HT or PAF (Table 4).
`
`[A] RBL Cells
`
`100
`
`so
`
`AL-4943A
`
`. .
`
`\ .
`
`
`
`~55
`
`-5.0
`
`4.5
`
`-4.0
`
`' p<0.05. Dunnett's Hut.
`-3.5
`-3.0
`.25
`
`Dose log(M)
`
`[B] Human Conjunctival Tissue Mast Cells
`
`%Inhibition
`
`%inhibition
`
`100
`
`80
`
`60
`
`ketotifen
`
`/
`
`'/
`
`
`
`
`'
`
`a
`
`_ AL-4943A
`
`’\
`
`2/-
`
`\ i
`
`' p<0.05, Dunnett's t-test.
`
`-3.5
`
`-3.0
`
`-25
`
`-2.0
`
`i
`i F
` -5.5
`
`-5.0
`
`4,5
`
`4.0
`
`Inhibition of Histamine Release from RBL Cells (A) and Human
`FIGURE 1. AL—4943A’s
`Conjunctival Tissue Mast Cells (B).
`
`Dose log(M)
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`TABLE 1
`
`Effect of AL-4943A on Passive Anaphylaxis in Rat or Guinea Pig Conjunctiva
`Following Intravenous Antigen Challenge'M
`
`Species
`
`Compound
`
`Conc.
`(96, w/v)
`
`Permeability
`Score
`(x 1 SD.)
`
`
`96 Change
`
`Regression
`Analysis
`
`Rat
`
`NaCl
`
`AL-4943A
`
`Guinea
`1
`p g
`
`NaCl
`AL-4943A
`
`0.9
`
`1.0
`0.1
`0.01
`
`0.9
`0.1
`0.01
`0.001
`
`308 1 48
`
`147 1 30 *
`185 1 37 *
`266 149
`
`506 1 124
`102 i 76 *
`264 1 161 *
`334 1133
`
`--
`
`-52
`-40
`-1 4
`
`-80
`~48
`-34
`
`y = -53x + 144
`slope t = p < 0.0001
`r - 0.821
`
`y = -11(>x - 0.48
`slope t = p < 0.0054
`r = 0.975
`
`* p < 0.05. Dunnett‘s t test
`a Interval between compound administration and antigen challenge: rat 20 min; guinea pig 30 min.
`
`TABLE 2
`
`Effect of AL—4943A on Passive Anaphylaxis in Guinea Pig Conjunctiva
`Following Topical Ocular Antigen Challenge
`
`
`Compound Pre-
`treatment
`Interval
`
`
`Conc.
`(96, w/v)
`
`Score
`(x1 S.D.)
`
`96
`Change
`
`EDso
`(96, w/v)
`
`Regression
`Analysis
`
`NaCl
`
`30 min
`
`AL—4943A
`
`NaCl
`
`4 hr
`
`AL-4943A
`
`NaCl
`
`8 hr
`
`AL-4943A
`
`0.9
`
`1.0
`0.1
`0.01
`0.001
`
`0.9
`
`1.0
`0.1
`0.01
`0.001
`
`0.9
`
`1.0
`0.1
`0.01
`0.001
`
`8.2 1 1.3
`
`3.8 1 0.8 *
`3.2 1 1.3 '
`4.5 1 1.1 "
`5.3 1 2.0 *
`
`7.2 1 1.0
`
`2.3 1 1.5 *
`3311.5”
`4.2 1 2.0 *
`5.3 11.2
`
`7.2 1 1.7
`
`1.7 10.8 *
`4.2 1 0.8 *
`5.4 11.1 *
`5.8 1 0.8
`
`--
`
`-53
`-61
`-45
`-3S
`
`--
`
`-67
`-53
`-42
`-2()
`
`--
`
`-77
`-41
`-25
`-19
`
`0.0170
`
`y - -l.08x + 2.17
`t slope = p < 0.020
`r =- 0.542
`
`0.0529
`
`y = —0.98x + 2.32
`tslope-p<0.0018
`r - 0.602
`
`0.100
`
`y - -l.37.\' + 2.17
`t slope = p < 0.0001
`r - 0.856
`
`* p < 0.05. Dunnett's t test
`——————\
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`TABLE 3
`
`The Effect of AL—4943A on Histamine-induced Vascular Permeability in Guinea Pigs
`
`Compound
`
`Conc.
`(96, w/v)
`
`Permeability
`Pre-
`Score
`treatment
`Interval
`(x1 S.D.)
`
`
`96
`Change
`
`EDso
`(96)
`
`Regression
`Analysis
`
`NaCl
`
`5 min
`
`AL-4943A
`
`0.9
`
`0.3
`0.03
`0.003
`
`324 1 S9
`
`94 1 65 *
`165 1 74 *
`197 1 56 *
`
`NaCl
`
`15 min
`
`0.9
`
`431 1 91
`
`AL-4943A
`
`0.1
`0.03
`0.01
`0.001
`0.0001
`0.00001
`
`NaCl
`
`30 min
`
`0.9
`
`AL-4943A
`
`AL-4943A
`
`2 hr
`
`NaCl
`
`4 hr
`
`AL-4943A
`
`NaCl
`
`8 hr
`
`AL-4943A
`
`NaCl
`
`24 hr
`
`AL-4943A
`
`0.1
`0.01
`0.001
`0.0001
`
`0.1
`0.01
`0.001
`0.0001
`
`0.9
`
`1.0
`0.1
`0.01
`0.001
`
`0.9
`
`1.0
`0.1
`0.01
`0.001
`
`0.9
`
`1.0
`0.1
`0.01
`
`98 1 87 *
`180 1 40 *
`238 1 49 *
`240 1 43 *
`302 1 SS *
`370 1 73
`
`228 1 25
`
`15 1 15 *
`69 1 33 *
`125 1 36 *
`195 1 S3
`
`17 1 3 *
`146 1 87 *
`201 1 105 *
`342 1 66
`
`423 1 47
`
`S 1 8 "
`66176"
`263 1 49 *
`240 1 69 *
`
`374 1 4S
`
`10 1 8 "
`152 1 33 *
`247 1 29 *
`389 1 S9
`
`347 1 27
`
`12 118 *
`2311 35 *
`302 1 69
`
`--
`
`-71
`-49
`-39
`
`--
`
`-77
`-58
`-45
`-44
`~30
`-14
`
`--
`
`-94
`~70
`~45
`-15
`
`-96
`-66
`-52
`-19
`
`~-
`
`-99
`-84
`~38
`-43
`
`--
`
`-97
`-60
`-34
`4
`
`-—
`
`-96
`-33
`-l 3
`
`0.019
`
`0.004
`
`y :- -52x + 73
`slope t = p < 0.013
`r = 0.571
`
`y = -59x + 73
`slope t = p < 0.0001
`r = 0.814
`
`0.002
`
`y = -61x - 56
`slope t - p < 0.0001
`r = 0.888
`
`0.0014
`
`y = -103x - 82
`slope t = p < 0.0001
`r = 0.850
`
`0.0056
`
`y = -90x + 8
`slopetx p<0.0001
`r = 0.826
`
`0.035
`
`y = -123x + 14
`slope t = p < 0.0001
`r - 0.969
`
`0.114
`
`y - -l45x + 36
`slope t = p < 0.0001
`r = 0.909
`
`* p < 0.05, Dunnett's t test
`
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`|PR2018-01020 and |PR2018-01021, Exhibit 1004, Page 8
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`396
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`

`TABLE 4
`
`The Effect of AL—4943A on Serotonin- and
`
`Platelet Activating Factor-induced Vascular Permeability
`
`Stimulus
`
`Compound
`
`Cone.
`(96, w/v)
`
`Permeability
`Score
`(x i S.D.)
`
`96 Change
`
`Serotonin
`
`NaCl
`
`AL—4943A
`
`cyproheptidine
`
`PAF
`
`NaCl
`
`AL-4943A
`
`* p < 0.05, Dunneu’s t test
`
`0.9
`
`0.01
`
`0.01
`
`0.9
`
`0.01
`
`385 i 59
`
`304 i 57
`
`118 1 52 "
`
`409 i 74
`
`404 i 49
`
`--
`
`-21
`
`-69
`
`--
`
`-1
`
`DISCUSSION
`
`The mast cell has long been considered to play a primary role in the pathogenesis of immediate
`hypersensitivity reactions. One therapeutic approach used in ocular allergic disease involves the use
`of mast cell stabilizers (13). Human conjunctiva] tissue is estimated to contain as many as 10,000
`mast cells per mm3 (14). Drugs developed as mast cell stabilizers for the treatment of asthma have
`been available for topical use in allergic conjunctivitis. Recently,
`the concept of mast cell
`heterogeneity has been illustrated and accepted (15,16). Mast cells from different species and from
`different tissues within the same animal differ with respect to their morphological, cytochemica], and
`functional properties.
`Importantly,
`the mast cell populations also differ in their responses to
`pharmacological agents (14,17). These findings suggest that efficacious anti-allergic drugs for treating
`conjunctivitis should have demonstrated efficacy on the target cell, the human conjunctiva] mast cell.
`Data presented clearly demonstrate AL-4943A’s concentration dependent inhibition of human
`conjunctiva] mast cell histamine release. Greater than 90% inhibition was achieved without observable
`histamine release due to cytotoxicity. AL-4943A, at concentrations 10 times greater than the
`maximally effective concentration, inhibited histamine release by greater than 90%. These data are
`in contrast to those obtained with ketotifen. Although ketotifen is more potent than AL-4943A at
`inhibiting mast cell mediator release, it causes pro-inflarmnatory mediator release at concentrations
`only 3 times greater than the maximally effective concentration. These data indicate that AL-4943A
`affords a larger safety margin than does ketotifen.
`The relevance of AL-4943A's effect on mast cells based upon the potency (IC,o= 559 pM)
`becomes apparent when the molar concentration is converted to percentage weight/volume (559 uM
`- 209 [Lg/ml - 0.0209952, w/v). Because the conjunctiva is the target tissue for this compound,
`penetration and transport into the eye do not confound drug delivery. For these reasons, and because
`of the efficacy observed following topical ocular administration, the potency on human conjunctiva]
`mast cells appears acceptable.
`Anti-allergic activity observed in vitro was confirmed in viva using models of passive
`anaphylaxis. Concentration dependent inhibition was noted following topical ocular administration
`of AL-4943A prior to antigen challenge. Significant activity was noted 8 hrs after administration of
`AL-4943A. The rat did not appear to be as responsive as the guinea pig to drug treatment. This can
`be partially explained by the two species' relative reactivity to the major vasoactive amines released
`
`|PR2018—01020 and |PR2018-01021, Exhibit 1004, Page 9
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`

`

`from activated mast cells (rat/serotonin; guinea pig/histamine) (18,19,20) and the activity of
`AL—4943A on histamine Hl receptors (1,4,5).
`The reported in vitro histamine H1 receptor antagonism was observed in vivo as well.
`AL—4943A applied topically onto the eye from 5 min to 24 hrs before histamine challenge
`significantly attenuated the vascular permeability response. A comparison of EDso values obtained
`in our laboratory at time intervals from 15 min to 4 hrs after dosing indicate that AL-4943A is
`equipotent to levocabastine (15 min EDsos: 0.004%, AL—4943A vs. 0.0072%, levocabastine; 4 hr:
`0.0056%, AL-4943A vs. 0.0080%, levocabastine). These results are consistent with the similar
`affinities of the two compounds observed in radioligand binding studies (5). Assessment of activity
`at 24 hrs following compound administration demonstrates AL—4943A’s long duration of action.
`These in viva results are supported by the data obtained in ex vivo experiments in which AL-4943A
`occupied 80% of lung histamine H. receptors within an hour of oral dosing, and approximately 50%
`of the receptors were still occupied 24 hrs later (4).
`AL-4943A’s ability to inhibit vascular permeability responses in the conjunctiva is agonist
`specific. No significant reductions in vascular permeability responses induced with serotonin or PAF
`were observed with concentrations of AL-4943A which inhibit histamine stimulated responses by
`70%. The specificity and selectivity of AL—4943A has been further characterized utilizing in vitra
`radioligand binding methodology (5). Data obtained from those studies confirm a lack of interaction
`at adrenergic, doparninergic and muscarinic receptors. This suggests that AL—4943A’s side-effect
`potential may be minimal.
`In addition, when AL-4943A was applied to one eye in a concentration
`which inhibited the response of that eye by 95%, no effect could be observed in the contralateral eye.
`This indicates that the potential for developing side effects is further reduced by the lack of systemic
`effect noted following topical ocular administration. AL-4943A's lack of side effects has been
`confirmed in clinical trials.
`
`Pharmacological intervention in allergic conjunctivitis has been primarily through the use of
`histamine Hl antagonists in combination with a-adrenergic agonists (22). Recently, Berdy et al. (23)
`demonstrated that Hl antagonists effectively relieve pruritis, conjunctival injection, and erythema in
`human volunteers. The other primary approach in managing ocular allergic diseases has been the use
`of mast cell stabilizers (24).
`The data presented demonstrate that AL-4943A combines both of these activities in one
`selective, topically effective, anti~allergiclantihistaminic agent. The compound possesses a rapid onset
`and long duration of action in preclinical models of allergic ocular disease. The anti-allergic and
`human conjunctival mast cell stabilizing activity of AL~4943A, coupled with its selective
`antihistaminic activity, suggests that the compound has potential utility when used prophylactically
`and therapeutically. This affords distinct advantages over other agents currently used for the
`treatment of ocular allergic diseases. Clinical trials with a topical ocular formulation of AL-4943A
`have been completed, and a New Drug Application (NDA) has been filed.
`
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