1

JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICSVolume XX, Number XX, 2010© Mary Ann Liebert, Inc.DOI: 10.1089/jop.2009.0098

Abstract

Purpose : To quantify the aqueous humor (AH) concentrations of bimatoprost (amide), travoprost (isopropyl ester), and their hydrolysis products, bimatoprost free acid (BFA) and travoprost free acid (TFA), after multiple topical ocular doses of LUMIGAN ® and TRAVATAN ® , respectively, in patients awaiting cataract surgery. Methods : In 2 separate open-label, sparse-sampling trials, glaucoma patients with cataracts received LUMIGAN ® (bimatoprost ophthalmic solution, 0.03%) or TRAVATAN ® (travoprost ophthalmic solution, 0.004%) bilaterally once daily for at least 21 days prior to cataract surgery. Anterior chamber paracentesis was performed at selected times up to 5 h after the last dose and an AH sample was collected. AH samples were assayed by an independent bioanalytical laboratory using a sensitive and validated tandem LC-MS/MS method. The assay lower limits of quantitation were 0.59 nM for bimatoprost, 0.29 nM for BFA, and 0.44 nM for TFA. Results : AH concentrations of BFA (17-phenyl-trinor PGF 2α ) were quantifi able in all but one sample at 0.5 h. The maximum concentration achieved ( C max ) of BFA was 30.9 ± 16.41 nM ( n =5), observed at 2 h postdose. AH con-centrations of bimatoprost amide were lower than BFA at all time points, with a C max of 6.81 ± 1.36 nM ( n = 7) at 1 h postdose. For TFA, measurable AH concentrations were obtained at all time points with a TFA C max of 3.91 ± 2.27 nM ( n = 5), which was observed at 3 h after the dose (all data are mean ± SEM). Conclusions : Once daily topical ocular administration of LUMIGAN ® or TRAVATAN ® for 3 weeks resulted in signifi cant concentrations of BFA and TFA in the AH. Quantifi able levels of bimatoprost amide were also mea-sured. Maximum concentrations of BFA (30.9 nM) and TFA (3.91 nM) in the anterior chamber are suffi cient to fully activate the FP prostanoid receptors in the target cells of the ciliary muscle and trabecular meshwork. Both bimatoprost in LUMIGAN ® and travoprost in TRAVATAN ® are essentially prodrugs that are rapidly hydrolyzed to their respective free acids that induce the IOP-lowering effect observed with both drugs in vivo .

Introduction

Of the various therapies for reducing high intraocular pressure (IOP), topical administration of prostaglandin

(PG) analogs of the FP class has been shown to be the most

potent and effi cacious therapy in the clinical management of glaucoma and ocular hypertension. 1–8 To date, 5 FP class prostaglandin analogs (FP prostanoids) are approved for the treatment of ocular hypertension and open-angle glaucoma in the United States, Europe, Japan, and other countries.

Aqueous Humor Concentrations of Bimatoprost Free Acid, Bimatoprost and Travoprost Free Acid in Cataract Surgical

Patients Administered Multiple Topical Ocular Doses of LUMIGAN ® or TRAVATAN ®

Robert Faulkner , 1 Najam A. Sharif , 1 Susan Orr , 1 Kenneth Sall , 2 Harvey DuBiner , 3 Jess T. Whitson , 4 Marlene Moster , 5 E. Randy Craven , 6 Michael Curtis , 1 Cynthia Pailliotet , 1 Kimberly Martens , 1 and David Dahlin 1

1 Alcon Research, Ltd., Fort Worth, Texas. 2 Sall Eye Research Medical Center, Bell Flower, California. 3 Eye Care Centers Management, Inc., Clayton Eye Center, Morrow, Georgia. 4 University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. 5 Wills Eye Hospital, Philadelphia, Pennsylvania. 6 Private Practice, Glaucoma Consultants of Colorado, Littleton, Colorado.

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FAULKNER ET AL.2

to their more potent free acid metabolites by esterases, aminopeptidases, and amidopeptidases that are present in the cornea and other anterior segment tissues. 6 , 14–17,20–22 The potent free acids activate FP receptors in both the trabecular meshwork and ciliary muscle leading to altera-tions of the extracellular matrix of the ciliary body muscle fi bers resulting in increased uveoscleral outfl ow of AH. 14–19 Figure 1 presents the chemical structures of the FP class of prostaglandin analogs and their free acid metabolites.

Travoprost is an isopropyl ester of the highly selective FP receptor agonist, (+)-fl uprostenol. 8 , 18 , 23 Studies have shown that compared to the other PG analogs, the free acid of travoprost possesses the highest functional potency at the FP prostanoid receptor. 23–28 Bimatoprost is the ethyl amide of 17-phenyl-trinor prostaglandin PGF 2α , a potent FP receptor agonist. 7 , 29 , 30 Both bimatoprost amide and bimatoprost free acid (BFA) bind to the FP receptor of various cell types with the bimatoprost amide having much lower affi nity than its free acid that has similar or slightly greater affi nity as latanoprost free acid. 18–30 Both the prodrug unoprostone isopropyl and its acid metab-olites have much less affi nity of the FP receptor. 19 , 24 , 25 , 28 , 31–33 Tafl uprost, the latest of the fl uorinated prostaglandins intro-duced on the market, has high affi nity for the FP receptor as

The fi rst to the US market was latanoprost (XALATAN ® ) in 1996 followed shortly thereafter by unoprostone iso-propyl (Rescula), travoprost (TRAVATAN ® ), bimatoprost (LUMIGAN ® ), and most recently, tafl uprost (TAFLOTAN ® ). The potent IOP-lowering effi cacy of this drug class has ren-dered them to be considered a fi rst-line treatment by many eye care professionals worldwide. 1 , 4 The proposed mecha-nism of action is to lower IOP by increasing the uveoscleral outfl ow of aqueous humor (AH) although activation of con-ventional outfl ow facility is also stimulated. 9–17 The ocular hypotensive effects of PGs are dependent upon their ability to reach the site of action (iris ciliary body and trabecular meshwork). PGs are polar compounds and have a low per-meability across the lipophilic corneal epithelium. 4 , 5 , 10 In the early 1980s, the natural prostaglandins PGE 2 and PGF 2α were shown to be effective in reducing IOP but they were not commercially viable as PGE 2 was labile in aqueous solu-tion and PGF 2α required high concentrations associated with severe ocular side effects to elicit the IOP-lowering effect. 4 , 7 Structural analogs (prodrugs) of PGF 2α were developed to maximize the corneal permeability and reduce ocular side effects. 6–8 The more lipophilic ester and amide prod-rugs rapidly penetrate the cornea and then are hydrolyzed

HO

HO HO

HO HO

HO

HO

HO

HO

HO

HO

HO F F

HO HO

HO

O

HO O

HOHO

HO

HO

HO

HO

HO

HO

O

F F

HO HO

HO HO

O

O

HO

Latanoprost LatanoprostAcid

Unoprostone UnoprostoneAcid

TravoprostAcid

BimatoprostAcid

Travoprost

Bimatoprost

Tafluprost TafluprostAcid

PGF2α

CO2CH(CH3)2

CO2CH(CH3)2

CO2CH(CH3)2

CO2CH(CH3)2

C(O)NHCH2CH3

CF3

CO2H

CO2H

CO2H

CO2H

CO2H

CO2H

O

CF3

FIG. 1. Chemical structures of prostaglandins.

jop.2009.0098.indd 2 3/11/2010 11:27:21 AM

BIMATOPROST ACID IN AQUEOUS HUMOR OF PATIENTS 3

before the scheduled cataract surgery. Patients returned to the clinic after 14 days of treatment for vital signs, visual acuity, IOP, medical history, and study medication compli-ance (ie, diaries).

After at least 21 days of treatment on the morning of sur-gery, each patient was asked to report any adverse events that occurred during treatment. Preoperative medication included administration of topical anesthetic within 5 min of topical PG and cycloplegic administered at least 1 h prior to surgery and a minimum of 15 min prior to instillation of the topical PG. Surgery was planned to accommodate the randomized AH sampling time. At the designated time, a limbal paracentesis incision was made and a sample (∼100 μL) of AH was collected using a 25-gauge cannula attached to a 1-mL tuberculin syringe. The time of collection was recorded. The AH samples were immediately placed on dry ice for transport to the contract bioanalytical laboratory and stored at −70°C until analysis.

Bioanalytical analysis

Aqueous humor samples were assayed in a masked manner by an independent bioanalytical laboratory (Alta Analytical, El Dorado Hills, CA). Concentrations of bimato-prost, BFA, and travoprost free acid (TFA) were determined using validated LC-MS/MS methods. The lower limits of quantitation were 0.20 ng/mL (0.59 nM) for bimatoprost, 0.20 ng/mL (0.29 nM) for BFA, and 0.10 ng/mL (0.44 nM) for TFA.

Tetradeuterated (d4) analogs of TFA or BFA were added to samples as internal standards. The samples were then extracted with 30:70 ethyl acetate: n -hexane and the extracts were evaporated to dryness and reconstituted in high per-formance liquid chromatography (HPLC) mobile phase. TFA and internal standard were resolved on an octadecyl-silica (C18) column under isocratic conditions using 69:4:27 methanol:acetonitrile:water containing 2 mM ammonium acetate. Bimatoprost and BFA were separated on a Clipeus phenyl column under ambient temperature using a linear gradient of H 2 O:acetonitrile (90:10 v/v) containing 0.005% for-mic acid (A) and H 2 O:acetonitrile (10:90) containing 0.005% formic acid (B). Column effl uents were subjected to nega-tive ion electrospray ionization and detection was by tan-dem mass spectrometry (Turbolon Spray™, Sciex, Concord, Ontario, Canada). The working ranges of the 2 methods were from 0.1 to 100 ng/mL for TFA and from 0.200 to 100 ng/mL for bimatoprost and BFA. Accuracy of the methods ranged from 93% to 105%.

Data analysis

The mean AH concentrations of TFA, bimatoprost, and BFA were determined using data from at least 5 patients at each time point. The primary pharmacokinetic parameters were the mean aqueous and plasma concentrations at each time point, maximum mean concentration ( C max ) and time to mean C max ( t max ), and the area under the aqueous/plasma concentration–time curve from time 0 to 3 or 5 h (ie, AUC 0–5 ). The mean concentrations for all values at a given time point were used for AUC 0–5 calculations using the linear trapezoi-dal method for sparse sampling and the variability across all time points allowed estimation of the standard error of the AUC. 36 Calculations were performed using SAS, Version 9.1.

well for the EP 3 receptor. 34 , 35 Thus, tafl uprost is less selective at the FP receptor than travoprost and latanoprost.

In the current report, we provide fi ndings from 2 sepa-rate clinical trials conducted to evaluate the corneal penetra-tion and in vivo hydrolysis to their respective free acids of travoprost and bimatoprost. These studies were conducted at several clinical investigative sites in the United States and involved the collection of AH samples from patients with ocular hypertension or glaucoma awaiting cataract surgery who had received topical ocular doses of either bimatoprost ophthalmic solution 0.03% (LUMIGAN ® ) or travoprost oph-thalmic solution 0.004% (TRAVATAN ® ) once daily for 21 days prior to their cataract surgery.

Methods

In both studies, the protocol and informed consent docu-ments were approved by an Institutional Review Board/Ethics Committee (IntegReview Inc., Austin, TX) or by the center’s local Institutional Review Board. Written informed consent was obtained prior to enrollment of patients in the studies. Both studies were conducted in accord with the pol-icies outlined under the Declaration of Helsinki.

Travoprost study design

This was an open-label, pharmacokinetic trial conducted at 4 clinical sites in the United States. Thirty open-angle glau-coma and ocular hypertension patients requiring cataract surgery in at least one eye were enrolled. The patients were randomized to 1 of 4 sampling times (0.5, 1, 2, or 3 h), with a target of 6 patients per time point. All subjects received tra-voprost 0.004% (TRAVATAN ® ) once daily for at least 21 days prior to the day of surgery.

Bimatoprost study design

This was an open-label, pharmacokinetic trial conducted at 4 clinical sites in the United States. Twenty-seven open-angle glaucoma and ocular hypertension patients requir-ing cataract surgery in at least one eye were enrolled. The patients were randomized to 1 of 5 sampling times (0.5, 1, 2, 3, or 5 h), with a target of 6 patients per time point. All sub-jects received bimatoprost 0.03% (LUMIGAN ® ) once daily for at least 21 days prior to the day of surgery.

Procedures

Both studies included patients over 18 years of age sched-uled for cataract extraction in one eye with a diagnosis of ocular hypertension and/or open-angle glaucoma in both eyes (IOP ≤ 24 mmHg) on treatment. All patients achieved 20/40 or better vision on a potential acuity meter test in the eye scheduled for surgery, and at least 0.6 logMAR in the fellow eye.

A presurgery eye examination was performed including demographic information, medical history, medication his-tory, heart rate, blood pressure, visual acuity, visual fi eld exam, slit lamp exam, dilated fundus exam, gonioscopy, and tonometry. After completion of the screening visit, qualifi ed patients were randomized to receive a bottle of LUMIGAN ® or TRAVATAN ® . Patients were instructed to self-administer 1 drop in each eye beginning at least 21 days

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FAULKNER ET AL.4

Of the 33 patients samples analyzed, BFA was quantifi -able (≥0.51 nM) in all but one sample collected at 0.5 h post-dose. The individual patient BFA concentrations at each time point are presented in Figure 3 . The BFA C max was 30.9 ± 16.41 nM (mean ± SEM, n = 5), which was observed at 2 h postdose ( Table 3 ). Quantifi able BFA concentrations in AH were still observed at 5 h postdose and averaged 9.86 ± 3.52 nM (mean ± SEM, n = 7). The AUC 0–5 (SEM) estimate was 73.4 (17.7) nM⋅h.

For the parent amide, quantifi able (≥0.48 nM) AH con-centrations were obtained in samples from 5/6 patients at 0.5 h, from 7/7 at 1 h, from 6/6 patients at 2 h, from 5/7 patients at 3 h, and from 4/7 patients at 5 h postdose ( Fig. 4 ). The bimatoprost amide C max was 6.81 ± 1.36 nM (mean ± SEM, n = 7), which was observed at 1 h postdose ( Table 4 ). The AUC 0–5 estimate and (SEM) for bimatoprost amide was 16.4 (3.25) nM⋅h.

Furthermore, the chemical structure of BFA in AH was confi rmed by an independent laboratory (Alta Analytical, El Dorado Hills, CA) based on the intensity ratios of 5 select fragment ions relative to a 6th intense fragment ion (ratios ± 10%) between samples and an authentic standard.

In order to ascertain a possible relationship between the max agonist concentration ( C max ) achieved in the AH

Results

Demographic details

Demographic data for the travoprost and bimatoprost studies are presented in Table 1 .

Travoprost study

Thirty patients were enrolled and completed the study. One patient discontinued the study due to hospitaliza-tion for an adverse event assessed to be not related to drug treatment.

Sparse-sampling analysis was performed on the AH data from 24 patients with suffi cient volume for analysis. Analysis showed that 9 samples were below the assay limit of quan-titation (<0.10 ng/mL or <0.44 nM) for TFA. Measurable TFA concentrations were obtained in AH samples from 1/6 patients at 0.5 h, from 3/7 at 1 h, from 6/6 at 2 h, and from 4/5 patients at 3 h postdose ( Table 2 ). The individual patient TFA concentrations in AH are presented in Figure 2 . The TFA C max (SD) was 3.91 ± 2.27 nM (mean ± SEM, n = 5), which was observed at 3 h postdose. The AUC 0–3 and stan-dard error (SEM) estimate was 5.86 (1.10) nM⋅h.

Bimatoprost study

Thirty-six patients were enrolled with 35 completing the study. One patient discontinued for uncontrolled IOP and data for 2 patients were not reportable.

T able 2. D escriptive S tatistics for T ravoprost F ree A cid AH C oncentrations

Time (h) N TFA Conc. (nM)

Mean ± SEM (range)

0.5 6 <0.44 (<0.44–1.38)1 7 0.77 ± 0.29 (<0.44–2.37)2 6 3.12 ± 0.28 (2.08–4.19)3 5 3.91 ± 2.27 (<0.44–14.0)

Data are mean ± SEM of the concentrations of travoprost free

acid (TFA) found in the aqueous humor of 5–7 human subjects

treated topical ocularly with TRAVATAN ® as described in the

Methods section.

0

0 0.5 1 1.5

Time (h)

TF

A C

oncentr

ation (

nM

)

2 2.5 3

2

4

6

8

10

12

14

TFA

Mean

FIG. 2. Individual patient travoprost free acid (TFA) con-centrations in aqueous humor.

Time (h)

0 1 2 3 4 5

BF

A C

oncentr

ation (

nM

)

0

20

40

60

80

100

120

BFA

Mean

FIG. 3. Individual patient bimatoprost free acid (BFA) con-centrations (nM) in aqueous humor.

T able 1. D emographics of the P atients I nvolved in the T ravoprost and B imatoprost S tudies

Travoprost Study Patient Demographics Diagnosis 24 Open-angle glaucoma 6 Ocular

hypertension Age 70.5 ± 8.1 years (53–84 years) Gender 15 Male, 15 female Race 17 White, 4 Black, 3 Asian, 5

Hispanic, 1 Other Iris color 20 Brown, 6 blue, 1 gray, 3 hazelBimatoprost Study Patient Demographics Diagnosis 20 Open-angle glaucoma 15

Ocular hypertension Age 71.4 ± 8.7 years (43–86 years) Gender 12 Male, 23 female Race 17 White, 4 Black, 1 Asian, 13 Hispanic Iris color 23 Brown, 6 blue, 1 green, 5 hazel

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BIMATOPROST ACID IN AQUEOUS HUMOR OF PATIENTS 5

Discussion

For the fi ve FP class prostaglandin analogs currently approved for the reduction of IOP in patients with ocular hypertension and open-angle glaucoma, the proposed mech-anism of action is to lower IOP by activating FP receptors in both the trabecular meshwork and ciliary muscle. 10–17 , 19 , 24–26 -, 28 , 30–34 Over the past decade, in both the scientifi c literature and the ophthalmic community there are confl icting views and reports surrounding the classifi cation of bimatoprost as a prostanoid receptor agonist, despite the fact that it is an amide analog of PGF 2α . 26 , 28 , 30 , 35–40 The controversy centers around 3 questions, 2 of which have been addressed previ-ously. The fi rst question is: which entity, the parent amide compound (bimatoprost) or the free acid metabolite (BFA), is responsible for the IOP-lowering effects of the drug? This leads into the second question: which receptors are acti-vated by the active agent(s)? Given that bimatoprost and

and the agonist potency (EC 50 ) in functionally activating the FP receptors in various cells or tissue, ratios of these parameters were plotted ( Fig. 5 ). For the majority of the compounds tested in various preparations, the ratios were ≥1 ( Fig. 5 ).

T able 3. B imatoprost F ree A cid AH C oncentrations

Time (h) N BFA Conc. (nM)

Mean ± SEM (range)

0.5 5 3.19 ± 1.75 (<0.51–10.1)1 7 11.1 ± 2.04 (1.34–16.0)2 5 30.9 ± 16.41 (5.97–95.8)3 7 16.4 ± 3.63 (3.71–30.9)5 7 9.86 ± 3.52 (1.71–28.1)

Data are mean ± SEM of the concentrations of bimatoprost

free acid (BFA) found in the aqueous humor of 5–7 human subjects

treated topical ocularly with LUMIGAN ® as described in the

Methods section.

Time (h)

Bimatoprost

Mean

Bim

ato

pro

st C

oncentr

ation (

nM

)

0

0

2

4

6

8

10

12

14

16

18

1 2 3 4 5

FIG. 4. Individual patient bimatoprost amide concentra-tions (nM) in aqueous humor.

T able 4. D escriptive S tatistics for B imatoprost A mide AH C oncentrations

Time (h) N Bimatoprost Amide Conc. (nM)

Mean ± SEM (range)

0.5 5 1.72 ± 0.83 (<0.48–4.96)1 7 6.81 ± 1.36 (1.73–11.9)2 5 6.16 ± 2.78 (1.33–16.7)3 7 1.91 ± 0.56 (<0.48–4.40)5 7 1.60 ± 1.00 (<0.48–7.56)

Data are mean ± SEM of the concentrations of bimatoprost

(amide) in the aqueous humor of 5–7 human subjects treated

topical ocularly with LUMIGAN ® as described in the Methods

section.

0.1

BFA (Alcon

)

BFA (Can

tor)

LFA (C

anto

r)

LFA (C

alisse

ndor

f)

LFA (S

joqu

ist)

TFA (Alcon

)

BFA (Cam

ras)

1

10

100HCB

h-CM

h-TM

Cat Iris

Cm

ax/E

C50 R

atio

FIG. 5. Cmax/EC50 ratios for free acids of bimatoprost, latanoprost, and travoprost in human aqueous humor after topical ocular dosing with LUMIGAN®, XALATAN®, and TRAVATAN®, respectively. BFA (Alcon) and TFA (Alcon) are from current study; BFA (Camras) from Ref. 51; BFA (Cantor) and LFA (Cantor) from Ref. 50; LFA (Calissendorf) from Ref. 55; and LFA (Sjöquist) from Ref. 56. Abbreviation: HCB, human ciliary body

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FAULKNER ET AL.6

The functional FP agonist potencies of these free acid metab-olites are in the nanomolar range, whereas for bimatoprost amide, travoprost ester, latanoprost ester, unoprostone iso-propyl ester, and its free acid metabolite (UFA), FP agonist potencies were much less, being in the micromolar range.

A proposed mechanism of action that bimatoprost is the receptor agonist rather than its acid metabolite is based on reports classifying bimatoprost amide as a prostamide acting on a prostamide receptor in the cat iris and cat lung. 29 , 30 , 46–49 The uniqueness of bimatoprost was further claimed due to its potent effect to contract the feline iris sphincter with-out promoting Ca 2+ signaling or DNA synthesis in Swiss 3T3 cells. 30 , 48 , 49 Initially, reports suggested that bimatoprost amide alone activated the prostamide receptor and was responsible for clinical IOP-lowering effi cacy. 29 , 47 Further, that bimatoprost alone was the only drug moiety that could be measured in the receiver chamber from in vitro cornea permeability experiments; in ocular tissues after topical ocular instillation in primates; or in the anterior chamber (eg, AH) following topical dosing in humans. 29 , 47 However, these reports confl ict with the results from in vitro studies showing bimatoprost and BFA have signifi cant potency on various animal and human cell types linked to the FP recep-tor including Swiss 3T3 and cat iris sphincter cells as well as the fi ndings reported in this article and others showing signifi cant concentrations of BFA in the anterior chamber after topical ocular administration of bimatoprost in cata-ract patients. 28 , 35–42 , 50–54

Proponents of the theory that IOP is lowered via the direct action of bimatoprost on prostamide receptors have

BFA are both PGF 2α analogs and have been shown to be FP receptor agonists in numerous cell types (ocular and non-ocular) from several animal species and in humans, 24–30 , 37 , 38 the third question asks: are the concentrations of bimato-prost or BFA in the AH adequate to activate FP receptors and thus lower IOP?

Several in vitro functional potency and receptor-binding studies have reported the concentrations of parent compound and active metabolite required to activate FP receptors for many of these FP class prostaglandin analogs including tra-voprost, latanoprost, bimatoprost, tafl uprost, cloprostenol, fl uprostenol, unoprostone, and PGF 2α . 19 , 23–31 , 34–42 These stud-ies have utilized various cell types and functional assays including feline and canine iris sphincter smooth muscle, rat vascular smooth muscle, mouse fi broblasts, human cili-ary muscle, and human trabecular meshwork. 19 , 23–31 , 34–42 The FP receptor has been found in the human ciliary body (HCB) and trabecular meshwork and was cloned from the HCB. 43–45 Recently, functional FP receptor studies with numerous FP class PG analogs via the cloned HCB have been reported. 25–28 Taken together, the agonist potencies of the FP prostanoids and the generation of second messengers by these com-pounds are highly correlated between species substanti-ating a high degree of homology of the FP receptor. 27 , 40 As listed in Table 5 , the agonist activity of these compounds at the FP receptor in the cloned HCB, human ciliary muscle cell (h-CM), human trabecular meshwork cell (h-TM), and feline iris sphincter assays showed a consistent trend that the TFA is the most potent compound followed by the free acid metabolites of latanoprost (LFA) and bimatoprost (BFA).

T able 5. E quilibrium F unctional FP Receptor A gonist P otency V alues in C loned H uman C iliary B ody (HCB), h-CM, h-TM, and C at I ris S phincter M uscle

Compound

Agonist potency (EC 50 ; nM) in cells and tissue

HCB 25 , 39 h-CM Cells 39 h-TM Cells 39 Cat Iris 29 , 41

PGF 2α (free acid) 29 104 62 18.6 41 58 29

Fluprostenol (free acid) 4.6 4.3 11 15.8 41 —

Latanoprost (ester) 173 313 564 11.7 41 1530 29

Latanoprost free acid 45.7 124 35 29.9 41 66 29

Bimatoprost (amide) 681 9,600 3,245 140 41 34 29

Bimatoprost free acid (BFA)

3.3 3.8 26 0.99 41 —

Travoprost (ester) 40.2 123 103 ——

Travoprost free acid (TFA)

2.4 1.4 3.6 0.46 41 —

Unoprostone isopropyl (ester)

9,100 8,420 2,310 ——

Unoprostone free acid 3,220 3,503 3,306 1,280 41 —

Data are the functional agonist potencies (EC 50 ; nM) for various PG analogs for activating FP receptors in various human ocular cells

and in cat iris sphincter muscle. These values can be used to compare with the concentrations of travoprost free acid (TFA), bimatoprost

free acid (BFA), and bimatoprost (amide) found in the aqueous humor after topical ocular dosing with TRAVATAN ® and LUMIGAN ® ,

respectively, in human subjects (see Tables 2–4 ). The superscripted numbers denote reference numbers.

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BIMATOPROST ACID IN AQUEOUS HUMOR OF PATIENTS 7

of action have been presented for tafl uprost, the latest FP prostanoid introduced to the market. 34 , 35

Evidence claiming the existence of prostamide receptor in the human eye has been extrapolated from studies con-ducted on cat lung and cat iris tissues in vitro . 29 , 30 , 47–49 These tissue contraction assays utilized cumulative dose–response paradigms spanning several minutes, thus there is a strong possibility of bimatoprost hydrolysis to BFA. The conclu-sions from these studies were that bimatoprost activates prostamide receptors in the cat iris and lung, and does not activate FP receptors in the same tissue. 29 , 30 , 49 However, the same authors reported that latanoprost and BFA fully acti-vated the FP receptors in the cat iris where [ 3 H]-BFA binding to FP receptors was also demonstrated, thus complicating and contradicting the latter conclusions. 29 , 30 , 49 Given that the existence of the prostamide receptor in the eye is based mostly on studies conducted in feline tissues, and that there now exist substantial FP receptor data in human tis-sues/cells, the more applicable human tissue results would appear to supercede the less relevant cat iris and cat lung study results. Nevertheless, a recent study comparing all the prostanoid prodrugs and their free acids in the cat iris sphincter contraction assay showed sub-nanomolar potency of BFA (0.99 nM) and TFA (0.46 nM). 41 In the cat iris sphinc-ter assay, the potency of LFA was reported to be 29.9–66 nM, which are above the observed AH levels reported in the literature. 56 As there is no debate that LFA is a FP pros-tanoid, the same argument for BFA for a separate receptor could be presented for LFA since the human AH levels are lower than the cat iris EC 50 values. These observations put to test the relevance of cat iris sphincter tissues for comparing the activity of these synthetic FP analogs and that the more relevant comparisons utilize human tissues. Furthermore, in the recent study, 41 the high potency of bimatoprost pre-viously reported in the cat iris sphincter muscle could not be confi rmed. 29 , 30 , 48 , 49 As such, this brings into question the earlier less extensive pharmacology studies of the PG recep-tor in the cat iris sphincter muscle and the validity of the presumed existence of a prostamide receptor. 29 , 30 , 48 , 49

More compelling evidence that bimatoprost is a FP recep-tor agonist comes from recent studies using transgenic mice bred to be devoid of the FP receptor (FP receptor knockout) treated with bimatoprost, latanoprost, travoprost, tafl uprost, or unoprostone. 68–71 Topical instillation of each of the 4 com-pounds signifi cantly lowered IOP in wild-type mice with the FP receptor but had no effect in the FP receptor knockout (FPKO) mice. 66–69 If there was a distinct prostamide recep-tor for bimatoprost, an IOP effect would have been seen in the knockout mice. Further, the same results were obtained in other transgenic knockout mouse studies with unopros-tone isopropyl casting doubt on the claimed existence of a different receptor for unoprostone. Of interest, in a recent knockout mouse studies, tafl uprost was shown to be less selective for the FP receptor than latanoprost, travoprost, bimatoprost, or unoprostone, as IOP reduction it was devoid of activity in FPKO and EP3KO mice. 71 Because of the rela-tively high affi nity of tafl uprost to prostanoid EP 3 receptors, it has been proposed that tafl uprost lowers IOP through the prostanoid FP receptor and that part of the IOP reduction may be through the EP3 receptor. 34 , 71

In terms of the signifi cance of the present fi ndings, the following discourse is relevant and important. Clearly, BFA

recently disputed the signifi cance of the levels of BFA in the AH studies reported by Cantor and coauthors. 50 and Camras and coauthors 51 as being insuffi cient alone to lower IOP. However, as previously mentioned, a review of the in vitro work published to date consistently demonstrates that BFA readily activates FP prostanoid receptors in ocular cells at low nanomolar concentrations (EC 50 = 0.99–26 nM) depending on the cells/tissues studied ( Table 5 ). 39 These concentrations are achieved in the AH after topical ocular dosing with bimatoprost amide (LUMIGAN ® ) in this study and others. 46 , 50–54 Comparison of the AH concentrations for TFA, BFA, and LFA to the functional FP agonist potencies reported in the literature shows that the AH concentrations achieved for all 3 compounds are above their respective EC 50 values, concentrations required to fully activate 50% of the total population of FP receptors. 25 , 29 , 37 , 39 , 50 , 51 , 54–56 For all 3 acids, the AH C max /EC 50 ratios are at or above 1.0. The similarity in these C max /EC 50 ratios is in-line with the comparable clinical IOP-lowering effi cacy of the 3 ophthalmic products reported in comparative clinical trials. 57–60 As the proposed mecha-nism of action for these compounds is to alter uveoscleral outfl ow through changes in the extracellular matrix of the ciliary muscle, the more relevant target tissue is the iris root and ciliary muscle. Ocular tissue distribution studies in the rabbit revealed that iris/ciliary body concentrations of the free acids are up to 10-fold higher than those in AH. 29 , 61 As the bimatoprost study compared total radioactivity concen-trations, the fraction of radioactivity in the ICB and AH rep-resenting either bimatoprost amide or BFA is not known. 61 From these cited results, the C max /EC 50 ratios presented in Figure 5 may be an underestimation of the C max /EC 50 values in the iris ciliary body.

The hydrolysis of bimatoprost to BFA by ocular tis-sues including the cornea, sclera, ciliary body, and iris has already been well demonstrated in vitro using ocular tis-sues of numerous species including bovine, porcine, rabbit, monkey, and human. 28 , 37–42 , 52 , 54 Although these studies dem-onstrate that the extent of hydrolysis to their free acids are somewhat greater for the ester analogs than for the amide analog, there was suffi cient BFA liberated by hydrolysis of bimatoprost amide to activate a suffi cient number of FP receptors. The lower conversion rate of amide to free acid and the lower agonist potency of the free acid explain why that the concentration of the commercial topical formula-tion of bimatoprost is much higher (0.03% LUMIGAN ® ) than for the ester analog formulations for latanoprost (0.005%, XALATAN ® ), tafl uprost (0.0015%, TAFLUTAN ® ), and travo-prost (0.004%; TRAVATAN ® ). The same can be said for uno-prostone isopropyl (Rescula) as this FP prostanoid has a low agonist potency and is rapidly and completely converted to a carboxylic acid metabolite, responsible for the major-ity of the pharmacological activity. 32 , 62 Even with a higher concentration in the ophthalmic formulation (0.12%) and employing a twice-daily regimen, the clinical IOP effi cacy of unoprostone is markedly less than the other prostaglandins on the market. 63–67 It is of interest that there were claims in the literature that the much lower potency of unoprostone vs. latanoprost and the other prostanoids was due to its dif-ferent structure (docosanoid) and possible interaction with a distinct receptor. 62 Subsequent citations now present uno-prostone as another prostaglandin analog acting on the FP receptor. 66 In contrast, no alternate claims on the mechanism

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FAULKNER ET AL.8

5. Madhu , C ., Rix , P ., Nguyen , T ., et al . Penetration of natural pros-

taglandins and their ester prodrugs and analogs across human

ocular tissues in vitro . J. Ocul. Pharmacol. Ther . 14 : 389 – 399 , 1998 .

6. Stjernschantz , J ., Selen , G ., Sjöquist , B ., et al . Preclinical

pharmacology of latanoprost, a phenyl-substituted PGF 2α

analogue . Adv. Prostaglandin. Thrombox. Leukotr. Res . 23 :

513 – 518 , 1995 .

7. Woodward , D ., Krauss , A ., and Chen , J . Replacement of the car-

boxylic acid group of prostaglandin PGF 2α with a hydroxyl or

methoxy substituent provides biologically unique compounds .

Br. J. Pharmacol . 130 : 1933 – 1943 , 2000 .

8. Whitson , J.T . Travoprost—a new prostaglandin analogue for the

treatment of glaucoma . Expert. Opin. Pharmacother . 3 : 965 – 977 ,

2002 .

9. Lim , K ., Nau , C ., O’Byrne , M ., et al . Mechanism of action of

bimatoprost, latanoprost and travoprost in healthy subjects .

Ophthalmology . 115 : 790 – 795 , 2008 .

10. Ocklind , A . Effect of latanoprost on the extracellular matrix

of the ciliary muscle. A study on cultured cells and tissue sec-

tions . Exp. Eye Res . 67 : 179 – 191 , 1998 .

11. Schachtschabel U ., Lindsey , J ., and Weinreb , R.N . The mecha-

nism of action of prostaglandins on uveoscleral outfl ow .

Glaucoma . 11 : 112 – 115 , 2000 .

12. Kashiwagi , J.M ., Suzuki , M ., Tanaka , Y ., et al . Isopropyl uno-

prostone increases the activities of matrix metalloproteinases

in cultured monkey ciliary muscle cells . J. Glaucoma . 10 : 271 – 276 ,

2001 .

13. Gabelt , B ., and Kaufman , P . Prostaglandin F2 alpha increases

uveoscleral outfl ow in the cynomologus monkey . Exp. Eye Res . 49 : 389 – 402 , 1989 .

14. Nilsson , S . The uveoscleral outfl ow routes . Eye . 11 : 149 – 154 ,

1997 .

15. Weinreb , R ., Toris , C ., Gabelt , B ., et al . Effects of prostaglandins

on the aqueous humor outfl ow pathways . Surv. Ophthalmol . 47 ( Suppl 1 ): S53 – S64 , 2002 .

16. Sagara , T ., Gaton , D ., Lindsey , J ., et al . Topical Prostaglandin F2

alpha treatment reduces collagen types I, III and IV in the mon-

key uveoscleral outfl ow pathway . Arch Ophthalmol . 117 : 794 – 801 .

17. Toris , C.B ., Camras , C ., Yablonski , M ., et al . Effects of exog-

enous prostaglandins on aqueous humor dynamics and blood-

aqueous barrier function . Surv. Ophthalmol . 41 ( Suppl 2 ): S69 – S75 ,

1997 .

18. Hellberg , M.R ., McLaughlin , M.A ., Sharif , N.A ., et al .

Identifi cation and characterization of the ocular hypotensive

effi cacy of travoprost, a potent and selective FP prostaglandin

receptor agonist, and AL-6598, a DP prostaglandin receptor

agonist . Surv. Ophthalmol . 47 ( Suppl #1 ): S13 – S33 , 2002 .

19. Sharif , N.A ., Kelly , C.R ., and Crider , J.Y . Human trabecular

meshwork cell responses induced by bimatoprost, travoprost,

unoprostone and other FP prostaglandin receptor agonist ana-

logues . Invest. Ophthalmol. Vis. Sci . 44 : 715 – 721 , 2003 .

20. Shen , Y.W ., and Tao , R.V . The presence of ceramide hydrolase

and synthethase in pig lens epithelium . Invest. Ophthalmol. Vis. Res . 22 : 734 – 743 , 1982 .

21. Matsuda , S ., Kanemitsu , N ., Nakamura , A ., et al . Metabolism

of anandamide, an endogenous cannabinoid receptor ligand, in

porcine ocular tissues . Exp. Eye Res . 64 : 707 – 711 , 1997 .

22. Startford , R.E ., Jr ., and Lee , V.H . Ocular aminopeptidase activ-

ity and distribution in the albino rabbit . Curr. Eye Res . 4 : 995 – 999 ,

1985 .

23. Sharif , N.A ., Davis , T.L ., and Williams , G.W . [ 3 H]AL-5848 (9-β-[+]

fl uprostenol): carboxylic acid of Travoprost (AL-6221), a novel

FP-prostaglandin to study the pharmacology and autoradio-

graphic localization of the FP receptor . J. Pharmacy Pharmacol . 51 : 685 – 594 , 1999 .

24. Sharif , N.A ., Crider , J.Y ., Husain , S ., et al . Human ciliary muscle

responses to FP-class prostaglandin analogs: phosphoinositide

hydrolysis, intracellular Ca2+ mobilization and MAP kinase

activation . J. Ocul. Pharmacol. Ther . 19 : 437 – 455 , 2003 .

was found at nanomolar concentrations in the AH of 27 cataract patients administered LUMIGAN ® . The observed maximal BFA AH concentration of 30.9 ± 16.4 nM is many-fold greater than necessary to fully occupy and activate FP receptors in the cat iris (half-max occupancy at 0.99 nM) or in the HCB (half-max occupancy at 5.8 nM) and promote IOP lowering. In support of this conclusion, it has been shown that the concentrations of BFA required to activate 50% of FP receptors in various tissues and cells were as follows: 0.7 nM in cat iris 6 , 41 ; 3.8 ± 0.9 nM in human ciliary muscle cells from several donors 39 ; 26 ± 10 nM in human trabecular meshwork cells from several donors 39 ; 3.3 ± 0.7 nM in HCB cloned human FP receptor cells 39 ; 2.8 ± 0.2 nM in mouse fi broblasts 39 ; and 2.8 ± 0.6 nM in rat vascular smooth mus-cle cells. 39 These results illustrate that the concentrations of BFA found in the human AH after LUMIGAN ® administra-tion are more than suffi cient to stimulate FP receptors and cause the IOP-lowering akin to that produced by travoprost and latanoprost. 6 , 19 In contrast, since the AH concentration of the bimatoprost amide was rather low in the current studies (<11 nM maximum at 1 h post-dosing; Table 3 ), it is unlikely that bimatoprost amide itself would have suffi cient activity by itself to cause the ocular hypotension, especially since the potencies in the cat iris (EC 50 = 0.14 μM) 41 and vari-ous cell types (EC 50 range from 0.68 to 9.6 μM; Table 5 ) are up to 1,000-fold less. At the concentrations of bimatoprost detected in the AH very little activation of the postulated prostamide receptors would be possible. Therefore, the only plausible explanation for the ocular hypotensive activity associated with bimatoprost is the hydrolysis to BFA by the ocular tissues and the activation of the FP receptors by the resultant BFA. 72

Conclusions

In summary, the majority of published evidence indicates that bimatoprost amide is a PG prodrug that liberates the FP agonist BFA in the AH as it is hydrolyzed by the cornea and other ocular tissues upon topical ocular administration, and thus bimatoprost acid is the active moiety acting on FP receptors to cause the IOP lowering.

Author Disclosure Statement

Authors who are not Alcon Research Ltd. employees have no proprietary interest in any of the materials used in this study.

References

1. Sharif, N.A., Klimko, P. CNS: Ophthalmic Agents, in

Comprehensive Medicinal Chemistry II., 2007; Vol.6, Chapter 12,

p297-320 (Eds: J.B. Taylor & D.J. Triggle), Elsevier, Oxford, UK

2. Martínez-Belló , C ., Chauhan , B.C ., Nicolela , M.T ., et al .

Intraocular pressure and progression of glaucomatous visual

fi eld loss . Am. J. Ophthalmol . 129 : 302 – 308 , 2000 .

3. Camras , C.B. Mechanism of the prostaglandin-induced reduc-

tion of intraocular pressure in humans . Adv. Prostaglandin Thromboxane Leukot. Res . 23 : 519 – 525 , 1995 .

4. Bito , L.Z ., and Baroody , R.A . The ocular pharmacokinetics of

eicosanoids and their derivatives. 1. Comparison of ocular

eicosanoid penetration and distribution following the topical

application of PGF2 alpha, PGF2 alpha-1-methyl ester, and PFG2

alpha-1-isopropyl ester . Exp. Eye Res . 44 : 217 – 226 , 1987 .

jop.2009.0098.indd 8 3/11/2010 11:27:23 AM

BIMATOPROST ACID IN AQUEOUS HUMOR OF PATIENTS 9

PGF 2α by human and rabbit ocular tissues . J. Ocul. Pharmacol. Ther . 19 : 97 – 103 , 2003 .

43. Abramovitz , M ., Boie , Y ., Nguyen , T ., et al . Cloning and expres-

sion of a cDNA for the human prostanoid FP receptor . J. Biol. Chem . 269 : 2632 – 2626 , 1994 .

44. Kunapuli , P ., Lawson , J ., Rokach , J ., et al . Functional character-

ization of the ocular prostaglandin F2alpha (PGF 2α ) receptor . J. Biol. Chem . 272 : 27147 – 27154 , 1997 .

45. Davis , T.L ., and Sharif , N.A . Quantitative autoradiographic

visualization and pharmacology of FP-prostaglandin receptors

in human eyes using the novel phosphor-imaging technology .

J. Ocul. Pharmacol. Ther . 15 : 323 – 336 , 1999 .

46. Cantor , L . Clinical pharmacology of bimatoprost . Expert Opin. Drug Metab. Toxicol . 1 : 151 – 157 , 2005 .

47. Cantor , L . Bimatoprost: a member of a new class of agents, the

prostamides, for glaucoma management . Exp. Opin. Invest. Drugs . 10 : 721 – 731 , 2001 .

48. Spada , C.S ., Krauss , A.H ., Woodward , D.F ., et al . Bimatoprost

and prostaglandin F2α selectively stimulate intracellular cal-

cium signaling in different cat iris sphincter cells . Exp. Eye Res . 80 : 135 – 145 , 2005 .

49. Krauss , A.H.P ., and Woodward , D.F . Update on the mechanism

of action of bimatoprost (Lumigan®): discussion of new evi-

dence . Surv. Ophthalmol . 49 :( Suppl. 1 ): S5 – S11 , 2004 .

50. Cantor , L.B ., Hoop , J ., Wudunn , D ., et al . Levels of bimatoprost

acid in the aqueous humor after bimatoprost treatment of

patients with cataracts . Br. J. Ophthalmol . 91 : 629 – 632 , 2007 .

51. Camras , C.B ., Toris , C.B ., Sjöquist , B ., et al . Detection of the

free acid of bimatoprost in aqueous humor samples from

human eyes treated with bimatoprost before cataract surgery .

Ophthalmol . 111 : 2193 – 2198 , 2004 .

52. Davies , S.S ., Ju , W-K ., Neufeld , A.H ., et al . Hydrolysis of bimato-

prost (Lumigan) to its free acid by ocular tissue in vitro . J. Ocul. Pharmacol. Ther . 19 : 45 – 54 , 2003 .

53. Dahlin , D ., Craven , E.R ., Moster , M ., et al . Human aqueous

humor concentrations of bimatoprost and bimatoprost free

acid following topical ocular dosing of Lumigan ([Bimatoprost;

17-phenyl-trinor PGF2α ethyl amide] 0.03% ophthalmic solu-

tion) . Assoc. Res. Vis. Ophthalmol. (ARVO) Abst. # 2096, 2004 .

54. Sharif , N.A ., Ke , T-L ., Haggard , K ., et al . Bimatoprost hydroly-

sis to 17-phenyl-trinor PGF 2α by human and rabbit ocular tis-

sues and agonist activity of bimatoprost and 17-phenyl-trinor

PGF2α . Assoc. Res. Vis. Ophthalmol . Abst. #4080, 2002 .

55. Calissendorff , B ., Sjöquist , B ., Högberg , G ., et al . Bioavailability

in the human eye of a fi xed combination of latanoprost and

timolol compared to monotherapy . J. Ocul. Pharmacol. Ther . 18 : 127 – 131 , 2002 .

56. Sjöquist , B ., and Stjernschantz , J . Ocular and systemic pharma-

cokinetic of latanoprost in humans . Surv. Ophthalmol . 47 ( Suppl

1 ): S6 – S12 , 2002 .

57. Lafuma , D ., Khoshnood , B ., Mimaud , V ., et al . A meta-analysis

of topical prostaglandin analogues intraocular pressure lower-

ing in glaucoma therapy . Curr. Med. Res. Opin . 23 : 601 – 608 , 2007 .

58. Parrish , R ., Palmberg , P ., and Sheu , W-P . A comparison of latano-

prost, bimatoprost and travoprost in patients with elevated

intraocular pressure: a 12-week, randomized, masked-evalua-

tor multicenter study . Am. J. Ophthalmol . 125 : 688 – 703 , 2003 .

59. Yildirim , N ., Sahin , A ., and Gultekin , S . The effect of latano-

prost, bimatoprost, and travoprost on circadian variation of

intraocular pressure in patients with open-angle glaucoma . J. Glaucoma . 17 : 36 – 39 , 2008 .

60. Maul , E ., Carrasco , F ., Costa , V ., et al . A 6-week, multicenter,

randomized, double-masked, parallel-group study comparing

travoprost 0.004% to latanoprost 0.005% followed by 6-week,

open-label treatment with travoprost 0.004% . Clin. Ther . 29 : 1915 –

1923 , 2007 .

61. Sjöquist , B ., Basu , S ., Byding , P ., et al . The pharmacokinetics of a

new antiglaucoma drug, latanoprost, in the rabbit . Drug Metab. Dispos . 26 : 745 – 754 , 1998 .

25. Sharif , N.A ., Kelly , C.R ., and Crider , J.Y . Agonist activity of

bimatoprost, travoprost, latanoprost, unoprostone isopropyl

ester and other prostaglandin analogs at the cloned human

ciliary body FP prostaglandin receptor . J. Ocul. Pharmacol. Ther . 18 : 313 – 324 , 2002 .

26. Sharif , N.A ., Kelly , C.R ., and Williams , G.W . Bimatoprost

(Lumigan®) is an agonist at the cloned human ocular FP pros-

taglandin receptor: real-time FLIPR-based intracellular Ca 2+

mobilization studies . Prost. Leukotr. Essent. Fatty Acids 68 : 27 – 33 ,

2003 .

27. Kelly , C.R ., Williams , G.W ., and Sharif , N.A . Real-time intracel-

lular Ca 2+ -mobilization by travoprost acid, bimatoprost, uno-

prostone and other analogs via endogenous mouse, rat and

cloned human FP prostaglandin receptors . J. Pharmacol. Expt. Ther . 304 : 238 – 245 , 2003 .

28. Sharif , N.A ., Williams , G.W ., and Kelly , C.R . Bimatoprost and

its free acid are prostaglandin FP receptor agonists . Eur. J. Pharmacol . 432 : 211 – 213 , 2001 .

29. Woodward , D.F ., Krauss , A.H.-P ., Chen , J ., et al . The pharmacol-

ogy of bimatoprost (Lumigan ™ ) . Surv. Ophthalmol . 45 ( Suppl. 1 ):

S337 – S345 , 2001 .

30. Woodward , D.F ., Krauss , A.H.-P ., Chen , J ., et al . Pharmacological

characterization of a novel antiglaucoma agent, bimatoprost

(AGN 192024) . J. Pharmacol. Exp. Ther . 305 : 772 – 785 , 2003 .

31. Shimura , M ., Yasuda , K ., Nakzawa , T ., et al . The effect of uno-

prostone isopropyl on Ca2+ release-activated Ca2+ current in

cultured monkey trabecular meshwork cells and ciliary muscle

cells . J. Ocul. Pharmacol. Ther . 22 : 219 – 226 , 2006 .

32. Kashiwagi , K ., Iizuka , Y ., and Tasukahara , S . Metabolites of iso-

propyl unoprostone as potential ophthalmic solutions to reduce

intraocular pressure in pigmented rabbits . Jpn. J. Pharmacol . 81 : 56 – 62 , 1999 .

33. Taniguchi , T ., Haque , M.S.R ., Sugiyama , K ., et al . Ocular

hypotensive mechanism of topical isopropyl unoprostone, a

novel prostaglandin metabolite-related drug, in rabbit . J. Ocul. Pharmacol . 12 : 489 – 498 , 1996 .

34. Takagi , Y ., Nakajima , T ., Shimazaki , A ., et al . Pharmacological

characteristics of AFP-168 (tafl uprost), a new prostanoid FP

receptor agonist, as an ocular hypotensive drug . Exp. Eye Res . 78 : 767 – 776 , 2004 .

35. Nakajima , T ., Matsugi , T ., Goto , W ., et al . New fl uoroprosta-

glandin F2 derivatives with prostanoid FP-receptor agonistic

activity as potent ocular-hypotensive agents . Biol. Pharm. Bull . 26 : 1691 – 1695 , 2003 .

36. Nedelman , J.R ., Gibiansky , E ., and Tau , D.T.W . Applying bail-

er’s method for AUC confi dence intervals to sparse sampling .

Pharm. Res . 12 : 124 – 128 , 1995 .

37. Kriatchko , A ., Zhan , G ., Cheruvu , N ., et al . In vitro hydrolysis of

bimatoprost in bovine cornea . Assoc. Res. Vis. Res . Abst. # 4422,

2003 .

38. Maxey , K.M ., Johnson , J.L ., and LaBreque , J . The hydroly-

sis of bimatoprost in corneal tissue generates a potent pros-

tanoid FP receptor agonist . Surv. Ophthalmol . 47 ( Suppl 1 ):

S34 – S40 , 2002 .

39. Sharif , N.A ., Kelly , C.R ., Crider , J.Y ., et al . Ocular hypotensive FP

prostaglandin (PG) analogs: PG receptor subtype binding affi n-

ities and selectivities, and agonist potencies at FP and other PG

receptors in cultured cells . J. Ocul. Pharmacol. Ther . 19 : 501 – 515 ,

2004 .

40. Bhattacherjee , P ., Smithson , M ., and Paterson , C.A . Generation

of second messengers by prostanoids in the iris-sphincter and

ciliary muscle of cows, cats and humans . Prostagland. Leukot. Essent. Fatty Acids . 56 : 443 – 449 , 1997 .

41. Sharif , N.A ., Kaddour-Djebbar , I ., & Abdel-Latif , A.A . Cat iris

sphincter smooth muscle contraction: comparison of FP-class

prostaglandin analog agonist activities . J. Ocul. Pharmacol. Ther . 24 : 152 – 163 , 2008 .

42. Hellberg , M.R ., Ke , T.-L ., Haggard , K ., et al . The hydrolysis of the

prostaglandin analog prodrug bimatoprost to 17-phenyl-trinor

jop.2009.0098.indd 9 3/11/2010 11:27:23 AM

FAULKNER ET AL.10

receptor knockout mice . Invest. Ophthalmol. Vis. Sci . 46 :

4571 – 4577 , 2005 .

69. Ota , T ., Aihara , M ., Narumiya , S ., et al . The effects of prostaglan-

din analogues on IOP in prostanoid FP-receptor-defi cient mice .

Invest. Ophthalmol. Vis. Sci . 46 : 4159 – 4163 , 2005 .

70. Crowston , J.G ., Lindsey , J.D ., Aihara , M ., et al . Effect of latano-

prost on intraocular pressure in mice lacking the prostaglan-

din FP receptor . Invest. Ophthalmol. Vis. Sci . 45 : 3555 – 3559 , 2004 .

71. Ota , T ., Aihara , M ., Saeki , T ., et al . The IOP-lowering effects and

mechanism of action of tafl uprost in prostanoid-receptor defi -

cient mice . Br. J. Ophthalmol . 91 : 673 – 676 , 2007 .

72. Sharif , N.A ., and Klimko , P . Update and commentary on the

prodrug bimatoprost and a putative “prostamide receptor.”

Expert Rev. Ophthalmol . 4 : 477 – 489 , 2009 .

62. Shimura , M ., Yasuda , K ., Nakzawa , T ., et al . The effect of uno-

prostone isopropyl on Ca 2+ release-activated Ca 2+ current in

cultured monkey trabecular meshwork cells and ciliary muscle

cells . J. Ocul. Pharmacol. Ther . 22 : 219 – 226 , 2006 .

63. Taniguchi , T ., Haque , M.S.R ., Sugiyama , K ., et al . Ocular

hypotensive mechanism of topical isopropyl unoprostone, a

novel prostaglandin metabolite-related drug, in rabbit . J. Ocul. Pharmacol . 12 : 489 – 498 , 1996 .

64. Sponsel , W ., Gianmarco , P ., Trigo , Y ., et al . Comparative effects

of latanoprost (Xalatan) and unoprostone (Rescula) in patients

with open-angle glaucoma and suspected glaucoma . Am. J. Ophthalmol . 234 : 552 – 559 , 2002 .

65. Susanna , R ., Giampani , J ., Borges , A ., et al . A double-masked,

randomized, clinical trial comparing latanoprost with unopro-

stone in patients with open-angle glaucoma or ocular hyperten-

sion . Ophthalmology . 108 : 259 – 263 , 2001 .

66. Tsukamoto , H ., Mishima , H ., Kitazawa , Y ., et al . A compara-

tive clinical study of latanoprost and isopropyl unoprostone in

Japanese patients with primary open-angle glaucoma and ocu-

lar hypertension . J. Glaucoma . 11 : 497 – 501 , 2002 .

67. Susanna , R ., Chew , P ., and Kitazawa , Y . Current status of prosta-

glandin therapy; latanoprost and unoprostone . Surv. Ophthalmol . 47 ( Suppl 1 ): S97 – S104 , 2002 .

68. Crowston , J.G ., Lindsey , J.D ., Morris , C.A ., et al . Effect of

bimatoprost on intraocular pressure in prostaglandin FP

Address correspondence to: Dr. Naj Sharif

Core Pharmacology & Imaging Alcon Research Ltd.

6201 S. Freeway Fort Worth, TX 76134

E-mail : naj.sharif@alconlabs.com

Received: August 31, 2009Accepted: December 17, 2009

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