Experimental Eye Research 78 (2004) 767–776
`
`www.elsevier.com/locate/yexer
`
`Pharmacological characteristics of AFP-168 (tafluprost), a new prostanoid
`FP receptor agonist, as an ocular hypotensive drug
`
`Yasutaka Takagia, Tadashi Nakajimaa, Atsushi Shimazakia, Masaaki Kageyamaa,
`Takeshi Matsugia, Yasushi Matsumurab, B’Ann T. Gabeltc, Paul L. Kaufmanc, Hideaki Haraa,*
`
`aGlaucoma Group, Research and Development Center, Santen Pharmaceutical Co., Ltd, 8916-16, Takayama-cho, Ikoma 630-0101, Nara, Japan
`bFine Chemicals Group, Chemicals Company, Asahi Glass Co., Ltd, Tokyo, Japan
`cDepartment of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA
`
`Received 9 July 2003; accepted in revised form 16 December 2003
`
`Abstract
`
`To evaluate the pharmacological characteristics of AFP-168 (tafluprost), a new prostaglandin (PG) F2a derivative, we examined its
`receptor-binding affinities, intraocular pressure (IOP)-lowering effect, effects on aqueous humor dynamics, and stimulating effect on
`melanogenesis. The receptor-binding profile for AFP-172, a carboxylic acid of AFP-168, was determined by measuring muscle contractions
`in an organ bath, inhibition of platelet aggregation, and competitive binding of a radio-labelled ligand. For the IOP-measurement study,
`ocular normotensive and laser-induced ocular hypertensive cynomolgus monkeys were used, and IOP was measured using a
`pneumatonograph. For the studies of aqueous humor dynamics, IOP (Goldmann applanation tonometry), fluorophotometry, two-level
`constant pressure perfusion, and isotope dilution and accumulation techniques were used in ocular normotensive monkeys. The melanin
`contents in the medium and in the cell bodies of cultured B16-F0 melanoma cells were measured. The affinity for the FP receptor shown by
`AFP-172 (Ki: 0·4 nM) was 12 times that of PhXA85 (Ki: 4·7 nM), a carboxylic acid of latanoprost. A single application of AFP-168 at
`0·0025% significantly lowered IOP in both ocular normotensive and hypertensive monkeys (3·1 and 11·8 mmHg, respectively, p , 0·01) and
`latanoprost at 0·005% significantly lowered IOP (2·1 mmHg, p , 0·01 and 9·5 mmHg, p ¼ 0·059; respectively). Once daily instillation of
`AFP-168 at 0·001, 0·0025, or 0·005% for 5 days in normotensive monkeys significantly reduced IOP not only for a few hours, but also at the
`drug-trough time 24 hr after application. Latanoprost at 0·005% also reduced IOP, but not at the drug-trough time. AFP-168 decreased IOP
`mainly by increasing uveoscleral outflow by 65% ðp , 0·05Þ and, as sometimes seen with other prostanoids, also increased total outflow
`facility (33% increase, p , 0·05). In cultured B16-F0 melanoma cells, AFP-172 (100 mM) did not stimulate melanogenesis, but PhXA85
`(100 mM) did. These findings indicate that AFP-168 has a high affinity for the prostanoid FP receptor, has potent IOP-lowering effects in both
`ocular normotensive and hypertensive monkeys that exceed those of latanoprost, and has less stimulating effect on melanogenesis in
`melanoma cells.
`q 2004 Elsevier Ltd. All rights reserved.
`
`Keywords: AFP-168; tafluprost; prostaglandin derivatives; FP agonist; receptor binding; intraocular pressure; aqueous humor dynamics; melanogenesis;
`cynomolgus monkey
`
`1. Introduction
`
`AFP-168 (tafluprost), 1-methylethyl (5Z)-7-[(1R,2R,
`3R,5S)-2-[(1E)-3,3-difluoro-4-phenoxy -1-butenyl]-3,5-
`dihydroxycyclopentyl]-5-heptenoate and AFP-172, a car-
`boxylic acid of AFP-168, are newly synthesized
`
`* Corresponding author. Dr Hideaki Hara, Glaucoma Group, Research
`and Development Center, Santen Pharmaceutical Co., Ltd, 8916-16,
`Takayama-cho, Ikoma 630-0101, Nara, Japan.
`E-mail address: hideaki.hara@santen.co.jp (H. Hara).
`
`0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
`DOI:10.1016/j.exer.2003.12.007
`
`prostaglandin (PG) F2a analogues (Fig. 1). AFP-168 is
`under development as an ocular hypotensive drug in the
`USA, Europe, and Japan. It is a pro-drug ester that facilitates
`corneal penetration and allows delivery of the active
`carboxylic acid form (AFP-172) to the aqueous humor, a
`similar situation to that seen with PGF2a-isopropyl ester and
`other ocular hypotensive derivatives of PGF2a, such as
`latanoprost.
`Glaucoma is a major cause of blindness worldwide, and
`many ocular hypotensive drugs are in existence. For over 20
`years, b-blockers such as timolol were the first-line therapy.
`
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`Y. Takagi et al. / Experimental Eye Research 78 (2004) 767–776
`
`unoprostone isopropyl, and PGF2a-isopropyl ester were
`purchased from Cayman Chemical Co. (Ann Arbor, MI,
`USA). Latanoprost ophthalmic solution (Xalatanw) was
`purchased from Pharmacia K. K. (Tokyo, Japan), pilocar-
`pine hydrochloride from Sigma-Aldrich Corp. (St Louis,
`MO, USA), and [3H] PGF2a (200 mCi/ml) from Amersham
`Biosciences Corp. (Piscataway, NJ, USA). [125I] and [131I]
`were obtained from PerkinElmer Life Sciences,
`Inc.
`(Boston, MA, USA). Timolol-gel forming ophthalmic
`solution (Timoptol-XEw) was a product of Santen Pharma-
`ceutical Co. Ltd (Osaka, Japan). PhXA85 and unoprostone
`(carboxylic acids of latanoprost and unoprostone isopropyl,
`respectively) were prepared at the laboratories of Santen
`Pharmaceutical Co. Ltd. Because latanoprost, unoprostone
`isopropyl, and AFP-168 are pro-drug esters, and each is
`converted to its respective acid by the esterase in the cornea,
`the carboxylic acid forms were used in vitro, while the ester
`forms were used in vivo.
`
`2.2. Receptor-binding assays
`
`2.2.1. Prostanoid FP receptor
`For the prostanoid FP receptor-binding study, we used a
`clonal cell line that stably expresses the recombinant human
`prostanoid FP receptor (Abramovitz et al., 1994). The
`radioligand-binding assay was performed according to the
`methods of Abramovitz et al. (1994) and Graves et al. (1995).
`In this conventional filtration assay, membranes expressing
`the receptor are incubated with a fixed concentration of the
`radioligand and increasing concentrations of the test
`compounds. After reaching equilibrium, the radioligand
`bound to the membranes (bound) is separated from the
`unbound (free) radioligand by filtration through glass-fiber
`filters. The amount of bound radioactivity is determined by
`liquid scintillation spectrometry (LS5000TD; Beckman
`Coulter Inc., Fullerton, CA, USA), and the amount of
`bound radioactivity is plotted as a function of the logarithm
`of the concentration of the competing test compound.
`Increasing concentrations of the competing test compound
`decrease the binding of the radioligand according to the laws
`of mass action, and the affinity (IC50) of the test compound
`for the receptor can be determined by non-linear regression
`analysis according to
`B ¼ non-specific binding
`þ ðtotal binding 2 non-specific bindingÞ
`1 þ 10log½CŠ2logðIC50Þ
`
`where B equals the amount of radioligand-binding at a given
`concentration of test compound [C], total binding equals the
`amount of radioligand-binding in the absence of test
`compound, and non-specific binding equals the residual
`amount of radioligand-binding after complete displacement
`of specific binding by a saturating concentration of the test
`compound.
`
`Fig. 1. Chemical structures of AFP-168 and AFP-172.
`
`Then, after the discovery of the intraocular pressure (IOP)-
`lowering effects of PGs (Camras and Bito, 1981), several
`PG-derivatives such as isopropyl unoprostone (Taniguchi
`et al., 1996), latanoprost (Stjernschantz et al., 1995; Camras
`et al., 1996), bimatoprost (Woodward et al., 2001), and
`travoprost (Sharif et al., 1999; Hellberg et al., 2001) were
`launched into the market as anti-glaucoma drugs. The IOP-
`lowering effect of latanoprost has been found to be greater
`than that of timolol in a number of clinical comparative
`studies (Alm, 1995; Mishima et al., 1996; Watson et al.,
`1996; Alm et al., 2000). Recently, because of its potent IOP-
`lowering effects and fewer general side effects, latanoprost
`has been used as first-line therapy in many countries,
`including the USA and Japan.
`Prostanoid FP-receptor agonists have potent IOP-low-
`ering efficacy, and effectively no general side effects.
`However, PG-related ocular hypotensive drugs do have
`local side effects, such as pigmentation of the iris (Wistrand
`et al., 1997; Yamamoto and Kitazawa, 1997; Sherwood and
`Brandt, 2001; Netland et al., 2001), palpebra or/and
`periocular skin (Wand et al., 2001), and abnormal eyelash
`growth (trichiasis) (Johnstone, 1997). Furthermore,
`the
`existing PG-related ocular hypotensive drugs,
`including
`latanoprost, do not produce satisfactory IOP control in all
`patients. We therefore tried to find a new candidate as an
`ocular hypotensive drug that exceeds latanoprost in terms of
`IOP-lowering efficacy, and has weaker local side effects.
`In this study,
`in a comparison with latanoprost, we
`examined the binding affinity of AFP-172 for recombinant
`human prostanoid FP receptors, the binding selectivity of
`AFP-172, the IOP-lowering effects of AFP-168 in ocular
`normotensive and laser-induced ocular hypertensive mon-
`keys, the effects of AFP-168 on aqueous humor dynamics in
`ocular normotensive monkeys, and the effects of AFP-172
`on melanogenesis in cultured B16-F0 melanoma cells.
`
`2. Materials and methods
`
`2.1. Materials
`
`AFP-168 and AFP-172 were synthesized at the labora-
`tories of Asahi Glass Co. Ltd (Tokyo, Japan). Latanoprost,
`
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`
`769
`
`2.2.2. Prostanoid DP, EP1, EP2, EP3, IP, and TP receptors
`Binding affinity for the prostanoid DP receptor was
`determined by measuring the inhibition of the adenosine
`diphosphate (ADP)-induced aggregation of rabbit platelets.
`Platelet-rich plasma was prepared from freshly obtained
`rabbit blood, and platelet aggregation was induced by 10 mM
`ADP (Eglen and Whiting, 1989). Binding affinities for the
`prostanoid EP1, EP2, and EP3 receptors were determined by
`measuring muscle contractions in an organ bath. The
`muscles, which were attached to an isometric transducer,
`were maintained at 378C in aerated (95% O2, 5% CO2)
`Krebs – Hensleit solution. For the prostanoid EP1 receptor,
`contraction of the guinea pig ileum was induced by 1 mM
`AFP-172 (Eglen and Whiting, 1989), and the contraction
`induced by AFP-172 was expressed as a percentage of the
`maximal response to 10 mM acetylcholine. For the prosta-
`noid EP2 receptor, contraction of the guinea pig trachea was
`induced by 1 mM carbachol, then inhibited by 1 mM AFP-
`172. For the prostanoid EP3 receptor, twitch contractions of
`the guinea pig vas deferens were induced by electrical
`stimulation (1 min interval, 5 sec trains of pulses at maximal
`voltage; pulse width, 1 msec; frequency, 5 Hz),
`then
`inhibited by AFP-172 (Savage et al., 1993). The binding
`studies for prostanoid IP and TP receptors were performed
`by a contract facility (Cerep, France) with standard binding
`procedures.
`
`2.2.3. Other receptors and transporters
`The binding studies for other receptors and transporters
`listed in Table 3 were performed by another contract facility
`(Daiichikagaku Pure Chemicals Co. Ltd, Toukaimura,
`Japan) with standard binding procedures.
`
`2.3. IOP measurement
`
`For the studies on ocular normotensive monkeys, 10
`adult male cynomolgus monkeys, each weighing 4·7 – 7·4 kg
`(Keari Co. Ltd, Osaka, Japan), were used for the single-
`application experiment, and another 10 adult monkeys, each
`weighing 4·5 – 8·1 kg (Animal Care Co. Ltd, Tokyo, Japan),
`were used for a repeated-application study. Only animals
`with normal eyes (in terms of anterior chamber, angles, IOP,
`ocular media, and optic nerve heads) were included in these
`studies. For the ocular hypertensive monkey study, 12 adult
`cynomolgus monkeys, each weighing 5·2 – 8·1 kg (Keari Co.
`Ltd, Osaka, Japan), were used. Elevated IOP was produced
`in the monkeys according to a previous report (Lee et al.,
`1985), with a minor modification. Briefly, ocular hyperten-
`sion was induced in the left eye by photocoagulating the
`trabecular meshwork using an argon laser (Ultima 2000;
`Coherent-Japan, Tokyo, Japan), applying 150 burns around
`360 degrees twice with a 1-week interval. The present study
`was carried out more than 4 weeks after
`the last
`photocoagulation. If the IOP in the photocoagulated eye
`was not 5 mmHg greater than that in the non-photocoagu-
`lated fellow eye, the animal was not used for this study.
`
`Before we measured IOP, all monkeys were trained for
`restraint in a monkey chair (CL-4535; Primate Products,
`Miami, FL, USA) and for measurements of IOP. For IOP
`measurements, the monkey was kept in a sitting position in
`the monkey chair, and the IOP was measured with a
`pneumatonograph (Model Classic 30R; Soran Ophthalmic,
`Jacksonville, FL, USA) without any general anesthesia or
`sedation. For corneal anesthesia, 0·4% oxybuprocain
`solution (Santen Pharmaceutical Co. Ltd, Osaka, Japan)
`was topically applied prior to IOP measurement. Drugs
`(20 ml) were applied to one eye in each monkey, with the
`contralateral eye remaining untreated. In the normotensive
`monkey study (single application), IOP was measured just
`before and at 2, 4, 6, 8, and 24 hr after drug application. In
`the normotensive monkey study (repeated application), IOP
`was measured just before and at 4, 6, 8, and 24 hr on days 1,
`3, and 5 of the drug-application protocol. In the ocular
`hypertensive monkey study, drugs (20 ml) were applied to
`the left eye with elevated IOP in each monkey, at 9 – 10 a.m.
`IOP was measured just before and at 2, 4, 6, 8, 12, and 24 hr
`after drug application. A masked observer (F. N.) measured
`IOP in all experiments. A crossover design was applied to
`these studies; each animal contributed to all groups.
`Prior to aqueous humor dynamics studies, IOP responses
`to AFP-168 (4 £ 5 ml of 0·005%), timolol (2 ml containing
`5 mg timolol) and PGF2a-isopropyl ester (2 £ 5 ml of
`
`0·02%) were confirmed in monkeys anaesthetized with
`ketamine HCl (10 mg kg21, i.m.) using a minified Gold-
`mann applanation tonometer (Kaufman and Davis, 1980).
`
`2.4. Aqueous humor dynamics assays
`
`For the aqueous humor dynamics study, we used a total
`of 28 adult male and female cynomolgus monkeys, each
`weighing 2·5 – 6·0 kg (the majority from Covance Inc.,
`Madison, WI, USA; two from the Coulston Foundation,
`Almagordo, NM, USA; one born at
`the University of
`Wisconsin, Madison, WI, USA). Aqueous humor formation
`(AHF) was measured in eight monkeys (i.m. ketamine
`anesthesia, 10 mg kg21 initial supplemented by 5 mg kg21
`as needed) by fluorophotometry (Gabelt et al., 1994); total
`outflow facility was measured in the same eight monkeys
`plus four additional ones (i.m. ketamine (10 mg kg21)
`(15 mg kg21
`followed by i.v. pentobarbital
`initial,
`supplemented with 5 – 10 mg kg21 as needed)) using two-
`level constant pressure perfusion (Ba´ra´ny, 1964; Gabelt
`et al., 1991); uveoscleral outflow and trabecular outflow
`were measured in the same 12 monkeys by means of isotope
`accumulation (Sperber and Bill, 1984; Gabelt et al., 2003)
`prior to outflow facility measurements on the same day.
`Only animals with normal eyes (in terms of anterior
`chamber, angles, IOP, ocular media, and optic nerve
`heads) were used. In these studies, 4 £ 5 ml of 0·005%
`AFP-168 was applied once daily for 3 – 5 days; 5 mg
`timolol (2 ml of timolol-gel forming solution diluted with
`saline) was applied once daily for 3 days; 2 £ 5 ml of 0·02%
`
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`
`PGF2a-isopropyl ester was applied twice daily for 4 or 5
`days to eight monkeys different from the previous 12;
`100 mg/10 ml pilocarpine HCl was applied intracamerally to
`yet another group of eight different monkeys.
`The rate of AHF was determined in ketamine anaes-
`thetized monkeys using a Coherent scanning ocular
`fluorophotometer (Fluorotron Master, Palo Alto, CA,
`USA). Fluorescein (10% fluorescite for injection, Alcon
`Laboratories, Inc., Fort Worth, TX, USA) was administered
`topically on the afternoon preceding the fluorophotometry
`experiments (usually five 2 ml drops of 5% solution diluted
`with 0·1 M sodium phosphate buffer, pH 7·4) 5 min after
`one drop of 0·5% proparacaine hydrochloride (Bausch and
`Lomb Pharmaceuticals, Inc., Tampa, FL, USA). This kept
`the corneal fluorescein concentration at 200 ng ml21 or
`more during the measurement period. On the experimental
`day, scans were recorded in each eye every hour for 5 hr.
`Isotopic determination of AHF was performed in
`pentobarbital anaesthetized monkeys beginning 3 hr after
`the last dose of AFP-168 (fourth or fifth dose of once daily
`treatments) or PGF2a-isopropyl ester (seventh or ninth dose
`twice daily treatments) by circulating [125I] or
`of
`[131I]monkey albumin through the anterior chamber and
`measuring the dilution of label with newly formed aqueous
`(by counting a portion of the circuit every 5 min over a 2 hr
`period in a well detector/multichannel analyzer system,
`(ASA-100 NaI multichannel analyzer, Genie-2000 Spec-
`troscopy Software, Canberra Industries, Inc., Meriden, CT,
`USA). Flow through the trabecular meshwork was deter-
`mined by taking blood samples every 10 min from a femoral
`artery and counting them in a gamma counter (COBRA
`model D5003, Packard Instrument Co., Downers Grove, IL,
`USA). Uveoscleral outflow was calculated as the difference
`between AHF and trabecular outflow.
`Total outflow facility was determined in pentobarbital
`anaesthetized monkeys by two-level constant pressure
`perfusion of the anterior chamber with Ba´ra´ny’s mock
`aqueous humor, correcting for the internal resistance of the
`perfusion apparatus as appropriate (Ba´ra´ny, 1964). For
`AFP-168 and PGF2a-isopropyl ester studies, total outflow
`facility was determined for approximately 45 min at the
`conclusion of isotope studies and were completed within
`hours 5 – 6 after the fourth or fifth dose of once daily
`treatments with AFP-168 or after the seventh or ninth dose
`of twice daily treatments with PGF2a-isopropyl ester. For
`pilocarpine,
`the anterior chambers of both eyes were
`cannulated with a branched needle (with one branch
`connected to a reservoir and the other to a pressure
`transducer). Baseline facility measurements were taken for
`35 – 45 min. Following baseline measurements, a bolus
`injection of 10 ml containing 100 mg pilocarpine HCl was
`made into the inflow tubing of one eye, Ba´ra´ny’s into the
`opposite eye tubing. Following a 5-min wash in period
`with the reservoirs elevated, cold air was blown on the
`corneas to mix the anterior chamber contents by convection.
`Post-drug outflow facility was then determined for another
`
`35 – 45 min. Facility was calculated by successive averaging
`(Ba´ra´ny, 1964).
`
`2.5. Melanogenesis assay
`
`B16-F0 melanoma cells were purchased from Dainippon
`Pharmaceutical Co. Ltd (Osaka, Japan). Melanin contents
`were measured as in previous reports (Siegrist and Eberle,
`1986; Kosano et al., 1995). Briefly, fourth- or fifth-passage
`B16-F0 melanoma cells (ATCC No. CRL6322; 5 £ 103
`cells/well) were seeded in a 24-well plate (Asahi Techno
`Glass Corp., Funabashi, Japan) in culture medium (Dulbec-
`co’s modified Eagles medium containing 10% fetal bovine
`serum and 50 mg ml21 gentamicin). Starting the day after
`seeding,
`the cells were cultured for 4 days in culture
`medium with or without drug (AFP-172 or PhXA85).
`The culture medium was collected, and the cells dissolved
`in lysis buffer (0·1N NaOH solution containing 10% Triton
`X-100) with 10-sec sonication. The melanin content of the
`medium and cell lysates was measured by absorbance at 490
`and 415 nm, respectively.
`
`2.6. Statistical analysis
`
`The following statistical analyses were performed. For
`the IOP measurement study, the maximal reduction in IOP
`was used for statistical analysis. A Student’s t-test was
`carried out following an F-test. Dunnett’s test and the
`Tukey – Kramer test were carried out following a one-way
`analysis of variance. For the aqueous humor dynamics
`study, a two-tailed paired t-test was carried out on the values
`obtained for treated/control ratios. p , 0·05 was taken as
`statistically significant.
`
`3. Results
`
`3.1. Receptor-binding assays
`
`3.1.1. Prostanoid FP receptor
`Prior to the FP binding study, we confirmed the
`expression of prostanoid FP receptors in the clonal cells.
`Immunofluorescence microscopy using an anti-FP receptor
`antibody, examination of the accumulation of inositol
`phosphates induced by fluprostenol, and radioligand-bind-
`ing competition curve analysis of PGF2a to [3H]PGF2a
`revealed that the clonal cell line used in this study did
`indeed express human FP receptors (data not shown). AFP-
`172 showed a high affinity for the human prostanoid FP
`receptor, with an EC50 of 0·53 nM. The affinity of AFP-172
`was 12 times that of PhXA85 and 1700 times that of
`unoprostone (Table 1). AFP-172 produced full displacement
`of specific [3H]PGF2a binding and competed in a
`manner consistent with a simple bimolecular reaction
`(Hill slope , 1), as did PhXA85 and unoprostone.
`
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`Y. Takagi et al. / Experimental Eye Research 78 (2004) 767–776
`
`771
`
`Table 1
`Fifty percent inhibition concentration (IC50) and affinities of AFP-172,
`PhXA85, and unoprostone for the human prostanoid FP receptor
`
`Table 3
`Inhibitory effects of AFP-172 on various receptors and transporters
`
`Compounds
`
`IC50 (nM)
`
`AFP-172
`PhXA85
`Unoprostone
`
`0·53 ^ 0·18
`6·3 ^ 1·3
`900 ^ 55
`
`Ki
`(nM)
`
`0·4
`4·7
`680
`
`Potency ratio
`
`(Ki value of AFP-172 ¼ 1)
`
`1
`12
`1700
`
`Affinity for the prostanoid FP receptor was determined by competitive
`binding to radiolabelled prostaglandin F2a. The 50% inhibition concen-
`tration (IC50) and Ki values were calculated from the following equations:
`B ¼ non-specific binding þ ðtotal binding 2 non-specific bindingÞ
`1 þ 10log½CŠ2logðIC50Þ
`Ki ¼
`IC50
`1 þ ½radioligandŠ=Kd
`Data represent the mean ^ S.E.M. from far to five experiments; B; amount
`of radioligand binding; C; concentration of test compound; Ki; equilibrium
`inhibition constant; Kd; equilibrium dissociation constant.
`
`;
`
`;
`
`3.1.2. Prostanoid DP, EP1, EP2, EP3, IP, and TP receptors
`The inhibitory or stimulatory effects of AFP-172 on
`the binding or pharmacological responses of prostanoid
`receptors are shown in Table 2. AFP-172 at 1 mM did not show
`evidence of inhibitory or stimulatory effects on any receptor
`binding, except that of the prostanoid EP3 receptor. The IC50
`of AFP-172 for the prostanoid EP3 receptor was 67 nM, and
`the binding affinity of AFP-172 was 126 times weaker for this
`receptor than for the prostanoid FP receptor (see Tables 1 and
`2). PGD2 (DP), PGE2 (EP1), butaprost (EP2), 17-phenyl
`trinol-PGE2 (EP3), iloprost (IP) and U44069 (TP) were used
`as positive controls in each experiment.
`
`3.1.3. Other receptors and transporters
`As shown in Table 3, the inhibitory effects of AFP-172 at
`1 mM on a variety of receptor and transporter bindings were
`all less than 15%.
`
`Table 2
`functional activities of AFP-172 to
`Receptor-binding affinities or
`prostanoid DP, EP1, EP2, EP3, IP, and TP receptors
`
`Prostanoid receptors Assays
`
`EC50 or IC50 (nM)
`
`DP
`
`EP1
`
`EP2
`
`EP3
`
`IP
`
`TP
`
`Inhibition of ADP-induced
`aggregation in
`rabbit platelets
`Contraction in
`guinea pig ileum
`Inhibition of carbachol-induced
`contraction in guinea
`pig trachea muscle
`Inhibition of electrical
`stimulation-induced
`contraction in guinea
`pig vas deferens
`Inhibition of [3H]iloprost
`binding in human platelets
`Inhibition of [3H]SQ29548
`binding in human platelet
`
`.1000
`
`.1000
`
`.1000
`
`67
`
`.1000
`
`.1000
`
`Data are averaged from two or four experiments.
`
`Receptors/
`transporters
`
`Radioligand/assay
`
`Inhibition (%)
`(1 mM AFP-172)
`
`Adenosine A1
`Adenosine A2a
`Adrenergic a1
`(non-selective)
`Adrenergic a2
`(non-selective)
`Adrenergic b1
`Angiotensin AT1
`Benzodiazepine
`Bradykinin B2
`Cannabinoid CB1
`Cannabinoid CB2
`Cholecystokinin
`CCKA
`Dopamine D1
`Dopamine D2
`Dopamine
`transporter
`Endothelin ETA
`g-Aminobutyric
`acid (GABA)A
`Glutamate
`(non-selective)
`Histamine H1
`Melanocortin MC4
`
`Muscarinic
`(non-selective)
`Neurokinin NK1
`Neuropeptide Y1
`Neuropeptide Y2
`Norepinephrine
`transporter
`Nicotinic Ni
`Opiate
`(non-selective)
`Orphanin ORL1
`Serotonin 5HT1
`(non-selective)
`Serotonin
`transporter
`Sigma (s)
`Vasopressin V1b
`Vasopressin V2
`
`[3H]dipropylcyclopentylxanthine , 15
`[3H]CGS21680
`, 15
`[3H]Prazosin
`, 15
`
`[3H]RX821002
`
`[3H]CGP12177
`[125I]Angiotensin II
`[3H]Flunitrazepam
`[3H]Bradykinin
`[3H]CP55940
`[3H]WIN55212-2
`[3H]L-364718
`
`[3H]SCH23390
`[3H]Spiperone
`[3H]WIN35428
`
`[125I]Endothelin-1
`[3H]Flunitrazepam
`
`[3H]Glutamic acid
`
`[3H]Pyrilamine
`[3H]4-Norleucine,
`7-D-phenylalanine-a-
`melanocyte stimulating
`hormone
`[3H]Quinuclidinyl benzilate
`
`[3H]Substance P
`[125I]Peptide YY
`[125I]Peptide YY
`[3H]Nisoxetine
`
`[3H]Epibatidine
`[3H]Naloxone
`
`[3H]Nociceptin
`[3H]Serotonin
`
`[3H]Imipramine
`
`[3H]1,3,-Di-o-tolylguanidine
`[3H]Arg-Vasopressin
`[3H]Arg-Vasopressin
`
`, 15
`
`, 15
`, 15
`, 15
`, 15
`, 15
`, 15
`, 15
`
`, 15
`, 15
`, 15
`
`, 15
`, 15
`
`, 15
`
`, 15
`, 15
`
`, 15
`
`, 15
`, 15
`, 15
`, 15
`
`, 15
`, 15
`
`, 15
`, 15
`
`, 15
`
`, 15
`, 15
`, 15
`
`Data are averaged from two experiments.
`
`3.2. Effects of a single application of AFP-168 on
`intraocular pressure in conscious normotensive monkeys
`
`Fig. 2 shows the maximum IOP reductions of a single
`application of either AFP-168 or latanoprost in conscious
`ocular normotensive monkeys. The maximal IOP reductions
`with AFP-168 (0·00002 – 0·0025%) were dose-dependent,
`and significance was reached at doses of 0·0005 and
`0·0025%. The maximal IOP reduction seen with AFP-168
`
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`

`

`772
`
`Y. Takagi et al. / Experimental Eye Research 78 (2004) 767–776
`
`in Fig. 3), AFP-168 at 0·0025 and 0·005% did significantly
`reduce IOP at each trough time-point from 0 hr on day 3
`(24 hr on day 2) through 0 hr on day 6 (24 hr on day 5). Even
`with AFP-168 at 0·001%, an IOP reduction at the trough
`time-point was observed after 0 hr on day 3 (24 hr on day 2),
`and this effect was significant on and after 0 hr on day 4
`(24 hr on day 3). Fig. 4 shows the daily maximal IOP
`reductions induced by AFP-168 and latanoprost. The daily
`maximal reductions induced by all doses of AFP-168 and
`latanoprost increased after day 1, but statistically significant
`increases vs. day 1 were observed only with AFP-168 at
`0·0025 and 0·005%. The maximal IOP reductions achieved
`with AFP-168 at 0·0025 (mean ^ S.E.M.: 4·7 ^ 0·2 and
`5·0 ^ 0·2 mmHg at day 3 and 5, respectively, n ¼ 10) and
`0·005% (6·1 ^ 0·2 and 5·8 ^ 0·3 mmHg at days 3 and 5,
`respectively, n ¼ 10) were greater than that achieved with
`latanoprost at 0·005% (4·2 ^ 0·3 and 4·2 ^ 0·4 mmHg at
`day 3 and 5, respectively, n ¼ 10).
`
`3.4. Effects of a single application of AFP-168 on
`intraocular pressure in conscious laser-induced ocular
`hypertensive monkeys
`
`Fig. 5 shows the maximal IOP reductions induced by
`applications of AFP-168 and latanoprost in laser-induced
`ocular hypertensive monkeys. The mean IOP values at the
`pre-treatment time in all groups were in the range 35·8 –
`38·7 mmHg, and there was no statistically significant
`difference among the groups. AFP-168 (0·00002 –
`0·0025%) induced a dose-dependent IOP reduction, the
`maximal reduction at 0·0025 and 0·005% (11·8 and
`10·5 mmHg, respectively,) being statistically significant
`compared to that in the vehicle-treated eye (6·2 mmHg).
`The effect of AFP-168 on laser-induced ocular hypertension
`seemed to peak at 0·0025%,
`then almost plateau. The
`maximal IOP reduction (9·5 mmHg) seen after application
`of latanoprost tended to be greater than that in the vehicle-
`treated eye, but with borderline significance ðp ¼ 0·057Þ·
`
`Fig. 2. Effects of a single application of AFP-168 or latanoprost (maximal
`reduction in intraocular pressure (IOP)) in conscious ocular normotensive
`monkeys. Data represent the mean ^ S.E.M. for 12 animals. **p , 0·01 vs.
`vehicle (Tukey– Kramer test).
`
`at 0·0025% was over 3 mmHg, greater than that induced by
`latanoprost at 0·005% (which was approximately 2 mmHg,
`statistically significant compared to that in the vehicle-
`treated eye). The potency of AFP-168 at 0·0005% was
`almost equal to that of latanoprost at 0·005%.
`
`3.3. Effects of repeated applications of AFP-168 on
`intraocular pressure in conscious normotensive monkeys
`
`Fig. 3 shows time – course data for the effects of repeated
`applications of AFP-168 or latanoprost on IOP in ocular
`normotensive monkeys. Mean IOP values at
`the pre-
`treatment time in all groups were within a narrow range
`(21·5 – 22·1 mmHg), and there was no statistically signifi-
`cant difference among the groups. All doses of AFP-168
`reduced IOP at 4, 6, and 8 hr after their application on days
`1, 3, and 5. Latanoprost at 0·005% caused significant IOP
`reductions at 4, 6, and 8 hr after its application on days 1, 3,
`and 5. While latanoprost did not reduce IOP at the trough
`time-point (24 hr after its application; i.e. 0 hr on days 2 – 6
`
`Fig. 3. Effects of once daily application of AFP-168 or latanoprost on intraocular pressure (IOP) in conscious ocular normotensive monkeys. IOP change was
`calculated from time 0 on day 1. Drugs were instilled at 10:30 a.m. on each day from day 1 to day 5. Data represent the mean ^ S.E.M. for 10 animals.
`*p , 0·05; **p , 0·01 vs. vehicle (Dunnett’s multiple-range test).
`
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`

`Y. Takagi et al. / Experimental Eye Research 78 (2004) 767–776
`
`773
`
`uveoscleral outflow (Fu) by 65%. In this experiment, the
`FTB value is assumed to represent outflow through the
`trabecular meshwork (conventional outflow).
`Twice daily application of PGF2a-isopropyl ester at
`0·02% for 4 or 5 days did not affect AHF, but significantly
`decreased FTB by 59% and tended to increase Fu (although
`not significantly due to variability in the data).
`Once daily application of AFP-168 at 0·005% for 4 or 5
`days significantly increased total outflow facility by 33%
`compared to contralateral control eyes, but twice daily
`application of PGF2a-isopropyl ester at 0·02% for 4 or 5
`days did not. However, the control eye values in AFP-168
`animals tended to be lower than those of the control eyes for
`PGF2a-isopropyl ester and pilocarpine; the absolute facility
`values for AFP-168-treated and PGF2a-isopropyl ester-
`treated eyes were almost exactly the same. Intracameral
`application of pilocarpine significantly increased total
`outflow facility by 184%.
`
`3.6. Effect of AFP-168 on melanogenesis in B16-F0
`melanoma cells
`
`Fig. 6 shows the effects of AFP-168 and latanoprost on
`melanogenesis in cultured B16-F0 melanoma cells. In these
`cells, a-melanocyte stimulating hormone increased the
`melanin content in both the culture medium and cell bodies
`(data not shown). AFP-172 at 1 – 100 mM and PhXA85 at 1
`and 10 mM did not increase the melanin content in either the
`culture medium or cell bodies. PhXA85 at 100 mM
`significantly increased the melanin content above that
`seen with vehicle.
`
`4. Discussion
`
`AFP-172, a carboxylic acid of AFP-168, showed a potent
`affinity for the prostanoid FP receptor, and its affinity was
`greater than that of either PhXA85, a carboxylic acid of
`latanoprost, or unoprostone. The binding affinity of AFP-
`172 was 126 times higher for the prostanoid FP receptor
`than for EP3 receptor. On the other hand, AFP-172 showed
`no or less affinity for other prostanoid receptors (DP, EP1,
`EP2, IP and TP) and other receptors and transporters. AFP-
`172 also contracted the isolated cat iris sphincter muscle and
`its efficacy was greater than that of PhXA85 (Nakajima
`et al., 2003). These findings indicate that the binding profile
`of AFP-172, like that of PhXA85, is that of a selective
`agonist for the prostanoid FP receptor (Stjernschantz et al.,
`1995). Since the structural characteristics of AFP-168 and
`AFP-172 involve two fluorine atoms in position 15 in the b-
`chain of the prostaglandin structure, its fluorine atoms may
`bestow a more potent affinity for the prostanoid FP receptor
`than that exhibited by other PG derivatives.
`We demonstrated ocular hypotensive effects of AFP-168
`in ocular normotensive
`and laser-induced ocular
`hypertensive monkeys. A single application of AFP-168
`
`Fig. 4. Effects of a once daily application of AFP-168 or latanoprost
`(maximal reduction in intraocular pressure (IOP)) in conscious ocular
`normotensive monkeys. Drugs were instilled at 10:30 a.m. on each day
`from day 1 to day 5. Data represent the mean ^ S.E.M. for 10 animals.
`*p , 0·05 vs. day 1 (Dunnett’s multiple-range test).
`
`The reduced IOP had returned almost to the initial value at
`24 hr after drug application.
`
`3.5. Effect of AFP-168 on aqueous humor dynamics in
`anaesthetized ocular normotensive monkeys
`
`timolol-gel
`Table 4 shows the effects of AFP-168,
`forming solution, PGF2a-isopropyl ester, and pilocarpine on
`each parameter relating to aqueous humor dynamics in
`ocular normotensive monkeys.
`By fluorophotometry, once daily application of AFP-168
`at 0·005% for 3 days did not affect AHF. On the other hand,
`once daily application of timolol for 3 days significantly
`decreased AHF