Evaluations of the Human COX-2 Inhibition for Amfenac, Bromfenac, Diclofenac, and Ketorolac

T. T. T.R. C.K. J.A. 1Research Laboratories, Senju Pharmaceutical Co. Ltd, Kobe, Japan; 2Senju USA, Inc., Encino, CA; 3ISTA Pharmaceuticals, Inc., Irvine, CA
1, Kida 2, Ogawa 3, McNamara 3, Song 3, Gow

INTRODUCTION
Currently, topical ophthalmic medications of nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used to treat external or anterior ocular inflammatory diseases and pain, and to prevent miosis in cataract surgery.
(Ohara 2004a, Ohara 2004b, Kawaguchi 2003, Miyake-Kashima 2004, Data on File)

COX-2 HYPOTHESIS
Arachidonic Acid COX-1 constitutive COX-2 inducibl e PGs
Inhibition of COX-2 (%)

RESULTS
Figure 2. Response Curves of Ophthalmic NSAIDs for COX-2
100 90 80 70 60 50 40 30 20 10 0 -11 -10 -9 -8 Log Conc. (M)
These data were obtained by triplicate assay

DISCUSSION
The lower the IC50 value: The less amount of drug required to exert a therapeutic effect The more potent the drug Bromfenac has the greatest activity against COX-2, the primary mediator for ocular inflammation and pain, (IC50=0.0075 mM) versus the other ophthalmic NSAIDs. In order of relative activity for COX-2 inhibition versus bromfenac (Table 1): Amfenac = 37% (0.0204 mM) Ketorolac = 27% (0.0279 mM) Diclofenac = 25% (0.0307 mM) Bromfenacs high potency may contribute to the rapid and substantial reduction in inflammation and pain following cataract extraction, seen in the pivotal, placebocontrolled, phase 3 US clinical trials with bromfenac.
(Donnenfeld 2005)

Ophthalmic NSAIDs containing 4 active pharmaceutical ingredients; amfenac (which is active metabolite of nepafenac), bromfenac, diclofenac and ketorolac is launched in U.S. and/or Japan markets (Figure 1).
NEVANAC (nepafenac ophthalmic suspension 0.1%, Alcon Laboratories, Inc., TX) XIBROM (bromfenac ophthalmic solution 0.09%, ISTA Pharmaceuticals, Inc., CA, and Senju pharmaceutical Co., Ltd, Japan) DICLOD (diclofenac sodium ophthalmic solution 0.1%, Wakamoto pharmaceutical Co., Ltd., Japan) ACULAR (ketorolac tromethamine ophthalmic solution 0.5%, Allergan, Inc., CA) and ACULAR LS (ketorolac tromethamine ophthalmic solution 0.4%, Allergan, Inc., CA)

PGs

Pharmacokinetic properties of a drug are determined to a great degree by the structure of the molecule itself. In general, compounds that contain a halogen, are more potent (Br-~I->Cl->F->H-). (Walsh 1984) Bromine (halogen) addition to bromfenac provides two significant advances: Increased potency and increased penetration. Cyclooxygenase-2 (COX-2) is expressed in response to insult and is the primary mediator for ocular inflammation. NSAIDs potency is measured as the concentration of drug required to inhibit COX enzyme activity by 50% (IC50). Inhibitory activities for all 4 commercially available ophthalmic NSAIDs, has not been directly compared in human COX-2 enzyme.

GI cytoprotection Platelet aggregation Renal function

(blood flow)

Inflammation Fever Pain Headache Carcinogenesis

Amfenac Bromfenac Diclofenac Ketorolac -7 -6 -5

PURPOSE
To compare the cyclooxygenase-2 (COX-2) inhibitory activity of the four commercially available ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs), amfenac, bromfenac, diclofenac, and ketorolac using recombinant human COX-2 enzymes.

Figure 1. Chemical structure of Amfenac, Bromfenac, Diclofenac, and Ketorolac

Br

NH2 O O NH2

NH2 O O OH
O

METHODS AND MATERIALS

NH2 OH O

Table 1. Relative IC50s: Rank Order Greatest to Least Activity IC50 (mM) Bromfenac Amfenac Ketorolac Diclofenac 0.0075 0.0204 0.0279 0.0307 Relative Potency
(vs. Bromfenac)

Adverse events associated with bromfenac in US clinical trials were predominantly mild and local (there were no clinically significant systemic adverse events). Bromfenacs excellent tolerability profile may be attributable to its twice-daily dosing regimen (decreased corneal exposure to the active ingredient and preservatives in the drop).

Nepafenac

Amfenac
(active metabolite)
Cl NH

Bromfenac

Cl O

OH

N O

OH

Diclofenac

Ketorolac

Specific COX-2 Inhibitor Binding to COX-2

Exploitation of the Side Pocket
CO X-2
S p e c if ic C O X - 2 in h ib it o r p h e n y l g ro u p b in d s t o h y d ro p h o b ic channel
C - t e rm in a l c o n t a in in g a c t iv e s it e s

H y d ro p h ilic s id e p o c k e t

A rg 5 1 3 , N - t e rm in a l H is t 9 0 f o rm s h y d ro g e n b o n d s w it h o x y g e n in s u lf o n a m id e s id e K u ru m b a il e t a l. N a t u r e 1 9 9 6 ; 3 8 4 : 6 4 4 - 6 4 8 c h a in

a Ar

d hi

ni o

i Ac

d
A rg 1 2 0

Compounds Sodium amfenac and sodium bromfenac were synthesized by ISTA Pharmaceuticals, Inc. (Irvine, CA) and Senju Pharmaceutical Co. Ltd. (Osaka, Japan), respectively. Sodium diclofenac and ketorolac tromethamine were purchased from Sigma-Aldrich Co. (St. Louis, MO). All other reagents used were reagent grade. Sources of COX-2 Enzyme Microsomal preparations of human recombinant COX-2 were prepared using insect Sf21 cells. Assay Condition The assay of COX-2 activity was conducted with a standard procedure by MDS Pharma Services (Taipei, R.O.C.). Microsomal enzyme preparations were diluted with buffer (100mM Tris-HCL, pH 7.7 at 37C) containing 1 mM Glutathione 1 M hematin and 0.5 mM phenol, and preincubated with vehicle (DMSO) or compounds in DMSO (1% DMSO in final assay) for 15 min at 37C. The reaction mixture was initiated by adding 0.3 M arachidonic acid (in final assay) and incubated for 5 min at 37C. Amount of prostaglandin E2 production in each mixture was conventionally quantified by Enzyme Immunoassay (EIA).

CONCLUSIONS
This is the first study directly comparing the relative potencies of the four commercially available ophthalmic NSAIDs. Bromfenac is approximately 3 to 4 times more potent than the other NSAIDs in COX-2 enzyme inhibition. This may explain why bromfenac is the only NSAID indicated for twice daily administration in the treatment of postREFERENCES pain. cataract ocular inflammation and
1. 2. Data on File, ISTA Pharmaceuticals; Xibrom US Phase III Trials. Donnenfeld ED, et al. ARVO Annual Meeting, Ft. Lauderdale, May 15, 2005. Kawaguchi T, et al. Folia Ophthalmol Jpn. 2003;54:276. Miyake-Kashima M, et al. Jpn J Ophthalmol. 2004;48:587-590. Ohara K, et al. Jpn J Cataract & Refractive Surgery 2004;18:1 -12(b). Ohara K, et al. Jpn J Clin Ophthalmol. 2004;58:1325-1328(a). Walsh D. J Medicinal Chem. 1984;11:1379-1388.

1.0 0.37 0.27 0.25

Clinical significance of these data is unknown

DISCUSSION
Comparative results are expressed as IC50s, the concentration of enzyme in micromoles (mM) necessary to inhibit 50% of the enzyme activity.

3. 4. 5. 6. 7.

Presented at ASCRS, April 27-May 2, 2007, San Diego, CA